IL-17 suppresses TNF-alpha-induced CCL27 production through induction of COX-2 in human keratinocytes

BACKGROUND: The chemokine CCL27 attracts skin-homing T cells. CCL27 production by keratinocytes is dependent on nuclear factor kappaB (NF-kappaB) activity and enhanced in lesions of patients with atopic dermatitis, psoriasis vulgaris, or allergic contact dermatitis. IL-17 is released from activated memory T cells and modulates skin inflammation. OBJECTIVE: We examined the in vitro effects of IL-17 on TNF-alpha-induced CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha, IL-17, or both. CCL27 secretion and mRNA levels were analyzed by means of ELISA and RT-PCR, respectively. COX-2 promoter and NF-kappaB activities were analyzed by using luciferase assays. COX-2 protein levels were analyzed by means of Western blotting. RESULTS: IL-17 suppressed TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity in keratinocytes. The COX-2 inhibitor NS398 counteracted the effects of IL-17, and prostaglandin E(2) prevented counteraction by NS398. IL-17 alone or synergistically with TNF-alpha increased prostaglandin E(2) release from keratinocytes, and the increase was suppressed by NS398. IL-17 alone or synergistically with TNF-alpha increased COX-2 mRNA and protein levels, promoter activity, and mRNA stability. The stimulatory effects of IL-17 on COX-2 expression were suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase. IL-17 alone or synergistically with TNF-alpha induced dual phosphorylation of p38 MAPK and ERK. CONCLUSION: IL-17 might suppress TNF-alpha-induced CCL27 production by inhibiting NF-kappaB through induction of COX-2. The induction of COX-2 might be mediated by activation of p38 MAPK and ERK. T cell-derived IL-17 might alleviate T-cell skin infiltration through inhibition of CCL27 production.     

Inhibition Of Ccl11, Ccl24, And Ccl26-induced Degranulation In Hl-60 Eosinophilic Cells By Specific Inhibitors Of Mek1/mek2, P38 Map Kinase,And Pi 3-Kinase

Eosinophilic leukocytes are the cellular hallmark of allergic inflammation. Apart from being potent eosinophils chemoattractants, the eotaxins CCL11, CCL24 and CCL26 are capable of activating eosinophils to generate reactive oxygen species, lipid mediators of inflammation and degranulation of toxic granule proteins. Due to their central role in eosinophil trafficking and activation, understanding the signal transduction mechanism of the eotaxin-induced eosinophil effector functions may provide an innovative therapeutic strategy for eosinophil-associated diseases. Thus, these investigations were conducted to delineate signal transduction mechanisms of CCL11, CCL24 and CCL26-induced eosinophil peroxidase (EPO) degranulation following pretreatment of cells with or without a specific inhibitor of MEK1/MEK2 (U0126), inhibitor of p38 MAP kinase (SB203580) or a specific inhibitor of PI 3-kinase (LY294002). Results have shown that CCR3-mediated eotaxin-induced eosinophilic degranulation was concentration-dependently reduced by specific inhibitors of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. However, the rank order of U0126 with respect to inhibition of chemokine-induced degranulation was CCL11=CCL24>CCL26. Potentiation of eotaxin-induced EPO degranulation by IL-5 was also seen. These investigations have not only confirmed the reported co-operativity between IL-5 and the eotaxins but also showed that the eosinophil-degranulating capabilities of the eotaxin CCL11, CCL24 and CCL26 is a consequence of activation of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. Thus, these signaling molecules may provide the biochemical basis for mechanism-based therapy of allergic inflammatory diseases.     

