Inflammatory disease in HLA-B27 transgenic rats

Summary: A spontaneous inflammatory disease in rats transgenic for HLAB27 resembles the B27-associated human spondyloarthropathies, Colitis and arthritis, the two most important features, require T cells, gut bacteria, and high expression of B27 in bone marrow-derived cells, Control rats with HLA-B7 remain healthy. Most rats with HLA-Cw6 (associated with psoriasis vulgaris) remain healthy; a minority develop mild and transient disease. Rats with a mutant B27 with a Cys67←Ser substitution resemble wild-type B27 transgenics, but with a lower prevalence of arthritis. A similar phenotype is seen in B2 7 rats co-expressing a viral peptide that binds B27. Disease-prone LEW but not F344 B27 rats develop high serum IgA levels concurrent with disease progression. Colitis is associated with high interferon-y, arthritis with high interleukin-6. Disease is similar in B27 LEW, F344, and PVG rats, but the DA background is protective. Conclusions: The spondyloarthropathy-like disease in rats is specific for HLA-B27 but does not require Cys67. Arthritis but not colitis is particularly sensitive to B27 peptide-binding specificity. Genetic background exerts a strong influence, but some phenotypic differences exist between permissive strains that do not influence disease susceptibility The data favor a role for B27 peptide presentation in arthritis, but other mechanisms to explain the role of B27 have not been excluded.     

Characterization of psoriasiform and alopecic skin lesions in HLA-B27 transgenic rats.

We have previously reported a multisystem inflammatory disease in transgenic rat lines with high expression of HLA-B*2705 and human beta 2 microglobulin. Skin disease in these rats includes two predominant lesions: 1) marked psoriasiform dermatitis of the tail and digits; and 2) progressive alopecia of face, neck, trunk, and extremities. Here we present the results of a systematic survey of these lesions. Tail and digit skin showed psoriasiform hyperplasia of the epidermis associated with parakeratosis, with marked dermal and epidermal inflammation. The alopecic skin showed perifollicular and follicular mononuclear infiltration and increased numbers of atrophic follicles. Immunohistochemical analysis revealed that B27 expression was prominent on keratinocytes in hyperplastic epidermis where lymphocytic infiltrates were prominent, but was absent in the absence of inflammation. In alopecic lesions, B27 was strongly expressed on follicular epithelium and dermal hair papillae associated with mononuclear infiltrates. T cells, both CD8 and CD4, were most prominent in inflammatory lesions and rat MHC-II expression on keratinocytes, and follicular epithelium was dramatically increased. This study suggests that T cell-mediated immune mechanisms participate in development of cutaneous lesions in HLA-B27 transgenic rats.     

Experimental autoimmune uveitis in HLA-B27 transgenic mice

The major histocompatibility complex (MHC) gene, HLA-B27 is strongly associated with autoimmune uveitis and spondyloarthropathies in humans. Experimental mouse models of autoimmune uveitis involve systemic immunization with the retinal autoantigen interphotoreceptor retinoid binding protein (IRBP). To assess possible roles of HLA-B27 in autoimmune uveitis, as well as to investigate a possible new animal model of human uveitis, inbred strains of C57BL/6 and C57BL/6 possessing the human HLA-B27 or HLA-A2 transgene were immunized with IRBP emulsified in complete Freund's adjuvant (CFA). Dilated eye examinations were performed to assess the timing and clinical course of any ensuing uveitis. Mice were sacrificed 3 to 4 weeks postinjection and the eyes submitted for histopathologic analysis.

