By
From * R.W. Johnson Pharmaceutical Research Institute, San Diego, California 92121; R.W. Johnson
Pharmaceutical Research Institute, Raritan, New Jersey 08869; and the § Department of Immunology
and Molecular Biology, Division of Virology, Scripps Research Institute, La Jolla, California 92037
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Abstract |
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Interleukin (IL)-18 is functionally similar to IL-12 in mediating T helper cell type 1 (Th1) response and natural killer (NK) cell activity but is related to IL-1 in protein structure and signaling, including recruitment of IL-1 receptor-associated kinase (IRAK) to the receptor and activation of c-Jun NH2-terminal kinase (JNK) and nuclear factor (NF)-B. The role of IRAK in
IL-18-induced responses was studied in IRAK-deficient mice. Significant defects in JNK induction and partial impairment in NF-
B activation were found in IRAK-deficient Th1 cells,
resulting in a dramatic decrease in interferon (IFN)-
mRNA expression. In vivo Th1 response
to Propionibacterium acnes and lipopolysaccharide in IFN-
production and induction of NK cytotoxicity by IL-18 were severely impaired in IRAK-deficient mice. IFN-
production by activated NK cells in an acute murine cytomegalovirus infection was significantly reduced despite
normal induction of NK cytotoxicity. These results demonstrate that IRAK plays an important
role in IL-18-induced signaling and function.
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Introduction |
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Interleukin (IL)-18 (previously known as IFN--inducing
factor, or IGIF) was cloned from the liver of mice primed
with Propionibacterium acnes, followed by challenge with LPS
(1). IL-18 induces production of IFN-
from Th1 cells and
NK cells (2). In addition, IL-18 enhances NK cell cytotoxicity and also synergizes with IL-12 in potentiating
IFN-
production and NK cell cytotoxicity (1, 3, 4). Thus,
cellular responses to IL-18 are similar to the biological functions known to be elicited by IL-12 (6).
Despite functional similarities, IL-12 and IL-18 differ in
their protein structure and exert these overlapping and synergistic biological effects via different mechanisms. IL-12
mediates Th1 responses and IFN- production via activation
of Janus kinases (JAKs),1 JAK2 and TYK2 (7), and transcription factors called signal transducer and activator of transcription (STAT), STAT3 and STAT4 (8). STAT4-deficient mice are defective in mounting Th1 responses and
IFN-
production (9, 10). Stimulation with IL-18 results in activation of c-Jun NH2-terminal kinase (JNK) and transcription factor nuclear factor (NF)-
B (2, 4).
IL-18 is similar in tertiary structure to proteins of the IL-1
family (11). Similar to IL-1, IL-18 is synthesized as an inactive precursor protein and is cleaved to the active form
by IL-1
-converting enzyme (ICE, also named caspase 1;
references 12, 13). Cleavage of IL-18 by ICE is essential for
the biological effects of IL-18. ICE-deficient mice exhibit
defects similar to those observed in IL-18
/
mice, such as
reduced IFN-
production in response to LPS injection (3,
12, 13). The IL-18 receptor was originally identified as IL-1
receptor-related protein (IL-1Rrp; references 14, 15). Receptors for IL-1, IL-18, and the recently identified mammalian Toll-like receptors (16) are evolutionarily conserved
and homologous to the Drosophila protein Toll (17). Toll
mediates activation of Dorsal, an NF-
B-like molecule, via
the serine threonine kinase Pelle and the adapter protein
Tube (17, 18). The IL-1 signaling pathway in mammals is
similar to the Toll pathway. NF-
B activation by IL-1 requires the interaction of the Pelle-like kinase IL-1 receptor-
associated kinase (IRAK) with the IL-1 receptor complex via the adapter protein MyD88 (19). IL-18 also triggers
phosphorylation of IRAK and its recruitment to the IL-18
receptor complex (4, 22). However, IL-1 and IL-18 act on
different cell types and lead to divergent cellular responses.
For example, IL-18 has been implicated primarily in inducing IFN-
from NK and Th1 cells (1), whereas IL-1 is a
potent inducer of IL-6 from fibroblasts and macrophages during inflammation (23). We have recently demonstrated that IRAK is required for optimal induction of IL-1 signaling, including JNK, p38, and NF-
B activation (25). Defective IL-1 signaling in IRAK-deficient fibroblasts results in
impaired IL-6 induction (25). Although IL-18 signaling is
known to involve activation of JNK and NF-
B (2, 4),
details of IL-18 signaling have not been well characterized.
