calcimycin and Autoimmune-Diseases

IL-22 is an immunoregulatory cytokine displaying pathological functions in models of autoimmunity like experimental psoriasis. Understanding molecular mechanisms driving IL-22, together with knowledge on the capacity of current immunosuppressive drugs to target this process, may open an avenue to novel therapeutic options. Here, we sought to characterize regulation of human IL22 gene expression with focus on the established model of Jurkat T cells. Moreover, effects of the prototypic immunosuppressant cyclosporin A (CsA) were investigated. We report that IL-22 induction by TPA/A23187 (T/A) or αCD3 is inhibited by CsA or related FK506. Similar data were obtained with peripheral blood mononuclear cells or purified CD3(+) T cells. IL22 promoter analysis (-1074 to +156 bp) revealed a role of an NF-AT (-95/-91 nt) and a CREB (-194/-190 nt) binding site for gene induction. Indeed, binding of CREB and NF-ATc2, but not c-Rel, under the influence of T/A to those elements could be proven by ChIP. Because CsA has the capability to impair IκB kinase (IKK) complex activation, the IKKα/β inhibitor IKKVII was evaluated. IKKVII likewise reduced IL-22 induction in Jurkat cells and peripheral blood mononuclear cells. Interestingly, transfection of Jurkat cells with siRNA directed against IKKα impaired IL22 gene expression. Data presented suggest that NF-AT, CREB, and IKKα contribute to rapid IL22 gene induction. In particular the crucial role of NF-AT detected herein may form the basis of direct action of CsA on IL-22 expression by T cells, which may contribute to therapeutic efficacy of the drug in autoimmunity.

Topics: Autoimmune Diseases; Calcimycin; Calcium Ionophores; Carcinogens; Cyclosporine; Female; Gene Expression Regulation; Humans; I-kappa B Kinase; Immunosuppressive Agents; Interleukin-22; Interleukins; Jurkat Cells; Lymphocyte Activation; Male; Response Elements; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription Factors

Neutrophil extracellular traps (NET) formation is part of the neutrophil response to infections, but excessive or inappropriate NETosis may trigger the production of autoantibodies and cause organ damage in autoimmune disorders. Spontaneously netting neutrophils are not frequent and induction of NET in vitro by selected stimuli is necessary to investigate their structure. In the present work, the protein composition and post-translational modifications of NET produced under different stimuli have been studied by means of proteomic analysis. Neutrophils from healthy donors were stimulated by PMA, A23187, Escherichia coli LPS or untreated; after three hours, cells were washed, treated with DNase and supernatants collected for mass spectrometry. Data were analyzed by unsupervised hierarchical clustering analyses. We identified proteins contained in NETs of any source or exclusive of one stimulus: LPS-induced and spontaneous NET diverge in protein composition, while PMA- and A23187-induced NET appear more similar. Among the post-translational modifications we examined, methionine sulfoxidation is frequent especially in PMA- and LPS-induced NETs. Myeloperoxidase is the protein more extensively modified. Thus, proteomic analysis indicates that NETs induced by different stimuli are heterogeneous in terms of both protein composition and post-translational modifications, suggesting that NET induced in different conditions may have different biological effects.

Topics: Autoantibodies; Autoimmune Diseases; Calcimycin; Chromatin; Cluster Analysis; Escherichia coli; Extracellular Traps; Gene Ontology; Histones; Humans; Lipopolysaccharides; Neutrophils; Peroxidase; Protein Processing, Post-Translational; Proteomics; Tetradecanoylphorbol Acetate

The Wiskott-Aldrich syndrome (WAS) is an inherited platelet/T-lymphocyte disease characterized by small platelets, thrombocytopenia and immunodeficiency. Because degradative events have a significant role, we directly examined calpain (Ca(2+)-dependent neutral protease), a prominent protease in the affected cells, by functional and antigenic quantitation. Calpain activity in platelets of seven WAS patients was decreased to 59 +/- 3.7% (P < 0.01) relative to platelets of 11 normals. Platelets of two patients with immune thrombocytopenia had normal calpain activity. By immunoblotting, mu-procalpain, the mu-calpain species in resting (unstimulated) blood cells, was decreased in platelets of nine WAS patients to 58 +/- 14.6% (P < 0.01) relative to paired normals. In contrast, mu-procalpain levels in lymphocytes of seven WAS patients did not differ from normal lymphocytes. Normal platelets and lymphocytes have different mechanisms for Ca(2+)-dependent mu-procalpain activation. On addition of ionophore and Ca2+ to stirred platelets, 80kD mu-procalpain was rapidly (0.5 min) and quantitatively converted to 76 kD active mu-calpain; this process was the same in WAS platelets. In lymphocytes, mu-procalpain activation was slow, only partially complete (40 min), and the active species was 78 kD. The marked depletion of calpain in WAS platelets demonstrated in this study may result from inappropriate stimulation of platelets and be related to the severe thrombocytopenia that characterizes this disease.

