The mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and p38, are activated in response to infectious agents and innate immune stimulators such as CpG DNA, and regulate the subsequent initiation and termination of immune responses. CpG DNA activates p38 and ERK with slightly different kinetics in monocytic cells. The present studies investigated the roles of these two key mitogen-activated protein kinases in regulating the CpG DNA-induced production of pro- and anti-inflammatory cytokines in the macrophage-like cell line RAW264.7. p38 activity was essential for the induction of both IL-10 and IL-12 expression by CpG DNA. In contrast, CpG DNA-mediated ERK activation was shown to suppress IL-12 production, but to be essential for the CpG DNA-induced IL-10 production. Studies using rIL-10 and IL-10 gene-deficient mice demonstrated that the inhibitory effect of ERK on CpG DNA-mediated IL-12 production is indirect, due to the role of ERK in mediating IL-10 production. These results demonstrate that ERK and p38 differentially regulate the production of pro- and anti-inflammatory cytokines in APCs that have been activated by CpG DNA. CpG DNA-induced p38 activity is required for the resulting innate immune activation. In contrast, ERK plays a central negative regulatory role in the CpG DNA-mediated Th1 type response by promoting production of the Th2 type cytokine, IL-10.

Exposure of macrophages to LPS induces a state of hyporesponsiveness to subsequent challenge with LPS. It has not been known whether previous exposure to CpG DNA induces a similar suppressive response to subsequent stimulation with CpG DNA. In the present study, we demonstrate that pretreatment with CpG DNA induces suppression of cytokine release in a murine macrophage-like cell RAW264.7 in response to subsequent challenge by CpG DNA. Additionally, CpG DNA-mediated activation of mitogen-activated protein kinases, including c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38, and activation of transcription factors AP-1, CREB, NF-κB, and STAT1 are greatly suppressed in the cells pre-exposed to CpG DNA. Pretreatment with CpG DNA also partially inhibited LPS-mediated production of cytokines and activation of mitogen-activated protein kinases and transcription factors. Neither LPS nor CpG DNA treatment inhibited Toll-like receptor 4, MD2, Toll-like receptor 9, myeloid differentiation factor 88, Toll/IL-1R domain-containing adaptor protein, Tollip, and TNF-α receptor-associated factor 6 expression. Interestingly, CpG DNA or LPS stimulation led to the inhibition of IL-1R-associated kinase expression. These results indicate that CpG DNA-induced refractory of RAW264.7 cells may be, at least in part, due to suppressed IL-1R-associated kinase expression.

Cyclooxygenase-2 (COX-2) is a key enzyme involved in the inflammatory process that is rapidly induced in macrophages in response to LPS. Carbon monoxide (CO), a byproduct of heme oxygnease-1, can suppress proinflammatory response in various in vitro and in vivo models of inflammation. This study was undertaken to examine whether CO can regulate (and if so, to delineate the mechanism by which CO regulates) LPS-induced COX-2 expression in macrophages. RAW 264.7 murine macrophages were stimulated with LPS (0-10 ng/ml) with or without CO (500 ppm). Northern and Western blot analysis was done. Progstaglandin E 2 and nitrite concentration was measured from cell culture supernatant. Electrophoretic mobility shift assay was performed to assess nuclear factor binding. CO downregulated LPS-induced COX-2 mRNA and protein expression. CO also inhibited LPS-induced prostaglandin E 2 secretion (P Ͻ 0.05). CO also decreased LPS-induced CCAAT/enhancer-binding protein (C/EBP) ␤ and ␦ protein expression in LPS-treated RAW 264.7 cells. Gel shift analysis revealed that CO treatment decreased LPSinduced activation of protein binding to C/EBP consensus oligonucleotides of murine cyclooxygenase-2 promoter. CO also decreased LPS-induced nitric oxide synthase-2 protein expression and nitrite production, and decreased LPS-induced activation of protein binding to C/EBP consensus oligonucleotides of murine nitric oxide synthase-2 promoter. CO may act as an important regulator of inflammation by virtue of its ability to regulate C/EBPs. Keywords: heme oxygenase; lipopolysaccharides; nitric oxide synthase Heme oxygenase-1 (HO-1) is a microsomal enzyme responsible for degradation of heme, generating biliverdin, iron, and carbon monoxide (CO) (1). HO-1 can be induced by a wide variety of stimuli, and the enzyme is involved in cellular and tissue defense against oxidative stress possessing potent anti-inflammatory properties (2, 3). There is growing interest in the role of CO in the anti-inflammatory and cytoprotective function of HO-1 (4-7), but the pathways involved in the anti-inflammatory effect of CO are poorly understood. CO can modulate mitogen-activated protein kinase (5) and guanylate cyclase/3Ј,5Ј-guanylate cyclic monophospate (cGMP) pathway (8) to inhibit secretion of proinflammatory cytokines. CO also modulates several transcription factors, including NF-B (4, 6) and activating protein-1 (4), which are involved in inflammation. But whether other pathways or molecules are involved in the anti-inflammatory effect of CO is not known.