Hypertonic Saline Activates Protein Tyrosine Kinases and Mitogen-activated Protein Kinase p38 in T-cells

Abstract

In previous in vitro studies, we have found that hypertonic saline (HTS) can augment T-cell proliferation and restore the function of suppressed T-cells. Our animal models have shown that HTS resuscitation reverses immunosuppression after hemorrhage and reduces mortality from sepsis. In the present study, we investigated if and how HTS may influence T-cell signaling and function on a subcellular level. Human peripheral blood mononuclear cells (PBMC) were used to determine the effect of HTS on T-cell interleukin 2 (IL-2) production and proliferation. Human Jurkat T-cells were used to study the effects of HTS on T-cell signal transduction, IL-2 mRNA transcription, and IL-2 expression. The effect of HTS on T-cell proliferation and IL-2 production was measured with PBMC and Jurkat T-cells. IL-2 mRNA transcription in HTS-treated Jurkat cells was measured by reverse transcriptase polymerase chain reaction. HTS-induced protein tyrosine phosphorylation in Jurkat T-cells was determined by immunoblotting with anti-phosphotyrosine antibodies. Expression in Jurkat cells of the mitogen-activated protein kinase p38 (MAPK p38), a signal transduction protein that is activated by osmotic stress, was determined by immunoblotting with anti-MAPK p38 antibodies. HTS-induced MAPK p38 activation in Jurkat cells was measured with an immune-complex kinase assay using ATF-2 as a substrate. Proliferation of activated human PBMC increased significantly upon addition of HTS to the culture medium. This effect of HTS was paralleled by enhanced IL-2 production of activated PBMC and Jurkat cells and IL-2 mRNA transcription of Jurkat cells. HTS exposure of Jurkat cells caused tyrosine phosphorylation of a number of cellular proteins. We found that Jurkat T-cells expressed MAPK p38 and that it was activated in the presence of HTS. All these effects of HTS on T-cell signaling and function were observed at NaCl concentrations that were within physiologically relevant levels (20-100 mmol/L hypertonicity). In T-cells, HTS triggers a signaling pathway that includes increased tyrosine phosphorylation of several cellular proteins and activation of MAPK p38. HTS alone does not result in IL-2 mRNA transcription, IL-2 expression, or T-cell proliferation. However, in combination with other stimuli, HTS augments T-cell IL-2 expression and proliferation. We speculate that HTS could "resuscitate" suppressed T-cells in trauma patients by circumvention of, or substituting for, blocked signaling pathways.