0021-972x/92/7404-0724$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
ARMIN E. HEUFELDER, AND REBECCA S. BAHN
JOHN
Detection and Localization Shock Protein in Autoimmune R. GOELLNER,
BJOERN
E. WENZEL,
Division of Endocrinology, Department of Internal Medicine, and Department Clinic, Rochester, Minnesota 55905; and Cell and Zmmunobiology Laboratory, Medizinische Uniuersitaet Luebeck (B.E. W.), 2400 Luebeck, Germany
ABSTRACT. Recently described immunological functions for heat shock proteins (HSPs) and our previous demonstration of site-selective HSP-72 expression in cultured fibroblasts derived from extrathyroidal manifestations of Graves’ disease (GD) prompted us to determine whether expression of the inducible 72-kilodalton HSP can be detected in human thyroid tissues. Immunohistochemistry was performed on formalin-fixed paraffin-embedded thyroid tissue specimens from patients with GD, Hashimoto’s thyroiditis (HD), and multinodular goiter (MNG) as well as on normal thyroid tissue. A mouse monoclonal antiHSP-72 antibody and an ultrasensitive avidin-biotin-peroxidase complex detection system were used for these studies. Striking differences in HSP-72 immunoreactivity were detected both between tissues from GD and HD compared with
MNG
of a
of Pathology (J.R.G.), Mayo Klinik fuer Znnere Medizin,
and
normal
thyroid
and
between
GD
thyroid
glands
treated preoperatively with antithyroid medication and untreated GD glands. Strong HSP-72 reactivity in GD and HD tissues was detected in thyroid follicles as well lymphocytic infiltrates. No HSP-72 reactivity was detected in MNG or normal thyroid tissue. HSP-72 immunoreactivity was markedly reduced in GD glands that received preoperative antithyroid drug treatment. In conclusion, high levels of HSP-72 expression in autoimmune
thyroid
disease
may
reflect
a state
of chronic
cellular
stress, but could also represent an immunomodulatory factor of relevance in the autoimmune process in GD. (J Clin Endocrirwl Metab 74: 724-731, 1992)
H
EAT shock proteins (HSPs) function to maintain basic cellular functions both under basal conditions and after cellular stress (1). Stress-related functions of HSPs include the refolding, translocation, and degradation of proteins, thus maintaining the cytoskeleta1 integrity and metabolic homeostasis of cells (2-4). Recently, various immunomodulatory functions have been attributed to products of the HSP-70 gene family (5, 6). These properties of HSP could play a role in the pathogenesis of autoimmune thyroid disease, in which the presentation of several autoantigens to the immune system results in the formation of organ-specific autoantibodies. Recently, a strong association between the presence of a particular HSP-70 allele and the development of Graves’ disease (GD) has been reported, suggesting a possible immunological role for HSP-70 in GD (7). In further support of this concept, we have demon-
strated previously that HSP-72 immunoreactivity is detected in retroocular and pretibial fibroblasts derived from patients with Graves’ ophthalmopathy and pretibial dermopathy and is not observed in abdominal fibroblasts from these same patients or in fibroblasts from any of these three anatomical sites of normal individuals (8). The current immunohistochemical study investigates the expression of HSP-72 in thyroid specimens from patients with GD, Hashimoto’s disease (HD), and multinodular goiter (MNG) as well as in normal thyroid tissues. We postulated that HSP-72 might be expressed only in the thyroid glands from patients with GD and HD, suggesting a function for this molecule in autoimmune thyroid disease.
Received June 4,199l. Address requests for reprints to: Dr. Rebecca S. Bahn, Division of Endocrinology, Mayo Clinic, Rochester, Minnesota 55905. * This work was supported by EY08819-01 from the National Eye Institute (to R.S.B.) and by Deutsche Forschungsgemeinschaft (Grant He 1485/2-l to A.E.H.).
Thyroid specimens were obtained at surgery from patients with GD (n = 9), HT (n = 5), and MNG (n = 5, two of which were hyperthyroid with toxic MNG). Normal thyroid tissue was obtained at surgery from the contralateral uninvolved thyroid lobes of patients undergoing total thyroid resection for
Materials
and Methods
Source of tissues
724
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Immunohistochemical 72-Kilodalton Heat Thyroid Disease*
Vol. 74, No. 4 Printed in U.S.A.
HSP-72 IN AUTOIMMUNE
Histology
Fresh tissue specimenswere fixed in 10% buffered formalin for 48 h, embeddedin paraffin, sectionedat 5 pm, mounted on microscopeslides, and heat dried at 60 C for 60 min. Representative sectionswere stainedroutinely with hematoxylin and eosin;consecutivesectionsunderwent the immunostainingprocedure.Parallel sectionswith the primary antibody replacedby unrelated monoclonal antibodies and nonimmune mouseIgG of these sameisotype and with the omissionof each layer in
DISEASE
725
turn were examined to assurespecificity and exclude crossreactivities between the antibodies and conjugatesemployed. Formalin-fixed paraffin-embedded cultured fibroblasts, harvested before and after in uitro heat shock, served as negative and positive controls. Other control tissuesincluded specimens from breast, liver, and lymph node. Immunodetection
of HSP-72
reactivity
Slides were deparaffinized in xylene, followed by absolute ethanol, 95% ethanol, and distilled water. Endogeneousperoxidase activity was blocked by incubating the sections for 30 min in methanol containing hydrogen peroxide (0.6%). This wasfollowed by treatment of sectionswith 0.4% pepsin in 0.01 N HCl, pH 2.25, for 45 s in a microwave oven and then rinsing with distilled water. Nonspecific binding was blocked by incubation for 30 min in phosphate-buffered saline (PBS), pH 7.4, containing normal horse serum (5%) and Tween-20 (0.05%). Mouse monoclonal anti-HSP-72 antibody [StressGenBiotechnology Corp., Sidney, British Columbia, Canada;1:500dilution in PBS, pH 7.4, containing normal horse serum (1%) and Tween-20 (0.05%)] was then applied for 30 min at room temperature. The sections were rinsed twice with tap water for 5 min each and once for 2 min in PBS containing 0.05% Tween20, and incubated for 1 h with a horse antimousebiotinylated antibody (Vector Laboratories, Burlingame, CA, 1:250dilution
FIG. 1. Normal human thyroid tissue demonstrating
the absence of HSP-72 immunoreactivity,
Downloaded from https://academic.oup.com/jcem/article-abstract/74/4/724/3004624 by Tulane University Medical Library user on 02 February 2019
thyroid cancer (n = 4) or at autopsy (n = 2). Five of the patients with GD and the two hyperthyroid patients with MNG had receivedmethimazolefor 3-8 months before surgery (specimens suppliedby B.E.W.). The remaining four hyperthyroid patients with GD had not received treatment with antithyroid drugs or iodine before thyroidectomy. In each caseof GD, the diagnosis was madeclinically and confirmed by the presenceof elevated thyroid hormone and thyroid-stimulating immunoglobulin (Ig) levels, suppressedTSH values, and high radioiodine uptake values. All patients with HD had a goiter, had high antithyroid peroxidase and antithyroglobulin antibody titers, and were euthyroid at the time of surgery. Three of these patients were receiving thyroid hormone replacement therapy. Surgery was performed for removal of thyroid nodulessuspiciousfor malignancy.
