HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sigal, A. Articles by Alon, R. Search for Related Content PubMed PubMed Citation Articles by Sigal, A. Articles by Alon, R. Pubmed/NCBI databases Substance via MeSH

The LFA-1 Integrin Supports Rolling Adhesions on ICAM-1 Under Physiological Shear Flow in a Permissive Cellular Environment¹

Alex Sigal^*, Diederik A. Bleijs, Valentin Grabovsky^*, Sandra J. van Vliet, Oren Dwir^*, Carl G. Figdor, Yvette van Kooyk and Ronen Alon^2,*

^* Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel; and Department of Tumor Immunology, University Hospital Nijmegen, Nijmegen, The Netherlands

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The LFA-1 integrin is crucial for the firm adhesion of circulating leukocytes to ICAM-1-expressing endothelial cells. In the present study, we demonstrate that LFA-1 can arrest unstimulated PBL subsets and lymphoblastoid Jurkat cells on immobilized ICAM-1 under subphysiological shear flow and mediate firm adhesion to ICAM-1 after short static contact. However, LFA-1 expressed in K562 cells failed to support firm adhesion to ICAM-1 but instead mediated K562 cell rolling on the endothelial ligand under physiological shear stress. LFA-1-mediated rolling required an intact LFA-1 I-domain, was enhanced by Mg²⁺, and was sharply dependent on ICAM-1 density. This is the first indication that LFA-1 can engage in rolling adhesions with ICAM-1 under physiological shear flow. The ability of LFA-1 to support rolling correlates with decreased avidity and impaired time-dependent adhesion strengthening. A ß₂ cytoplasmic domain-deletion mutant of LFA-1, with high avidity to immobilized ICAM-1, mediated firm arrests of K562 cells interacting with ICAM-1 under shear flow. Our results suggest that restrictions in LFA-1 clustering mediated by cytoskeletal attachments may lock the integrin into low-avidity states in particular cellular environments. Although low-avidity LFA-1 states fail to undergo adhesion strengthening upon contact with ICAM-1 at stasis, these states are permissive for leukocyte rolling on ICAM-1 under physiological shear flow. Rolling mediated by low-avidity LFA-1 interactions with ICAM-1 may stabilize rolling initiated by specialized vascular rolling receptors and allow the leukocyte to arrest on vascular endothelium upon exposure to stimulatory endothelial signals.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The recruitment of leukocytes into sites of inflammation and lymphoid tissues involves leukocyte interactions with vascular endothelium under shear flow. These interactions are initiated by weak reversible recognition of endothelial ligands followed by firm adhesion mediated by members of the integrin family and their respective Ig family ligands (1). ß₂ Integrins are the major cell-cell adhesion receptors on leukocytes, and they participate in versatile adhesive processes of these cells within the vasculature (1). These processes include the arrest of immune cells on vessel walls at sites of inflammation or lymphoid tissues, transendothelial migration, and final localization at sites of Ag presentation (1, 2). LFA-1 (_Lß₂) is the major ß₂ integrin on lymphocytes that mediates binding to ICAMs, Ig family integrin ligands expressed on venular endothelia and on APC, effector immune cells, and activated platelets (3, 4, 5, 6). ICAM-1 (CD54) is the major LFA-1 ligand; it is ubiquitously found on most leukocytes and endothelial cells, where it is up-regulated during inflammation. ICAM-1 is a bent rod and contains five Ig-like domains oriented head to tail (7). Domain 1 of ICAM-1, the most membrane-distal, contains the primary site of contact for LFA-1 (8).

LFA-1 has been postulated to require activation to support firm adhesion to its different ICAM ligands (9, 10). High-avidity LFA-1 binding to endothelial ICAM-1 is triggered on circulating leukocytes after they have established weak rolling adhesions on the vessel wall through selectins, CD44, or VAP-1 (1, 11). However, several integrins like ₄ß₁ and ₄ß₇ occur in constitutively adhesive states on circulating leukocytes and can also support rolling adhesions on their respective endothelial ligands (12, 13, 14, 15). The inability of ß₂ integrins to participate in similar rolling interactions between leukocytes and vascular endothelium has been attributed to the critical dependence of their adhesiveness on prior activation of chemoattractant receptors coupled to heterotrimeric G-proteins on circulating leukocytes (16, 17, 18).

Controlled flow assays performed in laminar flow chambers have been useful in the characterization of transient and weak adhesive interactions between cells and surface-immobilized ligands or Abs (19). Using these assays to generate low-range detachment forces, relatively weak adhesions between integrins on resting cells and immobilized ligands can be detected and quantified (20, 21, 22). Testing LFA-1 adhesiveness to ICAM-1 in the human leukemia cell Jurkat as a prototypic T cell model, we show in this paper that LFA-1 can spontaneously form weak but specific adhesion to immobilized ICAM-1. Subsets of PBL were also found to develop LFA-1 adhesions of considerable strength upon short contact with ICAM-1-coated surfaces. Under identical conditions, stable adhesion to ICAM-1 of K562 cells transfected with LFA-1 was not observed. Nevertheless, when exposed to physiological shear stresses, LFA-1-expressing K562 cells established persistent rolling on ICAM-1. These results extend previous findings, which showed that the isolated I-domain of LFA-1 can engage in rolling interactions with high-density ICAM-1 and ICAM-3 (23). This is the first indication that the intact LFA-1 integrin, when placed in a permissive cellular environment, can support rolling adhesions on physiological densities of ICAM-1 under physiological shear flow. Furthermore, this study provides evidence that cell-type-dependent differences in LFA-1 avidity states, but not intrinsic affinity properties, control LFA-1 ability to support rolling adhesions on ICAM-1 under physiological shear flow.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abs and reagents

All mAbs were used as purified IgGs. The function-blocking anti-LFA-1 mAb NKI-L15 and the nonblocking mAbs SPV-L7 and NKI-L16, directed against the subunit of LFA-1, were produced as previously described (24). TS1.22, a function-blocking anti-LFA-1 mAb directed against the I-domain of the integrin subunit (25), and mAb R 6.5, an anti-ICAM-1 mAb directed against the LFA-1 recognition site on the first Ig-like domain of ICAM-1 (26), were the generous gift of Dr. T. Springer, (Center for Blood Research, Harvard Medical School, Boston, MA). TS2.4, a nonblocking anti-LFA-1 mAb (25), was kindly provided by Dr. E. Martz. HP 1/2, a very late Ag-4 (VLA-4)³-specific mAb (27), was a kind gift of Dr. R. Lobb (Biogen, Cambridge, MA). The anti-ß₂ mAb KIM185 was used to activate LFA-1 (28). Purified mouse IgG (Zymed, South San Francisco, CA) was used as a control. Fluorescein isothiocyanate-conjugated goat anti-mouse IgG and goat F(ab')₂ anti-mouse IgG (Zymed), or goat anti-human Fc (Jackson Immunoresearch, West Grove, PA) were used as secondary Abs in FACS analysis and immunofluorescence microscopy. Recombinant soluble ICAM-1 (sICAM-1), encompassing the entire extracellular domain of human ICAM-1 was purchased from R&D Systems (Minneapolis, MN). ICAM-1-Fc, consisting of the entire extracellular portion of human ICAM-1 fused to human IgG Fc (29), was the generous gift of Dr. L. Klickstein (Brigham and Women’s Hospital, Boston, MA). A VCAM-1-Fc fusion protein, consisting of the two N-terminal domains of human VCAM-1, fused to human IgG Fc was a gift of Dr. R. Lobb. BSA- (fraction V), protein A-, Ca²⁺-, and Mg²⁺-free HBSS and Ficoll-Hypaque 1077 were obtained from Sigma (St. Louis, MO). Human serum albumin (HSA; fraction V) and PMA were purchased from Calbiochem (La Jolla, CA).

