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Acute activation of cells by tumor necrosis factor (TNF) has been well characterized, but little is known about later phases of TNF responses that are relevant for cells exposed to TNF for several days during inflammation. We found that prolonged exposure of human macrophages to TNF resulted in a wave of delayed but sustained activation of c-Jun and nuclear factor κB (NF-κB) proteins and of calcium oscillations that became apparent 1-3 d after TNF stimulation. These signaling events culminated in the induction and activation of the calcium-dependent transcription factor, nuclear factor of activated T cells (NFAT)c1, which mediated a gene expression program leading to cell fusion and osteoclast differentiation. TNF-induced NFATc1 activity primed macrophages for enhanced osteoclastogenesis in response to RANKL. High NFATc1 expression was apparent in synovial macrophages in a subset of patients with TNF-driven inflammatory arthritis. Thus, long-term exposure to TNF activates calcium-dependent signaling and an NFATc1-mediated gene activation program important for cell fusion and osteoclastogenesis. These findings identify a signaling pathway activated by TNF that is important for myeloid cell differentiation and suggest a role for TNF-induced calcium and NFAT signaling in chronic inflammation and associated bone resorption.

An important mechanism by which IFN-γ primes macrophages for enhanced innate immune responses is abrogation of feedback inhibitory pathways. Accordingly, IFN-γ abrogates endotoxin tolerance, a major negative feedback loop that silences expression of inflammatory cytokine genes in macrophages previously exposed to endotoxin/Toll-like receptor (TLR) ligands. Mechanisms by which IFN-γ inhibits endotoxin tolerance have not been elucidated. Here, we show that pretreatment with IFN-γ prevented tolerization of primary human monocytes and restored TLR4-mediated induction of various proinflammatory cytokines, including IL-6 and TNFα. Surprisingly, IFN-γ did not alter proximal TLR4 signaling defects in tolerized monocytes. Instead, IFN-γ blocked tolerance-associated down-regulation of IL6 and TNF transcription, RNA polymerase II recruitment, and NF-κB and CCAAT/enhancer-binding protein β transcription factor binding to the IL6 and TNF promoters in tolerized monocytes. The mechanism by which IFN-γ restored IL6 expression was by facilitating TLR4-induced recruitment of chromatin remodeling machinery to the IL6 promoter and promoting IL6 locus accessibility in tolerized monocytes. Our results suggest that IFN-γ overcomes endotoxin tolerance by facilitating TLR-induced chromatin remodeling to allow expression of proinflammatory genes. These results identify a mechanism by which IFN-γ promotes activation of macrophages and highlight the importance of chromatin remodeling and transcriptional control in the regulation of inflammatory cytokine production in tolerant and activated macrophages.

Glomerulonephritis is a major cause of morbidity in patients with systemic lupus erythematosus. Although substantial progress has been made in the identification of pathogenic triggers that result in autoantibody production, little is known about the pathogenesis of aggressive proliferative processes that lead directly to irreversible glomerular damage and compromise of renal function. In this study, we describe a model of polyinosinic: polycytidylic acid-accelerated lupus nephritis in NZB/W mice that is characterized by severe glomerular proliferative lesions with de novo crescent formation, findings that are linked with decreased survival and adverse outcomes in lupus. Proliferative glomerulonephritis was associated with infiltrating kidney macrophages and renal expression of IFN-inducible genes, matrix metalloproteinases (MMPs), and growth factors. Crescent formation and renal MMP and growth factor expression were dependent on renal macrophages that expressed Il10, MMPs, osteopontin, and growth factors, including Pdgfc and Hbegf. Infiltrating macrophages and renal MMP expression were induced by type I IFN. These findings reveal a role for type I IFNs and alternatively activated macrophages in aggressive proliferative lesions of lupus nephritis.

Cytokine signaling by the Jak-STAT pathway is subject to complex negative regulation that limits the amplitude and duration of signal transduction. Inhibition of signaling also mediates negative crosstalk, whereby factors with opposing biological activities crossinhibit each other's function. Here, we investigated a rapidly inducible mechanism that inhibited Jak-STAT activation by IFN-alpha, a cytokine that is important for antiviral responses, growth control, and modulation of immune responses. IFN-alpha-induced signaling and gene activation were inhibited by ligation of Fc receptors and Toll-like receptors 7 and 8 in a PKCbeta-dependent manner. Neither PKCbeta nor PKCdelta influenced responses of cells treated with IFN-alpha alone. Inhibition of IFN-alpha signaling correlated with suppression of IFN-alpha-dependent antiviral responses. PKC-mediated inhibition did not require de novo gene expression but involved the recruitment of PKCbeta to the IFN-alpha receptor and interaction with protein tyrosine phosphatase SHP-2, resulting in augmented phosphatase activity. PKC-mediated inhibition of IFN-alpha signaling was abolished in SHP-2-deficient cells, demonstrating a pivotal role for SHP-2 in this inhibitory pathway. Together, our data describe a rapidly inducible, direct mechanism of inhibition of Jak-STAT signaling mediated by a PKCbeta-SHP-2 signaling pathway.

