Mechanical induction of group V phospholipase A(2) causes lung inflammation and acute lung injury.

Ventilation at high tidal volume may cause lung inflammation and barrier dysfunction that culminates in ventilator-induced lung injury (VILI). However, the mechanisms by which mechanical stimulation triggers the inflammatory response have not been fully elucidated. This study tested the hypothesis that onset of VILI is triggered by activation of secretory group V phospholipase A(2) (gVPLA2) in pulmonary vascular endothelium exposed to excessive mechanical stretch. High-magnitude cyclic stretch (18% CS) increased expression and surface exposure of gVPLA2 in human pulmonary endothelial cells (EC). CS-induced gVPLA2 activation was required for activation of ICAM-1 expression and polymorphonuclear neutrophil (PMN) adhesion to CS-preconditioned EC. By contrast, physiological CS (5% CS) had no effect on gVPLA2 activation or EC-PMN adhesion. CS-induced ICAM-1 expression and EC-PMN adhesion were attenuated by the gVPLA2-blocking antibody (MCL-3G1), general inhibitor of soluble PLA2, LY311727, or siRNA-induced EC gVPLA2 knockdown. In vivo, ventilator-induced lung leukocyte recruitment, cell and protein accumulation in the alveolar space, and total lung myeloperoxidase activity were strongly suppressed in gVPLA2 mouse knockout model or upon administration of MCL-3G1. These results demonstrate a novel role for gVPLA2 as the downstream effector of pathological mechanical stretch leading to an inflammatory response associated with VILI.

Group V phospholipase A2 mediates barrier disruption of human pulmonary endothelial cells caused by LPS in vitro.

We examined the functional role of 14-kD secretory group V phospholipase A(2) (gVPLA(2)) on the barrier function of pulmonary endothelial cells (ECs) after LPS activation in vitro. Expression of gVPLA(2) was elicited by 20 ng/ml LPS as demonstrated by increased (1) mRNA, (2) protein content, and (3) cell surface expression of gVPLA(2) within 4 hours. The effect of LPS on EC barrier function was measured by transendothelial monolayer electrical resistance (TER). LPS increased permeability across EC monolayers at 2-3 hours, and was sustained for 10 hours or more. Blockade of gVPLA(2) with mouse monoclonal 3G1 (MCL-3G1) monoclonal antibody directed against gVPLA(2) inhibited EC barrier dysfunction elicited by LPS in a time- and concentration-dependent manner; control IgG had no effect on TER. Like LPS, exogenous gVPLA(2) caused increased EC permeability in a time- and concentration-dependent manner; neither gIIaPLA(2), a close homolog of gVPLA(2), nor W31A, an inactive mutant of gVPLA(2), caused a decrease in EC TER. Immunofluorescence analysis revealed comparable F-actin stress fiber and intercellular gap formation for ECs treated with either gVPLA(2) or LPS. Treatment with gVPLA(2) disrupted vascular endothelial-cadherin junctional complexes on ECs. Coincubation of ECs with MCL-3G1 substantially attenuated the structural changes caused by gVPLA(2) or LPS. We demonstrate that (1) gVPLA(2) is constitutively expressed in ECs and is up-regulated after LPS activation, (2) endogenously secreted gVPLA(2) from ECs after LPS increases EC permeability through F-actin and junctional complex rearrangement, and (3) inhibition of endogenous gVPLA(2) from ECs is sufficient to block disruption of the EC barrier function after LPS in vitro.

Blockade of inflammation and airway hyperresponsiveness in immune-sensitized mice by dominant-negative phosphoinositide 3-kinase-TAT.

