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*
The Wistar Institute, Philadelphia, PA 19104;
University of Pennsylvania, Veterinary School, Philadelphia, PA 19104; and
Johns Hopkins University School of Medicine, Baltimore, MD 21205
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-
, or nitric oxide, or the NF-
B
family members p50, p52, or RelB. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endotoxin tolerance is demonstrable both in vivo and in vitro, at the
level of whole organisms as well as in isolated cells. This apparent
tachyphylaxis to LPS has been shown to correlate with suppression of
proinflammatory cytokine production (5, 6, 10, 11).
Monocyte/macrophages have been the prime targets of research in
endotoxin tolerance. Tolerance in such cells clearly involves a
distinct functional state of activation or differentiation, not a
global inhibition of function. Although the production of several
proinflammatory cytokines (e.g., TNF-, IL-1, and IL-6) and
antiinflammatory cytokines (e.g., IL-10) is suppressed, the production
of other mediators (e.g., IL-1RA) remains unaltered (6, 12). Such observations have led to the concept that endotoxin
tolerance represents a reprogramming of macrophages as an adaptive
response to bacterial infection (13). It also has become
clear that LPS-driven tolerance is but a particular instance of a more
general phenomenon of activation-induced reprogramming of
monocyte/macrophages: similar effects are seen with other
bacterial products (e.g., macrophage-activating lipopeptides, 2
kDa (MALP-2)7)
and endogenous proinflammatory mediators (e.g., IL-1 plus
TNF-) (14, 15, 16). In addition to
monocyte/macrophages, other endotoxin-responsive cells also exhibit
endotoxin tolerance. In this regard, the likely relevance of microbial
product-induced changes in dendritic cell (DC) function and
localization to endotoxin tolerance in vivo recently has become clear
(17, 18, 19)
IL-12 is an immunoregulatory cytokine that is critical to the orchestration of cell-mediated immune responses in both the innate and adaptive immune systems. Produced largely by monocyte/macrophages and DCs, IL-12 is important for protection from diverse bacterial pathogens (20). LPS is a potent inducer of IL-12 secretion. In turn, IL-12 is central to pathological responses to LPS (21, 22). Production of IL-12 recently has been shown to be suppressed during endotoxin tolerance (17). Therefore, the likely relevance of endotoxin tolerance-related dysregulation of IL-12 production to the increased risk of bacterial superinfection in survivors of sepsis has been of considerable interest (17, 19, 23, 24, 25, 26, 27).
The mechanisms that underlie endotoxin tolerance and immunological
paralysis remain unclear. Secretion of soluble mediators, changes in
LPS receptor expression or function, alterations in LPS-driven
signaling pathways, and primary effects on transcription, mRNA
stability, and translational efficiency have all been implicated at one
time or another (6, 15, 28, 29). Despite considerable
experimental effort, no consensus has been achieved. In part, this may
be because of a lack of comparability of models: there are evident
species differences, and monocytic cell lines appear to be poor mimics
of primary monocytes when it comes to endotoxin tolerance
(6). A further reason is that distinct mechanisms appear
to be responsible for different aspects of the endotoxin tolerance
phenotype, even in single-cell types. For example, although IL-10 and
TGF- are central to TNF- suppression in tolerant human monocytes
(17, 30), these cytokines appear to play no role in
endotoxin tolerance-related IL-12 suppression in such cells
(17). Finally, the fact that the mechanisms responsible
for inducing endotoxin tolerance are not necessarily the same as the
mechanisms that are responsible for altered LPS-driven responses during
LPS tolerance is poorly appreciated. Thus, although many studies have
shown that endotoxin tolerance is associated with alterations in LPS
signal transduction at upstream levels (reviewed in Ref.
15), this is entirely compatible with a mechanism
involving LPS induction of soluble mediators that, secondarily, lead to
such aberrant signal transduction.
