Lipoteichoic acid induces nuclear factor-kappaB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages

We previously demonstrated that lipoteichoic acid (LTA) might activate phosphatidylcholine-phospholipase C (PC-PLC) and phosphatidylinositol-phospholipase C (PI-PLC) to induce protein kinase C activation, which in turn initiates nuclear factor-kappaB (NF-kappaB) activation and finally induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release in RAW 264.7 macrophages. In this study, we further investigated the roles of tyrosine kinase, phosphatidylinositiol 3-kinase (PI3K)/Akt, and p38 mitogen-activated protein kinase (MAPK) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Tyrosine kinase inhibitors (genistein and tyrphostin AG126), PI3K inhibitors (wortmannin and LY 294002), and a p38 MAPK inhibitor (SB 203580) attenuated LTA-induced iNOS expression and NO release in concentration-dependent manners. Treatment of RAW 264.7 macrophages with LTA caused time-dependent activations of Akt and p38 MAPK. The LTA-induced Akt activation was inhibited by wortmannin, LY 294002, genistein, and tyrphostin AG126. The LTA-induced p38 MAPK activation was inhibited by genistein, tyrphostin AG126, wortmannin, LY 294002, and SB 203580. The LTA-induced formation of an NF-kappaB-specific DNA-protein complex in the nucleus was inhibited by wortmannin, LY 294002, genistein, tyrphostin AG126, and SB 203580. Treatment of macrophages with LTA caused an increase in kappaB-luciferase activity, and this effect was inhibited by tyrphostin AG126, wortmannin, LY 294002, the Akt dominant negative mutant (AktDN), and SB 203580. Based on those findings, we suggest that LTA might activate the PI3K/Akt pathway through tyrosine kinase to induce p38 MAPK activation, which in turn initiates NF-kappaB activation, and ultimately induces iNOS expression and NO release in RAW 264.7 macrophages.     

Sesamin inhibits lipopolysaccharide-induced cytokine production by suppression of p38 mitogen-activated protein kinase and nuclear factor-kappaB

Sesame seed oil increases the survival after cecal ligation and puncture in mice and the increased IL-10 levels with non-lethal lipopolysaccharides (LPS) challenge. We showed that sesamin and sesamolin, major lignans of sesame oil, regulated LPS-induced nitric oxide production in the murine microglia and BV-2 cell line. In this study, we studied the effect of sesamin on cytokine production by LPS stimulation. The result showed that sesamin significantly inhibited LPS-stimulated IL-6 mRNA and protein, and to a lesser degree TNF-alpha, in BV-2 microglia. Sesamin and sesamolin also reduced LPS-activated p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB activations. Furthermore, SB203580, a specific inhibitor of p38 MAP kinase, specifically inhibited LPS-induced IL-6 production. These results suggest that sesamin inhibited LPS-induced IL-6 production by suppression of p38 MAPK signal pathway and NF-kappaB activation.     

p38 MAP kinase regulates TNFα-, IL-1α- and PAF-induced RANTES and GM-CSF production by human bronchial epithelial cells

BACKGROUND: RANTES and granulocyte macrophage-colony stimulating factor (GM-CSF) play an important role in the production of allergic inflammation of the airway through their chemotactic activity for eosinophils. Recent studies have indicated that p38 mitogen-activated protein (MAP) kinase regulates cytokine expression in various cells; however, the role of p38 MAP kinase in RANTES and GM-CSF production in human bronchial epithelial cells (BECs) has not yet been determined. OBJECTIVE: In the present study, we examined serine phosphorylation of MKK3 and MKK6 which is the upstream regulator of p38 MAP kinase and p38 MAP kinase activation in tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha and platelet-activating factor (PAF)-stimulated BECs and the effect of SB 203580 as the specific inhibitor for p38 MAP kinase activity on RANTES and GM-CSF expression in order to clarify the intracellular signal regulating RANTES and GM-CSF production by human BECs. RESULTS: The results showed that TNF alpha, IL-1 alpha and PAF induced serine phosphorylation of MKK3 and MKK6, and p38 MAP kinase activation in BECs. SB 203580 inhibited p38 MAP kinase activity and RANTES and GM-CSF production by TNF alpha-, IL-1 alpha- or PAF-stimulated human BECs. CONCLUSIONS: These results indicate that p38 MAP kinase plays an important role in TNF alpha-, IL-1 alpha- or PAF-activated signalling pathway which regulates RANTES and GM-CSF production by BECs and that the specific inhibitor for p38 MAP kinase activity might be useful for the treatment of allergic inflammation of the airway.     

