CHANGES IN CELLULAR AND PLASMA-MEMBRANE PHOSPHOLIPID-COMPOSITION AFTER LIPOPOLYSACCHARIDE STIMULATION OF HUMAN NEUTROPHILS, STUDIED BY P-31NMR

Lipopolysaccharide (endotoxin, LPS) exerts potent proinflammatory effe cts on neutrophils which may involve membrane phospholipid metabolism. The cellular and plasma membrane phospholipid composition of resting neutrophils and those stimulated with 50 mu g ml(-1) LPS were studied by P-31 NMR and chemical analysis. A rapid new method for plasma membr ane purification was employed, involving the direct lysis of cytoplast s. Chemical analyses showed that, although total cellular phospholipid content did not change with LPS stimulation, there was twice the amou nt of phospholipid present in plasma membranes isolated from stimulate d cells, resulting in a lowered cholesterol/phospholipid ratio. Since internal membranes have lower cholesterol content this result is consi stent with an origin from insertion of these membranes (most probably from the endoplasmic reticulum) into the plasma membrane, thereby incr easing its fluidity, The individual phospholipid classes of both cells and membranes were quantified by P-31-NMR spectroscopy after dissolut ion in sodium cholate without. prior extraction of lipids, allowing pa rtial resolution of the major phospholipid classes and ether-linked ph ospholipids. Ether-linked lipids were distinguished from diacyl phosph olipids by hydrolysis of lipid extracts with HCl and phospholipase A(1 ). There was a significant increase in phosphatidylserine in both cell s and plasma membranes after stimula tion, with a decrease in the phos phatidylethanolamine (diacyl and plasmalogen) content in the cells. Pl asma membranes from stimulated cells exhibited a significant decrease ina phospholipid tentatively identified as -arachidonoyl-1-alkyl-sn-gl ycero-3-phosphocholine, a precursor of the lipid inflammatory mediator , platelet-activating factor, This report is the first to elaborate th e changes in phospholipid composition in human neutrophils as a whole, and in plasma membranes separated from them, before and after stimula tion by the physiological activator, LPS.