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The Journal of Immunology, 2007, 179, 8172 -8179
Copyright © 2007 by The American Association of Immunologists, Inc.
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Critical Role for CXC Chemokine Ligand 16 (SR-PSOX) in Th1 Response Mediated by NKT Cells1

Takeshi Shimaoka*,{dagger}, Ken-ichiro Seino{ddagger},§, Noriaki Kume, Manabu Minami, Chiyoko Nishime||, Makoto Suematsu||, Toru Kita, Masaru Taniguchi{ddagger}, Kouji Matsushima{dagger} and Shin Yonehara2,*

* Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan; {dagger} Molecular Preventive Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan; {ddagger} Research Center for Allergy and Immunology, The Institute of Physical and Chemical Research, Tsurumi-ku, Yokohama, Kanagawa, Japan; § Institute of Medical Science, St. Marianna University School of Medicine, Miyamae-ku, Kawasaki, Kanagawa, Japan; Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan; and || School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The transmembrane chemokine CXCL 16 (CXCL16), which is the same molecule as the scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX), has been shown to mediate chemotaxis and adhesion of CXC chemokine receptor 6-expressing cells such as NKT and activated Th1 cells. We generated SR-PSOX/CXCL16-deficient mice and examined the role of this chemokine in vivo. The mutant mice showed a reduced number of liver NKT cells, and decreased production of IFN-{gamma} and IL-4 by administration of {alpha}-galactosylceramide ({alpha}GalCer). Of note, the {alpha}GalCer-induced production of IFN-{gamma} was more severely impaired than the production of IL-4 in SR-PSOX-deficient mice. In this context, SR-PSOX-deficient mice showed impaired sensitivity to {alpha}GalCer-induced anti-tumor effect mediated by IFN-{gamma} from NKT cells. NKT cells from wild-type mice showed impaired production of IFN-{gamma}, but not IL-4, after their culture with {alpha}GalCer and APCs from mutant mice. Moreover, Propionibacterium acnes-induced in vivo Th1 responses were severely impaired in SR-PSOX-deficient as well as NKT KO mice. Taken together, SR-PSOX/CXCL16 plays an important role in not only the production of IFN-{gamma} by NKT cells, but also promotion of Th1-inclined immune responses mediated by NKT cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
NKT cells, which coexpress NK1.1 and TCR on their surface, differ from conventional T and NK cells expressing TCR and NK1.1, respectively. NKT cells, most of which express an invariant TCR containing V{alpha}14-J{alpha}281, were shown to specifically recognize glycolipids such as {alpha}-galactosylceramide ({alpha}GalCer)3 and isoglobotrihexosylceramide (iGb3) presented by CD1d on APCs. NKT cells are generated in the thymus and distributed to the thymus, spleen, liver and bone marrow. NKT cells are highly concentrated in the liver where they account for ~20% of lymphocytes, compared with <1% in other organs (1, 2, 3). A hallmark of NKT cells is the rapid production of large amounts of IFN-{gamma} and/or IL-4 after stimulation with {alpha}GalCer, regulating the development of Th1 and Th2 cells, respectively (2).

NKT cells were reported to play an important role in host defense against infection with bacteria, virus and protozoan parasite (4) and NKT cell-induced IFN-{gamma} was shown to be important in host defense to several types of infection (4). {alpha}GalCer-stimulated NKT cells was also shown to exert an anti-tumor effect by inducing IFN-{gamma} (5, 6). These activities of NKT cells are mainly explained by the Th1 response induced by IFN-{gamma} production by NKT cells. On the contrary, {alpha}-GalCer treatment was reported to protect NOD mice from autoimmune type I diabetes through stimulating a polarized Th2-like response; suppressing IFN-{gamma} production and/or inducing IL-4 production by NKT cells (7, 8, 9). OCH, which is a truncated version of the prototypic NKT cell ligand {alpha}-GalCer, was reported to induce a predominant production of IL-4 by NKT cells, and to suppress experimental autoimmune encephalitis (EAE) through inducing Th2 bias of autoimmune T cells (10). Thus, NKT cells can control the development of Th1- and Th2-polarized immune responses by inducing Th1 and Th2 responses through the production of large amounts of IFN-{gamma} and IL-4, respectively (2). It would be important to investigate the molecular and cellular mechanism by which NKT cells differentially produce cytokines (IFN-{gamma} or IL-4). Recently, interaction of CD40 and CD154 on APCs and NKT cells, respectively, was reported to mediate production of IFN-{gamma} but not of IL-4 by {alpha}GalCer-stimulated NKT cells, while interaction between CD28 and CD80/CD86 on NKT cells and APCs, respectively, was shown to be important for the production of both IFN-{gamma} and IL-4 (11). LFA-1 was reported to function as a costimulatory molecule for {alpha}GalCer-activated NKT cells to produce IL-4 but not IFN-{gamma} (12). However, the molecular mechanism has not been fully understood how NKT cells are regulated to specifically produce IFN-{gamma} or IL-4 leading to generation of Th1 or Th2 cells, respectively.