Cells

K562 cells stably expressing LFA-1 (30) were maintained in RPMI 1640/Iscove’s medium (3:1 v/v) supplemented with 5% heat-inactivated FCS (Biological Industries, Beit Hahemek, Israel), 1% penicillin/streptomycin (Bio Lab, Jerusalem, Israel), and 2 mg/ml G418 (Calbiochem) to selectively maintain the transfected population. The ß₂ deletion mutant _Lß₂/724 generated by truncation of the ß₂ cytoplasmic tail at amino acid position 724 close to the transmembrane region (30) was stably expressed in K562 cells maintained as above. The Jurkat T lymphoblastoid cell line was maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS (Sigma), 2 mM L-glutamine, and 1% antibiotics. Human peripheral blood lymphocytes (obtained from healthy donors) were isolated from citrate-anticoagulated whole blood by dextran sedimentation and density separation over Ficoll-Hypaque (20). The mononuclear cells thus obtained were washed and further purified on nylon wool and by further panning on serum-coated dishes. The resulting purified PBL were more than 90% CD3-positive T lymphocytes. PBL were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS for 15–18 h after isolation and purification until use.

Flow cytometry

A total of 10⁶ cells were incubated for 1 h on ice in 20 µg/ml of the LFA-1 mAb TS1.22 and control mAb 4B9 in a solution of PBS with 1% BSA and 0.05% sodium azide (FACS buffer). Cells were washed three times in FACS buffer and incubated for 15 min on ice with 5 µg/ml FITC-conjugated goat anti-mouse IgG (Zymed) in FACS buffer. Cells were washed three times and analyzed by FACSort flow cytometer (Becton Dickinson).

ICAM-1-Fc binding to cells was determined by immunofluorescence by incubating 50,000 cells in TSA binding medium (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM Ca²⁺, 2 mM Mg²⁺, and 5 mg/ml BSA) with or without LFA-1 blocking mAb (20 µg/ml) for 10 min at room temperature (RT) in a 96-well V-bottom plate. Different concentrations of purified soluble ICAM-1-Fc were added, and the suspension was incubated for 30 min at 37°C. The cells were washed with TSA and incubated for 30 min at RT with FITC-conjugated goat-anti-human Fc-specific Ab, and then they were washed again, and the percentage of positively stained cells was measured by FACScan. Net binding values were depicted as: (percentage of positive cells stained in the absence of the LFA-1 blocking mAb (NKI-L15)) - (percentage of cells stained in the presence of the LFA-1 blocking mAb). The concentration of sICAM-1Fc that gave half-maximal net ICAM-1-Fc staining was determined for the different experimental groups.

Fluorescent beads adhesion assay

Cells were resuspended in TSA binding medium (5 x 10⁶ cells/ml). A total of 50,000 cells were preincubated with or without LFA-1-blocking mAb (20 µg/ml) for 10 min at RT in a 96-well V-bottom plate, alone or in the presence of 100 ng/ml PMA. Ligand-coated TransFluoSpheres (1 µm; Molecular Probes, Eugene, OR) were incubated with different experimental groups (at a ratio of 20 beads/cell) for 30 min at 37°C. The cells were washed with TSA, and LFA-1-mediated bead adhesion was measured by flow cytometry using FACScan as previously described (31). Values are depicted as integrin-specific adhesion, i.e., (percentage of cells binding to beads in the absence of the LFA-1 blocking mAb (NKI-L15)) - (percentage of cells binding to beads in the presence of the LFA-1 blocking mAb).

Confocal microscopy

Cells were fixed with 0.5% paraformaldehyde and stained with TS2/4 mAb (10 µg/ml) for 30 min at 37°C before incubation with FITC-labeled goat F(ab')₂ anti-mouse IgG mAb for 30 min at RT. Cells were attached to polyl-lysine-coated glass slides, and cell surface distribution of integrins was determined by confocal laser scanning microscopy (CLSM) at 488 nm with a krypton/argon laser (Bio-Rad 1000; Bio-Rad, Hercules, CA).

Laminar flow assays

Preparation of adhesive substrates. Recombinant sICAM-1 was dissolved in coating medium (PBS buffered with 20 mM bicarbonate (pH 8.5)) and directly coated to a polystyrene plate (60 x 15 mm petri dish; Becton Dickinson, Lincoln Park, NJ) for 2 h at 37°C. The plate was washed three times with PBS and blocked with HSA (20 mg/ml in PBS) overnight at 4°C. ICAM-1-Fc-coated substrates were prepared as previously described (32). Protein A (20 µg/ml in coating medium) was spotted onto a polystyrene plate, and the substrate was washed and blocked with HSA (20 mg/ml in PBS). The protein A-coated substrate was overlaid overnight at 4°C with 0.5–10 µg/ml of ICAM-1-Fc in PBS supplemented with HSA. The adsorbed chimeric ICAM-1 was shown to bind the protein A-coated substrate exclusively through its Fc region, as verified by the ability of excess human IgG to compete off all ICAM-1-Fc adsorption onto the substrate. LFA-1-specific mAbs were coated (5 µg/ml in PBS/HSA) on surfaces precoated with protein A and overlaid with IgG-Fc-specific rabbit anti-mouse (5 µg/ml in PBS/HSA). The density of functional sICAM-1 coated on the substrate was determined by saturation binding of ¹²⁵I-labeled anti-ICAM-1 mAb, R 6.5. The site density of ICAM-1-Fc adsorbed to the protein A substrates was determined from overlay experiments with ¹²⁵I-labeled ICAM-1-Fc. Labeled fusion protein or ICAM-1 mAb were prepared by the chloramine T method (33), using a ratio of 50 µg protein to 0.5 mCi of carrier-free [¹²⁵I]Na (Amersham, Buckinghamshire, U.K.).

