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* C.A.I.R. Institute, Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel;
Departments of Nephrology and Pathology, Rabin Medical Center, Golda Campus, Petah Tikva, Israel; and
Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1
was restored in
AS101-treated mice. The increased survival of AS101-treated mice was
due to the inhibition of IL-10, since cotreatment with murine rIL-10
abolished the protective activity of AS101. AS101 increased class II Ag
expression on peritoneal macrophages, severely depressed in control
mice, while it did not affect the expression of class I Ags. This was
accompanied by a significant elevation in the level of IFN-
secreted
by splenocytes. Moreover, AS101 ameliorated bacterial clearance in the
peritoneum and blood and decreased severe multiple organ damage, as
indicated by clinical chemistry. Furthermore, myeloperoxidase levels in
the liver and lung of AS101-treated mice, an indirect means of
determining the recruitment of neutrophils, were significantly
decreased. We suggest that nontoxic agents such as AS101, with the
capacity to inhibit IL-10 and stimulate macrophage functions, may have
clinical potential in the treatment of sepsis, provided they are
administered during the phase of sepsis characterized by immune
suppression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of cytokines in the pathogenesis of sepsis is complex, since
both deficient and excessive immune responses have been associated with
this syndrome (1). In models of severe systemic infection
or inflammation produced by i.v. administration of high doses of
bacteria or bacterial products such as endotoxin, excessive production
of proinflammatory cytokines significantly contributes to organ failure
and death, as reflected by findings that neutralization of either TNF
or IL-1 activity markedly reduced mortality in these systemic challenge
models (2, 3). However, in experiments in which an
initially localized infection was induced, including pneumonia and
peritonitis, the local activity of proinflammatory cytokines appeared
important for antibacterial host defense at the site of infection
(4, 5, 6). Together these data suggest that proinflammatory
cytokines are, on the one hand, required locally for effective
antibacterial effector mechanisms, but are, in contrast, potentially
toxic when secreted into the circulation. Similarly, several animal
studies indicated an important role for IL-10, the prototypical
antiinflammatory cytokine, in the pathogenesis of bacterial infection.
The administration of neutralizing anti-IL-10 Abs increases
endotoxin-induced mortality in mice, while the exogenous application of
IL-10 can prevent septic shock in mice by inhibiting the synthesis of
TNF-, IL-1, IFN-, and other proinflammatory mediators (7, 8). IL-10 gene-deficient mice demonstrated multiple organ
failure and an enhanced mortality after endotoxin injection or during
Escherichia coli peritonitis (9, 10), despite
accelerated bacterial clearance (10). Nevertheless, during
murine pneumonia, treatment with anti-IL-10 Abs inhibited bacterial
outgrowth in lungs and improved survival (11, 12).
However, during septic peritonitis induced by cecal ligation and
puncture (CLP),3
neutralizing IL-10 was associated with an increased mortality
(13, 14). In these experimental systems in which IL-10 or
anti-IL-10 Abs were administered, the protective or detrimental
effects were induced only if the treatments were applied shortly before
or immediately after LPS, CLP, or bacterial challenge. On the
basis of these observations, a strategy for inhibiting the synthesis of
proinflammatory cytokines was developed in an attempt to treat septic
patients. At present, it appears that this approach has not met with
the expected successes. Sepsis-induced mortality was not significantly
improved in any of the phase II/III studies that have been completed
(15, 16).
Recent studies demonstrated that systemic levels of both the proinflammatory cytokines and IL-10 are correlated directly with severity of illness (17). Several investigations on human infections indicated that elevated IL-10 predicts a poor prognosis in the presence of sepsis (18, 19). Recent data of Song et al. (20) suggest that at later time points after the onset of infection, IL-10 may have a net detrimental effect on host antimicrobial clearance mechanisms. In a cecal ligation model, anti-IL-10 Ab delivered at the time of CLP was ineffective in preventing mortality. However, when administered 12 h after CLP, this treatment was protective (20). These data indicate the critical importance of timing of manipulations that affect IL-10 activity in sepsis.