Phosphoinositide 3-kinase (PI3K) is thought to contribute to the pathogenesis of asthma by effecting the recruitment, activation, and apoptosis of inflammatory cells. We examined the role of class IA PI3K in antigen-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of Deltap85 protein, a dominant negative form of the class IA PI3K regulatory subunit, p85alpha, which was fused to HIV-TAT (TAT-Deltap85). Intraperitoneal administration of TAT-Deltap85 caused time-dependent transduction into blood leukocytes, and inhibited activated phosphorylation of protein kinase B (PKB), a downstream target of PI3K, in lung tissues in mice receiving intranasal FMLP. Antigen challenge elicited pulmonary infiltration of lymphocytes, eosinophils and neutrophils, increase in mucus-containing epithelial cells, and airway hyperresponsiveness to methacholine. Except for modest airway neutrophilia, these effects all were blocked by treatment with 3-10 mg/kg of TAT-Deltap85. There was also significant reduction in IL-5 and IL-4 secretion into the BAL. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Deltap85. We conclude that PI3K has a regulatory role in Th2-cell cytokine secretion, airway inflammation, and airway hyperresponsiveness in mice.

Attenuated translocation of group IVa phospholipase A2 and up-regulated annexin-1 synthesis by glucocorticoid blocks beta 2-integrin adhesion in neutrophils.

We examined the effect of glucocorticoid stimulation in blocking beta 2-integrin adhesion of polymorphonuclear leukocytes (PMNs) isolated from human subjects. Surface expression of CD11b and ERK-1/2-mediated gIVaPLA2 phosphorylation, which are required for beta 2-integrin adhesion, were not affected by treatment with < or =10(-6) M fluticasone propionate (FP) for PMNs activated by either 10(-7) M LTB4 or 30 ng/ml TNF-alpha and caused no significant blockade of beta 2-integrin adhesion in vitro. Baseline expression of annexin-1 (ANXA1) synthesis was increased only after 10(-6) M FP for PMNs; by contrast, comparable increase in ANXA1 expression was demonstrated in human eosinophils from the same subjects with 10(-8) M FP. Viability of PMNs was verified by propidium iodide and by the persistence of beta 2-integrin adhesion in treated groups. Exogenous administration of ANXA1 mimetic peptide fragment blocked significantly and comparably the beta 2-integrin adhesion in PMNs activated by LTB4 and TNF-alpha and in eosinophils activated by IL-5. Translocation of gIVaPLA2 from the cytosol to the nucleus also was refractory for activated PMNs treated with > or =10(-7) M FP; by contrast, complete blockade of nuclear translocation of cytosolic gIVaPLA2 was effected by 10(-9) M FP in eosinophils. Our data indicate that the cell surface ANXA1 synthesis is capable of blocking beta 2-integrin adhesion in both PMNs and eosinophils. However, in contrast to eosinophils, FP does not cause either substantial ANXA1 synthesis or nuclear transport of cytosolic gIVaPLA2 in PMNs and thus does not block beta2-integrin adhesion, a necessary step for granulocyte cell migration in vivo.

Blockade of avidity and focal clustering of beta 2-integrin by cysteinyl leukotriene antagonism attenuates eosinophil adhesion.

BACKGROUND: Cysteinyl leukotriene (cysLT) antagonism attenuates migration of eosinophils into airways during immune challenge in human subjects and animal models. The intracellular signaling mechanism by which this occurs has not been elucidated. OBJECTIVE: We sought to determine the relative efficacy and mechanism by which 5-lipoxygenase (5-LO) inhibition and cysLT(1) receptor (cysLT(1)R) antagonism block beta(2)-integrin adhesion in isolated human eosinophils in vitro. METHODS: Human blood eosinophils were isolated by means of immunomagnetic separation. Upregulation of CD11b expression, active conformation of CD11b, and focal clustering of beta(2)-integrin caused by IL-5, eotaxin-1 or leukotriene (LT) B(4) was assessed by means of flow cytometry and confocal microscopy. The effect and mechanism of cysLT(1)R or 5-LO blockade on these components of beta(2)-integrin adhesion were determined. RESULTS: Montelukast, a cysLT(1)R antagonist, and AA861, a 5-LO enzyme inhibitor, blocked (1) avidity of beta(2)-integrin, (2) beta(2)-integrin-mediated adhesion to intercellular adhesion molecule 1, and (3) focal clustering of CD11b elicited by LTB(4). However, adhesion caused by either IL-5 or eotaxin-1 was not attenuated for eosinophils pretreated with either montelukast or AA861. CONCLUSION: Our data demonstrate that (1) LTB(4) causes autocrine upregulation of adhesion through secretion of cysLTs, and (2) blockade of cysLT(1)R blocks the avidity and focal clustering of CD11b/CD18 for eosinophils activated by LTB(4) but not by IL-5 or eotaxin-1. CLINICAL IMPLICATIONS: Unlike cysLT-induced adhesion, adhesion caused by IL-5 or eotaxin-1 is not regulated through the cysLT(1)R, suggesting that cysLTs have specific but limited potential to upregulate eosinophil adhesion.