The chemokine superfamily consists of small proteins that trigger directed migration of various types of leukocytes by specifically interacting with a group of seven transmembrane G protein-coupled receptors. In addition to this migration-inducing activity, chemokines also mediate signals that cause the activation of integrins and differentiation of target cells (13, 14). Recently, CXCL 16 (CXCL16)/SR-PSOX (scavenger receptor that binds phosphatidylserine and oxidized lipoprotein) has been characterized as a membrane-anchored chemokine specific for the G-protein coupled receptor Bonzo/CXC chemokine receptor 6 (CXCR6) (15, 16) as well as a novel scavenger receptor for oxidized low density lipoprotein and bacteria (17, 18). SR-PSOX/CXCL16 is selectively expressed on APCs such as DCs and macrophages, and its receptor CXCR6 is expressed on NKT and activated Th1 cells (15, 16, 18, 19, 20). Furthermore, CXCR6-expressing effector T cells were shown to accumulate in type 1 inflammatory lesions such as rheumatoid joints and inflamed livers (20). In experimental autoimmune encephalitis (EAE), we showed that SR-PSOX/CXCL16 plays an important role in the elevation of the serum IFN-{gamma} level during the primary immune response as well as recruitment of inflammatory mononuclear cells into the CNS, and administration of anti-SR-PSOX mAb decreased disease activity in both acute and transfer EAE (21). In a model of graft-vs-host disease, CXCR6-deficient activated CD8 T cells were shown not to accumulate in the inflamed liver (22). Recently, Geissmann et al. reported that CXCR6-deficient mice showed a reduction in NKT cell numbers in the liver and decreased susceptibility to Con A-induced hepatitis (23). In addition, blocking of the interaction between CXCL16 and CXCR6 by anti-CXCL16 mAb was shown to result in the failure to maintain graft tolerance (24).

In this study, we have further explored the physiological role of SR-PSOX/CXCL16 by generating and analyzing SR-PSOX/CXCL16-deficient mice. SR-PSOX-deficient mice showed not only a reduced number of liver NKT cells, but also severely and moderately impaired production of IFN-{gamma} and IL-4, respectively, by administration of {alpha}GalCer. Moreover, Propionibacterium acnes (P. acnes)-induced Th1 responses were also impaired in both NKT-deficient and SR-PSOX-deficient mice. Taken together, SR-PSOX/CXCL16 was thus suggested to play a crucial role in not only the accumulation of liver NKT cells but also the enhancement of IFN-{gamma} production by NKT cells, and the promotion of Th1-polarized immunological responses mediated by NKT cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mutant Mice

SR-PSOX-deficient mice were generated in collaboration with Sankyo Co. Ltd. (Tokyo, Japan). Mouse genomic DNA containing the SR-PSOX gene was isolated from a mouse 129/SVJ genomic phage library (Stratagene). A targeting vector was constructed by replacing a genomic fragment containing exons 1, 2 and 3 with a DNA fragment containing a neomycin resistance gene (neo) (Fig. 1A). The targeting vector was introduced into ES cells (E14.1) by electroporation and the targeted clones were screened by Southern blot hybridization. SR-PSOX KO mice were backcrossed to C57BL6 mice for ten generations. All mice were bred and maintained in our animal facilities under specific pathogen-free conditions and examined at 6–12 wk of age. The genotyping of SR-PSOX KO mice was conducted by PCR using the following primers: 5'-taccgcagggtactttggatca-3' and 5'-ttgcgctcaaagcagtccacta-3', and 5'-ggatctcctgtcatctcaccttgc-3' and 5'-cggccacagtcgatgaatccagaa-3' for detection of the wild-type SR-PSOX allele (151 bp) and knockout allele (333 bp), respectively. The lack of SR-PSOX expression at the RNA level was confirmed by RT-PCR using the primers, 5'-atgaggcggggctttggacccttg-3' and 5'-agcaccggtacccaagctggtgtgc-3' (614 bp). NKT cell-deficient mice (V{alpha}14-deficient mouse) (25) were obtained from Research Center for Allergy and Immunology, RIKEN.


Figure 1
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  		FIGURE 1. Generation of SR-PSOX/CXCL16-deficient mice. (A) Structure of the SR-PSOX/CXCL16 genome (wild-type allele), the targeting vector and the predicted mutated SR-PSOX/CXCL16 gene (Mutated allele). Filled boxes represent exons. The targeting vector was designed to replace the SpeI and EcoRI fragment containing exon 1, 2 and 3 of SR-PSOX with a neomycin-resistance gene (Neo). A Herpes simplex virus 1-thymidine kinase (HSV-TK) gene was ligated to the 5' end of the targeting vector for negative selection. (B) PCR analysis of genomic DNA extracted from tails of SR-PSOX wild-type (+/+), heterozygous (+/–) and homozygous mutant (–/–) mice. The PCR product (151 bp) was amplified using wild-type SR-PSOX-specific primers (wild-type) from genomic DNA of the tails of the wild-type (+/+) and heterozygous (+/–) mice. The PCR product (333 bp) was amplified using knockout-specific primers (KO) from genomic DNA of the tails of the heterozygous (+/–) and homozygous (–/–) mice. (C) RT-PCR analysis of RNA extracted from thioglycolate-elicited macrophages and CD11c+ DCs from the wild-type (+/+) and homozygous mutant (–/–) mice. Peritoneal thioglycolate-elicited macrophages were prepared from 4 days after i.p. injection of 4% thioglycolate. CD11c+ DCs were prepared from spleen stimulated with LPS (1 µg/ml) for 2 days. Expression of SR-PSOX or translational elongation factor –1{alpha} (EF-1{alpha}) was examined by RT-PCR using total RNA from indicated cells. (D) Surface expression of SR-PSOX on thioglycolate-elicited macrophages and LPS-stimulated DCs. Flow cytometric analysis was conducted after staining with anti-SR-PSOX mAb (bold line) or control IgG (dotted line).

 
Reagents and antibodies

{alpha}GalCer (26) was kindly provided by Kirin Brewery Co. Ltd. (Takasaki, Japan). PE-labeled {alpha}GalCer/CD1d tetramer, prepared using a baculovirus expression system, was kindly provided by Dr M. Kronenberg (La Jolla Institute, La Jolla, CA). The following anti-mouse mAbs were purchased from BD Biosciences: anti-CD16/CD32 (2.4G2), anti-CD3 (2C11), anti-CD28 (37.51), PE-anti-NK1.1 (PK136), APC-anti-NK1.1, FITC-anti-TCRβ (H57–597), biotin-anti-TCRβ, APC-anti-TCRβ, biotin-anti-Ly49A (A1), PE-anti-CD11c (HL3) and FITC-anti-CD1d (1B1), FITC-anti-IFN-{gamma} (XMG1.2) and PE-anti-IL-4 (BVD4-1D11). Neutralizing polyclonal anti-IL-4 was purchased from R & D. Anti-mouse SR-PSOX Ab (12–81) was generated by Sankyo Co Ltd. (Tokyo, Japan) as described previously (27). Recombinant IL-2, IL-12 and SR-PSOX/CXCL16 were purchased from Peprotech.

