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Production in IFN Regulatory Factor-1 Knockout Mice During Endotoxemia Is Secondary to a Loss of Both IL-12 and IL-12 Receptor Expression1
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production. In response to LPS,
IRF-1-/- mice produced less IL-12 p40, IL-12 p35, and
IFN- mRNA in the liver than IRF-1+/+ mice. While
pulmonary IFN- mRNA levels were also mitigated in
IRF-1-/- mice, pulmonary IL-12 p40 and IL-12 p35 mRNA
were not dysregulated. Circulating IL-12 p70 and IFN- levels were
profoundly attenuated in LPS-challenged IRF-1-/- mice.
Further analysis revealed a major deficiency in hepatic IL-12Rß1 and
IL-12Rß2 mRNA expression as well as pulmonary IL-12Rß1 mRNA
expression in LPS-challenged IRF-1-/- mice. In vitro,
IFN- up-regulated IL-12Rß1 mRNA in macrophages from
IRF-1+/+, but not IRF-1-/-, mice.
IFN--induced IL-12Rß2 mRNA expression was also diminished in
macrophages from IRF-1-/- mice. In contrast to
IRF-1+/+ mice, administration of exogenous IL-12 to
IRF-1-/- mice resulted in reduced serum IFN- and
hepatic and pulmonary IFN- mRNA, demonstrating that loss of IL-12R
results in diminished IL-12 responsiveness. While LPS-challenged
IRF-1-/- mice also had reduced IL-15 mRNA levels, serum
IL-18 responses were intact. Finally, induction of IRF-1 mRNA by LPS in
livers of IFN- knockout mice were markedly attenuated, suggesting a
feedback amplification loop. These studies indicate that IRF-1
deficiency disrupts both IL-12-dependent and -independent pathways of
IFN- production and that IRF-1 is a critical transcription factor
involved in the regulation of not only IL-12, but also
IL-12R. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion from T cells, NK
cells, and macrophages (5, 6, 7, 8), IL-12 plays a critical role
in the development of a protective cell-mediated immune response to
many microbial pathogens (9, 10, 11). However, in endotoxin
shock, overproduction of IFN- contributes to mortality, and IL-12
production is deleterious, rather than protective, to the host
(12, 13). The predominant effect of IFN- in this
cytokine cascade induced by LPS is to up-regulate expression of
TNF-
, a principle mediator of tissue damage and lethality (14, 15). IFN- can further amplify this pathway by up-regulating
both IL-12 production and IL-12R expression (16, 17).
Bioactive IL-12 (p70) is a heterodimeric cytokine composed of two
independently regulated protein subunits, designated p35 and p40
(6). While IL-12 p40 is secreted in both monomeric and
homodimeric forms, secretion of IL-12 p35 has not been reported
(4). Both in vitro and in vivo, IL-12 p70 secretion is
accompanied by excess secretion of IL-12 p40 (16, 18),
which functions as an IL-12R antagonist (19). IL-12
p40 and IL-12 p35 expression is controlled primarily at the level of
transcription (20), and recent studies have begun to
delineate regulatory regions within the promoters and transcription
factors involved in their modulation by LPS and IFN-
(20, 21, 22, 23, 24). Recent studies using knockout mice have
demonstrated that IFN regulatory factor-1
(IRF-1),3 IRF-2, and
IFN consensus sequence binding protein (ICSBP), three members of the
IRF family of transcription factors, regulate inducible IL-12
expression. Following activation with LPS plus IFN-, macrophages
from IRF-1-deficient mice exhibit impaired synthesis of IL-12 p40 and
IL-12 p35 mRNA, resulting in profound reductions in IL-12 p40 and IL-12
p70 protein (16, 25). Impaired synthesis of IL-12 p40 mRNA
and protein, but not IL-12 p35 mRNA, was observed in LPS- plus
IFN--treated macrophages from IRF-2- and ICSBP-deficient mice
(16, 26). In vivo, IRF-1-deficient mice are resistant to
LPS-induced mortality, and this enhanced resistance was associated with
attenuated production of serum IL-12, IFN-, and TNF-
(27). Of interest, despite profound reductions in splenic
IFN- mRNA expression, diminished splenic IL-12 p40 mRNA expression
was not observed (27). Whether ameliorated IFN-
production in LPS-challenged IRF-1-deficient mice is affected by loss
of IL-12 through IL-12 p35 dysregulation, overproduction of IL-12 p40
(antagonist) relative to IL-12 p70 (agonist), or dysregulation of an
IL-12-independent pathway of IFN- induction has not been examined
to date.
