Neutrophil Proteinase 3-Mediated Induction of Bioactive IL-18 Secretion by Human Oral Epithelial Cells

Abstract

IL-18, a potent IFN-γ-inducing cytokine, is expressed by various nonimmune cells as well as macrophages, suggesting that it has important physiological and immunological roles. The present study focused on the mechanism of active IL-18 induction from human oral epithelial cells. The epithelial cells and the cell lines constitutively express IL-18 mRNA and the 24-kDa precursor form of IL-18. Bioactive IL-18 exhibiting IFN-γ-inducing activity was detected in the supernatant of the cells on costimulation with neutrophil proteinase 3 (PR3) and LPS for 24 h after IFN-γ-priming for 3 days. An active 18-kDa form of IL-18 was detected in lysate and supernatant of the cells only after the above treatment and the induction was inhibited by cycloheximide and by serine proteinase inhibitors. After the treatment, lactate dehydrogenase activity was not detected in the cell culture supernatant, and PR3 was detected only in the membrane and not in cytoplasm fractions of the cells. Caspase-1 was not detected in the cells even after the treatment and the IL-18 induction was not inhibited by a caspase-1 inhibitor. These results suggest that the PR3-mediated induction of bioactive IL-18 secretion from oral epithelial cells in combination with LPS after IFN-γ-priming occurred via a caspase-1-independent pathway, and provide new insight into the possible involvement of a neutrophil proteinase in the induction of bioactive IL-18 in oral inflammation such as periodontitis.

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2

“…A significant increase in production was observed from 8-h incubation, and a marked increase at 24 and 48 h. In contrast to PR3, two other neutrophil serine proteinases, HLE and Cat G, showed only marginal activity in oral epithelial cells as assessed by MCP-1 production (Fig. 1C), which was consistent with our recent observation (27). The PR3 from the two different sources showed the same results (Fig.…”

“…HSC-2 was grown in RPMI 1640 with 10% heat-inactivated FCS (Life Technologies, Grand Island, NY), and KB was grown in ␣-MEM with 10% FCS with a medium change every 3 days. Human gingival epithelial cells were prepared from explants of normal human gingival tissues with informed consent, as described previously (27). The experimental procedure was approved by the Ethical Review Board, Tohoku University School of Dentistry.…”

“…Random hexamer-primed reverse transcription was performed on 2.5 l total RNA in a 50-l reaction volume, and all PCR procedures were performed in a 20-l vol, as described previously (27). The primers used for PCR were as follows: IL-8, 5Ј-GATTGAGAGTGGACCACACT-3Ј, 5Ј-TCTC CCGTGCAATATCTAGG-3Ј; MCP-1, 5Ј-AACTGAAGCTCGCACTCT CG-3Ј, 5Ј-TCAGCACAGATCTCCTTGGC-3Ј; ICAM-1, 5Ј-CAGTCAC CTATGGCAACGAC-3Ј, 5Ј-ATTCAGCGTCACCTTGGCTC-3Ј; PAR-1,5Ј-AAGGAAGGCTGGAAGAGTGC-3Ј (27). The primers for IL-8, MCP-1, ICAM-1, PAR-1, PAR-2, PAR-3, PAR-4, and GAPDH were constructed to generate fragments of 422, 257, 243, 708, 1,066, 858, 725, and 527 bp, respectively.…”

“…In contrast to human colonic epithelial cells, human oral epithelial cells are shown to be basically unresponsive to many bacterial cell surface components, even in the presence of the soluble form of CD14, a bacterial pattern recognition receptor (26). We have recently shown that oral epithelial cells constitutively express a 24-kDa precursor form of IL-18, and that human neutrophil PR3 induced the secretion of a bioactive IL-18 from the epithelial cells in combination with LPS after priming with IFN-␥ (27). We extended the investigation of the underlying mechanism by which PR3 activates the epithelial cells, and in the present study, we obtained the evidence for the first time that PR3 by itself could activate the epithelial cells in culture to induce production of IL-8 and MCP-1 and expression of ICAM-1 via the PAR-2 pathway.…”

The inflammasomes in health and disease: from genetics to molecular mechanisms of autoinflammation and beyond

“…66,67 The immature form of IL-18 is constitutively expressed in some cells but only those with an active inflammasome, such as macrophages, dendritic cells and Kupffer cells, have the ability to release the mature form. 68 In the last decade, a number of groups have focused on the study of inflammasomes as initiators of immune disorders mediated by IL-1b and IL-18.…”