INFECTION AND IMMUNITY, Aug. 2002, p. 4441–4446 Vol. 70, No. 80019-9567/02/$04.00 0 DOI: 10.1128/IAI.70.8.4441–4446.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Interleukin-10 Controls the Onset of Irreversible Septic ShockSamir Q. Latifi,1 Mary Ann O’Riordan,1 and Alan D. Levine2*

Division of Pediatric Pharmacology and Critical Care Medicine, Department of Pediatrics,1 and Department of Medicine, Pathology,

and Pharmacology,2 Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4952

Received 3 April 2002/Accepted 29 April 2002

Lethality from sepsis is believed to be mediated by a proinflammatory cytokine cascade, yet blocking the proinflammatory cytokines tumor necrosis factor alpha (TNF- ) 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- 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.

Sepsis is a major cause of morbidity and mortality in our hospitals (2). A severe and uncontrolled systemic inflammatory response triggered by an invading microbe may lead to evolv-ing multiorgan dysfunction (7). Occasionally, despite appropri-ate treatment and support of the septic patient, death may still ensue if irreversible damage occurs to vital organs. The onset of this irreversible shock is not easily defined clinically, nor are the mechanisms regulating this transition known. Cytokines may be important mediators in the development of this lethal multiorgan damage. For example, inflammatory cytokines, such as tumor necrosis factor alpha (TNF- ), interleukin-1 (IL-1), and IL-6, are elevated in the sera of both adult and pediatric septic patients (6, 9, 20, 29) and affect symptoms such as hypotension, fever, and the production of acute-phase pro-teins (21). However, the contribution of these proinflammatory cytokines in directly mediating mortality from sepsis is not clear, since therapies utilizing neutralizing antibodies or solu-ble receptor antagonists against TNF- and IL-1 fail to show a significant benefit in outcome for patients with sepsis (5, 13). Possible explanations for the clinical failure of these cytokine-targeted therapies may be that the antibodies or antagonists were administered too late or that other unopposed inflamma-tory mediators continue to support the disease process.

In contrast to humans, the use of anticytokine therapies against TNF- (4) and IL-1 (22) in a murine model of endo-toxemia leads to dramatic improvement in animal mortality. When lipopolysaccharide (LPS), the endotoxin from gram-negative bacteria, is injected into susceptible animals a rapid systemic inflammatory cascade is initiated in the absence of

* Corresponding author. Mailing address: Department of Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4952. Phone: (216) 368-1668. Fax: (216) 368-1674. E-mail: adl4@po.cwru.edu.

bacteremia. The lack of ongoing injury after a single adminis-tration of endotoxin and the absence of bacteremia in this model highlight its artificial nature and may explain why many clinical trials, based on observations with this model, have failed. The kinetics and magnitude of cytokine production in LPS-induced sepsis are also dramatically different from that seen in a more clinically relevant model of peritonitis with bacteremia induced by cecal ligation and puncture (CLP) (25), which closely mimics the human disease of septic shock (14). In sepsis induced by CLP, neutralizing antibodies to TNF- are not protective against mortality (10), and C3H/HeJ mice that are resistant to the lethal effects of LPS are still susceptible to mortality from CLP (19). These differences between the LPS and CLP models suggest that the immunology of sepsis is far more complex than the cascade of events activated by endo-toxin and that the exact role of proinflammatory cytokines as mediators of mortality is unclear.

Besides stimulating the synthesis of proinflammatory cyto-kines, the septic response in patients and animal models also results in the production of antiinflammatory mediators, such as IL-10 (8, 11, 30, 31). IL-10 is a 35-kDa homodimeric protein, produced primarily by monocytes, T cells, and B cells, that inhibits the production of proinflammatory cytokines in vitro (16). IL-10 is completely protective in the LPS model of sepsis (3, 15), but in the CLP model the administration of neutraliz-ing antibodies to IL-10 at the time of CLP only partially exac-erbates mortality (28, 31). However, inhibition of IL-10 12 h after CLP actually improves survival (28). Thus, in this model IL-10 can be either protective or harmful depending on the time of intervention. We therefore postulate that IL-10 regu-lates the transition from reversible sepsis to irreversible shock. In the CLP model we define this transition as the time point when removal of the necrotic cecum by rescue surgery is no longer effective. Here we report the ability of IL-10 to regulate

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4442 LATIFI ET AL.

the progression of sepsis and control the onset of irreversible shock and mortality in the CLP model.