The biological response to IL-12 is mediated through specific binding
to the IL-12R. Like IL-12, the IL-12R consists of two independently
regulated subunits, IL-12Rß1 and IL-12Rß2 (28, 29, 30). In
murine cells, IL-12Rß1 binds IL-12 with high affinity, while
IL-12Rß2 binds IL-12 with low affinity and functions as the
signal-transducing component (29). Similar to IL-12
p40-deficient mice (31), IL-12Rß1-deficient mice exhibit
reduced levels of serum IFN- following endotoxin administration
(32), illustrating the importance of the IL-12/IL-12R
pathway to LPS-induced IFN- production in vivo. Whether IL-12R
expression is modulated by LPS or whether IL-12R is also regulated by
IRF-1 has yet to be explored.
The aim of the current study was to address more fully the impact of
IRF-1 deficiency on the IL-12/IFN- cytokine cascade following LPS
challenge. Results from this study indicate that the IL-12-mediated
pathway of IFN- production is disrupted at multiple levels. In
LPS-challenged mice, IRF-1 deficiency resulted in 1) ameliorated
production of both IL-12 p40 and IL-12 p35 mRNA in the liver, 2)
concomitant reductions in circulating IL-12 p40 and IL-12 p70, 3) an
even greater overproduction of IL-12 p40 (antagonist) relative to IL-12
p70 (agonist), and 4) impaired expression of IL-12Rß1 and IL-12Rß2
mRNA. Disruption of the IL-12/IL-12R pathway in
IRF-1-/- mice was paralleled by a significant
impairment in the ability to produce IFN- following LPS challenge or
administration of exogenous IL-12. These results clearly indicate that
IRF-1 plays a crucial role in endotoxicity not only by regulating IL-12
production, but also by controlling IL-12 responsiveness through
coordinate regulation of the IL-12R.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein-free (<0.008%) LPS was prepared from Escherichia coli K235 by phenol-water extraction (33). Recombinant murine IL-12 p70 (rmIL-12) was obtained from PharMingen (San Diego, CA).
Mice
IRF-1-/- mice were generated by targeted
disruption as described previously (34). Breeding pairs of
IRF-1-/- and IRF-1+/-
mice, which were backcrossed to C57BL/6 mice for three to five
generations, were a gift from Dr. Tak Mak (Amgen Institute, Toronto,
Canada). The IRF-1-/- and
IRF-1+/- mouse colonies used in this study were
bred in our facility as described previously (35).
IRF-1+/+ mice served as background-matched
controls (35). All IRF-1 mice were genotyped as described
previously (35, 36). IFN- knockout mice were a gift
from Genentech (San Francisco, CA) and were genotyped as previously
described (37). Mice were housed in cages with filter tops
in a laminar flow hood and were fed food and acid water ad libitum.
Mice were injected i.p. with either a sublethal (25 µg/mouse,
1.2–1.4 mg/kg) or a lethal (35 mg/kg) dose of LPS. In some
experiments mice were injected i.p. with 200 ng of rmIL-12 for 4
consecutive days; sera and tissue samples were harvested 3 h after
the final injection of rmIL-12.
The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council Department of Health, Education, and Welfare Publication (National Institutes of Health) 85-23.
Macrophage cultures
Peritoneal exudate macrophages were obtained by lavage 4 days
after injection of sterile 3% thioglycolate broth (3 ml, i.p.). Cells
were washed and resuspended in RPMI containing 2% FCS and standard
supplements (38). Macrophages were plated in six-well
tissue culture dishes (4 x 106 cells/well).
After overnight incubation to allow for adherence of macrophages,
monolayers were washed to remove nonadherent cells and were incubated
with medium or 5 U/ml of IFN- in a final volume of 2 ml. Samples
were harvested either 6 or 8 h after treatment for analysis of
IL-12Rß1 and IL-12Rß2, respectively, because preliminary
experiments using wild-type macrophages had established these times as
optimal for peak mRNA expression (data not shown).
Serum assays
Mice were bled by cardiac puncture at the indicated times, and
serum samples were stored at -70°C. IL-12 p70 and IFN- were
detected using the Ab pairs and standards provided in the OPT-EIA ELISA
kit (PharMingen) according to the manufacturer’s instructions.
