nitrophenols and Lupus-Erythematosus--Systemic

Caspase-1 is required for nephritis and robust autoantibody development in the pristane model of murine lupus. The objective of this study was to evaluate the immune response and to study the splenic B and T cell populations in wild-type (WT) and caspase-1-/- mice following pristane injection in order to develop an understanding of why absence of caspase-1 is protective in pristane-induced lupus.. Immunization responses to NP-Ficoll and NP-ovalbumin were assessed in WT and caspase-1-/- mice. In vitro IgM and IgG responses to R848 were measured by ELISA. Serum IgM anti-dsDNA and IL-1β were also measured by ELISA. B and T cell populations 2 weeks and 6 months following pristane injection were measured by flow cytometry in WT and caspase-1-/- mice.. Caspase-1-/- mice generate equivalent IgG responses to NP-Ficoll and NP-ova antigens when compared to wild-type mice. Additionally, they secrete IgM and IgG in response to TLR7 activation. Pristane injected WT and caspase-1-/- mice generate robust IgM anti-dsDNA responses. Caspase-1-/- mice have a significant reduction in marginal zone B cell populations compared to WT 6 months after pristane exposure whereas T cell responses are intact in these mice.. Caspase-1-/- mice have intact immune responses but do not develop an expanded marginal zone B cell population in response to pristane-induced lupus. This may be one explanation for reduced IgG autoantibody production in these mice.

Topics: Animals; Antibodies, Antinuclear; B-Lymphocytes; Caspase 1; Cells, Cultured; Disease Models, Animal; Ficoll; Genetic Predisposition to Disease; Imidazoles; Immunization; Immunoglobulin G; Immunoglobulin M; Lupus Erythematosus, Systemic; Mice, Inbred BALB C; Mice, Knockout; Nitrophenols; Ovalbumin; Phenotype; Phenylacetates; Spleen; T-Lymphocytes; Terpenes; Time Factors

Systemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of anti-inflammatory effects of glucocorticoids.. We conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice.. Reduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naïve B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naïve and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naïve B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli.. Our findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.

Topics: Adjuvants, Immunologic; Animals; Autoimmunity; B-Lymphocyte Subsets; B-Lymphocytes; Gene Expression Regulation; Germinal Center; Glucocorticoids; Hemocyanins; Histones; In Vitro Techniques; Leukocytes, Mononuclear; Lupus Erythematosus, Systemic; Lymphocyte Activation; Male; Mice; Mice, Knockout; Nitrophenols; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; T-Lymphocytes; Transcription Factors; Up-Regulation

Interferon-α (IFNα)-producing plasmacytoid dendritic cells (PDCs) are implicated in the pathogenesis of systemic lupus erythematosus (SLE). IFNα-related genes are highlighted among SLE susceptibility alleles and are characteristically expressed in the blood of patients with SLE, while in mouse models of lupus, PDC numbers and IFNα production are increased. This study was undertaken to investigate the effects of inhibitors that selectively target different antiapoptotic molecules on the survival of PDCs.. PDC numbers, in vitro survival, and expression of antiapoptotic molecules were evaluated in lupus-prone (NZB × NZW)F1 (NZB/NZW) mice. The impact of Bcl-2 antagonists and glucocorticoids on PDCs was evaluated in vitro and in vivo. IFNα production by NZB/NZW mice was evaluated before and after treatment with Bcl-2 antagonists.. PDCs, but not lymphoid tissue-resident conventional DCs, largely relied on the antiapoptotic protein Bcl-2 for survival. The enlarged PDC compartment in NZB/NZW mice was associated with selectively prolonged survival and increased Bcl-2 transcription. Functionally, this resulted in enhanced production of IFNα. Bcl-2 inhibitors selectively killed mouse and human PDCs, including PDCs from SLE patients, but not conventional DCs, dampened IFNα production by PDCs, and synergized with glucocorticoids to kill activated PDCs.. Enhanced PDC survival is a likely contributing factor to enhanced IFNα production by lupus PDCs. Bcl-2 antagonists potently and selectively kill PDCs and reduce IFNα production. Thus, we believe that they are attractive candidates for treating PDC-associated diseases.

Topics: Animals; Annexin A5; Antibodies, Antinuclear; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Survival; Cells, Cultured; Dendritic Cells; Flow Cytometry; Glucocorticoids; Humans; Interferon-alpha; Lupus Erythematosus, Systemic; Mice; Mice, Inbred C57BL; Mice, Inbred NZB; Mice, Transgenic; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Sulfonamides

Deficiencies in early complement components are associated with the development of systemic lupus erythematosus (SLE) and therefore early complement components have been proposed to influence B lymphocyte activation and tolerance induction. A defect in apoptosis is a potential mechanism for breaking of peripheral B cell tolerance, and we hypothesized that the lack of the early complement component C4 could initiate autoimmunity through a defect in peripheral B lymphocyte apoptosis. Previous studies have shown that injection of a high dose of soluble antigen, during an established primary immune response, induces massive apoptotic death in germinal centre B cells. Here, we tested if the antigen-induced apoptosis within germinal centres is influenced by early complement components by comparing complement C4-deficient mice with C57BL/6 wild-type mice. We demonstrate that after the application of a high dose of soluble antigen in wild-type mice, antibody levels declined temporarily but were restored almost completely after a week. However, after antigen-induced apoptosis, B cell memory was severely limited. Interestingly, no difference was observed between wild-type and complement C4-deficient animals in the number of apoptotic cells, restoration of antibody levels and memory response.

