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DEPARTMENT OF IMMUNOLOGY


STAFF

Richard J. Ulevitch, Ph.D. Member and Chairman
Carole L. Banka, Ph.D. Assistant Member
Richard Boismenu, Ph.D. Assistant Member
Gary M. Bokoch, Ph.D. Associate Member
Linda M. Bradley, Ph.D. Assistant Member
Dennis R. Burton, Ph.D.* Member
David A. Cheresh, Ph.D. Associate Member
Robert W. Chesnut, Ph.D. Adjunct Associate Member
Charles G. Cochrane, M.D. Member
Neil R. Cooper, M.D. Member
Linda K. Curtiss, Ph.D. Associate Member
Frank J. Dixon, M.D. Member, Director Emeritus, TSRI
Thomas S. Edgington, M.D. Member
Sao-Tah Fan, Ph.D. Assistant Member
Ann J. Feeney, Ph.D. Associate Member
Lily Feng, M.D. Assistant Member
Robert I. Fox, M.D., Ph.D. Adjunct Assistant Member
Nicholas R.J. Gascoigne, Ph.D. Associate Member
Elizabeth D. Getzoff, Ph.D.* Associate Member
Peter Ghazal, Ph.D.** Associate Member
Howard Gray, Ph.D. Adjunct Member
Jiahuai Han, Ph.D. Associate Member
Wendy L. Havran, Ph.D. Associate Member
Bessie Pei-Hsi Huang, Ph.D.* Associate Member
Tony E. Hugli, Ph.D. Member
Janis H. Jackson, M.D. Assistant Member
Jonathan G. Kaye, Ph.D. Associate Member
Norman R. Klinman, M.D., Ph.D. Member
Ulla Gissi Knaus, Ph.D. Assistant Member
Dwight H. Kono, M.D. Assistant Member
Vladimir V. Kravchenko, Ph.D. Assistant Member
Jiing-Dwan Lee, Ph.D. Assistant Member
David D. Lo, M.D., Ph.D. Associate Member
Nigel Mackman, Ph.D. Associate Member
David L. McElligott, Ph.D. Assistant Member
Anthony M. Montgomery, Ph.D. Assistant Member
Edward L. Morgan, Ph.D.*** Associate Member, Sidney Kimmel Cancer Center, La Jolla, CA
Donald E. Mosier, M.D., Ph.D. Member
Barbara M. Mueller, Ph.D. Assistant Member
Robert M. Nakamura, M.D.**** Member
Glen R. Nemerow, Ph.D. Associate Member
Anne M. O'Rourke, Ph.D. Assistant Member
Paul Parren, Ph.D. Assistant Member
Per A. Peterson, M.D., Ph.D. Adjunct Member
Mary Laurie Phillips, Ph.D. Adjunct Assistant Member
Eric R. Prossnitz, Ph.D. Assistant Member
Ralph A. Reisfeld, Ph.D. Member
Wolfram Ruf, M.D. Associate Member
Daniel R. Salomon, M.D. Adjunct Assistant Member
Nora Sarvetnick, Ph.D. Associate Member
David D. Schlaepfer, Ph.D. Assistant Member
Ingrid U. Schraufstatter, M.D. Assistant Member
Alessandro Sette, Ph.D. Adjunct Assistant Member
Linda A. Sherman, Ph.D. Associate Member
Jonathan Sprent, M.D. Member
Charles D. Surh, Ph.D. Assistant Member
Luc Teyton, M.D., Ph.D. Associate Member
Argyrios N. Theofilopoulos, M.D. Member
Marilyn Thoman, Ph.D.*** Assistant Member, Sidney Kimmel Cancer Center, La Jolla, CA
Peter S. Tobias, Ph.D. Associate Member
Bruce E. Torbett, Ph.D. Assistant Member
Ernest S. Tucker III, M.D.***** Member
Susan R. Webb, Ph.D. Associate Member
William O. Weigle, Ph.D. Member
Curtis B. Wilson, M.D. Member
Richard D. Ye, M.D., Ph.D. Associate Member


SENIOR RESEARCH ASSOCIATES

Syed Munir Alam, Ph.D.
Ana Angulo-Aguado, Ph.D.
William A. Boisvert, Ph.D.
Peter C. Brooks, Ph.D.*** University of Southern California, Los Angeles, CA
Mary Ann Campbell, Ph.D.*** La Jolla Pharmaceuticals, La Jolla, CA
Tsung-Hsien Chuang, Ph.D.
Surganghanie Dharmawardhane, Ph.D.
Pam Daffern, Ph.D.
Joanna Davies, Ph.D.
Craig Dickenson, Ph.D.
Henrik Ditzel, Ph.D.
Steven R. Duncan, M.D.
Julia A. Ember, Ph.D.
Colleen Fearns, Ph.D.
Shuang Huang, Ph.D.
Richard Klemke, Ph.D.
Tania Massamiri, Ph.D.+
Sally Orr, Ph.D.
Graham C. Parry, Ph.D.
Gaston Picchio, Ph.D.
M. Germana Sanna, Ph.D.
Bee-Cheng Sim, Ph.D.
David Tough, Ph.D.*** Edward Jenner Institute, Compton, England
R. Anthony Williamson, Ph.D.


RESEARCH ASSOCIATES

Isabelle Atencio, Ph.D.+
Balaji Balasa, Ph.D.
Dimitrios Balomenos, Ph.D.+
Valerie Benard, Ph.D.
Jan Bohuslav, Ph.D.
Nancy Boudreau, Ph.D.*** Virginia Commonwealth University, Richmond, VA
Ingo Brink, M.D.*** University of Freiburg, Freiburg, Germany
Darren Browning, Ph.D.
Zeling Cai, M.D., Ph.D.
Ta-Hsiang Chao, Ph.D.
Shi Zhong Chen, Ph.D.
Weiping Chen, M.D., Ph.D.
Yaping Chen, Ph.D.
Efthalia Chronopoulou, Ph.D.
Mary Crowley, Ph.D.
Robert H. Daniels, Ph.D.
Fernando Donate, Ph.D.
Torsten Dreier, Ph.D.
Brian P. Eliceiri, Ph.D.
Jonathan H. Erlich, Ph.D.
Marika Falcone, Ph.D.
Wolfgang Fischer, Ph.D.+
Paola Fisicaro, Ph.D.
Scott Gallichan, Ph.D.
Gabriella Garcia, Ph.D.
Julie Ann Gegner, Ph.D.
Jacqueline Glynn, Ph.D.*** Chiron, La Jolla, CA
Lothar Goretzki, Ph.D.
Carl Haarstad, Ph.D.
Javier Hernandez-Gil, Ph.D.
Robert C. Hoch, Ph.D.
Matthew H. Hsu, Ph.D.
Huang Huang, Ph.D.
Hui-Y Huang, Ph.D.
Inkyu Hwang, Ph.D.
Mark A. Jagels, Ph.D.
Yong Jiang, M.D.
Michael K. Jones, Ph.D.*** University of California, Irvine, CA
Archana Kapoor-Munchi, Ph.D.+
Yutaka Kato, M.D., Ph.D.
Curtis R. Kelly, Ph.D.
Cecelia M. King, Ph.D.
Charles King, Ph.D.
Hidehiro Kishimoto, M.D., Ph.D.
Gregory H. Kline, Ph.D.
Nobuhiko Kubo, Ph.D.
Lisa Dimolfotto Landon, Ph.D.
James Le Blanc, Ph.D.
Dong Sup Lee, M.D.
Jie Leng, Ph.D.
Erguang Li, Ph.D.
Li Li, Ph.D.
Cheng Liu, M.D., Ph.D.
Holger Lode, M.D.
Joseph Lustgarten, Ph.D.
Marion Marsh, Ph.D.
Toshiaki Maruyama, M.D., Ph.D.
Marielena McGuire, Ph.D.
Denise McKinney, Ph.D.
David John Morgan, Ph.D.
Yohei Miyagi, M.D., Ph.D.*** Yokohama University School of Medicine, Yokohama, Japan
Thomas M. Mohler, M.D.
Regular Mueller, Ph.D.+
Bertrand Nadel, Ph.D.
Naushaba Nayeem, Ph.D.
C. Thomas Nugent, Ph.D.
Ilka Ott, M.D., Ph.D.
Zhinxing Pan, M.D., Ph.D.*** La Jolla Institute for Allergy and Immunology, La Jolla, CA
James Pancook, Ph.D.+
Mary Pauza, Ph.D.
Eric Petitclerc, Ph.D.*** University of Southern California, Los Angeles CA
Philippe H. Pfeifer, Ph.D.*** BMA Biomedicals, Augst, Switzerland
Pascal R. Poignard, Ph.D.
Stella Redpath, Ph.D.
Helen Sabzevari, Ph.D.
Luraynne Sanders, Ph.D.
Urban Scheuring, M.D.
Deborah E. Schiff, M.D.
Jan Schimke, M.D.
Li Karine Schiotz, Ph.D.
Hui Shao, Ph.D.
Steven A. Silletti, Ph.D.
Mark Slifka, Ph.D.
Staffan Stromblad, Ph.D+
Dwayne G. Stupack, Ph.D.
Naoyuki Sugano, Ph.D.+
Siquan Sun, Ph.D.
Hiroshi Takamori, M.D., Ph.D.
Richard Tapping, Ph.D.
Sharon Tracy, Ph.D.+
Kimberly Victor, Ph.D.
Silvia Vidal, Ph.D.
Julia Voice, Ph.D.
Daniel Von Seggern, Ph.D.
Vincent R. Walker, Ph.D.
Kena Wang, Ph.D.
Mei-Ying Wang, Ph.D.
Meng Wang, M.D.
Catherine Werts-Larzilliere, Ph.D.
Karen Whitmer, Ph.D.*** Stanford University, Palo Alto CA
Yiyang Xia, M.D.
Rong Xiang, M.D., Ph.D.
Li Xu, Ph.D.
Jin Yao, M.D.
Mayra Yebra, Ph.D.
Tomasz Zal, Ph.D.
Frank T. Zenke, Ph.D.
Ming Zhao, M.D.
Qi Zhong, Ph.D.
Kemin Zhou, Ph.D.


SCIENTIFIC ASSOCIATES

Bonnie M. Bradt
John C. Mathison, Ph.D.
Jennifer Price
Susan D. Revak
Carole G. Romball


VISITING INVESTIGATORS

Martin J. Glennie, Ph.D. University of Southampton, Southampton, England
Tadhashi Yamamoto, M.D. Niigata University School of Medicine, Niigata, Japan
* Joint appointment in Department of Molecular Biology
** Joint appointment in Department of Neuropharmacology
*** Appointment completed; new location shown
**** Joint appointment in Department of Molecular and Experimental Medicine
***** Joint appointment in Scripps Reference Laboratory
+ Appointment completed

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Chairman's Overview

Richard J. Ulevitch, Ph.D.

One strength of the faculty of the Department of Immunology is being able to recognize great opportunities disguised as what others perceive as insoluble scientific problems. This approach to research is reflected by the accomplishments of the scientific staff of the department. We can look to many important achievements as shown by peer recognition. Publication of seminal findings, remarkable success in obtaining research funding, invitations to organize and speak at outstanding scientific meetings, and recruitment of the best postdoctoral fellows characterize the past year.

Our postdoctoral fellows deserve a special mention. They are the largest group of scientists--currently more than 100--within the department. The group includes trainees from the United States and from more than a dozen European and Asian countries. The fellows not only contribute to the scientific productivity and environment but also provide a unique opportunity for all of us to have colleagues with diverse cultural and ethnic backgrounds.

Among the notable achievements of this past year are publications documenting the research of our members that promises to provide new approaches to treat human disease. Research performed in the laboratory of Glen Nemerow has provided insights into how adenoviruses, important viral vectors for gene therapy, interact with cell membranes. The Nemerow group, together with members of the Cheresh laboratory, first showed how specific members of the integrin family promote viral uptake. Using information gained from these pioneering studies, this group has now shown how delivery of an antiviral ribozyme can inhibit processes essential for the proliferation of Epstein-Barr virus. This virus is thought to be responsible for several human malignant neoplasms and lymphoproliferative disease in immunocompromised patients. The results of these efforts, including demonstration of the in vivo efficacy of this approach, are described in a 1997 article in Proceedings of the National Academy of Sciences of the United States of America.

Dennis Burton and his colleagues have recently addressed issues related to understanding the structure of the prion protein. This work, done in collaboration with S.B. Prusiner, University of California, San Francisco, has been led by R.A. Williamson, with contributions from other members of the Burton laboratory. Prion diseases produce degenerative changes in the CNS by a unique mechanism that involves infectious prion particles. Although the exact nature of this process is not understood, apparently the fundamental lesion involves conversion of the cellular prion protein to an isoform that produces the disease. Antibodies directed to a range of conformational and linear epitopes are being used as probes to develop new structure-function relationships for prions. Results of these pioneering studies are described in another 1997 article in the Proceedings of the National Academy of Sciences of the United States of America.

Luc Teyton, a recent recruit, provides new strength to the department in structure-function relationships of essential receptors on immune system cells. In the past year, he has provided us with new information that markedly broadens our understanding of the function of T-cell receptors. The description in the journal Science of the crystal structure of a T-cell receptor heterodimer was the result of a collaborative effort between the Teyton group and the laboratory of Ian Wilson, Department of Molecular Biology. This seminal work provides a molecular description that will aid in understanding how the T-cell receptor functions in regulating various aspects of the immune response.

Appearing in the journal Nature are data from the Teyton laboratory that provide new insights into how another cell-surface protein, CD8, enhances formation of a stable complex consisting of the T-cell receptor and MHC class I molecules. This work reflects the ongoing commitment in this department to investigate various aspects of the immune system to acquire a full understanding of functional immune responses. In addition to providing new scientific information, this research on CD8, performed in collaboration with the Wilson laboratory, is an example of how the unique scientific environment of TSRI facilitates collaborations that would otherwise be difficult to put into practice.

I also wish to highlight several accomplishments that reflect the prominence that scientists in this department have achieved in the field of intracellular signaling. Work from the laboratory of Gary Bokoch continues to provide new information about small GTP-binding proteins and intracellular kinase cascades. Dr. Bokoch, together with Sandy Schmid, Department of Cell Biology, and other members of the Schmid laboratory, described an important role for small GTP-binding proteins in receptor-mediated apoptosis. In other work, reported in Science, the Bokoch group discovered an important role for a member of the p21-activated kinase family in apoptosis. Jiahuia Han, Shuang Huang, and their colleagues provided evidence in an article published in Immunity for an important role for the intracellular kinase MKK6b in apoptosis. Both of these papers provide seminal findings that will help us understand how programmed cell death is regulated.

Finally, in studies described in Nature, J. Han and R.J. Ulevitch found that a member of the myocyte-enhancing-factor family of transcription factors is an important downstream target of the MAP kinase known as p38. These data offer new insights on regulation of gene expression during inflammation.

Just as scientific progress is marked by transitions from one state of knowledge to another, the makeup of any research department also undergoes change. During the past several years, we have added many new staff members. The most recent addition is Philippe Gallay, who will implement a research program focused on the molecular mechanisms of HIV infection of mammalian cells. Finally, I want to note one additional change in the Department of Immunology and in TSRI: William Weigle, who has been a member of the staff of TSRI since 1961, announced his retirement. Bill Weigle, as he is known to his colleagues, leaves a permanent legacy in the many dozens of trainees who benefited from his mentoring, in the hundreds of publications in the scientific literature, and in his continuous efforts to ensure that the Department of Immunology and TSRI remain at the forefront of science. In his new role as professor emeritus, Bill will still be available to provide the benefit of his experience to all of us.


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Investigators' Reports


Lymphoepithelial Communication in the Intestine

R. Boismenu, Y. Chen, K. Chou, N. Chen

The intestinal mucosa shields the body from the external environment but still allows intake of essential nutrients. This epithelial barrier is continuously subjected to potentially toxic, infectious, and otherwise harmful agents. Distinct immune mechanisms have evolved to maintain the integrity of this compartment. In particular, T cells belonging to both the and the ß cell lineages are in close physical association with the mucosa. Previously, we showed that activated intestinal cells but not ß cells produce the epithelial growth factor FGF-7. Interestingly, FGF-7 appears to be the only cytokine expressed by intestinal cells that can support the survival of epithelial cell lines in tissue culture. In addition, we have shown that intestinal cells synthesize several chemokines whose function is to recruit inflammatory cells. These findings suggested to us that intestinal cells and the soluble factors they produce play important roles in maintaining intestinal homeostasis in normal and disease conditions.

ROLE OF CELLS IN INTESTINAL HOMEOSTASIS

We are evaluating the role of intestinal cells in a murine model of colitis induced by feeding the animals dextran sulfate sodium. In this model, focal areas of disease appear within the first 3 days of treatment with dextran sulfate sodium. Large numbers of activated cells accumulate in the affected intestinal segments. In contrast, marked inflammation occurs only after 5 days of treatment. We are pursuing the hypothesis that local production of chemokines by resident cells participate in the initiation of this inflammatory response. Using cellular and molecular approaches, we have shown that in this model system intestinal cells are a major source of FGF-7 and of several cytokines known to modulate immune responses. In ongoing projects, we will characterize the colitis induced by dextran sulfate sodium by using a variety of gene-knockout and transgenic mice that have perturbations in cytokine production and T-cell populations. In addition, we have begun studies to determine antigens recognized by intestinal cells.

ROLE OF CD81 IN THYMOCYTE DEVELOPMENT

In collaboration with W.L. Havran, Department of Immunology, we are continuing our investigation of the mechanisms that control the early stages of T-cell development. We previously showed that CD81, a surface protein normally expressed by thymic epithelial cells in the fetal thymus, is involved in the phenotypic conversion of immature CD4-CD8- thymocytes to more mature CD4+CD8+ cells. Currently, we are defining further the phenotype of immature thymocytes capable of interaction with CD81+ epithelial cells. This project includes analysis of defined cell-surface molecules, proteins associated with the cell cycle, and rearrangements of the genes for the T-cell receptor in immature thymocytes. In a related study, we are using biochemical and molecular techniques to isolate cell-surface proteins that interact with CD81.

PUBLICATIONS

Boismenu, R., Havran, W.L. T cells in host defense and epithelial cell biology. Clin. Immunol. Immunopathol., in press.

Boismenu, R., Havran, W.L. An innate view of T cells. Curr. Opin. Immunol. 9:57, 1997.

Boismenu, R., Hobbs, M.V., Boullier, S., Havran, W.L. Molecular and cellular biology of dendritic epidermal T cells. Semin. Immunol. 8:323, 1996.

Boismenu, R., Semeniuk, D., Murgita, R.A. Purification and characterization of human and mouse recombinant -fetoproteins expressed in Escherichia coli. Protein Expr. Purif. 10:10, 1997.

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Regulation of Cell Function by Rho GTPases

G.M. Bokoch, V. Benard, T.H. Chuang, R.H. Daniels, S. Dharmawardhane, C.C. King, T. Rudel, L.C. Sanders, F.T. Zenke, B.P. Bohl, P. Hall, Y. Wang

The Rho family of GTP-binding proteins (Rho, Rac, and Cdc42) has been a focus of research in our group. Rho GTPases control the assembly and activity of the actin cytoskeleton, the production of oxidants by leukocytes, the activity of regulatory kinase and phospholipase cascades, and signaling pathways that control cell growth. This spectrum of regulatory activities places the Rho GTPases as key components of such physiologic and pathologic processes as tumor growth and metastasis, wound healing, inflammatory responses, and even development. We have taken a variety of cellular, molecular, and biochemical approaches to understand how the activities of Rho GTPases are regulated, to determine the effector targets these proteins interact with to control cell function, and to determine how these regulatory processes are abnormal in various disease states.

CELLULAR REGULATION BY P21-ACTIVATED KINASES

During the course of studies on Rho GTPase regulation of human phagocyte function, we discovered that a family of serine/threonine kinases were targets for the active forms of Rac and Cdc42. Functional and structural analysis of the kinases, termed PAKs for p21-activated kinases, revealed their bifunctional nature. The C-terminal kinase domain phosphorylates substrates involved in regulating NADPH oxidase, stress responses, and the cellular actinomyosin system. The N terminus interacts with SH3 domain--containing proteins to modulate the actin cytoskeleton and direct polarized cell morphologies (Fig. 1). PAKs thus appear to be potentially important mediators of cell motility, wound healing, tumor metastasis, neurite outgrowth, and many other processes that depend on the dynamics of the actin cytoskeleton. Our current studies, in collaboration with U.G. Knaus, Department of Immunology, and others at TSRI, are directed at using molecular approaches to understand the function, regulation, and biology of PAKs.

RHO GTPASES AS COMPONENTS OF APOPTOSIS

Cells involved in formation of tissues and organs, development of the immune system, inflammatory responses, and even abnormally growing precancerous cells are normally removed from the body by a highly regulated death response termed apoptosis. Although initiated by diverse stimuli, the death response occurs via a well-defined program involving dramatic biochemical and morphologic events that have been investigated at only a basic level. Thus, the cytoplasm of the dying cells condenses toward the nucleus as the edges of the cell retract. Membrane blebbing occurs during and at the completion of the contraction phase. Finally, the cell breaks into fragments that can be effectively removed by noninflammatory phagocytic mechanisms. How this complicated series of cytoskeletal events is controlled is unknown.

We have found that Rho GTPases participate in apoptosis at both biochemical and morphologic levels. Cdc42 can induce apoptotic death through its ability to stimulate the Jun/p38 kinase cascades. In collaborative studies with K. Hahn, Department of Cell Biology, we have generated Rho GTPases tagged with a fluorescent marker. These tagged enzymes will be used to determine the role of GTPases in regulating apoptotic cytoskeletal dynamics. Preliminary findings suggest critical roles for Rho GTPases and their effectors in controlling the cell death response.

We have established that PAK2 is a particularly important target for apoptotic proteases. PAK2 is cleaved into active N- and C-terminal pieces that can regulate aspects of the apoptotic program, including morphologic and membrane changes (Fig. 2). Continuing studies will involve determining the importance of PAK2 cleavage during apoptosis of breast cancer cells and elucidating the molecular mechanisms of PAK action in the cell death program.

PUBLICATIONS

Bokoch, G.M., Wang, Y., Bohl, B.P., Sells, M.A., Quilliam, L.A., Knaus, U.G. Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J. Biol. Chem. 271: 25746, 1996.

Brzeska, H., Knaus, U.G., Wang, Z.-Y., Bokoch, G.M., Korn, E.D. p21-Activated kinase (PAK) has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I. Proc. Natl. Acad. Sci. U.S.A. 94:1092, 1997.

Chuang, T.H., Hahn, K., Lee, J.-D., Danley, D.E., Bokoch, G.M. The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes. Mol. Biol. Cell, in press.

Danley, D.E., Chuang, T.-H., Bokoch, G.M. Defective Rho GTPase regulation by IL-1b converting enzyme-mediated cleavage of D4 GDP dissociation inhibitor. J. Immunol. 157:500, 1996.

Dharmawardhane, S., Sanders, L.C., Daniels, R.H., Martin, S.S., Bokoch, G.M. Localization of p21-activated kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in stimulated cells. J. Cell Biol., in press.

Ding, J., Knaus, U.G., Bokoch, G.M., Badwey, J.A. The renaturable 69 and 63 kDa protein kinases that undergo rapid activation in chemoattractant-stimulated neutrophils are p21-activated kinases (Paks). J. Biol. Chem. 271:24869, 1996.

Ptasznik, A., Prossnitz, E.R., Yoshikawa, D., Smrcka, A., Traynor-Kaplan, A.E., Bokoch, G.M. A tyrosine kinase signaling pathway accounts for the majority of PIP3 formation in chemoattractant-stimulated human neutrophils. J. Biol. Chem. 271: 25204, 1996.

Rudel, T., Bokoch, G.M. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2. Science 276:1571, 1997.

Sells, M.A., Knaus, U.G., Bagrodia, S., Ambrose, D., Bokoch, G.M., Chernoff, J. Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 7:202, 1997.

Thomas, J.E., Venugopalan, M., Galvin, R., Wang, Y., Bokoch, G.M., Vlahos, C.J. Inhibition of MG-63 cell proliferation and PGDF-stimulated cellular processes by inhibitors of phosphatidylinositol 3-kinase. J. Cell. Biochem. 64:182, 1997.

Xu, X., Wang, Y., Barry, D.C., Chanock, S., Bokoch, G.M. Guanine nucleotide binding properties of Rac2 mutant proteins and analysis of responsiveness to guanine nucleotide dissociation stimulator. Biochemistry 36:626, 1997.

Zhou, K., Gorski, J.L., Chan, A., Kazusa cDNA group, Collard, J., Bokoch, G.M. Guanine nucleotide exchange factors dictate downstream signaling from Rac and Cdc42. J. Biol. Chem., in press.

Zigmond, S.H., Joyce, M., Borleis, J., Bokoch, G.M., Devreotes, P.N. Regulation of actin polymerization in cell-free systems by GTPgS and Cdc42. J. Cell Biol., in press.

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Regulation of T-Cell Memory

L.M. Bradley

A key feature of the immune system is its ability to respond quickly and specifically to previously encountered pathogens by mobilizing memory T and B lymphocytes generated in response to initial exposure to antigens. Despite the fundamental nature of immunologic memory, little is known about mechanisms that control its development, particularly within the T-cell population. CD4 cells regulate immune responses by producing cytokines that affect the differentiation and functions of T cells, B cells, and antigen-presenting cells.

Studies in this laboratory have identified four stages of differentiation of CD4 cells in vivo that are distinguished by a combination of functional, phenotypic, and temporal criteria: naive precursors, primary effectors, resting memory cells, and memory effector cells. These stages mark the progressive differentiation of peripheral CD4 cells during an immune response and show that development of memory is a multistep process that begins with the response of naive CD4 cells to antigen. Once activated, naive cells differentiate into transient effector cells that secrete cytokines. Although some effector cells are terminal cells that undergo apoptosis as the primary response subsides, many return to rest, becoming memory cells. Our current studies focus on the role of antigens, antigen-presenting cells, and cytokines in the development of memory CD4 subsets, as shown by secretion of cytokines associated with type 1 (IFN-, TNF-ß, and IL-2) and with type 2 (IL-4, IL-5, IL-6, and IL-10) T-helper cells in responses elicited by reexposure to antigen. Our studies indicate that memory CD4 cells influence their own development more so than do naive cells through autocrine use of IFN- and IL-4, cytokines that regulate differentiation of type 1 and type 2 T-helper cells, respectively.

Adhesion molecules such as CD44 and ß1, ß2, and ß7 integrins are upregulated after activation of naive CD4 cells, and on memory cells, the concentrations remain elevated, resulting in fundamental alterations in the biology and migratory behavior of antigen-primed CD4 lymphocytes. Greater expression of adhesion molecule contributes to decreased requirements for costimulation of memory cells compared with naive cells and to the capacity for activation with a broad range of antigen-presenting cells by lower doses of antigen.

Differences in adhesion molecules are also thought to underlie distinct recirculation pathways of memory and naive CD4 cells. Our studies of murine CD4 cells have shown that unlike naive cells, most memory cells lack L-selectin and do not home to lymphoid tissues where entry is controlled through high endothelial venules. Recirculation of naive cells in these sites can be abolished by a blockade of L-selectin and 4 integrins. In contrast, memory cells remain in the blood and tissues, where no adhesion requirements for entry of cells have been found. Many memory CD4 cells are found in the spleen, where they are ideally situated to encounter systemic antigens, and are recruited to sites of antigen exposure. We are using transgenic mouse models to study the regulation of infiltration of CD4 subsets into sites of inflammation such as the pancreas in autoimmune diabetes, lung allografts, and inflamed skin by cytokines, chemokines, and antigen.

PUBLICATIONS

Balasa, B., Deng, C., Lee, J., Bradley, L.M., Dalton, D.K., Christadoss, P., Sarvetnick, N. IFN-g is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice. J. Exp. Med., in press.

Bradley, L.M., Dalton, D.K., Croft. M. A direct role for IFN-g in regulation of Th1 development. J. Immunol. 157:1350, 1996.

Bradley, L.M., Malo, M.E, Tonkonogy, S., Watson, S.R. L-selectin is not essential for naive CD4 trafficking or development of primary responses in Peyer's patches. Eur. J. Immunol. 27:1140, 1997.

Bradley, L.M., Watson, S.R. Lymphocyte migration into tissue: The paradigm derived from CD4 subsets. Curr. Opin. Immunol. 8:312, 1996.

Dutton, R.W., Bradley, L.M., Swain, S.L. T-cell memory. Adv. Immunol., in press.

Swain, S.L., Croft, M., Dubey, C., Haynes, L. Rogers, P., Zhang, X., Bradley, L.M. From naive to memory T cells. Immunol. Rev. 150:143, 1996.

Yoshimoto, K., Swain, S.L., Bradley, L.M. Enhanced development of Th2-like primary CD4 effectors in response to sustained exposure to limited rIL-4 in vivo. J. Immunol. 156: 3267, 1996.

Zhang, X., Brunner, T., Carter, L., Dutton, R.W., Rogers, P., Bradley, L.M., Sato, T., Reed, J., Green, D., Swain, S.L. Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185:1837, 1997.

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Human Antibodies and Viral Infection

D.R. Burton, P.W.H.I. Parren, R.B. Bastidas, H.J. Ditzel, A. Hessell, P. Fisicaro, E. E. Karrer, T. Maruyama, P. Poignard, H. Sakurai, M. Wang, R.A. Williamson, C.F. Barbas III,* R.M. Chanock,** C.J. Peters,*** P.P. Sanna****

* Department of Molecular Biology, TSRI
** National Institute of Allergy and Infectious Diseases, Bethesda, MD
*** Centers for Disease Control and Prevention, Atlanta, GA
**** Department of Neuropharmacology, TSRI

The perception of humans as easy victors over microbes has changed dramatically in the last decade. Vaccination has offered protection against a number of viral pathogens, but it is increasingly recognized that the strategies used in the past will not be successful against all viruses. More understanding of viral pathogenesis and of the interaction of viruses with the immune system is required. We focus on the interplay of antibodies and viruses in humans. We make particular use of combinatorial antibody technology, which gives ready access to human antibodies. The viruses we are studying are HIV type 1 (HIV-1), respiratory syncytial virus (RSV), herpes simplex viruses 1 and 2 (HSV-1, HSV-2), and Ebola virus.

For HIV-1, we have generated a large panel of antibodies against the proteins gp120 and gp41, which in trimeric form make up the envelope spike of the virus. This panel has enabled us to map the accessible epitopes on gp120 and gp41 in some detail. We have shown that the overwhelming majority of human antibodies to these proteins do not neutralize the virus and do not bind to native trimeric forms of the envelope. These findings have led us to suggest that the predominant antibody response in HIV-1 infection may be to forms of the envelope protein produced during lysis of infected cells ("viral debris") rather than to native virions. This conclusion was supported when live virus was used to select antibodies from libraries of human antibodies obtained from HIV-seropositive donors. The number of antibodies obtained was much less than the number obtained with isolated envelope proteins.

However, some antibodies capable of neutralizing laboratory strains and primary isolates of HIV-1 have been derived. In a trial by the National Institutes of Health, one particularly potent antibody was one of only three antibodies generated worldwide that were capable of broad, potent neutralization of primary isolates of HIV-1. The antibody protected against infection with primary isolates of HIV in an animal model in which mice populated with human cells were challenged with HIV-1. This result was the first time that an antibody had protected against infection by a primary isolate of HIV in an animal model. Furthermore, we have shown that the antibody provides protection even when given several hours after exposure to HIV. This finding greatly enhances the possible usefulness of the antibody in prophylactic therapy given after accidental exposure to HIV and in the interruption of mother-to-child transmission of the virus. It is also of considerable encouragement in attempts to generate an antibody-based vaccine to HIV.

RSV is the most important cause worldwide of severe viral infection of the lower respiratory tract in young children. Antibodies are known to mediate resistance to RSV infection. We have isolated a potent human monoclonal Fab fragment that neutralizes diverse RSV isolates and is highly effective therapeutically when administered intranasally to infected mice at the time of peak viral replication in the lungs. These results suggest that human recombinant Fabs, possibly as an aerosol, might be useful in the treatment of serious RSV disease and in other viral diseases in which replication of the virus in vivo is limited primarily to the lumenal lining of the respiratory tract.

HSV-1 and HSV-2 are associated with a number of conditions of variable severity, which are only partially responsive to current therapies. We have prepared panels of Fabs to HSV-1 and HSV-2 from antibody phage display libraries. One Fab effectively neutralizes HSV-1 and HSV-2, and a whole antibody version of this molecule is a potent antiviral in animal models.

Ebola virus attracted much attention in 1995 after an outbreak of an infection caused by this virus in Kikwit, Zaire, killed more than 300 people, with a mortality rate of 80%. We are interested in whether treatment with antibody can control such a rampant virus. We obtained bone marrow from survivors of the epidemic, constructed phage libraries, and isolated a panel of human antibodies. A number of these antibodies bind effectively to native viral envelope on infected cells, and we plan to investigate the ability of these antibodies to protect against viral challenge in animal models.

PUBLICATIONS

Barbas, C.F. III, Burton, D.R. Selection and evolution of high-affinity human anti-viral antibodies. Trends Biotechnol. 14:230, 1996.

Binley, J.M., Ditzel, H.J., Barbas, C.F. III, Sullivan, N., Sodroski, J., Parren, P.W.H.I., Burton, D.R. Human antibody responses to HIV-1 glycoprotein 41 cloned in phage display libraries suggest three major epitopes are recognized and give evidence for conserved antibody motifs in antigen binding. AIDS Res. Hum. Retroviruses 12:911, 1996.

Burton, D.R. Immunoglobulin, functions. In: Encyclopedia of Immunology. Roit, I.M., Delves, P.J. (Eds). Academic Press, San Diego, in press.

Burton, D.R. A vaccine for human immunodeficiency virus type 1: The antibody perspective. Proc. Natl. Acad. Sci. U.S.A., in press.

