Expression of histocompatibility antigen HLA В ЭDR by salivary gland epithelial cells in Sjgren's syndrome.код для вставкиСкачать
1105 EXPRESSION OF HISTOCOMPATIBILITY ANTIGEN HLA-DR BY SALIVARY GLAND EPITHELIAL CELLS IN SJOGREN’S SYNDROME ROBERT I. FOX, THOMAS BUMOL, KICHARD FANTOZZI, ROBERT BONE, and ROBERT SCHREIBER Recent studies have suggested that the induction of HLA-DR antigens on epithelial cells plays an important role in the pathogenesis of autoimmune endocrine exocrine disorders. We found that salivary gland epithelial cells (i.e., acinar and ductal cells) in salivary gland biopsy specimens from patients with primary Sjogren’s syndrome (keratoconjunctivitis sicca) expressed high levels of HLA-DR antigen, which were detected by staining frozen tissue sections with monoclonai antibodies and immunoperoxidase technique. In contrast, Salivary gland epithelial cells from normal subjects did not express this antigen. Lymphocytes eluted from the salivary gland biopsy specimens of patients who had Sjogren’s syndrome produced a soluble factor that stimulated HLA-DR synthesis by a salivary gland-derived cell line (Sal-1). These tissue culture supernatants contained y-interferon, and their ability to induce HLA-DR synthesis was blocked by monoclonal anti-y-interferon antibody. These results Publication number 4078BCR from the Scripps Clinic and Research Foundation, La Jolla, California. From the Departments of Rheumatology, Basic and Clinical Research, Head and Neck Surgery, and Immunology, Scripps Clinic and Research Foundation, La Jolla, California, and the Eli Lilly Research Laboratory, Indianapolis, Indiana. Supported in part by grants Ah4-33294, AM-33983, and CA-34120 from the National Institutes of Health, a grant from the Eli Lilly Research Laboratories, and a grant from the Kieckhefer Foundation. Robert I. Fox, MD, PhD: Departments of Rheumatology and Basic and Clinical Research; Thomas Bumol, PhD: Eli Lilly Research Laboratory; Richard Fantozzi, MD: Department of Head and Neck Surgery; Robert Bone, MD: Department of Head and Neck Surgery; Robert Schreiber, PhD: Department of Immunology. Address reprint requests to Robert I. Fox, MD, PhD, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037. Submitted for publication October 2, 1985; accepted in revised form March 17, 1986. Arthritis and Rheumatism, Vol. 29, No. 9 (September 1986) demonstrate the presence of HLA-DR antigen on salivary gland epithelial cells and suggest that local production of y-interferon plays a role in this induction. During the past several years, the importance of histocompatibility antigens encoded by the HLA region of chromosome 6 in immune responsiveness and disease susceptibility has been established (1,2). The HLA--DR region encodes 2 polypeptides (molecular weights 34,000 and 28,000) that were initially recognized on macrophages and B cells. These molecules probably provide a structure that helps orient T helpedinducer cells during recognition of antigens (2-4). The HLA-DR molecules are highly polymorphic, and the HLA-DR3 allele has been associated with autoimmune endocrine-exocrine diseases such as Sjiigren’s syndrome (SS), Hashimoto’s thyroiditis, insulin-dependent diabetes, Addison’s disease, and primary biliary cirrhosis (5,6). However, the structural basis for the association of autoimmune diseases with HLA-DR alleles remains unknown. Investigators have recently demonstrated the presence of HLA-DR antigens on thyroid epithelial cells in patients with autoimmune thyroid disease, but not on normal thyroid tissues (6,7). It also has been proposed that autoantigens at the thyroid cell membrane might associate with the newly induced HLA-DR molecules and thus be recognized by immune T cells to initiate or perpetuate immune responses (6-8). To determine if similar mechanisms might be present in another autoimmune disease associated with HLA-DR3, we examined salivary gland specimens from patients who had SS, which is characterized by lymphocytic infiltration of the minor salivary glands (9). This disorder is particularly well-suited to 1106 FOX ET AL serve as a model for autoimmune endocrine-exocrine disease, since it is relatively common and the target organs (i.e., salivary glands) are safely accessible to biopsy (9,lO). Studies using monoclonal antibodies