THYROID
HEUFELDER
726
2. HSP-72
JCE & M -1992 Vol74.No4
immunoreactivityin thyroid tissuefrom a patient with GD (magnification,X400).
in PBS, pH 7.4, containing 1% normal horseserum).The tissue sectionswere washedas before and incubated for 30 min with avidin and biotinylated peroxidase(Vectastain PeroxidaseABC Elite kit, Vector Laboratories). The sectionswere again rinsed in tap water, PBS containing Tween-20, and distilled water. After incubation in sodium acetate buffer (pH 5.2) for 2 min, the reaction product was developed using 3-amino-g-ethylcarbazole [lo mg in 47.5 mL sodium acetate (0.1 M), pH 5.2, containing 2.5 mL dimethylformamide and 0.5 mL hydrogen peroxide (3%)] as the substrate and counterstained for 30 s with Schmitt’s hematoxylin before mounting with glycerinjelly. A reddish orange precipitate was indicative of HSP-72 immunoreactivity. For the detection of thyroglobulin and B- and T-lymphocytes, control sectionswere routinely stained using monoclonal antibodies against thyroglobulin, UHCL-1 (CD 45 RO), and LeuZ6(Dakopatts Corp., Santa Barbara, CA), respectively, in conjunction with the above-describedimmunostaining procedure.
Results In sections from normal thyroid (n = 6; Fig. 1) and nonthyroidal control tissues, no HSP-72 immunoreactivity was detected, nor was HSP-72 immunoreactivity detected in thyroid follicles of specimens from patients with
nonautoimmune thyroid disease, namely toxic (n = 2) or euthyroid (n = 3) MNG (data not shown). Minimal HSP-72 reactivity was observed in occasional cells of the interfollicular connective tissue in these specimens. In contrast, strong HSP-72 immunoreactivity was detected in the vast majority of thyroid follicles in all tissue specimens derived from patients with GD that had not received antithyroid medication before surgery (n = 4; Fig. 2). HSP-72 immunoreactivity was diffusely distributed throughout the cytoplasm of thyroid follicular epithelial cells and displayed sharp demarcation from the surrounding connective tissue. Focally, HSP-72 reactivity was also noted in the nuclei of thyrocytes (Fig. 3). The strongest HSP-72 expression was detected in thyroid follicles adjacent to areas of lymphocytic infiltration. In addition, cytoplasmic HSP-72 reactivity was observed in these tissues in scattered interstitial cells between thyroid follicles, within cells forming the lymphoplasmacytic infiltrate (Fig. 4), and in the walls of blood vessels (data not shown). Within the inflammatory infiltrates, HSP72 expression was noted in cells reactive with both Band T-cell markers. No HSP-72 reactivity was detected in sections treated with nonimmune mouse IgG of the same isotype or control monoclonal antibodies instead
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FIG.
ET AL.
HSP-72
IN AUTOIMMUNE
of the anti-HSP-72 antibody. Sections from thyroid glands of patients with GD (n = 5) who had been treated with methimazole for 3-8 months before surgery displayed HSP-72 immunoreactivity in only 10% of follicles, compared with its presence in the majority of follicles in specimens from untreated patients. HSP-72 reactivity within follicles was less intense than but of similar distribution to that in specimens from untreated patients with GD. Strong cytoplasmic HSP-72 immunoreactivity was detected in thyroid follicular epithelial cells of all patients with HD (n = 5) regardless of whether they were receiving thyroid hormone replacement (Fig. 5). HSP-72 reactivity was strongest in thyroid follicles adjacent to areas of lymphocytic infiltration and was also detected in high abundance in the cytoplasm of cells forming areas of diffuse lymphocytic infiltration. The results of this study are summarized in Table 1. Identity between the molecule immunoreactive with the anti-HSP-72 antibody and the 72-kilodalton (kDa) HSP was confirmed using immunoblotting techniques. Supernatants of crude thyroid homogenates prepared from the same surgical specimens that had been used for immunohistochemistry were subjected to sodium dodecyl
727
DISEASE
in thyroid epithelial cell nuclei.
sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with the monoclonal anti-HSP-72 antibody, as previously described (10). Strong immunoreactivity of approximately 70 kDa molecular size was detected in thyroid specimens from patients with untreated GD and HT. Immunoblot controls, using nonimmune mouse IgG of the same isotype on parallel lanes, were negative. In GD patients who had received preoperative treatment with methimazole, HSP-72 reactivity was detected in low abundance. No HSP-72 immunoreacitivity was detected in normal thyroid tissue or MNG (Fig. 6). Discussion HSPs are induced by a variety of stimuli of potential relevance to autoimmunity, including oxygen free radicals, bacterial or viral microorganisms, cellular transformation, and cytokines (1, 8-11). The expression of several HSPs as well as autoantibodies against these HSPs has been reported in a variety of infections and autoimmune diseases, including rheumatoid arthritis, ankylosing spondylitis, lupus erythematosis, scleroderma, and diabetes mellitus (5, 12-14). Genes encoding HSP-70 have been mapped within the human major histocompatibility complex between the loci for complement com-
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FIG. 3. HSP-72 immunoreactivity
THYROID
728
HEUFELDER
4. HSP-72
immunoreactivity
in lymphocytic
ponents and tumor necrosis factor (15). Studies from several laboratories have demonstrated epitope homologies among various HSPs and autoantigens of infectious and autoimmune diseases (5). In particular, members of the 70-kDa HSP family share homologies with bacterial antigens that show an immunological association with autoimmune thyroid diseases (16). Therefore, it is possible that cross-reactivity exists between the monoclonal anti-HSP-72 antibody and a mimicking molecule present in autoimmune thyroid glands, resulting in immunoreacitivity that does not reflect the true presence of HSP-72. However, the lack of HSP-72 immunoreactivity in normal thyroid tissue and multinodular goiter, as demonstrated by both immunohistochemistry and immunoblotting, argues against cross-reactivity with the native forms of thyroid antigens, such as thyroid peroxidase, thyroglobulin, or the TSH-receptor complex. Whether thyroid hormone levels influence HSP-72 expression in thyroid tissue remains to be established. However, based on our current data, elevated levels of thyroid hormone appear not to be involved in HSP-72 induction; HSP-72 reactivity was observed in both euthyroid HD and hyperthyroid GD, but not in toxic or
infiltrate
in thyroid
JCE & M .1992 Vol74.No4
tissue
from
a patient
with
GD.
euthyroid MNG. Therefore, the reduced level of HSP-72 expression detected in GD after antithyroid drug medication is more likely to be a consequence of the immunomodulatory effect of methimazole than its action on thyroid hormone synthesis and metabolism. The most obvious explanation for the selective presence of HSP-72 in thyroid tissues affected by an organspecific autoimmune process is the induction of its expression as a result of chronic cellular stress (5, 6). Further, the availability of 70-kDa and other HSPs in high abundance may be required for or associated with a state of increased cell proliferation or cellular activity (17). In this view, the presence of HSP-72 in autoimmune thyroid disease may reflect nonspecific immune activation, leading to cellular damage with consequent induction of HSPs to assist cells with their synthetic and metabolic functions. Alternatively, several recent observations support the view that products of the 70-kDa HSP gene family, in addition to their stress-related functions, play a role in a variety of immunological mechanisms (5). Such immunomodulatory functions of 70-kDa HSPs include the translocation and degradation of damaged cellular proteins (2-4), which may involve intracellular process-
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FIG.
ET AL.
HSP-72
TABLE
1. HSP-72 immunoreactivity GD (n = 4)”
GD (n = 5)*
5.
HSP-72 immunoreactivity
MNG (n = 5)
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729
in thyroid tissue from a patient with HD.
in thyroid specimens HD (n = 5)
THYROID
Normal izrz
Thyroid folli+++ + +++ cles Lymphocytic + + NA NA +Iinfiltrate Blood vessels ++ + ++ Connective tis+ + +Isue NA, Not applicable. a Patients received no antithyroid drug therapy before surgery. *Patients were treated with methimazole for 3-8 months before surgery.
ing and result in the surface presentation of altered selfproteins. In particular, HSP-70 molecules may play a role in the cell membrane anchoring of proteins and their presentation to the immune system in conjunction with MHC class II molecules (18). Further, 70-kDa HSPs facilitate the binding of Ig heavy chains and assist in the assembly of Ig molecules (19). Also, specific proliferation of T-cells in response to certain bacterial or cellular HSPs and a close functional interaction between y/6-
positive T-cell subsets and certain HSPs have been reported (20-24). In this capacity, HSPs or homologous epitopes shared with bacterial or cellular proteins have been implicated as potential targets in autoimmune diseases (5, 16, 25). In recent studies from our laboratory, HSP-72 expression was detected selectively in fibroblasts affected by the extrathyroidal manifestations of patients with GD, but not in unaffected anatomical areas of patients with GD or in normal individuals (8). For several reasons, our observations do not allow the conclusion that HSP-72 expression in thyroid follicular epithelial cells, lymphocytes, and connective tissue necessarily plays a role in the pathogenesis of thyroid autoimmunity. The inducible 72-kDa HSP is typically expressed in cells exposed to a variety of stressful environmental stimuli (1). Recent studies from this laboratory have demonstrated that changes in the local milieu of cultured retroocular fibroblasts enhance HSP-72 expression in these cells. In particular, cytokines, oxygen free radicals, and inflammatory mediators, probably released from inflammatory cells infiltrating the retroocular space in Graves’ ophthalmopathy, are potential mediators of HSP-72 expression in vivo (8,26,27). However, we found that baseline HSP-72 reactivity persists in cultured re-
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FIG.
IN AUTOIMMUNE
HEUFELDER
730
92
’
1 +70kDa HSP
i
I
ABCD
E
FIG. 6. Immunoblot analysis of HSP-72 immunoreactivity in thyroid tissue. Crude homogenates were prepared from the same tissue specimens that were used for HSP-72 immunohistochemistry. Cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with the anti-HSP-72 monoclonal antibody. Lane A, Untreated GD; lane B, normal thyroid tissue; lane C, methimazole-treated GD; lane D, MNG; lane E, HT.
troocular fibroblasts from patients with Graves’ ophthalmopathy for at least seven cell passages, suggesting that HSP-72 expression is not just a short-lived phenomenon induced by stress in vitro (8). Thus, thyrocyte HSP72 expression may be a consequence of chronic environmental stress in autoimmune thyroid disease or may represent an earlier step in the evolving autoimmune process. Further, as lymphocyte activation via the antigen receptor is known to induce HSP-70 expression (2% 30), HSP-72 expression by B- and T-cells in autoimmune thyroid disease may not only reflect activation of these cells, but could also have a function in the immunological signaling and proliferation of immunocompetent cells. In conclusion, the presence of HSP-72 immunoreactivity in GD and HD thyroid glands and its absence in MNG and normal thyroid tissue are demonstrated. High levels of thyrocyte HSP-72 reactivity in autoimmune thyroid disease may reflect immunological activation and chronic cellular stress, but this molecule may also have immunomodulatory functions pertinent to the immune processes in autoimmune thyroid disease. References 1. Lindquist S. The heat-shock 1986;55:1151-91.
response. Annu
Rev Biochem.
