The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheung, H.
Right arrow Articles by Yeh, W.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheung, H.
Right arrow Articles by Yeh, W.-C.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
*Substance via MeSH
The Journal of Immunology, 2005, 174: 5351-5357.
Copyright © 2005 by The American Association of Immunologists

Accessory Protein-Like Is Essential for IL-18-Mediated Signaling1

Heidi Cheung2,*, Nien-Jung Chen2,*, Zhaodan Cao{dagger}, Nobuyuki Ono*, Pamela S. Ohashi* and Wen-Chen Yeh3,*

* Advanced Medical Discovery Institute/Campbell Family Institute for Breast Cancer Research, University Health Network and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; and {dagger} Amgen, South San Francisco, CA 94080


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-18 is an essential cytokine for both innate and adaptive immunity. Signaling by IL-18 requires IL-18R{alpha}, which binds specifically to the ligand and contains sequence homology to IL-1R and TLRs. It is well established that IL-1R signaling requires an accessory cell surface protein, AcP. Other accessory proteins also exist with roles in regulating TLR signaling, but some have inhibitory functions. An AcP-like molecule (AcPL) has been identified with the ability to cooperate with IL-18R{alpha} in vitro; however, the physiological function of AcPL remains unknown. In this study, we demonstrate that IL-18 signals are abolished in AcPL-deficient mice and cells. Splenocytes from mutant mice fail to respond to IL-18-induced proliferation and IFN-{gamma} production. In particular, Th1 cells lacking AcPL fail to produce IFN-{gamma} in response to IL-18. AcPL-deficient neutrophils also fail to respond to IL-18-induced activation and cytokine production. Furthermore, AcPL is required for NK-mediated cytotoxicity induced by in vivo IL-18 stimulation. However, AcPL is dispensable for the activation or inhibition of IL-1R and the various TLR signals that we have examined. These results suggest that AcPL is a critical and specific cell surface receptor that is required for IL-18 signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-18, an IL-1-related cytokine, was identified in the liver of mice sequentially treated with Propionibacterium acnes and LPS (1, 2). Originally known as IFN-{gamma}-inducing factor, IL-18 is secreted by macrophages and induces IFN-{gamma} production from splenocytes, liver lymphocytes, and Th1 cells. In addition, IL-18 enhances NK cell cytotoxicity and synergizes with IL-12 to induce IFN-{gamma} production (3, 4). These studies suggest a versatile function for IL-18 in both innate and adaptive immunity. Recently, it has also been demonstrated that the function of IL-18 in innate immunity can extend to neutrophil activation. IL-18 therefore may be implicated in inflammatory disorders (5).

Signaling of IL-18 requires ligand-specific binding to IL-18R{alpha} (also known as IL-1R-related protein) (6). However, binding of IL-18 to IL-18R{alpha} alone does not trigger downstream signals such as NF-{kappa}B (7). In contrast, expression of a cell surface protein called accessory protein-like (AcPL)4 in the presence of IL-18 and IL-18R{alpha} is able to activate an NF-{kappa}B-dependent reporter in transient transfection assays (7). This is reminiscent of IL-1 signaling, which involves both IL-1R type I and IL-1R accessory protein (IL-1RAcP) (8, 9). Gene-targeting studies have revealed that IL-18R{alpha}–/– mice are defective in IL-18-mediated biological functions (6, 10). However, the physiological functions of AcPL remain to be investigated.

Both IL-18R{alpha} and AcPL contain an evolutionarily conserved domain called the TLR/IL-1R/plant R gene (TIR) domain in their cytoplasmic regions. The TIR domain is commonly found in the IL-1R and mammalian TLR family members and is required for receptor signaling (11, 12). One common signaling cascade induced by TIR-containing receptors involves the recruitment of the adaptor protein MyD88, which subsequently recruits IL-1R-associated kinase (IRAK)-1 and IRAK-4 to the receptor complex (13, 14, 15). After activation, IRAK-1 is translocated to a TNFR-associated factor 6-containing protein complex (16, 17), leading to the trigger of downstream signals including NF-{kappa}B and various MAPK pathways. Indeed, IL-18 signal transduction requires this common cascade as demonstrated by gene-targeting studies showing that MyD88, IRAK-1, and IRAK-4 play critical roles in IL-18 responses (15, 18, 19). Whether the signals are transmitted through IL-18R{alpha} or AcPL or both remains unclear.

Like IL-18R{alpha} or AcPL, T1/ST2 and single Ig IL-1R-related molecule (SIGIRR) are two other cell surface proteins of the IL-1R superfamily containing the TIR domain (12). T1/ST2 or SIGIRR may function as ligand-binding receptors, although no specific ligands have been reported to date. It is also possible that these two proteins function as receptor accessory proteins like AcPL. Intriguingly, a recent gene-targeting study suggested that T1/ST2 probably functions to inhibit IL-1R and TLR4 signaling (20). Although T1/ST2 cannot bind to IL-1, its TIR domain is capable of binding and probably sequestering common signaling molecules such as MyD88, preventing them from being used by IL-1R (20). Likewise, SIGIRR, which also cannot bind to IL-1, has been identified as a negative regulator of IL-1R, IL-18R, and TLR4 signaling (21). Given the inhibitory functions of T1/ST2 and SIGIRR, two AcPL-like proteins, we were interested in whether AcPL also plays a regulatory role in IL-1R/TLR signaling.

