iPLA2 Activation Mediates Granular Exocytosis and Corrects Microbicidal Defects in ROS-Deficient and CGD Human Neutrophils.

PURPOSE: The ubiquitous calcium-independent phospholipase A2 enzyme (iPLA2) is inhibited by calmodulin binding and known to be responsible for phospholipid remodeling housekeeping functions including granule exocytosis-associated membrane fusion in normal human neutrophils. We evaluate in human neutrophils the iPLA2 secretagogue effects using normal neutrophils, where reactive oxygen species (ROS) generation has been blocked by diphenyleneiodonium, as well as in neutrophils from chronic granulomatous disease (CGD) patients. METHODS: Neutrophils were pretreated with W7, a calmodulin inhibitor known to activate iPLA2 and exocytosis of granules, and vesicles as well as intra- and extra-microbicidal activity against Staphylococcus aureus and Aspergillus fumigatus were evaluated. RESULTS: W7 increases exocytosis of primary, secondary, and tertiary granules and vesicles and improves neutrophil microbicidal activity against S. aureus and A. fumigatus. CONCLUSIONS: In neutrophils, calmodulin-mediated iPLA2 inhibition controls granule and vesicle exocytosis in the phagosome and in the extracellular microenvironment. Relieving iPLA2 inhibition results in increased exocytosis of primary, secondary, and tertiary granules and secretory vesicles with correction of defective intracellular and extracellular microbicidal activity. In CGD patients presenting ROS defective production, this increase in the non-oxidative killing pathway partially corrects their microbicidal defects.

Eosinophils affect functions of in vitro-activated human CD3-CD4+ T cells.

BACKGROUND: The recent development of eosinophil-targeting agents has raised enthusiasm for management of patients with hypereosinophilic syndromes. Roughly half of anti-IL-5-treated patients with corticosteroid-responsive lymphocytic (L-HES) and idiopathic disease variants can be tapered off corticosteroids. Potential consequences of corticosteroid-withdrawal on clonal expansion of pre-malignant CD3⁻CD4⁺ T-cells associated with L-HES are a subject of concern. Indeed, corticosteroid treatment inhibits T-cell activation and may lower blood CD3⁻CD4⁺ cell counts. On the other hand, previous studies have shown that eosinophils support CD4 T-cell activation, suggesting that targeted eosinophil depletion may negatively regulate these cells. OBJECTIVES: Effects of eosinophils on CD4 T-cell activation in vitro were investigated as an indirect means of exploring whether treatment-induced eosinophil depletion may affect pathogenic T-cells driving L-HES. METHODS: Helper (CD4) T-cells and CD3⁻CD4⁺ cells from healthy controls and L-HES patients, respectively, were cultured in vitro in presence of anti-CD3/CD28 or dendritic cells. Effects of eosinophils on T-cell proliferation and cytokine production were investigated. RESULTS: Eosinophils enhanced CD3-driven proliferation of CD4 T-cells from healthy subjects in vitro, while inhibiting TCR-independent proliferation and IL-5 production by CD3⁻CD4⁺ T-cells. CONCLUSIONS: While this study confirms previous work showing that eosinophils support activation of normal helper T-cells, our in vitro findings with CD3⁻CD4⁺ T-cells suggest that eosinophil-depletion may favor activation and expansion of this pathogenic lymphocyte subset. With the ongoing development of eosinophil-targeted therapy for various eosinophilic conditions, the indirect consequences of treatment on the underlying immune mechanisms of disease should be investigated in detail in the setting of translational research programs.

Mechanisms and modulation of pituitary adenylate cyclase-activating protein-induced calcium mobilization in human neutrophils.

The neuropeptide pituitary adenylate cyclase-activating protein (PACAP) acts via the G protein-coupled receptor vasoactive intestinal peptide (VIP)/PACAP receptor-1 to induce phospholipase C (PLC)/calcium and mitogen-activated protein kinase (MAPK)-dependent proinflammatory activities in human polymorphonuclear neutrophils (PMNs). In this article, we evaluate other mechanisms that regulate PACAP-evoked calcium transients, the nature of the calcium sources, and the role of calcium in proinflammatory activities. Reduction in the activity of PMNs to respond to PACAP was observed after cell exposure to inhibitors of the cAMP/protein kinase A (PKA), protein kinase C (PKC), and PI3K pathways, to pertussis toxin (PTX), genistein, and after chelation of intracellular calcium or after extracellular calcium depletion. Mobilization of intracellular calcium stores was based on the fact that PACAP-associated calcium transient was decreased after exposure to (a) thapsigargin (Tg), (b) xestospongin C (XeC), and (c) the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; inhibition of calcium increase by calcium channel blockers, by nifedipine and verapamil, indicated that PACAP was also acting on calcium influx. Such mobilization was not dependent on a functional actin cytoskeleton. Homologous desensitization with nanomoles of PACAP concentration and heterologous receptors desensibilization by G protein-coupled receptor agonists were observed. Intracellular calcium depletion modulated PACAP-associated ERK but not p38 phosphorylation; in contrast, extracellular calcium depletion modulated PACAP-associated p38 but not ERK phosphorylation. In PACAP-treated PMNs, reactive oxygen species production and CD11b membrane upregulation in contrast to lactoferrin release were dependent on both intra- and extracellular calcium, whereas matrix metalloproteinase-9 release was unaffected by extracellular calcium depletion. These data indicate that both extracellular and intracellular calcium play key roles in PACAP proinflammatory activities.