A biphasic immunologic response in sepsis has been recently demonstrated: an initial hyperinflammatory phase, followed by a hypoinflammatory one. The latter stage is associated with an immunodeficient stage, which is characterized by monocyte deactivation (21). In this state, monocytic TNF secretion capacity as well as HLA-DR Ag expression and Ag-presenting capacity are all severely depressed, while, at least temporarily, the capacity to produce IL-10 is preserved. This switch to predominant release of antiinflammatory mediators is associated with a high risk of fatal outcome.
The nontoxic immunomodulator ammonium
trichloro(dioxoethylene-o,o')tellurate (AS101) first developed by us
has been shown to have beneficial effects in diverse preclinical and
clinical studies. Most of its activities have been primarily attributed
to the direct inhibition of the antiinflammatory cytokine IL-10,
followed by the simultaneous increase of specific cytokines. These
include IL-1, TNF-, IFN-, IL-2, IL-12, and GM-CSF
(22, 23, 24). These immunomodulatory properties were found to
be crucial for the clinical activities of AS101, demonstrating the
protective effects of AS101 in parasite- and viral-infected mice models
(25), in autoimmune diseases (26), and in a
variety of tumor models in which AS101 had a clear antitumoral effect
(27, 28, 29). AS101 has also been shown to have protective
properties against lethal and sublethal effects of irradiation and
chemotherapy (24, 27, 28, 29, 30, 31). These activities were also due
to the increased production of proinflammatory cytokines and were
associated with only minimal toxicity, thus enabling the use of the
compound as an adjuvant to chemotherapy in phase II studies
(32).
Given the ability of AS101 to decrease monocytic IL-10 production (23) and thereby stimulate the production of monocyte-activating cytokines, increase monocyte B-7 expression, followed by amelioration of Ag-presenting capacity (33), we examined its potential role in protecting mice from the lethal effects of sepsis. For this purpose, we used the model of CLP, which resembles the clinical syndrome of septic peritonitis, with spillage of polymicrobial flora into the peritoneal cavity, leading to systemic bacteremia and endotoxemia (34).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male and female BALB/c mice were bred at Bar Ilan University from strains obtained from The Jackson Laboratory (Bar Harbor, ME). Two-month-old male mice were used for experiments.
Animal experiments were performed in accordance with approved institutional protocols and approved by the Institutional Animal Care and Use Committee.
AS101
AS101 was supplied by M. Albeck from the chemistry department in Bar Ilan University, in a solution of PBS, pH 7.4, and maintained at 4°C. Before use, AS101 was diluted in PBS, and the appropriate concentrations in 0.2 ml vol were administered to mice by i.p. injections.
Cecal ligation and puncture
Animals were anesthetized using sodium pentobarbital (40 mg/kg)
administered i.p. CLP was performed by making a midline incision 
2.5
cm in length to expose the cecum. A 3-0 silk ligature was placed at the
base of the cecum without causing bowel obstruction. The cecum was then
punctured twice with a 21-gauge needle. The cecum was gently squeezed
to assure patency of the two holes as well as to express fecal material
into the peritoneal cavity. In the control animals (sham), the cecum
was located, but neither ligated nor punctured. The abdominal incision
was then closed in two layers using 3-0 Prolene and 4-0 Dexon,
respectively. All animals were fluid resuscitated with 1 ml normal
saline by s.c. injection.
IL-10 and anti-IL-10 Abs
Murine rIL-10 (rmIL-10; BD PharMingen, San Diego, CA) was injected s.c. at 5 µg/injection once daily starting 12 h following CLP. Anti-IL-10 (JESS 2A5; BD PharMingen)-neutralizing Abs were injected i.p. at 250 µg/mouse at 12 h post-CLP. Rat anti-mouse IgG1 served as control Abs.
Treatment regimen
For survival experiments, PBS or AS101 were injected i.p. daily at various concentrations starting either 24 h before or at 7 or 12 h following CLP until the end of experiment. For serum cytokine evaluation, AS101 at 10 µg/mouse or PBS was injected once 12 h following CLP.
