Involvement of Caspase-1 and Caspase-3 in the Production and Processing of Mature Human Interleukin 18 in Monocytic THP.1 Cells

Abstract

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 ...

CrmA gene expression protects mice against concanavalin-A-induced hepatitis by inhibiting IL-18 secretion and hepatocyte apoptosis

“…53 Although caspase-1 has greater specificity for pro-IL-18, other caspases, that is, caspase-3-like proteases, are able to process pro-IL-1b and pro-IL-18 in response to antigenic stimulation or FasL, providing an alternative pathway for cytokine generation. [53][54][55] Consistent with the above reports, we demonstrated the release of IL-18 and IFN-g in response to ConA-induced hepatitis. In addition to inhibiting apoptosis in the liver, mice transduced with CrmA inhibited IFN-g and IL-18 production induced by ConA (Figure 6).…”

Pentoxifylline inhibits the synthesis and IFN-γ-inducing activity of IL-18

“…In contrast to non-fractionated splenocytes ( [28], and the present study) or macrophages from P. acnes-infected mice [3], peritoneal or bone marrow-derived macrophages from healthy animals accumulated, but did not release IL-18 upon LPS stimulation in our study. Similar results were obtained after LPS stimulation of human monocyte line THP.1 [31], and murine macrophage lines RAW264 and J774 (our unpublished observation). Therefore, it seems that some additional signals, triggered by infectious agents or contact with other cells, are required for macrophage IL-18 release.…”

Activation of Intrinsic and Extrinsic Proapoptotic Signaling Pathways in Interleukin-18-mediated Human Cardiac Endothelial Cell Death

“…6). Of note, caspase-3 has been shown to degrade mature IL-18 to inactive metabolites (57). It is possible that once the apoptotic machinery is activated, persistent expression of IL-18 may not be necessary to induce cell death.…”