E. Hermann

Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD)

Hyporesponsiveness to a universe of bacterial and dietary antigens from the gut lumen is a hallmark of the intestinal immune system. Since hyperresponsiveness against these antigens might be associated with inflammation, we studied the immune response to the indigenous intestinal microflora in peripheral blood, inflamed and non‐inflamed human intestine. Lamina propria monocuclear cells (LPMC) isolated from inflamed intestine but not peripheral blood mononuclear cells (PBMC) of IBD patients with active inflammatory disease strongly proliferated after co‐culture with sonicates of bacteria from autologous intestine (BsA), Proliferation was inhibitable by anti‐MHC class II MoAb, suggesting that it was driven by antigen, LPMC from adjacent non‐inflamed intestinal areas of the same IBD patients and PBMC or LPMC isolated from non‐inflamed intestine of controls and patients with IBD in remission, in contrast, did not proliferate, PBMC or LPMC which had been tolerant to bacteria from autologous intestine, however, strongly proliferated after co‐culture with bacterial sonicates from heterologous intestine (BsH). This proliferation was associated with an expansion of CD8+ T cells, increased expression of activation markers on both CD4+ and CD8+ lymphocyte subsets, and production of IL‐12, interferon‐gamma (IFN‐γ), and IL‐10 protein. These results show that tolerance selectively exists to intestinal flora from autologous but not heterologous intestine, and that tolerance is broken in intestinal inflammation. This may be an important mechanism for the perpetuation of chronic IBD.

Reactivity of infiltrating T lymphocytes with microbial antigens in Crohn's disease

Intestinal T lymphocytes are normally unresponsive to microbial and recall antigens in vitro, whereas the same antigens induce strong immune responses in peripheral-blood-derived T cells. We obtained T lymphocytes from peripheral blood and from the non-inflamed and inflamed intestinal mucosa of 6 patients (3 male, 3 female; mean age 33 years) with Crohns disease. The T cells were stimulated in vitro with a range of microbial antigens. Whereas T cells from normal mucosa were unresponsive, those from inflamed mucosa had a proliferative response comparable to that of the peripheral-blood-derived T cells. These findings suggest that physiologic unresponsiveness to luminal antigens is abrogated in the inflammatory lesions of Crohns disease patients. Infiltrating T lymphocytes may therefore mediate chronic inflammation on encountering the many antigens present in the intestine.

Stimulation of synovial fluid mononuclear cells with the human 65-kD heat shock protein or with live enterobacteria leads to preferential expansion of TCR-γδ+ lymphocytes

T lymphocyte responses to heterologous or self 65‐kD heat shock protein (hsp) have been implicated in the pathogenesis of various forms of arthritis. To delineate the relationship of 65‐kD hsp to different synovial fluid (SF) T cell subsets, we stimulated synovial fluid (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with different inflammatory rheumatic diseases and from healthy controls with human or mycobacterial 65‐kD hsp, tetanus toxoid (TT), heat‐killed or live Yersinia enterocotitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR‐γδ+ lymphocytes in the 65‐kD hsp‐stimulatcd SF‐derived lines. This expansion of TCR‐γδ+ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR‐γδ+ cells was also shown after SFMC stimulation with live, but not with heat‐killed Yersinia or with TT. We conclude that a common mechanism is involved in the selective expansion of TCR‐γδ+ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65‐kD hsp. Out of a panel of TCR‐γδ+ T lymphocyte clones (TLC) derived from a human 65‐kD hsp‐stimulated line, only a minority of TLC proliferated weakly upon restimulation with this antigen in the presence of autologous monocytes, whereas TCR‐αβ+ TLC responded vigorously to the human 65‐kD hsp and in some cases also cross‐recognized the mycobacterial hsp homologue and/or heat‐killed Yersinia. This implies that additional factors or cells may be present in the milieu of SFMC cultures that propagate the expansion of TCR‐γδ+ cells in response to 65‐kD hsp or live bacteria.

Antibodies to cathepsin G in Crohn's disease.