IL-5-induced integrin adhesion of human eosinophils caused by ERK1/2-mediated activation of cPLA2.

We examined the mechanism by which interleukin (IL)-5 causes beta(2)-integrin adhesion of human eosinophils. IL-5 caused time-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38alpha in eosinophils as detected by their phosphorylation. Preincubation of eosinophils with U0126, a mitogen-activated protein kinase/ERK kinase inhibitor, suppressed IL-5-induced activation of cytosolic phospholipase A(2) (cPLA(2)) and eosinophil adhesion, and p38 inhibition by SB203580 had neither effect. ERK1/2 phosphorylation and eosinophil adhesion were blocked by inhibition of the src-family tyrosine kinase, Janus tyrosine kinase (JAK)2, or phosphoinositide-3 kinase (PI3K). Coimmunoprecipitation assay demonstrated that Lyn, a src-family tyrosine kinase, was constitutively associated with PI3K. Inhibition of src-tyrosine kinase but not JAK2 suppressed PI3K activation. Our data suggest that IL-5 induces beta(2)-integrin adhesion of human eosinophils by regulation of cPLA(2) activation caused by ERK1/2 phosphorylation. This phosphorylation results from activation of PI3K and protein tyrosine kinases. We also find that src-family tyrosine kinase, possibly Lyn, is the upstream kinase causing PI3K activation.

Quantitation of secretory group V phospholipase A(2) in human tissues by sandwich enzyme-linked immunosorbent assay.

We have developed a sensitive sandwich ELISA (sELISA) for quantitative determination of group V phospholipase A(2) (gVPLA(2)). This assay utilizes three monoclonal antibodies (mAbs) directed against human gVPLA(2) (MCL-1B7, MCL-2A5, and MCL-3G1), which recognize specifically different epitopes of gVPLA(2). A mixture of MCL-1B7 and MCL-2A5 was used as the capture mAb, and MCL-3G1 as the detector mAb; purified human gVPLA(2) was used as the standard protein. The limit of detection of the sELISA is 2 ng/ml; the intra- and inter-coefficients of variation were 4.97+/-0.81% and 8.42+/-3.4%. The validity of the sELISA was assured by the recovery of exogenous recombinant gVPLA(2), which was 99.7% to 102%, and demonstration of noninterference of the gVPLA(2) assay by a high concentrations of other protein from murine lung and heart. To assess the usefulness of this sELISA for tissue measurements, the amount of gVPLA(2) in cultured human epithelial cells and isolated human eosinophils was determined. Total gVPLA(2) mass in epithelial cells was 2.83+/-0.33 ng/10(7) cells; gVPLA(2) was not detected in eosinophils. The presence of high concentration of gVPLA(2) in epithelial cells was confirmed by immunoprecipitation/Western blot analysis and by flow cytometry. This assay allows for convenient differentiation between the highly homologous 14-kDa secretory PLA(2)s, gVPLA(2), gIIaPLA(2), gIbPLA(2) and gXPLA(2), and accurate quantitation of gVPLA(2) in biological samples.

Group V phospholipase A(2) increases pulmonary endothelial permeability through direct hydrolysis of the cell membrane.

