Francescopaolo Granata

Histamine Induces Exocytosis and IL-6 Production from Human Lung Macrophages Through Interaction with H1 Receptors

Increasing evidence suggests that a continuous release of histamine from mast cells occurs in the airways of asthmatic patients and that histamine may modulate functions of other inflammatory cells such as macrophages. In the present study histamine (10−9–10−6 M) increased in a concentration-dependent fashion the basal release of β-glucuronidase (EC50 = 8.2 ± 3.5 × 10−9 M) and IL-6 (EC50 = 9.3 ± 2.9 × 10−8 M) from human lung macrophages. Enhancement of β-glucuronidase release induced by histamine was evident after 30 min and peaked at 90 min, whereas that of IL-6 required 2–6 h of incubation. These effects were reproduced by the H1 agonist (6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane carboxamide but not by the H2 agonist dimaprit. Furthermore, histamine induced a concentration-dependent increase of intracellular Ca2+ concentrations ([Ca2+]i) that followed three types of response, one characterized by a rapid increase, a second in which [Ca2+]i displays a slow but progressive increase, and a third characterized by an oscillatory pattern. Histamine-induced β-glucuronidase and IL-6 release and [Ca2+]i elevation were inhibited by the selective H1 antagonist fexofenadine (10−7–10−4 M), but not by the H2 antagonist ranitidine. Inhibition of histamine-induced β-glucuronidase and IL-6 release by fexofenadine was concentration dependent and displayed the characteristics of a competitive antagonism (Kd = 89 nM). These data demonstrate that histamine induces exocytosis and IL-6 production from human macrophages by activating H1 receptor and by increasing [Ca2+]i and they suggest that histamine may play a relevant role in the long-term sustainment of allergic inflammation in the airways.

Activation of Cytokine Production by Secreted Phospholipase A2 in Human Lung Macrophages Expressing the M-Type Receptor

Secreted phospholipases A2 (sPLA2) are enzymes released in plasma and extracellular fluids during inflammatory diseases. Because human group IB and X sPLA2s are expressed in the lung, we examined their effects on primary human lung macrophages (HLM). Both sPLA2s induced TNF-α and IL-6 release in a concentration-dependent manner by increasing their mRNA expression. This effect was independent of their enzymatic activity because 1) the capacity of sPLA2s to mobilize arachidonic acid from HLM was unrelated to their ability to induce cytokine production; and 2) two catalytically inactive isoforms of group IB sPLA2 (bromophenacyl bromide-inactivated human sPLA2 and the H48Q mutant of the porcine sPLA2) were as effective as the catalytically active sPLA2s in inducing cytokine production. HLM expressed the M-type receptor for sPLA2s at both mRNA and protein levels, as determined by RT-PCR, immunoblotting, immunoprecipitation, and flow cytometry. Me-indoxam, which decreases sPLA2 activity as well as binding to the M-type receptor, suppressed sPLA2-induced cytokine production. Incubation of HLM with the sPLA2s was associated with phosphorylation of ERK1/2, and a specific inhibitor of this pathway, PD98059, significantly reduced the production of IL-6 elicited by sPLA2s. In conclusion, two distinct sPLA2s produced in the human lung stimulate cytokine production by HLM via a mechanism that is independent of their enzymatic activity and involves activation of the ERK1/2 pathway. HLM express the M-type receptor, but its involvement in eliciting cytokine production deserves further investigation.

Allergy and the cardiovascular system

The most dangerous and life‐threatening manifestation of allergic diseases is anaphylaxis, a condition in which the cardiovascular system is responsible for the majority of clinical symptoms and for potentially fatal outcome. The heart is both a source and a target of chemical mediators released during allergic reactions. Mast cells are abundant in the human heart, where they are located predominantly around the adventitia of large coronary arteries and in close contact with the small intramural vessels. Cardiac mast cells can be activated by a variety of stimuli including allergens, complement factors, general anesthetics and muscle relaxants. Mediators released from immunologically activated human heart mast cells strongly influence ventricular function, cardiac rhythm and coronary artery tone. Histamine, cysteinyl leukotrienes and platelet‐activating factor (PAF) exert negative inotropic effects and induce myocardial depression that contribute significantly to the pathogenesis of anaphylactic shock. Moreover, cardiac mast cells release chymase and renin that activates the angiotensin system locally, which further induces arteriolar vasoconstriction. The number and density of cardiac mast cells is increased in patients with ischaemic heart disease and dilated cardiomyopathies. This observation may help explain why these conditions are major risk factors for fatal anaphylaxis. A better understanding of the mechanisms involved in cardiac mast cell activation may lead to an improvement in prevention and treatment of systemic anaphylaxis.

Angiogenesis and lymphangiogenesis in bronchial asthma

To cite this article: Detoraki A, Granata F, Staibano S, Rossi FW, Marone G, Genovese A. Angiogenesis and lymphangiogenesis in bronchial asthma. Allergy 2010; 65: 946–958.