Preparation of leukocytes

Liver mononuclear cells were isolated as described previously (28, 29). In brief, the liver cell suspension was passed through stainless steel or nylon mesh, washed twice, re-suspended in a 33% percoll solution and centrifuged at 2,000 rpm for 10 min at room temperature. After lysing RBC, liver leukocytes were washed once and subjected to further analysis. CD11c+ and NK1.1+ cells were purified using MACS according to the manufacturer’s directions (Miltenyi Biotec). NKT cells were further purified as CD3+ and NK1.1+ cells by using cell sorter, EPICS Elite (Coulter). Splenocyte and thymocyte suspensions were passed through nylon mesh and washed twice. After the lysing of RBC, cells were washed once and subjected to further analysis. Thioglycolate-elicited peritoneal macrophages were prepared from mice 4 days after i.p. injection of 4% thioglycolate (2 ml/mouse).

Flow cytometry

After blocking with anti-CD16/CD32 mAb for 15 min on ice, thymus, spleen or liver leukocytes were stained with various mAbs for 30 min on ice. For CXCR6 staining, murine CXCL16-human Fc{gamma} fusion protein was used as described previously (15, 27). In brief, after blocking with anti-CD16/CD32 mAb, lymphocytes were incubated with CXCL16-Fc{gamma} fusion protein or control human Fc{gamma} for 30 min on ice, and the specific binding was detected by incubation with PE-conjugated anti-human Fc{gamma} (Jackson ImmunoResearch) for 30 min on ice. For SR-PSOX/CXCL16 staining, isolated liver leukocytes were cultured for 2 h in RPMI 1640 medium containing 10% FBS, blocked with anti-CD16/CD32 mAb for 15 min on ice, and stained with PE-anti-CD11c, FITC-anti-CD1d and biotin-anti-SR-PSOX mAbs for 30 min on ice. Thereafter, cells were stained with streptavidin-PBXL for 30 min on ice, and then analyzed by flow cytometry using EPICS Elite (Coulter).

{alpha}GalCer stimulation

For in vivo {alpha}GalCer stimulation, serum was obtained from wild-type and SR-PSOX KO mice at the indicated time points after i.p. administration of {alpha}GalCer (100 µg/kg). For in vitro {alpha}GalCer stimulation, NKT and CD11c+ liver cells were isolated from male wild-type and SR-PSOX KO mice. Then, the CD11c+ APCs (2 x 104 cells/well) and the CD3+NK1.1+ NKT cells (2 x 104 cells/well) were stimulated with {alpha}GalCer (100 ng/ml) in 96-well U-bottom plates for 72 h. The culture supernatant and cells were collected for quantification of produced cytokines and apoptosis, respectively. The amounts of IFN-{gamma} and IL-4 in the culture supernatant were determined using specific ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. For apoptosis detection assays, cells were stained with PE-anti-CD11c, followed by FITC-labeled Annexin-V according to the manufacturer’s instructions (MBL) (24). After staining, cells were washed and analyzed by EPICS Elite (Coulter).

Quantitative RT-PCR analysis

Cells were directly lysed on TRIzol (Invitrogen) and total RNA was extracted following the manufacturer’s instructions. Total cDNA was prepared using random primers (Promega Corporation) with PowerScript Reverse Transcriptase (Clontech) at 42°C for 90 min. The expression of T-bet and GATA-3 was analyzed by real time quantitative PCR using SYBR Green PCR Master Mix and ABI Prism 7500 Sequence Detection System (Applied Biosystems). Used primers were; T-bet, 5'-gttgacagttgggtccaggt-3' and 5'-cgccaggaagtttcatttgg-3'; GATA-3, 5'-tcggccattcgtacatggaa-3' and 5'-gagagccgtggtggatggac-3'; GAPDH, 5'-ccttcattgacctcaacta-3' and 5'-agtgatggcatggactgtggt-3'. The amounts of T-bet and GATA-3 in each sample were calculated with ABI Prism 7500 Sequence Detection System software (Applied Biosystems) and normalized to the amount of GAPDH.

Tumor growth assay

B16 melanoma cells (5 x 105 cells/mouse) was s.c. inoculated into male wild-type and SR-PSOX KO mice, and thereafter {alpha}GalCer (100 µg/kg) or vehicle was i.p. injected repeatedly at 4 days interval. Tumor volume was measured with a caliper every 2 days. Tumor volumes (mm3) were calculated by the following formula: Tumor volumes (mm3) = (longest diameter) x (shortest diameter) (2). The data shown represent the mean ± SEM.

Analysis of Th1 responses

To analyze in vitro cytokine-producing activity of Th1 cells generated in wild-type, SR-PSOX KO and NKT KO mice, CD4+ T cells were isolated from these mice seven days after i.p. injection of heat-killed P. acnes (200 µg/mouse) using MACS CD4 T cells isolation kit. The isolated CD4+ T cells, which were free of NKT cells, were then cultured in 2.5 µg/ml anti-CD3-coated plastic plates for 24 h, and culture supernatants were analyzed for IFN-{gamma} and IL-4 production by ELISA. RNA from cell lysates were analyzed for T-bet and GATA-3 mRNA expression by real time quantitative RT-PCR. To analyze in vivo cytokine-producing activity of P. acnes–injected mice, LPS (1.5 µg/mouse) was administrated i.v. into wild-type, SR-PSOX KO and NKT NO mice seven days after i.v. injection of heat-killed P. acnes (250 µg/mouse). At the indicated time points after the administration of LPS, IFN-{gamma} concentration and GPT activity in the serum were determined by ELISA and a GPT OA test Wako kit (Wako Pure Chemicals Industries), respectively.

