Inhibition of interferon (IFN) gamma-induced Jak-STAT1 activation in microglia by vasoactive intestinal peptide: inhibitory effect on CD40, IFN-induced protein-10, and inducible nitric-oxide synthase expression

Interferon (IFN)-gamma is one of the most important microglia stimulators in vivo participating in inflammation and Th1 activation/differentiation. IFN-gamma-mediated signaling involves the activation of the Jak/STAT1 pathway. The neuropeptides vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptide (PACAP) are two potent microglia-deactivating factors that inhibit the production of proinflammatory mediators in vitro and in vivo. The present study investigated the molecular mechanisms involved in the VIP/PACAP regulation of several IFN-gamma-induced microglia-derived factors, including IFN-gamma-inducible protein-10 (IP-10), inducible nitric-oxide synthase (iNOS), and CD40. The results indicate that VIP/PACAP inhibit Jak1-2 and STAT1 phosphorylation, and the binding of activated STAT1 to the IFN-gamma activated site motif in the IFN regulatory factor-1 and CD40 promoter and to the IFN-stimulated response element motif of the IP-10 promoter. Through its effect in the IFN-gamma-induced Jak/STAT1 pathway, VIP and PACAP are able to control the gene expression of IP-10, CD40, and iNOS, three microglia-derived mediators that play an essential role in several pathologies, i.e. inflammation and autoimmune disorders. The effects of VIP/PACAP are mediated through the specific receptor VPAC1 and the cAMP/protein kinase A transduction pathway. Because IFN-gamma is a major stimulator of innate and adaptive immune responses in vivo, the down-regulation of IFN-gamma-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response in the central nervous system by endogenous neuropeptides.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide prevent inducible nitric oxide synthase transcription in macrophages by inhibiting NF-kappa B and IFN regulatory factor 1 activation

High-output nitric oxide (NO) production from activated macrophages, resulting from the induction of inducible NO synthase (iNOS) expression, represents a major mechanism for macrophage cytotoxicity against pathogens. However, despite its beneficial role in host defense, sustained high-output NO production was also implicated in a variety of acute inflammatory diseases and autoimmune diseases. Therefore, the down-regulation of iNOS expression during an inflammatory process plays a significant physiological role. This study examines the role of two immunomodulatory neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), on NO production by LPS-, IFN-gamma-, and LPS/IFN-gamma-stimulated peritoneal macrophages and the Raw 264.7 cell line. Both VIP and PACAP inhibit NO production in a dose- and time-dependent manner by reducing iNOS expression at protein and mRNA level. VPAC1, the type 1 VIP receptor, which is constitutively expressed in macrophages, and to a lesser degree VPAC2, the type 2 VIP receptor, which is induced upon macrophage activation, mediate the effect of VIP/PACAP. VIP/PACAP inhibit iNOS expression and activity both in vivo and in vitro. Two transduction pathways appear to be involved, a cAMP-dependent pathway that preferentially inhibits IFN regulatory factor-1 transactivation and a cAMP-independent pathway that blocks NF-kappa B binding to the iNOS promoter. The down-regulation of iNOS expression, together with previously reported inhibitory effects on the production of the proinflammatory cytokines IL-6, TNF-alpha, and IL-12, and the stimulation of the anti-inflammatory IL-10, define VIP and PACAP as "macrophage deactivating factors" with significant physiological relevance.     

Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor.     

