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. 2008 Jan;99(1):113-20.
doi: 10.1111/j.1349-7006.2007.00636.x. Epub 2007 Oct 18.

Enhanced suppression of pulmonary metastasis of malignant melanoma cells by combined administration of alpha-galactosylceramide and interleukin-18

Shoji Nishio  1 , Naoko Yamada, Hideki Ohyama, Koji Yamanegi, Keiji Nakasho, Masaki Hata, Yoshiteru Nakamura, Satoru Fukunaga, Hiroyuki Futani, Shinichi Yoshiya, Haruyasu Ueda, Masaru Taniguchi, Haruki Okamura, Nobuyuki Terada
Affiliations

Enhanced suppression of pulmonary metastasis of malignant melanoma cells by combined administration of alpha-galactosylceramide and interleukin-18

Shoji Nishio et al. Cancer Sci. 2008 Jan.

Abstract

alpha-Galactosylceramide (alpha-GalCer) shows antitumor effects by activating natural killer (NK) cells indirectly through stimulation of the secretion of cytokines by NKT cells, whereas interleukin (IL)-18 shows antitumor effects by activating NK cells directly. In the present study, we examined the antitumor effect of the combined administration of alpha-GalCer and IL-18. An injection of NK cell-sensitive mouse B16 melanoma cells into a mouse tail vein produced pulmonary metastasis. The daily administration of alpha-GalCer or IL-18 alone for 4 days starting 1 day after the injection of B16 melanoma cells markedly suppressed the number of pulmonary metastatic foci, and their combined administration enhanced the antitumor effect compared with single administration. The antitumor effect of their combined administration was completely abolished by treatment of mice with anti-asialo GM1 serum, which depletes NK cells but not NKT cells. Combined administration of alpha-GalCer and IL-18 enhanced the cytotoxicity of NK cells and increased the number of NK cells in the lung. Analysis of NKT cell-dependent and NK cell-independent secretion of cytokines, to which NK cells can respond, showed that the administration of alpha-GalCer increased the secretion of IL-2, IL-4, interferon-gamma, IL-12, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and IL-10, and the combined administration of alpha-GalCer and IL-18 enhanced the secretion of IL-2, IL-4, interferon-gamma, and granulocyte-macrophage colony-stimulating factor further but only slightly. These results show that IL-18 in combination with alpha-GalCer exerts an antitumor effect on NK cell-sensitive tumors primarily by the direct stimulation of NK cells by IL-18 and the indirect stimulation of NK cells by alpha-GalCer through its activation of NKT cells.

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Figures

Figure 1
Figure 1
Schedules of the administration of α‐galactosylceramide (α‐GalCer), interleukin (IL)‐18, and anti‐asialo GM1 serum. B16 melanoma cells, α‐GalCer (2 µg), IL‐18 (2 µg), and anti‐asialo GM1 serum (20 µL) were injected into mice according to the protocols A, B and C. Protocol A was adopted for the quantitative analysis of pulmonary metastases of B16 melanoma cells, protocol B for the estimation of the cytotoxic activity of natural killer (NK) cells and the numbers of NK cells and NKT cells in the lung and spleen, and protocol C for the measurement of serum levels of cytokines.
Figure 2
Figure 2
Expression of interleukin (IL)‐18 receptor α and β mRNA in B16 melanoma cells and natural killer (NK) cells, and the effect of IL‐18 on the growth of B16 melanoma cells in vitro. (a) Reverse transcription–polymerase chain reaction analysis of mRNAs of the IL‐18 receptor subunits α and β. (b) Effect of IL‐18 on the growth of B16 melanoma cells. B16 melanoma cells (3 × 104 cells) were cultured in the presence or absence of IL‐18 (100 ng/mL) for 4 or 7 days. Each bar represents a mean + SE of six culture dishes.
Figure 3
Figure 3
The enhanced inhibitory effect caused by a combination of α‐galactosylceramide (α‐GalCer) and interleukin (IL)‐18 on the pulmonary metastasis of B16 melanoma cells through natural killer (NK) cells. (a) Effects of α‐GalCer, IL‐18, or their combination on pulmonary metastasis of B16 melanoma cells. Vehicle, IL‐18 alone, α‐GalCer alone, or their combination was injected into mice according to protocol A in Fig. 1 without treatment with anti‐asialo GM1 serum. (b) Effects of anti‐asialo GM1 serum on populations of NK cells and NKT cells in the spleen. Mice were injected with vehicle or anti‐asialo GM1 serum twice at an interval of 3 days and the percentages of NK cells and NKT cells in splenic lymphocytes were examined 24 h after the last injection. Dimer X indicates binding to α‐GalCer‐loaded CD1d. (c) The role of NK cells in the suppression of pulmonary metastasis by a combination of α‐GalCer and IL‐18. Vehicle or a combination of α‐GalCer and IL‐18 was injected into mice treated with anti‐asialo GM1 serum according to protocol A in Fig. 1. B16 melanoma cells were injected into mice at a dose of 3 × 104 cells/mouse. Each bar in (a) and (c) represents the mean + SE of the pulmonary metastatic foci of 10–16 mice. *P < 0.05, **P < 0.01.
Figure 4
Figure 4
Effects of interleukin (IL)‐18, α‐galactosylceramide (α‐GalCer), and their combination on the cytotoxic activity of natural killer (NK) cells. IL‐18, α‐GalCer, or their combination was injected into mice inoculated with B16 melanoma cells according to protocol B in Fig. 1. NK cells (effecter cells, E) were obtained from the spleens of these mice and mice without inoculation of B16 melanoma cells (B16 cells, –). Cytotoxic activity against B16 melanoma cells (target cells, T) was examined at E:T ratios of 12.5–100:1. Each point represents the mean ± SE of eight samples. a P < 0.05, significant differences from mice of four other groups. b P < 0.05, significant differences from mice injected with vehicle.
Figure 5
Figure 5
Effects of interleukin (IL)‐18, α‐galactosylceramide (α‐GalCer), and their combination on the numbers of natural killer (NK) cells, and NKT cells in the lung and spleen. IL‐18, α‐GalCer, or their combination was injected into mice according to protocol B in Fig. 1. Each bar represents the mean + SE of 4–5 samples. *P < 0.05, **P < 0.01.
Figure 6
Figure 6
Serum levels of interleukin (IL)‐2, IL‐4, interferon (IFN)‐γ, IL‐12, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), tumor necrosis factor (TNF)‐α and IL‐10. Normal mice (B6) and natural killer (NK) T cell‐deficient Jα18−/– mice with (▪) or without (□) treatment with anti‐asialo GM1 serum were injected with vehicle, IL‐18, α‐GalCer, or their combination, and 2 and 4 h later, the serum levels of cytokines were measured (Fig. 1, protocol C). α‐GC, α‐galactosylceramide. Each bar represents the mean + SE of 5–7 mice. a P < 0.05, significant differences from mice injected with vehicle only. *P < 0.05, **P < 0.01.
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