Human Polymorphonuclear Cell Death after Exposure to Resuscitation Fluids In Vitro

Abstract

BACKGROUND: Resuscitation fluids can have variable effects on key functions of circulating polymorphonuclear neutrophils (PMNs) such as oxidative burst, chemotaxis, and bacterial killing. We hypothesized that choice of resuscitation fluids will also affect the rate of PMN apoptosis. To test this, we studied cellular death (apoptosis and necrosis) in human PMNs after brief exposure to different hypertonic and isotonic fluids. METHODS: Blood from 12 volunteers was incubated for 1 hour at 37 degrees C in normal saline, 6.0% dextran-70, 7.5% hypertonic saline, and 7.5% hypertonic saline with 6% dextran-70. Isolated PMNs were double labeled with fluorescein-Annexin V and propidium iodide, and apoptosis and necrosis were measured using flow cytometry. Caspase activation was also detected with flow cytometry using pan-caspase inhibitor (carbobenzoxy-valyl-alanyl-aspartyl-fluoromethylketone) in non-isolated whole blood samples to determine apoptosis. Finally, cDNA macroarrays were used to evaluate the expression of 23 genes involved in the regulation of cell cycling and apoptosis. RESULTS: Exposure to hypertonic fluids (hypertonic saline and 7.5% hypertonic saline with 6% dextran-70) significantly (p < 0.05) increased necrosis in isolated PMNs. In whole blood samples, PMNs exposed to dextran demonstrated significant apoptosis as evidenced by increased caspase activation. Dextran was the only fluid that affected leukocyte gene expression, inducing significant up-regulation of Rb gene transcription. CONCLUSION: Hypertonic fluids and dextran decrease human polymorphonuclear cell survival through necrotic and apoptotic pathways, respectively.