Burton, D.R., Montefiori., D.C. The antibody response in HIV-1 infection. AIDS 11(suppl A):S87, 1997.

Ditzel, H.J., Itoh, K., Burton, D.R. Determinants of polyreactivity in a large panel of recombinant human antibodies from HIV-1 infection. J. Immunol. 157:739, 1996.

Ditzel, H.J., Parren, P.W.H.I., Binley, J.M., Sodroski, J., Moore, J.P., Barbas, C.F. III, Burton, D.R. Mapping the protein surface of HIV-1 gp120 using human monoclonal antibodies from phage display libraries. J. Mol. Biol. 267:684, 1997.

D'Souza, M.P., Livnat, D., Bradac, J.A., Bridges, S., the AIDS Clinical Trials Group Antibody Selection Working Group, collaborating investigators. Evaluation of monoclonal antibodies to HIV-1 primary isolates by neutralization assays: Performance criteria for selecting candidate antibodies for clinical trials. J. Infect. Dis. 175:1056, 1997.

Graus, Y.F., de Baets, M.H., Parren, P.W.H.I., Berrih-Aknin, S., Wokke, J., van Breda Vriesman, P.J., Burton, D.R. Human anti-nicotinic acetylcholine receptor recombinant Fab fragments isolated from thymus-derived phage display libraries from myasthenia gravis patients reflect predominant specificities in serum and block the action of pathogenic serum antibodies. J. Immunol. 158:1919, 1997.

Kessler, J.A. II, McKenna, P.M., Emini, E.A., Chan, C.P., Patel, M.D., Gupta, S.K., Mark, G.E. III, Barbas, C.F. III, Burton, D.R., Conley, A.J. Recombinant human monoclonal antibody IgG1b12 neutralizes diverse human immunodeficiency virus type 1 primary isolates. AIDS Res. Hum. Retroviruses 13:575, 1997.

Li, A., Baba, T.W., Sodroski, J., Zolla-Pazner, S., Gorny, M.K., Robinson, J., Posner, M.R., Katinger, H., Barbas, C.F. III, Burton, D.R., Chou, T.-C., Ruprecht, R.M. Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins. AIDS Res. Hum. Retroviruses 13:647, 1997.

Maruyama, T., Parren, P.W.H.I., Sanchez, A., Rensink, I., Rodriguez, L.L., Khan, A.S., Peters, C.J., Burton, D.R. Recombinant human monoclonal antibodies to Ebola virus. J. Infect. Dis., in press.

Parren, P.W.H.I., Burton, D.R. Antibodies against HIV-1 from phage display libraries: Mapping of an immune response and progress towards anti-viral immunotherapy. Chem. Immunol. 65:18, 1997.

Parren, P.W.H.I., Fisicaro, P., Labrijn, A., Ditzel, H.J., Yang, W.-P., Barbas, C.F. III, Burton, D.R. In vitro antigen challenge of human antibody libraries for vaccine evaluation: The human immunodeficiency virus type 1 envelope. J. Virol. 70:9046, 1996.

Parren, P.W.H.I., Gauduin, M.-C., Koup, R.A., Poignard, P., Fisicaro, P., Burton, D.R. Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design. Immunol. Lett., in press.

Parren, P.W.H.I., Sattentau, Q.J., Burton, D.R. HIV-1 antibody: Debris or virion? Nature Med. 3:366, 1997.

Seligman, S.J., Binley, J.M., Gorny, M.K., Burton, D.R., Zolla-Pazner, S., Sokolowski, K.A. Characterization by serial deletion competition ELISAs of HIV-1 V3 loop epitopes recognized by monoclonal antibodies. Mol. Immunol. 33:737, 1996.

Williamson, R.A., Lazzaroto, T., Sanna, P.P., Bastidas, R.B., Dalla Casa, B., Campisi, G., Burioni, R., Landini, M.P., Burton, D.R. Recombinant human antibody fragments for detection of cytomegalovirus antigenemia. J. Clin. Microbiol., in press.

Zeitlin, L., Whaley, K.J., Sanna, P.P., Moench, T.R., Bastidas, R., DeLogu, A., Williamson, R.A., Burton, D.R., Cone, R.A. Topically applied human recombinant monoclonal IgG1 antibody and its Fab and F(ab´)2 fragments protect mice from vaginal transmission of HSV-2. Virology 225:213, 1996.

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Antibody Recognition of DNA

D.R. Burton, R.A. Williamson, K.E. McLane,* J.F. LeBlanc, P. Ghazal

* Genentech, San Francisco, CA

A general method for the generation of molecules capable of recognizing double-stranded DNA with high affinity and specificity would have wide implications in chemistry, biology, and medicine. For instance, such reagents could be useful in genome mapping by blocking certain sites to enzyme action, in diagnostic procedures requiring identification of certain DNA sequences, and in therapeutic regimens requiring control of gene expression. Because they recognize a huge number of diverse molecular shapes, antibodies are promising candidates for high-affinity sequence-specific recognition of DNA. However, the generation of monoclonal antibodies to DNA by conventional immunization procedures has not been generally successful, because double-stranded DNA is ineffective in eliciting an immune response in normal animals.

To circumvent this problem, we have explored a number of strategies, including the affinity selection of high-affinity DNA binders from synthetic antibody libraries displayed on phage and the generation of binders from phage display libraries prepared from tissue from donors with the autoimmune disease systemic lupus erythematosus. In another approach, we have transplanted a 17 amino acid -helical domain from a transcription factor into a hypervariable part of the antibody molecule and have shown that the motif confers sequence-dependent DNA recognition on the mutant antibody molecule. Our most recent studies indicate that it is possible to generate high-affinity, specific DNA-binding antibodies. These molecules are being investigated for their ability to function as transcriptional repressors and activators.

In related studies, we are looking at the ability of antibodies to enter cells and target the nucleus when tagged with various motifs. The combination of sequence-specific recognition of DNA and nuclear targeting might provide useful reagents for control of gene expression.

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Probing the Structure of the Prion Protein

D.R. Burton, R.A. Williamson, R.B. Bastidas, D. Peretz,* S.B. Prusiner*

* University of California, San Francisco, CA

Prion diseases are degenerative illnesses of the CNS that affect animals and humans. These diseases may be inherited because of mutations in the chromosomal gene that encodes the prion protein (PrP), arise sporadically, or be acquired by transmission of infectious prion particles. The fundamental change underlying prion diseases is the conversion of cellular PrP into the scrapie isoform. Attempts to determine a chemical difference between the two forms have been unsuccessful. The scrapie isoform is formed from the cellular form after translation by a process that involves a profound conformational change, possibly the conversion of -helical structures to a ß-sheet.

For many years, investigators thought that scrapie and the related human prion diseases kuru and Creutzfeldt-Jakob disease were caused by slow viruses. Among the most puzzling features of these transmissible diseases was the lack of any detectable immune response to inoculated infectious particles. Neither scrapie-specific antibodies nor a cellular immune response has been detected.

Antibodies most likely would be valuable probes for exploring the conformation of prions, but because of tolerance, few monoclonal antibodies, and none to mouse prions, have been developed. We have solved this problem and have generated a large array of mouse monoclonal antibodies to mouse PrP. We immunized mice in which the gene for PrP had been ablated and then "rescued" antibodies by using phage display libraries.

Antibodies to a range of conformational and linear epitopes have been generated. The linear epitopes have been mapped by using overlapping PrP peptides and selection from PrP-fragment libraries expressed on the surface of phage. With these antibodies, we showed that a major difference in conformation between the cellular and the scrapie isoforms of PrP can be localized to the N-terminal part of the PrP molecule. In contrast, epitopes at the C-terminal part are conserved in the two forms of PrP. Nuclear magnetic resonance studies showed that the C-terminal part of cellular PrP has a well-defined -helical structure and that the N-terminal part has considerable flexibility. The antibody results suggest that a structural ordering in this region may be crucial in the acquisition of protein infectivity.

In another study, we have used the antibodies to show that recombinant cellular PrP undergoes a major conformational transition at low pH and only slowly returns to the native conformation. The possibility that this conformation is related to an intermediate form between the cellular and the scrapie isoforms of PrP is under study.

PUBLICATIONS

Peretz, D., Williamson, R.A., Matsunaga, Y., Serban, H., Pinilla, C., Bastidas, R.B., Rozenshteyn, R., James, T.L., Houghten, R.A., Cohen, F.E., Prusiner, S.B., Burton, D.R. A conformational transition at the N-terminus of the prion protein features in formation of the scrapie isoform. J. Mol. Biol., in press.

Williamson, R.A., Peretz, D., Smorodinsky, N., Bastidas, R., Blochberger, T., Serban, A., DeArmond, S., Prusiner, S.B., Burton, D.R. Circumventing tolerance in order to generate autologous monoclonal antibodies to the prion protein. Proc. Natl. Acad. Sci. U.S.A. 93:7279, 1996.

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Molecular and Biological Mechanisms Regulating Angiogenesis and Cellular Invasion

C. Andrews, P. Brooks, B. Eliceiri, M. Friedlander, R. Klemke, J. Leng, T. Möhler, R. Molander, G. Nemerow, E. Petitclerc, A. Reddy, M. Rosenfeld, L.C. Sanders, S. Silletti, S. Strömblad, C. Storgard, D. Stupack, T. von Schalscha, L. Yeh, D.A. Cheresh

BLOCKING OF ANGIOGENESIS BY ANTAGONISTS OF
V INTEGRINS

Antibodies, synthetic peptides, and organic antagonists directed to integrin vß3 or vß5 block angiogenesis in chicken embryos, mice, and rabbits. These antagonists cause tumor regression in these animals by disrupting the tumors' blood supply. In a rabbit model of rheumatoid arthritis, a cyclic peptide antagonist of vß3 not only blocked angiogenesis in the knee but also prevented the inflammatory events leading to a decrease in the losses of cartilage and bone associated with this disease. These antagonists produce no apparent side effects because they do not affect preexisting blood vessels. On the basis of these findings, a humanized form of a monoclonal antibody to vß3 is being evaluated in a phase I clinical trial in patients with late-stage cancer. Thus far, the antibody appears to be safe and is providing clinical benefit to a number of these patients. Additional trials are being planned to test small synthetic peptide and nonpeptide antagonists in patients with cancer, arthritis, and ocular diseases.

ROLE OF MATRIX METALLOPROTEINASE 2 IN ANGIOGENESIS IN VIVO

The collagenase matrix metalloproteinase 2 (MMP-2) binds to the surface of invasive cells by virtue of its ability to interact with integrin vß3. This binding depends on the C-terminal hemopexin domain of the enzyme, because removal of the domain makes MMP-2 incapable of binding to cells bearing vß3. A recombinant fragment consisting of the hemopexin domain of MMP-2 blocked binding of vß3 to intact MMP-2. This recombinant fragment also blocked ongoing angiogenesis on chick chorioallantoic membranes in response to cytokines or human tumors. Systemic administration of this hemopexin domain blocked the activation of MMP-2 in tissues undergoing angiogenesis. Therefore, inhibition of MMP-2 binding to vß3 not only prevents enzyme activation and matrix degradation but also is a novel mechanism for disrupting angiogenesis in vivo.

MAP KINASE ACTIVITY DURING ANGIOGENESIS INDUCED BY BASIC FIBROBLAST GROWTH FACTOR

Angiogenesis depends on growth factors and vascular cell adhesion. Integrins and growth receptors have been colocalized on the cell surface and appear to cooperate in activating the ras/MAP kinase pathway. On initiation of angiogenesis by basic fibroblast growth factor, vascular cell MAP kinase was activated within 5 minutes, and activation was sustained for 20 hours. The initial MAP kinase activity (<2 hr) was refractory to integrin antagonists, whereas the sustained activity (>4 hr) depended on ligation of vß3 and was essential for angiogenesis. A MAP kinase kinase inhibitor administered during this sustained vß3-dependent signal also blocked MAP kinase activity and angiogenesis. Thus, angiogenesis depends on ligation of vß3, which promotes sustained MAP kinase activity in blood vessels stimulated with angiogenic growth factors.

REGULATION OF CELL MIGRATION

Interaction of cells with extracellular matrix proteins, cytokines, or growth factors promotes signaling events that regulate cell migration. Ligation of integrin or cytokine receptors induced MAP kinase activity and cell locomotion on the extracellular matrix. Activated MAP kinase influenced the cells' motility machinery by directly phosphorylating myosin light chain kinase, a step that led to increased phosphorylation of myosin light chains. In addition, cytokine- and integrin-mediated cell migration involved the adaptor proteins c-crk and CAS. Expression of c-crk and CAS in cells promoted interaction between the two proteins and the induction of cell movement. Moreover, activation of these signaling pathways is associated with induction of tumor cell invasion and pulmonary metastasis in vivo.

ROLE OF INTEGRINS AND SIGNALING MOLECULES IN CELL MIGRATION

Videomicroscopy can be used to measure cell movement in response to a growth factor gradient. By introducing genes coupled to green fluorescent protein, we are able to study how signaling molecules modulate the cell actin-myosin cytoskeleton and motility in living cells. A camera enables us to follow these molecules inside the migratory cells in real time or with time-lapse videography. These studies address how cell motility is regulated at the molecular and cellular level.

INTERACTION OF INTEGRIN CYTOPLASMIC DOMAINS WITH MYOSIN HEAVY CHAINS

Ligation of integrins promotes coordination of the microfilament cytoskeleton during cell motility, and although originally indicated as the molecular motor of muscle cells, myosin has been implicated in other functions, including contractility during mitosis and collagen contraction. We detected a region of the conventional type II myosin heavy chain that interacts directly with the cytoplasmic domain of the subunits of ß integrins. This domain comprises the C terminus of the nonmuscle B isoform of myosin, a variant originally characterized as embryonic. This finding implies a role for this interaction in migration during embryogenesis. Differential specificity for distinct subunits of ß integrins suggests discriminate regulation of myosin-integrin coupling, and because this region is highly divergent among myosin isoforms, another level of specificity may exist. Thus, we have defined a novel linkage between integrins and the cytoskeleton that is potentially important in cellular migration and cell contractility.

LIGAND VALENCY AND INTEGRIN ACTIVATION

The extracellular matrix undergoes dynamic reorganization during a variety of processes, including wound healing and neovascularization. We examined the influence of ligand multimerization on hematopoietic cells expressing integrin vß3 and found that multimeric ligands induce cellular adhesion. Adhesion requires local valency but is not influenced by gross ligand avidity. Signaling studies have revealed a role for pp72 syk in directing attachment, but interactions with either ligand form can activate alternative pathways. These results indicate a significant role for the local extracellular environment in directing adhesion of hematopoietic cells and provide an alternative to modulation of activation by chemokine and antigen receptors.

PUBLICATIONS

Boudreau, N., Andrews, C., Srebrow, A., Ravanpay, A., Cheresh, D.A. Regulation of the angiogenic phenotype by Hox D3. J. Cell Biol., in press.

Brooks, P.C., Klemke, R.L., Schön, S., Lewis, J.M., Schwartz, M.A., Cheresh, D.A. Insulin-like growth factor receptor cooperates with integrin vß5 to promote tumor cell dissemination in vivo. J. Clin. Invest. 99:1390, 1997.

Filardo, E.J., Deming, S.L., Cheresh, D.A. Regulation of cell migration by the integrin ß subunit ectodomain. J. Cell Sci. 109:1615, 1996.

Friedlander, M., Theesfeld, C.L., Sugita, M., Fruttiger, M., Thomas, M.A., Chang, S., Cheresh, D.A. Involvement of integrins vß3 and vß5 in ocular neovascular diseases. Proc. Natl. Acad. Sci. U.S.A. 93:9764, 1996.

Klemke, R.L., Schuang, C., Giannini, A.L., Gallagher, P.J., de Lanerolle, P., Cheresh, D.A. Regulation of cell motility by mitogen-activated protein kinase. J. Cell Biol. 137:481, 1997.

Mousa, S.A., Cheresh, D.A. Recent advances in cell adhesion molecules and extracellular matrix proteins: Potential clinical implications. Drug Discovery Today 2:9, 1997.

Okada, Y., Copeland, B.R., Hamann, G.F., Koziol, J.A., Cheresh, D.A., del Zoppo, G.J. Integrin vß3 is expressed in selected microvessels following focal cerebral ischemia. Am. J. Pathol. 149:37, 1996.

Strömblad, S., Becker, J.C., Yebra, M., Brooks, P.C., Cheresh, D.A. Suppression of p53 and p21WAF1/CIP1 expression by vascular cell integrin vß3 during angiogenesis in vivo. J. Clin. Invest. 98:426, 1996.

Strömblad, S., Cheresh, D.A. Cell adhesion and angiogenesis. Trends Cell Biol. 6:462, 1996.

Strömblad, S., Cheresh, D.A. Integrins, angiogenesis and vascular cell survival. Chem. Biol. 3:881, 1996.

Varner, J.A., Cheresh, D.A. Integrins and cancer. Curr. Opin. Cell Biol. 8:724, 1996.

Yebra, M., Parry, G.C.N., Strömblad, S., Mackman, N., Rosenberg, S., Mueller, B.M., Cheresh, D.A. Requirement of receptor-bound urokinase-type plasminogen activator for integrin vß3-directed cell migration. J. Biol. Chem. 271:29393, 1996.

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The Therapeutic Effect of Dilute KL4-Surfactant in Inflammatory Disease of the Lung

C.G. Cochrane, S.D. Revak, T.A. Merritt,* I.U. Schraufstatter, R.C. Hoch, C. Henderson,**

* St. Charles Medical Center, Bend, OR
** University of California, San Diego, CA

Pulmonary surfactant is a complex of phospholipids and proteins that forms a monolayer along the alveolar surface of pulmonary epithelial cells. Its main function is to prevent collapse of the alveoli caused by the recoil of elastic tissue in the alveolar septa. The surfactant protein with greatest activity for this function is surfactant protein B. When mixed with appropriate phospholipids, surfactant protein B induces the greatest surfactant function. Antibodies to surfactant protein B inhibit activity of the surfactant and induce atelectatic collapse in the lungs of rabbits. In addition, in human infants and mice deficient in surfactant protein B, inflation of the lungs does not occur despite normal levels of the other surfactant proteins and phospholipids.

Surfactant protein B is a peptide of 80 amino acids and consists of stretches of hydrophobic residues with intermittent hydrophilic residues; 9 of 12 of the hydrophilic residues are basic and have a positive charge. Surfactant protein B is soluble in lipids or organic solvents but not in aqueous media. Synthetic peptides that mimic the pattern of surfactant protein B function the same as the native peptide in aqueous dispersions of phospholipids. These mimic peptides consist of arginine or lysine (K) residues along with leucines (L), representing the hydrophobic residues. An example of these is KLLLLKLLLLKLLLLKLLLLK (KL4).

When tryptophan was incorporated in the leucine stretches, the fluorescence emission maximum indicated that the peptide lies in the acyl side chains, close to the polar-head groups. Removal of the charged arginine or lysine residues markedly decreased surfactant activity of the peptides, and magnetic resonance assays indicated an electrostatic interaction between lysine residues and negatively charged residues of the polar-head groups. Raman vibrational spectroscopy suggested, over a temperature range of 6--46°C, that the peptides increase lateral stability of the phospholipid molecules. These data provide the basis for a theory on the mechanism of surfactant protein B (or its peptide mimics) in providing the stability essential for surfactant function by a phospholipid layer.

The combination of KL4 peptide and the phospholipids dipalmitylphosphatidylcholine and palmityloleoylphosphatidylglycerol provided strong surfactant activity in fetal rabbits; macaques; and, in a phase I/II trial, in 39 human infants. In the human infants, treatment with KL4-Surfactant was begun within 4 hours of birth. Average weight of the infants was 1148 ± 279 g, and the average ratio of arterial to alveolar oxygen concentration was 0.14 ± 0.06. After treatment, the average ratio increased into the normal range within 12 hours. Fractions of inspired oxygen, mean airway pressures, and oxygen index decreased in inverse proportion to the arterial-to-alveolar ratio. No fatalities related to respiratory distress syndrome occurred. A single instillation of KL4-Surfactant was sufficient in all but a few of the infants. No safety issues were detected.

A new method has been devised to administer KL4-Surfactant to inflamed lungs of adult and infant animals with respiratory distress. Two models have been used in this regard: meconium aspiration in 2.5-kg rabbits and newborn macaques and lipopolysaccharide-induced injury of the lung after saline lavage to remove intrinsic surfactant in 2.5-kg rabbits. After injury developed in response to the lipopolysaccharide or meconium in 2.5-kg rabbits or, with meconium, in newborn macaques, the lungs of the animals were lavaged with a diluted form of KL4-Surfactant. With two to three lavages, inflammatory exudate or meconium was mostly removed from the alveolar spaces, and the alveoli rapidly expanded with air. In control animals, saline lavages removed exudate or meconium, but the alveolar spaces did not expand. In addition, when a bolus of KL4-Surfactant was administered to adult rabbits with lipopolysaccharide-injured and inflamed lungs or to rabbits with meconium injury, various zones of the lungs showed partial expansion, but others showed continued collapse, and no loss of inflammatory exudate occurred.

The partial pressure of arterial oxygen (Pao2) in rabbits receiving lavage with dilute KL4-Surfactant and 100% oxygen increased from less than 100 mm Hg to more than 400 mm Hg in less than 1 hour, whereas the Pao2 in rabbits lavaged with saline did not increase to more than 100 mm Hg. In rabbits given a bolus of KL4-Surfactant, the Pao2 increased moderately (to 200--300 mm Hg) but, as the inflammation progressed, decreased to 100 mm Hg. Of interest, inflammation in the lungs of rabbits lavaged with dilute KL4-Surfactant did not develop further, whereas rabbits treated with saline lavage or a bolus of surfactant showed marked inflammation at the end of the study, 2--4 hours after treatment.

In collaboration with R.M. Smith, University of California, San Diego, 20-kg pigs were injured by using saline lavage to remove intrinsic surfactant. Dilute KL4-Surfactant was administered through a fiber-optic bronchoscope after lung injury had developed and Pao2 levels had been less than 100 mm Hg for more than 1 hour. Segmental lavage with the dilute surfactant resulted in expansion of the treated segments and removal of inflammatory exudate. When more than 50% of the lung was lavaged, Pao2 levels increased to more than 500 mm Hg within minutes.

With these experimental data available, clinical trials have been initiated in centers throughout the United States for two indications: acute respiratory distress syndrome in adults and meconium aspiration syndrome in newborn infants. The results of these phase I/II clinical trials will be forthcoming.

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Analysis of the Early Stages of Infection of Human B Cells by Epstein-Barr Virus

N. Sugano,* W. Chen , M.L. Roberts,** N.R. Cooper

* Nihon University, Tokyo, Japan
** ISIS Pharmaceuticals, Carlsbad, CA

Our studies address the early stages of infection of human B lymphocytes by Epstein-Barr virus (EBV), a transforming human herpesvirus with oncogenic potential. Infection is initiated by binding of the virus to CD21, a membrane glycoprotein that also serves as the cellular receptor for the C3dg activation and processing fragment of the third component of complement. Unlike most viruses, EBV infects nonactivated, noncycling, resting cells, although expression of the viral and cellular genes that mediate the earliest stages of infection depends on the cellular transcription machinery. We evaluated the hypothesis that an intracellular signaling pathway triggered by EBV binding to CD21 enables EBV to infect resting B cells.

In exploring the signaling events initiated by EBV binding to CD21, we found that the ubiquitous transcription factor NF-B was rapidly activated and reached peak levels 30 minutes after binding of the virus to the membrane of purified resting human B cells. Activation of NF-B was triggered by ligand binding to the receptor, because the viral glycoprotein mediating EBV binding to CD21, C3dg, and a monoclonal antibody to CD21 all activated the transcription factor. Activation of NF-B was relevant for infection, because different inhibitors of NF-B blocked transcription of the earliest expressed viral gene and subsequent infection.

The target of NF-B activated by EBV binding to CD21 was the initial viral promoter, Wp, which contains an NF-B--like sequence. Transfection studies showed that NF-B activated the native Wp sequence but not a Wp construct with an altered NF-B--binding sequence, a finding that shows functional relevance. After binding to resting uninfected cells, transcription from Wp was initiated within the period of marked NF-B activation. Thus, EBV binding to its cell-surface receptor activates an NF-B--dependent signaling pathway that mediates activation of the initial viral promoter. This finding is the first example of a crucial role for an intracellular signaling pathway triggered by a virus-receptor interaction in permitting viral infection.

Latent membrane protein 1 (LMP1), one of the EBV latent genes, is the only EBV protein that has the properties of an oncogene in vitro and in vivo. In studies to evaluate the mechanisms involved in EBV-induced malignant transformation, LMP1 activated the ERK 1/2 mitogen-activated protein kinases. Cotransfection approaches showed that LMP1-mediated activation of the kinases proceeded via a ras-dependent pathway. Transfection studies also revealed that malignant transformation of fibroblasts initiated by LMP1 was ras dependent.

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Complement Activation by ß-Amyloid in Alzheimer's Disease

B.M. Bradt, N.R. Cooper

Alzheimer's disease is the most common cause of senile dementia, a symptom complex characterized by the age-related progressive loss of cognitive functions. The disease is characterized pathologically by the presence of large numbers of neuritic (senile) plaques in the hippocampus and neocortex, regions of the brain concerned with cognition and memory. The plaques are largely composed of a highly insoluble fibrillar form of a 40- to 43-residue ß-amyloid peptide (Aß). Damaged (dystrophic) neuronal processes, loss of neurons and synapses, increased numbers of activated (reactive) astrocytes and microglia, and components of the proinflammatory complement system are also associated with neuritic plaques.

Complement activation in the vicinity of neuritic plaques could generate the cytokine-like C5a peptide and trigger the influx and activation of astrocytes and microglia, because these cells have receptors for C5a and react to this peptide with activation and directed motion. The cell-signaling and cytotoxic properties of the complement membrane attack complex (C5b-7, C5b-8, and C5b-9) could potentially damage neurons and activate glial cells. For complement to be biologically relevant in this manner to the progression of Alzheimer's disease, the Aß peptide must be able to activate complement, because activation is a prerequisite for all the biological actions of the complement system.

We used a novel definitive approach to show that Aß directly and independently activates the classical and alternative complement pathways, leading to the formation of covalent complexes of Aß with the C3b and iC3b activation fragments of the third component of complement. We also found that Aß-mediated triggering of complement activates the terminal proinflammatory part of the complement reaction sequence and generates C5a and the C5b-9 complex. The C5b-9 membrane attack complex was functionally competent, because it could insert itself into neuronal precursor cell membranes and make the cells permeable to small molecules. These findings provide inflammation-based mechanisms to account for the presence of complement components in neuritic plaques, for the damage to neurons near neuritic plaques, and for the influx and activation of glial cells. They also have important potential implications for the therapy of Alzheimer's disease.

PUBLICATIONS

Cooper, N.R. Complement and viruses. In: The Human Complement System in Health and Disease. Volanakis, J., Frank, M. (Eds.). Marcel Dekker, New York, in press.

Cooper, N.R. Complement-dependent pro-inflammatory properties of the Alzheimer's disease ß-amyloid peptide. In: Controlling the Complement System for Novel Drug Development. Mazarakis, H., Swart, S.J. (Eds.). International Business Communications, Southborough, MA, 1997, p. 3.

Cooper, N.R. Complement-dependent virus neutralization. In: The Complement System. Rother, K., Till, G., Hänsch, M. (Eds.). Springer-Verlag, New York, in press.

Cooper, N.R. Evasion of complement mediated damage by microorganisms. In: The Complement System. Rother, K., Till, G., Hänsch, M. (Eds.). Springer-Verlag, New York, in press.

Roberts, M.L., Luxembourg, A.T., Cooper, N.R. Epstein-Barr virus binding to CD21, the virus receptor, activates resting B cells via an intracellular pathway that is linked to B cell infection. J. Gen. Virol. 77:3077, 1996.

Sugano, N., Roberts, M.L., Cooper, N.R. EBV binding to CD21 activates the initial viral promoter via NF-B induction, J. Exp. Med., in press.

Webster, S., Bradt, B., Rogers, J., Cooper, N.R. Aggregation state-dependent activation of the classical complement pathway by the amyloid ß peptide (A ß). J. Neurochem. 69:388, 1997.

Webster, S., Lue, L.-F., Brachova, L., Tenner, A., McGeer, P., Walker, D., Bradt, B., Cooper, N.R, Rogers, J. Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer's disease. Neurobiol. Aging, in press.

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Apolipoprotein E, Inflammation, and Atherosclerosis

W.A. Boisvert, I.R. Nikoulin,* R. Terkeltaub,** L.K. Curtiss

* Lidak Pharmaceuticals, La Jolla, CA
** Veterans Affairs Medical Center, San Diego, CA

Apolipoprotein E (apoE) regulates the clearance of plasma cholesterol by mediating binding of lipoproteins to their cellular receptors. Previously, we determined that a synthetic peptide, which represents a linear dimeric repeat of apoE amino acid residues 141--155, binds the receptor for low density lipoprotein (LDL). To prepare a synthetic peptide that had high affinity for both cholesterol-rich lipoproteins and cellular receptors for lipoprotein, we synthesized an N-terminal acetylated dimer peptide. This peptide was soluble at neutral pH, formed stable peptide-LDL complexes in vitro, and enhanced the binding of LDL to cellular receptors for lipoprotein in vitro. When peptide-LDL complexes were injected into C57BL/6J apoE-deficient mice, the rate of removal of the complexes from the blood was threefold faster than that of LDL alone. Administration of free peptide resulted in a 30--40% reduction in total plasma cholesterol within 10 minutes, a finding that was accounted for by a 40% and a 26% reduction in plasma levels of very low density and intermediate density lipoproteins, respectively. Therefore, a synthetic peptide analog of apoE can selectively associate with cholesterol-rich lipoproteins and facilitate their immediate clearance from plasma.

ApoE also promotes efflux of cholesterol from foam cells, which are a hallmark of early atherosclerotic lesions. However, the role of another apolipoprotein, apoAI, in this process in vivo is unknown. We used mice deficient in both apoE and apoAI to test the hypothesis that lipoproteins containing apoAI both bind secreted cholesterol and transport it out of the lesions within the vessel wall.

We lethally irradiated mice deficient in apoE alone or in both apoE and apoAI and reconstituted them with bone marrow cells from wild-type mice. The rationale for this treatment was to generate two sets of animals, one with lesions that contained both apoAI and apoE (AI + E) and another with lesions that contained apoE only. This experimental manipulation was possible because the apoE in lesions was derived from the transplanted wild-type macrophages, whereas the apoAI was not.

Macrophage-derived apoE decreased the hypercholesterolemia of both experimental groups; however, the plasma levels of cholesterol were twofold to threefold higher in the AI + E animals than in the animals with lesions containing apoE only. Despite this higher concentration of plasma cholesterol, total cholesterol deposited in the aortas was twofold lower in the AI + E group, and the mean atherosclerotic lesion area was 45% smaller. Therefore, locally available apoAI participates in the efflux of free cholesterol and retards the progression of lesions.

Inflammation mediated by mononuclear leukocytes occurs in atherogenesis, and expression of the monocyte chemotactic C-C chemokine JE/MCP-1 has been described. However, the C-X-C chemokines such as IL-8 also are expressed in atherosclerotic lesions, although these mediators are best recognized as neutrophil chemotaxins. To understand the role of C-X-C chemokines in atherogenesis, we studied diet-induced atherosclerosis in LDL-receptor knockout mice that were repopulated with bone marrow cells from mice deficient in the mouse receptor for IL-8 (IL-8R). Analysis confirmed that the peripheral blood leukocytes in these mice did not express receptors for IL-8. The mice had splenomegaly and a lack of germinal centers in their spleens, known characteristics of IL-8R knockout mice.

After being fed an atherogenic diet, all LDL-receptor knockout mice had dramatically increased plasma levels of cholesterol, but the levels in the mice with receptors for IL-8 were about 30% higher than the levels in the mice that lacked receptors for IL-8. Of importance, the area of the lesion was double that of the area in IL-8R--deficient mice, a finding that strongly suggests that IL-8R expression plays a key role in the early onset of atherogenesis.

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Estrogen and Atherosclerosis

M.M. Marsh, V.R. Walker, J.C. Rutledge,* L.K. Curtiss, C.L. Banka

* University of California, Davis, CA

The decreased risk of heart disease associated with endogenous or exogenous estrogen has traditionally been attributed to a decrease in circulating levels of low density lipoprotein (LDL) associated with an increase in hepatic receptors for LDL. To examine alternative mechanisms of estrogen protection, we established a mouse model in which LDL levels do not respond to estrogen.

Mice deficient in receptors for LDL (LDLR-/- mice) were ovariectomized and randomly assigned to treatment groups that received physiologic levels of 17ß-estradiol, 17-estradiol, or a control substance implanted in slow-release pellets. Control LDLR-/- males and females had sham operations. Animals were fed a high-fat, high-cholesterol diet for 12 weeks. All mice were hypercholesterolemic at baseline (cholesterol, 218 ± 5 mg/dl compared with an average of 70 mg/dl in wild-type mice).

Although fasting levels of plasma cholesterol increased approximately fourfold in all treatment groups on the high-cholesterol diet, neither the sex of the animal nor the hormone treatment had any effect on cholesterol levels. Despite nearly identical plasma levels of total cholesterol, estrogen-treated ovariectomized mice (both 17- and 17ß-estradiol) had smaller aortic atherosclerotic lesions than did placebo-treated ovariectomized females and male sham-operated mice. These data suggest that estrogen can protect LDLR-/- female mice from atherosclerosis in the absence of LDLR-mediated changes in plasma levels of cholesterol. Studies are ongoing to determine the alternative mechanisms of estrogen protection, including the possible action of estrogen as an antioxidant in the LDLR-/- mice.

