Activation of human inflammatory cells by secreted phospholipases A2

Secreted phospholipases A2 (sPLA2s) are enzymes detected in serum and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Different isoforms of sPLA2s are expressed and released by human inflammatory cells, such as neutrophils, eosinophils, T cells, monocytes, macrophages and mast cells. sPLA2s generate arachidonic acid and lysophospholipids thus contributing to the production of bioactive lipid mediators in inflammatory cells. However, sPLA2s also activate human inflammatory cells by mechanisms unrelated to their enzymatic activity. Several human and non-human sPLA2s induce degranulation of mast cells, neutrophils and eosinophils and activate exocytosis in macrophages. In addition some, but not all, sPLA2 isoforms promote cytokine and chemokine production from macrophages, neutrophils, eosinophils, monocytes and endothelial cells. These effects are primarily mediated by binding of sPLA2s to specific membrane targets (heparan sulfate proteoglycans, M-type, N-type or mannose receptors) expressed on effector cells. Thus, sPLA2s may play an important role in the initiation and amplification of inflammatory reactions by at least two mechanisms: production of lipid mediators and direct activation of inflammatory cells. Selective inhibitors of sPLA2-enzymatic activity and specific antagonists of sPLA2 receptors are current being tested for pharmacological treatment of inflammatory and autoimmune diseases. © 2006 Elsevier B.V. All rights reserved

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

Secretory phospholipases A2 (sPLA2) 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) sPLA2 in human lung macrophages leading to cytokine/chemokine production. sPLA2 induced the production of cytokines (TNF-α, IL-6 and IL-10) and chemokines (CCL2, CCL3, CCL4 and CXCL8), whereas no effect was observed on IL-12, CCL1, CCL5 and CCL22. sPLA2 induced the phosphorylation of the MAPK p38 and ERK1/2, and inhibition of these kinases by SB203580 and PD98059, respectively, reduced TNF-α and CXCL8 release. Suppression of sPLA2 enzymatic activity by a site-directed inhibitor influenced neither cytokine/chemokine production nor activation of MAPK, whereas alteration of sPLA2 secondary structure suppressed both responses. GIA activated the phosphatidylinositol 3-kinase (PI3 K) /Akt system and a specific inhibitor of PI3 K (LY294002) reduced sPLA2-induced release of TNF-α and CXCL8. GIA promoted phosphorylation and degradation of IκB and inhibition of NF-κB by MG-132 and 6-amino-4-phenoxyphenylethylamino-quinazoline suppressed the production of TNF-α and CXCL8. These results indicate that sPLA2 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-κB. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Inhibition of secretory phospholipase A2-induced cytokine production in human lung macrophages by budesonide

Background: Secretory phospholipases A2 (sPLA2) are an emerging class of mediators of inflammation. These enzymes are released in vivo in patients with systemic inflammatory diseases and allergic disorders. sPLA2s may activate inflammatory cells by both enzymatic and nonenzymatic mechanisms. The aim of this study was to evaluate the effect of the inhaled glucocorticoid budesonide on sPLA2-induced activation of primary human macrophages. Methods: Macrophages isolated from human lung tissue were preincubated (3-18 h) with budesonide (1-1,000 nM) before stimulation with 2 distinct sPLA2s (group IA and group X). At the end of incubation the release of TNF-α, IL-6 and IL-8 was assessed by ELISA. Specific mRNA for these products was determined by quantitative RT-PCR. Activation of mitogen-activated kinases ERK 1/2 and p38 was assessed by Western blot. Results: Budesonide inhibited the release of TNF-α, IL-6 and IL-8 from sPLA 2-stimulated macrophages in a concentration-dependent manner. The inhibitory effect of budesonide was due to a reduction of gene expression and was complete after 18 h of preincubation. Budesonide had no effect on sPLA 2-induced arachidonic acid mobilization and exocytosis, assessed as β-glucuronidase release. Suppression of cytokine/chemokine production by budesonide was associated with inhibition of sPLA2-induced ERK 1/2 and p38 activation. Conclusions: Budesonide inhibits the production of proinflammatory cytokines/chemokines from human lung macrophages activated by sPLA2. Budesonide represents the first example of a drug able to block the nonenzymatic effects of sPLA2 on human inflammatory cells and, therefore, may provide a useful therapeutic options for diseases associated with enhanced release of sPLA2s in vivo. Copyright © 2009 S. Karger AG, Basel

Simplexide Induces CD1d-Dependent Cytokine and Chemokine Production from Human Monocytes

Monocytes are major effector cells of innate immunity and recognize several endogenous and exogenous molecules due to the expression of wide spectrum of receptors. Among them, the MHC class I-like molecule CD1d interacts with glycolipids and presents them to iNKT cells, mediating their activation. Simplexide belongs to a novel class of glycolipids isolated from marine sponges and is structurally distinct from other immunologically active glycolipids. In this study we have examined the effects of simplexide on cytokine and chemokine release from human monocytes. Simplexide induces a concentration- and time-dependent release of IL-6, CXCL8, TNF-α and IL-10 and increases the expression of IL6, CXCL8 and IL10 mRNA. Cytokine and chemokine release induced by simplexide from monocytes is dependent on CD1d since: i) a CD1d antagonist, 1,2-bis (diphenylphosphino) ethane [DPPE]- polyethylene glycolmonomethylether [PEG], specifically blocks simplexide-induced activation of monocytes; ii) CD1d knockdown inhibits monocyte activation by simplexide and iii) simplexide induces cytokine production from CD1d-transfected but not parental C1R cell line Finally, we have shown that simplexide also induces iNKT cell expansion in vitro. Our results demonstrate that simplexide, apart from activating iNKT cells, induces the production of cytokines and chemokines from human monocytes by direct interaction with CD1d

Production of vascular endothelial growth factors from human lung macrophages induced by group IIA and group X secreted phospholipases A2.