Distinct regulation of C3a-induced MCP-1/CCL2 and RANTES/CCL5 production in human mast cells by extracellular signal regulated kinase and PI3 kinase

Complement component C3a causes a robust degranulation in human mast cells. Whether C3a also stimulates chemokine production in human mast cells and what signaling pathway it activates is not known. In the present study, we demonstrate that CD34+ cell-derived primary mast cells and a human mast cell line LAD 2 express surface C3a receptors at similar levels. Furthermore, C3a caused approximately 50% internalization of cell surface C3a receptors in both cell types. We therefore used LAD 2 cells as a model to study C3a-induced biological responses and signaling in human mast cells. We found that C3a stimulated substantial degranulation and induced chemokine monocyte chemoattractant protein 1 (MCP-1/CCL2) and regulated upon activation, normal T-cell expressed and secreted (RANTES/CCL5) production in LAD 2 mast cells. C3a caused a rapid and sustained extracellular-signal-regulated kinase (ERK) phosphorylation and Akt phosphorylation in LAD 2 mast cells. Furthermore, U0126 and LY294002, which respectively inhibit MEK-induced ERK phosphorylation and PI3 kinase-mediated Akt phosphorylation had distinct effects on C3a-induced responses. Thus, U0126, which blocked C3a-induced RANTES/CCL5 production by 50.6+/-2.3%, inhibited MCP-1/CCL2 generation by 85.2+/-0.6%. In contrast, LY294002 had no effect on C3a-induced RANTES/CCL5 production but blocked MCP-1/CCL2 generation by 83.7+/-1.5%. These data demonstrate that C3a activates divergent signaling pathways to induce chemokine production in human mast cells.     

Inhibition of CCL11, CCL24, and CCL26-induced degranulation in HL-60 eosinophilic cells by specific inhibitors of MEK1/MEK2, p38 MAP kinase,and PI 3-kinase

Eosinophilic leukocytes are the cellular hallmark of allergic inflammation. Apart from being potent eosinophils chemoattractants, the eotaxins CCL11, CCL24 and CCL26 are capable of activating eosinophils to generate reactive oxygen species, lipid mediators of inflammation and degranulation of toxic granule proteins. Due to their central role in eosinophil trafficking and activation, understanding the signal transduction mechanism of the eotaxin-induced eosinophil effector functions may provide an innovative therapeutic strategy for eosinophil-associated diseases. Thus, these investigations were conducted to delineate signal transduction mechanisms of CCL11, CCL24 and CCL26-induced eosinophil peroxidase (EPO) degranulation following pretreatment of cells with or without a specific inhibitor of MEK1/MEK2 (U0126), inhibitor of p38 MAP kinase (SB203580) or a specific inhibitor of PI 3-kinase (LY294002). Results have shown that CCR3-mediated eotaxin-induced eosinophilic degranulation was concentration-dependently reduced by specific inhibitors of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. However, the rank order of U0126 with respect to inhibition of chemokine-induced degranulation was CCL11 = CCL24 > CCL26. Potentiation of eotaxin-induced EPO degranulation by IL-5 was also seen. These investigations have not only confirmed the reported co-operativity between IL-5 and the eotaxins but also showed that the eosinophil-degranulating capabilities of the eotaxin CCL11, CCL24 and CCL26 is a consequence of activation of ERK1/ERK2, p38 MAP kinase and PI 3-kinase. Thus, these signaling molecules may provide the biochemical basis for mechanism-based therapy of allergic inflammatory diseases.     

IL-18 enhances IFN-gamma-induced production of CXCL9, CXCL10, and CXCL11 in human keratinocytes

