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Regulation by Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 of -Galactosylceramide-Induced Antimetastatic Activity and Th1 and Th2 Responses of NKT Cells¹

Jun Okajo^*, Yoriaki Kaneko^*, Yoji Murata, Takeshi Tomizawa^*, Chie Okuzawa^*, Yasuyuki Saito^*, Yuka Kaneko, Tomomi Ishikawa-Sekigami^*, Hideki Okazawa, Hiroshi Ohnishi, Takashi Matozaki^2, and Yoshihisa Nojima^*

^* Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, Gunma, Japan; and Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan

	Abstract

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Interaction of -galactosylceramide (-GalCer) presented by CD1d on dendritic cells (DCs) with the invariant TCR of NKT cells activates NKT cells. We have now investigated the role of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), a transmembrane protein abundantly expressed on DCs, in regulation of NKT cells with the use of mice that express a mutant form of SHPS-1. The suppression by -GalCer of experimental lung metastasis was markedly attenuated in SHPS-1 mutant mice compared with that apparent in wild-type (WT) mice. The antimetastatic effect induced by adoptive transfer of -GalCer-pulsed DCs from SHPS-1 mutant mice was also reduced compared with that apparent with WT DCs. Both the production of IFN- and IL-4 as well as cell proliferation in response to -GalCer in vitro were greatly attenuated in splenocytes or hepatic mononuclear cells from SHPS-1 mutant mice compared with the responses of WT cells. Moreover, CD4⁺ mononuclear cells incubated with -GalCer and CD11c⁺ DCs from SHPS-1 mutant mice produced markedly smaller amounts of IFN- and IL-4 than did those incubated with -GalCer and CD11c⁺ DCs from WT mice. SHPS-1 on DCs thus appears to be essential for -GalCer-induced antimetastatic activity and Th1 and Th2 responses of NKT cells. Moreover, our recent findings suggest that SHPS-1 on DCs is also essential for the priming of CD4⁺ T cells by DCs.

	Introduction

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Natural killer T cells are characterized by the expression of an invariant TCR encoded by V14, J281, and V8, 7, or 2 gene segments. They are capable of producing large amounts of IFN- and IL-4, exhibit antitumor activities, and are implicated in suppression of the development of autoimmune diseases (1, 2, 3). Such functions of NKT cells are initiated by interaction of the invariant TCR with the glycolipid -galactosylceramide (-GalCer),³ which is presented by the MHC class I-like molecule CD1d on the surface of dendritic cells (DCs) and other APCs (1, 2, 3). In addition, interactions between costimulatory molecules expressed on DCs and NKT cells, such as that between CD40 and CD40L or that between CD80/86 and CD28, are thought to be essential for activation of NKT cells (4). IL-12, which is produced by DCs, is also required for the production of IFN- by NKT cells and the antimetastatic activity of NKT cells (5, 6). DCs are thus thought to be crucial for the biological functions of NKT cells, although the precise mechanisms by which DCs trigger the activation of NKT cells remain largely unknown.

Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) (7), also known as signal-regulatory protein (8, 9), is a transmembrane protein whose extracellular region comprises three Ig-like domains. The cytoplasmic region of SHPS-1 contains four tyrosine phosphorylation sites that mediate the binding of SHP-1 and SHP-2. Tyrosine phosphorylation of SHPS-1 is regulated by various growth factors and cytokines as well as by integrin-mediated cell adhesion to extracellular matrix proteins (7, 8, 10). SHPS-1 thus functions as a docking protein to recruit and activate SHP-1 or SHP-2 at the cell membrane in response to extracellular stimuli.

CD47 is a ligand for the extracellular region of SHPS-1 (11, 12). This protein, which was originally identified in association with _v3 integrin, is also a member of the Ig superfamily, possessing an Ig-V-like extracellular domain, five putative membrane-spanning segments, and a short cytoplasmic tail (13). Among hemopoietic cells, SHPS-1 is especially abundant in DCs, macrophages, and neutrophils (9, 10, 11, 14). In contrast, CD47 is expressed in a variety of hemopoietic cells, including RBCs and T cells (13). Indeed, the interaction of CD47 on RBCs with SHPS-1 on macrophages is thought to prevent phagocytosis of the former cells by the latter (15, 16, 17).

Ligation of SHPS-1 by a soluble CD47-Fc fusion protein suppressed the phenotypic and functional maturation of immature DCs and inhibited cytokine production by mature DCs (14), suggesting that SHPS-1 (on DCs), through its interaction with CD47 (on T cells), prevents activation of DCs. Moreover, the inhibitory effect of SHPS-1/MyD-1 ligation by the Abs is regulated by the ability of Abs to regulate levels of TNF- and thus T cell functions (18). In contrast, SHPS-1 is thought to play a stimulatory role in the production of NO by macrophages (19). These effects of SHPS-1 were demonstrated in vitro, however, and the physiological roles of SHPS-1 in the immune system remain largely unknown. Furthermore, as far as we are aware, the role of SHPS-1 in regulation of NKT cell functions has not previously been investigated.

