Mechanisms for flow-enhanced cell adhesion

Abstract

Cell adhesion is mediated by specific receptor-ligand bonds. In several biological systems, increasing flow has been observed to enhance cell adhesion despite the increasing dislodging fluid shear forces. Flow-enhanced cell adhesion includes several aspects: flow augments the initial tethering of flowing cells to a stationary surface, slows the velocity and increases the regularity of rolling cells, and increases the number of rollingly adherent cells. Mechanisms for this intriguing phenomenon may include transport-dependent acceleration of bond formation and force-dependent deceleration of bond dissociation. The former includes three distinct transport modes: sliding of cell bottom on the surface, Brownian motion of the cell, and rotational diffusion of the interacting molecules. The latter involves a recently demonstrated counterintuitive behavior called catch bonds where force prolongs rather than shortens the lifetimes of receptor-ligand bonds. In this article, we summarize our recently published data that used dimensional analysis and mutational analysis to elucidate the above mechanisms for flow-enhanced leukocyte adhesion mediated by L-selectin-ligand interactions.

FIGURE 1

Increasing wall shear stress in a flow chamber initially enhances L-selectin-mediated neutrophil tethering (a) and rolling adhesion (b) to a surface coated with P-selectin glycoprotein ligand 1 (PSGL-1) and reduces the neutrophil rolling velocity on PSGL-1 (c). After reaching an optimal level (∼1 dyn/cm²), further increase in wall shear stress decreases neutrophil tethering (a) and rolling adhesion (b) and increases the neutrophil rolling velocity (c). A suspension of 10⁶ cells/mL was used in these experiments. Tether rate in (a) was measured at each wall shear stress by counting the number of flowing cells that initially tethered to a chamber floor coated with <10 PSGL-1 molecules/μm² and dividing this number by the total number of cells flowing through the microscope field of view of in one minute. Neutrophil accumulation in (b) was measured at each wall shear stress as the number of cells per microscope field of view rolling on chamber floor coated with 140 PSGL-1 molecules/μm². Rolling velocity in (c) was measured from cells in (b) rolling for 1 s. Data are presented as mean ± s.e.m. of four or five independent experiments.

FIGURE 2

Parameters of cell tethering under flow. (a) The fluid velocity v of a Couette flow field is parallel to the surface and increases linearly with the distance from the surface (z direction). The shear rate $\stackrel{.}{\gamma }=\mathrm{d}v∕\mathrm{d}z$. The sphere bottom has a positive velocity V_s ≡ V - rΩ where V is the translational velocity of the sphere center, Ω is the angular velocity of the sphere, and r is the sphere radius. The sphere and the surface are respectively coated with receptors and ligands whose combined length l_m sets a contact threshold. When the gap distance l_i between the sphere bottom and the surface is less than l_m, the two are in contact with an area A_i = 2πr(l_m - l_i). (b) The sphere is susceptible to thermal excitations that cause Brownian motion. This produces fluctuations in sphere z-position, which are depicted by the wavy trajectory of the sphere shown in five different times and positions. Brownian motion randomly modulates the gap distance above and below the threshold, which causes discontinuous contacts between different portions of the sphere and different portions of the surface with alternating intervals of contact (t_i) and noncontact (t_j). A productive contact results in a tethering event, but many contacts are nonproductive. As schematically shown for one receptor and one ligand by the movements along the two-sided arrows (depicted by lighter colors), the binding sites of L-selectin and PSGL-1 can undergo rotational diffusion about anchor points such as interdomain hinges even though portions of the molecules are anchored to the respective sphere surface and chamber floor. To ensure that only first-time tethering events were observed, the chamber floor upstream to the microscope field of view was coated with HSA to allow measurement of the distance traveled by the sphere from the demarcation line to the location where tethering occurs. The cell, contact area and molecular sizes are not drawn to scale. Reproduced from Yago et al.

FIGURE 3

Adhesion probabilities per distance, p_ad, of L-selectin bearing microspheres (a—d) and neutrophils (e and f) were calculated from the tether rate data from Eq. (2), normalized by dividing by m_rm_lr, and plotted vs. wall shear rate $\stackrel{.}{\gamma }$ (a and e), wall shear stress $\sigma =\mu \stackrel{.}{\gamma }$ (b and f), product $r\stackrel{.}{\gamma }$ (c and e), and maximum tether force [F_t]_max = 13.2r² σ or $r\stackrel{.}{\gamma }∕D_{\mathrm{s}}$ where D_s = k_BT/(6πμr) is the sphere diffusivity according to the Stokes—Einstein relationship (where k_B is the Boltzmann constant and T is the absolute temperature) (d and f). Microspheres of three different radii and/or media of four different viscosities were used (indicated). The data were recorded at 250 frames per second using the method described in Fig. 1a legend. Reproduced from Yago et al.