and in vitro functional studies have shown that the saliv,arygland is infiltrated predominantly by T cells of the Leu-3a-positive (OKT4+) subset that have T helperhnducer cell function (10-12). The salivary glands of these patients are active sites of autoantibody and interleukin-2 (IL-2) production (13,14). We have extended those studies by demonstrating, with the use of monoclonal antibodies and immunohistologic techniques, the presence of HLA-DR antigen on salivary gland epithelial cells in biopsy specimens from SS patients. To understand the mechanisrri(s) responsible for antigen induction, we investigated the role of recombinant y-interferon (y-IFN) on salivary gland cells cultured in vitro, since recent reports have indicated the ability of this lymphokine to stimulate HLA-DR expression by epithelial cells (8). Also, we wished to determine whether lymphocytes eluted from salivary gland biopsy specimens of SS patients could be stimulated to secrete -/-IFN in vitro and tlius provide a signal for HLA-DR induction. PATIENTS AND METHODS Patients. Twelve patients with primary SS had keratoconjunctivitis sicca, xerostomia, and a minor salivary gland specimen classified as grade IV on the ChisholmMason1 scale (from I-IV) (9,10,15). These patients did not have associated rheumatoid arthritis and had not received corticosteroids or immunosuppressive drugs for at least 3 months before the biopsy was performed (10). Normal salivary gland tissue was obtained from autopsy samples and from the histologically normal margins of glands removed because of benign tumors. Three parotid tumors (Warthin's tumor or papillary cystadenoma lymphomatosum [PCL]) were studied (16). This type of parotid tumor is particularly interesting since it consists of columnar epithelial cells overlying a dense lymphoid infiltrate (16-18). Immunohistologicstudies. All specimens were placed in sterile saline until they were embedded in OCT medium (Miles, Napersville, IL), frozen in liquid nitrogen, and stored at -70°C. Frozen tissue sections (5p) were acetone-fixed and stained with monoclonal antibodies (MoAb) using immunoperoxidase methods previously described (19,20). Briefly, the tissue sections were incubated with specific MoAb or a control antibody of the same isotype and concentration. After rinsing, the sections were reacted with peroxiclase-conjugated goat anti-mouse IgG and substrate 3',3'-diaminobenzidine, resulting in an insoluble brown reaction product at sites where the original specific MoAb was bound. Monoclonal antibodies. Monoclonal antibody SC2, a murine IgG2 antibody produced in our laboratory, detects a nonpolymorphic determinant on HLA-DR molecules (2 1). MoAb Q513, produced by Dr. V. Quaranta, detects an antigenic epitope on HLA-DR molecules distinct from that recognized by MoAb SC2 (22). MoAb B539 recognizes the invariant ( y )chain that associates with the cytoplasmic form of HLA-DR (23,24). Antibody Leu-I0 (reactive with HLA-DQ), anti-Leu-MS (reactive with monocytes and tissue dendritic cells), and anti-cytokeratin (antibodies) were obtained from Becton-Dickinson (Mountain View, CA). Mature T cells were enumerated with anti-Leu-4 (BectonDickinson) and mature B cells with MoAbs B-1 (Coulter, Hialeah, FL) and Leu-I4 (Becton-Dickinson). A monoclonal antibody against y I F N (MoAb M125) was recently produced in one of our laboratories (25) and has been shown to block the effects of y I F N on monocytes (26). Preparation of salivary gland lymphocytes and detection of y-IFN production. Salivary gland lymphocytes (SGL) were obtained from the parotid glands of 4 SS patients who had undergone surgery to rule out lymphoma and/or infection (12). The SGL were eluted by cutting the tissues into 0.5 x 0.5-cm squares and gently pressing them through stainless steel mesh to obtain a suspension containing 107-109 SGL. After Ficoll-Hypaque centrifugation, the SGL were cultured in vitro at 37°C in RPMI 1640 (Flow Laboratories, Inglewood, CA) containing 5% fetal calf serum (FCS; Gibco, Grand Island, NY) that previously had been screened for the absence of endotoxin. Flat-bottom microtiter plates (Falcon Plastics, Oxnard, CA) containing 2 x lo5 lymphocytes received either media alone or mitogen stimulation with pretitered phytohemagglutinin (PHA; 0.1 pglml) plus 1% phorbol