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The lymphocyte function-associated antigen 1 I domain is a transient binding module for intercellular adhesion molecule (ICAM)-1 and ICAM-3 in hydrodynamic flow.

Knorr R, Dustin ML - J. Exp. Med. (1997)

Bottom Line: The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes.Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity.This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.

View Article: PubMed Central - PubMed

Affiliation: Center for Immmunology and Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
The I domain of lymphocyte function-associated antigen (LFA)-1 contains an intercellular adhesion molecule (ICAM)-1 and ICAM-3 binding site, but the relationship of this site to regulated adhesion is unknown. To study the adhesive properties of the LFA-1 I domain, we stably expressed a GPI-anchored form of this I domain (I-GPI) on the surface of baby hamster kidney cells. I-GPI cells bound soluble ICAM-1 (sICAM-1) with a low avidity and affinity. Flow cell experiments demonstrated a specific rolling interaction of I-GPI cells on bilayers containing purified full length ICAM-1 or ICAM-3. The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes. In contrast, the interaction of I-GPI cells with ICAM-3 was blocked by MEM-83. Rolling of I-GPI cells was dependent on the presence of Mg2+. Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity. This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.

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Stroboscopic images of  free, attached, and rolling cells. 16  images spanning 6 s digitized from  S-VHS tape (a, c, and d) or 8 images  spanning 4 s directly acquired with a  cooled CCD camera (40-ms exposure) (b) were added together such  that the motion of cells in the flow  field over time is represented in a  single image. (a) I-GPI–transfected  cells; (b) same as a at a higher magnification; (c) vector transfected cells;  (d) ICAM-1 GPI-transfected cells.  The bilayer membranes contained  500 molecules/μm2 ICAM-1 (a–c)  or 500 molecules/μm2 LFA-1 (d).  Scale bars = 50 μm.
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Figure 3: Stroboscopic images of free, attached, and rolling cells. 16 images spanning 6 s digitized from S-VHS tape (a, c, and d) or 8 images spanning 4 s directly acquired with a cooled CCD camera (40-ms exposure) (b) were added together such that the motion of cells in the flow field over time is represented in a single image. (a) I-GPI–transfected cells; (b) same as a at a higher magnification; (c) vector transfected cells; (d) ICAM-1 GPI-transfected cells. The bilayer membranes contained 500 molecules/μm2 ICAM-1 (a–c) or 500 molecules/μm2 LFA-1 (d). Scale bars = 50 μm.

Mentions: Laminar hydrodynamic flow produces a greater range of controlled shear stresses than can be applied by turbulent shear occurring in a 96-well plate adhesion assay. Therefore, adhesion of I-GPI E6 cells to a glass-supported planar bilayer reconstituted with 500 molecules/μm2 purified ICAM-1 or 1,000 molecules/μm2 purified ICAM-3 was examined in a parallel plate flow cell. The I-GPI E6 cells were allowed a 2-min static period to settle onto the bilayer before starting flow at a shear stress of 1 dyn/cm2. Surprisingly, upon starting flow, the I-GPI E6 began to roll on planar bilayers containing ICAM-1 (Fig. 3, A and B and Table 1) or ICAM-3 (not shown), in a manner reminiscent of selectin- or VLA-4-mediated rolling. In contrast, mock transfected cells showed neither rolling nor stable interaction with ICAM-1 in the glass-supported bilayer (Fig. 3 C and Table 1), whereas ICAM-1 GPI cells attached stably to bilayers containing 500 molecules/μm2 of purified LFA-1 (Fig. 3 D and Table 1).


The lymphocyte function-associated antigen 1 I domain is a transient binding module for intercellular adhesion molecule (ICAM)-1 and ICAM-3 in hydrodynamic flow.

Knorr R, Dustin ML - J. Exp. Med. (1997)

Stroboscopic images of  free, attached, and rolling cells. 16  images spanning 6 s digitized from  S-VHS tape (a, c, and d) or 8 images  spanning 4 s directly acquired with a  cooled CCD camera (40-ms exposure) (b) were added together such  that the motion of cells in the flow  field over time is represented in a  single image. (a) I-GPI–transfected  cells; (b) same as a at a higher magnification; (c) vector transfected cells;  (d) ICAM-1 GPI-transfected cells.  The bilayer membranes contained  500 molecules/μm2 ICAM-1 (a–c)  or 500 molecules/μm2 LFA-1 (d).  Scale bars = 50 μm.
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Related In: Results  -  Collection

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Figure 3: Stroboscopic images of free, attached, and rolling cells. 16 images spanning 6 s digitized from S-VHS tape (a, c, and d) or 8 images spanning 4 s directly acquired with a cooled CCD camera (40-ms exposure) (b) were added together such that the motion of cells in the flow field over time is represented in a single image. (a) I-GPI–transfected cells; (b) same as a at a higher magnification; (c) vector transfected cells; (d) ICAM-1 GPI-transfected cells. The bilayer membranes contained 500 molecules/μm2 ICAM-1 (a–c) or 500 molecules/μm2 LFA-1 (d). Scale bars = 50 μm.
Mentions: Laminar hydrodynamic flow produces a greater range of controlled shear stresses than can be applied by turbulent shear occurring in a 96-well plate adhesion assay. Therefore, adhesion of I-GPI E6 cells to a glass-supported planar bilayer reconstituted with 500 molecules/μm2 purified ICAM-1 or 1,000 molecules/μm2 purified ICAM-3 was examined in a parallel plate flow cell. The I-GPI E6 cells were allowed a 2-min static period to settle onto the bilayer before starting flow at a shear stress of 1 dyn/cm2. Surprisingly, upon starting flow, the I-GPI E6 began to roll on planar bilayers containing ICAM-1 (Fig. 3, A and B and Table 1) or ICAM-3 (not shown), in a manner reminiscent of selectin- or VLA-4-mediated rolling. In contrast, mock transfected cells showed neither rolling nor stable interaction with ICAM-1 in the glass-supported bilayer (Fig. 3 C and Table 1), whereas ICAM-1 GPI cells attached stably to bilayers containing 500 molecules/μm2 of purified LFA-1 (Fig. 3 D and Table 1).

Bottom Line: The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes.Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity.This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.

View Article: PubMed Central - PubMed

Affiliation: Center for Immmunology and Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
The I domain of lymphocyte function-associated antigen (LFA)-1 contains an intercellular adhesion molecule (ICAM)-1 and ICAM-3 binding site, but the relationship of this site to regulated adhesion is unknown. To study the adhesive properties of the LFA-1 I domain, we stably expressed a GPI-anchored form of this I domain (I-GPI) on the surface of baby hamster kidney cells. I-GPI cells bound soluble ICAM-1 (sICAM-1) with a low avidity and affinity. Flow cell experiments demonstrated a specific rolling interaction of I-GPI cells on bilayers containing purified full length ICAM-1 or ICAM-3. The LFA-1 activating antibody MEM-83, or its Fab fragment, decreased the rolling velocity of I-GPI cells on ICAM-1-containing membranes. In contrast, the interaction of I-GPI cells with ICAM-3 was blocked by MEM-83. Rolling of I-GPI cells was dependent on the presence of Mg2+. Mn2+ only partially substituted for Mg2+, giving rise to a small fraction of rolling cells and increased rolling velocity. This suggests that the I domain acts as a transient, Mg2+-dependent binding module that cooperates with another Mn2+-stimulated site in LFA-1 to give rise to the stable interaction of intact LFA-1 with ICAM-1.

Show MeSH
Related in: MedlinePlus