To investigate the physiological functions of AcPL, we generated AcPL-deficient mice by gene targeting. Mutant mice were generally healthy and did not exhibit any gross developmental abnormalities. However, AcPL–/– mice showed severely impaired IL-18-induced responses including NK cell activity, splenocyte proliferation, and splenocyte and Th1 cell IFN-{gamma} production. Furthermore, AcPL–/– neutrophils demonstrated a defect in IL-18-induced activation. In contrast, AcPL–/– fibroblasts and macrophages showed no significant defects in cytokine production in response to IL-1 and various TLR ligands, including LPS and poly(I:C). These results demonstrate that AcPL is essential and specific for mediating the IL-18 signaling cascade.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of AcPL-deficient mice

The mutant mice were originally generated by Tularik. The mouse AcPL gene in embryonic stem (ES) cells was disrupted by the replacement of a 152-bp region within exon 2–3 of the gene with a neomycin resistance gene. Chimeric mice were generated from embryos injected with mutant ES cells. Heterozygous mutant AcPL+/– mice were intercrossed to obtain homozygous AcPL–/– mice. The genotypes of the mutant mice were determined by PCR analysis of genomic DNA from tail biopsy samples. As shown in Fig. 1A, primers a and b were used to detect the wild-type allele, and primers a and c were used to detect the mutant allele. The sequences of these primers were as follows: primer a, 5'-TCTCCCATGCAAGTCAACTGTCACC-3'; primer b, 5'-CTTGATACAACAGGCCATATCCTGG-3'; primer c, 5'-GGGTGGGATTAGATAAATGCCTGCTCT-3'. RT-PCR were performed using RNA extracted from splenocytes, macrophages, fibroblasts, and neutrophils: AcPL (sense, 5'-GTTGATCAGACACTGAAGTTG-3'; antisense, 5'-TCTAGAAAGGCTCAATTTCTATCAG-3') or {beta}-actin (sense, 5'-GACTACCTCATGAAGATCCT-3'; antisense, 5'-CCACATCTGCTGGAAGGTGG-3'). Both primers for AcPL are from regions downstream of the deleted exons, and the expected PCR product is 394 bp.



View larger version (9K):
[in this window]
[in a new window]
  		FIGURE 1. Targeted disruption of the mouse AcPL gene. A, Targeting vector to create AcPL–/– mutation via homologous recombination. The genomic structure of the AcPL gene and the targeted exons are indicated. a, b, and c are positions of primers used for genotyping. WT, Wild type; MT, mutant. B, PCR genotyping of mouse tails. Primers a, b, and c were used in the same PCRs that yielded products of wild-type (260 bp; bottom) and knockout (478 bp; top) alleles. +/+, Wild type; +/–, heterozygous mutant; –/–, homozygous mutant. C, RT-PCR analysis of AcPL mRNA. Total RNA was isolated from primary embryonic fibroblasts and reverse-transcribed. The resulting cDNA was amplified by PCR using specific primers for AcPL and actin.

 
Reagents

Mouse recombinant IL-1{beta}, IL-2, IL-12, IL-18, and TNF-{alpha} were purchased from R&D Systems. Anti-CD-3 mAb and anti-IL-4 Ab were purchased from BD Pharmingen. LPS from Escherichia coli strain 055:B5 and human recombinant C5a were from Sigma-Aldrich. Poly(I:C) was from Amersham Biosciences. Splenocytes and neutrophils were cultured in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FBS (Invitrogen Life Technologies), 50 µM 2-ME (Invitrogen Life Technologies), 2 mM L-glutamine (Invitrogen Life Technologies), 50 U/ml penicillin, and 50 µg/ml streptomycin (Invitrogen Life Technologies). Embryonic fibroblasts were maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% FBS, 50 µM 2-ME, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin.

Flow-cytometric analyses

Single-cell suspensions were prepared from thymus, spleen, lymph nodes, and bone marrow. For each cytometric analysis, 5 x 105 cells were used. All Abs (including those specific for TCR{beta}, B220, CD4, CD8, CD25, CD44, CD43, IgM, and IgD) were purchased from BD Pharmingen. After staining with specific Abs for 20 min, cells were washed with PBS/0.2% FBS, and fluorescent signals on the cell surfaces were analyzed by FACS (FACSCalibur; BD Biosciences) using CellQuest software (BD Biosciences).

Generation of splenocytes and their in vitro response to IL-18

Spleens were harvested from AcPL-deficient mice and wild-type littermates and transferred to RPMI 1640. After dispersing spleen cells by grinding and filtering through a cell strainer, RBC were removed by ACK lysis buffer (8.29 g/L NH4Cl, 1 g/L KHCO3, and 37 mg/L Na2EDTA). Cells were washed with medium, transferred to a 96-well plate (105 cells/well), and incubated with varying concentrations of murine IL-18 in the absence, or presence of 1 ng/ml or 5 ng/ml murine IL-12 for 24 h. [3H]Thymidine (1 µCi/well) was then added for 4 h, and radioactivity was incorporated into dividing cells was measured using a TopCount Microplate scintillation counter (Packard). For IFN-{gamma} production, culture supernatants were collected and assayed using a mouse IFN-{gamma} ELISA kit (R&D Systems).

Preparation of Th1 cells

CD4+ T cells were purified from lymph node cells using a MACS CD4-positive selection kit (Miltenyi Biotec) following the manufacturer’s instructions. The purification of CD4+ T cells in different preparations was ~97%. Enriched CD4+ T cells were activated with immobilized anti-CD3 mAb, which had been coated overnight onto 24-well plates at 1 µg/ml. Th1 differentiation was induced by addition of 2 ng/ml IL-2, 5 ng/ml IL-12, and 5 µg/ml anti-IL-4 Ab in RPMI 1640. After 5 days, the cytokine profile of Th1 cells was determined by plating the cells at 105 cells/well in 96-well plates with varying concentrations of IL-18 in the absence or the presence of 5 ng/ml IL-12. Culture supernatants were collected after 24 h for cytokine detection by ELISA.