STAT5 induces macrophage differentiation of M1 leukemia cells through activation of IL-6 production mediated by NF-kappaB p65

We recently demonstrated that STAT5 can induce a variety of biological functions in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1, p21(WAF/Cip1), and suppressor of cytokine signaling-1/STAT-induced STAT inhibitor-1/Janus kinase binding protein is responsible for induction of proliferation, differentiation, and apoptosis, respectively. In the present study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5 induces macrophage differentiation of mouse leukemic M1 cells through a distinct mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced cells contained sufficient concentrations of IL-6 to induce macrophage differentiation of parental M1 cells, and STAT3 was phosphorylated on their tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6 transactivated the IL-6 promoter, which was mediated by the enhanced binding of NF-kappaB p65 (RelA) to the promoter region of IL-6. These findings indicate that STAT5A cooperates with Rel/NF-kappaB to induce production of IL-6, thereby inducing macrophage differentiation of M1 cells in an autocrine manner. In summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic functions. Cytokines     

General nature of the STAT3-activated anti-inflammatory response

Although many cytokine receptors generate their signals via the STAT3 pathway, the IL-10R appears unique in promoting a potent anti-inflammatory response (AIR) via STAT3 to antagonize proinflammatory signals that activate the innate immune response. We found that heterologous cytokine receptor systems that activate STAT3 but are naturally refractory (the IL-22R), or engineered to be refractory (the IL-6, leptin, and erythropoietin receptors), to suppressor of cytokine signaling-3-mediated inhibition activate an AIR indistinguishable from IL-10. We conclude that the AIR is a generic cytokine signaling pathway dependent on STAT3 but not unique to the IL-10R.     

Protection against progressive leishmaniasis by IFN-beta

Type I IFNs (IFN-alphabeta) exert potent antiviral and immunoregulatory activities during viral infections, but their role in bacterial or protozoan infections is poorly understood. In this study, we demonstrate that the application of low, but not of high doses of IFN-beta protects 60 or 100% of BALB/c mice from progressive cutaneous and fatal visceral disease after infection with a high (10(6)) or low (10(4)) number of Leishmania major parasites, respectively. IFN-beta treatment of BALB/c mice restored the NK cell cytotoxic activity, increased the lymphocyte proliferation, and augmented the production of IFN-gamma and IL-12 in the draining lymph node. Low, but not high doses of IFN-beta caused enhanced tyrosine phosphorylation of STAT1 and STAT4, suppressed the levels of suppressor of cytokine signaling-1, and up-regulated the expression of inducible NO synthase in vivo. The IFN-beta-induced increase of IFN-gamma production was dependent on STAT4. Protection by IFN-beta strictly required the presence of inducible NO synthase. In the absence of STAT4 or IL-12, IFN-beta led to an amelioration of the cutaneous and visceral disease, but was unable to prevent its progression. These results identify IFN-beta as a novel cytokine with a strong, dose-dependent protective effect against progressive cutaneous leishmaniasis that results from IL-12- and STAT4-dependent as well as -independent events.     

Macrophage Resistance to HIV-1 Infection Is Enhanced by the Neuropeptides VIP and PACAP

It is well established that host factors can modulate HIV-1 replication in macrophages, critical cells in the pathogenesis of HIV-1 infection due to their ability to continuously produce virus. The neuropeptides VIP and PACAP induce well-characterized effects on macrophages through binding to the G protein-coupled receptors VPAC1, VPAC2 and PAC1, but their influence on HIV-1 production by these cells has not been established. Here, we describe that VIP and PACAP reduce macrophage production of HIV-1, acting in a synergistic or additive manner to decrease viral growth. Using receptor antagonists, we detected that the HIV-1 inhibition promoted by VIP is dependent on its ligation to VPAC1/2, whereas PACAP decreases HIV-1 growth via activation of the VPAC1/2 and PAC1 receptors. Specific agonists of VPAC2 or PAC1 decrease macrophage production of HIV-1, whereas sole activation of VPAC1 enhances viral growth. However, the combination of specific agonists mimicking the receptor preference of the natural neuropeptides reproduces the ability of VIP and PACAP to increase macrophage resistance to HIV-1 replication. VIP and PACAP up-regulated macrophage secretion of the β-chemokines CCL3 and CCL5 and the cytokine IL-10, whose neutralization reversed the neuropeptide-induced inhibition of HIV-1 replication. Our results suggest that VIP and PACAP and the receptors VPAC2 and PAC1 could be used as targets for developing alternative therapeutic strategies for HIV-1 infection.     