It is also unclear whether IL-18 uses IRAK in pathways
similar to IL-1 signaling to elicit distinct cellular responses.
To determine the role of IRAK in IL-18-mediated responses, we analyzed IL-18-induced signaling and function
in IRAK-deficient mice.
In this report we showed that IRAK was essential for
IL-18-mediated activation of JNK and was also involved in
NF-B activation. Signaling defects in IRAK-deficient
Th1 cells resulted in a dramatic decrease in IFN-
expression. Serum IFN-
increase in response to P. acnes and LPS
treatment was severely impaired. IRAK-deficient mice also
exhibited defects in NK IFN-
production in an acute murine cytomegalovirus (MCMV) infection. NK cell cytotoxicity induced by IL-18 was defective, although its induction was normal in MCMV infection. These results suggest
that IRAK plays an important role in IL-18-mediated signaling and function.
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Materials and Methods |
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Generation of IRAK-deficient Mice.
The mouse IRAK gene in embryonic stem (ES) cells was disrupted by homologous recombination as described in our previous report (25). In brief, the mouse IRAK gene was disrupted by replacement of a 940-bp region covering exons 5-7 of the gene with a neomycin resistance gene. Chimeric mice were generated from embryos injected with ES cells. Germline mice were obtained from breeding of chimeric male mice with C57BL/6J females. Because the IRAK gene is on X chromosome (sequence data available from EMBL/GenBank/ DDBJ under accession No. U52112) and the ES cell line was derived from a male embryo, all the germline female mice were heterozygous for the disrupted IRAK gene. IRAK-deficient male mice carrying only the disrupted IRAK gene were obtained from breeding of heterozygous female mice with wild-type littermates. IRAK-deficient female mice were obtained from breeding of heterozygous females with IRAK-deficient males.Phenotypic Analysis of T Cells.
Thymocytes and splenocytes were stained with CD4- and CD8-specific antibodies (PharMingen). Enriched CD4+ T cells before and after 5 d of differentiation were stained with antibodies specific for CD25 and CD44 (PharMingen). Cells after antibody staining were analyzed using a FACScanTM (Becton Dickinson).Preparation of Th1/Th2 Cells.
CD4+ T cells were purified from lymph node and spleen cells by depletion of B cells and CD8+ T cells using guinea pig and rabbit complements and a combination of antibodies from hybridoma lines J11d, 28-16-8s, and 3-168. Purity of CD4+ T cells in different preparations was ~90%. Enriched CD4+ T cells were activated with immobilized anti-CD3 (PharMingen), which was coated overnight onto 6-well plates at 5 µg/ml. Differentiation of T cells towards Th1 cells was triggered by addition of 5 ng/ml IL-12 (R&D Systems) and 5 µg/ml anti-IL-4 (PharMingen) in RPMI medium with 10% FCS. Th2 cell differentiation was driven by supplementing culture medium with 5 ng/ml IL-4 (R&D Systems) and 5 µg/ml of anti-IFN-Cell Stimulation.
After 5 d of differentiation, Th1 cells were washed once with serum-free RPMI medium and starved for 3 h before stimulation. Cells were treated with different stimuli at 37°C. The different stimuli used in the assays include: IL-18 (PeproTech, Inc.), IL-12 (R&D Systems); IL-1In Vitro Kinase Assay.
Cell lysates (5 × 106 cells in 100 µl of lysis buffer) were immunoprecipitated with JNK1 kinase antibody (Santa Cruz Biotechnology). An in vitro kinase assay was performed using glutathione S-transferase (GST)-c-Jun (Santa Cruz Biotechnology) as a substrate in a reaction buffer containing 25 mM Hepes, pH 7.4, 25 µM ATP, 10 mM MgCl2, 1 µg GST- c-Jun, and 10 µCi [Western Blotting.
Western blot analyses were carried out as previously described (26). For detection of INF-B Mobility Shift Assay.
Northern Blot Analysis.
Total RNA was extracted from cells or spleens with RNAzol (Tel-Test Inc.). RNA was separated on 1% agarose-formaldehyde gels and transferred to nylon membranes (Amersham Pharmacia Biotech). Filters were hybridized to cDNA probes specific for IFN-Cytokine Detection.