IL-18 is involved in the pathogenesis of atopic dermatitis, psoriasis, and allergic contact dermatitis. CXCL9, CXCL10, and CXCL11 recruit type 1 T cells, and the production of these chemokines by keratinocytes is enhanced in these dermatoses. We examined the in vitro effects of IL-18 on IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production in human keratinocytes. IL-18 enhanced the IFN-gamma-induced secretion and mRNA expression of CXCL9, CXCL10, and CXCL11 in parallel to the activation of NF-kappaB, STAT1, and IFN-regulatory factor (IRF)-1. Antisense oligonucleotides against NF-kappaB p50, p65, or STAT1 suppressed CXCL9, CXCL10, and CXCL11 production, and antisense IRF-1 suppressed CXCL11 production. Inhibitors of PI3 K, p38 MAPK, and MEK suppressed IL-18 plus IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production and NF-kappaB, STAT1, and IRF-1 activities. IL-18 induced phosphorylation of ERK and Akt, while IFN-gamma induced phosphorylation of p38 MAPK. These results suggest that IL-18 may potentiate IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production in keratinocytes by activating NF-kappaB, STAT1, or IRF-1 through PI3 K/Akt and MEK/ERK pathways. These effects of IL-18 may promote the infiltration of type 1 T cells into lesions with inflammatory dermatoses and amplify the skin inflammation. IL-18 may act as a pro-inflammatory cytokine in these dermatoses and thus is a candidate therapeutic target.     

Signaling events involved in cytokine and chemokine production induced by secretory phospholipase A2 in human lung macrophages

Secretory phospholipases A(2) (sPLA(2)) are enzymes released during inflammatory reactions. These molecules activate immune cells by mechanisms either related or unrelated to their enzymatic activity. We examined the signaling events activated by group IA (GIA) and group IB (GIB) sPLA(2) in human lung macrophages leading to cytokine/chemokine production. sPLA(2) induced the production of cytokines (TNF-alpha, IL-6 and IL-10) and chemokines (CCL2, CCL3, CCL4 and CXCL8), whereas no effect was observed on IL-12, CCL1, CCL5 and CCL22. sPLA(2) induced the phosphorylation of the MAPK p38 and ERK1/2, and inhibition of these kinases by SB203580 and PD98059, respectively, reduced TNF-alpha and CXCL8 release. Suppression of sPLA(2) enzymatic activity by a site-directed inhibitor influenced neither cytokine/chemokine production nor activation of MAPK, whereas alteration of sPLA(2) secondary structure suppressed both responses. GIA activated the phosphatidylinositol 3-kinase (PI3 K)/Akt system and a specific inhibitor of PI3 K (LY294002) reduced sPLA(2)-induced release of TNF-alpha and CXCL8. GIA promoted phosphorylation and degradation of IkappaB and inhibition of NF-kappaB by MG-132 and 6-amino-4-phenoxyphenylethylamino-quinazoline suppressed the production of TNF-alpha and CXCL8. These results indicate that sPLA(2) induce the production of cytokines and chemokines in human macrophages by a non-enzymatic mechanism involving the PI3 K/Akt system, the MAPK p38 and ERK1/2 and NF-kappaB.     

Down-regulation of the PI3-kinase/Akt pathway by ERK MAP kinase in growth factor signaling

The ERK MAP kinase and PI3-kinase/Akt pathways are major intracellular signaling modules, which are known to regulate diverse cellular processes including cell proliferation, survival and malignant transformation. However, it has not been fully understood how these two pathways interact with each other. Here, we demonstrate that inhibition of the ERK pathway by the MEK inhibitor U0126 or PD98059 significantly potentiates EGF- and FGF-induced Akt phosphorylation at both Thr308 and Ser473. We also show that hyperactivation of the ERK pathway greatly attenuates EGF- and FGF-induced Akt phosphorylation. Furthermore, the enhanced Akt phosphorylation induced by U0126 is inhibited by the PI3-kinase inhibitor LY294002, and is accompanied by the up-regulation of Ras activity. These results suggest that the ERK pathway inhibition enhances Akt phosphorylation through the Ras/PI3-kinase pathway. Thus, our results demonstrate that the ERK pathway negatively modulates the PI3-kinase/Akt pathway in response to growth factor stimulation.     