JCE & M .1992 Vol74.No4
2. Beckmann RP, Mizzen LA, Welch WJ. Interaction of Hsp70 with newly synthesized proteins: implications for protein folding and assembly. Science 1990;248:850-4. 3. Anathan J, Goldberg AL, Voellmy R. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science. 1986;232:522-4. 4. Flynn GC, Chappell TG, Rothman JE. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 1989;245:385-90. 5. Kaufmann SHE. Heat shock proteins and the immune response. Immunol Today. 1990;11:129-36. 6. Young RA. Stress proteins and immunology. Annu Rev Immunol. 1990;8:401-20. 7. Ratanachaiyavong S, Demaine AG, Campbell RD, McGregor AM. Heat shock protein 70 (HSP70) and complement C4 genotypes in patients with hyperthyroid Graves’ disease. Clin Exp Immunol. 1989;84:48-52. 8. Heufelder AE, Wenzel BE, Gorman CA, Bahn RS. Detection, cellular localization, and modulation of heat shock proteins in cultured fibroblasts from patients with extrathyroidal manifestations of Graves’ disease. J Clin Endocrinol Metab. 1991;73:24839. 9. Young D, Lathigra R, Hendrix R, Sweetser D, Young RA. Stress proteins are immune targets in leprosy and tuberculosis. Proc Nat1 Acad Sci USA 1988,85:4267-70. 10. Nevins JR. Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus ElA gene product. Cell. 1982;29:913-9. 11. Fincato G, Polentarutti N, Sica A, Mantovani A, Colotta F. Expression of a heat-inducible gene of the HSP70 family in human myelomonocytic cells: regulation by bacterial products and cytokines. Blood. 1991;77:579-86. 12. Bahr GM, Rook GAW, Al-Saffar M, van Embden J, Stanford JL, Behbehani K. Antibody levels to mycobacteria in relation to HLA type: evidence for non-HLA-linked high levels of antibody to the 65 kDa heat shock protein of M. b0ui.s in rheumatoid arthritis. Clin Exp Immunol. 1988,74:211-5. 13. Minota S, Cameron B, Welch WJ, Winfield JB. Autoantibodies to the constitutive 73-kDa member of the hsp70 family of heat shock proteins in systemic lupus erythematosis. J Exp Med. 1988,168:1475-80. 14. Deguchi Y, Shibata N, Kishimoto S. Elevated transcription of heat shock protein gene in scleroderma fibroblasts. Clin Exp Immunol. 1990;81:97-100. 15. Sargent CA, Dunham I, Trowsdale J, Campbell RD. Human major hi&compatibility complex contains genes for the major heat shock nrotein HSP70. Proc Nat1 Acad Sci USA. 1989:86:1968-72. 16. Wenzel BE, Franke TF, Heufelder AE, Heesemann J. Autoimmune thyroid diseases and enteropathogenic yersinia enterocolitica. Autoimmunology. 1990;7:295-303. 17. Pechan PM. Heat shock proteins and cell proliferation. FEBS Lett. 1991;280:1-4. 18. VanBuskirk AM, DeNagel DC, Guagliardi LE, Brodsky FM, Pierce SX. Cellular and subcellular distribution of PBP72/74, a peptidebinding protein that plays a role in antigen processing. J Immunol. 1991;146:500-6. 19. Hass IG, Meo T. cDNA cloning of the immunoglobulin heavy chain binding protein. Proc Nat1 Acad Sci USA. 1988;85:2250-4. 20. Soederstroem K, Halapi E, Nilsson E, et al. Synovial cells responding to a 65-kDa mycobacterial heat shock protein have a high proportion of a TcR gamma/delta subtype uncommon in peripheral blood. Stand J Immunol. 1990;32:503-15. 21. Gaston JS, Life PF, Jenner PJ, Colston MJ, Bacon PA. Recognition of a mycobacteria-specific epitope in the 65-kDa heat-shock protein by synovial fluid-derived T cell clones. J Exp Med. 1990;171:831-41. 22. Born W, Hall L, Dallas A, et al. Recognition of a peptide antigen by heat-shock-reactive gamma-delta T lymphocytes. Science. 1990;249:67-9. 23. Koga T, Wand-Wuerttenberger A, DeBruyn J, Munk ME, Schoel B, Kaufmann SHE. T Cells against a bacterial heat shock protein recognize stressed macrophages. Science. 1989;245:1112-5.
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Sci. 1991;32:1327. 27. Santoro MG, Garaci E, Amici G. Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells. Proc Nat1 Acad Sci USA. 1989:86:8407-11. 28. Ferris DF, Harel-Bellan A, Morimoto RI, Welch WJ, Farrar WL. Mitogen and lymphokine stimulation of heat shock proteins in T lymphocytes. Proc Nat1 Acad Sci USA. 1988;85:3850-4. 29. Spector NL, Freedman AS, Greeman G, et al. Activation primes human B lymphocytes to respond to heat shock. J Exp Med. 1989;170:1763-8. 30. Ciavarra RP, Simeone A. T Lymphocyte stress response. Cell Immunol. 1990;131:11-26.