In these studies, we explored the mechanisms underlying IL-12
suppression during endotoxin tolerance in a murine system. We
demonstrate that decreased IL-12 production in vivo is clearly
multifactorial, involving both deletion of splenic
CD11chigh DCs as well as alterations in the
responsiveness of macrophages and remaining splenic DCs. We find no
mechanistic role for B or T lymphocytes, the soluble mediators IL-10,
TNF-, IFN-, or NO, or the NF-B family members p50, p52,
or RelB.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice were purchased from Harlan Sprague-Dawley
(Indianapolis, IN). Females of the following strains were purchased
from The Jackson Laboratory (Bar Harbor, ME): wild-type C57BL/6 mice;
C57BL/6 mice deficient in IL-10 (IL-10-/-;
C57BL/6-IL-10tm1Cgn); C57BL/6 mice deficient in nitric oxide synthase 2
(iNOS-/-); C57BL/6-Igh-6tm1Cgn mice deficient
in mature B cells (µMT); mice with a targeted disruption of NF-B
p50 (p50-/-; C57BL/6,129-NF-B1tm1Bal; and
their wild-type counterparts. BALB/c-IL-10-/-,
C57BL/6-NF-Bp52-/-, and C57BL/6-NF-B
RelB-/- mice were obtained from Dr. C. Hunter
(University of Pennsylvania, Philadelphia, PA). C57BL/6,129 TNF-
receptor-/- mice
(TNF-Rp55p75-/-) were obtained from Dr. P.
Scott (University of Pennsylvania, with permission from Dr. H.
Bluethmann). T cell-depleted (nude) mice were purchased from Charles
River Laboratories (Wilmington, MA).
IFN-R-/- mice on a 129,C57BL/6 background
were obtained from Dr. D. Griffin (Johns Hopkins University, Baltimore,
MD); wild-type counterparts were from The Wistar Institute
(Philadelphia, PA) breeding colony. In all experiments, mice were used
at 5–7 wk of age.
Reagents
LPS from Escherichia coli serotype 0127:B8 (used unless otherwise noted) and Salmonella typhimurium (protein content of both <1%) was purchased from Sigma (St. Louis, MO). Protein-free E. coli LPS K235 was generously provided by S. Vogel (Bethesda, MD). Human FLT3L was kindly provided by Immunex (Seattle, WA).
Induction of LPS tolerance in vivo
Unless otherwise indicated, mice were injected i.p. with 20 µg
of LPS or 0.1 ml of PBS, followed 26 h later by an i.v. injection
of 100 µg of LPS. Serum was obtained from blood collected at 1 or
3 h after LPS challenge to assess TNF- or IL-10 and IL-12
levels, respectively.
Induction of LPS tolerance in vitro
Single-cell suspensions from the spleens of two to three mice were cultured in 24-well plates at a density of 3 x 106 cells/well under LPS-free conditions in RPMI 1640 supplemented with 10% FCS (HyClone, Logan, UT), L-glutamine, nonessential amino acids, 1 mM sodium pyruvate, 1 mM HEPES, 2-ME, and penicillin. After 48 h, splenocytes were primed with 100 ng/ml LPS (or mock primed with medium) for 20 h. Cells were subsequently washed twice with PBS and incubated with a second dose of LPS (1 µg/ml) for a further 20 h, after which culture supernatants were harvested for cytokine analysis.
Peritoneal exudate macrophages were recovered by lavage 3 days after i.p. injection of 2 ml of thioglycollate (Difco, Detroit, MI). Cells were plated in 12-well plates at a density of 2 x 106 cells/well, and 4 h later, nonadherent cells were removed by washing. After resting overnight, cells were stimulated with 10 ng/ml LPS for 20 h, washed twice with PBS, and incubated with 10 ng/ml LPS for an additional 20 h, after which culture supernatants were harvested for cytokine analysis.
Induction of LPS tolerance ex vivo
Splenocytes from LPS- or PBS-injected mice (2–3 mice/group) were isolated as described above before secondary stimulation with 1 µg/ml LPS. Peritoneal macrophages from LPS- or PBS-injected mice (3–4 mice/group) were recovered by lavage, plated in 24-well plates at a density of 1 x 106 cells/well in supplemented RPMI 1640, and allowed to rest for 48 h before stimulation with 1 µg/ml LPS for an additional 20 h.