IFN-Cell Proliferation Assay.
Th1 cells were plated in 96-well plates at 105/well and treated with different cytokines for 24 h. [3H]thymidine (1 µCi/well) was then added for 16 h, and radioactivity incorporated in dividing cells was measured using a Topcount Microplate Scintillation Counter (Packard).In Vivo Th1 Response.
Mice were injected intraperitoneally with PBS as controls or PBS with 2 mg of heat-killed P. acnes (Van Kempen Group, Inc.). 7 d later, control mice were injected intravenously with PBS, whereas P. acnes-primed mice were injected with 1 µg LPS (Sigma Chemical Co.). Mice were bled 6 h after LPS challenge, and serum IFN-NK Cytotoxic Assay.
Mice were injected intraperitoneally daily with PBS alone as controls or PBS containing 1 µg IL-18 or 200 µg poly(I):poly(C) (Sigma Chemical Co.) for 2 d. Spleen cells prepared from these mice were incubated with 51Cr-labeled YAC-1 target cells for 4 h at 37°C at different E/T ratios. After 4 h of incubation, 51Cr released from target cells was counted using a gamma counter (Packard). Specific lysis was calculated as: (measured 51Cr releaseMCMV Infections.
The Smith strain of MCMV was obtained from the American Type Culture Collection (VR-1399). MCMV stocks were prepared in NIH 3T3 murine fibroblasts, and determination of viral titers was carried out by a standard plaque assay (27). Mice were intraperitoneally injected with 108 PFU of tissue culture-propagated MCMV in 400 µl DMEM. Animals were killed on day 3 after inoculation. Blood sera for cytokine detection were collected by bleeding mice daily starting from day 0 before viral inoculation. Spleens were collected after 71 h of infection for NK cytotoxic assays and RNA extraction. ![]() |
Results |
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IL-18 by itself has no effect on Th1 differentiation but can synergize IL-12-driven Th1 development (4).
In differentiated helper T cells, IL-18 exerts its biological
effects only on Th1 cells but not on Th2 cells (4). Since
IRAK is involved in IL-18 signaling (4, 22), we examined
helper T cell development and phenotype in IRAK-deficient mice. Development of T cells in the thymus and distribution of mature CD4+ and CD8+ T cells in the primary
and secondary lymphoid organs of IRAK-deficient mice appeared to be normal (Fig. 1 A). The vast majority of CD4+
T cells were characterized as CD25loCD44lo naive phenotype and were comparable to the cells harvested from control
wild-type mice (Fig. 1 B). Th1 and Th2 cells were prepared by in vitro differentiation of CD4+ T cells from IRAK-deficient and wild-type mice. CD4+ T cells were enriched from
lymph node cells and splenocytes by antibody and complement depletion as described in Material and Methods. Enriched CD4+ T cells were activated with immobilized anti-CD3 and differentiation towards Th1 cells was triggered by
coculture with IL-12 and anti-IL-4. Th2 cell differentiation was driven by IL-4 and anti-IFN- plus anti-IL-12.
Activation of IRAK-deficient CD4+ T cells appeared to be
normal, as indicated by the upregulation of the activation
marker CD25 and the memory T cell marker CD44 (Fig.
1 B). Numbers of blasting T cells after 5 d of differentiation were similar between IRAK-deficient and wild-type T cells
(data not shown), suggesting normal proliferation of activated IRAK-deficient T cells. Cytokines expressed by T cells
after 5 d of differentiation were characterized by ELISA.
Similar to wild-type Th1 cells, IRAK-deficient Th1 cells secreted predominantly IFN-
, whereas IL-4 was undetectable (Fig. 1 C). IRAK-deficient Th2 cells also showed a
typical Th2 cytokine profile similar to that of wild-type
Th2 cells, with high amounts of IL-4 but minimal levels of
IFN-
(Fig. 1 C). Our data suggest that IRAK-deficient T
cells can differentiate into Th1 or Th2 effector cells.
