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*
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel;
Department of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota Medical School, Minneapolis, MN 55455; and
Biogen, Inc., Cambridge, MA 02142
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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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4ß1) is a cell surface heterodimer that
is essential for the recruitment of leukocyte subsets and certain
malignant cells to endothelial sites that express VCAM-1 (2, 3, 4, 5).
Whereas most leukocyte integrins support stationary cell-cell or
cell-matrix adhesion, VLA-4 and the related
4ß7 integrin are unique in that they
can mediate versatile dynamic interactions under shear flow
with VCAM-1 and mucosal address in cell adhesion molecule-1 expressed
on specialized endothelial sites (6, 7, 8). 4 integrins can
overlap with both the primary selectin-mediated rolling adhesions and
the firm adhesion supported by ß2 integrins once
stationary contact on a vessel wall has been established (9, 10, 11).
4 integrins are constitutively expressed in partially
active forms on circulating lymphocytes, monocytes, and eosinophils and
can mediate tethering and rolling interactions on endothelial ligands
even before exposure to exogenous activating signals (7, 8, 12, 13).
Moreover, VLA-4 can facilitate the spontaneous development of firm
adhesion of lymphocyte subsets to VCAM-1 substrates, thus bypassing the
requirement for exogenous stimulation of adhesion by chemoattractants.
Nevertheless, phorbol esters can enhance VLA-4-mediated firm lymphocyte
adhesion under shear flow to VCAM-1 (7, 8); in addition, chemokine
binding to lymphoid cells that express high levels of seven spanner G
protein-coupled receptors can trigger VLA-4-mediated arrests on VCAM-1
(14). The molecular and cellular basis for support by VLA-4 of leukocyte rolling, spontaneous arrest, and rapid adhesion strengthening under the highly dynamic conditions of shear flow is poorly understood. Integrin-mediated adhesion strengthening is a multistep process. It can be regulated by the intrinsic affinity between the integrin and its ligand and by lateral clustering that is controlled by diffusion of the integrin or ligand on the interacting surfaces after the initial adhesive contact (15, 16, 17). Following ligand binding, rearrangement of the cytoskeleton at the initial site of integrin contact with ligand and cell spreading may further increase integrin-mediated adhesion without altering the original affinity of the integrin for its ligand (18, 19, 20). The kinetics of these diffusion-limited processes are probably slower than those of the rolling and arrest of circulating cells on blood vessels under physiological shear flow. Similar to other integrins, the adhesive properties of VLA-4 depend on its activation state, which is dependent on the cell type and is tightly regulated by cell stimulation and differentiation (3). The adhesiveness of VLA-4 is also regulated by its association with the cytoskeleton (17, 21). Elucidation of the mechanisms that contribute to VLA-4-mediated rolling and spontaneous arrest of leukocytes on VCAM-1 will further our understanding of the functional regulation of this key vascular integrin in different inflammatory contexts.
In this study the contribution of the intrinsic affinity of VLA-4 for soluble ligand to its involvement in initiation and propagation of lymphocyte adhesion to VCAM-1 under physiological shear flow was elucidated. A homogeneous cellular system, the lymphoblastoid T cell line Jurkat, was used along with freshly isolated PBL, to address this question. Jurkat cells express a high uniform level of VLA-4 and their integrin regulation and function resemble those in effector VLA-4high PBL subsets (22), which may comprise the major migratory populations at chronic sites of inflammation that express VCAM-1 (2). The Jurkat line is one of the most widely investigated T cell models for regulation of integrin function and T cell signaling (23, 24, 25, 26). The adhesive properties of VLA-4 were compared in wild-type (wt) Jurkat cells and in a Jurkat mutant with major defects in activation-dependent integrin adhesiveness (22). The mutant does not respond to activating signals downstream of PKC and expresses an altered form of the mitogen-activated protein kinase ERK1 (22). We show here that the mutant also lacks a subset of high affinity VLA-4 that can be found on wt cells. The absence of high affinity VLA-4 on mutant cells was associated with a markedly suppressed adhesion strengthening of the mutant cells on different VLA-4 ligands, but did not abrogate their VLA-4-mediated tethering and rolling adhesions on VCAM-1 in shear flow. High affinity VLA-4 subsets that are constitutively expressed on circulating lymphocytes may provide a regulatory mechanism for their arrest on inflamed VCAM-1-expressing endothelial sites independent of activation signals displayed on these sites.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purified Ig fractions of the following mAbs were used: 4B9,
which functionally blocks VCAM-1; HP 1/2, which blocks 4
subunit binding to VCAM-1 and fibronectin (FN) (27); B5G10, which binds
the 4 integrin subunit but does not block its binding to
ligand (28); TS2/16, which binds to and functionally activates
ß1 integrins (29); and 9EG7, which detects an activation
epitope on the ß1 integrin subunit (30). The A4-PUJ1, an
4 subunit-specific, VLA-4-blocking mAb (31) was used as
ascites. B5G10, 9EG7, TS2/16, and A4-PUJ1 were gifts from Dr. M. Hemler
(Dana-Farber Cancer Institute, Boston, MA). FITC-conjugated goat
anti-mouse and rabbit anti-mouse Ig (Zymed, South San
Francisco, CA) were used as secondary Abs. An eight-residue peptide
containing the tripeptide motif (leucine-asparatate-valine (LDV)),
EILDVPST, derived from the CS-1 region of FN, and its analogue,
EIDVLPST, were prepared by solid phase peptide synthesis using an
ABIMED AMS-422 automated peptide synthesizer (Langenfeld, Germany). A
VCAM-1-Ig fusion protein, consisting of the two N-terminal domains of
human VCAM-1, fused to human IgG Fc was prepared and purified as
previously described (32). BSA (fraction V), Ca2+- and
Mg2+-free HBSS, and Ficoll-Hypaque 1077 were obtained from
Sigma (St. Louis, MO). Human serum albumin (HSA; fraction V), PMA, and
cytochalasin B were purchased from Calbiochem (La Jolla, CA).
