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Related Collections Hemostasis, Thrombosis, and Vascular Biology Immunobiology Neoplasia Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 September 2002, Vol. 100, No. 5, pp. 1728-1733 IMMUNOBIOLOGY IFN--mediated inhibition of tumor angiogenesis by natural killer T-cell ligand, -galactosylceramide Yoshihiro Hayakawa, Kazuyoshi Takeda, Hideo Yagita, Mark J. Smyth, Luc Van Kaer, Ko Okumura, and Ikuo Saiki From the Department of Pathogenic Biochemistry, Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan; the Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan; the Cancer Immunology Program, Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Institute, East Melbourne, Victoria, Australia; and the Howard Hughes Medical Institute, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Alpha-galactosylceramide (-GalCer), which is a specific ligand for CD1d-restricted variable-14 chain (V14) natural killer T (NKT) cells, exerts a potent antitumor effect. We recently demonstrated that interferon- (IFN-) secreted by both NKT cells and NK cells plays a critical role in mediating the antimetastatic effect of -GalCer; however, the IFN--dependent antitumor mechanisms remain poorly defined. In the present study, we demonstrate IFN--dependent inhibition of tumor angiogenesis by -GalCer. In -GalCer-treated mice, subcutaneous tumor growth and tumor-induced angiogenesis were inhibited in an IFN--dependent manner. The -GalCer-activated splenic or hepatic mononuclear cells inhibited murine endothelial cell proliferation in vitro, and this inhibitory effect was mediated mostly by IFN- produced by NKT cells and NK cells. NK cell depletion resulted in significant but partial inhibition of tumor growth and angiogenesis in vivo. These results suggest that the IFN--mediated inhibition of tumor angiogenesis is critically involved in the effector mechanisms of antitumor effects evoked by -GalCer. (Blood. 2002;100:1728-1733) © 2002 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References Natural killer T (NKT) cells constitute a distinctive subpopulation of mature T cells that coexpress an NK cell marker, NK1.1 antigen (Ag), and a highly restricted T-cell receptor (TCR) repertoire composed of a single invariant variable-14-joining-281 (V14-J281) chain.1,2 Recent studies have revealed that the invariant TCR on V14 NKT cells recognizes glycolipid Ags, such as -galactosylceramide (-GalCer) presented by the major histocompatibility complex (MHC) class I-like molecule CD1d.1-4 The -GalCer specifically stimulates V14 NKT cells to produce large amounts of interferon- (IFN-) and interleukin 4 (IL-4), exhibit cytolytic activity, and exert antitumor effect in vivo.4-7 We recently demonstrated that NK cells and IFN-, but not perforin-mediated cytotoxicity, critically contributed to the antimetastatic effect of -GalCer.8,9 NK cells produce IFN- secondarily in response to IFN- secreted by -GalCer-activated V14 NKT cells.8-11 Although IFN- was critically important for the antimetastatic effect of -GalCer, the IFN--mediated antitumor effector mechanisms remain unclear. Tumor angiogenesis, new vessel formation draining the tumor mass, is critical for tumor progression, outgrowth, and metastatic spread of tumors.12,13 Tumor-induced angiogenesis is up-regulated/down-regulated by proangiogenic and antiangiogenic factors produced by tumor cells and host microenvironments.14-16 IFN- has been implicated in the regulation of angiogenesis during tumor development.17-21 In the present study, we demonstrated that -GalCer inhibited tumor-induced angiogenesis in vivo. IFN- secreted by -GalCer-activated NKT cells and secondary activated NK cells was critical for this antiangiogenic effect.