Functional Role of AGAP2/PIKE-A in Fcγ Receptor-Mediated Phagocytosis.

In phagocytes, cytoskeletal and membrane remodeling is finely regulated at the phagocytic cup. Various smaFll G proteins, including those of the Arf family, control these dynamic processes. Human neutrophils express AGAP2, an Arf GTPase activating protein (ArfGAP) that regulates endosomal trafficking and focal adhesion remodeling. We first examined the impact of AGAP2 on phagocytosis in CHO cells stably expressing the FcγRIIA receptor (CHO-IIA). In unstimulated CHO-IIA cells, AGAP2 only partially co-localized with cytoskeletal elements and intracellular compartments. In CHO-IIA cells, AGAP2 transiently accumulated at actin-rich phagocytic cups and increased Fcγ receptor-mediated phagocytosis. Enhanced phagocytosis was not dependent on the N-terminal GTP-binding protein-like (GLD) domain of AGAP2. AGAP2 deleted of its GTPase-activating protein (GAP) domain was not recruited to phagocytic cups and did not enhance the engulfment of IgG-opsonized beads. However, the GAP-deficient [R618K]AGAP2 transiently localized at the phagocytic cups and enhanced phagocytosis. In PLB-985 cells differentiated towards a neutrophil-like phenotype, silencing of AGAP2 reduced phagocytosis of opsonized zymosan. In human neutrophils, opsonized zymosan or monosodium urate crystals induced AGAP2 phosphorylation. The data indicate that particulate agonists induce AGAP2 phosphorylation in neutrophils. This study highlights the role of AGAP2 and its GAP domain but not GAP activity in FcγR-dependent uptake of opsonized particles.

Regulators and Effectors of Arf GTPases in Neutrophils.

Polymorphonuclear neutrophils (PMNs) are key innate immune cells that represent the first line of defence against infection. They are the first leukocytes to migrate from the blood to injured or infected sites. This process involves molecular mechanisms that coordinate cell polarization, delivery of receptors, and activation of integrins at the leading edge of migrating PMNs. These phagocytes actively engulf microorganisms or form neutrophil extracellular traps (NETs) to trap and kill pathogens with bactericidal compounds. Association of the NADPH oxidase complex at the phagosomal membrane for production of reactive oxygen species (ROS) and delivery of proteolytic enzymes into the phagosome initiate pathogen killing and removal. G protein-dependent signalling pathways tightly control PMN functions. In this review, we will focus on the small monomeric GTPases of the Arf family and their guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) as components of signalling cascades regulating PMN responses. GEFs and GAPs are multidomain proteins that control cellular events in time and space through interaction with other proteins and lipids inside the cells. The number of Arf GAPs identified in PMNs is expanding, and dissecting their functions will provide important insights into the role of these proteins in PMN physiology.

Regulation of inflammation by cannabinoids, the endocannabinoids 2-arachidonoyl-glycerol and arachidonoyl-ethanolamide, and their metabolites.

2-Arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA) are endocannabinoids that have been implicated in many physiologic disorders, including obesity, metabolic syndromes, hepatic diseases, pain, neurologic disorders, and inflammation. Their immunomodulatory effects are numerous and are not always mediated by cannabinoid receptors, reflecting the presence of an arachidonic acid (AA) molecule in their structure, the latter being the precursor of numerous bioactive lipids that are pro- or anti-inflammatory. 2-AG and AEA can thus serve as a source of AA but can also be metabolized by most eicosanoid biosynthetic enzymes, yielding additional lipids. In this regard, enhancing endocannabinoid levels by using endocannabinoid hydrolysis inhibitors is likely to augment the levels of these lipids that could regulate inflammatory cell functions. This review summarizes the metabolic pathways involved in the biosynthesis and metabolism of AEA and 2-AG, as well as the biologic effects of the 2-AG and AEA lipidomes in the regulation of inflammation.

2-Arachidonoyl-glycerol- and arachidonic acid-stimulated neutrophils release antimicrobial effectors against E. coli, S. aureus, HSV-1, and RSV.

