Effect of Hypertonic Saline Resuscitation on CD4+CD25+ Regulatory T Cells and γδ T Cells After Hemorrhagic Shock and Resuscitation in Relation to Apoptosis and iNOS

Background: Hemorrhagic shock and resuscitation induce immunosuppression. CD4 + CD25 + regulatory T cells and γδT cells may affect these immunosuppressive conditions. Hypertonic saline resuscitation reduces damage to organs and apoptosis and also restores immunosuppressive condition. We investigated how hypertonic saline resuscitation affected the induction of CD4 + CD25 + regulatory T cells and γδT cells, and their apoptosis after hemorrhagic shock and resuscitation, and its relationship to inducible nitric oxide synthase (iNOS) (nitric oxide production). Methods: Male inbred C57BL6/J mice 8-week to 12-week-old as wild type and iNOS gene knock out (iNOS―/―), weighing 20 g to 35 g, were used. Hemorrhagic shock model of ±40 mm Hg for 60 minutes was setup. Animals were randomly assigned to the following four resuscitation group: (1) wild HS: resuscitation with hypertonic saline (4 mL/Kg of 7.5% NaCl) and shed blood (SB), (2) wild 2LR: resuscitation with lactated Ringer's solution (two times the volume of the SB) and SB, (3) iNOS knockout HS, and (4) iNOS knockout 2LR. Untreated groups for wild and iNOS knockout mice were designated as control groups. Samples of thymus and spleen were harvested at 2 hours, 6 hours, 24 hours, and 48 hours after resuscitation. CD4 + CD25 + regulatory T cells and γδT cells were analyzed using three-color flow cytometry. Results: (1) γδ T cells increased earlier at 24 hours and CD4 + CD25 + regulatory T cells increased later at 48 hours compared with controls in spleen of wild type (p < 0.01). (2) Hypertonic saline resuscitation suppressed γδ T cells compared with 2LR at 24 hours in iNOS knockout mice in spleen (p < 0.05). Hypertonic saline resuscitation increased apoptosis of CD4 + CD25 + regulatory T cells at 48 hours in iNOS knockout mice in spleen (p < 0.01). (3) CD4 + CD25 + regulatory T cells of iNOS knockout both in HS and 2LR groups at 48 hours decreased compared with wild type both in HS and 2LR groups in spleen (p < 0.01). (4) Apoptotic γδ T cells both in spleen and thymus in iNOS knockout mice at 48 hours increased compared with those in wild type (p < 0.05, respectively, except γδ T cells 2LR in spleen: p = 0.058). Conclusion: γδ T cells increased earlier at 24 hours, whereas CD4 + CD25 + regulatory T cells increased later at 48 hours in spleen of wild type. Hypertonic saline was effective without the presence ofiNOS, i.e., decreased γδ T cells at 24 hours and increased apoptosis of CD4 + CD25 + regulatory T cells at 48 hours. CD4 + CD25 + regulatory T cells at 48 hours without iNOS decreased compared with those of wild type. γδ T cells at 48 hours induced apoptosis under the condition without iNOS in spleen and thymus. iNOS worked as an accelerating factor for immunosuppressive condition, affected apoptosis, and immunoenhancing effect by hypertonic saline.