Cytokine assays
IL-12p40, IL-12p70, TNF-, and IL-10 levels were measured by a
two-site radioimmunoassay as described previously (22)
with the following Ab pairs: C17.15 and C15.6 (sensitivity 50 pg/ml)
for IL-12 p40; C18.2 and C17.15 (sensitivity 5–10 pg/ml) for IL-12p70
(all IL-12 Abs were generated in our laboratory); XT22 (generously
provided by Dr. A. Sher, National Institute of Allergy and Infectious
Diseases, Bethesda, MD) and polyclonal anti-TNF- Ab
(sensitivity: 100 pg/ml; BD PharMingen, San Diego, CA) for TNF-;
Jess-2A5 (generously provided by Drs. J. Abrams and A. O’Garra, DNAX,
Palo Alto, CA) and Jess-16E-5 obtained from BD PharMingen,
(sensitivity,10 pg/ml) for IL-10. rIL-12 (kindly provided by Genetic
Institute, Boston, MA), rTNF- (Hoffman La Roche, Basel,
Switzerland), and rIL-10 (Endogen, Woburn, WA) were used as
standards.
Flow cytometry
For surface staining, splenocytes obtained from mechanically disrupted spleens were resuspended in PBS containing 0.1% gelatin, and incubated sequentially with anti-FcR Ab 2.4G2 (1 µg/ml) for 10 min and with CD11c-FITC or CD11b-FITC (1 µg/ml; all obtained from BD PharMingen) for 30 min (all incubations on ice). Cells were washed three times in PBS before analysis. Anti-hamster IgG-FITC and anti-rat IgG2b-FITC Abs were used as negative controls.
For intracellular IL-12 staining, splenocytes recovered from LPS-challenged mice were cultured for 5 h with monensin (BD PharMingen) followed by surface staining as described above. Cells then were fixed with 4% paraformaldehyde in PBS for 10 min, washed with PBS containing 1% gelatin, and permeabilized with buffer containing 0.1% saponin and 2% gelatin to reduce nonspecific binding. Cells were stained with PE-conjugated C15.6 Ab, which recognizes IL-12p40, and an IgG1 isotype control (BD PharMingen) for 30 min on ice in a buffer containing saponin. Cells were analyzed with an EPICS XL (Coulter Pharmaceutical, Palo Alto, CA) flow cytometer. For each condition, 150,000 events were collected andanalyzed by using WinMID software (http://scripps.edu/software.html;Scripps Institute, La Jolla, CA).
Statistical analysis
Comparisons were analyzed by using the unpaired Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inhibition
Mice exposed to LPS show a decreased ability to produce TNF-,
IL-12, and other proinflammatory cytokines in response to subsequent
LPS exposure (5, 24, 27, 31). In BALB/c mice primed with
1, 5, or 20 µg of LPS and challenged 26 h later with 100 µg of
LPS, serum TNF- levels were decreased by 87–90% compared with
nontolerized mice (Fig. 1
). Similar
treatment of C57BL/6 mice led to a 75–77% decrease in TNF-
production (data not shown). By contrast, priming with 1 µg of LPS
did not induce inhibition of IL-12 production on secondary challenge
with LPS in either strain of mice, and injection of 5 µg of LPS led
to only modest effects (either slight inhibition or enhancement) on
IL-12 production (shown for BALB/c in Fig. 1). However, priming with 20
µg of LPS, reproducibly induced significant inhibition of TNF-,
IL-12p40, and IL-12p70 production in both mouse strains (Fig. 1 and
data not shown). Serum IL-10 levels increased in an LPS dose-dependent
manner, with levels nearly 2-fold those in control mice after priming
with 20 µg of LPS (Fig. 1). In all subsequent experiments, a single
20-µg dose of LPS was used for tolerization.