CFA alone did not produce any clinical uveitis. Fifty percent of eyes from the background C57BL/6 strain developed uveitis as early as 10 days postinjection. Of the eyes demonstrating uveitis, an average clinical score of 2.5 was present. Pathologically, a moderate scleritis and anterior uveitis was present. Fifty percent of A2 transgenic eyes developed uveitis as early as 14 days postinjection with an average clinical score of 2.0. Pathologically, a mild vitriitis was present. Uveitis developed in only 20% of B27 transgenic mice and reached a peak on day 28. The average EAU score in diseased animals was 4.5. A dense retinitis and panuveitis was associated with severe vitritis. We conclude that the presence of the B27 gene is associated with a decreased incidence and slower rate of onset of EAU following immunization with IRBP; however, EAU may be more severe in the HLA-B27 expressing animals who do develop disease.     

Strong association of HLA-B27 heavy chain with beta 2-microglobulin

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with β₂-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with β₂-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/β₂-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.     

Reduced ratio of protective versus proinflammatory cytokine responses to commensal bacteria in HLA-B27 transgenic rats

Germ-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T- or B- cell-depleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-gamma, IL-12, TNF, IL-10 and TGF-beta production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-gamma, IL-12 and TNF than did non-TG MLN cells. In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-beta. T cell depletion abolished IFN-gamma and decreased IL-12 production, but did not affect IL-10 and TGF-beta production. Conversely, neither IL-10 nor TGF-beta was produced in cultures of B cell-depleted MLN. In addition, CD4(+) T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-gamma when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-beta, but not IFN-gamma, IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-gamma-producing CD4(+) T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation.     

Adoptive transfer of nontransgenic mesenteric lymph node cells induces colitis in athymic HLA-B27 transgenic nude rats

HLA-B27 transgenic (TG) rats develop spontaneous colitis when colonized with intestinal bacteria, whereas athymic nude (rnu/rnu) HLA-B27 TG rats remain disease free. The present study was designed to determine whether or not HLA-B27 expression on T cells is required for development of colitis after transfer of mesenteric lymph node (MLN) cells into rnu/rnu HLA-B27 recipients. Athymic nontransgenic (non-TG) and HLA-B27 TG recipients received MLN cells from either TG or non-TG rnu/+ heterozygous donor rats that contain T cells. HLA-B27 TG rnu/rnu recipients receiving either non-TG or TG MLN cells developed severe colitis and had higher caecal MPO and IL-1beta levels, and their MLN cells produced more IFN-gamma and less IL-10 after in vitro stimulation with caecal bacterial lysate compared to rnu/rnu non-TG recipients that remained disease free after receiving either TG or non-TG cells. Interestingly, proliferating donor TG T cells were detectable one week after adoptive transfer into rnu/rnu TG recipients but not after transfer into non-TG recipients. T cells from either non-TG or TG donors induce colitis in rnu/rnu TG but not in non-TG rats, suggesting that activation of effector T cells by other cell types that express HLA-B27 is pivotal for the pathogenesis of colitis in this model.     

Susceptibility of HLA-B27 transgenic mice to Yersinia enterocolitica infection

The majority of patients with reactive arthritis have the major histocompatibility complex class I gene HLA-B27. The development of arthritis in these patients often occurs following infection with one of several enteric bacteria, including Yersinia enterocolitica. In this study, transgenic mice expressing HLA-B27 and their negative full sibs were infected intravenously with Yersinia enterocolitica 0:8 WA in an attempt to develop an experimental model of reactive arthritis. To date, no reactive arthritis has been observed; however, a significantly higher incidence of paralysis was observed in the HLA-B27⁺ transgenic mice. Injection of 10⁵ organisms induced hind limb paralysis in 8 out of 30 of the HLA-B27 transgenic mice (27%) and in only 1 of the 24 negative siblings (4%). Paralysis occurred in 14 out of 30 HLA-B27⁺ mice (47%) at a dose of 10⁴ organisms. Only 2 of the 25 negative siblings (8%) were affected at this dose. Paraspinal abscesses were found in all of the paralyzed animals. At the 10⁴ dose most of the HLA-B27⁺ mice (70%) succumbled to the disease within 4 weeks, while the mortality in their B27⁻ full sibs was less than 10%. Thus, HLA-B27 transgenic mice have higher mortality and morbidity from infection with Y. enterocolitica 0:8 WA than corresponding HLA-B27⁻ littermates.     