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24. Lamb JR, Young DB. T Cell recognition of stress proteins. Mol Biol Med. 1990;7:311-21. 25. Kale AbB, Kiessling R, van Embden JDA, et al. Induction of antigen-specific CD4+ HLA-DR-restricted cytotoxic lymphocytes as well as nonspecific nonrestricted killer cells by the recombinant mycobacterial 65-kDa heat-shock protein. Eur J Immunol. 1990;20:369-77. 26. Heufelder AE, Wenzel BE, Bahn RS. Oxygen radical scavangers (ORS) inhibit the stress-induced expression of 70 kDa heat shock proteins (HSP) in human retroocular fibroblasts from patients with Graves’ ophthalmopathy [Abstract]. Invest Ophthalmol Vis
THYROID
ARMIN E. HEUFELDER, AND REBECCA S. BAHN
JOHN
Detection and Localization Shock Protein in Autoimmune R. GOELLNER,
BJOERN
E. WENZEL,
Division of Endocrinology, Department of Internal Medicine, and Department Clinic, Rochester, Minnesota 55905; and Cell and Zmmunobiology Laboratory, Medizinische Uniuersitaet Luebeck (B.E. W.), 2400 Luebeck, Germany
ABSTRACT. Recently described immunological functions for heat shock proteins (HSPs) and our previous demonstration of site-selective HSP-72 expression in cultured fibroblasts derived from extrathyroidal manifestations of Graves’ disease (GD) prompted us to determine whether expression of the inducible 72-kilodalton HSP can be detected in human thyroid tissues. Immunohistochemistry was performed on formalin-fixed paraffin-embedded thyroid tissue specimens from patients with GD, Hashimoto’s thyroiditis (HD), and multinodular goiter (MNG) as well as on normal thyroid tissue. A mouse monoclonal antiHSP-72 antibody and an ultrasensitive avidin-biotin-peroxidase complex detection system were used for these studies. Striking differences in HSP-72 immunoreactivity were detected both between tissues from GD and HD compared with
MNG
of a
of Pathology (J.R.G.), Mayo Klinik fuer Znnere Medizin,
and
normal
thyroid
and
between
GD
thyroid
glands
treated preoperatively with antithyroid medication and untreated GD glands. Strong HSP-72 reactivity in GD and HD tissues was detected in thyroid follicles as well lymphocytic infiltrates. No HSP-72 reactivity was detected in MNG or normal thyroid tissue. HSP-72 immunoreactivity was markedly reduced in GD glands that received preoperative antithyroid drug treatment. In conclusion, high levels of HSP-72 expression in autoimmune
thyroid
disease
may
reflect
a state
of chronic
cellular
stress, but could also represent an immunomodulatory factor of relevance in the autoimmune process in GD. (J Clin Endocrirwl Metab 74: 724-731, 1992)
H
EAT shock proteins (HSPs) function to maintain basic cellular functions both under basal conditions and after cellular stress (1). Stress-related functions of HSPs include the refolding, translocation, and degradation of proteins, thus maintaining the cytoskeleta1 integrity and metabolic homeostasis of cells (2-4). Recently, various immunomodulatory functions have been attributed to products of the HSP-70 gene family (5, 6). These properties of HSP could play a role in the pathogenesis of autoimmune thyroid disease, in which the presentation of several autoantigens to the immune system results in the formation of organ-specific autoantibodies. Recently, a strong association between the presence of a particular HSP-70 allele and the development of Graves’ disease (GD) has been reported, suggesting a possible immunological role for HSP-70 in GD (7). In further support of this concept, we have demon-
strated previously that HSP-72 immunoreactivity is detected in retroocular and pretibial fibroblasts derived from patients with Graves’ ophthalmopathy and pretibial dermopathy and is not observed in abdominal fibroblasts from these same patients or in fibroblasts from any of these three anatomical sites of normal individuals (8). The current immunohistochemical study investigates the expression of HSP-72 in thyroid specimens from patients with GD, Hashimoto’s disease (HD), and multinodular goiter (MNG) as well as in normal thyroid tissues. We postulated that HSP-72 might be expressed only in the thyroid glands from patients with GD and HD, suggesting a function for this molecule in autoimmune thyroid disease.
Received June 4,199l. Address requests for reprints to: Dr. Rebecca S. Bahn, Division of Endocrinology, Mayo Clinic, Rochester, Minnesota 55905. * This work was supported by EY08819-01 from the National Eye Institute (to R.S.B.) and by Deutsche Forschungsgemeinschaft (Grant He 1485/2-l to A.E.H.).
Thyroid specimens were obtained at surgery from patients with GD (n = 9), HT (n = 5), and MNG (n = 5, two of which were hyperthyroid with toxic MNG). Normal thyroid tissue was obtained at surgery from the contralateral uninvolved thyroid lobes of patients undergoing total thyroid resection for
Materials
and Methods
Source of tissues
724
Downloaded from https://academic.oup.com/jcem/article-abstract/74/4/724/3004624 by Tulane University Medical Library user on 02 February 2019
Immunohistochemical 72-Kilodalton Heat Thyroid Disease*
Vol. 74, No. 4 Printed in U.S.A.
HSP-72 IN AUTOIMMUNE
Histology
Fresh tissue specimenswere fixed in 10% buffered formalin for 48 h, embeddedin paraffin, sectionedat 5 pm, mounted on microscopeslides, and heat dried at 60 C for 60 min. Representative sectionswere stainedroutinely with hematoxylin and eosin;consecutivesectionsunderwent the immunostainingprocedure.Parallel sectionswith the primary antibody replacedby unrelated monoclonal antibodies and nonimmune mouseIgG of these sameisotype and with the omissionof each layer in
DISEASE
725
turn were examined to assurespecificity and exclude crossreactivities between the antibodies and conjugatesemployed. Formalin-fixed paraffin-embedded cultured fibroblasts, harvested before and after in uitro heat shock, served as negative and positive controls. Other control tissuesincluded specimens from breast, liver, and lymph node. Immunodetection
of HSP-72
reactivity
Slides were deparaffinized in xylene, followed by absolute ethanol, 95% ethanol, and distilled water. Endogeneousperoxidase activity was blocked by incubating the sections for 30 min in methanol containing hydrogen peroxide (0.6%). This wasfollowed by treatment of sectionswith 0.4% pepsin in 0.01 N HCl, pH 2.25, for 45 s in a microwave oven and then rinsing with distilled water. Nonspecific binding was blocked by incubation for 30 min in phosphate-buffered saline (PBS), pH 7.4, containing normal horse serum (5%) and Tween-20 (0.05%). Mouse monoclonal anti-HSP-72 antibody [StressGenBiotechnology Corp., Sidney, British Columbia, Canada;1:500dilution in PBS, pH 7.4, containing normal horse serum (1%) and Tween-20 (0.05%)] was then applied for 30 min at room temperature. The sections were rinsed twice with tap water for 5 min each and once for 2 min in PBS containing 0.05% Tween20, and incubated for 1 h with a horse antimousebiotinylated antibody (Vector Laboratories, Burlingame, CA, 1:250dilution
FIG. 1. Normal human thyroid tissue demonstrating
the absence of HSP-72 immunoreactivity,
Downloaded from https://academic.oup.com/jcem/article-abstract/74/4/724/3004624 by Tulane University Medical Library user on 02 February 2019
thyroid cancer (n = 4) or at autopsy (n = 2). Five of the patients with GD and the two hyperthyroid patients with MNG had receivedmethimazolefor 3-8 months before surgery (specimens suppliedby B.E.W.). The remaining four hyperthyroid patients with GD had not received treatment with antithyroid drugs or iodine before thyroidectomy. In each caseof GD, the diagnosis was madeclinically and confirmed by the presenceof elevated thyroid hormone and thyroid-stimulating immunoglobulin (Ig) levels, suppressedTSH values, and high radioiodine uptake values. All patients with HD had a goiter, had high antithyroid peroxidase and antithyroglobulin antibody titers, and were euthyroid at the time of surgery. Three of these patients were receiving thyroid hormone replacement therapy. Surgery was performed for removal of thyroid nodulessuspiciousfor malignancy.
