Mitsukiyo Fujii

Purification and Characterization of the Human Interleukin-18 Receptor

Interleukin (IL)-18 was identified as a molecule that induces IFN-γ production and enhances NK cell cytotoxicity. In this paper, we report upon the purification and characterization of human IL-18 receptor (hIL-18R). We selected the Hodgkin’s disease cell line, L428, as the most strongly hIL-18R-expressing cell line based on the results of binding assays. This binding was inhibited by IL-18 but not by IL-1β. The dissociation constant (K d ) of125I-IL-18 binding to L428 cells was about 18.5 nm, with 18,000 binding sites/cell. After immunizing mice with L428 cells and cloning, a single monoclonal antibody (mAb) against hIL-18R was obtained (mAb 117-10C). Sequentially, hIL-18R was purified from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-extracted L428 cells by wheat germ lectin-Sepharose 4B chromatography and mAb 117-10C-Sepharose chromatography. The internal amino acid sequences of hIL-18R all matched those of human IL-1 receptor-related protein (IL-1Rrp), the ligand of which was unknown to date. When expressed in COS-1 cells, the cDNA of IL-1Rrp conferred IL-18 binding properties on the cells and the capacity for signal transduction. From these results, we conclude that a functional IL-18 receptor component is IL-1Rrp.

Involvement of Caspase-1 and Caspase-3 in the Production and Processing of Mature Human Interleukin 18 in Monocytic THP.1 Cells

Recently, human interleukin 18 (hIL-18) cDNA was cloned, and the recombinant protein with a tentatively assigned NH2-terminal amino acid sequence was generated. However, natural hIL-18 has not yet been isolated, and its cellular processing is therefore still unclear. To clarify this, we purified natural hIL-18 from the cytosolic extract of monocytic THP.1 cells. Natural hIL-18 exhibited a molecular mass of 18.2 kDa, and the NH2-terminal amino acid was Tyr37. Biological activities of the purified protein were identical to those of recombinant hIL-18 with respect to the enhancement of natural killer cell cytotoxicity and interferon-γ production by human peripheral blood mononuclear cells. We also found two precursor hIL-18 (prohIL-18)-processing activities in the cytosol of THP.1 cells. These activities were blocked separately by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. Further analyses of the partially purified enzymes revealed that one is caspase-1, which cleaves prohIL-18 at the Asp36-Tyr37 site to generate the mature hIL-18, and the other is caspase-3, which cleaves both precursor and mature hIL-18 at Asp71-Ser72 and Asp76-Asn77 to generate biologically inactive products. These results suggest that the production and processing of natural hIL-18 are regulated by two processing enzymes, caspase-1 and caspase-3, in THP.1 cells.

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Abstract Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4+ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4+ cells.

Antitumor Effects on Mouse Melanoma Elicited by Local Secretion of Interleukin-12 and Their Enhancement by Treatment with Interleukin-18

Abstract To investigate the mechanism of the antitumor effect of locally secreted interleukin-12 (IL-12), we introduced the IL-12 p35 and p40 cDNAs into mouse B16 melanoma cells. IL-12 gene-transfected B16 melanoma (B16/IL12) showed marked retardation of tumor growth when implanted subcutaneously into syngeneic mice. In these mice, depletion of not only Natural Killer (NK) cells but also CD8+ T cells diminished the antitumor effect of locally secreted IL-12. Immunohistochemical analysis showed that NK cells and macrophages accumulated more densely at the center and periphery of B16/IL12 tumors than that of parental B16 tumors, whereas CD4+ T cells and CD8+ T cells accumulated sparsely only at the periphery of both transfected and untransfected tumors. Systemic treatment with interleukin-18 (IL-18) markedly inhibited the growth of B16/IL12 but did not influence the tumor growth of parental B16 cells in vivo. These results suggest that local IL-12 secretion can retard the growth of B16 melanoma mediated primarily by NK cells and indirectly by CD8+ T cells and that its antitumor effect is augmented by systemic treatment with the novel cytokine IL-18.

Oral use of interferon-alpha delays the onset of insulin-dependent diabetes mellitus in nonobese diabetes mice.

Insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetes (NOD) mouse model is thought to be an autoimmune CD4 Th1-like cell-mediated disease. We tested the efficacy of oral use of interferon-alpha (IFN-alpha) therapy on IDDM in NOD mice. Using urine and blood sugar levels as indicators of IDDM, oral administration of murine IFN-alpha (100 IU/body) to NOD mice significantly delayed the onset of symptomatic diabetes. However, oral use of IFN-alpha did not prevent diabetic NOD mice from losing weight once NOD mice were symptomatic, suggesting that orally administered IFN-alpha is a prophylactic rather than therapeutic approach to the management of IDDM.

