Endotoxins Stimulate Neutrophil Adhesion Followed by Synthesis and Release of Platelet-activating Factor in Microparticles

Abstract

Lipopolysaccharides and triacyl-cysteine-modified proteins of Gram-negative and positive organisms are potent endotoxins. Animal models show that the receptor for platelet-activating factor (PAF) is responsible for many of the deleterious effects of endotoxin, where regulated, localized PAF production localizes the inflammatory response. In contrast, biologically active analogs of PAF (PAF-like lipids) are generated by oxidative attack on phospholipids by chemical reactions that are unregulated and unlocalized. The identity and distribution of the PAF receptor ligand in endotoxemia is unknown. We found human polymorphonuclear leukocytes (PMNs) were a significant source of PAF receptor agonists after stimulation by either class of endotoxin. Production of PAF receptor agonists required that the PMN adhere to a surface, and adhesion (and therefore accumulation of PAF-like bioactivity) in response to endotoxic stimulation was delayed for several minutes. PAF-like oxidized phospholipids were found by mass spectroscopy, but biosynthetic PAF accounted for most of the phospholipid agonists arising from endotoxic stimulation. A significant portion of the PAF made by PMNs was secreted, in contrast to its near complete retention by other inflammatory cells. Endotoxic stimulation induced a respiratory burst with the production of superoxide and the formation and shedding of microparticles. Free and microparticle-bound PAF appeared in the media, and blocking microvesiculation with calpeptin blocked PAF release. The released material activated platelets, and platelets co-aggregated with endotoxin-stimulated PMNs. Adherent PMNs therefore behave differently than suspended cells and are a significant source of free PAF after endotoxin exposure. Leukocytes can couple endotoxic challenge to the widespread circulatory and inflammatory effects of endotoxin.

Intracellular PAF catabolism by PAF acetylhydrolase counteracts continual PAF synthesis

“…We confirmed these data by quantifying PAF accumulation by HPLC MS/MS to find 1.8, 17.4, 21.8, 32.8, and 53.2 pg of PAF per 10 6 cells after treatment by buffer or 10, 25, 50, or 100 mM MAFP, respectively. This is consistent with our previous determination of 58.8 pg of PAF in LPS-stimulated cells (40).…”

“…The amount of PAF generated by A23187-stimulated neutrophils was sufficient to induce a sustained increase in intracellular Ca 21 in the test cells, whereas the PAF produced by LPS or TNF-a-stimulated cells was significantly less because it caused only transient stimulation when added to naïve cells. These results are consistent with the 30-fold difference in PAF accumulation between LPS-and A23187-stimulated cells we documented previously by mass spectrometry (40).…”

An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

“…Harrison, Clay, and Murphy (34) demonstrated detection of 500 pg of PAF with a good signal-tonoise ratio. It also should be noted that a more recent report by those authors achieves better sensitivity, on the order of a few picograms (47). However, we sought to develop a more sensitive method, because in our hands negative-ion detection of PAF was insufficiently sensitive for many biological applications, even in the presence of ammonium acetate, an additive reported to enhance the negative ionization of choline-containing phospholipids (48).…”

Changing world of neutrophils

“…These microparticles expressed tissue factor and supported thrombin generation [79]. In another study, stimulation of neutrophils with bacterial endotoxin resulted in shedding of PAF containing microparticles which activated platelets [118]. More recently, the active form of the β2 integrin Mac-1was detected on the surface of EVs released from activated neutrophils, and the active integrin played a central role in binding to and activation of resting platelets [84].…”