myristate acetate (PMA; Sigma, St. Louis, MO), as previously described (27). After 72 hours, the tissue culture supernatants were harvested and stored at -70°C until analyzed by solid-phase enzyme-linked immunosorbent assay (ELISA; Centocor, Malverne, PA) for ./-IFN. Tonsillar lymph node lymphocytes from immunologically normal adults were prepared and cultured in a manner similar to that used for the SGL. Tissue culture of Sal-1 cells and induction of HLA-DR antigens by y-IFN. A continuous cell line (Sal-I) was established by collagenase digestion of a PCL parotid tumor, followed by culture of adherent cells in RPMI 1640 (Gibco) plus 10% FCS. This cell line has been maintained in culture for over 16 months and continues to have epithelial cell morphology, extracellular matrix proteins (defined by monoclonal antibodies), and electron microscopic features suggestive of an epithelial cell (Bumol T, Fox RI: manuscript in preparation). In certain experiments, the Sal-1 cells were grown on sterile multichamber wells (Labtek, Napersville, IL) in the presence of either: (a) 50 international reference units (IRU)/ml recombinant ./-IFN (Cetus, San Francisco, CA) in RPMI 1640 plus 5% FCS; (b) 1,000 IU/ml of recombinant a-IFN (Hoffman-La Roche, Nutley, NJ) in RPMI 1640 plus 5% FCS; (c) cell supernatants (previously stimulated in vitro as described above) from SGL of SS patients; or (d) cell supernatants derived from stimulated SGL that contained a pretitered anti-y-IFN monoclonal antibody. After 5 days of incubation at 37"C, the slide chambers were fixed in cold HLA-DR ON SALIVARY GLAND CELLS 1107 Figure 1. Immunohistologic evaluation of salivary gland specimens. A, Minor salivary gland biopsy specimen from a Sjogren’s syndrome (SS) patient, stained with monoclonal antibody (MoAb) anti-HLA-DR, demonstrating reactivity of the acinar and ductal cells (arrows). B, Serial section of the salivary gland biopsy specimen from an S S patient, which was reacted with a control murine myeloma antibody. C, Normal minor salivary gland biopsy specimen, which was tested with MoAb anti-HLA-DR. D, Serial section of a normal salivary gland (shown at lower magnification) reacted with MoAb Leu-MS, which detects monocytes and dendritic cells. E, Frozen tissue section of parotid tumor stained with MoAb anti-HLA-DR, which reacts with cells in the lymphoid region (brown stain); the epithelial cells were unreactive (only methylene blue counterstain). F, Serial section of parotid tumor stained with anti-cytokeratin MoAb, showing staining of the epithelial cells (arrow); the lymphoid cells were unreactive. (A, B, C, E, and F, original magnification x 400;D, original magnification x 100.) acetone, and stained with monoclonal antibodies using immunoperoxidase techniques. Microscopy. Photographs of immunoperoxidasestained slides were taken with a Nikon Optiplot microscope. For scanning electron microscopy, Sal-1 cells were plated on circular (12-mm) glass coverslips in Linbro 24-well culture vessels. Established cultures were rinsed in phosphate buffered saline (PBS), pH 7.4, and subsequently fixed in 1% glutaraldehyde in PBS, pH 7.4, for 15 minutes at room temperature. After fixation, the samples were subjected to a graded series of ethanol dehydration, followed by critical point drying from Freon 13 and carbon shadowing. These samples were then evaluated by a Hitachi scanning electron microscope. RESULTS Immunohistologic characterization of HLA-DR expression in salivary gland biopsy specimens. Salivary gland epithelial cells in specimens from all 12 S S patients showed staining with anti-HLA-DR antibody 1 1013 FOX ET AL Figure 2. Induction of HLA-DR antigen on salivary gland-derived cell line. A, Scanning electron microscopic view of the salivary gland-derived cell line (Sal-l), showing its dendritic morphology (original magnification x 1,000). B, Sal-1 cells cultured for 5 days at 37°C in the preisence of recombinant yinterferon (y-IFN) (50 international reference unitsiml). The cells were fixed with acetone and stained with monocllonal antibody (MoAb) anti-HLA-DR using irnmunoperoxidase methods (arrows). C, Sal-1 cells cultured in the absence of yIFN, which fail to show staining with MoAb anti-HLA-DR (arrows). D, Sal-1 cells cultured in the presence of tissue culture supernatant derived