Clinically relevant hypertonicity prevents stored blood- and lipid-mediated delayed neutrophil apoptosis independent of p38 MAPK or caspase-3 activation

Background. Delayed apoptosis of primed neutrophils (PMNs) may facilitate PMN-mediated tissue injury leading to postinjury multiple organ failure. Aged (42-day-old) stored red blood cells (RBC42) delay PMN apoptosis through proinflammatory phospholipids such as platelet-activating factor (PAF) and lyso-phosphatidylcholine (LPC). Hypertonic saline (HTS) attenuates PMN cytotoxic functions. We hypothesized that clinically relevant HTS would provoke PMN apoptosis, as well as prevent stored blood and lipid-mediated delayed PMN apoptosis through activation of p38 mitogen-activated protein kinase (MAPK) and caspase-3. Methods. PMNs harvested from healthy volunteers were incubated (5% CO2, 37degrees C, 24 hr) with RBC42 plasma, PAF (20 muM), or LPC (4.5 muM), with or without the p38 MAPK inhibitor SB 203580, the caspase-3 inhibitor zDEVD-fmk (10 mumol/L) or the pan-caspase inhibitor zVAD-fmk (20 mumol/L). Duplicate samples were preincubated in HTS (Na [180 mM]). Apoptotic index (% PMNs undergoing apoptosis) was assessed morphologically. p38 AMPK activation was assessed by Western blotting. Caspase-3 activity was measured colorimetrically. Results. PAF, LPC, and RBC42 plasma delayed apoptosis; HTS increased apoptosis compared with controls. HTS prevented PAF, LPC, and RBC42-delayed apoptosis. p38 MAPK was not activated by HTS; its inhibition had no effect on the actions of HTS. Caspase inhibition attenuated the ability of HTS to increase apoptosis, but it did not affect the ability of HTS to restore healthy PMN apoptosis in the presence of RBC42. Conclusion. HTS increases PMN apoptosis and prevents stored blood- and lipid-mediated delayed PMN apoptosis. HTS may activate caspase-3, but alternative signaling pathways appear to be involved in modulating the effects of lipids on PMN apoptosis.