Luminal bacterial antigen-specific CD4+ T-cell responses in HLA-B27 transgenic rats with chronic colitis are mediated by both major histocompatibility class II and HLA-B27 molecules

Rats transgenic (TG) for the human major histocompatibility complex (MHC) class I HLA-B27 and beta2-microglobulin genes develop chronic colitis under specific pathogen-free (SPF) but not sterile (germ-free, GF) conditions. We investigated the role of antigen-presenting molecules involved in generating immune responses by CD4+ mesenteric lymph node (MLN) cells from colitic HLA-B27 TG rats to commensal enteric micro-organisms. All TG MLN cells expressed HLA-B27. A higher level of MHC class II was expressed on cells from TG rats, both SPF and GF, compared to non-TG littermates. In contrast, rat MHC class I expression was lower on TG than non-TG cells. Both TG and non-TG antigen presenting cells (APC) pulsed with caecal bacterial antigens induced a marked interferon-gamma (IFN-gamma) response in TG CD4+ T lymphocytes but failed to stimulate non-TG cells. Blocking MHC class II on both TG and non-TG APC dramatically inhibited their ability to induce TG CD4+ T cells to produce IFN-gamma. Blocking HLA-B27 on TG APC similarly inhibited IFN-gamma responses. When the antibodies against MHC class II and HLA-B27 were combined, no APC-dependent IFN-gamma response was detected. These data implicate both native rat MHC class II and TG HLA-B27 in CD4+ MLN T-cell IFN-gamma responses to commensal enteric microflora in this colitis model.     

Dysregulated luminal bacterial antigen-specific T-cell responses and antigen-presenting cell function in HLA-B27 transgenic rats with chronic colitis

HLA-B27/beta2 microglobulin transgenic (TG) rats spontaneously develop T-cell-mediated colitis when colonized with normal commensal bacteria, but remain disease-free under germ-free conditions. We investigated regulation of in vitro T-cell responses to enteric bacterial components. Bacterial lysates prepared from the caecal contents of specific pathogen-free (SPF) rats stimulated interferon-gamma (IFN-gamma) production by TG but not non-TG mesenteric lymph node (MLN) cells. In contrast, essentially equivalent amounts of interleukin-10 (IL-10) were produced by TG and non-TG cells. However, when cells from MLNs of non-TG rats were cocultured with TG MLN cells, no suppression of IFN-gamma production was noted. Both non-TG and TG antigen-presenting cells (APC) pulsed with caecal bacterial lysate were able to induce IFN-gamma production by TG CD4+ cells, although non-TG APC were more efficient than TG APC. Interestingly, the addition of exogenous IL-10 inhibited non-TG APC but not TG APC stimulation of IFN-gamma production by cocultured TG CD4+ lymphocytes. Conversely, in the presence of exogenous IFN-gamma, production of IL-10 was significantly lower in the supernatants of TG compared to non-TG APC cultures. We conclude that commensal luminal bacterial components induce exaggerated in vitro IFN-gamma responses in HLA-B27 TG T cells, which may in turn inhibit the production of regulatory molecules, such as IL-10. Alterations in the production of IFN-gamma, and in responses to this cytokine, as well as possible resistance of TG cells to suppressive regulation could together contribute to the development of chronic colitis in TG rats.     

Systemic amyloidosis of beta 2 microglobulin type.

A patient receiving haemodialysis for 15 years developed systemic amyloidosis of beta 2 microglobulin type. Noticeable deposits of amyloid were present in the myocardium, intervertebral discs, joint cartilages and tendons. Less conspicuous amounts were present in blood vessel walls in the lungs, liver, adrenal glands and brain, and within the stroma of the prostate, testis and kidney, often with foci of calcification.     

