Abstract
Nitric oxide (NO) has frequently been shown to inhibit leukocyte adherence to activated endothelium thus displaying anti-adhesive and immunosuppressive activities. A molecular mechanism contributing to this effect is described. Primary murine aortic endothelial cells were activated with interleukin (IL)-1beta to express intercellular adhesion molecule-1 (ICAM-1) mRNA in the presence or absence of the physiological spontaneous NO-donor S-nitrosocysteine. Subsequently, semiquantitative RT-PCR and gel shift assays with nuclear extracts were performed to analyse the effects of NO on ICAM-1 mRNA expression and on the activity of transcription factors involved in ICAM-1 transcription. In addition, luciferase reporter gene activity of cytokine-activated cells transiently transfected with an ICAM-1 promoter-luciferase construct and cultured in the presence of the slow-releasing NO-donor DETA/NO was determined. NO at subtoxic concentrations decreases IL-1beta-induced endothelial ICAM-1 mRNA expression. This inhibition occurs at the transcriptional level, as NO affects IL-1b-induced ICAM-1 promoter activity in transiently transfected cells. Using gel-shift assays and double-stranded oligonucleotide consensus sequences of the known transcription factor binding sites of the ICAM-1 promoter, Sp1 and AP-1 were identified as transcriptional activators of IL-1beta-driven ICAM-1 expression. The DNA binding of both of these transcription factors to specific binding sites of the ICAM-1 promoter was decreased in MAEC exposed to NO. Our studies indicate that the anti-adhesive effect of NO concentrations equivalent to high-output NO synthesis is mediated, at least in part, by inhibition of ICAM-1 expression via a concerted action of NO on the redox-sensitive transcriptional activators Sp1 and AP-1. This molecular mechanism may contribute to the anti-inflammatory actions of NO synthesized by the inducible NO synthase.
Highlights
Leukocyte recruitment to sites of inflammation entails a sequence of interactions between leukocytes moving in the bloodstream and the microvasculature lining endothelium
The effect of exogenously added Nitric oxide (NO) on the IL-1 driven intercellular adhesion molecule-1 (ICAM-1) mRNA expression was investigated in primary murine aortic endothelial cells (MAEC)
After activation of Murine aortic endothelial cells (MAEC) with 200 U/ml IL-1, ICAM1 mRNA could be detected by Reverse transcription (RT)-PCR as early as 1 h after addition of IL-1
Summary
Leukocyte recruitment to sites of inflammation entails a sequence of interactions between leukocytes moving in the bloodstream and the microvasculature lining endothelium. Contact between these two cell types is initiated when leukocytes reversibly adhere to the endothelial cells and start rolling along the endothelium [1,2]. Leukocytes are able to emigrate out of the vasculature and to enter the inflammatory site This sequence of events is orchestrated by activated microvascular endothelial cells in the inflamed tissue via expression of a number of molecules with specific functions in capturing and activating leukocytes. Materials and Methods: Primary murine aortic endothelial cells were activated with interleukin (IL)-1 to express intercellular adhesion molecule-1 (ICAM-1) mRNA in the presence or absence of the physiological spontaneous NOdonor S-nitrosocysteine. Luciferase reporter gene activity of cytokine-activated cells transiently transfected with an ICAM-1 promoter-luciferase construct and cultured in the presence of the slow-releasing NOdonor DETA/NO was determined
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