Acute lung injury (ALI) is characterized by inflammatory disruption of the alveolar-vascular barrier, resulting in severe respiratory compromise. Inhibition of the intercellular messenger protein, Group V phospholipase A(2) (gVPLA(2)), blocks vascular permeability caused by LPS both in vivo and in vitro. In this investigation we studied the mechanism by which recombinant gVPLA(2) increases permeability of cultured human pulmonary endothelial cells (EC). Exogenous gVPLA(2) (500 nM), a highly hydrolytic enzyme, caused a significant increase in EC permeability that began within minutes and persisted for >10 hours. However, the major hydrolysis products of gVPLA(2) (Lyso-PC, Lyso-PG, LPA, arachidonic acid) did not cause EC structural rearrangement or loss of barrier function at concentrations <10 µM. Higher concentrations (≥ 30 µM) of these membrane hydrolysis products caused some increased permeability but were associated with EC toxicity (measured by propidium iodide incorporation) that did not occur with barrier disruption by gVPLA(2) (500 nM). Pharmacologic inhibition of multiple intracellular signaling pathways induced by gVPLA(2) activity (ERK, p38, PI3K, cytosolic gIVPLA(2)) also did not prevent EC barrier disruption by gVPLA(2). Finally, pretreatment with heparinase to prevent internalization of gVPLA(2) did not inhibit EC barrier disruption by gVPLA(2). Our data thus indicate that gVPLA(2) increases pulmonary EC permeability directly through action as a membrane hydrolytic agent. Disruption of EC barrier function does not depend upon membrane hydrolysis products, gVPLA(2) internalization, or upregulation of downstream intracellular signaling.

Cytosolic group IVa phospholipase A2 mediates IL-8/CXCL8-induced transmigration of human polymorphonuclear leukocytes in vitro.

BACKGROUND: Cytosolic gIVaPLA2 is a critical enzyme in the generation of arachidonate metabolites and in induction of beta2-integrin adhesion in granulocytes. We hypothesized that gIVaPLA2 activation also is an essential downstream step for post adhesive migration of PMN in vitro. METHODS: Migration of PMNs caused by IL-8/CXCL8 was assessed using a transwell migration chamber. PMNs were pretreated with two structurally unrelated inhibitors of gIVaPLA2, arachidonyl trifluoromethylketone (TFMK) or pyrrophenone, prior to IL-8/CXCL8 exposure. The fraction of migrated PMNs present in the lower chamber was measured as total myeloperoxidase content. GIVaPLA2 enzyme activity was analyzed using [14C-PAPC] as specific substrate F-actin polymerization and cell structure were examined after rhodamine-phalloidin staining. RESULTS: IL-8/CXCL8-induced migration of PMNs was elicited in concentration- and time-dependent manner. Time-related phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus was observed for PMNs stimulated with IL-8/CXCL8 in concentration sufficient to cause upstream phosphorylation of MAPKs (ERK-1/2 and p38) and Akt/PKB. Inhibition of gIVaPLA2 corresponded to the magnitude of blockade of PMN migration. Neither AA nor LTB4 secretion was elicited following IL-8/CXCL8 activation. In unstimulated PMNs, F-actin was located diffusely in the cytosol; however, a clear polarized morphology with F-actin-rich ruffles around the edges of the cell was observed after activation with IL-8/CXCL8. Inhibition of gIVaPLA2 blocked change in cell shape and migration caused by IL-8/CXCL8 but did not cause F-actin polymerization or translocation of cytosolic F-actin to inner leaflet of the PMN membrane. CONCLUSION: We demonstrate that IL-8/CXCL8 causes a) phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus, b) change in cell shape, c) polymerization of F-actin, and d) chemoattractant/migration of PMN in vitro. Inhibition of gIVaPLA2 blocks the deformability and subsequent migration of PMNs caused by IL-8/CXCL8. Our data suggest that activation of gIVaPLA2 is an essential step in PMN migration in vitro.

Secretory group V phospholipase A2 regulates acute lung injury and neutrophilic inflammation caused by LPS in mice.