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The stress-activated protein kinase (SAPK) family of mitogen-activated protein (MAP) kinases JNK and p38 are rapidly activated by proinflammatory cytokines such as IL-1 and
TNF- (24, 25, 28). IL-18 stimulation of Th1 cells also results in JNK activation (2). JNK plays a role in induction of
activator protein (AP)-1-dependent genes via phosphorylation of the transcription factor c-Jun (28, 29). We have shown
previously that IRAK-deficient fibroblasts are defective in
IL-1-mediated JNK activation (25). To determine the role of
IRAK in IL-18-induced JNK activation, JNK activity in Th1
cells was studied by immunoprecipitating JNK1 with a specific antibody followed by an in vitro kinase assay using the
GST-c-Jun protein as a substrate. Activation of JNK was
minimal in IL-18-treated IRAK-deficient Th1 cells as opposed to the significant JNK activation observed in wild-type
cells (Fig. 2). In contrast, TNF-
induced similar levels of
JNK activity in both wild-type and IRAK-deficient Th1
cells, whereas IL-1
did not have any effects on either samples (Fig. 2). IL-18 and IL-1 have been suggested to act on
Th1 and Th2 cells, respectively, to induce signaling and
cellular responses (4). In our studies, Th1 cells do not respond
to IL-1
in JNK activation and this is consistent with a previous observation (4), which suggested that IL-1 does not induce signaling in Th1 cells due to the lack of IL-1 receptor
expression. Our results indicate that the defect in JNK activation observed in IRAK-deficient Th1 cells is IL-18 specific.
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IL-18
binding to its receptor mediates activation of NF-B (4, 22).
In unstimulated cells, NF-
B is present in the cytoplasm as
an inactive complex sequestered by its inhibitory partners, I
B (30). Upon activation, I
B proteins are phosphorylated
by I
B kinases IKK1 and IKK2, and subsequently degraded
by proteosomes to allow nuclear translocation and activation of NF-
B (30). To determine the role of IRAK
in IL-18-induced NF-
B activation, I
B-
protein levels
were determined in Th1 cells stimulated with IL-18 for
different time courses (Fig. 3 A). Reduced levels of I
B-
due to protein degradation were observed in both wild-type and IRAK-deficient cells after IL-18 treatment (Fig.
3 A). Maximum degradation of I
B protein occurred 15 min
after stimulation and returned to 80% of original levels within
60 min of stimulation. The extent of I
B-
protein degradation mediated by IL-18 appeared to be less in IRAK-deficient Th1 cells as compared with that in wild-type cells.
Similar results were observed when wild-type and IRAK-deficient Th1 cells were stimulated with different concentrations of IL-18 (data not shown). This result was further
confirmed by NF-
B DNA binding activity in nuclear
extracts determined by mobility shift assay. IL-18-induced
NF-
B activation in both IRAK-deficient and wild-type
cells, but NF-
B DNA binding activity was slightly lower
in IRAK-deficient cells than in wild-type cells (Fig. 3 B).
Stimulation with TNF-
or phorbol ester plus ionomycin
induced comparable levels of NF-
B activation in both cell
types, indicating that the partial defect in NF-
B activation
observed in IRAK-deficient cells is restricted to IL-18 stimulation. These results suggest that involvement of IRAK in
IL-18-mediated NF-
B activation is dispensable.
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IL-18 induces IFN- production from Th1 cells (1, 5).
Stimulation of Th1 cells with a combination of IL-18 and
IL-12 results in synergistic induction of IFN-
production
(4). To determine whether IRAK is required for induction
of IFN-
by IL-18 itself or in combination with IL-12,
IFN-
mRNA expression was determined by Northern
blot analysis. IFN-
was significantly induced by IL-18 in
wild-type cells but its induction in IRAK-deficient cells
was minimal (Fig. 4 A). A suboptimal dose of IL-12 was
used in our experiments, which induced minimal amounts
of IFN-
, and no difference was observed between IRAK-deficient and wild-type cells. Synergistic induction of IFN-
expression by a combination of IL-18 and IL-12 was substantially decreased in IRAK-deficient cells as compared with
wild-type cells. This result shows that IRAK is required for
optimal induction of IFN-
by IL-18.
