Endocrinology, doi:10.1210/en.2006-0018
Endocrinology Vol. 147, No. 5 2411-2416
Copyright © 2006 by The Endocrine Society
The Substrate-Binding Domain of 21-Hydroxylase, the Main Autoantigen in Autoimmune Addison’s Disease, Is an Immunodominant T Cell Epitope
Eystein S. Husebye,
Eirik Bratland,
Geir Bredholt,
Mati Fridkin,
Molly Dayan and
Edna Mozes
Departments of Immunology (E.S.H., M.D., E.M.) and Organic Chemistry (M.F.), The Weizmann Institute of Science, Rehovot 76100, Israel; and Division of Endocrinology, Institute of Medicine (E.S.H., E.B), University of Bergen and Department of Medicine (E.S.H., E.B.) and Center for Organ-Specific Autoimmune Diseases (G.B.), Haukeland University Hospital, N-5021 Bergen, Norway
Address all correspondence and requests for reprints to: Eystein Husebye, M.D., Ph.D., Division of Endocrinology, Department of Medicine, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. E-mail: Eystein.Husebye{at}med.uib.no.
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Abstract
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The steroidogenic enzyme 21-hydroxylase (21OH) is the main autoantigen in autoimmune primary adrenal failure (Addison’s disease). Autoantibodies against 21OH are immunological markers of an ongoing autoimmune process but are not directly involved in the tissue destruction. Autoreactive T cells are thought to mediate tissue damage, but the T cell antigen(s) has not been identified. To find out whether 21OH contains important immunodominant epitopes for T cells, we first immunized BALB/c and SJL inbred mouse strains with recombinant 21OH and showed that lymph node cells proliferated effectively following in vitro stimulation with recombinant 21OH (stimulation indices (SI) 20–40). We further synthesized a series of peptides based on 21OH with amino acid sequences with propensity to bind to major histocompatibility complex class II molecules. Only a few peptides could trigger lymphocytes of 21OH-primed mice to proliferate. One of these, 21OH (342–361), stimulated effectively 21OH-primed lymph node cells of SJL mice (SI = 4–8) and also, although to a lesser extent, of BALB/c mice (SI = 2.5). When SJL mice were immunized with 21OH (342–361), the immunizing peptide as well as peptide 21OH (346–361) triggered a significant proliferative response (SI = 24). A peptide from another part of 21OH, namely 21OH (191–202), did not stimulate the 21OH (342–361)-primed cells. Moreover, stimulation of lymph node cells of mice immunized with 21OH (342–361) with 21OH resulted in a significant proliferative response. We conclude that 21OH (342–361) is an immunodominant determinant for T cells in SJL and probably BALB/c mice. 21OH (342–361) corresponds to the substrate binding site of the enzyme. The p342–361 region may be involved in the pathogenesis of autoimmune adrenal failure in humans.
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Introduction
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PRIMARY ADRENOCORTICAL insufficiency (Addison’s disease), is most often caused by autoimmune tissue destruction involving T and B lymphocytes (1), at least in developed countries. The main autoantigen is 21-hydroxylase (21OH), one of the enzymes participating in cortisol and aldosterone biosynthesis (2, 3). Cytotoxic T lymphocytes are thought to be the most important effector cells in mediating the autoimmune tissue destruction (4, 5), because autoantibodies have not been shown to be directly involved in the pathogenesis (6). However, little is known about the T cell-mediated destruction of the adrenal cortex. Nerup et al. showed that patients with Addison’s disease had reactive T cells when exposed to fetal adrenal extracts (7) or to a mitochondrial fraction from adrenals (8). However, the mitochondrial fraction was not able to induce blast transformation of lymphocytes (8). Freeman and Weetman (4) in contrast, were able to demonstrate T cell proliferation when cells were stimulated with adrenal proteins fractionated according to molecular weight. However, individual antigens were not identified. Moreover, an increased expression of circulating Ia-positive T cells among patients with recent onset of Addison’s disease compared with controls have been reported (9). Recently, patients with autoimmune polyendocrine syndrome type II (Addison’s disease being one of the components), but not individuals with isolated Addison’s disease, were found to have CD4+CD25+ regulatory T cells with defective suppressive capacity (10), pointing at a role of T cells in the pathogenesis of autoimmune adrenal failure.
