The Effect of Hypertonic Saline on mRNA of Proinflammatory Cytokines in Lipopolysaccharide-Stimulated Polymorphonuclear Cells

BACKGROUND: Hypertonic saline is often used to resuscitate patients experiencing shock. In such conditions, polymorphonuclear cells and Toll-like receptors (TLRs) form an essential part of early induced innate immunity. OBJECTIVE: To investigate the immunomodulatory effect of hypertonic saline on polymorphonuclear cells by evaluating the changes in TLR-4 receptors and proinflammatory cytokines. METHODS: Polymorphonuclear cells were isolated from whole blood using Polymorphprep (Axis-Shield, Oslo, Norway). The isolated polymorphonuclear cells were plated at a density of 1 × 10(6) cells/mL in 6-well flat-bottomed culture plates and were stimulated with 1 μg/mL lipopolysaccharide or N-formyl-methionyl-leucyl-phenylalanine. The stimulated polymorphonuclear cells were cultured in hypertonic saline at 10, 20, or 40 mmol/L above isotonicity. After that, the changes in TLR-4 and cytokines were measured by quantitative real-time polymerase chain reaction and flow cytometry. RESULTS: The level of TLR-4 mRNA expression decreased after stimulation with N-formyl-methionyl-leucyl-phenylalanine, but hypertonic saline did not affect the TLR-4 mRNA expression. TLR-4 mRNA expression was clearly induced upon stimulation with lipopolysaccharide, and the addition of hypertonic saline restored TLR-4 mRNA expression in polymorphonuclear cells. The interleukin-1β mRNA expression was decreased in the hypertonic environment. On the other hand, the tumor necrosis factor-α value was not influenced by the addition of hypertonic saline. CONCLUSIONS: Hypertonic saline has an immunomodulatory effect on polymorphonuclear cells through the TLR-4 pathway, and the interleukin–1β-associated pathway is influenced more by hypertonic saline than is the tumor necrosis factor–α-associated pathway.