Limits...
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.

Show MeSH

Related in: MedlinePlus

Competition of sICAM-1 with 125I TS1/22 Fab fragments  for binding to I-GPI (A) and static adhesion assay of SKW3 cells and I-GPI  cells to ICAM-1 coated on plates (B). (A) I-GPI E6 cells were incubated  with the indicated concentrations of sICAM-1 and 125I TS1/22 Fab fragments (2 nM) for 20 min at room temperature. Cells and supernatant  were separated by centrifugation through an oil cushion. The binding of  TS1/22 Fab fragments is expressed as a fraction of I domain sites. Protein  concentration in the assay was kept constant with ovalbumin. The result  represents one experiment out of three. (B) Unstimulated SKW3 cells  (open squares), SKW3 cells preincubated with 50 ng/ml PMA (filled  squares), and I-GPI E6 cells (closed circles) or I-GPI E5 cells (open circles)  were tested for adhesion to ICAM-1–coated plastic at 37°C in RPMI-1640, 2% BSA. Purified ICAM-1 was coated on polystyrene at the indicated density as determined by immunometric assay. SKW3 cells were labeled with calcein AM and BHK cells with BCECF.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199009&req=5

Figure 2: Competition of sICAM-1 with 125I TS1/22 Fab fragments for binding to I-GPI (A) and static adhesion assay of SKW3 cells and I-GPI cells to ICAM-1 coated on plates (B). (A) I-GPI E6 cells were incubated with the indicated concentrations of sICAM-1 and 125I TS1/22 Fab fragments (2 nM) for 20 min at room temperature. Cells and supernatant were separated by centrifugation through an oil cushion. The binding of TS1/22 Fab fragments is expressed as a fraction of I domain sites. Protein concentration in the assay was kept constant with ovalbumin. The result represents one experiment out of three. (B) Unstimulated SKW3 cells (open squares), SKW3 cells preincubated with 50 ng/ml PMA (filled squares), and I-GPI E6 cells (closed circles) or I-GPI E5 cells (open circles) were tested for adhesion to ICAM-1–coated plastic at 37°C in RPMI-1640, 2% BSA. Purified ICAM-1 was coated on polystyrene at the indicated density as determined by immunometric assay. SKW3 cells were labeled with calcein AM and BHK cells with BCECF.

Mentions: In initial experiments to determine the affinity of the I domain, the binding of radiolabeled sICAM-1 dimers to I-GPI E6 cells was not detectable (data not shown). Subsequently, we measured the inhibition of binding of radiolabeled TS1/22 Fab fragments to I-GPI E6 cells by titrating in sICAM-1 (Fig. 2 A). Half maximal reduction of TS1/22 Fab binding was achieved at ∼150 μM sICAM-1. Using the formula of Chen and Prusoff (29), the Kd for the I domain/ ICAM-1 interaction was calculated to be in the range of 100–200 μM, as determined in three independent experiments. This result indicates that the affinity between sICAM-1 and the I domain is low and similar to the affinity of sICAM-1 and intact LFA-1 on unstimulated T cells (∼100 μM) (33).


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)

Competition of sICAM-1 with 125I TS1/22 Fab fragments  for binding to I-GPI (A) and static adhesion assay of SKW3 cells and I-GPI  cells to ICAM-1 coated on plates (B). (A) I-GPI E6 cells were incubated  with the indicated concentrations of sICAM-1 and 125I TS1/22 Fab fragments (2 nM) for 20 min at room temperature. Cells and supernatant  were separated by centrifugation through an oil cushion. The binding of  TS1/22 Fab fragments is expressed as a fraction of I domain sites. Protein  concentration in the assay was kept constant with ovalbumin. The result  represents one experiment out of three. (B) Unstimulated SKW3 cells  (open squares), SKW3 cells preincubated with 50 ng/ml PMA (filled  squares), and I-GPI E6 cells (closed circles) or I-GPI E5 cells (open circles)  were tested for adhesion to ICAM-1–coated plastic at 37°C in RPMI-1640, 2% BSA. Purified ICAM-1 was coated on polystyrene at the indicated density as determined by immunometric assay. SKW3 cells were labeled with calcein AM and BHK cells with BCECF.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Competition of sICAM-1 with 125I TS1/22 Fab fragments for binding to I-GPI (A) and static adhesion assay of SKW3 cells and I-GPI cells to ICAM-1 coated on plates (B). (A) I-GPI E6 cells were incubated with the indicated concentrations of sICAM-1 and 125I TS1/22 Fab fragments (2 nM) for 20 min at room temperature. Cells and supernatant were separated by centrifugation through an oil cushion. The binding of TS1/22 Fab fragments is expressed as a fraction of I domain sites. Protein concentration in the assay was kept constant with ovalbumin. The result represents one experiment out of three. (B) Unstimulated SKW3 cells (open squares), SKW3 cells preincubated with 50 ng/ml PMA (filled squares), and I-GPI E6 cells (closed circles) or I-GPI E5 cells (open circles) were tested for adhesion to ICAM-1–coated plastic at 37°C in RPMI-1640, 2% BSA. Purified ICAM-1 was coated on polystyrene at the indicated density as determined by immunometric assay. SKW3 cells were labeled with calcein AM and BHK cells with BCECF.
Mentions: In initial experiments to determine the affinity of the I domain, the binding of radiolabeled sICAM-1 dimers to I-GPI E6 cells was not detectable (data not shown). Subsequently, we measured the inhibition of binding of radiolabeled TS1/22 Fab fragments to I-GPI E6 cells by titrating in sICAM-1 (Fig. 2 A). Half maximal reduction of TS1/22 Fab binding was achieved at ∼150 μM sICAM-1. Using the formula of Chen and Prusoff (29), the Kd for the I domain/ ICAM-1 interaction was calculated to be in the range of 100–200 μM, as determined in three independent experiments. This result indicates that the affinity between sICAM-1 and the I domain is low and similar to the affinity of sICAM-1 and intact LFA-1 on unstimulated T cells (∼100 μM) (33).

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