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It has been demonstrated that treatment with LPS in
P. acnes-sensitized mice results in a significant increase in
serum IFN- (33). IL-18-deficient mice exhibited a minimal increase in serum IFN-
under these conditions (3). To
determine the role of IRAK in IL-18-dependent IFN-
production in vivo, IRAK-deficient mice were tested in
this experimental system. Mice were injected intraperitoneally
with heat-killed P. acnes and 7 d later were injected intravenously with LPS. IFN-
in the serum was detected by
ELISA 6 h after LPS treatment. Serum IFN-
levels were
significantly lower in IRAK-deficient mice as compared with
wild-type animals (Fig. 4 B). Consistent with these data,
IFN-
mRNA expression in the spleen was substantially
reduced in IRAK-deficient mice. In contrast, induction of
IL-18 mRNA expression was comparable between wild-type and IRAK-deficient animals (Fig. 4 C). These results
suggest that the reduced IFN-
production in IRAK-deficient mice is not due to a change in IL-18 levels but rather
originated from defects in IL-18 signaling.
Similar
to induction in IFN- production, proliferation of Th1
cells was also enhanced by IL-18 or IL-12, and synergized by the combination of both (4). The effect of IL-18 and its synergism with IL-12 on proliferation of IRAK-deficient
Th1 cells was studied. Wild-type and IRAK-deficient Th1
cells were treated with different concentrations of IL-18,
IL-12, or IL-18 plus IL-12. Proliferation of Th1 cells after
24 h of stimulation was determined by [3H]thymidine uptake. As shown in Fig. 5, proliferation of wild-type Th1
cells was significantly enhanced by IL-18 in a dose-dependent manner, whereas the effect of IL-18 on proliferation
of IRAK-deficient cells was minimal. At concentrations as
low as 2 ng/ml, IL-12 stimulated proliferation of both wild-type and IRAK-deficient cells to maximal extent. Combination of IL-18 and IL-12 resulted in a synergistic proliferative
response in both cell types, and no significant difference
was observed. Consistent with the defects shown in IFN-
expression, IRAK-deficient cells are also impaired in proliferative response to IL-18, confirming the role of IRAK in IL-18-mediated cellular responses.
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IL-18 has been shown to enhance NK cell cytotoxicity (1). Reduced NK activity was reported in IL-18- deficient mice (3). To determine whether IRAK is involved in IL-18-induced NK activity, mice were injected intraperitoneally with IL-18 for 2 d consecutively, and NK activity in splenocytes was assayed using 51Cr-labeled YAC-1 as target cells. Basal NK cell activities in PBS injected wild-type and IRAK-deficient mice were comparable (Fig. 6). IL-18 injection resulted in significant increase in NK activity in wild-type animals but its effect in IRAK-deficient mice was minimal (Fig. 6). However, injection of the double-stranded RNA poly(I):poly(C), an inducer of IFNs, resulted in a pronounced NK activity in both IRAK-deficient and wild-type animals (Fig. 6), indicating that IRAK is required specifically for IL-18-mediated induction of NK cytotoxic activity.
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NK cells are the major effector
cells in the early defense against viral infections. Both NK
cytotoxicity and IFN- production by NK cells are induced in mice upon MCMV infection (34). IL-12 has been
shown to be responsible for induction of IFN-
in NK cells
(35), whereas possible roles of IL-18 in NK activities during MCMV infection have not been reported. IRAK-deficient mice were infected with MCMV to study the involvement of IRAK in NK activity during viral infection.
Splenocytes obtained from mice on day 3 of MCMV infection were assayed for NK cytolytic activity using 51Cr-
labeled YAC-1 cells as targets. Dramatic increase in NK cytotoxicity was observed in both MCMV-infected wild-type
and IRAK-deficient mice and no significant difference was
found between the two types of mice (Fig. 7 A). IFN-
produced by NK cells during MCMV infection can be detected as an increase in serum IFN-
levels. Kinetics of
IFN-
increase in sera during the first 3 d of MCMV infections was studied. In both wild-type and IRAK-deficient
mice, IFN-
levels peaked at 45 h of infection (Fig. 7 B).
However, maximal IFN-
levels in IRAK-deficient mice
were significantly lower than those in wild-type control
mice (Fig. 7 B). Similar results were obtained in studies of
IFN-
mRNA expression. IFN-
mRNA in the spleens
was undetectable in uninfected mice but was induced significantly in MCMV infected mice (Fig. 7 C). IFN-
mRNA
induced by MCMV was less significant in IRAK-deficient
mice than that in wild-type mice. IL-18 expression in response to MCMV infection was also studied. IL-18 mRNA
was expressed at low levels in the spleens of uninfected
mice and was induced strongly on day 3 of MCMV infection (Fig. 7 C). IL-18 expression in IRAK-deficient mice
was comparable to that in wild-type mice under healthy
and viral-infected conditions (Fig. 7 C). Thus, impairment in
viral-induced IFN-
production in IRAK-deficient mice is
not due to the levels of IL-18 expression, suggesting that
other mechanisms, such as IL-18 signaling, may be responsible for the defects.