Paeoniflorin suppresses IL-33 production by macrophages

Abstract

Objective: Interleukin (IL)-33 has been attracting more and more attention as a new member of theIL-1 cytokine family in recent years. However, the underlying mechanisms referred to the regulation of endogenous IL-33 production are not fully illustrated. Paeoniflorin (PF) has been reported to possess multiple pharmacological activities, including anti-inflammation and anti-allergy. In this study, we aimed to investigate the effect of PF on IL-33 production by macrophages and explore the underlying mechanisms.

Methods: In vivo, IL-33 production in mice after lipopolysaccharide (LPS) injection together with PF application was detected by enzyme-linked immunosorbent assay (ELISA). In vitro, MTT, Real-time PCR, ELISA, Calcium (Ca²⁺) imaging and Western blot were used to assess the cytotoxicity of PF, IL-33 expression at mRNA and protein levels, Ca²⁺ influx, protein kinase C (PKC) activity, nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) activation in LPS-stimulated RAW264.7 macrophages with PF administration.

Results: Our results indicated that PF (5 and 25?mg/kg) significantly reduced the production of TNF-a, IL-1β, and IL-33 in the peritoneal exudate of LPS-treated mice. In vitro assay, upregulation of PF concentration (≥ 20?μM) showed an increased cytotoxicity in RAW264.7 cells during the 24-h cell culture. PF (10?μM) inhibited IL-33 production, Ca²⁺ influx, PKC activity, NF-κB (p65) activation, and P38MAPK phosphorylation in LPS-treated macrophages. Notably, NF-κB inhibitor (BAY 11-7085), P38MAPK inhibitor (SB203580), and Ca²⁺ blocker (NiCl₂) also curbed LPS-induced IL-33 production, respectively.

Conclusions: PF suppresses IL-33 production by macrophages via inhibiting NF-κB and P38MAPK activation associated with the regulation of Ca²⁺ mobilization.     

Inhibitory effect of linomide on lipopolysaccharide-induced proinflammatory cytokine tumor necrosis factor-alpha production in RAW264.7 macrophages through suppression of NF-kappaB, p38, and JNK activation

Linomide is an immunomodulator that can effectively inhibit the development of several autoimmune diseases in animal models. Previously, linomide was shown to influence macrophage function, although the mechanism was elusive. In this study, we investigated the effect of linomide on the macrophage inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), production induced by lipopolysaccharide (LPS) in vitro on the murine macrophage cell line, RAW264.7. Linomide exposure reduced LPS-evoked TNF-alpha production in a dose-dependent manner. Gel shift and reporter gene analyses revealed linomide inhibited LPS-induced NF-kappaB binding to the NF-kappaB consensus oligonucleotide and NF-kappaB-mediated reporter gene expression. Immunoblot analysis showed that linomide inhibited phosphorylation of p38 kinase and c-jun N terminal kinase (JNK) in LPS-stimulated RAW264.7 cells. Taken together, these results suggest that linomide inhibits TNF-alpha production by suppressing the activation of NF-kappaB and mitogen-activated protein kinase (MAPK), which might, at least in part, contribute to the beneficial effects of linomide in the treatment of autoimmune diseases.     

