Raf1 plays a pivotal role in lipopolysaccharide-induced activation of dendritic cells

Activation of extracellular-regulated kinases 1/2 (ERK) is involved in lipopolysaccharide (LPS)-induced cellular responses such as the increased production of proinflammatory cytokines. However, mitogen-activated protein kinases (MAPKs) such as p38 are also activated by LPS and have been postulated to be important in the control of these end points. Therefore, establishing the relative contribution of MAPKs in each cell type is important, as is elucidating the molecular mechanisms by which these MAPKs are activated in LPS-induced signaling cascades. We demonstrated in DC2.4 dendritic cells that ERK regulates tyrosine phosphorylation of phosphatidyl-inositol-3-kinase (PI3-K) and the production of TNF-alpha. We also demonstrated that Raf1 is phosphorylated and involved in the production of TNF-alpha and tyrosine phosphorylation of PI3-K via ERK. Raf1 also regulates the activation of NF-kappaB. We propose that Raf1 plays a pivotal role in LPS-induced activation of the dendritic cells.     

Polycomb group protein Bmi1 negatively regulates IL-10 expression in activated macrophages

Macrophages exert a wide variety of functions, which necessitate a high level of plasticity on the chromatin level. In the work presented here, we analyzed the role of the polycomb group protein Bmi1 during the acute response of bone marrow derived macrophages (BMDM) to lipopolysaccharide (LPS). Unexpectedly, we observed that Bmi1 was rapidly induced at the protein level and transiently phosphorylated upon LPS treatment. The induction of Bmi1 was dependent on MAP-kinase signaling. LPS treatment of BMDM in the absence of Bmi1 resulted in a pronounced increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Our results identify Bmi1 as a repressor of IL-10 expression during macrophage activation.     

GM-CSF plays a key role in zymosan-stimulated human dendritic cells for activation of Th1 and Th17 cells

In this study, we compared the effects of zymosan and LPS on human monocyte-derived dendritic cells. The specific effects of zymosan on the expression of several key cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins (IL-1α, IL-1β and IL-12 p70) were quite distinct from the effects of LPS. Unlike activation with LPS, DCs activated by zymosan expressed little or no IL-12 p70 due to lack of expression of the p35 subunit. However, treatment with zymosan resulted in a substantial increase in Th1 and Th17 cell-polarizing capacity of DCs. Furthermore, the GM-CSF secreted by zymosan-activated DCs enhanced IL-23 production, resulting in activation of a Th17 response. GM-CSF and IL-27, rather than IL-12 p70, were both major direct contributors to the activation of a Th1 response. This signaling mechanism is distinct and yet complementary to LPS-mediated T-cell activation. We suggest that this novel zymosan-induced GM-CSF-mediated signaling network may play a key role in regulating specific immune cell type activities.     

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.     

ERK and p38 MAPK signaling pathways negatively regulate CIITA gene expression in dendritic cells and macrophages

The CIITA is a master regulator for MHC class II expression, but the signaling events that control CIITA expression remain poorly understood. In this study, we report that both constitutive and IFN-gamma-inducible expression of CIITA in mouse bone marrow-derived dendritic cells (DC) and macrophages, respectively, are regulated by MAPK signals. In DC, the inhibitory effect of LPS on CIITA expression was prevented by MyD88 deficiency or pharmacological MAPK inhibitors specific for MEK (U0126) and p38 (SB203580), but not JNK (SP600125). In macrophages, LPS inhibited IFN-gamma-inducible CIITA and MHC class II expression without affecting expression of IFN regulatory factor-1 and MHC class I. Blocking ERK and p38 by MAPK inhibitors not only rescued LPS-mediated inhibition, but also augmented IFN-gamma induction of CIITA. Moreover, the induction of CIITA by IFN-gamma was enhanced by overexpressing MAPK phosphatase-1 that inactivates MAPK. Conversely, CIITA expression was attenuated in the absence of MAPK phosphatase-1. The down-regulation of CIITA gene expression by ERK and p38 was at least partly due to decreased histone acetylation of the CIITA promoter. Our study indicates that both MAPK and phosphatase play an important role for CIITA regulation in DC and macrophages.     