Cell purification
Spleens were removed and placed in PBS. Spleen cells were passed through stainless steel mesh nets, treated with hypotonic solution to lyse erythrocytes, and washed three times. Cell counts were obtained using a hemocytometer. Viability, as assessed by trypan blue exclusion method, was always found to be >95%.
Adherent splenocytes were obtained following adherence of cells for 1 h in plastic dishes in RPMI 1640 culture medium supplemented with 10% FCS.
Peritoneal macrophages were obtained by peritoneal washing with PBS and adherence of cells for 1 h.
Induction of cytokine secretion in vitro
Splenocytes (5 x 106/ml) were
suspended in enriched RPMI 1640 culture medium supplemented with 10%
FCS and seeded in 24-well culture plates in the presence of 2.5 µg/ml
Con A (for IFN-). Adherent splenocytes or peritoneal macrophages
were incubated with 1 µg/ml LPS (for IL-1, IL-10, and TNF-).
The cultures were incubated at 37°C for 24 h. Supernatants were
collected and evaluated for cytokine content.
Quantitation of cytokine levels
The R&D Systems (Minneapolis, MN) IL-1, TNF-, IL-10, and
IFN- ELISA kits were used for the quantitative measurement of these
cytokines either in mice sera or in spleen and peritoneal exudate cell
(PEC) supernatants.
Immunofluorescence staining
Peritoneal cells were incubated with FITC and PE directly labeled Abs against MHC molecules on ice for 60 min and then washed. The mAbs used included FITC-labeled mouse IgG2a anti-mouse H-2Dd, rat IgG2a anti-mouse I-Ad/I-Ed, rat IgG2a anti-mouse I-Ad, and PE-labeled rat IgG1 anti-mouse CD14 (BD PharMingen); purified rat IgG2 Abs (Genzyme, Cambridge, MA) were used as isotype controls. For each sample, 104 cells were analyzed on a FACScan (BD Biosciences, Mountain View, CA) evaluating the percentage of double-stained CD14 and class I- or class II-positive cells.
Determination of bacterial CFU in the CLP model
Mice were killed 24 h following CLP. The peritoneal cavities were washed with 2 ml sterile PBS, and the lavage fluids were harvested under sterile conditions. A total of 10 µl peritoneal lavage fluids and peripheral blood from each mouse was placed on ice and serially diluted with sterile PBS. A total of 10 µl of each dilution was asceptically plated on tryptose soy agar blood agar plates (Difco, Detroit, MI) and incubated overnight at 37°C, after which the number of colonies was counted. Data were expressed as CFU per microliter.
Clinical chemistry
Serum levels of aspartate transaminase (AST), alanine transaminase (ALT), amylase, blood urea nitrogen (BUN), and creatinine were measured using standardized techniques.
Measurement of myeloperoxidase
Myeloperoxidase (MPO) in tissue extracts was measured by ELISA kit (Calbiochem-Novabiochem, La Jolla, CA), according to the manufacturer’s instructions.
Preparation of organ extracts
A total of 0.1 g excised organs was placed in 1 ml PBS containing complete protease inhibitors (Calbiochem) and homogenized. The homogenates were subsequently freeze-thawed twice. The homogenates were centrifuged at 6000 x g for 20 min at 4°C, and the cleared supernatants were used for measurement of MPO. The levels of MPO in organ extracts were expressed as ng/mg protein.
Statistical analysis
Survival curves were statistically analyzed by comparing the cumulative percentage of survival using the Gehan-Wilcoxon test. For comparisons of means of the various groups, the pairwise t test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first determined whether AS101 may rescue mice from
sepsis-induced death. Peritonitis induced by CLP with a 21-gauge needle
results in 100% lethality at day 9 (Fig. 1
). Treatment with AS101 had a salutary
effect only if injected after sepsis induction (Fig. 1A).
