Annunziata Frattini

Production of Vascular Endothelial Growth Factors from Human Lung Macrophages Induced by Group IIA and Group X Secreted Phospholipases A2

Angiogenesis and lymphangiogenesis mediated by vascular endothelial growth factors (VEGFs) are main features of chronic inflammation and tumors. Secreted phospholipases A2 (sPLA2s) are overexpressed in inflammatory lung diseases and cancer and they activate inflammatory cells by enzymatic and receptor-mediated mechanisms. We investigated the effect of sPLA2s 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 sPLA2s (group IIA and group X) induced the expression and release of VEGF-A and VEGF-C from macrophages. Enzymatically-inactive sPLA2s were as effective as the active enzymes in inducing VEGF production. Me-Indoxam and RO092906A, two compounds that block receptor-mediated effects of sPLA2s, inhibited group X-induced release of VEGF-A. Inhibition of the MAPK p38 by SB203580 also reduced sPLA2-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 sPLA2 in the presence of adenosine analogs induced a synergistic increase of VEGF-A release and inhibited TNF-α production through a cooperation between A2A and A3 receptors. These results demonstrate that sPLA2s induce production of VEGF-A and VEGF-C in human macrophages by a receptor-mediated mechanism independent from sPLA2 catalytic activity. Thus, sPLA2s may play an important role in inflammatory and/or neoplastic angiogenesis and lymphangiogenesis.

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.

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

The Na+/Ca2+ exchanger (NCX) is a membrane transporter that can switch Na+ and Ca2+ in either direction to maintain the homeostasis of intracellular Ca2+. Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na+‐free medium induced a significant increase in intracellular calcium concentration ([Ca2+]i) in both cell types. This response was completely abolished by the NCX inhibitor 5‐(N‐4‐chlorobenzyl)‐20,40‐dimethylbenzamil (CB‐DMB). Moreover, inhibition of NCX activity during Ca2+‐signaling induced by histamine caused a delay in restoring baseline [Ca2+]i. Na+‐free medium induced TNF‐α expression and release in HLM comparable to that caused by LPS. TNF‐α release induced by Na+‐free medium was blocked by CB‐DMB and greatly reduced by RNAi‐mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca2+]i, to generate Ca2+‐signals, and to induce TNF‐α production. Therefore, NCX may contribute to regulate Ca2+ homeostasis and proinflammatory functions in human macrophages and monocytes.

Secreted phospholipases A2: A proinflammatory connection between macrophages and mast cells in the human lung

Secretory phospholipases A(2) (sPLA(2)) are an emerging class of mediators of inflammation. These enzymes accumulate in plasma and other biological fluids of patients with inflammatory, autoimmune and allergic diseases. sPLA(2)s are secreted at low levels in the normal airways and tend to increase during inflammatory lung diseases (e.g. bronchial asthma, chronic obstructive pulmonary disease, interstitial lung fibrosis, and sarcoidosis) as the result of plasma extravasation and/or local production. Such immune resident cells as macrophages and mast cells can be a source of sPLA(2)s in the lung. However, these cells are also targets for sPLA(2)s that sustain the activation programs of macrophages and mast cells with mechanism related to their enzymatic activity as well as to their capacity to interact with surface molecules (e.g., heparan sulfate proteoglycans, M-type receptor, mannose receptor). Recent evidence suggests that mast cells are a better source of extracellular sPLA(2)s than macrophages. On the other hand, macrophages appear to be a preferential target for sPLA(2)s. Anatomical association between macrophages and mast cells in the airways suggest that sPLA(2)s released by mast cells may activate in a paracrine fashion several macrophage functions relevant to the modulation of lung inflammation. Thus, sPLA(2)s may play a major role in inflammatory lung diseases by acting as a proinflammatory connection between macrophages and mast cells.

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 sPLA2-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 sPLA2-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.

Benzene Metabolites Inhibit the Release of Proinflammatory Mediators and Cytokines from Human Basophils

Benzene and its metabolites have been involved in the pathogenesis of chronic lung inflammation and allergic disorders such as bronchial asthma. However, the effects of these xenobiotics on human basophils, key cells in the development of respiratory allergy, have not been investigated. We examined the effects of hydroquinone (HQ) and benzoquinone (BQ), two important chemicals implicated in benzene toxicity, on the release of preformed (histamine) and de novo synthesized mediators (cysteinyl leukotriene C4, LTC4, and IL-4) from human basophils. Preincubation of basophils purified from normal donors with HQ (3–100 μM) inhibited up to 30% histamine release induced by anti-IgE and up to 55% of that induced by the Ca2+ ionophore A23187. HQ had no effect on histamine release induced by formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). Preincubation of basophils with BQ (3–100 μM) resulted in the concentration-dependent inhibition of histamine release (up to 70%) induced by anti-IgE, A23187 and f-Met-Leu-Phe. HQ completely suppressed the de novo synthesis of LTC4 from basophils challenged with anti-IgE or f-Met-Leu-Phe and the production of IL-4 in cells stimulated with anti-IgE. These results indicate that two major benzene metabolites, HQ and BQ, inhibit the release of proinflammatory mediators and Th2-promoting cytokines from basophils activated by different stimuli. These results suggest that benzene metabolites interfere with multiple intracellular signals involved in the activation of human basophils.

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.