Determination of cell adhesion to ICAM-1 in stasis and under flow. A polystyrene coated with an adhesive ligand was assembled in a parallel-plate laminar flow chamber (260-µm gap) and mounted on the stage of an inverted phase contrast microscope (Diaphot 300; Nikon, Tokyo, Japan) as previously described (13, 34). Cell images were videotaped with a long-integration LIS-700 CCD video camera (Applitech, Holon, Israel) and a Panasonic AG-6730 SVHS video recorder (Panasonic, Osaka, Japan). Cultured cells were washed twice with cation-free H/H medium (HBSS containing BSA (2 mg/ml) and 10 mM HEPES (pH 7.4)) containing 5 mM EDTA, resuspended in H/H medium, and kept at RT until use. Cells were suspended at RT in binding medium (H/H supplemented with 1 mM each of Ca²⁺ and Mg²⁺; also referred to as physiological binding medium). Alternatively, cells were suspended in H/H supplemented with 2 mM Mg²⁺, alone or with 0.5 mM EGTA (Mg²⁺/EGTA). Suspended cells (10⁶ cells/ml) were perfused through the flow chamber and allowed to settle on the plate for different periods in the absence of flow or were perfused through the chamber for 1 min at a constant low shear flow (0.25 dyn/cm², unless otherwise indicated) before being subjected to an incremental increase in shear stress. Shear flow was generated and automatically controlled with an automated syringe pump (Harvard Apparatus, Natick, MA) attached to the outlet side of the flow chamber. The wall shear stress was increased step-wise every 5 s (by a programmed set of flow rates) until it reached 10 dyn/cm². At the end of each 5-s interval at a particular shear stress, the number of cells that remained bound (stationary or rolling; see below) was determined relative to the number of cells originally settled in stasis or relative to the number of cells that had accumulated at low shear flow on the ICAM-1-coated field. Cellular interactions were determined on two representative fields of view (each typically 0.34 mm² of area). For Ab inhibition studies, cells (10⁷/ml) were preincubated (5 min; 4°C) in H/H medium with LFA-1 blocking mAb (20 µg/ml). The cells were diluted 1:10 into binding medium without washing out the Abs, and the suspension was perfused into the flow chamber. To selectively block high-affinity LFA-1 on compared cells, cells were suspended in binding medium containing soluble ICAM-1-Fc (0.5–1 µg/ml) or control VCAM-1-Fc for 5 min at 24°C and then perfused through the flow chamber without diluting out the soluble ligands. For metabolic energy inhibition experiments, cells (2 x 10⁷/ml) were treated (10 min; 22°C) with 0.1% sodium azide (Sigma) and 50 mM 2-deoxyglucose (2-DOG; Sigma) in H/H medium and then diluted (1:5) with binding medium and immediately perfused into the flow chamber.

Determination of rolling fractions and rolling velocities. Cells interacting with ICAM-1 were defined as arrested if they adhered to the substrate and remained motionless throughout the shear-flow interval examined. Cellular motions were defined as rolling if they persisted for at least 4 s at a mean velocity >2 µm/s that was reduced at least 20-fold compared with a freely flowing cell at a given shear stress. Some cells that arrested after tethering to high-density ICAM-1 under low shear flow began rolling when subjected to elevated shear stresses. The rolling fraction at a given shear stress was defined as: [(no. of rolling cells) x 100]/(no. of rolling cells + no. of stationary cells). The mean rolling velocities of cell populations at a given shear stress were determined by measuring the displacements of 15–20 cells over 5-s shear intervals.

Image analysis. Quantitative analysis of cell displacements was performed with the WSCAN-Array-3 imaging software (Galai, Migdal-Ha’emek, Israel). Video frame images were digitized using a Matrox Pulsar frame grabber (Matrox Graphics, Dorval, Quebec, Canada) and processed by software running on an Atlas Pentium MMX-200 work station. The program output provided the coordinates of the center point of each cell with time, and analysis was performed with a dedicated software run under Matlab 5.2 (developed in collaboration with the lab of Prof. David Malah, Signal and Image Processing Lab, Technion, Haifa, Israel). Selected video images were also digitized on a G3 Macintosh using a Media100 frame grabber, and cellular motions were tracked using the DIAS imaging software (Solltech, Oakdale, IA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
LFA-1 expressed in K562 cells supports rolling adhesions on ICAM-1 under physiological shear flow

To assess the magnitude of adhesiveness spontaneously developed by LFA-1 during brief cell contact with ICAM-1, we used a controlled flow assay to study weak LFA-1-dependent adhesion of various cell types to ICAM-1 substrates. LFA-1-mediated adhesion of the T cell leukemia line Jurkat was compared with that of K562 cells transfected with LFA-1 cDNA (K562-LFA-1 cells). The level of LFA-1 expressed on both cell types was comparable, although it was somewhat more heterogeneous in the K562 transfectants (Fig. 1). Substrates were coated with a recombinant human ICAM-1-Fc fusion protein by overlaying the protein on plastic precoated with protein A. Unstimulated Jurkat and K562-LFA-1 cells failed to develop firm adherence to immobilized ICAM-1 in conventional tip plate adhesion assays (data not shown). Nevertheless, when allowed to briefly settle on ICAM-1-coated substrates assembled in a flow chamber, significant fractions of both cell types developed considerable LFA-1-specific adhesion to ICAM-1 even without stimulation (Fig. 2A). Adhesion of both cell types was entirely LFA-1-dependent because it was blocked in the presence of the LFA-1-blocking mAb TS1.22 or by integrin inhibition in the presence of EDTA (Fig. 2A and data not shown). VLA-4-expressing K562 cells failed to adhere to identical ICAM-1 substrates (Fig. 2A). Replacement of Ca²⁺ by Mg²⁺, which induces a high-affinity state on LFA-1 without dependence on intracellular signaling events, increased LFA-1 adhesiveness to ICAM-1 in both cell types to a comparable degree (Fig. 2A). The fraction of Jurkat cells that spontaneously adhered to ICAM-1 was markedly higher than that of K562-LFA-1 cells, both in medium containing Ca²⁺ and Mg²⁺ and in medium containing Mg²⁺ alone.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Expression of LFA-1 on K562-LFA-1, Jurkat, and PBL. Cells were stained with the anti-LFA-1 mAb TS1.22 and then with FITC-labeled goat anti-mouse and were analyzed by FACS (open histograms). Negative control staining with murine IgG is shown in the filled histograms.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. LFA-1-mediated adhesion to ICAM-1 in different unstimulated LFA-1-expressing cells. A, K562-LFA-1 cells or Jurkat cells were introduced into the flow chamber in the indicated medium and allowed to settle for 1 min on a substrate coated with ICAM-1-Fc as described in Materials and Methods. The substrate contained 375 sites/µm2 of ICAM-1-Fc molecules/µm2 (750 ICAM-1 sites/µm2). Cells were then subjected to a shear stress of 0.25 dyn/cm2 for 10 s before incremented shear stresses were applied, as indicated in the figure. Each increment lasted for 5 s, and the number of cells remaining adherent to ICAM-1 at the end of each interval of incremented shear was determined in two representative fields. The mean values ± range are shown in the figure. Reduction in the number of LFA-1 K562 cells remaining bound in the field was a result of cell detachment from the substrate and not of cells rolling out of the field of view. No adhesion of K562-LFA-1 or Jurkat cells was detected on substrates coated with protein A or with HSA alone. One of six similar independent experiments. B, Effect of shear stress and cation composition in the binding medium on the ability of LFA-1-expressing cells to roll on ICAM-1. Fractions of adherent cells within each cell type that maintained steady rolling on the ICAM-1 substrate at the indicated shear stresses are depicted. Rolling fractions were determined in two representative fields at a shear stress of 1.5 dyn/cm2 or 3.5 dyn/cm2. Average values ± range are shown. C, Images of K562-LFA-1 cells rolling on ICAM-1-Fc coated at 950 ICAM-1 sites/µm2 at a shear stress of 3.5 dyn/cm2. The figure shows superposition of sequential images of cells captured from videotaped cellular motions at 0.28-s intervals. Cell images analyzed by the imaging software DIAS (see Materials and Methods) are represented as filled superimposed circles. Representative cells rolling with either a persistent or a jerky pattern are shown in medium containing either Ca2+ and Mg2+ (top panel) or Mg2+ alone (bottom panel). The arrow in the top panel indicates the position of a representative rolling cell at the position where it began to detach from the ICAM-1-coated substrate. The velocity of freely flowing cells at a shear stress of 3.5 dyn/cm2 was >400 µm/s.

