INFECTION AND IMMUNITY, Aug. 2002, p.

44414446
0019-9567/02/$04.000 DOI: 10.1128/IAI.70.8.44414446.2002
Copyright 2002, American Society for Microbiology. All Rights Reserved.

Vol. 70, No. 8

Interleukin-10 Controls the Onset of Irreversible Septic Shock

Samir Q. Latifi,1 Mary Ann ORiordan,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.
bacteremia. The lack of ongoing injury after a single administration 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 endotoxin and that the exact role of proinflammatory cytokines as
mediators of mortality is unclear.
Besides stimulating the synthesis of proinflammatory cytokines, 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 neutralizing antibodies to IL-10 at the time of CLP only partially exacerbates 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 regulates 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

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 evolving multiorgan dysfunction (7). Occasionally, despite appropriate 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 proteins (21). However, the contribution of these proinflammatory
cytokines in directly mediating mortality from sepsis is not
clear, since therapies utilizing neutralizing antibodies or soluble 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 cytokinetargeted therapies may be that the antibodies or antagonists
were administered too late or that other unopposed inflammatory 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 endotoxemia leads to dramatic improvement in animal mortality.
When lipopolysaccharide (LPS), the endotoxin from gramnegative 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.
4441

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INFECT. IMMUN.

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 chloroformisoamyl 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 (Stratagene). 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 amplification conditions were 94C for 5 min, 94C for 1 min, 55C for 1 min, and
72C for 1 min for 40 cycles, followed by 72C 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 abdomen 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 punctured 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 subcutaneous 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 injection 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 70C 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 recommended by the manufacturer (R&D Systems, Minneapolis, Minn.). The sensitivity of the IL-6 and the IL-10 ELISA kits was 15 pg/ml, and for the TNF- kit
it was 23 pg/ml.

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, Kenilworth, 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 described 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. Proportions alive at specific time points were compared by using Fishers 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 response, we compared the time of onset of mortality and the
subsequent kinetics of disease progression between IL-10-deficient 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 (Fishers 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 modest 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 between IL-10/ and IL-10/ mice (FET, P 0.52).

between IL-10/ and IL-10/ mice was not statistically significant (FET, P 0.20). These results indicate that the absence 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 demonstrate that endogenous IL-10 has little influence on longterm 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 inflammation. We therefore predicted that the absence of IL-10 would
alter the kinetics of production of the proinflammatory cytokines TNF- and IL-6 after CLP. IL-10/ mice produced
significantly greater levels of TNF- and IL-6 in serum compared 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 underwent 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 measured 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 compared to IL-10/ mice are not unexpected. Therefore, we
investigated the kinetics of serum IL-10 production in IL10/ 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 proinflammatory 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 lethality of CLP could be reversed by a second surgery to remove the
ligated necrotic cecum (1). Therefore, we defined the transition from reversible sepsis to irreversible shock in the CLP
model as the time when rescue surgery by removal of the

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LATIFI ET AL.

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.

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 intervention (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 transition from reversible sepsis to irreversible shock.

INFECT. IMMUN.

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 18gauge 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 (IL10/, 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).

Treatment with IL-10 improves survival and delays the onset 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 therapeutic 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

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

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.

Mouse straina

rhIL-10
treatmentb

% Survival at 30 h
after CLP

IL-10/
IL-10/
IL-10/
IL-10/

26 ]
75 ]
80 ]
100

Pc

0.002
0.94
0.07

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 Fishers exact
test.

VOL. 70, 2002

IL-10 CONTROLS THE ONSET OF IRREVERSIBLE SEPSIS

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 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 significant 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 hypothesized 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 surgery 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 compared to IL-10/ mice after CLP is similar to data obtained by
using blocking antibodies to IL-10 (28, 31). However, the eventual 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 previous reports, indicate that endogenous IL-10 alters the kinetics of the septic response in the CLP model but probably does
not dramatically affect long-term survival.
Increased synthesis of proinflammatory cytokines in IL-

4445

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 IL10/ mice was associated with an accelerated bacterial clearance 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 irreversible damage to vital organs and hastens the onset of irreversible 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.

INFECT. IMMUN.

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 administered 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 benefit, 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 intervention 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 therapeutic window for rescue surgery.

13.

14.

15.
16.

17.
18.