Secretory Phospholipases A2 Induce β-Glucuronidase Release and IL-6 Production from Human Lung Macrophages

Secretory phospholipases A2 (sPLA2s) are a group of extracellular enzymes that release fatty acids at the sn-2 position of phospholipids. Group IIA sPLA2 has been detected in inflammatory fluids, and its plasma level is increased in inflammatory diseases. To investigate a potential mechanism of sPLA2-induced inflammation we studied the effect of group IA (from cobra venom) and group IIA (human synovial) sPLA2s on human macrophages. Both sPLA2s induced a concentration- and Ca2+-dependent, noncytotoxic release of β-glucuronidase (16.2 ± 2.4% and 13.1 ± 1.5% of the total content with groups IA and IIA, respectively). Both sPLA2s also increased the rate of secretion of IL-6 and enhanced the expression of IL-6 mRNA. Preincubation of macrophages with inhibitors of the hydrolytic activity of sPLA2 or cytosolic PLA2 did not influence the release of β-glucuronidase. Incubation of macrophages with p-aminophenyl-mannopyranoside-BSA (mp-BSA), a ligand of the mannose receptor, also resulted in β-glucuronidase release. However, while preincubation of macrophages with mp-BSA had no effect on β-glucuronidase release induced by group IIA sPLA2, it enhanced that induced by group IA sPLA2. A blocking Ab anti-mannose receptor inhibited both mp-BSA- and group IIA-induced β-glucuronidase release. Taken together, these data indicate that group IA and IIA sPLA2s activate macrophages with a mechanism independent from their enzymatic activities and probably related to the activation of the mannose receptor or sPLA2-specific receptors. The secretion of enzymes and cytokines induced by sPLA2s from human macrophages may play an important role in inflammation and tissue damage associated with the release of sPLA2s.

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.

Secretory phospholipases A2 activate selective functions in human eosinophils.

Secretory phospholipases A2 (sPLA2s) are released in large amounts in the blood of patients with systemic inflammatory diseases and accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils or eosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA2. We have examined the effects of two structurally different sPLA2s (group IA and group IIA) on several functions of eosinophils isolated from normal donors and patients with hypereosinophilia. Both group IA and IIA sPLA2 induced a concentration-dependent release of β-glucuronidase, IL-6, and IL-8. Release of the two cytokines was associated with the accumulation of their specific mRNA. In addition, sPLA2s induced the surface expression of CD44 and CD69, two major activation markers of eosinophils. In contrast, none of the sPLA2s examined induced the production of IL-5, the de novo synthesis of leukotriene C4 and platelet-activating factor, or the generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of the sPLA2s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes’ effects. In addition, inactivation of sPLA2 enzymatic activity by bromophenacyl bromide did not influence the release of β-glucuronidase or of cytokines. Stimulation of eosinophils by sPLA2s was associated with activation of extracellular signal-regulated kinases 1/2. These results indicate that sPLA2s selectively activate certain proinflammatory and immunoregulatory functions of human eosinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.

Secretory phospholipases A2 induce cytokine release from blood and synovial fluid monocytes.

Secretory phospholipases A2 (sPLA2) are released in the blood of patients with various inflammatory diseases and exert proinflammatory activities by releasing arachidonic acid (AA), the precursor of eicosanoids. We examined the ability of four sPLA2 to activate blood and synovial fluid monocytes in vitro. Monocytes were purified from blood of healthy donors or from synovial fluid of patients with rheumatoid arthritis by negative immunoselection and by adherence to plastic dishes, respectively. The cells were incubated with group IA, IB, IIA and III sPLA2 and the release of TNF‐α, IL‐6 and IL‐12 was determined by ELISA. Group IA, IB and IIA sPLA2 induced a concentration‐dependent release of TNF‐α and IL‐6 from bloodmonocytes. These sPLA2 activated IL‐12 production only in monocytes preincubated with IFN‐γ. Group IA and IIA sPLA2 also induced TNF‐α and IL‐6 release from synovial fluid monocytes. TNF‐α and IL‐6 release paralleled an increase in their mRNA expression and was independent from the capacity of sPLA2 to mobilize AA. These results indicate that sPLA2 stimulate cytokine release from blood and synovial fluid monocytes by a mechanism at least partially unrelated to their enzymatic activity. This effect may concur with the generation of AA in theproinflammatory activity of sPLA2 released during inflammatory diseases.

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.

Treatment of mastocytosis: pharmacologic basis and current concepts

Mastocytosis is a rare, heterogeneous disorder characterized by a marked increase in mast cell density in various tissues. Mast cells from different human tissues are heterogeneous. So far, there is no cure for systemic mastocytosis. Conventional therapy is based on agents that antagonize mediators released from mast cells, drugs that inhibit the release of mediators and agents that modulate mast cell proliferation. This pharmacologic approach is satisfactory in the majority of patients with indolent mastocytosis. At the beginning of the new millennium, the therapy of severe forms of aggressive mastocytosis remains a challenge for students of this intriguing disorder.