We previously generated mice that express a mutant version of SHPS-1 that lacks most of the cytoplasmic region (16, 17). This mutant protein does not undergo tyrosine phosphorylation or form a complex with SHP-1 or SHP-2. Furthermore, the abundance of this mutant protein in mouse cells is markedly reduced compared with that of the full-length native protein in wild-type (WT) mice. With the use of these SHPS-1 mutant mice, we have now examined the possible roles of SHPS-1 in prevention of experimental cancer metastasis by, and in the Th1 and Th2 responses of, NKT cells stimulated with -GalCer.

	Materials and Methods

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Abs and other reagents

-GalCer ((2S,3S,4R)-1-o-(-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol) was provided by Kirin Brewery and dissolved in distilled water supplemented with 0.5% (w/v) polysorbate-20. Hybridoma cells producing the rat P84 mAb to SHPS-1 and those producing a rat mAb to mouse CD47 (miap301) were provided by C. F. Lagenaur (University of Pittsburgh, Pittsburgh, PA) and P.-A. Oldenborg (Umeå University, Umeå, Sweden), respectively. A mAb to mouse CD16/32 (2.4G2), a FITC-conjugated mAb to mouse CD3 (145.2C11), a FITC-conjugated mAb to mouse CD11c (HL3), a PE-conjugated mAb to mouse CD8 (53-6.7), a FITC-conjugated mAb to the mouse TCR chain (H57-597), a PE-conjugated mAb to mouse IgG1, a biotin-conjugated mAb to mouse CD40 (3/23), a biotin-conjugated rat IgG to TNP (a biotin-conjugated control mAb), PerCP-conjugated streptavidin, PE-conjugated streptavidin, and dimeric mouse CD1d-Ig were from BD Pharmingen. Biotin-conjugated mAbs to mouse NK1.1 (PK136), to mouse CD1d (1B1), to mouse MHC class I (28-14-8), to mouse MHC class II (M5/114.15.2), to mouse CD80 (16-10A1), and to mouse CD86 (GL1) were from eBioscience; normal rat IgG was from Sigma-Aldrich; and PE-conjugated goat Abs to rat IgG were from Caltag Laboratories.

Animals

The generation of mice that express a mutant version of SHPS-1 that lacks most of the cytoplasmic region was described previously (16, 17). Mice were bred and maintained in the Institute of Experimental Animal Research of Gunma University under specific pathogen-free conditions. The mutant mice were backcrossed onto the C57BL/6N background for five generations. All animal experiments were performed in accordance with institutional and national guidelines and were approved by the Experimental Animal Committee of Gunma University (Gunma, Japan).

Cell preparation

Cell suspensions were prepared from spleen, liver, thymus, and bone marrow of WT (C57BL/6N) and SHPS-1 mutant mice, as previously described (20, 21). For preparation of splenocytes or thymocytes, the spleen or thymus was ground gently with autoclaved frosted-glass slides in PBS. RBCs were subjected to hypotonic lysis by incubation of the tissue suspension with Gey’s solution, and the remaining cells were washed twice with PBS. CD4⁺ mononuclear cells (MNCs) were purified from splenocytes with the use of magnetic beads coated with a mAb to CD4 (Miltenyi Biotec). For preparation of bone marrow cell suspensions, marrow was flushed from the femur with PBS and the constituent cells were washed with PBS, suspended in Gey’s solution, and then again washed twice with PBS. For preparation of hepatic MNCs, liver tissue was pressed through a 180-gauge stainless steel mesh. The liver cells in the filtrate were suspended in RPMI 1640 medium (Sigma-Aldrich), isolated by centrifugation at 500 x g for 5 min, and resuspended in RPMI 1640 containing 40% Percoll (Amersham Biosciences). The cell suspension was gently overlaid on a solution of 70% Percoll in PBS and then centrifuged at 750 x g for 20 min at room temperature. Cells were collected from the interface of the two Percoll solutions, resuspended in Gey’s solution, and washed twice with PBS.

Preparation of a DC-enriched low-density fraction (LDF) of splenic cells and that of CD11c⁺ DCs was performed, as previously described (22). In brief, the spleen was homogenized and treated with collagenase (WAKO), and the released splenocytes were suspended in 2 ml of Ca²⁺- and Mg²⁺-free HBSS (Invitrogen Life Technologies) containing 17% Optiprep (Nycomed Pharma). The cell suspension was overlaid consecutively with 2 ml of a solution containing 12% Optiprep and with 2 ml of Ca²⁺- and Mg²⁺-free HBSS. The gradient was then centrifuged at 700 x g for 15 min at 20°C, and the cells at the interface between the top two layers were collected, washed twice with PBS, and used as the DC-enriched LDF. CD11c⁺ DCs were further isolated from the splenic LDF with the use of magnetic beads coated with a mAb to CD11c (Miltenyi Biotec).