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Effect of ICAM-1 type and site density on K562-LFA-1 accumulation and rolling efficiency. Rolling attachments formed by K562-LFA-1 cells on different ICAM-1 substrates under shear flow. Cells were perfused in the presence of 2 mM Mg2+ and allowed to accumulate at 0.25 dyn/cm2 for 60 s. The shear stress was increased to 0.5 dyn/cm2 for 5 s. The number of cells remaining adherent at the end of this interval is depicted by the hatched bars, and the fraction of adherent cells rolling on ICAM-1 at 0.5 dyn/cm2 is shown in parentheses on top of each bar graph. Cells remaining bound at 0.5 dyn/cm2 were subjected to incremented shear stresses of 0.75, 1.0, and 1.5 dyn/cm2, with each incremented shear lasting for 5 s. The number of cells remaining adherent at 1.5 dyn/cm2 (average ± range of two representative fields) is depicted by the filled bars, and the fraction of rolling cells at this shear stress is shown in parentheses on each bar. One of four similar experiments.

Agonists of VLA-4 affinity to VCAM-1 such as Mn²⁺ or Mg²⁺ have been shown to slow integrin-mediated rolling (13). Therefore, we asked how LFA-1 rolling on ICAM-1 is regulated by divalent cations. Although rolling adhesions of K562-LFA-1 cells increased in the presence of Mg²⁺ alone (Fig. 2, A and B), the velocities of cells rolling on ICAM-1 in the presence of this cation alone were not significantly different from those measured in physiological concentrations of Ca²⁺ and Mg²⁺ (Fig. 4A). LFA-1 binding to ICAM-1 in the presence of Mg²⁺ can be increased by chelation of residual Ca²⁺ in the presence of EGTA because Ca²⁺ exclusion stabilizes LFA-1 in a conformation with high affinity for ICAM-1 (36). Indeed, in the presence of Mg²⁺/EGTA, the fraction of adherent K562-LFA-1 cells rolling on ICAM-1 was sharply reduced (Fig. 4B, inset), and the cells that could still roll on ICAM-1 did so at 5- to 7-fold slower velocities than in the presence of Mg²⁺ alone (Fig. 4B). Increasing LFA-1 affinity to ICAM-1 in the presence of Mg²⁺/EGTA thus resulted in the majority of K562-LFA-1 cells developing firm adherence to ICAM-1 substrates. Elevation of ICAM-1 density caused all cells to arrest on ICAM-1 in the presence of Mg²⁺/EGTA (data not shown). Thus, increasing LFA-1 affinity restricts rolling interactions and leads to arrest of K562 cells interacting with ICAM-1 under shear flow.

View larger version (39K): [in this window] [in a new window]   FIGURE 4. Effect of cations and shear stress on LFA-1-mediated rolling dynamics on ICAM-1. A, Velocities of K562-LFA-1 cells rolling on ICAM-1-Fc coated at 950 ICAM-1 sites/cm2 in physiological medium or medium containing 2 mM Mg2+. Cells were tethered to the substrate at low shear stress of 0.25 dyn/cm2 for 1 min and then subjected to incremented shear stresses as described in Fig. 2. Displacement of rolling cells was determined over 5-s periods at the indicated shear stresses. Velocities were determined only for persistently rolling cells that remained adherent throughout the entire shear stress interval, indicated below each bar. Each bar shows the mean rolling velocity (and SEM) for 15–20 cells. B, Velocities of K562-LFA-1 cells rolling on ICAM-1-Fc coated at 750 ICAM-1 sites/cm2 in Mg2+ or Mg2+/EGTA. The fractions of adherent cells rolling at each indicated shear stress are shown in the inset. C, Displacement in the direction of flow of representative K562-LFA-1 cells rolling on high-density ICAM-1-Fc (at 1900 ICAM-1 sites/cm2) in physiological medium at incremented shear stresses. Cells were allowed to accumulate for 1 min on the ICAM-1 substrate at 0.25 dyn/cm2 and then were subjected to incremented shear stresses as described in Fig. 2. The wall shear stress was increased at the indicated time points of the observation period. Cell positions were determined at 0.2-s intervals by WSCAN Array-3 image analysis software, as explained in Materials and Methods. Only cells remaining bound over the entire assay period (up to a shear stress of 8.25 dyn/cm2) were analyzed. Results shown in A–C are representative of three independent experiments.

LFA-1 tail-deletion mutant expressed in K562 cells exhibits high avidity to ICAM-1 concomitant with loss of rolling

To study how increased LFA-1 avidity to ICAM-1 can modulate interactions between this integrin ligand pair under shear flow, we compared the dynamic properties of wild-type (wt) LFA-1 and an LFA-1 cytoplasmic tail deletion mutant. The ß₂ subunit tail deletion mutant _Lß₂/724 exhibits constitutive high avidity to ICAM-1 and high expression of the activation epitope M24 concomitant with ligand-independent clustering (30, 37), yet it binds soluble ICAM-1-Fc with affinity similar to that of wt LFA-1 (37). K562 cells expressing the deletion mutant at levels identical to those of K562-LFA-1 cells (not shown) accumulated on both high- and low-density ICAM-1 at strikingly higher levels than did K562-LFA-1 under shear flow (Fig. 5A). The ß deletion mutant-expressing cells immediately arrested upon tethering to ICAM-1 and remained arrested on the ligand without ability to roll even when exposed to elevated shear stresses that caused their detachment from ICAM-1 (Fig. 5A). The resistance to detachment from ICAM-1 of LFA-1 mutant-expressing cells was markedly higher than that of K562-LFA-1 cells on both bivalent (high-density) or monovalent (low-density) ICAM-1 (Fig. 5). No rolling was supported by the mutant LFA-1 on ICAM-1 at any ligand concentration or shear stress tested, in contrast to highly efficient rolling mediated by wt LFA-1 (Fig. 5A). These findings are consistent with the notion that enhanced LFA-1 avidity to ICAM-1 results in immediate adhesion strengthening under shear flow at the expense of reversible rolling interactions. Thus, even in a cellular environment permissive for LFA-1-mediated rolling like that of K562 cells, interference with the cytoskeletal association of LFA-1 through tail deletion abrogates the ability of the integrin to support rolling adhesions under physiological flow.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. LFA-1 tail deletion mutant expressed in K562 cells exhibits increased avidity to ICAM-1 and loss of rolling. Accumulation under flow on ICAM-1 and resistance to detachment by elevated shear stresses of K562 cells transfected with wt or ß2 tail deletion mutant of LFA-1. K562 cells expressing either wt LFA-1 or the LFA-1 cytoplasmic tail mutant Lß2/724 were allowed to accumulate for 1 min on either ICAM-1-Fc (950 ICAM-1 sites/µm2; A) or sICAM-1 (100 sites/µm2; B) at 0.25 dyn/cm2 and then were subjected to incremented shear stresses as described in Fig. 2. The different ICAM-1 forms were coated as described in Materials and Methods. The number of cells originally accumulated and the number of cells remaining adherent at the end of each shear stress interval were determined in two fields of view. Rolling fractions of wt and tail deletion LFA-1 mutant at a shear stress of 0.5 dyn/cm2 are shown in parentheses. One of three independent experiments.