Total IL-12 (p40 monomer, p40 dimer, and p70) was detected by ELISA as
previously described (16) using Ab pairs obtained from
Genzyme (Cambridge, MA). To calculate IL-12 p40 protein levels, the
values in the IL-12 p70 ELISA were subtracted from those obtained in
the total IL-12 ELISA. IL-18 was quantified using the Ab pairs and
standard provided in the Quantikine M ELISA kit (R&D Systems,
Minneapolis, MN).
Analysis of mRNA
At the indicated time after LPS injection, liver and lung
samples from individual mice were isolated and frozen at -70°C.
Total RNA was isolated, and the relative quantities of mRNA for
hypoxanthine-guanine phosphoribosyl transferase (HPRT), IL-12 p35,
IL-12 p40, IL-12Rß1, IL-12Rß2, IL-15, IFN-, and IRF-1 were
determined by RT-PCR as described previously (39). The
primers and probe combinations for IL-12Rß2 were 5'-AAG ACT CAT GGC
ACA GAC TGT TAG (sense), 5'-GAG TTG CTA CAG TTT AGC TTG CAG
(antisense), and 5'-AGT CAC CAA CCT GTC CCT TG (probe). All other probe
and primer combinations have been published (11, 40, 41, 42, 43).
Amplified products were electrophoresed and transferred to Hybond
N+ membranes (Amersham, Arlington Heights, IL) in
10x SSC by standard Southern blotting techniques. DNA was cross-linked
by exposure to UV light, baked onto the nylon membrane, and hybridized
with an internal oligonucleotide probe. Labeling of the probe and
subsequent detection of bound probe were conducted using an enhanced
chemiluminescence system (Amersham).
Statistics
Results were analyzed using Student’s t test for comparisons between two groups and ANOVA. p < 0.05 was accepted as the level of significance. All experiments were repeated at least twice with similar results.
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Results |
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In vitro studies with murine macrophages have demonstrated that
IRF-1 mRNA expression is increased by LPS in a
cycloheximide-independent manner (41). Thus, we initially
examined the in vivo regulation of IRF-1 mRNA following LPS challenge.
IRF-1 mRNA expression was increased in liver and lung as early as
1 h after LPS injection (Table I
).
Heightened levels of IRF-1 mRNA persisted in both liver and lung for at
least 8–12 h.
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production are impaired in LPS-challenged
IRF-1-deficient mice
In a previous study we demonstrated that macrophages from
IRF-1-/- mice exhibited impaired production of
IL-12 p35 and IL-12 p40 mRNA as well as IL-12 p40 and IL-12 p70 protein
following treatment with either LPS or LPS plus IFN-
(16). To examine the extent to which IRF-1 deficiency
dysregulated IL-12 production in vivo, IL-12 p35 and IL-12 p40 mRNA and
IL-12 protein expression were analyzed at multiple time points in
IRF-1+/+ and IRF-1-/-
mice injected with 25 µg of LPS. As shown in Fig. 1, IRF-1-/- mice
produced 70–80% less hepatic IL-12 p40 mRNA by 1, 3, and 6 h
after LPS challenge than IRF-1+/+ mice. Hepatic
IL-12 p35 mRNA expression in LPS-challenged
IRF-1-/- mice also was reduced 60–89%
compared with that in IRF-1+/+ mice at all time
points examined. In contrast, neither IL-12 p40 nor IL-12 p35 mRNA
levels were dysregulated in the lungs of LPS-challenged
IRF-1-/- mice. Untreated
IRF-1-/- mice had lower basal levels of
circulating IL-12 p40 than untreated IRF-1+/+
mice, and LPS-injected IRF-1-/- mice
consistently produced less IL-12 p40 in serum than LPS-injected
IRF-1+/+ controls (Table II). In contrast to
IRF-1+/+ mice, IRF-1-/-
mice produced no detectable IL-12 p70 following LPS challenge. Because
IL-12 drives IFN- production following LPS challenge (18, 31), we examined whether impaired IL-12 production in vivo in
IRF-1-/- mice was associated with impaired
production of IFN-. Following LPS challenge, both hepatic and
pulmonary IFN- mRNA levels were reduced by >90% in
IRF-1-/- mice (Fig. 1). These substantial
reductions in pulmonary and hepatic IFN- mRNA expression paralleled
attenuated levels of circulating IFN- (Table II).