Glucocorticoids (GCs) are potent anti-inflammatory agents that block cytokine production. We investigated whether GCs also block cytokine signaling via the Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway. Dexamethasone inhibited IL-2-induced DNA binding, tyrosine phosphorylation, and nuclear translocation of Stat5 in primary T cells. Inhibition of Stat5 correlated with inhibition of expression of IL-2-inducible genes and T cell proliferation. The mechanism of inhibition involved suppression of IL-2 receptor and Jak3 expression. Signaling by IL-4, IL-7, and IL-15, which use IL-2 receptor components, also was inhibited, indicating a block in T cell responses similar to that seen in immunodeficient patients lacking the IL-2 receptor gamma chain or Jak3. IL-2 signaling also was blocked in patients after treatment with GCs, suggesting that inhibition of cytokine signaling contributes to the clinical efficacy of these agents. These results identify inhibition of Jak-STAT signaling by IL-2 and related cytokines as a novel mechanism of GC action and suggest that inhibition of both cytokine production and signaling contribute to their therapeutic potency.

Gene activation and cellular differentiation induced by interleukin-6 (IL-6) and transcription factor Stat3 are suppressed by several factors, including ionomycin, granulocyte/macrophage-colony-stimulating factor, and phorbol 12-myristate 13-acetate (PMA), that block IL-6-induced Stat3 activation. These inhibitory agents activate mitogen activated protein kinases (MAPKs), and thus the role of MAPKs in the mechanism of inhibition of Stat3 activation was investigated. Inhibition of IL-6-induced Stat3 activation by PMA and ionomycin was rapid (within 5 min) and did not require new RNA or protein synthesis. Inhibition of Stat3 DNA-binding activity and tyrosine phosphorylation by PMA, ionomycin, and granulocyte/macrophage-colony-stimulating factor was reversed when activation of the extracellular signal-regulated kinase (ERK) group of MAPKs was blocked by using specific kinase inhibitors. Expression of constitutively active MEK1, the kinase that activates ERKs, or overexpression of ERK2, but not JNK1, inhibited Stat3 activation. Inhibition of Stat3 correlated with suppression of IL-6-induction of a signal transducer and activator of transcription (STAT)-dependent reporter gene. In contrast to IL-6, activation of Stat3 by interferon-alpha was not inhibited. MEKs and ERKs inhibited IL-6 activation of Stat3 harboring a mutation at serine-727, the major site for serine phosphorylation, similar to inhibition of wild-type Stat3, and inhibited Janus kinases Jak1 and Jak2 upstream of Stat3 in the Jak-STAT-signaling pathway. These results demonstrate an ERK-mediated mechanism for inhibiting IL-6-induced Jak-STAT signaling that is rapid and inducible, and thus differs from previously described mechanisms for downmodulation of the Jak-STAT pathway. This inhibitory pathway provides a molecular mechanism for the antagonism of Stat3-mediated IL-6 activity by factors that activate ERKs.

An important component of cytokine regulation of cell growth and differentiation is rapid transcriptional activation of genes by the JAK-STAT (signal transducer and activator of transcription) signaling pathway. Ligation of cytokine receptors results in tyrosine phosphorylation and activation of receptor-associated Jak protein tyrosine kinases and cytoplasmic STAT transcription factors, which then translocate to the nucleus. We describe the interruption of cytokine triggered JAK-STAT signals by cAMP, the calcium ionophore ionomycin, and granulocyte/macrophage colony-stimulating factor. Jak1 kinase activity, interleukin 6-induced gene activation, Stat3 tyrosine phosphorylation, and DNA-binding were inhibited, as was activation of Jak1 and Stat1 by interferon gamma. The kinetics and requirement for new RNA and protein synthesis for inhibition of interleukin 6 by ionomycin and GM-CSF differed, but both agents increased the association of Jak1 with protein tyrosine phosphatase ID (SH2-containing phosphatase 2). Our results demonstrate that crosstalk with distinct signaling pathways can inhibit JAK-STAT signal transduction, and suggest approaches for modulating cytokine activity during immune responses and inflammatory processes.