Flow cytometry

For determination of the expression of SHPS-1 or CD1d on CD8⁺CD11c⁺ or CD8^–CD11c⁺ DCs, a DC-enriched LDF of splenic cells was first incubated with a mAb to mouse CD16/32 to prevent nonspecific binding of labeled mAbs to FcRs. The cells were washed and then incubated with biotin-conjugated mAbs to SHPS-1 or to mouse CD1d as well as with a FITC-conjugated mAb to mouse CD11c and a PE-conjugated mAb to mouse CD8. The cells were washed again and then incubated with PerCP-conjugated streptavidin before analysis by three-color flow cytometry with a FACSCalibur instrument and CellQuest software (BD Biosciences). The expression of CD47 and SHPS-1 on CD3⁺NK1.1⁺ splenocytes was similarly determined by three-color flow cytometry.

The distribution of NKT cells in various organs was determined as described (21, 23). In brief, either -GalCer or vehicle was mixed with dimeric CD1d-Ig in PBS and incubated overnight at room temperature. Cell suspensions from various organs were first incubated with a mAb to mouse CD16/32 to prevent nonspecific binding of labeled mAbs to FcRs. The cells were washed and then incubated with the mixtures of -GalCer or vehicle with dimeric CD1d-Ig as well as with a FITC-conjugated mAb to the mouse TCR chain. The cells were again washed and then incubated with a PE-conjugated mAb to mouse IgG1 and analyzed by two-color flow cytometry.

Experimental lung metastasis

Cultured B16-BL6 melanoma cells (provided by K. Takeda and K. Okumura, Juntendo University, Tokyo, Japan) were harvested, washed three times with serum-free RPMI 1640 medium, and suspended in PBS. The cells (4 x 10⁴) were then injected i.v. into WT or SHPS-1 mutant mice, which were also injected i.p. with 2 µg of -GalCer or with vehicle on days 0, 4, and 8. For adoptive transfer of -GalCer-pulsed DCs (24), CD11c⁺ DCs were prepared from the spleen of WT or SHPS-1 mutant mice, as described above, and then incubated overnight under a humidified atmosphere of 5% CO₂ and at 37°C with -GalCer (100 ng/ml) in RPMI 1640 supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate, 10 mM HEPES-NaOH (pH 7.4), penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM L-glutamine (RPMI 1640 complete medium). The cells (1 x 10⁵) were then injected together with B16-BL6 melanoma cells i.v. into WT mice on day 0. Alternatively, CD11c⁺ DCs prepared from the spleen of WT mice were pulsed with -GalCer, as described above, and then the cells (1 x 10⁵) were injected together with B16-BL6 cells i.v. into either WT or SHPS-1 mutant mice on day 0. In all experiments, mice were killed on day 14 and the number of tumor colonies formed in the lungs was counted with the use of a dissection microscope (MZ9.5; Leica Microsystems).

In vitro stimulation of splenocytes or hepatic MNCs with -GalCer

Freshly prepared splenocytes (5 x 10⁵) or hepatic MNCs (2.5 x 10⁵) were cultured for 72 h with various concentrations of -GalCer or vehicle in RPMI 1640 complete medium in 96-well U-bottom plates (Corning). The culture supernatants were then collected for measurement of the concentrations of IFN- and IL-4 with the use of ELISA kits (BD Pharmingen). For assay of cell proliferation, cells were incubated with various concentrations of -GalCer or vehicle for 72 h, and 1 µCi of [³H]TdR (PerkinElmer) was added to each well for the final 16 h of incubation. The cells were then collected on glass fiber filters with the use of an automated sample harvester (PerkinElmer), and the incorporated radioactivity was measured with a scintillation spectrometer (PerkinElmer).

In vitro stimulation of CD4⁺ MNCs with CD11c⁺ DCs and -GalCer

CD4⁺ MNCs (2 x 10⁵) prepared from the spleen of WT mice, as described above, were cultured together with CD11c⁺ DCs (1 x 10⁵) isolated from WT or SHPS-1 mutant mice as well as with -GalCer (100 ng/ml) or vehicle. Alternatively, CD4⁺ MNCs prepared from the spleen of WT or SHPS-1 mutant mice were cultured together with CD11c⁺ DCs isolated from WT mice and with -GalCer (100 ng/ml). After culture for 72 h, culture supernatants were collected for measurement of IFN- and IL-4.