We have approached this problem by investigating the possibility that 21OH, the main B cell autoantigen, also contains the immunological determinants for autoreactive T cells in autoimmune Addison’s disease. The rationale behind this assumption is the information available on related diseases, in which B and T cells were shown to react against the same protein, albeit at different epitopes. Thus, in myasthenia gravis, the autoantibodies are directed against the acetylcholine receptor (AChR), and T cells of both patients (11) and mouse strains that are susceptible to the induction of experimental autoimmune myasthenia gravis (12) recognize defined epitopes on the 
-subunit of the AChR. In the case of autoimmune thyroid disease, both thyroid peroxidase and thyroglobulin are the common denominators for B and T cell autoreactivity (13).
To find possible T cell epitopes in 21OH, we expressed 21OH in the baculovirus insect cell expression system to obtain a pure preparation of recombinant 21OH (r21OH) (14), and designed and synthesized a series of peptides based on 21OH. Immunization of BALB/c and SJL mouse strains with either r21OH or peptides derived from 21OH followed by in vitro cross-stimulation with either the r21OH or the peptides enabled us to identify an immunodominant T cell epitope in 21OH.
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Materials and Methods
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Mice
Female mice of the inbred strains SJL (The Jackson Laboratory, Bar Harbor, ME) and BALB/c (Olac, Bichester, UK) were used at the ages between 8–12 wk. Animal experiments were conducted in accordance with accepted standards of humane animal care. The study was approved by the Animal Care and Use Committee of The Weizmann Institute of Science.
Expression and purification of his-21OH
Full-length human 21OH (a kind gift from Prof. Holger Luthman, Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden), fused to a N-terminal 6x histidine affinity tag, was expressed and purified in insect cells by using the baculovirus expression system bac-to-bac (Life Technologies, Inc., Gaithersburg, MD) as described in detail by Bøe et al. (14). SDS-PAGE and Western blot were performed as described (14). The purified protein preparation contained one dominating band on SDS-PAGE (Fig. 1A
), identified as 21OH by Western blot analysis using serum from a patient with autoimmune Addison’s disease with anti-21OH antibodies. (Fig. 1B).
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FIG. 1. SDS-PAGE (A) and Western blot analysis (B) of the purified 21OH preparation. For SDS-PAGE analysis (A), 10% polyacrylamide gels were used. A, Lane 1, SeeBlue prestained protein standard; lane 2, whole cell lysate of baculovirus-infected Sf-9 cells (15 µg protein); lane 3, extract containing the insoluble protein fraction of baculovirus-infected Sf-9 cells (10 µg protein); lane 4, purified and concentrated 21OH (5 µg protein). Proteins in the gel were visualized with Coomassie R-250 by conventional procedures. B, Western blot analysis was used to confirm the true identity of the purified protein as 21OH. B, Lane 1, SeeBlue prestained protein standard; lane 2, purified 21OH probed with a monoclonal antibody toward the his-tag; lane 3, purified 21OH probed with Addison’s disease patient serum containing antibodies against 21OH; lane 4, purified 21OH probed with serum from a healthy blood donor. Secondary antibodies were goat antimouse IgG (B, lane 2) and goat antihuman IgG (B, lanes 3 and 4). Secondary antibodies were conjugated with alkaline phosphatase, and bands were visualized by adding 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chromogenic substrate. The protein contents in lanes 2–4 were approximately 2 µg.
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Synthetic peptides
Using the database (http://www.uni-tuebingen.de/uni/kxi/) (15) and information about binding motives to major histocompatibility complex (MHC) II (16), peptides of human 21OH were synthesized with an automated synthesizer (model AMS 422; Abimed Analyses-Technik, Langenfeld, Germany) (17) (Table 1). Crude peptides were purified to homogeneity by semipreparative HPLC on a Lichrosorb RP-8 column (7 µM, 250 x 10 mm; Merck, Darmstadt, Germany) by using a linear gradient established between 0.1% trifluoroacetic acid in double-distilled water and 75% acetonitrile in double-distilled water containing 0.1% trifluoroacetic acid. Purity of the peptides was confirmed by analytical HPLC (RP-18, 125 x 4 mm; Merck) using the above gradient and by amino acid analysis, after exhaustive acid (6 M HCl) hydrolysis. The sequences of the different peptides are given in Table 1.
Immunizations
For priming of lymph node cells, mice were injected intradermally into the hindfoot pads with 2.5–5 µg of r21OH or 20 µg peptide (if not stated otherwise) in complete Freund’s adjuvant (Difco Laboratories, Sparks, MD; 100 µl total volume) and lymph node cells were harvested 10–12 d later. Three to five animals were used for each experiment, and each experiment was repeated at least twice.