The optimal dose of AS101 was 10 µg/mouse, injected 12 h
following CLP and thereafter every day until the end of experiments
(Fig. 1). Under these conditions, 60% of treated mice survived
(p < 0.01; increase vs CLP + PBS). This
concentration of AS101 was previously found by us to be optimal in
various preclinical studies in which AS101 had beneficial effects. A
concentration of 30 µg/mouse AS101 was less effective, although this
dose also significantly increased the rate of survival
(p < 0.05). A lower concentration of 5
µg/mouse was ineffective (Fig. 1B). Injection of AS101
7 h post-CLP was also effective, although to a lesser extent
(20%; p < 0.05). No significant difference was
observed when AS101 was injected at the time of injury (time zero).
Notably, treatment with AS101 24 h before CLP decreased survival,
compared with PBS-treated mice. Thus, it appears that AS101 could
protect mice from sepsis-induced death only if injected after, but not
before, or at the time of sepsis induction.
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Organ injury observed in sepsis is due to the explosive release of
cytokines into the plasma. We therefore sought to determine the plasma
levels of cytokines folowing CLP and their response to AS101 treatment.
Two of the classic cytokines produced in the initial period of an
inflammatory insult are TNF- and IL-1, which are followed by
overproduction of IL-10. It can be seen that TNF- and IL-1 were
undetectable in the serum at 1 h post-CLP (Fig. 2). Maximal levels of these cytokines
were found at 6 and 12 h. The inflammatory response was not
persistent and it gradually declined, until at 24–48 h after CLP it
was only minimal. Evaluation of plasma levels of proinflammatory
cytokines in the AS101-treated mice revealed no significant differences
in plasma TNF- and IL-1 levels between AS101- and PBS-treated
mice (Fig. 2).
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Quantitation of IL-10 plasma levels revealed a gradual increase in
plasma content of this antiinflammatory cytokine that peaked at 24
h following CLP. These levels remained elevated until 96 h after
CLP (Fig. 3), although they gradually
decreased (data not shown). AS101 injected 12 h after CLP promptly
and significantly (p < 0.01) inhibited IL-10
levels at both 24 and 48 h (Fig. 3). At 72–96 h, IL-10 levels in
the AS101 group were negligible (data not shown).
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The primary effect of AS101 has been shown previously to involve
the direct inhibition of IL-10 in human and mouse macrophages, at the
transcription level (23). Moreover, most of the beneficial
activities of AS101 observed in preclinical studies have been primarily
attributed to the direct inhibition of IL-10 (25, 26, 27). We
therefore examined the role of IL-10 inhibition in the increased
survival of AS101-treated mice subjected to CLP. To this end, we
injected AS101-treated mice with rmIL-10 (5 µg/injection) once daily
starting 12 h after the induction of CLP. Fig. 4 shows that treatment with rmIL-10
significantly reduced the salutary effect of AS101. The percentage of
survival decreased from 60 to 10% (p < 0.01).
Treatment of PBS-treated mice with rmIL-10 starting 12 h following
CLP resulted in enhanced mortality, 100% of mice dying 7 days
following CLP. Alternatively, injection of neutralizing anti-IL-10
Abs 12 h after CLP resulted in 50% survival
(p < 0.01 vs control Ab).
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Recently, it has been suggested that septic patients demonstrate a state in which monocytic TNF secretion capacity as well as HLA-DR Ag expression are severely depressed, while the capacity to produce IL-10 is at least temporarily preserved (35). This predominantly antiinflammatory state, in contrast to the assumption of persistent hyperinflammation, was associated with a high risk of death from persistent infection and multiple organ failure.
Table I shows that peritoneal macrophages
obtained from CLP-induced mice gradually lose the ability to secrete
TNF- and IL-1. This loss of function is significantly reflected
at 24–48 h following CLP. At 48 h, peritoneal macrophages
completely lose their ability to secrete those proinflammatory
cytokines (Table I). This capacity is significantly restored
(p < 0.01) in AS101-treated mice at all time
points following AS101 injections. Similar results were obtained when
TNF- and IL-1 in supernatants from adherent splenocytes were
quantitated (data not shown). In contrast to the loss of macrophage
ability to secrete TNF- and IL-1, the capacity of PEC cells to
secrete IL-10 at 24–48 h was preserved. Nevertheless, PEC cells from
AS101-treated mice secreted significantly decreased amounts of IL-10 at
24–48 h post-CLP (p < 0.01) (Table I).