In vitro generation of Th1 cells

For in vitro Th1 cell differentiation, CD4+ naive T cells were isolated from spleen of male wild-type, SR-PSOX KO and NKT KO mice, and cultured in plastic plates coated with 2.5 µg/ml anti-CD3 mAb and 10 µg/ml anti-CD28 mAb in the presence of 4 ng/ml IL-2, 5 ng/ml IL-12 and 2.5 µg/ml neutralizing anti-IL-4 mAb for 7 days. Th1 differentiated CD4 T cells in wild-type, SR-PSOX KO and NKT KO mice were stained by anti-IFN-{gamma} (XMG1.2) and anti-IL-4 Ab (BVD4-1D11) for intracellular cytokine staining.

Statistical Analysis

Statistical analyses between two groups were performed by two-sample t test. p values <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of SR-PSOX/CXCL16-deficient mice

To explore the role of SR-PSOX/CXCL16 in vivo, we generated SR-PSOX/CXCL16-deficient mice by replacing exon 1, 2, and 3 of the mouse SR-PSOX gene with a neomycin-resistance gene (Fig. 1A). The correct targeting of the SR-PSOX/CXCL16 locus was confirmed by genomic PCR analysis (Fig. 1B). SR-PSOX KO mice were born according to the expected Mendelian ratio when SR-PSOX+/– mice were intercrossed. SR-PSOX KO mice were fertile and macroscopically normal (data not shown). Because SR-PSOX/CXCL16 was described to be expressed on thioglycolate-elicited peritoneal macrophages or LPS-stimulated-CD11c+ splenic DCs (15, 17), loss of SR-PSOX/CXCL16 expression was confirmed at both the RNA and protein level in these cells from SR-PSOX KO mice by RT-PCR and flow cytometry (Fig. 1C and D).

SR-PSOX plays an important role in {alpha}GalCer-induced IFN-{gamma} production in vivo

{alpha}GalCer presented by CD1d on APCs is a specific ligand of NKT cells. {alpha}GalCer-stimulated NKT cells produce large amounts of both IFN-{gamma} and IL-4, and the balance between the production of IFN-{gamma} and IL-4 was reported to determine the type of immunological environment, Th1 or Th2 responses (2). CXCR6 was reported to be expressed on NKT cells and we confirm the expression of CXCR6 on liver NKT cells and the chemotactic and adhesion activity of SR-PSOX/CXCL16 against liver NKT cells (data not shown). To evaluate the contribution of SR-PSOX/CXCL16 to the activation of NKT cells by {alpha}GalCer in vivo, {alpha}GalCer was injected into wild-type and SR-PSOX KO mice, and serum levels of IFN-{gamma} and IL-4 were quantified (Fig. 2A). In wild-type mice, serum IFN-{gamma} levels were remarkably increased by the administration of {alpha}GalCer, peaked at 12 h and decreased to the basal level by 24 h. In contrast, the levels of IFN-{gamma} in SR-PSOX KO mice were reduced to ~20% of those in wild-type mice. Serum IL-4 levels in wild-type mice also increased on administration of {alpha}GalCer, peaked at 3 h and decreased to the basal level by 6 h. The increased levels of serum IL-4 in SR-PSOX KO mice, however, were decreased to only 60% of those in wild-type mice. In SR-PSOX KO mice, the {alpha}GalCer-induced production of IFN-{gamma} was more severely impaired than the production of IL-4.


Figure 2
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  		FIGURE 2. Requirement of SR-PSOX for IFN-{gamma} production in {alpha}GalCer-administered mice. (A) Determination of serum levels of IFN-{gamma} and IL-4 after i.p. injection of {alpha}GalCer. The amounts of serum IFN-{gamma} and IL-4 were determined using specific ELISA kits (R&D Systems) in wild-type and SR-PSOX KO mice at the indicated time points after i.p. administration of {alpha}GalCer (100 µg/kg). Amounts of serum IFN-{gamma} and IL-4 in vehicle-injected wild-type and SR-PSOX KO mice were under the detection limit. Statistical analysis was performed by two-sample t test between two groups, wild-type and SR-PSOX KO mice (*, p < 0.05). (B) In vivo antitumor effect of {alpha}GalCer. B16 melanoma cells (5 x 105 cells/mouse) were s.c. inoculated into male wild-type and SR-PSOX KO mice, and thereafter {alpha}GalCer or vehicle was injected repeatedly to wild-type and SR-PSOX KO mice at 4 days interval. Tumor volume was measured every 2 days. The data shown represent the mean ± SEM.

 
{alpha}GalCer has been shown to exert an antitumor effect by inducing IFN-{gamma} from NKT cells. In {alpha}GalCer-treated mice, s.c. tumor growth was reported to be inhibited in an IFN-{gamma} dependent manner (5). To examine whether SR-PSOX is involved in in vivo antitumor effect of {alpha}GalCer, B16 melanoma cells were s.c. inoculated in wild-type and SR-PSOX KO mice and thereafter {alpha}GalCer was injected repeatedly. As reported previously, {alpha}GalCer treatment in wild-type mice significantly inhibited the outgrowth of s.c. inoculated B16 cells, while the anti-tumor effect was not observed in SR-PSOX KO mice (Fig. 2B). These results indicate that SR-PSOX/CXCL16 also plays an important role in IFN-{gamma} production from {alpha}GalCer-treated NKT cells in vivo, which exerts a potent antitumor effect.

