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 ...