Leukotriene B4 enhances tumour necrosis factor-α-induced CCL27 production in human keratinocytes

BACKGROUND: A chemokine CCL27 recruits skin-homing T cells. CCL27 production by epidermal keratinocytes is dependent on nuclear factor-kappaB (NF-kappaB) activity and is enhanced in lesions with atopic dermatitis or allergic contact dermatitis. A lipid mediator leukotriene B(4) (LTB(4)) may be involved in the development of these allergic dermatoses. LTB(4) acts on cell surface G-protein-coupled receptors, BLT1 and BLT2. OBJECTIVE: The aim of this study was to investigate the in vitro effects of LTB(4) on CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha and LTB(4). CCL27 secretion and mRNA levels were analysed by ELISA and RT-PCR, respectively. NF-kappaB activities were analysed by luciferase assays. Protein levels or phosphorylation status were analysed by cell-based ELISA. RESULTS: LTB(4) alone did not enhance CCL27 production and modestly enhanced NF-kappaB activity in human keratinocytes. However, LTB(4) potently enhanced TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity. LTB(4) alone or together with TNF-alpha, induced phosphorylation and degradation of inhibitory NF-kappaB alpha (IkappaBalpha) and phosphorylation of NF-kappaB p65. These effects of LTB(4) were suppressed by BLT1 antagonist U75302, pertussis toxin, phosphoinositide-3 kinase (PI3K) inhibitor LY294002 and extracellular signal-regulated kinase (ERK) kinase inhibitor U0126, but not by BLT2 antagonist LY255283. LTB(4) induced phosphorylation of ERK and Akt, downstream kinase of PI3K; LY294002 suppressed phosphorylation of both kinases while U0126 suppressed only the former. CONCLUSION: These results suggest that LTB(4) may enhance TNF-alpha-induced CCL27 production by activating NF-kappaB via the BLT1/G(i/o)/PI3K/ERK pathway in human keratinocytes. LTB(4) may contribute to the enhanced CCL27 production of keratinocytes in lesions with atopic dermatitis or allergic contact dermatitis.     

ANP inhibits TNF-alpha-induced endothelial MCP-1 expression--involvement of p38 MAPK and MKP-1

Atrial natriuretic peptide (ANP) has been shown to reduce tumor necrosis factor-alpha (TNF-alpha)-induced activation of endothelial cells via inhibition of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB pathways. The aim of this study was to determine whether ANP is able to inhibit TNF-alpha-induced expression of monocyte chemoattractant protein-1 (MCP-1) in endothelial cells and to elucidate the mechanisms involved. Pretreatment of human umbilical vein endothelial cells (HUVEC) with ANP significantly reduced TNF-alpha-induced expression of MCP-1 protein and mRNA. The effects of ANP were shown to be mediated via the guanylyl-cyclase (GC)-coupled A receptor. Activation of the other GC-coupled receptor (natriuretic peptide receptor-B) by the C-type natriuretic peptide as well as activation of soluble GC with S-nitroso-L-glutathione (GSNO) exerted similar effects as ANP, supporting a role for cyclic guanosine monophosphate (cGMP) in the signal transduction. Antisense experiments showed a requirement of MAPK phosphatase-1 (MKP-1) induction and therefore, inhibition of p38 MAPK in the ANP-mediated inhibition of TNF-alpha-induced expression of MCP-1. To investigate a potential interplay between TNF-alpha-induced activation of p38 MAPK and NF-kappaB, the p38 MAPK inhibitor SB203580 and a dominant-negative p38 MAPK mutant were used. The results indicated that the blockade of p38 MAPK activity leads to an increased activation of NF-kappaB and therefore, suggest a counter-regulatory action of p38 MAPK and NF-kappaB. As antisense experiments revealed a pivotal role for MKP-1 induction and therefore, p38 MAPK inhibition in ANP-mediated attenuation of MCP-1 expression, this action seems to be rather independent of NF-kappaB inhibition.     

C-X-C and C-C chemokine expression and secretion by the human colonic epithelial cell line, HT-29: differential effect of T lymphocyte-derived cytokines

Differential chemokine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected population of leukocytes cells in inflammatory bowel disease. We have previously demonstrated that IL-13 enhances IL-1alpha-induced IL-8 secretion by the colonic epithelial cell line HT-29. We have now explored the C-C chemokine expression and modulation in this system. The combination of TNF-alpha and IFN-gamma was the minimal stimulation required for regulated on activation, normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein (MCP-1) mRNA expression and secretion by HT-29 cells. The same stimulation induced a stronger IL-8 mRNA expression and secretion. Pretreatment with IL-13 or IL-4, reduced significantly the RANTES, and MCP-1, but not IL-8 mRNA expression and secretion. In contrast, IL-10 had no effect on either MCP-1, or RANTES, or IL-8 generation. Pretreatment of HT-29 cells with wortmannin suggested that the IL-13-induced inhibition of C-C chemokine expression is via activation of a wortmannin-sensitive phosphatidylinositol 3-kinase. These data demonstrate that colonic epithelial cell chemokine production can be differentially regulated by T cell-derived cytokines and suggest an interplay between epithelial cells and T lymphocytes potentially important in the intestinal inflammation.     

