RESUMO
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.
Assuntos
Células Endoteliais/citologia , Fosfolipases A2 do Grupo V/metabolismo , Lipopolissacarídeos/metabolismo , Pulmão/enzimologia , Actinas/metabolismo , Anticorpos Monoclonais/química , Células Cultivadas , Dextranos/química , Humanos , Técnicas In Vitro , Microcirculação , Microscopia de Fluorescência/métodos , Mutação , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Fatores de TempoRESUMO
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.