MATERIALS AND METHODS

Mice. Inbred C57BL/6J mice deficient for the IL-10 gene (IL-10 / ) and wild-type

littermates (IL-10 / ) were used for experimentation between the ages of 8 to 12 weeks. The mice were bred and maintained in microisolator cages in our specific-

pathogen-free barrier facility under a 12-h light-dark cycle. While IL-10 / mice are known to develop colitis, in our facility no signs or symptoms of colitis are observed until 14 weeks of age. All experiments described were performed in adherence to the National Institutes of Health guidelines on the use of experimental animals, and approval was obtained from the Institutional Animal Care and Use Committee of Case Western Reserve University.

Genotyping of IL-10 / mice. The genotype of each mouse was determined by PCR analysis from tail clip DNA (12). Briefly, tail clips 0.25 cm in length were digested with proteinase K (Gibco-BRL, Gaithersburg, Md.) in tail buffer (50 mM Tris buffer, pH 8.0; 100 mM EDTA; 100 mM NaCl; 1% sodium dodecyl sulfate) overnight. The following day, digested tails were extracted twice with phenol (Gibco-BRL), followed by an extraction once with 24:1 chloroform-isoamyl alcohol (Sigma, St. Louis, Mo.). DNA was precipitated with 100% isopropanol, washed in 70% ethanol, air dried, and dissolved in 100 l of TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0). A total of 1 l of DNA was added to a 25- l PCR containing 1 Taq extender buffer (Stratagene, La Jolla, Calif.), 0.25 mM deoxynucleoside triphosphates (Gibco-BRL), 2 U of Taq polymerase (Boehringer Mannheim, Indianapolis, Ind.), and 2 U of Taq Extender (Strat-agene). Then, 1 l of a three-primer cocktail (1.6 g/ml) was added (primer 1, 5 -GCCTTCAGTATAAAAGGGGGAC-3 ; primer 2, 5 -CAGTTCATTCAGG GCACCGG-3 ; primer 3, 5 -CTTCAAACCCCCAAACCTCTG-3 ). PCR am-plification conditions were 94°C for 5 min, 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for 40 cycles, followed by 72°C for 5 min. The products were analyzed on a 2.0% agarose gel in 0.5 Tris-borate-EDTA buffer. Samples were loaded in buffer containing bromophenol blue and xylene cyanol. The gel was stained with 1 g of ethidium bromide/ml for 30 min and then destained in distilled water for an additional 30 min. The product size for the wild-type and disrupted IL-10 genes were 700 and 600 bp, respectively.

Cecal ligation and puncture. Anesthesia in the mice was induced with an intraperitoneal injection of 2.5% tribromoethanol at 0.02 ml/g (23). The abdo-men was opened, the cecum identified and ligated with a 3-O silk suture at its base below the ileocecal junction. Patency of the bowel lumen from the ileum to the colon was maintained. The antimesenteric border of the cecum was punc-tured once with either a 25- or 18-gauge needle, and the cecum was gently squeezed to extrude a small amount of stool. The cecum was returned to the abdomen, which was closed in two layers with 5-O silk sutures. After surgery mice were resuscitated with 0.5 ml of normal saline given by subcutaneous injection. Mice received 25 mg of the broad-spectrum antibiotic imipenem (Merck, West Point, Pa.)/kg in 0.5 ml of normal saline twice daily by subcuta-neous injection for 3 days. Mice were observed for a minimum of 120 h after CLP, after which a live animal was considered a long-term survivor. For mice undergoing sham surgery, the procedure for CLP was followed as described above except the cecum was not ligated and punctured.

