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The mechanisms involved in anti-inflammatory action of transforming growth factor beta (TGFbeta) have been examined by evaluating its effect on chemokine gene expression in mouse macrophages. Lipopolysaccharide (LPS)-stimulated expression of the CXC chemokines KC and MIP-2 was selectively reduced by TGFbeta in a time- and protein synthesis-dependent process. While TGFbeta had a modest effect on transcription of the KC and MIP-2 mRNAs as measured by nuclear run-on, it had no effect on LPS-stimulated luciferase expression driven by the KC promoter nor on the activation of nuclear factor kappaB (NFkappaB) DNA-binding activity and transactivation function. Interestingly, KC mRNA levels were markedly reduced by TGFbeta treatment in cells transfected with KC genomic or cDNA constructs driven from either the KC or cytomegalovirus (CMV) promoters, demonstrating the importance of sequences within the mature mRNA and suggesting that suppression may involve a posttranscriptional mechanism. In support of this possibility, LPS stimulation prolonged the half-life of KC mRNA and this stabilization response was blocked in cells treated with TGFbeta. Examination of KC mRNA expressed under control of a tetracycline-responsive promoter demonstrated that TGFbeta prevented stabilization of KC mRNA, in response to LPS but did not alter KC mRNA half-life directly. KC mRNA stabilization by LPS was dependent on activation of p38 mitogen-activated protein kinase (MAPK) activity, and TGFbeta treatment inhibited p38 MAPK activation. These findings support the hypothesis that TGFbeta-mediated suppression of chemokine gene expression involves antagonism of LPS-stimulated KC mRNA stabilization via inhibition of p38 MAPK.

Tissue factor, which is expressed in vascular lesions, increases thrombin production, blood coagulation, and smooth muscle cell proliferation. We demonstrate that oxidized low-density lipoprotein (LDL) induces surface tissue factor pathway activity (ie, activity of the tissue factor:factor VIIa complex) on human and rat smooth muscle cells. Tissue factor messenger RNA (mRNA) was induced by oxidized LDL or native LDL; however, native LDL did not markedly increase tissue factor activity. We hypothesized that oxidized LDL mediated the activation of the tissue factor pathway via an oxidant-dependent mechanism, because antioxidants blocked the enhanced tissue factor pathway activity by oxidized LDL, but not the increased mRNA or protein induction. We separated total lipid extracts of oxidized LDL using high-performance liquid chromatography (HPLC). This yielded 2 major peaks that induced tissue factor activity. Of the known oxysterols contained in the first peak, 7alpha- or 7beta-hydroxy or 7-ketocholesterol had no effect on tissue factor pathway activity; however, 7beta-hydroperoxycholesterol increased tissue factor pathway activity without induction of tissue factor mRNA. Tertiary butyl hydroperoxide also increased tissue factor pathway activity, suggesting that lipid hydroperoxides, some of which exist in atherosclerotic lesions, activate the tissue factor pathway. We speculate that thrombin production could be elevated via a mechanism involving peroxidation of cellular lipids, contributing to arterial thrombosis after plaque rupture. Our data suggest a mechanism by which antioxidants may offer a clinical benefit in acute coronary syndrome and restenosis.

Interleukin-10 (IL-10) selectively inhibited lipopolysaccharide (LPS)-induced chemoattractant cytokine gene expression: levels of IP-10 mRNA were markedly suppressed in IL-10-treated mouse peritoneal macrophages, whereas the expression of the RANTES mRNA was only modestly reduced. IL-10 inhibited IP-10 mRNA accumulation by reducing IP-10 gene transcription as demonstrated by nuclear run-on analysis. Interestingly, the ability of IL-10 to inhibit expression of IP-10 was dependent on the inducing stimulus; IL-10 did not suppress interferon gamma (IFNgamma)- or IFNbeta-stimulated IP-10 transcription or mRNA accumulation. These results suggested that IL-10 might act indirectly to suppress IP-10 expression by inhibiting LPS-induced class I IFN production. This hypothesis was supported by the following observations. First, LPS-induced IP-10 mRNA expression was blocked in cells cotreated with cycloheximide. Second, IL-10 inhibited the production of IFN/beta-mediated antiviral activity. Finally, the IL-10-mediated suppression of LPS-stimulated IP-10 production could be rescued by cotreatment with IFNbeta.