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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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Ion dependency of the interaction of I-GPI E6 cells with  ICAM-1–containing membranes in the absence (A) and presence (B) of  MEM-83. I-GPI E6 cells in HBS/BSA buffer containing 2 mM MgCl2  (black), 0.2 mM MnCl2 (stipled), 1 mM CaCl2 (diagonal stripes), or 2 mM  EDTA (vertical stripes) were not treated (A) or pretreated with MEM-83  (0.1 mg/ml) (B), washed and assayed on bilayer membranes with 500  ICAM-1 molecules/μm2 at a flow rate of 1.1 dyn/cm2. Error bars represent 95% confidence interval on the mean of free, rolling, or attached cells.
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Figure 6: Ion dependency of the interaction of I-GPI E6 cells with ICAM-1–containing membranes in the absence (A) and presence (B) of MEM-83. I-GPI E6 cells in HBS/BSA buffer containing 2 mM MgCl2 (black), 0.2 mM MnCl2 (stipled), 1 mM CaCl2 (diagonal stripes), or 2 mM EDTA (vertical stripes) were not treated (A) or pretreated with MEM-83 (0.1 mg/ml) (B), washed and assayed on bilayer membranes with 500 ICAM-1 molecules/μm2 at a flow rate of 1.1 dyn/cm2. Error bars represent 95% confidence interval on the mean of free, rolling, or attached cells.

Mentions: In the presence of 2 mM Mg2+, 72% of I-GPI E6 cells rolled on ICAM-1–containing bilayers at a flow rate of 1.1 dyn/cm2 (Fig. 6 A). When Mg2+ was replaced by 0.2 mM Mn2+, rolling of the I-GPI E6 cells was still supported. However, the percentage of rolling cells dropped to 16%, a fourfold reduction compared with Mg2+. Rising the concentration of Mn2+ from 0.2 to 1 mM did not affect the percentage of rolling cells or the rolling velocity (data not shown). In the presence of 2 mM Ca2+, no rolling was detectable (Fig. 6 A).


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)

Ion dependency of the interaction of I-GPI E6 cells with  ICAM-1–containing membranes in the absence (A) and presence (B) of  MEM-83. I-GPI E6 cells in HBS/BSA buffer containing 2 mM MgCl2  (black), 0.2 mM MnCl2 (stipled), 1 mM CaCl2 (diagonal stripes), or 2 mM  EDTA (vertical stripes) were not treated (A) or pretreated with MEM-83  (0.1 mg/ml) (B), washed and assayed on bilayer membranes with 500  ICAM-1 molecules/μm2 at a flow rate of 1.1 dyn/cm2. Error bars represent 95% confidence interval on the mean of free, rolling, or attached cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199009&req=5

Figure 6: Ion dependency of the interaction of I-GPI E6 cells with ICAM-1–containing membranes in the absence (A) and presence (B) of MEM-83. I-GPI E6 cells in HBS/BSA buffer containing 2 mM MgCl2 (black), 0.2 mM MnCl2 (stipled), 1 mM CaCl2 (diagonal stripes), or 2 mM EDTA (vertical stripes) were not treated (A) or pretreated with MEM-83 (0.1 mg/ml) (B), washed and assayed on bilayer membranes with 500 ICAM-1 molecules/μm2 at a flow rate of 1.1 dyn/cm2. Error bars represent 95% confidence interval on the mean of free, rolling, or attached cells.
Mentions: In the presence of 2 mM Mg2+, 72% of I-GPI E6 cells rolled on ICAM-1–containing bilayers at a flow rate of 1.1 dyn/cm2 (Fig. 6 A). When Mg2+ was replaced by 0.2 mM Mn2+, rolling of the I-GPI E6 cells was still supported. However, the percentage of rolling cells dropped to 16%, a fourfold reduction compared with Mg2+. Rising the concentration of Mn2+ from 0.2 to 1 mM did not affect the percentage of rolling cells or the rolling velocity (data not shown). In the presence of 2 mM Ca2+, no rolling was detectable (Fig. 6 A).

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