Emression of 65-kd Heat Sh&k Proteins in the Inflammatory Myopathies Reinhard Hohlfeld, MD,* and Andrew G. Engel, MD

In normal muscle, 65-kd heat shock proteins (hsp) were detected on capillary endothelial cells, the mural elements of larger vessels, and some intracellular organelles, probably mitochondria. In the inflammatory myopathies, the 65-kd hsp were detected on inflanunaxory cells, degenerating and regenerating fibers, and on many but not all nonnecrotic muscle fibers invaded by T cells. The expression of the 65-kd hsp may be an immunenonspecific response to cellular “stress,” but hsp determinants could possibly also serve as autoantigen(s) recognized by autoreactive T cells. Hohlfeld R, Engel AG. Expression of 65-kd heat shock proteins in the inflammatory myopathies. Ann Neurol 1992;32:821-823

Heat shock proteins (hsp) constitute a heterogeneous family of molecules that serve important functions in the folding, assembly, and transport of cellular proteins 11-31. The majority of hsp are expressed constitutively in various cells [1-3]. In addition, many hsp are induced under conditions of stress including heat shock, nutritional deprivation, and on exposure to cytokines or inflammatory mediators {l-31. Hsp may serve as antigens recognized by T cells expressing either the common a / p or the uncommon $6 T-cell receptor [4-61. This has led to speculation that hsp might be involved in T-cell-mediated autoimmune reactions [4-61. In this communication, we document the distribution of 65-kd hsp in polymyositis, inclusion body myositis, dermatomyositis, and granulomatous myopathy.

Materials and Methods Muscle specimens were used from 23 patients with inflammatory myopathies (polymyositis,6 patients; inclusion body myositis, 6 patients; dermatomyositis, 6 patients; granulomatous myopathy, 5 patients) and from 2 subjects without mus-

From the Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic and Mayo Foundation, Rochester, MN. Received Apr 28, 1992. Accepted for publication Jun 17, 1992. Address correspondence to Dr Engel, Neuromuscular Research Laboratory, Guggenheim Building G801, Mayo Clinic, Rochester, MN

55905. *Present address: Department of Neurology and Department of Neuroimmunology, University of Munich and Max Planck Institute, Martinsried, Germany.

cle disease. Of the 5 patients with granulomatous myopathy, 3 had systemic manifestations of sarcoidosis and 2 had no associated illness. Four-micrometer-thick acetone-fixed cryostat sections were used for immunoperoxidase or paired immunofluorescence studies. The 65-kd hsp was localized with the mouse monoclonal antibody HS 60 (ML30)(a gift from Dr J. Ivanyi 17, 81) applied at a concentration of 10 pg/ml for 1 hour or overnight. This was followed by treatment with a biotinlabeled second antibody and then with either peroxidaselabeled streptavidin and JSarnovsky’s diaminobenzidine medium, or avidin labeled with fluorescein isothiocyanate. T cells were localized with an affinity-purified antiLCD3 antibody raised in rabbits (Dako) applied at a concentration of 5 pg/ml for 1 hour. This was followed by treatment with rhodamine-labeled goat anti-rabbit IgG. For negative controls, identical concentrations of nonimmune isotypematched mouse IgG or nonimmune rabbit IgG were substituted for the primary antibodies. The paired immunolocalization of CD3+ T cells and $8 T cells was previously described 197.

Results Nomal Muscle When the primary antibody was applied for 1 hour, reactivity for the 65-kd hsp was detected only in capillaries and in mural elements of larger vessels. Overnight application of the primary antibody immunostained the intermyofibrillar membranous network of the muscle fibers in transverse sections and fine granular material adjacent to the I bands in longitudinal sections. The staining pattern resembled the distribution of NADH dehydrogenase reactivity in adjacent sections with most intense staining of type 1 and least intense staining of type 2B fibers.

Inj’ammatory Myopathies In all cases, after either 1 hour or overnight incubation with the primary antibody, hsp reactivity was detected

on most inflammatory cells and on regenerating (Fig A, B) or degenerating muscle fibers. In polymyositis and inclusion body myositis, after even 1 hour of incubation with the primary antibody, increased hsp reactivity was detected on the surface and frequently in subsarcolemmal and intermyofibrillar regions of many but not all nonnecrotic muscle fibers invaded by CD3’ T cells (Fig D, E). Further, hsp expression was detected in neighboring fibers not invaded by mononuclear cells in the plane of section. In the granulomatous myopathies, increased surface and often cytoplasmic reactivity was observed on muscle fibers abutting on the granulomas and on some nonnecrotic muscle fibers invaded by mononuclear cells. There was no obvious relationship between hsp expression by the muscle fibers and the few y/S T cells present in these specimens.