Metabolic energy is essential for optimal integrin function (38). Energy depletion of Jurkat cells by sodium azide and 2-deoxyglucose enhances VLA-4-mediated rolling on VCAM-1 at the expense of firm adhesion associated with reduction of integrin affinity to ligand (22). Therefore, we asked whether a similar energy depletion treatment of resting T cells would enhance their LFA-1 rolling on ICAM-1. However, metabolic energy depletion of Jurkat cells and PBL by azide/2-deoxyglucose greatly decreased LFA-1 adhesion to ICAM-1 without promoting LFA-1 rolling (Fig. 6). Phorbol esters like PMA are strong agonists of integrin adhesion in leukocytes (13, 39, 40, 41, 42). PMA treatment enhances LFA-1 adhesiveness in T cells by augmenting integrin clustering rather than affinity to ligand (43, 44). Therefore, we set out to determine whether PMA stimulation of T cell LFA-1 can render a fraction of LFA-1-expressing cells capable of rolling on ICAM-1 under shear flow. PMA stimulation of PBL increased the number of PBL that arrested on ICAM-1 under low flow by 3-fold, but did not render any subset of treated cells capable of rolling on ICAM-1 (Fig. 6B).

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Effect of PMA stimulation and energy depletion of T cells on LFA-1 adhesiveness to ICAM-1 under shear flow. Resting, energy-depleted (azide-treated), or PMA-activated Jurkat (A) or PBL (B) were perfused into the chamber at low shear stress of 0.25 dyn/cm2 in physiological binding medium containing 1 mM each of Ca2+ and Mg2+. Cells were allowed to accumulate for 1 min on ICAM-1-Fc coated at 1900 ICAM-1 sites/µm2. Accumulated cells were subjected to incremented shear stresses as described in Fig. 2, and the number of cells remaining adherent at the end of each shear stress interval was determined. Values in B represent the mean of two fields of view. Insets, rolling fractions of untreated or treated cells at a shear stress of 1.5 dyn/cm2, determined as in Fig. 2B. The rolling fraction of K562-LFA-1 cells under identical experimental conditions is shown for comparison. One of three independent experiments.

The markedly stronger adhesion developed by T cell LFA-1, together with its inability to support rolling, suggests that LFA-1 avidity to ICAM-1 is higher in Jurkat T cells, as well as in PBL, than in K562 cells. Consistent with such a possibility, Jurkat cells could bind ICAM-1-coated beads better than K562 cells could in physiological medium in the absence of shear flow, even without cell stimulation (Fig. 7A). Thus, markedly enhanced adhesiveness of Jurkat cell LFA-1 to surface-bound ICAM-1 could be observed even in shear-less conditions. Integrin-mediated adhesion to physiological ligands is dynamically regulated after ligand binding (29, 42, 44, 45). LFA-1 adhesion strengthening is diffusion-limited and depends strongly on contact time with the immobilized ligand (43). Consistent with the inherent defect in adhesion strengthening of wt LFA-1 on K562 cells, K562-LFA-1 transfectants could not develop firm adhesion to ICAM-1 even after prolonged contact with ligand (Fig. 7B). When statically adhered to low- or high-density ICAM-1, only a small fraction of K562 cells developed detectable adhesion to ICAM-1 during 1 min of contact, and a 3-fold longer contact period with the ligand did not increase the fraction of K562-LFA-1 cells firmly adherent to ICAM-1 (Fig. 7B). Notably, even though Jurkat cell adhesion developed over 1 min of contact was much higher than that of the K562-LFA-1 cells, it could be further enhanced by a 3-fold or 10-fold prolonged contact period with ICAM-1, in contrast to K562-LFA-1 adhesion (Fig. 7B and data not shown). These experiments collectively suggest that Jurkat LFA-1 and PBL develop firmer adhesion to surface-bound ICAM-1 than do K562-LFA-1 cells because of higher constitutive avidity of LFA-1 and higher contact-dependent adhesion strengthening of the integrin expressed in these T cells compared with K562 cells.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. LFA-1 avidity to ICAM-1 is higher in resting Jurkat T cells than in K562 cells. A, Binding of ICAM-1-coated beads to K562-LFA-1 and Jurkat cells. Cells were suspended in the presence of the indicated agonists for 10 min with fluorescent beads coated with ICAM-1 at a cell:bead ratio of 1:20. After washing off unbound beads, the fraction of cells with bound beads was determined by FACS. Background bead binding was determined by including an LFA-1 blocking mAb in the reaction suspension and was less than 5%. Results are expressed as the mean percentage of cells with bound beads from two independent experiments, each conducted in triplicate. Mean values ± SD of LFA-1-specific adhesion are depicted. PMA was present at 100 ng/ml, and the LFA-1 activating mAb KIM185 was used at 5 µg/ml. ND, not determined because of cell aggregation induced by the mAb. B, Development of adhesion over contact time by LFA-1 in K562 cells and Jurkat. Cells were allowed to settle in physiological binding medium for 1 or 3 min in stasis on sICAM-1 coated at 200 sites/cm2 and then subjected to detachment by shear force of 0.25 dyn/cm2 for 10 s and then 0.5 dyn/cm2 for an additional 5 s. The fraction of originally settled cells remaining bound at the end of the assay is shown for cells settled for different periods. Results represent the mean ± range of two fields of view. Results are representative of three independent experiments.

View larger version (19K): [in this window] [in a new window]   FIGURE 8. Susceptibility of LFA-1-dependent adhesion of K562 cells and PBL to inhibition by soluble ICAM-1-Fc. Cells were suspended in physiological binding medium containing soluble ICAM-1-Fc at the indicated concentrations for 5 min at 24°C and then perfused unwashed at 0.25 dyn/cm2 for 1 min over a substrate coated with sICAM-1 at 200 sites/µm2. Accumulated cells were subjected to incremented shear stresses as described in Fig. 2, and the number of cells remaining adherent at the end of each shear stress interval was determined. Values represent the mean (± range) of two fields of view. Control VCAM-1-Fc had no inhibitory effect on cell accumulation or on adhesion to the ICAM-1 substrates at the indicated concentrations (data not shown). A representative experiment of three.