Preparation of immobilized adhesive substrates
Soluble, affinity-purified, recombinant seven-domain human VCAM-1 (sVCAM-1) (33), human FN (Life Technologies, Gaithersburg, MD), or the 40-kDa CS-1-containing fragment of human FN (FN40; Life Technologies) was dissolved in PBS buffered with 20 mM bicarbonate, pH 8.5, and incubated on a polystyrene plate (60- x 15-mm petri dish; Becton Dickinson, Lincoln Park, NJ) for 2 h at 37°C. The plate were then washed three times with PBS and blocked with HSA (20 mg/ml in PBS) overnight at 4°C.
Cells
The Jurkat E6–1 T lymphoblastoid cell line and the mutant 11.1,
which was derived by gamma irradiation and was selected for inability
to undergo activation-dependent up-regulation of its integrin function,
have been described previously (22). The wild-type and mutant Jurkat
cells were maintained in RPMI 1640 supplemented with 10%
heat-inactivated FCS (Sigma), 2 mM L-glutamine, and
penicillin/streptomycin (Bio Lab, Jerusalem, Israel). CHO cells
transfected with full-length human VCAM-1 were maintained in -MEM
supplemented with 10% dialyzed FCS, 4 mM L-glutamine, and
200 nM methotrexate (Sigma). Human PBL (obtained from healthy donors)
were isolated from citrate-anticoagulated whole blood by dextran
sedimentation and density separation over Ficoll-Hypaque (34). The
mononuclear cells thus obtained were washed and further purified on
nylon wool. The resulting purified PBL were >90% CD3+ T
lymphocytes.
Immunofluorescence flow cytometry
For indirect immunofluorescence, washed cells were resuspended in PBS supplemented with 5% FCS and 5 mM EDTA (PBS-EDTA) and incubated at 4°C for 60 min with A4-PUJ4 ascites fluid (diluted 1/200), HP1/2 mAb (at 10 µg/ml), or preimmune mouse IgG (a control for background staining; Zymed, South San Francisco, CA). The cells were then washed and incubated for an additional 30 min at 4°C with FITC-conjugated goat anti-mouse Ig (Zymed). Washed cells were resuspended in PBS supplemented with 0.05% sodium azide and immediately analyzed in a FACScan flow cytometer (Becton Dickinson, Erembodegem, Belgium).
The induction of ligand-induced binding site (LIBS) epitopes by soluble
ligand was tested by incubating (5 min, 24°C) PBS-EDTA-washed cells
in binding medium (HBSS containing 2 mg/ml BSA and 10 mM HEPES, pH 7.4,
supplemented with Ca2+ and Mg2+, each at 1 mM)
in the presence of LDV- or DVL-containing octapeptides. High
affinity recognition of LDV coincides with high VLA-4 affinity for
VCAM-1: both ligands bind the integrin at nearly identical sites, and
their binding properties are similarly modulated by integrin
activation. The LIBS-specific anti-ß1 mAb, 9EG7, was
added at 10 µg/ml for a short incubation period (5 min, 24°C),
followed by a second incubation of 60 min at 4°C. Unbound primary mAb
was removed by washing the cells with PBS supplemented with 5% FCS,
and the cells were stained with FITC-labeled goat anti-mouse Ig as
described above. Induction of the 9EG7 epitope (determined in mean
fluorescence units and corrected for background 9EG7 staining that was
detected in cells suspended in binding medium in the absence of soluble
VLA-4 ligands) was expressed as a percentage of the mean fluorescence
intensity of 4-specific staining with mAb HP1/2.