Cytotoxicity assay

The -GalCer-induced cytolytic activity of NKT cells against NK-sensitive YAC-1 cells or NK-resistant P815 cells (both provided by K. Takeda and K. Okumura, Juntendo University, Tokyo, Japan) was determined with the use of DELFIA europium (Eu) 2,2^'':6',2^''-terpyridine-6,6^''-dicarboxylic acid (TDA) cytotoxicity assay reagents (Wallac Oy), as previously described (25, 26). Hepatic MNCs as effector cells were isolated from WT or SHPS-1 mutant mice injected i.p. 24 h previously with 2 µg of -GalCer or vehicle. Each type of target cell was cultured under a humidified atmosphere of 5% CO₂ at 37°C in RPMI 1640 complete medium. The target cells were then washed with PBS, labeled with TDA, washed again, and resuspended in culture medium. They (1 x 10⁴ per well) were plated in 96-well round-bottom plates and incubated for 2 h with various numbers of effector cells. The plates were then centrifuged, and 20 µl of each supernatant was transferred to a well of a 96-well microtiter plate containing 200 µl of DELFIA Eu solution. The Eu forms a stable complex with TDA released from lysed target cells into the medium and generates fluorescence, which was measured with a time-resolved fluorometer (ARVO SX1420; Wallac Oy). The percentage of target cell lysis was calculated as: 100 x (experimental release – spontaneous release)/(maximal release – spontaneous release). Maximal release was determined by lysis of target cells with DELFIA lysis buffer; spontaneous release was measured by incubation of target cells in the absence of effector cells.

Assays for -GalCer-induced DC maturation and the serum level of IL-12

For assay of -GalCer-induced DC maturation, a DC-enriched LDF of splenic cells was prepared from -GalCer-treated WT or SHPS-1 mutant mice 0, 3, and 6 h after injection. The cells were then incubated with a biotin-conjugated mAb to either MHC class I, to MHC class II, to CD80, to CD86, to CD40, or a biotin-conjugated control mAb and with a FITC-conjugated mAb to mouse CD11c. The cells were further incubated with PE-conjugated streptavidin and analyzed by two-color flow cytometry. WT or SHPS-1 mutant mice were injected i.p. with 2 µg of -GalCer or with vehicle. The level of serum IL-12p70 was measured by ELISA kits (BD Pharmingen) 6 h after injection with -GalCer.

Statistical analysis

Data are presented as means ± SE and were analyzed by Student’s t test. A p value of <0.05 was considered statistically significant.

	Results

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Expression of SHPS-1 and CD1d on CD11c⁺ DCs of WT and SHPS-1 mutant mice

SHPS-1 is expressed at a high level on the surface of DCs and macrophages, but at a low level in T and B cells (9, 11, 12, 16, 27). We therefore first examined the expression of SHPS-1 on splenic CD11c⁺ DCs of WT or SHPS-1 mutant mice. The level of expression of SHPS-1 on CD8^–CD11c⁺ cells of WT mice was much greater than that on the corresponding CD8⁺CD11c⁺ cells (Fig. 1A). During the course of the present study, a similar expression pattern of SHPS-1 was also demonstrated by Naik et al. (28). In contrast, the extent of SHPS-1 expression on CD8⁺CD11c⁺ or CD8^–CD11c⁺ DCs of SHPS-1 mutant mice was greatly reduced compared with that apparent for the corresponding cells of WT mice (Fig. 1B). Moreover, the percentage of CD8^–CD11c⁺ DCs from SHPS-1 mutant mice was markedly lower than the value for WT mice (5.39 ± 0.09 vs 17.15 ± 2.09%, respectively; means ± SE, n = 3, p < 0.05) (Fig. 1). We also examined the expression of CD1d on CD11c⁺ DCs of WT or SHPS-1 mutant mice. The expression of CD1d was greater on CD8⁺CD11c⁺ DCs of WT mice than on the corresponding CD8^–CD11c⁺ cells (Fig. 1A), as previously described (29). The level and pattern of CD1d expression on CD11c⁺ DCs of SHPS-1 mutant mice were similar to those apparent for the corresponding cells of WT mice (Fig. 1B).

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Expression of SHPS-1 and CD1d on CD8-positive or -negative CD11c+ DCs or CD8-negative CD11c– DCs of WT or SHPS-1 mutant mice. A DC-enriched LDF of splenic cells from WT (A) or SHPS-1 mutant (MT) (B) mice was incubated with a biotin-conjugated mAb to SHPS-1 (thick trace), a biotin-conjugated mAb to mouse CD1d (thick trace), or a biotin-conjugated control mAb (thin trace), as indicated; with a FITC-conjugated mAb to mouse CD11c; and with a PE-conjugated mAb to mouse CD8. The cells were then incubated with PerCP-conjugated streptavidin and analyzed by three-color flow cytometry. The relative numbers of cells in each region were expressed as a percentage of all viable splenocytes, as indicated on each plot. All results are representative of three separate experiments.