Copyright 0 1992 by the American Neurological Association 821

Localization of the G k d heat shock protein (hsp) with the immunoperoxidase method (A, C ) and of hsp and the CD3 T-cell marker by paired immunoJuorescence (D, E ) in polymyositis (A, D, E) and dermatomyositis (C). In A, there is dzjjfuse ueactivity for hsp in a regenerating muscle fiber (upper center) and in scattered inflammatory cells (arrowhead). B is a nonadjacent serial section stained with hematoxylin and eosin. In dermatomyositis (C), there are diffuse or focal increases of hsp expression in the abnormal perzyascicularfibers. The mural elements of small blood vessels are also immunostained. D and E image nonnecrotic mascle fibers invaded by T cells; hsp is visualized with green jluorescence and the CD3 T-cell marker with red fluorescence. In both D and E, hsp expression is incremed over that segment of the fiber surface invaded by T cells. In E, sarcoplasmic hsp is also increased in the invadedfiber. (A and B, x 265; C, x 125; D and E, x 400.)

In dermatomyositis, there was pronounced cytoplasmic and surface hsp reactivity in the abnormal fibers after even 1 hour of incubation with the primary antibody (Fig C). This was particularly obvious in cases with perifascicular atrophy. There was no obvious relationship between hsp expression by the muscle fibers and the presence of mononuclear cells.

Discussion The fiber type-specific distribution of hsp immunoreactivity in normal muscle revealed by prolonged incuba-

tion with the primary antibody probably reflects the known association of hsp with mitochondria [8]. The increased 65-kd hsp expression observed even after 1 hour of incubation with the primary antibody in muscle in different inflammatory myopathies could have at least three functions. First, the hsp could act as cellular “chaperones.” It is well established that hsp, which are upregulated in stressed cells, recognize and stabilize partially folded polypeptides during folding, assembly, and disassembly [l-61. This ubiquitous immune-nonspecific function of hsp is consistent with our observation that increased hsp expression was present on all regenerating and degenerating muscle fibers in all myopathies studied. Second, hsp could play a role in antigen presentation, processing, or both. It is noteworthy that the distribution of 65-kd hsp in inflammatory myopathies resembles the distribution of major histocompatibility complex (MHC) class I molecules described earlier [lo, 113. This may indicate that hsp and human leukocyte antigen (HLA) class I expression in muscle are regulated by similar or identical signals. One major function of HLA class I molecules is to bind endogenous peptides and to present them to CD8+ T cells 1127. Conceivably, hsp might play a similar role. However, whereas increased MHC class I expression can be detected on all nonnecrotic muscle fibers invaded

822 Annals of Neurology Vol 32 No 6 December 1992

by mononuclear cells, increased hsp expression was not present on all such fibers. Also, although members of the 70-kd family of hsp are encoded in the HLA class I11 region 1131 and may play a role in antigen processing [I-61, there is no evidence that 65-kd hsp are involved in antigen presentation or processing [ 1-61. Third, hsp could act as an autoantigen detected by autoreactive cytotoxic T cells 13-61. CD4+ T cells and CD8’ T cells expressing the a / p receptor can recognize 65-kd hsp determinants associated with MHC molecules 15, 6, 14, 151, and there is now evidence that peptides derived from endogenous hsp can bind directly to class I MHC molecules [161. In polymyositis and in inclusion body myositis, the distribution of 65-kd hsp, MHC class I molecules and CD8+ cells is consistent with the hypothesis that hsp-derived peptides bound to MHC class I molecules may act as an autoantigen, or an additional autoantigen, recognized by autoreactive CD8+ T cells in some patients in vivo [I71 and in vitro 1181. However, hsp could not play such a role in dermatomyositis where the increased hsp expression is not accompanied by T-cell-mediated myocytotoxicity. Clearly, further studies are needed to examine the role of hsp as potential autoantigen(s) in the inflammatory myopathies. Hsp are recognized not only by the CD4+ and CD8+ T cells expressing the conventional alp receptor, but also by CD4- and CD8-T cells expressing the uncommon yI6 receptor 13-61. In contrast to hspspecific CD4+ and CD8+ T cells, hsp-reactive y/6 T cells kill hsp-expressing target cells without MHC restriction 1191. y/6 T cells were exceedingly rare in the 25 muscle specimens studied here, but comprised 54% of the endomysial T cells in a recently recognized form of polymyositis [91. In that case, the distribution of 65-kd hsp and y/6 T cells suggested the possibility that autoaggressive y/6 T cells recognized hsp determinants expressed on muscle fibers [9]. Hsp-reactive y/6 T cells might play a similar role in other diseases, as exemplified by recent observations in multiple sclerosis