SR-PSOX/CXCL16 functions as a costimulatory factor to induce IFN-{gamma} production by liver NKT cells

Recently, Geissmann et al. reported CXCR6 (a specific ligand for SR-PSOX/CXCL16)-deficient mice exhibit a selective reduction of the number of liver NKT cells (23). To examine the physiological roles of SR-PSOX/CXCL16 especially in regulation of the number and/or function of NKT cells in various organs, we examined the thymus, spleen and liver of SR-PSOX KO mice (Fig. 3). The absolute numbers of mononuclear cells in these organs of SR-PSOX KO mice were not significantly changed when compared with those of wild-type mice. Although the numbers of T (NK1.1–TCRβ+) and NK cells (NK1.1+TCRβ–) in these organs were essentially the same in SR-PSOX KO and wild-type mice, the number of liver NKT cells (NK1.1+TCRβ+ and CD1d tetramers+TCRβ+) was reduced in SR-PSOX KO mice compared with wild-type mice. The numbers of NKT cells in thymus and spleen of SR-PSOX KO mice were not decreased.


Figure 3
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  		FIGURE 3. Characterization of NKT cells in the liver of SR-PSOX-deficient mice. (A) Quantification of NKT cells in the liver (upper), thymus (middle) and spleen (lower) from wild-type and SR-PSOX KO mice. Mononuclear cells were prepared from the indicated organs and stained with NK1.1 and TCRβ. The percentage of NKT cells (NK1.1+ and TCR+) among the mononuclear cells is shown. (B) Quantification of V{alpha}14i NKT cells in the liver (upper), thymus (middle) and spleen (lower) from wild-type and SR-PSOX KO mice. Mononuclear cells were stained with CD1d-tetramer and TCRβ. In the case of splenocytes, cells were stained with CD1d-tetramer and TCRβ after B lymphocytes were eliminated. The percentage of V{alpha}14i NKT cells among the mononuclear cells is shown. (C) Absolute numbers of T, NK and NKT cells in the liver of wild-type and SR-PSOX KO mice. Liver mononuclear cells were stained with NK1.1 and TCRβ. Absolute numbers of T (NK1.1–TCRβ+), NK (NK1.1+TCRβ–), NKT (NK1.1+TCRβ+) and CD1d-tetramer+TCRβ+ cells were determined by flow cytometry. Statistical analysis was performed by two-sample t test (*, p < 0.05).

 
The impaired accumulation of liver NKT cells would be reflected by the overall reduction of {alpha}GalCer-induced production of cytokines including IFN-{gamma} and IL-4, but cannot explain the greater defect in IFN-{gamma} production compared with IL-4 production in {alpha}GalCer-administrated SR-PSOX KO mice. To examine the contribution of SR-PSOX to the production of cytokines by NKT cells in detail, we first investigated the expression of SR-PSOX in the liver. Because 1) SR-PSOX is expressed on activated APCs such as macrophages and DCs (Fig. 1C and D), 2) liver CD11c+ cells were reported as liver DCs (30) and 3) CD1d is necessary for {alpha}GalCer to be presented to NKT cells, we investigated whether the expression of CD1d and CD11c is related to that of SR-PSOX/CXCL16. Liver CD1d+CD11c+ cells were found to express SR-PSOX/CXCL16 (Fig. 4A), while CD1d–CD11c+, CD1d+CD11c– or CD1d–CD11c– cells did not express it (data not shown). SR-PSOX/CXCL16 was thus shown to be expressed on liver APCs. Interestingly, cytofluorometric analyses revealed the expression of SR-PSOX to be increased by stimulation with {alpha}GalCer (Fig. 4B). We confirmed loss of SR-PSOX/CXCL16 expression in these CD1d+CD11c+ cells from SR-PSOX KO mice (Fig. 4A). In contrast, the numbers of CD11c+ cells and CD1d+CD11c+ cells from the liver were shown to be almost equivalent between wild-type and SR-PSOX KO mice, and the costimulatory molecules CD80 and CD86 were equivalently expressed on CD1d+CD11c+ cells from SR-PSOX KO and wild-type mice (Fig. 4A). Thus, we did not detect any differences in the number and characteristics of liver DCs from wild-type and SR-PSOX KO mice without the expression of SR-PSOX.


Figure 4
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  		FIGURE 4. Expression of SR-PSOX/CXCL16 on liver CD11c+CD1d+ cells. (A) Surface expression of SR-PSOX, CD80 and CD86 on liver CD11c+CD1d+ cells. Mononuclear cells were isolated from the liver from wild-type or SR-PSOX KO mice, and stained with mAbs for CD11c, CD1d and SR-PSOX, CD80 or CD86. Upper panels: Total liver mononuclear cells were stained with CD11c and CD1d. CD11c+ cells were 4.1 (3.5 + 0.6)% and 3.7 (3.2 + 0.5)% of total liver mononuclear cells from wild-type and SR-PSOX KO mice, respectively. CD11c+CD1d+ cells were 0.6% and 0.5% of total liver mononuclear cells from wild-type and SR-PSOX KO mice, respectively. Lower panels: Liver CD11c+CD1d+ cells were stained with anti-SR-PSOX mAb, CD80 or CD86. (B) Surface expression of SR-PSOX on liver CD11c+CD1d+ cells after in vitro cultivation with {alpha}GalCer. Mononuclear cells were isolated from the liver of wild-type or SR-PSOX KO mice, cultured in vitro for 48 h with {alpha}GalCer (100 ng/ml), and then stained with CD11c, CD1d and SR-PSOX. Histogram shows expression levels of SR-PSOX on liver CD11c+CD1d+ cells from wild-type and SR-PSOX KO mice.

