Dexamethasone inhibits interleukin-1beta-induced corneal neovascularization: role of nuclear factor-kappaB-activated stromal cells in inflammatory angiogenesis

Dexamethasone, a synthetic corticosteroid, is widely used as a potent anti-inflammatory drug in various diseases including corneal angiogenesis. However, dexamethasone's impact on interleukin (IL)-1beta-dependent inflammatory angiogenesis is unknown. Here, we show that dexamethasone inhibits IL-1beta-induced neovascularization and the expression of the angiogenesis-related factors, vascular endothelial growth factor-A, KC, and prostaglandin E(2) in the mouse cornea 2 days after IL-1beta implantation. IL-1beta caused IkappaB-alpha phosphorylation in corneal stromal cells but not in infiltrated CD11b(+) cells 2 days after IL-1beta implantation. In contrast, both cell types were positive for phosphorylated IkappaB-alpha 4 days after IL-1beta implantation. Dexamethasone significantly inhibited IkappaB-alpha phosphorylation 2 and 4 days after IL-1beta implantation. Furthermore, dexamethasone inhibited IL-1beta-induced expression of vascular endothelial growth factor-A, KC, and prostaglandin E(2), and signaling of nuclear factor (NF)-kappaB in corneal fibroblasts in vitro. A selective NF-kappaB inhibitor attenuated IL-1beta-induced corneal angiogenesis. These findings suggest that NF-kappaB activation in the corneal stromal cells is an important early event during IL-1beta-induced corneal angiogenesis and that dexamethasone inhibits IL-1beta-induced angiogenesis partially via blocking NF-kappaB signaling.     

Saxatilin inhibits TNF-alpha-induced proliferation by suppressing AP-1-dependent IL-8 expression in the ovarian cancer cell line MDAH 2774

Tumor necrosis factor-alpha (TNF-alpha)-induced proliferation of cancer cell line MDAH 2774 was significantly suppressed by treating the cells with saxatilin, a snake venom disintegrin. The suppressed proliferation was found to be associated with the level of interleukin-8 (IL-8) expression in the cells. TNF-alpha-induced IL-8 promoter activation that is inhibited by saxatilin treatment was dependent on activating protein-1 (AP-1) instead of nuclear factor-kappa B (NF-kappaB). Coexpression of dominant negative p38 (DN-p38) suggested that p38 is involved in the IL-8 promoter activity which is regulated by saxatilin or TNF-alpha. Experimental evidence clearly indicated that saxatilin inhibits TNF-alpha-induced proliferation of the ovarian cancer cells by suppressing IL-8 expression in AP-1-dependent manner.     

IL-1beta acts in synergy with endogenous IL-1beta in A375-S2 human melanoma cell apoptosis through mitochondrial pathway

Interleukin-1beta (IL-1beta) is a pivotal proinflammatory cytokine. To investigate the mechanism of IL-1beta-induced cell death in human malignant melanoma A375-S2 cells, MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, radioimmunoassay and Western blot analysis were carried out. IL-1beta did not only induce nuclear condensation and DNA fragmentation, but also increased degradation of two substrates of caspase-3, poly ADP-ribose polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Simultaneously, release of precursor of IL-1beta (pro-IL-1beta) and endogenous IL-1beta production were involved in the apoptotic process. IL-1beta enhanced the ratio of Bax/Bcl-2 and Bax/Bcl-xL expression and up-regulated apoptosis inducing factor (AIF) expression, which required the activation of downstream caspases. These results suggest that IL-1beta induces endogenous IL-1beta production, enhances cleavage of caspase downstream substrates and promotes mitochondria mediated apoptosis in A375-S2 cells.     

Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes

Recent studies indicate that nitric oxide (NO) or related compounds may regulate the production of interleukin (IL)-8, a potent proinflammatory chemokine. Here we report that peroxynitrite (ONOO(-)) formed by a reaction of NO with superoxide mediates IL-8 gene expression and IL-8 production in IL-1beta- and TNF-alpha-stimulated human leukocytes in whole blood. The NO synthase inhibitors aminoguanidine and N(G)-nitro-L-arginine methyl ester blocked nuclear accumulation of activator protein-1 (AP-1) and nuclear factor (NF)-kappaB in both polymorphonuclear (PMN) and mononuclear leukocytes and inhibited IL-8 mRNA expression and IL-8 release by approximately 90% in response to IL-1beta and TNF-alpha. Enhanced ONOO(-) formation was detected in granulocytes, monocytes, and lymphocytes after challenge with IL-1beta or TNF-alpha. The addition of ONOO(-) (0.2-80 microM) to whole blood increased nuclear accumulation of AP-1 and NF-kappaB in PMN and mononuclear leukocytes and augmented IL-8 mRNA expression and IL-8 production in a concentration-dependent fashion. Pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB activation, attenuated approximately 70% of IL-8 release evoked by IL-1beta, TNF-alpha, or ONOO(-). These results indicate that ONOO(-) formation may underlie the action of cytokines towards IL-8 gene expression in human leukocytes.     