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A, the proportion of
CD14+ peritoneal cells expressing I-A Ag
gradually decreased following CLP from 26.2 ± 2 to 4 ± 2%
at 48 h post-CLP. Administration of AS101 12 h after CLP
significantly restored the percentage of
I-A+-expressing CD14+
cells. This was reflected by an increase amounting to 17 ± 1%
CD14+I-A+ cells at 24
h post-CLP and 23 ± 2%
CD14+I-A+ cells at 48
h after the insult. Similar results were obtained when the proportion
of CD14+ cells expressing both I-A and I-E was
evaluated. While the percentage of these cells decreased from 61
± 5% to 6 ± 1% at 48 h post-CLP, they amounted at that
time point to 55 ± 6% in mice treated with AS101 (Fig. 5B).
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c). Treatment with AS101 did not
alter H-2D Ag expression on CD14+ cells.
Effect of AS101 on IFN- production by spleen cells
The increased expression of class II Ags on
CD14+ cells from AS101-treated mice led us to
examine the level of IFN- production and secretion in these mice due
to the potent ability of this cytokine to induce class II Ag expression
on CD14+ cells. As can be seen in Fig. 6, Con A-stimulated splenocytes
significantly decrease their ability to secrete IFN- at 24–48 h
following CLP. A considerable and significant increase
(p < 0.01) in the levels of IFN- were
obtained at all time points following AS101 injections when splenocytes
from AS101-treated mice were used (Fig. 6).
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IL-10 has been shown recently to impair bacterial clearance from
the peritoneal cavity and to facilitate dissemination of bacteria to
distant organs (10). The significant decrease in serum
IL-10 levels in CLP-induced mice treated with AS101, as well as the
increased survival of these mice led us to evaluate the role of AS101
in the bacterial clearance of mice subjected to CLP. To this end, we
examined the bacterial load in the peritoneal fluids and blood of
AS101- and PBS-treated mice. No bacteria were recovered from the
peritoneum or blood of mice that were not subjected to CLP (data not
shown). The peritoneal fluids at 24 h after CLP in PBS-injected
mice contained a significant number of bacteria in the peritoneum (Fig. 7). At this time point, the bacterial
load in the peritoneum of AS101-treated mice was significantly
decreased (p < 0.01). At 72 h,
AS101-treated mice who shivered and had bristled hair, clinical
symptoms of active sepsis, had increased peritoneal bacterial load
(13,000–40,000 CFU/10 µl). These amounted to 40%. In the remaining
mice, the peritoneal bacterial load did not change significantly as
compared with that at 24 h after CLP (data not shown). Likewise,
the bacterial load recovered from peripheral blood of AS101-treated
mice was significantly lower than that in control PBS-treated mice
(p < 0.05) (Fig. 7).
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Sepsis frequently causes multiple organ failure, a condition that
leads to death (36). To understand the mechanism whereby
AS101-treated mice were resistant to CLP, experiments were conducted to
assess the organ damage induced by CLP and its response to AS101. For
this purpose, we measured biochemical markers of liver damage (AST,
ALT), pancreas damage (amylase), and renal failure (creatinine, BUN) at
24 h following CLP. Fig. 8 shows
that control PBS-treated mice demonstrated biochemical evidence of more
severe multiple organ damage than AS101-treated mice, as reflected by
higher ALT, AST, amylase, creatinine, and BUN concentrations (all
p < 0.05). The levels in AS101-treated mice were
comparable with those in mice not subjected to CLP, suggesting that
organ injury was avoided in AS101-treated mice. Nevertheless, in 40%
of mice in the AS101 group, in which the bacterial load was elevated at
72 h, the levels of AST and ALT increased to 100–120 U/L and
65–80 U/L, respectively, at that time (data not shown), suggesting
that in these mice organ damage was only delayed and not entirely
avoided.