To examine the primary effects of estrogen on the artery wall, we have collaborated with J. Rutledge at the University of California, Davis, to examine the effect of female sex hormones on lipid accumulation in a model system of isolated, perfused, rat carotid arteries. When oxidatively modified LDL or native LDL was perfused separately through isolated arteries, oxidatively modified LDL accumulated in the artery wall to a greater extent than did native LDL. Exposure of the artery to estradiol before perfusion with native or modified LDL had no effect on the rate of accumulation of normal LDL in the artery wall but significantly decreased the rate of accumulation of oxidatively modified LDL. Because perfusion with oxidatively modified LDL increased endothelial permeability, we are examining the possibility that estrogens may act as membrane antioxidants to protect the endothelium from damage that results in arterial accumulation of lipid, an early feature of atherosclerosis.

PUBLICATIONS

Banka, C.L. Antioxidant properties of estrogen: Selective protection of high density lipoprotein. In: Proceedings of the Workshop on Hormonal, Metabolic and Cellular Influences on Cardiovascular Disease. Forte, T.E. (Ed). Futura, New York, 1997, p. 193.

Banka, C.L. Non-genomic actions of estrogens: Estrogens as antioxidants. In: Estrogen and the Vessel Wall. Rubanyi, G. (Ed.). Harwood Academic, Reading, England, in press.

Boisvert, W.A., Spangenberg, J., Curtiss, L.K. Role of leukocyte-specific LDL receptors on plasma lipoprotein cholesterol distribution and atherosclerosis in mice. Arterioscler. Thromb. Vasc. Biol. 17:340, 1997.

Choi, S.Y., Pang, L., Kern, P.A., Kayden, H.J., Curtiss, L.K., Vanni-Reyes, T.M., Goldberg, I.J. Dissociation of LPL and LDL: Effects of lipoproteins and anti-apoB antibodies. J. Lipid Res. 38:77, 1997.

Exner, M., Susani, M., Witztum, J.L., Hovorka, A., Curtiss, L.K., Spitzauer, S., Kerjaschki, D. Lipoproteins accumulate in immune deposits and are modified by lipid peroxidation in passive Heymann nephritis. Am. J. Pathol. 149:1313, 1996.

Massamiri, T., Tobias, P.S., Curtiss, L.K. Structural determinants for the interaction of lipopolysaccharide binding protein with purified human high density lipoproteins: Role of apolipoprotein A-I. J. Lipid Res. 38:516, 1997.

Phillips, M.L., Pullinger, C., Kroes, I., Kroes, J., Hardman, D.A., Chen, G., Curtiss, L.K., Gutierrez, M.M., Kane, J.P., Schumaker, V.N. A single copy of apoprotein B-48 is present on the human chylomicron remnant. J. Lipd Res. 38:1170, 1997.

Rutledge, J.C., Woo, M.M., Rezai, A.A., Curtiss, L.K., Goldberg, I.J. Lipoprotein lipase increases lipoprotein binding to the artery wall and increases endothelial layer permeability by formation of lipolysis products. Circ. Res., 80:819, 1997.

Sorci-Thomas, M.G., Curtiss, L.K., Parks, J.S., Thomas, M.J., Kearns, M.W. Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity. J. Biol. Chem. 272:7278, 1997.

Spangenberg, J., Curtiss, L.K. Influence of macrophage-derived apoprotein E on plasma lipoprotein distribution of apolipoprotein A-I in apoprotein E-deficient mice. Biochem. Biophys. Acta, in press.

Spencer, T.A., Clark, D.S., Johnson, G.A., Erickson, S.K., Curtiss, L.K. Feasibility of an immunoassay for mevalonolactone. Bioorg. Med. Chem. 5:873, 1997.

Webb, N., deBeer, M.C., Kindy, M., van der Westhuyzen, D.R., Banka, C.L., Rader, D.J., deBeer, F.C. Adenoviral vector-mediated over-expression of SAA in apo-AI-deficient mice. J. Lipid Res. 39:45, 1997.

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The Molecular Biology of Protease Receptors

T.S. Edgington, W. Ruf, C.D. Dickinson, P.E. Thorpe,* S. Dittmar,** P. Carmeliet,*** D. Collen,*** D.C. Altieri,**** A.C. Nicholson,***** R.L. Nachman,***** D.P. Hajjar,***** W. Risau+

* University of Texas Southwestern Medical Center, Dallas, TX
** Behringwerke AG--Hoechst Marion Roussel, Marburg, Germany
*** Center for Molecular and Vascular Biology, Leuven, Belgium
**** Yale University School of Medicine, New Haven, CT
***** Cornell University Medical College, New York, NY
+ Max-Planck Institute, Bad Nauheim, Germany

Tissue factor (TF), a structural member of the cytokine receptor superfamily, is the most extensively characterized protease receptor and a prototypic model for receptor-cofactor control of enzyme function. Apart from its role as the in vivo cell-surface initiator of the hemostatic and thrombogenic cascades and its role in the initiation of effector inflammatory responses associated with both innate immunity and antigen-driven cellular immune responses, recent evidence suggests that TF may have currently unknown ligands and resultant unanticipated biological functions. For example, TF expressed on the surface of tumor cells plays an important role in successful implantation and growth of hematogenous metastases, gene knockout studies indicate a role for TF in embryonic vascular development, and a role of TF as a recognitive molecule that can signal cellular responses has been advanced.

Furthermore, we have created soluble mutant forms of TF that have been fused to monoclonal antibodies, creating a "switchable" targetable thrombogen. In vivo, once it is switched on by docking on the vascular endothelium of a tumor, the thrombogen can eradicate solid tumors in mice by selective thrombosis of tumor vasculature and infarctive necrosis of tumor. Our studies of the three-dimensional structures of TF and of TF in complex with cognate ligands and specific monoclonal antibodies provide the knowledge needed to relate structure to function for this prototypic protease receptor and regulatory cofactor.

We have also studied other protease receptors, including effector protease receptor-1 (EPR-1), the receptor for coagulation factor Xa. The gene for EPR-1 is an activation-responsive gene that encodes expression of the receptor on endothelial cells and vascular smooth muscle cells after injury. Assembly of factor Xa on EPR-1 results in cellular responses important in the pathobiology of vascular injury and repair. We are determining the structures of the complex consisting of TF and factor VIIa and of the ternary complex consisting of TF, factor VIIa, and factor X. Elucidation of the structure of the ternary complex will provide insight to the cellular initiation of thrombosis and molecular detail. This information will facilitate more effective and safe molecular solutions to treatment of thrombotic diseases, the major cause of death in advanced societies.

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Transcriptional Regulation of the Gene for TNF-

S.-T. Fan, J. Yao, N. Mackman, T.S. Edgington

The cytokine TNF- has been implicated in a variety of inflammatory responses. The trimeric structure of the protein has been described, its cell-surface receptors have been determined, and the sometimes beneficial effects of antibody neutralization in vivo have been investigated. However, information is limited on the regulation of transcription of the gene for TNF- in cells of monocytic lineage, which are predominantly responsible for production of this cytokine in vivo.

We have now mapped and characterized various cis-acting regulatory elements in the promoter of the gene that are involved in initiation and control of transcription. Bacterial lipopolysaccharide was used as the agonist for induction of transcription of the gene for human TNF- in THP-1 cells of monoblastic leukemia origin. The 5´ aspect of the gene was mapped by transfecting the cells with plasmids containing various lengths of the putative promoter region and measuring the response of the reporter gene in the plasmid to stimulation with lipopolysaccharide.

The region from -182 to -37 had multiple regions that influenced the transcriptional response. Region I (-182 to -162) contained an overlapping Sp1/Egr-1 site. Region II (-119 to -88) contained CRE and NF-B sites; the NF-B site is the B3 site in the 5´ region of the gene. The results also showed constitutive occupancy of the CRE site by the proteins CREB and, to a lesser extent, c-Jun. Cell stimulation led to increased occupancy of the CRE site by c-Jun, with consequent displacement of CREB. The response to stimulation also resulted in occupancy of the EGR-1 site by EGR-1 protein and of the B3 site by the heterodimer p50/p65.

The CRE and B3 sites together conferred strong responsiveness to lipopolysaccharide to a heterologous promoter, although individually they were ineffective. The spacing in the DNA between CRE and B3 sites was critical, inferring cooperation between c-Jun and p50/p65 in the effective initiation and amplification of transcription of the gene for TNF- in these cells representative of cells of the monocytic lineage. Additional control elements have been suggested; however, the B3 and CRE elements appear to provide the major control of transcription in cells of monocytic lineage and to differ from control of this gene in cells of lymphocytic lineage.

PUBLICATIONS

Carmeliet, P., Mackman, N., Moons, L., Luther, T., Gressens, P., Van Vlaenderen, I., Demunck, H., Kasper, M., Breier, G., Evrard, P., Müller, M., Risau, W., Edgington, T., Collen, D. Role of tissue factor in blood vessel development. Nature 383:73, 1996.

Carmeliet, P., Moons, L., Dewerchin, M., Mackman, N., Luther, T., Breier, G., Ploplis, V., Müller, M., Nagy, A., Plow, E., Gerard, R., Edgington, T., Risau, W., Collen, D. Insights in vessel development and vascular disorders using targeted inactivation and transfer of vascular endothelial growth factor, the tissue factor receptor, and the plasminogen system. Ann. N. Y. Acad. Sci. 811:191, 1997.

Dittmar, S., Ruf, W., Edgington, T.S. Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation. Biochem. J. 321:787, 1997.

Edgington, T.S. More inflammatory effects of protease receptors. J. Clin. Invest. 99:2299, 1997.

Edgington, T.S., Dickinson, C.D., Ruf, W. The structural basis of function of the TF*VIIa complex in the cellular initiation of coagulation. Thromb. Haemost. 78:401, 1997.

Huang, X., Molema, G., King, S., Watkins, L., Edgington, T.S., Thorpe, P.E. Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature. Science 275:547, 1997.

Morrissey, J.H., Agis, H., Albrecht, S., Carson, S.D., Dignat-George, F., Edgington, T.S., Luther, T., Müller, M., Mutin, M., Nakamura, S., Valnet, P., Vercellotti, G.M. CD142 (tissue factor) workshop panel report. In: Leucocyte Typing VI. Garland Publishing, London, in press.

Nicholson, A.C., Nachman, R.L., Altieri, D.C., Summers, B.D., Ruf, W., Edgington, T.S., Hajjar, D.P. Effector cell protease receptor-1 is a vascular receptor for coagulation factor Xa. J. Biol. Chem. 271:28407, 1996.

Yao, J., Mackman, N., Edgington, T.S., Fan, S.-T. Lipopolysaccharide induction of the tumor necrosis factor- promoter in human monocytic cells: Regulation by Egr-1, c-Jun, and NF-B transcription factors. J. Biol. Chem., in press.

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Formation of B- and T-Cell Repertoires in Mice and Humans

A.J. Feeney, B. Nadel, K. Victor, G. Escuro, G. Lugo

The diversity of antibodies and T-cell receptors is due to both combinatorial and junctional diversity. The antigen-binding parts of each heterodimeric receptor are encoded by two V, one D, and two J segments, and many such germline segments are present at each receptor locus. These gene segments are far apart from each other in the genome, and in each lymphocyte precursor, a unique combination of V, D, and J gene segments recombines at the DNA level to form a continuous exon encoding the antigen-binding domain for that cell's receptor. In addition to this vast combinatorial diversity, significant diversity is created at the junctions of the V, D, and J segments by the removal of various numbers of nucleotides from the ends of the coding regions and the addition of nontemplated N nucleotides. The main focus of our laboratory is the analysis of factors that influence the composition of the primary repertoires of B and T cells. We are particularly interested in revealing biases imposed by the V(D)J recombination mechanism itself on nonrandom junctional diversity and gene utilization.

Although each receptor locus has many V, D, and J genes, the genes are not used equally. We are analyzing use of V genes in the human locus. We first analyzed the peripheral B-cell repertoire of newborn infants. Seventy-five percent of this repertoire was composed of only 9 V genes, and only 22 V genes of the 37 potentially functional V genes were detected at all. To determine if V genes recombine at different rates or whether this nonrandom use was due to antigenic selection, we are examining use of V genes in pre-B cells isolated from bone marrow. We also found nonrandom gene utilization in these pre-B cells, indicating that V genes do recombine at different rates. Strikingly, the V genes in the distal half of the locus were rarely used.

One factor likely to play a major role in this nonrandom recombination is the naturally occurring variation in the recombination signal sequences (RSSs) that flank each V, D, and J gene segment. The RSS is the site where the recombinase binds. Each change from the consensus reduces the efficiency of recombination of the adjacent gene segment to a variable degree. We have constructed plasmid-based competition recombination substrates to analyze the relative efficiency of recombination of various natural RSSs.

For example, the V gene A2 is the predominantly used V gene in the protective antibody response to the bacterium Haemophilus influenzae type b. Navajos have a 10-fold higher incidence of H. influenzae disease than other populations, and we previously found a new allele of A2, called A2b, in more than 50% of Navajos but not in control subjects. The A2b allele recombines much less frequently than the A2a allele does in vivo. Because one of the changes in the A2b allele is in the RSS, we tested the relative rate of recombination of the A2a and A2b alleles in these substrates. The A2b allele showed a fourfold to fivefold reduction in recombination in our in vitro system. Thus, the altered RSS of this gene is likely to be the reason for the gene's inability to recombine efficiently in vivo and thus may play a role in disease susceptibility.

During the past year, we have extended our analysis of junctional diversity through the use of these recombination substrates. Previously we observed that each V, D, and J coding end analyzed ex vivo had a unique pattern of nucleotide deletion. To further dissect the reasons for this nonrandom processing of coding ends, we put several of these natural coding ends, and also variants of them, into recombination substrates. The data derived from these studies directly show that the coding-end sequence dramatically influences the extent and pattern of nucleotide deletion. Because coding ends transiently undergo hairpin formation before being joined, we propose that the sequence of each coding end dictates a unique hairpin structure and that this structure determines how the hairpin is resolved. Thus, the sequence of each coding end directly influences the junctional diversity created from that gene segment.

PUBLICATIONS

Bain, G., Maandag, E.C.R., te Riele, H.P.J., Feeney, A.J., Sheehy, A., Schlissel, M., Shinton, S.A., Hardy, R.R., Murre, C. Both E12 and E47 allow commitment to the B cell lineage. Immunity 6:145, 1997.

Feeney, A.J., Lugo, G., Escuro, G. The human cord blood repertoire. J. Immunol. 158:3761, 1997.

McKercher, S.C., Torbett, B.E., Andersen, K.L., Henkel, G.W., Vestal, D.J., Baribault, H., Klemsz, M., Feeney, A.J., Wu, G., Paige, C., Maki, R.A. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15:5647, 1996.

Nadel, B., Feeney, A.J. Nucleotide deletion and P addition in V(D)J recombination: A determinant role of the coding end sequence. Mol. Cell. Biol. 17:3768, 1997.

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Potential Effects of Leptin on the Interaction of Neuroendocrines

S. Chen, Y. Xia, C.B. Wilson, L. Feng

The protein OB (leptin) is produced by adipocytes in proportion to the mass of stored fat. Infusion of OB into the lateral ventricles of normal or obese mice suppressed appetite and stimulated metabolism, suggesting a negative feedback and CNS action of the protein. One potential site of OB's action is the hypothalamus, where the functional form of its receptor is prominently expressed, but other regions may also be involved. Binding of OB to its receptor activates the JAK/STAT signaling pathway; however, little is known about the downstream events and the mediating molecules. A number of neuroendocrines have been implicated in the control of appetite and metabolic rate and may be potential mediators of the effects produced by binding of OB to its receptor.

In collaboration with L. Mucke's group at the University of California, San Francisco, we found that receptors for OB are expressed not only in the hypothalamus but also in the amygdala and the pituitary gland and that recombinant murine OB elicited release of corticotropin releasing factor from the hypothalamus and amygdala and release of ACTH from the pituitary gland. These results suggest that OB has specific effects in other brain regions in addition to the hypothalamus and that corticotropin releasing factor and ACTH are potential central mediators of the effects of OB.

Because ACTH-induced elevation of plasma glucocorticoids can inhibit release of corticotropin releasing factor via negative feedback, the effects of OB on the pituitary release of ACTH may be pertinent to human obesity (Fig. 1) and may explain the paradox of high OB levels in obese humans. Two additional factors may enhance the effects of OB in obesity. First, the pituitary gland is not shielded from the systemic circulation by the blood-brain barrier. Second, OB is transported across the blood-brain barrier via a saturable system, which may become increasingly inefficient when the plasma level of OB increases above a critical level. Our findings therefore emphasize the need to consider the differential effects of OB on multiple regions of the CNS in the design of OB-targeted interventions to treat obesity.

PUBLICATIONS

Raber, J., Chen, S., Mucke, L., Feng, L. Corticotropin-releasing factor and adrenocorticotrophic hormone: Potential central mediators of OB effects. J. Biol. Chem. 272:15057, 1997.

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Expression and Regulation of Chemokines and Chemokine Receptors in Inflammation

Y. Xia, S. Chen, G.E. Garcia, L. Feng

Chemokines play essential roles in mediating leukocyte trafficking. Our laboratory has been studying the expression and regulation of chemokines and chemokine receptors. Expression of chemokines is thought to be regulated in part by the action of members of the NF-B/Rel family of transcription factors. In RelB-deficient mice, inflammation occurs in multiple organs. We speculated that chemokines might be involved in the infiltration of inflammatory cells into these inflamed organs.

To investigate the role of chemokines in mice, in collaboration with D. Lo, Department of Immunology, we studied fibroblasts and macrophages isolated from RelB-deficient and wild-type mice. We showed that RelB is an important regulator of chemokines. Stimulated fibroblasts showed dramatic persistent induction of nine chemokines. The persistent overexpression of chemokines in fibroblasts stimulated with lipopolysaccharide correlated with increased binding of NF-B and with increased levels of p50 and P65/RelA. Transfection of RelB cDNA into RelB-deficient fibroblasts reversed lipopolysaccharide-induced expression of chemokines. Further analysis showed that induction of the chemokines was at the transcriptional level. In contrast, expression of chemokines in RelB-deficient macrophages was reduced compared with the expression in wild-type macrophages, suggesting that the regulation of chemokine expression by RelB is cell specific. In vivo, activated RelB-deficient fibroblasts dramatically increased recruitment of granulocytes into tissues. In conclusion, expression of chemokines is negatively regulated by RelB in fibroblasts.

We have also studied another chemokine, SDF-1. Unlike the other inducible chemokines, SDF-1 is constitutively expressed at high levels in a broad range of organs. We found that SDF-1 was downregulated by lipopolysaccharide, IL-1ß, and TNF- but not by other chemokines. Our data indicate that SDF-1 and other inducible chemokines are differentially regulated by lipopolysaccharide and cytokines. The downregulation of SDF-1 coupled with the upregulation of the other chemokines might contribute to the disturbance of immune surveillance and homeostasis during inflammation and infection.

In collaboration with J. Harrison, University of Florida, Gainesville, we have isolated rat genes orthologous to those that code for human chemokine receptors CXCR1, CXCR2, CCR2, and CCR5 and have shown that these receptors are members of the rhodopsin G-protein--coupled receptor superfamily. Macrophage inflammatory protein-2 increases intracellular levels of calcium in HEK 293 cells expressing rat CXCR2, and CXCR2 mRNA can be detected in the lungs, spleen, and neutrophils in adult rats. In addition, mRNA homologous to that for human CXCR1 can be detected in rat lung, native rat macrophages, and a rat alveolar macrophage cell line. These data indicate cellular and tissue targets of rat CXC chemokine peptides.

Both CCR2 and CCR5 can be detected in rat spleen, lung, kidney, thymus, macrophages, and brain. Primary cultures of rat microglia expressed CCR5, and both cultured astrocytes and microglia contained mRNA for CXCR4 and for RBS11, another chemokine receptor--like gene, although we have not yet been able to identify the RBS11 by using immunostaining. Examination of cultured rat microglia treated with IFN- revealed dose-dependent upregulation of steady-state levels of CCR5 mRNA. Induction of experimental allergic encephalomyelitis in rats was accompanied by increased levels of CCR2, CCR5, CXCR4, and RBS11 mRNAs in the lumbar spinal cords of animals with clinical signs of the disease. These data indicate the rat orthologs of chemokine receptors and show that brain, spinal cord, and cultured glial cells express chemokine receptors that can be regulated in vitro and in vivo under pathologic conditions.

PUBLICATIONS

Dunstan, C.-A.N., Salafranca, M.N., Adhikari, S., Xia, Y., Feng, L., Harrison, J.K. Identification of two rat genes orthologous to the human interleukin-8 receptors. J. Biol. Chem. 271:32770, 1996.

Xia, Y., Pauza, M.E., Feng, L., Lo, D. RelB regulation of chemokine expression modulates local inflammation. Am. J. Pathol. 151:375, 1997.

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T-Cell Receptor Recognition of Antigen and Superantigen

N.R.J. Gascoigne, S.M. Alam, C. Haarstad, C. Lin, M.V. McGuire, S. Redpath, B.-C. Sim, T. Zal, P.J. Travers*

* University of London, London, England

Our group is interested in the structural basis of recognition by T cells during T-cell development and antigen recognition. The T-cell receptor (TCR) recognizes foreign antigens as fragments of protein presented on self-MHC proteins. During development in the thymus, T cells are subjected to both positive and negative selection by similar ligands.

AFFINITY OF THE TCR FOR POSITIVE- AND NEGATIVE-SELECTING PEPTIDES

MHC-peptide ligands that are antigenic for a particular T cell cause negative selection in the thymus of immature cells bearing the same TCR. Other peptide ligands related to the antigenic peptide can cause partial activation of the T cell. In mature T cells, these ligands act as partial agonists or antagonists, but in thymus cell cultures, they can cause positive selection. We previously determined the kinetics of binding of some of these MHC-peptide complexes to TCRs in solution. We found a threefold difference in affinity between the negative- and positive-selecting peptides, and the range from full activation to no activation covered about a 50-fold range in affinity. We have extended these studies to a natural ligand that causes positive selection but is too weak to act as an antagonist. This ligand has a measurable affinity that is lower than that of the known antagonists but higher than that of completely null ligands. Most interestingly, we have shown that strong agonists promote dimerization of the TCR, indicating a mechanism for enhancing signal transduction.

SELECTION OF VARIABLE REGIONS OF THE -CHAIN OF THE TCR

Individual variable regions of the -chain (V) of the TCR are expressed mainly in either CD4 or CD8 subsets of peripheral T cells. This situation could be due to preferential recognition of the MHC class I or class II molecules or to interaction between the V region and the CD4 or CD8 coreceptor. We used mutants of an -chain transgene to show that the selection specificity for MHC class I or class II molecules can be determined by a single amino acid residue in either complementarity-determining region 1 or complementarity-determining region 2. The position of these residues suggests that they interact with MHC and makes it unlikely that they interact with coreceptors.

We have sequenced the expressed members of the V3 and V11 families from mice hemizygous for the TCR -chain. These mice are the progeny of a cross between normal mice and -chain knockout mice and therefore can make only a single species of -chain per cell. With these mice, we can avoid the problem of lack of allelic exclusion in the expression of -chain mRNA. We found that individual members of each family showed preferential expression in CD4 or CD8 cells. The position of sequence differences between the V regions strengthens our conclusion that "skewed" expression of V regions is due to preferential recognition of MHC contact residues.

In collaboration with D. Lo's laboratory, Department of Immunology, we have investigated the effects of V polymorphism on the T-cell repertoire. It has long been known that the CD4/CD8 ratio in mice (and, indeed, in humans) is polymorphic. We analyzed the genetics of this polymorphism and found that a major contribution maps closely to the locus of the TCR -chain. This finding suggests that the V polymorphisms that determine a preference for MHC restriction have a wider effect on the relative size of the CD4 and CD8 T-cell pools.

Using a combination of mouse strains with mutant MHC molecules, we have evidence that the orientation of the TCR relative to the MHC may, in some cases, be 180° rotated from that found in the crystal structure.

ALLELIC EXCLUSION OF THE TCR -CHAIN

Allelic exclusion of the TCR -chain is poor. Many mature T cells express two mRNAs capable of encoding -chain proteins, and many express two proteins. However, expression of two -chains on the cell surface is quite rare. We showed that functional allelic exclusion is attained in the thymus at the time that the cell-surface expression of TCR is upregulated, after the start of positive selection. We have now shown that this exclusion is posttranslationally regulated. It is not caused by downregulation of one of the -chains at either the mRNA or the protein level. This finding suggests that phenotypic allelic exclusion depends on competition by the -chain proteins for pairing with the ß-chain.

RECOGNITION AND EXPRESSION OF SUPERANTIGEN

The superantigens are a functionally defined group of proteins that activate a large percentage of T cells. These proteins recognize one of the relatively low number of variable regions of the TCR ß-chain (Vß), rather than the almost infinite number of regular TCR combining sites. Superantigens are presented to the TCR after binding to MHC class II molecules. Different superantigens recognize different Vß elements, causing most or all of the cells bearing that particular Vß to be activated and then to die, with diverse pathologic effects.

Many different superantigens have been described, including those made by bacteria (e.g., staphylococci), mycoplasma, and viruses such as mouse mammary tumor virus. We have expressed fragments of the superantigen of mouse mammary tumor virus in yeast. Some of these recombinant proteins activate T cells in vitro. We are currently trying to measure the affinity of these superantigens for MHC class II molecules and for TCRs. The results will enable us to determine which sites on the superantigen are involved in the interaction with its ligands.

Additionally, we have expressed bacterial superantigens on the surface of bacteriophage. We have shown that the recombinant phage bind to MHC class II proteins and can activate T cells. We are using this technology to clone novel superantigens and to select for superantigens with novel binding properties.

PUBLICATIONS

Gascoigne, N.R.J. Natural killer cells: Influence of the home environment. Curr. Biol., in press.

Sim, B.-C., Travers, P.J., Gascoigne, N.R.J. V 3.2 selection in MHC class I mutant mice: Evidence for an alternate orientation of TCR-MHC class I interaction. J. Immunol., in press.

Wung, J.L., Gascoigne, N.R.J. Antibody screening for secreted proteins expressed in Pichia pastoris. BioTechniques 21:808, 1996.

Wung, J.L., Gascoigne, N.R.J. Selection of phage-displayed superantigen by binding to cell-surface MHC class II. J. Immunol. Methods 204:33, 1997.

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Homeostatic Control of Viruses

A. Angulo, H. Huang, J. LeBlanc, E. Staite, F. Garcia del Rey, D. Foster, J. Wittig, P. Ghazal

The goal of our work is to understand, from the molecular to the organismal level, the principles that drive and sustain lifelong infection by viruses. These infectious agents live in a dynamic equilibrium (homeostasis) with their hosts in which both immune and nonimmune pathways contribute to viral homeostasis. Disruption of these pathways can have dramatic consequences on pathogenesis. Indeed, those viruses that establish an incurable lifelong infection are often the most devastating in hosts that are immunosuppressed. For instance, human cytomegalovirus (CMV) rarely causes disease in healthy persons but is life threatening in immunodeficient patients and in infants congenitally infected with the virus. Approximately 80% of the adult population is infected with CMV. Our research focuses on natural and synthetic approaches for control of viral growth, and we use CMV infection as a clinically relevant model. This work primarily involves mechanistic studies on transcriptional control of the expression of viral genes and the application of cell biology and molecular genetics to viral pathogenesis.

CONTROL OF VIRAL GROWTH BY LIGANDS TO NUCLEAR HORMONE RECEPTORS

One pathway that influences CMV growth is the vitamin A (retinoic acid) signaling pathway. Retinoic acid transmits its information to a family of specific proteins in the nucleus of the cell by directly penetrating the membrane. We are carrying out detailed investigations of the mechanisms by which this hormone receptor family sets in motion biochemical reactions that modulate the growth of CMV. In this regard, chemistry has provided us with synthetic agonists, antagonists, and inverse agonists to the retinoid receptors that we use to probe the biology of CMV infection. At the biological level, we have developed in vitro and in vivo systems that range from analyzing molecular interactions in a test tube to working with models of infectious disease and transgenic animals. This work is providing new insights into growth-control mechanisms of viruses outside immunoregulatory pathways and potentially new ways of controlling viral disease.

A COUNTERBALANCE TO NATURAL IMMUNITY

The immune system provides an extremely hostile environment to viruses. Those viruses that establish a lifelong infection must evolve mechanisms to evade immune attack. In collaboration with Dr. Früh and colleagues at the R.W. Johnson Pharmaceutical Institute, we have shown that CMV encodes glycoproteins resident in the endoplasmic reticulum that sequentially prevent intracellular transport of MHC class I molecules. Moreover, this work suggests that CMV may use different T-cell escape strategies at different times during the infectious cycle. Use of specific drugs that interfere with the function of these virally encoded proteins should allow the immune system to better protect against CMV infection.

SPECIFIC RECOGNITION OF DNA BY ANTIBODIES

In collaboration with D. Burton's group at TSRI, we are exploiting the diversity and specificity of the immune system by molecularly evolving antibodies to recognize specific sequences of DNA. We have generated antibodies with a significant degree of base recognition. These results show that the modular nature of immunoglobulins and transcription factors can be exploited to create unique DNA-binding proteins by molecular design. Ongoing studies are aimed at determining whether these antibodies can block transcription of genes selected on the basis of our studies with a viral repressor protein. In the future, these antibodies could be used to inhibit viral growth.

PUBLICATIONS

Ahn, K., Angulo, A., Ghazal, P., Peterson, P.A., Yang, Y., Früh, K. Human cytomegalovirus inhibits antigen presentation by a sequential multistep process. Proc. Natl. Acad. Sci. U.S.A. 93:10990, 1996.

Ghazal, P., Leblanc, J.F., Angulo, A. Vitamin A regulation of viral growth. Rev. Med. Virol. 7:21, 1997.

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The p38 MAP Kinase Pathway in the Innate Immune System

Y. Jiang, M. Zhao, L. New, J. Han

The p38 MAP kinase signaling pathway is thought to play an important role in cells of the innate immune system during maturation and responses to infection. During the past year, we cloned three new p38 isoforms that have more than 60% sequence identity to p38. All four p38 isoforms have been categorized as p38 group MAP kinases. This group of kinases is thought to have evolved from a single yeast gene, which regulates cellular responses to extracellular stress, including hyperosmolarity and oxidation. In mammalian cells, these closely related p38 isoforms appear to be often activated coordinately by a broad panel of stimuli that include physicochemical stresses and proinflammatory cytokines. Two activators of the p38 group MAP kinases, MKK3 and MKK6, which catalyze the dual phosphorylation at the canonical threonine-glycine-tyrosine site, have been cloned simultaneously by our group and by others. We view the p38 pathway as a discrete cassette consisting of MKK3 and MKK6 and the four p38 isoforms.

Although the p38 isoforms share a high level of amino acid sequence identity and respond to similar stimuli, our findings of different tissue distributions and different substrate specificities suggest the functional differentiation of this group of kinases in mammals. The biological consequence of the activation of different p38 isoforms is under investigation.

Studies with the p38-specific inhibitors (bicyclic imidazole compounds) and studies measuring p38 activation associated with cytokine production provided data that support a role of p38 in cytokine biosynthesis. It is thought that p38 is involved in both transcriptional and translational regulation of the expression of cytokine genes. Research has shown that a group of serine/threonine protein kinases, including MAPKAPK2, MAPKAPK3 (3pK), Mnk1, and Mnk2, are substrates for p38. In addition, p38 may regulate transcription factors, such as ATF2 and CHOP10.

We have shown by several approaches that the transcription factor MEF2C is a physiologic substrate for p38. Phosphorylation of MEF2C increases its transactivation activity. One consequence of MEF2C activation is an increase in the transcription of the gene for c-Jun. p38-Enhanced expression of c-Jun might contribute to the binding activity of activator protein-1 (AP-1) or the AP-1/CRE-like element. Thus, p38 may, via MEF2C, play an important role in controlling the balance of cytokine production, because cytokine genes typically have AP-1/CRE-like elements.

We have also examined the potential role of the p38 pathway in T-cell apoptosis. We have shown that activation of MKK6 is necessary and sufficient for Fas-induced apoptosis. MKK6 activation occurs downstream of proteases similar to interleukin-1ß convertase. Activation of MKK6 leads to activation of proteases similar to CPP32and of other proteases similar to interleukin-1ß convertase. Although p38 appears to be activated by MKK6, its activity has no significant effect on T-cell apoptosis, suggesting the existence of other protein substrates downstream of MKK6. These findings indicate a divergence of signals downstream of MKK6; one that activates p38 and regulates gene expression and one that activates the interleukin-1ß convertase/Ced-3 family of proteases and leads to cell death. Currently, part of our focus is the mechanism of signal transduction between kinases and proteases.

PUBLICATIONS

Chen, C., Zhang, B., Han, J., Lin, S.-C. Characterization of a novel mammalian RGS protein that binds to G proteins and inhibits pheromone signaling in yeast. J. Biol. Chem. 272:8679, 1997.

Han, J., Jiang, Y., Li, Z., Kravchenko, V.V., Ulevitch, R.J. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 386:296, 1997.

Han, J., Wang, X., Jiang, Y., Ulevitch, R.J., Lin, S. Identification and characterization of a predominant isoform of human MKK3. FEBS Lett. 403:19, 1997.

Huang, S., Jiang, Y., Li, Z., Nishida, E., Mathias, P., Lin, S., Ulevitch, R.J., Nemerow, G.R., Han, J. Apoptosis signaling pathway in T cells is composed of ICE/Ced-3 family proteases and MAP kinase kinase 6b. Immunity 6:739, 1997.

Jiang, Y., Li, Z., Schwarz, E.M., Lin, A., Guan, K., Ulevitch, R.J., Han, J. Structure-function studies of p38 mitogen-activated protein kinase. J. Biol. Chem. 272:11096, 1997.

Li, Z., Jiang, Y., Ulevitch, R.J., Han, J. The primary structure of p38: A new member of p38 group of MAP kinases. Biochem. Biophys. Res. Commun. 228:334, 1996.

Su, Y.-C., Han, J., Xu, S., Cobb, M.. Skolnik, E.Y. NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain. EMBO J. 16:1279, 1997.

