Immunopathologic responses to non-lethal 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.

Differential local and systemic regulation of the murine chemokines KC and MIP2.

We characterized the relative biological activity and expression of two murine chemokines that may serve as functional homologues for human IL-8, KC, and macrophage inflammatory protein 2 (MIP2). Recombinant chemokines were produced in bacterial expression systems and antibodies specific for KC or MIP2 were raised. In vitro assays showed that KC elicited 4-fold greater neutrophil chemotaxis compared with MIP2, while MIP2 elicited significantly greater release of elastase. Lipopolysaccharide- (LPS) stimulated macrophages (8 h) secreted more MIP2 (approximately 10 ng/mL) compared with KC (approximately 4 ng/ml) and expression of either murine chemokine was independent of TNFalpha or IL-1beta production. Thioglycollate (thio) and glycogen (gly) induced peritonitis produced more KC (thio = 7.1 and gly = 2.5 ng/mL) in the peritoneum compared with MIP2 (thio = 4.5 and gly = 0.3 ng/mL). Plasma KC levels were very high after either challenge (approximately 24 ng/mL), which was >50-fold more than the systemic increase in MIP2 (approximately 0.3 ng/mL). Our data demonstrate that while KC and MIP2 have similar in vitro production characteristics, KC appears to be a more potent and systemically distributed chemokine during acute in vivo inflammation, while MIP2 expression appears limited to localized expression.

Immunopathology of a two-hit murine model of acid aspiration lung injury.

In a two-hit model of acid aspiration lung injury, mice were subjected to nonlethal cecal ligation and puncture (CLP). After 48 h, intratracheal (IT) acid was administered, and mice were killed at several time points. Recruitment of neutrophils in response to acid was documented by myeloperoxidase assay and neutrophil counts in bronchoalveolar lavage (BAL) fluid and peaked at 8 h post-IT injection. Albumin in BAL fluid, an indicator of lung injury, also peaked at 8 h. When the contributions of the two hits were compared, neutrophil recruitment and lung injury occurred in response to acid but were not greatly influenced by addition of another hit. Neutrophil sequestration was preceded by elevations in KC and macrophage inflammatory protein-2alpha in plasma and BAL fluid. KC levels in BAL fluid were higher and peaked earlier than macrophage inflammatory protein-2alpha levels. When KC was blocked with specific antiserum, neutrophil recruitment was significantly reduced, whereas albumin in BAL fluid was not affected. In conclusion, murine KC mediated neutrophil recruitment but not lung injury in a two-hit model of aspiration lung injury.

Critical role of CD14 for production of proinflammatory cytokines and cytokine inhibitors during sepsis with failure to alter morbidity or mortality.

We investigated the immunopathophysiologic responses during sepsis induced by cecal ligation and puncture (CLP) in CD4-deficient (CD14 knockout [CD14KO]) mice. Our studies were designed to specifically test the role of CD14 in the inflammatory response to sepsis and to ascertain if alterations would improve morbidity or mortality. Sepsis was induced using the CLP model with appropriate antibiotic treatment. The severity of sepsis increased in the CD14KO mice with increasing puncture size (18 gauge [18G], 21G, and 25G). Following CLP, body temperature (at 12 h) and gross motor activity levels of the sham and 25G CLP groups recovered to normal, while the 21G and 18G CLP groups exhibited severe hypothermia coupled with decreased gross motor activity and body weight. There were no significant differences in survival, temperature, body weight, or activity levels between CD14KO and control mice after 21G CLP. However, CD14KO mice expressed two- to fourfold less pro-inflammatory (interleukin-1beta [IL-1beta], tumor necrosis factor [TNF], and IL-6) and anti-inflammatory (IL-10, IL-1 receptor antagonist, and TNF receptors I and II) cytokines in the blood after 21G CLP. Plasma levels of the chemokines macrophage inflammatory protein 2alpha and KC were similarly reduced in CD14KO mice. A similar trend of decreased cytokine and cytokine inhibitor levels was observed in the peritoneal cavity of CD14KO mice. Our results indicate that the CD14 pathway of activation plays a critical role in the production of both pro-inflammatory cytokines and cytokine inhibitors but has minimal impact on the morbidity or mortality induced by the CLP model of sepsis.

Ratio of local to systemic chemokine concentrations regulates neutrophil recruitment.

CXC chemokines are important regulators of local neutrophil recruitment. In this study, we examined the role of the ratio of local to systemic chemokine concentrations as a significant factor determining local neutrophil recruitment. Thioglycollate was injected intraperitoneally into BALB/c mice resulting in a dose-dependent increase in neutrophil recruitment and local inflammation, as measured by peritoneal levels of interleukin 6. At the high dose of 3% thioglycollate, antibody inhibition of the murine chemokines KC and macrophage inflammatory protein-2 caused a reduction in peritoneal neutrophil recruitment by as much as 93%. A paradoxical effect was observed with a 0.3% thioglycollate intraperitoneal challenge. In this situation, inhibition of KC resulted in a significant increase in peritoneal neutrophils, and inhibition of macrophage inflammatory protein-2 also resulted in increased peritoneal neutrophils. These results were consistent with a reverse chemotactic gradient as described by the ratio of peritoneal to plasma KC levels. A higher ratio (ie, increased peritoneal chemokines compared to plasma) resulted in increased neutrophil recruitment after either the 3% or 0.3% thioglycollate challenge. Our results demonstrate that whereas sufficient local concentrations of chemokines are necessary, a critical factor dictating local neutrophil recruitment is the ratio of the local to the systemic chemokine concentrations.

Combination immunotherapy with soluble tumor necrosis factor receptors plus interleukin 1 receptor antagonist decreases sepsis mortality.

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.