from mitogem-stimulated salivary gland lymphocytes (SGL) and then stained with MoAb anti-HLA-DR (arrows). E, Sal-1 cells cultured in the presence of monoclonal anti-yIFN antibody plus cell supernatant from stimulated SGL and then stained with MoAb anti-HLA-DR antibody (arrows). F, Sal-1 cells cultured as in E, but stained with control murine myeloma protein (arrow). (B, C, and D, original magnification X 100; E and F, original magnification X 400.) (Figure 1A). The staining was most marked in areas of lymphoid infiltration and was barely detectable in adjacent areas. No staining was noted when a control myeloima antibody was used in place of the specific MoAb (Figure IB). A similar pattern of staining also was noted when MoAb Q513 (which detects distinct epitopes on HLA-DR) and MoAb B539 (which detects the invariant y chain associated with the cytoplasmic HLA-DR ON SALIVARY GLAND CELLS 1109 form of HLA-DR) were used (Fox RI et al: unpublished observation). In contrast, no detectable staining of the epithelial cells was noted in normal salivary glands (Figure lC), although some reactive cells were present between the epithelial cells. Examination of serial tissue sections of normal salivary glands revealed that these HLA-DR positive cells also were stained by anti-Leu-MS, but were not stained with antibodies against B cells or against complement receptors CR1 or CR2. These cells are distributed throughout the normal salivary gland (Figure 1D). In comparison, we found that the epithelial cells in a PCL parotid tumor were not stained by antiHLA-DR antibodies (Figure lE), in contrast to the underlying lymphoid regions. We have recently shown that the intense staining of the lymphoid component of this parotid tumor results from a high content of dendritic-type cells which are reactive with MoAb Leu-MS and from a few B cells which are reactive with MoAb B-1 or Leu-12 (16). The epithelial cells in the parotid tumor could be stained with anti-cytokeratin antibody (Figure 1F). The underlying lymphoid cells were not reactive with this MoAb. A similar pattern of epithelial cell staining (i.e., absence of HLA-DR and presence of anti-cytokeratin) was noted in 3 of 3 PCL biopsies. Induction of HLA-DR on salivary gland epithelial cells by y-IFN. In order to investigate the role of soluble factors in HLA-DR induction by salivary gland epithelial cells, a cell line (Sal-1) was established from a PCL salivary gland tumor. These cells have been maintained in continuous culture for over 12 months. They exhibit a dendritic morphology under scanning electron microscopy (Figure 2A) and contain features at the biochemical and ultrastructural level which indicate that they are epithelial in origin (Bumol T, Fox RI: manuscript in preparation). The Sal-1 cell line can be induced to express HLA-DR by culturing in the presence of y I F N (50 IRU/ml for 5 days), as indicated by cytoplasmic staining with MoAb SC-2 (Figure 2B). No cytoplasmic staining was noted when Sal-1 cells were cultured in the absence of yIFN or with other lymphokines (a-IFN, P IF N , or IL-2) (Figure 2C). Cell supernatants obtained from stimulated SGL were able to stimulate synthesis of this class I1 histocompatibility antigen (Figure 2D). When the Sal-1 cells were cultured with monoclonal anti-yIFN antibody plus the SGL supernatant, the induction of HLA-DR was abolished since no cytoplasmic staining was detected (Figure 2E). These results suggest that y IFN is sufficient and necessary for the induction of this histocompatibility antigen by Sal- 1 cells. Production of y-IFN by salivary gland lymphocytes. The content of yinterferon in each of the supernatants from salivary gland lymphocytes was measured by solid-phase ELISA (Table 1). The mean k SD a-IFN content was 67 5 11 IRU/ml. The production of y I F N by normal tonsillar lymphocytes (64 5 18 IRU/ml) was not significantly different from that of stimulated salivary gland lymphocytes. In the absence of mitogen stimulation, neither SGL nor tonsillar lymphocytes produced detectable y I F N (<1 IRU/ml) (Table 1). DISCUSSION Epithelial cells lining the ducts and acini of salivary gland biopsy specimens from SS patients show intense staining with monoclonal antibodies against HLA-DR. This reactivity was confirmed using several different anti-HLA-DR monoclonal