Werner-Johannes Mayet

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.

Expression of intercellular adhesion molecule 1 (ICAM-1, CD54) in colonic epithelial cells.

The expression of intercellular adhesion molecule-1 (ICAM-1, CD54) was examined in 16 surgically removed colonic tumours and two colonic carcinoma cell lines. Immunohistochemistry showed a varying percentage of ICAM-1 positive colonic carcinoma cells in 9/16 tissue specimens, while normal colonic tissue (apart from a slight reactivity of endothelial cells) was not stained. The presence of the ICAM-1 molecule on the cell surface and the expression of ICAM-1 mRNA were investigated for two colonic carcinoma cell lines. It was possible to enhance the expression of ICAM-1 considerably by incubating the cells in the presence of inflammatory cytokines in HT-29 and CaCo-2 cells. The responsiveness to either interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha), or interleukin 1 beta (IL-1 beta) treatment was different in each cell line. Interestingly, ICAM-1 is shed by colonic carcinoma cells because soluble sICAM-1 was detected in the cell culture supernatants. In comparison with normal serum samples, the mean value of sICAM-1 in 63 samples of patients with colonic carcinoma and in 20 cases of active inflammatory bowel disease is raised about twofold. It remains to be clarified what part both forms of ICAM-1 play in the course of colonic cancer, ulcerative colitis, and Crohns disease.

Mycophenolate Mofetil Versus Azathioprine in Patients With Chronic Active Ulcerative Colitis: A 12-Month Pilot Study

OBJECTIVE:Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) of unknown etiology frequently requiring long-term therapy for control of symptoms and prevention of relapse. Azathioprine (AZA) has been shown to be effective and safe in the treatment of chronic active UC. However, the alternatives to treatment with AZA are limited. Our aim was to compare the efficacy and safety of treatment with mycophenolate mofetil (MMF)/prednisolone versus standard immunosuppressive treatment with azathioprine (AZA)/prednisolone in patients with chronic active UC.METHODS:The study was designed as an open comparison of MMF versus AZA. Twenty-four patients with active UC (Rachmilewitz score ≥6 points) were randomly assigned to the MMF (20 mg/kg)/prednisolone or AZA (2 mg/kg)/prednisolone group. The initial prednisolone dosage was 50 mg and was tapered according to a standard protocol. Treatment was scheduled for 1 yr.RESULTS:The rates of remission were higher in the AZA/prednisolone group (n = 12) than in the MMF/prednisolone group (n = 12) throughout the study. Remission rates were 92% versus 67% after 4 wk, 92% versus 67% after 3 months, 92% versus 67% after 6 months, 83% versus 78% after 9 months, and 100% versus 88% after 1 yr. The number of patients not requiring steroids was higher in the AZA/prednisolone group than in the MMF/prednisolone group. Moreover, in the AZA/prednisolone group no severe adverse events were recorded, whereas in the MMF/prednisolone group two severe adverse events were observed: one patient discontinued MMF after 6 months because of recurrent upper airway infections, and one patient exhibited a bacterial meningitis after 9 months.CONCLUSIONS:Treatment with AZA/prednisolone appears to be more effective and safe compared to MMF/prednisolone in patients with chronic active UC. MMF might be an alternative treatment for patients with contraindications to AZA. To further evaluate the effects of MMF in active UC, a placebo-controlled double-blinded study appears warranted.

Antibodies to proteinase 3 mediate expression of vascular cell adhesion molecule-1 (VCAM-1).

VCAM‐1 was first identified as an adhesion molecule induced on human endothelial cells (HEC) by inflammatory cytokines such as IL‐1, tumour necrosis factor (TNF), and lipopolysaccharide (LPS). The molecule binds to a variety of leucocytes, including B cells, T cells, basophils, eosinophils and monocytes. Vascular expression of VCAM‐1 has been associated with a number of disease states, including rheumatoid arthritis and vasculitis. The detection of antineutrophil cytoplasmic antibodies (ANCA), especially to proteinase 3 (PR3), has become important in the diagnosis of Wegener’s granulomatosis (WG) and related vasculitides. Recently we were able to demonstrate a direct effect of anti‐PR3 antibodies on neutrophil–endothelial interactions (Blood 1993; 82:1221). Binding of anti‐PR3 antibodies to their antigen translocated into the membrane of HEC leads to an enhanced adhesion of neutrophils via induction of E‐selectin (Clin Exp Immunol 1993; 94:440). The aim of this study was to investigate the effect of anti‐PR3 antibodies on the expression of VCAM‐1. HEC was isolated from umbilical vein and cultured on microtitre plates. After preincubatiion with purified anti‐PR3 antibody, purified control antibodies (SS‐A, SS‐B, RNP) (IgG and F(ab′)2 fragments) or different cytokines (controls), VCAM‐1 was detected on the surface of unfixed HEC by cyto‐ELISA and polymerase chain reaction analysis. Incubation of HEC with anti‐PR3 antibodies led to a marked increase of endothelial VCAM‐1 expression with a peak after 8 h. Incubation with TNF‐α also led to maximal VCAM‐1 expression after 4–6 h (control). Increased adhesion of T lymphocytes to HEC after binding of anti‐PR3 antibodies to their antigen could be confirmed by performing adherence assays. This effect could be inhibited by antibodies to VLA‐4. In conclusion, we have been able to show that cytokine‐like effects of anti‐PR3 antibodies on HEC are not limited to induction of neutrophil adhesion. Anti‐PR3 antibodies may thus contribute to the regulation of T lymphocyte migration from the blood by HEC in ANCA‐related vasculitides.