Cecal removal (CR) surgery. After CLP some mice underwent a second rescue surgery, wherein the necrotic cecum was excised to remove the infecting nidus. Before the septic mice were anesthetized for their second laparotomy, they were further fluid resuscitated by a subcutaneous injection of 0.5 ml of normal saline. A 30% lower dose of tribromoethanol was administered by intraperitoneal in-jection to ensure recovery from anesthesia by all animals. The abdomen of each animal was opened, and the necrotic cecum was excised with scissors, while ensuring that the silk tie at the base of the cecum remained intact to prevent leakage of bowel contents. The peritoneal cavity was then lavaged with 1 ml of normal saline, and the abdomen was closed in two layers with 5-O silk sutures. After recovery from surgery, observation of the mice continued as before.

Blood sample collection for cytokine analysis. For cytokine analysis, blood was collected either by tail vein bleed or cardiac puncture in the euthanized animal. No mouse was bled more than twice; only 50 to 100 l was taken each time to minimize iatrogenic blood loss. Serum was stored at 70°C for later cytokine assay.

Cytokine ELISAs. The concentrations of TNF- , IL-6, and IL-10 in serum were measured by enzyme-linked immunosorbant assay (ELISA) as recom-mended by the manufacturer (R&D Systems, Minneapolis, Minn.). The sensi-tivity of the IL-6 and the IL-10 ELISA kits was 15 pg/ml, and for the TNF- kit it was 23 pg/ml.

INFECT. IMMUN.

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).

Treatment of mice with IL-10. In specific experiments, 1 g of recombinant human IL-10 (rhIL-10 [a gift from Schering-Plough Research Institute, Ken-ilworth, N.J.]) was administered after CLP by subcutaneous injection in 0.25 ml of normal saline, whereas control mice received vehicle alone. The total daily fluid administered to the mice after CLP remained the same by an appropriate decrease in the volume of the subsequent dose of antibiotic. The dose of IL-10 used here has previously been shown to be protective in the LPS model of sepsis (15).

Data analysis. The overall survival for lethal and sublethal injury was de-scribed

by using Kaplan-Meier plots. The difference between the survival curves for IL-10 /

and IL-10 / mice was tested by using the Logrank test. Propor-tions alive at specific time points were compared by using Fisher’s exact test. Cytokine data were

transformed to the log10 scale, and the results were plotted as means and standard deviations of the data. Two-way analysis of variance (ANOVA) was used to test for

the main effects of time after CLP and group (IL-10 / versus IL-10 / ), as well as the interaction between time and group. Analyses for TNF- and IL 6 were performed separately. Time of death of the animals used in these experiments was included in the model as a covariate. All analyses were done by using the SAS System (v8.1; SAS Institute, Carey, N.C.).

RESULTS

Endogenous IL-10 delays the onset of lethality induced by CLP and protects against mortality. To test our hypothesis that IL-10 functions as a temporal regulator of the septic re-sponse, we compared the time of onset of mortality and the subsequent kinetics of disease progression between IL-10-de-ficient mice on a C57BL/6J background (IL-10 / ) and their wild-type littermates (IL-10 / ) after CLP with an 18-gauge needle (Fig. 1). Onset of lethality in IL-10 / mice occurred at 10 h after CLP, but in IL-10 /

mice it was delayed until 30 h, by which time the IL-10 / mice had a significant 67% greater mortality (Fisher’s exact test [FET], P 0.0001). The overall mortality in the IL-10 / mice was also significantly more rapid when we compared the survival curves between both strains of mice (Logrank test, P 0.0001). However, the mod-est 15% difference in long-term survival at 120 h after CLP

VOL. 70, 2002 IL-10 CONTROLS THE ONSET OF IRREVERSIBLE SEPSIS 4443

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 be-tween IL-10 / and IL-10 /

mice (FET, P 0.52).

between IL-10 / and IL-10 / mice was not statistically sig-nificant (FET, P 0.20). These results indicate that the ab-sence of endogenous IL-10 leads to a more rapid onset of death but does not affect the overall mortality.

