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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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Rolling interaction of I-GPI cells with ICAM-1– (A and B)  or ICAM-3– (C) containing bilayers. The rolling velocity of I-GPI E6  cells treated with no IgG (solid square), MEM-83 IgG (solid circle), or  MEM-83 Fab (open circle) on membranes containing 500 molecules/μm2  (A) or 150 molecules/μm2 ICAM-1 (B) or 1,000 molecules/μm2 ICAM-3  (C) as function of shear stress is shown. Error bars represent 95% confidence interval on the mean rolling velocity.
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Figure 5: Rolling interaction of I-GPI cells with ICAM-1– (A and B) or ICAM-3– (C) containing bilayers. The rolling velocity of I-GPI E6 cells treated with no IgG (solid square), MEM-83 IgG (solid circle), or MEM-83 Fab (open circle) on membranes containing 500 molecules/μm2 (A) or 150 molecules/μm2 ICAM-1 (B) or 1,000 molecules/μm2 ICAM-3 (C) as function of shear stress is shown. Error bars represent 95% confidence interval on the mean rolling velocity.

Mentions: The relative kinetics of the I-GPI/ICAM interaction were examined by measuring the rolling velocity of I-GPI E6 cells on ICAM-1– or ICAM-3–containing bilayers, respectively (Fig. 5). On bilayers containing 500 molecules/μm2 of ICAM-1, an increasing shear stress resulted in a linear increase in the rolling velocity of I-GPI E6 cells in the presence of 2 mM Mg2+ that was paralleled by a decrease in the number of rolling cells (data not shown). The maximal average velocity was ∼40 μm/s at a shear stress of 12 dyn/cm2 (Fig. 5 A). In the presence of MEM-83 or its Fab fragment, the rolling velocity of I-GPI E6 cells dropped considerably, especially at higher shear stresses, and reached maximal levels of ∼10 and ∼15 μm/s, respectively. To investigate whether the MEM-83–induced increase of avidity is sufficient to support rolling at low site densities, we reconstitued ICAM-1 into phosphatidylcholine at a site density of 150 ICAM-1 molecules/μm2. In the absence of the activating antibody, very few I-GPI E6 cells rolled. In the presence of MEM-83 or MEM-83 Fab fragment, however, I-GPI E6 cells rolled on bilayers containing 150 molecules/ μm2 of ICAM-1 (Fig. 5 B). Altogether, it appears that MEM-83 increased the avidity of the I-GPI/ICAM-1 interaction about fourfold.


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)

Rolling interaction of I-GPI cells with ICAM-1– (A and B)  or ICAM-3– (C) containing bilayers. The rolling velocity of I-GPI E6  cells treated with no IgG (solid square), MEM-83 IgG (solid circle), or  MEM-83 Fab (open circle) on membranes containing 500 molecules/μm2  (A) or 150 molecules/μm2 ICAM-1 (B) or 1,000 molecules/μm2 ICAM-3  (C) as function of shear stress is shown. Error bars represent 95% confidence interval on the mean rolling velocity.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199009&req=5

Figure 5: Rolling interaction of I-GPI cells with ICAM-1– (A and B) or ICAM-3– (C) containing bilayers. The rolling velocity of I-GPI E6 cells treated with no IgG (solid square), MEM-83 IgG (solid circle), or MEM-83 Fab (open circle) on membranes containing 500 molecules/μm2 (A) or 150 molecules/μm2 ICAM-1 (B) or 1,000 molecules/μm2 ICAM-3 (C) as function of shear stress is shown. Error bars represent 95% confidence interval on the mean rolling velocity.
Mentions: The relative kinetics of the I-GPI/ICAM interaction were examined by measuring the rolling velocity of I-GPI E6 cells on ICAM-1– or ICAM-3–containing bilayers, respectively (Fig. 5). On bilayers containing 500 molecules/μm2 of ICAM-1, an increasing shear stress resulted in a linear increase in the rolling velocity of I-GPI E6 cells in the presence of 2 mM Mg2+ that was paralleled by a decrease in the number of rolling cells (data not shown). The maximal average velocity was ∼40 μm/s at a shear stress of 12 dyn/cm2 (Fig. 5 A). In the presence of MEM-83 or its Fab fragment, the rolling velocity of I-GPI E6 cells dropped considerably, especially at higher shear stresses, and reached maximal levels of ∼10 and ∼15 μm/s, respectively. To investigate whether the MEM-83–induced increase of avidity is sufficient to support rolling at low site densities, we reconstitued ICAM-1 into phosphatidylcholine at a site density of 150 ICAM-1 molecules/μm2. In the absence of the activating antibody, very few I-GPI E6 cells rolled. In the presence of MEM-83 or MEM-83 Fab fragment, however, I-GPI E6 cells rolled on bilayers containing 150 molecules/ μm2 of ICAM-1 (Fig. 5 B). Altogether, it appears that MEM-83 increased the avidity of the I-GPI/ICAM-1 interaction about fourfold.

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