Immunoelectron microscopy
Localization of VLA-4 was assessed by immunoelectron microscopy, as previously described (35). Briefly, cultured Jurkat cells were washed with PBS containing 5 mM EDTA and resuspended in PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2. The following fixation and immunolabeling steps were conducted at 22°C. The cell suspension was diluted in 0.1 M phosphate buffer, pH 7.4, containing 2% p-formaldehyde and 0.05% glutaraldehyde. After 20 min, the cells were washed (three times) with HBSS containing 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES, pH 7.4. They were then incubated (30 min) with the anti-VLA-4 mAb B5G10 (10 µg/ml) in HBSS containing 1% BSA (HBSS/BSA). After washing (three times), cells were incubated (30 min) with rabbit anti-mouse Ig (10 µg/ml) in HBSS/BSA. The cells were washed (three times) and incubated (45 min) with 5-nm gold particle-conjugated protein A (Zymed). After extensive washing with HBSS/BSA, the cells were subjected to an additional fixation (30 min) in 0.1 M sodium cacodylate buffer containing 1.5% glutaraldehyde and 1% sucrose. The cells were then washed in 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, and stained with aqueous uranyl acetate. Next, the cells were embedded in Epon, sectioned, and stained with uranyl acetate and lead nitrate. The sectioned cells (40–60 cells/experiment) were examined with a Phillips 410 electron microscope (Phillips, Eindhoven, The Netherlands); only representative cells were photographed.
Laminar flow assays
Analysis of cell tethering. A polystyrene plate on which purified ligand had been adsorbed 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 (7, 36). The cells to be analyzed 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 (107 cells/ml, 4°C) in H/H medium, and kept at 4°C until used. Cells were diluted with room temperature binding medium to a concentration of 106 cells/ml and perfused through the flow chamber at the desired shear stress. This stress was generated with an automated syringe pump (Harvard Apparatus, Natick, MA) attached to the outlet side of the flow chamber. Cellular interactions on two different fields of view (each one 0.17 mm2 in area) were visualized with a x10 objective. Cell images were videotaped with a long integration LIS-700 CCD video camera (Applitech, Holon, Israel) and a Sony SLV E400 video recorder (Sony, Tokyo, Japan). Cell images were manually quantitated by analysis of images directly from the monitor screen.
All adhesive interactions between the flowing cells and the
ligand-coated or uncoated (control) substrates were tracked and
quantified. Tethering events were defined as adhesive interactions
between freely flowing cells that moved closest to the lower wall of
the flow chamber coated with the substrate. Since the majority of
tethered cells that spontaneously arrested on VCAM-1 did so within
10 s after its initial adhesive attachment (tethering), the motion
of each tethered cell was monitored for 10 s following the initial
tether, and four categories of cell tethers were defined. Tethers were
defined as transient if cells attached briefly (
2 s) to the substrate
but did not continue to roll on it. Tethers were defined as rolling
tethers if they were followed by rolling motions 
5 s with a velocity
of at least 1 µm/s but not more than 10% of the hydrodynamic
velocity of a cell freely flowing at the given shear stress.
Spontaneous arrests (also referred to as rolling-associated arrests)
were defined as cells rolling for 1–10 s post-tethering before coming
to a full arrest on the substrate, whereas a fully arrested cell was
defined as a cell that remained adherent and stationary for at least
20 s. The last category of tethers, termed immediate arrests,
consisted of cells that upon tethering arrested within <0.5 s on the
ligand-coated substrate and remained stationary for 20 s. Successive
transient tethering events separated by <200 µm of cell motion at
the hydrodynamic velocity were counted as a single tethering event. The
number of tethers for each category that occurred within the field of
view during a 60-s period of continuous flow was divided by the flux of
freely flowing cells that moved through the same field without
exhibiting any adhesive interactions. This flux was assessed by running
parallel experiments on the same substrates in the presence of EDTA,
which inhibited all integrin-mediated interactions with the substrate.
For calculations of cell flux, only the fraction of perfused cells that
came into close proximity with the substrate, and therefore were
potentially capable of interacting with the substrate, were considered.
Analysis of cell resistance to detachment and rolling at elevated
shear stress.
The resistance of an adherent cell to detachment by shear force is a
function of the adhesive forces generated by the integrin:ligand bonds
at the cell-substrate contact zone (15). Detachment assays were
performed with cells that had tethered and accumulated at low shear
flow (0.5 dyn/cm2) for 30 s on VCAM-1-coated plates or
that had bound at stasis to ligand-coated plates for various periods.
After cells had bound to the various substrates, the wall shear stress
was increased stepwise every 5 s (by a programmed set of flow
rates delivered by the syringe pump) to 40 dyn/cm2. At the
end of each 5-s interval of the increase in shear stress, the number of
cells that remained bound was expressed relative to the number of cells
that accumulated on VCAM-1 in flow or that bound on FN in stasis. The
contribution of cells rolling into the observation field from upstream
fields was minimized by locating the field at the upstream edge of the
spot of adsorbed ligands. Reduction in the number of cells remaining
bound in the field at elevated shear stresses was due both to cell
detachment from the substrate and to cells rolling out of the field of
view without being detached. During the detachment experiments, cell
fractions that rolled, i.e., cells that moved 0.5 µm/s for at least
3 s, were assessed at selected shear stresses. Unless otherwise
indicated, all assays were performed at room temperature, which
minimized spontaneous cell activation or change in shape. Rolling
velocities were determined for cells tethered under flow by analysis of
cell displacements over 5- to 10-s intervals, typically 2–3 s after
the initial tethering event.
