I-domain of lymphocyte function-associated antigen-1 mediates rolling of polystyrene particles on ICAM-1 under flow

Abstract

In their active state, beta(2)-integrins, such as LFA-1, mediate the firm arrest of leukocytes by binding intercellular adhesion molecules (ICAMs) expressed on endothelium. Although the primary function of LFA-1 is assumed to be the ability to mediate firm adhesion, recent work has shown that LFA-1 can contribute to cell tethering and rolling under hydrodynamic flow, a role previously largely attributed to the selectins. The inserted (I) domain of LFA-1 has recently been crystallized in the wild-type (wt) and locked-open conformations and has been shown to, respectively, support rolling and firm adhesion under flow when expressed in alpha(L)beta(2) heterodimers or as isolated domains on cells. Here, we report results from cell-free adhesion assays where wt I-domain-coated polystyrene particles were allowed to interact with ICAM-1-coated surfaces in shear flow. We show that wt I-domain can independently mediate the capture of particles from flow and support their rolling on ICAM-1 surfaces in a manner similar to how carbohydrate-selectin interactions mediate rolling. Adhesion is specific and blocked by appropriate antibodies. We also show that the rolling velocity of I-domain-coated particles depends on the wall shear stress in flow chamber, I-domain site density on microsphere surfaces, and ICAM-1 site density on substrate surfaces. Furthermore, we show that rolling is less sensitive to wall shear stress and ICAM-1 substrate density at high density of I-domain on the microsphere surface. Computer simulations using adhesive dynamics can recreate bead rolling dynamics and show that the mechanochemical properties of ICAM-1-I-domain interactions are similar to those of carbohydrate-selectin interactions. Understanding the biophysics of adhesion mediated by the I-domain of LFA-1 can elucidate the complex roles this integrin plays in leukocyte adhesion in inflammation.

FIGURE 1

Fluorescence histogram for SuperAvidin microspheres (9.95 μm) incubated with 5 μg/mL solutions containing 0% (control), 30%, 50%, 70%, and 100% I-domain. Primary antibody: TS2/6 (1:10 dilution). Secondary antibody: FITC-labeled anti-mouse IgG (25 μg/mL).

FIGURE 2

Rolling and binding flux of beads coated with 1730 (100%) and 824 (70%) sites of I-domain, 2800 sites/μm² of anti-ICAM-1 (100%), and 1321 sites/μm² of sLe^X (100%) on 210 sites/μm² ICAM-1 surfaces (n ≥ 4) at 130 s⁻¹. Beads were suspended in PBS⁺ (1% BSA, 1 mM Ca²⁺, 2 mM Mg²⁺). The experiment with EDTA was with PBS⁺ w/o ions. Field of view = 0.32 mm², time = 1 min, and TS2/6 concentration = ∼20 μg/mL. *Baseline flux = 264 beads/mm²×min.

FIGURE 3

Rolling velocity as a function of wall shear rate for I-domain microspheres on surfaces coated with (A) 1.25, (B) 2.5, and (C) 5.0 μg/mL of ICAM-1. Beads were suspended in PBS⁺ (1% BSA, 1 mM Ca²⁺, 2 mM Mg²⁺). Error bars = standard error (n ≥ 4); ^#no significant increase from previous shear rate (P > 0.05). (Inset) (A) Rolling velocity of I-domain microspheres at 70 s⁻¹ and 180 s⁻¹ shear rates (*P < 0.0001, ^#P > 0.05).

FIGURE 4

Contour plots showing the effect of (A) I-domain site density (at 1.25 μg/mL ICAM-1 coating) and (B) ICAM-1 substrate coating concentration (at 824 sites/μm² of I-domain) on microsphere rolling velocity sensitivity to wall shear stress in flow chamber. (Legend) Average microsphere rolling velocity range.

FIGURE 5

Comparison of instantaneous velocity traces of I-domain-coated SuperAvidin microspheres. Top panel compares traces for microspheres coated with 100% I-domain interacting with ICAM-1 surfaces (2.5 μg/mL coating) at shear rates of (A) 70 s⁻¹ and (B) 210 s⁻¹. Middle panel compares traces of microspheres coated with (C) 100% and (D) 70% I-domain interacting with ICAM-1 surfaces (2.5 μg/mL coating) at a shear rate of 180 s⁻¹. Bottom panel compares traces of microspheres coated with 100% I-domain interacting on surfaces coated with (E) 5 μg/ml and (F) 1.25 μg/ml ICAM-1 at 130 s⁻¹.

FIGURE 6

Average RMS velocities of microspheres (n = 3) (A) as a function of shear rate for particles with different I-domain site density interacting with surfaces coated with 2.5 μg/mL ICAM-1, (B) as a function of ICAM-1 substrate coating concentration for 100% I-domain particles (∼1730 sites/μm) at 130 s⁻¹, and (C) as a function of average particle rolling velocity for all sites of I-domain and ICAM-1 densities tested at different wall shear stress. Error bar represents standard error calculations.

FIGURE 7

Simulated and experimental rolling velocity data for I-domain microspheres on (A) 210, (B) 122, and (C) 67 sites/μm² ICAM-1 surfaces. γ = 0.1 Å; ; and Error = standard deviation (n ≥ 7).

FIGURE 8

Effect of I-domain site density and shear stress on simulated bead instantaneous velocity distribution. Instantaneous velocity traces of simulated microspheres rolling on 122 sites/μm² ICAM-1 surfaces. Simulated microspheres with 252 sites/μm² I-domain interacting ICAM-1 at a shear rate of (A) 70 s⁻¹ and (B) 180 s⁻¹. Panel C depicts a simulated trace for microspheres with 94 sites/μm² I-domain at 180 s⁻¹ shear rate. γ = 0.1 Å;