Pathways Regulation by STAT1Dependent Pamidronate in Murine Macrophages: Expression by IFN{gamma} and Modulation of TNF{alpha} Gene (original) (raw)
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The Journal of Immunology, 2005
Aminobisphosphonates are drugs used in the treatment of hypercalcemia, Paget's disease, osteoporosis, and malignancy. Some patients treated with aminobisphosphonates have a transient febrile reaction that may be caused by an increased serum concentration of proinflammatory cytokines. Aminobisphosphonates induce the production of certain proinflammatory cytokines in vitro, especially in cells of monocytic lineage. A unique feature of aminobisphosphonates is that they bind the V␥2V␦2 class of T cells, which are found only in primates, and stimulate cytokine production. The effects of aminobisphosphonates on other cells, including macrophages, are incompletely understood. We show in this study that treatment of murine macrophages with pamidronate, a second generation aminobisphosphonate,
Journal of Immunology, 1997
IFN signaling is mediated by binding of IFNs to their receptors and subsequent activation of Janus tyrosine kinase (JAK)-STAT signaling pathway. Stimulation of cells with IFN-a leads to the assembly of IFN-stimulated gene factor 3 transcription factor complex formed by STAT1 , STATZ, and p48 protein. IFN-y signaling is mediated by homodimeric STATl protein. Although these signaling molecules are expressed constitutively, there is also evidence of transcriptional regulation by IFNs. We have characterized the expression of STAT and IFN regulatory factor (IRF) family transcription factors in primary human blood mononuclear cells and macrophages in response to IFN-a and IFN-y stimulation. We show that IFN-a and IFN-y rapidly and efficiently enhanced STATI, STAT2, p48, and IRF-1 gene expression. IFN-y induced IRF-1 gene expression more strongly than IFN-a. Stimulation experiments in the presence of protein synthesis inhibitor, cycloheximide, suggested that these genes were activated directly by IFNs. IRF-2 gene was apparently only weakly responsive to IFNs in these cells. When macrophages were pretreated with low doses of IFN-y and then stimulated with IFN-a, clearly enhanced formation of specific transcription factor complexes was detected. This suggests that higher intracellular levels of STAT1, STATZ, and p48 protein may result in enhanced signal transduction for cytokines utilizing these transcription factors. C ytokine signal transduction from the cell surface receptor to the nucleus is mediated by receptor-associated Janus family tyrosine kinases (JAKs)' and STAT proteins (the JAK-STAT pathway). To date, four JAK kinase genes (JAKI, JAK2, JAK3, and TYK2) and seven STAT genes (STATI, 2, 3, 4, 5a, 5b, and 6) have been molecularly cloned. Receptor-associated ligand-activated JAKs phosphorylate STAT proteins on tyrosine residues, which leads to STAT protein activation, dimerization, translocation into the nucleus, and transcriptional activation of target genes. The IFN signaling system has functioned as a model for cytokine signaling in general (for reviews, see Refs. 1-3). IFNs are classified into type I (IFN-a,-p, and-m) and type I1 (IFN-y) IFNs and they have antiviral, antiproliferative, and immunomodulatory functions (4, 5). The two types of IFNs use distinct cell surface receptors, but their signal transduction pathways are partially overlapping. IFN-aP receptor-associated JAKl and TYK2 tyrosine phosphorylate STATl and STAT2 proteins. For full transcriptional activation, STATl also needs to be phosphorylated on a serine residue (6) by a serine-threonine kinase (mitogen-activated protein kinase) (7). Activated STATl and STAT2
British Journal of Pharmacology, 2001
Inducible NO synthase (iNOS) expression and activity were measured in the mouse macrophage cell line J774 after exposure to bacterial lipopolysaccharide (LPS) with or without interferon-g (IFN-g). 2 Inhibition of NOS activity by concomitant N G-monomethyl-L-arginine (L-NMMA) treatment further increased iNOS protein levels, with a substantial increase in iNOS half-life. 3 Western blotting and ELISA demonstrated that several cell proteins were tyrosine-nitrated when iNOS activity was high. 4 Rapid IFN-g-induced phosphorylation of STAT1 was reduced by about 40% when cells were pretreated to induce iNOS, unless L-NMMA was present during the pretreatment period. 2D gel electrophoresis demonstrated the presence of nitrotyrosine in STAT1 after iNOS induction, and con®rmed the reduction in phospho-STAT1 on subsequent stimulation with IFN-g for 15 min and its partial restoration when L-NMMA was present during the pretreatment period. 5 We did not detect tyrosine nitration of the upstream kinase JAK2 in LPS+IFN-g pretreated cells, but JAK2 activity was also impaired, and was partially restored by concomitant L-NMMA pretreatment. 6 We conclude that endogenous production of NO induces feedback inhibition of signalling pathways activated by IFN-g, at least in part by nitrating tyrosine residues in STAT1 which prevents phosphorylation.