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The pattern of cytokines secreted ex vivo by splenocytes from
LPS-primed mice closely resembled that observed in vivo in
LPS-tolerized mice. Splenocytes from BALB/c mice sensitized in vivo
with LPS produced 47% less IL-12p40 and 75% less IL-12p70 and TNF-
after secondary in vitro stimulation with LPS, compared with
mock-tolerized mice (Fig. 2A).
Similarly, IL-10 secretion was increased 
2.5-fold in splenocytes
from BALB/c mice sensitized in vivo with LPS, suggesting a potential
role for IL-10 in controlling IL-12 production in this model.
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production (Fig. 2
B). IL-12p70 production by peritoneal
macrophages was undetectable in these experiments, as they had not been
pretreated with IFN-
, a necessary requirement for the production of
IL-12p70 by these cells (20). In addition to providing a cellular counterpart for the in vivo data, these results suggest that IL-10 production is differentially regulated in disparate cell types in response to LPS. They also suggest that cells other than macrophages are likely to be responsible for augmented IL-10 levels in the serum of LPS-tolerized mice.
Down-modulation of IL-12 in LPS-tolerized mice does not require
IL-10, NO, TNF-, IFN-, or T and B cells
IL-10-/- mice could not be primed with our
standard tolerogenic dose of 20 µg of LPS, because doses higher than
5 µg are lethal to these mice (31). Thus,
IL-10-/- C57BL/6 and
IL-10-/- BALB/c mice were primed and challenged
with 1 and 5 µg of LPS, respectively, according to a previously
published protocol (31). This resulted in a 2-fold
decrease in TNF- levels in both strains of mice (data not shown).
Serum levels of IL-12p40 and IL-12p70 were, on average, 5-fold higher
than in control wild-type mice in the absence of LPS priming. Such
IL-12 levels were either not affected or only slightly decreased
(10–15%) by sensitization with LPS at these doses (data not shown),
similar to data reported in Fig. 1 for BALB/c mice. Thus, in vitro
experiments were conducted in which splenocytes from
IL-10-/- and wild-type C57BL/6 mice were primed
with 100 ng/ml LPS for 20 h, followed by a challenge dose of 1
µg/ml LPS for an additional 20 h. Notably, this priming dose
reproducibly induces IL-12 suppression in the splenocytes of all mouse
strains tested to date (data not shown). LPS priming of
IL-10-/- splenocytes led to 90% inhibition of
IL-12p40, IL-12p70, and TNF- production in response to secondary LPS
challenge (Fig. 3A).
Similarly, wild-type C57BL/6 splenocytes demonstrated an 80% decrease
in IL-12 production. Consistent with reported in vivo data
(32), IL-10-/- splenocytes
produced higher levels of IL-12 in response to LPS than did splenocytes
from wild-type controls. Although splenocytes from wild-type mice
produced increased levels of IL-12 and TNF- when endogenous IL-10
was neutralized with Ab, genetic deletion or Ab-mediated neutralization
of IL-10 did not prevent the development of endotoxin
tolerance-mediated inhibition of these cytokines (Fig. 3A).
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production in these mice
(Fig. 3B).
TNF- recently has been shown to exert an inhibitory effect on IL-12
production (39, 40). Although itself a target of
inhibition during endotoxin tolerance, TNF- is produced earlier than
IL-12 and thus might be of mechanistic importance in endotoxin
tolerance-driven inhibition of IL-12 (22). However,
LPS-primed TNF-Rp55p75-/- mice produced
decreased serum levels of IL-12 and TNF- in response to secondary
LPS exposure (Fig. 3C), suggesting that TNF- controls
neither itself nor IL-12 production in LPS-induced tolerance.
IFN- inhibits IL-12 production stimulated by bacterial and viral
infection. In vitro data suggest inhibition of IL-12 production by both
monocyte/macrophages and DCs (Refs. 41, 42, 43, 44 ; C. L.
Karp, unpublished observations). IFN- production also is
triggered by LPS (45). However, mice with a genetic
disruption in the IFN- receptor
(IFN-R-/-) produced decreased levels of
IL-12 and TNF- in response to secondary LPS exposure (Fig. 3D), suggesting the lack of a significant role for
IFN- in inhibiting the production of these proinflammatory
cytokines in this model.