Topics: Autoimmune Diseases; Blood Platelets; Calcimycin; Calcium; Calpain; Enzyme Precursors; Humans; Immunoblotting; Lymphocytes; Male; Thrombocytopenia; Wiskott-Aldrich Syndrome

Patients with hepatitis have multiple immunologic abnormalities, which may be related to cytokine production.. We examined the in vitro production of interleukins (IL-2, IL-4, IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in purified peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B virus positive (HBV), acute viral hepatitis (A-HBV), HBV + chronic active hepatitis (HBV-CAH), and autoimmune-type chronic active hepatitis (AI-ACH).. IFN-gamma and TNF-alpha production were characteristically higher in patients with A-HBV than in healthy control subjects (p < 0.001). However, patients with AI-CAH produced highly elevated levels of IL-4 and IL-6 compared with patients with A-HBV and HBV-CAH and healthy control subjects. The cytokine profile (PBMC-induced IL-2, IL-4, IL-6, IFN-gamma, and TNF-alpha production) is different in A-HBV, HBV-CAH, and AI-CAH disease. The increased cytokine secretion (IFN-gamma and TNF-alpha in A-HBV and IL-4 and IL-6 in AI-CAH) could reflect altered relative frequencies of different cell phenotypes in these diseases.. Specific cytokine production may be important in the pathophysiology associated with diverse inflammatory states in patients with hepatitis.

Topics: Adult; Autoimmune Diseases; Calcimycin; Concanavalin A; Cytokines; Female; Hepatitis B; Hepatitis, Chronic; Humans; In Vitro Techniques; Interferon-gamma; Interleukin-2; Interleukin-4; Interleukin-6; Leukocytes, Mononuclear; Male; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Mice homozygous for the lymphoproliferation (lpr) gene spontaneously develop autoimmune syndrome. These mice were characterized by the massive accumulation of double negative (DN) T cells. Although peripheral T cells in normal mice do not express J11d antigen, those abnormal DN T cells in autoimmune-prone mice express J11d antigen. In this study, the mechanisms that control the expression of J11d antigen are analyzed. High concentration of calcium ionophore alone induces the expression of J11d antigen, but not of CD4, CD8, and activation antigens such as interleukin 2 receptor as well as transferrin receptor by J11d- DN T cells from lpr mice. The expression of J11d antigen is primarily regulated at the transcription level rather than the post transcription level. Experiments using metabolic inhibitors reveal that the induction of J11d antigen requires the activation of not only a Ca2+/calmodulin- but also protein kinase C-dependent signaling pathway. Furthermore, J11d- DN thymocytes from control mice share the similar functional property with DN lpr T cells in J11d antigen inducibility.

Topics: Animals; Antigens, CD; Antigens, Differentiation; Autoimmune Diseases; Base Sequence; Calcimycin; CD24 Antigen; CD4 Antigens; CD8 Antigens; Female; Ionomycin; Lymphoproliferative Disorders; Membrane Glycoproteins; Mice; Molecular Sequence Data; T-Lymphocytes