We also examined the expression of SHPS-1 as well as that of the SHPS-1 ligand CD47 on CD3⁺NK1.1⁺ NKT cells. The expression of SHPS-1 was virtually undetectable on NKT cells of either WT or SHPS-1 mutant mice (Fig. 2A). In contrast, the expression of CD47 on NKT cells was readily detected, but was similar for WT and SHPS-1 mutant mice (Fig. 2A). Similarly, the expression of SHPS-1 was minimal on NK cells (CD3^–NK1.1⁺ cells) of either WT or SHPS-1 mutant mice, whereas the expression of CD47 on NK cells was readily detected, but was similar for WT and SHPS-1 mutant mice (Fig. 2A). We then determined the percentage of NKT cells (those identified by flow cytometric analysis with both a CD1d-Ig:-GalCer complex and a mAb to TCR) among cells isolated from the spleen, thymus, bone marrow, or liver of WT and SHPS-1 mutant mice. The total cell number in each organ did not differ substantially between WT and SHPS-1 mutant mice (data not shown). Furthermore, the percentage of NKT cells in each organ was virtually indistinguishable between mice of the two genotypes (Fig. 2B), although the percentage of NKT cells in the spleen of SHPS-1 mutant mice was slightly higher than the value for WT mice (2.86 ± 0.25 vs 1.61 ± 0.34%, respectively; means ± SE, n = 3, p < 0.05).

View larger version (46K): [in this window] [in a new window]   FIGURE 2. Expression of SHPS-1 and CD47 on NKT or NK cells and the distribution of NKT cells in various organs of WT or SHPS-1 mutant mice. A, Freshly isolated splenocytes of WT or SHPS-1 mutant mice were incubated with a mAb to SHPS-1 (thick trace), a mAb to mouse CD47 (thick trace), or a control mAb (thin trace); with a biotin-conjugated mAb to mouse NK1.1; and with a FITC-conjugated mAb to mouse CD3. The cells were then incubated with PE-conjugated goat Abs to rat IgG and PerCP-conjugated streptavidin. The expression of SHPS-1 or CD47 was then analyzed by flow cytometry on electronically gated CD3+NK1.1+ cells for NKT cells and CD3–NK1.1+ cells for NK cells, respectively. The relative numbers of cells in each region were expressed as a percentage of all viable splenocytes, as indicated on each plot. B, Cells from the indicated organs (BM, bone marrow) of WT or SHPS-1 mutant mice were labeled with -GalCer-bound CD1d-Ig dimers (or CD1d-Ig dimers incubated with vehicle) and with a FITC-conjugated mAb to the mouse TCR chain. They were then incubated with a PE-conjugated mAb to mouse IgG1 and analyzed by two-color flow cytometry. The numbers indicate the percentage of total cells positive for labeling with both CD1d-Ig dimers and the mAb to TCR. All results are representative of three separate experiments.

Loss of -GalCer-induced antimetastatic activity in SHPS-1 mutant mice

NKT cells activated by -GalCer exhibit antimetastatic activity against various cancer cell lines in a manner dependent, at least in part, on NK cells (1, 2). To investigate the possible role of SHPS-1 in regulation of NKT cell functions, we therefore examined the effect of -GalCer on metastasis of B16-BL6 melanoma cells to the lungs of WT and SHPS-1 mutant mice. The numbers of metastatic nodules formed in the lungs were similar for vehicle-treated WT and SHPS-1 mutant mice (Fig. 3A). However, whereas treatment with -GalCer markedly inhibited the metastasis of B16-BL6 melanoma cells to the lungs of WT animals, it had no such effect in SHPS-1 mutant mice (Fig. 3A), suggesting that SHPS-1 is essential for the -GalCer-induced antimetastatic activity of NKT cells.

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Impairment of -GalCer-induced antimetastatic activity in SHPS-1 mutant mice. A, B16-BL6 melanoma cells (4 x 104) were injected i.v. into WT or SHPS-1 mutant mice on day 0, and -GalCer (2 µg) or vehicle was administered i.p. on days 0, 4, and 8. B, CD11c+ DCs prepared from the spleen of WT or SHPS-1 mutant donor mice were pulsed overnight with -GalCer (100 ng/ml) or vehicle and were then injected i.v. (1 x 105 cells) together with B16-BL6 cells (4 x 104) into WT mice on day 0. C, CD11c+ DCs prepared from the spleen of WT donor mice were pulsed overnight with -GalCer (100 ng/ml) or vehicle and were then injected i.v. (1 x 105 cells) together with B16-BL6 cells (4 x 104) into either WT or SHPS-1 mutant mice on day 0. In all experiments, mice were examined on day 14 for the number of tumor colonies in the lungs. Data are means ± SE of values from four or five recipients in individual experiments and are representative of three separate experiments. *, p < 0.05; **, p < 0.01 for the indicated comparisons.