Proliferative responses of lymph node cells
Popliteal lymph node cells (0.5 x 106) obtained from immunized mice were cultured in enriched RPMI 1640 medium (18) supplemented with 1% normal mouse serum, in the presence of various concentrations of antigen, for 96 h. Then 0.5 µCi of [3H]thymidine was added, and 16 h later, plates were harvested onto filter paper and radioactivity was counted. In some experiments monoclonal antimouse CD8a (clone 53-6.7, isotype rat IgG2a) and CD4 (clone GK 1.5, isotype rat IgG2b) antibodies were used (Ebioscience, San Diego, CA).
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Results
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Immunization with recombinant 21OH
BALB/c and SJL mice were immunized with 2.5–5 µg full-length r21OH. Lymph node cells harvested after 9–11 d were stimulated with different concentrations of r21OH. Figure 2A shows a representative experiment with SJL mice. A clear concentration-dependent stimulatory response (maximal stimulation index (SI) between 25–30) was seen after immunization with both 2.5 and 5 µg r21OH. The stimulation was evident at a quite low concentration of 0.1 µg r21OH per well. A similar result was obtained with BALB/c mice using 5 µg of r21OH for immunization (Fig. 2B). Immunization with ovalbumin followed by stimulation with r21OH did not lead to T cell proliferation, confirming that the stimulation by 21OH is a specific response to the immunizing antigen r21OH (Fig. 2C).
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FIG. 2. The proliferation responses of lymph node cells of mice immunized with r21OH or ovalbumin. Lymph node cells of the immunized mice were removed 10 d after immunization and were stimulated in vitro. A, BALB/c mice immunized with r21OH (2.5 µg, black columns; 5 µg, gray columns). Lymph node cells were stimulated with different concentrations of 21OH. B, SJL mice were immunized with r21OH (5 µg/mouse). Lymph node cells were stimulated with different concentrations of 21OH. C, BALB/c mice immunized with ovalbumin (20 µg/mouse). Lymph node cells were stimulated with different concentrations of either ovalbumin (open columns) or 21OH (gray columns). Results are expressed as counts per minute of triplicates ± SD.
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Identification of immunodominant peptides of recombinant 21OH
In an attempt to identify immunodominant T cell epitopes in the r21OH molecule, a number of peptides were synthesized and tested to find out whether any of them acted as T cell epitopes. Mice were immunized with r21OH and subsequently stimulated with r21OH (as a positive control) and the various 21OH-derived peptides. Table 2 summarizes the SI observed for all peptides used. A SI above 2 was considered positive. As can be seen, some of the peptides gave a positive proliferative response, some stimulated better SJL cells, others stimulated those of BALB/c mice. However, the best proliferative responses were observed with 21OH (342–361). This peptide triggered a SI of 8 and 2.44 in lymph nodes of SJL and of BALB/c mice, respectively (Table 2). Figure 3 shows the proliferation responses observed with lymph node cells of SJL mice that were immunized with r21OH and stimulated by various peptides. 21OH (342–361) triggered a proliferative response with a maximal SI of 8 at 20 µg in the culture medium, whereas the peptides truncated in either end, i.e. 21OH (342–357) and 21OH (346–361), did not stimulate prominent proliferative responses. Based on these results, further studies were performed using the peptide 21OH (342–361).
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FIG. 3. Proliferative responses of lymph node cells of mice immunized with 21OH and stimulated with various 21OH-derived peptides. Lymph node cells of the immunized mice were removed 10 d after immunization and stimulated in vitro with various concentrations of 21OH-derived peptides. 21OH (342–361), black columns; 21OH (346–361), light gray columns; 21OH (342–357), white columns; 21OH (346–361) and 21OH (342–357) incubated together, dark gray columns. Results are expressed as counts per minute of triplicates ± SD.
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When SJL mice were immunized with peptide 21OH (342–361), both the immunizing peptide and the peptide lacking four amino acids at the N terminal [21OH (346–361)], triggered prominent proliferative responses (Fig. 4). The peptide 21OH (342–357), lacking four amino acids at the C-terminal stimulated a significant but somewhat weaker proliferation (Fig. 4). A peptide from another part of the protein, 21OH (191–202), was ineffective in triggering lymph node cells of mice immunized with either r21OH (Table 2
) or 21OH (342–361) (Fig. 4). 21OH (191–202) in itself was immunogenic, because immunization of mice with this peptide followed by in vitro stimulation elicited a clear proliferative response [SI of 5.4 at 0.1 µg 21OH (191–202)]. When SJL mice were immunized with 21OH (342–361), r21OH stimulated their lymph node cells to proliferate efficiently with a SI of 7.0.
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FIG. 4. Proliferative responses of lymph node cells of mice immunized with peptide 21OH (342–361). SJL mice were immunized (20 µg/mouse) with peptide 21OH (342–361). Ten days later, their lymph node cells were removed and stimulated in vitro with different concentrations of the 21OH-derived peptides. 21OH (342–361), black columns; 21OH (346–361), light gray columns; 21OH (342–357), white columns; 21OH (191–202), dark gray columns. Results are expressed as counts per minute of triplicates ± SD.
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To elucidate whether the proliferative response was mediated by CD4- or CD8-positive cells, SJL mice were immunized with 5 µg 21OH as described above. Isolated lymph node cells were stimulated by r21OH or 21OH (342–361), alone or in the presence of antibodies against either CD4 or CD8. Figure 5 reveals that anti-CD4 inhibited the proliferative response to both r21OH and 21OH (342–361). Anti-CD8 had only negligible effects on the proliferative responses.
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FIG. 5. The effect of anti-CD4 or anti-CD8 monoclonal antibodies on the proliferative responses of lymph node cells of mice immunized with r21OH. SJL mice were immunized with r21OH (5 µg/mouse). Lymph node cells were removed 10 d later and stimulated in vitro with 0.5 µg/well r21OH (gray bars) and 10 µg/well 21OH (342–361) (black bars) alone or in the presence of blocking monoclonal antibodies (50 ng/well) to the CD4 or CD8 coreceptor. Results are expressed as counts per minute of triplicates ± SD.
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Discussion
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The main findings of the present study were that 21OH triggered significant T cell proliferative responses in lymph node cells of immunized mice and that the 21OH-derived peptide 21OH (342–361) is an immunodominant T cell epitope in SJL and possibly BALB/c mice.
The pathogenesis of autoimmune Addison’s disease is still largely unknown. Autoantibodies against 21OH are markers of an ongoing autoimmune process, and assays of these autoantibodies are in clinical use to determine the cause of primary adrenal failure. There is no evidence as yet to support a direct pathogenic role in vivo for autoantibodies (6). T cells are considered to be responsible for the actual destruction of the adrenal cortex. However, very little is known about this process. In particular, the T cell antigens and the triggering processes involved have not been characterized.
Based on the assumption that 21OH is the autoantigen of both B and T cells, we have looked for murine T cell responses to 21OH. We have demonstrated in the present study, that when SJL and BALB/c mice were immunized with 21OH, T cell proliferation could be demonstrated after challenge with 21OH. Furthermore, the 21OH-derived peptide 21OH (342–361) was capable of triggering T cell proliferation of lymph node cells of SJL mice immunized with 21OH. In contrast, when SJL mice were immunized with 21OH (342–361), 21OH induced prominent T cell proliferation, suggesting that 21OH (342–361) is an immunodominant T cell epitope of 21OH. Thus, T cells from SJL mice proliferate in response to 21OH by reacting toward the peptide 21OH (342–361), although it cannot be excluded that other parts of the 21OH molecule are immunogenic as well.
It is noteworthy that, when mice were immunized with peptide 21OH (342–361), their lymph node cells could be triggered to proliferate in vitro, either with the immunizing peptide or with peptides within the same region, namely 21OH (346–361) and (342–357). However, a peptide from another part of the protein, 21OH (191–202), was unable to elicit proliferative responses in 21OH (342–361)-primed lymph node cells. This finding suggests that 21OH (342–361) is important in determining the immunogenicity of the protein, and demonstrates the specificity of the proliferative responses to the 21OH-derived immunodominant peptide.
21OH (342–361) has a propensity to bind to DR4 molecules as judged by information about binding motifs (15, 16). It turns out that DR4, and especially the DR4 subtype DR4*0404 together with DR3, are the two main susceptibility haplotypes for autoimmune Addison’s disease (19). The presentation of peptides from exogenous antigens (e.g. extracellular pathogens) has been regarded as the main function of MHC class II molecules in humans and mice. As 21OH is an intracellular enzyme, the role of MHC class II epitopes in the pathogenesis in autoimmune Addison’s disease may be questionable, at least in the initiation of the pathogenic events. However, many cell types appear to be capable of presenting cytoplasmic antigens via MHC class II molecules, including B cells, dendritic cells, tumor cells, epithelial cells, and thymic epithelial cells (20). Given the ligand diversity and the variety of cell types capable of presenting endogenous antigens via MHC class II, it is becoming more widely accepted that CD4+ T lymphocytes can be activated by cytosolic antigens in this context (20, 21). Intriguingly, an immunodominant epitope of the type 1 diabetes autoantigen glutamate decarboxylase (naturally expressed in the cytoplasm of islet ß-cells in the pancreas), has been shown to be presented in the context of HLA-DR4 molecules on human B cell lines transfected with glutamate decarboxylase encoding cDNA (22). This phenomenon has not yet been demonstrated in adrenocortical cells, but the ability of these cells (especially the zona reticularis) to express MHC class II molecules is well known (23, 24). Indeed, we were able to show that the proliferative response observed after immunization by both 21OH and 21OH (342–361) were restricted to CD4-positive cells (Fig. 5), a strong indication of MHC class II presentation. Thus, autoreactivity toward 21OH may be triggered without hypothesizing an insult to the adrenocortical cells (e.g. by a virus infection) giving rise to leakage of 21OH extracellularly.
It is noteworthy that multiple immunizations with 21OH of several SJL mice during several months resulted only in some mononuclear infiltration of the adrenal cortex. However, no signs of adrenal failure could be observed (results not shown). Sera were also collected after the latter immunizations to evaluate any humoral responses to 21OH. Using a sensitive RIA (with protein G) (19), we could not detect significant levels of anti-21OH, but some reactivity was observed when Western blots were performed (results not shown). These results may suggest that the conformational folding pattern of the recombinant 21OH was different in the two assays, and that antibodies of mice to 21OH were evoked mainly toward linear epitopes. This might also indicate that the protein preparation used for immunization consisted of partially denatured 21OH. Ma and co-workers (25) have presented evidence that changes in native antigen structure may disrupt presentation of immunodominant epitopes. However, the peptide 21OH (342–361) was predicted in silico (15) to represent a naturally processed and cleaved MHC class II ligand from the native structure of 21OH. Therefore, we are confident that 21OH (342–361) does in fact represent an immunodominant epitope in the experimental context presented here.
The immunodominant peptide 21OH (342–361) is part of the steroid binding site of 21OH, which is in a relatively conserved part of the molecule (26). In this particular region there is high sequence homology, both between 21OH from different species (27) and between the different hydroxylases participating in steroid biosynthesis, especially 21OH, 17-hydroxylase, and 11ß-hydroxylase (26). The substrate binding region is highly conserved and only one amino acid differs between human and mouse; the asparagine at position 346 in human 21OH is exchanged for a methionine at the equivalent position in murine 21OH (26). One may speculate that the peptide 21OH (342–361) is immunodominant also in patients with autoimmune Addison’s disease. Preliminary data indicate that this can be the case in some patients with autoimmune Addison’s disease (data not shown), but since the number of autoreactive T cell clones in the peripheral circulation is probably very low, this may be difficult to prove. Evidence in support for such a concept is the demonstration that two peptides representing sequences of the human AChR -subunit, namely p195–212 and p259–271, were able to stimulate peripheral blood lymphocytes of patients with the autoimmune disease myasthenia gravis, and were determined to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively (11, 12).
In conclusion, we have identified an immunodominant peptide in 21OH in mice. The peptide is located in the steroid-binding site of the protein, an area that is well conserved between species and between different P450 enzymes participating in steroid biosynthesis. It remains to be seen whether reactivity toward this and other parts of 21OH may be pathogenic in the autoimmune destruction of human adrenal cortices in autoimmune Addison’s disease.
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Footnotes
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The study was supported by The Weston Visiting Scholarship Program at The Weizmann Institute of Science, Innovest, Haukeland University Hospital, and The Research Council of Norway.
Disclosure Statement: E.H., E.B., G.B., M.F., M.D., and E.M. have nothing to declare.
First Published Online February 23, 2006
Abbreviations: AChR, Acetylcholine receptor; 21OH, 21-hydroxylase; r21OH, recombinant 21OH; SI, stimulation index.
Received January 5, 2006.
Accepted for publication February 13, 2006.
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References
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Abstract
Introduction