We investigated the regulatory role of 14-kDa secretory group V phospholipase A(2) (gVPLA(2)) in the development of acute lung injury (ALI) and neutrophilic inflammation (NI) caused by intratracheal administration of LPS. Experiments were conducted in gVPLA(2) knockout (pla2g5(-/-)) mice, which lack the gene, and gVPLA(2) wild-type littermate control (pla2g5(+/+)) mice. Indices of pulmonary injury were evaluated 24 h after intratracheal administration of LPS. Expression of gVPLA(2) in microsections of airways and mRNA content in lung homogenates were increased substantially in pla2g5(+/+) mice after LPS-administered compared with saline-treated pla2g5(+/+) mice. By contrast, expression of gVPLA(2) was neither localized in LPS- nor saline-treated pla2g5(-/-) mice. LPS also caused 1) reduced transthoracic static compliance, 2) lung edema, 3) neutrophilic infiltration, and 4) increased neutrophil myeloperoxidase activity in pla2g5(+/+) mice. These events were attenuated in pla2g5(-/-) mice exposed to LPS or in pla2g5(+/+) mice receiving MCL-3G1, a neutralizing MAb directed against gVPLA(2), before LPS administration. Our data demonstrate that gVPLA(2) is an inducible protein in pla2g5(+/+) mice but not in pla2g5(-/-) mice within 24 h after LPS treatment. Specific inhibition of gVPLA(2) with MCL-3G1 or gene-targeted mice lacking gVPLA(2) blocks ALI and attenuates NI caused by LPS.

Future treatment to lessen exacerbations of chronic obstructive pulmonary disease.

Therapies currently used to reduce exacerbations of chronic obstructive pulmonary disease (COPD) are compounds used almost entirely for asthma therapy. A notable exception is tiotropium, a long-acting parasympatholytic agent. This compound and its precursor, iprotropium, are only occasionally used for asthma therapy. Likewise, leukotriene-modifying drugs are used occasionally for the treatment of COPD. In neither circumstance is there agency-approved indication for these particular cross-over therapies, but the use of long-acting beta(2)-adrenergic compounds and high-solubility inhaled steroids is a mainstay for therapy in both asthma and COPD. Similarly, theophylline, although less often used for either process, is therapeutically applicable to both asthma and COPD. Although overlap syndromes point to the occurrence of a common pathway in some cases, the inflammatory process for asthma and chronic obstructive pulmonary disease (COPD) differs substantially in most cases. Hence, the application of therapies designed to relax airway smooth muscle and ameliorate asthmatic inflammation lacks a therapeutic rationale for a disease characterized by predominant neutrophilic inflammation occurring in the small airways and alveoli. By definition, COPD is poorly reversible airflow obstruction; hence, the use of drugs designed to relax airway smooth muscle is somewhat counterintuitive and does not address the pathophysiological process of the disease.

Deletion of secretory group V phospholipase A2 attenuates cell migration and airway hyperresponsiveness in immunosensitized mice.

We investigated the role of group V phospholipase A2 (gVPLA2) in OVA-induced inflammatory cell migration and airway hyperresponsiveness (AHR) in C57BL/6 mice. Repeated allergen challenge induced biosynthesis of gVPLA2 in airways. By aerosol, gVPLA2 caused dose-related increase in airway resistance in saline-treated mice; in allergic mice, gVPLA2 caused persistent airway narrowing. Neither group IIa phospholipase A2, a close homolog of gVPLA2, nor W31A, an inactive gVPLA2 mutant with reduced activity, caused airway narrowing in immune-sensitized mice. Pretreatment with MCL-3G1, a blocking Ab against gVPLA2, before OVA challenge blocked fully gVPLA2-induced cell migration and airway narrowing as marked by reduction of migrating leukocytes in bronchoalveolar lavage fluid and decreased airway resistance. We also assessed whether nonspecific AHR caused by methacholine challenge was elicited by gVPLA2 secreted from resident airway cells of immune-sensitized mice. MCL-3G1 also blocked methacholine-induced airway bronchoconstriction in allergic mice. Blockade of bronchoconstriction by MCL-3G1 was replicated in allergic pla2g5-/- mice, which lack the gene encoding gVPLA2. Bronchoconstriction caused by gVPLA2 in pla2g4-/- mice was comparable to that in pla2g4+/+ mice. Our data demonstrate that gVPLA2 is a critical messenger enzyme in the development of AHR and regulation of cell migration during immunosensitization by a pathway that is independent of group IVa phospholipase A2.

Regulation of eosinophil adhesion by lysophosphatidylcholine via a non-store-operated Ca2+ channel.