Reduced production of IFN- and IL-4 and cell proliferation in response to -GalCer in splenocytes or hepatic MNCs from SHPS-1 mutant mice

The antimetastatic activity of NKT cells activated by -GalCer is dependent on the production of IFN- by NKT cells or NK cells (6, 30, 31). We thus next examined the effects of -GalCer on cytokine production and cell proliferation in splenocytes in vitro. The production of both IFN- and IL-4 in response to -GalCer was markedly reduced in splenocytes from SHPS-1 mutant mice compared with that in such cells from WT mice (Fig. 4A). In addition, the proliferative response of splenocytes to -GalCer was substantially reduced for those from SHPS-1 mutant mice compared with that apparent for those from WT mice (Fig. 4A). We also found that the -GalCer-induced production of IFN- or IL-4 was markedly reduced in hepatic MNCs from SHPS-1 mutant mice compared with that in the corresponding cells from WT mice (Fig. 4B).

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Reduced production of IFN- and IL-4 and cell proliferation in response to -GalCer in splenocytes or hepatic MNCs from SHPS-1 mutant mice. A, Splenocytes (5 x 105) freshly isolated from WT or SHPS-1 mutant mice were stimulated with various concentrations of -GalCer for 72 h, after which culture supernatants were harvested and assayed for IFN- and IL-4. Similarly, splenocytes were incubated with various concentrations of -GalCer for 72 h and [3H]TdR was also added during the last 16 h; the radioactivity incorporated into the cells was then measured with a scintillation spectrometer. B, Hepatic MNCs (2.5 x 105) isolated from WT or SHPS-1 mutant mice were stimulated with various concentrations of -GalCer for 72 h, after which culture supernatants were harvested and assayed for IFN- and IL-4. All data are means ± SE of values from triplicate determinations in individual experiments and are representative of three separate experiments.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Stimulation of CD4+ MNCs with CD11c+ DCs and -GalCer. A, CD4+ MNCs (2 x 105) prepared from the spleen of WT mice were cultured both with -GalCer (100 ng/ml) or vehicle and with CD11c+ DCs (1 x 105) from the spleen of either WT or SHPS-1 mutant mice. B, CD4+ MNCs (2 x 105) from the spleen of WT or SHPS-1 mutant mice were cultured both with -GalCer (100 ng/ml) or vehicle and with CD11c+ DCs (1 x 105) from WT mice. In all experiments, culture supernatants were harvested after 72 h and assayed for IFN- and IL-4. Data are means ± SE of triplicate determinations in individual experiments and are representative of three separate experiments. N.D., Not detected; *, p < 0.05; **, p < 0.01 for the indicated comparisons.

-GalCer-induced cytotoxic activity of hepatic MNCs from WT and SHPS-1 mutant mice

Activation of NKT cells by -GalCer increases cytotoxic activity against tumor cells in a manner dependent on NK cells and IFN- (6, 30). We therefore examined the -GalCer-induced cytotoxic activity of hepatic MNCs from WT and SHPS-1 mutant mice. Compared with hepatic MNCs from vehicle-treated WT mice, those from -GalCer-treated WT animals exhibited a marked increase in cytotoxic activity against both NK-sensitive YAC-1 cells and NK-resistant P815 cells (Fig. 6). However, treatment of SHPS-1 mutant mice with -GalCer also induced a marked increase in the cytotoxic activity of hepatic MNCs against both YAC-1 cells and P815 cells (Fig. 6).

View larger version (18K): [in this window] [in a new window]   FIGURE 6. -GalCer-induced cytotoxic activity of hepatic MNCs from WT and SHPS-1 mutant mice. Hepatic MNCs as effector cells were isolated from WT or SHPS-1 mutant mice 24 h after the injection of -GalCer (2 µg) or vehicle. YAC-1 (A) or P815 (B) cells as target cells were labeled with TDA and then incubated for 2 h with various numbers of effector cells at the indicated E:T ratios. The extent of target cell lysis was then determined on the basis of the amount of TDA released into the medium. Data are means ± SE of triplicate determinations in individual experiments and are representative of three separate experiments.

-GalCer-induced DC maturation and IL-12 production in WT and SHPS-1 mutant mice

We next tried to investigate what function of DCs for -GalCer-induced activation of NKT cells was indeed impaired in SHPS-1 mutant mice. Treatment of mice with -GalCer induces functional maturation of DCs as well as activation of NKT cells (22). However, the -GalCer-induced up-regulation of the expression of MHC class I, MHC class II, CD80, CD86, and CD40 on the surface of CD11c⁺ DCs in SHPS-1 mutant mice was not impaired, compared with that apparent with those from WT mice (Fig. 7A), suggesting that -GalCer-induced maturation of DCs is not defective in SHPS-1 mutant mice. The production of IL-12 by DCs is also thought to be important for -GalCer-induced activation of NKT cells and their antimetastatic activity (5, 6, 32, 33). However, the in vivo production of IL-12p70 in response to -GalCer treatment in SHPS-1 mutant mice was also similar to that apparent in WT animals (Fig. 7B).