Stat1 combines signals derived from IFN-γ and LPS receptors during macrophage activation
The EMBO Journal, 1998
Complete activation of macrophages during immune responses results from stimulation with the activating cytokine interferon-γ (IFN-γ) and a second stimulus, usually a microbial product. Bacterial infection of macrophages, or treatment with bacterial lipopolysaccharide (LPS), resulted in rapid Stat1 phosphorylation on Ser727 (S727) independently of concomitant tyrosine phosphorylation. IFN-γ also caused rapid phosphorylation of S727. In both situations, S727 phosphorylation was reduced by pre-treatment of cells with the serine kinase inhibitor H7. When macrophages were treated sequentially or simultaneously with LPS and IFN-γ, the pool of molecules phosphorylated on both Tyr701 (Y701) and S727 was strongly increased. Consistently, Stat1-dependent transcription in response to IFN-γ was significantly enhanced if the cells were pre-treated with bacterial LPS. The relative amount of S727-phosphorylated Stat1 in the non-tyrosine phosphorylated fraction was considerably smaller than that in the tyrosine-phosphorylated fraction. No evidence was found for an effect of S727 phosphorylation on the phosphorylation of Y701 by IFN-γ. Thus, serine and tyrosine phosphorylation of Stat1 are caused independently of each other, but the serine kinase may recognize tyrosine-phosphorylated Stat1 preferentially in the course of an IFN-γ response. The data suggest Stat1 to be a convergence point for immunological stimuli in a macrophage proinflammatory response.
Biologic consequences of Stat1-independent IFN signaling
Proceedings of the National Academy of Sciences, 2001
Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFN␥ functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFN␥ and IFN␣͞ regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFN␥ exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFN␥ receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences.
IFN-γ Suppresses STAT6 Phosphorylation by Inhibiting Its Recruitment to the IL-4 Receptor
The Journal of Immunology, 2005
Polarized Th1 cells show a stable phenotype: they become insensitive to IL-4 stimulation and lose the potential to produce IL-4. Previously, we reported that IFN-γ played a critical role in stabilizing Th1 phenotype. However, the mechanism by which IFN-γ stabilizes Th1 phenotype is not clear. In this study, we compared STAT6 phosphorylation in wild-type (WT) and IFN-γ receptor knockout (IFNGR−/−) Th1 cells. We found a striking diminution of STAT6 phosphorylation in differentiated WT Th1 cells, but not in differentiated IFNGR−/− Th1 cells. The impairment of STAT6 phosphorylation in differentiated WT Th1 cells was not due to a lack of IL-4R expression or phosphorylation. Jak1 and Jak3 expression and phosphorylation were comparable in both cell types. No differential expression of suppressor of cytokine signaling 1 (SOCS1), SOCS3, or SOCS5 was observed in the two cell types. In addition, Src homology 2-containing phosphatase mutation did not affect IL-4-induced STAT6 phosphorylation in...
Cellular Immunology, 2002
The L L -tryptophan catabolite, picolinic acid (PA), is an activator of macrophage effector functions and an inducer of macrophage inflammatory protein-1a (MIP-1a) and -1b (MIPs). We have investigated the regulation of PA-induced MIPs production in mouse macrophages. We demonstrated a dose-and time-dependent downregulation of MIPs mRNA by the Th1 cytokine, IFN-c, that was associated with inhibition of intracellular chemokine production and secretion. This effect was IFN-c-specific because MIPs induction was unaffected by the Th2 cytokines, IL-10 and IL-4, or the proinflammatory stimulus, LPS. Moreover, MIPs downregulation by IFN-c was dependent on both mRNA destabilization and gene transcription inhibition. These results demonstrate that MIP-1a=b production by macrophages is a tightly regulated process resulting from the interaction between inhibitory stimuli derived from the immune system and stimulatory signals of non-immunologic origin. The antagonistic effect of PA and IFN-c on MIPs production may be important for the regulation of the inflammatory responses in vivo. (M.C. Bosco).
The Journal of Immunology, 2000
The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two immunomodulatory neuropeptides that affect both innate and acquired immunity, down-regulate IL-12 p40 and inducible NO synthase expression in LPS/IFN-␥-stimulated macrophages. We showed previously that VIP/PACAP inhibit NF-B nuclear translocation through the stabilization of IB and reduce IFN regulatory factor-1 (IRF-1) binding to the regulatory elements found in the IL-12 p40 and inducible NO synthase promoters. In this paper we studied the molecular mechanisms involved in the VIP/PACAP regulation of IRF-1 transactivating activity. Our studies indicate that the inhibition in IRF-1 binding correlates with a reduction in IRF-1 protein and mRNA in IFN-␥-treated Raw 264.7 macrophages. In agreement with the described Janus kinase (Jak)1/ Jak2/STAT1/IRF-1 activation pathway, VIP/PACAP inhibit Jak1/Jak2, STAT1 phosphorylation, and the binding of STAT1 to the GAS sequence motif in the IRF-1 promoter. The effects of VIP/PACAP are mediated through the specific VIP/PACAP receptor-1 and the cAMP/protein kinase A (PKA) transduction pathway, but not through the induction of suppressor of cytokine signaling-1 or suppressor of cytokine signaling-3. Because IFN-␥ is a major stimulator of innate immune responses in vivo, the downregulation of IFN-␥-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response by endogenous neuropeptides.