We examined the mechanism by which lysophosphatidylcholine (LPC) regulates beta2-integrin-mediated adhesion of eosiniophils. Eosinophils were isolated from blood of mildly atopic volunteers by negative immunomagnetic selection. beta2-integrin-dependent adhesion of eosinophils to plated bovine serum albumin (BSA) was measured by residual eosinophil peroxidase activity. LPC caused maximal adhesion of eosinophils to plated BSA at 4 microM. Lysophosphatidylinositol, which has a similar molecular shape, mimicked the effect of LPC on eosinophil adhesion, while neither lysophosphatidylserine nor lysophosphatidylethanolamine had any effect. Phosphatidylethanolamine, a lipid that has a molecular orientation that is the inverse of LPC, blocked eosinophil adhesion caused by LPC. Unlike platelet-activating factor, a G-protein-coupled receptor agonist, LPC did not cause Ca2+-store depletion, but caused increased Ca2+ influx upon addition of Ca2+ to extracellular medium. This influx was not inhibited by U73122, a phospholipase C inhibitor, demonstrating independence from the G protein-activated phospholipase C pathway. Ca2+ influx was inhibited by either preincubation of phosphotidylethanolamine or La3+, a broad spectrum blocker of cation channels. LPC induced up-regulation of the active conformation of CD11b, which was blocked by preincubation with phosphatidylethanolamine. These data suggest that LPC causes a non-store-operated Ca2+ influx into eosinophils, which subsequently activates CD11b/CD18 to promote eosinophil adhesion.

Highly purified selective isolation of eosinophils from human peripheral blood by negative immunomagnetic selection.

Eosinophils are a minority constituent in human peripheral blood. The study of eosinophils has been limited by difficulty in achieving sufficient cell number and purity. We describe a modified protocol for immunomagnetic cell separation for efficient isolation of human peripheral blood eosinophils. We employ a mixture of monoclonal antibodies (mAbs) directed against cell-surface antigens on human hematopoietic cells combined with secondary labeling with a colloidal suspension of magnetic dextran-iron particles for negative selection of eosinophils. Unwanted labeled cells are retained in the magnetized column, permitting high recovery (70%) and purity (>98%) of eosinophils while retaining cell viability. Eosinophils remain quiescent after isolation, and stimulation caused by cytokines upregulates (i) cell-cell or cell-extracellular matrix protein adhesion, (ii) secretion of bioactive mediators and (iii) cell-surface adhesion molecules. This method for purified isolation is accomplished in < or = 4 h and preserves eosinophils in a quiescent, viable state.

Transcellular secretion of group V phospholipase A2 from epithelium induces beta 2-integrin-mediated adhesion and synthesis of leukotriene C4 in eosinophils.

We examined the mechanism by which secretory group V phospholipase A(2) (gVPLA(2)) secreted from stimulated epithelial cells activates eosinophil adhesion to ICAM-1 surrogate protein and secretion of leukotriene (LT)C(4). Exogenous human group V PLA(2) (hVPLA(2)) caused an increase in surface CD11b expression and focal clustering of this integrin, which corresponded to increased beta(2) integrin-mediated adhesion. Human IIaPLA(2), a close homolog of hVPLA(2), or W31A, an inactive mutant of hVPLA(2), did not affect these responses. Exogenous lysophosphatidylcholine but not arachidonic acid mimicked the beta(2) integrin-mediated adhesion caused by hVPLA(2) activation. Inhibition of hVPLA(2) with MCL-3G1, a mAb against gVPLA(2), or with LY311727, a global secretory phospholipase A(2) (PLA(2)) inhibitor, attenuated the activity of hVPLA(2); trifluoromethylketone, an inhibitor of cytosolic group IVA PLA(2) (gIVA-PLA(2)), had no inhibitory effect on hVPLA(2)-mediated adhesion. Activation of beta(2) integrin-dependent adhesion by hVPLA(2) did not cause ERK1/2 activation and was independent of gIVA-PLA(2) phosphorylation. In other studies, eosinophils cocultured with epithelial cells were stimulated with FMLP/cytochalasin B (FMLP/B) and/or endothelin-1 (ET-1) before LTC(4) assay. FMLP/B alone caused release of LTC(4) from eosinophils, which was augmented by coculture with epithelial cells activated with ET-1. Addition of MCL-3G1 to cocultured cells caused approximately 50% inhibition of LTC(4) secretion elicited by ET-1, which was blocked further by trifluoromethylketone. Our data indicate that hVPLA(2) causes focal clustering of CD11b and beta(2) integrin adhesion by a novel mechanism that is independent of arachidonic acid synthesis and gIVA-PLA(2) activation. We also demonstrate that gVPLA(2), endogenously secreted from activated epithelial cells, promotes secretion of LTC(4) in cocultured eosinophils.