Role of CXCL1 in tumorigenesis of melanoma

The CXC chemokine, CXCL1 (melanoma growth-stimulatory activity/growth-regulated protein alpha), plays a major role in inflammation, angiogenesis, tumorigenesis, and wound healing. Recently, chemokines have been extensively related to cellular transformation, tumor growth, homing, and metastasis. CXCL1 and its mouse homologue MIP-2 have been shown to be involved in the process of tumor formation. When chemokines such as CXCL1 and CXCL8 (IL-8) become disregulated so that they are chronically expressed, tissue damage, angiogenesis, and tumorigenesis can follow. This up-regulation of chemokines has been attributed to constitutive activation of NF-kappaB. The constitutive NF-kappaB activation is an emerging hallmark in various types of tumors including breast, colon, pancreatic, ovarian, as well as melanoma. Previous findings from our laboratory and other laboratories have demonstrated the role of endogenous activation of NF-kappaB in association with enhanced metastatic potential of malignant melanoma cells and suggest that targeting NF-kappaB may have potential therapeutic effects in clinical trials. An important step in this direction would be to delineate the important intracellular pathways and upstream kinases involved in up-regulation of NF-kappaB in melanoma cells. In this review, the signaling pathways involved in the disregulation of NF-kappaB and chemokine expression are discussed.     

CpG oligodeoxynucleotides induce IL-8 expression in CD34+ cells via mitogen-activated protein kinase-dependent and NF-kappaB-independent pathways

To elucidate the role of Toll-like receptor 9 (TLR9) activation along with the intracellular signaling pathways triggered by CpG DNA in CD34+ cells, we investigated whether synthetic oligodeoxynucleotides (ODNs), containing unmethylated CpG motifs, could induce IL-8 expression in CD34+ cells through mitogen-activated protein kinase (MAPK) or nuclear factor-kappaB (NF-kappaB) pathway. We demonstrated evidence for the first time that CD34+ cells constitutively expressed TLR9. Exposure of the cells to CpG ODN resulted in a time- and dose-dependent increase of IL-8 expression, and activation of phosphorylated ERK1/2 and phosphorylated p38. In addition, CpG ODN stimulated AP-1, but not NF-kappaB, signals. Moreover, inhibitors of MAPK (U0126 and SB203580) significantly reduced the IL-8 production, while the inhibition of NF-kappaB (pyrrolidinedithiocarbamate and retrovirus containing dominant-negative IkappaB alpha plasmid) did not affect the IL-8 expression increased by CpG ODN. Moreover, co-stimulation with LPS and CpG synergistically up-regulates IL-8 in CD34+ cells. These results suggest that CpG DNA, acting on TLR9, activates CD34+ cells to express IL-8 through MAPK-dependent and NF-kappaB-independent pathways.     

Role of nitric oxide in the activation of NF-κB, AP-1 and NOS II expression in articular chondrocytes

OBJECTIVE AND DESIGN: Determine the sources of nitric oxide (NO) and evaluate its role in the activation of nuclear Factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) and in the expression of NO synthase II (NOS II), induced by interleukin-1beta (IL-1). MATERIAL OR SUBJECTS: Primary cultures of bovine articular chondrocytes. TREATMENT: The cells were treated with IL-1, 5 ng/ml with or without the NO donor S-nitroso-N-acetylpenicillamine (SNAP), in concentrations ranging from 10 to 300 microM. METHODS: NF-kappaB and AP-1 activation were evaluated by electrophoretic mobility shift assay. Northern blot was used to detect NOS II mRNA levels and western blot to evaluate IkappaB-alpha, NOS I and NOS II protein levels. RESULTS: Under basal conditions, chondrocytes expressed NOS I, which was lost upon IL-I treatment. SNAP inhibited IL-I-induced NF-kappaB activation and NOS II expression. When added alone, SNAP induced AP-1 activation to approximately the same extent as IL-I. CONCLUSIONS: These results suggest that, in chondrocytes, NO is a key regulator of the signaling pathways leading from IL-I to NF-kappaB and AP-1 activation and to the expression of genes that are involved in the pathophysiology of arthritic diseases.