Detection of IFN-{gamma} mRNA levels in response to IL-18 by RT-PCR

Splenocytes and Th1 cells were treated with 10 ng/ml IL-18 in the absence or the presence of 5 ng/ml IL-12 for 2 h, and total cellular RNA was prepared using TRIzol reagent (Invitrogen Life Technologies). Ten micrograms of total RNA was then reversed transcribed for 1 h at 50°C in 20 µl of total reaction mix (200 U of Superscript III reverse transcriptase; 150 µg/ml random primers; 1 mM DTT; 1 mM dNTPs; 4 µl first-strand buffer; all final concentrations; all Invitrogen Life Technologies). Three microliters of cDNA products were used to amplify out IFN-{gamma} using the following primer pair (1 mM): sense, 5'-CATTGAAAGCCTAGAAAGTCT-3'; antisense, 5'-ACTCATGAATGCATCCTTTTTCG-3'. {beta}-Actin was also amplified as control using the primers described previously. We used HotStar Taq DNA polymerase (1 U; Qiagen) in a 30-µl reaction mix (3 µl of 10x PCR buffer containing 0.5 mM dNTPs; all final concentrations; all Qiagen). The reaction conditions were 95°C for 15 min followed by 35 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 2.5 min. The expected size for the RT-PCR production for IFN-{gamma} was 267 bp.

Generation of neutrophils and in vitro stimulation assay

Bone marrow cells were harvested from AcPL-deficient mice and wild-type littermates and transferred to PBS. After separating the pellet fraction (containing neutrophils and RBCs) by using Histopaque-1083 (Sigma-Aldrich) according to the manufacturer’s instructions, RBCs were lysed using ACK buffer. Bone marrow cells were then washed with medium, transferred to a 96-well plate (1 x 106 cells/well), and incubated with 10 ng/ml IL-18, 0.1 µg/ml LPS, or 0.1 µg/ml C5a (as positive control). To assay activation, neutrophils were harvested after 1 h of stimulation, washed with PBS/0.2% BSA, and then stained with Gr-1- and Mac-1-specific Abs (BD Pharmingen) for 20 min. After staining, cells were washed with PBS/0.2% BSA and the percentage of viable cells remaining were analyzed by 7-amino-actinomycin D staining using FACS). To assay cytokine production, culture supernatants were collected after 24 h and analyzed for IL-6 production by ELISA (BD Biosciences).

NK cytotoxicity assay

Mice were injected i.p. with PBS alone as control or with PBS containing 1 µg of IL-18 for 2 consecutive days. 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 follows: ((measured 51Cr release – spontaneous 51Cr release)/(maximum 51Cr release – spontaneous 51Cr release)) x 100. Maximum release was determined based on acid-lysed target cells. Spontaneous release was determined by incubating target cells in the absence of effector cells.

Generation of macrophage and in vitro stimulation assays

Bone marrow cells harvested from mice were transferred to RPMI 1640. RBCs were lysed by ACK buffer. Cells in the pellet fraction were washed and plated at 5 x 106 cells per 10-cm dish with 6 ml of RPMI 1640 and 25 ng/ml murine M-CSF. After 24 h, 3 ml of the above medium was added fresh to the culture. After 3–4 days, adherent macrophages were transferred to a 96-well plate (2 x 104 cells/well), and incubated with various concentrations of LPS or poly(I:C) (as indicated in the figures) for 24 h. Culture supernatants were collected and assayed for IL-6 production by ELISA. To assay in vitro LPS tolerance, purified macrophages were cultured in a 96-well plate (2 x 104 cells/well), and incubated with 10 µg/ml LPS for 24 h. Culture supernatants were removed and assayed for IL-6 and TNF-{alpha} production by ELISA (IL-6 kit from BD Biosciences; TNF-{alpha} kit from eBioscience). Cells were then washed with PBS and restimulated with various concentrations of LPS (as indicated in the figures) for 24 h. Culture supernatants were again assayed for cytokine production by ELISA.

Generation of mouse embryonic fibroblasts (MEFs) and in vitro stimulation assay

MEFs were isolated from embryos at day 14 of gestation. Cell suspensions were prepared by trypsin treatment of minced embryonic tissues and cultured in DMEM. For in vitro stimulation, MEFs were transferred to a 96-well plate (5 x 103 cells/well), and incubated with medium alone or stimulated with IL-1{beta} (10 ng/ml) or TNF-{alpha} (10 ng/ml) for 24 h. Culture supernatants were collected and assayed for IL-6 production by ELISA (BD Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of AcPL-deficient mice

The mouse AcPL gene was disrupted by homologous recombination in ES cells. A targeting vector was designed to replace a region of the genomic DNA covering exons 2 and 3 of the AcPL gene, which encodes the transmembrane region, with the neomycin cassette (Fig. 1A). AcPL–/– mice were born at the expected Mendelian ratio and were phenotypically normal and fertile. PCR analysis for the presence of the AcPL gene from wild-type, heterozygous, and homozygous mutant mice are shown in Fig. 1B. No expression of AcPL mRNA in AcPL–/– MEFs was detectable by RT-PCR analyses, confirming that this is a null mutation (Fig. 1C).

