Involvement of oxidative and nitrosative stress in modulation of gene expression and functional responses by IFNγ

International Immunology, Vol. 19, No. 7, pp. 867–879 doi:10.1093/intimm/dxm058 ª The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: Involvement of oxidative and nitrosative stress in modulation of gene expression and functional responses by IFNg S. Jyothi Prasanna, Banishree Saha and Dipankar Nandi Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Keywords: cytokine, growth suppression, inflammation, redox, transcriptional profiling Abstract IFNg is a potent immunomodulator which plays important roles in host defense. IFNg modulates transcription of growth-related genes [N-myc downstream regulator 1, growth arrest and DNA damage inducible g and inhibitor of DNA binding 2 (Id2)], which is followed by increased growth suppression in the mouse hepatoma cell line, H6. Further studies revealed modulation of genes involved in oxidative and nitrosative stress (iNos, gp91phox and Catalase) and increased generation of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNIs) upon IFNg treatment. High amounts of ROS and RNI are responsible for IFNg-mediated reduction in cell growth as this process is blocked, using either diphenylene iodonium (DPI), an inhibitor of flavin-containing NADPH oxidases, or N-methyl L-arginine (LNMA), an inhibitor of nitric oxide synthase. Based on studies with LNMA and DPI, IFNg-modulated genes can be categorized into two distinct sets: oxidative and nitrosative stress independent (transporter associated with antigen processing 2, Cd80, Lmp10 and Icosl) and oxidative and nitrosative stress dependent (iNos, gp91phox, Catalase and Id2). In addition, DPI or LNMA blocked IFNg-induced activation of Ras, demonstrating the involvement of oxidative and nitrosative stress. Manumycin A, a farnesyl transferase inhibitor, blocked Ras activation and reduced NADPH oxidase activity and ROS amounts leading to increased cell growth in the presence of IFNg. Notably, the IFNg-induced MHC class I levels are not modulated in cells treated with DPI, LNMA or manumycin A. Together, these results delineate the role of high amounts of ROS, RNI and Ras activation in modulating expression of some genes and, thereby, function by IFNg. The implications of these results during modulation of immune responses by IFNg are discussed. Introduction IFNc is a type II IFN produced, primarily, by NK cells and T cells which plays important roles during host defense. Ifnc / mice are very sensitive to infection by several pathogens, including Mycobacterium tuberculosis. Also, humans lacking IFNc or its receptor display a similar phenotype and are susceptible to mycobacterial infections (1). Apart from modulating host immunity, IFNc causes pleiotropic effects and modulates inflammatory responses, cell growth and survival (2, 3). IFNc employs the Janus kinase (Jak)–signal transducers and activators of transcription coactivator (Stat) pathway to signal from the cell-surface receptor to modulate transcriptional activation of several genes. Jak1, a non-receptor protein tyrosine kinase, phosphorylates IFNc receptors which generate a binding site for Stat1, a transcriptional coactivator (4, 5). After phosphorylation, Stat1 dimers dissociate from the receptor and translocate to the nucleus and bind to Correspondence to: D. Nandi; E-mail: Transmitting editor: D. Wallach a motif known as the gamma-IFN-activated site (Gas): TTNCNNNAA. Gas sequences are present in several primary responsive IFNc-modulated genes and Stat1 enhances transcriptional activation by recruiting several transcriptional coactivators (6–8). Cellular responses mediated by IFNc are, primarily, due to modulation of gene expression. Therefore, identification and studying roles of IFN-stimulated genes (ISGs) during immune responses are an active area of investigation (2, 9–13). IFN-modulated genes can be further classified into primary responsive or secondary responsive genes. Primary responsive genes are induced early due to the binding of the Stat1 dimer to Gas elements present in promoters of genes, e.g. Irf1. The secondary responsive genes are induced following the binding of Irf1 to their promoters (2). Although IFNc signaling has been studied for several years, accumulating evidences clearly demonstrate the roles of Received 6 July 2006, accepted 18 April 2007 Advance Access publication 2 July 2007 868 Free radicals regulate IFNc responses multiple pathways that coordinate to generate functional responses (4). Most IFNc-responsive genes are dependent on Stat1 although some genes are Stat1 independent (14). Breast cancer 1 (BRCA1), the tumor suppressor, together with Stat1 differentially activates a subset of ISGs (7). Also, IFNc has been shown to activate a subset of genes in an IjB kinase-dependent manner (15). Further studies are required to fully comprehend IFNc signaling and the cross-talk that occurs between several pathways. One of the potent effects of IFNc is its growth-suppressive effect. It induces the cyclin-dependent kinase (CDK) inhibitor p21waf1/CIP1 and p27Kip1 which interfere with the actions of CDKs and prevents hyperphosphorylation of Rb and entry into the S phase of the cell cycle (16–19). Also, recruitment of BRCA1 by Stat1 results in activation of the CDK inhibitor, p21 waf1, which may be involved in the induction of the growthsuppressive effects of IFNc (7). Another mechanism may involve reduction in telomerase activity and telomerase reverse transcriptase (RT) (20). The role of IFNc in inhibiting cellular proliferation may be physiologically important. For example, in a model of experimental autoimmune encephalomyelitis, Ifnc / mice accumulate 10- to 16-fold more activated T cells compared with wild type (21). Also, mice lacking IFNc develop more tumors compared with wild-type mice (22–24). IFNc enhances the immunogenicity of tumor cells that are recognized and eliminated by the host defense. This may be due to increased immune responses, e.g. by enhancing expression of MHC class I (MHC-I) antigen-processing pathway or increased NK activity; in fact, basal NK activity is lowered in mice lacking IFNc (25). Alternately, IFNc may directly inhibit tumors due to its growth-suppressive effect (26). Despite numerous studies on the growth-suppressive actions of IFNc, the role of oxidative and nitrosative stress in this process is not well appreciated. We studied IFNc-induced gene expression and functional responses in the mouse hepatoma cell line, H6, which greatly enhances the expression of genes involved in the MHC-I assembly pathway (27). In this study, the crucial role of oxidative and nitrosative stress in modulating expression of distinct sets of genes involved in some functions mediated by IFNc is demonstrated. Methods Cell culture H6 cells (hepatoma, H-2a) were cultured in RPMI 1640 medium containing 25 mM HEPES (Sigma, St Louis, MO, USA), 5% heat-inactivated FCS (Sigma), 5 lM b-mercaptoethanol (Sigma), 10 (...truncated)