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HEUFELDER
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2. HSP-72
JCE & M -1992 Vol74.No4
immunoreactivityin thyroid tissuefrom a patient with GD (magnification,X400).
in PBS, pH 7.4, containing 1% normal horseserum).The tissue sectionswere washedas before and incubated for 30 min with avidin and biotinylated peroxidase(Vectastain PeroxidaseABC Elite kit, Vector Laboratories). The sectionswere again rinsed in tap water, PBS containing Tween-20, and distilled water. After incubation in sodium acetate buffer (pH 5.2) for 2 min, the reaction product was developed using 3-amino-g-ethylcarbazole [lo mg in 47.5 mL sodium acetate (0.1 M), pH 5.2, containing 2.5 mL dimethylformamide and 0.5 mL hydrogen peroxide (3%)] as the substrate and counterstained for 30 s with Schmitt’s hematoxylin before mounting with glycerinjelly. A reddish orange precipitate was indicative of HSP-72 immunoreactivity. For the detection of thyroglobulin and B- and T-lymphocytes, control sectionswere routinely stained using monoclonal antibodies against thyroglobulin, UHCL-1 (CD 45 RO), and LeuZ6(Dakopatts Corp., Santa Barbara, CA), respectively, in conjunction with the above-describedimmunostaining procedure.
Results In sections from normal thyroid (n = 6; Fig. 1) and nonthyroidal control tissues, no HSP-72 immunoreactivity was detected, nor was HSP-72 immunoreactivity detected in thyroid follicles of specimens from patients with
nonautoimmune thyroid disease, namely toxic (n = 2) or euthyroid (n = 3) MNG (data not shown). Minimal HSP-72 reactivity was observed in occasional cells of the interfollicular connective tissue in these specimens. In contrast, strong HSP-72 immunoreactivity was detected in the vast majority of thyroid follicles in all tissue specimens derived from patients with GD that had not received antithyroid medication before surgery (n = 4; Fig. 2). HSP-72 immunoreactivity was diffusely distributed throughout the cytoplasm of thyroid follicular epithelial cells and displayed sharp demarcation from the surrounding connective tissue. Focally, HSP-72 reactivity was also noted in the nuclei of thyrocytes (Fig. 3). The strongest HSP-72 expression was detected in thyroid follicles adjacent to areas of lymphocytic infiltration. In addition, cytoplasmic HSP-72 reactivity was observed in these tissues in scattered interstitial cells between thyroid follicles, within cells forming the lymphoplasmacytic infiltrate (Fig. 4), and in the walls of blood vessels (data not shown). Within the inflammatory infiltrates, HSP72 expression was noted in cells reactive with both Band T-cell markers. No HSP-72 reactivity was detected in sections treated with nonimmune mouse IgG of the same isotype or control monoclonal antibodies instead
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of the anti-HSP-72 antibody. Sections from thyroid glands of patients with GD (n = 5) who had been treated with methimazole for 3-8 months before surgery displayed HSP-72 immunoreactivity in only 10% of follicles, compared with its presence in the majority of follicles in specimens from untreated patients. HSP-72 reactivity within follicles was less intense than but of similar distribution to that in specimens from untreated patients with GD. Strong cytoplasmic HSP-72 immunoreactivity was detected in thyroid follicular epithelial cells of all patients with HD (n = 5) regardless of whether they were receiving thyroid hormone replacement (Fig. 5). HSP-72 reactivity was strongest in thyroid follicles adjacent to areas of lymphocytic infiltration and was also detected in high abundance in the cytoplasm of cells forming areas of diffuse lymphocytic infiltration. The results of this study are summarized in Table 1. Identity between the molecule immunoreactive with the anti-HSP-72 antibody and the 72-kilodalton (kDa) HSP was confirmed using immunoblotting techniques. Supernatants of crude thyroid homogenates prepared from the same surgical specimens that had been used for immunohistochemistry were subjected to sodium dodecyl
727
DISEASE
in thyroid epithelial cell nuclei.
sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with the monoclonal anti-HSP-72 antibody, as previously described (10). Strong immunoreactivity of approximately 70 kDa molecular size was detected in thyroid specimens from patients with untreated GD and HT. Immunoblot controls, using nonimmune mouse IgG of the same isotype on parallel lanes, were negative. In GD patients who had received preoperative treatment with methimazole, HSP-72 reactivity was detected in low abundance. No HSP-72 immunoreacitivity was detected in normal thyroid tissue or MNG (Fig. 6). Discussion HSPs are induced by a variety of stimuli of potential relevance to autoimmunity, including oxygen free radicals, bacterial or viral microorganisms, cellular transformation, and cytokines (1, 8-11). The expression of several HSPs as well as autoantibodies against these HSPs has been reported in a variety of infections and autoimmune diseases, including rheumatoid arthritis, ankylosing spondylitis, lupus erythematosis, scleroderma, and diabetes mellitus (5, 12-14). Genes encoding HSP-70 have been mapped within the human major histocompatibility complex between the loci for complement com-
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FIG. 3. HSP-72 immunoreactivity
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HEUFELDER
4. HSP-72
immunoreactivity
in lymphocytic
ponents and tumor necrosis factor (15). Studies from several laboratories have demonstrated epitope homologies among various HSPs and autoantigens of infectious and autoimmune diseases (5). In particular, members of the 70-kDa HSP family share homologies with bacterial antigens that show an immunological association with autoimmune thyroid diseases (16). Therefore, it is possible that cross-reactivity exists between the monoclonal anti-HSP-72 antibody and a mimicking molecule present in autoimmune thyroid glands, resulting in immunoreacitivity that does not reflect the true presence of HSP-72. However, the lack of HSP-72 immunoreactivity in normal thyroid tissue and multinodular goiter, as demonstrated by both immunohistochemistry and immunoblotting, argues against cross-reactivity with the native forms of thyroid antigens, such as thyroid peroxidase, thyroglobulin, or the TSH-receptor complex. Whether thyroid hormone levels influence HSP-72 expression in thyroid tissue remains to be established. However, based on our current data, elevated levels of thyroid hormone appear not to be involved in HSP-72 induction; HSP-72 reactivity was observed in both euthyroid HD and hyperthyroid GD, but not in toxic or
infiltrate
in thyroid
JCE & M .1992 Vol74.No4
tissue
from
a patient
with
GD.
euthyroid MNG. Therefore, the reduced level of HSP-72 expression detected in GD after antithyroid drug medication is more likely to be a consequence of the immunomodulatory effect of methimazole than its action on thyroid hormone synthesis and metabolism. The most obvious explanation for the selective presence of HSP-72 in thyroid tissues affected by an organspecific autoimmune process is the induction of its expression as a result of chronic cellular stress (5, 6). Further, the availability of 70-kDa and other HSPs in high abundance may be required for or associated with a state of increased cell proliferation or cellular activity (17). In this view, the presence of HSP-72 in autoimmune thyroid disease may reflect nonspecific immune activation, leading to cellular damage with consequent induction of HSPs to assist cells with their synthetic and metabolic functions. Alternatively, several recent observations support the view that products of the 70-kDa HSP gene family, in addition to their stress-related functions, play a role in a variety of immunological mechanisms (5). Such immunomodulatory functions of 70-kDa HSPs include the translocation and degradation of damaged cellular proteins (2-4), which may involve intracellular process-
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ET AL.
HSP-72
TABLE
1. HSP-72 immunoreactivity GD (n = 4)”
GD (n = 5)*
5.
HSP-72 immunoreactivity
MNG (n = 5)
DISEASE
729
in thyroid tissue from a patient with HD.
in thyroid specimens HD (n = 5)
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Normal izrz
Thyroid folli+++ + +++ cles Lymphocytic + + NA NA +Iinfiltrate Blood vessels ++ + ++ Connective tis+ + +Isue NA, Not applicable. a Patients received no antithyroid drug therapy before surgery. *Patients were treated with methimazole for 3-8 months before surgery.
ing and result in the surface presentation of altered selfproteins. In particular, HSP-70 molecules may play a role in the cell membrane anchoring of proteins and their presentation to the immune system in conjunction with MHC class II molecules (18). Further, 70-kDa HSPs facilitate the binding of Ig heavy chains and assist in the assembly of Ig molecules (19). Also, specific proliferation of T-cells in response to certain bacterial or cellular HSPs and a close functional interaction between y/6-
positive T-cell subsets and certain HSPs have been reported (20-24). In this capacity, HSPs or homologous epitopes shared with bacterial or cellular proteins have been implicated as potential targets in autoimmune diseases (5, 16, 25). In recent studies from our laboratory, HSP-72 expression was detected selectively in fibroblasts affected by the extrathyroidal manifestations of patients with GD, but not in unaffected anatomical areas of patients with GD or in normal individuals (8). For several reasons, our observations do not allow the conclusion that HSP-72 expression in thyroid follicular epithelial cells, lymphocytes, and connective tissue necessarily plays a role in the pathogenesis of thyroid autoimmunity. The inducible 72-kDa HSP is typically expressed in cells exposed to a variety of stressful environmental stimuli (1). Recent studies from this laboratory have demonstrated that changes in the local milieu of cultured retroocular fibroblasts enhance HSP-72 expression in these cells. In particular, cytokines, oxygen free radicals, and inflammatory mediators, probably released from inflammatory cells infiltrating the retroocular space in Graves’ ophthalmopathy, are potential mediators of HSP-72 expression in vivo (8,26,27). However, we found that baseline HSP-72 reactivity persists in cultured re-
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FIG.
IN AUTOIMMUNE
HEUFELDER
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92
’
1 +70kDa HSP
i
I
ABCD
E
FIG. 6. Immunoblot analysis of HSP-72 immunoreactivity in thyroid tissue. Crude homogenates were prepared from the same tissue specimens that were used for HSP-72 immunohistochemistry. Cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with the anti-HSP-72 monoclonal antibody. Lane A, Untreated GD; lane B, normal thyroid tissue; lane C, methimazole-treated GD; lane D, MNG; lane E, HT.
troocular fibroblasts from patients with Graves’ ophthalmopathy for at least seven cell passages, suggesting that HSP-72 expression is not just a short-lived phenomenon induced by stress in vitro (8). Thus, thyrocyte HSP72 expression may be a consequence of chronic environmental stress in autoimmune thyroid disease or may represent an earlier step in the evolving autoimmune process. Further, as lymphocyte activation via the antigen receptor is known to induce HSP-70 expression (2% 30), HSP-72 expression by B- and T-cells in autoimmune thyroid disease may not only reflect activation of these cells, but could also have a function in the immunological signaling and proliferation of immunocompetent cells. In conclusion, the presence of HSP-72 immunoreactivity in GD and HD thyroid glands and its absence in MNG and normal thyroid tissue are demonstrated. High levels of thyrocyte HSP-72 reactivity in autoimmune thyroid disease may reflect immunological activation and chronic cellular stress, but this molecule may also have immunomodulatory functions pertinent to the immune processes in autoimmune thyroid disease. References 1. Lindquist S. The heat-shock 1986;55:1151-91.
response. Annu
Rev Biochem.