View larger version (48K): [in this window] [in a new window]   FIGURE 9. Constitutive LFA-1 distribution on Jurkat and K562-LFA-1 cells determined by confocal microscopy. K562 cells (top panel) or Jurkat cells (bottom panel) were prefixed in medium containing 1 mM each of Ca2+ and Mg2+, and cells were stained with the anti-LFA-1 mAb TS2/4 and then with FITC-labeled goat F(ab')2 anti-mouse IgG. Single representative cells from each experimental group are depicted. The majority of LFA-1 is dispersed, but small patches can be seen on both cell types.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

To support reversible rolling adhesions, major rolling receptors like selectins need to rapidly and reversibly bind a small number of ligands at the adhesive contact zone (47). To engage in rolling interactions with its endothelial ligand, LFA-1 might also need to be prevented from engaging in highly multivalent interactions with ICAM-1. We have shown that a ß₂ tail-truncated LFA-1 mutant with conserved affinity to ICAM-1 but impaired cytoskeletal constraints in the K562 cell system exhibited enhanced ability to undergo rapid adhesion strengthening on ICAM-1 under shear flow. These results suggest that the inability of wt LFA-1 to mediate rolling in Jurkat cells and PBL subsets may also result in reduced cytoskeletal constraints and hence increased adhesion strengthening of wt LFA-1 in T cells relative to those imposed on the integrin in K562 cells. Ligand-induced LFA-1 clustering in T cells requires the release of cytoskeletal restraints on LFA-1, which restricts its lateral clustering (42, 43, 44). Notably, release of cytoskeletal constraints of LFA-1 and adhesion strengthening is facilitated upon T cell activation by PMA without alteration of LFA-1 affinity (36). PMA fails to induce adhesion strengthening to ICAM-1 in K562 cells (37, 42), which supports major reduced susceptibility of LFA-1 association with the actin cytoskeleton to PMA stimulation in these cells. Consistent with an inherent defect in LFA-1 release of cytoskeletal constraints in K562 cells, cytochalasin D treatment has been reported to induce LFA-1 clustering in resting PBL but not in K562 cells (30, 42). Higher cytoskeletal restraints of LFA-1 in K562 cells and impaired adhesion strengthening may keep LFA-1:ICAM-1 interactions at low avidity and may allow reversible fast-breaking rolling interactions to form under constant shear flow. Thus, a cell-type-dependent restriction in LFA-1-mediated adhesion strengthening, rather than intrinsic integrin properties, appears to control the ability of LFA-1 to promote rolling adhesions of circulating leukocytes on its endothelial ligand. Enhanced constitutive clustering of LFA-1 before ligand binding has been shown to correlate with increased avidity to ICAM-1 in various cellular systems (42, 43). However, the impaired ability of K562-LFA-1 to support high-avidity adherence was not because of impaired constitutive clustering of LFA-1 in K562 cells relative to Jurkat T cells or PBL (Fig. 9 and Refs. 30, 42). Taken together, these observations suggest that the deficient adhesion strengthening properties of LFA-1 in K562 cells and the acquisition of a rolling phenotype by LFA-1 in these cells reflect a defect in postligand binding clustering, rather than a constitutive, i.e., preligand binding, LFA-1 clustering defect.

LFA-1-mediated rolling requires an intact I-domain, suggesting the critical contribution of this module to recognition of ICAM-1 under both static and dynamic conditions (48). The I-domain of LFA-1 has recently been shown to act as a transient Mg²⁺-dependent binding module that supports transient rolling adhesions on substrates containing ICAM-1 or ICAM-3 (23). Unstimulated T cell LFA-1 binds ICAM-1 with a K_d of 100 µM (49); a slightly lower affinity (K_d of 100–200 µM) was estimated for the binding of ICAM-1 to the isolated I-domain (23). This affinity is similar to the recently determined affinity between L-selectin and its endothelial ligand, glycosylation-dependent cell adhesion molecule-1 (50). Nevertheless, the ability of LFA-1 to mediate rolling does not depend on its affinity to ligand because the affinity of LFA-1 to ICAM-1 in the T cell systems that failed to roll on ICAM-1 was not higher than LFA-1 affinity in the K562 cells that efficiently rolled on the LFA-1 ligand. ICAM-1-Fc bound saturably to K562-LFA-1 and Jurkat cells with comparable half-saturation concentrations (10 nM; data not shown), and comparable levels of high-affinity LFA-1 contributed to adhesion of both T cells and K562-LFA-1 cells to ICAM-1 because both cells exhibited similar susceptibility to inhibition by soluble ICAM-1. Therefore, our results suggest that affinity-independent mechanisms of integrin clustering control LFA-1 ability to undergo adhesion strengthening in T cells and also engage weak reversible rolling interactions on ICAM-1 in K562 cells.

Cell adhesive bonds that support rolling adhesions have been suggested to share fast kinetics of formation and dissociation (47, 51). Cellular environment may alter the kinetics of tether bond formation under flow, and faster kinetics of bond formation by LFA-1 can be favored by increased cell-surface availability of the integrin (52, 53, 54). ß₂ Integrins are generally excluded from microvilli in most leukocytes (55). A recent study showed that about 30% of LFA-1 is located on microvilli in transfected K562 cells (55). Our electron microscopy studies confirm that more than 20% of the cell-surface LFA-1 in K562 cells is localized to microvilli, whereas a diminished fraction of LFA-1 on Jurkat cells or PBL is distributed to these cellular projections (Y.V.K., unpublished observations). Nevertheless, exclusion of selectins from microvilli does not preclude their ability to support rolling (54), suggesting that exclusion of LFA-1 from these surface extensions in T cells could not have accounted for the complete loss of rolling activity of LFA-1 expressed in these cells. Indeed, although excluded from microvilli, LFA-1 on Jurkat cells was still able to tether to ICAM-1 at comparably high shear stresses relative to those permissive for LFA-1-mediated tethering of K562 cells to ICAM-1 (data not shown). Therefore, the ability of LFA-1 expressed in K562 cells to mediate rolling appears to be primarily because of a defect in LFA-1 avidity to ICAM-1 rather than because of markedly higher topographical availability of the integrin for rapid interactions with ICAM-1.