View larger version (34K): [in this window] [in a new window]   FIGURE 7. -GalCer-induced DC maturation and IL-12 production in WT and SHPS-1 mutant mice. A, For assay of -GalCer-induced DC maturation, a DC-enriched LDF of splenic cells was prepared from -GalCer (2 µg)-treated WT or SHPS-1 mutant mice 0, 3, and 6 h after injection. The cells were then incubated with a biotin-conjugated mAb to either MHC class I, to MHC class II, to CD80, to CD86, to CD40 (thick trace), or a biotin-conjugated control mAb (thin trace), as indicated; and with a FITC-conjugated mAb to mouse CD11c. The cells were further incubated with PE-conjugated streptavidin and analyzed by two-color flow cytometry. All results are representative of three separate experiments. B, WT or SHPS-1 mutant mice were injected i.p. with 2 µg of -GalCer or with vehicle. The level of serum IL-12p70 was measured by ELISA 6 h after injection of -GalCer. Data are means ± SE of triplicate determinations in individual experiments and are representative of three separate experiments. N.D., Not detected.

	Discussion

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The -GalCer-induced antimetastatic activity of NKT cells is dependent on the production of IFN- by NKT and NK cells (6, 31). We have now shown that the production of both IFN- and IL-4 as well as cell proliferation in response to -GalCer in vitro were greatly attenuated in splenocytes or hepatic MNCs derived from SHPS-1 mutant mice, compared with the corresponding responses of cells from WT mice. These results suggest that SHPS-1 is required for the -GalCer-induced Th1 and Th2 responses of NKT cells. We have also shown that incubation of CD4⁺ splenic MNCs from WT mice with -GalCer and CD11c⁺ DCs from SHPS-1 mutant mice was associated with a marked decrease in the production of IFN- and IL-4 compared with that apparent on incubation of these MNCs with -GalCer and CD11c⁺ DCs from WT mice. In contrast, incubation of CD4⁺ splenic MNCs from SHPS-1 mutant mice with -GalCer and CD11c⁺ DCs from WT mice did not result in a decrease, but rather an increase, in the production of IFN- and IL-4 compared with that apparent with CD4⁺ splenic MNCs from WT mice. These results again suggest that the impaired Th1 and Th2 responses of splenic MNCs from SHPS-1 mutant mice to -GalCer are attributable to dysfunction of CD11c⁺ DCs, not to that of NKT or NK cells. Moreover, the impairment of -GalCer-induced antimetastatic activity in SHPS-1 mutant mice appears to be due, at least in part, to the reduced production of IFN- by NKT cells in response to -GalCer.

The production of both IFN- and IL-4 in the presence of -GalCer and CD11c⁺ DCs from WT mice was greater with CD4⁺ splenic MNCs from SHPS-1 mutant mice than with those from WT mice. The results suggest a notion that NKT cells from SHPS-1 mutant mice (in the presence of DCs) are markedly activated compared with those from WT mice. The notion also corresponds to the result in Fig. 3C, in which the numbers of metastatic nodules formed in SHPS-1 mutant mice that received -GalCer-pulsed DCs from WT donor mice were smaller than the corresponding values for WT recipients. The mechanism underlying such activation of NKT cells in SHPS-1 mutant mice is unknown. However, we have found recently that the TCR-stimulated production of either IL-2 or IFN- from CD4⁺ T cells of SHPS-1 mutant mice was markedly increased compared with that apparent with WT cells (Y. Kaneko and T. Matozaki, unpublished data). Thus, SHPS-1 might negatively regulate activation of NKT cells as well as of CD4⁺ T cells.

We have shown in this study that SHPS-1 is especially abundant on CD11c⁺ DCs and that its expression on CD8^–CD11c⁺ DCs is much greater than that on CD8⁺CD11c⁺ DCs, as described previously (28). Both CD8⁺CD11c⁺ and CD8^–CD11c⁺ DCs of SHPS-1 mutant mice express the mutant form of this protein, but its abundance is greatly reduced compared with that of the full-length protein on the corresponding cells of WT mice. The mutant SHPS-1 protein lacks the cytoplasmic region and therefore does not undergo tyrosine phosphorylation or form a complex with SHP-1 or SHP-2 (15, 16). It is thus likely that the impairment of -GalCer-induced activation of NKT cells in SHPS-1 mutant mice is attributable to elimination of functional SHPS-1 from CD11c⁺ DCs. Moreover, the percentage of CD8^–CD11c⁺ DCs from SHPS-1 mutant mice was markedly lower than that of those from WT mice. Such reduction of numbers of CD8^–CD11c⁺ DCs could participate in the impairment of -GalCer-induced activation of NKT cells in SHPS-1 mutant mice. The result also suggests that SHPS-1 regulates the number of CD11c⁺ DCs in vivo. Because there is still a defect when DC numbers were standardized, multiple effects must be involved.

