Neutrophil-bead collision assay: pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime

Abstract

Visualization of flowing neutrophils colliding with adherent 1-mum-diameter beads presenting P-selectin allowed the simultaneous measurement of collision efficiency (epsilon), membrane tethering fraction (f), membrane tether growth dynamics, and PSGL-1/P-selectin binding lifetime. For 1391 collisions analyzed over venous wall shear rates from 25 to 200 s(-1), epsilon decreased from 0.17 to 0.004, whereas f increased from 0.15 to 0.70, and the average projected membrane tether length, L(tether)(m), increased from 0.35 mum to approximately 2.0 mum over this shear range. At all shear rates tested, adhesive collisions lacking membrane tethers had average bond lifetimes less than those observed for collisions with tethers. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters (consistent with single bond Monte Carlo simulation) were zero-stress off-rate, k(off)(0) = 0.56 s(-1) and reactive compliance, r = 0.10 nm, similar to published atomic force microscopy (AFM) measurements. For all adhesion events (+/- tethers), the bond lifetime distributions were more similar to those obtained by rolling assay and best simulated by Monte Carlo with the above Bell parameters and an average of 1.48 bonds (n = 1 bond (67%), n = 2 (22%), and n = 3-5 (11%)). For collisions at 100 s(-1), pretreatment of neutrophils with actin depolymerizing agents, latrunculin or cytochalasin D, had no effect on epsilon, but increased L(tether)(m) by 1.74- or 2.65-fold and prolonged the average tether lifetime by 1.41- or 1.65-fold, respectively. Jasplakinolide, an actin polymerizing agent known to cause blebbing, yielded results similar to the depolymerizing agents. Conversely, cholesterol-depletion with methyl-beta-cyclodextrin or formaldehyde fixation had no effect on epsilon, but reduced L(tether)(m) by 66% or 97% and reduced the average tether lifetime by 30% or 42%, respectively. The neutrophil-bead collision assay combines advantages of atomic force microscopy (small contact zone), aggregometry (discrete interactions), micropipette manipulation (tether visualization), and rolling assays (physiologic flow loading). Membrane tether growth can be enhanced or reduced pharmacologically with consequent effects on PSGL-1/P-selectin lifetimes.

FIGURE 1

Images of neutrophils colliding with and forming tethers on P-selectin-coated beads. The length of tether pulled from the neutrophil is measured from the top of the bead to the lagging edge of the neutrophil (A). This method of measurement imposes a minimum tether length equivalent to the radius of the bead (0.5 μm). A montage of a neutrophil flowing over a bead at γ_w = 100 s⁻¹ is given (B), along with the trajectory of the cell (D). A force diagram is constructed to determine the force on the bond, F_B (E), as described in the text. Small stress lines were detected in the neutrophil membrane, suggesting deformation of the neutrophil. Two representative collisions at γ_w = 50 and 75 s⁻¹ are shown (C), with the stress lines indicated with white arrows. Neutrophil treated with 0.3-μM latrunculin assumed a teardrop-shaped formation under flow (F) as compared to the spherical shape assumed by untreated cells (B).

FIGURE 2

Dynamics of neutrophil interaction with P-selectin-coated beads. We calculated the adhesion collision efficiency ɛ (A) and the membrane tethering fraction f (B) at each shear rate. Error bars for calculated parameters are for N = 4–10 donors, except in the case at γ_w = 200 s⁻¹, where N = 2 donors. Projected tether lengths were measured for all neutrophils making adhesive collisions (C). The average lifetimes for all the adhesive and nonadhesive events were calculated (E), and the lifetimes were then binned according to whether they formed discernible membrane tethers (D). Each standard deviation error bar for the measured parameters is for n > 89 interactions, and N = 9 donors, except in the case of γ_w = 200 s⁻¹, where n = 20 interactions.

FIGURE 3

Tether growth rates for neutrophils colliding with P-selectin-coated beads. Membrane tether lengths and lifetimes were used to calculate the membrane tether growth rate for untreated cells at γ_w = 25–200 s⁻¹. Our results for neutrophil collision with beads (solid squares) were compared with prior results for neutrophils tethering onto platelets (open circles; (18)). Error bars are for standard deviation.

