Our studies have demonstrated that hemorrhage produces a marked depression in cell-mediated immunity which persists despite fluid resuscitation and increases susceptibility to sepsis. Preliminary studies indicate that: a) ATP levels (by 31P-NMR) in splenocytes are barely detectable after hemorrhage and remain depressed even 2 hrs after resuscitation; b) hemorrhage increases splenic macrophage (Mphi) intracellular Ca2+ while decreasing the response to stimulant; c) after exposure to hypoxic environment (without hemorrhage), Mphi antigen presentation (AP) function is depressed and PGE2 production is increased. Our hypothesis, therefore, is that hemorrhage produces regional hypoxia which causes cell ATP to decrease, inducing alterations in calcium homeostasis/second messenger systems, translocation of bacteria (i.e. endotoxin release), and stimulation of Kupffer cells to produce inflammatory cytokines. The net results of the above event(s) is increased PGE2 production, which mediates the depression of Mphi (AP and associated processes) and splenocyte function (proliferation and lymphokine generation), producing immunodepression and increasing susceptibility to sepsis. Studies are proposed to determine whether or not: 1) hemorrhage and resuscitation with and without immunomodulation (with agents such as ATP-MgCl2, calcium antagonists, anti-endotoxin, IL-6 or TNF antibodies, chloroquine), as compared to hypoxia, produce alterations in splenocyte and Mphi ATP/calcium levels and PGE2 production; 2) hemorrhage or hypoxia produces alterations in the second messenger system; 3) the alterations in Mphi cytokine production after hemorrhage and resuscitation with and without the above immunomodulators are transcriptional and/or translational in nature. Such studies will determine whether or not the gene expression for TNF and IL-6 is altered after hemorrhage/immunomodulation. Additionally, in situ hybridization will be carried out to localize the cellular compartments of altered cytokine production; and 4) depletion of arachidonic acid stores before hemorrhage using omega-3 fatty acid diet will prevent the depression of Mphi and splenocyte functions following hemorrhage. The use of biochemical, physiological, cellular and molecular biological techniques to determine the mechanism responsible for immunodepression and increased susceptibility to sepsis following hemorrhage and resuscitation should provide useful information for the treatment and care of patients with major blood loss.
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