Agonistic Monoclonal Antibody Against CD40 Receptor Decreases Lymphocyte Apoptosis and Improves Survival in Sepsis

Abstract

Sepsis causes a marked apoptosis-induced depletion of lymphocytes. The degree of lymphocyte apoptosis during sepsis strongly correlates with survival. CD40, a member of the TNFR family, is expressed on APCs and has potent antiapoptotic activity. In this study we determined whether an agonistic Ab against CD40 could protect lymphocytes from sepsis-induced apoptosis. Secondly, we examined potential antiapoptotic mechanisms of the putative protection. Lastly, we aimed to determine whether anti-CD40 treatment could improve survival in sepsis. CD1 mice were made septic by the cecal ligation and puncture method and treated postoperatively with anti-CD40 Ab. Treatment with anti-CD40 completely abrogated sepsis-induced splenic B cell death and, surprisingly, decreased splenic and thymic T cell death as well (p < 0.001). To investigate the mechanism of protection of anti-CD40 therapy on T cells, CD40 receptor expression was examined. As anticipated, the CD40 receptor was constitutively expressed on B cells, but, unexpectedly, splenic and thymic T cells were found to express CD40 receptor during sepsis. Furthermore, CD4+CD8− T cells were the predominant subtype of T cells expressing CD40 receptor during sepsis. Additionally, the antiapoptotic protein Bcl-xL was found to be markedly increased in splenic B and T cells as well as in thymic T cells after treatment with anti-CD40 Ab (p < 0.0025). Lastly, mice that were made septic in a double injury model of sepsis had improved survival after treatment with anti-CD40 as compared with controls (p = 0.05). In conclusion, anti-CD40 treatment increases Bcl-xL, provides nearly complete protection against sepsis-induced lymphocyte apoptosis, and improves survival in sepsis.

Adoptive transfer of dying cells causes bystander-induced apoptosis

“…In accordance with our results, Iwata et al reported that mice which were transgenic for Bcl-2 in their myeloid-derived cells had decreased sepsis-induced apoptosis in their intestinal epithelial cells [8]. The mechanism of this cross-protection is unknown but has been reported by other groups [5][6][7][8][9][10]. Iwata et al speculated, however, that the protective action of Bcl-2 over expressing cells was via a "trans" effect, i.e., Bcl-2 over expressing cells released an unknown cytoprotective factor which prevented death in non-Bcl-2 over expressing cells of distinct and distant lineages.…”

“…The cecal ligation and puncture (CLP) technique as developed by Chaudry et al [16] and modified by our laboratory was used to induce septic peritonitis as previously described [4][5][6][7]. In brief, mice were anesthetized and an abdominal incision was made.…”

“…Apoptosis was quantified by two independent methods. A commercially available phycoerythrin-labeled terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) kit (Phoenix Flow Systems, Inc.) was used to detect DNA strand breaks, and, alternatively, staining for active caspase 3 with a primary rabbit anti-caspase 3 antibody (Cell Signaling Technology, Inc.) and a phycoerythrin-labeled donkey anti-rabbit secondary antibody was performed as previously described [5,10]. T and B cells were identified using cyanine 5-labeled anti-mouse CD3 and cyanine 5-labeled anti-mouse b220 antibodies, respectively (BD Pharmingen).…”

“…Furthermore, it is well established in animal models that blocking lymphocyte apoptosis by a number of diverse methods improves survival in this highly lethal disorder. One particularly efficacious method is the overexpression of the anti-apoptotic Bcl-2 protein [4][5][6][7]. A surprising finding in studies involving apoptosis has been the observation that protection against cell death in a particular type of cell can confer protection in a different phenotypic cell.…”

Role of programmed cell death in the immunopathogenesis of sepsis

“…With respect to the inhibition of lymphocyte apoptosis, it has also been shown that activating CD40, a potent anti-apoptotic member of the TNF receptor family, provides extensive protection against sepsis-induced lymphocyte apoptosis and improves survival in sepsis by a mechanisms involving Bcl-xL [57]. The cell membrane permeable TAT-Bcl-X L peptide construct as well as a TAT-conjugated peptide of the anti-apoptotic BH4 domain of Bcl-X L (TAT-BH4) decreased sepsis-induced splenocyte T-and B-cell apoptosis to near sham levels in experimental murine sepsis.…”