Abstract-Nitric oxide (NO) is known to have antiatherogenic and anti-inflammatory properties, but its effects on the cytokine-induced nuclear factor-kappa B (NF-B) activation pathway in relation to the regulation of inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs) remain elusive. To elucidate the roles of NO in the regulation of cytokine-induced NF-B activation and consequent iNOS gene expression, we studied the effects of synthases (NOSs). Three distinct isozymes of NOS have been identified to date: 2 Ca 2ϩ /calmodulin-dependent constitutive isozymes dominantly expressed in the brain and endothelium, and a Ca 2ϩ -independent, cytokine-inducible isozyme (iNOS). 1 iNOS produces large amounts of NO in response to bacterial lipopolysaccharides (LPS) and certain cytokines in a variety of cells, including vascular smooth muscle cells (VSMCs). 2 NO possesses diverse physiological properties, such as vasodilation, neurotransmission, and mediation of immune responses. 1 High-output NO produced by iNOS in VSMCs not only causes inhibition of cell proliferation but apoptosis of VSMCs as well. 3,4 Therefore, regulation of iNOS gene expression has been implicated in the pathogenesis of vascular remodeling and atherosclerosis. 5 Many of the biological effects of NO have been attributed to cGMP generation via the stimulation of soluble guanylate cyclase, although a cGMP-independent mechanism is also involved in its diverse actions.The promoter region of the rat and mouse iNOS gene contains several potential cis-elements for the binding of different transcription factors, among which 2 putative binding sites for nuclear factor-kappa B (NF-B) exist in the upstream (GGGGATTTTCC, nucleotides Ϫ965 to Ϫ955: NF-Bu) and downstream (GGGGACTCTCC, nucleotides Ϫ107 to Ϫ97: NF-Bd) regions. [6][7][8] The sequence of NF-Bd is unique in that it is found only in murine and human iNOS genes. It has been shown that a key region of the promoter activity in mediation of LPS inducibility resides in the NF-Bd region in mouse macrophages. 6,7 However, its role in mediation of iNOS expression in response to cytokines in VSMCs remains largely unknown.NF-B complexes function as a pleiotropic regulator of many genes modulating immunologic and inflammatory pro-

IGFBP-2, -7, -8 and -10 are produced by normal and injured glomerular podocytes and may regulate local IGF-I actions in podocytes and/or cortical tubular cells in the kidney.

Glucocorticoids and nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory and immune diseases. Nitric oxide (NO) has a diversity of physiological functions, but its excess production has been implicated in the inflammatory process. The present study was designed to elucidate the mechanisms by which glucocorticoids and NSAIDs affect inducible nitric oxide synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMCs). Both interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha potently stimulated nitrite/nitrate (NOx) production with a concomitant expression of iNOS mRNA and protein as demonstrated by Northern and Western blot analysis, respectively. Both IL-1beta and TNF-alpha activated nuclear factor (NF)-kappaB as demonstrated by electrophoretic mobility shift assay. Dexamethasone, salicylate and aspirin, but not indomethacin, dose dependently inhibited cytokine-stimulated NOx production and iNOS protein expression. Dexamethasone decreased cytokine-induced NF-kappaB activation and iNOS mRNA expression, but neither salicylate nor aspirin affected NF-kappaB activation or iNOS mRNA expression. IL-1beta caused a rapid increase in phosphorylated IkappaB-alpha levels and subsequent transient decrease in IkappaB-alpha levels, an inhibitor of NF-kappaB, as revealed by Western blot analysis using specific antibodies for phosphorylated and nonphosphorylated IkappaB-alpha. These effects were blocked by pretreatment with dexamethasone. Aspirin dose dependently inhibited iNOS enzymatic activity, whereas salicylate and dexamethasone had limited effect. The present study demonstrates that 1) inhibitory effect of dexamethasone on cytokine-induced iNOS expression and NO production in rat VSMCs, although potentially acting at multiple levels, is partly mediated by inhibition of NF-kappaB activation resulting from decreased phosphorylation and degradation of IkappaB-alpha, 2) both salicylate and aspirin inhibit cytokine-stimulated NO production at translational and/or posttranslational levels without affecting NF-kappaB- mediated iNOS gene expression, and 3) aspirin directly inhibits iNOS enzyme activity. These data suggest the differential inhibitory mechanisms of iNOS-mediated NO synthesis by glucocorticoids and NSAIDs in the vasculature.

The depletion of CD8+ cells has been shown to prevent the initiation and progression of antiglomerular basement membrane (GBM) crescentic glomerulonephritis (GN) in Wistar-Kyoto (WKY) rats. In this study, we asked whether CD8+ cells produce their effects by perforin/granzyme-mediated or by Fas ligand (FasL)-mediated pathways. The glomerular mRNA expression of perforin and granzyme B corresponded with the number of CD8+ cells, whereas that of granzyme A, Fas, and FasL did not. The enhanced mRNA level of perforin and granzyme B was not evident in CD8+-depleted rats. The number of apoptotic cells in the glomeruli was significantly increased at day 3. Perforin mRNA was found in cells infiltrating the glomerulus by in situ hybridization and by using dual-staining immunohistochemistry perforin protein was found in glomerular CD8+ cells. We found that perforin was readily visualized at the inner surface of the glomerular capillaries by immunoelectron microscopy. Based on these results, we treated animals with a perforin antibody in vivo and found that it significantly reduced the amount of proteinuria, frequency of crescentic glomeruli, and the number of glomerular monocytes and macrophages, although the number of glomerular CD8+ cells was not changed. Our results suggest that CD8+ cells play a role in glomerular injury as effector cells in part through a perforin/granzyme-mediated pathway in the anti-GBM WKY rat model of crescentic GN.