Role of Group V Phospholipase A2 in Zymosan-induced Eicosanoid Generation and Vascular Permeability Revealed by Targeted Gene Disruption

Abstract

Conclusions regarding the contribution of low molecular weight secretory phospholipase A 2 (sPLA 2 ) enzymes in eicosanoid generation have relied on data obtained from transfected cells or the use of inhibitors that fail to discriminate between individual members of the large family of mammalian sPLA 2 enzymes. To elucidate the role of group V sPLA 2 , we used targeted gene disruption to generate mice lacking this enzyme. Zymosan-induced generation of leukotriene C 4 and prostaglandin E 2 was attenuated ϳ50% in peritoneal macrophages from group V sPLA 2 -null mice compared with macrophages from wild-type littermates. Furthermore, the early phase of plasma exudation in response to intraperitoneal injection of zymosan and the accompanying in vivo generation of cysteinyl leukotrienes were markedly attenuated in group V sPLA 2 -null mice compared with wild-type controls. These data provide clear evidence of a role for group V sPLA 2 in regulating eicosanoid generation in response to an acute innate stimulus of the immune response both in vitro and in vivo, suggesting a role for this enzyme in innate immunity.The first step in the biosynthesis of eicosanoids is the release of arachidonic acid from cell membrane phospholipids by phospholipase A 2 . Several classes of phospholipase A 2 have been described in mammals (1, 2). Cytosolic phospholipase A 2 (cPLA 2 ) 1 ␣ is an 85-kDa cytosolic enzyme that uses a catalytic serine residue and preferentially cleaves arachidonic acid from cell membrane phospholipids (3). The Ca 2ϩ -dependent translocation of cPLA 2 -␣ from the cytosol to the nuclear envelope (4), a prominent site of eicosanoid biosynthesis, is dependent on a Ca 2ϩ -dependent lipid binding (C-2) domain. Paralogues of cPLA 2 -␣ (cPLA 2 -␤ and cPLA 2 -␥) have been described previously (5, 6). cPLA 2 -␤ has a M r of 110,000 and shares 30% identity with cPLA 2 -␣, including a functional C-2 domain. cPLA 2 -␥ has a M r of 61,000, shares 29% sequence identity with cPLA 2 -␣, lacks a C-2 domain, and is Ca 2ϩ -independent. Mammalian low molecular weight secretory phospholipase A 2 (sPLA 2 ) enzymes, which are now 10 in number, are characterized by a conserved motif containing a catalytic histidine residue, by their relatively small size of ϳ14 kDa, and by their highly disulfide-linked tertiary structures (7-13). They are distinguished from one another by their structures, their biochemical properties, and their tissue distribution. Calcium-independent phospholipase A 2 enzymes have been described in myocardium and in leukocytes (14, 15). They have been implicated in membrane remodeling, regulation of store operated calcium channels, apoptosis, and release of arachidonic acid. The fourth group of phospholipase A 2 enzymes comprises the acetyl hydrolases of platelet activating factor (16).Given the complexity and size of the phospholipase A 2 family, targeted gene disruption is a suitable approach to elucidating the role(s) of individual enzymes and proved fruitful in determining the role of cPLA 2 -␣ in regulating eicosan...

Arachidonic Acid Release from Mammalian Cells Transfected with Human Groups IIA and X Secreted Phospholipase A2 Occurs Predominantly during the Secretory Process and with the Involvement of Cytosolic Phospholipase A2-α

“…Note Added in Proof-Recently, Arm and co-workers have shown that disruption of the mouse group V sPLA 2 gene leads to an ϳ50% reduction in the amount of arachidonic acid liberated from zymosanstimulated peritoneal macrophages (58), and yet earlier studies showed that the disruption of the cPLA 2 -␣ gene leads to complete loss of arachidonic acid from these cells (59). Thus, it appears that group V sPLA 2 action somehow augments the function of cPLA 2 -␣ in peritoneal macrophages.…”

Group V Phospholipase A2 in Bone Marrow-derived Myeloid Cells and Bronchial Epithelial Cells Promotes Bacterial Clearance after Escherichia coli Pneumonia

“…Construction of the GV sPLA 2 -deficient mouse line, backcrossed to the C57BL/6 background for 10 generations, was carried out as described (16). Heterozygous breeding pairs (GV ϩ/Ϫ ) were used to generate GV Ϫ/Ϫ and GV ϩ/ϩ littermates.…”

“…The functional importance of GV sPLA 2 in eicosanoid biosynthesis is established by the observations that LTB 4 and PGE 2 production is ϳ50% lower in macrophages from mice with targeted deletion of GV sPLA 2 (GV Ϫ/Ϫ mice) than from GV ϩ/ϩ mice (16) and that BAL fluid from mice that overexpress GV sPLA 2 contains about 2-fold more PGE 2 than wild type mice (14). sPLA 2 enzymes also regulate biological responses by generating 1-Oalkyl-lysophosphatylcholine, the precursor of platelet activating factor (17), and through binding to specific mannose-type sPLA 2 receptors (18,19).…”

Transcellular Secretion of Group V Phospholipase A2 from Epithelium Induces β2-Integrin-Mediated Adhesion and Synthesis of Leukotriene C4 in Eosinophils

“…The concentration of LTC 4 secreted during eosinophil activation was assayed by competitive enzyme immunoassay (Cayman Chemicals) using a method that we have reported previously (20,33,34). A total of 21 eosinophil isolations from different donors were used in this protocol.…”