Antitumor activity of interleukin-18 against the murine T-cell leukemia/lymphoma EL-4 in syngeneic mice.

Interleukin (IL)-18 induces interferon (IFN)-&ggr; production by T cells and natural killer (NK) cells, and augments NK cell activity in mouse spleen cell cultures. It has recently been demonstrated that in vivo administration of IL-18 to mice results in considerable antitumor effects against syngeneic Meth A sarcoma. In this study, the antitumor effects of IL-18 against murine T-cell leukemia (EL-4) were evaluated. EL-4 proliferation was resistant in vitro to IL-18 and IFN-&ggr;. When 4 × 106 EL-4 cells were transplanted intravenously, the antitumor effects of IL-18 were not pronounced, and only a slight prolongation of the mean survival times was observed. The antitumor effects of IFN-&ggr; were even less apparent than those of IL-18. However, when mice were transplanted intravenously with 5 × 105 EL-4 cells, the extent of experimental visceral dissemination of EL-4 was markedly reduced in mice treated subcutaneously with IL-18, resulting in an increase in survival time with some mice even cured. Although IL-18 was highly effective at inhibiting the development of EL-4 lymphoma dissemination in C57BL/6 mice, it could not inhibit the development of dissemination in mutant C57BL/6 beige (bg/bg) mice lacking NK cell activity. The efficacy of IL-18 was also significantly reduced in nude mice lacking T cells. These results suggest that antitumor efficacy of IL-18 is mediated primarily by NK cells, but that T cells are also required for the complete antitumor efficacy of IL-18.

Inhibitory Effects of 2-Amino-3H-phenoxazin-3-one on the Melanogenesis of Murine B16 Melanoma Cell Line

Hyperpigmentations are a serious concern addressed by both the medical community and the cosmetic industry through the development of agents that block melanin biosynthesis. In this study, we found that 2-amino-3H-phenoxazin-3-one (APO), isolated from extracts of the edible mushroom Agaricus bisporus Imbach, exhibited potent inhibitory effects on melanogenesis in B16 cells, a murine melanoma cell line. APO inhibited melanin biosynthesis at 1,000 times lower concentrations (IC50=1.31±0.08 μM) than kojic acid (IC50=1.31±0.13 mM), without causing cellular toxicity. APO did not directly inhibit the enzyme activity of tyrosinase, the rate-limiting melanogenic enzyme. Further study showed that APO inhibited the protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF), a melanogenic transcription factor that regulates the expression of tyrosinase. These results suggest that APO is a promising depigmenting agent with both therapeutic and cosmetic value in preventing melanogenesis.

Effects of Oral Administration of Interferon-alpha on Antibody Production in Mice with Induced Tolerance

In vivo systemic effects and the immunomodulating potential of the oral administration of murine interferon-alpha (IFN-alpha) were investigated through mRNA expression of both IFN-alpha-inducible factors, interferon regulatory factor-1 (IRF-1) and 2,5-adenylate synthetase [2-5(A) synthetase] and 2-5(A) synthetase enzymatic activity in spleen and antibody production. The daily administration of IFN-alpha (0.1, 1, 10, and 100 IU/body) for 1 week augmented IRF-1 and 2-5(A) synthetase mRNA expression levels, as well as 2-5(A) synthetase enzymatic activity in spleen cells but not in cervical lymph nodes. The in vivo immunomodulating potential of the oral administration of IFN-alpha was also evaluated through antibody production in mice with induced tolerance. Ovalbumin (OVA) was administered intraperitoneally (i.p.) to induce systemic antibody production on day 0 when OVA feeding was initiated. The OVA was fed every 2-3 days for a total of 14 doses to suppress serum antibody levels. Oral administration of murine IFN-alpha was initiated on day 0 and was continued for 5 consecutive days weekly for 5 weeks (24 doses). On every sampling date (days 10, 17, 24, and 32), specific antibody levels in the IFN-alpha-administered groups were significantly higher than those in the control (nonadministered) group. This was especially noted in early phases (days 10 and 17) of antibody production when the levels of antibody in serum from the IFN-alpha-administration groups were equivalent to those of the nontolerance group. Altogether, it is suggested that oral use of IFN-alpha can elicit immunomodulating actions (e.g., antibody levels) by affecting the systemic immune system(s).