FIGURE 4

Kinetics of dissociation for the P-selectin/PSGL-1 bond. The linear fit of the curve through ln(N) versus t, where N is the number of events lasting at least t s, yields a slope of −k_off. Presented are the slopes −k_off for all data (± tethers) at all shear rates (A). Values for k_off obtained in this manner were used to determine the unloaded off-rate k_off(0) and reactive compliance, r, by determining a least-squares fit of an exponential curve through the plot of k_off versus F_B for all data (± tethers) (B) and interactions without membrane tether formation (C). Because the data at γ_w = 200 s⁻¹ was sparse and appeared to be an outlier when compared with a global Monte Carlo simulation of collisions at all shear rates (MC in B and C), we regressed the plot including (SPE 2) or neglecting (SPE 1) k_off at 200 s⁻¹.

FIGURE 5

Bell model parameters for the PSGL-1/P-selectin bond. The Bell model parameters for the PSGL-1/P-selectin bond were compared against several previous studies (–7,9,10,14,33) by fitting each with the Bell model and graphing k_off versus the force on the bond, F_B. Data of Heinrich et al. (Fig. 7 in (14)) was plotted (top panel) for k_off = (attachment lifetime)⁻¹ as a function of F_bond where F_bond was set equal to f_∞ (the nearly constant rupture force) calculated from the applied pulling rate (ν_pull in μm/s) by f_∞ = 60 pN(ν_pull)^0.25 that was used to generate the initial loading rate (r_f in pN/s, a value that rapidly plummets), thereby allowing a determination (bottom panel) of Bell parameters (R² = 0.98) for 71.4 < f_∞ < 210 pN. (Nomenclature: d/d, dimeric PSGL-1/dimeric P-selectin interaction; m/m, monomeric PSGL-1/monomeric P-selectin interaction; dPSGL1, dimeric PSGL-1; dP-selectin, dimeric P-selectin; SPE, statistical point estimate; and MC, Monte Carlo simulation.)

FIGURE 6

Adhesion dynamics for chemically treated neutrophils. The average primary adhesion collision efficiencies ɛ (A) and the membrane tethering fractions f (B) between treated neutrophils and selectin-coated beads were calculated over N donors. Membrane tether lengths were measured and averaged for all neutrophils making adhesive collisions (C), and the average lifetimes for all the adhesive and nonadhesive events were also calculated (E). The lifetimes were then binned according to whether discernible membrane tethers were formed (D). Error bars refer to standard deviations for N donors or n collisions.

FIGURE 7

Tether growth rates for neutrophils under several conditions. Tether growth rates were calculated for neutrophils treated with membrane or cytoskeleton altering reagents, and perfused over selectin-coated beads at γ_w = 100 s⁻¹. Tether growth rates were calculated based on averages over all adhesive events (solid bars in A), and also specifically for membrane-tether-forming events (open bars in A). These results were compared versus untreated data at the same shear rate (A). Stars represent data for which the treated data set was significantly different (p < 0.05) than control data. Results returned from a two-tailed Student's t-test are shown for all data that differed significantly from control. Averages are for n > 60 collisions and N ≥3 donors, and error bars given are for standard deviation. Tether growth rates for treated neutrophils (v_tether) were then normalized by the tether growth rate of untreated cells at the same wall shear rate (v₀). These were then compared to normalized adhesion lifetimes (t/t₀) for all adhesive events (B).

FIGURE A1

Dependence on number of bonds. The number of initial bonds present affects the simulation because of the division of forces between them. The distribution weighting factor (d) describes how the initial number of bonds were allocated according to the exponential probability of exp(−dn) for n = 1–5. These distribution weighting factors indicate the best fit at d = 1.1 (solid line, left scale) with a mean number of bonds of 1.48 (dashed line, right scale). The Bell parameters used were and r = 0.10 nm. Two-thousand simulations were averaged at each data point shown.