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.


CONCLUSIONS
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.
From the Department of Acute Critical Care and Disaster Medicine (M.K., Y.O.), Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University; Trauma and Acute Critical Care Medical Center (J.A.), Tokyo Medical and Dental University Hospital of Medicine; Department of Bioscience and Engineering (M.S.), Shibaura Institute of Technology; and Department of Biology (T.K.), Faculty of Science, Ochanomizu University, Tokyo, Japan.
Mitsuaki
M
Kojima
Junichi
J
Aiboshi
Masahiro
M
Shibata
Tetsuyuki
T
Kobayashi
Yasuhiro
Y
Otomo
D000818:Animals; D002450:Cell Communication; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D008297:Male; D008643:Mesentery / Q000648:ultrastructure*; D009504:Neutrophils / Q000378:metabolism; D054512:Phospholipases A2, Calcium-Independent / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D012380:Role; D012680:Sensitivity and Specificity
C493463:PITPNA protein, human; D049294:Phospholipid Transfer Proteins; D054512:Phospholipases A2, Calcium-Independent