1995; Smith et al., 1999). Plasmon resonance measure- ments of the affinity and kinetics of the bond between soluble monomeric human P-selectin and immobilized PSGL-1 from human neutrophils yielded values of kon 0 5 4.4 3 106 M21 s21, ko0ff 5 1.4 s21, and Kd 5 320 nM (Mehta et al., 1998). In contrast, atomic force microscopy mea- surements of single complexes between a PSGL-1/IgG chi- mera immobilized on the tip of an atomic force micros- copy cantilever with a surface coated with P-selectin/IgG chimera (Fritz et al., 1998) displayed higher avidity (Kd 5 60 nM) than the native P-selectin/PSGL-1 bond (Kd 5 320 nM), and resulted in off rates from 0.02 s21 under zero force up to 15 s21 under applied forces. Proposed mechanisms of neutrophil detachment during rolling and adhesion under fluid shear forces involve the following: (1) the dissociation of the receptor bond (Alon et al., 1995); (2) the extraction of membrane receptors (Evans et al., 1991; Shao and Hochmuth, 1999); and/or (3) the failure of the membrane. Of these mechanisms, force- induced bond dissociation is the favored model of cell de- tachment. Microvilli rheology may also have a role during neutrophil rolling (Shao et al., 1998; Chen and Springer, 1999). Shao and Hochmuth (1996) using micropipette ma- nipulation and antibody-coated beads demonstrated that microvilli on neutrophils, under a pulling force, can be ex- tended (microvillus extension, 1 mm) or can form long thin membrane cylinders (tether formation, up to 20 mm) de- pending on the magnitude of the force (Shao et al., 1998). Although these measurements were made under nonphys- iological conditions, it is predicted that tether formation could also occur as a result of a bond between PSGL-1 on the neutrophil microvilli and platelet (or endothelial) P-selectin under physiological hemodynamic conditions. Similarly, recent work by Chen and Springer (1999) sup- ports the role for microvilli extension during rolling adhe- sion by demonstrating neutrophil recoil upon sudden flow deceleration, while Alon et al. (1997) detected a moment arm upon flow reversal; both observations are suggestive of a tether structure. In this study, we investigated the initial adhesive inter- actions between neutrophils and platelets using high speed, high resolution differential interference contrast (DIC) videomicroscopy. Under physiological flow condi- tions, we found that when flowing neutrophils collided with immobilized platelets or rolled on P-selectin–coated surfaces, membrane tethers were pulled from the neutro- phil surface. These results are the first reported direct vi- sualization of such tethers forming under venous flow conditions, and may have significant implications in the understanding of bond loading and neutrophil rolling dy- namics. Materials and Methods Antibodies and Reagents Human P-selectin, purified from outdated platelet lysates (Lawrence et al., 1997), was provided by Dr. Michael B. Lawrence (University of Vir- ginia, Charlottesville, VA). Function-blocking anti–PSGL-1 mAb PL1, anti-CD18 mAb IB4, and anti–P-selectin mAb G1 were purchased from Ancell, whereas human serum albumin (HSA) was obtained from Golden West Biologicals. Neutrophil Isolation Venous human blood for neutrophil isolation was obtained from healthy donors by venipuncture and anticoagulated with EDTA (nine parts blood to one part K3EDTA). Neutrophils were isolated by centrifugation over neutrophil isolation medium (Cardinal Associates). After isolation, neu- trophils were resuspended in either a Mg21-free Hepes-buffered Tyrode’s solution, pH 7.35, containing BSA (3.5 mg/ml), 137 mM NaCl, 2.7 mM KCl, 3.8 mM NaH2PO4, 3.8 mM Hepes, 2 mM CaCl2, and 5.5 mM glucose or in Mg21-free Hank’s balanced salt solution (HBSS; Gibco Laborato- ries) supplemented with 2% HSA and 2 mM CaCl2, pH 7.4. Immediately after