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Mice Wild-type C57BL/6 (B6) mice, BALB/c mice, and DBA/2 mice were purchased from Japan SLC (Hamamatsu, Japan). IFN--deficient (IFN-/) B6 mice and CD1-deficient (CD1/) B6 mice were generated as previously described.22,23 All mice were maintained under specific pathogen-free conditions and used at 6 to 7 weeks of age according to the institutional guidelines. Tumor cells B6-derived melanoma (B16-BL6) and lung carcinoma (3LL) cell lines, BALB/c-derived colon carcinoma (colon 26-L5) cell line, and DBA/2-derived mastocytoma (P815) cell line were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS). These cells, except for P815, were collected by brief trypsin treatment and resuspended in phosphate-buffered saline (PBS) before inoculation. Reagents The -GalCer [(2S, 3S, 4R)-1-o-(-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetiol] was provided by Y. Koezuka and K. Motoki (Kirin Brewery, Gumma, Japan) and prepared as previously described.5,6 For in vivo stimulation of NKT cells, mice were intraperitoneally administered 2 µg/200 µL -GalCer or 200 µL vehicle.1 To deplete NK cells in vivo, 150 µg antiasialo GM1 (anti-AGM1) antibody (Ab) per mouse (Wako, Osaka, Japan) was intravenously injected 2 days before the -GalCer treatment.24 To deplete NK cells and NKT cells in vivo, anti-NK1.1 monoclonal Ab (mAb) (PK136; BD Pharmingen, San Diego, CA) was intravenously injected 2 days before the -GalCer treatment.24 Depletion of respective cells was confirmed by flow cytometric analysis. The number of NKT cells was not affected by the anti-AGM1 Ab treatment (data not shown). Purified mAb (no azide/low endotoxin grade) against mouse IFN- (R4-6A2) was purchased from BD Pharmingen. Control rat immunoglobulin G (IgG) was purchased from Sigma Chemical (St Louis, MO). Angiogenesis assay Tumor-induced angiogenesis was assessed according to the previously described methods with minor modifications.25 Mice were inoculated intradermally with B16-BL6 cells (1 × 105/50 µL), colon 26-L5 cells (8 × 104/50 µL), 3LL cells (1 × 105/50 µL), or P815 cells (2 × 105/50 µL) on the back. At appropriate time periods after tumor inoculation, mice were killed, and the tumor-inoculated skin was separated from the underlying tissues. Angiogenesis was quantified by counting only the vessels directly supplying the tumor under a dissecting microscope. Matrigel angiogenesis assay17 was conducted by intradermally inoculating with B16-BL6 (2 × 106/100 µL) cells containing 10 µg Matrigel (Collaborative Research, Bedford, MA). At 4 days after inoculation, the tumor-inoculated skin was isolated from the underlying tissues, and the vessels drawn into the tumor-containing gel were counted. To evaluate the serum IFN- levels, sera were collected from vehicle- or -GalCer-treated mice at 16 hours after the first administration. IFN- levels in the serum were evaluated with the use of specific enzyme-linked immunosorbent assay (ELISA) kits (Endogen, Boston, MA) according to the manufacturer's instructions. Tumor growth assay Mice were inoculated intradermally with B16-BL6 cells (1 × 105/50 µL), colon 26-L5 cells (8 × 104/50 µL), 3LL cells (1 × 105/50 µL), or P815 cells (2 × 105/50 µL) on the back and observed every 4 days for tumor growth by measuring with a caliper square along the longer axes (a) and the shorter axes (b). Tumor volumes (mm3) were calculated by the formula: tumor volume (mm3) = ab2/2. Endothelial cell cultures Murine hepatic sinusoidal endothelial (HSE) cells were kindly provided by Dr G L Nicolson (The University of Texas, M D Anderson Cancer Center, Houston). HSE cells were maintained on Attachment Factorcoated (Cell Systems, Kirkland, WA) culture flasks in Dulbecco Modified Eagle medium (DMEM)/F12 medium supplemented with 5% FBS and 100 µg/mL endothelial mitogen (Biomedical Technologies, Stoughton, MA). All cultures were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. Coculture of HSE cells and mononuclear cells in membrane-separated wells Coculture experiments were performed in Intercell (equipped with 0.4- µm-pore membrane) (Kurabo, Osaka, Japan), with murine HSE (1 × 104) cells being plated at the bottom of a 24-well culture plate (Costar, Cambridge, MA). Splenic (3 × 106/200 µL) or hepatic (5 × 105/200 µL) mononuclear cells (MNCs) isolated from vehicle- or -GalCer-treated mice were placed onto the inserts. The splenic or hepatic MNCs were isolated 16 hours after vehicle or -GalCer administration as described previously.8 In the blocking experiments, anti-IFN- mAb (R4-6A2) or control rat IgG was added at 10 µg/mL to the cultures. After 72-hour incubation, proliferation of HSE cells in the bottom wells was measured by evaluating cell number by crystal violet staining. Briefly, cells were washed twice