Interleukin-10 controls the onset of irreversible septic shock

Abstract

Lethality from sepsis is believed to be mediated by a proinflammatory cytokine cascade, yet blocking the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) fails to prevent mortality in human disease and a mouse model of sepsis induced by cecal ligation and puncture (CLP). The role of the antiinflammatory cytokine IL-10 in the CLP model of sepsis is unclear, with either protective or harmful effects demonstrated, depending upon the time of intervention. We therefore hypothesize that IL-10 functions as a temporal regulator of the transition from early reversible sepsis to the late phase of irreversible shock. Transition from reversible sepsis to irreversible shock in the CLP model was defined as the time when removal of the necrotic cecum by rescue surgery is no longer effective. We subjected IL-10-deficient (IL-10(-/-)) and wild-type (IL-10(+/+)) mice to CLP and monitored the progression of sepsis, the onset of irreversible shock, and mortality. Onset of lethality in IL-10(-/-) mice occurred significantly earlier than in IL-10(+/+) mice and was associated with 15-fold-higher serum levels of TNF-alpha and IL-6. Consistent with these findings, the efficacy of rescue surgery after lethal CLP is lost 10 h earlier in IL-10(-/-) mice than in IL-10(+/+) mice. Treatment with recombinant human IL-10 5 h after CLP significantly improved survival and lengthened the therapeutic window for rescue surgery in both strains of mice. These results demonstrate that IL-10 controls the onset of irreversible septic shock after CLP.

FIG. 1.

In the absence of IL-10 mortality is more rapid after lethal CLP. IL-10^−/− (dashed line) and IL-10^+/+ (solid line) C57BL/6J mice underwent CLP with an 18-gauge needle (n = 30 mice per strain). The time of death within 10-h intervals was recorded, and the data are expressed as the cumulative percentage of mice still alive within each interval. The overall mortality in the IL-10^−/− mice was significantly more rapid when the survival curves between both strains of mice were compared (Logrank test, P < 0.0001), but the mortality at 120 h was statistically indistinguishable between the two strains (FET, P = 0.20).

FIG. 2.

Delayed onset of mortality in the presence of IL-10 after sublethal CLP. IL-10^−/− (dashed line) and IL-10^+/+ (solid line) mice underwent CLP with a 25-gauge needle, and the time of death after surgery was recorded within 10-h intervals (n = 20 mice per strain). At the onset of lethality in IL-10^+/+ mice at 60 h there was a 35% significantly greater mortality in the IL-10^−/− mice (FET, P < 0.01). However, there was no statistical difference in long-term survival between IL-10^+/+ and IL-10^−/− mice (FET, P = 0.52).

FIG. 3.

The absence of IL-10 led to a greater elevation in TNF-α and IL-6 in serum during sepsis initiated by CLP. IL-10^+/+ (•) and IL-10^−/− (○) mice that underwent lethal CLP had sera collected at the indicated times, and TNF-α (A) and IL-6 (B) concentrations were determined by ELISA (n = 8 mice per time point). The data are shown as the log transformation of the mean ± the standard deviation.

FIG. 4.

The IL-10 concentrations in serum are elevated and maintained from 5 to 20 h during sepsis initiated by CLP. Groups of IL-10^+/+ mice underwent lethal CLP and had sera collected at the indicated times (n = 8 mice per time point). IL-10 concentrations in serum were determined by ELISA. The data are shown as the log transformation of the mean ± the standard deviation.

FIG. 5.

Rescue surgery with CR must occur earlier in IL-10^−/− mice to improve survival. Groups of IL-10^+/+ (▪, n = 10) and IL-10^−/− (□, n = 10) mice underwent CR at 5, 10, 15, and 20 h after CLP, and the survival at 120 h was recorded. Compared to CLP-treated animals with no additional intervention, CR at 5 h in IL-10^−/− mice and at 5, 10, and 15 h in IL-10^+/+ mice led to improved survival. However, intervention in IL-10^−/− mice at 10 h and in IL-10^+/+ mice at 20 h was not successful (FET, P ≥ 0.15). ND, not done.

FIG. 6.

Treatment with rhIL-10 delayed the onset of lethality and improved long-term survival in both IL-10^+/+ and IL-10^−/− mice. Groups of IL-10^−/− and IL-10^+/+ mice underwent CLP with an 18-gauge needle. At 5 h after CLP-treated mice (IL-10^−/−, dashed line; IL-10^+/+, solid broken line) received 1 μg of rhIL-10 in 0.25 ml of normal saline (n = 20) by subcutaneous injection. Control mice (IL-10^−/−, dotted line; IL-10^+/+, solid thin line) were injected with normal saline alone (n = 30). The time of death within 10-h intervals was recorded, and the data were expressed as the cumulative percentage of mice alive within each interval. Survival of treated mice in both strains, as determined by the Logrank test, was significantly improved compared to untreated animals (P ≤ 0.007).

FIG. 7.

rhIL-10 treatment extends the therapeutic window for rescue surgery after CLP. Groups of IL-10^−/− and IL-10^+/+ mice received either no treatment (□, n = 30), CR alone at 10 h or 20 h, respectively (▪, n = 10); or 1 μg of rhIL-10 5 h after lethal CLP and CR at 10 h or 20 h (░⃞, n = 10). In rhIL-10-treated mice, the efficacy of rescue surgery by CR was now significantly improved at the time point when it had previously failed in both strains of mice (FET, P ≤ 0.02 [✽]).