Annexin 1 negatively regulates IL-6 expression via effects on p38 MAPK and MAPK phosphatase-1

Annexin 1 (Anx-1) is a mediator of the anti-inflammatory actions of glucocorticoids, but the mechanism of its anti-inflammatory effects is not known. We investigated the role of Anx-1 in the regulation of the proinflammatory cytokine, IL-6. Lung fibroblast cell lines derived from Anx-1(-/-) and wild-type (WT) mice were treated with dexamethasone and/or IL-1. IL-6 mRNA and protein were measured using real-time PCR and ELISA, and MAPK pathway activation was studied. Compared with WT cells, unstimulated Anx-1(-/-) cells exhibited dramatically increased basal IL-6 mRNA and protein expression. In concert with this result, Anx-1 deficiency was associated with increased basal phosphorylated p38, JNK, and ERK1/2 MAPKs. IL-1-inducible phosphorylated p38 was also increased in Anx-1(-/-) cells. The increase in IL-6 release in Anx-1(-/-) cells was inhibited by inhibition of p38 MAPK. Anx-1(-/-) cells were less sensitive to dexamethasone inhibition of IL-6 mRNA expression than WT cells, although inhibition by dexamethasone of IL-6 protein was similar. MAPK phosphatase-1 (MKP-1), a glucocorticoid-induced negative regulator of MAPK activation, was up-regulated by dexamethasone in WT cells, but this effect of dexamethasone was significantly impaired in Anx-1(-/-) cells. Treatment of Anx-1(-/-) cells with Anx-1 N-terminal peptide restored MKP-1 expression and inhibited p38 MAPK activity. These data demonstrate that Anx-1 is an endogenous inhibitory regulator of MAPK activation and IL-6 expression, and that Anx-1 is required for glucocorticoid up-regulation of MKP-1. Therapeutic manipulation of Anx-1 could provide glucocorticoid-mimicking effects in inflammatory disease.     

Inhibition of interleukin-12 p40 transcription and NF-kappaB activation by nitric oxide in murine macrophages and dendritic cells

Nitric oxide (NO), an important effector molecule of the innate immune system, can also regulate adaptive immunity. In this study, the molecular effects of NO on the toll-like receptor signaling pathway were determined using interleukin-12 (IL-12) as an immunologically relevant target gene. The principal conclusion of these experiments is that NO inhibits IL-1 receptor-associated kinase (IRAK) activity and attenuates the molecular interaction between tumor necrosis factor receptor-associated factor-6 and IRAK. As a consequence, the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibits lipopolysaccharide (LPS)-induced IL-12 p40 mRNA expression, protein production, and promoter activity in murine macrophages, dendritic cells, and the murine macrophage cell line RAW 264.7. Splenocytes from inducible nitric-oxide synthase-deficient mice demonstrate markedly increased IL-12 p40 protein and mRNA expression compared with wild type splenocytes. The inhibitory action of NO on IL-12 p40 is independent of the cytokine IL-10. The effects of NO can be directly attributed to inhibition of NF-kappaB activation through IRAK-dependent pathways. Accordingly, SNAP strongly reduces LPS-induced NF-kappaB DNA binding to the p40 promoter and inhibits LPS-induced IkappaB phosphorylation. Similarly, NO attenuates IL-1beta-induced NF-kappaB activation. These experiments provide another example of how an innate immune molecule may have a profound effect on adaptive immunity.     

Differential effects of CpG DNA on IFN-beta induction and STAT1 activation in murine macrophages versus dendritic cells: alternatively activated STAT1 negatively regulates TLR signaling in macrophages

Classical STAT1 activation in response to TLR agonists occurs by phosphorylation of the Y701 and S727 residues through autocrine type I IFN signaling and p38 MAPK signaling, respectively. In this study, we report that the TLR9 agonist CpG DNA induced Ifn-beta mRNA, as well as downstream type I IFN-dependent genes, in a MyD88-dependent manner in mouse myeloid dendritic cells. This pathway was required for maximal TNF and IL-6 secretion, as well as expression of cell surface costimulatory molecules. By contrast, neither A- nor B-type CpG-containing oligonucleotides induced Ifn-beta in mouse bone marrow-derived macrophages (BMM) and a CpG-B oligonucleotide did not induce IFn-beta in the macrophage-like cell line, J774. In BMM, STAT1 was alternatively activated (phosphorylated on S727, but not Y701), and was retained in the cytoplasm in response to CpG DNA. CpG DNA responses were altered in BMM from STAT1(S727A) mice; Il-12p40 and Cox-2 mRNAs were more highly induced, whereas Tlr4 and Tlr9 mRNAs were more repressed. The data suggest a novel inhibitory function for cytoplasmic STAT1 in response to TLR agonists that activate p38 MAPK but do not elicit type I IFN production. Indeed, the TLR7 agonist, R837, failed to induce Ifn-beta mRNA and consequently triggered STAT1 phosphorylation on S727, but not Y701, in human monocyte-derived macrophages. The differential activation of Ifn-beta and STAT1 by CpG DNA in mouse macrophages vs dendritic cells provides a likely mechanism for their divergent roles in priming the adaptive immune response.     