with PBS, fixed with 2.5% glutaraldehyde for 20 minutes at room temperature, and stained with 0.5 mL crystal violet (0.1% in 20% methanol solution). After washes, color was dissolved in 30% acetic acid and read at 590 nm on a microplate reader (Immuno Mini NJ-2300; Nippon Inter-Med, Tokyo, Japan). A calibration curve was set up with known numbers of cells, and a linear correlation between absorbance and cell number was established from 1 × 104 cells to 4 × 105 cells. To evaluate cytokine levels in the cultures, cell-free culture supernatants were harvested from the bottom wells. IFN- levels in the culture supernatants were evaluated with the use of specific ELISA kits (OptEIA; BD Pharmingen). Statistical analysis Data were analyzed by means of the the Mann-Whitney U test. P < .05 was considered significant.     Results Top Abstract Introduction Materials and methods Results Discussion References Effect of -GalCer on subcutaneous tumor growth and tumor-induced angiogenesis We first investigated the effect of -GalCer treatment on the outgrowth of subcutaneously inoculated B16-BL6 melanoma cells in wild-type B6 mice. Administration of -GalCer significantly inhibited the subcutaneous outgrowth of B16-BL6 cells during the early time points (days 8-16) but not later (Figure 1A). A similar inhibition of subcutaneous tumor growth by -GalCer treatment was also observed with colon 26-L5, 3LL, and P815 cells (data not shown). These results suggested that -GalCer could inhibit the early formation of solid tumor mass. View larger version (35K): [in this window] [in a new window]   Figure 1. Effects of -GalCer on subcutaneous tumor growth and tumor angiogenesis. Wild-type B6 mice were intradermally inoculated with B16-BL6 (1 × 105) cells and intraperitoneally administered -GalCer (2 µg/200 µL) or vehicle (200 µL) from day 0 and then every 4 days for 28 days. (A) Tumor size was measured every 4 days after tumor inoculation. Data are represented as the mean ± SD of 5 mice in each group. Similar results were obtained in 2 independent experiments. *P < .05 compared with vehicle. (B) The tumor-inoculated sites in vehicle- or -GalCer-treated mice at day 10. (C) Tumor-inoculated sites were isolated from vehicle- or -GalCer-treated mice when the tumor size reached the indicated volumes and tumor-supplying vessels were counted. The mean tumor sizes of vehicle- and -GalCer-treated groups were not different within any one range described. (At 100 to 200 mm3, vehicle 153 ± 24, -GalCer 161 ± 22. At 300 to 400 mm3, vehicle 365 ± 25, -GalCer 358 ± 34. At greater than 500 mm3, vehicle 622 ± 56, -GalCer 608 ± 70.) Data are represented as the mean ± SD of 5 mice in each group. Similar results were obtained in 2 independent experiments. *P < .01. When the tumor-inoculated sites were inspected, we noticed that a smaller size of tumor mass in the -GalCer-treated mice was associated with a reduced number of vessels around the tumor mass (tumor-induced angiogenesis) as represented in Figure 1B. To examine whether the reduced angiogenesis was a result or a cause of the tumor growth inhibition, we compared the number of blood vessels supplying tumors of the same volume in vehicle-versus- -GalCer-treated mice. As shown in Figure 1C, -GalCer treatment significantly reduced the number of blood vessels when tumor volumes were less than 400 mm3. This suggested that the reduced angiogenesis was not a result, but a cause, of the inhibition of tumor growth in -GalCer-treated mice. -GalCer inhibits tumor growth and tumor-induced angiogenesis in an IFN--dependent manner Our previous studies demonstrated that -GalCer-induced antimetastatic activity was mediated by IFN-, but not perforin-mediated cytolysis.8,9 Thus, we next examined the involvement of IFN- in the inhibition of subcutaneous tumor growth and angiogenesis by -GalCer using IFN-/ mice. As shown in Figure 2A-B, -GalCer treatment in wild-type B6 mice significantly inhibited the growth of subcutaneously inoculated B16-BL6 cells and the tumor angiogenesis as estimated at day 10. In contrast, no significant inhibition of either tumor growth or tumor angiogenesis by -GalCer treatment was observed in IFN-/ mice or CD1/ mice lacking V14 NKT cells (Figure 2A-B). To further examine the antiangiogenic effect