Leukotriene D4 activates {beta}2-integrin adhesion in human polymorphonuclear leukocytes.

We examined the functional role and mechanisms by which activation of cysteinyl leukotriene-1 receptor (cysLT(1)R) regulates beta(2)-integrin adhesion to intercellular adhesion molecule (ICAM)-1 in human polymorphonuclear leukocytes (PMNs) in vitro. Human peripheral blood PMNs and eosinophils were isolated separately from the same mildly atopic donors. Surface expression of cysLT(1)R was identified both in PMNs and in eosinophils by immunofluorescence analysis. Total cysLT(1)R protein was substantially greater in eosinophils than in PMNs as determined by Western blot analysis. However, leukotriene D(4) (LTD(4)) upregulated beta(2)-integrin adhesion of PMNs to ICAM-1 with high efficacy in a time- and concentration-dependent manner. Upregulated beta(2)-integrin adhesion of PMNs was related temporally and quantitatively to phosphorylation of 85-kDa cytosolic group IVa phospholipase A2 (gIVaPLA2). Augmented LTD(4)-induced adhesion was blocked significantly by montelukast, a cysLT(1)R antagonist. Trifluoromethylketone (a gIVaPLA2 inhibitor) blocked beta(2)-integrin adhesion caused by LTD(4) activation, as did anti-CD18 monoclonal antibody directed against beta(2)-integrin on the PMN surface. Our data demonstrate that LTD(4) causes phosphorylation of gIVaPLA2 and upregulation of beta(2)-integrin adhesion to ICAM-1 or ICAM-1 surrogate through cysLT(1)R activation. Activation of gIVaPLA2 is a critical step through which beta(2)-integrin adhesion is upregulated by the cysLT(1)R expressed on the surface membrane of human PMN.

Phosphodiesterase 4 inhibition of beta2-integrin adhesion caused by leukotriene B4 and TNF-alpha in human neutrophils.

Phosphodiesterase (PDE)4 inhibition attenuates neutrophilic inflammation in chronic obstructive pulmonary disease. The objective of the present study was to examine the efficacy and mechanism by which PDE4 inhibition blocks adhesion of beta(2)-integrin to an endothelial counterligand. Neutrophils (polymorphonuclear leukocytes (PMNs)) were isolated from humans receiving no medication. Adhesion was analysed by myeloperoxidase activity. The effects of cilomilast+/-salmeterol on the following were determined: 1) surface CD11b expression; 2) adhesion; 3) intracellular cyclic adenosine monophosphate (cAMP) concentration; and 4) extracellular signal-regulated kinase (ERK)-1/2-mediated group IVA-phospholipase A(2) (gIVA-PLA(2)) phosphorylation caused by leukotriene (LT)B(4) or tumour necrosis factor (TNF)-alpha activation. Either cilomilast or rolipram+/-salmeterol caused concentration-related blockade of LTB(4)-induced adhesion to counterligand, but had no effect on TNF-alpha-activated PMNs. A comparable increase in intracellular cAMP concentration for PMNs activated with LTB(4) and TNF-alpha was caused by 1 muM cilomilast and 0.1 microM salmeterol. Upregulation of surface CD11b expression and ERK-1/2 phosphorylation were blocked by cilomilast or rolipram+/-salmeterol for PMNs activated by LTB(4), but not for cells stimulated by TNF-alpha. Cilomilast+/-salmeterol also blocked gIVA-PLA(2) phosphorylation caused by LTB(4) but not TNF-alpha. In conclusion, the current study demonstrates that both leukotriene B(4) and tumour necrosis factor-alpha upregulate cyclic adenosine monophosphate. However, cyclic adenosine monophosphate does not block beta(2)-integrin adhesion caused by tumour necrosis factor-alpha. It was concluded that tumour necrosis factor-alpha prevents inhibition of extracellular signal-regulated kinase-1/2-mediated group IVA-phospholipase A(2) activation, which is essential for beta(2)-integrin adhesion in polymorphonuclear leukocytes.

Additive blockade of beta 2-integrin adhesion of eosinophils by salmeterol and fluticasone propionate.

Migration of human eosinophils is regulated by integrin expression, conformational change, and activation of cytosolic phospholipase A2 (cPLA2). Corticosteroids have been shown to inhibit cPLA2 hydrolysis in human eosinophils. The objective of this study was to determine the mechanisms of fluticasone propionate (FP) alone or in combination with salmeterol (SM) in blocking adhesion mediated by beta 2-integrin in human eosinophils. Human eosinophils were isolated by negative magnetic selection. beta 2-integrin-mediated eosinophil adhesion was measured by residual eosinophil peroxidase activity. Eosinophils were pretreated for 12 h to 24 h with FP and with or without SM for 30 min. Both SM alone and FP alone inhibited eosinophil adhesion in concentration- and time-dependent manner. SM alone modestly (approximately 30%) inhibited interleukin (IL)-5-induced eosinophil adhesion. Blockade of IL-5-induced eosinophil adhesion caused by 10(-7) M FP at 24 h was augmented by 10(-7) M SM from 41.5% to 72.5%. Similar blockade was also observed for eotaxin-induced eosinophil adhesion. Neither SM, FP, nor FP + SM blocked either: 1) upregulation of CD11b surface expression; or 2) phosphorylation of cPLA2. Blockade of beta 2-integrin-mediated eosinophil adhesion by fluticasone propionate is augmented by salmeterol. Decreased adhesion results from augmented blockade of nuclear translocation of cytosolic phospholipase A2 caused by addition of salmeterol to fluticasone.