Delay of disease onset by IL-10 is similarly observed after a sublethal injury. The more rapid onset of mortality in the IL-10 /

mice compared to IL-10 / animals after CLP with an 18-gauge needle may be due to the severity of the insult. We therefore evaluated a sublethal injury with a 25-gauge needle to puncture the cecum and then monitored survival (Fig. 2). The onset of mortality at 10 h after CLP in IL-10 / mice was identical between

a lethal and sublethal injury. In IL-10 / mice the onset of mortality after sublethal injury was delayed for 60 h, by which time there was a 35% significantly greater mortality in the IL-10 / mice (FET, P 0.01). Similar to the findings from the more lethal CLP, overall survival at 120 h was not significantly different (FET, P 0.52). These results dem-onstrate that endogenous IL-10 has little influence on long-term survival but delays the onset of mortality.

Kinetics of proinflammatory cytokine production after CLP.An IL-10 deficiency has greater impact on mortality earlier in the disease process, suggesting that IL-10 functions in the CLP model of sepsis to regulate the timeline of systemic inflamma-tion. We therefore predicted that the absence of IL-10 would alter the kinetics of production of the proinflammatory cyto-kines TNF- and IL-6 after CLP. IL-10 / mice produced significantly greater

levels of TNF- and IL-6 in serum com-pared to IL-10 / animals at each time point from 5 to 20 h (ANOVA, P 0.0001) after CLP with an 18-gauge needle (Fig. 3). In comparison, IL-10 / and IL-

10 / mice that un-derwent sham surgery had no detectable TNF- in their serum and only a small peak of IL-6, to 1 ng/ml, at 5 h after laparotomy (data not shown). There was no significant change from 5 to 20 h after CLP in the concentrations of either TNF-or IL-6 in the sera in both strains of mice (ANOVA, P 0.07).

FIG. 3. The absence of IL-10 led to a greater elevation in TNF-and IL-6 in serum during sepsis initiated by CLP. IL-10 / (F) and IL-10 / (E) mice that underwent lethal CLP had sera collected at the indicated times, and TNF- (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.

The 15-fold-greater concentrations of TNF- and IL-6 mea-sured in the sera of IL-10 / mice after CLP may contribute to the earlier onset of lethality.

IL-10 levels in serum are elevated after CLP. The lower TNF- and IL-6 levels seen after CLP in IL-10 / mice com-pared to

IL-10 / mice are not unexpected. Therefore, we investigated the

kinetics of serum IL-10 production in IL-10 / mice after lethal CLP to determine whether it paralleled the proinflammatory cytokine profile. Levels of IL-10 in serum were maximal at 5 h after CLP and remained elevated until 20 h after injury (Fig. 4), matching the kinetics of proinflam-matory cytokine production. As expected, IL-10 / mice did not have any detectable IL-10.

Absence of IL-10 shortens the therapeutic window for rescue surgery after lethal CLP. Our findings thus far indicate that IL-10 regulates the onset of mortality, suggesting that IL-10 may also modulate the transition from early reversible sepsis to late irreversible shock. It was shown previously that the lethal-ity of CLP could be reversed by a second surgery to remove the ligated necrotic cecum (1). Therefore, we defined the transi-tion from reversible sepsis to irreversible shock in the CLP model as the time when rescue surgery by removal of the

4444 LATIFI ET AL. INFECT. IMMUN.

FIG. 4. The IL-10 concentrations in serum are elevated and main-tained 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.

necrotic cecum is no longer effective. At the time of CR all mice exhibited similar signs of murine sepsis, such as ruffled fur, periorbital exudates, tremor, and lethargy. IL-10 / mice could be partially rescued by CR at 5 h after lethal CLP (18-gauge needle), with 60% surviving long term compared to 3% after CLP alone (FET, P 0.001; Fig. 5). At 10 h CR was ineffective in IL-10 /

mice, with the 20% survival being not significantly different to that after CLP alone (FET, P 0.15), whereas IL-10 / mice that underwent CR at 10 h still showed 100% survival compared to the 17% survival with no interven-tion (FET, P 0.0001). However, by 20 h CR in IL-10 / mice was no longer efficacious (FET, P 1.0). These findings show that in lethal sepsis induced by CLP the success of rescue surgery is time dependent. Furthermore, the earlier failure of CR in IL-10 / mice suggests that IL-10 regulates the transi-tion from reversible sepsis to irreversible shock.

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 g 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 com-pared to untreated animals (P 0.007).

