Factors Responsible for Peritoneal Granulocyte Apoptosis during Sepsis

Abstract

Apoptosis (Ao), is a process by which cells undergo a form of non-necrotic cellular suicide, the control of which may have significant impact on host immunoresponsiveness to a septic challenge. The aim of this study was to determine (1) if Aois evident in granulocytes harvested from the blood or peritoneum of septic animals and to what extent this was associated with cell activation and (2) whether thein vivoadministration of the TNF inhibitor (TNFbp) alters this process. To assess the first aim, C3H/HeN male mice were subjected to polymicrobial sepsis [cecal ligation and puncture (CLP)] or sham-CLP (Sham) and sacrificed at 4 or 24 hr following CLP. Blood leukocytes and peritoneal exudate cells were harvested, stained with monoclonal fluorochrome-conjugated antibodies to granulocytes (Gr1), the activation marker ICAM-1, and either the cell cycle dye, 4′,6-diamidino-2-phenylindole, or TUNEL assay (to assess the %Ao+), was used for two- and three-color flow cytometric analysis. Peritoneal exudate cells exhibited increased %Ao+cells at both 4 and 24 hr post-CLP, while blood leukocytes showed a decrease in %Ao+only at 24 hr. The increase in Aoin the peritoneum was evident only in the Gr1−cell population at 4 hr but was present in both Gr1+and Gr1−cells at 24 h. Furthermore, the increase in the %Ao+cells was associated with an increased % of ICAM-1 positive cells. To the extent that TNF affects the 24 hr induction of Aoin peritoneal exudate cells, mice were treated with either 250 μg TNFbp/mouse (s.c.) or vehicle control immediately following CLP. The results indicate that administration of TNFbp markedly decreased Gr1+but not Gr1−cell Ao. Thus, not only does polymicrobial sepsis induce a marked early rise in phagocyte Aoassociated with cell activation, but the increase in peritoneal granulocyte Ao, unlike macrophage Ao, is mediated by TNF and/or an agent released by TNF.