Pretreatments of cells and substrates for the flow experiments
For Ab inhibition studies, cells (107/ml) were preincubated (5 min, 4°C) in H/H medium with VLA-4-blocking mAb (20 µg/ml). The cells were diluted 1/10 into binding medium (H/H medium containing 1 mM Ca2+ and 1 mM Mg2+) without washing out the Ab, and the suspension was perfused into the flow chamber. For metabolic inhibition experiments, cells (107/ml) were treated (10 min, 22°C) with 0.1% NaN3 (azide) and 50 mM 2-deoxyglucose (2-DOG) in H/H medium and then diluted (1/10) with binding medium containing 0.05% azide and 25 mM 2-DOG. To interfere with the integrity of their actin cytoskeleton, leukocytes were suspended in binding medium containing 20 µM cytochalasin B for 10 min at 24°C, before being perfused, unwashed, through the flow chamber. To study peptide inhibition, cells were suspended in binding medium with or without 0.1–1 mM of the octapeptide EILDVPST or its control analogue EIDVLPST and were incubated for 5 min at 24°C before perfusion through the flow chamber. The effect of cell activation on cell binding to substrates was assessed by preincubation (3 min, 24°C) in binding medium containing either PMA (100 ng/ml) or the ß1 activating mAb TS2/16 (5 µg/ml); the cells were not washed before perfusion through the chamber. For assessment of cation-induced integrin activation, H/H medium containing 0.2 mM Mn2+, rather than binding medium, was used during cell perfusion through the chamber.
The functional activity of VCAM-1 on the ligand-coated plates used in the flow chamber was blocked by pretreatment with the anti-VCAM-1 mAb 4B9. Medium containing the mAb (20 µg/ml) was perfused (for 20 min) through the flow chamber. mAb (5 µg/ml) was also added to the binding medium in which cells were perfused into the chamber. Background adhesive interactions of the cells were determined by analyzing cell tethering on plates coated with HSA (20 mg/ml). The adhesive interactions of differently pretreated cells were assessed on identical microscopic fields; this ensured that variance in distribution of the immobilized ligands in each test field could not affect the adhesive properties of the compared cells. Both VCAM-1 and FN remained functionally intact throughout the experiment, as verified by comparing the behavior of control cells in multiple tests performed either at the start or at the end of each series of experiments.
Statistical analysis
Two-tailed Student’s t test was used, where indicated, to determine the level of significance of differences in mean values of paired experimental groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The VLA-4-mediated adhesion to VCAM-1 of wt and activation mutant
Jurkat cells termed 11.1 was characterized by controlled detachment
assays using a parallel plate flow chamber. The shear resistance
developed by wt and mutant cells briefly adhered to VCAM-1 substrates
was analyzed by subjecting the adherent cells to incremental increases
in shear stress. PMA treatment significantly increased the resistance
to detachment of wt Jurkat cells from VCAM-1, but only slightly
augmented the shear resistance of the mutant cells (Fig. 1
, A and B).
Mn2+ dramatically enhanced the resistance to detachment
from VCAM-1 of both wt and activation mutant cells (Fig. 1, A and B), indicating that the ability of VLA-4 to
undergo cation-induced activation remained intact on the mutant cells.
Interestingly, the adhesions to VCAM-1 of unactivated Jurkat cells were
more shear resistant than those of the unactivated mutant cells (Fig. 1). Both wt and mutant cells expressed comparable levels of
4 and ß1 integrin chains on their surface,
as previously shown (22). Thus, besides the inability of the mutant
cells to up-regulate their VLA-4 adhesiveness by exogenous cellular
activation, VLA-4 on these cells exhibited a defect in constitutive
adhesive activity.
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A). The shear resistance of
the resting mutant cells or of energy-depleted Jurkat cells was much
lower than that of the wt Jurkat resistance on both high and low VCAM-1
densities (Fig. 2A and data not shown). The shear resistance
of wt, mutant, and energy-depleted cells dropped rapidly as the VCAM-1
concentration adsorbed on the substrate was reduced (data not shown).
This higher resistance of wt T cells occurred regardless of whether
they had accumulated under low shear flow or they had adhered to VCAM-1
in stasis. Furthermore, prolonged accumulation on VCAM-1 did not rescue
the adhesion deficiency of the mutant cells, suggesting that it did not
result from a slower kinetics of adhesion strengthening of the mutant
cells (data not shown). The strong VLA-4-mediated adhesion of resting
wt Jurkat on VCAM-1 was associated with reduced rolling relative to
that of resting mutant cells. Although the majority of mutant cells
could roll on the VCAM-1 substrate at shear stresses >0.5
dyn/cm2, most wt Jurkat cells, remained bound and
stationary on the VCAM-1 substrate under similar conditions (Fig. 2B). Surprisingly, energy-depleted wt Jurkat cells also
exhibited persistent rolling adhesions on VCAM-1. Similar patterns of
increased mutant rolling were observed on VCAM-1-transfected CHO cells
and on cytokine-stimulated VCAM-1-expressing endothelial cell
monolayers (data not shown).