We have examined the effects of FK-506 and of the struturally related macrolide rapamycin, which bind with high affinity to a specific binding protein (FKBP), to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized inflammatory mediators (sulfidopeptide leukotriene C4 and prostaglandin D2) from mast cells isolated from human lung parenchyma. FK-506 (0.1 to 300 nM) concentration dependently inhibited histamine release from lung parenchymal mast cells activated by anti-IgE. FK-506 was more potent in lung mast cells than in basophils (IC50 = 1.13 +/- 0.46 nM vs 5.28 +/- 0.88 nM; p less than 0.001), whereas the maximal inhibitory effect was higher in basophils than in lung mast cells (88.4 +/- 2.5% vs 76.4 +/- 3.8%; p less than 0.01). FK-506 had little or no inhibitory effect on histamine release from lung mast cells challenged with compound A23187, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 also inhibited the de novo synthesis of 5-lipoxygenase (sulfidopeptide leukotriene C4) and cyclo-oxygenase (prostaglandin D2) metabolites of arachidonic acid from mast cells challenged with anti-IgE. Unlike in basophils, Il-3 (3 to 30 ng/ml) did not modify anti-IgE- or A23187-induced histamine release from lung mast cells nor did it reverse the inhibitory effect of FK-506. Rapamycin (3 to 300 nM) had little or no effect on the release of histamine from lung mast cells, but it was a competitive antagonist of the inhibitory effect of FK-506 on anti-IgE-induced histamine release from human mast cells with a dissociation constant of about 12 nM. These data indicate that FK-506 is a potent anti-inflammatory agent that acts on human lung mast cells presumably by binding to a receptor site (i.e., FKBP).

Topics: Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Autoimmune Diseases; Calcimycin; Cells, Cultured; Histamine Release; Humans; Immunoglobulin E; Interleukin-3; Leukotrienes; Mast Cells; Polyenes; Sirolimus; Tacrolimus

Autoimmune-susceptible, MRL-lpr/lpr (lpr) mice develop a profound lymphadenopathy resulting from the accumulation of CD4-CD8- (double-negative, DN) cells in peripheral lymphoid organs. The source and the mechanism of this abnormal accumulation of cells is still unknown. Recently, we reported that a significant number (approximately 35%) of the CD4-CD8- cells expressed J11d, a marker expressed by immature thymocytes but not by mature functional peripheral T cells. In the present study, we investigated the phenotype, growth requirements, and functional properties of purified J11d+ and J11d- subpopulations. Using the mAb, F23.1, which recognizes a TCR determinant encoded by the V beta 8 gene family, it was observed that approximately 30% of the J11d+ and J11d- DN cells expressed this determinant. Further studies on the thymus revealed that J11d+ DN cells from lpr thymus also contained F23.1+ cells (approximately 25%), whereas, similar cells from normal MRL(-)+/+mice were all F23.1-, consistent with earlier reports in other normal strains. Further phenotypic studies revealed that the peripheral J11d+ and J11d- cells from lpr mice were similar in expressing CD3, Ly-5 (B220), and Ly-24 (Pgp-1) determinants. When stimulated with phorbol myristic acetate (PMA) and recombinant IL-2 (rIL-2), only J11d- cells but not J11d+ cells responded by proliferation. However, in the presence of calcium ionophore (A23187) and PMA, both J11d+ and J11d- subpopulations proliferated by producing and responding to endogenous IL-2 but not IL-4. The lymph node T cells from 1-month-old MRL-lpr/lpr mice responded strongly when stimulated with PMA + rIL-4 or PMA + rIL-6. In contrast both J11d+ and J11d- subpopulations failed to respond when similarly stimulated. The J11d+ but not J11d- cells demonstrated spontaneous cytotoxic activity against the NK-sensitive YAC-1 tumor targets. The J11d- cells did not exhibit cytotoxic potential in spite of culture with PMA + rIL-2. Even after repeated culture in vitro with PMA + A23187 or PMA + rIL-2, both J11d+ and J11d- subpopulations failed to express the mature phenotype bearing CD4 and/or CD8 antigens. The present study demonstrates the expansion of unique J11d+, alpha beta-TCR+, DN T cells with cytotoxic potential in lpr mice and further suggests the existence of phenotypic and functional heterogeneity among the abnormal lpr DN cells.