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Discussion |
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We have demonstrated IL-18 signaling defects in IRAK-deficient Th1
cells. Impairment is significant in JNK activation but much
less obvious in NF-B induction. The partial activation of NF-
B suggests that other mechanisms can compensate
for the function of IRAK. However, the role of IRAK in
JNK activation is essential. We have previously reported
that IRAK-deficient fibroblasts are defective in both NF-
B
and JNK/p38 pathways induced by IL-1 (25). The defects
in IL-18 signaling that we observed here are similar to the
defects in IL-1 signaling (25). Impairment in NF-
B activation can be overcome by high concentrations of IL-1, but
similar treatment cannot correct defects in JNK activation
(25). Taken together, our results suggest that IRAK is used
similarly by both IL-18 and IL-1 in mediating intracellular signaling.
IL-1 signaling leading to NF-B activation has been relatively well characterized. Upon IL-1 binding to its receptor, IRAK is rapidly recruited to the receptor complex via
MyD88 (20). Activated IRAK interacts with TNF receptor-associated factor (TRAF)6, which in turn activates
NF-
B-inducing kinase (NIK) (36, 37). NIK is involved
in the NF-
B pathway by activating I
B kinases (IKKs)
(31, 32, 38, 39). Activated IKKs phosphorylate I
B for degradation, allowing nuclear translocation of NF-
B for gene
induction (30). However, details of IL-1-mediated signaling leading to JNK activation are still unclear. It has been
reported that only MyD88 but not TRAF6 is essential for
JNK activation (40). Since IRAK is positioned between
MyD88 and TRAF6 in the signaling cascade, IRAK could
be the bifurcating molecule for both NF-
B and JNK
pathways. IL-18 stimulation also induces the stress-activated MAP kinase JNK (2). The defects in JNK activation in IRAK-deficient cells indicate that IRAK is essential for
JNK activation but intermediary molecules linking IRAK
to JNK have not been identified.
Recent reports on IL-1 signaling also suggest additional
complexity and divergence in this pathway. In addition to
IRAK, other proteins, including IRAK homologue IRAK2,
are reported to interact with the IL-1 receptor (19, 41).
IRAK2 also interacts with MyD88 and TRAF6 (19). A
dominant negative form of IRAK2 mutant blocks MyD88-induced NF-B activation (19). Although overexpression of
IRAK in transfection studies has been reported to activate NF-
B but not JNK (42), our studies demonstrate that
IRAK is essential for IL-18-mediated activation of JNK
but its role in NF-
B pathway is less critical. In the absence
of IRAK, NF-
B activation can still occur, possibly mediated by other related kinases such as IRAK2. The relative
role of IRAK and IRAK2 in inducing JNK and NF-
B
downstream of MyD88 is still unclear and the involvement
of IRAK2 in IL-18 signaling remains to be elucidated.
IL-18 alone does not support Th1 differentiation, but it can
potentiate Th1 development driven by IL-12 (4). In differentiated Th1 cells, IFN- production can be induced by
IL-18 or IL-12 alone, and this effect can be further synergized by a combination of both cytokines (4). We observed
no defect in Th1 differentiation of IRAK-deficient cells
mediated by IL-12, confirming that IL-18 signaling does
not play an essential role in Th1 development. IL-18-induced
IFN-
expression in IRAK-deficient Th1 cells is significantly decreased, although NF-
B activation is only partially impaired. Increase in serum IFN-
levels as a result
of a Th1 response to P. acnes and LPS treatment, or an NK
response in the early phase of MCMV infection, was also
reduced significantly in IRAK-deficient mice. The results
suggest that optimal gene induction by IL-18 requires
proper activation of multiple signaling pathways including
NF-
B and JNK. Dramatic decrease in IFN-
production in IRAK-deficient cells may have resulted from the impairment in JNK activation even though NF-
B activity was
not severely affected. We also observed similar phenomena
in our previous studies; IL-6 induction in IRAK-deficient
fibroblasts in response to IL-1 treatment was significantly
reduced due to defects in JNK/p38 activation despite minimal impacts on NF-
B activation (25). The involvement
of both JNK and NF-
B pathways may imply that optimal
gene expression depends on the binding of multiple transcription factors including NF-
B and AP-1 onto the promoter regions of the target genes. It is also possible that the
transactivation potential of NF-
B is modulated by the kinase activity of JNK and the other MAP-related kinase
p38, as suggested in studies of TNF-induced IL-6 gene expression (43).
