Giorgio Giannattasio

Secretory Phospholipases A2 as Multivalent Mediators of Inflammatory and Allergic Disorders

Phospholipases A2 (PLA2s) are enzymes responsible for mobilization of fatty acids, including arachidonic acid (AA), from phospholipids. These enzymes are classified as high-molecular-weight cytosolic PLA2s (cPLA2s) and low-molecular-weight secretory PLA2s (sPLA2s). There is increasing evidence that large quantities of sPLA2s are released in the plasma of patients with systemic inflammatory and autoimmune diseases. In addition, high levels of sPLA2s can be detected at sites of allergic inflammation including the upper airways of patients with rhinitis and the lower airways of patients with asthma. These extracellular enzymes play an important role in inflammation by releasing AA, which can be subsequently converted to proinflammatory prostaglandins and leukotrienes. Generation of AA mediated by sPLA2s occurs through different mechanisms, including (1) the direct hydrolysis of outer cell membrane phospholipids, (2) internalization and transfer of sPLA2s to intracellular pools of phospholipids enriched in AA, and (3) activation of cPLA2s. In addition, sPLA2s induce degranulation and production of cytokines and chemokines from a variety of cells involved in inflammatory and immune responses. These effects are exerted by mechanisms that are independent of the enzymatic activity and are mediated by the interaction of sPLA2s with specific or promiscuous membrane receptors. Therefore, sPLA2s may have an important role in inflammatory and allergic reactions by activating multiple mechanisms within inflammatory and immune cells, leading to the production of eicosanoids, cytokines and chemokines.

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.

Group V Secretory Phospholipase A2 Reveals Its Role in House Dust Mite-Induced Allergic Pulmonary Inflammation by Regulation of Dendritic Cell Function

We have previously shown that group V secretory phospholipase A2 (sPLA2) regulates phagocytosis of zymosan and Candida albicans by a mechanism that depends on fusion of phagosomes with late endosomes in macrophages. In this study, we report that group V sPLA2 (Pla2g5)-null mice exposed to an extract of house dust mite Dermatophagoides farinae had markedly reduced pulmonary inflammation and goblet cell metaplasia compared with wild-type (WT) mice. Pla2g5-null mice had also impaired Th2-type adaptive immune responses to D. farinae compared with WT mice. Pla2g5-null bone marrow-derived dendritic cells (BMDCs) activated by D. farinae had delayed intracellular processing of allergen and impaired allergen-dependent maturation, a pattern recapitulated by the native lung DCs of D. farinae-challenged mice. Adoptively transferred D. farinae-loaded Pla2g5-null BMDCs were less able than D. farinae-loaded WT BMDCs to induce pulmonary inflammation and Th2 polarization in WT mice. However, Pla2g5-null recipients transferred with WT or Pla2g5-null D. farinae-loaded BMDCs exhibited significantly reduced local inflammatory responses to D. farinae, even though the transfer of WT BMDCs still induced an intact Th2 cytokine response in regional lymph nodes. Thus, the expression of group V sPLA2 in APCs regulates Ag processing and maturation of DCs and contributes to pulmonary inflammation and immune response against D. farinae. Furthermore, an additional yet to be identified resident cell type is essential for the development of pulmonary inflammation, likely a cell in which group V sPLA2 is upregulated by D. farinae, and whose function is also regulated by group V sPLA2.

Expression of phospholipases A2 in primary human lung macrophages Role of cytosolic phospholipase A2-α in arachidonic acid release and platelet activating factor synthesis

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA(2)) and secreted phospholipases A(2) (sPLA(2)) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA(2)s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA(2) (iPLA(2)). Two structurally-divergent inhibitors of group IV cPLA(2) completely block arachidonic acid release by macrophages in response to non-physiological (Ca(2+) ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA(2)s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA(2)s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA(2) inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA(2)-alpha, iPLA(2) and several sPLA(2)s. Cytosolic PLA(2)-alpha is the major enzyme responsible for lipid mediator production in human macrophages.

Differential modulation of mediator release from human basophils and mast cells by mizolastine

Background Basophils and mast cells play a major role in the pathogenesis of allergic disorders by releasing several proinflammatory mediators. Some histamine H1 receptor antagonists exert anti‐inflammatory activities by modulating mediator release from basophils and mast cells.