Regulation of interleukin-5-induced beta2-integrin adhesion of human eosinophils by phosphoinositide 3-kinase.

We examined the role of phosphoinositide 3-kinase (PI3K) in integrin-mediated eosinophil adhesion. Deltap85, a dominant-negative form of the class IA PI3K adaptor subunit, was fused to an HIV-TAT protein transduction domain (TAT-Deltap85). Recombinant TAT-Deltap85 inhibited interleukin (IL)-5-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. beta(2)-Integrin-dependent adhesion caused by IL-5 to the plated intracellular adhesion molecule-1 surrogate, bovine serum albumin, was inhibited by TAT-Deltap85 in a concentration-dependent manner. Similarly, two PI3K inhibitors, wortmannin and LY294002, blocked eosinophil adhesion to plated bovine serum albumin. By contrast, beta(1)-integrin-mediated eosinophil adhesion to vascular cell adhesion moelcule-1 was not blocked by TAT-Deltap85, wortmannin, or LY294002. Rottlerin, a protein kinase C (PKC)-delta inhibitor, also blocked beta(2)-integrin adhesion of eosinophils caused by IL-5, whereas beta(1) adhesion to vascular cell adhesion molecule-1 was not affected. IL-5 caused translocation of PKCdelta from the cytosol to cell membrane; inhibition of PI3K by wortmannin blocked translocation of PKCdelta. Western blot analysis demonstrated that extracellular signal-regulated kinase phosphorylation, a critical intermediary in adhesion elicited by IL-5, was blocked by inhibition of either PI3K or PKC-delta. These data suggest that extracellular signal-regulated kinase-mediated adhesion of beta(2)-integrin caused by IL-5 is mediated in human eosinophils by a class IA PI3K through activation of a PKCdelta pathway.

Glucocorticoid-induced surface expression of annexin 1 blocks beta2-integrin adhesion of human eosinophils to intercellular adhesion molecule 1 surrogate protein.

BACKGROUND: Glucocorticoids attenuate the population of eosinophils and T lymphocytes in asthmatic airways. The decrease in airway eosinophilia is caused both by accelerated cell death and by induction of blockade of integrin adhesion. In this study, we examined the hypothesis that annexin 1 surface expression, which is upregulated by the glucocorticoid receptor, prevents integrin adhesion essential to cell migration by blocking intracellular translocation of cytosolic group IV phospholipase A2 (cPLA2). OBJECTIVE: To examine the relationship of the glucocorticoid on annexin 1 expression and the effect of blockade of annexin 1 activity on adhesion of human eosinophils in vitro. To determine the relationship between annexin 1surface expression and nuclear membrane translocation of cPLA2. METHODS: Eosinophils isolated from human peripheral blood were pretreated with fluticasone propionate (FP), and beta2-integrin adhesion was measured after stimulation with IL-5 or eotaxin. Effects of FP on cPLA2 expression, phosphorylation, and translocation were determined. The role of annexin 1 was examined by using annexin 1 blocking antibody and/or mimetic peptides. RESULTS: Fluticasone propionate decreased stimulated eosinophil adhesion and caused 4-fold increase in annexin 1 expression on the plasma membrane. Inhibition of adhesion by FP was blocked with annexin 1 blocking antibody. Annexin 1 N-terminal mimetic peptide also blocked beta2-integrin adhesion. Translocation of cPLA2 to the nuclear membrane was significantly blocked by incubation with FP. Blockade was reversed with annexin 1 blocking antibody. CONCLUSION: Blockade of beta2-integrin adhesion by glucocorticoid is regulated by annexin 1, which blocks cPLA2 translocation to nuclear membrane.

Blockade of focal clustering and active conformation in beta 2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras.

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to beta(2)-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for beta(1)-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for beta(2)-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.