International audienceAngiogenesis and lymphangiogenesis mediated by vascular endothelial growth factors (VEGFs) are main features of chronic inflammation and tumors. Secreted phospholipases A(2) (sPLA(2)s) are overexpressed in inflammatory lung diseases and cancer and they activate inflammatory cells by enzymatic and receptor-mediated mechanisms. We investigated the effect of sPLA(2)s on the production of VEGFs from human macrophages purified from the lung tissue of patients undergoing thoracic surgery. Primary macrophages express VEGF-A, VEGF-B, VEGF-C, and VEGF-D at both mRNA and protein level. Two human sPLA(2)s (group IIA and group X) induced the expression and release of VEGF-A and VEGF-C from macrophages. Enzymatically-inactive sPLA(2)s were as effective as the active enzymes in inducing VEGF production. Me-Indoxam and RO092906A, two compounds that block receptor-mediated effects of sPLA(2)s, inhibited group X-induced release of VEGF-A. Inhibition of the MAPK p38 by SB203580 also reduced sPLA(2)-induced release of VEGF-A. Supernatants of group X-activated macrophages induced an angiogenic response in chorioallantoic membranes that was inhibited by Me-Indoxam. Stimulation of macrophages with group X sPLA(2) in the presence of adenosine analogs induced a synergistic increase of VEGF-A release and inhibited TNF-alpha production through a cooperation between A(2A) and A(3) receptors. These results demonstrate that sPLA(2)s induce production of VEGF-A and VEGF-C in human macrophages by a receptor-mediated mechanism independent from sPLA(2) catalytic activity. Thus, sPLA(2)s may play an important role in inflammatory and/or neoplastic angiogenesis and lymphangiogenesis

Effect of increasing concentrations of natural or synthetic simplexide on IL-6 (panel A) and CXCL8 (panel B) release from monocytes.

<p>Polystyrene plates were coated with indicated concentrations of glycolipids dissolved in methanol. Solvent was dried under nitrogen immediately before the addiction of cells. After 24 hours of incubation, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). Cytokines and chemokines were determined by ELISA. The values are expressed as ng of IL-6 or CXCL8 per mg of total proteins. Data are the mean ± SEM of six experiments. * p<0.05 <i>vs</i>. control.</p

Effect of simplexide on C1R and C1R-CD1d cell lines.

<p>C1R and C1R-CD1d were incubated (37°C, 24 h) with X-VIVO alone (unstimulated), simplexide (10 µM), KRN7000 (1 µM) or PMA (100 ng/ml). At the end of incubation, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). CXCL8 was determined by ELISA. The values are expressed as pg of CXCL8 per mg of total proteins. Data are the mean ± SEM of six experiments. * p<0.05 <i>vs</i>. the respective unstimulated.</p

Effect of polymyxin B on simplexide- and LPS-induced release of IL-6, CXCL8 and TNF-α from monocytes.

<p>Monocytes were incubated for 8 h (TNF-α) or 24 h (IL-6 and CXCL8) with simplexide (10 µM) or LPS (100 ng/ml) either in the absence (Control) or the presence of polymyxin B (50 µg/ml). Data are the mean ± SE of three experiments.</p><p>* p<0.05 vs. respective untreated.</p>†<p>p<0.05 vs. respective control.</p><p>Effect of polymyxin B on simplexide- and LPS-induced release of IL-6, CXCL8 and TNF-α from monocytes.</p

Effect of DPPE-PEG on simplexide-induced IL-6 (panel A) and CXCL8 (panel B) release from monocytes.

<p>Monocytes were preincubated (37°C, 5 min) with X-VIVO alone, or with the indicated concentrations of DPPE-PEG (1–30 µg/ml) and then stimulated (37°C, 24 h) with simplexide (•; 10 µM), α-GalCer (▪; 50 µM) or LPS (♦; 1 µg/ml). At the end of incubation, the supernatant was collected and centrifuged (1000×<i>g</i>, 4°C, 5 min). Cytokines and chemokines were determined by ELISA. The values are expressed as ng of IL-6 or CXCL8 per mg of total proteins. Data are expressed as percent inhibition of the maximum response induced by simplexide or LPS alone calculated as (R−R_b)/(R_max−R_b) ×100, where R is the release in samples treated with the agonists plus DPPE-PEG, R_b is the release in unstimulated samples and R_max is the release in samples stimulated with agonists alone. Data are the mean ± SEM of five experiments. The lines represent the best fit for inhibition of simplexide, α-GalCer or LPS.</p