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in mice injected i.p. with a much higher dose of LPS (35
mg/kg; Fig. 2). This dose of LPS was
lethal for 80% (8 of 10) IRF-1+/+ mice, whereas
all IRF-1-/- mice (10 of 10) survived. Both
IRF-1+/+ and IRF-1-/-
mice produced very high levels of IL-12 p40 in the serum. The
differences observed in serum IL-12 p40 levels between
IRF-1+/+ and IRF-1-/-
mice were less apparent following this high dose of LPS than with low
dose LPS challenge (Table II
vs Fig. 2 and data not shown). In contrast
to IL-12 p40, the level of serum IL-12 p70 was reduced by >90% in
IRF-1-/- mice lethally challenged with LPS
(100–200 pg/ml). A concomitant reduction in the level of serum
IFN- was observed in lethally challenged
IRF-1-/- mice.
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Several observations suggested that the observed loss of IFN-
in LPS-treated IRF-1-/- mice may not be due
entirely to impaired IL-12 p70 production. First, both hepatic IL-12
p40 and IL-12 p35 mRNA were induced, albeit to lower levels, in
IRF-1-/- mice by 1 and 3 h after LPS
injection, yet IFN- mRNA levels remained uninduced at these time
points. Second, in the lungs of IRF-1-/- mice,
no dysregulation of either IL-12 p35 or IL-12 p40 was observed at any
time point despite ablation of LPS-induced IFN- mRNA expression.
Studies have demonstrated that IL-12R is up-regulated by IL-12
(44, 45) and that IL-12Rß1 and IL-12Rß2 expression can
be selectively regulated, either positively or negatively, during
parasitic infection or endotoxin tolerance (46, 47, 48). Thus,
IL-12Rß1 and IL-12Rß2 mRNA levels were examined to ascertain
whether lRF-1-/- mice exhibited altered IL-12R
mRNA levels. Both hepatic IL-12Rß1 and IL-12Rß2 mRNA as well
as pulmonary IL-12Rß1 mRNA were up-regulated in
IRF-1+/+ mice following LPS challenge; however,
the ability of LPS to induce these IL-12R mRNA species in
IRF-1-/- mice was largely abrogated (Fig. 3). Of note, both basal and inducible
levels of hepatic IL-12Rß1 mRNA in IRF-1-/-
mice were 90% less than those in
IRF-1+/+ mice.
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-induced IL-12Rß1 mRNA expression is IRF-1 dependent in
vitro
To date, our data indicate that LPS-induced IL-12, IFN-, and
IL-12R expression are all dysregulated in
IRF-1-/- mice. To test the hypothesis that
IFN- modulates IL-12R expression in an IRF-1-dependent manner,
peritoneal exudate macrophages from IRF-1+/+ and
IRF-1-/- mice were treated with medium or
IFN- (5 U/ml), and steady state levels of IL-12R mRNA
expression were assessed by RT-PCR. IFN- up-regulated IL-12Rß1
(Fig. 4A) and IL-12Rß2 (Fig. 4B) mRNA expression in macrophages from
IRF-1+/+ mice (90- and 5-fold, respectively)
and in RAW 264.7 cells. In contrast to IRF-1+/+
macrophages, IRF-1-/- macrophages produced very
low levels of IL-12Rß1 mRNA. To detect IL-12Rß1 mRNA in
IRF-1-/- macrophages, the cycle number had to
be increased from 27, the optimum cycle number for detection in
IRF-1+/+ macrophages, to 32. Even at this
increased cycle number, it was clear that
IRF-1-/- macrophages failed to up-regulate
IL-12Rß1 mRNA expression in response to IFN- treatment. In
contrast to IL-12Rß1, IFN--induced IL-12Rß2 mRNA expression in
IRF-1-/- macrophages was dysregulated to a
lesser extent. Nonetheless, expression of IL-12Rß2 mRNA was increased
by IFN- about 5-fold in IRF-1+/+ macrophages
compared with only an 2-fold increase in
IRF-1-/- macrophages.
