News and Publications

Molecular Mechanisms of Chemoattractant-Induced Leukocyte Activation

R.D. Ye, D.D. Browning, M.H. Hsu, J.Q. Cao, M. Wang, W.C. Diehl, N.D. Windes

Leukocyte chemoattractants can be exogenous, such as the bacterially derived fMet-Leu-Phe, or endogenous, such as chemokines. These molecules induce positive chemotaxis at subnanomolar concentrations. Chemoattractants play an important role in transendothelial migration of leukocytes to inflammatory sites. At higher concentrations, chemoattractants stimulate other leukocyte functions, including degranulation, generation of superoxide anions, and activation of integrins. In addition to these well-established functions, some chemokines can block entry of HIV type 1 into cells and regulate differentiation of stem cells.

Our laboratory is interested in the interaction of chemoattractants with their receptors and the signaling events caused by such interactions. These receptors are G protein--coupled cell-surface membrane proteins that typically contain 7 transmembrane helices. To study agonist binding and receptor signaling, we developed model systems that use both peripheral blood leukocytes and transfected cells. The receptor for the N-formyl peptide fMet-Leu-Phe was expressed in transfected cells for studies of binding kinetics. We found that peptides with bulky N-terminal modification can interact effectively with the receptor, findings that do not agree with the previous assumption that the binding pocket of the receptor can hold only a small molecule. N-terminal--modified agonists also activated the receptor and produced a similar signal transduction profile, as with the much smaller tripeptide fMet-Leu-Phe. These preliminary results should lead to a better understanding of the requirements for agonist structure that induces receptor activation.

Chemoattractants such as fMet-Leu-Phe and the activated fifth component of complement (C5a) can stimulate release of inflammatory cytokines from leukocytes. Some chemokines also can induce gene expression and cytokine synthesis, but because the production of these chemokines is regulated at the transcriptional level, autocrine regulation is most likely. We investigated the possibility of autocrine regulation by using IL-8, a CXC chemokine.

Immobilized IL-8 is functional and can be separated from newly produced IL-8 when incubated with peripheral blood mononuclear cells. Using this system, we observed IL-8 induction of IL-8 production from mononuclear cells but not neutrophils. Cells at various stages of differentiation seem to respond to IL-8 quite differently. Undifferentiated HL-60 cells responded to both IL-8 and a related chemokine, MGSA/Gro, with IL-8 production, whereas monocytes responded only to IL-8 and not to MGSA/Gro. Recent results suggest that this regulation may be at the level of MAP kinase activation. Additional studies are needed to investigate whether other chemokines can also regulate their biosynthesis in an autocrine fashion.

Role of G Protein--Coupled Receptors in Lung Inflammation

R.D. Ye, T.-H. Chao, M.P. Ero, P.A. Dulin, L. Feng, J.A. Ember, T.E. Hugli, Z.K. Pan,* B.L. Zuraw*

* Department of Molecular and Experimental Medicine, TSRI

In the lungs, G protein--coupled receptors are present on alveolar macrophages, epithelial cells, endothelial cells, and leukocytes in circulation. Binding of agonists to these receptors induces cellular functions ranging from release of granule contents to cytokine secretion. We are interested in the signaling mechanisms that lead to these cell functions and in the effects of these agonists on lung inflammation.

Eosinophils are attracted to the lungs during allergic inflammation. Subsequent activation of the cells can cause the damage to lung tissue commonly seen in allergic subjects. Eosinophils respond to a number of chemoattractants, including activated components of complement (C5a and C3a) and chemokines such as eotaxin. We are collaborating with other investigators to study the structure and function of C3aR, the receptor for C3a, and CCR3, the receptor for eotaxin. Although both C3aR and CCR3 are G protein--coupled receptors with 7 transmembrane domains, C3aR contains a large extracellular loop between the fourth and fifth transmembrane domains. Whereas C3aR is known to interact potently with only a single agonist, CCR3 can bind 6 chemokines. Understanding the structural basis for this functional diversity should be useful in the design of antagonists for potential therapeutic use.

To determine whether and how C3aR interacts with its agonist C3a, we constructed a large number of mutant and chimeric receptors and tested their binding properties in transfected cells. The results indicate that the large extracellular loop of C3aR is important for agonist binding, because replacement of the loop with its counterpart from the receptor for C5a completely abolished C3a binding. A large part of the loop, however, may not be required for agonist binding, because removal of up to 62% of it did not affect binding of C3a and receptor signaling. The remaining parts of the loop, especially the segments next to the transmembrane domains, are critical for high-affinity binding of C3a. These segments of the receptor contain negatively charged amino acids that may interact with positively charged residues in the C3a molecule near the carboxy-terminus of the molecule.

Unlike C3aR, which interacts with only a single agonist, human CCR3 interacts with at least 6 chemokines, including eotaxin. To understand the structural basis for the less specific binding profile of CCR3, we are preparing chimeric receptors in which part of the CCR3 domain has been replaced by a sequence from other receptors that have the same partial agonist binding profile. By testing these receptors, we hope to determine the CCR3 domains responsible for binding to each of the 6 chemokines.

PUBLICATIONS

Becker, E.L., Forouhar, F.A., Grunnet, M.L., Boulay, F., Tardif, M., Bornamm, B.-J., Sodja, D., Ye, R.D., Woska, J., Jr., Murphy, P.M. Broad immunocytochemical localization of the formyl peptide receptor in human organs, tissues and cells. Cell Tissue Res. 292:129, 1998.

Hsu, M.M., Chang, S., Ye, R.D., Prossnitz, E.R. Phosphorylation of the N-formyl peptide receptor is required for receptor internalization but not chemotaxis. J. Biol. Chem. 272:29426, 1997.

Hsu, M.M., 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 47:64, 1997.

Pan, Z.K., Ye, R.D., Christiansen, S.C., Jagels, M.A., Bokoch, G.M., Zuraw, B.L. Role of the Rho GTPase in bradykinin stimulated NF-B activation and interleukin-1ß gene expression in cultured human epithelial cells. J. Immunol. 160:3038, 1998.

Quehenberger, O., Pan, Z.K., Prossnitz, E.R., Cavanagh, S.L., Cochrane, C.G., Ye, R.D. Identification of an N-formyl peptide receptor ligand binding domain by a gain-of-function approach. Biochem. Biophys. Res. Commun. 238: 377, 1997.