Wang, Z., Harkins, P.C., Ulevitch, R.J., Han, J. Cobb, M.H., Goldsmith, E.J. The structure of mitogen-activated protein kinase p38 at 2.1-Å resolution. Proc. Natl. Acad. Sci. U.S.A. 94:2327, 1997.

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Development and Function of Intraepithelial T Cells

W.L. Havran, K. Albers, Y. Chen, T. Wu

This laboratory has a long-term interest in interactions between intraepithelial T cells and neighboring epithelial cells. We have focused our studies on interactions in the thymus, skin, and intestine. We are investigating the development, specificity, and function of these T cells. Our results have defined unique properties of these cells and support a specialized role for epithelial T cells in immune surveillance, wound repair, inflammation, and protection from malignant tumors.

CD81 IN T-CELL DEVELOPMENT AND FUNCTION

CD81 is a recently described member of the four transmembrane spanning family of membrane proteins. It is broadly expressed and can associate with other cell-surface molecules in a cell type--specific manner. No clear function has been defined for CD81 or other members of this family. We have shown that interactions between CD81 on thymic epithelial cells and immature thymocytes occur during a discrete stage of development. In addition, CD81 is expressed by epithelial cells in the skin and intestine and plays a role in the activation of intraepithelial T cells. CD81 is also expressed by mature ß T cells in lymphoid organs and provides costimulatory activity when coligated with the antigen receptor. These results indicate that CD81 can play diverse roles in T-cell development and function by tissue- and cell type--specific associations. Our future studies will focus on unraveling some of the complexities of this molecule.

ANTIGENS FOR T CELLS

Close physical interactions between T cells and neighboring epithelial cells suggested the possibility of functional interactions between these two populations. We showed that T cells in the skin monitor neighboring keratinocytes for signs of damage or disease. If a problem is detected, the T cells secrete growth factors and chemokines that contribute to tissue repair and direct recruitment of other cell types to sites of trauma. We have isolated the antigen for the skin T cells from damaged keratinocytes. Most ß T cells recognize peptide antigens derived from foreign proteins and presented by self-MHC molecules. In contrast, the antigen is nonpeptidic and does not require MHC molecules for presentation. Experiments are in progress to further define this novel antigen. The recognition of unique antigens and the ability to perform specialized functions support a novel immunologic role for intraepithelial T cells.

A ROLE FOR INTRAEPITHELIAL T CELLS IN EPITHELIAL INFLAMMATION

Our previous studies indicated that intraepithelial T cells in the skin and intestine inducibly produce the epithelial growth factor FGF-7. In addition, these cells produce a panel of cytokines and chemokines that may recruit inflammatory cells and modulate the growth and function of neighboring cells. We proposed that intraepithelial T cells may play an important role in epithelial homeostasis in normal and disease conditions.

In collaboration with R. Boismenu, Department of Immunology, we analyzed the role of intestinal T cells in a murine model of colitis. In this model system, focal areas of inflammation appear in the large intestine after animals are fed dextran sodium sulfate. We found increased numbers of T cells at these sites and have evidence that the cells are activated and are producing cytokines and chemokines locally. In particular, using in situ hybridization, we showed that the T cells in the animals with colitis produce higher levels of FGF-7 than do T cells in control animals.

In collaboration with the Strohm Inflammatory Bowel Disease Center at Scripps Clinic, we analyzed tissue and blood samples from patients with ulcerative colitis or Crohn's disease. We found that these patients have greater numbers of T cells both in blood and at sites of active disease than do healthy subjects or patients with cancer. In addition, we found that levels of FGF-7 in the patients with ulcerative colitis or Crohn's disease were higher at sites of inflammation than at uninvolved sites. These results validate the mouse model of colitis. Our future studies should provide information to further define the role of T cells in epithelial inflammatory disorders and may be useful in designing therapies.

PUBLICATIONS

Boismenu, R., Havran, W.L. T cells in host defense and epithelial cell biology. Clin. Immunol. Immunopathol., in press.

Boismenu, R., Havran, W.L. An innate view of T cells. Curr. Opin. Immunol. 9:57, 1997.

Boismenu, R., Hobbs, M.V., Boullier, S., Havran, W.L. Molecular and cellular biology of dendritic epidermal T cells. Semin. Immunol. 8:323, 1996.

DeKoning, J., DiMolfetto, L., Reilly, C., Wei, Q., Havran, W.L., Lo, D. Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice. J. Immunol. 158:2558, 1997.

Havran, W.L. Dendritic epidermal T cells. Semin. Immunol. 8:313, 1996.

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Molecular Cloning of Guinea Pig and Rat C3a Receptors

Y. Fukuoka,* J.A. Ember, T.E. Hugli

* Tohoku University, Sendai, Japan

The anaphylatoxin C3a is a potent spasmogen and mediates many inflammatory reactions. The gene for the receptor for human C3a (C3aR) was recently cloned. Previously, we showed a C3a-specific receptor on guinea pig platelets. We isolated cDNA clones homologous to human C3aR from guinea pig spleen and rat lung. A human C3aR probe was used to screen each cDNA library. The guinea pig and rat C3aRs consisted of 475 and 473 amino acids, respectively.

Hydropathy analysis of the deduced protein sequence of guinea pig and rat C3aR clones indicated seven transmembrane domains with a large extracellular loop between domains 4 and 5. The large second extracellular loop is a unique feature of human C3aR; it is not found in other G-protein--coupled receptors. Comparison of guinea pig and rat C3aR with human C3aR revealed 59% sequence identity. The sequence of the large second extracellular loop in guinea pig and rat C3aR was only 26% and 19% identical, respectively, with the loop in human C3aR. L cells stably transfected with a mammalian expression vector encoding the guinea pig or rat C3aR showed specific binding of C3a. Cloning of C3aR from different species should help determine critical extracellular regions or residues required for ligand binding and identify residues on the cytoplasmic C-terminal domain of C3aR involved in G-protein interactions.

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Immunomodulatory Effects of C3a on Purified Peripheral Blood Monocytes

M.A. Jagels, W.H. Fischer, T.E. Hugli

The anaphylatoxin C3a has both stimulatory and suppressive effects on a number of lymphoid cells, depending on the cell type and the parameters of activation measured. C3a suppresses polyclonal and antigen-specific antibody responses in peripheral blood mononuclear cells and in isolated resting tonsilar B cells but enhances the production of inflammatory cytokines by peripheral blood mononuclear cells stimulated with lipopolysaccharide.

Using a rabbit polyclonal antibody generated against the C3a receptor, we found that expression of the receptor is limited to CD14+ monocytes in peripheral blood mononuclear cells. We therefore investigated the effects of C3a on isolated peripheral blood monocytes, both alone and in combination with lipopolysaccharide. C3a alone had no effect on cytokine production by isolated monocytes. When monocytes were stimulated with lipopolysaccharide, both intact C3a and its circulating desArg form suppressed production of the proinflammatory cytokines IL-1, TNF-, and IL-6 in a dose-dependent manner. In contrast, lipopolysaccharide-induced production of the antiinflammatory cytokine IL-10 was enhanced in monocytes by cotreatment with C3a. Suppression of the production of inflammatory cytokines was not secondary to the enhanced production of IL-10, because blocking antibodies to IL-10 did not reverse the effects of C3a.

Because C3a reportedly enhances production of prostaglandin E2 by monocytes, and because prostaglandin E2 can suppress production of cytokines by monocytes, we investigated the potential role of arachidonate metabolites in C3a-mediated immunosuppression. Neither indomethacin, a cyclooxygenase inhibitor, nor 3-(4-octadecyl)-benzoylacrylic acid, a phospholipase A2 inhibitor, reversed the effects of C3a, suggesting that the actions of C3a are independent of prostaglandin formation by isolated monocytes. Results of Northern blot analysis and kinetic studies of cytokine release suggest that the effects of C3a may occur at both transcriptional and posttranscriptional levels. These studies suggest that C3a may play an important immunomodulatory role after its generation during acute inflammatory reactions.

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Regulation of B-Cell Functions by C3a and C3adesArg

W.H. Fischer, T.E. Hugli

Regulation of the humoral immune response by fragments of complement component C3 has been shown, indicating that this molecule may play an important role in host defense. We investigated whether human tonsil-derived B cells express the receptor for C3a (C3aR) and examined the effects of C3a and C3adesArg on the production of IgG, TNF-, and IL-6 by B cells activated by Staphylococcus aureus Cowan strain I (SAC)/IL-2.

We found that human B cells express a C3aR that is indistinguishable at the protein or the mRNA level from the C3aR found in human neutrophils and in C3aR-transfected mouse L cells. Incubation of SAC/IL-2--
activated B cells with C3a or C3adesArg resulted in a dose-dependent suppression of the polyclonal immune response. Under the same experimental conditions, the release of IL-6 and TNF- was also suppressed in a dose-dependent manner. Kinetic studies with SAC/IL-2--activated B cells revealed that C3a must be present at the start of the culture to exert its suppressive effect on the production of IgG and IL-6. These results show that tonsil-derived B cells express C3aR and that both C3a and C3adesArg have a direct immunomodulatory effect on these cells.

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Enhancement of Neutrophil Survival by Multiple Factors Derived from Epithelial Cells

P.J. Daffern, M.A. Jagels, W.H. Fischer, T.E. Hugli

Neutrophils are predominant cells in acute inflammatory sites and may have enhanced functional properties and prolonged survival in these sites. We used primary cultures of respiratory epithelial cells to examine the factors that contribute to neutrophil activation and survival in the airways. Supernatants derived from unstimulated or TNF---stimulated epithelial cells were cultured with peripheral blood neutrophils isolated from healthy donors. Survival of neutrophils cultured in the presence of supernatants derived from both unstimulated and stimulated epithelial cells was significantly greater than that of neutrophils cultured in medium alone.

Epithelial cells were examined for the production of factors that may influence neutrophil survival. Of the factors that enhance neutrophil apoptosis, neither TNF- nor IL-10 transcripts were detected in unstimulated epithelial cells or in the supernatants from TNF---stimulated epithelial cells. Of the factors that enhance survival, mRNA for IL-1ß, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF), but not IFN- or granulocyte colony-stimulating factor, was detected, and production of the corresponding proteins was confirmed by enzyme-linked immunosorbent analysis. Of two additional factors that enhance survival, platelet-activating factor was not detected in the supernatants, whereas as prostaglandin E2 was produced in modest amounts. When neutrophils were tested for enhancement of survival in the presence of purified factors alone, IL-6, GM-CSF, and prostaglandin E2, but not IL-1, promoted survival over the range of concentrations detected in the supernatants. Additional studies confirmed that these factors delay neutrophil apoptosis. A combination of neutralizing antibody to GM-CSF and IL-6 inhibited enhancement of neutrophil survival induced by the epithelial cell supernatant. When used separately, antibody to GM-CSF inhibited the majority of the enhanced survival activity, whereas neutralizing antibody to IL-6 alone had only modest effects.

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Characterization of C3a Receptors from Various Species

J.A. Ember, P.J. Daffern, Y. Fukuoka,* M. Hsu, R.D. Ye, T.E. Hugli

* Tohoku University, Sendai, Japan

The gene for the receptor for human C3a (C3aR) has been cloned. It encodes a G-protein--coupled heptahelical receptor with a unique extracellular loop of approximately 170 amino acids between the fourth and fifth transmembrane domains. Our recent investigations focused on expression and function of C3aR on various human cell types, such as neutrophils and eosinophils.

Both neutrophils and eosinophils have high levels of expression of C3aR. Ligand-binding assays indicated a Kd of 1.37 ± 0.66 nM and 9000--23,000 binding sites per cell for neutrophils and a Kd of 4.45 ± 6.57 nM and 63,000--85,000 binding sites per cell for eosinophils. Both cell types reacted to C3a and synthetic C3a analogs in the calcium mobilization assay, but only eosinophils reacted to these factors in oxygen production and in Mac-1 upregulation assays.

The difference in functional behavior for these two cell types cannot be fully explained on the basis of the modest differences in receptor number or Kd values we observed. These observations suggest significant differences in functional reactivity of these two cell types to C3a that may be due to differences in their signaling mechanisms. Our further investigations will explore this possibility.

The molecular cloning, sequencing, and functional analyses of C3aR and of the receptor for C5a (C5aR) from various species has been completed. In collaboration with R.D. Ye's group, Department of Immunology, mouse C3aR has been cloned and analyzed. In collaboration with Y. Fukuoka, Tohoku University, Sendai, Japan, rat and guinea pig C3aR and rat and guinea pig C5aR have been cloned.

All these C3aR receptors contain the unique extracellular loop of approximately 170 amino acids between the fourth and fifth transmembrane domains described first for human C3aR. In addition, sequence-homology analysis comparing the five known C3aRs enabled us to determine structurally and functionally important regions of these receptors. Similar advantages are expected from the recent molecular cloning of rat and guinea pig C5aR.

PUBLICATIONS

Akatsu, H., Miwa, T., Sakudara, C., Fukuoka, Y., Ember, J.A., Hugli, T.E., Okada, H. cDNA cloning and characterization of rat C5a anaphylatoxin receptor. Microbiol. Immunol. 41:575, 1997.

Ember, J.A., Jagels, M.A., Hugli, T.E. Characterization of complement anaphylatoxins and their biological responses. In: The Human Complement System in Health and Disease. Volanakis, J., Frank, M. (Eds.). Marcel Dekker, New York, in press.

Fischer, W.H., Hugli, T.E. Regulation of B cell functions by C3a and C3adesArg: Suppression of TNF-, IL-6, and the polyclonal immune response. J. Immunol., in press.

Hetland, G., Hugli, T.E. Effect of 125I-labeled C5a on U937 cells in vitro: A model for cytotoxicity. Cancer Lett. 110:97, 1996.

Hsu, M.H., Ember, J.A., Wang, M., Prossnitz, E.R., Hugli, T.E., Ye, R.D. Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene. Immunogenetics, in press.

Hugli, T.E. Complement and inflammation: Inhibiting complement proteases/factors. Trends Biotechnol. 14:409, 1996.

Mulligan, M.S., Schmid, E., Till, G.O., Friedl, H.P., Hugli, T.E., Johnson, K.J., Ward, P.A. Requirement and role of C5a in acute lung inflammatory injury in rats. J. Clin. Invest. 98:503, 1996.

Mulligan, M.S., Schmid, E., Till, G.O., Hugli, T.E., Friedl, H.P., Roth, R.A., Ward, P.A. C5a-dependent upregulation in vivo of lung vascular P-selectin. J. Immunol. 158:1857, 1997.

Pfeifer, P.H., Hugli, T.E., Davie, E.W., Fujikawa, K. Complement activation in EDTA blood/plasma samples may be caused by coagulation proteases. In: Techniques in Protein Chemistry VIII. Crabb, J.W., Marshak, D.R. (Eds.). Academic Press, San Diego, in press.

Schmid, E., Warner, R.L., Crouch, L.D., Friedl, H.P., Till, G.O., Hugli, T.E., Ward, P.A. Neutrophil chemotactic activity and C5a following systemic activation of complement in rats. Inflammation 21:325, 1997.

Tanaka, F., Dannenberg, A.M., Jr., Higuchi, K., Nakamura, M., Pula, P.J., Hugli, T.E., DiScipio, R.G., Wagner, J.L., Kreutzer, D.L. Chemotactic factors are continuously released by cultured intact developing and healing skin lesions produced in rabbits by sulfur mustard. Inflammation 21:251. 1997.

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Mechanisms of Cellular Signaling by Ras and Rac Proteins

J.H. Jackson, M.K. Jones, J.K. Voice, D. Warnock, V. Hill, Q. Zhang, Y. Fu

MECHANISMS OF RAS SIGNALING

Ras proteins lie at the heart of signal transduction pathways that link cell-surface receptors to the nucleus. Diverse extracellular ligands, including growth factors, hormones, antigens, interleukins, and trophic factors, activate their respective receptors. The activated receptors induce a guanine nucleotide exchange factor (GEF) to convert inactive GDP-bound Ras to active GTP-bound Ras, activated Ras subsequently binds one or more effector proteins, and the activated effector initiates a signaling cascade that ultimately induces cell growth or differentiation. After stimulation, active Ras-GTP is normally hydrolyzed back to inactive Ras-GDP. However, mutant Ras proteins that have lost the ability to hydrolyze bound GTP to GDP are constitutively active and are thought to contribute to carcinogenesis.

Although many of the components involved in Ras-mediated signal transduction have been elucidated, many critical questions remain. For instance, human cells contain four homologous Ras proteins: H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B. Despite many years of intensive study, it is still not known whether these four homologs have different biological functions or participate in distinct signaling pathways.

Recently, we showed that one GEF, Ras-GRF, selectively activates H-Ras but not N-Ras or K-Ras 4B in vivo. Our studies provide the first biochemical evidence that a Ras GEF can selectively activate a single Ras homolog in vivo and suggest that extracellular ligands that exclusively bind Ras-GRF--dependent receptors can transduce signals through H-Ras but not N-Ras or K-Ras 4B. In addition, we have preliminary evidence that the four Ras homologs have substantially different oncogenic potencies. Our combined results are consistent with the notion that each Ras homolog may participate in distinct signaling cascades and may have different biological functions. We are assessing whether extracellular ligands or other Ras GEFs, such as SOS, show Ras homolog specificity in vivo, and we are investigating whether each of the Ras homologs activates a different effector or a different transcription factor in vivo.

In addition, because the four Ras proteins are essentially identical except for their 25 C-terminal amino acids, the hypervariable domain, we are evaluating whether residues within this domain dictate which Ras homolog is activated by, or activates, a given GEF or effector protein, respectively. Preliminary studies suggest that a stretch of charged residues within the hypervariable domain of K-Ras 4B is crucial for K-Ras 4B biological activity, GEF specificity, and effector activation.

Future studies should greatly improve our understanding of Ras-mediated signal transduction and could enable us to discern whether each Ras homolog has unique biological functions. In addition, our studies could form the theoretical basis for the development of Ras homolog--specific inhibitors that could be useful in the treatment of cancer and many other medical disorders.

MECHANISMS OF RAC SIGNALING

Rac 1 is a member of the Ras superfamily of GTP-binding proteins. Rac 1 regulates the activity of the superoxide anion--generating NADPH oxidase system of phagocytes, plays a central role in organization of the actin cytoskeleton, and is essential for Ras-induced transformation. In addition, mutant, constitutively active Rac-1 can induce cellular transformation, invasion, and metastasis. Similar to Ras proteins, Rac 1 is activated by an upstream GEF and binds an effector protein that signals downstream. Human cells contain two homologous Rac proteins: Rac 1 and Rac 2. Like Ras proteins, these Rac proteins are essentially identical except for their hypervariable C-terminal domains.

In collaboration with U. Knaus, Department of Immunology, we recently found that Rac 1 activates the effector protein PAK 65 far more efficiently than Rac 2 does. Interestingly, Rac 1, but not Rac 2, contains a stretch of charged residues within its hypervariable domain that is virtually identical to the aforementioned stretch of charged residues in the hypervariable domain of K-Ras 4B. In collaboration with P. Heyworth, Department of Molecular and Experimental Medicine, M. Schwartz, Department of Vascular Biology, and U. Knaus, we found that similar to the case with K-Ras 4B, these residues are crucial for Rac 1 biological activity and effector activation. Moreover, we found that these charged residues directly account for the discrepant ability of Rac 1 vs Rac 2 to activate PAK 65.

We are now assessing whether the two Rac homologs have different biological functions, selectively activate different effectors, or are activated by different GEFs in vivo. These studies should enable us to determine whether Rac proteins participate in homolog-specific signal transduction pathways and could ultimately facilitate the development of Rac homolog--specific inhibitors useful in the treatment of cancer, inflammatory diseases, and so forth.

PUBLICATIONS

Jackson, J.H., Vollenweider, M., Hill, J., Rodriquez, H., Schwabacher, A.W., Mitra, G., Kuo, C. Stimulated human leukocytes cause activating mutations in the k-ras proto-oncogene. Oncogene 14:2803, 1997.

Jones, M.K., Jackson, J.H. Ras-GRF activates H-Ras, but not N-Ras or K-Ras 4B, protein in vivo. J. Biol. Chem., in press.

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Regulation of T-Cell Development in the Thymus

J. Kaye, H. Shao, L.-Y. Chen, E.M. Rubin, D.H. Kono

Although many cell-surface markers have been described that delineate the stages of T-cell development in the thymus, little is known about the underlying mechanisms that regulate these events. This situation is particularly true for positive selection, a process in which developing thymocytes are driven to differentiate. In this process, antigen-specific T-cell receptors mediate recognition of MHC-encoded molecules expressed by thymic epithelial cells. Our laboratory is interested in the early events of this differentiation, both at the level of the cellular interaction between thymocytes and epithelial cells and at the molecular level, where we are investigating signaling and changes in gene expression in responding thymocytes.

To facilitate the study of positive selection, we developed a precursor CD4+CD8+ thymocyte cell line, designated DPK, from cells obtained from a mouse transgenic for T-cell receptors. This cell line can be induced to differentiate by stimulation with specific peptide antigen and antigen-presenting cells or by thymic epithelial cells in the absence of an exogenous source of antigen. We are using this system to investigate various aspects of T-cell development in the thymus.

ROLE OF THYMIC EPITHELIAL CELLS IN THYMOCYTE DIFFERENTIATION

Positive selection requires the interaction of immature thymocytes with resident cortical epithelial cells. We have established culture conditions in which isolated thymic epithelial cells can induce the differentiation of DPK cells. We have also isolated a conditionally immortalized thymic epithelial cell line that maintains the ability to induce differentiation of DPK cells. Evidence suggests that non-MHC proteins are involved in this process. We are using various approaches, including subtractive hybridization and the production of monoclonal antibodies, to take advantage of this system in identifying proteins on the surface of thymic epithelial cells that play a role in positive selection.

REGULATION OF GENE EXPRESSION DURING THYMIC SELECTION

Little is known about the regulation of gene expression during the differentiation of immature thymocytes. We have used representational difference analysis to detect early changes in gene expression during the differentiation of DPK cells. With this method, we have determined that the immediate-early gene Egr-1, which encodes a zinc-finger transcription factor, is a possible mediator between signals on the cell surface and changes in gene expression during positive selection. We have further shown that the Egr family of transcriptional regulators is coordinately upregulated in normal thymocytes as a result of activation and differentiation mediated by T-cell receptors. We found that induction of the Egr gene family depends on Ras and calcineurin signaling pathways, as does positive selection. We are exploring the functional role of these transcription factors in thymic selection and peripheral T-cell activation and are using this system to detect other genes that regulate this receptor-mediated differentiation event.

SIGNALING PATHWAYS IN POSITIVE SELECTION

Evidence suggests that both the Ras and the calcineurin signaling pathways are required for positive selection. Using the cell culture system described here, we have been investigating the specific role of these signaling pathways in regulating the changes in gene expression that accompany maturation of thymocytes mediated by T-cell receptors. Expression of dominant negative and constitutively active mutant forms of Ras in DPK cells has enabled us to determine the specific downstream consequences of Ras signaling during positive selection. The results showed a role for Ras in the downregulation of CD8 coreceptors, the induction of early markers of positive selection, and the upregulation of integrin expression. Other experiments are under way to determine the contribution of these pathways to the commitment of thymocytes to CD4 and CD8 lineages.

PUBLICATIONS

Shao, H., Kono, D., Chen, L.-Y., Rubin, E., Kaye, J. Induction of the early growth response (Egr) family of transcription factors during thymic selection. J. Exp. Med. 185:731, 1997.

DeKoning, J., Kaye, J. Requirements for differentiation of a CD4+8+ T-cell line. Dev. Immunol., in press.

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Regulation of B-Cell Development in Young and Aged Mice

N.R. Klinman, G.H. Kline, M.K. Slifka, N.B. Leaf, S.J. Zaharevitz, T.A. Hayden, M. Klyusner

IMMUNOGLOBULIN HEAVY CHAIN--SPECIFIC SELECTION IN B-CELL DEVELOPMENT

Development of B cells depends on a highly regulated progression of sequential molecular and cellular events. Initially, on commitment of early progenitor cells to the B-cell lineage, cells express a variety of cell-surface and intracellular proteins. Among the intracellular proteins are Ig and Igß, which ultimately form part of the cell surface--transmembrane triggering complex, and VpreB and lambda5, two polypeptides that cojointly bind nascent heavy (H) chains to form a light chain--like complex called the surrogate light chain (SL).

Expression of immunoglobulin is initiated by the rearrangement on both chromosomes of D and JH segments of the gene for the variable (V) region of the H chain; this step is followed by VH to DJH rearrangement. As a consequence of the recombination of these gene segments to form the V region, only one third of all VH-D-JH rearrangements are productive, that is, yield an intact H chain. However, among the productive VH-D-JH rearrangements, enormous diversity is created by the assortment of 102--103 structurally distinct VH gene segments, 13 distinct D gene segments, and 4 JH gene segments and a recombinational process that both adds and deletes nucleotides at both the D-JH and the VH-D junctions. Production of a nascent H chain is a pivotal event in B-cell development, because a complex of H chains, SLs, Ig, and Igß acts as a pre-B cell receptor that is essential for clonal maturation and expansion.

Several years ago, we found that certain categories of H chains were apparently less efficient than others in fostering clonal maturation. This finding led us to predict that because of the great structural variability created by the imprecise joining of such diverse sets of gene segments, many H chains would fold improperly and be dysfunctional in their ability to foster clonal maturation. We also suggested that the ability to assemble with SLs might be a quality control mechanism for H chains. This mechanism would ensure that only those B cells with H chains that could fold properly and ultimately assemble with light chains would clonally expand.

Recently, H.-M. Jäck and colleagues at Loyola University, Chicago, described two H chains that could not assemble with SLs. In collaboration with Dr. Jäck, we generated transgenic mice that express one of these H chains. Our findings showed that cells that express the transgene-encoded H chain as their only H chain could not mature beyond the pro-B cell stage. Thus, the SL does act both as an integral component of the pre-B cell receptor and as a quality control mechanism that ensures that only H chains that can assemble with light chains can foster clonal maturation.

B-CELL DEVELOPMENT IN AGED MICE

Aged mice maintain normal levels of mature peripheral B cells, but the population of pre-B cells in the bone marrow is markedly reduced. Some researchers have suggested that despite the reduction in pre-B cells, the populations of newly generated bone marrow and splenic B cells are not markedly reduced. To investigate this issue, we gave aged and young mice bromodeoxyuridine (BrdU) in their drinking water for 3--8 days and determined the number of cells in various B-cell subsets that had divided (incorporated BrdU) during that time.

Our results indicated that the proportion of total BrdU-labeled cells in the spleen was substantially less in aged mice than in young mice even after 8 days. This finding suggests that even among mature B cells, turnover is much slower in aged mice than in young mice. In addition, although spleen cells that phenotypically appear to be comparable to newly generated splenic B cells in young mice (newly emerged from the bone marrow and highly BrdU labeled) are poorly labeled in aged mice, suggesting that newly generated splenic B cells are rare in aged mice.

Consistent with previous findings, the population of pre-B cells was more markedly reduced in aged mice than was the population of immature sIg+ B cells. However, whereas in young adult mice most cells within the immature B-cell compartment accumulated BrdU within 2--3 days, most of these cells in aged mice were not labeled until after 6 days. Thus, it appears that in aged mice most peripheral B cells are very long-lived and replenishment from the bone marrow is very slow. Whether the decrease in the rate of generation and peripheralization of B cells in aged mice is due to feedback from the extremely stable mature B-cell pool or alternatively is responsible for the slow turnover of peripheral cells has yet to be determined.

PUBLICATIONS

Klinman, N.R. B-cell activation: Immunology. In: Encyclopedia of Human Biology, 2nd ed., vol. 2. Dulbecco, R. (Ed.). Academic Press, Orlando, 1997, p. 683.

Klinman, N.R. The cellular origins of memory B cells. Semin. Immunol., in press.

Klinman, N.R. The 'clonal selection hypothesis' and current concepts of B-cell tolerance. Immunity 5:189, 1996.

Klinman, N.R. Repertoire diversification of primary vs memory B cell subsets. Curr. Top. Microbiol. Immunol., in press.

Linton, P.-J., Haynes L., Klinman, N.R., Swain, S.L. Antigen dependent and independent changes in CD4 T cells with aging. J. Exp. Med. 184:1891, 1996.

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Cellular Functions of Kinases Regulated by GTP-Binding Proteins

U.G. Knaus, A. Reilly, Y. Wang, P. Hall

Protein kinases and phosphatases play critical roles in regulating and coordinating aspects of gene expression, cellular growth and motility, differentiation, and division. As a result, normal cellular function depends not only on the catalytic activity of protein kinases and phosphatases but also on regulatory proteins or second messengers capable of switching the kinases and phosphatases on and off. A class of serine/threonine protein kinases known as p21-activated kinases (PAKs) are activated in response to the Rho family GTPases Rac and Cdc42. Rac and Cdc42 regulate signaling pathways involved in cytoskeletal reorganization, stress-induced activation of kinases, transformation of cells, and so forth. Our laboratory studies how GTPase effector protein kinases such as PAKs mediate the functions of Rac and Cdc42.

GTPase-dependent and GTPase-independent activation of PAKs has been one focus of our recent work. In collaboration with J. Jackson, Department of Immunology, we have detected critical regions on Rac GTP-binding proteins and on PAK itself that determine the interaction of both proteins and are crucial for biological activity. Structural studies of unstimulated and activated PAK have been started in collaboration with G.S. Prasad, Department of Molecular Biology.

We are also interested in processes in phagocytic leukocytes that are regulated by GTP-binding proteins and that can compromise the human immune response in inflammatory or infectious diseases. Rac and PAKs are implicated in a variety of leukocyte functions, ranging from production of reactive oxygen species to chemotaxis and phagocytosis. Stimulation of leukocytes by chemotactic factors or phagocytic stimuli leads to the generation of superoxide via a Rac-dependent NADPH oxidase.

We have detected several PAKs in leukocytes and have established the activation of these kinases by chemoattractants as well as by activated Rac. In collaboration with J. Johnson and B. Babior, Department of Molecular and Experimental Medicine, we are investigating the role of PAK in the NADPH oxidase activation leading to the generation of superoxide. Determination of (1) PAK-dependent phosphorylation of components of NADPH oxidase and (2) possible PAK-dependent protein-protein interactions that precede the production of superoxide will enable us to define relevant PAK targets. Further studies are directed at the involvement of PAKs in other neutrophil-specific functions such as adhesion and phagocytosis.

PAKs are also integral elements of MAPK cascades leading to the activation of transcription factors. Preliminary data suggest a loss of regulatory control in these kinase cascades during tumor pathogenesis in breast cancer cells. Investigations on the contribution of PAK hyperactivation to transformation and invasiveness are under way. PAK is involved in cytoskeletal rearrangements and actin polymerization, which are prerequisites for shape changes and cell motility. Consequently, during malignant events, PAK might be involved not only in tumor progression but also in tumor metastasis. These issues will be addressed in ongoing collaborative efforts with G. Bokoch, Departments of Immunology and Cell Biology.

PUBLICATIONS

Bokoch, G.M., Wang, Y., Bohl, B.P., Sells, M.A., Quilliam, L.A., Knaus, U.G. Interaction of the Nck adapter protein with p21-activated kinase (PAK1). J. Biol. Chem. 271:25746, 1996.

Brzeska, H., Knaus, U.G., Wang, Z.-Y., Bokoch, G.M., Korn, E.D. p21-Activated kinase (PAK) has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I. Proc. Natl. Acad. Sci. U.S.A. 94:1092, 1997.

Ding, J., Knaus, U.G., Bokoch, G.M., Badwey, J.A. The renaturable 69 and 63 kDa protein kinases that undergo rapid activation in chemoattractant-stimulated neutrophils are p21-activated kinases (Paks). J. Biol. Chem. 271:24869, 1996.

Sells, M.A., Knaus, U.G., Bagrodia, S., Ambrose, D., Bokoch, G.M., Chernoff, J. Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 7:202, 1997.

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Lymphocyte Development and Immune Regulation In Vivo

D. Lo, C. Reilly, N. Aftahi, H. Neal, Q. Wei, M. Crowley, J. DeKoning, L. DiMolfetto, L. Li, M. Pauza, A. Wu

GENETICS OF CD4 T-CELL EFFECTOR FUNCTION AND AUTOIMMUNITY

The susceptibility to autoimmune diseases such as diabetes is a multigenic phenomenon due to contributions from several polymorphic, but otherwise normal, genetic loci. Our interest is in determining which genetic components lead to susceptibility to autoimmune disease, with specific attention to the genes that regulate the function of T lymphocytes.

We have been using a transgenic mouse model in which a well-defined antigen, influenza hemagglutinin, is expressed in pancreatic islet ß cells and a hemagglutinin-specific receptor is expressed on CD4 T cells. When mice with these transgenes were backcrossed to different mouse strains (B10.D2 and BALB/c), we found striking differences in the development of spontaneous autoimmune disease that appear to be related to the expression of IFN- and IL-4 in CD4 T cells. We are analyzing genetic backcrosses to map the gene or genes that influence this cytokine regulation.

SELECTION OF THE T-CELL RECEPTOR REPERTOIRE IN THE THYMUS

Our previous studies showed that thymic medullary epithelium is specialized for negative but not positive selection of CD4 T cells. We used mice deficient in relB (relB knockout mice) to examine the issue of compartmentalization of thymic function. These mice lack both thymic dendritic cells and the UEA-1+ subset of medullary epithelial cells, leaving mainly thymic cortex. In collaboration with P. Naquet, Centre d'Immunologie, Marseille Luminy, France, we found that because of the absence of UEA-1+ cells, which express class II MHC molecules, a second population of medullary epithelial cells lost its ability to organize into thymic medullary structures. Instead, the cells of this second population were scattered throughout the thymic cortex. This result indicates that development of the thymic medulla is critically dependent on the organizing ability of UEA-1+ cells.

We have also studied T-cell development in the thymus of relB knockout mice and in the progeny of mice transgenic for the T-cell receptor backcrossed with relB knockout mice. We found that functional T cells, both ß and , were generated quite efficiently. This finding suggests that T-cell development and maturation require only cortical epithelium, because these mice lack thymic medullary stromal elements. Our results support a model of thymic selection in which selection of T cells is separated both temporally and spatially into cortical (positive) and medullary (negative) compartments (Fig. 1).

