Changes in Cellular and Plasma Membrane Phospholipid Composition after Lipopolysaccharide Stimulation of Human Neutrophils, Studied by³¹P NMR

Abstract

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 pg ml-' LPS were studied by "P 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 "P-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 froin diacyl phospholipids by hydrolysis of lipid extracts with HCl and phospholipase A , . 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-l-alkyl-slz-glycero-3-phosphocho~ine, 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.Keywm-ds: neutrophil ; membrane ; phospholipid: "P-NMR; lipopolysaccharide.Stimulation of human neutrophils with bacterial lipopolysaccharide (LPS, endotoxin). 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

Characteristic Oxidation Products of Choline Plasmalogens are Detectable in Cattle and Roe Deer Spermatozoa by MALDI‐TOF Mass Spectrometry

“…In order to prove the sensitivity of alkenyl-acyl species to acid treatment, a flask with the dried lipids obtained from roe deer spermatozoa was exposed to HCl fumes for 10 min by keeping the inverted flask over an open bottle of concentrated HCl [14].…”

Combined application of TLC and matrix-assisted laser desorption and lonisation time-of-flight mass spectrometry (MALDI-TOF MS) to phospholipid analysis of brain

“…In order to clarify the assignment of individual PE species, the total brain extract was digested on the one hand with phospholipase A2 (PLA2) and on the other hand treated with HC1 fumes [16]. The comparison of both treatments should allow further conclusions on the fatty acid composition of the individual PL since PLA2 cleaves selectively the fatty acid in sn-2 position, whereas HC1 fumes hydrolyse the ether bond in alkenyl-acyl-PL, i.e.…”

“…To make assignment of alkenyl-acyl-PL unambiguous, dried PL samples were exposed 10 min to HC1 fumes by keeping the inverted flask over an open bottle of concentrated HC1 [16].…”

Parallel Secretion of Pancreatic Phospholipase A2, Phospholipase A1, Lipase, and Colipase in Children with Exocrine Pancreatic Dysfunction

“…Quantitative analysis of the concentrations of phospholipids was performed as previously described (17)(18)(19)(20).…”