Development of T and B lymphocytes were examined using flow-cytometric analysis of various cell surface markers (TCR{beta}, B220, CD4, CD8, CD25, CD44, CD43, IgM, and IgD). As shown in Fig. 2A, early and late AcPL–/– thymocyte development was comparable to wild type. Development of B cells and the ratios of T and B cells in the periphery were all normal when AcPL–/– bone marrow cells (Fig. 2B) and splenocytes (C) were examined.



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 2. Normal lymphocyte composition and development in AcPL–/– mice. A, Flow cytometry analysis of thymocyte surface marker expression levels. Total thymoctyes isolated from wild-type and AcPL–/– mice were analyzed for the expression of CD4 and CD8 (top). Cells that were double negative for CD4 and CD8 were gated and analyzed for the expression of CD25 and CD44 (bottom, as shown by arrows). Numbers indicate the percentages of total or gated cells. B, Profiles of surface maker expression in bone marrow cells. Total bone marrow cells isolated from wild-type and AcPL–/– were analyzed for the expression of B220, CD43, and IgM. Numbers indicate the percentage of total cells. C, Profiles of surface marker expression in splenocytes. Total splenocytes from wild-type and AcPL–/– mice were analyzed from the expression of TCR{beta}, B220, CD4, CD8, IgM, and IgD. Numbers indicate the percentage of total cells.

 
Requirement of AcPL for IL-18-induced IFN-{gamma} production and splenocyte proliferation

IFN-{gamma} production by immune cells such as NK and Th1 cells can be induced by IL-18 and further enhanced by the combination of IL-18 and IL-12 (3). To determine the role of AcPL in IL-18-induced production of IFN-{gamma}, we stimulated splenocytes from wild-type and AcPL–/– mice with varying concentrations of IL-18, or IL-18 plus 1 or 5 ng/ml IL-12, and the production of IFN-{gamma} was measured by ELISA. In wild-type splenocytes, a significant induction of IFN-{gamma} stimulated by IL-18 alone was observed. This induction was further enhanced when cells were treated with both IL-18 and IL-12 (Fig. 3A). However, in AcPL–/– splenocytes, induction of IFN-{gamma} by either IL-18 alone or IL-18 plus IL-12 was completely abolished (Fig. 3A). RT-PCR analysis also showed that IFN-{gamma} mRNA level is enhanced after IL-18 or IL-12/IL-18 stimulations in wild-type cells but not in AcPL–/– splenocytes (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 3. Impaired IL-18-induced IFN-{gamma} production and proliferation in AcPL–/– splenocytes. A, IFN-{gamma} production by splenocytes in response to IL-18. Total splenocytes harvested from AcPL-deficient and wild-type littermates and stimulated with the indicated concentrations of IL-18 alone or IL-18 with IL-12 for 24 h. Culture supernatants were collected and assayed for IFN-{gamma} production by ELISA. B, Proliferation of splenocytes in response to IL-18 stimulation. Total splenocytes harvested from mice were stimulated with the indicated concentrations of IL-18 alone, or IL-18 in combination with IL-12 for 24 h. Cell proliferation was measured by [3H]thymidine incorporation assay.

 
We next examined the effect of IL-18 and its synergism with IL-12 on stimulating splenocyte proliferation. Total splenocytes from wild-type and AcPL–/– mice were stimulated with varying concentrations of IL-18 or IL-18 with IL-12, and cell proliferation was determined by [3H]thymidine incorporation. As shown in Fig. 3B, proliferation of wild-type splenocytes, but not AcPL–/–, was significantly enhanced by IL-18 in a concentration-dependent manner. IL-12 alone induced a mild degree of proliferation, which was similar in both wild-type and AcPL–/– splenocytes. The synergistic effect of IL-18 and IL-12 on cell proliferation, however, was observed only in wild-type and not in AcPL–/– splenocytes. Taken together, these results suggest that AcPL is essential for in vitro IL-18-induced cell proliferation and IFN-{gamma} production.

Requirement of AcPL for IL-18-induced IFN-{gamma} production by Th1 cells

To further determine the role of AcPL in Th1 cells, Th1 cells from wild-type and AcPL–/– mice were stimulated with varying concentrations of IL-18, or IL-18 with IL-12, and the production of IFN-{gamma} was measured by ELISA. In wild-type Th1 cells, there was a significant induction of IFN-{gamma} by IL-18 alone or IL-18 with IL-12. In AcPL–/– cells, however, IFN-{gamma} induction was negligible (Fig. 4). RT-PCR analysis also detected increased IFN-{gamma} mRNA levels in wild-type cells after IL-18 or IL-12/IL-18 stimulation, but AcPL–/– Th1 cells failed to exhibit any significant increase after the same treatment (data not shown). These data suggest that AcPL plays an essential role in IL-18-induced IFN-{gamma} production in Th1 cells.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4. Impaired IL-18 signaling in AcPL–/– Th1 cells. Wild-type and AcPL–/– Th1 cells were stimulated with varying concentrations of IL-18 alone or IL-18 with IL-12 for 24 h. Culture supernatants were collected and assayed for IFN-{gamma} production by ELISA.