“I. In conclusion, we have observed increased expression of 65-kd hsp in all inflammatory myopathies investigated. Although this may largely reflect an immunenonspecific “stress” response, the intriguing possibility that intramuscular hsp may act as autoantigen(s) in some forms of inflammatory myopathy deserves further investigation.

References 1. Gething M-J, Sambrook J. Protein folding in the cell. Nature 1991;355:33-45 2. Hightower LE. Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 1991;66:191- 197 3. Schlesinger MJ. Heat shock proteins. J Biol Chem 1990;265: 12111-12114 4. Young RA. Stress proteins and immunology. Annu Rev Immuno1 1990;8:401-420 5. Kaufmann SHE. Heat shock proteins and the immune response. Immunol Today 1990;129-136 6. Born W, Happ MP, Dallas A, et al. Recognition of heat shock proteins and gamma-delta function. Immunol Today 1990; 11:40-43 7. Ivanyi J, Sinha S, Aston R, et al. Definition of species specific and cross-reactive antigenic determinants of Myrobartevium leprae using monoclonal antibodies. Clin Exp Immunol 1983;52: 528-536 8. Evans DJ, Norton P, Ivanyi J. Distribution in tissue sections of the human groEL stress-protein homologue. Acta Pathol Microbiol Immunol Scand 1992;98:437-441 9. Hohlfeld R, Engel AG, Ii K, Harper MC. Polymyositis mediated by T lymphocytes that express the gamma-delta receptor. N Engl J Med 1991;324:877-881 10. Karpati G, Pouliot Y, Carpenter S. Expression of immunoreactive major histocompatibility complex products in human muscles. Ann Neurol 1988;23:64-72 1. Emslie-Smith AM, Arahata K, Engel AG. Major histocompatibility complex class I antigen expression, immunolocalization of interferon subtypes, and T-cell mediated cytotoxicity in myopathies. Hum Pathol 1989;20:224-23 1 2. Hohlfeld R. Neurological autoimmune disease and the trimolecular complex of T lymphocytes. Ann Neurol 1989;25:531-538 13. Trowsdale J, Ragoussis J, Campbell RD. Map of the human MHC. Immunol Today 1991;12:443-446 14. Munk ME, Scoel B, Modrow S, et al. T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein. J Immunol 1989;143:2844-2849 15. Koga T, Wand-Wurttenberger A, DeBruyn J, et al. T cells against a bacterial heat shock protein recognize stressed macrophages. Science 1989;245:1112-1115 16. Jardetzky TS, Lane WS, Robinson RA, et al. Identification of self peptides bound to purified HLA-B27. Nature 1991;353: 326-329 17. Engel AG, Arahata K. Mononuclear cells in myopathies: quantitation of functionally distinct subsets, recognition of antigenspecific cell mediated cytotoxicity in some diseases, and implications for the pathogenesis of the different inflammatory myopathies. Hum Pathol 1986;17:704-721 18. Hohlfeld R, Engel AG. Coculture with autologous myotubes of cytotoxic T cells isolated from muscle in inflammatory myopathies. Ann Neurol 1991;29:498-507 19. Fisch P, Malkovsky M, Kovats S, et al. Recognition by human V,9//V82 T cells of a GroEL homolog on Daudi Burkitt’s lymphoma cells. Science 1990;250:1269-1273 20. Selmaj K, Brosnan C , Rake CS. Colocalization of lymphocytes bearing gamma-delta T cell receptor and heat shock protein hsp65 oligodendrocytes in multiple sclerosis. Proc Natl Acad Sci USA 1991;82:6452-6456 +

This work was supported by Grant NS-6277 from the National Institutes of Health and a research grant from the Muscular Dystrophy Association. We are grateful to Dr J. Ivanyi for providing the monoclonal antibody ML30.

Brief Communication: Hohlfeld and Engel: Heat Shock Proteins 823

Expression of 65-kd heat shock proteins in the inflammatory myopathies.

In normal muscle, 65-kd heat shock proteins (hsp) were detected on capillary endothelial cells, the mural elements of larger vessels, and some intrace...
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