 
To examine the contribution of SR-PSOX to the production of cytokine by NKT cells in detail, we next investigated {alpha}-GalCer-induced production of IFN-{gamma} and IL-4 in vitro using total liver mononuclear cells isolated from wild-type and SR-PSOX KO mice. The liver leukocytes form wild-type mice stimulated with {alpha}-GalCer for 72 h showed vigorous production of IFN-{gamma} and IL-4. In contrast, the liver leukocytes from SR-PSOX KO mice showed decreased production of both IFN-{gamma} and IL-4 (Fig. 5A). These results would be caused by the decreased number of liver NKT cells in SR-PSOX KO mice compared with wild-type mice (Fig. 3). To examine the role of SR-PSOX/CXCL16 as a costimulatory factor, NKT cells were isolated from the liver of wild-type mice and cultured with isolated CD11c+ cells from the liver of wild-type and SR-PSOX KO mice in the presence of {alpha}GalCer (100 ng/ml) for 72 h (Fig. 5B). {alpha}GalCer was clearly shown to induce production of IFN-{gamma} and IL-4 by the NKT cells only when cocultured with CD11c+ APCs from wild-type mice. Interestingly, the wild-type NKT cells, cocultured with SR-PSOX KO CD11c+ APCs and stimulated with {alpha}GalCer, produced equal amounts of IL-4 but only about half the amount of IFN-{gamma} as those cocultured with wild-type APC (Fig. 5B). In contrast, NKT cells from SR-PSOX KO mice were shown to be able to produce equivalent amounts of IFN-{gamma} and IL-4 compared with wild-type NKT cells after cultivation with wild-type APC in the presence of {alpha}GalCer. We then examined the mRNA expression of T-bet (Th1-specific transcription factor) and GATA-3 (Th2-specific transcription factor) by quantitative real time RT-PCR. Wild-type NKT cells, cocultured with SR-PSOX KO CD11c+ APCs and stimulated with {alpha}GalCer, showed similar amounts of GATA-3 mRNA expression and significantly reduced amounts of T-bet mRNA expression, compared with those cocultured with wild-type APC (Fig. 5C). NKT cells from SR-PSOX KO mice showed similar levels of GATA-3 and T-bet mRNA expressions by {alpha}GalCer stimulation.


Figure 5
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  		FIGURE 5. Costimulatory activity of SR-PSOX/CXCL16 to induce IFN-{gamma} production by {alpha}GalCer-stimulated liver NKT cells. (A) {alpha}GalCer-induced in vitro responses in liver leukocytes. Freshly isolated liver leukocytes (2 x 105 cells/well) from male wild-type and SR-PSOX KO mice were stimulated with or without (vehicle) {alpha}GalCer (100 ng/ml) in 96-well U-bottom plates for 72 h. Then, the culture supernatant was collected and the amounts of IFN-{gamma} and IL-4 in the culture supernatant were determined by ELISA. Statistical analysis was performed by two-sample t test (*, p < 0.05). (B) {alpha}GalCer-induced responses in reconstituted liver NKT and liver CD11c+ APCs in vitro. CD3+ and NK1.1+ NKT cells and CD11c+ liver cells were isolated from male wild-type and SR-PSOX KO mice. Then, the CD11c+ APCs (2 x 104 cells/well) and the NKT cells (2 x 104 cells/well) were stimulated with {alpha}GalCer (100 ng/ml) in 96-well U-bottom plates for 72 h. Then, the culture supernatant was collected and the amounts of IFN-{gamma} and IL-4 produced were determined by ELISA. Statistical analysis was performed by two-sample t test (*, p < 0.05). (C) {alpha}GalCer-induced responses in reconstituted liver NKT and liver CD11c+ APCs in vitro. NKT and CD11c+ liver cells were isolated as APCs from male wild-type and SR-PSOX KO mice. Then, the CD11c+ APCs (2 x 104 cells/well) and the NKT cells (2 x 104 cells/well) were stimulated with {alpha}GalCer (100 ng/ml) in 96-well U-bottom plates for 72 h and isolated total RNA. Expression of T-bet and GATA-3 mRNA was determined by quantitative RT-PCR. The amount of each mRNA was normalized to GAPDH. Statistical analysis was performed by two-sample t test (*, p < 0.05). (D) {alpha}GalCer-induced apoptosis in liver NKT cocultured with liver CD11c+ APCs in vitro. NKT and CD11c+ liver cells were isolated from male wild-type and SR-PSOX KO mice. The CD11c+ APCs (2 x 104 cells/well) and the NKT cells (2 x 104 cells/well) were cocultured and stimulated with {alpha}GalCer (100 ng/ml) for 72 h. Cells were collected and stained with PE-anti-CD11c and FITC-labeled Annexin-V. The numbers of apoptotic NKT cells were quantified as CD11c-negative and annexin-V-positive cells and expressed as percentages to the total number of CD11c-negative cells. Statistical analysis was performed by two-sample t test (*, p < 0.05).

 
Geissmann et al. demonstrated a deficiency of CXCR6 leading to reduced survival of NKT cells (23). We quantified apoptosis in liver NKT cells from wild-type and SR-PSOX-deficient mice after in vitro cultivation of liver mononuclear cells for 0, 7 and 14h, indicating that the numbers of annexin V-positive apoptotic NKT cells (NK1.1+TCRβ+) to be the same in both groups (data not shown). We also quantified apoptosis in {alpha}GalCer-stimulated NKT cells. Purified liver NKT cells from wild-type or SR-PSOX KO mice were cultured with wild-type or SR-PSOX KO CD11c+ APCs in the presence of {alpha}GalCer for 72h. Apoptotic NKT cells were then quantified as CD11c-negative and annexin-V-positive cells, indicating that the numbers of apoptotic NKT cells did not differ among all the groups (Fig. 5D). These results are not inconsistent with those of CXCR6-deficient mice, because the reduced survival of liver NKT cells in CXCR6-deficient mice was indicated to be independent of its ligand SR-PSOX/CXCL16 (23) and SR-PSOX-deficient NKT cells express CXCR6 (data not shown). Taken together, the reduced production of IFN-{gamma} in SR-PSOX KO mice cannot simply explained by increased death of stimulated NKT cells, and SR-PSOX/CXCL16 was shown to function as a costimulatory factor for liver NKT cells expressing CXCR6 to produce IFN-{gamma} but not IL-4. The costimulatory function of SR-PSOX may contribute the establishment of a Th1-polarized immune environment mediated by NKT- cells.

