Interleukin 6 (IL-6) was established as a transcriptional inducer of the rat a2-macroglobulin gene, a prototype liver acute-phase gene. Maximum induction occurred when the 5' flanking sequences of this gene (position -209 to -43) directed expression from the gene's own TATA box and transcription start site. Removal of the hexanucleotide CTGGGA (position -164 to -159) abolished 60-70% of the hormonal induction in FAO1 rat hepatoma cells. This hexanucleotide was dermed as the IL-6 response element (IL-6-RE).The IL-6-RE is well conserved in the cytokine-responsive regions of other acute-phase genes and serves as a binding site for nuclear proteins. A characteristic DNA-protein complex (complex I) was formed with nuclear proteins from normal rat livers. A different, hormone-inducible complex (complex II) was assembled specifically with nuclear proteins from acutephase rat livers or from IL-6-treated human Hep 3B hepatoma cells. Complex II was competitively inhibited by oligonucleotides representing the conserved IL-6-RE sequence from other acute-phase genes. Thus, the proteins building complex II likely participate in a general signal transduction mechanism mediating the transcriptional activation by IL-6 of several acute-phase genes.Interleukin 6 (IL-6) coordinates several important reactions in inflammation and the host defense against tissue damage and infections (1). It induces the synthesis of protective plasma proteins (acute-phase proteins) in the liver. It also modulates the immune response and regulates body temperature and fever (2-5). We have chosen the rat a2-macroglobulin (a2M) gene to study gene activation by because this gene is dramatically induced by IL-6. a2M contributes to the host defense as a broad-range proteinase inhibitor and as a carrier of various hormones (6-13). During an acute inflammatory reaction, a2M plasma concentrations rise several 100-fold within 48 hr, following a corresponding elevation of hepatic a2M mRNA levels (6,7,14). Transcription rates of the gene were reportedly increased up to 9-fold (6, 7, 15, 16). In living rats and most hepatic cell culture systems, both IL-6 and glucocorticoids are required for the full induction of the a2M gene (3,17,18). Here we have evaluated the respective contributions of IL-6 and glucocorticoids to the transcriptional induction of the a2M gene by using a hepatoma cell line stably transfected with an a2M gene promoter region construct. In this assay system, IL-6 alone induced transcription of the a2M gene, and glucocorticoids enhanced its effect. Using mutagenesis and transfection into hepatoma cells, we have mapped an IL-6-responsive cis-acting transcriptional control element in the 5' flanking region ofthe a2M gene. This element serves as a binding site for specific nuclear proteins, and we have observed a hormone-induced characteristic protein-DNA complex between this element and nuclear proteins from acute-phase rat livers and IL-6-treated cultured hepatoma cells. MATERIALS AND METHODSCell Cultures, DNA Transfections, and Luciferas...

Interleukin 6 (IL-6) induces the acute phase response, differentiation of B cells, proliferation of T cells, thymocytes, hematopoietic progenitors, hybridoma and plasmacytoma cells. Monocytes, T cells, fibroblasts, epithelial and endothelial cells secrete IL-6. Since IL-6 responsive cell-types may participate in the pathogenesis of glomerular inflammation, we studied the secretion of IL-6 by rat MCs, using the IL-6 dependent hybridoma cell line B9. The results of our studies indicate that MCs secrete IL-6 with a molecular weight of 17-42 kDa and isoelectric point of 4.0 to 5.3 MC-IL-6 activity could be blocked by a polyclonal antimurine-IL-6 antibody. MC express IL-6 mRNA as determined by Northern blot. Furthermore, our data demonstrate that IL-6 acts as an autocrine growth factor for MC. Incubation of subconfluent MC with recombinant IL-6 results in a dose-dependent increase of 3H-thymidine incorporation and number of MCs. Moreover, reverse phase HPLC fractions of MC-CM containing IL-6 activity increase 3H-thymidine incorporation by MC. In addition to its possible paracrine role in mediating the immune response in the glomerulus, MC-IL-6 may also be one of the autocrine signals leading to mesangial cell proliferation in vivo.

We investigated the presence of autoantibodies to baculovirusexpressed human recombinant 65-and 67-kD isoforms of glutamate decarboxylase (GAD65 and GAD67) in insulin-dependent diabetes mellitus (IDDM). In the immunoprecipitation test using I35Slmethionine-labeled GADs antibodies to GAD65 were detected in 13/15 (87%) islet cell antibody (ICA)-positive and in 1/35 (2.9%) ICA-negative first-degree relatives of patients with IDDM, in 6/11 (54.5%) ICA-positive nondiabetic schoolchildren, and in 35/50 (70%) patients with newly diagnosed IDDM. GAD67 antibodies were positive only in five (33%) of the ICA-positive relatives (P < 0.05) and in nine (18%) IDDM patients at onset (P < 0.00001). After onset of IDDM antibodies to GAD65 and GAD67 declined but were still positive in 25 and 9.4% of subjects with long-standing IDDM (> 10 yr). In all study groups antibodies to GAD67 were only detected in GAD65 antibody-positive sera. An immunotrapping enzyme activity assay for GAD65 antibodies was positive in 64/75 (85.3%) of sera that were GAD antibody positive in the immunoprecipitation test (r = 0.870, P < 0.0001). In two (2.7%) sera GAD65 antibodies that block GAD enzyme activity were found. Our data suggest that antibodies to GAD65 but not to GAD67 represent sensitive markers for preclinical and overt IDDM. The immunotrapping assay here described represents a valuable technique for specific and sensitive screening for GAD antibodies. (J.

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