Katherine Chang

Patients with sepsis have impaired host defenses that contribute to the lethality of the disorder. Recent work implicates lymphocyte apoptosis as a potential factor in the immunosuppression of sepsis. If lymphocyte apoptosis is an important mechanism, specific subsets of lymphocytes may be more vulnerable. A prospective study of lymphocyte cell typing and apoptosis was conducted in spleens from 27 patients with sepsis and 25 patients with trauma. Spleens from 16 critically ill nonseptic (3 prospective and 13 retrospective) patients were also evaluated. Immunohistochemical staining showed a caspase-9-mediated profound progressive loss of B and CD4 T helper cells in sepsis. Interestingly, sepsis did not decrease CD8 T or NK cells. Although there was no overall effect on lymphocytes from critically ill nonseptic patients (considered as a group), certain individual patients did exhibit significant loss of B and CD4 T cells. The loss of B and CD4 T cells in sepsis is especially significant because it occurs during life-threatening infection, a state in which massive lymphocyte clonal expansion should exist. Mitochondria-dependent lymphocyte apoptosis may contribute to the immunosuppression in sepsis by decreasing the number of immune effector cells. Similar loss of lymphocytes may be occurring in critically ill patients with other disorders.

Sepsis induces extensive lymphocyte apoptosis, a process which may be beneficial to host survival by down-regulating the inflammatory response or, alternatively, harmful by impairing host defenses. To determine the beneficial vs. adverse effects of lymphocyte apoptosis in sepsis, we blocked lymphocyte apoptosis either by N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl) fluoromethyl ketone (z-VAD), a broad-spectrum caspase inhibitor, or by use of Bcl-2 Ig transgenic mice that selectively overexpress the antiapoptotic protein Bcl-2 in a lymphoid pattern. Both z-VAD and Bcl-2 prevented lymphocyte apoptosis and resulted in a marked improvement in survival. z-VAD did not decrease lymphocyte tumor necrosis factor-alpha production. Considered together, these two studies employing different methods of blocking lymphocyte apoptosis provide compelling evidence that immunodepression resulting from the loss of lymphocytes is a central pathogenic event in sepsis, and they challenge the current paradigm that regards sepsis as a disorder resulting from an uncontrolled inflammatory response. Caspase inhibitors may represent a treatment strategy in this highly lethal disorder.

Abstract In sepsis there is extensive apoptosis of lymphocytes, which may be beneficial by down-regulating the accompanying inflammation. Alternatively, apoptosis may be detrimental by impairing host defense. We studied whether Bcl-2, a potent antiapoptotic protein, could prevent lymphocyte apoptosis in a clinically relevant model of sepsis. Transgenic mice in which Bcl-2 was overexpressed in T cells had complete protection against sepsis-induced T lymphocyte apoptosis in thymus and spleen. Surprisingly, there was also a decrease in splenic B cell apoptosis in septic Bcl-2 overexpressors compared with septic HeJ and HeOuJ mice. There were marked increases in TNF-α, IL-1β, and IL-10 in thymic tissue in sepsis in the three species of mice, and the increase in TNF-α and IL-10 in HeOuJ mice was greater than that in Bcl-2 mice. Mitotracker, a mitochondrial membrane potential indicator, demonstrated a sepsis-induced loss of membrane potential in T cells in HeJ and HeOuJ mice but not in Bcl-2 mice. Importantly, Bcl-2 overexpressors also had improved survival in sepsis. To investigate the potential impact of loss of lymphocytes on survival in sepsis, Rag-1−/− mice, which are totally deficient in mature T and B cells, were also studied. Rag-1−/− mice had decreased survival compared with immunologically normal mice with sepsis. We conclude that overexpression of Bcl-2 provides protection against cell death in sepsis. Lymphocyte death may be detrimental in sepsis by compromising host defense.

Patients with sepsis are immune compromised, as evidenced by their failure to clear their primary infection and their propensity to develop secondary infections with pathogens that are often not particularly virulent in normal healthy individuals. A potential mechanism for immunosuppression in sepsis is lymphocyte apoptosis, which may occur by either a death receptor or a mitochondrial-mediated pathway. A prospective study of blood samples from 71 patients with sepsis, 55 nonseptic patients, and 6 healthy volunteers was undertaken to quantitate lymphocyte apoptosis and determine cell death pathways and mechanisms of apoptosis. Apoptosis was evaluated by flow cytometry and Western blotting. Lymphocyte apoptosis was increased in CD4 and CD8 T cells, B cells (CD20), and NK cells (CD56) in septic vs nonseptic patients. Samples taken sequentially from 10 patients with sepsis showed that the degree of CD3 T cell apoptosis correlated with the activity of his/her sepsis. In septic patients, apoptotic lymphocytes were positive for active caspases 8 and 9, consistent with death occurring by both mitochondrial-mediated and receptor-mediated pathways. In support of the concept that both death pathways were operative, lymphocyte apoptosis occurred in cells with markedly decreased Bcl-2 (an inhibitor of mitochondrial-mediated apoptosis) as well as cells with normal concentrations of Bcl-2. In conclusion, apoptosis occurs in a broad range of lymphocyte subsets in patients with sepsis and correlates with the activity of the disease. Lymphocyte loss occurs by both death receptor and mitochondrial-mediated apoptosis, suggesting that there may be multiple triggers for lymphocyte apoptosis.

IL-15 is a pluripotent antiapoptotic cytokine that signals to cells of both the innate and adaptive immune system and is regarded as a highly promising immunomodulatory agent in cancer therapy. Sepsis is a lethal condition in which apoptosis-induced depletion of immune cells and subsequent immunosuppression are thought to contribute to morbidity and mortality. This study tested the ability of IL-15 to block apoptosis, prevent immunosuppression, and improve survival in sepsis. Mice were made septic using cecal ligation and puncture or Pseudomonas aeruginosa pneumonia. The experiments comprised a 2 x 2 full factorial design with surgical sepsis versus sham and IL-15 versus vehicle. In addition to survival studies, splenic cellularity, canonical markers of activation and proliferation, intracellular pro- and antiapoptotic Bcl-2 family protein expression, and markers of immune cell apoptosis were evaluated by flow cytometry. Cytokine production was examined both in plasma of treated mice and splenocytes that were stimulated ex vivo. IL-15 blocked sepsis-induced apoptosis of NK cells, dendritic cells, and CD8 T cells. IL-15 also decreased sepsis-induced gut epithelial apoptosis. IL-15 therapy increased the abundance of antiapoptotic Bcl-2 while decreasing proapoptotic Bim and PUMA. IL-15 increased both circulating IFN-gamma, as well as the percentage of NK cells that produced IFN-gamma. Finally, IL-15 increased survival in both cecal ligation and puncture and P. aeruginosa pneumonia. In conclusion, IL-15 prevents two immunopathologic hallmarks of sepsis, namely, apoptosis and immunosuppression, and improves survival in two different models of sepsis. IL-15 represents a potentially novel therapy of this highly lethal disorder.