The endocannabinoid 2-AG is highly susceptible to its hydrolysis into AA, which activates neutrophils through de novo LTB(4) biosynthesis, independently of CB activation. In this study, we show that 2-AG and AA stimulate neutrophils to release antimicrobial effectors. Supernatants of neutrophils activated with nanomolar concentrations of 2-AG and AA indeed inhibited the infectivity of HSV-1 and RSV. Additionally, the supernatants of 2-AG- and AA-stimulated neutrophils strongly impaired the growth of Escherichia coli and Staphylococcus aureus. This correlated with the release of a large amount (micrograms) of α-defensins, as well as a limited amount (nanograms) of LL-37. All the effects of AA and 2-AG mentioned above were prevented by inhibiting LTB(4) biosynthesis or by blocking BLT(1). Importantly, neither CB(2) receptor agonists nor antagonists could mimic nor prevent the effects of 2-AG, respectively. In fact, qPCR data show that contaminating eosinophils express ∼100-fold more CB(2) receptor mRNA than purified neutrophils, suggesting that CB(2) receptor expression by human neutrophils is limited and that contaminating eosinophils are likely responsible for the previously documented CB(2) expression by freshly isolated human neutrophils. The rapid conversion of 2-AG to AA and their subsequent metabolism into LTB(4) promote 2-AG and AA as multifunctional activators of neutrophils, mainly exerting their effects by activating the BLT(1). Considering that nanomolar concentrations of AA or 2-AG were sufficient to impair viral infectivity, this suggests potential physiological roles for 2-AG and AA as regulators of host defense in vivo.

Leukotriene D4 and interleukin-13 cooperate to increase the release of eotaxin-3 by airway epithelial cells.

INTRODUCTION: Airway epithelial cells play a central role in the physiopathology of asthma. They release eotaxins when treated with T(H)2 cytokines such as interleukin (IL)-4 or IL-13, and these chemokines attract eosinophils and potentiate the biosynthesis of cysteinyl leukotrienes (cysLTs), which in turn induce bronchoconstriction and mucus secretion. These effects of cysLTs mainly mediated by CysLT(1) and CysLT(2) receptors on epithelial cell functions remain largely undefined. Because the release of inflammatory cytokines, eotaxins, and cysLTs occur relatively at the same time and location in the lung tissue, we hypothesized that they regulate inflammation cooperatively rather than redundantly. We therefore investigated whether cysLTs and the T(H)2 cytokines would act in concert to augment the release of eotaxins by airway epithelial cells. METHODS: A549 cells or human primary bronchial epithelial cells were incubated with or without IL-4, IL-13, and/or LTD(4). The release of eotaxin-3 and the expression of cysLT receptors were assessed by ELISA, RT-PCR, and flow cytometry, respectively. RESULTS: IL-4 and IL-13 induced the release of eotaxin-3 by airway epithelial cells. LTD(4) weakly induced the release of eotaxin-3 but clearly potentiated the IL-13-induced eotaxin-3 release. LTD(4) had no effect on IL-4-stimulated cells. Epithelial cells expressed CysLT(1) but not CysLT(2). CysLT(1) expression was increased by IL-13 but not by IL-4 and/or LTD(4). Importantly, the upregulation of CysLT(1) by IL-13 preceded eotaxin-3 release. CONCLUSIONS: These results demonstrate a stepwise cooperation between IL-13 and LTD(4). IL-13 upregulates CysLT(1) expression and consequently the response to cysLTs This results in an increased release of eotaxin-3 by epithelial cells which at its turn increases the recruitment of leukocytes and their biosynthesis of cysLTs. This positive amplification loop involving epithelial cells and leukocytes could be implicated in the recruitment of eosinophils observed in asthmatics.

The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis.

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.

An essential role for phospholipase D in the recruitment of vesicle amine transport protein-1 to membranes in human neutrophils.

