Novel role of group VIB Ca²⁺-independent phospholipase A₂γ in leukocyte-endothelial cell interactions: An intravital microscopic study in rat mesentery

BACKGROUND Phospholipase A₂ (PLA₂) is associated with a variety of inflammatory processes related to polymorphonuclear neutrophil (PMN)-endothelial cell interactions. However, the cellular and molecular mechanisms underlying the interactions and the causative isoform(s) of PLA₂ remain elusive. In addition, we recently showed that calcium-independent PLA₂γ (iPLA₂γ), but not cytosolic PLA₂ (cPLA₂), is responsible for the cytotoxic functions of human PMN including respiratory bursts, degranulation, and chemotaxis. We therefore hypothesized that iPLA₂γ is a prerequisite for the PMN recruitment cascade into the site of inflammation. The aim of this study was to elucidate the roles of the three major phospholipases A₂, iPLA₂, cPLA₂ and secretory PLA₂, in leukocyte rolling and adherence and in the surface expression of β₂-integrins in vivo and in vitro in response to well-defined stimuli. METHODS Male Wistar rats were pretreated with PLA₂ inhibitors selective for iPLA₂β, iPLA₂γ, cPLA₂, or secretory PLA₂. Leukocyte rolling/adherence in the mesenteric venules superfused with platelet-activating factor (PAF) were quantified by intravital microscopy. Furthermore, isolated human PMNs or whole blood were incubated with each PLA₂ inhibitor and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or PAF. PMN adherence was assessed by counting cells bound to purified fibrinogen, and the surface expression of lymphocyte function-associated antigen 1 and macrophage antigen 1 (Mac-1) was measured by flow cytometry. RESULTS The iPLA₂γ-specific inhibitor almost completely inhibited the fMLP/PAF-induced leukocyte adherence in vivo and in vitro and also decreased the fMLP/PAF-stimulated surface expression of Mac-1 by 60% and 95%, respectively. In contrast, the other inhibitors did not affect these cellular functions. CONCLUSION iPLA₂γ seems to be involved in leukocyte/PMN adherence in vivo and in vitro as well as in the up-regulation of Mac-1 in vitro in response to PAF/fMLP. This enzyme is therefore likely to be a major regulator in the PMN recruitment cascade.