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MPO level is an indirect means of determining the recruitment of
neutrophils to the infected organs. As can be seen in Fig. 9, MPO levels in the liver and lung,
24 h after CLP, were elevated in PBS-treated mice as compared with
AS101-treated mice. This increase was significant in both organs
(p < 0.01; p < 0.05). The
level of MPO in both organs in control PBS-treated mice was >10-fold
increased as compared with normal untreated mice. MPO levels in the
kidney of PBS-treated mice were only slightly elevated as compared with
untreated mice (2-fold). AS101 also decreased the level of MPO in the
kidney, but not significantly (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1, 6 h
after CLP. Nevertheless, the proinflammatory response was not
consistent and it gradually decreased over 24–48 h. This was followed
by an immune suppressed state characterized by overproduction of IL-10.
Treatment with AS101 12 h following CLP did not result in
important changes in serum TNF- or IL-1 levels. However, the
antiinflammatory response was profoundly decreased in AS101-treated
mice, as reflected by profound suppression of serum IL-10 levels. The
switch to an antiinflammatory state in late sepsis was also reflected
by the relatively preserved ability of peritoneal macrophages from
untreated CLP-induced mice to secrete IL-10, while their ability to
secrete TNF- and IL-1 was totally lost. Treatment of mice with
AS101 decreased IL-10 secretion by macrophages and restored their
ability to produce and secrete proinflammatory cytokines. AS101 has previously been shown to directly inhibit IL-10 production by both mice and human macrophages, at the transcriptional level (23). This property accounted for most of the beneficial effects of AS101 in preclinical studies associated with IL-10 overexpression.
In some of those models, such as systemic lupus erythematosus, the beneficial effect of AS101 was directly due to the inhibition of the high levels of IL-10, resulting in decreased production of autoantibodies and delayed development of lupus-like autoimmunity (26). In other models such as tumor-bearing (27) or parasite- and viral-infected mice (25), the salutary effect of AS101 was due to its ability to switch predominant type II responses to type I responses, thus augmenting the cell-mediated immune response to cancer, viral, and parasitic infections. Moreover, inhibition by AS101 of IL-10 production by macrophages results in up-regulation of the costimulatory molecule B7-1, and increased Ag-presenting ability of these cells (33). Most importantly, AS101 decreases IL-10 production only in models associated with overproduction of this cytokine, resulting in nearly normal IL-10 levels, followed by increased, yet normal, levels of proinflammatory and Th1 cytokines. This probably accounts for AS101’s lack of toxicity, enabling the use of the compound as an adjuvant to chemotherapy in phase II studies (32).
The cytokine network plays a pivotal role in the orchestration of
inflammatory responses to bacterial infection. The balance between pro-
and anti-inflammatory cytokines critically influences the function of
immunocompetent cells and the resistance against infection. While
proinflammatory cytokines such as IL-12 and TNF- have been shown to
enhance bacterial clearance in several bacterial infection models
including septic peritonitis (37, 38), antiinflammatory
cytokines such as IL-10 and IL-13 have been shown to play a protective
role in this model of sepsis, by down-regulating the excessive
inflammatory response (13, 39), while IL-10 was not
successful as a therapeutic strategy in a cecal ligation model of
intraabdominal sepsis (40). The development of an adequate
immune response to a bacterial challenge relies on the complex
interplay between the inate and the specific immune system, reflected
by intact monocyte/T cell/neutrophil interactions. Monocyte and
macrophage functions include the recognition, uptake, and killing of
invading organisms, and Ag presentation to initiate an immune response.
