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In order to elucidate the pathogenic role(s) of autoantibodies in connective tissue disease (CTD), we examined whether autoantibodies against U1-ribonucleoprotein (RNP) and double-stranded (ds) DNA can up-regulate ICAM-1, ELAM-1 and class I and II MHC molecule expression on pulmonary artery endothelial cells (HPAEC). ICAM-1, ELAM-1 and class II MHC molecule expression on HPAEC cultured in the presence of anti-U1-RNP-containing and anti-dsDNA-containing IgG from CTD patients was up-regulated significantly in comparison with that on HPAEC cultured with IgG from normal healthy volunteers. Affinity chromatographic enrichment and depletion of the anti-U1-RNP antibody content of anti-U1-RNP-containing IgG confirmed that the anti-U1-RNP antibody did up-regulate ICAM-1, ELAM-1 and class II MHC molecule expression. The finding that an IgG F(ab')2-purified anti-U1-RNP antibody also up-regulated expression of these molecules may indicate that mechanisms other than Fc receptor-mediated stimulation are involved. These in vitro findings suggest that autoantibodies against U1-RNP and dsDNA play important roles in the immunopathological processes leading to the proliferative pulmonary arterial vasculopathy observed in CTD patients with pulmonary hypertension by up-regulating adhesion and class II MHC molecule expression on endothelial cells.

Propofol appears to inhibit both IL-6 and IL-10 production by LPS-stimulated PBMCs in vitro. Further study is required to clarify the mechanism of the suppressive effect of propofol.

SummaryAssociations of Epstein-Barr virus (EBV) and autoimmune diseases have been hypothesized. We have analysed IgG antibodies to EBV nuclear antigen (EBNA)-2 in sera from Japanese patients with autoimmune systemic connective tissue diseases (CTD), exemplified by systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis (SSc) and secondary SS (classical CTDs complicated with SS). An enzyme-linked immunosorbent assay (ELISA) which uses glutathione-Stransferase polypeptides fused to EBV nuclear antigen (EBNA)-2 and EBNA-1 was developed. Ratios of IgG antibody reactivity to whole IgG concentrations of sera were calculated to normalize EBNA-2 and EBNA-1 antibody levels to the hypergammaglobulinaemia that occurs in CTD. The ELISA optical density OD 450 readings of IgG antibodies to both the amino-terminal aa 1-116 of EBNA-2 and carboxyl-terminal aa 451-641 of EBNA-1 were elevated significantly in patients with SLE, primary SS, RA, SSc and secondary SS when compared to EBNA-1. The OD readings were divided by serum IgG concentrations to normalize for the hypergammaglobulinaemia. The specific levels of IgG antibodies to the amino-terminal region of EBNA-2 were elevated in patients with SLE, primary SS or RA, as well as those with secondary SS complicated with SLE or RA. The EBNA-2 amino-terminal region contains a polyproline tract and a proline-rich sequence and has considerable amino acid sequence homology with many cellular proline-rich proteins . High ratios of EBNA-2 aa 1-116 to EBNA-1 aa 451-641 IgG antibody levels which probably suggest reactivation of EBV latent infection were associated significantly with pulmonary involvement in SS patients. These results are consistent with the hypothesis that the sequence similarity between the amino-terminal region of EBNA-2 and proline-rich cellular proteins is associated with pathogenesis in a subpopulation of CTD patients, possibly by the molecular mimicry-epitope shift mechanism.

SUMMARYIn order to elucidate the immunological properties of anti-U1-ribonucleoprotein (RNP) antibody, one of the autoantibodies detected in patients with connective tissue diseases (CTDs), we tested the endothelial cell-binding by anti-U1-RNP antibodies and epitopes on human pulmonary artery endothelial cells (HPAECs) to which the autoantibody bound. IgG fractions positive for anti-U1-RNP from patients with CTDs bound to the HPAECs. Furthermore, intact and F(ab H ) 2 IgG anti-U1-RNP purified by affinity chromatography also bound to endothelial cells. The binding activity of IgG fractions positive for anti-U1-RNP to the endothelial cells could be effectively absorbed by U1-RNP-Sepharose. An immunoblotting assay of purified IgG anti-U1-RNP antibodies showed that these antibodies could bind to various membrane proteins of NP40-treated HPAECs such as 68, 48, 43,38,33,29,28 and 24 kDa. Some bands, 68, 33, 28 and 24 kDa, seemed to correspond to components of U1-RNP, i.e. 68 kDa, A, B H and C peptides, respectively. We confirmed that the anti-U1-RNP antibody from patients with CTDs can directly recognize a variety of antigens on the endothelial surface of the pulmonary artery, including the components of U1-RNP or other unknown polypeptides. These results suggest that binding to pulmonary artery endothelial cells of this autoantibody may be one of the triggers of endothelial cell inflammation in CTDs.