of -GalCer, we used the Matrigel angiogenesis assay.17 Mice were intradermally inoculated with a larger number (2 × 106) of B16-BL6 cells embedded in Matrigel matrix, and the tumor-induced angiogenesis was assessed after a short period (4 days). As shown in Figure 2C, -GalCer treatment markedly inhibited the angiogenesis in wild-type mice but not in IFN-/ mice or CD1/ mice. These results indicated that -GalCer inhibited subcutaneous tumor growth and angiogenesis in an IFN--dependent manner. View larger version (32K): [in this window] [in a new window]   Figure 2. IFN--dependent inhibition of tumor growth and tumor-induced angiogenesis by -GalCer. Wild-type, IFN-/, or CD1/ B6 mice were intradermally inoculated with B16-BL6 cells (1 × 105) and then intraperitoneally administered -GalCer (2 µg/200 µL) or vehicle (200 µL) on days 0, 4, and 8. At 10 days after tumor inoculation, mice were killed and the tumor-inoculated skin was isolated. Tumor size was measured (panel A) and tumor-supplying vessels were counted (panel B). (C) Wild-type, IFN-/, or CD1/ B6 mice were intradermally inoculated with B16-BL6 (2 × 106) cells embedded in Matrigel (10 µg/100 µL) and then intraperitoneally administered -GalCer (2 µg/200 µL) or vehicle (200 µL). At 4 days after inoculation, vessels drawn into the gel were counted. All data are represented as the mean ± SD of 5 mice in each group. Similar results were obtained in 2 independent experiments. *P < .01. Splenic and hepatic mononuclear cells from -GalCer-treated mice inhibit proliferation of murine endothelial cells in vitro To further explore the mechanisms by which -GalCer inhibits tumor angiogenesis, we devised an in vitro assay system for estimating the effects of -GalCer-activated NKT and/or NK cells on proliferation of endothelial cells by a transmembrane coculture. Murine HSE cells were seeded on the bottom wells, and splenic or hepatic MNCs from vehicle- or -GalCer-treated mice were placed on the upper wells, which were separated from the bottom wells by a 0.4-µm-pore membrane. After 72-hour coculture, proliferation of HSE cells in the bottom wells was estimated by determining the cell numbers by staining with crystal violet. As represented in Figure 3, splenic and hepatic MNCs from -GalCer-treated mice markedly inhibited the proliferation of HSE cells as compared with those from vehicle-treated mice. Direct cell-cell contact was not required to inhibit the HSE cell proliferation by -GalCer-activated MNCs, because proliferation was observed when these cells were cultured in the membrane-separated wells. View larger version (24K): [in this window] [in a new window]   Figure 3. Inhibition of HSE cell proliferation by transmembrane coculture with MNCs from -GalCer-treated mice. Wild-type B6 mice were intraperitoneally administered vehicle (200 µL) or -GalCer (2 µg/200 µL). At 16 hours later, isolated splenic (3 × 106) or hepatic (5 × 105) MNCs were cocultured with murine HSE (1 × 104) cells in membrane-separated wells for 72 hours. The number of HSE cells in the bottom wells was determined by crystal violet staining. Data are indicated as the mean ± SD of triplicate samples. Similar results were obtained in 3 independent experiments. *P < .01. Critical contribution of IFN- produced by both NKT and NK cells to the inhibition of endothelial cell proliferation in vitro We next examined the involvement of IFN- in the inhibition of endothelial cell proliferation by splenic MNCs from -GalCer-treated mice. As shown in Figure 4, -GalCer-activated splenic MNCs produced a large amount of IFN- (Figure 4B) and markedly inhibited the proliferation of HSE cells (Figure 4A). This inhibitory effect on the proliferation of HSE cells was completely abolished by addition of neutralizing antimouse IFN- mAb (Figure 4A). These results indicated that the inhibition of HSE cell proliferation by -GalCer-activated MNCs was totally mediated by IFN-. We further examined the relative contributions of NK cells and NKT cells to the inhibition of HSE cell proliferation by -GalCer-activated MNCs, since we had previously observed a substantial contribution of secondarily activated NK cells to -GalCer-induced IFN- production.8-11 Specific depletion of NK cells by anti-AGM1 Ab before -GalCer administration partially