Topics: Animals; Antigens; Apoptosis; B-Lymphocytes; Complement C4; Enzyme-Linked Immunosorbent Assay; Female; Germinal Center; Immune Tolerance; Immunologic Memory; Lupus Erythematosus, Systemic; Mice; Mice, Inbred C57BL; Nitrophenols; Phenylacetates

To disclose the mechanism of aberrant function of peripheral blood lymphocytes (PBL) in SLE, we focused on the catalytic function of CD45, and determined the CD45 PTPase activity in SLE patients. The sample population consisted of 32 SLE patients with different disease activity. PTPase activity of cell lysates immunoprecipitated by anti-CD45 MoAb was assayed against phosphotyrosine analogue PNPP, followed by measuring the release of para-nitro phenol at 410 nm. CD45 PTPase activity of PBL was significantly decreased in SLE patients, compared with that of normal controls and patients with systemic sclerosis (964 +/- 265, 1202 +/- 172, 1210 +/- 125, respectively; SLE versus normal, P<0.05). It was correlated with SLE Disease Activity Index (SLEDAI) score (r = 0.597, P = 0.0006), but not with the dose of prednisolone (r = 0.214, P = 0.2657), indicating that CD45 PTPase activity became reduced when the disease was active, but it was not affected by prednisolone. Moreover, it was not corrected by in vitro culture with or without stimulation. The expression of CD45 on PBL was comparable between normal and SLE, raising a possibility that it may be due to aberrant regulation of catalytic function of CD45 in SLE. Given the evidence that tyrosine phosphorylation of cellular proteins by tyrosine kinases and phosphatases is one of the key biochemical events in the signal transduction pathway, the decreased CD45 PTPase activity in SLE may account for the defective signal transduction via TCR/CD3, leading to dysregulated effector function of the lymphocytes.

Topics: Adult; Aniline Compounds; Anti-Inflammatory Agents; Female; Fluorescent Antibody Technique, Indirect; Humans; Leukocyte Common Antigens; Lupus Erythematosus, Systemic; Lymphocytes; Middle Aged; Nitrophenols; Organophosphorus Compounds; Phosphorylation; Precipitin Tests; Prednisolone; Protein Tyrosine Phosphatases; Scleroderma, Systemic; Signal Transduction

The antigen-binding selectivity of 2 sets of anti-DNA antibodies from autoimmune mice and from normal mice was examined. Eighteen affinity-purified anti-DNA auto-antibodies from MRL-lpr/lpr mice were examined for binding to the haptens azobenzenearsonate, phosphorylcholine, (4-hydroxy-3-nitrophenyl)acetyl and (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP). Five of these autoantibodies bound to NIP-protein conjugates. In contrast, none of 12 monoclonal antibodies to single-stranded DNA or left-handed Z-DNA induced by immunization of BALB/c and C57BL/6 mice with nucleic acid antigens reacted with the tested haptens. In a reciprocal test of the relationship between anti-DNA and anti-NIP binding, we examined 24 monoclonal antibodies to NIP, from various strains of mice, for binding to DNA. One such antibody from a BALB/c mouse also bound to DNA. These results are discussed in the context of the mechanisms underlying autoantibody hyperproduction.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Autoantibodies; Cross Reactions; DNA; DNA, Single-Stranded; Haptens; Immunization; Immunoglobulin Isotypes; Lupus Erythematosus, Systemic; Mice; Mice, Mutant Strains; Nitrohydroxyiodophenylacetate; Nitrophenols; p-Azobenzenearsonate; Phenylacetates; Phosphorylcholine

An enzyme-linked immunosorbent assay is described for the assay of anti-DNA antibodies. The method employs plastic surface for immobilization of the antigen and alkaline phosphatase-linked rabbit anti-human IgG for the detection of immune complex using PNP-P and 4MU-P as substrates. The sensitivity of the assay increased by as much as 16-fold when fluorogenic substrate was used instead of conventional PNP-P and could therefore be employed for the detection of low avidity antibodies. Using PNP-P as substrate 57% of SLE patients were positive for DNA antibody, but if 4MU-P was introduced as substrate, 71% gave a positive response. Moreover, using a fluorogenic substrate, it was possible to minimise the amount of antigen (2 nM bp). The technique is simple, reproducible and of high sensitivity.

Topics: Alkaline Phosphatase; Animals; Antibodies, Antinuclear; Cattle; Chromogenic Compounds; DNA; Enzyme-Linked Immunosorbent Assay; Humans; Hymecromone; Lupus Erythematosus, Systemic; Nitrophenols; Rabbits; Substrate Specificity; Umbelliferones

Topics: Adipates; Adult; Alcohols; Female; Glucuronidase; Hexosaminidases; Humans; Hyaluronoglucosaminidase; Lupus Erythematosus, Systemic; Male; Methods; Middle Aged; Nitrophenols; Rheumatic Diseases; Spectrophotometry