RESUMO
Proinflammatory cell activation via the receptor for advanced glycation end products (RAGE) pathway may play a central pathogenetic role in atherosclerosis. Since S100A8/A9 was recently identified as ligand of RAGE, we determined the effects of proinflammatory cytokines on RAGE-mediated induction of gene expression of S100A8 and S100A9. mRNA levels of S100A8 and S100A9 were upregulated following cytokine stimulation with IL-6 (1, 10, 100 ng/ml) or TNFα (10 ng/ml) in human THP-1 cells. Preincubation of cells with 2000 ng/ml AGE (advanced glycation end products) before cytokine stimulation resulted in upregulation of RAGE. Pretreatment of THP-1 with AGE followed by stimulation with IL-6 (10 ng/ml) or TNFα (10 ng/ml) further increased S100A8 and S100A9 mRNA expression and S100A8/A9 release into cell culture supernatant, as compared to pretreatment with non-glycated albumin as control. Binding of AGE to RAGE was blocked with a neutralizing anti-RAGE antibody. Normal mouse IgG served as control. Cytokine-stimulated induction of S100A8 and S100A9 mRNA levels as well as of S100A8/A9 release after preincubation of cells with AGE were significantly suppressed by RAGE blockade, indicating a RAGE-dependent pathway of AGE-mediated S100A8/A9 expression.The cytokine-induced potentiated S100A8 and S100A9 expression under conditions with a high AGE burden is able to aggravate proinflammatory conditions via activation of the RAGE pathway.
Assuntos
Calgranulina A/genética , Calgranulina B/genética , Leucemia/patologia , Receptores Imunológicos/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/farmacologia , Leucemia/genética , Leucemia/metabolismo , Camundongos , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Cellular adhesion molecules play a pivotal role in the pathogenesis of atherosclerosis by mediating the adherence of blood leukocytes. Since hyperhomocysteinemia appears to be an independent risk factor for the development of atherosclerosis, in this study we investigated the effect of homocysteine on basal and TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell-adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) on human umbilical-vein endothelial cells. Incubation of endothelial cells with homocysteine resulted in dose-dependent reduction in TNF-alpha-induced (5 ng/ml) expression of VCAM-1, E-selectin, and ICAM-1 (the latter less pronounced). This effect was found to be specific since other thiol compounds-cysteine and glutathione-did not mimic homocysteine activity. Homocysteine attenuated TNF-alpha-stimulated U-937 adhesion to the endothelial monolayer and reduced TNF-alpha-induced activation of the transcription factor NF-kappaB, indicating that NF-kappaB inhibition may play a role in inhibiting expression of adhesion molecules in endothelial cells.
Assuntos
Endotélio/metabolismo , Homocisteína/metabolismo , Monócitos/citologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adesão Celular , Células Cultivadas , Cisteína/farmacologia , Relação Dose-Resposta a Droga , Selectina E/biossíntese , Endotélio Vascular/citologia , Ensaio de Imunoadsorção Enzimática , Glutationa/metabolismo , Glutationa/farmacologia , Homocisteína/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Monócitos/metabolismo , NF-kappa B/fisiologia , Reação em Cadeia da Polimerase , Células U937 , Cordão Umbilical/citologia , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
The cellular adhesion molecule E-selectin is expressed on activated endothelial cells, and is involved in the process of adherence of blood cells to vessel endothelium in inflammatory events such as atherosclerosis. In a recent study we found a Ser128Arg mutation in the EGF domain as well as a Leu554Phe mutation in the membrane domain of E-selectin. We also established increased frequencies of both mutations among young patients with severe coronary atherosclerosis. In the present study we investigated the influence of these mutations on cell adhesion and on the release of soluble E-selectin. Mutants were created by site-directed mutagenesis and COS cells were transfected with E-selectin, either wild-type or mutant. Antibody-binding studies and cell-adhesion assays were performed on transfected COS cells and on interleukin-1 beta-stimulated HUVECs. Soluble E-selectin in supernatants of wild type and Leu554Phe mutant-transfected COS cells was measured by ELISA. We discovered significant differences in the strength of HL-60 cell adhesion for the Ser128Arg mutant: in comparison with the wild type, the strength of adhesion to the mutant was reduced on transfected COS cells (P < 0.01) as well as on stimulated HUVECs (P < 0.01). Significantly diminished release of soluble E-selectin was detected for the Leu554Phe membrane domain mutant, in comparison with the wild type. In summary, the mutations studied here influence the E-selectin function in vitro and may be considered as one of the risk factors involved in the complex pathogenesis of atherosclerosis.