Group V phospholipase A2 induces leukotriene biosynthesis in human neutrophils through the activation of group IVA phospholipase A2.

We reported previously that exogenously added human group V phospholipase A(2) (hVPLA(2)) could elicit leukotriene B(4) (LTB(4)) biosynthesis in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism of the hVPLA(2)-induced LTB(4) biosynthesis in neutrophils, we thoroughly examined the effects of hVPLA(2) and their lipid products on the activity of group IVA cytosolic PLA(2) (cPLA(2)) and LTB(4) biosynthesis under different conditions. As low as 1 nm exogenous hVPLA(2) was able to induce the release of arachidonic acid (AA) and LTB(4). Typically, AA and LTB(4) were released in two phases, which were synchronized with a rise in intracellular calcium concentration ([Ca(2+)](i)) near the perinuclear region and cPLA(2) phosphorylation. A cellular PLA(2) assay showed that hVPLA(2) acted primarily on the outer plasma membrane, liberating fatty acids and lysophosphatidylcholine (lyso-PC), whereas cPLA(2) acted on the perinuclear membrane. Lyso-PC and polyunsaturated fatty acids including AA activated cPLA(2) and 5-lipoxygenase by increasing [Ca(2+)](i) and inducing cPLA(2) phosphorylation, which then led to LTB(4) biosynthesis. The delayed phase was triggered by the binding of secreted LTB(4) to the cell surface LTB(4) receptor, which resulted in a rise in [Ca(2+)](i) and cPLA(2) phosphorylation through the activation of mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2. These results indicate that a main role of exogenous hVPLA(2) in neutrophil activation and LTB(4) biosynthesis is to activate cPLA(2) and 5-lipoxygenase primarily by liberating from the outer plasma membrane lyso-PC that induces [Ca(2+)](i) increase and cPLA(2) phosphorylation and that hVPLA(2)-induced LTB(4) production is augmented by the positive feedback activation of cPLA(2) by LTB(4).

Regulation of adhesion of AML14.3D10 cells by surface clustering of beta2-integrin caused by ERK-independent activation of cPLA2.

We examined the role of cell surface clustering of beta2-integrin caused by protein kinase C (PKC)-activated-cPLA2 in adhesion of eosinophilic AML14.3D10 (AML) cells. Phorbol 12-myristate 13-acetate (PMA) caused time- and concentration-dependent adhesion of AML cells to plated bovine serum albumin (BSA), which was blocked by anti-CD11b or anti-CD18 monoclonal antibodies (mAb) directed against beta2-integrin. Inhibition of PKC with Ro-31-8220 or rottlerin blocked PMA-induced cell adhesion in a concentration-dependent fashion. Inhibition of cytosolic phospholipase A2 (cPLA2) with trifluoromethyl ketone or methyl arachidonyl fluorophosphonate also blocked PMA-induced cell adhesion. PMA caused time-dependent p42/44 mitogen-activated protein kinase (MAPK) (ERK) phosphorylation in these cells. U0126, a MAPK/extracellular signal-regulated protein kinase kinase (MEK) inhibitor, at the concentrations that blocked PMA-induced ERK phosphorylation, had no effect on PMA stimulated AML cell adhesion. Neither p38 MAPK nor c-Jun N-terminal kinase (JNK) was phosphorylated by PMA. PMA also caused increased cPLA2 activity, which was inhibited by Ro-31-8220, but not U0126. Confocal immunofluorescence microscopy showed that PMA caused clustering of CD11b on the cell surface, which was blocked by either PKC or cPLA2 inhibition. PMA stimulation also caused up-regulation of CD11b on the AML cell surface. However, this up-regulation was not affected by cPLA2- or PKC-inhibition. Using the mAb, CBRM1/5, we also demonstrated that PMA does not induce the active conformation of CD11b/CD18. Our data indicate that PMA causes AML cell adhesion through beta2-integrin by PKC activation of cPLA2. This pathway is independent of MEK/ERK and does not require change of CD11b/CD18 to its active conformation. We find that avidity caused by integrin surface clustering - rather than conformational change or up-regulation of CD11b/CD18 - causes PMA stimulated adhesion of AML cells.