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The preferential presentation of 4 integrins on
lymphocyte microvilli (8, 37) has been suggested to facilitate
leukocyte tethering under high shear flow to endothelial
counter-receptors. Therefore, the VLA-4 distribution on mutant and wt
cells was compared by transmission electron microscopy. The cells were
prefixed in the presence of physiological concentrations of
Ca2+ and Mg2+, and immunogold staining of
4 was performed with mAb directed against epitope distal
from the ligand binding site. The surface topology was similar with
regard to microvilli number and dimensions of the wt and mutant cells
(Fig. 3). VLA-4 was localized
predominantly to the microvilli in both cell types. A small amount of
the 4 molecules of both cell types was present in small
clusters that consisted of two or more gold particles. Thus, the
deficient VLA-4 adhesiveness of the mutant could not be due to an
altered surface distribution of VLA-4.
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B and data not shown),
suggesting that VLA-4 on both wt and mutant cells is accessible to the
same extent for interaction with an immobilized VLA-4-specific mAb
under shear flow. In contrast, at shear stresses >1.2
dyn/cm2, energy-depleted wt Jurkat cells were captured less
efficiently than untreated cells by the immobilized mAb (Fig. 4A). Cell capture by the anti-VLA-4 mAb was abrogated by
cytochalasin B treatment (Fig. 4A). Since at elevated shear
flow, cell capture becomes more sensitive to the surface accessibility
of the tethering receptor (38, 40), these results suggested that the
surface accessibility of VLA-4 was partially reduced by energy
depletion of wt cells, but was fully retained on the 11.1 mutant.
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B). Importantly, LIBS staining
was uniformly distributed on the entire population of cells, ruling out
the existence of a subset of wt cells with an exclusive expression of
high affinity VLA-4 (data not shown). The LDV peptide induced
significantly weaker LIBS staining on the 11.1 mutant cells, and a
significantly weaker LDV dose-response of LIBS staining was observed in
these cells (Fig. 4B). In energy-depleted wt cells, LIBS
induction by LDV was almost nonexistent (Fig. 4B). These
results indicate that VLA-4 on resting 11.1 mutant and energy-depleted
wt cells recognize the LDV peptide with a considerably lower affinity
than VLA-4 on resting wt cells. Tethering, rolling, and adhesion strengthening of wt and activation mutant Jurkat cells on VCAM-1 and FN
VLA-4 adhesions to VCAM-1 in shear flow can be divided into three distinct adhesive stages: tethering, rolling, and spontaneous cell arrest. Spontaneous cell arrest is established either immediately upon the initial cell tethering or after a period of rolling on the ligand. To learn about the molecular basis of these distinct processes, different types of adhesive interactions between wt and mutant Jurkat cells with different densities of VCAM-1 were examined at representative physiological shear stresses. Cells were individually tracked for the first 10 s after their initial tethering to the substrate. Tethers were categorized as transient, rolling, rolling followed by spontaneous arrests, and immediate arrests. The spontaneous arrest category included rolling cells that spontaneously arrested on VCAM-1 within less than 10 s. Cells that immediately arrested upon tethering and remained stationarily bound to ligand for at least 20 s were assigned to the arrest category.
The accumulation of wt Jurkat cells at a medium shear stress on a high
density VCAM-1 field (coated at 2 µg/ml) was 3 times that of the 11.1
mutant (58 ± 10 vs 20 ± 4 cells/field.min) and of
energy-depleted wt cells (22 ± 6 cells/field/min). The vast
majority of the tethered mutant and energy-depleted cells continued to
roll after tethering on VCAM-1. In contrast, as much as 40% of the wt
Jurkat cells immediately arrested upon tethering to the ligand in shear
flow (Fig. 5A). Another 25%
of the tethered wt cells arrested spontaneously within <10 s following
tethering. The fraction of immediately or spontaneously arresting T
cells diminished on substrates coated at lower coating concentrations
even when considerable tethering and rolling were still observed,
consistent with previous findings that spontaneous arrest of
lymphocytes on VCAM-1 increases with VCAM-1 density (43). When the
VCAM-1 substrate was blocked by 4B9 mAb, which is directed against
domain 1 of VCAM-1, no T cell tethers occurred (data not shown). This
indicates that the tethers were VCAM-1 specific and that domain 4 on
VCAM-1 is not involved in VLA-4-mediated tethering under flow. When
tethering was measured at a high shear stress (2.5
dyn/cm2), its frequency decreased similarly for both wt and
11.1 mutant cells, the tethers became more labile, and most wt cells
failed to immediately arrest on VCAM-1, but, rather, arrested after
short rolling periods (Fig. 5B). Notably, wt Jurkat and its
activation mutant tethered to VCAM-1 at identical rates even at these
extreme shear stresses. Essentially none of the mutant cells
spontaneously arrested on VCAM-1 (Fig. 5B), but they could
do so in the presence of the VLA-4-activating reagents mAb TS2/16 or
Mn2+ (data not shown). High fractions of wt, but not
mutant, cells spontaneously arrested in a VLA-4-dependent fashion on a
monolayer of VCAM-1-transfected CHO cells, although similar fractions
of wt and 11.1 mutant cells could initiate tethers on the cell-based
VCAM-1 (data not shown). When VCAM-1 was coated below a threshold
concentration of 0.1 µg/ml, it could no longer support rolling or
arrests. Under these conditions, wt, 11.1 mutant, and energy-depleted
Jurkat cells tethered at identical efficiencies to low density VCAM-1
(Fig. 5C).