FIGURE 4

Analysis of optimal values of tether rate curves. (a) Peak locations of the p_ad/(m_rm_lr) vs. $r\stackrel{.}{\gamma }$ curves (optimal $r\stackrel{.}{\gamma }$) were plotted against the sphere diffusivity D_s. (b) Peak locations of the p_ad/(m_rm_lr) vs. $D_{\mathrm{s}}∕r\stackrel{.}{\gamma }$ curves (optimal $D_{\mathrm{s}}∕r\stackrel{.}{\gamma }$) where plotted against the sphere diffusivity D_s. (c) Maximum p_ad/(m_rm_lr) values were plotted against the molecular diffusivity D_m. (d) Reciprocal of maximum p_ad/(m_rm_lr) values were plotted against reciprocal of the molecular diffusivity. Positive correlations were evident in all plots for both microspheres (open symbols) and neutrophils (solid circles). A straight line was fit to each set of the data for microspheres or neutrophils in (b) and (d). The best-fit equations are indicated along with the R² values. Reproduced from Yago et al.

FIGURE 5

Collapse of multiple data curves by proper scaling of the contributions by three transport mechanisms. When the normalized adhesion probabilities per distance for microspheres (a) and neutrophils (b), p_ad/(m_rm_lr), were plotted vs. $r\stackrel{.}{\gamma }(1+C_1∕D_{\mathrm{s}})$, a variable that combines sphere transport mechanisms for both relative sliding and Brownian motion, the ranges of all curves were aligned. When the p_ad/(m_rm_lr) values were further multiplied by (1/D_m - C₂) to obtain a variable that combines molecular diffusion and molecular docking, all 12 microsphere curves collapsed into a single curve (c). Similarly, all four neutrophil curves collapsed into a single curve (d). Reproduced from Yago et al.

FIGURE 6

Parameters of cell rolling under flow. (a) Balance of forces acting on a rolling cell. Shear stress applies a resultant force F_s and a torque T_s to the cell, which are balanced by a tether force F_t on the receptor—ligand bonds at the trailing edge of a tethered cell and by a compressive force F_c at the sphere bottom. Elevating the viscosity by addition of 6% Ficoll increases shear stress by 2.6-fold and increase F_t on the sphere of the same size, as illustrated by the comparative lengths of the thin and thick vectors for F_t on the small sphere on the right. At the same shear stress, F_t is 9-fold greater for a sphere of 3-μm radius then for a sphere of 1-μm radius, as illustrated by the comparative lengths of the thin vectors for F_t on the large and small spheres. The conversion of wall shear stress into F_t is described in Yago et al. (b) Decomposition of a cyclic rolling step. See text for a detailed description. Reproduced from Yago et al.

FIGURE 7

Mean velocities of L-selectin microspheres (a—c) or of unfixed or fixed neutrophils (d—f) rolling on PSGL-1 in the absence or presence of 6% Ficoll (which increased the viscosity by 2.6 fold) were measured as described in Fig. 1c legend and plotted against wall shear rate (a and d), wall shear stress (b and e), and tether force (c and f). Reproduced from Yago et al.

FIGURE 8

Off-rates (k_off) derived from lifetimes of transient tethers of L-selectin-bearing microspheres of 3- and 1-μm radii (a—c) or of unfixed or fixed neutrophils (d—f) to low-density sPSGL-1 (<10 sites/μm²) in the absence or presence of 6% Ficoll (which increased the medium viscosity by 2.6 fold) were plotted against wall shear rate (a and d), wall shear stress (b and e), and tether force (c and f). Low ligand density favors (but does not guarantee) single bonds. Tether lifetimes (∼100 measured at 4-ms temporal resolution under any given condition) are distributed as single exponential decays, consistent with first-order dissociation of single bonds. Plotting the data in semi-log scale linearizes the distribution. k_off can be evaluated from the negative slope of the linear fit to the data or from the average lifetime. Reproduced from Yago et al.

FIGURE 9

Changing features of instantaneous velocities of L-selectin-bearing mocrospheres freely flowing over HSA (1st panel) or continuously rolling on PSGL-1 (other panels) at indicated wall shear rates (s⁻¹) and corresponding tether forces (pN). The microsphere radius was 3 μm and the media did not contain Ficoll. Data were derived using particle tracking software from images recorded at 4-ms temporal resolution. Reproduced from Yago et al.

FIGURE 10

Tether force governs stop frequency below and above the flow optimum. The stop frequency (number of stops in a 1-s time course of instantaneous velocities like those shown in Fig. 9) of L-selectin-bearing microspheres of 3- or 1-μm radii (a—c) and fixed or unfixed neutrophils (d—f) rolling on sPSGL-1 in the absence or presence of 6% Ficoll (corresponding to viscosity of 1 or 2.6 cP) were plotted against wall shear rate (a and d), wall shear stress (b and e), and tether force (c and f). Reproduced from Yago et al.

