Interleukin (IL)-18 was identified as a molecule that induces IFN-␥ 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. Murine interleukin-18 (mIL-18) 1 was identified in the livers of mice sequentially injected with heat-killed Propionibacterium acnes and with lipopolysaccharide (1). Murine IL-18 cDNA was cloned from murine liver mRNA, and the factor was provisionally termed IFN-␥-inducing factor because it was first identified as an IFN-␥ inducer in mice. Consequently, human interleukin-18 (hIL-18) was cloned from normal human liver mRNA (2). IL-18 is a non-N-linked, glycosylated, 18.3-kDa cytokine in its mature form and exhibits biologic activities in the monomeric form.IL-18 has been found to have a variety of biologic actions, including the stimulation of the proliferation of activated T cells, enhancement of the lytic activity of NK cells, induction of interferon-␥ (IFN-␥), and granulocyte-macrophage colony-stimulating factor production by activated T cells and promotion of Th1-type helper (Th1) clone responses (1-4). It has also been reported that IL-18 inhibits osteoclast-like multinucleated cell formation in co-cultures of osteoblasts and hemopoietic cells of spleen or bone marrow origin (5). Thus, it is very obvious that IL-18 plays an important role in the immune system.IL-18 shares some of its biologic activities with IL-12, although the primary structures of the two cytokines show no homology (2). In addition, in the experiments using murine Th1 clones and enriched human T cells, IL-18 and IL-12 acted on the T cells synergistically to induce IFN-␥ production (1, 4). Interestingly, the amino acid sequence of IL-18 includes the IL-1 signature-like sequence (2) and has been shown to have 15% homology at the amino acid level with the IL-1␤ protein, but does not bear significant functional resemblance to the IL-1 family (2).The identification of the receptor for IL-18 is important for investigation of the physiological role of IL-18 in nature. In this report, we describe the purification and identification of hIL-18R from a Hodgkin's disease-derived cell line, L428, and present some characterization of this molecule. EXPERIMENTAL PROCEDURES Cell Lines and ReagentsC5/MJ, CCRF-HSB-2, HPB-ALL, JM, MOLT-3, MOLT-4, MOLT-16, PEER, SKW-3 (human T cell leukemia), ARH-77, BALL-1 (human B cell leukemia), KG-1, HL-60, U-937 (human myelomonocytic cell leukemia), NALM-16, HEL (human non-T, non-B cell leukemia), and L-428 and HDLM (human Hodgkin's disease) cell lines were maintained in culture at 37°C, in a 5% CO 2 air mixture in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (BioWhittaker Inc.). Recombinant human IL-1␤ (R&D Systems) and 125 -I-IL-1␤ (Amersham) were obtained commercially. Recombinant IL-18Recombinant human IL-18 (rhIL-18) was produced by cu...

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 Still's 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‐γ (IFNγ) 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, Sjögren's 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 IFNγ 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.

We isolated and characterized the second major allergen (Cry j II) from Japanese cedar pollen. We found that most patients with this pollinosis had IgE antibody to this protein in addition to IgE antibody to Cry j I; however, some sera reacted only with Cry j I or Cry j II. IgE-ELISA inhibition studies revealed that Cry j I and Cry j II had no cross-allergenicity. Cry j II did not react with anti-Cry j I monoclonal antibodies. In SDS-PAGE under a non-reducing condition, Cry j II showed a band at the 37 kDa position, compared with the 45-50 kDa bands of Cry j I. N-terminal amino acid sequence of Cry j II was completely different from that of Cry j I.

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-Q Q production from KG-1 cells effectively. These results suggest that the interleukin-18 binding protein may possess interleukine-18 antagonist activity.z 1999 Federation of European Biochemical Societies.

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.