The ability of the LFA-1:ICAM-1 pair to participate in rolling adhesions under physiological conditions is clearly more limited than that of the ₄ integrins. First, tethering of LFA-1 to ICAM-1 in the presence of physiological cation concentrations was not as efficient as the tethering of VLA-4 to VCAM-1 or of ₄ß₇ to mucosal addressin cell adhesion molecule-1 (13, 14). Second, LFA-1-mediated rolling required higher-density ICAM-1 than was required for ₄ integrin-mediated rolling on VCAM-1 (data not shown). Third, in contrast to LFA-1, the ability of the VLA-4 integrin to support rolling on its endothelial ligand VCAM-1 is not cell-type-restricted, in that this integrin supports rolling of several types of cells, including T cells, polymorphonuclear cells, and hematopoietic progenitors (12, 13, 14, 15, 56, 57). Therefore, we predict that rolling through LFA-1 may take place mainly in a context of another rolling interaction initiated by a selectin or ₄ integrin. Indeed, recent studies implicate the LFA-1:ICAM-1 interaction in rolling adhesions promoted by P- and L-selectin. The stability of leukocyte rolling mediated by these selectins on inflamed vessel walls is significantly reduced in ICAM-1 knockout mice (58) after either trauma or cytokine-induced inflammation. Reduction in physiologic shear rates has been shown in another study to enhance CD11/CD18-dependent, selectin-independent leukocyte rolling in vivo, suggesting that LFA-1 mediates weak rolling interactions on inflamed vessel walls under particular conditions (59). Our results would predict that to engage in rolling interactions with endothelial ICAM-1, LFA-1 must be restricted from forming multivalent receptor:ligand clusters. Such restriction may be regulated by the cytoskeletal associations of the integrin. Small subsets of circulating leukocytes may regulate the cytoskeletal associations of LFA-1 such that they can engage in reversible rolling adhesions on ICAM-1 in vivo. Taken together, the multistep paradigm of leukocyte adhesion to specific endothelial sites may be more complex than previously realized. Rather than a sequential process conducted by distinct adhesive steps, which are exclusively mediated by either selectins or integrins, leukocytes may use weak interactions of both ₄ integrins and LFA-1 with their respective ligands to stabilize selectin-initiated rolling. The LFA-1-mediated rolling demonstrated in the present study further increases the complexity and functional diversity of adhesive cascades, which determine the cell-type-specific recruitment of circulating leukocytes at sites of inflammation and at specialized hematopoietic beds.

	Acknowledgments

 
We thank Drs. L. Klickstein, T. Springer, R. Lobb, and E. Martz for providing reagents. We also thank Dr. S. Schwarzbaum for editorial assistance; Moshe Miller, Shahar Feldman, Nimrod Peleg, Renato Kresch-Keshet, and Prof. D. Malah of the Signal and Image Processing Lab (Technion) for their help in the development of the computerized imaging software used for cell motion analysis; and Nurit Lichtenstein for assistance in sampling video images for motion analysis.

	Footnotes

 
¹ This work was supported in part by the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities and by The Israel Cancer Research Fund (to R.A.) as well as by a grant (900-512-143) from the Netherlands Organization for Scientific Research (to Y.V.K). R.A. is the Incumbent of the Tauro Career Development Chair in Biomedical Research.

² Address correspondence and reprint requests to Dr. Ronen Alon, Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100 Israel.

³ Abbreviations used in this paper: VLA-4, very late Ag-4; sICAM-1, soluble ICAM-1; HSA, human serum albumin; RT, room temperature; wt, wild type; CLSM, confocal laser scanning microscopy.