REGULATION OF DENDRITIC CELL FUNCTION AND THE TRANSITION FROM INNATE TO ADAPTIVE IMMUNITY

RelB knockout mice have defects in the production of mature lymphoid dendritic cells, although stem cells from these mice generate cells with dendritic cell--like properties when cultured in the presence of granulocyte-macrophage colony-stimulating factor. However, whereas dendritic cells from normal mice can aggregate with T cells through some mechanism that does not require low-affinity T-cell receptors, dendritic cells from relB knockout mice show significant defects in this aggregation. Thus, although cells from relB knockout mice can commit to the dendritic cell lineage, they cannot develop into mature lymphoid dendritic cells. Consistent with this hypothesis, relB knockout mice lack organized lymphoid tissues such as lymph nodes and Peyer's patches, and the animals' cellular immune responses are impaired.

In collaboration with L. Feng, Department of Immunology, we have also discovered that relB induced in nonhemopoietic cells such as fibroblasts has an important role in suppressing the expression of inflammatory cytokines and chemokines. Although relB is a transcriptional activator in hemopoietic cells such as macrophages, it appears to suppress chemokine expression in endotoxin-stimulated fibroblasts. Our findings show that relB has a critical role in both the function of mature dendritic cells and the expression of inflammatory cytokines. These results suggest a model (Fig. 2) in which induction of relB by alarm or danger signals is important in mediating the transition from innate immunity (acute inflammation) to adaptive immunity (cellular immune responses).

PUBLICATIONS

Carson, M.J., Reilly, C.R., Sutcliffe, J.G., Lo, D. Mature microglia resemble immature antigen presenting cells. Glia, in press.

DeKoning, J., DiMolfetto, L., Reilly, C., Wei, Q., Havran, W., Lo, D. Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice. J. Immunol. 158:2558, 1997.

DiMolfetto, L., Reilly, C., Wei, Q., Lo, D. Dendritic-like cells from relB mutant mice. In: Dendritic Cells in Fundamental and Clinical Immunology, Vol. 3. Ricciardi-Castagnoli, P. (Ed.). Plenum, London, in press.

Laufer, T.M., DeKoning, J., Markowitz, J.S., Lo, D., Glimcher, L.H. Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex. Nature 382:81, 1996.

Lo, D., Reilly, C., Burkly, L., DeKoning, J., Laufer, T., Glimcher, L. Thymic stromal cell specialization and the T-cell receptor repertoire. Immunol. Res. 16:3, 1997.

Naspetti, M., Aurrand-Lions, M., DeKoning, J., Malissen, M., Galland, F., Lo, D., Naquet, P. Thymocytes and relB-dependent medullary epithelial cells provide respectively growth-promoting and organization signals to thymic medullary stromal cells. Eur. J. Immunol. 27:1392, 1997.

Xia, Y., Pauza, M., Feng, L., Lo, D. RelB regulation of chemokine expression modulates local inflammation. Am. J. Pathol. 151:375, 1997.

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Cross-Talk Between Signaling Pathways

G. Parry, N. Mackman

Activation of the protein kinase A (PKA) signaling pathway leads to phosphorylation of the transcription factor CREB, recruitment of the coactivator CREB-binding protein (CBP), and induction of genes such as c-fos that contain CRE-binding sites. CBP binds both to transcription factors and to the basal complex protein TF11B and thus functions as an adaptor molecule that stabilizes assembly of the basal transcription complex. We showed that activation of the PKA pathway by elevated levels of cAMP inhibited transcription mediated by the transcription factor NF-B in human monocytes and endothelial cells. However, inhibition did not prevent nuclear translocation of NF-B/Rel complexes.

We hypothesized that activation of the PKA pathway inhibited NF-B--mediated transcription by competition for limiting amounts of the coactivator CBP. In vitro binding studies indicated that the N-terminal region of CBP (amino acids 1--450) bound to p65 (RelA) but not to p50 or c-Rel. In vivo binding studies with the yeast two-hybrid assay showed that an interaction between the N-terminus of CBP and the C-terminus of p65 (amino acids 286--551) induced expression of the GAL4-responsive ß-galactosidase reporter gene in the yeast strain Y190. Functional studies with human endothelial cells showed that overexpression of CBP rescued cAMP inhibition of NF-B--mediated transcription in a dose-dependent manner.

These results strongly suggest that activation of the PKA signaling pathway inhibits NF-B--mediated transcription by competition for limiting amounts of CBP (Fig. 1). Finally, overexpression of CBP enhanced
NF-B--mediated transcription, consistent with the idea that CBP acts as a coactivator in NF-B--mediated transcription by binding to the C-terminal region of p65.


Regulation of Inducible Expression in Monocytes by the Transcription Factor c-Rel/p65

M. O'Connell, J. Erlich, G. Parry, N. Mackman

We have shown that the transcription factor c-Rel/p65 selectively binds to B sites in the genes for tissue factor, IL-8, intracellular adhesion molecule-1, and granulocyte-macrophage colony-stimulating factor. The sequence of these sites matches the following consensus: 5´-HGGARNYYCC-3´, where R indicates adenine or guanine, Y indicates cytosine or thymine, H indicates adenine, cytosine, or thymine, and N indicates any base. Recently, the gene for c-Rel was inactivated by homologous recombination. Studies with c-Rel-/- macrophages indicate that c-Rel acts as both a positive and a negative regulator of transcription of genes that encode inflammatory mediators.

We have obtained c-Rel-/- mice and will analyze lipopolysaccharide induction of the c-Rel/p65--regulated tissue factor gene and the p50/p65-regulated TNF- gene. In addition, we are using the yeast two-hybrid assay and screening of lambdagt11 cDNA libraries to determine proteins that bind to the C-terminal region of c-Rel and participate in c-Rel--mediated transcription. These studies should elucidate how c-Rel/p65 heterodimers activate transcription of a distinct set of genes in monocytes.


Regulation of a Human Tissue Factor Minigene in Transgenic Mice

G. Parry, J. Erlich, P. Carmeliet,* T. Luther,** N. Mackman

* Center for Transgene Technology and Gene Therapy, Leuven, Belgium
** Technical University, Dresden, Germany

We have made a human tissue factor (TF) minigene that includes human TF cDNA expressed from the human TF promoter. Human TF mRNA was expressed in a tissue-specific manner in transgenic mice. Lipopolysaccharide induced human TF mRNA and protein expression in peritoneal macrophages from transgenic mice. Transgenic mice containing the human TF minigene were crossed with mice heterozygous for the targeted disruption of the murine TF gene (TF+/-) to determine if expression of human TF could substitute for murine TF. We showed that most TF null embryos die at embryonic day 10.5. The presence of the human TF minigene prevented the death of the embryos (Fig. 1).

Immunohistochemical studies indicated that human TF was expressed in a cell type--specific manner in rescued mice. These results indicate that the human TF promoter directs cell type--specific expression, lipopolysaccharide-inducible expression, and embryonic expression of human TF. Thus, human TF expressed from the human TF minigene can substitute for the presence of murine TF in rescued mice. These mice will be used to analyze the role of TF in various biological processes, including embryogenesis, angiogenesis, and inflammatory responses.

PUBLICATIONS

Bierhaus, A., Zhang, Y., Quehenberger, P., Luther, T., Haase, M., Müller, M., Mackman, N., Ziegler, R., Nawroth, P.P. The dietary pigment curcumin reduces endothelial tissue factor gene expression by inhibiting binding of AP-1 to the DNA and activation of NF-*B. Thromb. Haemost. 77:772, 1997.

Brand, K., Eisele, T., Kreusel, U., Page, M., Page, S., Haas, M., Gerling, A., Kalschmidt, C., Mackman, N., Neumann, F.-J., Baeuerle, P.A., Walli, A.K., Neumeier, D. Dysregulation of monocytic nuclear factor-*B by oxidized low density lipoprotein. Arterioscler. Thromb. Vasc. Biol., in press.

Carmeliet, P., Mackman, N., Moons, L., Luther, T., Gressens, P., Vlaenderen, I., Demunck, H., Kasper, M., Breier, G., Evrard, P., Müller, M., Risau, W., Edgington, T., Collen, D. Role for the cellular receptor tissue factor in embryonic blood vessel development. Nature 783:73, 1996.

Cobb, R.R., Felts, K.A., Parry, G.C.N., Mackman, N. D609, a phosphatidylcholine specific phospholipase C inhibitor, blocks IL-1ß induced VCAM-1 gene expression in human endothelial cells. Mol. Pharmacol. 49:998, 1996.

Lin, M.-C., Shyy, J.Y.-J., Almus-Jacobs, F., Chen, H.-H., Parry, G.C.N., Mackman, N., Chien, S. The shear stress-induced tissue factor gene expression is mediated through the Sp1 cis-element. J. Clin. Invest. 99:737, 1997.

Luther, T., Flossel, C., Mackman, N., Bierhaus, A., Kasper, M., Albrecht, S., Sage, E.H., Iruela-Arispe, L., Großmann, H., Ströhlein, A., Zhang, Y., Nawroth, P.P., Carmeliet, P., Loskutoff, D.J., Müller, M. Tissue factor expression during human and mouse development. Am. J. Pathol. 149:101, 1996.

Mackman, N. Regulation of the tissue factor gene. Thromb. Haemost. 78:747, 1997.

Oeth, P., Parry, G.C.N., Mackman, N. Regulation of the tissue factor gene in human monocytic cells: Role of AP-1, NF-B/Rel and Sp1 proteins in uninduced and lipopolysaccharide-induced expression. Arterioscler. Thromb. Vasc. Biol. 17:365, 1997.

Ollivier, V., Parry, G.C.N., Cobb, R.R., de Prost, D., Mackman, N. Elevated cyclic AMP inhibits NF-B--mediated transcription in human monocytic cells and endothelial cells. J. Biol. Chem. 271:20828, 1996.

Parry, G.C.N., Erlich, J.H., Luther, T., Mackman, N. Low levels of human tissue factor rescue murine tissue factor null embryos. J. Clin. Invest., in press.

Parry, G.C.N., Mackman, N. NF-B--mediated transcription in human monocytic and endothelial cells. Trends Cardiovasc. Med., in press.

Parry, G.C.N., Mackman, N. Role of CREB-binding protein in cyclic AMP inhibition of NF--B-mediated transcription. J. Immunol., in press.

Yao, J., Mackman, N., Edgington, T.S., Fan, S.-T. Lipopolysaccharide induction of the tumor necrosis factor- promoter in human monocytic cells: Regulation by Egr-1, c-Jun, and NF-B transcription factors. J. Biol. Chem. 272:17795, 1997.

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Role of the Cell Adhesion Molecule L1 in Vascular, Immune, and Tumorigenic Processes

A.M.P. Montgomery, N. Nayeem, B. Felding-Habermann, J.D. Pancook

The cell adhesion molecule L1 has received much attention for its role in neural development and neuritogenesis. However, L1 is also expressed on nonneural cells of diverse origin. Defining the functional significance of such expression in tumor progression and in immune or vascular processes is the ultimate objective.

We recently described a novel heterophilic interaction between L1 and the integrin vß3. This work has been extended to show that a single immunoglobulin-like domain of human L1 can function as a ligand for multiple additional integrins, including vß1, 5ß1, and IIbß3. Interaction of L1 with vß3 and vß1 supported attachment and migration of endothelial cell, whereas interaction with active IIbß3 supported attachment of human platelets. Site-directed mutagenesis confirmed that a single arginine--glycine--aspartic acid motif in human L1 is required for integrin recognition. Significantly, physiologic levels of calcium preferentially inhibited binding of ß1 integrins, favoring an interaction with active IIbß3 or vß3. On the basis of these findings and a recent observation that L1 is expressed on blood vessels in certain diseases, we propose a role for L1 in vascular and thrombogenic processes.

We recently reported the expression and regulation of L1 on cells of myelomonocytic and lymphoid origin. Significantly, expression of L1 is associated with activated myelomonocytic cells. Thus, little or no L1 expression was detected on circulating monocytes, tissue macrophages, or monocyte-derived dendritic cells. However, stimulation of monocytes with IFN- or functional activation of dendritic cells with lipopolysaccharide induced significant expression of L1. Constitutive expression of L1 was further evident on a subset of circulating B cells and on follicular dendritic cells. This pattern of expression may indicate a role for L1 in cell-cell interactions involved in antigen presentation or cell trafficking.

L1 is shed by many neuroectodermal tumors and can be found in inflammatory exudates. We found that soluble L1 fragments induced a significant angiogenic response in an avian model. Remarkably, we have recently observed that a 15-mer peptide derived from L1 can also induce neovascularization. This same peptide interacts with endothelial cells via both vß1 and vß3. Although our findings suggest that soluble L1 will affect angiogenic (and immune) processes, the mechanism by which L1 is shed is poorly defined. We now have evidence that a number of serine proteases can cleave L1 from the cell surface.

PUBLICATIONS

Altevogt, P., Kadmon, G., Montgomery, A.M.P. L1 makes immunological progress by expanding its relations. Dev. Immunol., in press.

Brooks, P.C., Montgomery, A.M.P., Cheresh, D.A. Use of the avian model for studying angiogenesis. Methods Mol. Biol., in press.

Pancook, J.D., Reisfeld, R.A., Varki, N., Vitiello, A., Fox, R.I., Montgomery, A.M.P. Expression and regulation of the neural cell adhesion molecule L1 on human cells of myelomonocytic and lymphoid origin. J. Immunol. 158:4413, 1997.

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Viral and Host Factors in the Pathogenesis of AIDS

D.E. Mosier, I. Atencio, A. Beernink, J.M. Glynn, R.J. Gulizia, M. Kohls, Y. Lin, D. McElligott, D. McKinney, P. Poignard, G. Picchio, B. Sabbe, M. McGrath,* B. Herndier,* D. Trono,** B. Chesebro***

* University of California, San Francisco, CA
** Salk Institute, La Jolla, CA
*** Rocky Mountain Laboratories, Hamilton, MT

Infection of humans with HIV type 1 (HIV-1) leads to progressive depletion of CD4 T cells until AIDS develops. HIV-1 isolates differ extensively in their target cells and in their ability to kill infected cells. We are studying how different HIV-1 isolates cause disease in a unique small animal model of HIV-1 infection. In this model, human peripheral blood leukocytes (PBLs) transplanted to mice with severe combined immunodeficiency (SCID) survive to generate hu-PBL-SCID mice.

Most patients infected with HIV-1 initially harbor macrophage-tropic (M-tropic), non--syncytial-inducing viruses that may evolve into T cell--tropic (T-tropic), syncytial-inducing viruses after several years, often passing through an intermediate step of dual tropism (M/T-tropic). The cell tropism of HIV-1 is closely linked to the use of chemokine receptors for viral entry into cells. M-tropic viruses enter cells by binding CD4 and CCR5, the chemokine receptor for macrophage inflammatory proteins 1 and 1ß and RANTES. T-tropic viruses use CD4 and CXCR4, the chemokine receptor for stromal cell--derived factor 1, for cell entry, and M/T-tropic viruses can use either CCR5 or CXCR4 as a coreceptor. The envelope glycoprotein gp120 of the virus determines cell tropism. The appearance of T-tropic, syncytial-inducing viruses is associated with more rapid progression to AIDS. The reasons for the more efficient transmission of M-tropic, non--syncytial-inducing viruses and the slow evolution of T-tropic, syncytial-inducing viruses remain unclear.

We examined plasma viral RNA levels and the extent of CD4+ T-cell depletion in hu-PBL-SCID mice infected with M-, dual-, or T-tropic HIV-1 isolates. We found that cell tropism determined the course of viremia. M-tropic viruses produced sustained high viral RNA levels and spared some CD4+ T cells, dual-tropic viruses produced a transient viral RNA spike and extremely rapid depletion of CD4+ T cells, and T-tropic viruses caused lower viral RNA levels and intermediate rates of CD4+ T-cell depletion. A single amino acid change in the V3 loop region of gp120 (glutamine to glutamic acid at position 25) was sufficient to cause one isolate to switch from M-tropism to dual-tropism and acquire the ability to rapidly deplete all CD4+ T cells (Fig. 1). These results suggest that HIV-1 has evolved into (1) M-tropic variants that are optimized for transmission and that produce high viral loads because they are relatively slow to kill all infected cells (i.e., produce sustained infection) and (2) M/T- and T-tropic variants that kill infected (and probably uninfected bystander) cells more rapidly.

The CCR5 coreceptor for M-tropic virus is expressed on memory T cells, which appear to be the primary target for viral replication throughout most of the course of disease. The CXCR4 coreceptor is expressed on naive T cells, which require activation to become permissive for HIV-1 replication. Subjects with a 32-bp deletion in the gene for CCR5 are naturally resistant to HIV-1 infection by M-tropic viruses, and our studies show that hu-PBL-SCID mice derived by transplantation of cells from these subjects have much reduced replication of M/T-tropic viruses, suggesting that CCR5 is the most important viral coreceptor for viral entry.

We have also examined the effect of deleting the HIV-1 accessory gene nef (negative effect factor) in M-, M/T-, and T-tropic isolates. This gene is known to contribute to viral infectivity as well as immune escape by downregulating CD4 and MHC class I molecules. Deletion of nef slowed the kinetics of viral replication in hu-PBL-SCID mice with all HIV-1 isolates tested, but the impact of its deletion was greatest in T-tropic isolates and less in M- and M/T-tropic isolates. Deletion of nef from a highly pathogenic M/T-tropic isolate delayed the peak viral level and the onset of CD4 T-cell loss by 1 week, whereas deletion of nef from a T-tropic isolate prevented loss of CD4 T cells. In this model, nef appears to contribute to two distinct aspects of HIV-1 infection: the efficiency of primary infection and the rate at which infected cells die.

HIV-1 infection of hu-PBL-SCID continues to be a valuable model for the testing of vaccine approaches for preventing viral transmission. Both neutralizing antibodies and cytotoxic T lymphocytes show efficacy in preventing infection, although neither one has significant impact on established infection. These findings underscore what is obvious in most infected patients: HIV-1 has evolved to survive despite a vigorous immune response. A vaccine capable of blocking primary infection remains the best hope of containing the spreading worldwide epidemic of HIV-1 infection.

PUBLICATIONS

Gulizia, R.J., Collman, R.G., Levy, J.A., Trono, D., Mosier, D.E. Deletion of nef slows but does not prevent CD4-positive T-cell depletion in human immunodeficiency virus type 1-infected human-PBL-SCID mice. J. Virol. 71:4161, 1997.

Picchio, G.R., Gulizia, R.J., Mosier, D.E. Chemokine receptor CCR5 genotype influences the kinetics of human immunodeficiency virus type 1 infection in human PBL-SCID mice. J. Virol., in press.

Picchio, G.R., Nakatsuno, M., Boggiano, C., Sabbe, R., Corti, M., Daruich, J., Perez-Bianco, R., Tezanos-Pinto, M., Kokka, R., Wilber, J., Mosier, D.E. Hepatitis C (HCV) genotype and viral titer distribution among Argentinean hemophilic patients in the presence and absence of human immunodeficiency virus (HIV) co-infection. J. Med. Virol. 52:219, 1997.

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Protease Receptors in Metastasis

B.M. Mueller, L. Goretzki, M. Yebra

Protease receptors modulate proteolytic activities on the cell surface and are thought to be crucially involved in normal and pathologic processes of tissue remodeling, including tumor cell invasion and metastasis. Our studies focus on receptors that regulate pericellular plasminogen activation, namely the receptor for the urokinase-type plasminogen activator (uPAR) and the endocytic receptor low density lipoprotein receptor--related protein (LRP).

The receptor uPAR enables invading cells to degrade the extracellular matrix by directing the proteolytic activity of urokinase-type plasminogen activator (uPA), by enhancing the catalytic activity of the activator and protecting it from inactivation by protease inhibitors. Binding of uPA to uPAR, independent of uPA's catalytic activity, also initiates signal transduction events that lead to the activation and migration of tumor cells. Because uPAR is anchored with a glycosylphosphatidylinositol moiety in the cell membrane and lacks an intracellular domain, it has been postulated that the receptor must interact with a transmembrane adapter protein to transduce signals into the cytoplasm.

In studies on the biological consequences of the expression of uPAR in human prostate cancer cells, we analyzed cell migration on a fibronectin matrix, which depends on the integrin 5ß1. We found that uPA binding to uPAR results in (1) formation of complexes consisting of uPAR and the ß1 integrin subunit in detergent-insoluble fractions of the cell membrane; (2) enhanced tyrosine phosphorylation of two intracellular proteins implicated in integrin-mediated cell migration, focal adhesion kinase and crk-associated substrate p130cas; and (3) enhanced cell migration in haptotactic migration assays. These data indicate that signal transduction by uPA/uPAR involves extracellular interactions of uPAR with the ß subunit of integrins that result in enhanced activation of signaling pathways downstream of the integrin.

The two receptors uPAR and LRP cooperate to mediate endocytosis of uPA and of complexes formed by uPA and protease inhibitors, another important process in the regulation of pericellular proteolysis. Using various inhibitors for intracellular protein kinases, we found that the internalization of uPA, and of other uPAR-independent LRP ligands, requires the activity of the cAMP-dependent protein kinase. The known pathway of activation of this kinase involves adenylate cyclase and heterotrimeric GTP-binding proteins. We are therefore investigating the possibility that LRP is a receptor coupled to GTP-binding protein. This idea is supported by our observation that ligation of LRP with uPA causes suppression of ADP ribosylation mediated by cholera toxin and the activation of the cAMP-dependent protein kinase. Thus, our data suggest that the endocytosis of LRP, which has been thought to be a constitutive, ligand-independent process, is regulated by a heterotrimeric GTP-binding protein.

PUBLICATIONS

Goretzki, L., Mueller, B.M. Receptor-mediated endocytosis of urokinase-type plasminogen activator is regulated by cAMP-dependent kinase. J. Cell Sci., in press.

Ruf, W., Mueller, B.M. Tissue factor in cancer angiogenesis and metastasis. Curr. Opin. Hematol. 3:379, 1996.

Stahl, A., Mueller, B.M. Melanoma cell migration on vitronectin: Regulation by components of the plasminogen activation system. Int. J. Cancer 71:116, 1997.

Yebra, M., Parry, G.C.N., Stromblad, S., Mackman, N., Rosenberg, S., Mueller, B.M., Cheresh, D.A. Requirement of receptor bound urokinase-type plasminogen activator for integrin vß5-directed cell migration. J. Biol. Chem. 271:29393, 1996.

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Integrin-Mediated Signaling Events Involved in Adenovirus Internalization and Cell Motility

E. Li, D.G. Stupack, R. Klemke, D.A. Cheresh, G.R. Nemerow

Integrin-mediated signaling events mediate a variety of diverse cellular functions, including adhesion, motility, and cell growth. Integrins have also been co-opted by a number of human viruses to allow infection of susceptible host cells. However, the precise mechanisms underlying integrin-mediated viral infection are poorly understood.

Previous studies in our laboratory showed that human adenoviruses use integrins vß3 and vß5 to promote internalization. In recent studies, we investigated the mechanisms by which v integrins promote viral uptake by cells. We found that several signaling molecules, including focal adhesion kinase and phosphatidylinositol-3-kinase, become phosphorylated during interaction of the adenovirus penton base with v integrins. Kinase phosphorylation is also associated with increased activation of phosphatidylinositol-3-kinase. Pharmacologic agents and genetic dysregulation of phosphatidylinositol-3-kinase blocked internalization of adenovirus and infection yet had no effect on integrin-mediated cell adhesion or motility, a cellular process regulated by the Erk1/Erk2 MAP kinase pathway. These studies indicate that integrin ligation by the extracellular matrix or by viral proteins can lead to the engagement of distinct signaling pathways that promote viral endocytosis or cell movement (Fig. 1).

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Retargeting of Recombinant Adenoviral Vectors for Gene Therapy

D.J. Von Seggern, J. Kehler, R. Endo, G.R. Nemerow

Adenoviral vectors are currently being used in a number of clinical trials to deliver therapeutic genes to a variety of cell and tissue types. Unfortunately, several obstacles to the widespread clinical use of adenoviral vectors remain, including the host immune response to virally encoded proteins and the lack of selectivity in tissue targeting. We are attempting to address some of these problems through the development of modified viral vectors.

We have developed mammalian cell lines that stably express the adenovirus type 5 fiber protein in the fiber's native trimeric form. This protein mediates viral attachment to a 46-kD receptor that is expressed on human epithelial cells. The packaging cells can complement a temperature-sensitive mutant adenovirus type 5 that lacks a functional fiber protein and allow incorporation of the adenovirus type 5 fiber protein into viral particles of a different serotype. Further studies are in progress to determine whether a replication-defective adenovirus lacking the fiber gene can be propagated in the fiber-packaging cell lines and to determine whether fiber proteins with different receptor specificities can be incorporated into modified viral vectors.

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Inhibition of a Latent Viral Infection and Cell Proliferation by Adenovirus-Delivered Ribozymes

S. Huang, D.G. Stupack, P. Mathias, Y. Wang, G.R. Nemerow

Epstein-Barr virus (EBV), a member of the herpesvirus family, persists in latently infected B cells for the life of the host. Under certain conditions, infection is associated with the development of several human malignant tumors and lymphoproliferative diseases in immunocompromised patients such as those with AIDS or in transplant recipients. Currently, little, if any, effective treatment is available for EBV-induced lymphoproliferative diseases. As an approach for treating EBV infections, we examined the capacity of recombinant adenoviral vectors to deliver antiviral ribozymes directed against the EBV nuclear antigen 1 (EBNA-1). EBNA-1 is uniquely expressed in all latently infected B cells and plays an essential role in persistence of the EBV genome. EBNA-1 also reportedly has oncogenic potential.

In contrast to normal B lymphocytes, EBV-transformed B lymphoblastoid cells expressed v integrins and were also susceptible to adenovirus-mediated gene delivery. Adenovirus delivery of an EBNA-1 specific ribozyme to lymphoblastoid cell lines suppressed EBNA-1 mRNA and protein expression. The ribozyme also significantly reduced the number of EBV genomes in latently infected B cells and greatly inhibited B-cell proliferation in medium containing low concentrations of serum.

In recent studies to investigate the use of adenovirus-delivered ribozymes in preventing EBV-induced lymphoproliferative disease in vivo, we used a model system in which mice with severe combined immunodeficiency are reconstituted with human cells. Pretreatment of EBV-transformed B cells with adenovirus-encoded EBNA-1 ribozymes delayed the outgrowth of B-cell tumors and reduced tumor mass in these mice. These studies indicate the potential use of adenovirus-encoded ribozymes in the treatment of EBV-induced lymphoproliferative disorders. Adenoviral vectors are also valuable tools for investigating the precise events in EBV-induced B-cell transformation.

PUBLICATIONS

Huang, S., Jiang, Y., Li, Z., Mathias, P., Lin, S., Ulevitch, R.J., Nemerow, G.R., Han, J. Apoptosis signaling pathways in T cells is composed of ICE/Ced-3 family proteases and MAP kinase 6b. Immunity 6:739, 1997.

Huang, S., Stupack, D., Mathias, P., Wang, Y., Nemerow, G. Growth arrest of Epstein-Barr virus immortalized B lymphocytes by adenovirus delivered ribozymes. Proc. Natl. Acad. Sci. U.S.A. 94:8156, 1997.

Stewart, P., Chiu, C., Huang, S., Muir, T., Zhao, Y., Chait, B., Mathias, P., Nemerow, G.R. Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization. EMBO J. 16:1189, 1997.

Stewart, P.L., Nemerow, G.R. Recent structural solutions for antibody neutralization of viruses. Trends Microbiol. 5:22, 1997.

Von Seggern, D.J., Nemerow, G.R. Adenoviral vectors for protein expression. In: Nature: The Palette for the Art of Expression. Fernandez, J., Hoeffler, J. (Eds.). Academic Press, New York, in press.

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Regulation of T-Cell Adhesion and Signal Transduction

A.M. O'Rourke, M. Buda, A. Brunmark*

* R.W. Johnson Pharmaceutical Research Institute, La Jolla, CA

Activation of T lymphocytes by antigen-presenting cells (APCs) involves a number of receptor-ligand interactions. Recognition of specific antigenic MHC molecules by the T-cell receptor initiates a series of events that increase stabilized binding between T-cell adhesion receptors and their ligands on the APCs. These interactions also elicit signaling events that contribute to secretion of cytokines by CD4+ cells and cytolysis by CD8+ cells. Our research is aimed at understanding how the function of individual adhesion molecules is regulated by the T cell, both at the level of the signals necessary to increase adhesion and by the events that occur after ligand binding. To simplify the complex multivalent nature of the interaction between T cells and APCs, we use purified APC ligands so that the T-cell response to defined ligands can be analyzed in isolation.

In the past year, we completed a study of the minimal activation requirements necessary to trigger previously activated T cells to proliferate and secrete cytokines. We showed that high densities of antigenic peptide--MHC complexes alone were sufficient for activation and that additional engagement of accessory receptors made significant contributions to the magnitude of the T-cell response only when engagement of T-cell receptors was limiting. We are extending these studies to examine responses of naive T cells to isolated MHC complexes and costimulatory ligands such as intracellular adhesion molecule-1 and B7-1.

In addition, we are studying the signaling events that upregulate the adhesive function of T-cell integrins such as lymphocyte function--associated antigen-1. Although many factors act on T cells to increase the affinity or avidity of lymphocyte function--associated antigen-1, the biochemical events that occur during upregulation triggered by T-cell receptors remain unclear. Our work thus far implicates a number of signals that likely act synergistically, including activation of the enzymes protein kinase C and phosphatidylinositol-3-kinase. In collaboration with J. Kaye, Department of Immunology, we are also examining the role of the p21ras GTP-binding protein in controlling thymocyte adhesion to isolated ligands.

We recently began a study of integrin function in T cells from aged mice. The T-cell pool in aged humans and mice is composed mainly of memory cells, and one hypothesis suggests that the age-associated decline in the function of the immune system may reflect impaired responses by these memory T cells. Our aim is to determine whether the expression and function of T-cell integrins are significantly altered during the animals' life span.

PUBLICATIONS

Brunmark, A., O'Rourke, A.M. Augmentation of mature CD4+ T cell responses to isolated antigenic class II proteins by fibronectin and intercellular adhesion molecule-1. J. Immunol. 159:1676, 1997.

Ybarrondo, B., O'Rourke, A.M., McCarthy, J.B., Mescher, M.F. CTL interaction with fibronectin: Activated adhesion and cosignaling. Immunology 91:186, 1997.

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Cancer Immunotherapy With Immunocytokines

R. Reisfeld, H.N. Lode, R. Xiang, J.C. Becker,* T. Dreier, N. Varki,** K. Furukawa,*** S.D. Gillies****

* University of Würzburg, Würzburg, Germany
** Cancer Center, University of California, San Diego, CA
*** Nagasaki University School of Medicine, Nagasaki, Japan
**** Fuji ImmunoPharmaceuticals Corp., Lexington, MA

Our major goal is to induce cell-mediated immune responses that can eradicate established, disseminated tumor metastases in tumor-bearing hosts and provide a persistent tumor-protective immunity. We achieved this goal with a genetically engineered fusion protein that provides an effective concentration of the cytokine IL-2 in the tumor microenvironment. This fusion protein consists of (1) a human-mouse chimeric monoclonal antibody against ganglioside GD2 and (2) recombinant human IL-2.

Treatment with this immunocytokine (ch14.18-IL2) resulted in (1) eradication of established, experimental pulmonary and hepatic metastases in a syngeneic murine melanoma model and (2) induction of a long-lived tumor-protective immunity that prevented tumor growth in the majority of successfully treated mice challenged with tumor cells up to 4 months later. This effect was tumor specific, because cross-protection was not observed against challenge with syngeneic, ganglioside GD2+ EL-4 thymoma cells. Both the initial eradication of established metastases and the tumor-specific protection depend on CD8+ T cells. Thus, tumor-specific protection can be transmitted horizontally to naive, syngeneic mice with severe combined immunodeficiency by passive transfer of CD8+ T lymphocytes derived from immune animals. These results suggest that antibody-targeted delivery of IL-2 provides a means of eliciting effective tumor-protective immune responses against established tumors in the immunotherapy of malignant melanoma.

Immunocytokines can also effectively induce an immune response that eradicates established experimental hepatic and pulmonary metastases of colon carcinoma in a syngeneic murine tumor model. In this model, an immunocytokine with a different specificity, a fusion protein consisting of (1) recombinant human IL-2 and (2) the humanized monoclonal antibody KS1/4 directed against KSA, a human epithelial cell adhesion molecule, effectively targeted IL-2 to the tumor microenvironment. The efficacy of the immunocytokine in eradicating tumor metastases was established, because mixtures of the monoclonal antibody KS1/4 and IL-2 at equivalent dose levels were ineffective and only partially reduced tumor loads. Tumor-bearing mice treated with the immunocytokine were cured of colon cancer. Quantification of KSA by reverse transcriptase polymerase chain reaction detected no residual tumor cells, and the life span of treated animals was three times longer than that of control mice given saline or a mixture of the monoclonal antibody and recombinant human IL-2.

The effector mechanisms involved depend on T cells, because targeted IL-2 therapy was ineffective in mice with severe combined immunodeficiency that are deficient in T cells. In contrast, the eradication of micrometastases is independent of natural killer (NK) cells; the antitumor response induced by the immunocytokine was equally effective in mice depleted of NK cells. The essential effector cell population was characterized as CD8+ T cells by in vivo depletion studies. CD8+ cells isolated from tumor-bearing mice after immunocytokine therapy elicited MHC class I--restricted cytotoxic effects against colon carcinoma target cells. These results indicate that immunocytokine therapy induces a T cell--dependent host cellular immune response that is followed by eradication of established colon cancer metastases in a clinically relevant animal tumor model.