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IRF-1+/+ and
IRF-1-/- mice were injected with IL-12 to
ascertain whether apparent defects in IL-12R expression observed in
vivo resulted in an impaired ability to produce IFN- as well as to
examine the potential role of IL-12 in the regulation of IL-12Rß1,
IL-12Rß2, and IRF-1 mRNA expression. Mice were injected i.p. with 200
ng of rmIL-12 for 4 sequential days, and levels of serum IFN- and
tissue IFN- mRNA were monitored 3 h after the final injection
of rmIL-12. Administration of exogenous IL-12 positively regulated the
expression of IFN-, IL-12Rß1, IL-12Rß2, and IRF-1 mRNA in livers
(Fig. 5) and lungs (data not shown) of
IRF-1+/+ mice. In contrast to
IRF-1+/+ mice, IRF-1-/-
mice produced substantially less basal and IL-12-induced IFN-,
IL-12Rß1, and IL-12Rß2 mRNA than IRF-1+/+
mice; an identical pattern of gene expression was observed in the lungs
of IRF-1-/- mice (data not shown). Serum
IFN- levels were significantly reduced in rmIL-12-treated
IRF-1-/- mice (290 ± 56 pg/ml for
IRF-1+/+ mice; 41 ± 9 pg/ml for
IRF-1-/- mice). We also examined the ability of
IL-12 to up-regulate IL-12R, IFN-, and IRF-1 mRNA expression in
macrophages from IRF-1+/+ and
IRF-1-/- mice. In these studies peritoneal
exudate macrophages were treated with 10 ng/ml rmIL-12 for 8 h.
Analogous to our in vivo findings, IL-12 treatment up-regulated
IL-12Rß1, IFN-, and IRF-1 mRNA expression in
IRF-1+/+, but not
IRF-1-/-, macrophages (data not shown).
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In addition to IL-12, both IL-15 and IL-18 have been shown to
up-regulate IFN- production in vivo (49, 50). Thus, we
next examined whether these IL-12-independent pathways of IFN-
production were also dysregulated in LPS-challenged
IRF-1-/- mice. As shown in Fig. 6, IRF-1-/- mice
challenged with 25 µg of LPS had 80–90% lower levels of IL-15 mRNA
expression in both the liver and lung than
IRF-1+/+ mice. Following injection with 25 µg
of LPS, neither hepatic nor pulmonary IL-18 mRNA levels were modulated,
and serum IL-18 was not detected (data not shown). While serum IL-18
levels increased to about 500 pg/ml in mice challenged with a lethal
dose of LPS (35 mg/kg), there was no significant difference between
IRF-1+/+ and IRF-1-/-
mice (data not shown).
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dependent
Having demonstrated that IRF-1 is critical for regulation of both
IL-12-dependent and -independent pathways of IFN- production, we
next examined the potential role of endogenous IFN- in the
regulation of IRF-1 mRNA expression in vivo. IFN- knockout mice and
C57BL/6 controls were injected with 25 µg of LPS, and IRF-1 mRNA
expression in the liver was assessed. As shown in Fig. 7, LPS-injected IFN- knockout mice
produced significantly less hepatic IRF-1 mRNA than C57BL/6 controls,
illustrating the importance of endogenous IFN- in the autocrine
regulation of IRF-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induction, as indicated not only by the
loss of LPS-induced IFN-, but also by the inability of exogenous
IL-12 to drive IFN- production in
IRF-1-/- mice.
These in vivo findings extend our previous in vitro work that
demonstrated that IRF-1-/- macrophages produced
substantially less IL-12 p40 and IL-12 p35 mRNA than
IRF-1+/+ macrophages following activation with
LPS or LPS plus IFN- (16). Reduced IL-12 mRNA in
IRF-1-/- macrophages in our earlier study was
also paralleled by impaired production of both IL-12 p70 and IL-12 p40.
In both our in vitro and in vivo studies IL-12 p70 was more profoundly
dysregulated than its antagonist, IL-12 p40. Although IL-12 p40 is
typically produced in excess of IL-12 p70, there is a further increase
in the ratio of IL-12 p40 to IL-12 p70 following administration of LPS
to IRF-1-/- mice. In vivo, administration of
IL-12 p40 homodimers has been shown to ameliorate serum IFN- levels
and block lethality following LPS challenge (51). Thus,
this shift in the availability of antagonist relative to agonist may
also contribute to the loss of LPS-induced IFN- observed both in
vivo (Figs. 1 and 2) and in vitro (16) as well as
the enhanced resistance of IRF-1-/- mice to
LPS-induced lethality.
