Tadao Tanimoto

Levels of interleukin‐18 and its binding inhibitors in the blood circulation of patients with adult‐onset Still's disease

OBJECTIVE Interleukin-18 (IL-18) is a proinflammatory cytokine that is involved in immunologically mediated tissue damage, but its bioactivity is regulated in vivo by its soluble decoy receptor, IL-18 binding protein (IL-18BP). This study was undertaken to determine levels of IL-18 and IL-18 binding inhibition in the blood of patients with adult-onset Stills disease (ASD). METHODS Serum concentrations of IL-18 in ASD patients were compared by enzyme-linked immunosorbent assay (ELISA) with those in patients with other systemic rheumatic diseases and healthy controls. The biologically active mature protein of IL-18 was detected by Western blot analysis with anti-IL-18 antibody and its induction of interferon-gamma (IFNgamma) secretion from IL-18-responding human myelomonocytic KG-1 cells. The inhibitory activity on IL-18 binding to its receptor was determined by 125I-IL-18 binding inhibition assay using the Chinese hamster ovary cell line transfected with a murine IL-18 receptor (CHO-K1/mIL-18R). RESULTS Concentrations of serum IL-18 were extremely elevated in patients with active ASD compared with those in patients with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, polymyositis/dermatomyositis, Sjogrens syndrome, or healthy individuals. Levels of IL-18 were found to correlate with serum ferritin values and disease severity in ASD. Western blot analysis revealed that serum samples from patients with active ASD contained an 18-kd polypeptide of IL-18, corresponding in size to the mature form. Accordingly, the samples were able to induce IFNgamma secretion from KG-1 cells, which was largely abolished by neutralizing anti-IL-18 antibody. However, the level of IL-18 bioactivity was more than 10-fold weaker than the concentration of IL-18 protein measured by ELISA. Serum samples from patients with active ASD showed an inhibitory effect on the binding of 125I-IL-18 to CHO-K1/mIL-18R cells, and this activity was associated with elevation of IL-18. CONCLUSION These data indicate that systemic overproduction of IL-18 may be closely related to the pathogenesis of ASD, despite the restriction on its inflammatory activity by IL-18 binding inhibitors such as IL-18BP. The disease activity appears to be determined on the basis of the relative levels of IL-18 and its specific inhibitors.

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.

Interferon‐γ–inducing activity of interleukin‐18 in the joint with rheumatoid arthritis

Objective To examine the levels of interleukin-18 (IL-18) bioactivity within the rheumatoid arthritis (RA) joint, and the differential effects of IL-12 and IL-18 on interferon-γ (IFNγ) production by T cell infiltrates. Methods Expression of IL-18 protein and messenger RNA (mRNA) was determined by enzyme-linked immunosorbent assay and reverse transcriptase–polymerase chain reaction, respectively. The biologic activity of IL-18 was detected on the basis of IFNγ secretion from IL-18–responding human myelomonocytic KG-1 cells. To determine the extent of inhibitory activity on binding of IL-18 to its receptor, a [125I]–IL-18 binding inhibition assay was performed, using a Chinese hamster ovary cell line transfected with a murine IL-18 receptor. Results The amount of IL-18 protein detected in both the serum and synovial fluid of RA patients was markedly larger than that detected in the serum and synovial fluid of osteoarthritis (OA) patients, and serum IL-18 levels correlated with the levels of serum C-reactive protein. IFNγ production by KG-1 cells was more strongly stimulated in synovial fluid samples from RA patients than in samples from OA patients, and this activity was largely diminished in the presence of anti–IL-18 antibody. In contrast, the activity of IL-18 binding inhibition in the serum and synovial fluid of RA patients was not significantly elevated compared with that in OA patients. RA synovial tissues showed increased expression of IL-18 mRNA and increased IL-18 protein synthesis compared with that in OA tissues. Purified CD14+ macrophages, but not activated fibroblast cell lines, from RA synovium were able to release mature IL-18, although both cell types expressed its transcripts. IL-18 alone showed a negligible effect on IFNγ production by RA synovial tissue cells, in contrast to IL-12, which was directly stimulatory. However, IL-12–induced IFNγ production was synergistically enhanced by IL-18, and yet was >50% reduced by neutralization of endogenous IL-18 with anti–IL-18 antibody. Conclusion These results indicate that IL-18, produced predominantly by tissue macrophages, primarily potentiates IL-12–induced IFNγ production by T cell infiltrates in RA synovium. Detection of significant IL-18 bioactivity in the joints, despite the presence of IL-18 binding inhibitors, supports an integral role of this cytokine in perpetuating the IFNγ-dominant T cell cytokine response in RA.

Interleukin-18/interferon-gamma-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells.

