Induction of cytosolic phospholipase A₂ in human leukocytes by lipopolysaccharide

Abstract

Stimulation of peripheral blood leukocytes with lipopolysaccharide results in the synthesis of inflammatory cytokines including interleukin-1/3, interleukin-6, tumor necrosis factor-a and prostaglandin E, correlating with an increase in phospholipase A, activity. Mammalian cells contain several phospholipase A, isoforms including the 14-kDa secretory isoform and the more recently described high-molecular-mass cytosolic isoform. It is commonly believed that during inflammatory responses secretory phospholipase A, becomes activated. However, we could not detect secretory phospholipase A, nor its corresponding mRNA after lipopolysaccharide-induced activation. By contrast, we found increased mRNA levels for cytosolic phospholipase A, following activation of peripheral blood leukocytes when levels were compared to non-stimulated controls. Our results demonstrate that cytosolic phospholipase A,, rather than the secretory isoform may be the mediator of the lipopolysaccharide-induced inflammatory cascade in human peripheral blood leukocytes.Phospholipase A, is a lipolytic enzyme that hydrolyzes the fatty acyl ester at the sn-2 position of glycerophosphatides producing equimolar amounts of free fatty acids and lysophosphatides. The observation that arachidonic acid (A,Ach) is predominantly found esterified at the sn-2 position suggests that the action of this enzyme is the rate-limiting step in the synthesis of leukotrienes and prostaglandins. Furthermore, phospholipase A, is involved in the formation of platelet-activating factor [ 1, 21. These observations indicate that phospholipase A, plays a significant role in the synthesis of potent mediators of inflammation. Therefore, studies were initiated to correlate phospholipase A, levels in patients with different pathophysiological conditions. Evidence now indicates that elevated phospholipase A, levels are associated with diseases like acute pancreatitis, acute and chronic colitis, rheumatoid arthritis, allergic shock, septic shock, psoriasis and thrombotic cardiovascular diseases [3]. Experiments are therefore in progress to evaluate phospholipase A, activity and immunoreactivity as a diagnostic or prognostic marker for the above-mentioned diseases To date, the different phospholipase A, have been subdivided into two main classes, the secreted forms, including type I (pancreatic) and type I1 (secretory), and the nonsecreted forms, namely type IV (cytosolic) [7].Pharmacological research has focused primarily on secretory and cytosolic phospholipase A,, two isoforms which

Cyclooxygenase-2-Issued Prostaglandin E2 Enhances the Production of Endogenous IL-10, Which Down-Regulates Dendritic Cell Functions

“…These data are consistent with previous studies, which reported induction of cPLA 2 by LPS in human leukocytes (44,45). Analysis of COX expression shows that resting BM-DC express only COX-1 and produce low levels of PGE 2 .…”

Electrogenic H+ pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A2

“…One of them is a 14 kDa secretory-type enzyme (sPLA # ) that requires high (probably millimolar) calcium concentrations for the catalysis [22][23][24]. The other one is a cytosolic PLA # (cPLA # ), with an apparent molecular mass of 85 kDa [25][26][27]. This enzyme is specific for AA in the sn-2 position of phospholipids and requires submicromolar Ca# + concentrations for translocation to membranes [28][29][30][31].…”

Changes in Cellular and Plasma Membrane Phospholipid Composition after Lipopolysaccharide Stimulation of Human Neutrophils, Studied by 31P NMR

“…a major lipid component of gramnegative bacterial cell walls, results in the synthesis of prostaglandin E2 and inflammatory cytokines including interleukin lp, interleukin-6 and tuinour necrosis factor-rx (Rodewald et al, 1994). In addition, LPS may prime neutrophils for an enhanced…”

“…LPS also stimulates expression of the adhesion molecule p2 integrin (CDI 1 b/ CD18) in neutrophils (Lynn et al, 1991). The expression of CDI 1WCD18 in human neutrophils is regulated by phospholipase A, (PLA,: Jacobson and Schrier, 1993), and LPS triggers activation of the high-molecular-mass cytosolic isoform (Forehand et al, 1993;Rodewald et al, 1994). We sought to investigate the changes to the phospholipid composition of human neutrophils and their plasma membranes when stimulated with LPS, since the substrates for PLAL activity under these conditions have not been investigated.…”

“…Ether-linked lipids were identified by their response to hydrolysis with HCI and phospholipase A, (PLA,). We also developed a simple plasma membrane purification method in which the cells were rapidly disrupted at the end, rather than at the beginning of the procedure, which is more usual (Rodewald et al, 1994;MacDonald and Sprecher, 1989). Our results indicate that LPS stimulation (50 pg ml -I ) increases levels of both cellular and plasma membrane PtdSer and decreases membrane levels of a lipid tentatively identified as 2-arachidonoyl-I-alkyl-sn-glycero-3-phosphocholine (d,AchRGroPCho).…”