The Purinergic G Protein-Coupled Receptor 6 Inhibits Effector T Cell Activation in Allergic Pulmonary Inflammation

We show that the P2Y6 receptor, a purinergic G protein-coupled receptor with a high affinity for the nucleotide uridine diphosphate, is an important endogenous inhibitor of T cell function in allergic pulmonary inflammation. Mice conditionally deficient in P2Y6 receptors [p2ry6 (flox/flox);cre/+ mice] exhibited severe airway and tissue pathology relative to P2Y6-sufficient [p2ry6 (flox/flox)] littermates (+/+ mice) when treated intranasally with an extract of the dust mite Dermatophagoides farinae (Df). P2Y6 receptors were inducibly expressed by lung, lymph node, and splenic CD4+ and CD8+ T cells of Df-treated +/+ mice. Df-restimulated P2Y6-deficient lymph node cells produced higher levels of Th1 and Th2 cytokines, and polyclonally stimulated P2Y6-deficient CD4+ T cells proliferated faster than comparably stimulated P2Y6-sufficient cells. The absence of P2Y6 receptors on CD4+ cells, but not APCs, was sufficient to amplify cytokine generation. Thus, P2Y6 receptors protect the lung against exuberant allergen-induced pulmonary inflammation by inhibiting the activation of effector T cells.

Fas-Activated Serine/Threonine Phosphoprotein Promotes Immune-Mediated Pulmonary Inflammation

We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST−/−) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST−/− mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST−/− mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-α and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST−/− neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.

The translational repressor T-cell intracellular antigen-1 (TIA-1) is a key modulator of Th2 and Th17 responses driving pulmonary inflammation induced by exposure to house dust mite

T-cell intracellular antigen-1 (TIA-1) is a translational repressor that dampens the production of proinflammatory cytokines and enzymes. In this study we investigated the role of TIA-1 in a mouse model of pulmonary inflammation induced by exposure to the allergenic extract (Df) of the house dust mite Dermatophagoides farinae. When intranasally challenged with a low dose of Df, mice lacking TIA-1 protein (Tia-1(-/-)) showed more severe airway and tissue eosinophilia, infiltration of lung bronchovascular bundles, and goblet cell metaplasia than wild-type littermates. Tia-1(-/-) mice also had higher levels of Df-specific IgE and IgG(1) in serum and ex vivo restimulated Tia-1(-/-) lymph node cells and splenocytes transcribed and released more Th2/Th17 cytokines. To evaluate the site of action of TIA-1, we studied the response to Df in bone marrow chimeras. These experiments revealed that TIA-1 acts on both hematopoietic and non-hematopoietic cells to dampen pulmonary inflammation. Our results identify TIA-1 as a negative regulator of allergen-mediated pulmonary inflammation in vivo. Thus, TIA-1 might be an important player in the pathogenesis of bronchial asthma.

Lysophospholipid Transacetylase in the Regulation of Paf Levels in Human Monocytes and Macrophages

The transacetylase (TA), reported to be identical to platelet‐activating factor (PAF) acetylhydrolase (II), is a multifunctional enzyme with three catalytic activities: lysophospholipid transacetylase (TAL), sphingosine transacetylase (TAS), and acetylhydrolase (AH). We report that TAL activity participates in the control of PAF levels in monocytes and macrophages and that its regulation differs in these two types of cells. In monocytes, LPS or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) specifically increased the TAL activity. Western blot analysis and enzyme assays on immunoprecipitates revealed that the increased activity can be ascribed to PAF‐AH (II) and that both translocation from cytosol to membranes and p38/ERKs‐mediated phosphorylation regulate the enzyme activation. Instead, in macrophages differentiated in vitro from monocytes by incubation with FCS, an increase of both TAL and AH activities was observed. Moreover, activation of ERKs and p38 MAP kinase was not required for the up‐regulation of PAF‐AH (II) in differentiated macrophages. The differences observed in macrophages as compared to monocytes can be explained by 1) p38/ERKs‐independent phosphorylation of PAF‐AH (II) and 2) appearance of plasma PAF‐AH in the course of macrophage differentiation.—Servillo L., Balestrieri C., Giovane A., Pari P., Palma D., Giannattasio G., Triggiani M., Balestrieri M. L. Lysophospholipid transacetylase in the regulation of PAF levels in human monocytes and macrophages. FASEB J. 20, E222–E234 (2006)

Remodeling of arachidonic acid in inflammatory cells of the human lung

Arachidonic acid (AA) is a key molecule in the modulation of several pathophysiological events in mammalian cells, including gene expression, membrane signal transduction, cell differentiation and apoptosis, exocytosis and generation of eicosanoids [1, 2, 3, 4]. It has been now convincingly demonstrated that the intracellular levels of free AA within mammalian cells are crucial for most of these events and, therefore, they are carefully regulated by complex biochemical reactions [5, 6]. These reactions are catalyzed by enzymes involved in both AA mobilization and reesterification into the storage sites and transfer from one intracellular pool to another [7, 8].