An enzyme linked immunosorbent assay (ELISA) was used to estimate the production of intracellular and extracellular interleukin-1 (IL-1) a and 13 by peripheral blood monocytes from 26 patients with various connective tissue diseases (CTD), including 19 with systemic lupus erythematosus, four with progressive systemic sclerosis, two with mixed connective tissue disease, and one with Sj6gren's syndrome. Monocytes obtained from patients with CTD with serum antibodies to nuclear ribonucleoprotein (nRNP) released significantly higher concentrations of extracellular IL-la and IL-1(3, whereas intracellular IL-la and IL-,8 production was similar to that by monocytes from patients with CTD without antibodies to nRNP. Furthermore, the concentrations of extracellular IL-la correlated significantly with those of extracellular IL-1j3. There was no significant correlation between the concentrations of extracellular and intracellular IL-la, and those of extracellular and intracellular IL-1,B, indicating that synthesis and secretion of IL-1 by human monocytes may be two distinct biological events. It seems that enhanced extracellular release of both IL-la and IL-11 contributes to the excessive anti-nRNP production in CTD.Interleukin-l (IL-1) is a cellular product with diverse amplifying effects on immunological cell reactions. 1 2 Interleukin-l was originally identified and subsequently routinely measured by a mouse thymocyte comitogenesis assay, in which IL-I increased the proliferation of thymocytes in response to suboptimal concentrations of T cell mitogens such as phytohaemagglutinin.3 4 Cytotoxic agents mask IL-I activity, however, whereas other substances, such as mitogens, can mimic or synergise with IL-I on target cells. Complementary DNA cloning, protein purification, and sequencing studies have shown the presence of two distinct types of IL-I molecules:an acidic form (pl 5)-IL-la and a neutral form (pl 7)-IL-10.6 These two proteins are distantly related to the primary sequence levels in humans and have similar biological functions.7 Recently, specific monoclonal and polyclonal antibodies against IL-la or IL-1, have become available. Here we describe a sandwich ELISA for IL-la and IL-1lB which uses a rabbit polyclonal antibody and murine monoclonal antibody, both of which recognise human IL-la and IL-1lB respectively. We used an ELISA to examine the production of intracellular and extracellular IL-la and IL-13 by peripheral blood monocytes from patients with connective tissue diseases (CTD). Materials and methods CYTOKINESRecombinant IL-la (rIL-la) was donated by Dai-nippon Pharmaceutical Co (Osaka, Japan).8 Recombinant IL-1, (rIL-11) was donated by Dr Y Hirai (Otsuka Pharmaceutical Co, Tokushima, Japan).9 Recombinant IL-2, recombinant IL-6, and recombinant interferon-y were obtained from Genzyme. Recombinant IL4 was obtained from Boehringer Mannheim. ANTIBODIES TO IL-la AND IL-16The ELISA for IL-la or IL-13 used a sandwich technique with a rabbit polyclonal antibody to rIL-la (Dai-nippon Pharmaceutical Co) or ...

SUMMARYAntibodies to dsDNA are characteristieally present in serum from patients with systemic lupus erythematosus (SLE), and have been shown to have ihe capaeily lo reaei with various molecules bearing repeating negative eharges. After a number of polymeric or monomerie molecules with differently charged groups and hydrophobic molecules had been eoupled eovalently as ligands on cellulose gel, the adsorption capaeities ofthe ligands for anti-dsDNA antibodies were evaluated. It was found that gels coupled with polyanionie dextran sulphate (DXS) and polyaerylieacid(PA)and monoanionic sulphanilic aeid (SA) absorbed anti-dsDNA antibodies effeetively. DXS gel also adsorbed antibodies to ssDNA and heparan sulphate, antigens with repeating negatively eharged moieties, while no ligand was able to adsorb anti-nRNP antibodies. The fmding that DXS gels adsorbed anti-dsDNA antibody in proportion to their charge density, and that ihe interaction between anti-dsDNA and DXS gel is broken readily by an increase in ionic strength, indicated that the binding is ionic in nature. Moreover, virtually all F(ab )-anti-dsDNA beeame adsorbed onto the DXS gels, suggesting that the binding occurred via speeifie antigen-binding sites on the antibody molecule. Binding of these polyanion-binding autoantibodies with anionic sites in the glomerular basement membrane may therefore eaiise the tissue damage observed in SLE.

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