Finally, both T cell-deficient (nude) and B cell-deficient (µMT) mice
demonstrated decreased IL-12 and TNF- production in response to
secondary LPS challenge (Fig. 3E), indicating that
inhibition of these cytokines in this model of experimental endotoxin
tolerance is independent of T and B cells and of downstream factors
produced by these cells.
NF-B p50, p52, and RelB family members are not responsible for
LPS-induced tolerance
In vitro studies have suggested that formation of
transcriptionally inactive NF-B p50 homodimers may be responsible
for endotoxin tolerance in some systems (46, 47). Most
recently, Bohuslav et al. reported that peritoneal macrophages from
mice deficient in the NF-B family member p50
(p50-/-) were incapable of undergoing endotoxin
tolerance-mediated suppression of TNF- production (48).
To address the role of this transcription factor in IL-12 suppression
during LPS tolerance, p50-/- mice were injected
with LPS in vivo. Surprisingly, LPS-primed
p50-/- mice (along with wild-type controls)
exhibited decreased serum levels of IL-12 and TNF- in response to
LPS challenge (Fig. 4). Of note,
p50-/- mice produced significantly more TNF-
than wild-type mice, suggesting a basal role for the p50 in TNF-
regulation.
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production; again, the overall production of TNF- by
p50-/- splenocytes was higher than that of
wild-type splenocytes (Fig. 5A). Finally, and in direct
contrast to the findings of Bohuslav, et al. (48), we
found that elicited peritoneal macrophages from
p50-/- mice were indeed capable of endotoxin
tolerance-mediated inhibition of both IL-12 and TNF- production.
Priming of macrophages with 10 ng/ml LPS from E. coli strain
0127:B8 consistently induced inhibition of IL-12 and TNF- production
in response to secondary LPS exposure (Fig. 5B). This
inhibition of IL-12 and TNF- production by
p50-/- and wild-type macrophages was
independent of the source of LPS used for priming and challenge, as
similar data were obtained with LPS from S. typhimurium or
with protein-free, phenol/water-extracted LPS from E. coli
strain K3562 (Ref. 49 ; data not shown). Because of the
surprising nature of these findings, all mice used in these studies
were carefully genotyped.
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B family
members p52 and RelB, failed to implicate these transcription factors
in LPS-induced suppression of IL-12 and TNF- as well, as splenocytes
from both knockout strains were able to undergo endotoxin tolerance
induction in vitro (Fig. 5C). Interestingly, LPS-stimulated
splenocytes from p52-/- mice produced
significantly more IL-12p40 and IL-12p70 than wild-type mice,
suggesting a regulatory role for p52 in IL-12 production in response
to LPS. LPS-dependent depletion of CD11chigh DCs leads to decreased IL-12 production
It previously has been shown that LPS not only induces maturation
and migration of splenic DCs, but also triggers apoptosis in those
cells (19, 50, 51). These data, together with the fact
that DCs are major producers of IL-12 (52, 53, 54), have
suggested that endotoxin tolerance-related decreases in IL-12 levels in
serum and in the supernatants of splenocytes predominantly reflect loss
of splenic DCs rather than the induction of a tolerant state in these
cells. Flow cytometric analysis was used to examine the effect of LPS
on splenic populations of DCs and macrophages. Such analysis revealed a
profound decrease in the number of CD11chigh DCs
in the spleens of mice primed with LPS 26 h earlier (Fig. 6A). A particularly dramatic
loss of splenic DCs was seen in FLT3L-treated mice primed with LPS
(Fig. 6B). FLT3L, which stimulates production of DCs de novo
from precursors (55, 56), increased the number of DCs in
spleen but did not prevent the rapid disappearance of such
CD11chigh cells from spleens after in vivo
administration of LPS. This correlated with markedly decreased
production of IL-12 and TNF- ex vivo by LPS-stimulated splenocytes
(data not shown).
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, top). Endotoxin priming
thus leads to loss of splenic DCs as well as suppression of the IL-12
productive capacity of remaining DCs.