The early stages of such a response may be mediated by secretion of
proinflammatory mediators such as TNF, IL-1, IL-12, and a direct
Ag-presenting capacity reflected by the expression of HLA class II and
CD80/86. These monocyte functions are up-regulated by immunostimulatory
cytokines such as IFN- and suppressed by IL-10. This functional
monocytic depression is closely associated with a loss of type I T cell
response, which can be characterized by an increase in Th cell
production of IL-4 and IL-10 and a decreased IFN--producing Th cell
phenotype. Indeed, Song et al. (20) have recently shown
that IL-10 is involved in the immune dysfunction in splenic lymphocytes
from septic animals, as reflected by their decreased ability to release
IL-2 and IFN-. Furthermore, the role of IL-10 in depressing monocyte
functions important in sepsis was recently demonstrated
(41). These data are in line with our results showing that
treatment with AS101, through inhibition of IL-10, restores moncytic
functions, as reflected by preservation of their ability to secrete
TNF- and IL-1 and by the prompt increase of their class II Ag
expression. Moreover, treatment with AS101 restores the ability of
lymphocytes to secrete IFN-, a known up-regulator of monocyte
functions. Taken together, it appears that sepsis is characterized by a
biphasic immunologic response: an initial systemic proinflammatory
mediator response that is the motor for developing the
anti-inflammatory response causing immune cell dysfunction. Therefore,
a number of investigators have suggested that inhibition of this
proinflammatory response may provide a survival benefit. However, this
often does not take into consideration that the degree of
proinflammation produced in response to a polymicrobial insult, such as
CLP, is typically modest compared with that seen in lethal endotoxemia.
A number of studies have documented that the levels of proinflammatory
mediators such as TNF and IL-1 produced in CLP may actually be required
to initiate a protective immune response and that anti-TNF Ab
therapy fails to prevent lethality after CLP (42, 43).
Our results, demonstrating the beneficial effects of AS101 in CLP-induced sepsis, illustrate not only the significance of the immunosuppressive mediator IL-10 in the development of sepsis, but also the importance of correct timing of the potential intervention. Most studies demonstrating the beneficial effect of IL-10 in sepsis either injected IL-10 before or immediately after the induction of sepsis, or used IL-10 knockout mice to demonstrate the role of endogenous IL-10 in sepsis. In the first case, IL-10 was injected in mice in which an excessive hyperinflammatory state dominated. Therefore, this treatment balanced hyperinflammation. However, in IL-10 KO mice, some degree of hyperinflammation exists before the induction of sepsis. In this case, after the insult, hyperinflammation is more excessive than in normal mice subjected to sepsis. Alternatively, we show in the present study that the response of sepsis-induced mice to treatment with an agent that decreases IL-10 levels depends on the time of therapeutic intervention. When AS101 was injected before CLP, a significant decrease in the percentage of survival was obtained. Only when AS101 was injected at the time in which the anti-inflammatory response predominated was the resulting decrease in IL-10 beneficial to the septic mice. Treatment with AS101 at that stage of sepsis eventually restored the balance between the pro- and antiinflammatory arms of the cytokine network, ameliorating the bacterial clearance on the one hand and diminishing organ damage in contrast. AS101 protects 60% of mice from sepsis-induced death. In the remaining 40%, the level of bacterial load and that of organ damage is only delayed and is increased later on until the mice die. These data are in line with studies (20) that showed that treatment with anti-IL-10 Abs during the immune suppressive state (12 h after CLP) markedly increased survival. Our data are also consistent with studies (44) showing that inhibition of mitogen-activated protein kinase p38 in vivo results in suppression of IL-10 and in increased survival, provided the inhibitor is injected 12 h post-CLP.
Our results may explain why clinical trials with septic patients aimed
at down-regulating proinflammatory cytokines using Abs against TNF-,
antagonists of IL-1, or platelet-derived growth factor have proved to
be uniformly disappointing. Not only have these agents been found to be
ineffective, but they may also increase mortality. It appears that
these trials all addressed the proinflammatory phase of sepsis when
there was no evidence that the proinflammatory phase was dominant.
Taken together, clinical and experimental evidence suggests that septic patients are not necessarily in an overactivated immunological state. Therefore, any immunomodulatory therapy should be based on measurable immune functions to determine which patients may benefit from such therapies. We suggest that the use of the nontoxic compound AS101, currently undergoing phase II clinical trials, has potential in the clinical treatment of sepsis if given during the immune suppressive state of septic patients.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Benjamin Sredni, C.A.I.R. Institute, Faculty of Life Sciences, Bar Ilan University, Ramat Gan, 52900 Israel.
