survival and attenuates pro-inflammatory cytokine production in CLP. However, these studies also demonstrated that monocyte expression of CD80 and CD86 were differentially regulated in sepsis. Specifically, sepsis was associated with an increase in CD80 expression, while there appeared to be a downregulation of constitutive CD86 expression. Further, recent studies suggest CD80 and CD86 have differential regulation in the inflammatory response in vivo in diseases regulated by the adaptive immune response, including allergic rhinitis and graft rejection. Combined, these data imply a possible differential role for CD80 and CD86 in regulating mortality and inflammation in the innate immune response as well. Consequently, the goals of this study were to determine the individual contribution of CD28, CD80 and CD86 to the inflammatory response in murine sepsis, and to better correlate expression of these molecules with outcome in humans with sepsis and septic shock. Human monocytes derived macrophages were isolated from healthy volunteers and prepared as previously described.Cells harvested and washed with PBS63 and total cell 869113-09-7 66547-09-9 manufacturer lysate prepared using NP40 lysis buffer as previously described. Lysate was then incubated with 10 mg antibodies to CD80 or CD86. One ml of extract was then incubated with end-over-end mixing for 2 hours at 4uC. Fifty ml of Protein A/G Sepharose were washed with 1 ml of lysis buffer. This was repeated 3 times and then excess buffer was carefully removed. After the 2 hour incubation, the washed Protein A/G Sepharose was added to the immunoprecipitates and mixed for 1 hour at 4uC. The immunoprecipitates were washed 3X with 1 ml of lysis buffer. Samples then assayed by Western blot as previously described. One of the major findings of this paper is the observation of improved survival in CD282/2 mice after CLP. The ability of CD28 to regulate inflammation in the innate immune response to sepsis is consistent with prior reports documenting CD28 expression on PMNs and the ability of CD28, either soluble or in PMN lipid rafts, to induce NF-kB and pro-inflammatory cytokine production via CD80/CD86. Our results are also consistent with earlier reports suggesting an important role for CD28 in regula