ERK1/2和PI3-K通路在蛛网膜下腔出血大鼠海马区对神经细胞自噬的调控作用

目的:探讨细胞外信号调节激酶(extracellular regulated protein kinases,ERK1/2)和磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3-K)通路对大鼠蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后神经细胞自噬的调控作用。方法:雄性SD大鼠160只,分为假手术(Sham)组、模型(SAH)组、ERK1/2抑制剂U0126组和PI3-K抑制剂LY294002组。采用二次注血法制作SAH大鼠模型,HE染色观察海马区神经细胞的形态变化;免疫组织化学染色法检测海马区磷酸化ERK1/2、PI3-K、自噬相关蛋白Beclin-1和微管相关蛋白1轻链(LC3)-II的表达实时荧光定量PCR检测海马区ERK1/2、PI3-K、自噬相关蛋白Beclin-1和微管相关蛋白LC3的表达。结果:SAH组海马区神经细胞存活率低于Sham组,ERK1/2、PI3-K、Beclin-1和LC3的表达水平高于Sham组(P0.05);U0126组和LY294002组海马区神经细胞存活率均高于SAH组,ERK1/2、PI3-K、Beclin-1和LC3表达均低于SAH组(P0.05);U0126组和LY294002组两组间海马区神经细胞存活率差异无统计学意义(P0.05),U0126组ERK1/2、Beclin-1和LC3表达水平低于LY294002组(P0.05),PI3-K表达水平高于LY294002组(P0.05)。结论:ERK1/2和PI3-K通路激活共同参与SAH后神经细胞自噬的调节,且以PI3-K通路更为主要。     

LY294002 inhibits LPS-induced NO production through a inhibition of NF-kappaB activation: independent mechanism of phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinase (PI3K) is critical player in cell proliferation and survival. The effects of LY294002 and wortmannin, inhibitors of PI3K, on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipoploysaccharide (LPS)-induced Raw 264.7 cells were investigated. Significant inhibition of LPS-induced protein kinase B (PKB, Akt) phosphorylation occurred at 25 microM LY294002 or 0.5 microM wortmannin. At the same concentrations, LY294002, but not wortmannin, significantly inhibited NO production and iNOS expression. LY303511, an inactive analogue of LY294002, also inhibited NO production and iNOS expression. In addition, LY294002 and LY303511 significantly inhibited the DNA binding activity of NF-kappaB and NF-kappaB dependent reporter gene expression. These results suggest that LY294002 inhibits iNOS expression at least in part via inhibition of NF-kappaB activation, independent of PI3K.     

Involvement of JAK2, but not PI 3-kinase/Akt and MAP kinase pathways, in anti-apoptotic signals of GM-CSF in human eosinophils.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) transmits anti-apoptotic signals in eosinophils and is involved in tissue eosinophilia at the site of allergic inflammation. We determined whether phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein kinase (MAP kinase) are involved in anti-apoptotic signals of GM-CSF in eosinophils. GM-CSF phosphorylated Akt, a downstream component of PI 3-kinase, and MAP kinases (ERK1 and ERK2) at 10 min after stimulation in eosinophils. GM-CSF prevented eosinophil apoptosis and sustained its survival during the 5-day culture. However, neither two PI-3 kinase inhibitors, wortmannin and LY294002, nor MEK inhibitor PD98059 inhibited GM-CSF-induced survival of eosinophils, although wortmannin and PD98059 inhibited GM-CSF-induced Akt phosphorylation and MAP kinase activation in eosinophils, respectively. In contrast, JAK2 inhibitor AG-490 inhibited both GM-CSF-induced JAK2 phosphorylation and cell survival in eosinophils. These results indicate that activation of JAK2, but not activation of PI 3-kinase/Akt and MAP kinase pathways, is critical for anti-apoptotic signals of GM-CSF in human eosinophils. Our findings suggest that manipulation of JAK2 activation would be useful for the treatment of allergic disorders.     