Intercellular adhesion molecule‐1 (ICAM‐1, CD54) is a membrane glycoprotein and a member of the immunoglobulin superfamily. It plays a central role in cell to cell‐mediated immune responses and is a ligand for leukocyte function‐associated antigen‐1 (LFA‐1). We report here that a newly discovered cytokine, interferon‐γ‐inducing factor (IGIF) [H. Okamura et al. (1995) Nature 378, 88] recently proposed to be designated as IL‐18, selectively up‐regulates ICAM‐1 expression in KG‐1 cells, a human myelomonocytic cell line, in which IL‐18 also enhances interferon‐γ production. IL‐18 induced heterotypic aggregation between KG‐1 and Peer T cells, which was blocked by anti‐ICAM‐1 and/or LFA‐1 antibodies. Anti‐interferon‐γ antibody did not block the IL‐18‐induced up‐regulation of ICAM‐1 on KG‐1 cells. These results thus show that IGIF/IL‐18, enhances ICAM‐1 expression in KG‐1 cells in an interferon‐γ‐independent pathway, up‐regulates ICAM‐1 functions, and that IL‐18 might play a potential role in immunoregulation by mediating immune cell infiltration into the tissues. J. Leukoc. Biol. 64: 519–527; 1998.

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.

Elevation of serum interleukin-18 levels and activation of kupffer cells in biliary atresia

BACKGROUND/PURPOSE Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel proinflammatory cytokine that can induce interferon gamma (IFN-gamma). In addition, IL-18 enhances intracellular adhesion molecule-1 (ICAM-1) expression as well as Fas ligand (FasL) expression, and induces apoptosis in hepatic injury. The aim of this study was to clarify the potential role of IL-18 in the pathogenesis of the progressive inflammation and fibrosis in biliary atresia (BA). METHODS Six children with BA before hepatic portoenterostomy (HPE), 13 with BA including 7 without jaundice and 6 with persistent jaundice after HPE, and 16 healthy controls were examined. Blood samples were obtained preoperatively from 6 patients, after HPE from 13, and after liver transplantation from 4. The IL-18 level was determined by an enzyme-linked immunosorbent assay (ELISA). Immunohistochemically, liver specimens from BA patients were studied using a monoclonal antibody to macrophage-associated antigen (CD68). RESULTS IL-18 levels were elevated in the patients before HPE compared with those of the controls (349+/-54 pg/mL v. 138+/-13 pg/mL, P<.0001). After HPE, extremely high concentrations of IL-18 were observed in patients with persistent jaundice (532+/-95 pg/mL, P<.0001), and the IL-18 levels were significantly high even in the patients without jaundice (249+/-29 pg/mL, P<0.005). The high IL-18 level lasted for a long time even in the patients without jaundice after HPE. In contrast, the IL-18 levels immediately decreased after liver transplantation. Immunohistochemically, the number of CD68-positive Kupffer cells was significantly higher, and the size was larger in the livers of the patients than in the controls. The proliferation of CD68-positive cells was much more conspicuous in the liver specimens obtained during liver transplantation than in those at the time of HPE. CONCLUSIONS Our findings showed elevation of serum IL-18 levels and activation of Kupffer cells in BA. IL-18 released from activated Kupffer cells might play an important role in the pathophysiology of the progressive inflammation and fibrosis in BA. Furthermore, IL-18 level may be related to the prognosis in patients with BA.

Serum gamma-interferon-inducing factor (IL-18) and IL-10 levels in patients with acute hepatitis and fulminant hepatic failure.

Background and Aims: The aim was to determine the role of T‐helper (Th)1/Th2 cytokine responses in the clinical outcome of patients with acute liver injury.

A simple and sensitive bioassay for the detection of human interleukin-18/interferon-γ-inducing factor using human myelomonocytic KG-1 cells

Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel cytokine which plays an important role in Th1 responses. Here we describe a simple, sensitive bioassay for human IL-18 using the human myelomonocytic cell line, KG-1, which produces IFN-gamma in response to human IL-18. IFN-gamma production induced by human IL-18 was completely blocked by an antibody against human IL-18. Human IL-18 could be measured in a concentration range from approximately 100 to 10,000 pg/ml, and intra- and inter-assay coefficient variations were both below 15%. It was possible to measure human IL-18 in human serum, cell lysate or culture supernatant by this bioassay. Thus, the human IL-18 bioassay can be expected to be useful in the investigation of the relationship between human IL-18 and various diseases or in analyzing the mechanisms of human IL-18 secretion from IL-18 producing 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.

Involvement of interleukin-18 (IL-18) in mixed lymphocyte reactions (MLR).

The in vitro mixed lymphocyte reaction (MLR) is a useful model to study alloresponsiveness to histocompatibility antigens. Secretion of different cytokine proteins in the supernatant of allo-MLR cultures has been reported in a few studies. We studied the levels of the cytokines interferon gamma (IFN-gamma) and interleukin-6 (IL-6), IL-10, IL-12, and IL-18 in the supernatant in allo-MLR by ELISA assay. Supernatant levels of IFN-y, IL-6, IL-10, and IL-18 were detected at 12 h after MLR and markedly increased thereafter. In contrast, secretion of IL-12 was detected after 48-72 h. These results suggested that IFN-gamma production depended on IL-18 in the early phase of MLR and depended on both IL-18 and IL-12 in the late phase. An antibody (Ab) neutralizing test was also performed. The levels of IFN-gamma were significantly downregulated after the addition of anti-IL-18 Ab, anti-IL-12 Ab, or anti-IFN-y Ab, and the levels of IL-12 were significantly downregulated after the addition of anti-IL-12 Ab and anti-IL-18 Ab. Treatment with these Ab did not suppress IL-6 production at all. The two-way MLR showed the same tendency as the one-way MLR. These results suggest the importance of IL-18 and IL-12 in allogeneic cell interactions and also suggest the usefullness of these Ab as regulators of alloresponsiveness.