The objective of the present study was to determine whether prolonged inhibition of nitric oxide synthesis in endothelial cells increased the surface adhesion of these cells for neutrophils. Human umbilical vein endothelial cells (HUVECs) were grown to confluence in 48-well microtiter plates. Exposure of HUVECs to the nitric oxide synthesis inhibitor N0-nitro-L-arginine methyl ester (L-NAME) did not cause neutrophil adhesion at 1 hour but increased adhesion at 4 hours in a dose-dependent manner. The increased adhesion was prevented with L-arginine or nitric oxide donors but not an analogue of cGMP. The increased adhesion was inhibited by monoclonal antibodies directed against the f32-integrin CD18 and endothelial cell adhesion molecule ICAM-1. Plateletactivating factor (PAF) receptor antagonist WEB 2086 also prevented the L-NAME-induced neutrophil adhesion. Intracellular oxygen radical scavengers (dimethyl sulfoxide, butylated hydroxytoluene, and a,a'-dipyridyl), the iron chelator desferrioxamine, and the mitochondrial inhibitor azide inhibited the L-NAME-induced neutrophil adhesion, whereas extracellular oxygen radical scavengers (superoxide dismutase and catalase) had no effect. HUVECs were loaded with 2',7'-dichlorodihydrofluorescein diacetate, and oxidation to the fluorescent dichlorodihydrofluorescein (DCHF) was monitored. Fluorescence was enhanced in the L-NAME-treated HUVECs throughout the 4-hour incubation, an event inhibitable by an antioxidant and azide. The magnitude of the intracellular oxidation of DCHF was equivalent to -0.8 ,umol/L H202. These data suggest that prolonged nitric oxide synthesis inhibition in HUVECs causes an oxidant-and PAFassociated rise in adhesion on the surface of these endothelial cells for neutrophils. (Circ Res. 1994;74:1133-1140 Key Words * integrin * neutrophil * N0-nitro-L-arginine methyl ester E ndothelium-derived relaxing factor (nitric oxide [NO]) is synthesized by vascular endothelial cells from the terminal guanidino nitrogen atoms of L-arginine.' This biosynthetic process is inhibited by analogues of L-arginine such as NG-nitro-L-arginine methyl ester (L-NAME) and N0-monomethyl-L-arginine (L-NMMA) as described by Rees et or reperfused vessels to agonist-mediated NO production but not exogenous NO donors.5-8A hallmark feature of hypercholesterolemia, hypertension, and ischemia/reperfusion is the adhesion of leukocytes as well as platelets to vascular endotheli-UM.6,9-12 It remains unclear whether the early alteration in endothelial NO production is a cause of subsequent leukocyte adhesion in these vascular pathologies; however, circumstantial evidence would support this notion. Inhibition of NO synthesis results in increased leukocyte adherence in postcapillary venules, a response that could be prevented by adding exogenous NO13 or high concentrations of L-arginine.4 Moreover, impairment of basal NO production in postischemic vessels correlated temporally with increased leukocyte adhesion, and the latter was reduced by administering exogenous NO.14 On the ...

Recent work has demonstrated that inhibition of nitric oxide production with various nitric oxide synthesis inhibitors (L-NAME, L-NMMA) initiate leukocyte adhesion to postcapillary venules. The objective of this study was to elucidate the mechanism (or mechanisms) that promote the L-NAME-induced leukocyte response. Intravital microscopy was used to examine 25-40 microns venules in the rat mesentery. Nitric oxide synthesis was inhibited with L-NAME and leukocyte adhesion was observed over the first 60 min. The fourfold increase in leukocyte adhesion was independent of alterations in venular red blood cell velocity. The adhesion was superoxide-mediated inasmuch as superoxide dismutase (SOD) abolished the rise in leukocyte adhesion associated with nitric oxide synthesis inhibition. Ketotifen, a mast cell stabilizer, also abolished the rise in leukocyte adhesion induced by L-NAME. Histology revealed that mast cell degranulation occurred only in animals treated with L-NAME but not in animals pretreated with SOD or ketotifen. This observation suggests that mast cells become activated in the absence of nitric oxide production and superoxide contributes to the mast cell activation. The L-NAME-induced leukocyte adhesion could be reproduced by infusing hypoxanthine/xanthine oxidase (a superoxide generating system) or compound 48/80 (an activator of mast cells) and both responses were attenuated by ketotifen. These data suggest that inhibition of nitric oxide synthesis results in a superoxide and mast cell-dependent leukocyte adhesion.

It is generally accepted that the beta 2-integrin is restricted to mononuclear leukocytes. The objective of this study was to determine whether neutrophils can also express beta 1-integrin (specifically alpha 4 beta 1) and whether this can support neutrophil adhesion to endothelial cells and to extracellular matrix. We stimulated neutrophils with dihydrocytochalasin B (DHCB) and various chemotactic stimuli and observed that chemotactic stimuli induced neutrophil adhesion via beta 2-integrin (CD18), whereas DHCB and either fMLP, PAF, or IL-8 induced adhesion to endothelium or protein-coated plastic that was not inhibitable by anti-CD18 antibody. beta 2-integrin-deficient cells, which did not respond to chemotactic stimuli alone, also adhered avidly in the presence of chemotactic stimuli and DHCB. The induced neutrophil adhesion was inhibited by antibody to beta 1- or alpha 4-integrin chains, but only if an anti-beta 2-integrin antibody was also present. Flow cytometry revealed increased expression of both beta 1 and alpha 4 in the presence of fMLP plus DHCB. Transendothelial migration of neutrophils induced by chemotactic stimuli alone also increased expression of beta 1 and alpha 4. Transmigration across deendothelialized membranes induced a similar beta 1 expression on neutrophils suggesting that events other than an endothelial signal elicited beta 1-integrin expression. Transmigration-induced beta 1-dependent expression translated into only modest adhesion to protein-coated plastic. These data suggest that both a pharmacological (DHCB) and a physiological (transmigration) stimulus can invoke expression of alpha 4 and beta 1 on human neutrophils to mediate adhesion.

Taken together, these results suggest that exogenous NO can modulate leukocyte recruitment and microvascular permeability alterations elicited by mast cell activation and raises the possibility that the use of NO donors may be a reasonable therapeutic approach to reducing mast cell-dependent inflammation.

Nitric oxide (NO) synthesis inhibition causes neutrophil adhesion to endothelium via a mast cell– and oxidant-dependent mechanism. The objective of this study was to delineate the cascade of events in the mast cell– and oxidant-induced neutrophil-endothelium interactions after NO synthesis inhibition. Mast cells were isolated and purified from the rat peritoneal cavity and coadministered with neutrophils to wells of endothelium. This system was treated with an NO synthesis inhibitor ( N G -nitro- l -arginine methyl ester; L-NAME) for 60 minutes. L-NAME did not induce neutrophil-endothelium interactions in the absence of mast cells, but the addition of mast cells in a ratio as low as 1:50 mast cells to neutrophils was sufficient to induce a large increase in neutrophil adhesion to endothelium within 20 to 25 minutes. l -arginine, NO donors, and 8-bromo-cGMP reversed the L-NAME effect, whereas N G -nitro- d -arginine methyl ester alone had no proadhesive effect. The adhesion was inhibited by an anti-CD18 or an anti–intracellular adhesion molecule-1 antibody and a platelet-activating factor-receptor antagonist. Inhibition of NO in isolated endothelial monolayers induced oxidant release (reduction of cytochrome C) into extracellular fluid. The endothelium-derived superoxide contributed to the mast cell–induced adhesion, inasmuch as the extracellular antioxidant superoxide dismutase reduced the neutrophil adhesion response as did disruption of endothelial function. There was some direct activation of mast cells with L-NAME (independent of endothelium) inasmuch as intracellular calcium and oxidative stress increased within mast cells after L-NAME treatment, and this translated into increased neutrophil adhesion to nonendothelial substrata. These data demonstrate that depletion of NO increases oxidative stress within mast cells and endothelium and together these events promote neutrophil adhesion within the vasculature.