FIGURE 11

Tether force governs mean stop time below and above the flow optimum. The mean stop times (durations of stops in the time courses of instantaneous velocities like those shown in Fig. 9) of L-selectin-bearing microspheres of 3- or 1-μm radii (a—c) and fixed or unfixed neutrophils (d—f) rolling on sPSGL-1 in the absence or presence of 6% Ficoll (corresponding to viscosity of 1 or 2.6 cP) were plotted against wall shear rate (a and d), wall shear stress (b and e), and tether force (c and f). Reproduced from Yago et al.

FIGURE 12

Pathways of sliding-rebinding mechanism. (a—d and f) Sequential SMD-simulated structures of the N-terminal region of PSGL-1 (pink for the peptide and green for the glycan) dissociating from P-selectin lectin-EGF domains (blue) at indicated times. (e) Separate structures of P-selectin lectin-EGF domains and the N-terminal region of PSGL-1 indicating complete dissociation. The thick purple arrows indicate the hypothetical sequence of events along dissociation pathways. The interdomain angles are marked by arched double-sided red arrows. The inclinations of the binding interface are marked by inclined red lines. (a) The initial bound state. (b) Force-induced opening of the hinge angle. (c) Rupture of pre-existing interactions. (d) Sliding and formation of new interactions. (e) Dissociation from fast pathway 1. (f) Dissociation from slow pathway 2. When a small force f (short black arrows) is applied, the complex may detach by dissociation of all noncovalent interactions that pre-existed in the bound state. An intermediate force (long black arrows) may open the hinge angle, tilt the binding interface, and promote sliding of PSGL-1 over the lectin-domain binding interface after pre-existing atomic-level interactions dissociate. This provides an opportunity for new interactions to form, which would replace those that are disrupted, or for the original interactions to reform, which would return the system back to its previously bound state, before the ligand fully dissociates. Reproduced from Lou and Zhu.

FIGURE 13

Greater flexibility of the L-selectinN138G hinge increases tethering by enhanced rotational diffusion. (a, b) The tether-rate dependence for L-selectin or L-selectinN138G on the product $r\stackrel{.}{\gamma }$ of the microsphere radius r and the wall shear rate $\stackrel{.}{\gamma }$. The data were measured by the same method as described in Fig. 1a legend and represent the mean ± the SD from three experiments. The microspheres were coated with 750 molecules μm⁻² of either L-selectin or L-selectinN138G, except in one case in (b), where they were coated with 1500 molecules μm⁻² of L-selectinN138G (blue squares). The flow chamber floor in (a) was coated with PSGL-1 at either 120 (red triangles and green diamonds) or 240 (blue squares) molecules μm⁻². The flow chamber floor in (b) was coated with a constant density of 6-sulfo-sLe^x. (c) Optimal $r\stackrel{.}{\gamma }$ (peak locations of the p_ad/(m_rm_lr) vs. $r\stackrel{.}{\gamma }$ curves) vs. microsphere diffusivity k_BT/(6πμr)of 3-μm radius microspheres bearing L-selectin (red triangle) or L-selectinN138G (green diamond) tethering to PSGL-1 were plotted for comparison. The open circles are microsphere data from Fig. 4a for calibration. (d) Maximum p_ad/(m_rm_lr) vs. molecular diffusivity k_BT/(6πμl) data for L-selectin bearing microspheres tethering to PSGL-1 from Fig. 4c were replotted to provide calibration (open circles), which assumed the characteristic length for molecular diffusion as l = 100 nm. The same l value was used for the L-selectin datum (red triangle), which matched the calibration curve well. The molecular diffusivity for L-selectinN138G is predicted to be larger. Using the measured maximum p_ad/(m_rm_lr) value, the L-selectinN138G datum point (green diamond) was located at the intercept of y = [p_ad/(m_rm_lr)]_max (green dashed horizontal line) and the extrapolation of the calibration curve (red line). The increased molecular diffusivity for L-selectinN138G could be calculated from the x-axis value of this datum point (green dashed vertical line), which is 85% over the value for L-selectin. Reproduced from Lou et al.

FIGURE 14

Greater flexibility of the L-selectinN138G hinge augments catch bonds and flow-enhanced rolling. Micro-spheres coated with matched densities of L-selectin or L-selectinN138G were perfused through a flow chamber containing immobilized PSGL-1 (a, c, e, and g) or 6-sulfo-sLe^x (b, d, f, and h). The force dependence of the tether lifetime is shown in (a and b); the solid lines indicated the pseudoatom model fits of the sliding-rebinding mechanism., The mean rolling velocities shown in (c and d) represent the mean ± SD from five experiments. The mean stop time (e and f) or fractional stop time (g and h) represent analyses of thousands of rolling steps collected from 10−15 microspheres rolling for 1 s at each wall shear stress for each pair of selectin—ligand interactions. Reproduced from Lou et al.