Hakuo Ikegami

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.

Cloning and expression of interleukin-18 binding protein

Interleukin‐18 binding protein is a novel glycoprotein that we successfully cloned and expressed. First, murine interleukin‐18 binding protein was purified from the sera of mice with endotoxin shock using ligand affinity chromatography. The murine interleukin‐18 binding protein cDNA was cloned after RT‐PCR using mixed primer pair sequences based on partial murine interleukin‐18 binding protein amino acid sequence analysis. Subsequently, human interleukin‐18 binding protein cDNA was cloned from cDNA libraries of normal human liver using murine interleukin‐18 binding protein cDNA as a probe. Next, we transiently expressed recombinant human and murine interleukin‐18 binding proteins in COS‐1 cells and purified them from culture supernatants. Both recombinant interleukin‐18 binding proteins did not exhibit species specificity and prevented interleukin‐18 binding to its receptor. In addition, they inhibited interleukine‐18 dependent IFN‐γ production from KG‐1 cells effectively. These results suggest that the interleukin‐18 binding protein may possess interleukine‐18 antagonist activity.

INTRACELLULAR PRODUCTION OF INTERLEUKIN-18 IN HUMAN EPITHELIAL-LIKE CELL LINES IS ENHANCED BY HYPEROSMOTIC STRESS IN VITRO

Abstract Interleukin-18 is a novel multifunctional cytokine, which enhances natural killer cell activity and promotes the induction of cytokine production, including that of interferon-γ by T cells and antitumor effects. Interleukin-18 is produced by cells of several different tissues (e.g., macrophages, keratinocytes, osteoblasts, and intestinal epithelium); however, it is unclear what physiological conditions or stimuli induce interleukin-18 production. To determine physiological conditions for the production of interleukin-18, we have examined the effect of mannitol-induced hyperosmotic conditions on normal human umbilical vein endothelial cells (HUVEC) and eight established human epithelial-like cell lines (Intestine 407, Caco-2, A253, HeLa, SCC25, HT1197, ACHN, A549). Hyperosmotic conditions induced interleukin-18 immunoreactivity in all the human cell lines tested, as detected by immunocytochemistry. The enhanced interleukin-18 production was also observed when mannitol was replaced with NaCl as the inducer of hyperosmotic stress. Enzyme-linked immunosorbent assays revealed that interleukin-18 concentrations in cell extracts were significantly increased by hyperosmotic conditions. Reporter gene assays also revealed that hyperosmotic conditions stimulated transcriptional activity of the interleukin-18 promoter. These results show for the first time that hyperosmotic stress is a stimulator of interleukin-18 production in epithelial-like cells.

IL-18 Prevents the Development of Chronic Graft-Versus-Host Disease in Mice

The development of chronic graft-versus-host disease (GVHD), which is induced by the transfer of DBA/2 spleen cells into (C57BL/6 × DBA/2)F1 (BDF1) mice, is closely related to diminished donor anti-host CTL activity and host B cell hyperactivation. Therefore, an approach which activates donor CD8+ T cells or suppresses donor CD4+ T cell-host B cell interaction may have clinical utility in the treatment of chronic GVHD. We have previously demonstrated that IL-18 induces the development of naive CD8+ T cells into type I effector cells in DBA/2 anti-BDF1 MLC. In this paper we examined the effect of IL-18 administration on the development of chronic GVHD in mice. The treatment was started before or after the onset of clinical evidence of the disease. Regardless of the treatment schedule, IL-18 significantly decreased immunological parameters indicative of chronic GVHD, such as elevated serum IgG antinuclear Abs, IgG1, and IgE levels, and host B cell numbers and their activation. Importantly, IL-18-treated mice did not show the same acute GVHD-like symptoms reported for IL-12 treatment, because there was no weight loss, death, or severe immunodeficiency as indicated by a decrease in IL-2 and IFN-γ production by Con A-stimulated spleen cells. In contrast, IL-18 treatment partially but significantly restored the production of these cytokines. Data further suggested that these IL-18-mediated therapeutic effects may be due to the induction of donor CD8+ CTL, the decrease in donor CD4+ T cell numbers, and a down-regulation of host B cell MHC class II expression. Thus, our results suggest that IL-18 has beneficial effects in the prevention and treatment of chronic GVHD.

Analysis of the Antiviral Activities of Natural IFN-α Preparations and Their Subtype Compositions

Here we report on the antiviral effects of two commercially available natural interferon-alpha (IFN-alpha) preparations, their subtype compositions, and the effects of combinations of pairs of the subtypes on virally infected cells. Our results show that the antiviral effects of these preparations depend on the target cell and on the infecting virus. The component subtypes vary with the preparations, and combinations of pairs of IFN-alpha subtypes may have synergistic or competitive effects. Our results suggest that optimal preparations of synergistically acting subtypes may provide more therapeutic benefit to patients.

Different antiviral activities of IFN-α subtypes in human liver cell lines : synergism between IFN-α2 and IFN-α8

Abstract We have studied the antiviral activities of five recombinant interferon-alpha (IFN-α) subtypes, namely IFN-α1, -α2, -α5, -α8 and -α10, in eight human liver cancer cell lines. The relative antiviral activities, expressed in terms of the mean 50% inhibitory concentration (IC50), were different for each cell line. In general, IFN-α8 was the most potent, IFN-α2, -α5, and -α10 were intermediately active, and IFN-α1 was the least potent in the all cell lines. The observed differences between the IC50s of IFN-α1 and -α8 ranged from 250- to 2200-fold in these cell lines. Thus, the ranking order of relative antiviral activity was similar but the sensitivity to the subtypes was different among these cell lines. The relative antiviral activities of the subtypes were associated with the induction of 2′,5′-oligoadenylate synthetase (2′,5′-OAS) in the typical hepatocellular carcinoma cell line HAK-3 but not in the cell line KYN-3. Next, we examined for synergistic antiviral activity induced by IFN-α2 and -α8 that has been reported for the hepatocellular carcinoma cell line, HepG2. Synergism was observed in three of the eight liver cell lines at an IFN-α2 to -α8 ratio of 60:40, and is considered to reflect the synergistic induction of 2′,5′-OAS.