In contrast to negative roles of SHPS-1 in regulation of the immune and hemopoietic systems (14, 15, 16, 17, 34, 35), our present results indicate that SHPS-1 plays a positive role in the activation of NKT cells by DCs. It remains to be determined what function of DCs is actually impaired in SHPS-1 mutant mice and thus what function of DCs SHPS-1 indeed participates in for activation of NKT cells, however. We showed that the level of expression of CD1d on CD8⁺CD11c⁺ or CD8^–CD11c⁺ DCs did not differ substantially between WT and SHPS-1 mutant mice. The -GalCer-induced maturation of DCs is not defective in these animals. Moreover, the in vivo production of IL-12 in response to -GalCer treatment in SHPS-1 mutant mice was also similar to that apparent in WT animals. Migration of epidermal Langerhans cells in response to haptens has been shown to be markedly attenuated in SHPS-1 mutant mice (36), suggesting that SHPS-1 promotes the migration of these cells. SHP-1 and SHP-2, both of which bind to tyrosine-phosphorylated SHPS-1, are implicated in regulation of the migration of neutrophils as well as in that of other cell types (37, 38, 39). Given that DCs with CD1d-bound -GalCer must migrate to the site of NKT cells to activate these cells, it is possible that SHPS-1 is required for such migration of DCs. Furthermore, SHPS-1 has recently been shown to promote NO production by macrophages in a manner dependent on PI3K (19). It is therefore also possible that SHPS-1 is required for the production by -GalCer-pulsed DCs of cytokines that are essential for the activation of NKT cells and their antimetastatic effect. It is not yet determined whether SHPS-1 is required for the prevention by -GalCer of metastasis of other cancer cell lines as well as B16-BL6 melanoma cells. However, we have found recently that SHPS-1 mutant mice fail to develop experimental autoimmune encephalomyelitis (T. Tomizawa, Y. Kaneko, T. Matozaki, manuscript submitted for publication). We further showed that SHPS-1 positively regulates the priming of CD4⁺ T cells by DCs (T. Tomizawa, Y. Kaneko, T. Matozaki, manuscript submitted for publication). Thus, SHPS-1 on DCs might mediate activation of NKT cell as well as CD4⁺ T cells (presumably through interaction of SHPS-1 with CD47 on NKT cells or CD4⁺ T cells), which occurs in a manner functionally similar to that apparent for costimulatory molecules on DCs such as CD80/86 or CD40 (4). Furthermore, a defect in CD4⁺ T cells could contribute to the reduced antitumor effect of -GalCer in SHPS-1 mutant mice.

Whereas -GalCer-induced antimetastatic activity was defective in SHPS-1 mutant mice, -GalCer-induced cytotoxic activity of hepatic MNCs against either NK-sensitive or NK-resistant tumor cell lines was not. The production of IFN- by both NKT and NK cells is essential for -GalCer-induced antimetastatic activity, but not for -GalCer-induced cytotoxicity, whereas perforin produced by NK cells is essential for -GalCer-induced cytotoxic activity, but not for antimetastatic activity (6). It is thus possible that SHPS-1 is required for some, but not all, -GalCer-induced activities of NKT and NK cells.

Overall, our present results suggest that SHPS-1 on DCs positively regulates the -GalCer-induced antimetastatic activity of NKT cells. Modulation of the ligand binding site of SHPS-1 with specific Abs or recombinant forms of CD47, combined with treatment with -GalCer, might therefore represent a potential new therapeutic approach to cancer metastasis.

	Acknowledgments

 
We thank C. F. Lagenaur for the p84 mAb to SHPS-1; P.-A. Oldenborg for the miap301 mAb to CD47; K. Takeda and K. Okumura for B16-BL6, YAC-1, and P815 cells; N. Honma for -GalCer; I. Serizawa and Y. Koezuka for helpful discussion; as well as A. Morita, R. Koitabashi, and Y. Niwayama for technical assistance.

	Disclosures

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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 a Grant-in-Aid for Scientific Research on Priority Areas Cancer, a Grant-in-Aid for Scientific Research (B) and (C), and a grant from the 21st Century Center of Excellence Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

² Address correspondence and reprint requests to Dr. Takashi Matozaki, Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8512, Japan. E-mail address: matozaki{at}showa.gunma-u.ac.jp

³ Abbreviations used in this paper: -GalCer, -galactosylceramide; DC, dendritic cell; Eu, europium; LDF, low-density fraction; MNC, mononuclear cell; SHP, Src homology 2 domain-containing protein tyrosine phosphatase; SHPS-1, SHP substrate-1; TDA, 2,2'':6',2''-terpyridine-6,6''-dicarboxylic acid; WT, wild type.

Received for publication August 14, 2006. Accepted for publication March 2, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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