Studying human airway pharmacology in microsections: application of videomicrometry.

The influence of endogenously-released mediators and activated eosinophils on the airway lumen and the effect of passive sensitization on anti-immunoglobulin (Ig)-E-induced contractile responses was investigated by videomicrometry. Human bronchial sections of 2-3 mm internal diameter, placed in 250 microL Hank's balanced salt solution on microtitre plates, were monitored and recorded by digitized image analysis. Airway preparations exhibited a spontaneous narrowing (mean+/-SEM -33+/-5% of the luminal area). Removal of the bronchial epithelium almost completely prevented the development of spontaneuous narrowing (-6+/-3%; p<0.001). The addition of platelet-activating factor stimulated human eosinophils to the bronchial sections led to significant narrowing of the airway lumen (-39+/-9%; p<0.05). Passive sensitization induced hyperresponsiveness to polyclonal anti-IgE (-35+/-8%; p<0.01). It is concluded that videomicrometry is suitable for studying interactions between human airways and inflammatory cells, as well as the effect of passive sensitization on smooth muscle reactivity in vitro, without the imposition of preload. Under these conditions, human airways exhibited a spontaneous decrease of the airway lumen over time suggesting a role for epithelium-derived mediators because the development of spontaneous tone was epithelium dependent.

Regulation of eosinophil function by phosphatidylinositol-specific PLC and cytosolic PLA(2).

We examined the regulatory role of cytosolic phospholipase A(2) (cPLA(2)) and phosphatidylinositol (PI)-specific phospholipase C (PLC) in the degranulation of human eosinophils and leukotriene (LT) C(4) synthesis. Activation with formyl-Met-Leu-Phe + cytochalasin B (fMLP/B) caused a time-dependent release of eosinophil peroxidase (EPO) and LTC(4), which was inhibited by pertussis toxin. By immunoblotting, eosinophil PLC-beta2 and -gamma2 isoforms were identified, and PLC activation was measured as a function of inositol 1,4,5-trisphosphate concentration. Stimulated release of EPO and intracellular Ca(2+) concentration was inhibited by ET-18-OCH(3), a PI-PLC inhibitor, whereas trifluoromethylketone (TFMK), a cPLA(2) blocker, had no inhibitory effect. Both TFMK and ET-18-OCH(3) attenuated stimulated arachidonate release and LTC(4) secretion, suggesting that activation of both PLC and cPLA(2) is essential for LTC(4) synthesis caused by fMLP/B. The structurally unrelated protein kinase C inhibitors bisindolylmaleimide, Ro-31-8220, and Go-6976 all blocked fMLP/B-induced EPO release but not LTC(4) secretion. 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethyl ester, an intracellular Ca(2+) chelator, suppressed both EPO release and LTC(4) secretion. We found that fMLP/B-induced LTC(4) secretion from human eosinophils is regulated by PI-PLC through calcium-mediated activation of cPLA(2). However, cPLA(2) does not regulate eosinophil degranulation.

Role of mitogen-activated protein kinase-mediated cytosolic phospholipase A2 activation in arachidonic acid metabolism in human eosinophils.

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A(2) (PLA(2)) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA(2) activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC(50) = 8.5 nM)- and time-dependent (t(1/2) = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C(4) (LTC(4)) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA(2) (cPLA(2)) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A(2) by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca(2+)-independent phospholipase A(2) inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC(4) release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1-5 min, and declined thereafter. fMLP stimulation also increased cPLA(2) activity in eosinophils, which was inhibited completely by 30 microM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA(2) activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA(2) activation causes AA hydrolysis and LTC(4) secretion. We also find that cPLA(2) activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA(2) isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC(4).

Blockade of eosinophil migration and airway hyperresponsiveness by cPLA2-inhibition.

We examined the role of a cytosolic phospholipase A2 (cPLA2) in antigen-induced eosinophil infiltration of airways and in airway hyperresponsiveness to methacholine. Inhibition of cPLA2, or blockade of the platelet-activating factor (PAF) receptor, blocked antigen-induced airway hyperresponsiveness and suppressed eosinophil infiltration. Neither cyclooxygenase nor 5-lipoxygenase inhibition had either effect. We show here that, in antigen-sensitized guinea pigs, cPLA2 inhibition prevents both eosinophilic infiltration and subsequent airway hyperresponsiveness after antigen challenge. We also show that this effect is mediated by first-step hydrolysis of membrane phospholipid into lysophospholipid rather than by prostanoid or leukotriene metabolites of arachidonate.