Abstract. Antibodies directed against antigens in human neutrophils have proved to be of great diagnostic value in certain systemic vasculitides. Recent reports have focused the attention on these antigens as targets of antibodies in sera of patients with inflammatory bowel disease. We investigated the sera drawn from 60 patients suffering from biopsy proven Crohns disease and 15 patients with active ulcerative colitis. Using sensitive enzyme‐linked immunosorbent assays with purified antigens and Western blotting the following antibodies could be demonstrated: cathepsin G (cat‐G) antibodies IgG 38.3%, IgM 13.3%, IgA 23.3% and antibodies against human leucocyte elastase (HLE) IgG, IgA, IgM 3.3%. Low but significant correlations could be found for cat‐G antibodies (IgG) and the van HEES index of activity. 73.9% of the cat‐G (IgG) positive patients had colon involvement. In the sera of patients with ulcerative colitis no antibodies to cat‐G or HLE were detectable. Only 8.3% of the patients with Crohns disease had antibodies against proteinase 3 (C‐ANCA). Our data indicate that cat‐G among other myeloid lysosomal enzymes seems to be an important target antigen of antibodies in sera of patients with Crohns disease. Cat‐G antibodies might be helpful to distinguish Crohns disease from ulcerative colitis.

A human renal cancer line as a new antigen source for the detection of antibodies to cytoplasmic and nuclear antigens in sera of patients with Wegener's granulomatosis.

Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA), especially proteinase 3 (C-ANCA), have proved to be a useful clinical tool to support the diagnosis or to monitor disease activity in Wegeners granulomatosis (WG). Till now, human neutrophil granulocytes have represented the major antigen source used to detect antibodies in WG by the immunofluorescence technique (IFT). We have tested serum samples of 164 patients with different connective tissue diseases (50 suffering from clinically active WG) performing IFT on a human renal cancer line (SK-RC11) and have found antibodies against the nuclear and cytoplasmic antigens in 39 patients. C-ANCA+ sera displayed a characteristic diffuse cytoplasmic staining pattern. Antibody titers measured with human granulocytes were comparable to titers obtained using culture cells. Antibody binding could be inhibited by preabsorption with an extract of human granulocytes or purified proteinase 3. A protein of 29 kDa MW could be isolated by affinity purification using a SK-RC11 extract and a high-titer C-ANCA+ serum and antigenic identity was further confirmed by IFT using a monoclonal antibody to proteinase 3. Treatment of tumor cells with cytokines (interferon, tumor necrosis factor) led to a time dependent translocation of the antigen into the nucleus and back to the cytoplasm. The antigen was also expressed on the surface of live cells colocalized with MHC II. In addition, 21 WG patients had antibodies to cytoplasmic organelles identified by laser scanning microscopy as secretory vesicles of the Golgi complex, and five had antibodies to nuclear antigens. This is, to the best of our knowledge, the first report of proteinase 3 in human non-leukemic cells. Our data demonstrate, that the repertoire of antigens recognized by antibodies in WG sera is not limited to human neutrophils and monocytes and indicates a possible functional role of the antigenic proteins.

T cells in reactive arthritis

T cells appear to play a major role in the development, maintenance and also resolution of reactive arthritis (ReA). Recent advances in understanding the processes involved in T cell activation now allow us to examine the peripheral blood and synovial fluid T cell responses to given “arthritogenic” microorganisms in terms of antigen specificity, epitope identification, cytokine secretion patterns, HLA restriction and the role of different T cell subsets in ReA. Peripheral blood bulk proliferation and limiting dilution studies provide evidence that the peripheral T cell response against arthritis‐associated gram‐negative bacteria is decreased in patients developing immunological sequelae such as ReA after gastrointestinal infection. Using clonal analysis of synovial fluid CD4+ T cells it has been shown that a polyclonal rather than an oligoclonal response to a variety of bacterial antigens is induced at the site of synovitis and that these CD4+ T cells produce a Thl‐type of cytokine. 65 kD heat shock protein may represent one of the possible linkages of anti‐infectious and autoimmune reactions. Furthermore, a spectrum of killer cells is present in the synovial fluid of patients with ReA. This spectrum of cytotoxic T cells includes antigen‐specific, class I‐restricted αβ‐TCR+CD8+ lymphocytes, antigen‐specific, apparently non‐MHC‐restricted αβ‐TCR+CD8+ lymphocytes and γδ‐TCR+ cells with broad cytolytic activity directed against bacteria‐infected target cells. HLA‐B27‐restricted Yersinia‐ or Salmonella‐specific synovial fluid CD8+ T cells may provide the missing link between genetic disposition (HLA‐B27) and extra‐articular infection with arthritogenic bacteria in these patients.

Salmonella-reactive Synovial Fluid T-cell Clones in a Patient with Post-infectious Salmonella Arthritis

From a patient with reactive arthritis following Salmonella typhimurium enteritis, synovial fluid T-lymphocytes were cloned and expanded in vitro. Seven out of 74 clones showed a marked proliferative response to antigens of heat-killed Salmonella typhimurium with autologous T-cell-depleted peripheral blood mononuclear cells as antigen-presenting cells. The Salmonella-reactive clones were of the CD4+ phenotype, antigen-induced proliferation could be inhibited by a monoclonal antibody to HLA class II. One clone recognized both Salmonella and Campylobacter jejuni antigens in the proliferation assay. The multiclonality of Salmonella-reactive synovial fluid T-cells indicates that the microorganisms have been present, at least transiently, within the affected joint and thus recruited specific T-lymphocytes that might initiate synovitis.

Antibodies to cytoskeletal proteins in patients with Crohn's disease

Abstract. The immunologic basis of inflammatory bowel disease has been the focus of interest of a series of studies on Crohns disease and the process of immune sensitization at the gastrointestinal mucosal level is functionally poorly understood. To date only few contradictory reports concerning the incidence of autoantibodies in patients with this disease exist. The aim of this study was to investigate the sera drawn from 60 patients suffering from biopsy‐proven Crohns disease to evaluate the prevalence of autoantibodies against nuclear antigens and cytoskeletal proteins. Using standard methods, no anti‐nuclear antibodies or antibodies to extractable nuclear antigens could be detected. All sera were also negative for antibodies to double‐stranded DNA, anti‐mitochondrial antibodies, and antibodies to gastric parietal cells. Using sensitive enzyme‐linked immunosorbent assays with purified antigens and Western blotting with cytoskeletal proteins of human intestinal cells, the following antibodies could be demonstrated: cytokeratin 18 autoantibodies (IgG 20.0%; IgM 6.7%; IgA 13.3%), actin antibodies (IgG 36.7%; IgM 48.3%, IgA 26.7%), desmin antibodies (IgG 6.7%; IgM 15.08%; IgA 50%), vimentin antibodies (IgG 3.3%; IgM 16.7%; IgA 10.0%) and tropomyosin antibodies (IgG 3.3%; IgM 3.3%, IgA 5.0%). Statistically significant correlations could be found for levels of cytokeratin 18 antibodies (IgM‐type) and the BEST index of activity, and for levels of desmin antibodies (IgM‐type) and the van HEES index of activity. Highest levels could be measured for actin antibodies (IgG‐type) in patients with isolated disease manifestation in the colon. The mechanism of induction of autoantibodies against cytoskeletal components in Crohns disease still remains obscure. Unmasking of hidden antigens after cell injury during the inflammatory process of disease might lead to sensitization and antibody production. The pattern of antibodies in patients with Crohns disease seems to be different compared with that of connective tissue diseases.

Thymoma and Pure Red Cell Aplasia in a Patient with Systemic Lupus Erythematosus

We present the case of a female patient with a diagnosis of systemic lupus erythematosus (SLE) at the age of 54 years. At the age of 63 years, she suffered from malignant thymoma and 3 years after removal of the thymoma a diagnosis of pure red cell aplasia (PRCA) was established. This is, to our knowledge, the first report of the occurrence of SLE, thymoma and PRCA in the same patient. The case is discussed with regard to the already known associations between these diseases.

Bacteria-specific Cytotoxic CD8+ T Cells: A Missing Link in the Pathogenesis of the HLA-B27-associated Spondylarthropathies

The term seronegative spondylarthropathies is used for an entity of rheumatic syndromes of peripheral joints and the spine (ankylosing spondylitis, reactive arthritis, Reiters syndrome, arthritis in psoriasis and in inflammatory bowel disease) which are strongly associated with the MHC class I molecule HLA-B27. However, the mechanisms whereby HLA-B27 confers disease susceptibility have so far remained unknown. There is strong evidence that gut inflammation and infection with gram-negative bacteria play a role in the induction of B27-associated disease. HLA-B27, like other MHC class I molecules, physiologically binds antigenic peptides in its binding groove and presents them to CD8+ T lymphocytes. Consequently, if the disease association with HLA-B27 arises from its role as a T-cell restriction element, synovial fluid CD8+ rather than CD4+ T cells should play a prominent pathogenetic role and should be detectable within the affected joints. In this paper, recent studies on bacteria-specific cytotoxic T cells and on peptide binding to HLA-B27 are reviewed. Particular emphasis is laid on the role of HLA-B27 restricted synovial CD8+ T cells with specificity for bacterial antigens or autoantigens. These cytotoxic T cells could provide a missing link in the pathogenesis of the spondylarthropathies and could now serve as tools to identify the critical antigenic epitopes of bacterial and self peptides which are involved in disease induction.