Effect of hypertonic saline on apoptosis of polymorphonuclear cells

The function of polymorphonuclear (PMN) cells can be influenced by the choice of resuscitation fluids in hemorrhagic shock. Widespread interest in the use of hypertonic solutions for resuscitation has led to extensive investigation of their immune-modulating properties. Hypertonic saline (HTS) is known to modulate immune reactions, preventing the multiorgan failure mediated by immune reactions in trauma and hemorrhagic shock. PMN cells play a key role in such immune-mediated inflammatory processes, and HTS is believed to affect these PMN cells. However, how these events influence the actual event of apoptosis has not yet been described. Thus, in the present study, we aimed to investigate the differences in the apoptosis of PMN cells when exposed to isotonic and hypertonic environments and the temporal relations between the interval of administration of HTS after the stimulation of PMN cells.

Whole blood was sampled from healthy volunteers, and the PMN cells were isolated. The isolated layer of PMN cells was washed twice with phosphate-buffered saline to yield the PMN cells. The number of cells was kept uniform, and an overall survival rate greater than 95% was maintained. After stimulation of the isolated PMN cells with N-formyl-methionyl-leucyl-phenylalanine, the PMN cells were allocated into 3 study groups (i.e., 1 isotonic group and 2 hypertonic groups with an osmolarity of 160 mM and 180 mM each). The extent of apoptosis was investigated in each group after culturing the PMN cells for 0, 1, 3, 6, 12, 15, 18, and 24 h. Depending on whether the PMN cells were stimulated with N-formyl-methionyl-leucyl-phenylalanine, they were also divided into stimulated and nonstimulated groups. In the stimulated group, the hypertonic environment was fostered immediately (HTS 0 h) and 6 h (HTS 6 h) after stimulation, which was accomplished after allocating the cells into an isotonic group (140 mM) and a hypertonic group (180 mM), according to the concentration of the culture medium. The PMN cells were then cultured at 37°C for 15 h with 5% carbon dioxide incubation. Each PMN suspension was labeled with Annexin V-fluorescein isothiocyanate and propidium iodide. Each sample underwent immediate flow cytometric analysis. PMN cells with high propidium iodide uptake were considered nonviable (necrotic). Among the viable PMN cells, those with no Annexin V uptake were considered normal and those with Annexin V uptake were considered apoptotic.

Decreased apoptosis was observed in the PMN cells stimulated with N-formyl-methionyl-leucyl-phenylalanine. Increased apoptosis was observed in the stimulated PMN cells incubated in hypertonic condition compared with the cells incubated in isotonic condition. Early HTS administration demonstrated increased apoptosis compared with late administration.

HTS treatment resulted in increased PMN apoptosis and an anti-inflammatory effect. Decreased apoptosis (prolonged lifespan) has been implicated in neutrophil-mediated tissue damage. HTS, by increasing the apoptosis of PMN cells, attenuates the postinjury inflammatory response. Also, early treatment with HTS was more efficient than delayed treatment.