 
Impairment of IL-18-induced NK cytotoxicity in AcPL–/– mice

IL-18 has been shown to augment NK cell cytotoxicity (3). To determine whether AcPL plays a role in IL-18-induced NK activity, mice were injected i.p. with PBS or IL-18 daily for 2 consecutive days. Splenocytes were then harvested and incubated with 51Cr-labeled YAC-1 NK target cells at the indicated E:T ratios, and 51Cr release was measured. Basal NK cell activities in PBS-injected wild-type and AcPL–/– mice were comparable (data not shown). In contrast, when animals were challenged with IL-18, the killing activity of splenocytes from AcPL–/– mice was ~25% of the activity from splenocytes derived from wild-type mice (Fig. 5). Similar results have been reported for both IL-18–/– and IL-18R{alpha}–/– mice (6). This demonstrates that AcPL is required for in vivo IL-18-mediated NK cytotoxic activity.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 5. Impaired IL-18-induced NK cytotoxicity in AcPL–/– mice. Wild-type and AcPL–/– mice were injected i.p. with IL-18 (1 µg) for 2 consecutive days. Splenocytes were then harvested and assayed for NK lytic activity against 51Cr-labeled YAC-1 target cells.

 
Requirement of AcPL for IL-18-induced neutrophil activation and cytokine production

Recently, IL-18 has been shown to activate neutrophils during early innate immune responses (5). To determine whether AcPL is essential for IL-18-mediated neutrophil activation, we stimulated neutrophils from wild-type and AcPL–/– mice with IL-18, and up-regulation of Mac-1 was measured by flow cytometry. Enhanced Mac-1 expression in response to IL-18 stimulation was observed in wild-type but not in AcPL–/– neutrophils (Fig. 6B). In contrast, Mac-1 up-regulation was comparable between wild-type and AcPL–/– neutrophils when cells were stimulated with C5a or LPS (Fig. 6, A and C).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 6. Impaired IL-18-induced cell activation and cytokine production in AcPL–/– neutrophils. Up-regulation of Mac-1 expression in response to C5a, IL-18, and LPS stimulations. Neutrophils isolated from wild-type and AcPL–/– mice were stimulated with C5a (0.1 µg/ml) (A), IL-18 (10 ng/ml) (B), and LPS (0.1 µg/ml) (C) for 1 h. Cells were stained and analyzed for Mac-1 expression by flow cytometry. Numbers indicate the mean fluorescence corresponding to Mac-1 expression on cells. D, IL-6 production of neutrophils in response to IL-18. Neutrophils were stimulated with IL-18 and LPS for 24 h. Culture supernatants were collected and assayed for IL-6 production by ELISA.

 
We next investigated whether AcPL is critical for IL-18-mediated cytokine production by neutrophils, which is another measure of neutrophil function after activation. Neutrophils were stimulated with IL-18 for 24 h, and the culture supernatants were collected and measured for IL-6 production by ELISA. As shown in Fig. 6D, IL-6 production was significantly induced in wild-type neutrophils stimulated with IL-18, whereas this induction was completely abolished in AcPL–/– neutrophils. In contrast, AcPL–/– neutrophils show no defect in IL-6 production when stimulated with LPS. These results demonstrate that AcPL is essential for the IL-18-induced activation and cytokine production of neutrophils.

IL-1R/TLR signaling in AcPL-deficient mice and cells

In addition to partnering with IL-18R{alpha} to promote IL-18 signaling, AcPL may function as a non-ligand-binding cell surface protein of the IL-1R superfamily similar to T1/ST2 and SIGIRR, two inhibitory proteins that play a physiological role in regulating IL-1R and TLR signals (20, 21). Furthermore, IL-18 is thought to be essential in down-regulating LPS-induced TNF production (22), but the receptor mediating this regulatory effect remains unknown. We investigated whether deficiency in AcPL also affects the signals induced by IL-1 or other TLR ligands. First, we examined IL-6 production induced by IL-1 in MEFs, and found that this response was comparable between wild-type and AcPL–/– cells (Fig. 7A). Similar results in response to IL-1 stimulation were also observed in wild-type and AcPL–/– macrophages (data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 7. AcPL is not required for other TIR signaling pathways. A, Primary embryonic fibroblasts isolated from wild-type and AcPL–/– mice were stimulated with IL-1 and TNF-{alpha} for 24 h. IL-6 production was determined by ELISA. B and C, Macrophages isolated from wild-type and AcPL–/– mice were stimulated with LPS (B) or poly(I:C) (C) for 24 h. Culture supernatants were collected and analyzed for IL-6 production by ELISA. D, Purified macrophages were stimulated with 10 µg/ml LPS (1st LPS) for 24 h. Culture supernatants were removed and assayed for IL-6 and TNF-{alpha} production by ELISA. Cells were restimulated with various concentrations of LPS (2nd LPS) for 24 h, and culture supernatants were assayed for cytokine production by ELISA. The concentrations of cytokines in each sample were normalized to the concentration of the sample after the first LPS (10 µg/ml) stimulation, and data are presented as percentages of cytokine production in samples after receiving the first stimulation.

 
Similarly, when the TLR3 and TLR4 ligands, poly(I:C) and LPS, respectively, were used to stimulate macrophages, AcPL–/– macrophages did not exhibit an enhanced or reduced response compared with the wild-type counterparts (Fig. 7, B and C). LPS-induced IL-6 production was also comparable between wild-type and AcPL–/– neutrophils (Fig. 6B) and MEFs (data not shown). Furthermore, LPS challenge to wild-type and AcPL–/– mice in vivo resulted in similar survival outcomes and serum cytokine production (data not shown). The phenomenon of LPS tolerance provides a sensitive assay of TLR4 signal negative regulation, because tolerance is often defective when critical negative regulators of LPS signaling, such as IRAK-M and T1/ST2, are deleted. We therefore examined the cytokine response of AcPL–/– macrophages to a second stimulation of LPS after an initial challenge and found that this was also comparable to wild type (Fig. 7D). Taken together, our results demonstrate that AcPL is not required for mediating other TIR signals, at least in IL-1R, TLR3, and TLR4 systems.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we described the generation and characterization of AcPL–/– mice. We found that IL-18-mediated immune responses were defective in these mutant mice. Splenocytes lacking AcPL failed to produce IFN-{gamma} or proliferate in response to IL-18. Th1 cells lacking AcPL also failed to produce IFN-{gamma}. NK cell cytotoxicity was severely defective in IL-18-challenged AcPL–/– mice, and AcPL–/– neutrophils showed impaired IL-18-induced activation and IL-6 production. Although AcPL shares structural similarity to other TIR-containing cell surface proteins of IL-1R family, the absence of AcPL did not cause significant impact on IL-1R or the TLR responses that we examined. These results suggest that AcPL is an essential and specific mediator of IL-18 signals.

