Inflammation in sepsis is controlled primarily by the degree of macrophage activation. Costimulatory molecules are a class of receptors capable of macrophage activation in adaptive immunity. Ligation of macrophage expressed CD40, CD80/CD86 and ICAM by CD154, CD28/CTLA4 and LFA-1 on activated T- cells, increases NF-kB DNA binding and the ratio of stimulatory/inhibitory isoforms of C/EBPb. This results in increased production of numerous inflammatory cytokines. However, the role for costimulatory molecules in the innate immune response during polymicrobial sepsis is not well described. We recently described CD40- /- mice have delayed mortality with polymicrobial sepsis with attenuations in NF-kB activity and the ratio of stimulatory/inhibitory C/EBPb. We now present data that neutrophils, a main component of innate immune response in sepsis, can directly activate macrophages via engagement of the costimulatory molecules CD40, CD80/CD86 and ICAM in vitro. In vivo, CD80/CD86-/- mice have improved survival compared to WT mice after CLP. This was associated with a reduction in NF-kB, the ratio of stimulatory/inhibitory C/EBPb and levels of IL-6 and IL-10 in BAL and plasma. Similar to what has been reported in moels of adaptive immunity, inhibtion for multiple costimulatory molecules provided additional benefit. Both CD40/CD80/CD86-/- mice and ICAM neutralization in CD80/CD86-/- mice improved survival compared to CD80/86-/- mice after CLP. In humans with sepsis, we found upregulation of PMNexpressed CD28, CTLA4 and CD154, and monocyte expressed CD40, CD80/CD86 and ICAM compared to healthy controls. In addition, the soluble isoforms of CD154, CD28, ICAM and CTLA4 were upregulated in septic patients comparedto healthy controls. Furthermore, levels of sCD28 and sCD154 were only upregulated in non-survivors, suggesting a potential role as a biomarker of disease activity. Together, these results suggest an important role for costimulatory molecules in sepsis. In this proposal we plan to extend these observations to: 1. Characterize the kinetics and activity of the CD40-CD154, CD80/CD86-CD28/CTLA4 and ICAM-LFA-1 systems in a murine model of polymicrobial sepsis, 2. Test the effect of inhibition of multiple costimulatory molecules in sepsis via a novel CD40/CD80/CD86-/- mouse, and 3. To fully assess the significance of costimulatory molecule expression and activity both in vivo and ex vivo in humans with sepsis and septic shock.