The anti-tumor activities of interferon (IFN)-α in chronic myelogenous leukaemia (CML)-derived cell lines depends on the IFN-α subtypes

Abstract Here we report on the anti-tumor effects of five interferon (IFN)-α subtypes, α1, α2, α5, α8, and α10 in chronic myelogenous leukaemia (CML)-derived cell lines. All of the CML cells can respond to IFN-α although the anti-tumor effects of IFN-α depend on the target cell and on the type of IFN-α subtype used. Proliferation assays showed that IFN-α8 was substantially more effective than the other four IFN-α subtypes. IFN-α8 was the most potent at upregulating immunomodulatory molecule expression while IFN-α1 was least potent. These data indicate in vitro distinctions between IFN-α subtypes that should be appreciated more in the clinic.

Characterization of the Antitumor Activities of IFN-α8 on Renal Cell Carcinoma Cells In Vitro

Interferon-alpha (IFN-alpha) has a number of therapeutic applications in the treatment of various human cancers and diseases of viral origin. IFN-alpha includes several subtypes, and little has been reported on the biologic properties of the individual subtypes. Here, we report on the individual antitumor effects of five IFN-alpha subtypes, alpha1, alpha2, alpha5, alpha8, and alpha10, against six renal cell carcinoma (RCC) cell lines in vitro. Among the subtypes, IFN-alpha8 most potently inhibited cell proliferation and delayed the G(1)/S transition. Synergistic induction of apoptosis was shown in two of the RCC cell lines when treated with the combination of IFN-alpha and IFN-gamma rather than with either IFN-alpha or IFN-gamma alone. IFN-alpha8 was most effective in the induction of apoptosis when combined with IFN-gamma. In addition, IFN-alpha8 had the strongest ability to upregulate HLA class II antigen expression in the subtypes examined. These data indicate that subtypes of IFN-alpha have disparate antitumor effects in vitro, and in vitro distinctions among the IFN-alpha subtypes should be appreciated more in clinical application.

Simultaneous exposure to interleukin-18 and interleukin-10 in vitro synergistically augments murine spleen natural killer cell activity.

Abstract Interleukin-18 (IL-18) enhances interferon γ (IFNγ) production and natural killer (NK) cell activity, and elicits protective antitumor effects in vivo. IL-18 and IL-12 synergistically augment IFNγ production reportedly because IL-12 enhances IL-18 receptor (IL-18R) expression. We now show that IL-18 also synergizes with IL-10 to augment murine splenic NK activity against Yac-1 cells in a standard 4-h chromium-release assay, but IFNγ production is only slightly enhanced. This pattern of NK activity was also observed with severe combined immunodeficient (SCID) mouse spleen cells indicating that the cytokines were not acting on T or B cells. The cytokines had no priming activity on the spleen cells and, when cells were left unstimulated for 24 h in culture, little NK activity was induced when IL-18 was added for the next 24 h. The reverse transcriptase/polymerase chain reaction revealed that IL-18 receptor (IL-18R) mRNA was expressed early during in vitro spleen cell culture but none was expressed after culture for 24 h regardless of the stimulus. Binding of 125I-labeled IL-18 revealed that exposure to IL-10 only slightly increased IL-18R expression. Expression of perforin mRNA was constitutive and was unaffected by the cytokines; however, Fas ligand (FasL) mRNA expression was strong in cultures with IL-18 alone or combined with IL-10. When Fas-expressing cells and their parental cells were used as targets, weak Fas-mediated cytolytic activity was observed after exposure to IL-18, and this was further enhanced by combination with IL-10. Finally, the augmentation of NK activity was abrogated by the inhibitor concanamycin A, indicating that the enhanced NK activity is perforin-dependent.

EFFECTS OF INTERFERON-ALPHA SUBTYPES ON THE TH1/TH2 BALANCE IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH HEPATITIS VIRUS INFECTION–ASSOCIATED LIVER DISORDERS

SummaryInterferon-alpha (IFN-α) has recently been shown to modulate in vitro T helper (Th) 1-driven responses in the peripheral blood mononuclear cells (PBMC) of patients with hepatitis B virus or C virus infection. In this study, we examined the in vitro effects of IFN-α subtypes (IFN-α1, −α2, −α5, −α8, and −α10) on the Th1/Th2 balance in PBMC obtained from patients with hepatitis virus infection-associated liver disorders and chronic hepatitis (CH), in comparison with the effect on healthy control volunteer PBMC. The Th1-type cell percentages and Th1/Th2 ratios were significantly higher in the PBMC of patients when compared with controls both before and after cultivation in vitro, with the IFN-α subtypes. The IFNα-5 induced an increase in the Th2-type cell percentages in both control and patient PBMC, resulting in that IFN-α5 lowered the Th1/Th2 ratio in patients with CH. Furthermore, statistical analysis revealed that IFN-α8 significantly promoted an increase in the Th1/Th2 ratios of PBMC from patients with CH and liver cirrhosis (LC) but not that of PBMC from patients with LC-hepatocellular carcinoma (HCC) and HCC. These findings imply that hepatitis virus infection and its disease status modify the effects of IFN-α subtypes on Th1 and Th2 immune balance in patients. Our findings should help to elucidate the mechanisms underlying successful IFN therapy for hepatitis virus infection and prevention of hepatocellular carcinogenesis.