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     

Cytokine expression and AIF-1-mediated activation of Rac2 in vascular smooth muscle cells: a role for Rac2 in VSMC activation

Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein involved in vascular smooth muscle cell (VSMC) migration and proliferation. The objective of this study is to characterize AIF-1 functional protein interactions that may regulate VSMC activation. Through use of a bacterial two-hybrid screen, we identified a molecular interaction between AIF-1 and the small GTPase, Rac2, which was verified by pull-down and colocalization experiments. This was unexpected in that Rac2 expression had been considered to be restricted to hematopoietic cells. The Rac2/AIF-1 interaction is functional, in that a loss-of-function, point-mutated AIF-1 does not interact with Rac2; Rac2 colocalizes with AIF-1 in the cytoplasm of VSMC and cotranslocates to lamellopodia upon platelet-derived growth factor stimulation; and AIF-1 expression in VSMC leads to Rac2 activation. Because Rac2 function in VSMC had not been described, we focused on characterization of its function in these cells. Rac2 protein expression in VSMC is inducible by inflammatory cytokines, and Rac2 activation in VSMC is also responsive to inflammatory cytokines. Rac2 expression and activation patterns differ from the ubiquitously expressed Rac1. We hypothesized that Rac2 participates in VSMC activation. Retroviral overexpression of Rac2 in primary VSMC leads to increased migration, activation of the NADPH oxidation cascade, and increased activation of the Rac2 effector protein Pak1 and its proximal effectors, ERK1/2, and p38 (P < 0.05 for all). The major points of this study indicate a functional interaction between AIF-1 and Rac2 in VSMC leading to Rac2 activation and a potential function for Rac2 in inflammation-driven VSMC response to injury. allograft inflammatory factor-1; signal transduction     

Thrombin induces NF-kappaB activation and IL-8/CXCL8 expression in lung epithelial cells by a Rac1-dependent PI3K/Akt pathway

We previously showed that thrombin induces interleukin (IL)-8/CXCL8 expression via the protein kinase C (PKC)α/c-Src-dependent IκB kinase α/β (IKKα/β)/NF-κB signaling pathway in human lung epithelial cells. In this study, we further investigated the roles of Rac1, phosphoinositide 3-kinase (PI3K), and Akt in thrombin-induced NF-κB activation and IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by a PI3K inhibitor (LY294002), an Akt inhibitor (1-L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), and the dominant negative mutants of Rac1 (RacN17) and Akt (AktDN). Treatment of cells with thrombin caused activation of Rac and Akt. The thrombin-induced increase in Akt activation was inhibited by RacN17 and LY294002. Stimulation of cells with thrombin resulted in increases in IKKα/β activation and κB-luciferase activity; these effects were inhibited by RacN17, LY294002, an Akt inhibitor, and AktDN. Treatment of cells with thrombin induced Gβγ, p85α, and Rac1 complex formation in a time-dependent manner. These results imply that thrombin activates the Rac1/PI3K/Akt pathway through formation of the Gβγ, Rac1, and p85α complex to induce IKKα/β activation, NF-κB transactivation, and IL-8/CXCL8 expression in human lung epithelial cells.     

RelB/p50 regulates TNF production in LPS-stimulated dendritic cells and macrophages

Dendritic cells (DCs) and macrophages are effective antigen-presenting cells, and DCs, once matured, have the ability to potently activate naïve T cells. While the canonical p65/p50 NF-κB pathway seems to have an important role during LPS-stimulation of these cells, the specific contribution of the non-canonical RelB/p50 subunits is not clear yet. We aimed to investigate the relevance of this pathway in DCs and macrophages by using replication-deficient adenoviruses overexpressing RelB and p50 subunits to test their effect on cytokine production. In both cells, after LPS treatment, overexpression of RelB and p50 inhibited the production of some pro-inflammatory cytokines e.g., TNF, but not of others e.g. IL6. Anti-inflammatory IL10 was not affected. Moreover, when overexpressing p50 alone, IL10 was increased in LPS-activated macrophages.We thus demonstrated that the dimer RelB/p50 rather than the p50/p50 complex inhibits TNF production in LPS-stimulated DCs and macrophages. This implies that the non-canonical RelB/p50 could modulate the canonical p65/p50 pathway.