suspension in buffer, cells were counted electronically by a Coulter counter (model Z2) and used in the flow experiments at 105 cells/ml. All subsequent procedures were performed at room temperature. Microcapillary Flow Chambers Flow chambers were constructed with rectangular glass capillaries (micro- capillary; VitroCom) with a cross-section of 0.2 3 2.0 mm and a wall thick- ness of 0.15 mm. Microcapillaries were cleaned by overnight incubation in 20% nitric acid, followed by extensive washing with deionized water. The microcapillaries were rinsed with 100% dry ethanol and dried under ar- gon. The ends of the clean microcapillary (length 5 7 cm) were inserted into silastic tubing (I.D. 1.47 mm 3 OD 1.96 mm) and a hermetic seal be- tween the glass capillary and the silastic tubing was established by cover- ing the joint with adhesive lined, heat shrinkable tubing (3M Electronics). Fibrinogen and P-selectin–coated Surfaces To promote platelet adhesion, the clean microcapillary flow chambers were incubated with a human fibrinogen solution (100 mg/ml; Enzyme Re- search Labs) for 1 h at room temperature. After incubation, each flow chamber was washed of excess fibrinogen solution by perfusing Ca21- and Mg21-free HBSS containing HSA for at least 20 min. Human platelets were sparsely adsorbed on the fibrinogen before binding studies with neu- trophils. In some experiments, monolayers of platelets were prepared by perfusing washed platelets into the chamber and allowing them to settle for at least 30 min. P-selectin–coated surfaces were prepared by incubat- ing clean microcapillary flow chambers with affinity-purified human plate- let P-selectin (z1 mg/ml final concentration) in Ca21- and Mg21-free HBSS for at least 3 h at room temperature. The flow chamber was washed by perfusing Ca21- and Mg21-free HBSS containing 2% HSA through the chamber for at least 30 min, to remove any excess or unbound protein. For site density determinations, clean microcapillary chambers were incu- bated with the dilute P-selectin solution for 3 h, followed by washing/ blocking with the 2% HSA/HBSS for 30 min. The glass microcapillaries were incubated with a solution of 125I-labeled anti–P-selectin antibody clone G1 (3.95 3 106 cpm/mg; Ancell) and diluted in 1% BSA/PBS. After antibody incubation, the tubes were washed with PBS, crushed, and counted in triplicate with a gamma counter. Cell Treatments For blocking P-selectin on the surface of platelets, flow chambers coated with platelets were filled with mAb G1 at 20 mg/ml and incubated at room temperature for 20 min before perfusion of neutrophils. To block bonding via PSGL-1 or b2-integrins, neutrophils were incubated with either 20 mg/ ml mAb PL1 (anti–PSGL-1) or 20 mg/ml mAb IB4 (anti-CD18) in 2% HSA/HBSS medium containing 2 mM Ca21 for 30 min before perfusion. The neutrophil suspension was diluted in a 3–10-fold volume of HSA/ HBSS medium and immediately perfused into the flow chamber. Energy- depleted neutrophils were prepared by incubating isolated neutrophils with 30 mM sodium azide for 30 min at room temperature before perfu- sion. High Resolution, High Speed Imaging under Flow The flow chamber was mounted on a Zeiss Axiovert 135 microscope. DIC microscopy was conducted with a 633 (NA 1.40) oil immersion objective lens (Plan-Apochromate) and an oil immersion condenser (NA 1.4). Dur- ing perfusion, neutrophils were withdrawn from a reservoir through the flow chamber at controlled flow rates by a syringe pump (Harvard Appa- ratus). The wall shear stress imposed on the surface was calculated from the equation tw 5 (6Qm)/(B2W), which is the solution of the Navier- Stokes equation for laminar flow of a Newtonian liquid between parallel plates, where tw represents the wall shear stress (dynes/cm2), Q represents The Journal of Cell Biology, Volume 149, 2000 720