Complete congenital heart block is associated with increased autoantibody titers against calreticulin

Complete congenital heart block (CCHB) is associated with anti‐Ro/SS‐A and anti‐La/SS‐B antibodies. Calreticulin, a calcium‐binding, multifunctional protein of the endoplasmic reticulum with C‐terminal KDEL‐sequence, is not part of the Ro/SS‐A ribonucleoprotein complex. In this study anti‐calreticulin autoantibody responses in serum samples from 18 infants with CCHB, their mothers and in a control group of 11 anti‐Ro/SS‐A or anti‐La/SS‐B positive infants without heart block and their mothers were analysed. Specific enzyme‐linked immunosorbent assays were performed. Nine out of 18 sera with CCHB contained IgG anti‐calreticulin antibodies. Four sera of those with IgG antibodies also had IgM antibodies. One serum contained anti‐calreticulin IgM antibodies only. In the non‐CCHB group two sera were positive for IgG and one serum was positive for IgM anti‐calreticulin antibodies. Sera of healthy infants were negative both for anti‐IgG and anti‐IgM calreticulin antibodies. Calreticulin is involved in calcium storage and therefore anti‐calreticulin antibodies might influence the development of CCHB. The new finding of IgM autoantibodies and the observed differences in antibody response in infants and mothers support the hypothesis of a fetally mediated and passively acquired autoimmune disease.

Endothelin-1 modulates the expression of adhesion molecules on fibroblast-like synovial cells (FLS)

Endothelin-1 is known to possess various biological properties. In the present study we have investigated the effects of Endothelin-1 (Et-1) on the expression of adhesion molecules ICAM-1, VCAM-1 and CD-44 by fibroblast-like synoviocytes. Cultured fibroblast-like synoviocytes were treated with Et-1 in the absence or presence of C1306, a specific endothelin-A-receptor antagonist. Cell surface expression of ICAM-1, VCAM-1 and CD-44 was determined by immunofluorescence studies, Cyto-ELISA and FACS-analysis. ICAM-1, VCAM-1 and CD-44 were constitutively expressed on cultured FLS. After incubation with Et-1 the expression of ICAM-1, VCAM-1 and CD-44 increased. The level of expression of adhesion molecules after Et-1 stimulation was similar to cytokine mediated effects (IL-1 beta, TNF-alpha). IL-1 beta showed the strongest stimulatory effect on the expression of ICAM-1, TNF-alpha preferentially induced the expression of VCAM-1 and CD-44, and Et-1 strongly stimulated the upregulation of CD-44 expression. In addition, Et-1 enhanced significantly the IL-1 beta mediated upregulation of VCAM-1 expression, whereas TNF-alpha mediated expression of VCAM-1 was downregulated by Et-1. Furthermore, the Et-1 induced expression of adhesion molecules on FLS was mediated via the endothelin-A-receptor (EtA-receptor), since C-1306, a selective endothelin-A-receptor antagonist, could block this effect. These results indicate that Et-1 has stimulating effects on FLS in vitro. The expression of adhesion molecules can be upregulated by Et-1 similar to proinflammatory cytokines. The modulating effect of Et-1 can be inhibited by the pretreatment with a selective EtA-receptor antagonist. Thus, Et-1 may have immunoregulatory functions in the recruitment of cells infiltrating the inflamed tissue.

