The mechanism underlying the differentiation of CD4+ T cells into functionally distinct subsets (Th1 and Th2) is incompletely understood, and hitherto unidentified cytokines may be required for the functional maturation of these cells. Here we report the cloning of a recently identified IFN-gamma-inducing factor (IGIF) that augments natural killer (NK) activity in spleen cells. The gene encodes a precursor protein of 192 amino acids and a mature protein of 157 amino acids, which have no obvious similarities to any peptide in the databases. Messenger RNAs for IGIF and interleukin-12 (IL-12) are readily detected in Kupffer cells and activated macrophages. Recombinant IGIF induces IFN-gamma more potently than does IL-12, apparently through a separate pathway. Administration of anti-IGIF antibodies prevents liver damage in mice inoculated with Propionibacterium acnes and challenged with lipopolysaccharide, which induces toxic shock. IGIF may be involved in the development of Th1 cells and also in mechanisms of tissue injury in inflammatory reactions.

The novel cytokine interferon-gamma-inducing factor (IGIF) augments natural killer (NK) cell activity in cultures of human peripheral blood mononuclear cells (PBMC), similarly to the structurally unrelated cytokine interleukin (IL)-12. IGIF has been found to enhance the production of interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF) while inhibiting the production of IL-10 in concanavalin A (Con A)-stimulated PBMC. In this study, when anti-CD3 monoclonal antibody (mAb)-stimulated human enriched T cells were exposed to IGIF, the cytokine dose-dependently enhanced the proliferation of the cells and this could be completely inhibited by a neutralizing antibody against IL-2 at lower concentrations of IGIF. Neutralizing antibody against IFN-gamma had only insignificant inhibitory effects on T cell proliferation at higher concentrations of IGIF. Enzyme-linked immunosorbent assays (ELISA) revealed that, like PBMC, T cells exposed to IGIF produced large amounts of IFN-gamma; however, changes in the production of IL-4 and IL-10 were minimal. IGIF, but not IL-12, significantly enhanced IL-2 and GM-CSF production in T cell cultures, as determined by CTLL-2 bioassay and ELISA, respectively; however, both IGIF and IL-12 enhanced IFN-gamma production by the T cells. When T cells were exposed to a combination of IGIF and IL-12, a synergistic effect was observed on the production of IFN-gamma, but not on production of IL-2 and GM-CSF. In conclusion, IGIF enhances T cell proliferation apparently through an IL-2-dependent pathway and enhances Th1 cytokine production in vitro and exhibits synergism when combined with IL-12 in terms of enhanced IFN-gamma production but not IL-2 and GM-CSF production. Based on structural and functional differences from any known cytokines, it was recently proposed that this cytokine be designated interleukin-18.

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...

IL-18 is a proinflammatory cytokine that plays an important role in NK cell activation and T(h)1 response. IL-18 has a structural homology to IL-1, particularly IL-1beta. IL-18R, composed of IL-1R-related protein (IL-18Ralpha) and IL-1R accessory protein-like (IL-18Rbeta), belongs to the IL-1R family. Furthermore, IL-18R at least partly shares the signal transducing system with IL-1R. Thus, the IL-18-IL-18R system has a striking similarity to the IL-1-IL-1R system. For this reason, we regarded it important to investigate whether, like IL-18, IL-1beta synergizes with IL-12 in inducing IFN-gamma production from human T cells and plays an important role in the T(h)1 response. Here we show that IL-12 and IL-1beta synergistically induce T cells to proliferate and produce IFN-gamma without their TCR engagement. IL-12 stimulation induced an increase in the proportion of T cells positive for IL-18R. Then, IL-12-stimulated T cells responded to IL-18 or IL-1beta by their proliferation and IFN-gamma production, although levels of IL-1beta-induced responses were lower. CD4(+)CD45RA(+) T cells, although they constitutively expressed IL-18Rbeta mRNA, did not express IL-18Ralpha mRNA. Phytohemagglutinin (PHA) stimulation alone induced IL-18Ralpha mRNA without affecting the expression of IL-18Rbeta mRNA. T(h)1-inducing conditions (PHA, IL-12 and anti-IL-4) further increased this expression. We also show that T(h)1 cells but not T(h)2 cells have increased expression of IL-18R and IL-1R, and produce IFN-gamma in response to IL-18 and/or IL-1beta.

Recently, human interleukin 18 (hIL-18) cDNA was cloned, and the recombinant protein with a tentatively assigned NH 2 -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 NH 2 -terminal amino acid was Tyr 37 . 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-␥ 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 Asp 36 -Tyr 37 site to generate the mature hIL-18, and the other is caspase-3, which cleaves both precursor and mature hIL-18 at Asp 71 -Ser 72 and Asp 76 -Asn 77 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. Interleukin (IL)1 -18 (originally called IGIF, interferon-␥-inducing factor) is a novel cytokine with multiple biological functions. In 1995 we purified murine IL-18 from the liver extracts of mice sensitized with Propionibacterium acnes followed by elicitation with lipopolysaccaride (1). The cDNA of murine IL-18 was cloned from cDNA libraries prepared from the livers of mice with endotoxin shock (2). Using this as a probe, human IL-18 cDNA was also cloned from a human normal liver cDNA library (3). The recombinant human IL-18 with a tentatively assigned NH 2 -terminal amino acid based on its homology with the natural murine IL-18 sequence was expressed in Escherichia coli, and its biological activities were examined (3).IL-18 has an interleukin 1 (IL-1) signature-like sequence (3) as reported and is similar to the IL-1 family and fibroblast growth factor in terms of their trefoil structures (4, 5). Despite their similarities, IL-18 and IL-1␤ exhibit different biological activities (2, 3, 6), transmitted through their specific receptors.2 Genetic information suggested that IL-18 is synthesized as an inactive precursor form (prohIL-18) and that this prohIL-18 has no known signal peptide sequence. Therefore, proteolytic cleavage is required for its maturation like IL-1␤ (2, 3, 7, 8). Gu et al. (7) reported that IL-1␤-converting enzyme (ICE)/ caspase-1 cleaved murine proIL-18 at the authentic processing site, Asp 35 -Asn 36 , to generate biologically active mature murine IL-18. However, natural hIL-18 had not yet been isolated, and its maturation site remained unclear.In this report, we screened for hIL-18 mRNA-expressing cell lines and purified natural hIL-18 from ...