Modulators of inflammation use nuclear factor-kappa B and activator protein-1 sites to induce the caspase-1 and granzyme B inhibitor,proteinase inhibitor 9

Proteinase inhibitor 9 (PI-9) inhibits caspase-1 (interleukin (IL)-1beta-converting enzyme) and granzyme B, thereby regulating production of the pro-inflammatory cytokine IL-1beta and susceptibility to granzyme B-induced apoptosis. We show that cellular PI-9 mRNA and protein are induced by IL-1beta, lipopolysaccharide, and 12-O-tetradecanoylphorbol-13-acetate. We identified functional imperfect nuclear factor-kappaB (NF-kappaB) sites at -135 and -88 and a consensus activator protein-1 (AP-1) site at -308 in the PI-9 promoter region. Using transient transfections in HepG2 cells to assay PI-9 promoter mutations, we find that mutational ablation of the AP-1 site or of either NF-kappaB site reduces IL-1beta-induced expression of PI-9 by approximately 60%. Mutational ablation of the two NF-kappaB sites and of the AP-1 site nearly abolishes both basal and IL-1beta-induced expression of PI-9. Nuclear extracts from IL-1beta-treated HepG2 cells exhibited strong, IL-1beta-inducible binding to the NF-kappaB sites and to the AP-1 site. Electrophoretic mobility shift assays show that after IL-1beta treatment c-Jun/c-Fos and JunD bind to the AP-1 site, whereas the p50/p65 heterodimer binds to the two NF-kappaB sites. Estrogens induce PI-9, but induction of PI-9 by estrogens and IL-1beta is not synergistic. In transiently transfected, estrogen receptor-positive HepG2ER7 cells, estrogens do not interfere with IL-1beta induction, whereas IL-1beta exhibits dose-dependent repression of estrogen-inducible PI-9 expression. Our surprising finding that the pro-inflammatory cytokine IL-1beta strongly induces PI-9 suggests a novel mechanism for regulating inflammation and apoptosis through a negative feedback loop controlling expression of the anti-inflammatory and anti-apoptotic protein, PI-9.     

Interleukin-1beta stimulates macrophage inflammatory protein-1alpha and -1beta expression in human neuronal cells (NT2-N)

Chemokines are important mediators in immune responses and inflammatory processes of neuroimmunologic and infectious diseases. Although chemokines are expressed predominantly by cells of the immune system, neurons also express chemokines and chemokine receptors. We report herein that human neuronal cells (NT2-N) produce macrophage inflammatory protein-1alpha and -1beta (MIP-1alpha and MIP-1beta), which could be enhanced by interleukin (IL)-1beta at both mRNA and protein levels. The addition of supernatants from human peripheral blood monocyte-derived macrophage (MDM) cultures induced MIP-1beta mRNA expression in NT2-N cells. Anti-IL-1beta antibody removed most, but not all, of the MDM culture supernatant-induced MIP-1beta mRNA expression in NT2-N cells, suggesting that IL-1beta in the MDM culture supernatants is a major factor in the induction of MIP-1beta expression. Investigation of the mechanism(s) responsible for IL-1beta-induced MIP-1alpha and -1beta expression demonstrated that IL-1beta activated nuclear factor kappa B (NF-kappaB) promoter-directed luciferase activity in NT2-N cells. Caffeic acid phenethyl ester, a potent and specific inhibitor of activation of NF-kappaB, not only blocked IL-1beta-induced activation of the NF-kappaB promoter but also decreased IL-1beta-induced MIP-1alpha and -1beta expression in NT2-N cells. These data suggest that NF-kappaB is at least partially involved in the IL-1beta-mediated action on MIP-1alpha and -1beta in NT2-N cells. IL-1beta-mediated up-regulation of beta-chemokine expression may have important implications in the immunopathogenesis of inflammatory diseases in the CNS.     

Interleukin-1beta induces macrophage inflammatory protein-1beta expression in human hepatocytes

The investigation of factors that regulate expression of CC-chemokines, the important mediators in immune responses and inflammation processes, has an important significance in understanding the immunopathogenesis of liver diseases. We examined the role of interleukin-1beta (IL-1beta), a multifunctional cytokine, in regulating the expression of macrophage inflammatory protein (MIP)-1beta in human hepatocytes (Huh7 and HepG2). IL-1beta significantly enhanced MIP-1beta expression in these cells at both the mRNA and protein levels. Cytokine-enriched supernatants from monocyte-derived macrophage (MDM) cultures also induced MIP-1beta expression. IL-1beta is responsible for MDM supernatant-mediated up-regulation of MIP-1beta since the antibody to IL-1beta abolished MDM supernatant action. Investigation of the mechanism involved in MIP-1beta induction by IL-1beta showed that IL-1beta activated the nuclear factor kappa B (NF-kappaB) promoter in Huh7 cells. In addition, caffeic acid phenethyl ester (CAPE), a specific inhibitor of the activation of NF-kappaB, not only abolished IL-1beta-mediated NF-kappaB promoter activation, but also blocked IL-1beta-induced MIP-1beta expression. These observations suggest that IL-1beta-mediated up-regulation of MIP-1beta production in the hepatic cells may contribute a critical mechanism for continuous recruitment of inflammatory cell to liver and maintenance of inflammation.