Treatment with IL-10 improves survival and delays the on-set of mortality and irreversible shock. As IL-10 appears to modulate the therapeutic window for rescue surgery, then treatment with exogenous IL-10 should delay the onset of irreversible shock. We therefore treated both IL-10 / and IL-10 /

mice with 1 g of rhIL-10 after CLP to determine whether the onset of lethality would be delayed and the ther-apeutic window for rescue surgery extended. The time chosen for administration of the rhIL-10 was 5 h after CLP because this was the earliest time point at which we found maximal IL-10 levels in the sera of IL-10 / mice (Fig. 4). In both strains of mice after CLP with an 18-gauge needle, rhIL-10 treatment delayed the onset of lethality compared to mice who received no treatment (Fig. 6 and Table 1). Furthermore, for both IL-10 / and IL-10 / mice treated with rhIL-10 5 h after CLP overall survival was significantly improved compared to that of untreated mice of the same strain (Logrank test, P 0.007). Consistent with previous reports (17, 24), treatment of

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.

TABLE 1. IL-10 Treatment delays the onset of lethality

Mouse strainarhIL-10 b % Survival at 30 h Pc

treatment after CLP

IL-10 / 26 ] 0.002IL-10 / 75 ] 0.94IL-10 / 80 ]

IL-10 / 100 0.07

a Each group of mice on a C57BL/6J background underwent CLP with an 18-gauge needle.

b Five hours later the mice were injected subcutaneously with 1 g of rhIL-10 in 0.25 ml of normal saline (n 20) or vehicle alone (n 30).

c Statistical differences between groups was determined by using Fisher’s exact test.

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FIG. 7. rhIL-10 treatment extends the therapeutic window for res-cue 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 g of rhIL-10 5 h after lethal CLP and CR at 10 h or 20 h (u, 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 [❋]).

mice with rhIL-10 at the time of CLP did not lead to a signif-icant improvement in survival (data not shown).

Since the onset of mortality was delayed in IL-10 / and IL-10 /

mice treated with rhIL-10 5 h after CLP, we hypoth-esized that the efficacy of rescue surgery would be significantly improved (Fig. 7). Treatment of IL-10 / mice with rhIL-10 increased the effectiveness of CR at 10 h from a survival rate of 20 to 80% (FET, P 0.02). Similarly rhIL-10 treatment in IL-10 / mice significantly improved survival after rescue sur-gery at 20 h from 20 to 90% (FET, P 0.006). Thus, IL-10 treatment extends the therapeutic window for rescue surgery and delays the onset of irreversible shock.

DISCUSSION

In this report we demonstrate that in sepsis triggered by CLP the transition from reversible sepsis to irreversible septic shock, defined as the time point at which rescue surgery fails, is regulated by IL-10. The absence of IL-10 leads to a more rapid onset of mortality after CLP and an earlier transition from reversible to irreversible sepsis. In contrast, treatment of mice with rhIL-10 after CLP delays the onset of lethality, improves survival, and extends the therapeutic window for rescue surgery. We therefore propose that IL-10 regulates a “point of no return” for recovery from severe sepsis.

The more-rapid onset of mortality in IL-10 / mice com-pared to

IL-10 / mice after CLP is similar to data obtained by using blocking antibodies to IL-10 (28, 31). However, the even-tual survival in anti-IL-10-treated mice was not that much lower compared to that of control mice and, in the study by Song et al., was not significantly different (28). Similarly, in our study the ultimate lower survival in IL-10 / mice was not significantly different to that of IL-10 / mice after either lethal or sublethal CLP. Our results, together with these pre-vious reports, indicate that endogenous IL-10 alters the kinet-ics of the septic response in the CLP model but probably does not dramatically affect long-term survival.

Increased synthesis of proinflammatory cytokines in IL-

10 / compared to IL-10 / mice after CLP is not unexpected since IL-10 is known to suppress the production of TNF- and IL-6 (16). The 15-fold-greater levels of TNF- and IL-6 after CLP in the IL-10 / mice may contribute to their more rapid onset of mortality. It

is unlikely that the more rapid lethality of IL-10 / mice is due to increased bacteremia, since in an Escherichia coli peritonitis model increased lethality of IL-10 / mice was associated with an accelerated bacterial clear-ance but greater organ dysfunction (27). Therefore, it could be that the exaggerated proinflammatory cytokine response in the IL-10 / mice after CLP leads to a more rapid onset of irre-versible damage to vital organs and hastens the onset of irre-versible shock.