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2. Beckmann RP, Mizzen LA, Welch WJ. Interaction of Hsp70 with newly synthesized proteins: implications for protein folding and assembly. Science 1990;248:850-4. 3. Anathan J, Goldberg AL, Voellmy R. Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science. 1986;232:522-4. 4. Flynn GC, Chappell TG, Rothman JE. Peptide binding and release by proteins implicated as catalysts of protein assembly. Science. 1989;245:385-90. 5. Kaufmann SHE. Heat shock proteins and the immune response. Immunol Today. 1990;11:129-36. 6. Young RA. Stress proteins and immunology. Annu Rev Immunol. 1990;8:401-20. 7. Ratanachaiyavong S, Demaine AG, Campbell RD, McGregor AM. Heat shock protein 70 (HSP70) and complement C4 genotypes in patients with hyperthyroid Graves’ disease. Clin Exp Immunol. 1989;84:48-52. 8. Heufelder AE, Wenzel BE, Gorman CA, Bahn RS. Detection, cellular localization, and modulation of heat shock proteins in cultured fibroblasts from patients with extrathyroidal manifestations of Graves’ disease. J Clin Endocrinol Metab. 1991;73:24839. 9. Young D, Lathigra R, Hendrix R, Sweetser D, Young RA. Stress proteins are immune targets in leprosy and tuberculosis. Proc Nat1 Acad Sci USA 1988,85:4267-70. 10. Nevins JR. Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus ElA gene product. Cell. 1982;29:913-9. 11. Fincato G, Polentarutti N, Sica A, Mantovani A, Colotta F. Expression of a heat-inducible gene of the HSP70 family in human myelomonocytic cells: regulation by bacterial products and cytokines. Blood. 1991;77:579-86. 12. Bahr GM, Rook GAW, Al-Saffar M, van Embden J, Stanford JL, Behbehani K. Antibody levels to mycobacteria in relation to HLA type: evidence for non-HLA-linked high levels of antibody to the 65 kDa heat shock protein of M. b0ui.s in rheumatoid arthritis. Clin Exp Immunol. 1988,74:211-5. 13. Minota S, Cameron B, Welch WJ, Winfield JB. Autoantibodies to the constitutive 73-kDa member of the hsp70 family of heat shock proteins in systemic lupus erythematosis. J Exp Med. 1988,168:1475-80. 14. Deguchi Y, Shibata N, Kishimoto S. Elevated transcription of heat shock protein gene in scleroderma fibroblasts. Clin Exp Immunol. 1990;81:97-100. 15. Sargent CA, Dunham I, Trowsdale J, Campbell RD. Human major hi&compatibility complex contains genes for the major heat shock nrotein HSP70. Proc Nat1 Acad Sci USA. 1989:86:1968-72. 16. Wenzel BE, Franke TF, Heufelder AE, Heesemann J. Autoimmune thyroid diseases and enteropathogenic yersinia enterocolitica. Autoimmunology. 1990;7:295-303. 17. Pechan PM. Heat shock proteins and cell proliferation. FEBS Lett. 1991;280:1-4. 18. VanBuskirk AM, DeNagel DC, Guagliardi LE, Brodsky FM, Pierce SX. Cellular and subcellular distribution of PBP72/74, a peptidebinding protein that plays a role in antigen processing. J Immunol. 1991;146:500-6. 19. Hass IG, Meo T. cDNA cloning of the immunoglobulin heavy chain binding protein. Proc Nat1 Acad Sci USA. 1988;85:2250-4. 20. Soederstroem K, Halapi E, Nilsson E, et al. Synovial cells responding to a 65-kDa mycobacterial heat shock protein have a high proportion of a TcR gamma/delta subtype uncommon in peripheral blood. Stand J Immunol. 1990;32:503-15. 21. Gaston JS, Life PF, Jenner PJ, Colston MJ, Bacon PA. Recognition of a mycobacteria-specific epitope in the 65-kDa heat-shock protein by synovial fluid-derived T cell clones. J Exp Med. 1990;171:831-41. 22. Born W, Hall L, Dallas A, et al. Recognition of a peptide antigen by heat-shock-reactive gamma-delta T lymphocytes. Science. 1990;249:67-9. 23. Koga T, Wand-Wuerttenberger A, DeBruyn J, Munk ME, Schoel B, Kaufmann SHE. T Cells against a bacterial heat shock protein recognize stressed macrophages. Science. 1989;245:1112-5.
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Sci. 1991;32:1327. 27. Santoro MG, Garaci E, Amici G. Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells. Proc Nat1 Acad Sci USA. 1989:86:8407-11. 28. Ferris DF, Harel-Bellan A, Morimoto RI, Welch WJ, Farrar WL. Mitogen and lymphokine stimulation of heat shock proteins in T lymphocytes. Proc Nat1 Acad Sci USA. 1988;85:3850-4. 29. Spector NL, Freedman AS, Greeman G, et al. Activation primes human B lymphocytes to respond to heat shock. J Exp Med. 1989;170:1763-8. 30. Ciavarra RP, Simeone A. T Lymphocyte stress response. Cell Immunol. 1990;131:11-26.
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24. Lamb JR, Young DB. T Cell recognition of stress proteins. Mol Biol Med. 1990;7:311-21. 25. Kale AbB, Kiessling R, van Embden JDA, et al. Induction of antigen-specific CD4+ HLA-DR-restricted cytotoxic lymphocytes as well as nonspecific nonrestricted killer cells by the recombinant mycobacterial 65-kDa heat-shock protein. Eur J Immunol. 1990;20:369-77. 26. Heufelder AE, Wenzel BE, Bahn RS. Oxygen radical scavangers (ORS) inhibit the stress-induced expression of 70 kDa heat shock proteins (HSP) in human retroocular fibroblasts from patients with Graves’ ophthalmopathy [Abstract]. Invest Ophthalmol Vis
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