Human polymorphonuclear cell death after exposure to resuscitation fluids in vitro: apoptosis versus necrosis.
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.


CONCLUSIONS
Hypertonic fluids and dextran decrease human polymorphonuclear cell survival through necrotic and apoptotic pathways, respectively.
Stanton
Kathleen
K
Department of Surgery, Uniformed Services University of Health Sciences, Bethesda, Maryland 20814, USA.
Alam
Hasan B
HB
Rhee
Peter
P
Llorente
Orlando
O
Kirkpatrick
John
J
Koustova
Elena
E
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D016923:Cell Death / Q000187:drug effects* / Q000235:genetics; D003911:Dextrans / Q000008:administration & dosage; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006982:Hypertonic Solutions / Q000008:administration & dosage*; D066298:In Vitro Techniques; D009336:Necrosis; D009504:Neutrophils / Q000187:drug effects* / Q000473:pathology*; D009994:Osmolar Concentration; D010952:Plasma Substitutes / Q000008:administration & dosage; D012151:Resuscitation / Q000379:methods*
D003911:Dextrans; D006982:Hypertonic Solutions; D010952:Plasma Substitutes