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Objective: Inhibition of tumor necrosis factor (TNF) or interleukin 1 (IL-1) alone has not improved sepsis survival in human clinical trials; therefore, it has been suggested that blockade of both may be successful. We tested whether combination immunotherapy would improve survival in mice subjected to a lethal lipopolysaccharide (LPS) challenge or the sepsis model of cecal ligation and puncture., Design: Mice were treated with the combination immunotherapy and challenged with either a lethal dose of lipopolysaccharide or a septic challenge induced by cecal ligation and puncture., Setting: University research laboratory., Subjects: Adult, female Balb/c mice., Interventions: Mice were treated with the combination of the IL-1 receptor antagonist plus a polyethylene glycol-linked dimer of the TNF soluble receptor., Measurements and Main Results: LPS lethality was reduced in the treated mice with a decrease in biologically active TNF in the plasma and peritoneal fluid. In the cecal ligation and puncture (CLP) model of sepsis, this combination immunotherapy for 1 day decreased plasma and peritoneal levels of IL-6 and the murine chemokines KC and MIP-2. However, treatment did not result in a reduction in the hypothermia or peripheral blood alterations that occur after CLP, and the 1-day therapy did not result in an improvement in survival. In contrast, when combination immunotherapy was extended to 3 days there was a significant improvement in survival., Conclusions: These data demonstrate that inhibition of both TNF and IL-1 will decrease the lethality of sepsis initiated by CLP if the combination immunotherapy is provided for a sufficient amount of time.

Sepsis remains a serious clinical problem despite intense efforts to improve survival. Experimental animal models of sepsis have responded dramatically to immunotherapy blocking the activity of cytokines. Despite these preclinical successes, human clinical trials have not demonstrated any improvement in survival. We directly compared the mortality, morbidity, and immunopathology in two models of sepsis, one due to lipopolysaccharide (LPS) and the other to cecal ligation and puncture (CLP). BALB/c mice were injected intraperitoneally with 250 microg of LPS or subjected to CLP with an 18-gauge needle. Both models yielded similar mortality (> 85%) and morbidity. Additionally, neutropenia and lymphopenia developed in both groups. Plasma and peritoneal levels of cytokines (TNF, IL-1, IL-6, and the chemokines KC and MIP-2) were measured at 1.5, 4, and 8 h after challenge. LPS induced substantially higher levels of cytokines in both compartments with peak levels between 1.5 and 4 h that began to decline at 8 h. In contrast, cytokine levels in the CLP model were continuing to increase at the 8 h-time point and often exceeded the LPS-induced values at this time. Our data demonstrate that the LPS and CLP models have similar mortality but significant differences in the kinetics and magnitude of cytokine production. Immunotherapy for sepsis based on cytokine production after LPS challenge is misdirected because the LPS model does not accurately reproduce the cytokine profile of sepsis.

Although sepsis causes significant morbidity and mortality, its basic pathology is still not well understood. We investigated the inflammatory and physiologic alterations of non-lethal sepsis using cecal ligation and puncture (CLP), a model that induces peritonitis due to mixed intestinal flora, reproducing the complex immunology of sepsis. Groups of mice were subjected to CLP (25G needle) or sham surgery, had minimitters implanted to continuously monitor temperature and activity, and were sacrificed daily for 6 days. There was significant hypothermia (6-13 hrs post-surgery), and decreases in activity (to day 4) and weight (to day 3) but no mortality in the CLP group. Blood analyses of the CLP-treated mice showed reduced hemoglobin, platelets, lymphocytes, monocytes, and neutrophils, compared to sham animals. Both groups had nearly equivalent neutrophil influx into the peritoneum. Plasma and peritoneal G-CSF, IL-6, as well as the murine chemokines KC and MIP2-alpha were significantly higher in the CLP-treated mice at day 1. Plasma and peritoneal TNF were low (<70 pg/mL). While there was elevated IL-1beta in the peritoneum of the CLP-treated mice, this cytokine was not detected in the plasma in either treatment group. Cytokines were not detected in the pulmonary airspace of the CLP-treated mice and PMNs were not recruited to this site. Our data shows altered immunopathology in non-lethal sepsis with significant blood and cytokine alterations. Since there was 100% survival, the inflammatory response was appropriate and probably even protective.

Objective: To determine if exogenous interleukin (IL)-10 will decrease the morbidity or mortality of sepsis induced by cecal ligation and puncture., Design: Prospective, randomized, controlled study., Setting: University research laboratory., Subjects: Adult, female, Balb¿c mice., Interventions: Balb¿c mice were subjected to cecal ligation and puncture with an 18- or 23-gauge needle and treated with triple antibiotics. Mice were injected subcutaneously with recombinant human IL-10 (diluted in normal saline with 0.1% mouse serum albumin) and followed until death. In a separate experiment, IL-10 was also injected subcutaneously and lipopolysaccharide (LPS) injected intraperitoneally and plasma tumor necrosis factor concentrations measured 90 mins later., Measurements and Main Results: In the LPS experiments, IL-10 decreased tumor necrosis factor (TNF) production by nearly 90%. For the cecal ligation and puncture experiments, temperature and movement were recorded continuously via implanted transmitters. Studies on mortality indicated that exogenous IL-10 given at 0, +6 and +12 hrs after surgery failed to increase survival when using an 18-gauge needle (alive:total cecal ligation and puncture alone 4:21; IL-10 10 microg/mouse 2:12; 1 microg/mouse 8:25; 0.1 microg/mouse 1:12) or a 23-gauge needle (cecal ligation and puncture alone 13:29; IL-10 1 microg 18:30). There was no difference in the number of hours to death between the groups. IL-10 did not prevent the hypothermia after cecal ligation and puncture or increase the animals' activity. To examine parameters of inflammation, mice were killed 8 hrs after 18-gauge cecal ligation and puncture. IL-10 (1 microg/mL) failed to reduce pulmonary neutrophil sequestration (lung myeloperoxidase, cecal ligation and puncture 107 +/- 10 [SEM], IL-10 107 +/- 5) or recruitment of neutrophils to the peritoneum (neutrophils x 10(6), cecal ligation and puncture 3.72 +/- 0.62; IL-10 3.49 +/- 0.37). IL-10 also failed to reduce the appearance of TNF or IL-6 in the plasma or peritoneal fluid. The chemokine KC was reduced in the peritoneal fluid but not the plasma and endogenous IL-10 production was not reduced in the peritoneum., Conclusion: Our data indicate that exogenous IL-10 fails to improve morbidity or mortality in the clinically relevant cecal ligation and puncture model of sepsis.