inhibited the production of IFN- (Figure 4B) and partially abolished the inhibition of HSE cell proliferation (Figure 4A). The residual inhibitory effect on HSE cell proliferation was completely abolished by neutralization of IFN- (Figure 4A). In contrast, depletion of both NK cells and NKT cells by anti-NK1.1 mAb completely abolished the -GalCer-induced IFN- production (Figure 4B) and the inhibition of HSE cell proliferation (Figure 4A). Similar results were obtained with hepatic MNCs (data not shown). These results indicated that both -GalCer-activated NKT cells and secondarily activated NK cells contributed to the inhibition of endothelial cell proliferation via their IFN- production. View larger version (25K): [in this window] [in a new window]   Figure 4. Critical contribution of IFN- and both NKT and NK cells to inhibition of HSE cell proliferation by splenic MNCs from -GalCer-treated mice. Untreated, anti-AGM1 Ab-treated, or anti-NK1.1 mAb-treated wild-type B6 mice were intraperitoneally administered -GalCer (2 µg/200 µL) or vehicle (200 µL). Splenic MNCs were isolated 16 hours later and cocultured with HSE cells as in Figure 3 in the presence of 10 µg/mL anti-IFN- mAb (+) or control IgG (). The number of HSE cells was determined by crystal violet staining (panel A) and the production of IFN- by splenic MNCs was determined by ELISA (panel B). Data are indicated as the mean ± SD of triplicate samples. Similar results were obtained in 3 independent experiments. *P < .05. **P < .01. Relative contribution of NKT cells and NK cells to the antiangiogenic effect of -GalCer in vivo Finally, we determined the relative contribution of NKT cells and NK cells to the antiangiogenic effect of -GalCer in vivo. Specific depletion of NK cells by anti-AGM1 Ab prior to -GalCer administration partially reduced the -GalCer-induced serum IFN- elevation (Figure 5C) and also partially reduced the -GalCer-induced inhibition of B16-BL6 tumor growth (Figure 5A) and tumor angiogenesis (Figure 5B) as compared with control mice. Similar results were obtained with colon 26-L5 (Figure 5A-B, right panels), 3LL, and P815 (data not shown). In contrast, the depletion of both NK cells and NKT cells by anti-NK1.1 mAb completely abolished all these -GalCer-induced effects (Figure 5A-B, left panels). These results indicated that both primary activated NKT cells and secondary activated NK cells contributed to the antitumor and antiangiogenic effects of -GalCer in vivo. View larger version (51K): [in this window] [in a new window]   Figure 5. Partial contribution of NK cells to inhibition of tumor growth and tumor-induced angiogenesis by -GalCer. Untreated, anti-AGM1 Ab-treated, or anti-NK1.1 mAb-treated wild-type B6 or BALB/c mice were intradermally inoculated with B16-BL6 (1 × 105) or colon 26-L5 (8 × 104) cells, and then intraperitoneally administered -GalCer (2 µg/200 µL) or vehicle (200 µL) on days 0, 4, and 8. At 10 days after tumor inoculation, mice were killed and the tumor-inoculated skin was isolated. Tumor size was measured (panel A) and tumor-supplying vessels were counted (panel B). Serum IFN- level at 16 hours after the first administration of -GalCer in B6 mice was also evaluated by ELISA (panel C). Data are represented as the mean ± SD of 5 mice in each group. Similar results were obtained in 2 independent experiments. *P < .05. **P < .01.     Discussion Top Abstract Introduction Materials and methods Results Discussion References The present study demonstrated that -GalCer inhibited the early stage of subcutaneous tumor growth and the tumor-induced angiogenesis, and that these inhibitory effects were totally mediated by IFN- produced by primary activated NKT cells and secondary activated NK cells. The outgrowth and spread of tumors depend on an adequate blood supply achieved through angiogenesis.12-14,26,27 Our present results suggest that the IFN--dependent antiangiogenic effect may be one mechanism for the antitumor effects of -GalCer. It has been reported that the antiangiogenic effect of IFN- in vivo was mediated mainly by IFN--inducible antiangiogenic chemokines, such as IFN--inducible protein of 10 kDa (IP-10) and monokine induced by IFN- (Mig).28,29 In the present study, the inhibitory effect of -GalCer on tumor-induced