The immunocytokine ch14.18-IL2 also completely suppressed the growth of experimental and spontaneous metastases of murine neuroblastoma to bone marrow and liver in a syngeneic animal model. This immunocytokine has a distinct therapeutic advantage over a mixture of recombinant human IL-2 and the monoclonal antibody ch14.18. The mixture was ineffective in suppressing tumor growth; at best, it partially reduced tumor load. Only mice treated with the ch14.18-IL2 immunocytokine showed a significant, twofold prolongation in life span and lacked residual tumor cells. Because this immunocytokine suppresses metastases, despite heterogenous expression of the targeted GD2 antigen, this therapy may deal effectively with this type of heterogeneity in cancer patients.

The effector mechanism involved in this tumor model is mediated by NK cells. The immunocytokine was effective in scid/scid mice, which have NK cells but not T cells, and was ineffective in scid/beige mice, which lack both T cells and NK cells. Our neuroblastoma model is clinically relevant, because it is pathophysiologically similar to neuroblastoma in humans. The murine tumor naturally expresses ganglioside GD2, produces dopamine, and has dense core vesicles. In addition, the components involved in metastasis include bone marrow, liver, the adrenal glands, and lymph nodes, which reflect the pathophysiologic characteristics of human neuroblastoma.

The involvement of NK cells in the observed antitumor response may be of advantage in a clinical setting in which effective T cell--mediated responses are often lacking because of the immunosuppressive effects of tumors and treatment with chemotherapy or radiotherapy. Because NK cells recover before T cell--dependent immunity does, an initial NK cell response may be advantageous. The NK cells can suppress growth of metastases and thereby provide a chance for the eventual induction of an effective T cell--mediated immune response to eradicate metastases and provide tumor-protective immunity.

PUBLICATIONS

Albertini, M.R., Hank, J.A., Schiller, J.H., Khorsand, M., Borchert, A.A., Gan, J., Bechhofer, R., Storer, B., Reisfeld, R.A., Sondel, P.M. A phase IB trial of chimeric anti-GD2 antibody plus interleukin 2 for melanoma patients. Clin. Cancer Res., in press.

Becker, J.C., Varki, N., Gillies, S.D., Furukawa, K., Reisfeld, R.A. An antibody-interleukin 2 fusion protein overcomes tumor heterogeneity by induction of a cellular immune response. Proc. Natl. Acad. Sci. U.S.A. 93:7826, 1996.

Becker, J.C., Varki, N., Gillies, S.D., Furukawa, K., Reisfeld, R.A. Long-lived and transferable tumor immunity in mice following targeted interleukin 2 therapy. J. Clin. Invest. 98:2801, 1996.

Deryugina, E.I., Bourdon, M.A., Luo, G.-X., Resifeld, R.A., Strongin, A. Matrix metalloproteinase-2 activation modulates glioma cell migrationl J.Cell Sci., in press.

Liu, L., Barth, R.F., Adams, D.M., Soloway, A.H., Reisfeld, R.A. Critical evaluation of bispecific antibodies as targeting agents for boron neutron capture therapy of brain tumors. Anticancer Res. 16:2581, 1996.

Lode, H.N., Xiang, R., Varki, N., Dolman, C.S., Gillies, S.D., Reisfeld, R.A. Targeted interleukin 2 therapy of spontaneous neuroblastoma metastases to bone marrow. J. Natl. Cancer Inst., in press.

Reisfeld, R.A., Gillies, S.D., Becker, J.C. Immunocytokines: A new approach to immunotherapy of melanoma. Melanoma Res., in press.

Sen, G., Chakraborty, M., Foon, K.A., Reisfeld, R.A., Bhattacharya-Chatterjee, M. Preclinical evaluation in nonhuman primates of murine monoclonal anti-idiotype antibody that mimicks the disialoganglioside GD2. Clin. Cancer Res., in press.

Xiang, R., Lode, H.N., Dolman, C.S., Dreier, T., Varki, N.M., Qian, X., Lo, K.-M., Lan, Y., Super, M., Gillies, S.D., Reisfeld, R.A. Elimination of established murine colon carcinoma metatases by antibody-interleukin 2 fusion protein therapy. Cancer Res., in press.

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Dual Function of Tissue Factor in Metastasis

B.M. Mueller, W. Ruf

Tissue factor (TF), the cell-surface receptor that initiates the coagulation cascade, supports hematogenous spread of tumor cells. It is controversial whether the proteolytic function of the complex formed by TF bound to its serine protease ligand, coagulation factor VIIa, is required for metastasis. Using a model for TF-dependent experimental metastasis, we showed that TF prometastatic function involves both proteolytic activity and signaling functions of the TF cytoplasmic domain. Using site-directed mutagenesis, we showed that binding of factor VIIa to TF is required for metastasis. However, binding of catalytically inactive factor VIIa is insufficient to support metastasis, emphasizing that proteolytic activity is necessary for the metastatic process.

Because local generation of protease on the surface of tumor cells may serve solely to activate the cytoplasmic domain of TF by serine phosphorylation, we analyzed a TF mutant that mimicked phosphorylation of the cytoplasmic domain. Metastasis of cells expressing this mutant still depended on the formation of a catalytically active TF-VIIa complex, showing cooperation of proteolysis and specific functions of the TF cytoplasmic domain in hematogenous dissemination of tumor cells. These data support the emerging model that multiple and overlapping functions of cell-surface receptors coordinate the behavior of invasive cells in the extracellular environment of the cells.

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Cell Biology of Tissue Factor

I. Ott, Y. Miyagi, H.-Y. Huang, E.G. Fischer, B.M. Mueller, W. Ruf

Tissue factor (TF), the protease receptor that initiates the coagulation system, functions in the development of extraembryonic blood vessels, tumor angiogenesis, and hematogenous spread of tumor cells. However, the molecular mechanisms of TF in these processes are poorly defined. To gain insight into cellular functions of TF, we identified molecules that interact with the 21 amino acids of the cytoplasmic domain of TF. We found that actin-binding protein 280 specifically binds to the TF cytoplasmic domain and is recruited to TF-dependent adhesion contacts, resulting in a reorganization of the subcortical actin filament network. Immobilized ligands for TF specifically support cellular adhesion, migration, and spreading, generating intracellular signals, such as focal adhesion kinase phosphorylation. The cellular signaling through TF is distinct from integrin-mediated signaling cascades, as shown by the lack of recruitment of cytoskeletal adaptor molecules to the TF adhesion sites. These data define TF as a cell-surface receptor that participates in adhesive and migratory functions of cells, suggesting novel functional roles by which TF influences organization of vessel walls and dissemination of tumor cells.

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Allosteric Activation of the Serine Protease Factor VIIa

C.D. Dickinson, C.R. Kelly, W. Ruf

The catalytic function of the serine protease factor VIIa is allosterically regulated by interaction with tissue factor (TF), its cell-surface receptor. Using alanine scanning mutagenesis, we have defined residues of factor VIIa that interact with TF. The light chain of VIIa appears to serve as a tether to allow specific activating interactions of the protease domain with TF. The mutagenesis data suggest that the free protease VIIa exists in a "zymogen-like" state and that VIIa is activated by receptor-cofactor interactions through a global allosteric switch that occurs spontaneously in the cofactor-independent digestive serine proteases after proteolytic zymogen cleavage.

Mediating the activating conformational switch, certain residue side chains in the interface of VIIa with TF selectively affect the active-site cleft, whereas others predominantly influence regions for extended recognition of macromolecular substrates. Conversely, covalent binding of inhibitors to the catalytic cleft of VIIa allosterically affects one specific residue in the interface with TF. These data begin to shed light on the specific conformational interdependencies of the catalytic cleft of VIIa with the exosites for interaction with cofactor and for binding of macromolecular substrate. Future studies will probe the conformational transitions of the VIIa protease domain as a model that provides insight into fundamental mechanisms of allosteric activation of cofactor-dependent serine proteases.

PUBLICATIONS

Dickinson, C.D., Kelly, C.R., Ruf, W. Identification of surface residues mediating cofactor binding and catalytic function of the serine protease factor VIIa. Proc. Natl. Acad. Sci. U.S.A. 93:14379, 1996.

Dickinson, C.D., Ruf, W. Active site modification of factor VIIa affects interactions of the protease domain with tissue factor. J. Biol. Chem. 272:19875, 1997.

Dittmar, S., Ruf, W., Edgington, T.S. Fine specificity of macromolecular substrate recognition by factor VIIa in complex with mutants of tissue factor. Biochem. J. 321:787, 1997.

Kelly, C.R., Dickinson, C.D., Ruf, W. Ca2+ binding to the first epidermal growth factor module of coagulation factor VIIa is important for cofactor interaction and proteolytic function. J. Biol. Chem. 272:17467, 1997.

Mueller, B., Ruf, W. Tissue factor in cancer angiogenesis and metastasis. Curr. Opin. Hematol. 3:379, 1996.

Nicholson, A.C., Nachman, R.L., Altieri, D.C., Summers, B.D., Ruf, W., Edgington, T.S., Hajjar, D.P. Effector cell protease receptor-1 (EPR-1) is a vascular receptor for coagulation factor Xa. J. Biol. Chem. 271:28407, 1996.

Stura, E.A., Ruf, W., Wilson, I.A. Crystallization and preliminary crystallographic data for a ternary complex between tissue factor, factor VIIa and BPTI-derived inhibitor. J. Cryst. Growth 168:260, 1996.

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Regulation of Autoimmunity

N. Sarvetnick, J. Davies, B. Balasa, M. Falcone, S. Gallichan, A. Good, M. Horwitz, E. Jones, C. King, T. Krahl, M. Krakowski, J. Lee, R. Mueller, G. Patstone, L. Schiotz, L. Xu

During the past year, the laboratory has expanded in terms of projects and expertise. We have continued our commitment to understanding the factors that regulate autoimmune responses. Our focus is on insulin-dependent diabetes mellitus, a disease caused by the cell-mediated destruction of pancreatic islets. We have also continued to do some work on myasthenia gravis, a humoral autoimmune disease that affects the neuromuscular junction. We wish to understand the role of proinflammatory and immunoregulatory molecules in eliciting or preventing T-cell responses to self-tissues and self-antigens.

We are interested in the effects of IL-4 on autoimmune disease in nondiabetic obese (NOD) mice, a model for autoimmunity. We are examining the pathway whereby IL-4 counterregulates autoreactive processes. The results thus far suggest a less than classical immune-deviation pathway, where type 2 T-helper cells mediate protective effects. Previously, we reported that transgenic expression of IL-4 completely protects NOD mice from insulitis and diabetes. We have shown the presence of autoreactivity but lack of pathogenicity of the IL-4--regulated lymphocytes. The importance of T-cell diversity for the protective effect of IL-4 was shown by breeding NOD mice with BDC2.5 mice transgenic for the T-cell receptor, which have an almost exclusively monoclonal T-cell repertoire. Limitation of T-cell diversity in the offspring of these two strains of mice abrogated the protection by IL-4. We suggest that immune deviation in NOD mice transgenic for IL-4 is mediated by the pancreatic tissue itself, which causes activation of nonpathogenic T cells that have distinct specificities.

Recently, we have been studying how viruses trigger autopathogenic processes that lead to self-reactivity. We have attempted to elucidate the underlying mechanism of Coxsackie virus--induced autoreactivity of pancreatic islet cells. This autoreactivity is implicated in the etiology of insulin-dependent diabetes mellitus in humans. Mechanistic hypotheses are being tested by using a murine system. Each hypothesis tested has some attractive features and, to at least some degree, could explain the available data on humans and rodents. More than one avenue toward pancreatic damage and autoreactivity could occur, and such collaborative interactions might be heightened in genetically predisposed individuals. The knowledge acquired from the proposed studies will expand the understanding of the relationship between Coxsackie virus and the development of insulin-dependent diabetes mellitus.

Experimental autoimmune myasthenia gravis is an animal model of human myasthenia gravis. In mice, the disease is induced by immunization with the receptor for acetylcholine (AChR). Because experimental autoimmune myasthenia gravis is mediated by antibodies, the prevailing notion is that cytokines associated with type 2 but not type 1 T-helper cells play a role in the pathogenesis of this disease. To test the hypothesis that IFN-, a cytokine associated with type 1 T-helper cells, plays a role in the development of experimental autoimmune myasthenia gravis, we immunized IFN- knockout (-/-) mice and wild-type (+/+) mice with AChR.

We observed that AChR-primed lymph node cells from the knockout mice but not the wild-type mice proliferated normally in response to AChR and to its dominant pathogenic sequence. However, the knockout mice had no signs of muscle weakness and remained resistant to clinical experimental autoimmune myasthenia gravis at a time when the wild-type mice had severe muscle weakness and some died. The resistance of the knockout mice was associated with the level of circulating antibodies to AChR, which was much less than the level in wild-type mice. However, priming of knockout mice with keyhole limpet hemocyanin readily elicited both T-cell and antibody responses, suggesting that IFN- regulates the humoral immune response distinctly to self-antigens (AChR) vs foreign antigens (keyhole limpet hemocyanin). We conclude that IFN- is required for the generation of a pathogenic anti-AChR humoral immune response.

It is known that the affinity of T cells responding to self-antigens is generally lower than that of T cells responding to foreign antigens. During cognate T-cell--B-cell interactions, these AChR-specific low-affinity T cells may require a higher level of costimulation to achieve the threshold for contact-dependent signals to cause stimulation and differentiation of B cells and subsequent production of antibodies, Therefore, we speculate that mounting an effective humoral immune response to this self-antigen (AChR) is IFN-g--dependent, because the cytokine regulates the expression of costimulatory molecules.

PUBLICATIONS

Allison, J., Miller, J., Sarvetnick, N. Insulin-dependent diabetes mellitus. In: Role of Cytokines in Autoimmunity. Feldmann, M., Brennan, F.M. (Eds.). R.G. Landes, Austin, TX, in press.

Balasa, B., Deng, C., Lee, J., Bradley, L., Dalton, D., Christadoss, P., Sarvetnick, N. IFN- is necessary for the genesis of acetylcholine receptor-induced clinical experimental autoimmune myasthenia gravis in mice. J. Exp. Med., in press.

Balasa, B., Krahl, T., Sarvetnick, N. CD40 ligand-CD40 interactions are necessary for the initiation of insulitis and diabetes in nonobese diabetic mice. J. Immunol., in press.

Geiger, K., Bloom, F.E., Sarvetnick, N. Apoptosis techniques and protocols. In: Neuromethods. Boulton, A.A., Baker, G.B., Poirier, J. (Eds.). Humana Press, Clifton, NJ, in press.

Geiger, K.D., Sarvetnick, N. Transgenic expression of IFN- in the eye as a model to study ocular inflammatory disease. In: NeuroProtocols. Campbell, I. (Ed.). Academic Press, San Diego, 1996, p. 392.

Gu, D.-L., Arnush, M., Sarvetnick, N. Endocrine-exocrine intermediate cells in STZ-treated transgenic mice. Pancreas, in press.

Horwitz, M., Sarvetnick, N. Viruses and autoimmunity. In: Annales de l'Institut Pasteur: Actualités. Cohen, G.N. (Ed.). Elsevier, New York, 1996, p. 81.

Jones, E.M., Sarvetnick, N. Balance between autoimmune destruction and regeneration: Dual activities of IFN- in wound healing and inflammation. In: Pancreatic Growth and Regeneration. Sarvetnick, N. (Ed.). R.G. Landes, Austin, TX, in press.

Jones, E.M., Sarvetnick, N. Islet regeneration in IFN- transgenic mice. Horm. Metab. Res., in press.

Lee, M.-S., Mueller, R., Wicker, L.S., Peterson, L.B., Sarvetnick, N. IL-10 is necessary and sufficient for autoimmune diabetes in conjunction with NOD MHC homozygosity. J. Exp. Med. 183:2663, 1996.

Lee, M.-S., Sarvetnick, N. Fetal exposure to interferon- leads to induction of antigen-presenting molecules and leukocyte recruitment. Autoimmunity, in press.

Lee, M.-S., Sarvetnick, N. Cytokine transgenic mice and autoimmunity. In: Genetic Models of Immune and Inflammatory Diseases: Proceedings from Serono Symposia, Savannah, GA. Abbas, A.K., Flavel, R.A. (Eds.). Springer-Verlag, New York, 1996, p. 121.

Lee, M.-S., Wicker, L.S., Peterson, L.B., Sarvetnick, N. Pancreatic IL-10 induces diabetes in NOD-B6-Idd3-Idd10 mice even in the presence of I-E. Autoimmunity, in press.

Morgan, D.J., Liblau, R., Scott, B., Fleck, S., McDevitt, J.O., Sarvetnick, N., Lo, D., Sherman, L.A. CD8+ cell-mediated spontaneous diabetes in neonatal mice. J. Immunol. 157:978, 1996.

Mueller, R., Bradley, L.M., Krahl, T., Sarvetnick, N. Mechanism underlying counter-regulation of autoimmune diabetes by IL-4. Immunity, in press.

Mueller, R., Davies, J.D., Krahl, T., Sarvetnick, N. Interleukin-4 expression by grafts from transgenic mice fails to prevent allograft rejection. J. Immunol., in press.

Mueller, R., Krahl, T., Sarvetnick, N. Pancreatic expression of interleukin-4 abrogates insulitis and autoimmune diabetes in nonobese diabetic mice. J. Exp. Med. 184:1093, 1996.

Mueller, R., Krahl, T., Sarvetnick, N. Tissue-specific expression of IL-4 induces extracellular matrix accumulation and in vivo migration of naive B cells. Lab. Invest. 76:171, 1997.

O'Reilly, L.A., Gu, D., Sarvetnick, N., Cooke, A. cell neogenesis in an animal model of IDDM. Diabetes 46:599, 1997.

Sarvetnick, N. IFN-, IGIF and IDDM. J. Clin. Invest. 99:371, 1997.

Sarvetnick, N. Mechanisms of cytokine-mediated localized immunoprotection. J. Exp. Med. 184:1597, 1996.

Shaw, M.K., Lorens, J.B., Dhawan, A., DalCanto, R., Tse, H.Y., Tran, A.B., Bonpane, C., Eswaran, S.L., Brooke, S., Sarvetnick, N., Steinman, L., Nolan, G.P., Fathman, C.G. Local delivery of interleukin-4 by retrovirus-transduced lymphocytes ameliorates experimental autoimmune encephalomyelitis. J. Exp. Med. 185:1711, 1996.

Tian, J., Clare-Salzler, M., Herschenfeld, A., Middleton, B., Newman, D., Mueller, R., Arita, S., Evans, C., Atkinson, M.A., Mullen, Y., Sarvetnick, N., Tobin, A.J., Lehmann, P.V., Kaufman, D.L. Glutamate decarboxylase administration inhibits disease progression in pre-diabetic mice and prolongs survival of transplanted islets in diabetic mice. Nature Med. 2:1348, 1996.

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Role of Nonreceptor Protein-Tyrosine Kinases in Integrin Signaling

D.D. Schlaepfer, K.C. Jones

Activation of cell-surface receptors for integrins can promote intracellular signal transduction that affects proliferation and migration of cells. Because transmembrane integrin receptors do not have catalytic activity, these signaling events must be mediated by associated proteins. We are studying the role of focal adhesion kinase (FAK) and C-Src nonreceptor protein-tyrosine kinases in integrin-stimulated signaling to downstream targets such as ERK2/MAP kinase.

FAK associates with integrin receptors, and its in vitro kinase activity is enhanced by cell binding to extracellular matrix proteins such as fibronectin. We found that fibronectin-induced activation of FAK promotes the transient Src homology 2 (SH2) domain-dependent binding of the C-Src protein-tyrosine kinase to the FAK autophosphorylation site at Tyr 397. Subsequent C-Src phosphorylation of FAK at Tyr 925 promotes the SH2-dependent binding of the Grb2 adaptor protein to FAK, and this pathway may be one through which integrins can generate signals to ERK2.

Studies showed that Grb2 binding to FAK and fibronectin-induced activation of ERK2 in Src-deficient (Src-) cells were significantly less than in Src- cells engineered to reexpress C-Src. Expression of a dominant-negative fragment of C-Src in the Src- cells blocked Grb2 binding to FAK. However, fibronectin-stimulated signaling to ERK2 was slightly greater in cells containign the dominant-negative Src fragment than in Src- cells. In this context, we found that expression of the Src fragment promoted the association and FAK-mediated phosphorylation of another protein, p130Cas. Fibronectin-stimulated binding of SH2 domain--containing adaptor proteins such as Nck to p130Cas may provide alternative routes to ERK2 activation.

To determine the importance of FAK in fibronectin-stimulated signaling, we overexpressed wild-type FAK in cells and then stimulated the cells with fibronectin. FAK overexpression enhanced fibronectin-stimulated C-Src protein-tyrosine kinase activity, Shc tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation. Significantly, expression of the autophosphorylation and C-Src binding mutant of FAK blocked fibronectin-stimulated signaling to ERK2. Our studies reaffirm the importance of SH2-dependent binding interactions with FAK for the generation of signaling events and also support the hypothesis that it may be the summation of multiple integrin-initiated signaling pathways that provides maximal stimulation to targets such as ERK2.

PUBLICATIONS

Schlaepfer, D.D., Broome, M.A., Hunter, T. Fibronectin-stimulated signaling from a FAK and c-Src complex: Involvement of the Grb2, p130Cas, and Nck adaptor proteins. Mol. Cell. Biol. 17:1702, 1997.

Schlaepfer, D.D., Hunter, T. Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase (FAK) by Src-family protein-tyrosine kinases. Mol. Cell. Biol. 16:5623, 1996.

Schlaepfer, D.D., Hunter, T. FAK overexpression enhances Ras-dependent integrin signaling to ERK2/mitogen-activated protein kinase through interactions with and activation of c-Src. J. Biol. Chem. 272:13189, 1997.

Schlaepfer, D.D., Hunter, T. Signal transduction from the extracellular matrix: A role for the focal adhesion protein-tyrosine kinase FAK. Cell Struct. Funct. 21:445, 1996.

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Importance of the Carboxyl Terminus of the IL-8 Receptor 2 for Signal Transduction

I.U. Schraufstätter, R.C. Hoch, Z.G. Oades, M. Burger, C.G. Cochrane

The IL-8 receptor 2 (CXCR2), expressed in two hematopoietic cell lines, appears as a wide band of molecular weight 60,000. The receptor is phosphorylated within 15 seconds after the addition of IL-8 and slightly later when stimulated with melanoma growth stimulatory activity. A truncated form of the receptor missing the last 12 amino acids (T3) had normal IL-8 binding affinity but was no longer phosphorylated. Individual alanine replacement indicated that Ser346 and Ser348 were the primary sites of phosphorylation. Translocation of calcium was prolonged in phosphorylation-deficient cells and resulted in a greater than 200% increase in the release of ß-hexosaminidase. In studies of the importance of this phosphorylation in receptor desensitization, cells expressing the T3 mutant that were pretreated with IL-8 retained 60% of their capacity to induce GTPS exchange when exposed to IL-8 a second time; under the same conditions, wild-type IL-8 receptor 2 did not show GTPS binding above the basal rate. In contrast, internalization of the receptor was not affected by the loss of phosphorylation of the T3 mutant.

Truncated forms of the receptor missing 17 or 22 amino acids from the carboxy terminus had decreased binding affinities for IL-8 and melanoma growth stimulatory activity, a requirement for increased concentrations of ligand for calcium mobilization, and a decreased rate of GTPS exchange after addition of ligand. These results suggest that the region between amino acids 334 and 343 of the receptor may be important for coupling to GTP-binding proteins.

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Macrophage Inflammatory Protein-X, A Novel CC Chemokine

H. Takamori, J. Han, I.U. Schraufstätter

The cDNA for a novel CC chemokine, macrophage inflammatory protein-X (MIP-X), was cloned from a human lung library. Studies indicated that MIP-X is expressed by monocytes, peripheral lymphocytes, and bone marrow cells. Comparison of the deduced amino acid sequence of the mature protein showed a 61% sequence homology with macrophage inflammatory protein-1. MIP-X also shares certain motifs with the CXC family of chemokines, specifically a glutamic acid--leucine sequence in the NH2-terminus that is not seen in any other CC chemokine but is similar to the glutamic acid--leucine--arginine binding site shared by the CXC chemokines that bind to the receptor CXCR2. MIP-X mobilizes calcium in human peripheral monocytes and neutrophils and causes chemotaxis of these two cell types.

PUBLICATIONS

Hoch R.C., Schraufstätter I.U., Cochrane C.G. In vivo, in vitro, and molecular aspects of interleukin-8 and the interleukin-8 receptors. J. Lab. Clin. Med. 128:134, 1996.

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Recognition of Tumor Antigens by Cytotoxic T Lymphocytes

L. Sherman, M. Theobald,* D.J. Morgan, J. Lustgarten, J. Hernandez, T. Nugent, J. Biggs, S. Fleck, K. Holst

* Johannes Gutenberg University, Mainz, Germany

Inactivation of the p53 tumor-suppressor protein, through mutation or deletion, occurs in most human tumors. Often this inactivation involves a missense mutation at one of several defined mutational hot spots on the molecule. The altered p53 protein accumulates to high levels in the cytoplasm and has been used as a marker for cellular transformation. Previous studies, in both murine and human models, have shown the preferential susceptibility of transformed cells to lysis by cytotoxic T lymphocytes (CTLs) specific for peptides of the processed p53 protein presented on the cell surface in association with class I MHC molecules.

Using transgenic mice that express the HLA A2.1 molecule, we previously determined two peptide sequences, 149--156 and 264--272, from the human p53 molecule that bind A2.1 and serve as targets for CTL recognition and lysis. Of importance, CTLs specific for these peptide sequences can recognize and lyse a broad range of human tumors that express A2.1. In contrast, nontransformed cells that express A2.1 are not killed, presumably because of insufficient levels of presentation of the p53 peptides. These peptide epitopes also can stimulate A2.1-restricted CTLs from human peripheral blood, suggesting the presence of precursors with specificity for p53 that may potentially be mobilized to destroy tumor cells expressing high levels of p53 peptides for CTL recognition.

Although A2-restricted CTLs specific for the 264--273 epitope were able to lyse a variety of human tumor cells, a tumor cell line containing a mutation at amino acid residue 273 of the p53 molecule was not susceptible to lysis by A2.1-restricted CTLs specific for the flanking peptide 264--272. These results suggest that the mutation of arginine to histidine at position 273 of the protein may have prevented formation of the 264--272 peptide epitope.

Peptides presented in association with class I MHC molecules on the cell surface for recognition by the immune system are usually derived by proteolytic processing of proteins expressed within the cell by the proteasome complex. Accordingly, one mechanism that could interfere with the availability of the 264--272 peptide would be the inability of the proteasome to produce this peptide from the mutant sequence. This condition could occur if cleavage at the carboxyl side of the V at position 272 were abrogated by the change from arginine to histidine at residue 273. To test this hypothesis, we synthesized 27-mer peptides containing the 264--272 epitope flanked by either the wild-type or the mutant residue at position 273 and used the peptides as substrates for digestion by purified 20S proteasomes. These experiments were done in collaboration with U.H. Koszinowski, Ludwig Maximilians University, Munich, Germany, and P.-M. Kloetzel, Humboldt University, Berlin, Germany.

The results indicated that a change at amino acid position 273 from arginine to histidine, a known hot spot for missense mutation in p53, alters proteasome-mediated processing of the protein by inhibiting proteolytic cleavage between residues 272 and 273. This alteration prevents release of a flanking peptide epitope that spans residues 264--272 and makes cells resistant to killing by HLA A2.1--restricted CTLs specific for this epitope. This finding is significant because it suggests that in addition to contributing to malignant transformation, some p53 mutations may also affect immune recognition of tumor cells. Knowledge of the immunologic consequences of mutation in p53 is significant in the design of cancer vaccines and immunotherapies.

One of the major barriers to the development of T cells specific for epitopes from self-proteins such as p53 is the fact that tolerance has dampened the ability of the immune system to respond to many self-proteins. The degree to which self-tolerance debilitates the T-cell response varies for different epitopes. In studying a transgenic murine model in which the hemagglutinin (HA) of influenza virus is expressed in the pancreas, we observed a 100-fold reduction in the avidity of the CTL response to an antigenic peptide epitope of the HA molecule. This reduction correlated with the inability of these transgenic mice to spontaneously reject tumor cells expressing HA.

Surprisingly, despite such profound tolerance, tumors expressing HA could not grow if mice were vaccinated with a viral vector containing a peptide epitope of HA before receiving the tumor cells. This finding suggests that despite self-tolerance, for some tumor epitopes, the residual T-cell repertoire can be enlisted to prevent tumor growth. Furthermore, the fact that the T-cell response in these mice did not result in destruction of the pancreatic islets that express HA indicates that the level of immunity can be sufficient to eliminate tumor cells but not harm normal tissue expressing the same protein. These results suggest that it may be possible to develop tumor-specific vaccines that target proteins expressed not only at high levels in the tumor but also in normal tissue.

PUBLICATIONS

Lustgarten, J., Theobald, M., Labadie, C., Sherman. L.A. Identification of Her-2neu CTL epitopes using double transgenic mice expressing HLA-A2.1 and human CD8. Hum. Immunol. 52:109, 1996.

Malarkannan, S., Serwold, T., Nguyen, V., Sherman, L.A., Shastri, N. The MMTV env gene is the source of a CD8+ T-cell stimulating peptide/MHC class I complex in a murine thymoma. Proc. Natl. Acad. Sci. U.S.A. 93:13991, 1996.

Sherman, L.A., Theobald, M., Lustgarten, J. The use of HLA transgenic mice in identifying and targeting human tumor cells. Symp. Immunol. 6:41, 1997.

Sherman, L.A., Theobald, M., Morgan, D., Hernandez, J., Bacik, I., Yewdell, J., Bennink, J., Biggs, J. Strategies for tumor elimination by cytotoxic T lymphocytes. Crit. Rev. Immunol., in press.

Theobald, M., Biggs, J., Hernandez, J., Lustgarten, J., Labadie, C., Sherman,L.A. Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes. J. Exp. Med. 185.883, 1997.

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The Thymus and T-Cell Specificity

J. Sprent, I. Hwang, H. Kishimoto, S. Sun, D.F. Tough,* X. Zhang, A. Brunmark,** M. Jackson,** P. Peterson,** C. Beard,*** R. Jaenisch,*** P. Jones.****

* The Edward Jenner Institute for Vaccine Research, Compton, England
** R.W. Johnson Pharmaceutical Research Institute, La Jolla, CA
*** Whitehead Institute for Biomedical Research and Massachusetts Institute
of Technology, Cambridge, MA
**** University of Southern California, Los Angeles, CA

The specificity of mature T cells is shaped in the thymus by a process of positive and negative selection directed to self-MHC molecules. We are interested in defining the mechanisms involved in thymic selection and the factors that control the response of mature T cells in the extrathymic environment.

NEGATIVE SELECTION

The thymic medulla plays a key role in negative selection (induction of self-tolerance) and contains differentiated T cells en route to the extrathymic environment. However, being relatively mature, medullary T cells are thought to be beyond the stage of tolerance induction. This paradox is resolved by the finding that medullary T cells (CD4+CD8- thymocytes) comprise two distinct subsets. Medullary thymocytes expressing a fully mature (HSAlo) phenotype are strongly resistant to tolerance induction, whereas cells with a semimature (HSAhi) phenotype are tolerance susceptible. These findings suggest that the differentiated T cells that reach the medulla from the cortex remain sensitive to tolerance induction for a brief period before acquiring a fully mature tolerance-resistant phenotype. The semimature subset of medullary T cells has unique requirements for tolerance induction; depending on the conditions used, tolerizing these cells can involve either a Fas (CD95)-dependent or a Fas-independent pathway.

TRANSFECTED DROSOPHILA CELLS AS ANTIGEN-PRESENTING CELLS

Stimulation of naive T cells by antigen-presenting cells (APCs) is thought to involve two qualitatively different signals: signal 1 is due to recognition by T-cell receptors (TCRs) of antigenic peptides bound to MHC molecules, whereas signal 2 reflects contact with one or more costimulatory molecules. We studied the requirements for stimulating naive T cells by using MHC class I--restricted CD8+ T cells from a TCR transgenic line, defined peptides as antigen, and transfected Drosophila cells as APCs. Three main findings emerged from these studies. First, stimulation of naive T cells via signal 1 alone (MHC plus peptide) was essentially nonimmunogenic. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule-1 (ICAM-1), converted class I+ Drosophila cells to potent APCs capable of inducing strong T-cell proliferative responses and production of IL-2. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal 2 is complex. Transfected Drosophila cells are thus a useful tool for examining the minimal APC requirements of naive T cells.

TCR DOWNREGULATION ON NAIVE CD8+ T CELLS

We investigated the requirements for inducing downregulation of ß TCR molecules on naive MHC class I--restricted T cells by using 2C TCR transgenic mice and defined peptides as antigen. Confirming previous results, activation of 2C T cells in response to specific peptides required expression of CD8 on the responder cells and was heavily dependent on costimulation provided by either B7-1 or ICAM-1 on APCs. These stringent requirements did not apply to TCR downregulation. Thus, TCR downregulation seemed to depend solely on interactions between TCR and peptide and did not require expression of either CD8 or B7-1; ICAM-1 potentiated TCR downregulation, but only with limiting doses of peptides. TCR downregulation was most prominent with high-affinity peptides and appeared to be neither obligatory nor sufficient for T-cell activation. In marked contrast to T-cell activation, TCR downregulation was resistant to various metabolic inhibitors. Although its biological significance is unclear, TCR downregulation could be a device for protecting T cells against excessive signaling.