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), energy
depletion of PBL reduced their accumulation on high density VCAM-1
substrates at physiological shear stresses by 55–60%. This reduction
was accompanied by a destabilization of T cell resistance to detachment
at elevated shear stresses (data not shown). As seen with the Jurkat T
cell line, a substantial number of PBL immediately arrested shortly
after tethering on high density VCAM-1 in flow, and that arrest
required cellular energy (Fig. 6A). Higher VCAM-1 densities
were required to immediately arrest tethered PBL than to arrest Jurkat
cells (Figs. 5A and 6A). Of the energy-depleted
PBL tethered to high density VCAM-1, almost all lymphocytes failed to
arrest and continued to roll on VCAM-1 at elevated shear stresses. Some
energy-depleted PBL also spontaneously arrested after a short period of
rolling on VCAM-1, but these PBL readily detached from the VCAM-1
substrate at elevated shear stresses (data not shown). The majority of
untreated PBL, including those that rolled for short periods after
their initial tethering to VCAM-1, came to arrest and remained
stationary even at incremented shear stresses (Fig. 6B).
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,
A and B). Blocking experiments revealed that
binding of resting Jurkat T cells to FN was primarily mediated by VLA-4
(data not shown). Interestingly, the 11.1 mutant tethered at a
subphysiological shear stress to FN40 with an efficiency similar to
that of wt Jurkat cells (Fig. 7C). Energy-depleted wt Jurkat
cells tethered at this low flow as efficiently as untreated cells to
FN40, similar to their near-identical tethering rates to low density
VCAM-1 (Fig. 5C). Nevertheless, the majority of tethers by
wt Jurkat cells, but not those of the 11.1 mutant and energy-depleted
wt cells, were followed by immediate cell arrest on the ligand. Thus,
the initial VLA-4-mediated tethering on both VCAM-1 and FN CS-1 in
shear flow is not impaired in the mutant or by energy depletion of wt
cells, but the spontaneous translation of tethers into firm arrests is.
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The ability of wt T cells to spontaneously arrest upon tethering
to VCAM-1 in shear flow (Figs. 5 and 6) and the presence of high
affinity VLA-4 on these T cells suggested that this high affinity VLA-4
form is essential for rapid development of adhesion strengthening. To
monitor the function of different affinity states in cells interacting
with surface-bound ligand under flow, we developed a peptide-blocking
assay in which high affinity VLA-4 states become preferentially
occupied by soluble LDV-containing peptide. To verify the specificity
of the assay for high affinity states, either low or high affinity
VLA-4 states were artificially induced on Jurkat cells in medium
containing either Ca2+ or Mg2+, respectively.
Although at stasis, VLA-4 supported weaker adhesions in the presence of
Ca2+ alone on both VCAM-1 and FN (data not shown), VLA-4
tethering to VCAM-1 in shear flow was comparable in the presence of
either cation (Fig. 8A). This
suggested that both low affinity (Ca2+-dependent) and high
affinity (Mg2+-dependent) VLA-4 tether T cells to VCAM-1
under flow with comparable efficiencies. In agreement with our
assumption, the LDV peptide, but not the control DVL-containing
peptide, could block tethering of wt Jurkat cells to low density VCAM-1
in the presence of Mg2+, but not in the presence of
Ca2+ (Fig. 8A). Accordingly, cell preincubation
with mAb TS2/16, a ß1 integrin affinity-stimulating mAb,
rendered VLA-4-mediated tethering to VCAM-1 more susceptible to
inhibition by the LDV peptide (data not shown). The LDV peptide
inhibition of tethering was dose dependent, with a maximum reached at
0.5 mM (data not shown). These collective results confirmed our
hypothesis that LDV can selectively block tethers supported by high
affinity VLA-4 states. In physiological medium containing equimolar
levels of both Ca2+ and Mg2+, inhibition by the
LDV peptide of VLA-4-mediated tethering of wt Jurkat cells to low
density VCAM-1 was half that obtained in medium containing
Mg2+ alone (25–30 vs 50%; Fig. 8B). In
contrast, VLA-4-mediated tethering of the 11.1 activation mutant as
well as that of energy-depleted wt Jurkat cells to VCAM-1 were
absolutely insensitive to inhibition by the LDV peptide in
physiological medium (Fig. 8B).