Differential calcium regulation of proinflammatory activities in human neutrophils exposed to the neuropeptide pituitary adenylate cyclase-activating protein.

The neuropeptide pituitary adenylate cyclase-activating protein (PACAP) acts via the G protein-coupled receptor vasoactive intestinal peptide/PACAP receptor-1 to induce phospholipase C/calcium and MAPK-dependent proinflammatory activities in human polymorphonuclear neutrophils (PMNs). In this study, we evaluate other mechanisms that regulate PACAP-evoked calcium transients, the nature of the calcium sources, and the role of calcium in proinflammatory activities. Reduction in the activity of PMNs to respond to PACAP was observed after cell exposure to inhibitors of the cAMP/protein kinase A, protein kinase C, and PI3K pathways, to pertussis toxin, genistein, and after chelation of intracellular calcium or after extracellular calcium depletion. Mobilization of intracellular calcium stores was based on the fact that PACAP-associated calcium transient was decreased after exposure to 1) thapsigargin, 2) Xestospongin C, and 3) the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenyl hydrazone; inhibition of calcium increase by calcium channel blockers, by nifedipine and verapamil, indicated that PACAP was also acting on calcium influx. Such mobilization was not dependent on a functional actin cytoskeleton. Homologous desensitization with nanomoles of PACAP concentration and heterologous receptors desensibilization by G protein-coupled receptor agonists were observed. Intracellular calcium depletion modulated PACAP-associated ERK but not p38 phosphorylation; in contrast, extracellular calcium depletion modulated PACAP-associated p38 but not ERK phosphorylation. In PACAP-treated PMNs, reactive oxygen species production and CD11b membrane up-regulation in contrast to lactoferrin release were dependent on both intra- and extracellular calcium, whereas matrix metalloproteinase-9 release was unaffected by extracellular calcium depletion. These data indicate that both extracellular and intracellular calcium play key roles in PACAP proinflammatory activities.

Regulation of human polymorphonuclear leukocytes functions by the neuropeptide pituitary adenylate cyclase-activating polypeptide after activation of MAPKs.

Anti-inflammatory activities of pituitary adenylate cyclase-activating protein (PACAP) are mediated in part through specific effects on lymphocytes and macrophages. This study shows that in human polymorphonuclear neutrophils (PMNs), PACAP acts as a proinflammatory molecule. In PMNs, vaso-intestinal peptide/PACAP receptor 1 (VPAC-1) was the only receptor found to be expressed by RT-PCR. Using VPAC-1 Ab, we found that VPAC-1 mRNA was translated into proteins. In PMNs, PACAP increases cAMP, inositol triphosphate metabolites, and calcium. It activates two of the three members of the MAPK superfamily, the ERK and the stress-activated MAPK p38. U73122, an inhibitor of phospholipase C (PLC), inhibits PACAP-induced ERK activation, whereas p38 MAPK phosphorylation was unaffected. Using specific pharmalogical inhibitors of ERK (PD098059) and p38 MAPK (SB203580), we found that PACAP-mediated calcium increase was ERK and PLC dependent and p38 independent. PACAP primes fMLP-associated calcium increase; it also primes fMLP activation of the respiratory burst as well as elastase release, these last two processes being ERK and PLC dependent and p38 MAPK independent. PACAP also increases membrane expression of CD11b and release of lactoferrin and metallo proteinase-9 (MMP-9). These effects were PLC dependent (CD 11b, lactoferrin, MMP-9), ERK dependent (CD 11b, lactoferrin, MMP-9), and p38 dependent (CD11b, lactoferrin). We conclude that PACAP is a direct PMN activator as well as an effective PMN priming agent that requires PLC, ERK, and p38 MAPK activities.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) modulate several biochemical pathways in human leukemic myeloid cells.

The neuropeptides Vasoactive-intestinal peptide (VIP) and Pituitary adenylate-cyclase activating protein (PACAP) increased cAMP levels in three out of five human myeloid leukemic cell lines tested while an increased in calcium intracytoplasmic levels was seen only in one cell line (HEL). This increase was phospholipase C, Pertussis toxin dependent and associated with an increase in c-fos and c-jun protein expression together with the formation of functional AP-1 transcriptional factor complex. Cell exposure to VIP or PACAP resulted in a decrease in HEL cell proliferation associated with a down-regulation of the erythroid marker, Glycophorin A. Both peptides were found to increase intra-cytoplasmic calcium levels in blasts isolated from patients with myeloid leukemia. Thus VIP and PACAP are involved in the physiology and pathophysiology of human myeloid cells.