Cancer of the breast and beta 2 microglobulin

In carcinoma of the breast the disease stage at diagnosis determines therapy and is closely related to prognosis. To date no single biochemical marker of disseminated breast cancer has been described although beta 2 microglobulin values in the serum have been suggested as a discriminant between localized and systemic disease. Using a new method of beta 2 microglobulin estimation, we carried out a prospective study of levels in 53 patients with breast cancer who were studied repetitively over a 2 yr period. No relationship could be determined between beta 2 microglobulin values and the stage of the disease. Moreover beta 2 microglobulin levels, even when elevated, did not predict early metastasis. Repeated beta 2 microglobulin estimations during treatment of metastatic disease had limited usefulness in that patients with responsive disease usually showed a fall in beta 2 microglobulin, whereas there was generally no change in non-responsive patients. These changes were, however, often within the normal range and seemed to offer a marginal improvement in assessment.     

Rheumatoid factors bind beta 2 microglobulin. Detection of beta 2 microglobulin-complexes in rheumatoid arthritis.

beta 2 Microglobulin binding rheumatoid factors and occurrence of complexed form of beta 2 microglobulin were found in rheumatoid arthritis (RA) by radioimmunological methods. 1. As related to the appropriate values of control sera and osteoarthritic synovial fluids, one of the six sera and five of the six synovial fluids of RA patients exhibited increased binding activity for 125I-beta 2 microglobulin, 2. Rheumatoid factors (RF) purified by means of immunoadsorbent bound beta 2 microglobulin; 3. Both 19S and 7S components of sera, synovial fluids and purified RF samples had beta 2 microglobulin binding activity, which was inhibited by an excess of unlabeled beta 2 microglobulin; 4. Complexed (3% PEG 6000 insoluble) beta 2 microglobulin was found in three of fourteen sera and in twelve of fourteen synovial fluids of RA patients. Level of complexed beta 2 microglobulin was independent of its total concentration in the original samples; 5. In the fraction of synovial fluids enriched immune complex the amount of beta 2 microglobulin was in positive correlation with quantity of RF. Presumable immunological and pathological significance of "anti-beta 2 microglobulin"-like cross-reactive RF antibodies is discussed.     

Peptide binding alpha1alpha2 domain of HLA-B27 contributes to the disease pathogenesis in transgenic mice

Human spondyloarthropathies are strongly associated with a major histocompatibility complex (MHC) class I allele, HLA-B27. HLA-B27 transgenic mice and rats demonstrate many features of these diseases further confirming the role of HLA-B27 in disease. Yet the exact role of this molecule in disease pathogenesis is not clearly understood. We have previously reported spontaneous arthritis and nail disease in HLA-B27 transgenic mice lacking beta2-microglobulin (B27+beta2m(o)). These observations along with binding studies of B27 derived peptides to HLA-B27 molecule itself led to two hypotheses: (i) HLA-B27 derived peptide as a source of autoantigen; and (ii) HLA-B27 functions as an antigen presenting molecule. In this report, we confirm spontaneous disease in transgenic mice expressing a hybrid B27 molecule with alpha1alpha2 domain of B27 and alpha3 domain of mouse H-2Kd. These mice developed spontaneous arthritis and nail disease when transferred from specific pathogen free barrier facility to the conventional area. Other control mice with MHC class I transgene (e.g., HLA-B7, HLA-Cw3, and H2-Dd) did not develop such disease. In a MHC reassembly assay, binding of similar peptides to both wild type and hybrid B27 molecules was observed. In addition, the hybrid B27 molecule lacks at least one of the 3 proposed peptides from the third hypervariable (HV3) region of HLA-B27. These data strongly suggest that HLA-B27 molecule is an antigen presenting molecule rather than a peptide donor in the disease pathogenesis.     

Immunoturbidimetric assay of beta 2 microglobulin using latex particles in microplates

A latex particle immunoturbidimetric assay in microplates has been developed for the quantitation of beta 2 microglobulin. The assay which involves three pipetting steps and one incubation, has a clinically useful range of 0.4-16 mg/l. A comparison of the method with a commercial radioimmunoassay in the analysis of 125 sera revealed a high degree of correlation. Although the assay was performed manually, it showed considerable potential for full automation.     

