Michael Manns

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.

HLA DRw8 and complement C4 deficiency as risk factors in primary biliary cirrhosis.

HLA class I, II, and III alleles were investigated in 25 consecutive unrelated German patients with primary biliary cirrhosis and in two families with two primary biliary cirrhosis patients in each. In primary biliary cirrhosis patients, HLA class I antigens did not differ significantly from in health controls. For HLA class II antigens, a highly significant increase of HLA DRw8 was found in patients with primary biliary cirrhosis compared with controls. Thirty-six percent vs. 3.6% were DRw8 positive [relative risk = 15.28; P (corrected) = 0.00013]. The genetic typing of HLA class III alleles revealed an increased incidence for C4AQ0 alleles [72% vs. 34.5%, relative risk = 4.89: P (corrected) = 0.0056]. A highly significant proportion of primary biliary cirrhosis patients carrying both DRw8 and C4A-Q0 alleles (relative risk = 183.75; P = 9.7 x 10(-7)) were found. In one family, a mother and her daughter had primary biliary cirrhosis, both sharing the major histocompatibility complex haplotype HLA-A1, -B8, -DR3, -C4AQ0B1. In the other family, two sisters with primary biliary cirrhosis shared the major histocompatibility complex haplotype HLA-A24, -B8, -DRw8, -C4A4B2. These studies contribute to the further elucidation of the immunogenetic background of primary biliary cirrhosis.

The designer cytokine hyper-interleukin-6 is a potent activator of STAT3-dependent gene transcription in vivo and in vitro.

Interleukin-6 (IL-6) triggers pivotal pathwaysin vivo. The designer protein hyper-IL-6 (H-IL-6) fuses the soluble IL-6 receptor (sIL-6R) through an intermediate linker with IL-6. The intracellular pathways that are triggered by H-IL-6 are not defined yet. Therefore, we studied the molecular mechanisms leading to H-IL-6-dependent gene activation. H-IL-6 stimulates haptoglobin mRNA expression in HepG2 cells, which is transcriptionally mediated as assessed by run-off experiments. The increase in haptoglobin gene transcription correlates with higher nuclear translocation of tyrosine-phosphorylated STAT3 and its DNA binding. As H-IL-6 stimulates STAT3-dependent gene transcription, we compared the molecular mechanism between IL-6 and H-IL-6. Transfection experiments were performed with a STAT3-dependent luciferase construct. The same amount of H-IL-6 stimulated luciferase activity faster, stronger, and for a longer period of time. Dose response experiments showed that a 10-fold lower dose of H-IL-6 stimulated STAT3-dependent gene transcription comparable with the higher amount of IL-6. Cotransfection with the gp80 and/or gp130 receptor revealed that the effect of H-IL-6 on STAT3-dependent gene transcription is restricted to the gp80/gp130 receptor ratio. High amounts of gp130 increased and high amounts of gp80 decreased the effect on H-IL-6-dependent gene transcription. To investigate the in vivo effect of H-IL-6 on gene transcription in the liver, H-IL-6 and IL-6 were injected into C3H mice. H-IL-6 was at least 10-fold more effective in stimulating the DNA binding and nuclear translocation of STAT3, which enhances haptoglobin mRNA and protein expression. Thus H-IL-6 stimulates STAT3-dependent gene transcription in liver cells in vitro and in vivo at least 10-fold more effectively than IL-6. Our results provide evidence that H-IL-6 is a promising designer protein for therapeutic intervention during different pathophysiological conditions also in humans.

Identification of cytochrome P450IA2 as a human autoantigen

Autoantibodies occurring in a patient with idiopathic autoimmune type chronic active hepatitis (CAH) were found to react with purified rabbit cytochrome P450IA2 and to a much lesser extent with P450IA1. Both cytochrome P450s are known to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rabbit, and the expression of the microsomal protein recognized by the patient serum was induced in adult rabbit livers after treatment with TCDD. This protein is only weakly detected in liver microsomes from neonatal rabbits exposed to TCDD in utero, which is consistent with the age-dependent induction of P450IA2 by TCDD. The serum specifically reacted with a protein of similar size in microsomes prepared from COS-1 cells transfected with an expression vector containing the full length human P450IA2 cDNA. This reactivity was not detected in the cells transfected with the vector alone, indicating that the antibody recognizes human P450IA2. In addition, the serum extensively inhibited 7-ethoxyresorufin O-deethylation catalyzed by isolated human liver microsomes. This catalytic activity is associated with class IA P450s in other species. A screen of sera from patients with various hepatic and nonhepatic diseases indicates that the autoantibody to P450IA2 occurs rarely in CAH. Cytochrome P450IA2 becomes the third P450 identified as an autoantigen in inflammatory liver diseases.