The function of AcPL/IL-18R{alpha} in response to IL-18 stimulation is likely similar to IL-1RAcP and IL-1RI in IL-1 signaling (9, 11). IL-1RAcP lacks ligand-binding ability but is essential for mediating IL-1 signaling. The docking of IL-1RAcP to IL-1RI increases the binding affinity of IL-1 to the IL-1R complex (9). Similarly, AcPL alone does not appear to bind to IL-18, but AcPL/IL-18R{alpha} complex affords a high-affinity binding capacity for IL-18 interaction (23). Although both AcPL and IL-18R{alpha} are required for IL-18 responses, it remains unclear whether both TIR-containing proteins are capable of recruiting signaling adaptors or whether there is exclusive dependence on one protein for signal transduction. This may be interesting for a future study whether a specific and partial modulation of IL-18 signals is useful for regulating immune responses.

One well-characterized player regulating the potent response of IL-18 is the soluble IL-18 binding protein, which antagonizes IL-18 by preventing access to its receptor (24, 25). Numerous studies have shown that perturbation of such a control system can contribute to the pathogenesis of inflammatory and infectious diseases and various cancers (26), particularly endotoxin-induced liver injury and Th1-mediated autoimmune disorders (2, 27, 28, 29). The blockade of the IL-18 ligand-receptor interaction has proven to be an effective strategy in the development of IL-18 antagonists. For example, anti-IL-18 Abs protect mice primed with P. acnes and challenged with LPS from liver damage, and prevent progression of experimental acute encephalomyelitis in rats (2, 29). The soluble IL-18 binding protein inhibits bacterial-induced IFN-{gamma} production (24, 29). Moreover, anti-IL-18R{alpha} Ab reduces Th1 responses to LPS (30). Recently, anti-AcPL mAbs have been generated and are found to inhibit IL-18 responses effectively in vitro (23). Our data show that deletion of AcPL in mice causes severe impairment in IL-18 responses, and therefore suggest the potential for anti-AcPL Abs in treating Th1-mediated pathologies.

Although IL-18 is well characterized in mediating NK and Th1 cell responses, recent studies have shown that IL-18 also participates in neutrophil activation. In fact, it is hypothesized that the function of IL-18 in neutrophil activation may play a role in several autoimmune diseases, such as inflammatory bowl disease and rheumatoid arthritis (31, 32, 33). IL-18 may amplify acute inflammation through promotion of neutrophil adhesion and migration, accompanied by cytokine and chemokine production, and the eventual release of granules. These effects are relevant in vivo because IL-18 administration induces peritoneal neutrophil recruitment (5). In our study, we showed that IL-18 signaling through AcPL is required to up-regulate Mac-1 expression and cytokine production by neutrophils. The effect of AcPL on neutrophil response is likely specific to IL-18 stimulation, because thioglycolate administration in vivo recruited a normal amount of activated neutrophils in AcPL–/– mice (our unpublished results). Additionally, recent studies by Gutzmer et al. (34) have suggested that macrophages and dendritic cells may also respond to IL-18 as part of the inflammatory response. Taken together, AcPL–/– mice can potentially serve as an animal model for the study of autoimmune diseases and their progression, and may provide insight in targeting IL-18 signals for therapeutic applications in these diseases.


   Acknowledgments
 
We thank Wen-Jye Lin for excellent technical assistance. We also thank Billie Au and Kip Wigmore for manuscript editing.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by an operating grant awarded to W.-C.Y. by Canadian Institute of Health Research (CIHR; MOP57734 and by Canadian Network for Vaccines and Immunotherapeutics. N.O. was supported by a CIHR fellowship. P.S.O. was supported by a National Cancer Institute of Canada grant with funds from the Canadian Cancer Society. Back

2 H.C. and N.-J.C. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Wen-Chen Yeh, Department of Medical Biophysics, University of Toronto, 620 University Avenue, Number 706, Toronto, Ontario M5G 2C1, Canada. E-mail address: wyeh{at}uhnres.utoronto.ca Back

4 Abbreviations used in this paper: AcPL, accessory protein-like; IL-1RAcP, IL-1R accessory protein; TIR, TLR/IL-1R/plant R gene; IRAK, IL-1R-associated kinase; SIGIRR, single Ig IL-1R-related molecule; ES, embryonic stem; MEF, mouse embryonic fibroblast. Back