Differential regulation of mast cell cytokines by both dexamethasone and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580

Activated mast cells generate multiple cytokines but it is not known if these can be differentially regulated by pharmacological agents. We report here that the glucocorticoid dexamethasone (DEX) preferentially inhibited Ag-induced expression of IL-4 and IL-6 mRNA relative to TNF-alpha mRNA in RBL-2H3 cells. Likewise, the drug more readily inhibited release of IL-4 than TNF-alpha protein. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), enhanced Ag-induced TNF-alpha mRNA expression without affecting IL-4 or IL-6 mRNA. At the protein level, SB203580 exerted little effect on TNF-alpha release but inhibited IL-4 release; notably, the ratio of TNF-alpha : IL-4 increased markedly with the concentration of SB203580, confirming the differential regulation of these cytokines. PD98059, an inhibitor of MAPK kinase (MEK), a component of the p44/42 MAPK pathway, partially inhibited Ag-induced expression of mRNA for all three cytokines while cyclosporin A inhibited Ag-induced IL-4 and IL-6 mRNA more readily than TNF-alpha mRNA. Ag activation of the cells led to phosphorylation of p38 and p44/42 MAPK but this was not influenced by DEX. In conclusion, mast cell cytokines can be differentially regulated pre- and post-translationally by DEX and SB203580 but there does not appear to be a direct mechanistic link between the actions of these two drugs.     

ERK-MAP-kinases differentially regulate expression of IL-23 p19 compared with p40 and IFN-beta in Theiler's virus-infected RAW264.7 cells

Theiler's murine encephalomyelitis virus (TMEV) infection of macrophages induces a demyelinating disease (DD) in certain strains of mice that is similar to human multiple sclerosis. In contrast to IFN-beta, expression of IL-23 p19 and p40 subunits by macrophages in response to TMEV may contribute to DD. TMEV infection of macrophages likely induces IL-23 and IFN-beta by activating p38 or ERK MAP-kinases (MAPK) and the p38 substrate ATF-2 within 30 min. To determine the role of MAPKs in TMEV-induced IL-23 and IFN-beta expression, RAW264.7 cells were pretreated with SB203580 or U0126, inhibitors of p38 and ERK MAPKs, respectively. SB203580 significantly increased TMEV-induced p19 but decreased p40 expression. In contrast, U0126 decreased p19 and increased TMEV-induced p40 and IFN-beta expression. Interestingly, U0126 prolonged TMEV-induced ATF-2 activation to at least 3h. Thus ERK MAPKs regulate expression of TMEV-induced p19 differently than p40 and IFN-beta suggesting the benefits of U0126 in treatment of DD.     

The p38 mitogen-activated protein kinases can have opposing roles in the antigen-dependent or endotoxin-stimulated production of IL-12 and IFN-gamma.

The p38 mitogen-activated protein kinases (p38 MAPK) are activated in lymphocytes and acessory cells during innate and antigen-specific responses. We show that an inhibitor of two isoforms of p38 MAPK, SB 203580, inhibited the antigen-initiated production of IL-12, and IFN-gamma by cultures of splenic APC and naive CD4(+) T cells. Paradoxically, SB 203580 enhanced the LPS plus IFN-gamma-initiated production of IL-12 by peritoneal exudate macrophages, and the LPS-initiated of the production of both IL-12 and IFN-gamma by non-T non-B (scid) splenocytes. The enhancing effect of SB 203580 on the production of IL-12 by peritoneal exudate macrophages stimulated by LPS and IFN-gamma was dose dependent (EC(50) 0.3 microM), was only seen at lower concentrations of IFN-gamma and was due, at least in part, to a dose-dependent (IC(50) 0.3 microM) inhibition of the production of IL-10. These results indicate first, that p38 MAP kinase activity is required for the production of IL-10, as well as that of proinflammatory cytokines such as IL-12 and IFN-gamma, and, second, that the net effects of SB 203580 on the production of IL-12 and IFN-gamma can be positive or negative, depending on stimuli, cell populations, and levels of cytokines such as IFN-gamma and IL-10.     