3 Abbreviations used in this paper: CLP, cecal ligation and puncture; ALT, alanine transaminase; AS101, ammonium trichloro(dioxoethylene-o,o')tellurate; AST, aspartate transaminase; BUN, blood urea nitrogen; MPO, myeloperoxidase; PEC, peritoneal exudate cell; rmIL-10, murine rIL-10.
Received for publication December 5, 2001. Accepted for publication April 11, 2002.
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 I. Sinuani, Z. Averbukh, I. Gitelman, M. J. Rapoport, J. Sandbank, M. Albeck, B. Sredni, and J. Weissgarten Mesangial cells initiate compensatory renal tubular hypertrophy via IL-10-induced TGF-beta secretion: effect of the immunomodulator AS101 on this process Am J Physiol Renal Physiol, August 1, 2006; 291(2): F384 - F394. [Abstract] [Full Text] [PDF]
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Z. H. Nemeth, B. Csoka, J. Wilmanski, D. Xu, Q. Lu, C. Ledent, E. A. Deitch, P. Pacher, Z. Spolarics, and G. Hasko Adenosine A2A Receptor Inactivation Increases Survival in Polymicrobial Sepsis J. Immunol., May 1, 2006; 176(9): 5616 - 5626. [Abstract] [Full Text] [PDF]
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 A. Naldini, C. Bernini, A. Pucci, and F. Carraro Thrombin-mediated IL-10 up-regulation involves protease-activated receptor (PAR)-1 expression in human mononuclear leukocytes J. Leukoc. Biol., September 1, 2005; 78(3): 736 - 744. [Abstract] [Full Text] [PDF]
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A. von Knethen, A. Tautenhahn, H. Link, D. Lindemann, and B. Brune Activation-Induced Depletion of Protein Kinase C{alpha} Provokes Desensitization of Monocytes/Macrophages in Sepsis J. Immunol., April 15, 2005; 174(8): 4960 - 4965. [Abstract] [Full Text] [PDF]
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 Y. Kalechman, U. Gafter, T. Weinstein, A. Chagnac, I. Freidkin, A. Tobar, M. Albeck, and B. Sredni Inhibition of Interleukin-10 by the Immunomodulator AS101 Reduces Mesangial Cell Proliferation in Experimental Mesangioproliferative Glomerulonephritis: ASSOCIATION WITH DEPHOSPHORYLATION OF STAT3 J. Biol. Chem., June 4, 2004; 279(23): 24724 - 24732. [Abstract] [Full Text] [PDF]
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 B. Sredni, M. Weil, G. Khomenok, I. Lebenthal, S. Teitz, Y. Mardor, Z. Ram, A. Orenstein, A. Kershenovich, S. Michowiz, et al. Ammonium Trichloro(dioxoethylene-o,o')tellurate (AS101) Sensitizes Tumors to Chemotherapy by Inhibiting the Tumor Interleukin 10 Autocrine Loop Cancer Res., March 1, 2004; 64(5): 1843 - 1852. [Abstract] [Full Text] [PDF]
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 B. M. Schaaf, F. Boehmke, H. Esnaashari, U. Seitzer, H. Kothe, M. Maass, P. Zabel, and K. Dalhoff Pneumococcal Septic Shock Is Associated with the Interleukin-10-1082 Gene Promoter Polymorphism Am. J. Respir. Crit. Care Med., August 15, 2003; 168(4): 476 - 480. [Abstract] [Full Text] [PDF]
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 Y. Kalechman, B. Sredni, T. Weinstein, I. Freidkin, A. Tobar, M. Albeck, and U. Gafter Production of the Novel Mesangial Autocrine Growth Factors GDNF and IL-10 Is Regulated by the Immunomodulator AS101 J. Am. Soc. Nephrol., March 1, 2003; 14(3): 620 - 630. [Abstract] [Full Text] [PDF]
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