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Consistent with previous findings (51), an increased
number of CD11bhigh macrophages was observed in
the spleen and peritoneal cavity after LPS priming (Fig. 8). No significant intracellular staining
for IL-12 was observed in CD11bhigh cells, likely
because of the poor response of this population to LPS
(53) and the low threshold of sensitivity of the FACS
assay (Fig. 7, bottom).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Our
major findings with these models are 2-fold. First, IL-12 suppression
appears to be a function both of splenic DC loss and of altered
monocyte/macrophage and residual DC function. Second, by using knockout
mice, we find no mechanistic role for lymphocytes, the soluble
mediators IL-10, TNF-, IFN-, or NO, or the NF-B family
members p50, p52, or RelB in endotoxin tolerance-mediated suppression
of IL-12. These latter findings include data that are both confirmatory
of (for IL-10; Ref. 30) and in direct opposition to (for
NF-B p50; Ref. 48) previously reported
observations.
With the mechanisms underlying endotoxin tolerance remaining obscure
even after reductive approaches involving single-cell types in vitro,
the analysis of such phenomena in vivo adds an unavoidable further
layer of complexity. The origin of the cytokines present in serum after
LPS challenge presumably represents an integration of total body
cytokine production, but the actual origin of the cytokines produced is
unclear. In the experiments reported here, serum cytokine levels
closely paralleled splenocyte responses. The discrepancies between
spleen and serum on the one hand and purified, elicited peritoneal
macrophages on the other are instructive. Like purified human
monocyte/macrophages (but unlike murine serum and splenocytes): 1)
IL-10 production by peritoneal macrophages was suppressed by LPS
priming; and 2) IL 12p70 in such cells was not inducible without
IFN- priming. However, peritoneal macrophages clearly undergo
endotoxin tolerance-mediated suppression of IL-12 production.
In the spleen, in vivo priming with LPS led to an increase in macrophage numbers, but IL 12 production (even of the p40 subunit) by such cells was not easily demonstrable, as previously reported by Reis e Sousa et al. (54). However, splenic DC numbers plummeted after LPS priming. Of note, the kinetics of splenic DC repopulation closely paralleled that of increasing IL-12 production by splenic leukocytes after secondary stimulation ex vivo. IL-12 production by residual DCs was also significantly impaired. These findings integrate data previously reported in human and murine systems. By using blood-derived human DCs, we and others have reported endotoxin tolerance-mediated suppression of IL-12 production in vitro (17, 57). Langenkamp et al. (18) have referred to this as an exhaustion of cytokine production, pointing out the likely role of a defined temporal pattern of cytokine release by DCs after microbial stimulation in the maintenance of immune response class homeostasis. In none of these studies was death of DCs seen. However, in vivo exposure to LPS has been shown to trigger apoptotic death of DCs (not, e.g., simply down-regulation of identifying surface markers) in the murine spleen (19, 50, 51). In our hands, the process leading to DC disappearance is very rapid, with marked depletion within 24 h after LPS administration. Although FLT3L treatment of mice increased baseline numbers of DCs in the spleen, it did not protect these cells from the effects of LPS. Whether the differences in DC fate in these various studies is attributable to fundamental differences between human and murine DCs or, more likely, is attributable to the biological context of an intact splenic microenvironment in vivo (19) remains to be seen.
The ability of isolated cell populations to undergo endotoxin tolerance
suggests that, if soluble mediators are mechanistically important in
the genesis or maintenance of this state, they can be autocrine. The
relevant pathways in vivo may be quite different, however, as suggested
by the relative resistance of adrenalectomized mice to the induction of
endotoxin tolerance (58) as well as the critical
effects of the splenic microenvironment on DC "paralysis"
attributable to microbial stimuli other than LPS (19).