Identification and characterization of a monoclonal antibody to the membrane fatty acid binding protein

A monoclonal antibody to the rat liver membrane fatty acid binding protein (MFABP) was prepared by immunizing mice with purified MFABP isolated from solubilized rat liver plasma membrane proteins by oleate-agarose affinity chromatography technique. The monoclonal antibody K15/6 identified a single 40 kDa protein in rat liver plasma membranes with pI values of 8.5, 8.8 and 9.0, which is identical to the authentic MFABP, but clearly distinct from rat mitochondrial GOT. The antibody K15/6 selectively inhibited cellular influx as well as membrane binding of fatty acids, but not of cholesterol or vitamin E. The same antibody was used in immunofluorescence, ELISA and Western blot analysis to determine the subcellular and organ distribution pattern of MFABP. The protein was identified in rat liver plasma membranes and mitochondria, but in no other cell compartment. It was detectable in homogenates of rat liver but not in homogenates of other organs. Therefore, the monoclonal antibody K15/6 represents an organ specific antibody to MFABP which reveals inhibitory action on membrane binding/transport of fatty acids.

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.

Discontinuation of immunosuppressive therapy in hepatitis B surface antigen-positive chronic hepatitis: effect on viral replication and on liver cell damage.

Immunosuppressive therapy was stopped in 12 individuals positive for hepatitis Be antigen (HBeAg) and hepatitis B surface antigen (HBsAg) and in 4 individuals positive for HBsAg but negative for HBeAg. Discontinuation of immunosuppressive therapy in HBeAg-positive patients was always associated with a bout of hepatitis and elimination of HBeAg in 8/12 patients. One patient died from liver failure and 2 patients experienced a decompensation of their liver disease indicating that this approach might be harmful if used therapeutically. A bout of hepatitis was not noted in any of the individuals negative for HBeAg when the immunosuppressive therapy was stopped, implying that this event is not potentially harmful to the patient.

In vitro production of anti-neutrophilocyte-cytoplasm-antibodies (ANCA) by Epstein-Barr virus-transformed B-cell lines in Wegener's granulomatosis.

The frequent detection of anti-neutrophilocyte-cytoplasm-antibodies (ANCA) in patients with Wegeners granulomatosis (WG) led to the supposition that this disease might be of autoimmune nature. For some authors assume that Epstein-Barr virus (EBV) infection of human B-lymphocytes besides polyclonal activation could reveal the cryptic immune status against different autoantigens in patients with autoimmune diseases we investigated EBV-transformed B-lymphocytes from patients with Sjögrens syndrome, mixed connective tissue disease, WG and healthy blood donors. Two stable B-cell lines (Ho3, We1) could be established. Inhibition experiments showed that antibodies produced by transformed B-lymphocytes and serum ANCA (C-ANCA type) of 10 WG patients recognized the identical antigen. Stimulation of one clone (Ho3) with interleukin 6 (IL-6) led to a switch from IgM to IgG production. Antibodies produced by this clone also stained glomeruli of human frozen kidney sections. Western blot analysis using immunoaffinity purified antigen prepared from human granulocytes revealed a reaction with a protein of approx. 29 kD MW. Our data underscore some new aspects concerning the direct pathogenicity of C-ANCA confirming the hypothesis that the autoimmune B-cell repertoire in WG not only reflects a polyclonal B-cell activation but is shaped by antigen driven responses.

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.

Chromosomal aberrations in patients with primary biliary cirrhosis

SummaryChromosomal aberrations in untreated lymphocyte cultures, bleomycin (BLM)-induced aberrations and sister chromatid exchanges (SCE) in the peripheral blood lymphocytes of 11 patients suffering from primary biliary cirrhosis (PBC) and 14 matched control individuals were analysed. The lymphocytes of the PBC patients had on average a lower mitotic index (2.3) compared with controls (3.5) in the untreated cultures. The mean baseline rate of aberrations of the cultured lymphocytes of the patients was 5.3 aberrations per 100 metaphases (%); this was significantly different (P=0.0291) from that of the controls with a mean of 2.3%. In lymphocytes of the patients and controls, most of the aberrations observed took the form of gaps; there was an almost equal breakage rate in both groups (0.5% and 0.4%, respectively). The average number of mitoses with aberrations in the PBC patients studied was double that of the controls (4.9% and 2.3% respectively, P=0.0323). The mean number of the BLM-induced aberrations was 54.0% and 27.7% for the lymphocytes of the patients and controls, respectively. The mean number of the aberrant mitoses in the BLM cultures was 6 times higher than that of the untreated cultures for both groups, 25.7% and 14.6% respectively (P=0.018). The chromosomal distribution of baseline and induced aberrations was not random. The PBC patients had a mean number of 8.7 SCE per mitosis, which was significantly higher than the SCEs in the controls (6.3 SCE per mitosis; P=0.0156). The evidence suggests that the chromosomes of the lymphocytes of PBC patients may be less stable than those of the control individuals in this study.