Inhibition of nitric oxide synthase (NOS) conversion of L-arginine to nitric oxide (NO) decreases low density mononuclear cell (LD MNC) trans-endothelial migration and cytokine output.

BACKGROUND: Biochemical, molecular, and cellular events at the micro-vascular endothelial interface determine the integrity of the vascular system. Disruption of these events has been described to occur in accordance with ischemic/reperfusion injury leading to inflammation, cell adhesion, and endothelial permeability changes. It has also been suggested that nitric oxide (NO) participates in these events. However, the manner in which it does is debated. The purpose of this study was to investigate the effects of exogenous L-arginine, an NO precursor, and L-N (G) nitroarginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, upon inflammatory events at the endothelial interface.
MATERIAL AND METHODS: Fresh cultures of human umbilical vein endothelial cells were established and used to seed Transwell chemotaxic chambers, and then grown to confluence. Whole blood was obtained from the same healthy volunteer and processed for light density mononuclear cells. Following per-stimulation of the endothelial monolayer with IL-1beta or antigen-antibody complex, known numbers of mononuclear cells were seeded to the endothelium. Incubation with and without exogenous L-arginine or L-NAME for 48 h was done. Lower chamber supernatant was then collected, cell numbers and viability determined and levels of inflammatory cytokines TNF-alpha and INF-lambda determined via ELISA assay.
RESULTS: Tran-endothelial cellular migration was nil lacking pre-stimulation, regardless of the addition of exogenous L-arginine. With pre-stimulation trans-endothelial migration increased significantly, a response that was greatly enhanced by L-arginine. With the further addition of L-NAME cellular migration decreased substantially. Pro-inflammatory cytokine levels of TNF-alpha and INF-lambda followed levels of cellular migration.
CONCLUSIONS: In vitro there was little to no trans-endothelial migration of inflammatory cells across an unstimulated monolayer of vascular endothelium. Pre-stimulation of the same endothelial monolayer with either a cytokine or antigen-antibody complex resulted in a significant trans-endothelial migration of inflammatory cells. This latter response was associated with a concurrent increase in the secretion of the pro-inflammatory cytokines TNF-alpha and INF-gamma. The presence of the NO precursor L-arginine greatly enhanced the observed inflammatory response. Conversely, L-NAME, an inhibitor of NOS, depressed the inflammatory response.