T-CELL STIMULATION IN VIVO BY LIPOPOLYSACCHARIDE

Under in vivo conditions, lipopolysaccharide (LPS) induces strong stimulation of T cells. As manifested by CD69 upregulation, injection of LPS stimulates both CD4+ and CD8+ T cells and at high doses stimulates naive (CD44lo) cells as well as memory (CD44hi) cells. In terms of cell division, however, the response of T cells to injection of LPS is limited to the CD44hi subset of CD8+ cells. In contrast to findings with B cells, proliferative responses of CD44hi CD8+ cells require only very low doses of LPS (10 ng). According to studies with LPS-nonresponder and knockout mice, LPS-induced proliferation of CD44hi CD8+ cells appears to operate via an indirect pathway involving LPS stimulation of APCs and release of type I (,ß) interferon. Similar selective stimulation of CD44hi CD8+ cells occurs in viral infections and after injection of type I interferon, implying a common mechanism. Hence, intermittent exposure to pathogens (gram-negative bacteria and viruses) could contribute to the high "background" proliferation of memory-phenotype CD8+ cells found in normal animals.

MITOGENICITY OF DIFFERENT ORGANISMS FOR MURINE B CELLS

Extending previous studies on bacteria and insects, we found that the capacity of DNA to stimulate B cells correlates closely with hypomethylation of DNA CpG dinucleotide motifs. Thus, DNA from yeasts, nematodes, mollusks, bacteria, and insects--organisms with little or no methylation of CpG motifs--showed strong stimulation of B cells. DNA from these organisms induced nearly all B cells (including small resting B cells) to upregulate the activation marker CD69 and caused many B cells to enter the cell cycle, indicative of polyclonal activation. These effects were not seen after selective methylation of CpG motifs. In contrast, DNA from various vertebrates (mammals, fish, frogs) and plants (corn)--organisms with heavily methylated CpG motifs--caused no stimulation of B cells. Despite this correlation, DNA prepared from two murine cell lines with hypomethylation of CpG motifs caused little or no stimulation of B cells. Thus, the notion that the stimulatory properties of DNA correlate solely with the presence of unmethylated CpG motifs may be an oversimplification.

PUBLICATIONS

Cai, Z., Brunmark, A., Jackson, M.R., Loh, D., Peterson, P.A., Sprent, J. Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells. Proc. Natl. Acad. Sci. U.S.A. 93:14736, 1996.

Cai, Z., Kishimoto, H., Brunmark, A., Jackson, M.R., Peterson, P.A., Sprent, J. Requirements for peptide-induced TCR downregulation on naive CD8+ T cells. J. Exp. Med. 185:641, 1997.

Kishimoto, H., Cai, Z., Brunmark, A., Jackson, M.R., Peterson, P.A., Sprent, J. Differing roles for B7 and ICAM-1 in negative selection of thymocytes. J. Exp. Med. 184:531, 1996.

Kishimoto, H., Sprent, J. Negative selection in the thymus includes semi-mature T cells. J. Exp. Med. 185:263, 1997.

Sprent, J. Immunological memory. Curr. Opin. Immunol. 9:371, 1997.

Sprent, J. The mixed lymphocyte reaction (MLR). In: Encyclopedia of Immunology, 2nd ed. Delves, P.J., Roitt, I.M. (Eds.). Academic Press, London, in press.

Sprent, J. Lymphocyte migration in vivo assessed with radioisotopes. In: Weir's Handbook of Experimental Immunology, 5th ed. Herzenberg, L.A., et al. (Eds.). Blackwell Science, Cambridge, England, 1997, p. 142.1.

Sprent, J. T cells and memory lapses. Trends Microbiol. 5:259, 1997.

Sprent, J., Cai, Z., Brunmark, A., Jackson, M.R., Peterson, P.A. Constructing artificial antigen-presenting cells from Drosophila cells. In: Dendritic Cells in Fundamental and Clinical Immunology, Vol. 3. Ricciardi-Castagnoli, P. (Ed.). Plenum, New York, in press.

Sprent, J., Kishimoto, H. Negative selection in the thymus. In: Immune Tolerance. Libert, V.A. (Ed.) . Elsevier, New York, 1996, p. 27.

Sprent, J., Kishimoto, H. T-cell tolerance and the thymus. Ann. N. Y. Acad. Sci., in press.

Sprent, J., Kishimoto, H., Cai, Z., Surh, C.D., Brunmark, A., Jackson, M.R., Peterson, P.A. The thymus and T cell death. Adv. Exp. Med. Biol. 406:191, 1996.

Sprent, J., Surh, C.D. Thymus. In: Encyclopedia of Immunology, 2nd ed. Delves, P.J., Roitt, I.M. (Eds.). Academic Press, London, in press.

Sprent, J., Tough, D.F. Immunological memory. In: Frontiers in Biology, Vol. 6. Lanzavecchia, A., Malissen, B., Sitia, R. (Eds.). Istituto della Enciclopedia Italiana Treccani, Rome, in press.

Sprent, J., Tough, D.F., Sun, S. Factors controlling the turnover of T memory cells. Immunol. Rev. 156:79, 1997.

Sun, S., Beard, C., Jaenisch, R., Jones, P., Sprent, J. Mitogenicity of DNA from different organisms for murine B cells. J. Immunol., in press.

Surh, C.D., Kosaka, H., Sprent, J. Rat stem cells developing in irradiated SCID mice fail to become tolerized and cause lethal graft-versus-host disease. Am. J. Pathol. 151:591, 1997.

Surh, C.D., Lee, D.-S., Fung-Leung, W.-P., Karlsson, L., Sprent, J. Thymic selection by a single MHC/peptide ligand produces a semi-diverse repertoire of CD4+ T cells. Immunity 7:209, 1997.

Tough, D.F., Sprent, J. Bystander stimulation of T cells in vivo by cytokines. Vet. Immunol. Immunopathol., in press.

Tough, D.F., Sun, W., Sprent, J. T-cell stimulation in vivo by LPS. J. Exp. Med. 185:2089, 1997.

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Thymic T-Cell Development

C.D. Surh, D.-S. Lee, B.B. Ernst, C. Ahn,* W. Chang, J. Chang, B. Lau, S.R. Webb, W.-P. Fung-Leung,** L. Karlsson**

* Seoul National University Hospital, Seoul, Korea
** R.W. Johnson Pharmaceutical Research Institute, La Jolla, CA

ROLE OF SELF-PEPTIDES IN T-CELL SELECTION

In normal animals, the thymus selects a T-cell repertoire that is capable of recognizing an infinite variety of foreign antigens presented by self-MHC molecules. To determine the role of individual peptides in selection of the T-cell repertoire, we examined the development of T cells in H2-M--deficient mice in which positive selection is directed to a single peptide, the class II--associated invariant chain peptide (CLIP), bound to H2-Ab.

Using two sensitive in vivo approaches, we showed that approximately 75% of CD4+ T cells produced by selection directed to the CLIP--H2-Ab complex have self-reactivity to the various peptides expressed on wild-type syngeneic antigen-presenting cells. When these self-reactive T cells were depleted, the residual CD4+ T cells had a polyclonal repertoire in terms of alloreactivity, responses to foreign protein antigens, and use of the Vß T-cell receptor. Reconstitution experiments with irradiated H2-M--deficient mice, however, showed that CLIP could not positively select T cells from two lines of T-cell receptor transgenic mice, suggesting that the repertoire of CD4+ cells generated by CLIP was only semidiverse. Production of a fully diverse T-cell repertoire thus requires positive selection against multiple peptides.

ABERRANT THYMIC TOLERANCE IN RAT-MOUSE CHIMERAS

Graft-versus-host disease does not develop in irradiated hosts given allogeneic bone marrow cells depleted of T cells; under these conditions, the mature T cells arising from the donor stem cells become tolerant to host antigens in the thymus. A radically different result can occur when hosts are reconstituted with xenogeneic stem cells. When reconstituted with fetal liver cells from Lewis rats, lightly irradiated, adult C.B-17 mice with severe combined immunodeficiency (SCID) have nearly total repopulation with rat-derived lymphohemopoietic cells, but severe and often lethal chronic graft-versus-host disease develops. The rat T cells in these rat-mouse chimeras have limited tolerance to host mouse antigens, as shown by in vitro functional assays and adoptive transfer of T cells to secondary SCID hosts.

The failure of newly formed rat T cells in these chimeras to become fully tolerant to host mouse antigens appears to be due to depletion of host antigen-presenting cells by irradiation. Thus, graft-versus-host disease does not develop in rat-mouse chimeras generated by transplanting rat fetal liver cells into unirradiated neonatal SCID mice, and the rat T cells show self-tolerance. Because chimeras generated by reconstituting irradiated SCID mice with fetal liver cells from allogeneic mice have strong self-tolerance, presumably through recognition of host antigens on thymic epithelial cells, the implication is that mouse thymic epithelial cells are tolerogenic only for mouse and not for rat immature T cells.

PUBLICATIONS

Ernst, B.B., Surh, C.D., Sprent, J. Bone-marrow-derived cells fail to induce positive selection in thymus reaggregation cultures. J. Exp. Med. 183:1235, 1996.

Song, E.S., Lee, V., Surh, C.D., Lynn, A., Brumm, D., Jolly, D.J., Warner, J.F., Chada, S. Antigen presentation in retroviral mediated gene transfer in vivo. Proc. Natl. Acad. Sci. U.S.A. 94:1943, 1997.

Sprent, J., Kishimoto, H., Cai, Z., Surh, C.D., Brunmark, A., Jackson, M.R., Peterson, P.A. The thymus and T-cell death. Adv. Exp. Med. Biol. 406:191, 1996.

Surh, C.D., Kosaka, H., Sprent, J. Rat stem cells developing in irradiated SCID mice fail to become tolerized and cause lethal graft-versus-host disease. Am. J. Pathol., in press.

Surh, C.D., Lee, D.-S., Fung-Leung, W.-P., Karlsson, L., Sprent, J. Thymic selection by a single MHC/peptide ligand produces a semidiverse repertoire of CD4+ T cells. Immunity, in press.

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Toward a Structural Understanding of T-Cell Recognition and MHC Class II Presentation of Antigen

L. Teyton, M. Bondad, H. Celia, C. Garcia, G. Luo, C. Scott, R. Stefanko

IN VITRO T-CELL RECOGNITION

T-cell activation is the result of a productive interaction between the T-cell receptor (TCR) at the surface of a T cell and an MHC molecule at the surface of an antigen-presenting cell. This interaction is governed by the rules of MHC restriction, which limit the recognition of a T cell to a single specific ligand. This restriction is the result of thymic maturation through negative and positive selections. The structural basis of the TCR-MHC interaction is still unclear. Our group has focused its efforts on determining the structural parameters that govern MHC restriction, specificity, and T-cell activation.

We have expressed a large number of soluble TCRs and MHC molecules in both class I-- and class II--restricted systems. Plasmon resonance and purified molecules have been used to measure the strength of TCR-MHC interactions. In most cases, the affinity of TCRs for MHC molecules is low, from 10 to 100 mM. To determine which parts of the interface are the most important in providing the energy of binding, we are using two complementary routes. First, in collaboration with D. Kranz, University of Illinois, we are using plasmon resonance to examine the binding of alanine mutants of each of the complementarity-determining regions of both the - and the ß-chains of TCRs to the peptide-MHC complex. Second, these results are rationalized by using the high-resolution three-dimensional structure of two MHC-TCR complexes that were solved in I. Wilson's laboratory, Department of Molecular Biology. These structures are our guide to additional mutagenesis. The goal of this work is to determine the basis of T-cell specificity and MHC restriction.

To complement this project, we have also characterized the role of accessory molecules in T-cell recognition. For instance, it was shown that CD8 increases the affinity of TCRs for MHC molecules. For studies to determine the mechanisms of this effect, recombinant CD8 and ß molecules were purified and used in crystallization trials with TCRs and MHC molecules. A similar study will be done for CD4 and class II--restricted TCRs.

Finally, in collaboration with E. Kubalek and R. Milligan, Department of Cell Biology, we are using a new technique to reconstitute a functional TCR complex in vitro from recombinant molecules. We are taking advantage of an engineered histidine tag at the C-terminus of our TCR and accessory molecules (CD3, CD4/CD8) to capture these components with lipids retaining a chelating cage at the hydrophilic pole. The capture orients the extracytoplasmic part of the molecule to have the same topology as a transmembrane molecule. Liposomes made with this lipid can be fused to hydrophobic surfaces to reconstitute bilayers that mimic T-cell membranes. Surface plasmon resonance measurements are carried out on such surfaces.

MHC CLASS II PRESENTATION

Cell-mediated autoimmunity is restricted to MHC class II molecules. Some MHC class II molecules linked to autoimmunity have peculiar sequence features such as position 57 of the ß-chain of the MHC molecule associated with diabetes in mice and humans. We are evaluating the functional impact of these structural differences. Murine MHC class II molecules have been expressed, purified, and crystallized in collaboration with I. Wilson, and their structures will be determined. These structures and the analysis of peptide-binding motifs, done in collaboration with A. Kang, Department of Molecular Biology, will enable us to determine peptides derived from autoantigens. In addition, in collaboration with D. Mathis, INSERM, Paris, two TCRs in nonobese diabetic mice are being expressed and characterized in terms of specificity and binding. The structure of TCR-MHC complexes involved in autoimmunity will show if unusual structural features cause autoreactivity.

In addition, in collaboration with A. Brünmark, R.W. Johnson Pharmaceutical Research Institute, peptide binding to MHC class II molecules is being studied in vitro by using (1) complexes composed of MHC class II molecules and the invariant chain and (2) accessory molecules such as HLA-DM. We have shown that at acidic pH, the invariant chain drives conformational changes of the complexes, leading to the loading of peptides onto MHC class II molecules. This method of loading peptides is now being used to screen peptide phage-display libraries and complex peptide mixtures. Structural studies of the complexes composed of class II molecules and invariant chains are also being pursued.

PUBLICATIONS

Garcia, K.C., Degano, M., Stanfield, R.L., Scott, C., Brünmark, A., Peterson, P.A., Wilson, I.A., Teyton, L. Structural basis of T-cell recognition. In: Vaccine 97. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1997, p. 51.

Garcia, K.C., Scott, C., Brünmark, A., Peterson, P.A., Teyton, L., Wilson, I.A. Crystal structure of an -ß T-cell receptor heterodimer at 2.5 Å. Science 274:209, 1996.

Garcia, K.C., Scott, C.A., Brünmark, A., Peterson, P.A., Wilson, I.A., Teyton, L. CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes. Nature 384:577, 1996.

Huang, J.-F., Teyton, L., Harper, J.F. Activation of a Ca2+-dependent protein kinase (CDPK) involves intra-molecular binding of a calmodulin-like regulatory domain. Biochemistry 35:13222, 1996.

Lang, J., Jackson, M., Teyton, L., Brünmark, A., Kane, K., Nemazee, D. B cells are exquisitely sensitive to central tolerance and receptor editing induced by ultralow affinity, membrane-bound antigen. J. Exp. Med. 184:1685, 1996.

Scott, C., Garcia, C., Carbone, F.R., Wilson, I.A., Teyton, L. Role of chain pairing for the production of functional soluble IA MHC class II molecules. J. Exp. Med. 183:2087, 1996.

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Genes and Genetics of Systemic Autoimmunity

A.N. Theofilopoulos, D.H. Kono, S.R. Duncan, D. Balomenos, H. Sabzevari, U. Scheuring, S. Alcorisa-Vidal

Propensity to autoimmune diseases unequivocally has a strong genetic basis, but the exact genes that confer this propensity remain largely unknown. Determination of the genes will catalyze an understanding of the etiology and pathogenesis of autoimmune diseases, provide better means for diagnosis and prevention, and ultimately lead to the design of specific therapies. Because these diseases appear to have a polygenic basis, their study in humans is complicated. We have therefore concentrated our initial efforts on the study of inbred mouse strains with uniform genetic compositions. Clinical characteristics similar to those of human systemic lupus erythematosus, the prototypic systemic autoimmune disease, develop spontaneously in several of these strains. We are using gene modifications and genomewide searches of specific genes to determine predisposing loci.

REQUIREMENT FOR IFN-GAMMA IN LUPUS DISEASE

Cytokines play a critical role in regulating the quantitative and qualitative characteristics of cellular and humoral immune responses. As such, these molecules would be expected to inhibit or promote the initiation and perpetuation of autoimmune diseases, including lupus disease.

Previous studies with blockers or stimulators of IFN- suggested that this major cytokine plays an important role in the pathogenesis of lupus disease. To conclusively assess the role of IFN-, we created congenic MRL-lpr mice homozygous and heterozygous for the deletion of the gene for this cytokine. Early death was prevented and glomerulonephritis was severely reduced in both types of mice compared with control animals. In mice homozygous for the deletion, hyperglobulinemia was maintained, but with a switch from IgG2a to IgG1 predominance, and the predominantly IgG2a autoantibodies to double-stranded DNA were severely reduced without concomitant increases in other IgG isotypes. Despite the reduction in glomerulonephritis in mice heterozygous for the deletion (50% reduction in IFN- levels compared with the levels in controls), the levels of autoantibody were unaffected, indicating the importance of additional locally exerted disease-promoting effects of IFN-. Mice homozygous for the deletion also had reduced lymphadenopathy associated with decreases in the dominant double-negative (CD4-CD8-) T-cell population. Moreover, macrophages (antigen-presenting cells) from the homozygous mice expressed markedly lower levels of MHC class I and II molecules than did control mice.

These findings indicate that hyperproduction of IFN- is required for the development of lupus disease. The high levels of this cytokine presumably increase MHC expression and autoantigen presentation to otherwise quiescent, nontolerant, low-affinity, anti-self T cells and promote local inflammatory processes. These results suggest that even partial reduction of IFN- may inhibit or reduce immunopathologic changes associated with lupus disease.

ROLE OF P59FYN IN DOUBLE-NEGATIVE T-CELL ACTIVATION AND EXPANSION

The severe spleen and lymph node hyperplasia of MRL-lpr lupus mice that have a defect in the Fas gene (apoptosis gene) is characterized by expansion of double-negative (CD4-CD8-) T cells. These T cells appear to be derived from CD8+ precursors and are thought to be functionally inert. The latter conclusion, however, is not supported by the activation phenotype of double-negative cells.

To further characterize the functional status of the lpr double-negative T cells, we performed in vivo labeling experiments in wild-type MRL-+/+ and mutant MRL-lpr as well as p59fyn-/- MRL-lpr mice. Fyn transduces T-cell receptor--derived signals by association with the CD3 zeta-chain, whereas the other major proximal tyrosine kinase, p56lck, transduces signals by association with CD4 or CD8. Therefore, the only kinase available for activation of double-negative cells is Fyn.

Analysis of T cells from 8-week-old mice given a DNA precursor in drinking water for 9 days showed that higher proportions of CD4+ and CD8+ lymph node cells were dividing in lpr (15%) than in +/+ mice (3%), and the proportion of cycling cells was even higher in the double-negative (71%) and CD4+B220+ (54%) lpr subsets. The CD4+B220+ cells also expand in lpr mice, but to a much smaller degree than do the double-negative cells. Radionuclide chase experiments showed that activation and division in most double-negative cells were initiated subsequent to the cells' precursor CD8+ stage. Lymphadenopathy and other manifestations of disease were greatly reduced in Fyn-/- lpr mice concomitant with decreased proportions of double-negative cells (from 77% to 20%). Division of double-negative cells was also severely reduced in Fyn-/- lpr mice, whereas all other subsets were unaffected. A similar phenomenon was observed in double-negative liver cells of Fyn-/- mice of normal background.

We conclude the following: (1) Double-negative lpr T cells, rather than being end-stage, retain the capacity to be activated and repeatedly divide before reaching an anergic or replicative senescence stage. (2) The Fyn kinase associated with the CD3 zeta-chain is important for double-negative cell signal transduction. (3) Double-negative cells are highly dependent on Fas participation for apoptosis. (4) Double-negative cells contribute to the early development of the serologic and histologic features of the MRL-lpr lupus disease.

CYCLIN KINASE INHIBITORS AND REPLICATIVE SENESCENCE

A general characteristic of lupus-prone mice (and humans) is the expedited accumulation of large numbers of presumably self-reactive T cells with the phenotypes of activated and memory cells. The mechanisms by which these cells escape apoptosis have not been elucidated. We used CD4+CD44hi cells, which have the phenotypes of activated and memory cells, obtained from male BXSB mice with early-life severe lupuslike disease to investigate the status of the cell cycle and susceptibility to apoptosis and to determine the role of corresponding genes in the survival of these cells.

Most of the rapidly accumulating memory phenotype CD4+CD44hi cells of 4-month-old male BXSB mice were arrested in G1 of the cell cycle. With advanced age, the CD4+CD44hi cells switched from predominantly cycling to predominantly noncycling. Moreover, the CD4+CD44hi cells of older mice were refractory to proliferation and apoptosis induced by antibodies to CD3. These cells also expressed high levels of certain cyclin-dependent kinase inhibitors.

We propose that in autoimmunity, self-reactive T cells are subjected to successive rounds of activation and division that eventually lead to a buildup in cyclin-dependent kinase inhibitors. High levels of these inhibitors cause refractoriness to further activation, impaired entry into the cell cycle, and resistance to apoptosis, a situation akin to replicative senescence. These findings are important not only in systemic autoimmunity but also in many other situations in which the immune system is subjected to repetitive immune responses against foreign antigens or self-antigens, as in AIDS, aging, rheumatoid arthritis, and other conditions.

QUANTITATIVE TRAIT LOCI

Previous studies in our laboratory determined several autoimmune susceptibility loci for the (NZB X NZW)F1 hybrid, one of the major animal models of systemic lupus erythematosus, which also include the MRL-Faslpr and BXSB strains. We have now identified non-Faslpr susceptibility loci in mice homozygous for the apoptosis-defective Faslpr mutation by using MRL-Faslpr (severe autoimmune disease) and C57BL/6-Faslpr (B6-Faslpr; minimal late-onset disease) mice. A panel of 274 (MRL-Faslpr x B6-Faslpr)F2 intercross mice was used to study pathologic features associated with systemic lupus erythematosus or inflammatory arthritis. We found significant differences between the two parental backgrounds in lymphadenopathy, splenomegaly, antibodies to double-stranded DNA, glomerulonephritis, and arthritis.

Using interval mapping, we found that four quantitative trait loci, designated Lmb1, Lmb2, Lmb3, and Lmb4, on chromosomes 4, 5, 7, and 10, respectively, were linked with lymphadenopathy and/or splenomegaly. Lmb1, Lmb2, and Lmb3 were also linked to the production of antibodies to double-stranded DNA but not to glomerulonephritis; Lmb4 was associated with glomerulonephritis. Several other loci that met the criteria of suggestive significance were also detected, including a single locus associated with arthritis on chromosome 1 and two others associated with glomerulonephritis on chromosomes 6 and 19. Lmb2, Lmb3, and Lmb4 were inherited from the MRL background, but interestingly, Lmb1 was derived from the B6 background. Each locus acted in an additive manner regardless of its strain of origin, although certain combinations were more effective. Thus, the mild disease in the B6 background may be attributable to the presence of only a single major susceptibility locus.

These findings clearly show that genetic heterogeneity for lupus susceptibility genes may sometimes include genes from so-called normal strains, provide an explanation for why crosses of the same strain to different backgrounds may yield different sets of susceptibility loci, and suggest that analysis of F2 intercrosses may be preferable for autoimmune susceptibility studies because genetic contributions from both parental strains can be defined. The exact nature of genes corresponding to these susceptibility loci must await the development of congenic animals and further refinements of the chromosomal locations of the loci. The determination of quantitative trait loci with highly significant linkage to disease manifestations in Fas-lpr strains will make it possible to map and clone new genetic defects that contribute to autoimmunity.

PUBLICATIONS

Baccala, R., Gonzalez-Quintial, R., Kono, D.H., Theofilopoulos, A.N. Multiprobe RNase protection assay and sequence enrichment nuclease assay for TCR analysis. In: The Human Antigen T Cell Receptor: Selected Protocols and Applications. Oksenberg, J. (Ed.). R.G. Landes, Georgetown, TX, 1997, p. 86.

Balomenos, D., Rumold, R., Theofilopoulos, A.N. The proliferative in vivo activities of lpr double-negative T cells and the primary role of p59fyn in their activation and expansion. J. Immunol. 159:2265, 1997.

Beijleveld, L.J.J., Damoiseaux, J.G.M.C., Roglic, M., Theofilopoulos, A.N., van Breda Vriesman, P.J.C. Effector cells in cyclosporin-A-Induced autoimmunity: Both CD4 and CD8 T cells generate autoimmune disease upon adoptive transfer. Transplantation 62:1468, 1996.

Chowers, Y., Marsh, M.N., deGrandpre, L., Nyberg, A., Theofilopoulos, A.N., Kagnoff, M.F. Increased proinflammatory cytokine expression in the colonic mucosa of coeliac disease patients in the early period after gluten challenge. Clin. Exp. Immunol. 107:141, 1997.

Duncan, S.R., Elias, D.J., Roglic, M., Pekny, K.W., Theofilopoulos, A.N. T-cell receptor biases and clonal proliferations in blood and pleural effusions of patients with lung cancer. Hum. Immunol. 53:39, 1997.

Hui, S., Kono, D.H., Chen, L.-Y., Rubin, E.M., Kaye, J. Induction of the early growth response (Egr) family of transcription factors during thymic selection. J. Exp. Med. 185:731, 1997.

Kono, D.H., Owens, D.G., Wechsler, A.R. Jak3 maps to chromosome 8. Mamm. Genome 7:476, 1996.

Kono, D.H., Theofilopoulos, A.N. Genetics of murine SLE. In: Dubois' Lupus Erythematosus, 5th ed. Wallace, D.J., Hahn, B.H. (Eds.). Williams & Wilkins, Baltimore, 1996, p. 119.

Roglic, M., MacPhee, R.D., Duncan, S.R., Sattler, F.R., Theofilopoulos, A.N. T cell receptor BV gene repertoires and clonal expansions of CD4 cells in patients with HIV infections. Clin. Exp. Immunol. 107:21, 1997.

Sabzevari, H., Propp, S., Kono, D.H., Theofilopoulos, A.N. G1 arrest and high expression of cyclin kinase and apoptosis inhibitors in accumulated memory phenotype CD4+ cells of lupus mice. Eur. J. Immunol. 27:1901, 1997.

Theofilopoulos, A.N. Mechanisms and genetics of autoimmunity. In: The Role of Immune Mechanisms in Cardiovascular Disease. Schultheiss, H.-P., Schwimmbeck, P.L. (Eds.). Springer-Verlag, New York, 1997, p. 3.

Theofilopoulos, A.N. Systemic lupus erythematosus: Experimental models. In: Encyclopedia of Immunology, 2nd ed. Delves, P.J., Roitt, I.M. (Eds.). Saunders, Philadelphia, in press.

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Hematopoietic Regulation and HIV Type 1--Induced Dysfunction

B.E. Torbett, D.E. Mosier, K.A. Smith, D.R. Salomon,* K. Conners. L. Crisa,* K. Anderson,** R.A. Maki**

* Division of Experimental Hemostasis and Thrombosis, TSRI
** The Burnham Institute, La Jolla, CA

Our research interests are in virology and hematology. We are investigating (1) the cellular and viral mechanisms in hematopoietic and thymic dysfunction induced by HIV type 1 (HIV-1), (2) the use of human-mouse chimeras as a model of human hematopoiesis, and (3) the role of the ets-family transcription factor PU.1 in regulating hematopoiesis in mice.

HIV-1 DISRUPTION OF HEMATOPOIESIS AND THYMOPOIESIS

Anemia, neutropenia, and thrombocytopenia are common in patients with AIDS. Disruption of thymopoiesis is also a consequence of HIV-1 infection. We are investigating whether HIV-1 dysregulates hematopoiesis by infecting and disrupting the function of bone marrow stromal cells or by infecting and disrupting the development of stem or progenitor cells. HIV-1 can infect thymocytes and macrophages, but it is less clear at what stage thymocytes can be infected and whether thymic stroma can be infected. The infection and disruption of thymic stroma would be detrimental for thymopoiesis.

We found that human primary bone marrow or thymic stromal cells cannot be infected (1) directly by HIV-1 isolates that use CD4 and the CC or CXC chemokine family of receptors for infection or (2) via T cells infected by these isolates. Bone marrow or thymic stromal cells do not appear to express known CC or CXC receptors on the surface. Thus, we conclude that stromal cells might not normally become dysfunctional as the result of direct HIV-1 infection. Our studies also suggest that primitive bone marrow or thymic CD34+ cells are not directly infected by HIV-1. We are now investigating (1) whether viral proteins or infected T cells or macrophages contribute to hematopoietic disruption and (2) what effect the expression of chemokine receptors on thymocytes has on HIV-1 infection.

HEMATOPOIETIC STUDIES IN HUMAN-MOUSE CHIMERAS

We have developed a human-mouse model to study human hematopoiesis. Purified human cord blood cells are transplanted to nonobese diabetic mice that have severe combined immunodeficiency. Hematopoiesis of human cells is maintained in these mice for 6 months, with production of B cells and myeloid cells and expansion of CD34+/CD38-/lo cells. Primitive human progenitor cells egress from the mouse bone marrow space to the periphery when induced with human growth factors. This model should allow us to study the mechanisms responsible for cytokine-mediated migration of bone marrow cells. Transplantation of fetal or neonatal thymus cells into these chimeras allows T-cell development and enables us to test whether infected CD4+ T cells contribute to HIV-1--induced hematopoietic dysfunction.

THE PU.1 TRANSCRIPTION FACTOR IN HEMATOPOIETIC DEVELOPMENT

The role of transcription factors in regulating development has contributed insights to understanding hematopoiesis. PU.1 is a hematopoietic-specific transcription factor critical for the development of myeloid and lymphoid cells. Mice homozygous for the disrupted PU.1 gene have multiple hematopoietic abnormalities. They are devoid of B lymphocytes, monocytes and macrophages, dendritic cells, and mature neutrophils but not T cells or immature neutrophils. We recently showed that disruption of the PU.1 gene results in loss of cell-surface receptors for granulocyte, granulocyte-macrophage, and macrophage colony-stimulating factors. These myeloid cytokines are required for normal myeloid development. We are characterizing how and where PU.1 dictates commitment to neutrophil, macrophage, and dendritic cell lineages.

PUBLICATIONS

McKercher, S.R., Torbett, B.E., Anderson, K.L., Henkel, G.W., Vestal, D.J., Baribault, H., Klemsz, M., Feeney, A.J., Wu, G.E., Paige, C.J., Maki, R.A. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15:5647, 1996.

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Molecular Mechanisms of Host-Pathogen Interactions

R.J. Ulevitch, P.S. Tobias, J.D. Lee, V.V. Kravchenko, J.C. Mathison, J. Bohuslav, C. Fearns, J. Gegner,* W. Guo, Y. Jiang, Y. Kato, J. Lee, L. Mira-Arbibe, S. Orr, B. Richter, G. Sanna, D. Schiff, J. Schimke, R. Tapping, C. Werts, Q. Zhong, T. Kirkland,** T.R. Martin***

* University of San Diego, San Diego, CA
** University of California, San Diego, CA
*** University of Washington, Seattle, WA

Attacks by pathogens often set in motion chains of events that cause severe injury to the host, and nowhere is this phenomenon illustrated more dramatically than in the response by humans to infection by gram-negative bacteria. In his book Lives of a Cell, Lewis Thomas characterizes the host response to the endotoxin, or lipopolysaccharide (LPS), of gram-negative bacteria as being "read by our tissues as the very worst of bad news. . . . There is nothing intrinsically poisonous about endotoxin, but it must look awful, or feel awful, when sensed by cells. Cells believe that it signifies the presence of gram-negative bacteria, and they will stop at nothing to avoid this threat."

Clearly, much human suffering could be eased if such overzealous host responses could be tempered. However, such responses, when not overzealous, are a normal part of the host's homeostatic mechanisms, designed to respond to the threat of gram-negative bacterial infection. Accordingly, we are attempting to (1) provide a molecular description of the events that enable cells to recognize LPS and (2) learn how to control these responses without compromising host defenses against pathogens. Because LPS is a prototypic activator of cells of the immune, inflammatory, and vascular systems, information derived from our studies has broad implications for all areas of biomedical research and for understanding how other types of microbial pathogens cause human disease.

A major part of our effort is to define the membrane receptor and intracellular signaling pathways used by LPS during septicemia. Through studies of LPS-binding protein (LBP) and CD14, our research has provided an entirely new basis for understanding how components of microbial pathogens initiate host responses. LBP is a plasma protein; CD14 is a protein found both on the membranes of cells of the myeloid lineage and soluble in plasma.

LPS INTERACTIONS WITH LBP AND CD14

The biochemical basis for targeting CD14 to intercede in the development of endotoxic shock is derived from our understanding of the mechanism by which CD14, together with LBP, enables cells to respond to LPS. LBP binds to LPS whether the LPS is in the outer membranes of gram-negative bacteria or free in solution. The key activity of LBP is that it promotes the interaction of LPS with CD14.

If the CD14 is present as an extracellular membrane protein of myeloid cells, LBP enhances phagocytosis of intact gram-negative bacteria and enables cellular responses to concentrations of LPS that would not otherwise initiate the activation of these inflammatory cells. If the CD14 is the soluble form, the LPS-CD14 complexes may be recognized by receptors on a wide variety of cells, including endothelial, epithelial, dendritic, and smooth muscle cells, and the recognition initiates the inflammatory responses of the affected cells. Normally, these inflammatory responses are local and result in resolution of an infection before the infection can spread to be life threatening. However, the response to LPS can also, paradoxically, become life threatening when it is uncontrolled; it spreads throughout the body and causes endotoxic shock.

Interestingly, LBP and CD14 also participate in antiinflammatory responses to LPS and gram-negative bacteria. For example, when LPS binds with LBP to CD14 on the monocyte membrane, the LPS partitions into two pathways, one that activates inflammatory responses and one that causes internalization of the LPS without initiating an inflammatory response. How the partitioning of LPS into these two pathways is controlled is not understood and is a focus of our studies.

The clinical syndrome of septic shock is still a serious medical problem; the 40% mortality rates have not decreased during the past two decades. Studies completed in animal models of septic shock in subhuman primates and in rabbits provide evidence for the benefit of blocking LPS binding to CD14 by using monoclonal antibodies to epitopes in the CD14-ligand binding pocket. A key element in treatment of septic shock is the ability to treat patients therapeutically. Antibodies to CD14 that are protective when administered prophylactically are also effective when given to experimental animals after the initiation of changes leading to septic shock.