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A). In medium alone or in the
presence of the control peptide, 34–42% of initially tethered wt
cells arrested immediately upon tethering to VCAM-1, and an additional
32–35% of tethered cells were arrested after a short rolling period
(Fig. 9A). In the presence of saturating levels of the LDV
peptide, on the other hand, total tethering of wt Jurkat cells to
VCAM-1 was reduced by about 25% (Fig. 9A). Strikingly, the
remaining wt Jurkat cells tethered to VCAM-1 could not immediately
arrest on the VLA-4 ligand, but continued to roll on it (Fig. 9A), indicating that the LDV-sensitive, high affinity VLA-4
subset supports all immediate arrests of wt cells tethered to VCAM-1
under shear flow. The LDV peptide also partially interfered with the
ability of tethered cells to spontaneously arrest during rolling on
VCAM-1, which suggests that this category of tethers is mediated by a
heterogeneous VLA-4 subset with overall lower affinity than that of the
VLA-4 subset responsible for immediate cell arrest on VCAM-1.
|
A). Another soluble ligand to VLA-4,
VCAM-1-Ig fusion protein, at saturating levels not only blocked all
immediate arrests, but also completely interfered with the ability of
tethered cells to spontaneously arrest during rolling on VCAM-1 (Fig. 9B). VCAM-1-Ig also reduced the total tethering of wt cells
by 70%. The residual transient tethers and tethers followed by rolling
adhesions were still completely insensitive to sVCAM-1 (Fig. 9B). Similar selectivity of inhibition of cell arrest, but
not of rolling adhesions, by soluble LDV and VCAM-1-Ig was observed
with Jurkat cells perfused over substrates cocoated with both VCAM-1
and the endothelial L-selectin ligand, peripheral node addressin (44)
(data not shown).
By sharp contrast to wt cells, the tethering of the 11.1 mutant Jurkat
and of energy-depleted wt Jurkat cells to high density VCAM-1 was not
susceptible to inhibition by the LDV peptide (Fig. 9A).
Energy-depleted wt Jurkat cells initiated fewer total tethers than wt
or 11.1 mutant cells on VCAM-1 (Fig. 9A) at physiological
shear stresses, consistent with previous findings (
Figs. 4–6 and 8).
As shown above (Fig. 5), both mutant and energy-depleted cells
established efficient rolling, but usually failed to arrest upon
tethering to high density VCAM-1. The small fraction of mutant cells
that did arrest after a short period of rolling was insensitive to
inhibition by LDV (Fig. 9A), but could be blocked by
sVCAM-1-Ig (Fig. 9B). As with wt cells, neither LDV nor
VCAM-1-Ig interfered with the rolling or transient tether categories of
the 11.1 mutant or the energy-depleted wt cells. The LDV peptide did
not affect the rolling velocities of the wt Jurkat fraction that
initiated rolling after tethering to VCAM-1 (Fig. 9C),
further indicating that rolling adhesions are mediated primarily by low
affinity, LDV-insensitive VLA-4 states. Although the rolling dynamics
of the 11.1 mutant and those of energy-depleted Jurkat cells were as
insensitive to the presence of LDV peptide as those of wt cells, both
types of cells rolled slightly faster than wt cells (Fig. 9C). High affinity, LDV-sensitive states of VLA-4 also
contributed to the development of high resistance of wt Jurkat cells to
detachment by elevated shear forces from VCAM-1; in the presence of the
LDV peptide, wt cells detached more readily from VCAM-1 (data not
shown). Similar to Jurkat cells, peripheral blood CD4+ T
cells could spontaneously arrest on high density VCAM-1, either
immediately upon tethering or after short periods of rolling. About
half the individual PBL examined could establish either immediate or
rolling-associated arrests after tethering to high density VCAM-1 (Fig. 9D). As with the Jurkat cells, all immediate arrests and
most of the rolling-associated arrests could be inhibited by saturating
levels of the LDV peptide, but not by the control DVL peptide (Fig. 9D). Rolling and transient tethers of PBL on VCAM-1 were
insensitive to the LDV peptide. These findings indicate that low
affinity VLA-4 states preferentially mediate the transient tethering
and rolling of PBL that fail to spontaneously arrest on VCAM-1. The
high affinity VLA-4 states of PBL preferentially support all types of
spontaneous arrests on VCAM-1 in a homologous manner to the high
affinity VLA-4 subsets of wt Jurkat cells.