Gram-positive and gram-negative bacteria do not trigger monocytic cytokine production through similar intracellular pathways

Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) and Staphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1alpha (IL-1alpha), IL-1ra, and tumor necrosis factor alpha (TNF-alpha) production, whereas blocking of the ERK1/2 pathways inhibited IL-1alpha, IL-1beta, and IL-1ra but not TNF-alpha production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1alpha production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.     

Macrophages produce IL-33 by activating MAPK signaling pathway during RSV infection

It has been reported that RSV infection can enhance IL-33 production in lung macrophages. However, little is known about specific signaling pathways for activation of macrophages during RSV infection. In the present study, by using real-time RT-PCR as well as western blot assay, it became clear that RSV infection can enhance not only the expression of mRNAs for MAPK molecules (including p38, JNK1/2, and ERK1/2), but also the levels of MAPK proteins in lung macrophages as well as RAW264.7 cells. Furthermore, infection with RSV resulted in an increased level of phosphorylated MAPK proteins in RAW264.7 cells, suggesting that MAPK signaling pathway may participate in the process of RSV-induced IL-33 secretion by macrophages. In fact, the elevated production of IL-33 in RAW264.7 was attenuated significantly by pretreatment of the cells with special MAPK inhibitor before RSV infection, further confirming the function of MAPKs pathway in RSV-induced IL-33 production in macrophages. In contrast, the expression of NF-κB mRNA as well as the production of NF-κB protein in lung macrophages and RAW264.7 cells was not enhanced markedly after RSV infection. Moreover, RSV infection failed to induce the phosphorylation of NF-κB in RAW264.7 cells, suggesting that NF-κB signaling pathway may be not involved in RSV-induced IL-33 production in macrophages. Conclusion, these results indicate that RSV-induced production of IL-33 in macrophages is dependent on the activation of MAPK signaling pathway.     

Induction of high mobility group box 1 release from serotonin-stimulated human umbilical vein endothelial cells

High mobility group box 1 (HMGB1) is a non-histone nuclear protein which is released from the nucleus of activated macrophages into the extracellular space in response to stimuli such as endotoxin or necrosis. The HMGB1 functions as a potent proinflammatory cytokine in the extracellular spaces. Recently, HMGB1 has been implicated in the progression of atherosclerosis. However, the association between HMGB1 and the development of atherosclerosis is poorly understood. Therefore, we examined whether serotonin (5-HT), a key factor involved in the development of atherosclerosis, induced HMGB1 release in human umbilical vein endothelial cells (HUVECs). We found that 5-HT induced the release of HMGB1 but not of ERK1/2 and JNK from HUVECs via the 5-HT receptor (5-HT1B)/p38 mitogen-activated protein kinase (MAPK) signaling pathway. The p38MAPK inhibitor SB203580 and the 5-HT1B antagonist GR55526 markedly inhibited HMGB1 release from 5-HT-stimulated HUVECs. The vascular endothelial growth factor (VEGF) derived from activated macrophages in atherosclerotic lesions also plays an important role in the progression of atherosclerosis. We found that HMGB1 induced VEGF production in macrophage-like RAW264.7 cells. HMGB1 induced the activation of p38MAPK, ERK1/2 and Akt. The PI3-kinase inhibitor LY294002 significantly inhibited VEGF production in HMGB1-stimulated macrophages, while other kinase inhibitors did not. These results suggest that HMGB1 release may contribute as a risk factor in the development and progression of atherosclerosis.     

p-Cymene Modulates In Vitro and In Vivo Cytokine Production by Inhibiting MAPK and NF-κB Activation

The present study was designed to investigate the effects of p-cymene on lipopolysaccharide (LPS)-induced inflammatory cytokine production both in vitro and in vivo. The production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) in LPS-stimulated RAW 264.7 cells and C57BL/6 mice was evaluated by sandwich ELISA. Meanwhile, the mRNA levels of cytokine genes were examined in vitro by semiquantitative RT-PCR. In a further study, we analyzed the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways by western blotting. We found that p-cymene significantly regulated TNF-α, IL-1β, and IL-6 production in LPS-stimulated RAW 264.7 cells. Furthermore, the levels of relative mRNAs were also found to be downregulated. In in vivo trail, p-cymene markedly suppressed the production of TNF-α and IL-1β and increased IL-10 secretion. We also found that p-cymene inhibited LPS-induced activation of extracellular signal receptor-activated kinase 1/2, p38, c-Jun N-terminal kinase, and IκBα. These results suggest that p-cymene may have a potential anti-inflammatory action on cytokine production by blocking NF-κB and MAPK signaling pathways.     

