IL-18 is an essential cytokine for both innate and adaptive immunity. Signaling by IL-18 requires IL-18Rα, 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-18Rα 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-γ production. In particular, Th1 cells lacking AcPL fail to produce IFN-γ 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.

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-γ-inducing factor, IL-18 is secreted by macrophages and induces IFN-γ production from splenocytes, liver lymphocytes, and Th1 cells. In addition, IL-18 enhances NK cell cytotoxicity and synergizes with IL-12 to induce IFN-γ 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-18Rα (also known as IL-1R-related protein) (6). However, binding of IL-18 to IL-18Rα alone does not trigger downstream signals such as NF-κB (7). In contrast, expression of a cell surface protein called accessory protein-like (AcPL)4 in the presence of IL-18 and IL-18Rα is able to activate an NF-κB-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-18Rα−/− mice are defective in IL-18-mediated biological functions (6, 10). However, the physiological functions of AcPL remain to be investigated.

Both IL-18Rα 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-κB 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-18Rα or AcPL or both remains unclear.

Like IL-18Rα 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-γ 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.

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. 1 A, 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 β-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.

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.

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.

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Mouse recombinant IL-1β, IL-2, IL-12, IL-18, and TNF-α 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.

Single-cell suspensions were prepared from thymus, spleen, lymph nodes, and bone marrow. For each cytometric analysis, 5 × 105 cells were used. All Abs (including those specific for TCRβ, 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).

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-γ production, culture supernatants were collected and assayed using a mouse IFN-γ ELISA kit (R&D Systems).

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.

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-γ using the following primer pair (1 mM): sense, 5′-CATTGAAAGCCTAGAAAGTCT-3′; antisense, 5′-ACTCATGAATGCATCCTTTTTCG-3′. β-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 10× 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-γ was 267 bp.

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 × 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).

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)) × 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.

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 × 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 × 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 × 104 cells/well), and incubated with 10 μg/ml LPS for 24 h. Culture supernatants were removed and assayed for IL-6 and TNF-α production by ELISA (IL-6 kit from BD Biosciences; TNF-α 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.

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 × 103 cells/well), and incubated with medium alone or stimulated with IL-1β (10 ng/ml) or TNF-α (10 ng/ml) for 24 h. Culture supernatants were collected and assayed for IL-6 production by ELISA (BD Biosciences).

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. 1,A). 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. 1,B. No expression of AcPL mRNA in AcPL−/− MEFs was detectable by RT-PCR analyses, confirming that this is a null mutation (Fig. 1 C).

Development of T and B lymphocytes were examined using flow-cytometric analysis of various cell surface markers (TCRβ, B220, CD4, CD8, CD25, CD44, CD43, IgM, and IgD). As shown in Fig. 2,A, 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. 2 B) and splenocytes (C) were examined.

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 TCRβ, B220, CD4, CD8, IgM, and IgD. Numbers indicate the percentage of total cells.

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 TCRβ, B220, CD4, CD8, IgM, and IgD. Numbers indicate the percentage of total cells.

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IFN-γ 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-γ, 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-γ was measured by ELISA. In wild-type splenocytes, a significant induction of IFN-γ stimulated by IL-18 alone was observed. This induction was further enhanced when cells were treated with both IL-18 and IL-12 (Fig. 3,A). However, in AcPL−/− splenocytes, induction of IFN-γ by either IL-18 alone or IL-18 plus IL-12 was completely abolished (Fig. 3 A). RT-PCR analysis also showed that IFN-γ 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).

FIGURE 3.

Impaired IL-18-induced IFN-γ production and proliferation in AcPL−/− splenocytes. A, IFN-γ 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-γ 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.

FIGURE 3.

Impaired IL-18-induced IFN-γ production and proliferation in AcPL−/− splenocytes. A, IFN-γ 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-γ 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.

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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. 3 B, 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-γ production.

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-γ was measured by ELISA. In wild-type Th1 cells, there was a significant induction of IFN-γ by IL-18 alone or IL-18 with IL-12. In AcPL−/− cells, however, IFN-γ induction was negligible (Fig. 4). RT-PCR analysis also detected increased IFN-γ 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-γ production in Th1 cells.

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-γ production by ELISA.

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-γ production by ELISA.

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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-18Rα−/− mice (6). This demonstrates that AcPL is required for in vivo IL-18-mediated NK cytotoxic activity.

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.

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.

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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. 6,B). 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).

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.

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.

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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. 6 D, 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.

In addition to partnering with IL-18Rα 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. 7 A). Similar results in response to IL-1 stimulation were also observed in wild-type and AcPL−/− macrophages (data not shown).

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-α 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-α 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.

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-α 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-α 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.

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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. 6,B) 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. 7 D). Taken together, our results demonstrate that AcPL is not required for mediating other TIR signals, at least in IL-1R, TLR3, and TLR4 systems.

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-γ or proliferate in response to IL-18. Th1 cells lacking AcPL also failed to produce IFN-γ. 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-18Rα 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-18Rα complex affords a high-affinity binding capacity for IL-18 interaction (23). Although both AcPL and IL-18Rα 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-γ production (24, 29). Moreover, anti-IL-18Rα 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.

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

The authors have no financial conflict of interest.

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.

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.

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