A. Kyriatsoulis

CHARACTERISATION OF A NEW SUBGROUP OF AUTOIMMUNE CHRONIC ACTIVE HEPATITIS BY AUTOANTIBODIES AGAINST A SOLUBLE LIVER ANTIGEN

Autoantibodies against a soluble liver antigen (SLA) were detected in 23 patients with HBsAg-negative chronic active hepatitis (CAH) but not in 502 patients with various other hepatic and non-hepatic disorders or 165 healthy blood donors. Anti-SLA-positive serum samples were negative for antinuclear and liver-kidney-microsomal antibodies, markers of two subgroups of autoimmune-type CAH, 6 anti-SLA-positive patients were negative for all autoantibodies sought. Most of the anti-SLA-positive patients were young women (2 men, 21 women; mean age 37 years) with hypergammaglobulinaemia (mean 3.2 g/l, range 1.8-5.3 g/l); 18 of the 23 patients had received immunosuppressive treatment and all responded well. Anti-SLA titres declined during therapy, corresponding to disease activity. Anti-SLA cannot be detected by immunofluorescence. SLA is not organ-specific or species-specific, but the highest concentrations were found in liver and kidney. Anti-SLA autoantibodies characterise a third subgroup of autoimmune-type CAH and will allow a better differentiation of HBsAg-negative CAH which has therapeutic consequences.

Clonal analysis of liver-infiltrating T cells in patients with LKM-1 antibody-positive autoimmune chronic active hepatitis

Autoantibodies against microsomal antigen of liver and kidney (LKM‐1) are diagnostic markers for a subgroup of autoimmune chronic active hepatitis (AI‐CAH). Cytochrome P4S0dbl, now classified as cytochrome P450 IID6, is the major antigen of LKM‐1 antibodies. Immunohistological studies suggest that hepatic injury in AI‐CAH is mediated by liver‐infiltrating T cells. In the present study the specificity and function of liver‐infiltrating T cells was analysed at the clonal level. Phenotypical characterization of 189 T cell clones isolated from four liver biopsies of LKM‐1 antibody‐positive patients showed an enrichment of CD4+CD8‐ T cells. Five CD4+CD8‐ T cell clones proliferated specifically in the presence of recombinant human LKM‐1 antigen (rLKM). This reaction was restricted to autologous antigen‐presenting cells and to HLA class II molecules. In order to see whether rLKM was also recognized by peripheral blood T lymphocytes (PBL) we tested the proliferative response of PBL from several individuals. PBL from three of the four patients with LKM‐1 antibody‐positive AI‐CAH proliferated to rLKM, whereas no response was seen with PBL from patients with LKM‐1 antibody‐negative chronic liver diseases and from healthy blood donors. These data demonstrate that the LKM‐1 antigen is recognized by liver‐infiltrating T cells in LKM‐1 antibody‐positive AI‐CAH. For further functional characterization, liver‐derived T cell clones were tested for their cytotoxic activity. In the presence of phytohacmagglutinin 24 out of 26 CD4‐CD8+ but also 20 out of 63 CD4+CD8‐ T cell clones lysed autologous as well as allogenic EBV‐transformed B cell lines or K562 cells. Five CD4‐CD8+ T cell clones lysed autologous but not allogenic B cell lines spontaneously in a HLA class I‐restricted manner. Although the antigen specificity of these clones is still unknown the data show the presence of autoreactive T cells at the site of inflammation that could contribute in the pathogenesis of AI‐CAH.

Characterization of liver cytokeratin as a major target antigen of anti-SLA antibodies

Anti-SLA antibodies characterize a newly defined subgroup of patients with autoimmune chronic active hepatitis. The aim of the present study was the immunochemical characterization of the target antigen(s) of anti-SLA antibodies. Anti-SLA-positive sera were found to contain high titres of anti-cytokeratin antibodies. In immunoblotting analyses with 100,000 x g supernatants of human liver homogenates (S-100) these sera recognized various proteins with a molecular mass of 40-60 kDa. These proteins were also recognized by monoclonal anti-cytokeratin antibodies. Two-dimensional co-electrophoresis and immunoblotting analysis of S-100 and liver cytokeratins showed that anti-SLA antibodies were primarily directed against cytokeratins, particularly against liver cytokeratin types 8 and 18. This was supported by affinity chromatography, immunofluorescence and absorption methods using anti-SLA sera.

Autoantibodies in experimental autoimmune hepatitis

Experimental autoimmune hepatitis (EAH) can be induced in mice by immunization with syngeneic soluble liver antigens in complete Freunds adjuvant. It has previously been shown that autoreactive T cells play an important role in this animal model of autoimmune hepatitis. We have studied the occurrence of liver autoantibodies in EAH. Characteristic autoantibodies appeared several weeks after disease induction and antibody titres continued to rise when histological and biochemical signs of disease activity had already regressed. Autoantibodies in EAH seemed to recognize autoantigens other than those present in autoimmune chronic active hepatitis patients. We conclude that autoantibodies arise in experimental autoimmune hepatitis but that these autoantibodies do not play a critical role in the pathogenesis of the disease.

In vitro secretion of specific antimitochondrial antibodies in primary biliary cirrhosis

Antimitochondrial antibodies are present in the serum of virtually all patients with primary biliary cirrhosis. They have a well-defined antigen reactivity that is diagnostic for the disease. The role of these autoantibodies in the disease process remains to be defined. In this study we show that antimitochondrial antibodies can be produced in vitro by peripheral blood lymphocytes, that the cells producing antimitochondrial antibodies are present in the peripheral blood in a high frequency and seem to be maximally activated. Stimulation with pokeweed mitogen did not augment the in vitro production of antimitochondrial antibodies in patients nor did it induce the production of these antibodies by control lymphocytes. Thus, antimitochondrial antibodies are not simply an expression of polyclonal B-cell stimulation. The high frequency of maximally activated B-cells producing antimitochondrial antibodies suggests active antigenic stimulation.

Untersuchungen zum Verteilungsmuster von Prä S und S-kodierten viralen Hüllproteinen des Hepatitis B Virus (HBV) auf molekularer Ebene im Serum und Lebergewebe von chronischen HBsAg-Trägern

Die biologische Bedeutung der PraS-kodierten Hepatitis B Virus (HBV) — Hullproteine — insbesondere ihre Regulation innerhalb der Leberzelle — ist noch unklar. Daher war es Ziel unserer Untersuchungen, die PraS- und S-kodierten Hullproteine vergleichend im Serum und im Lebergewebe auf molekularer Ebene zu untersuchen.