Topics: Animals; Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; Antigens, Ly; Autoimmune Diseases; Calcimycin; CD4 Antigens; CD8 Antigens; Cell Differentiation; Cytotoxicity, Immunologic; Flow Cytometry; Interleukin-2; Interleukin-4; Interleukin-6; Lymphocyte Activation; Mice; Mice, Mutant Strains; Receptors, Antigen, T-Cell; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Interleukin-2; T-Lymphocytes; Tetradecanoylphorbol Acetate

Peripheral blood lymphocytes from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), Reiter's disease, osteoarthritis, and from healthy volunteers were investigated for interferon-gamma (IFN-gamma) production after mitogen activation. Phytohaemagglutinin stimulation revealed an impaired IFN-gamma production in RA, SLE, and PSS but normal levels in Reiter's disease and osteoarthritis. In RA this deficiency was also seen after pokeweed mitogen, OKT3, and concanavalin A activation. No major differences were found in interleukin 2 (IL-2) production and cell proliferation. The IL-2 receptor expression was reduced on stimulated RA lymphocytes. The deficient IFN-gamma production was compensated in RA by co-stimulation of PHA or OKT3 with phorbol myristic acetate (PMA). In addition, the combination of the calcium ionophore A 23187 and PMA induced a strong IFN-gamma secretion in all patient groups and in the controls.

Topics: Adolescent; Adult; Aged; Arthritis, Reactive; Arthritis, Rheumatoid; Autoimmune Diseases; Calcimycin; Drug Synergism; Humans; Interferon-gamma; Lectins; Lupus Erythematosus, Systemic; Lymphocyte Activation; Middle Aged; Osteoarthritis; Receptors, Immunologic; Receptors, Interleukin-2; Scleroderma, Systemic; Tetradecanoylphorbol Acetate

The major population of cells that accumulate abnormally in MRL/Mp-lpr/lpr lymphoid tissue is Thy-1+, L3T4-, and Lyt-2-. To clarify the functional potential of these cells, we examined their proliferation, interleukin 2 (IL 2) receptor expression, and IL 2 secretion by using as stimulants the combination of 12-O-tetradecanoylphorbol-2-acetate and A23187 (a calcium ionophore). Although the lpr T cells were capable of responding to these stimulants, the nature of the response and of the concentrations of ligand required differed sharply from the responses of normal adult T cells, and of adult L3T4-Lyt-2- thymocytes. There was a strong similarity but not identity when responses of 16 day fetal thymocytes were compared with those of lpr L3T4-Lyt-2- cells. The unusual functional properties of the lpr cells, such as high A23187 dose requirement for maximal proliferation, low percentage of IL 2 receptor-expressing cells, and low levels of IL 2 secretion, suggested that these cells are arrested at a stage of development similar to that of 16-day fetal thymocytes and before adult L3T4-/Lyt-2- thymocytes.

Topics: Animals; Autoimmune Diseases; Calcimycin; Interleukin-2; Lymphocyte Activation; Mice; Mice, Mutant Strains; Receptors, Immunologic; Receptors, Interleukin-2; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thymus Gland

Treatment of splenocytes and lymph node cells of 5 month-old MRL/lpr mice with TPA induced IL-2-dependent proliferation of the cells in the presence of CA++. The induced response was inhibited completely by a monoclonal antibody to IL-2 receptor. The combination of TPA and A23187 in the lpr cells induced both proliferation and production of IL-2 in a Ca++-dependent fashion. The proliferative response of the lpr cells was equivalent to that of congenic (MRL/+/+) or normal cells, but the quantity of IL-2 secreted from the lpr cells was significantly less than that of the controls. Actinomycin D, but not mitomycin C, blocked IL-2 secretion from the treated lpr cells indicating de novo synthesis of IL-2 mRNA in the cells. Thus the lpr lymphocytes can be activated to proliferate in response to IL-2, yet they do not secrete IL-2 at a normal level even if activation signals are transmitted into the cells.

Topics: Animals; Autoimmune Diseases; Calcimycin; Calcium; Calcium Channel Blockers; Dactinomycin; Egtazic Acid; Interleukin-2; Lymph Nodes; Lymphocyte Activation; Lymphocytes; Mice; Mice, Mutant Strains; Mitomycin; Mitomycins; Receptors, Immunologic; Receptors, Interleukin-2; Spleen; Tetradecanoylphorbol Acetate

Topics: Aging; Animals; Autoimmune Diseases; Calcimycin; Concanavalin A; DNA; Gene Expression Regulation; Interleukin-2; Lymphocyte Activation; Male; Mice; Proto-Oncogenes; Receptors, Immunologic; Receptors, Interleukin-2; Tetradecanoylphorbol Acetate