angiogenesis was not observed in IFN-/ mice (Figure 2), and neutralization of IFN- completely abrogated the inhibition of endothelial cell proliferation by -GalCer-activated MNCs in vitro (Figure 4). Moreover, recombinant IFN- inhibited the proliferation of HSE cells in a dose-dependent manner in vitro (data not shown). These results suggest that the antiangiogenic effect of -GalCer might be mediated by IFN--dependent inhibition of endothelial cell proliferation at the tumor site, which was required for tumor angiogenesis. However, it remains to be determined whether the IFN--dependent inhibition of endothelial cell proliferation was a direct effect of IFN- or mediated by other inducible cytokines or chemokines produced by IFN--stimulated endothelial cells. It is also possible that IFN- might inhibit the tumor-induced angiogenesis by suppressing the production of angiogenic factors by tumor cells or by inducing the production of antiangiogenic factors by tumor cells or host stroma cells in vivo. Further studies are needed to determine the detailed mechanisms of IFN--mediated antiangiogenic effect. It has recently been reported that NK cells could lyse endothelial cells in a cell contact-dependent manner.30 Although a direct contact with -GalCer-activated MNCs was not required for the inhibition of HSE cell proliferation in vitro (Figure 3), NKT and/or NK cell-mediated injury of endothelial cells might contribute to the antiangiogenic effect of -GalCer in vivo. However, this seems unlikely, because we previously demonstrated that the antitumor effect of -GalCer was not mediated by perforin- or Fas ligand (FasL)-dependent cytotoxicity.8 Since some members of the tumor necrosis factor (TNF) family other than FasL, including TNF- and vascular endothelial cell growth inhibitor (VEGI), have been also implicated in the inhibition of tumor angiogenesis,31-33 the possible contribution of these molecules to the IFN--dependent antiangiogenic effect of -GalCer remains to be explored in further studies. The present study indicated that NK cells played a partial but substantial role in the inhibition of subcutaneous tumor growth, angiogenesis, and endothelial cell proliferation by -GalCer. Although we previously reported that the systemic administration of recombinant IFN- failed to inhibit tumor-induced angiogenesis,34 it was also reported that local production of IFN- by gene transfer inhibited tumor angiogenesis in vivo.19 We recently demonstrated a rapid depletion of primary activated NKT cells but a prolonged production of IFN- by secondary activated NK cells after -GalCer treatment.8 Therefore, the prolonged production of IFN- by secondary activated NK cells might play a substantial role in sustaining the antiangiogenic effect of -GalCer. It was previously reported that IL-12 also inhibited tumor-induced angiogenesis and endothelial cell functions in an IFN--dependent manner20,35-37 and that this effect was mediated by NK cells.38 However, it has been also demonstrated that IL-12 preferentially activated NKT cells rather than NK cells,39,40 and that IL-12 played a critical role in -GalCer-induced NKT cell activation.41 We also observed that IL-12 partially contributed to the -GalCer-induced IFN- production42 and to the inhibition of endothelial cell proliferation by -GalCer-activated splenic MNCs (data not shown). Therefore, both IL-12 secreted by -GalCer-presenting dendritic cells and IFN- secreted by -GalCer-activated NKT cells might contribute to the secondary IFN- production by NK cells, which augmented the antitumor effect of an NKT cell-specific ligand, -GalCer.     Acknowledgments We thank Yasuhiko Koezuka and Kazuhiro Motoki (Pharmaceutical Research Laboratory, Kirin Brewery) for generously providing -GalCer, and Mitsuhiro Matsuo and Kazuko Hayashi for technical assistance.     Footnotes Submitted November 8, 2001; accepted April 5, 2002. Supported by research grant from Human Frontier Science Program Organization; Y.H. was supported by the Uehara Foundation Research Fellowship. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Yoshihiro Hayakawa, Cancer Immunology, Peter MacCallum Cancer Institute, Locked Bag 1, A'Beckett St, Victoria 8006, Australia; e-mail: y.hayakawa{at}pmci.unimelb.edu.au.