Activation of p38 MAPK by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells

Feline infectious peritonitis (FIP) is an invariably fatal disease of cats caused by systemic infection with a feline coronavirus (FCoV) termed feline infectious peritonitis virus (FIPV). The lethal pathology associated with FIP (granulomatous inflammation and T-cell lymphopenia) is thought to be mediated by aberrant modulation of the immune system due to infection of cells such as monocytes and macrophages. Overproduction of pro-inflammatory cytokines occurs in cats with FIP, and has been suggested to play a significant role in the disease process. However, the mechanism underlying this process remains unknown. Here we show that infection of primary blood-derived feline mononuclear cells by FIPV WSU 79-1146 and FIPV-DF2 leads to rapid activation of the p38 MAPK pathway and that this activation regulates production of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). FIPV-induced p38 MAPK activation and pro-inflammatory cytokine production was inhibited by the pyridinyl imidazole inhibitors SB 203580 and SC 409 in a dose-dependent manner. FIPV-induced p38 MAPK activation was observed in primary feline blood-derived mononuclear cells individually purified from multiple SPF cats, as was the inhibition of TNF-alpha production by pyridinyl imidazole inhibitors.     

p38 MAPK在周期性机械牵张诱导肺泡巨噬细胞表达HMGB1中的作用

目的： 研究p38 MAPK在周期性机械牵张诱导肺泡巨噬细胞(AM)表达高迁移率族蛋白B1(HMGB1)中的作用。方法：大鼠AM随机分为A、B、C 3组,A组为对照组;B组细胞施加20%牵张应变,牵张时间为4 h;C组细胞的牵张模式与B组相同,在牵张前用p38 MAPK特异性抑制剂SB203580(40 μmol/L)预处理2 h。利用RT-PCR法检测HMGB1 mRNA的表达,Western blotting检测HMGB1蛋白表达和p38 MAPK的活性。结果：与对照组相比,AM施加20%牵张应变可诱导HMGB1蛋白和mRNA表达明显增加、p38 MAPK活性明显增高(均P<0.05),SB203580可显著抑制牵张应变的这种诱导作用(均P<0.05)。结论：周期性机械牵张可能通过p38 MAPK信号通路,调节肺泡巨噬细胞HMGB1 mRNA和蛋白的表达。     

Modulation of TLR signalling by the C-terminal Src kinase (Csk) in macrophages

In macrophages and monocytes, lipopolysaccharide (LPS) triggers the production of pro-inflammatory cytokine through Toll-like receptor (TLR) 4. Although major TLR signalling pathways are mediated by serine or threonine kinases including IKK, TAK1, p38 and JNKs, a number of reports suggested that tyrosine phosphorylation of intracellular proteins is involved in LPS signalling. Here, we identified several tyrosine-phosphorylated proteins using mass spectrometric analysis in response to LPS stimulation. Among these proteins, we characterized C-terminal Src kinase (Csk), which negatively regulates Src-like kinases in RAW 264.7 cells using RNAi knockdown technology. Unexpectedly, LPS-induced CD40 activation and the secretion of pro-inflammatory cytokine such as IL-6 and TNF-alpha, was down-regulated in Csk knockdown cells. Furthermore, overall cellular tyrosine phosphorylation and TLR4-mediated activation of IkappaB-alpha, Erk and p38 but not of JNK, were also down-regulated in Csk knockdown cells. The protein expression levels of a tyrosine kinase, Fgr, were reduced in Csk knockdown cells, suggesting that Csk is a critical regulator of TLR4-mediated signalling by modifying the levels of Src-like kinases.