Thus, we examined the role of various known soluble inhibitors of IL-12
in endotoxin tolerance in vivo in knockout mice. T and B lymphocytes
produce multiple IL-12 down-regulatory mediators. However,
experiments with T and B cell-deficient mice suggested that such cells
are unnecessary for the induction of endotoxin tolerance-mediated
suppression of IL-12 in vivo. IL-10 is a prime autocrine and paracrine
regulator of IL-12 production. In human monocyte/macrophages,
neutralization of IL-10 ablates endotoxin tolerance-mediated inhibition
of TNF- (17, 30) though not IL-12 (17).
Replicating previously published data (31), we found that
IL-10-/- mice undergo tolerance for TNF-
production both in vivo and in vitro. The LPS doses required for in
vivo induction of tolerance for IL-12 production are lethal for
IL-10-/- mice. However, such tolerance is
exhibited by the splenocytes of such mice and is easily demonstrated in
the presence of Ab-mediated neutralization of IL-10 in wild-type
splenocytes. Increased levels of IL-10 were seen after LPS priming in
serum and in the supernatants of splenocytes, but not in those from
peritoneal macrophages, suggesting that cells others than macrophages
may be a significant source of IL-10 in vivo. The inverse correlation
between serum IL-12 and IL-10 levels in LPS-tolerized mice suggest
tight, reciprocal regulation of the production of these cytokines, as
has been previously reported in in vitro studies (13, 59, 60). Further, splenocytes from IL-10-/-
mice exhibit enhanced baseline production of IL-12 in response to LPS,
clearly indicating a tonic down-regulatory role for IL-10 in IL-12
release.
Type I IFNs are potent inhibitors of IL-12 production in human and
murine systems (Refs. 42, 43, 44 ; C. L. Karp, unpublished
observation). Indeed, exogenous IFN- has been shown to protect
against endotoxin toxicity in mice (61). However, our
findings with IFN-R-/- mice indicate that
type I IFNs are not important in IL-12 suppression during endotoxin
tolerance. Studies with TNFR-/- and
iNOS-/- mice similarly were negative. One
notable finding of these studies, unlike experiments with human
monocyte/macrophages (17), is that none of them
functionally distinguished IL-12 from TNF- regulation. However, such
in vivo experiments have clear limitations. First, it is unclear which
cells are primarily responsible for the serum cytokines being measured.
It is possible, e.g., that IFN- is important mechanistically in
the induction of tolerance in peritoneal macrophages, but that serum
cytokines derive largely from splenic DCs (hepatic macrophages,
intravascular neutrophils; Ref. 62) that have no such
dependence on IFN-. Second, there may be extensive redundancy in
mechanism; single-knockout mice may be insufficient.
Recently, two potential mechanistic explanations for endotoxin
tolerance have emerged in the literature: alterations in NF-B family
member activity (46, 47, 48) and down-regulation of surface
expression of Toll-like receptor 4 (TLR4; Refs.16, 29).
The NF-B/Rel family of transcription factors play important roles in
proinflammatory cytokine production. Five mammalian family members are
known: p50, p65 (RelA), p52, c-Rel, and RelB. DNA binding by family
members occurs after homo- or heterodimeric complex formation, and/or
higher-order complex formation with other transcription factors.
Although suppressed transcription of TNF- has been associated with
increased nuclear mobilization of transcriptionally inactive NF-B
p50 homodimers in some murine and human cell lines (46, 47), endotoxin tolerance has largely seemed to correlate with
inhibition of NF-B nuclear translocation in primary human and murine
monocyte/macrophages (63, 64). However, Bohuslav et al.