SR-PSOX-deficient mice are impaired in Propionibacterium acnes–induced Th1 responses

In several reports, IFN-{gamma} production from NKT cells was shown to play important roles in host defense against bacteria (4). Expression of SR-PSOX/CXCL16 mRNA was reported to be increased in the mouse liver after administration of Propionibacterium acnes (P. acnes) (31), which is known to be an agent that induces Th1 responses in vivo (32, 33). Therefore, we examined P. acnes-induced Th1 responses in NKT-KO and SR-PSOX KO mice. After 7 days i.p. injection of heat-killed P. acnes, splenic CD4 T cells were isolated and stimulated with anti-CD3 mAb for 24 h. And then, culture supernatants were analyzed for IFN-{gamma} and IL-4 production (Fig. 6A). Elevated production of IFN-{gamma}, but not IL-4, was observed in P. acnes-primed wild-type CD4 T cells, however, P. acnes-primed CD4 T cells of NKT KO and SR-PSOX KO mice produced less than half the amounts of IFN-{gamma} compared with those from wild-type mice (Fig. 6A). Of note, although CXCR6 was reported to be expressed on activated T cells prepared by in vitro cultivation with anti-CD3 and anti-CD28 (27), P. acnes-primed CD4 T cells did not express CXCR6 (data not shown), indicating that SR-PSOX on APCs and CXCR6 on NKT cells would play an important role in P. acnes-primed CD4 T cells to produce IFN-{gamma}. We next examined the mRNA expression of T-bet and GATA-3 by quantitative real time RT-PCR. Although expression of T-bet mRNA, but not GATA-3 mRNA, was increased in P. acnes-primed wild-type CD4 T cells, P. acnes-primed CD4 T cells from NKT KO and SR-PSOX KO mice exhibited significantly lower T-bet expression than those from wild-type mice (Fig. 6B). We also analyzed serum IFN-{gamma} level after administration of LPS into P. acnes-sensitized mice (Fig. 6C). Seven days after i.v. injection of heat-killed P. acnes, small amount of LPS (1.5 µg/mouse) was i.v. injected. Serum IFN-{gamma} levels were remarkably increased and peaked at 6 h after LPS injection in wild-type mice, whereas serum IFN-{gamma} level was not increased in NKT-KO and SR-PSOX KO mice (Fig. 6C). The activity of GPT, a marker of damage to the liver, was also not increased in NKT KO and SR-PSOX KO mice (Fig. 6C). All the data show that both NKT cells and SR-PSOX/CXCL16 are involved in in vivo Th1 responses induced by P. acnes.


Figure 6
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  		FIGURE 6. Impairment of in vivo Th1 responses in SR-PSOX KO and NKT KO mice. (A) Th1 responses induced by P. acnes. Male wild-type, SR-PSOX KO and NKT KO mice were i.p. injected with 200 µg/mouse heat-killed P. acnes. Seven days after injection, CD4+ T cells from the spleen were stimulated with anti-CD3 for 24 h. Culture supernatants were analyzed for IFN-{gamma} and IL-4 production by ELISA. Statistical analysis was performed by two-sample t test (*, p < 0.05). (B) Expression of T-bet and GATA-3 mRNA in P. acnes-primed CD4+ T cells. Male wild-type, SR-PSOX KO and NKT KO mice were i.p. injected with 200 µg/mouse heat-killed P. acnes. Seven days after injection, CD4+ T cells from the spleen were stimulated with anti-CD3 for 24h and isolated total RNA. Expression of T-bet and GATA-3 mRNA was determined by quantitative RT-PCR. The amount of each mRNA was normalized to GAPDH. Statistical analysis was performed by two-sample t test (*, p < 0.05). (C) LPS challenge in P. acnes-sensitized mice. Seven days after i.v. administration of heat-killed P. acnes (250 µg/mouse) into wild-type, SR-PSOX KO and NKT KO mice, 1.5 µg LPS was injected i.v. Serum IFN-{gamma} concentration and GPT activity were determined at the indicated time points after the administration of LPS by ELISA and a GPT OA test Wako kit (Wako Pure Chemicals Industries), respectively. The data shown represent the mean ± SEM. (D) In vitro generation of Th1 cells from wild-type, SR-PSOX KO and NKT KO mice. Th1 cells were generated in Th1-skewed condition as described in Materials and Methods. Th1 differentiated CD4 T cells in wild-type, SR-PSOX KO and NKT KO mice were stained by anti-IFN-{gamma} and anti-IL-4 Ab for intracellular cytokine staining.

 
To confirm the role of NKT cell-induced cytokine in SR-PSOX/CXCL16-mediated in vivo Th1 responses, we further examined whether SR-PSOX/CXCL16 affected in vitro generation of Th1 cells in the absence of NKT cells. Splenic CD4 T cells from wild-type, NKT KO and SR-PSOX KO mice were cultured with anti-CD3 and anti-CD28 mAbs in the presence of IL-2, IL-12 and anti-IL-4 for 7 days, and then intracellular cytokine staining were performed. Significant differences were not found in IFN-{gamma} and IL-4 production by in vitro generated Th1 cells from wild-type, NKT KO and SR-PSOX KO mice (Fig. 6D). Thus, SR-PSOX/CXCL16 and NKT cells were involved in neither in vitro generation of Th1 cells by cultivation with exogenous IL-2, IL-12 and anti-IL-4 nor production of IFN-{gamma} from previously differentiated Th1 cells.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, {alpha}GalCer-administrated SR-PSOX KO mice showed severely and slightly impaired production of IFN-{gamma} and IL-4, respectively (Fig. 2A). In SR-PSOX KO mice, the number of liver NKT cells was revealed to be specifically reduced (Fig. 3). This reduction in NKT cell numbers might be reflected by the overall reduced production of cytokines including IFN-{gamma} and IL-4, but cannot fully explain the greater defect in IFN-{gamma} production than IL-4 production in {alpha}GalCer-administrated SR-PSOX KO mice (Fig. 2A). We also demonstrated that SR-PSOX/CXCL16 is expressed on CD1d+CD11c+ cells and functions as a costimulatory factor to induce liver NKT cells to produce IFN-{gamma} but not IL-4 through reduction of T-bet mRNA expression (Th1-specific transcription factor) (Fig. 5). Taken together, severe impairments of {alpha}GalCer-induced IFN-{gamma} production in SR-PSOX KO mice in vivo would be reflected by both reduction of the number of liver NKT cells and defect of the costimulatory function of APCs to induce IFN-{gamma} from NKT cells. We also investigated IFN-{gamma} production by NKT cells in SR-PSOX KO and wild-type mice after stimulation with iGb3, a commercially available physiological ligand for NKT cells; however significant amounts of IFN-{gamma} were not detected in either serum of iGb3-injected mice nor culture supernatant of iGb3-stimulated total liver leukocytes (data not shown).

