Stefania Loffredo

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.

Lung mast cells are a source of secreted phospholipases A2

BACKGROUND Secreted phospholipases A(2) (sPLA(2)s) are released in plasma and other biologic fluids of patients with inflammatory, autoimmune, and allergic diseases. OBJECTIVE We sought to evaluate sPLA(2) activity in the bronchoalveolar lavage fluid (BALF) of asthmatic patients and to examine the expression and release of sPLA(2)s from primary human lung mast cells (HLMCs). METHODS sPLA(2) activity was measured in BALF and supernatants of either unstimulated or anti-IgE-activated HLMCs as hydrolysis of oleic acid from radiolabeled Escherichia coli membranes. Expression of sPLA(2)s was examined by using RT-PCR. The release of cysteinyl leukotriene (LT) C(4) was measured by means of enzyme immunoassay. RESULTS Phospholipase A(2) (PLA(2)) activity was higher in the BALF of asthmatic patients than in the control group. BALF PLA(2) activity was blocked by the sPLA(2) inhibitors dithiothreitol and Me-Indoxam but not by the cytosolic PLA(2) inhibitor AZ-1. HLMCs spontaneously released a PLA(2) activity that was increased on stimulation with anti-IgE. This PLA(2) activity was blocked by dithiothreitol and Me-Indoxam but not by AZ-1. HLMCs constitutively express mRNA for group IB, IIA, IID, IIE, IIF, III, V, X, XIIA, and XIIB sPLA(2)s. Anti-IgE did not modify the expression of sPLA(2)s. The cell-impermeable inhibitor Me-Indoxam significantly reduced (up to 40%) the production of LTC(4) from anti-IgE-stimulated HLMCs. CONCLUSIONS sPLA(2) activity is increased in the airways of asthmatic patients. HLMCs express multiple sPLA(2)s and release 1 or more of them when activated by anti-IgE. The sPLA(2)s released by mast cells contribute to LTC(4) production by acting in an autocrine fashion. Mast cells can be a source of sPLA(2)s in the airways of asthmatic patients.

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.

Mast cells and basophils in inflammatory and tumor angiogenesis and lymphangiogenesis

Angiogenesis, namely, the growth of new blood vessels from pre-existing ones, is an essential process of embryonic development and post-natal growth. In adult life, it may occur in physiological conditions (menstrual cycle and wound healing), during inflammatory disorders (autoimmune diseases and allergic disorders) and in tumor growth. The angiogenic process requires a tightly regulated interaction among different cell types (e.g. endothelial cells and pericytes), the extracellular matrix, several specific growth factors (e.g. VEGFs, Angiopoietins), cytokines and chemokines. Lymphangiogenesis, namely, the growth of new lymphatic vessels, is an important process in tumor development, in the formation of metastasis and in several inflammatory and metabolic disorders. In addition to tumors, several effector cells of inflammation (mast cells, macrophages, basophils, eosinophils, neutrophils, etc.) are important sources of a wide spectrum of angiogenic and lymphangiogenic factors. Human mast cells produce a large array of angiogenic and lymphangiogenic molecules. Primary human mast cells and two mast cell lines constitutively express several isoforms of angiogenic (VEGF-A and VEGF-B) and the two lymphangiogenic factors (VEGF-C and VEGF-D). In addition, human mast cells express the VEGF receptor 1 (VEGFR-1) and 2 (VEGFR-2), the co-receptors neuropilin-1 (NRP1) and -2 (NRP2) and the Tie1 and Tie2 receptors. Immunologically activated human basophils selectively produce VEGF-A and -B, but not VEGF-C and -D. They also release Angiopoietin1 that activates Tie2 on human mast cells. Collectively, these findings indicate that human mast cells and basophils might participate in the complex network involving inflammatory and tumor angiogenesis and lymphangiogenesis.

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.

Are Mast Cells MASTers in Cancer

Prolonged low-grade inflammation or smoldering inflammation is a hallmark of cancer. Mast cells form a heterogeneous population of immune cells with differences in their ultra-structure, morphology, mediator content, and surface receptors. Mast cells are widely distributed throughout all tissues and are stromal components of the inflammatory microenvironment that modulates tumor initiation and development. Although canonically associated with allergic disorders, mast cells are a major source of pro-tumorigenic (e.g., angiogenic and lymphangiogenic factors) and antitumorigenic molecules (e.g., TNF-α and IL-9), depending on the milieu. In certain neoplasias (e.g., gastric, thyroid and Hodgkin’s lymphoma) mast cells play a pro-tumorigenic role, in others (e.g., breast cancer) a protective role, whereas in yet others they are apparently innocent bystanders. These seemingly conflicting results suggest that the role of mast cells and their mediators could be cancer specific. The microlocalization (e.g., peritumoral vs intratumoral) of mast cells is another important aspect in the initiation/progression of solid and hematologic tumors. Increasing evidence in certain experimental models indicates that targeting mast cells and/or their mediators represent a potential therapeutic target in cancer. Thus, mast cells deserve focused consideration also as therapeutic targets in different types of tumors. There are many unanswered questions that should be addressed before we understand whether mast cells are an ally, adversary, or innocent bystanders in human cancers.