Received for publication November 30, 2004. Accepted for publication February 19, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Dinarello, C. A., D. Novick, A. J. Puren, G. Fantuzzi, L. Shapiro, H. Muhl, D. Y. Yoon, L. L. Reznikov, S. H. Kim, M. Rubinstein. 1998. Overview of interleukin-18: more than an interferon-{gamma} inducing factor. J. Leukocyte Biol. 63:658.[Abstract]
  2. Okamura, H., H. Tsutsi, T. Komatsu, M. Yutsudo, A. Hakura, T. Tanimoto, K. Torigoe, T. Okura, Y. Nukada, K. Hattori. 1995. Cloning of a new cytokine that induces IFN-{gamma} production by T cells. Nature 378:88.[Medline]
  3. Okamura, H., S. Kashiwamura, H. Tsutsui, T. Yoshimoto, K. Nakanishi. 1998. Regulation of interferon-{gamma} production by IL-12 and IL-18. Curr. Opin. Immunol. 10:259.[Medline]
  4. Takeda, K., H. Tsutsui, T. Yoshimoto, O. Adachi, N. Yoshida, T. Kishimoto, H. Okamura, K. Nakanishi, S. Akira. 1998. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 8:383.[Medline]
  5. Leung, B. P., S. Culshaw, J. A. Gracie, D. Hunter, C. A. Canetti, C. Campbell, F. Cunha, F. Y. Liew, I. B. McInnes. 2001. A role for IL-18 in neutrophil activation. J. Immunol. 167:2879.[Abstract/Free Full Text]
  6. Hoshino, K., H. Tsutsui, T. Kawai, K. Takeda, K. Nakanishi, Y. Takeda, S. Akira. 1999. Cutting edge: generation of IL-18 receptor-deficient mice: evidence for IL-1 receptor-related protein as an essential IL-18 binding receptor. J. Immunol. 162:5041.[Abstract/Free Full Text]
  7. Born, T. L., E. Thomassen, T. A. Bird, J. E. Sims. 1998. Cloning of a novel receptor subunit, AcPL, required for interleukin-18 signaling. J. Biol. Chem. 273:29445.[Abstract/Free Full Text]
  8. Greenfeder, S. A., P. Nunes, L. Kwee, M. Labow, R. A. Chizzonite, G. Ju. 1995. Molecular cloning and characterization of a second subunit of the interleukin 1 receptor complex. J. Biol. Chem. 270:13757.[Abstract/Free Full Text]
  9. Dinarello, C. A.. 1996. Biologic basis for interleukin-1 in disease. Blood 87:2095.[Abstract/Free Full Text]
  10. Torigoe, K., S. Ushio, T. Okura, S. Kobayashi, M. Taniai, T. Kunikata, T. Murakami, O. Sanou, H. Kojima, M. Fujii, et al 1997. Purification and characterization of the human interleukin-18 receptor. J. Biol. Chem. 272:25737.[Abstract/Free Full Text]
  11. Sims, J. E.. 2002. IL-1 and IL-18 receptors, and their extended family. Curr. Opin. Immunol. 14:117.[Medline]
  12. O’Neill, L. A.. 2000. The interleukin-1 receptor/Toll-like receptor superfamily: signal transduction during inflammation and host defense. Sci. STKE 44:RE1.
  13. Suzuki, N., S. Suzuki, G. S. Duncan, D. G. Millar, T. Wada, C. Mirtsos, H. Takada, A. Wakeham, A. Itie, S. Li, et al 2002. Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. Nature 416:750.[Medline]
  14. Wesche, H., W. J. Henzel, W. Shillinglaw, S. Li, Z. Cao. 1997. MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 7:837.[Medline]
  15. Kanakaraj, P., K. Ngo, Y. Wu, A. Angulo, P. Ghazal, C. A. Harris, J. J. Siekierka, P. A. Peterson, W. P. Fung-Leung. 1999. Defective interleukin (IL)-18-mediated natural killer and T helper cell type 1 responses in IL-1 receptor-associated kinase (IRAK)-deficient mice. J. Exp. Med. 189:1129.[Abstract/Free Full Text]
  16. Cao, Z., J. Xiong, M. Takeuchi, T. Kurama, D. V. Goeddel. 1996. TRAF6 is a signal transducer for interleukin-1. Nature 383:443.[Medline]
  17. Lomaga, M. A., W. C. Yeh, I. Sarosi, G. S. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, et al 1999. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13:1015.[Abstract/Free Full Text]
  18. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami, K. Nakanishi, S. Akira. 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9:143.[Medline]
  19. Thomas, J. A., J. L. Allen, M. Tsen, T. Dubnicoff, J. Danao, X. C. Liao, Z. Cao, S. A. Wasserman. 1999. Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase. J. Immunol. 163:978.[Abstract/Free Full Text]
  20. Brint, E. K., D. Xu, H. Liu, A. Dunne, A. N. McKenzie, L. A. O’Neill, F. Y. Liew. 2004. ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat. Immunol. 5:373.[Medline]
  21. Wald, D., J. Qin, Z. Zhao, Y. Qian, M. Naramura, L. Tian, J. Towne, J. E. Sims, G. R. Stark, X. Li. 2003. SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat. Immunol. 4:920.[Medline]
  22. Sakao, Y., K. Takeda, H. Tsutsui, T. Kaisho, F. Nomura, H. Okamura, K. Nakanishi, S. Akira. 1999. IL-18-deficient mice are resistant to endotoxin-induced liver injury but highly susceptible to endotoxin shock. Int. Immunol. 11:471.[Abstract/Free Full Text]
  23. Debets, R., J. C. Timans, T. Churakowa, S. Zurawski, R. de Waal Malefyt, K. W. Moore, J. S. Abrams, A. O’Garra, J. F. Bazan, R. A. Kastelein. 2000. IL-18 receptors, their role in ligand binding and function: anti-IL-1RAcPL antibody, a potent antagonist of IL-18. J. Immunol. 165:4950.[Abstract/Free Full Text]