Actin is a target antigen of anti-neutrophil cytoplasmic antibodies (ANCA) in autoimmune hepatitis type-1

BACKGROUND/AIM Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies first associated with Wegeners granulomatosis and microscopic polyangiitis. The significance of ANCA in autoimmune hepatitis remains uncertain; the nature of the antigen or antigens has not been defined yet. The purpose of this study was to identify the target antigen of ANCA in patients with autoimmune hepatitis. METHODS/RESULTS Sera from 32 type-1 autoimmune hepatitis patients were used in the present study. ANCA were detected in 24 of 32 sera (75%). A diffuse cytoplasmic staining pattern (C-ANCA) was detected in 14 patients; the P-ANCA pattern was observed in 10 patients. An extract of human neutrophils was prepared and subjected to SDS-PAGE and Western Blot analysis. A 43-kD dominant immunoreactive protein was found in 20 (63%) autoimmune hepatitis patients. Aminoacid sequence analysis of the 43 kD protein identified actin. Cytoplasmic or perinuclear staining pattern could be reduced after absorption of sera with actin and after removing anti-actin antibodies by affinity chromatography. This was observed for all C-ANCA and for 8 out of 10 P-ANCA. Moreover in double-staining indirect immunofluorescence, the same type of diffuse cytoplasmic staining was observed with autoimmune hepatitis-sera and anti-actin antibodies. In Western Blot analysis with actin, 17 (53%) patients gave a positive result, while 15 (47%) patients had a positive actin-ELISA. CONCLUSION This is the first report to identify the cytoskeletal protein actin as an ANCA antigen.

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.

A novel rat model of chronic fibrosing cholangitis induced by local administration of a hapten reagent into the dilated bile duct is associated with increased TNF-α production and autoantibodies

BACKGROUND/AIM The cholangiopathies represent hepatobiliary diseases in which bile-duct epithelial cells are targets for destructive processes, including immune-mediated damage. We describe a novel rat model of chronic fibrosing cholangitis induced by administration of the hapten reagent 2,4,6-trinitrobenzenesulfonic acid (TNBS) into the dilated bile duct. METHODS The common bile duct was dilated due to a mild stenosis in 8-week-old female Lewis rats. TNBS (50 mg/kg) was injected during a second laparotomy. RESULTS TNBS-treatment reproducibly resulted in chronic fibrosing cholangitis. In retrograde cholangiography the bile ducts showed irregularities, beading and strictures. Alkaline phosphatase levels remained abnormal throughout the study period. Immunohistochemical staining showed an increased number of macrophages, CD3+ T-lympbocytes and MHC class II antigen upregulation. The spontaneous interferon-gamma, tumor necrosis factor-alpha and interleukin-10 production of liver-derived mononuclear cells was increased. Anti-neutrophil cytoplasmic antibodies with specificity against myeloperoxidase, catalase and actin were found between 1 and 12 weeks after TNBS injection. CONCLUSIONS We established a novel rat model of chronic fibrosing cholangitis with histologic, cholangiographic, serologic and immunologic similarities to human primary sclerosing cholangitis. This model may be used to study pathomechanisms of chronic cholangitis without concomitant inflammatory bowel disease.

Effects of Th1 and Th2 cytokines on cytokine production and ICAM-1 expression on synovial fibroblasts.

OBJECTIVES--To investigate the influence of the Th1 and Th2 lymphokines interleukins (IL)-4 and IL-13, interferon gamma (IFN gamma), and several monokines on the adhesion of mononuclear cells to synovial fibroblasts and intercellular adhesion molecule-1 (ICAM-1) expression and cytokine production of synovial fibroblasts in patients with osteoarthritis. METHODS--Synovial fibroblasts were isolated from patients with osteoarthritis and stimulated with IL-1 beta, IL-4, IL-6, IL-10, IL-12, IL-13, tumour necrosis factor alpha (TNF alpha), and IFN gamma. Subsequently, we determined the production of IL-1 alpha, IL-1 beta, IL-6, IL-10, IL-12, IFN alpha and TNF alpha, and the expression of ICAM-1 lymphocyte function associated antigen 3 (LFA-3), BB7, and major histocompatibility complex class II molecules on these cells. Furthermore, the adhesion of freshly isolated mononuclear cells from the peripheral blood was tested using a colourimetric cell-cell adhesion assay. RESULTS--Only production of IL-6 and the expression of ICAM-1 were observed. IL-1 beta and TNF alpha were the most potent stimulatory mediators of both cytokine production and ICAM-1 expression. IL-4 and IL-13 had differential effects as they upregulated cytokine production but downregulated IFN gamma induced ICAM-1 expression. In functional adhesion assays, TNF alpha, IL-1 alpha and, to a lesser extent, IFN gamma led to increased adhesion of mononuclear cells, whereas IL-4 and IL-13 had no effect. CONCLUSIONS--Our data indicate that Th1 and Th2 lymphokines can modulate the function (cytokine production and expression of adhesion molecules) of synovial fibroblasts.