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Human polymorphonuclear cell death after exposure to resuscitation fluids in vitro: apoptosis versus necrosis
Kathleen Stanton1, Hasan B Alam, Peter Rhee
1Department of Surgery, Uniformed Services University of Health Sciences, Bethesda, Maryland 20814, USA.
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.
Insights
Resuscitation fluids impact polymorphonuclear neutrophil (PMN) survival. Hypertonic saline increased necrosis, while dextran induced apoptosis in human PMNs, affecting cell death pathways.
Area of Science:
- Immunology
- Cell Biology
- Critical Care Medicine
Background:
- Resuscitation fluids can alter polymorphonuclear neutrophil (PMN) functions, including oxidative burst, chemotaxis, and bacterial killing.
- The impact of different resuscitation fluids on PMN apoptosis rates was investigated.
Purpose of the Study:
- To determine the effects of various hypertonic and isotonic resuscitation fluids on human PMN apoptosis and necrosis.
- To assess the influence of these fluids on cellular death pathways and gene expression.
Main Methods:
- Human blood samples were incubated with normal saline, dextran-70, hypertonic saline, or hypertonic saline with dextran.
- Apoptosis and necrosis were quantified using Annexin V and propidium iodide staining via flow cytometry.
- Caspase activation and gene expression related to cell cycling and apoptosis were analyzed.
Main Results:
- Hypertonic fluids significantly increased necrosis in isolated PMNs.
- Dextran exposure led to significant apoptosis in whole blood PMNs, indicated by increased caspase activation.
- Dextran uniquely altered leukocyte gene expression, up-regulating Rb gene transcription.
Conclusions:
- Hypertonic fluids and dextran reduce human PMN survival.
- Hypertonic fluids induce necrosis, while dextran promotes apoptosis in PMNs.
- These findings highlight differential effects of resuscitation fluids on immune cell viability.