  24. Novick, D., S. H. Kim, G. Fantuzzi, L. L. Reznikov, C. A. Dinarello, M. Rubinstein. 1999. Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response. Immunity 10:127.[Medline]
  25. Aizawa, Y., K. Akita, M. Taniai, K. Torigoe, T. Mori, Y. Nishida, S. Ushio, Y. Nukada, T. Tanimoto, H. Ikegami, et al 1999. Cloning and expression of interleukin-18 binding protein. FEBS Lett. 445:338.[Medline]
  26. Lebel-Binay, S., A. Berger, F. Zinzindohoue, P. Cugnenc, N. Thiounn, W. H. Fridman, F. Pages. 2000. Interleukin-18: biological properties and clinical implications. Eur. Cytokine Network 11:15.[Medline]
  27. Rothe, H., T. Hibino, Y. Itoh, H. Kolb, S. Martin. 1997. Systemic production of interferon-{gamma} inducing factor (IGIF) versus local IFN-{gamma} expression involved in the development of Th1 insulitis in NOD mice. J. Autoimmun. 10:251.[Medline]
  28. Ludwiczek, O., A. Kaser, D. Novick, C. A. Dinarello, M. Rubinstein, W. Vogel, H. Tilg. 2002. Plasma levels of interleukin-18 and interleukin-18 binding protein are elevated in patients with chronic liver disease. J. Clin. Immunol. 22:331.[Medline]
  29. Wildbaum, G., S. Youssef, N. Grabie, N. Karin. 1998. Neutralizing antibodies to IFN-{gamma}-inducing factor prevent experimental autoimmune encephalomyelitis. J. Immunol. 161:6368.[Abstract/Free Full Text]
  30. Xu, D., W. L. Chan, B. P. Leung, D. Hunter, K. Schulz, R. W. Carter, I. B. McInnes, J. H. Robinson, F. Y. Liew. 1998. Selective expression and functions of interleukin 18 receptor on T helper (Th) type 1 but not Th2 cells. J. Exp. Med. 188:1485.[Abstract/Free Full Text]
  31. Pizarro, T. T., M. H. Michie, M. Bentz, J. Woraratanadharm, M. F. Smith, Jr, E. Foley, C. A. Moskaluk, S. J. Bickston, F. Cominelli. 1999. IL-18, a novel immunoregulatory cytokine, is up-regulated in Crohn’s disease: expression and localization in intestinal mucosal cells. J. Immunol. 162:6829.[Abstract/Free Full Text]
  32. Gracie, J. A., R. J. Forsey, W. L. Chan, A. Gilmour, B. P. Leung, M. R. Greer, K. Kennedy, R. Carter, X. Q. Wei, D. Xu, et al 1999. A proinflammatory role for IL-18 in rheumatoid arthritis. J. Clin. Invest. 104:1393.[Medline]
  33. Reuter, B. K., T. T. Pizarro. 2004. Commentary: the role of the IL-18 system and other members of the IL-1R/TLR superfamily in innate mucosal immunity and the pathogenesis of inflammatory bowel disease: friend or foe?. Eur. J. Immunol. 34:2347.[Medline]
  34. Gutzmer, R., K. Langer, S. Mommert, M. Wittmann, A. Kapp, T. Werfel. 2003. Human dendritic cells express the IL-18R and are chemoattracted to IL-18. J. Immunol. 171:6363.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
I. A. Parish, S. Rao, G. K. Smyth, T. Juelich, G. S. Denyer, G. M. Davey, A. Strasser, and W. R. Heath
The molecular signature of CD8+ T cells undergoing deletional tolerance
Blood, May 7, 2009; 113(19): 4575 - 4585.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
J. S. Haring and J. T. Harty
Interleukin-18-Related Genes Are Induced during the Contraction Phase but Do Not Play Major Roles in Regulating the Dynamics or Function of the T-Cell Response to Listeria monocytogenes Infection
Infect. Immun., May 1, 2009; 77(5): 1894 - 1903.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
L. L.E. Koskinen, E. Einarsdottir, E. Dukes, G. A. R. Heap, P. Dubois, I. R. Korponay-Szabo, K. Kaukinen, K. Kurppa, F. Ziberna, S. Vatta, et al.
Association study of the IL18RAP locus in three European populations with coeliac disease
Hum. Mol. Genet., March 15, 2009; 18(6): 1148 - 1155.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. C. Siracusa, M. G. Overstreet, F. Housseau, A. L. Scott, and S. L. Klein
17{beta}-Estradiol Alters the Activity of Conventional and IFN-Producing Killer Dendritic Cells
J. Immunol., February 1, 2008; 180(3): 1423 - 1431.
[Abstract] [Full Text] [PDF]

Home page
 Ann Rheum DisHome page
S.-M. Dai, Z.-Z. Shan, H. Xu, and K. Nishioka
Cellular targets of interleukin-18 in rheumatoid arthritis
Ann Rheum Dis, November 1, 2007; 66(11): 1411 - 1418.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
H. Blumberg, H. Dinh, E. S. Trueblood, J. Pretorius, D. Kugler, N. Weng, S. T. Kanaly, J. E. Towne, C. R. Willis, M. K. Kuechle, et al.
Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation
J. Exp. Med., October 29, 2007; 204(11): 2603 - 2614.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cheung, H.
Right arrow Articles by Yeh, W.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cheung, H.
Right arrow Articles by Yeh, W.-C.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS