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Firm adhesion of rolling neutrophils on inflamed endothelium is dependent on beta(2) (CD18)-integrins and activating stimuli. LFA-1 (CD11a/CD18) appears to be more important than Mac-1 (CD11b/CD18) in neutrophil emigration at inflammatory sites, but little is known of the relative binding characteristics of these two integrins under conditions thought to regulate firm adhesion. The present study examined the effect of chemoattractants on the kinetics of LFA-1 and Mac-1 adhesion in human neutrophils. We found that subnanomolar concentrations of interleukin-8, Gro-alpha, and leukotriene B(4) (LTB(4)) induced rapid and optimal rates of LFA-1-dependent adhesion of neutrophils to intercellular adhesion molecule (ICAM)-1-coated beads. These optimal rates of LFA-1 adhesion were transient and decayed within 1 min after chemoattractant stimulation. Mac-1 adhesion was equally rapid initially but continued to rise for >/=6 min after stimulation. A fourfold higher density of ICAM-1 on beads markedly increased the rate of binding to LFA-1 but did not change the early and narrow time window for the optimal rate of adhesion. Using well-characterized monoclonal antibodies, we showed that activation of LFA-1 and Mac-1 by Gro-alpha was completely blocked by anti-CXC chemokine receptor R2, but activation of these integrins by interleukin-8 was most effectively blocked by anti-CXC chemokine receptor R1. The topographical distribution of beads also reflected significant differences between LFA-1 and Mac-1. Beads bound to Mac-1 translocated to the cell uropod within 4 min, but beads bound to LFA-1 remained bound to the lamellipodial regions at the same time. These kinetic and topographical differences may indicate distinct functional contributions of LFA-1 and Mac-1 on neutrophils.

This study demonstrated that exposure of cultured human aortic smooth muscle cells (SMC) to fluid flow resulted in nitric oxide (NO) production, monitored by nitrite and guanosine 3',5'-cyclic monophosphate production. A rapid burst in nitrite production rate was followed by a more gradual increase throughout the period of flow exposure. Neither the initial burst nor the prolonged nitrite production was dependent on the level of shear stress in the range of 1.1-25 dyn/cm2. Repeated exposure to shear stress after a 30-min static period restimulated nitrite production similar to the initial burst. Ca(2+)-calmodulin antagonists blocked the initial burst in nitrite release. An inhibitor of nitric oxide synthase (NOS) blocked nitrite production, indicating that changes in nitrite reflect NO production. Treatment with dexamethasone or cycloheximide had no effect on nitrite production. Monoclonal antibodies directed against the inducible and endothelial NOS isoforms showed no immunoreactivity on Western blots, whereas monoclonal antibodies directed against the neuronal NOS gave specific products. These findings suggest that human aortic SMC express a constitutive neuronal NOS isoform, the enzymatic activity of which is modulated by flow.