NKT cells produce large amounts of IFN-{gamma} and/or IL-4 soon after stimulation with {alpha}GalCer, and regulate the development of Th1 and Th2 cells (2). CD40, CD28 and LFA-1 on APC are reported to work as costimulatory factors in IFN-{gamma} and/or IL-4 production by {alpha}GalCer-stimulated NKT cells (11, 12). CD40 mediates production of IFN-{gamma} but not of IL-4, while CD28 is important for both (11). Interestingly, in {alpha}GalCer administrated LFA-1-deficient mice, the production of IL-4 was increased twice as compared with wild-type, although the production of IFN-{gamma} was almost normal (12). GATA-3 expression but not T-bet was increased in LFA-deficient NKT cells by in vitro {alpha}GalCer-stimulation. In contrast, our data indicate that T-bet expression in NKT cells is important for SR-PSOX-induced IFN-{gamma} production. Taken together, LFA-1 acts as a negative regulator of NKT cell-regulated Th2-like immunity induced by {alpha}GalCer, while SR-PSOX acts as a positive regulator of NKT cell-regulated Th1-like immunity by {alpha}GalCer (12). LFA-1-deficient mice also show a reduction in the number of NKT cells only in the liver. Given that chemokines presented on endothelial cells are known to mediate the activation of integrin, and both chemokine and integrin are necessary for leukocytes to be arrested in the secondary lymphoid organs (13, 14), SR-PSOX and LFA-1 may play an important role not only in the control of the liver-specific accumulation of NKT cells but also in the regulation of IFN-{gamma} and IL-4 production as costimulatory molecules.

CXCR6 was shown to be expressed on IFN-{gamma}-producing Th1 cells as well as NKT cells (15, 16, 18, 20), and Th1 cells generated in vitro also showed chemotaxis and adhesion activities toward SR-PSOX/CXCL16 (data not shown). We also found severely impaired P. acnes-induced in vivo Th1 response in NKT KO and SR-PSOX KO mice (Fig. 6A–C). However, Th1 cells from splenocytes and in vitro stimulated Th1 cells were normal in both NKT KO and SR-PSOX KO mice (Fig. 6D). All these results indicate that SR-PSOX/CXCL16 is involved in P. acnes-induced Th1 response by stimulating NKT cells to produce IFN-{gamma}, likely by acting as a costimulatory molecule. However, it remains to be determined how NKT cells and their produced IFN-{gamma} induce in vivo generation of Th1 cells by P. acnes administration.

In conclusion, SR-PSOX plays a crucial role in in vivo IFN-{gamma} production by administration of {alpha}GalCer and SR-PSOX on liver CD11c+CD1d+ dendritic cells enhances in vitro IFN-{gamma} production from NKT cells as a costimulatory factor. SR-PSOX also plays important roles in inducing Th1 responses in vivo. Potent cytokine producing activity of NKT cells by {alpha}GalCer stimulation has been recognized to be an attractive target for various immunotherapies, because regulation of NKT cell-produced cytokine (IFN-{gamma} or IL-4) has been shown to control Th1- or Th2-inclined immune responses involved in infection immunity, anti-tumor immunity, autoimmune disease and tolerance (1, 2, 20, 21, 34). Importantly, activation of NKT cells was reported to protect NOD mice and other mice from the onset and recurrence of autoimmune type I diabetes (7, 8, 9) and the onset of EAE (10), respectively, by suppressing IFN-{gamma} production and/or enhancing IL-4 production. Modification of SR-PSOX-stimulated IFN-{gamma} production from NKT cells may be useful in stimulation or suppression of NKT cell-mediated immunotherapy as well as immune reactions.


   Acknowledgment
 
We thank Drs. K. Sakamaki, K. K. Lee, O. Yoshie, S. Ueha, K. Matsuno, and M. Harada for generous help.


   Disclosures
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 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 in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from Solution Oriented Research for Science and Technology of Japan Science and Technology Corporation. Back

2 Address correspondence and reprint requests to Dr. Shin Yonehara, Graduate School of Biostudies, Kyoto University, South Campus Research Building (Building G), Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. E-mail address: yonehara{at}lif.kyoto-u.ac.jp Back

3 Abbreviations used in this paper: {alpha}GalCer, {alpha}-galactosylceramide; GPT, glutamic pyruvic transaminase; SR-PSOX, scavenger receptor that binds phosphatidylserine and oxidized lipoprotein. Back

Received for publication March 22, 2007. Accepted for publication October 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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E. Germanov, L. Veinotte, R. Cullen, E. Chamberlain, E. C. Butcher, and B. Johnston
Critical Role for the Chemokine Receptor CXCR6 in Homeostasis and Activation of CD1d-Restricted NKT Cells
J. Immunol., July 1, 2008; 181(1): 81 - 91.
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