Monoclonal antibody (B27M2) subdividing HLA-B27

Because HLA-B27 shows strong association with ankylosing spondylitis, it was of interest to produce murine monoclonal antibodies to study this antigen in detail. The first such anti-B27 antibody, anti-B27M1, reacted with 100% of B27 + cells and cross-reacted with homozygous B7 cells. In the present report a second monoclonal antibody, anti-B27M2, is described which subdivides HLA-B27 into two variants. Among healthy B27⁺ individuals, 87% were B27M1⁺ and B27M2⁺, and 13% were B27M1⁺ but B27M2⁺ by standard lymphocytotoxicity assays. The specificity of B27M2 antibody for the HLA-B27 molecule was confirmed with blocking studies using F(ab′)₂ fragments of HLA alloantibodies. Both the B27M2⁺ and B27M2 variants of HLA-B27 bred true in family studies. Unlike B27M1, B27M2 antibody did not react with B7 but did react with the rare Bw47 allele.For antibody binding studies Epstein-Barr virus-transformed B lymphoblastoid cell lines were derived from normal donors KCA (by lymphocytotoxicity shown to be B27⁺, B27M2⁺. VC (B27⁺, B27M1⁺, B27M2^?), and WH (B27^?, B27M1^?, B27M2^?). Each cell line bound equivalent amounts of W6/32 (monoclonal anti-HLA-ABC): KCA and VC bound similar amounts of anti-B27M1, but only KCA bound substantial anti-B27M2 antibody. These data are consistent with a model in which all B27 antigens possess a B27M1 epitope: whereas most, but not all, possess an additional and distinct epitope, B27M2. Although the relation of these genetic variants to disease susceptibility remains to be determined, the availability of epitope-specific monoclonal antibodies should help to refine our understanding of the structure and function of HLA molecules.     

Triggering of lymphocytes by antibodies against beta2 microglobulin.

beta2 microglobulin (beta2m), structurally related to "domains" of immunoglobulin molecules, is associated with products of the major histocompatibility system on cell surfaces. Heteroantibodies against beta2m are mitogenic to a specific subpopulation of human and mouse B lymphocytes. This subpopulation is present in human blood which makes the antibody convenient and clinically useful as a functional marker for peripheral B lymphocytes. Absorption and elution experiments, as well as tests showing mitogenic activity of Fab monomers of anti-beta2m indicate that the interaction of the binding site of the antibody and relevant cell surface structures, probably beta2m itself, is directly responsible for lymphocyte activation. The relevance of these findings for cell receptors involved in lymphocyte activation is discussed.     

Spontaneous inflammatory disease in HLA-B27 transgenic mice does not require transporter of antigenic peptides

HLA-B27 is strongly linked with a group of human diseases called spondyloarthropathies. Even though HLA-B27 as an MHC class I molecule would be expected to present endogenously processed peptides such as cytosolic or viral proteins, many of the B27-linked diseases begin after an infection with an enterobacteria, an exogenous antigen. In our previous studies, we have described development of spontaneous inflammatory disease in HLA-B27 transgenic mice expressing beta(2)m free heavy chains on the cell surface. In order to address the role of endogenous versus exogenous antigens and a role for Tap genes in the development of spontaneous diseases, mice lacking Tap-1 (knockout) were mated to HLA-B27/human beta(2)m transgenic mice. B27(+)/human beta(2)m(+) double-transgenic mice (without mouse beta(2)m) lacking the Tap-1 gene developed spontaneous inflammatory disease similar to wild-type Tap-1 gene-expressing counterparts. Our data demonstrate that peptide transporters (Tap) were not involved in the development of spontaneous inflammatory disease in B27(+)/human beta(2)m transgenic animals.