While IL-12 p40 and IL-12 p35 mRNA were strongly up-regulated in liver,
this appears not to be the case in lung. Pulmonary IL-12 p40 mRNA
increased only 3- to 6-fold, while IL-12 p35 mRNA was constituitively
expressed at relatively high levels and was slightly down-regulated
after LPS challenge. This less modulated pattern of IL-12 mRNA
inducibility in lung vs liver has also been observed following
polymicrobial sepsis and injection of parasite Ags (11, 52). Dysregulation of IL-12 p40 and IL-12 p35 mRNA was not
observed in either the lungs (Fig. 1) or spleen (27) of
LPS-challenged IRF-1-/- mice, suggesting that
IL-12 regulation may be IRF-1 independent in these organs.
Our in vivo data demonstrate that both subunits of the IL-12R are
up-regulated during endotoxemia, illustrating the presence of an
autocrine pathway for amplification of IL-12 responsiveness. This
finding is consistent with that of Thibodeaux et al. (45),
who demonstrated up-regulation of both IL-12R subunits in the lymph
nodes of IL-12-injected mice. While it is well established that
secondary production of IFN- mediates many of the activities of
IL-12, studies using IFN- knockout mice have illustrated that
autocrine regulation of the IL-12R by IL-12 is IFN- independent in
the lymph node (45). Increased IL-12Rß1 and IL-12Rß2
mRNA expression following LPS administration is very likely the result
of endogenous production of both LPS-induced IL-12 and IFN-, as both
receptor subunits are up-regulated in the liver and lungs following
administration of exogenous IL-12 and in murine macrophages treated
with either IL-12 or IFN- (Figs. 4 and 5 and data not shown).
While macrophages have been shown to produce IFN- in response to
IL-12 (8), relatively little is known about the regulation
of macrophage IL-12R expression. Results from this study indicate that
IL-12 and IFN- play an important role in the up-regulation of both
IL-12Rß1 and IL-12Rß2 mRNA in macrophages. This finding is
consistent with the observation that IFN- positively regulates
IL-12Rß2 expression in T cells (17). IL-12
responsiveness in T cells appears to be regulated primarily through the
expression of IL-12Rß2 subunit, as it is selectively expressed on
Th1, but not Th2, cells, while IL-12Rß1 is expressed on both
populations of Th cells. In contrast to T cells, the IL-12Rß1
subunit was more profoundly up-regulated than the IL-12Rß2 subunit in
macrophages, suggesting that T cells and macrophages may control IL-12
responsiveness by selectively up-regulating different IL-12R
subunits.
Results from this study indicate that IRF-1 regulates IL-12
responsiveness not only by controlling IL-12 production, but also by
regulating IL-12R expression in vivo and in macrophage cultures.
Consistent with the losses in IL-12Rß1 and IL-12Rß2 mRNA expression
in the liver and lung, IRF-1-/- mice had an
impaired ability to up-regulate tissue IFN- mRNA expression and
reduced levels of circulating IFN- in response to exogenous IL-12
administration. In addition, peritoneal exudate macrophages from
IRF-1-/- mice did not up-regulate IFN- mRNA
following treatment with IL-12 (data not shown). Our findings are
further supported by the work of Duncan et al. (53), who
reported that NK cell activity from IRF-1-/-
mice could not be augmented by IL-12 and postulated that
IRF-1-/- mice had defects in IL-12-mediated
signaling. The inability of either IL-12 or IFN- to up-regulate
IL-12Rß1 in murine macrophage cultures suggests that IRF-1 may be
directly involved in regulation of the IL-12Rß1
gene. In the future, promoter analysis studies will be required to
address more fully the role of IRF-1 in transcriptional regulation of
IL-12Rß1 and IL-12Rß2
genes. Moreover, whether IRF-1 regulates IL-12Rß1 and IL-12Rß2 mRNA
expression in other IL-12R-expressing cells, such as T cells, NK cells,
or B cells, has yet to be determined.