IL-18 and IL-12 share many biological properties, although
different signaling pathways are used by the two cytokines.
Transcription factor STAT4 is activated by IL-12 whereas
NF-B and AP-1 are activated by IL-18. The binding sites
of these transcription factors within the IFN-
promoter
region have been identified (44). IL-18 alone can directly
induce IFN-
promoter activity via AP-1, whereas IL-12-
mediated induction of the promoter activity requires both
AP-1 and STAT4 (44). Synergistic expression of IFN-
by IL-18 and IL-12 probably results from the interplay of multiple transcription factors in differential regulation of IFN-
promoter activity. A strong synergistic effect in IFN-
induction and cell proliferation was observed in IRAK-deficient Th1 cells when treated with combination of IL-12 and
IL-18. The results suggest that minimal activation of NF-
B
and JNK by IL-18 in IRAK-deficient cells is sufficient to
function synergistically with IL-12 to achieve a significant
synergistic response.
MCMV infection in mice results in a strong NK response in the early phase
of infection before the onset of T and B cell responses (34).
Induction of NK cell IFN- production and cytotoxicity peaks on day 2-5 of infection (34, 45). IFN-
production
by NK cells is the major defense mechanism in controlling
MCMV replication (34). It has been well characterized that
in MCMV infection IL-12 plays a major role in NK cell
IFN-
production, whereas induction of NK cytotoxicity
is regulated by IFN-
/
(35, 46). Although IL-18 is known
to be involved in NK cell function, the role of IL-18 in
NK responses during MCMV infection has not been investigated. We have undertaken this study to understand the importance of IRAK in IL-18-mediated responses in a viral
infection. MCMV-infected IRAK-deficient mice have a
normal induction of IL-18 expression but exhibited a significant decrease in IFN-
induction and in serum IFN-
.
The results in this study suggest that IL-18 plays a distinct
role in IFN-
production during the NK response to
MCMV infection and that its function cannot be overcome
completely by IL-12 or other mechanisms. However, we
cannot rule out the possibility that IRAK may also play a
role in viral infections via its involvement in other related
receptors in addition to the IL-18 receptor. Although we
have demonstrated a defect in IL-18-induced NK cytotoxicity in IRAK-deficient mice, MCMV infection or poly(I):
poly(C) injection triggers a normal NK cytotoxicity in these
mice. Our results suggest that the role of IL-18 in NK cytotoxicity can be compensated by IFN-
/
or other mechanisms during MCMV infection. Studies are underway in
our laboratory to understand the mechanisms of MCMV-induced immune responses in IRAK-deficient mice.
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Footnotes |
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Address correspondence to Wai-Ping Fung-Leung, 3535 General Atomics Ct., Suite 100, R.W. Johnson Pharmaceutical Research Institute, San Diego, CA 92121. Phone: 619-450-2016; Fax: 619-450-2070; E-mail: wleung{at}prius.jnj.com
Received for publication 19 November 1998 and in revised form 3 February 1999.
We thank Julie Culver and Michelle Courtney for their excellent technical assistance, and Lars Karlsson for critical review of the manuscript.
This work was supported in part by grants from the National Institutes of Health to P. Ghazal (CA66167 and AI30627). P. Ghazal is a Scholar of the Leukemia Society of America. A. Angulo is a Fellow from the University of California Universitywide AIDS research program.
Abbreviations used in this paper
AP-1, activator protein 1;
ES, embryonic
stem;
GST, glutathione S-transferase;
ICE, IL-1 converting enzyme;
I
B, inhibitor of NF-
B;
IKK, I
B kinase;
IRAK, IL-1 receptor-associated kinase;
JAK, Janus kinase;
JNK, c-Jun NH2-terminal kinase;
MAP, mitogen-activated protein;
MCMV, murine cytomegalovirus;
NF, nuclear factor;
STAT, signal transducer and activator of transcription;
TRAF, TNF receptor-associated factor.
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