News and Publications

Division of Research Rheumatology

W.M. Keck Autoimmune Disease Center

Eng M. Tan, M.D., Division Head

Autoimmune Antibodies as Unique Reagents in Clinical Medicine and Cell Biology

W. Hassfeld,* G. Steiner,* D. Portman,** G. Dreyfuss,** K. Kiyosawa,*** G. Reimer,**** C. Luderschmidt,***** K. Furuta, B. Hildebrandt, S. Matsuoka, D.A. Mathison, E.K.L. Chan, E.M. Tan

* Lainz Hospital, Vienna, Austria
** Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA
*** Shinshu University School of Medicine, Matsumoto, Japan
**** University of Erlangen, Erlangen, Germany
***** University of Munich, Munich, Germany

Sera from patients with autoimmune diseases contain autoantibodies to many intranuclear and intracytoplasmic proteins. Some of these antigens have been characterized and include proteins associated with the snRNP family of splicing factors, such as the Sm antigens, and proteins associated with DNA replication, such as proliferating cell nuclear antigen. However, many of the target antigens detected either by Western blotting or by immunohistochemistry have not been identified. We continue to use these human autoimmune antibodies as unique reagents for the identification and characterization of intracellular proteins; we use the antibodies in immunoscreening of cDNA expression libraries.

We isolated a full-length cDNA that encodes a protein with 3 consecutive consensus sequence RNA-binding domains followed by a glycine-arginine--rich region termed an RGG box, which is another type of RNA-binding motif. These and other features of the protein, which has a calculated molecular weight of 82 kD, indicate that it is identical to heterogeneous nuclear ribonucleoprotein (hnRNP) R, a component of the hnRNP family of proteins that hitherto had been identified only by 2-dimensional gel chromatography. The availability of the full-length cDNA encoding this protein opens the way to study of the protein's function.

A second autoimmune serum was used to isolate a cDNA clone encoding a heterochromatin protein of approximately 25 kD that we have termed HP1^Hsß or p25ß. Heterochromatin-associated protein HP1 of Drosophila was originally thought to function in modifying chromatin via the chromodomain region and in this way play a role in position-effect variegation. The p25ß we have isolated appears to be the human analog of Drosophila HP1.

Human autoantibodies to p25ß can be directed primarily at the C-terminal or the N-terminal region, and in immunofluorescent assays with tissue cells, only affinity-purified antibodies from the C-terminal epitope bound to chromatin. This finding presumably reflects differences in the N- and C-terminal regions. The C-terminal epitope is more surface exposed, whereas the N-terminal epitope may be buried.

We showed that p25ß was localized to the centromeric regions of metaphase chromosomes and that this association continued into anaphase. Autoantibodies to p25ß and to centromere proteins CENP-A, CENP-B, and CENP-C co-occur in human autoimmune sera. Co-occurrence of autoantibodies to different intracellular proteins suggests that the target antigens might be proteins that are present as complexes and that the proteins come together to perform certain cellular functions. In an extension of these studies, we found that patients with systemic scleroderma associated with pulmonary fibrosis had autoantibodies to heterochromatin p25ß.

Autoimmune Response and Autoimmunity in Cancer

J.-Y. Zhang, W. Zhu, J.B. Rattner,* M.J. Fritzler,* R.L. Humbel,** K. Conrad,*** C.A. Casiano, E.K.L. Chan, E.M. Tan

* University of Calgary, Calgary, Alberta
** Centre Hospitalier de Luxembourg, Luxembourg
*** Dresden Technical University, Dresden, Germany

CENP-F is a protein associated with the centromere, and autoantibodies to CENP-F have been detected in 36 patients. This autoantibody is readily detectable by immunoblotting and has a characteristic pattern in immunofluorescent assays, particularly in cells in the mitotic phase of the cell cycle. Of the 36 patients with autoantibodies to CENP-F, 22 had malignant neoplasms; breast (9 patients) and lung (5 patients) cancers were the most common tumors. Others included stomach cancer, ovarian cancer, hepatocellular carcinoma, and tracheal cancer. Thus, a high proportion of persons with CENP-F antibodies have malignant neoplasms. CENP-F antigens appear to be highly expressed in malignant tissues, and this observation supports the notion that the immune response in cancer is in large part antigen driven.

As part of a study in progress, we examined sera from a group of 95 patients in Henan Province in China who have hepatocellular carcinoma. Twenty of these patients (21%) had autoantibodies to a 62-kD cytoplasmic antigen that we named HCC2. The protein contains 2 types of RNA-binding motifs. One is the consensus sequence RNA-binding domain; the other is 4 repeats of the homology domain of heterogeneous nuclear ribonucleoprotein K. Sera from patients in the United States who had cancer other than hepatocellular carcinoma did not have autoantibodies to HCC2. When sera from patients in other countries, such as Italy, Japan, and South Korea, who had hepatocellular carcinoma were examined, the percentages of patients who had autoantibodies to HCC2 were significantly lower than the percentage of patients from Henan Province who did.

Epidemiologic studies of hepatocellular carcinoma in Henan Province revealed that the populace of this region has high dietary exposure to aflatoxin B1. Further studies are being done to confirm the presence of autoantibodies to HCC2 in persons in other regions in China where exposure to aflatoxin B1 is high. Aflatoxin B1 may be an agent that makes HCC2 immunogenic. This possibility is supported by observations that aflatoxin B1 is mutagenic for certain proteins involved in tumorigenesis, such as p53 and ras.

PUBLICATIONS

Casiano, C.A., Ochs, R.L., Tan, E.M. Differences in autoantigen cleavage in apoptosis and necrosis. Cell Death Differ. 5:183, 1998.

Furuta, K., Chan, E.K.L., Kiyosawa, K., Reimer, G., Luderschmidt, C., Tan, E.M. Heterochromatin protein HP1^HSß (p25ß) and its localization with centromeres in mitosis. Chromosoma 106:11, 1997.

Furuta, K., Matsuoka, S., Hildebrandt, B., Kiyosawa, K., Reimer, G., Luderschmidt, C., Chan, E.K.L., Tan, E.M. Immunologic characterization of heterochromatin protein p25ß autoantibodies and relationship with centromere autoantibodies and pulmonary fibrosis in systemic scleroderma. J. Mol. Med. 76:54, 1998.

Hassfeld, W., Chan, E.K.L., Mathison, D.A., Portman, D., Dreyfuss, G., Steiner, G., Tan, E.M. Molecular definition of heterogeneous nuclear ribonucleoprotein R (hnRNP R) using autoimmune antibody: Immunological relationship with hnRNP P. Nucleic Acids Res. 26:439, 1998.

Rattner, J.B., Rees, J., Whitehead, C.M., Casiano, C.A., Tan, E.M., Humbel, R.L., Conrad, K., Fritzler, M.J. High frequency of neoplasia in patients with autoantibodies to centromere protein CENP-F. Clin. Invest. Med. 20:308, 1997.