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, Cleveland, Ohio) and CA-77717 (A.D.L.) from the NIH.

19.
20.

21.
22.

REFERENCES
1. Baker, C. C., I. H. Chaudry, H. O. Gaines, and A. E. Baue. 1983. Evaluations
of factors affecting mortality rate after sepsis in a murine cecal ligation and
puncture model. Surgery 94:331335.
2. Balk, R. A. 2000. Severe sepsis and septic shock: definitions, epidemiology,
and clinical manifestations. Crit. Care Clin. 16:179192.
3. Berg, D. J., K. Kuhn, K. Rajewsky, W. Muller, S. Menon, N. Davidson, G.
Grunig, and D. Rennick. 1995. Interleukin-10 is a central regulator of the
response to LPS in murine models of endotoxic shock and the Shwartzman
reaction but not endotoxin tolerance. J. Clin. Investig. 96:23392347.
4. Beutler, B., I. W. Milsark, and A. C. Cerami. 1985. Passive immunization
against cachectin/tumor necrosis factor protects mice from lethal effect of
endotoxin. Science 229:869871.
5. Cain, B. S., D. R. Meldrum, A. H. Harken, and R. C. McIntyre. 1998. The
physiologic basis for anticytokine clinical trials in the treatment of sepsis.
J. Am. Coll. Surg. 186:337351.
6. Damas, P., J.-L. Canivet, D. de Groote, Y. Vrindts, A. Albert, P. Franchimont, and M. Lamy. 1997. Sepsis and serum cytokine concentrations. Crit.
Care Med. 25:405412.
7. Despond, O., F. Proulx, J. A. Carcillo, and J. Lacroix. 2001. Pediatric sepsis
and multiple organ dysfunction syndrome. Curr. Opin. Pediatr. 13:247253.
8. Doughty, L., J. A. Carcillo, S. Kaplan, and J. Janosky. 1998. The compensatory anti-inflammatory cytokine interleukin 10 response in pediatric sepsisinduced multiple organ failure. Chest 113:16251631.
9. Doughty, L. A., S. S. Kaplan, and J. A. Carcillo. 1996. Inflammatory cytokine
and nitric oxide responses in pediatric sepsis and organ failure. Crit. Care
Med. 24:11371143.
10. Eskandari, M. K., G. Bolgos, C. Miller, D. Nguyen, L. E. DeForge, and D. G.
Remick. 1992. Anti-tumor necrosis factor antibody therapy fails to prevent
lethality after cecal ligation and puncture or endotoxemia. J. Immunol.
148:27242730.
11. Friedman, G., S. Jankowski, A. Marchant, M. Goldman, R. J. Kahn, and
J. L. Vincent. 1997. Blood interleukin 10 levels parallel the severity of septic
shock. J. Crit. Care 12:183187.
12. Gazzinelli, R. T., M. Wysocka, S. Hieny, T. Scharton-Kersten, A. Cheever, R.

Editor: A. D. OBrien

23.
24.
25.
26.

27.

28.
29.
30.

31.

32.