Reduction of myeloid-derived suppressor cells and induction of M1 macrophages facilitate the rejection of established metastatic disease

More than 60% of STAT6(-/-) mice immunologically reject spontaneous metastatic mammary carcinoma and survive indefinitely if their primary tumors are removed, whereas 95% of STAT6-competent BALB/c mice succumb to metastatic disease. BALB/c and STAT6-deficient mice with primary tumors have elevated levels of Gr1(+)CD11b(+) myeloid suppressor cells (MSCs), which inhibit T cell activation. After removal of primary tumor, MSC levels revert to baseline in STAT6-deficient mice, but remain elevated in BALB/c mice. The decrease is IFN-gamma dependent, as is the reduction in metastatic disease. Neither BALB/c nor STAT6-deficient MSCs produce inducible NO synthase; however, both produce arginase and reactive oxygen species. STAT6-deficient mice produce M1 macrophages, which contain high levels of NO and are tumoricidal, whereas BALB/c mice produce M2 macrophages, which make arginase and are not tumoricidal. Immunity in STAT6-deficient mice requires the activation of NO-producing M1 macrophages that are tumoricidal, the reduction in MSC levels to baseline after surgical removal of primary tumor, and the activation of tumor-specific T cells. These mechanisms occur in STAT6(-/-) mice because STAT6 deficiency prevents signaling through the type 2 IL-4Ralpha, thereby blocking the production of arginase and promoting the synthesis of NO.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide: players in innate and adaptive immunity.

Recent reports identified and described neural pathways, both hard-wiring and soluble mediators, that control and adjust the peripheral immune response. Immune organs are innervated by fibers rich in neurotransmitters and neuropeptides released in inflammatory conditions. Here we focus on the immunomodulatory role of two peptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP). VIP/PACAP are present and released from both innervation and immune cells, particularly Th2 cells, and immune cells express receptors for VIP/PACAP. VIP/PACAP have a general anti-inflammatory effect, both in innate and adaptive immunity. In innate immunity, VIP/PACAP inhibit the production of pro-inflammatory cytokines and chemokines from macrophages, microglia and dendritic cells. In addition, VIP/PACAP reduce the expression of costimulatory molecules (particularly CD80 and CD86) on the antigen-presenting cells, and therefore reduce stimulation of antigen-specific CD4+ T cells. In terms of adaptive immunity, VIP/PACAP promote Th2-type responses, and reduce the pro-inflammatory Th1-type responses. Several of the molecular mechanisms involved in the inhibition of cytokine and chemokine expression, and in the preferential development and/or survival of Th2 effects, are discussed.     

Vasoactive intestinal peptide inhibits cytokine production in T lymphocytes through cAMP-dependent and cAMP-independent mechanisms.

Previous reports indicate that VIP and the structurally related peptide PACAP, inhibit IL-2 and IL-10 production in antigen-stimulated T lymphocytes. Intracellular cAMP elevation appears to be the primary transduction pathway involved. However, in the lower concentration range, an additional, cAMP-independent transduction pathway appears to mediate the VIP inhibition of cytokine production. Here, we address this question by using VIP agonists and antagonists which act through cAMP-dependent and -independent pathways. The antagonists based on the neurotensin-VIP hybrid molecule did not affect the inhibitory effect of VIP/PACAP on IL-2 and IL-10 production, confirming that astrocytes and T lymphocytes express different receptors. A lipophilic antagonist with increased membrane permeability, partially reversed the inhibitory effect of VIP/PACAP, forskolin, prostaglandin E2, and 8-bromo-cAMP without significantly affecting cAMP levels, suggesting that it acts downstream of cAMP. Two VIP agonists inhibit IL-2 and IL-10 production. One of the agonists increases cAMP, whereas the second one does not induce cAMP/cGMP. Our results indicate that VIP inhibits cytokine production in stimulated CD4+ T cells through two separate mechanisms, which involve both cAMP-dependent and cAMP-independent transduction pathways.     

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.