MAP kinase subtypes and Akt regulate diosgenin-induced apoptosis of rheumatoid synovial cells in association with COX-2 expression and prostanoid production

In the present study, we investigated the signalling pathways involved in diosgenin-induced apoptosis in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) in vitro with particular interest on Akt and MAPKs activation in relation to arachidonic acid metabolism via COX-2 pathway. MAPK activation was measured by ELISA quantification in diosgenin-treated human RA FLS. Expression of Akt and phospho-Akt was analyzed by Western blot analysis. Nuclear factor-kappaB (NF-kappaB) translocation was evaluated by electromobility shift assay. The prostanoid production (COX-2 activity) was measured by quantitative ELISA. Diosgenin-induced apoptosis in the presence of MAPK or Akt inhibitors was detected by a quantitative determination of DNA fragmentation. Treatment of human RA FLS with 40 microM diosgenin caused an activation of p38 and JNK and an inhibition of ERK phosphorylation. Akt and NF-kappaB are potentially required for diosgenin-induced apoptosis in human RA FLS because 40 microM diosgenin abrogated Akt phosphorylation which correlated with an inhibition of NF-kappaB nuclear translocation. SB203580 and SP600125 (p38 and JNK inhibitors) reduced diosgenin-induced DNA fragmentation whereas U0126 and LY294002 (MEK and PI3 kinase/Akt inhibitors) caused an amplification of proapoptotic effect of diosgenin. Diosgenin increased COX-2 activity resulting in PGE2 and 6-keto-PGF1alpha overproduction in human RA FLS. All MAPK inhibitors markedly reduced diosgenin-induced PGE2 and 6-keto-PGF1alpha synthesis except for SP600125 on 6-keto-PGF1alpha production. These results provide, for the first time, strong evidence that a combined association implicating a MEK inhibitor (U0126) and diosgenin is the most effective in inducing very strong apoptosis with down-regulation of COX-2 expression and activity in human RA FLS.     

Fcgamma receptor signaling in primary human microglia: differential roles of PI-3K and Ras/ERK MAPK pathways in phagocytosis and chemokine induction

Cryptococcus neoformans monoclonal antibody immune complex (IC) induces beta-chemokines and phagocytosis in primary human microglia via activation of Fc receptor for immunoglobulin G (FcgammaR). In this report, we investigated microglial FcgammaR signal-transduction pathways by using adenoviral-mediated gene transfer and specific inhibitors of cell-signaling pathways. We found that Src inhibitor PP2 and Syk inhibitor piceatannol inhibited phagocytosis, macrophage-inflammatory protein-1alpha (MIP-1alpha) release, as well as phosphorylation of extracellular-regulated kinase (ERK) and Akt, consistent with Src/Syk involvement early in FcgammaR signaling. Constitutively active mitogen-activated protein kinase kinase (MEK) induced MIP-1alpha, and Ras dominant-negative (DN) inhibited IC-induced ERK phosphorylation and MIP-1alpha production. These results suggest that the Ras/MEK/ERK pathway is necessary and sufficient in IC-induced MIP-1alpha expression. Neither Ras DN nor the MEK inhibitor U0126 inhibited phagocytosis. In contrast, phosphatidylinositol-3 kinase (PI-3K) inhibitors Wortmannin and LY294002 inhibited phagocytosis without affecting ERK phosphorylation or MIP-1alpha production. Conversely, Ras DN or U0126 did not affect Akt phosphorylation. Together, these results demonstrate distinct roles played by the PI-3K and Ras/MEK/ERK pathways in phagocytosis and MIP-1alpha induction, respectively. Our results demonstrating activation of functionally distinct pathways following microglial FcgammaR engagement may have implications for human central nervous system diseases.     

