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First published online 6 May 2003
doi: 10.1242/jcs.00465

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Contributions of molecular binding events and cellular compliance to the modulation of leukocyte adhesion

Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL 33136, USA

View larger version (18K): [in a new window]   Fig. 1. Schematics of AFM force measurements. The principal events of cell-substrate interaction during an AFM force versus displacement measurement are approach of the 3A9 cell onto a surface coated with ICAM-1, contact between cell and substrate, retraction of the 3A9 cell and separation of the cell from the substrate. Arrows indicate the direction of cantilever movement.

View larger version (31K): [in a new window]   Fig. 2. Force versus displacement traces of the interaction between 3A9 cells and immobilized ICAM-1. The measurements were carried out with a resting cell (first trace), a Mg2+-treated cell (second trace) and a PMA-stimulated cell (third trace). The measurements were acquired with a compression force of 200 pN, 5 seconds contact and a cantilever retraction speed of 2 µm/second. The fourth trace corresponds to a measurement acquired from a PMA-stimulated cell in the presence of LFA-1 (20 µg/ml FD441.8) and ICAM-1 (20 µg/ml BE29G1) function-blocking antibodies (inh. mAbs). The shaded area shows an estimate of the work of de-adhesion, and is the detachment force. Arrows point to breakage of LFA-1–ICAM-1 bond(s).

View larger version (20K): [in a new window]   Fig. 3. Work of de-adhesion (A) and detachment forces (B) of resting and stimulated 3A9 cells bound to immobilized ICAM-1/Fc. The inhibitory monoclonal antibodies used were FD441.8 (anti-LFA-1; 20 µg/ml) and BE29G1 (anti-ICAM-1; 20 µg/ml). The numbers given here were derived from measurements that were acquired with a compression force of 200 pN, 5 seconds contact and a cantilever retraction speed of 2 µm/second. The error bar is the standard deviation and N>15 in each case (inh. mAbs, inhibitory antibodies).

View larger version (27K): [in a new window]   Fig. 4. Histograms of breakage forces of LFA-1–ICAM-1 bond(s) from the force-displacement traces of (A) resting and (B) PMA-stimulated 3A9 cells. The breakage forces were derived from the magnitude of the force transitions acquired in measurements obtained with a compression force of 200 pN, 5 seconds contact and a cantilever retraction speed of 2 µm/second. The y-axis plots the number of force transitions detected.

View larger version (26K): [in a new window]   Fig. 5. Work of de-adhesion of resting (open bars), PMA-stimulated (gray bars), Mg2+-treated (black bars) 3A9 cells bound to immobilized antibodies against LFA-1. The FD441.8 mAb recognizes an epitope formed by both and ß subunits of LFA-1. The M17/4.2 mAb recognizes an epitope on L. The numbers given here were derived from measurements that were acquired with a compression force of 200 pN, 2 seconds contact and a cantilever retraction speed of 2 µm/second. The error bar is the standard error.

View larger version (27K): [in a new window]   Fig. 6. (A) Measurements of unitary LFA-1–ICAM-1 unbinding forces. A series of AFM force measurements are shown. Traces 2 and 5 show molecular adhesion. Measurements of LFA-1–ICAM-1 unbinding forces were obtained under conditions that minimized contact between the 3A9 cell and the ICAM-1-coated surface. An adhesion frequency of less than 30% in the force measurements ensured that there is a >85% probability that the adhesion event is mediated by a single LFA-1–ICAM-1 complex (Tees et al., 2001). The specificity of the molecular interaction was confirmed by examining the frequency of adhesion in test and control experiments (Tees et al., 2001; Evans et al., 2001). Under identical experimental conditions, the addition of monoclonal antibodies against either LFA-1 or ICAM-1 significantly lowered the frequency of adhesion of both resting and activated cells. Moreover, both resting and stimulated 3A9 cells exhibited lower frequency of adhesion to immobilized bovine albumin than to immobilized ICAM-1. (B) Force histograms of unitary LFA-1–ICAM-1 unbinding forces of resting, PMA-stimulated and Mg2+/EGTA-treated 3A9 cells at low (73-98 pN/second) intermediate (1050-1500 pN/second) and high (21,000-35,000 pN/second) loading rates.

View larger version (22K): [in a new window]   Fig. 7. Average unbinding force of individual LFA-1–ICAM-1 complexes as a function of force loading rate. Measurements were acquired using resting (open circle), PMA-stimulated (square) and Mg2+/EGTA-activated (closed circle) 3A9 cells at loading rates between 20 and 50,000 pN/second. This range of loading rates was achieved by varying the retraction rate of the cantilever from 0.1 to 15 µm/second and as a result of variations in the local compliance of the cell. This allowed for the effective spring constant of the cell-cantilever combination to have a range of values between 0.1 and 5 mN/meter. At fast cantilever retraction speeds (>1 µm/second), the hydrodynamic drag on the cantilever resulted in smaller forces recorded than were actually applied to rupture the LFA-1–ICAM-1 complex (Evans et al., 2001). The damping coefficient of the cantilever in the culture medium was 2 pN-second/µm. Each of the force spectra were acquired using five cells with an average of 100 measurements/cell. The error bar is the standard error.

View larger version (17K): [in a new window]   Fig. 8. (A) Force versus indentation traces of resting, PMA-stimulated and Mg2+-treated 3A9 cells. The fitted curves derived from the Hertz model are overlaid on the measurements. (B) Histograms showing distribution of Young's modulus values for resting, PMA-stimulated and Mg2+-treated 3A9 cells. (C) Young's modulus of resting, PMA-stimulated and Mg2+-treated 3A9 cells. The error bar is the standard error.

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