Effect of cycloheximide on the expression of LPS‐inducible iNOS, IFN‐β, and IRF‐1 genes in J774 macrophages

Abstract

The effect of cycloheximide (CHX) on the gene expression for inducible NO synthase (iNOS), interferon (IFN)-~ and IFN regulatory factor (IRF)-I was examined in LPSstimulated J774 macrophages. LPS caused increased expression of mRNAs specific for iNOS, IFN-I~, and IRF-1 with different kinetics. Addition of CHX resulted in inhibition of the LPSinduced iNOS gene expression and parallel decrease in NO production. In contrast, expression of IFN-I~ and IRF-1 genes in response to LPS was potentiated in the presence of CHX. These results indicate that de novo protein synthesis is not required for IFN-13 and IRF-1 gene expression and that ongoing protein synthesis including IFN-13 and IRF-1 may be involved in the induction process of iNOS in mouse macrophages.We report here that J774 macrophages respond to LPS treatment by increased expression of mRNAs specific for IFN-I~, IRF-1 and iNOS with the kinetics unique for each gene. Addition of protein synthesis inhibitor cycloheximide (CHX) inhibits the LPS-induced iNOS gene expression while expression of IFN-fl and IRF-1 genes in response to LPS is clearly increased in the presence of CHX. These findings may suggest that LPS acts to induce iNOS via synthesis or release of intermediary cytokines and/or regulatory proteins in J774 macrophages. MATERIALS AND METHODSCell culture: The mouse macrophage cell line J774 was cultured and prepared as described previously (12). J774 macrophages were seeded into 96-well plates for nitrite assay and cytotoxic assay and into 10-era dishes for RNA preparation. To induce NO synthase in the cells, fresh culture medium containing LPS (E. coli, serotype No. 0127: B8, Sigma Chemical Co., Saint Louis, MO, USA)) was added at a concentration of 1 yg/ml. To assess the effects of cyeloheximide (CHX: 0.01 -10/~g/ml, Sigma Chemical Co.), the agent was added to the cells simultaneously with LPS except time-cx)urse study of CHX addition.

IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways

“…The J774 cell line has been used as an in vitro model to study macrophage activation by LPS (Fujihara et al ., 1994; Briend et al ., 1995; Hattori et al ., 1996). Treatment with LPS promotes differentiation and activation, manifested by up‐regulated expression of specific cell surface antigens, production of reactive nitrogen intermediates, chemokines and cytokines including TNFα and IL‐1β.…”

The expression of inducible nitric oxide synthase in the human fallopian tube during the menstrual cycle and in ectopic pregnancy

“…However, higher concentrations of NO have been shown to decrease embryo development, possibly through the production of free radicals (43). Nitric oxide has also been shown to up-regulate the receptors for growth factors such as epidermal growth factor and those for anchoring proteins and integrins (44,45). The authors suggested that NO may induce the embryo to develop these growth-regulating and -anchoring proteins on the cell surface.…”

Gene Regulatory Network Inference of Immunoresponsive Gene 1 (IRG1) Identifies Interferon Regulatory Factor 1 (IRF1) as Its Transcriptional Regulator in Mammalian Macrophages

“…It is already known that IRF1 has a significant role in antibacterial and antiviral responses, induction of apoptosis and tumorigenesis, with several of these processes known to be mediated by iNOS and NO [ 58 ]. Thus, from our results, it is tempting to conclude that IRF1 regulates these processes also through the induction of Irg1 and itaconic acid production.…”