Received for publication June 28, 1999. Accepted for publication April 19, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
2. Dustin, M. L., O. Carpen, T. A. Springer. 1992. Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors. J. Immunol. 148:2654.[Abstract]
3. Dustin, M. L., T. A. Springer. 1991. Role of lymphocyte adhesion receptors in transient interactions and cell locomotion. Annu. Rev. Immunol. 9:27.[Medline]
4. Campanero, M. R., M. A. del Pozo, A. G. Arroyo, P. Sanchez Mateos, T. Hernandez Caselles, A. Craig, R. Pulido, F. Sanchez Madrid. 1993. ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway. J. Cell Biol. 123:1007.[Abstract/Free Full Text]
5. Diacovo, T. G., S. J. Roth, J. M. Buccola, D. F. Bainton, T. A. Springer. 1996. Neutrophil rolling, arrest, and transmigration across activated, surface-adherent platelets via sequential action of P-selectin and the ß₂-integrin CD11b/CD18. Blood 88:146.[Abstract/Free Full Text]
6. Katada, Y., T. Tanaka, H. Ochi, M. Aitani, A. Yokota, H. Kikutani, M. Suemura, T. Kishimoto. 1996. B cell-B cell interaction through intercellular adhesion molecule-1 and lymphocyte functional antigen-1 regulates immunoglobulin E synthesis by B cells stimulated with interleukin-4 and anti-CD40 antibody. Eur. J. Immunol. 26:192.[Medline]
7. Staunton, D. E., M. L. Dustin, H. P. Erickson, T. A. Springer. 1990. The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus. Cell 61:243.[Medline]
8. Marlin, S. D., D. E. Staunton, T. A. Springer, C. Stratowa, W. Sommergruber, V. J. Merluzzi. 1990. A soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection. Nature 344:70.[Medline]
9. Dustin, M. L., T. A. Springer. 1989. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341:619.[Medline]
10. van Kooyk, Y., P. van de Wiel-van Kemenade, P. Weder, T. W. Kuijpers, C. G. Figdor. 1989. Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes. Nature 342:811.[Medline]
11. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
12. Jones, D. A., L. V. McIntire, C. W. Smith, L. J. Picker. 1994. A two-step adhesion cascade for T cell/endothelial cell interactions under flow conditions. J. Clin. Invest. 94:2443.
13. Alon, R., P. D. Kassner, M. W. Carr, E. B. Finger, M. E. Hemler, T. A. Springer. 1995. The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J. Cell Biol. 128:1243.[Abstract/Free Full Text]
14. Berlin, C., R. F. Bargatze, J. J. Campbell, U. H. von Andrian, M. C. Szabo, S. R. Hasslen, R. D. Nelson, E. L. Berg, S. L. Erlandsen, E. C. Butcher. 1995. ₄ Integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[Medline]
15. Johnston, B., T. B. Issekutz, P. Kubes. 1996. The ₄-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo. J. Exp. Med. 183:1995.[Abstract/Free Full Text]
16. Bargatze, R. F., M. A. Jutila, E. C. Butcher. 1995. Distinct roles of L-selectin and integrins ₄ß₇ and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined. Immunity 3:99.[Medline]
17. Campbell, J. J., E. P. Bowman, K. Murphy, K. R. Youngman, M. A. Siani, D. A. Thompson, L. Wu, A. Zlotnik, E. C. Butcher. 1998. 6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3ß receptor CCR7. J. Cell Biol. 141:1053.[Abstract/Free Full Text]
18. Campbell, J. J., G. Haraldsen, J. Pan, J. Rottman, S. Qin, P. Ponath, D. P. Andrew, R. Warnke, N. Ruffing, N. Kassam, et al 1999. The chemokine receptor CCR4 in vascular recognition by cutaneous but not intestinal memory T cells. Nature 400:776.[Medline]
19. Chen, S., R. Alon, R. C. Fuhlbrigge, T. A. Springer. 1997. Rolling and transient tethering of leukocytes on antibodies reveal specializations of selectins. Proc. Natl. Acad. Sci. USA 94:3172.[Abstract/Free Full Text]
20. Carr, M. W., R. Alon, T. A. Springer. 1996. The C-C chemokine MCP-1 differentially modulates the avidity of ß₁ and ß₂ integrins on T lymphocytes. Immunity 4:179.[Medline]
21. Weber, C., C. F. Lu, J. M. Casasnovas, T. A. Springer. 1997. Role of _Lß₂ integrin avidity in transendothelial chemotaxis of mononuclear cells. J. Immunol. 159:3968.[Abstract]
22. Chen, C., J. L. Mobley, O. Dwir, F. Shimron, V. Grabovsky, R. L. Lobb, Y. Shimizu, R. Alon. 1999. High affinity VLA-4 subsets expressed on T cells are mandatory for spontaneous adhesion strengthening but not for rolling on VCAM-1 in shear flow. J. Immunol. 162:1084.[Abstract/Free Full Text]
23. Knorr, R., M. L. Dustin. 1997. 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. J. Exp. Med. 186:719.[Abstract/Free Full Text]
24. Keizer, G. D., W. Visser, M. Vliem, C. G. Figdor. 1988. A monoclonal antibody (NKI-L16) directed against a unique epitope on the -chain of human leukocyte function-associated antigen 1 induces homotypic cell-cell interactions. J. Immunol. 140:1393.[Abstract]
25. Huang, C., T. A. Springer. 1995. A binding interface on the I domain of lymphocyte function-associated antigen-1 (LFA-1) required for specific interaction with intercellular adhesion molecule 1 (ICAM-1). J. Biol. Chem. 270:19008.[Abstract/Free Full Text]
26. Smith, C. W., S. D. Marlin, R. Rothlein, C. Toman, D. C. Anderson. 1989. Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion molecule-1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J. Clin. Invest. 83:2008.
27. Sanchez-Madrid, F., M. O. De Landazuri, G. Morago, M. Cebrian, A. Acevedo, C. Bernabeu. 1986. VLA-3: a novel polypeptide associated with the VLA molecular complex: cell distribution and biochemical characterization. Eur. J. Immunol. 16:1343.[Medline]
28. Andrew, D., A. Shock, E. Ball, S. Ortlepp, J. Bell, M. Robinson. 1993. KIM185, a monoclonal antibody to CD18 which induces a change in the conformation of CD18 and promotes both LFA-1- and CR3-dependent adhesion. Eur. J. Immunol. 23:2217.[Medline]
29. Dransfield, I., C. Cabanas, J. Barrett, N. Hogg. 1992. Interaction of leukocyte integrins with ligand is necessary but not sufficient for function. J. Cell Biol. 116:1527.[Abstract/Free Full Text]
30. Lub, M., S. J. van Vliet, S. P. Oomen, R. A. Pieters, M. Robinson, C. G. Figdor, Y. van Kooyk. 1997. Cytoplasmic tails of ß₁, ß₂, and ß₇ integrins differentially regulate LFA-1 function in K562 cells. Mol. Biol. Cell 8:719.[Abstract]
31. Geijtenbeek, T. B. H., Y. van Kooyk, S. J. van Vliet, M. H. Renes, R. A. P. Raymakers, C. G. Figdor. 1999. High frequency of adhesion defects in B-lineage acute lymphoblastic leukemia. Blood 94:754.[Abstract/Free Full Text]
32. Fuhlbrigge, R. C., R. Alon, K. D. Puri, J. B. Lowe, T. A. Springer. 1996. Sialylated, fucosylated ligands for L-selectin expressed on leukocytes mediate tethering and rolling adhesions in physiologic flow conditions. J. Cell Biol. 135:837.[Abstract/Free Full Text]
33. Hunter, W. M., F. C. Greenwood. 1962. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194:495.[Medline]
34. Lawrence, M. B., T. A. Springer. 1991. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65:859.[Medline]
35. Miller, J., R. Knorr, M. Ferrone, R. Houdei, C. P. Carron, M. L. Dustin. 1995. Intercellular adhesion molecule-1 dimerization and its consequences for adhesion mediated by lymphocyte function associated-1. J. Exp. Med. 182:1231.[Abstract/Free Full Text]
36. Stewart, M. P., C. Cabanas, N. Hogg. 1996. T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1. J. Immunol. 156:1810.[Abstract]
37. van Kooyk, Y., S. J. van Vliet, C. G. Figdor. 1999. The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes. J. Biol. Chem. 274:26869.[Abstract/Free Full Text]
38. O’Toole, T. E., Y. Katagiri, R. J. Faull, K. Peter, R. Tamura, V. Quaranta, J. C. Loftus, S. J. Shattil, M. H. Ginsberg. 1994. Integrin cytoplasmic domains mediate inside-out signal transduction. J. Cell Biol. 124:1047.[Abstract/Free Full Text]
39. Faull, R. J., N. L. Kovach, J. M. Harlan, M. H. Ginsberg. 1994. Stimulation of integrin-mediated adhesion of T lymphocytes and monocytes: two mechanisms with divergent biological consequences. J. Exp. Med. 179:1307.[Abstract/Free Full Text]
40. Lawrence, M. B., E. L. Berg, E. C. Butcher, T. A. Springer. 1995. Rolling of lymphocytes and neutrophils on peripheral node addressin and subsequent arrest on ICAM-1 in shear flow. Eur. J. Immunol. 25:1025.[Medline]
41. Laudanna, C., J. J. Campbell, E. C. Butcher. 1996. Role of Rho in chemoattractant-activated leukocyte adhesion through integrins. Science 271:981.[Abstract]
42. Lub, M., Y. van Kooyk, S. J. van Vliet, C. G. Figdor. 1997. Dual role of the actin cytoskeleton in regulating cell adhesion mediated by the integrin lymphocyte function-associated molecule-1. Mol. Biol. Cell 8:341.[Abstract]
43. Kucik, D. F., M. L. Dustin, J. M. Miller, E. J. Brown. 1996. Adhesion-activating phorbol ester increases the mobility of leukocyte integrin LFA-1 in cultured lymphocytes. J. Clin. Invest. 97:2139.[Medline]
44. Stewart, M. P., A. McDowall, N. Hogg. 1998. LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca²⁺-dependent protease, calpain. J. Cell Biol. 140:699.[Abstract/Free Full Text]
45. Yauch, R. L., D. P. Felsenfeld, S. K. Kraeft, L. B. Chen, M. P. Sheetz, M. E. Hemler. 1997. Mutational evidence for control of cell adhesion through integrin recruitment, independent of ligand binding. J. Exp. Med. 186:1.[Free Full Text]
46. van Kooyk, Y., P. Weder, K. Heije, C. G. Figdor. 1994. Extracellular Ca²⁺ modulates leukocyte function-associated antigen-1 cell surface distribution on T lymphocytes and consequently affects cell adhesion. J. Cell Biol. 124:1061.[Abstract/Free Full Text]
47. Alon, R., S. Chen, K. D. Puri, E. B. Finger, T. A. Springer. 1997. The kinetics of L-selectin tethers and the mechanics of selectin-mediated rolling. J. Cell Biol. 138:1169.[Abstract/Free Full Text]
48. Landis, R. C., A. McDowall, C. L. Holness, A. J. Littler, D. L. Simmons, N. Hogg. 1994. Involvement of the "I" domain of LFA-1 in selective binding to ligands ICAM-1 and ICAM-3. J. Cell Biol. 126:529.[Abstract/Free Full Text]
49. Lollo, B. A., K. W. Chan, E. M. Hanson, V. T. Moy, A. A. Brian. 1993. Direct evidence for two affinity states for lymphocyte function-associated antigen 1 on activated T cells. [Published erratum appears in 1994 J. Biol. Chem. 269:10184.]. J. Biol. Chem. 268</