As studies have shown that NK1.1+
TCR-ß+ T (NK1+ T)
cells and NK cells are reduced in number in
IRF-1-/- mice (54), we cannot
exclude their possible contribution to the loss of IL-12R expression as
potential contaminating cells in our in vitro studies with primary
macrophages. However, the finding that IFN- up-regulated both
IL-12Rß1 and IL-12Rß2 mRNA in the macrophage cell line, RAW 264.7,
indicates that both IL-12R subunits are IFN--inducible genes in
macrophages. These data in conjunction with the relatively low cycle
number required to detect basal IL-12R mRNA levels in
IRF-1-/- peritoneal exudate preparations, the
finding that basal levels of IL-12Rß1, but not IL-12Rß2, were
reduced, and the inability of either IL-12 or IFN- to up-regulate
IL-12Rß1 in IRF-1-/- macrophages suggest that
impaired IL-12Rß1 inducibility was very likely a direct result of the
lack of IRF-1 regulation. Conversely, it is likely that loss of
NK1+ T cells and NK cells contribute at least in
part to the reduced expression of IL-12Rß1 and IL-12Rß2 in vivo.
NK1+ T cells, while rare in the periphery, are
found in markedly higher numbers in the liver (55). Both
NK1+ T cell and NK cell numbers are reduced in
the livers of IRF-1-/- mice (54).
The defect in NK cell number, however, was not observed in
IRF-1-/- mice injected with
polyinosinic:polycytidylic acid (53), indicating that the
appropriate in vivo signals in IRF-1-/- mice
can overcome these deficiencies. This is analogous to the finding that
there is a recovery of the number of NK1+ T cells
and NK cells following incubation of hepatic mononuclear cells from
IRF-1-/- mice with IL-15 (54).
Whether these subpopulations increase in number in
IRF-1-/- mice after LPS challenge, as observed
following polyinosinic:polycytidylic acid injection, or are impacted by
the low levels of endogenously produced IL-15 in
IRF-1-/- mice remains to be established.
In vivo, LPS up-regulates levels of steady state IRF-1 mRNA in the
livers and lungs of wild-type control mice (Table I), extending our
previous in vitro studies that demonstrated that LPS induces IRF-1 mRNA
in murine macrophages in a cycloheximide-independent manner
(41). This study also demonstrates that both IL-12 and
IFN- are important regulators of IRF-1 mRNA expression. Both IFN-
and IL-12 increased IRF-1 mRNA expression in murine macrophages, while
hepatic and pulmonary IRF-1 mRNA expression increased following the
administration of IL-12. The diminution in IRF-1 mRNA expression in the
livers of IFN- knockout mice suggests that secondary production of
IFN- is necessary to sustain heightened levels of IRF-1 mRNA in
vivo. Thus, IRF-1 is not only involved in the direct and/or indirect
regulation of IL-12 and IFN-, but these cytokines feed back to
up-regulate IRF-1 expression, further amplifying the IL-12/IFN-
pathway.
While IL-12 plays a prominent role in driving production following LPS
challenge, the strength of the IFN- response is positively
influenced by other cytokines. In this context, both IL-15 and IL-18
have been shown to synergize with IL-12 to induce IFN- production
from T cells, NK cells, and macrophages (56, 57, 58) as well
as contribute to IFN- production in vivo (49, 50). We
found that the IL-15 mRNA response in both liver and lung was
attenuated in IRF-1-/- mice, while the IL-18
response remained intact. Thus, it is likely that loss of IL-15 in
IRF-1-/- mice also contributes to the impaired
production of IFN- observed in vivo. Whether IRF-1 directly or
indirectly regulates IL-15 expression remains a possibility to be
explored.
In summary, studies in IRF-1 knockout mice have illustrated that the
transcription factor IRF-1 plays an essential role in the regulation of
the LPS-induced cytokine cascade leading to IFN- production and that
this regulation occurs at multiple levels. IRF-1 controls both the
IL-12-dependent and -independent pathways of IFN- production by
regulating IL-12, IL-12R, and IL-15 expression as well as the
maturation of the predominant populations of IFN--producing cells.
Moreover, these studies in conjunction with work by other investigators
illustrate a complex pathway of gene regulation (Fig. 8) by which IRF-1 regulates IL-12 and
IL-12R expression and ultimately IFN- production. IFN- functions
in an autocrine manner, up-regulating the transcription factor, IRF-1,
and its target genes, IL-12 and IL-12R. Similarly, IL-12 feeds back to
up-regulate IFN-, IRF-1, and IL-12R. The result is the amplification
of the IFN--dependent arm of the immune response to LPS.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814.
3 Abbreviations used in this paper: IRF, IFN regulatory factor; ICSBP, IFN consensus sequence binding protein; rmIL-12, recombinant murine IL-12; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
Received for publication May 2, 2000. Accepted for publication July 13, 2000.
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References |
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