In the CLP model it is well described that survival can be significantly improved by the use of antibiotics (18) and fluid resuscitation (18, 32), both treatments being utilized in our experimental CLP protocol. Removal of the necrotic cecum after CLP has also been shown to improve outcome (1). The results from our therapeutic approach that involves removal of the necrotic cecum at various times after CLP has a striking clinical correlate. In human disease the mainstay of clinical management for bacterial peritonitis is to operate and remove the infectious nidus. With timely intervention this leads to resolution of the peritonitis and recovery of the patient in the majority of cases. However, some patients go on to die from unremitting sepsis and multiorgan failure despite appropriate surgery and supportive care (26). At the time of presentation these critically ill patients appear alike in their level of acuity as those who recover but are presumably further advanced in the disease process. Similarly, as presented here, the efficacy of CR after CLP decreases the longer it is delayed, and the transition into irreversible shock in both IL-

10 / and IL-10 / mice does not correlate with any obvious clinical signs nor any changes in the serum cytokine profile. However, our finding that the therapeutic window for rescue surgery is extended

by 10 h in IL-10 / compared to IL-10 / mice suggests that IL-10 may have a significant role in regulating the onset of the “point of no return” and irreversible shock.

If IL-10 does control the onset of irreversible shock in severe

sepsis, then the treatment of IL-10 / and IL-10 / mice with IL-10 should delay the onset of mortality and lengthen the therapeutic window for rescue surgery. Our data show that for both strains of mice treated with rhIL-10 after CLP the onset of mortality was delayed, and the efficacy of CR was now extended beyond the time at which it was previously ineffective in untreated mice. Thus, the combination therapy of rhIL-10 and late CR after lethal CLP can lead to increased recovery of septic mice.

The modest improvement in survival in IL-10 / mice after rhIL-10 treatment alone 5 h after CLP is consistent with a previous report in which a 50% improvement was seen when IL-10 was given as a single dose 6 h after CLP (17). Similarly, our findings that treatment with rhIL-10 at the time of CLP does not increase survival (data not shown) are in agreement with reports showing that initiation of IL-10 treatment at the time of CLP does not improve outcome (17, 24). It may be that IL-10 is not critical at the initiation of the septic response, as evidenced by the ability of early surgical intervention with CR at 5 h to partially reverse CLP-induced lethality in IL-10 / mice. However, soon after the septic response is initiated, the

4446 LATIFI ET AL.

absence of IL-10 supports a more rapid onset of mortality, yet the ultimate impact of the absence of IL-10 on long-term survival is not that great. In contrast, exogenous IL-10 admin-istered during the reversible phase of sepsis improves outcome. We also hypothesize that administering IL-10 too late during the irreversible phase of shock has a limited therapeutic ben-efit, since blocking endogenous IL-10 late after CLP improves survival (28). Therefore, therapy with IL-10 once irreversible shock has begun may be harmful.

Not surprisingly, the role of IL-10 in sepsis is complex, with potentially opposite effects depending on the timing of inter-vention and whether endogenous versus exogenous IL-10 is manipulated. The findings presented here, however, help to clarify the confusion by demonstrating that IL-10 functions to regulate the onset of irreversible septic shock and the thera-peutic window for rescue surgery.

ACKNOWLEDGMENTS

We thank Scott Fulton for providing the tribromoethanol anesthetic, David Spencer, Doug Kou, Robin Jump, and Kelly Miller for their technical assistance. Critical discussions and review of the manuscript by Giovanni Latella, Fred Heinzel, Leslie T. Webster, Jr., and Jeffrey L. Blumer are much appreciated.

This study was supported by grants HD-31323 (Jeffrey L. Blumer, Department of Pediatrics, Case Western Reserve University, Cleve-land, Ohio) and CA-77717 (A.D.L.) from the NIH.

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Editor: A. D. O’Brien