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of cytosolic phospholipase A2 is essential for human eosinophil adhesion to fibronectin.

We examined the role of p38, p42, and p44 mitogen-activated protein kinase (MAPK) isoforms and cytosolic phospholipase A(2) (cPLA(2)) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 microg/ml FN at 30 min, and decreased after 60-90 min. Western blot analysis demonstrated that p44/42 MAPK (extracellular signal-regulated kinase (ERK)1/2) and cPLA(2) were phosphorylated during adhesion to FN, whereas p38 MAPK phosphorylation was unchanged. Preincubation of eosinophils with U0126 or PD98059, two structurally unrelated MAPK kinase inhibitors, or arachidonic trifluoromethyl ketone, a cPLA(2) inhibitor, blocked eosinophil adhesion to FN. By contrast, eosinophil adhesion was unaffected by SB203580, a p38 MAPK inhibitor. Pretreatment of eosinophils with okadaic acid, a serine/threonine phosphatase inhibitor, at the concentrations that induced ERK1/2 and cPLA(2) phosphorylation caused an increase in maximal eosinophil adhesion to FN for >60 min. MAPK kinase inhibition but not p38 inhibition also blocked FN-mediated F-actin redistribution in eosinophils and prevented cPLA(2) phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA(2) activation is essential for eosinophil adhesion to FN.

Mechanism of human group V phospholipase A2 (PLA2)-induced leukotriene biosynthesis in human neutrophils. A potential role of heparan sulfate binding in PLA2 internalization and degradation.

Human group V phospholipase A(2) (hVPLA(2)) has been shown to have high activity to elicit leukotriene production 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 by which hVPLA(2) interacts with cell membranes to induce leukotriene formation, we mutated surface cationic residues and a catalytic residue of hVPLA(2) and measured the interactions of mutants with model membranes, immobilized heparin, and human neutrophils. These studies showed that cationic residues, Lys(7), Lys(11), and Arg(34), constitute a part of the interfacial binding surface of hVPLA(2), which accounts for its moderate preference for anionic membranes. Additionally, hVPLA(2) binds heparin with high affinity and has a well defined heparin-binding site. The site is composed of Arg(100), Lys(101), Lys(107), Arg(108), and Arg(111), and is spatially distinct from its interfacial binding surface. Importantly, the activities of the mutants to hydrolyze cell membrane phospholipids and induce leukotriene biosynthesis, when enzymes were added exogenously to neutrophils, correlated with their activities on phosphatidylcholine membranes but not with their affinities for anionic membranes and heparin. These results indicate that hVPLA(2) acts directly on the outer plasma membranes of neutrophils to release fatty acids and lysophospholipids. Further studies suggest that products of hVPLA(2) hydrolysis trigger the cellular leukotriene production by activating cellular enzymes involved in leukotriene formation. Finally, the temporal and spatial resolution of exogenously added hVPLA(2) and mutants suggests that binding to cell surface heparan sulfate proteoglycans is important for the internalization and clearance of cell surface-bound hVPLA(2).

Involvement of protein tyrosine kinases in activation of human eosinophils by platelet-activating factor.

Activation of human eosinophils by platelet-activating factor (PAF) involves multiple signal transduction pathways. Among these, protein kinase C has been demonstrated both to mediate respiratory burst and to suppress an alternative pathway of activation of respiratory burst and arachidonic acid metabolism in eosinophils. We utilized inhibitors of protein tyrosine kinases (PTK) to elucidate the role of PTK in PAF-induced activation of eosinophils. Eosinophils were isolated from peripheral blood of atopic donors and stimulated with PAF in the absence or presence of broad-spectrum PTK inhibitors-genistein or lavendustin A; an inhibitor of mitogen-activated protein (MAP) kinase activation-tyrphostin AG126; or an inhibitor of Janus kinase 2 (Jak2)-tyrphostin B42 (AG490). PAF induced superoxide anion (O2-*) generation, leukotriene C4 (LTC4) release, intracellular calcium ion mobilization and tyrosine phosphorylation of multiple eosinophil proteins in a concentration-dependent manner. All of these responses were concentration-dependently inhibited by genistein; lavendustin A also exhibited potent inhibition of PAF-induced LTC4 release. AG126 had no effect on either O2-* generation or LTC4 release, while AG490 inhibited both responses, albeit less effectively than genistein. We conclude that PAF activates PTK in human eosinophils and that this signalling pathway is involved in eliciting respiratory burst and leukotriene production. The specific PTK(s) involved are unknown but may include Jak2.