antibodies and using an antibody against the invariant ( y ) chain associated with HLA-DR molecules. In contrast, no significant staining of epithelial cells from normal salivary glands or from parotid PCL tumors was noted. The presence of HLA-DR antigens in salivary gland epithelial cells in SS biopsy specimens could be due either to synthesis by these cells or to passive acquisition from other cells. The induction of HLA-DR on a salivary gland cell line by y I F N suggests that the presence of this antigen is due, at least in part, to synthesis. The induction of HLA-DR Table 1. Comparison of production of ?interferon (yIFN) by salivary gland and lymph node lymphocytes yIFN, international reference unitshl Stimulated ~ Salivary gland lymphocytes from Sjogren’s syndrome patients* Patient 1 Patient 2 Patient 3 Patient 4 Mean ? SD Tonsillar lymph node lymphocytes from normal subjects (n =: 3), mean SD * ~~~ Unstimulated ~ 71.3 76 48.3 74 67 k l l t <1 <1 Cl <1 645 18 <I <It * In the absence of mitogen, or after incubation for 72 hours at 37°C with phytohemagglutinin plus phorbol myristate acetate. t P < 0.001. 11 1 0 antigens may be important in pathogenesis, since epithelial cells bearing these molecules may be able to “present” antigens to T lymphocytes in order to generate immune responses (2,3). Our findings on SS specimens are similar to those observed in biopsies from patients with Hashimoto’s thyroiditis, where epithelial cells were found to express HLA-DR antigens (6,7). It is intriguing tbat patients with these autoimmune diseases have an increased prevalence of HLA-DR3 histocompatibility antigen. Perhaps extrinsic antigens (such as a virus) or an intrinsic autoantigen(s) may associate with the HLA-DR present on salivary gland epithelial cells to initiate or perpetuate immune reactions. The induction of HLA-DR-like molecules on non-hematopoietic cells was first demonstrated with conditioned media from activated T cells, and the enhancing substance was shown to be y I F N (27-29). Therefore, we examined supernatants from stimulated SGL from S S patients and found that these fluids could stimulate HLA-DR synthesis of a salivary glandderived cell line. The active substance again appears to be yIFN, since the enhancing activity was abolished by monoclonal anti-yIFN antibody. Furthermore, the induction of HLA-DR could be reproduced by recombinant yIFN. Thus, this lymphokine appears to be necessary and sufficient for HLA-DR induction by salivary gland cells. Other substances, including mitogens (PHA, PMA) , recombinant a-IFN , recombinant ki-IFN, and recombinant IL-2 were examined, but no stiinulatory activity was noted. In summary, the present study demonstrates the presence of HLA-DR antigens on salivary gland epithelial cells and shows that this induction may be caused by yIFN. The HLA-DR molecules on the epithelial cells may allow these cells to present antigens to immune T cells, and thus hitiatelperpetuate immune responses. These results extend previous observations on induction of HLA-DR in human autoimmune disease (Hashimoto’s thyroiditis) (649, by demonstrating similar induction in a different disease state, thereby suggesting that this may be a common feature shared by autoimmune endocrineexocrine disorders. Further, we have demonstrated the production of y I F N by lymphocytes at the disease site, iridicating the potential of these cells to mediate the induction of HLA-DR antigens. Addendum. After this manuscript was submitted, we became aware of a report by Lindahl G, Hedfors E, Klareskog L, Forsum U: Epithelial HLA-DR expression in FOX ET AL T lymphocyte subsets in salivary glands in Sjogren’s syndrome. Clin Exp Immunol 61:475482, 1985 REFERENCES 1. Sasazuki T, McDevitt HO: The association between genes in the major histocompatibility complex and disease susceptibility. Annu Rev Med 28:425452, 1977 2. Unanue E: Antigen-presenting function of the macrophage. Annu Rev Immunol2:395428, 1984 3. Mengle-Gaw L, McDevitt HO: Genetics and expression of mouse Ia antigens. Annu Rev Immunol 3:367-396, 1985 4. Katz D, Hamaoka T, Dorf M, Benaceraf B: Cell interactions between histocompatible T and B lymphocytes: the H-2 gene complex determines successful physiologic lymphocyte interactions. 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