Although phosphatidic acid (PA) regulates a wide variety of physiological processes, its targets remain poorly characterized in human neutrophils. By co-sedimentation with PA-containing vesicles we identified several PA-binding proteins including vesicle amine transport protein-1 (VAT-1), Annexin A3 (ANXA3), Rac2, Cdc42 and RhoG in neutrophil cytosol. Except for ANXA3, protein binding to PA-containing liposomes was calcium-independent. Cdc42 and RhoG preferentially interacted with PA whereas VAT-1 bound to PA or phosphatidylserine with the same affinity. VAT-1 translocated to neutrophil membranes upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation. Inhibition of fMLF-induced PLD activity with the Src kinase inhibitor PP2, the selective inhibitor of PLD FIPI, or of PA formation with primary alcohols reduced VAT-1 translocation. In contrast, inhibition of PA hydrolysis with propranolol enhanced fMLF-mediated VAT-1 recruitment to membranes. PMA also redistributed VAT-1 to membranes in a PKC- and PLD-dependent manner. Though fMLF and PMA increased VAT-1 phosphorylation, different kinases appear to be involved. Cell fractionation revealed that a pool of VAT-1 was co-localized with primary, secondary and tertiary granules and plasma membrane markers in resting neutrophils. Stimulation with fMLF enhanced VAT-1 co-localization with CD32a, a plasma membrane marker. Confocal microscopy revealed that VAT-1 decorates granular structures at the cell periphery and double labeling with VAT-1/lactoferrin antibodies showed a partial co-localization with secondary granules in control and fMLF-stimulated cells. Characterization of these putative PA-binding proteins constitutes another step forward for a better understanding of the role of PLD-derived PA in neutrophil physiology.

Characterisation of degranulation and phagocytic capacity of a human neutrophilic cellular model, PLB-985 cells.

Exocytosis of neutrophil granules is a major event that converts circulating neutrophils into fully activated cells capable of chemotaxis, phagocytosis and destruction of pathogens. The PLB-985 cell line is a suitable neutrophilic cellular model which is utilised to study the different functional responses of neutrophils. In this study, we characterised the differentiation of PLB-985 cells toward the granulocytic pathway, using three different inducing agents: dbcAMP, DMSO and DMF. The differentiation efficiency was monitored by observation of cell morphology with electron microscopy, and by analysis of the expression of receptors such as FPRL1 and FcgammaRIIA, the distribution or release of granule markers, phagocytic capacity, as well as measurement of fMLF-induced calcium fluxes. Exocytosis and phagocytosis in differentiated cells were weaker as compared to neutrophils. fMLF stimulated primary granule exocytosis in cells differentiated with dbcAMP, DMSO and DMF, whereas the release of the contents of tertiary granules, as well as that of secretory vesicles, was only observed in dbcAMP-differentiated cells. DMSO-differentiated cells exhibited the highest phagocytic capacity. Altogether our results reinforce the fact that depending on the differentiating agent used, PLB-985 cells represent a useful model to study neutrophil functions and to bypass difficulties inherent to these primary cells.

Crucial implication of protein kinase C (PKC)-delta, PKC-zeta, ERK-1/2, and p38 MAPK in migration of human asthmatic eosinophils.

Asthma is associated with an eosinophil infiltration into the bronchial mucosa. 5-Oxo-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemotactic factor, activates cell motility, adherence, and proteolysis, notably, by promoting CD11b expression, matrix metalloproteinase (MMP)-9 secretion, and plasmin generation. We investigated the intracellular signaling pathways implicated in these various steps by using different, selective inhibitors. Human eosinophil migration through a reconstituted basement membrane in response to 5-oxo-ETE was greatly inhibited (>or=72%) by the protein kinase C (PKC)-delta, PKC-zeta, ERK-1/2, and p38 inhibitors. Our findings indicate that PKC-delta mediates cell motility, CD11b expression, and MMP-9 granule release. PKC-zeta is also largely involved in eosinophil migration, although its specific targets remain undefined. ERK-1/2 and p38 modulate CD11b expression; ERK-1/2 is also involved in long-term MMP-9 secretion and p38 in the plasmin activation system. We demonstrated the crucial implication of PKC-delta, PKC-zeta, ERK-1/2, and p38 in human blood eosinophil migration through extracellular matrix components. Targeting specific pathways may have therapeutic potential for the treatment of allergic airway inflammation.