Figure 9. Deposition of membrane tethers on P-selectin–coated surfaces. Tethers could be deposited on the surface (a) and dis- play a beadlike appearance (b). Neutrophil string formation on an ultralong-deposited and beaded neutrophil tether (c). tether that was greater than four fields of view (.100 mm), illustrates that these tethers could grow to be extremely long, and demonstrated the tremendous excess of neutro- phil membrane that can supply tether growth. Finally, it was observed that free-flowing neutrophils interacted and rolled on these deposited and beaded tethers, possibly through L-selectin/PSGL-1 interactions (Fig. 9 c). Discussion The recruitment of neutrophils to sites of inflamed endo- thelium or to platelets localized on the vessel wall during thrombosis is viewed as a multistep adhesion process that involves initial cell contact, rolling, firm adhesion, and transmigration. The initial contact made by the neutrophil is most likely to occur at the tip of the microvilli that pro- trude from its surface. Previous studies have shown that, unlike most other cell-surface molecules that are uniformly distributed, the molecules responsible for mediating initial neutrophil adhesion with other cells (i.e., L-selectin and PSGL-1) are localized at the tips of the neutrophil mi- crovilli. It appears that localization of L-selectin and PSGL-1 at the microvilli tips, facilitates neutrophil tether- ing interactions by increasing their concentration and mak- ing them more accessible to their counter ligands. There- fore, a key question regarding the initial adhesion of neutrophils via these bonds is what happens when a force is imposed on them. Does the receptor–ligand bond break? Is either the ligand or receptor extracted from the cell mem- brane? Or does the cell membrane deform or break as the bond is loaded to reduce the force on the bond? In this study, we investigated the initial interactions be- tween neutrophils and platelets and report the first direct observation of the formation of elongated membrane teth- ers by neutrophils under physiological flow conditions. Elongated tethers were pulled from the neutrophil surface when free-flowing neutrophils were captured by spread platelets and when neutrophils rolled on P-selectin–coated surfaces. Tethering was completely blocked with mAbs against P-selectin and PSGL-1, whereas the mAb against CD18 had no effect. These observations extend the follow- ing recent measurements of: tether growth between neu- trophils and beads coated with antibody using micro- pipette aspiration (Shao et al., 1998); moment arms of attached neutrophils in reversing flow (Alon et al., 1997); and neutrophil recoil in rapidly decelerated flow (Chen and Springer, 1999). Direct observation of the formation of these tethers under physiological flow was made achiev- able by using high speed, high resolution microscopy, a technique that makes it possible to directly observe cellu- lar events with characteristic times as short as z2 ms and length scales of z100 nm. In agreement with previous studies, the molecular bond responsible for the initial attachment and tethering of flowing neutrophils to platelets was the P-selectin/PSGL-1 interaction. This fact was demonstrated by the tethering being completely abolished after preincubation of plate- lets with anti–P-selectin or neutrophils with anti–PSGL-1, whereas incubation of neutrophils with anti-CD18 had no effect (Fig. 5). Similarly, preincubation of neutrophils with the metabolic inhibitor sodium azide, to prevent neu- trophil activation during neutrophil–platelet interactions, also did not prevent tether formation, demonstrating that cell activation was not required. These observations, in conjunction with the tethers displaying lifetimes and strengths that are the signature of the P-selectin/PSGL-1 bond (Figs. 3 b and 4 d) and the reproduction of tethering in a purified system with P-selectin–coated surfaces, pro- vide confirmation of the molecular mechanism of tether attachment. Since cell–cell attachment depends on the balance be- tween the dispersive hydrodynamic forces and the adhe- sive forces generated by the interactions between mem- brane bound receptors and their ligands, the effect of shear on tether formation was investigated. Both the tether growth rate and the lifetime of the tether were func- tions of the wall shear rate, while the average tether length of z6 mm appeared as a plateau between 100 s21 , gw , 250 s21. Transient membrane tethers were not observed at shear rates #50 or $300 s21. During neutrophil tethering to adherent platelets, shear forces generated at a shear rate of 50 s21 appeared insufficient to pull membrane teth- ers fast enough or long enough to be observed in our sys- tem, which could only resolve membrane tethers that were pulled beyond the cell’s diameter (as opposed to microvilli extension). At a shear rate of 300 s21, the frequency of neutrophil capture was extremely low, and the force load- ing of the tether likely caused bond breakage before teth- ers could grow to an observable length beyond the cell’s diameter. The lack of observable membrane tethering below 50 s21 or above 300 s21 in our system is consistent with observa- tions of Shao et al. (1998), Alon et al. (1997), and Chen and Springer (1999). Shao et al. (1998) estimated mi- crovilli lengths between 0.35 and 1.0 mm at a shear rate of 22 s21 (0.22 dyne/cm2) since, at forces ,45 pN, microvilli become extended but do not form tethers. Alon et al. (1997) detected lever arms between 2 and 4 mm corre- sponding to a tether length of 1.0 6 0.32 mm at revers- ing flows of 0.3–0.8 dyne/cm2 (gw z 30–80 s21) during neutrophil rolling on P-selectin. For neutrophil rolling on E-selectin at a high wall shear stress of 16 dyne/cm2 Schmidtke and Diamond Neutrophil Tethering under Flow 727