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Bendelac A, Rivera MN, Park SH, Roark JH. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu Rev Immunol. 1997;15:535-562[CrossRef][Medline] [Order article via Infotrieve]. 2. MacDonald HR. NK1.1+ T cell receptor-+ cells: new clues to their origin, specificity, and function. J Exp Med. 1995;182:633-638[Free Full Text]. 3. Brossay L, Burdin N, Tangri S, Kronenberg M. Antigen-presenting function of mouse CD1: one molecule with two different kinds of antigenic ligands. Immunol Rev. 1998;163:139-150[CrossRef][Medline] [Order article via Infotrieve]. 4. Burdin N, Kronenberg M. CD1-mediated immune responses to glycolipids. Curr Opin Immunol. 1999;11:326-331[CrossRef][Medline] [Order article via Infotrieve]. 5. Kawano T, Cui J, Koezuka Y, et al. CD1d-restricted and TCR-mediated activation of V14 NKT cells by glycosylceramides. Science. 1997;278:1626-1629[Abstract/Free Full Text]. 6. Kawano T, Cui J, Koezuka Y, et al. Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated V14 NKT cells. Proc Natl Acad Sci U S A. 1998;95:5690-5693[Abstract/Free Full Text]. 7. Burdin N, Brossay L, Koezuka Y, et al. Selective ability of mouse CD1 to present glycolipids: -galactosylceramide specifically stimulates V 14+ NK T lymphocytes. J Immunol. 1998;161:3271-3281[Abstract/Free Full Text]. 8. Hayakawa Y, Takeda K, Yagita H, et al. Critical contribution of IFN- and NK cells, but not perforin-mediated cytotoxicity, to anti-metastatic effect of -galactosylceramide. Eur J Immunol. 2001;31:1720-1727[CrossRef][Medline] [Order article via Infotrieve]. 9. Street SEA, Crentney E, Smyth MJ. Perforin and interferon- activities independently control tumor initiation, growth, and metastasis. Blood. 2001;97:192-197[Abstract/Free Full Text]. 10. Carnaud C, Lee D, Donnars O, et al. Cross-talk between cells of the innate immune system: NKT cells rapidly activate NK cells. J Immunol. 1999;163:4647-4650[Abstract/Free Full Text]. 11. Eberl G, MacDonald HR. Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells. Eur J Immunol. 2000;30:985-992[CrossRef][Medline] [Order article via Infotrieve]. 12. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27-31[CrossRef][Medline] [Order article via Infotrieve]. 13. Zetter BR. Angiogenesis and tumor metastasis. Annu Rev Med. 1998;49:407-424[CrossRef][Medline] [Order article via Infotrieve]. 14. Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86:353-364[CrossRef][Medline] [Order article via Infotrieve]. 15. Mantovani A, Bottazzi B, Colotta F, Sozzani S, Ruco L. The origin and function of tumor-associated macrophages. Immunol Today. 1992;13:265-270[CrossRef][Medline] [Order article via Infotrieve]. 16. Fukumura D, Xavier R, Sugiura T, et al. Tumor induction of VEGF promoter activity in stromal cells. Cell. 1998;94:715-725[CrossRef][Medline] [Order article via Infotrieve]. 17. Beatty G, Paterson Y. IFN--dependent inhibition of tumor angiogenesis by tumor-infiltrating CD4+ T cells requires tumor responsiveness to IFN-. J Immunol. 2001;15:2276-2282. 18. Qin Z, Blankenstein T. CD4+ T cell-mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN- receptor expression by nonhematopoietic cells. Immunity. 2000;12:677-686[CrossRef][Medline] [Order article via Infotrieve]. 19. Fathallah-Shaykh HM, Zhao LJ, Kafrouni AI, Smith GM, Forman J. Gene transfer of IFN- into established brain tumors represses growth by antiangiogenesis. J Immunol. 2000;164:217-222[Abstract/Free Full Text]. 20. Coughlin CM, Salhany KE, Gee MS, et al. Tumor cell responses to IFN affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity. 1998;9:25-34[CrossRef][Medline] [Order article via Infotrieve]. 21. Ruegg C, Yilmaz A, Bieler G, Bamat J, Chaubert P, Lejeune FJ. Evidence for the involvement of endothelial cell integrin V3 in the disruption of the tumor vasculature induced by TNF and IFN-. Nat Med. 1998;4:408-414[CrossRef][Medline] [Order article via Infotrieve]. 22. Tagawa Y, Sekikawa K, Iwakura Y. Suppression of concanavalin A-induced hepatitis in IFN-(/) mice, but not in TNF-(/) mice: role for IFN- in activating apoptosis of hepatocytes. J Immunol. 1997;159:1418-1428[Abstract]. 23. Mendiratta SK, Martin WD, Hong S, Boesteanu A, Joyce S, Van Kaer L. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity. 1997;6:469-477[CrossRef][Medline] [Order article via Infotrieve]. 24. Ogasawara K, Takeda K, Hashimoto W, et al. Involvement of NK1+ T cells and their IFN- production in the generalized Shwartzman reaction. J Immunol. 1998;160:3522-3527[Abstract/Free Full Text]. 25. Saiki I, Murata J, Makabe T, Nishi N, Tokura S, Azuma I. Inhibition of tumor angiogenesis by a synthetic cell-adhesive polypeptide containing the Arg-Gly-Asp (RGD) sequence of fibronectin, poly(RGD). Jpn J Cancer Res. 1990;81:668-675[CrossRef][Medline] [Order article via Infotrieve]. 26. Holash J, Maisonpierre PC, Compton D, et al. Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science. 1999;284:1994-1998[Abstract/Free Full Text]. 27. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275:964-967[Abstract/Free Full Text]. 28. Sgadari C, Angiolillo AL, Cherney BW, et al. Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo. Proc Natl Acad Sci U S A. 1996;93:13791-13796[Abstract/Free Full Text]. 29. Sgadari C, Farber JM, Angiolillo AL, et al. Mig, the monokine induced by interferon-, promotes tumor necrosis in vivo. Blood. 1997;89:2635-2643[Abstract/Free Full Text]. 30. Yoneda O, Imai T, Goda S, et al. Fractalkine-mediated endothelial cell injury by NK cells. J Immunol. 2000;164:4055-4062[Abstract/Free Full Text]. 31. Boldrini L, Calcinai A, Samaritani E, et al. Tumour necrosis factor-alpha and transforming growth factor-beta are significantly associated with better prognosis in non-small cell lung carcinoma: putative relation with BCL-2-mediated neovascularization. Br J Cancer. 2000;83:480-486[CrossRef][Medline] [Order article via Infotrieve]. 32. Zhai Y, Yu J, Iruela-Arispe L, et al. Inhibition of angiogenesis and breast cancer xenograft tumor growth by VEGI, a novel cytokine of the TNF superfamily. Int J Cancer. 1999;82:131-136[CrossRef][Medline] [Order article via Infotrieve]. 33. Zhai Y, Ni J, Jiang GW, et al. VEGI, a novel cytokine of the tumor necrosis factor family, is an angiogenesis inhibitor that suppresses the growth of colon carcinomas in vivo. FASEB J. 1999;13:181-189[Abstract/Free Full Text]. 34. Saiki I, Sato K, Yoo YC, et al. Inhibition of tumor-induced angiogenesis by the administration of recombinant interferon-gamma followed by a synthetic lipid-A subunit analogue (GLA-60). Int J Cancer. 1992;51:641-645[Medline] [Order article via Infotrieve]. 35. Voest EE, Kenyon BM, O'Reilly MS, Truitt G, D'Amato RJ, Folkman J. Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst. 1995;87:581-586[Abstract/Free Full Text]. 36. Sgadari C, Angiolillo AL, Tosato G. Inhibition of angiogenesis by interleukin-12 is mediated by the interferon-inducible protein 10. Blood. 1996;87:3877-3882[Abstract/Free Full Text]. 37. Strasly M, Cavallo F, Geuna M, et al. IL-12 inhibition of endothelial cell functions and angiogenesis depends on lymphocyte-endothelial cell cross-talk. J Immunol. 2001;166:3890-3899[Abstract/Free Full Text]. 38. Yao L, Sgadari C, Furuke K, Bloom ET, Teruya-Feldstein J, Tosato G. Contribution of natural killer cells to inhibition of angiogenesis by interleukin-12. Blood. 1999;93:1612-1621[Abstract/Free Full Text]. 39. Takeda K, Hayakawa Y, Atsuta M, et al. Relative contribution of NK and NKT cells to the anti-metastatic activities of IL-12. Int Immunol. 2000;12:909-914[Abstract/Free Full Text]. 40. Smyth MJ, Taniguchi M, Street SE. The anti-tumor activity of IL-12: mechanisms of innate immunity that are model and dose dependent. J Immunol. 2000;165:2665-2670[Abstract/Free Full Text]. 41. Kitamura HK, Iwakabe T, Yahata S, et al. The natural killer T (NKT) cell ligand -galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J Exp Med. 1999;189:1121-1127[Abstract/Free Full Text]. 42. Hayakawa Y, Takeda K, Yagita H, et al. Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways. J Immunol. 2001;166:6012-6018[Abstract/Free Full Text]. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: S. Motohashi, K. Nagato, N. Kunii, H. Yamamoto, K. Yamasaki, K. Okita, H. Hanaoka, N. Shimizu, M. Suzuki, I. Yoshino, et al. A Phase I-II Study of {alpha}-Galactosylceramide-Pulsed IL-2/GM-CSF-Cultured Peripheral Blood Mononuclear Cells in Patients with Advanced and Recurrent Non-Small Cell Lung Cancer J. 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