Kuhn, W. Muller, G. Trinchieri, and A. Sher. 1996. In the absence of

endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to
a lethal immune response dependent on CD4 T cells and accompanied by
overproduction of IL-12, IFN- and TNF-. J. Immunol. 157:798805.
Group, T. A. S. S. 2001. Randomized, placebo-controlled trial of the antitumor necrosis factor antibody fragment afelimomab in hyperinflammatory
response during severe sepsis: the RAMSES study. Crit. Care Med. 29:765
769.
Hollenberg, S. M., A. Dumasius, C. Easington, S. A. Colilla, A. Neumann,
and J. E. Parrillo. 2001. Characterization of a hyperdynamic murine model
of resuscitated sepsis using echocardiography. Am. J. Respir. Crit. Care
Med. 164:891895.
Howard, M., T. Muchamuel, S. Andrade, and S. Menon. 1993. Interleukin 10
protects mice from lethal endotoxemia. J. Exp. Med. 177:12051208.
Huhn, R. D., E. Radwanski, S. M. OConnell, M. G. Sturgill, L. Clarke, R. P.
Cody, M. B. Affrime, and D. L. Cutler. 1996. Pharmacokinetics and immunomodulatory properties of intravenously administered recombinant human
interleukin-10 in healthy volunteers. Blood 87:699705.
Kato, T., A. Murata, H. Ishida, H. Toda, N. Tanaka, H. Hayashida, M.
Monden, and N. Matsuura. 1995. Interleukin 10 reduces mortality from
severe peritonitis in mice. Antimicrob. Agents Chemother. 39:13361340.
Lundblad, R., and K.-E. Giercksky. 1995. Effect of volume support, antibiotic therapy, and monoclonal antiendotoxin antibodies on mortality rate and
blood concentrations of endothelin and other mediators in fulminant intraabdominal sepsis in rats. Crit. Care Med. 23:13821390.
McMasters, K. M., J. C. Peyton, D. J. Hadjiminas, and W. G. Cheadle. 1994.
Endotoxin and tumor necrosis factor do not cause mortality from caecal
ligation and puncture. Cytokine 6:530536.
Meduri, G. U., S. Headley, G. Kohler, F. Stentz, E. Tolley, R. Umberger, and
K. Leeper. 1995. Persistent elevation of inflammatory cytokines predicts a
poor outcome in ARDS, plasma IL-1 and IL-6 levels are consistent and
efficient predictors of outcome over time. Chest 107:10621073.
Murphy, K., S. B. Haudek, M. Thompson, and B. P. Giroir. 1998. Molecular
biology of septic shock. New Horiz. 6:181193.
Ohlsson, K., P. Bjork, M. Bergenfeldt, R. Hageman, and R. C. Thompson.
1990. Interleukin-1 receptor antagonist reduces mortality from endotoxin
shock. Nature 348:550552.
Papaioannou, V. E., and J. G. Fox. 1993. Efficacy of tribromoethanol anesthesia in mice. Lab. Anim. Sci. 43:189192.
Remick, D. G., S. J. Garg, D. E. Newcomb, G. Wollenberg, T. K. Huie, and
G. L. Bolgos. 1998. Exogenous interleukin-10 fails to decrease the mortality
or morbidity of sepsis. Crit. Care Med. 26:895904.
Remick, D. G., D. E. Newcomb, G. L. Bolgos, and D. R. Call. 2000. Comparison of the mortality and inflammatory response of two models of sepsis:
lipopolysaccharide versus cecal ligation and puncture. Shock 13:110116.
Riche, F. C., B. P. Cholley, Y. H. Panis, M.-J. C. Laisne, C. G. Briard, A.-M.
Graulet, J. L. Gueris, and P. D. Valleur. 2000. Inflammatory cytokine response in patients with septic shock secondary to generalized peritonitis.
Crit. Care Med. 28:433437.
Sewnath, M. E., D. P. Olszyna, R. Birjmohun, F. J. ten Kate, D. J. Gouma,
and T. van der Poll. 2001. IL-10 deficient mice demonstrate multiple organ
failure and increased mortality during Escherichia coli peritonitis despite an
accelerated bacterial clearance. J. Immunol. 166:63236331.
Song, G. Y., C.-S. Chung, I. H. Chaudry, and A. Ayala. 1999. What is the role
of interleukin 10 in polymicrobial sepsis: anti-inflammatory agent or immunosuppressant? Surgery 126:378383.
Sullivan, J. S., L. Kilpatrick, J. Costarino, A. T., S. C. Lee, and M. C. Harris.
1992. Correlation of plasma cytokine elevations with mortality rate in children with sepsis. J. Pediatr. 120:510515.
van der Poll, T., R. Malefyt, S. M. Coyle, and S. F. Lowry. 1997. Antiinflammatory cytokine responses during clinical sepsis and experimental endotoxemia: sequential measurements of plasma soluble interleukin (IL)-1 receptor
type II, IL-10, and IL-13. J. Infect. Dis. 175:118122.
van der Poll, T., A. Marchant, W. A. Buurman, L. Berman, C. V. Keogh,
D. D. Lazarus, L. Nguyen, M. Goldman, L. L. Moldawer, and S. F. Lowry.
1995. Endogenous IL-10 protects mice from death during septic peritonitis.
J. Immunol. 155:53975401.
Wilson, M. A., M. C. Chou, D. A. Spain, P. J. Downard, Q. Qian, W. G.
Cheadle, and N. Garrison. 1996. Fluid resuscitation attenuates early cytokine mRNA expression after peritonitis. J. Trauma 41:622627.