During the past year, we did extensive studies in a rabbit model of endotoxic shock with a monoclonal antibody that blocks binding of LPS to both soluble and membrane-bound CD14. Injury in this animal model is provoked by an initial injection of LPS that is followed by two additional injections 5 and 24 hours later. The mortality rate is 40% at 48 hours, and more than 90% of the surviving animals have extensive organ injury characterized by cellular infiltrates and marked accumulation of fibrin. Treatment with antibodies to CD14 1 hour before the third LPS injection still provides complete protection against lethality and tissue injury. Thus, we are optimistic about the potential use of antibodies to CD14 during septic shock in humans when therapeutic intervention is a necessity.

CELLULAR ACTIVATION PATHWAYS INVOLVING MAP KINASES

Transfer of information from the extracellular environment to the nucleus often proceeds through intracellular kinase cascades initiated by binding of a ligand to the ligand's membrane receptor. Members of the MAP kinase family of serine/threonine kinases play a central role in many cellular responses to extracellular stimuli. These kinases are activated by phosphorylation of adjacent threonine and tyrosine residues. The sequence motif threonine-Xaa-tyrosine defines subgroups of MAP kinases; the Xaa may be glutamate, glycine, or proline.

We discovered one member of this family, p38, and have now determined three additional isoforms of this enzyme. These isoforms have distinct biochemical and biological properties. A variety of experimental studies link activation of the p38 enzyme group to transcriptional and posttranscriptional events associated with production of important mediators of inflammation, and this group of enzymes is a potentially important target for antiinflammatory drugs.

During the past year, we focused on determining downstream targets of the p38 group. We found that members of the myocyte-enhancing factor family of transcription factors are p38 substrates. The p38 mediates phosphorylation of myocyte-enhancing factor-2, and this step increases the transactivation activity of the transcription factor. One consequence of this increase is increased expression of c-Jun; these findings provide a novel link between myocyte-enhancing factor-2 and inflammatory responses where activation of the p38 pathway is involved.

PUBLICATIONS

Abe, J., Takahashi, M., Ishida, M., Lee, J.-D., Berk, B.C. c-Src is required for oxidative stress-mediated activation of big mitogen-activated protein kinase I (BMK1). J. Biol. Chem. 272:20389, 1997.

Chuang, T.-H., Hahn, K.M., Lee, J.-D., Danley, D.E., Bokoch, G.M. The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes. Mol. Biol. Cell 8:1687, 1997.

Dubin, W., Martin, T.R., Swoveland, P., Leturcq, D.J., Moriarty, A.M., Tobias, P.S., Bleecker, E.R., Goldblum, S.E., Hasday, J.D. Asthma and endotoxin: Lipopolysaccharide-binding protein and soluble CD14 in bronchoalveolar compartment. Am. J. Physiol. 270:L736, 1996.

Han, J., Li, Z., Kravchenko, V.V., Ulevitch, R.J. MEF2C participates in inflammatory responses via p38 mediated activation. Nature 386:296, 1997.

Han, J., Wang, X., Jiang, Y., Ulevitch, R.J., Lin, S. Identification and characterization of a predominant isoform of human MKK3. FEBS Lett. 403:19, 1997.

Huang, S., Jiang, Y., Li, Z., Nishida, E., Mathias, P., Lin, S., Ulevitch, R.J., Nemerow, G.R., Han, J. Apoptosis signaling pathway in T cells is composed of ICE/Ced-3 family proteases and MAP kinase kinase 6b. Immunity 6:739, 1997.

Jiang, Y., Li, Z., Schwarz, E.M., Lin, A., Guan, K., Ulevitch, R.J., Han, J. Structure-function studies of p38 MAP kinase: Loop 12 influences substrate specificity and autophosphorylation but not upstream kinase selection. J. Biol. Chem. 272:11096, 1997.

Kravchenko, V.V., Steinemann, S., Kline, L., Feng, L., Ulevitch, R.J. Endotoxin tolerance is induced in Chinese hamster ovary cell lines expressing human CD14. Shock 5:194, 1996.

Massamiri, T., Tobias, P.S., Curtiss, L.K. Structural determinants for the binding of lipopolysaccharide binding protein to purified human high density lipoproteins: Role of apolipoprotein A-1. J. Lipid Res. 38:516, 1997.

Tapping, R.I., Tobias, P.S. Cellular binding of soluble CD14 requires lipopolysaccharide (LPS) and LPS binding proteins. J. Biol. Chem. 272:23157, 1997.

Tobias, P.S., Gegner, J., Tapping, R., Orr, S., Mathison, J., Lee, J.-D., Kravchenko, V., Han, J., Ulevitch, R.J. Lipopolysaccharide dependent cellular activation. J. Periodontal Res. 32:99, 1997.

Tobias, P.S., Soldau, K., Iovine, N., Elsbach, P., Weiss, J. Lipopolysaccharide (LPS) binding proteins BPI and LBP form different types of complexes with LPS. J. Biol. Chem. 272:18682, 1997.

Wang, Z., Harkins, P., Ulevitch, R.J., Han, J., Cobb, M.H., Goldsmith, E.J. The structure of the MAP kinase p38 at 2.1 Å resolution. Proc. Natl. Acad. Sci. U.S.A. 94:2327, 1997.

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Regulation of Mature T-Cell Responses

S.R. Webb, M.-A. Campbell, M.E. Ozaki, B.A. Coren, E. Chronopoulou, D.J. Redondo, J.B. Ferrer, L. Karlsson,* O. Winqvist,* M.R. Jackson,* P.A. Peterson*

*R.W. Johnson Pharmaceutical Research Institute, La Jolla, CA

The majority of self-reactive T cells are purged from the repertoire during negative selection in the thymus. Nevertheless, CD4+ T cells with autoreactivity can be found in peripheral lymphoid tissues and can, under some conditions, be activated to become autodestructive. Clearly, understanding how these cells are normally regulated is important in attempting to control autoimmune diseases. During the past several years, we have focused on defining the accessory-molecule interactions that positively and negatively regulate the responses of mature CD4+ T cells.

The specificity of T-cell responses is dictated by interactions between the antigen-specific T-cell receptor and peptide-bound MHC molecules. For naive T cells, interactions via the T-cell receptor alone are not sufficient to induce T-cell activation and effector function. Interactions between additional molecules, termed accessory molecules, are required. Although costimulation via CD28 binding to B7 molecules is well defined, it is increasingly clear that many accessory molecules promote T-cell proliferative responses. During the past year, one of our major goals was to determine whether the particular accessory molecules engaged during T-cell priming dictate not only whether T cells become activated but also the type of effector cell activity displayed by the activated T cells.

In collaboration with investigators at R.W. Johnson Pharmaceutical Research Institute, we used Drosophila cells transfected with MHC class II molecules and various combinations of accessory molecules to test this model. Transfected Drosophila cells expressing class II molecules and the accessory molecule B7.1 or B7.2 stimulated strong peptide-specific proliferative responses in naive T cells and production of IL-2 and IL-4. The production of large amounts of IL-4 by naive T cells is surprising, because naive T cells reacting to peptide presented by splenic antigen-presenting cells (APCs) generally produce little of this cytokine on primary stimulation. This discrepancy is at least partially resolved by the finding that coexpression of intracellular adhesion molecule-1 with B7 on the Drosophila APCs inhibits production of IL-4. Given the broad expression of intracellular adhesion molecule-1 on normal APCs and the constitutive expression of its ligand, lymphocyte function--associated antigen-1, on T cells, it follows that the generation of IL-4 is limited during typical interactions between T cells and APCs.

Molecules belonging to the TNF receptor family are well known for their ability to influence T-cell responses and survival. In particular, interactions involving Fas and the Fas ligand and TNF and the TNF receptor play a key role in activation-driven apoptosis. In earlier studies, we showed that another member of this family, CD27, also appears to regulate T-cell responses. Drosophila cells expressing CD27 ligand costimulate peptide-specific proliferative responses, but in the absence of B7, these responses are not accompanied by production of IL-4 or IL-10. These findings suggest that CD27 costimulation may play a role in promoting inflammatory-type T-cell responses.

In other studies, we examined the role of interactions between CD40 and CD40 ligand in the regulation of CD4+ T-cell responses. The requirement for these interactions in T-cell activation is variable and depends on the antigen used for activation. T-cell responses to Mlsa superantigens encoded by mouse mammary tumor virus require CD40--CD40 ligand interactions, whereas T-cell responses to conventional peptide antigens or alloantigens do not. Thus far, our studies appear to exclude the possibility that the affinity of the interactions between T-cell receptors and antigen bound to MHC molecules are responsible for this difference. We are assessing the possibility that the requirement is dictated by the type of APC involved.

Ultimately, our aim is to understand how the accessory molecules involved in regulating T-cell responses determine the pathogenicity of autospecific T cells. For this aim, we developed a transgenic model of diabetes by using mice transgenic for a T-cell receptor recognizing an ovalbumin peptide. T-cell receptor transgenic T cells are activated in vitro by using Drosophila APCs that express defined accessory molecules and are adoptively transferred into transgenic mice that express ovalbumin under the control of the rat insulin promoter.

In this system, priming T cells with splenic APCs induces marked autoimmune diabetes characterized by highly elevated glucose levels and destruction of both the islet cells and the exocrine tissue of the pancreas. Stimulation with Drosophila APCs expressing only B7 does not induce disease, whereas stimulation with Drosophila APCs expressing the ligand for CD27 induces rapid-onset diabetes. Although preliminary, these findings confirm our prediction that accessory-molecule interactions that occur during T-cell priming dictate the effector activity of the activated T cells and begin to define the conditions of activation associated with autoimmunity.

PUBLICATIONS

Ozaki, M.E., Karlsson, L., Peterson, P.A., Webb, S.R. Antigen specificity of dual reactive T hybridomas determines the requirement for CD40L-CD40 interactions. J. Immunol. 159:214, 1997.

Webb, S.R., Hayden, K.A. T cell responses to viral superantigens. In: Viral Superantigens. Tomonari, K. (Ed.). CRC Press, Boca Raton, FL, 1997, p. 113.

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Tolerance in B-Cell Subsets

C.G. Romball, K.J. Whitmer, W.O. Weigle

It is well established that injections of monomeric preparations of human gamma globulin (HGG) result in tolerance in both type 1 and type 2 T-helper cell subsets (Th1 and Th2 cells, respectively) of CD4+ cells. CD4+ cells obtained from mice immunized with monomeric HGG and challenged in vivo with HGG do not produce cytokines associated with either Th1 or Th2 cells. Recently, we showed that the limiting dose of monomeric HGG for the induction of tolerance was identical for tolerizing the cells responsible for both the Th1- and the Th2-like responses to HGG. These two observations strongly suggest that tolerance is induced in a T precursor cell for both subsets.

We also showed previously that injection of generators of cytokines (e.g., lipopolysaccharide, IL-1, and TNF-) along with monomeric HGG converts a tolerogenic signal to an immunogenic one. That is, serum antibodies to HGG are produced, and subsequent injection of aggregated HGG results in further production of antibodies to HGG. More recently, we showed that injection of lipopolysaccharide along with monomeric HGG induced production of both IgG2a (Th1-like) and IgG1 (Th2-like) antibodies to HGG, whereas injection of IL-1 along with monomeric HGG induced production of IgG1 antibodies only. Thus, it appears that in this model the tolerogenic signal may allow transient development of the CD4 subsets from precursor T cells, but in the absence of an appropriate cytokine response, these cells cannot expand. IL-1 and possibly other cytokines along with IL-4 may allow expansion of the Th2 subsets, and a combination of other cytokines may result in expansion of the Th1 subsets.

In the past, the question arose whether the Fc receptor plays a role in the induction of tolerance to HGG. Monomeric immunoglobulins can generate a transducing signal through the high-affinity FcRI but not through other Fc receptors. Furthermore, it is well documented that immune complexes can downregulate B-cell activation through the FcII receptor. These concerns were dismissed by the ability to induce tolerance in both Th1- and Th2-like CD4+ cells after injection of monomeric HGG in mice deficient in FcRI or FcRII (generously supplied by J. Ravetch, Memorial Sloan-Kettering Cancer Institute).

The finding that tolerance to HGG can be readily induced in mice in which the Fc chain has been deleted indicates that no signal transduction to the high-affinity FcRI by monomeric HGG is required to induce tolerance. Conventional C57BL/6 mice and mice deficient in FcRI had similar responses to both soluble aggregated HGG and HGG incorporated into adjuvant. Both kinds of mice showed tolerance at all levels of T- and B-cell function. Most likely the tolerogenic properties of monomeric HGG for T cells lie in (1) its ability to equilibrate between intravascular and extravascular spaces and thus persist in body fluids and (2) its inability to be processed aggressively by antigen-presenting cells, leading to its failure to activate the cytokine cascade.

Although FcR-deficient mice have presumably lost all functional FcRI and FcRIII responses, expression of FcRII can be detected in these mice. Downregulation of antibody responses by cross-linking of FcRII in antigen-specific B-cell receptors is a well-characterized event, and the possibility that cross-linking may occur between the B-cell receptor via a specific epitope and the FcRII Fc piece on monomeric HGG is likely. Therefore, we attempted to tolerize FcRII knockout mice after injecting the mice with monomeric HGG. As expected, production of serum antibodies to HGG was higher in FcRII knockout mice given a single injection of aggregated HGG than in similarly injected conventional C57BL/6 mice. On the other hand, FcRII knockout mice pretreated with monomeric HGG did not respond to a subsequent injection of aggregated HGG.

To determine if competent B cells were present, we injected FcRII knockout mice tolerized to monomeric HGG with both lipopolysaccharide and aggregated HGG. This treatment did not induce an antibody response, indicating that tolerance in these mice was induced at the B-cell level as well as the T-cell level. Possibly, B-cell tolerance in FcRII mice results in deletion of B cells, and thus B cells are not available for activation through the combination of lipopolysaccharide plus aggregated HGG.

The idea that deletion may be the mechanism of B-cell tolerance in the HGG mouse model has been suggested in studies that used mice transgenic for the B-cell receptor and a high-affinity rheumatoid factor (IgM) specific for human IgG (see following report). Injection of such mice with deaggregated (monomeric) human IgG results in the deletion of the transgenic cells specific for this antigen. Such deletion is due to activated cell death and occurred only in the absence of T cells. Because such deletion also occurs with nonimmunoglobulin antigens, the case for FcRII downregulation as a mechanism for the induction of tolerance to HGG does not appear to be justified.

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T-Cell Tolerance by Antigen-Activated Cell Death

H. Tighe,* K. Warnatz,* D. Brinson,* M. Corr,* W.O. Weigle, S.M. Baird,* D.A. Carson*

* University of California, San Diego, CA

The studies described here were done in collaboration with H. Tighe and others in the laboratory of D. Carson at the University of California, San Diego. Exposure of transgenic mice expressing a human IgM rheumatoid factor to soluble human IgG (monomeric HGG) results in the deletion of B cells that express the human rheumatoid factor. The kinetic pattern is identical to that observed with the deletion of antigen-specific B cells after tolerization of conventional mice with monomeric HGG.

In more recent studies, exposure of such transgenic mice to monomeric HGG in the absence of T-cell help resulted in antigen-specific B-cell deletion in 2--3 days. This deletion was independent of the Fas/Fas-ligand pathway of apoptosis and was preceded by a phase of partial activation involving an increase in cell size, an increase in expression of B7 and intracellular adhesion molecule-1, and a transient release of low levels of immunoglobulin. Complete B-cell activation involving the formation of germinal centers and secretion of high levels of rheumatoid factor only occurred if T-cell help was provided. In the absence of T-cell help, death of the B cells specific for rheumatoid factor was not preceded by anergy. Abortive activation or deletion of B cells by antigen in the absence of T-cell--derived survival signals may represent the major mechanisms for maintaining peripheral tolerance in B cells expressing the higher affinity rheumatoid factor in humans and may have significant implications for rheumatoid arthritis.

PUBLICATIONS

Bandeira, A., Dembic, Z., Fuchs, E., Green, D., Langman, R., Schaffner, K., Weigle, W.O. Models of immunologic tolerance: Sense of self. In: HMS Beagle: A BioMedNet Publication [On-line serial]. Issue 12 (June 27), 1997. Available at http://biomednet.com/hmsbeagle/1997/12/cutedge/

Blossom, S., Chu, E.B., Weigle, W.O., Gilbert, K.M. CD40L expressed on B cells in the BXSB mouse model of systemic lupus erythematosus. J. Immunol., in press.

Gilbert, K.M., Thoman, M.L., Bauche, K., Pham, T., Weigle, W.O. TGF-ß1 induces anergy in naive T cells. Immunol. Invest., in press.

Tighe, H., Warnatz, K., Brinson, D., Corr, M., Weigle, W.O., Baird, S.M., Carson, D.A. Peripheral deletion of rheumatoid factor B cells after abortive activation by IgG. Proc. Natl. Acad. Sci. U.S.A. 94:646, 1997.

Weigle, W.O. Advances in basic concepts of autoimmune disease. Clin. North Am., in press.

Weigle, W.O. Overview of the immune system. In: Comprehensive Toxicology. Vol. 5: Immune System Toxicology. Lawrence, D.A. (Ed.). Elsevier, New York, in press.

Weigle, W.O. Tolerance: Models. In: Encyclopedia of Immunology, 2nd ed. Roitt I.M., Delves, P.J. (Eds.). Academic Press, San Diego, in press.

Weigle, W.O., Romball, C.G. CD4+ T cell subsets and cytokines involved in peripheral tolerance. Immunol. Today, in press.

Whitmer, K.J., Romball, C.G., Weigle, W.O. Induction of tolerance to human -globulin in FcR - and Fc RII-deficient mice. J. Immunol. 159:644, 1997.

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Molecular Immunopathology and Pathophysiology of Inflammatory Injury

L. Feng, Y. Xia, S. Chen, G.E. Garcia, S. Ishizuka, R.C. Blantz,* T. Yamamoto,** C.B. Wilson

* University of California, San Diego, and Veterans Affairs Medical Center, San Diego, CA
** Niigata University School of Medicine, Niigata, Japan

A major focus of our laboratory is the cascade of molecular events that lead to the induction, mediation, and often incomplete resolution of inflammatory injury with sclerosis, leading to organ failure. Most often, the kidney is used as a model organ for study.

The processes that lead to sclerosis after immune injury in the kidney are set in motion during the acute inflammatory stages of the injury via cellular and humoral mediation pathways. In WKY rats, administration of antibodies to glomerular basement membrane causes a severe crescent form of antibody-mediated glomerulonephritis that leads to glomerulosclerosis. CD8+ lymphocytes and monocytes/macrophages dominate the lesion, suggesting their role in the glomerulosclerotic process. Monocytes/macrophages are major players in the proteinase cascades of interest to our studies and are an important focus of study. Accumulation of CD8+ lymphocytes parallels the expression of intracellular adhesion molecule-1, and depletion of CD8+ lymphocytes decreased the accumulation of monocytes/macrophages and expression of intracellular adhesion molecule-1, TNF-, IFN-, macrophage inflammatory protein-1, and monocyte chemoattractant protein-1, suggesting a role for CD8+ lymphocytes in the recruitment of macrophages and monocytes.

In separate studies in the WKY model, expression of the monocyte chemoattractant protein-1 was observed 4 days after the administration of antibodies to glomerular basement membrane. Treatment with antibodies to the chemoattractant resulted in a decrease in the number of monocytes/macrophages and a decrease in proteinuria without altering the numbers of CD8+ lymphocytes found. Antibodies against the ß T-cell receptor also reduced proteinuria in this model, which could be enhanced by passive transfer of sensitized lymphocytes.

The roles of perforin and granzyme B were evaluated in the WKY model of glomerular injury. Levels of mRNA for these mediators of cellular injury were elevated early in the evolution of the lesion. Expression of the mediators was almost completely suppressed by depletion of CD8+ lymphocytes and was accompanied by a reduction in proteinuria and crescent formation. A neutralizing antibody against perforin reduced proteinuria and crescent formation, suggesting that perforin plays an important role in the glomerular injury mediated by CD8+ lymphocytes in this model.

We cloned IFN---inducing factor and showed that it is expressed in the WKY model, peaking on days 3 and 5 after administration of antibodies to glomerular basement membrane and declining by days 7 and 9. Variants of the factor with different lengths of the 5´ untranslated region were expressed, suggesting alternative splicing. Expression of IFN---inducing factor appears to be regulated at multiple steps and may contribute to the production of IFN- in the WKY model of crescentic glomerular injury.

The rat homolog of the macrophage-specific gene-1 (MSG-1) has been cloned in our laboratory and is under study. MSG-1 was expressed in a time-dependent manner in the glomeruli of rats with glomerulonephritis induced by antibodies to glomerular basement membrane. It was first detected at 30 minutes after antibody injection, peaked at 6 hours, and persisted through 24 hours, when macrophages and monocytes were the predominant infiltrating cells. MSG-1 expression was also evident in rats with tubulointerstitial nephritis induced to produce antibodies against their tubular basement membranes. MSG-1 was detected 9--16 days after immunization, with a peak at 12 days, again when monocytes/macrophages were accumulating. In situ hybridization localized MSG-1 to mononuclear infiltrative cells. Studies now in progress suggest that antibodies to MSG-1 may be beneficial in manipulating the lesions caused by antibodies to glomerular basement membrane.

In continuing studies on the factors responsible for proteinase expression in renal inflammation, we studied the direct effects of monocytes/macrophages in vitro. Coculture of rat mesangial cells with macrophages in the form of the mouse cell line Raw 264.7 induced expression of the 72-kD type IV collagenase by the rat cells. Expression was maximal at 24--48 hours. Contact of monocytes/macrophages with intrinsic glomerular cells, leading to expression of proteases, may contribute to injury and subsequently to the accumulation and remodeling of extracellular matrix.

We also examined the role of production of nitric oxide in glomerular injury caused by bacterial lipopolysaccharide, which can induce a glomerular coagulopathy. Injury was enhanced by the presence of a nonspecific inhibitor of nitric oxide synthase (NOS), L-NAME, but not by administration of specific inhibitors of inducible NOS. Lipopolysaccharide decreased the response of isolated glomeruli to carbamylcholine, an index of the activity of endothelial NOS. These findings suggest that the decrease in glomerular filtration rate after injection of lipopolysaccharide is due to local inhibition of endothelial NOS by inducible NOS, possibly via autoinhibition of nitric oxide.

PUBLICATIONS

Blantz, R.C., Wilson, C.B., Gabbai, F.B. Pathophysiology of acute immune injury. In: Immunologic Renal Diseases. Neilson, E.G., Couser, W.G. (Eds.). Lippincott-Raven, Philadelphia, 1997, p. 237.

Fujinaka, H., Yamamoto, T., Feng, L., Kawasaki, K., Yaoita, E., Hirose, S., Goto, S., Wilson, C.B., Uchiyama, M., Kihara, I. A crucial role of CD8-positive lymphocytes in glomerular expression of ICAM-1 and cytokines in crescentic glomerulonephritis of WKY rats. J. Immunol. 158:4978, 1997.

Fujinaka, H., Yamamoto, T., Takeya, M., Feng, L., Kawasaki, K., Yaoita, E., Kondo, D., Wilson, C.B., Uchiyama, M., Kihara, I. Suppression of anti-GBM nephritis by administration of anti-MCP-1 antibody in WKY rats. J. Am. Soc. Nephrol., in press.

Gabbai, F.B., De Nicola, L., Peterson, O.W., Obagi, S., Thomson, S.C., Tucker, B.J., Keiser, J.A., Wilson, C.B., Blantz, R.C. Renal response to blood pressure elevation in normal and glomerulonephritic rats. J. Am. Soc. Nephrol. 7:2590, 1996.

Schwartz, D., Mendonca, M., Schwartz, I., Xia, Y., Satriano, J., Wilson, C.B., Blantz, R.C. Inhibition of constitutive nitric oxide synthase (NOS) by nitric oxide generated by inducible NOS after lipopolysaccharide administration provokes renal dysfunction in rats. J. Clin. Invest., in press.

Wilson, C.B. Immune models of glomerular injury. In: Immunologic Renal Diseases. Neilson, E.G., Couser, W.G. (Eds.). Lippincott-Raven, Philadelphia, 1997, p. 729.

Wilson, C.B. Immune models of tubulointerstitial injury. In: Immunologic Renal Diseases. Neilson, E.G., Couser, W.G. (Eds.), Lippincott-Raven, Philadelphia, 1997, p. 775.

Wilson, C.B., Feng, L., Ward, D.M. Renal diseases. In: Medical Immunology, 9th ed. Stites, D.P., Terr, A.I., Parslow, T.G. (Eds.). Appleton & Lange, Stamford, CT, 1997, p. 549.

Yamamoto, T., Fujinaka, H., Kawasaki, K., Yaoita, E., Feng, L., Wilson, C.B., Kihara, I. CD8+ lymphocytes play a central role in the development of anti-GBM nephritis through induction of ICAM-I and chemokines in WKY rats. Contrib. Nephrol. 118:109, 1996.

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Activation of Inflammatory Cells by Receptors Coupled to GTP-Binding Proteins

R.D. Ye, E.R. Prossnitz, D.D. Browning, M.H. Hsu, T.-H. Chao, M. Wang, W.C. Diehl, L. Feng, T.E. Hugli, Z.K. Pan,* B.L. Zuraw**

* La Jolla Institute of Allergy and Immunology, La Jolla, CA
** Department of Molecular and Experimental Medicine, TSRI

Receptors coupled to GTP-binding protein (G-protein) play an important role in the activation of inflammatory cells. Receptors of this type expressed on leukocytes respond to chemoattractants and chemokines, resulting in directed cell migration, release of degradation enzymes, generation of superoxide anions, and activation of adhesion molecules. G-protein--coupled receptors expressed on epithelial and fibroblast cells in the lungs and various tissues mediate activation of these cells by promoting the synthesis and secretion of cytokines and growth factors important in inflammation and tissue remodeling. A major focus of our work is the mechanisms that underlie G-protein--mediated signaling events leading to activation of inflammatory cells.

CHEMOATTRACTANT STIMULATION OF TRANSCRIPTIONAL ACTIVITY IN LEUKOCYTES

Using the G-protein--coupled N-formyl peptide receptor as a model, we have characterized agonist-stimulated release of inflammatory cytokines in both monocytes and neutrophils. Formyl peptide stimulation of both types of cells results in secretion of IL-8, a potent chemokine that binds two types of G-protein--coupled receptors. Stimulation of monocytes with formyl peptide leads to activation of NF-B, a transcription factor responsible for the synthesis of a large number of cytokines, including IL-8. In neutrophils, however, activation of NF-B is hardly detectable, and the mechanism for expression of IL-8 remains unclear.

Myeloid cells appear to respond differently to formyl peptide and several other chemoattractants, depending on the differentiation stage of the cells. No activation of NF-B occurs in promyelocytic HL-60 cells when the cells are stimulated with formyl peptide, regardless of the expression of the N-formyl peptide receptor. After cellular differentiation induced by dimethyl sulfoxide, formyl peptide stimulation of HL-60 cells readily leads to activation of NF-B. These results suggest the presence of an inducible factor that is necessary for the cells to respond to chemoattractants.

PRODUCTION OF INFLAMMATORY CYTOKINES IN LUNG FIBROBLASTS AND EPITHELIAL CELLS

Inflammation of the lungs often involves activation of epithelial cells, endothelial cells, and fibroblasts. Along with activated leukocytes, these cells are responsible for the local production of cytokines and other inflammatory mediators. We have started a study to understand the mechanisms by which G-protein--coupled receptors activate cellular functions associated with inflammation of the airways.

Our study has shown a novel mechanism for bradykinin, a nonapeptide proinflammatory factor, in mediation of inflammation. Binding of bradykinin to the B2-type receptor in lung-derived epithelial and fibroblast cells results in activation of NF-B and synthesis of IL-1ß, IL-6, and IL-8. This response is mediated by a G-protein--coupled mechanism, because pertussis toxin inhibits the secretion of these cytokines. Platelet-activating factor, a lipid mediator of inflammation and a chemoattractant, also stimulates activation of NF-B and synthesis of cytokines in these cells. Current effort is focused on the involvement of small GTPases, phosphatidylinositol 3-kinases, and other signaling molecules in G-protein--coupled activation of transcription factors.

STRUCTURE AND FUNCTION OF THE C3A ANAPHYLATOXIN RECEPTOR

Our laboratory recently reported the cloning of a G-protein--coupled receptor with a large (~170 amino acids) extracellular loop. This receptor, which is the receptor for the activated C3 component of complement (C3a), is the only G-protein--coupled receptor that has an extended extracellular loop. We are interested in knowing whether the loop structure is part of the binding domain for C3a.

After our initial cloning of the cDNA for the human receptor for C3a, we now have characterized the mouse gene that encodes the receptor for C3a. The large loop is conserved in the mouse receptor; however, sequence homology of the loop is lower (44%) than the overall homology between the human and mouse receptors (65%). To determine the role of the loop in C3a binding, we have generated a set of chimeric receptors that contain fragments from both C3a receptor and C5a receptor. The receptor for C5a is known for the function of its N-terminus in binding to C5a. Our preliminary results suggest that the equivalent of the receptor for C3a is not critical for C3a binding, because replacement of this domain with that of the C5a receptor has little effect on agonist binding and signaling. A series of deletion mutants has been generated to further examine the role of the large extracellular loop on C3a binding.

In other related studies, we are investigating the mechanisms for C3a activation of eosinophils but not neutrophils. These studies focus on G-protein activation and the involvement of other signaling molecules, with emphasis on the difference between the two types of granulocytes.

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Activation of Chemotactic Receptors Coupled to GTP-Binding Proteins

E.R. Prossnitz, M.H. Hsu, R.D. Ye

Leukocytes play an important role in host defense against invading microorganisms. Their ability to generate superoxide radicals and release degradative enzymes after migration to sites of inflammation is essential for this function. However, these same responses can also participate in the development of pathologic conditions such as ischemia-reperfusion injury. Ischemia is associated with numerous conditions, including myocardial thrombosis, stroke, and frostbite. Reperfusion, specifically the reintroduction of neutrophils to ischemic tissues, can cause damage to the host. Chemoattractants elicit their effects on neutrophils by binding to cell-surface receptors coupled to guanine nucleotide--binding regulatory proteins. The goal of our work is to understand the molecular mechanisms involved in the activation and regulation of neutrophil function by chemoattractant receptors.

To accomplish this goal, we are using the N-formyl peptide receptor, a major neutrophil chemotactic receptor for small peptides. One of the first events after the binding of ligand is presumed to be a conformational change in the receptor that transmits information to GTP-binding proteins (G-proteins). We have studied this process by using mutants of the N-formyl peptide receptor that bind ligand but do not initiate signaling. These mutants have a defect in the ability to couple to G-proteins. Interestingly, a mutant in which an arginine at position 123 has been replaced by a glycine can induce phosphorylation and internalization of the receptor, whereas a mutant in which an aspartic acid at position 71 has been replaced by a glycine cannot. These findings indicate that the mechanisms responsible for the defect in coupling to G-proteins are most likely distinct. Whereas the arginine most likely is directly and solely involved in G-protein interactions, the aspartic acid is involved in the conformational switch that takes place on ligand binding.

After activation, neutrophils rapidly become unresponsive to continued or repeated stimulation, a process termed desensitization. The aim of this change most likely is to prevent excessive damage to the host. We recently showed that phosphorylation of the N-formyl peptide receptor is required for desensitization. A mutant form of the receptor lacking all the potential phosphorylation sites within the carboxy terminus could not undergo ligand-induced desensitization. Furthermore, we showed that one of these sites had a critical role in desensitization. This site corresponded to one we previously determined was a phosphorylation site for the kinase GRK2. We hope that a better understanding of the mechanisms that lead from ligand binding to cellular activation and culminate in receptor desensitization will lead to new possibilities for therapeutic intervention.

PUBLICATIONS

Browning, D.D., Pan, Z., Prossnitz, E.R., Ye, R.D. Cell type- and developmental stage-specific activation of NF-B by fMet-Leu-Phe in myeloid cells. J. Biol. Chem. 272:7995, 1997.

Darrow, A.L., Fung-Leung, W.-P., Ye, R.D., Santulli, R.J., Cheung, W.-M., Derian, C.K., Burns, C.L., Damiano, B.P., Zhou, L., Keenan, C.M., Peterson, P.A., Andrade-Gordon, P. Biological consequences of thrombin receptor deficiency in mice. Thromb. Haemost. 76:860, 1996.

Hsu, M.H., Ember, J.A., Wang, M., Prossnitz, E.R., Hugli, T.E., Ye, R.D. Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene. Immunogenetics, in press.

Kew, R.R., Peng, T., DiMartino, S.J., Madhavan, D., Weinman, S.J.,

Cheng, D., Prossnitz, E.R. Undifferentiated U937 cells transfected with chemoattractant receptors: A model system to investigate chemotactic mechanisms and receptor structure/function relationships. J. Leuk. Biol. 61:329, 1997.

Pan, Z., Zuraw, B.L., Lung, C.-C., Prossnitz, E.R., Browning, D.B., Ye, R.D. Bradykinin stimulates NF-B activation and interleukin-1ß gene expression in cultured human fibroblasts. J. Clin. Invest. 98:2042, 1996.

Prossnitz, E.R. Desensitization of N-formyl peptide receptor-mediated activation is dependent upon receptor phosphorylation. J. Biol. Chem. 272:15213, 1997.

Prossnitz, E.R., Ye, R.D. The N-formyl peptide receptor: A model for the study of chemoattractant receptor structure and function. Pharmacol. Ther. 74:73, 1997.

Ye, R.D., Boulay, F. Structure and function of the leukocyte chemoattractant receptors. Adv. Pharmacol. 39:221, 1997.

Ye, R.D., Kravchenko, V.V., Pan, Z., Feng, L. Stimulation of NF-B activation and gene expression by platelet-activating factor. Adv. Exp. Med. Biol. 416:143, 1997.

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