Mutation of the angiopoietin-1 gene (ANGPT1) associates with a new type of hereditary angioedema

Background: Hereditary angioedema (HAE) is a rare genetic disease usually caused by mutation in the C1 inhibitor or the coagulation Factor XII gene. However, in a series of patients with HAE, no causative variants have been described, and the pathophysiology of the disease remains unknown (hereditary angioedema with yet unknown genetic defect [U‐HAE]). Identification of causative genes in patients with U‐HAE is valuable for understanding the cause of the disease. Objective: We conducted genetic studies in Italian patients with U‐HAE to identify novel causative genes. Methods: Among patients belonging to 10 independent families and unrelated index patients with U‐HAE recruited from the Italian Network for C1‐INH‐HAE (ITACA), we selected a large multiplex family with U‐HAE and performed whole‐exome sequencing. The angiopoietin‐1 gene (ANGPT1) was investigated in all patients with familial or sporadic U‐HAE. The effect of ANGPT1 variants was investigated by using in silico prediction and plasma and transfected cells from both patients and control subjects. Results: We identified a missense mutation (ANGPT1, c.807G>T, p.A119S) in a family with U‐HAE. The ANGPT1 p.A119S variant was detected in all members of the index family with U‐HAE but not in asymptomatic family members or an additional 20 patients with familial U‐HAE, 22 patients with sporadic U‐HAE, and 200 control subjects. Protein analysis of the plasma of patients revealed a reduction of multimeric forms and a reduced ability to bind the natural receptor tunica interna endothelial cell kinase 2 of the ANGPT1 p.A119S variant. The recombinant mutated ANGPT1 p.A119S formed a reduced amount of multimers and showed reduced binding capability to its receptor. Conclusion: ANGPT1 impairment is associated with angioedema, and ANGPT1 variants can be the basis of HAE.

Immune Cells as a Source and Target of Angiogenic and Lymphangiogenic Factors

Angiogenesis and lymphangiogenesis are distinct and complex processes requiring a finely tuned balance between stimulatory and inhibitory signals. Immune and inflammatory cells can contribute to these processes by multiple mechanisms: directly by producing a broad array of angiogenic growth factors, and indirectly by secreting several cytokines, chemokines and other mediators able to coordinate the cell-cell interactions. Immune cells can stimulate or inhibit angiogenesis/lymphangiogenesis, depending on their activation status and subset specificity. We summarize recent findings reporting the expression and activity of angiogenic and lymphangiogenic factors and their receptors and coreceptors in immune cells. It is evident that modulation of angiogenesis and lymphangiogenesis by the innate and adaptive immune cells (mast cells, macrophages, dendritic cells, basophils, eosinophils, and some subsets of T cells) is a highly complex process not yet completely understood.

Human lung-resident macrophages express CB1 and CB2 receptors whose activation inhibits the release of angiogenic and lymphangiogenic factors

Macrophages are pivotal effector cells in immune responses and tissue remodeling by producing a wide spectrum of mediators, including angiogenic and lymphangiogenic factors. Activation of cannabinoid receptor types 1 and 2 has been suggested as a new strategy to modulate angiogenesis in vitro and in vivo. We investigated whether human lung‐resident macrophages express a complete endocannabinoid system by assessing their production of endocannabinoids and expression of cannabinoid receptors. Unstimulated human lung macrophage produce 2‐arachidonoylglycerol, N‐arachidonoyl‐ethanolamine, N‐palmitoyl‐ethanolamine, and N‐oleoyl‐ethanolamine. On LPS stimulation, human lung macrophages selectively synthesize 2‐arachidonoylglycerol in a calcium‐dependent manner. Human lung macrophages express cannabinoid receptor types 1 and 2, and their activation induces ERK1/2 phosphorylation and reactive oxygen species generation. Cannabinoid receptor activation by the specific synthetic agonists ACEA and JWH‐133 (but not the endogenous agonist 2‐arachidonoylglycerol) markedly inhibits LPS‐induced production of vascular endothelial growth factor‐A, vascular endothelial growth factor‐C, and angiopoietins and modestly affects IL‐6 secretion. No significant modulation of TNF‐α or IL‐8/CXCL8 release was observed. The production of vascular endothelial growth factor‐A by human monocyte‐derived macrophages is not modulated by activation of cannabinoid receptor types 1 and 2. Given the prominent role of macrophage‐assisted vascular remodeling in many tumors, we identified the expression of cannabinoid receptors in lung cancer‐associated macrophages. Our results demonstrate that cannabinoid receptor activation selectively inhibits the release of angiogenic and lymphangiogenic factors from human lung macrophage but not from monocyte‐derived macrophages. Activation of cannabinoid receptors on tissue‐resident macrophages might be a novel strategy to modulate macrophage‐assisted vascular remodeling in cancer and chronic inflammation.