Phosphatidylinositol 3-kinase-mediated regulation of IL-10 and IL-12 production in macrophages stimulated with CpG oligodeoxynucleotide

The aim of this study was to clarify the role of phosphatidylinositol 3-kinase (PI3K) on the production of interleukine-10 (IL-10) and IL-12 in mouse peritoneal macrophages stimulated with CpG-ODN. CpG-ODN-induced IL-10 mRNA expression and protein production decreased following the treatment of macrophages with wortmannin and LY294002, specific inhibitors for PI3K. In contrast, IL-12 p40 mRNA expression and p70 protein production increased. Neutralizing anti-IL-10 monoclonal antibody (anti-IL-10 mAb) exactly mimicked the effects of PI3K inhibitors to enhance IL-12 p70 production. The enhancement effect of PI3K inhibitors on IL-12 p70 production almost completely disappeared by the treatment with anti-IL-10 mAb. PI3K inhibitors suppressed the activation of extracellular-regulated kinase (ERK), a member of the mitogen-activated protein kinases, by CpG-ODN. A specific ERK inhibitor, U0126, as well as PI3K inhibitors, differentially regulated IL-10 and IL-12 p70 productions. These results suggest that PI3K positively and negatively regulates the production of CpG-ODN-induced IL-10 and IL-12 p70, respectively, and negatively regulates IL-12 p70 production in macrophages through ERK-mediated IL-10 induction.     

Phosphoinositol 3-kinase, a novel target molecule for the inhibitory effects of juglone on TPA-induced cell transformation

Juglone (5-hydroxy-1,4-naphthalenedione) from black walnut trees induces apoptosis and inhibits proliferation of various malignant cells. Here, we investigated whether juglone affects 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation through the phosphoinositol 3-kinase (PI3K) pathway. The results showed that TPA- and endothelial growth factor (EGF)-induced anchorage-independent colony formation were suppressed in a dose-dependent manner by treatment of JB6 CI41 mouse skin epidermal cells with juglone (2.5 and 5 μM). We demonstrated that juglone suppressed PI3K activity via direct binding to PI3K by sepharose 4B pull-down assay and western blot analysis. Juglone significantly suppressed TPA-induced protein kinase B (AKT) and c-Jun phosphorylation and c-fos activation, but not mitogen-activated protein-kinase kinase (MEK), extracellular signaling-regulated kinase (ERK) or 90 kDa ribosomal protein S6 kinase (RSK) phosphorylation. Juglone significantly blocked activator protein-1 (AP-1) and cyclooxygenase-2 (COX-2) activation more than the PI3K inhibitors LY294002 and wortmannin. Overall, these results showed the anticancer efficacy of juglone targeting PI3K to prevent TPA-induced tumorigenesis.     

PHOSPHATIDYLINOSITOL 3-KINASE REGULATES PMA-INDUCED DIFFERENTIATION AND SUPEROXIDE PRODUCTION IN HL-60 CELLS

ABSTRACT

Treatment of the human promyelocytic leukemia cell line HL-60 with phorbol myristate acetate (PMA) is associated with induction of monocytic or myelocytic differentiation. Since phosphatidylinositol 3-kinase (PI3-kinase) is a critical player in cell proliferation, survival, and differentiation, we studied the role of PI3-kinase during induction of the differentiated monocytic phenotype and superoxide production. In treatment of HL-60 cells with PMA, the PI3-kinase inhibitors LY294002 and wortmannin inhibited cell adhesion and spreading and phagocytic activity. LY294002 and wortmannin also inhibited the proliferation of HL-60 cells. During PMA-induced monocytic differentiation, LY294002 induced apoptosis in a dose dependent manner. The phosphorylation of p85α derived from PMA-stimulated HL-60 cells was shown in the time dependent manner. However, p70 S6 kinase inhibitor, rapamycin, did not inhibit PMA-induced monocytic differentiation. During PMA-induced monocytic differentiation, LY294002 inhibited c-jun protein expression and decrease of c-myc protein level. In contrast, LY294002 induced production of superoxide in the HL-60 cells stimulated with forskolin. Moreover, staurosporine and H7, PKC inhibitors, enhanced superoxide production in dibutyryl cAMP-induced HL-60 cells. These results suggest that PI3-kinase may regulate PMA-induced differentiation signal and provide a crucial link between PKC and cAMP in HL-60 cells.