Quantifying the effects of contact duration, loading rate, and approach velocity on P-selectin-PSGL-1 interactions using AFM. roles in regulating PMN adhesion on endothelium. LFA-1 dominates PMN adhesion on ICAM-1 and ICAM-2, while Mac-1 mediates PMN adhesion on RAGE, JAM-A, and JAM-C, which is consistent with their bond strength. All ligands can trigger PMN spreading and polarization, in which Mac-1 seems to induce outside-in signaling more effectively. LFA-1CICAM-1 and LFA-1/Mac-1CJAM-C bonds can accelerate PMN crawling under high shear stress, presumably due to their high mechanical strength. This work provides new insight into basic molecular mechanisms of physiological ligands of 2 integrins in PMN recruitment. INTRODUCTION Polymorphonuclear leukocyte (neutrophil, PMN) recruitment from blood circulation to the sites of infection or sterile injury, where PMNs exhibit numerous effector functions such as phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs), is the first line of host defense against invading pathogens and tissue injury (Zarbock and Ley, 2009 ; Robert is observed when LFA-1- or Mac-1Ccoated tips contact their ligands but the adhesion is significantly abrogated to 1 1.2C12.9% in the presence of LFA-1 or Mac-1 blocking mAbs (Figure 1, D and E). These data demonstrate that the observed adhesion is specifically mediated by the bindings between LFA-1/Mac-1 and their ligands. Moreover, all ligands are found to present direct interactions with both LFA-1 and Mac-1. Open in a separate window FIGURE 1: AFM tests for 2 integrin-ligand bonds. (A) Schematic of AFM setup. A PZT was used to drive the movement of an AFM cantilever. Adhesion events and forced bond rupture signals were collected from a QPD that measures the deflection of a laser beam reflected on the cantilever. (B) AFM functionalization. Recombinant human LFA-1s or Mac-1s were adsorbed onto the AFM tip and treated with TS1/18 mAbs or Mn2+ to obtain low- or high-affinity conformation integrins, respectively. Soluble ligand (ICAM-1, ICAM-2, RAGE, JAM-A, or JAM-C)CIgG Fc chimeras were coated via anti-IgG Fc secondary antibodies precoated on a Petri dish. (C) Typical forceCdisplacement curves. An LFA-1C or Mac-1Ccaptured AFM tip was driven to approach to (from left to right, blue lines), contact, and retract from (from right to left, reddish lines) a ligand-coated Petri dish. Adhesion was visualized from cantilever deflection and rupture push ( 0.05; **, 0.01; ***, 0.001 between each ligand and negative control, by #, 0.05; ##, 0.01; ###, 0.001 between each blocking group and isotype control, or by $, 0.05; $$, 0.01; $$$, 0.001 among various ligands. (F) Standard rupture push distributions for the relationships between LFA-1 and ICAM-1 with TS1/18 in the indicated loading rates. Total 251C394 single-bond rupture push data at each loading rate were collected and analyzed using a push bin of 50 pN. We next measured the dependence of relationship rupture Apigenin-7-O-beta-D-glucopyranoside causes on assorted retraction velocities (related loading rate ranging from 3171C114,852 pN/s). In all the measurements, an Apigenin-7-O-beta-D-glucopyranoside adhesion rate of recurrence of 36.7% ensures that there is a 78.9% probability the adhesion is achieved by single-bond events with Poisson distribution statistics (Tees from Number 2A (Supplemental Table 3). (C) Lifetime like a function of applied push (points), from the Apigenin-7-O-beta-D-glucopyranoside variance of rupture push distribution using SELPLG Eq. 4. The fitted curves (lines) were acquired using Eq. 1. Measurements were acquired between LFA-1 (reddish squares and lines) or Mac pc-1 (blue circles and lines) and the indicated ligand with TS1/18 (closed points, solid lines) or Mn2+ (open points, dashed lines). All the data (Number 3A) were further fitted with DFS theory (Eq. 3; observe that characterize the spontaneous force-free dissociation and the width of the energy well of the LFA-1/Mac pc-1Cligand relationship were substituted into Eq. 1 and then used to obtain linear dependence of the logarithm of lifetimes (1/ 0.05; **, 0.01; ***, 0.001 among various ligands, by #, 0.05; ##, 0.01; ###, 0.001 between each blocking group and isotype control, or by $, 0.05; $$, 0.01; $$$, 0.001 between LFA-1C and Mac pc-1Cblocking organizations. Resting or fMLF-activated PMNs were allowed to abide by numerous ligands for 5 min without circulation, washed at a wall shear stress of = 0.301). LFA-1CICAM-1 and Mac pc-1CICAM-1 relationships mediate specific adhesion of resting PMN cooperatively (Number 4A), but, after.
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