The lipid fraction of post-hemorrhagic shock mesenteric lymph (PHSML) inhibits neutrophil apoptosis and enhances cytotoxic potential

Dysfunctional neutrophil (PMN) apoptosis facilitates hyperinflammatory tiss ue injury. Previous work has demonstrated that post-hemorrhagic shock mesen teric lymph (PHSML) provokes PMN-mediated acute lung injury in animal model s, but the mechanism remains unclear. We have documented that the lipid fra ction of PHSML is responsible for PMN priming of the respiratory burst. In this study, we hypothesized that PHSML lipids delay PMN apoptosis and there by further enhance PMN cytotoxic potential. Mesenteric lymph was collected from rats (n = 5) before (control), during non-lethal hemorrhagic shock (MA P 40 mmHg, 30 min), and during resuscitation (shed blood + 2x crystalloid). Human PMNs were incubated with control, PHSML, PHSML lipid extracts, and h eat-treated PHSML (60 degreesC, 30 min.) at 1-10% (v:v) in RPMI 1640 for 24 h. Apoptosis was assessed using acridine orange/ethidium bromide staining and fluorescence microscopy. Priming of the respiratory burst was evaluated by incubating PMNs with (a) control PHSML or (6) PHSML lipid extracts for 24 h and by activating with fMLP (1 mu mol/L). PHSML and PHSML lipid extrac ts (5-10%) inhibited PMN apoptosis. Heat denaturing the PHSML (to eliminate cytokines and complement) had no effect on the inhibition of PMN apoptosis . Similarly, incubation with polymixin B at a concentration that binds endo toxin had no effect. Both the PHSML and PHSML lipids (5%) following 24-h in cubation primed the fMLP-activated oxidase. At physiologic concentrations, both PHSML and the lipid fraction of PHSML delay PMN apoptosis and prime th e NADPH oxidase. These data further implicate the lipid components of mesen teric lymph as central in the pathogenesis of hemorrhagic shock induced PMN -mediated acute lung injury.