槲皮素改善大鼠骨髓来源内皮祖细胞生物学功能及其机制研究

目的:探讨槲皮素(quercetin,QUE)对大鼠骨髓来源内皮祖细胞(endothelial progenitor cells,EPCs)生物学功能的影响与机制。方法:密度梯度离心法分离大鼠骨髓单个核细胞,条件培养基EGM-2诱导分化后,进行双荧光染色及免疫表型鉴定。将培养14 d的细胞用PI3K抑制剂BYL719(3μmol/L)和ERK抑制剂FR180204(15μmol/L)预处理2 h后,再加入不同浓度QUE(0、10、20、40、80和100μmol/L)处理。MTT法检测细胞活力,Transwell法检测细胞迁移,Western blot法检测AKT、内皮型一氧化氮合酶(eNOS)和ERK蛋白表达及磷酸化水平。结果:QUE呈浓度依赖性提高EPCs活力,促进EPCs迁移;PI3K抑制剂BYL719能抑制QUE诱导的EPCs活力和迁移能力,ERK抑制剂FR180204能抑制QUE诱导的EPCs活力,而对EPCs迁移能力并没有影响。QUE能激活AKT、eNOS和ERK蛋白;BYL719能同时抑制AKT和ERK蛋白的激活,FR180204仅能抑制ERK的激活,而未能抑制AKT的激活,但两者对QUE诱导的eNOS蛋白表达均有抑制作用。结论:QUE能部分通过PI3K/AKT/eNOS和ERK/eNOS信号转导通路,提高EPCs活力及迁移能力,促进EPCs发挥心血管保护作用。     

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.     

CARMA3 mediates lysophosphatidic acid-stimulated cytokine secretion by bronchial epithelial cells

NF-kappaB activation in bronchial epithelial cells is important for the development of allergic airway inflammation, and may control the expression of critical mediators of allergic inflammation such as thymic stromal lymphopoietin (TSLP) and the chemokine CCL20. Members of the caspase recruitment domain (CARD) family of proteins are differentially expressed in tissue and help mediate NF-kappaB activity in response to numerous stimuli. Here we demonstrate that CARMA3 (CARD10) is specifically expressed in human airway epithelial cells, and that expression of CARMA3 in these cells leads to activation of NF-kappaB. CARMA3 has recently been shown to mediate NF-kappaB activation in embryonic fibroblasts after stimulation with lysophosphatidic acid (LPA), a bioactive lipid-mediator that is elevated in the lungs of individuals with asthma. Consistent with this, we demonstrate that stimulation of airway epithelial cells with LPA leads to increased expression of TSLP and CCL20. We then show that inhibition of CARMA3 activity in airway epithelial cells reduces LPA-mediated NF-kappaB activity and the production of TSLP and CCL20. In conclusion, these data demonstrate that LPA stimulates TSLP and CCL20 expression in bronchial epithelial cells via CARMA3-mediated NF-kappaB activation.     

The role of PTEN in allergic inflammation

Bronchial asthma is a chronic inflammatory disease of the airways, characterized by airway eosinophilia, goblet cell hyperplasia with mucus hyper-secretion, and hyper-responsiveness to inhaled allergens and to non-specific stimuli. Eosinophil accumulation and subsequent activation in bronchial tissues play critical roles in the pathophysiology of bronchial asthma. Many inflammatory mediators attract and activate eosinophils via signal transduction pathways involving an enzyme phosphatidylinositol 3-kinase (PI3-kinase). Studies using wortmannin, a specific inhibitor of PI3-kinase, have revealed the involvement of PI3-kinase in the biochemical transduction of activation signals generated by many inflammatory mediators in eosinophils. Wortmannin prevents the development of airway inflammation, either by inhibiting the eosinophil infiltration of bronchial tissues or their activation on arrival. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is part of a complex signaling system that affects a variety of important cell functions. PTEN opposes the action of PI3-kinase by dephosphorylating the signaling lipid phosphatidylinositol 3,4,5-triphosphate. Recently we have demonstrated that PTEN expression is diminished in airway epithelial cells of antigen-sensitized and -challenged mice. Administration of PI3-kinase inhibitors or adenoviruses carrying PTEN complementary DNA remarkably reduces eosinophil levels and inflammation. One likely mechanism for this reduction is PTEN-mediated eosinophil degranulation and suppression of interleukin (IL)-4 and IL-5.These findings indicate that use of PTEN may be a good therapeutic strategy for the management of allergic inflammation.