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*
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel; and
Department of Tumor Immunology, University Hospital Nijmegen, Nijmegen, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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, B and C). Rolling
required a threshold of ICAM-1 density because it diminished on
substrates coated at densities below 80
sites/µm2 (data not shown). The median strength
of LFA-1-transfected K562 cell adhesions i.e., the shear stress causing
the detachment of half of originally adherent cells, also varied with
ICAM-1 density. The median strength dropped from 2.5
dyn/cm2 on ICAM-1-Fc coated at 750
sites/µm2 to a median strength of 1
dyn/cm2 on ICAM-1-Fc coated at 190
sites/µm2. Mg2+ was
obligatory for LFA-1-mediated rolling of K562 cells on ICAM-1 because,
in the presence of Ca2+ alone, LFA-1 adhesiveness
was abolished on any ICAM-1 substrate tested (Fig. 2A).
Replacement of Ca2+ with
Mg2+ rendered essentially all adherent K562 cells
rolling on the ligand (Fig. 2B). Recently, ICAM-1
dimerization has been shown to strengthen LFA-1-mediated adhesions
(35). ICAM-1 dimerization within the ICAM-1-Fc chimera did
not appear obligatory for efficient LFA-1-mediated rolling because
LFA-1 adhesiveness to monovalent ICAM-1, sICAM-1, directly immobilized
on the substrate was comparable to LFA-1 adhesiveness to the dimeric
ICAM-1-Fc when both were present at high site densities (Fig. 3). Notably, the ability of K562-LFA-1
cells to roll on ICAM-1 did not depend on a prior contact with
immobilized ICAM-1. Cells tethered at shear flow to high-density ICAM-1
substrates (Fig. 3) continued to efficiently roll on the substrates,
similarly to cells statically adhered and then subjected to shear flow
(Fig. 2).
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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.
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4 integrins is that rolling velocity increases
with increasing shear forces (13, 34). A remarkable
feature of LFA-1-mediated rolling demonstrated by our experiments was
the invariance of rolling velocity with elevated shear stress (Fig. 4, A and B). To rule out the possibility that cells
with higher resistance to detachment from ICAM-1, and therefore higher
avidity of LFA-1 and slower rolling dynamics, were enriched within the
population of cells remaining rolling on ICAM-1 at elevated shear
stresses, single cells were analyzed for their displacement with time
at continuously incremented shear stress. Surprisingly, rolling
velocities of individual K562-LFA-1 remained constant (Fig. 4C, open symbols) or increased only moderately with elevated
shear stress (Fig. 4C, 
). This peculiar dynamic behavior
was seen only in cell subsets that exhibited higher resistance to
detachment by elevated shear stresses. Thus, highly adhesive
LFA-1-expressing K562 cells could overcome the destabilizing effects of
elevated shear stress, which lead to faster rolling and enhanced
detachment in the more weakly adhered K562-LFA-1 cells. 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.
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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).
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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.
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). The similar ICAM-1-Fc dose dependence
of LFA-1 adhesion seen with both cell types suggested that LFA-1
molecules with comparable levels of affinity contributed to LFA-1
adhesiveness in both PBL and K562-LFA-1 cells.
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).
Rather, LFA-1 was found either evenly diffused or in small patches on
the entire cell surface of both cell types, but no major difference in
the number or size of LFA-1 patches between the two cell types was
detected (Fig. 9). Consistent with the CLSM analysis, the levels of
NKL16, a Ca2+-dependent LFA-1
clustering-associated epitope (46), were similar on both
cell types; a comparable ratio of L16 staining to LFA-1 staining was
observed in K562-LFA-1 (0.36 ± 0.06) and in Jurkat cells
(0.25 ± 0.03). These results suggest that before ligand binding,
LFA-1 on both Jurkat cells and K562 cells as well as on PBL
(42) is largely unclustered. Thus, differences in
constitutive preligand binding of LFA-1 in resting T cells and K562
cells cannot account for the firm adhesion to ICAM-1 rapidly developed
by these cells but not by K562-LFA-1 cells under dynamic conditions of
shear flow.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 Kd of 100 µM (49); a slightly lower affinity (Kd 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.
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Acknowledgments |
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Footnotes |
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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.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 Integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[Medline]
4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo. J. Exp. Med. 183:1995.4ß7 and LFA-1 in lymphocyte homing to Peyer’s patch-HEV in situ: the multistep model confirmed and refined. Immunity 3:99.[Medline]
Lß2 integrin avidity in transendothelial chemotaxis of mononuclear cells. J. Immunol. 159:3968.[Abstract]
-chain of human leukocyte function-associated antigen 1 induces homotypic cell-cell interactions. J. Immunol. 140:1393.[Abstract]
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