Changes in cellular and plasma membrane phospholipid composition after lipopolysaccharide stimulation of human neutrophils, studied by 31P NMR.

Lipopolysaccharide (endotoxin, LPS) exerts potent proinflammatory effects on neutrophils which may involve membrane phospholipid metabolism. The cellular and plasma membrane phospholipid composition of resting neutrophils and those stimulated with 50 microg ml(-1) LPS were studied by 31P NMR and chemical analysis. A rapid new method for plasma membrane purification was employed, involving the direct lysis of cytoplasts. Chemical analyses showed that, although total cellular phospholipid content did not change with LPS stimulation, there was twice the amount of phospholipid present in plasma membranes isolated from stimulated cells, resulting in a lowered cholesterol/phospholipid ratio. Since internal membranes have lower cholesterol content this result is consistent with an origin from insertion of these membranes (most probably from the endoplasmic reticulum) into the plasma membrane, thereby increasing its fluidity. The individual phospholipid classes of both cells and membranes were quantified by 31P-NMR spectroscopy after dissolution in sodium cholate without prior extraction of lipids, allowing partial resolution of the major phospholipid classes and ether-linked phospholipids. Ether-linked lipids were distinguished from diacyl phospholipids by hydrolysis of lipid extracts with HCl and phospholipase A1, There was a significant increase in phosphatidylserine in both cells and plasma membranes after stimulation, with a decrease in the phosphatidylethanolamine (diacyl and plasmalogen) content in the cells. Plasma membranes from stimulated cells exhibited a significant decrease in a phospholipid tentatively identified as 2-arachidonoyl-1-alkyl-sn-glycero-3-phosphocholine, a precursor of the lipid inflammatory mediator, platelet-activating factor. This report is the first to elaborate the changes in phospholipid composition in human neutrophils as a whole, and in plasma membranes separated from them, before and after stimulation by the physiological activator, LPS.