(48) have reported that endotoxin tolerance (as measured
by the suppression of TNF- production) is not inducible in
peritoneal macrophages derived from mice with a genetic deletion of
NF-B p50. We are unable to replicate these findings. We provide data
demonstrating that: 1) the peritoneal macrophages and splenocytes of
such mice are unaltered in their ability to undergo endotoxin
tolerance-mediated suppression of both TNF- and IL-12; and 2)
suppression of TNF- and IL-12 production in response to systemic LPS
challenge is part of the in vivo phenotype of endotoxin tolerance in
such mice. Inhibition occurred regardless of the source (or purity) of
the LPS used. Consistent with the finding that p50 homodimer
overexpression leads to the inhibition of TNF- gene expression
(48), we found that p50-/- mice
produce more TNF- (though not IL-12) in response to LPS than their
wild-type counterparts, suggesting a tonic regulatory role for p50 in
TNF- gene expression. This several-fold increase in TNF-
production was seen in serum and in the supernatants of splenocytes
stimulated with LPS. However, in accord with previous studies, TNF-
levels produced by p50-/- peritoneal
macrophages stimulated in vitro with LPS were not dramatically
increased (46, 65). Because LPS stimulation induces
TNF- production in both macrophages and DCs, our results support the
possibility that the activation of NF-B p50 is differentially
regulated in these different cell types. Studies with mice deficient in
other subunits of the NF-B transcription factors family also have
supported this notion (66, 67, 68). Splenocytes from
p52-/-, though not
RelB-/-, mice produced increased levels of
IL-12 in response to LPS compared with wild-type splenocytes,
suggesting a regulatory role for pathways involving p52 in limiting
IL-12 production similar to that suggested by the experiments in IL-10
knockout mice. Nevertheless, splenocytes from
p52-/- and RelB-/- mice
clearly undergo endotoxin tolerance-mediated inhibition of TNF- and
IL-12 production.
The most recent candidate mechanism for endotoxin tolerance focuses on TLR expression. The endotoxin-tolerant state is known to be associated with alterations in LPS signal transduction at upstream levels (reviewed in Ref. 15). Before identification of the mammalian TLRs (69, 70), down-regulation of surface expression of the LPS-binding receptor CD14 was investigated and excluded as an underlying mechanism for tolerance (6, 71). With recognition of the central role of TLR4 in LPS signaling, data have been presented suggesting that down-regulation of surface expression of TLR4 (or of surface expression of TLR4/MD-2 complexes) may be responsible for LPS tolerance (16, 29, 72). We report no data bearing directly on this assertion in the present paper. However, these data are somewhat difficult to relate causally to the fact that only a subset of LPS-driven responses are suppressed during endotoxin tolerance. Furthermore: 1) such data, derived with a mAb of unclear specificity, are at variance with TLR4 mRNA data (15, 29); 2) LPS sensitization leads to down-regulation of TLR2-driven signaling in the absence of apparent effects on TLR2 expression, something mirrored by TLR2-mediated tolerance induction itself (16); and 3) TLR4 overexpression fails to obviate the induction of endotoxin tolerance (S. Vogel, unpublished observations). It would appear that the mechanisms that underlie endotoxin tolerance-mediated alterations in cytokine production will bear further investigation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence to Dr. Maria Wysocka, University of Pennsylvania, 238 CRB, 415 Curie Boulevard, Philadelphia, PA 19104. E-mail address: Mwysocka{at}mail.med.upenn.edu
3 Current address: Center for Rheumatic Diseases, University Department of Medicine, Glasgow Royal Infirmary 10, Alexandra Parade, Glasgow, G 312ER U.K.
4 Current address: University of Munster, 56 Von-Esmarch strasse, 48149 Munster, Germany.
5 Current address: Schering-Plough, 27 Chemin des Peupliers-B.P.-11, 69571 Dardilly Cedex, France.
6 Current address: Children’s Hospital Research Foundation, University of Cincinnati, TCHRF 1566, 3333 Burnet Avenue, Cincinnati, OH 45229
7 Abbreviations used in this paper: MALP-2, macrophage-activating lipopeptides, 2 kDa; DC, dendritic cell; TLR, Toll-like receptor; iNOS, inducible NO synthase.
Received for publication January 16, 2001. Accepted for publication April 2, 2001.
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) or medium (
) for
20 h. B, BALB/c peritoneal macrophages (pooled from
four mice per group) isolated 26 h after similar LPS- or
mock-priming, and similarly restimulated in vitro with LPS (
) and challenged i.v. 26 h later with 100 µg of
LPS. Data represent means (+SD) of five mice per group from single
experiment, representative of two performed. D,
IFN-
:
NF-