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the activation mutant was originally selected for its
inability to respond to exogenous signals associated with TCR- and
PKC-dependent signaling (22), it also exhibits impaired VLA-4
adhesiveness in its resting state despite expressing VLA-4 at levels
comparable to those of wt Jurkat cells. The mutant could tether
efficiently to different VLA-4 ligands and establish rolling adhesions
on VCAM-1 under shear flow, but it failed to arrest on ligand and could
not develop firm adhesion (Figs. 2 and 5). Immunolocalization of VLA-4
by electron microscopy indicated similar distribution of the integrin
on the surfaces of wt and mutant cells. VLA-4 on mutant cells was as
available for binding ligand as VLA-4 on wt cells, as indicated by the
similar efficiencies by which wt and mutant cells could tether to a
substrate-bound anti-VLA-4 mAb under shear flow (Fig. 4). Moreover,
in the presence of nonphysiological VLA-4 agonists, such as
Mn2+ or the affinity-stimulating mAb TS2/16, which
artificially stabilized the mutant’s VLA-4 at high affinity state, the
mutant rapidly developed firm adhesions and spread on VCAM-1. This
observation not only suggested that the VLA-4 in the mutant remained
structurally intact with respect to its ability to acquire high
affinity conformation if properly activated, but it also indicated that
the cytoskeletal associations of VLA-4 were not impaired in the mutant.
We therefore reasoned that the reduced adhesiveness of VLA-4 in the
mutant, in both its unstimulated and stimulated states, may result from
an inability of the VLA-4 on the mutant to acquire high affinity to
ligand. VLA-4 affinity to ligand is very low in physiological medium
and cannot be monitored by direct cell binding assays using monovalent
physiological ligands of VLA-4, because such ligands are readily washed
out from the cell surface during these assays. The affinity to LDV of
the high affinity VLA-4 subset characterized here on wt Jurkat cells
was enhanced by at least 10-fold in the presence of Mn2+
(50) (data not shown). Even under these conditions, direct binding of
sVCAM-1 or CS-1 fragments could not be assessed. VLA-4 affinity states
were therefore assessed by their ability to express a ligand-induced
epitope (LIBS) in the presence of a soluble LDV-containing peptide or
VCAM-1 (50). This approach identified a small subset of high affinity
VLA-4 receptors that are constitutively expressed on unstimulated wt
Jurkat cells, but are depleted in the mutant cells. Strikingly, the
ability of VLA-4 to support cell tethering to immobilized VCAM-1 or FN
CS-1 did not require the presence of the high affinity subset, as
suggested by the identical tethering rates of both the activation
mutant and wt Jurkat cells observed on both ligands. The wt Jurkat
tethering to VCAM-1 was also comparable in the presence of
Mg2+-depleted Ca2+-containing medium, which is
known to suppress VLA-4 affinity, and as in high affinity-inducing
binding medium that contained Mg2+ only (45). These results
further suggest that low affinity VLA-4 and high affinity VLA-4 support
similar cell tethers on ligand under the shear flow conditions tested.
The distribution of LDV-induced LIBS epitopes on Jurkat cells suggested
that VLA-4 occurs in heterogeneous affinity states on resting T cells.
Heterogeneity of integrin affinity states on individual leukocytes and
fibroblasts has previously been reported (18, 51, 52). We assumed that
high affinity VLA-4 states could be distinguished from low affinity
states by their preferrential occupancies of and inhibition by a
soluble ligand, as was previously shown with other integrins (18, 19).
Indeed, the LDV peptide could selectively inhibit tethers mediated by
high affinity VLA-4, artificially induced by Mg2+ or by an
integrin-activating mAb. The peptide failed to inhibit tethers
supported by low affinity VLA-4, exclusively expressed on
energy-depleted T cells or on wt cells in Mg2+-depleted,
Ca2+-containing medium (Fig. 8). This differential
susceptibility of high and low affinity VLA-4 to inhibition by soluble
ligand allowed us to dissect the contribution of each of these states
to initial tethering, rolling, and spontaneous arrests of different T
cells on VCAM-1-coated substrates under shear flow. Using this
approach, we determined that the presence of high affinity VLA-4 is
mandatory for wt T cells to rapidly develop spontaneous arrests upon
tethering to high density VCAM-1; selective inhibition by soluble
ligand eliminated the ability of wt cells to arrest on VCAM-1
immediately after tethering to the ligand under shear flow. In
contrast, the peptide did not block any transient tethers or rolling
adhesions initiated by wt cells on high density VCAM-1, suggesting that
low affinity VLA-4 states are exclusively associated with these labile
adhesive interactions between flowing wt cells and surface-bound
VCAM-1. Similarly, high affinity (LDV-sensitive) VLA-4 on freshly
isolated PBL was indispensable for their spontaneous adhesion
strengthening on high density VCAM-1, whereas low affinity
(LDV-insensitive) VLA-4 states supported rolling adhesions on the
ligand. In both Jurkat cells and PBL, the overall number of initial
tethers to VCAM-1, in addition to the extent of spontaneously arrested
tethers, was reduced in the presence of the LDV peptide. This indicates
that once bound to a high affinity VLA-4 receptor, the peptide
not only blocked the ability of the integrin to support cellular
arrests on VCAM-1, but also interfered with its ability to promote
initial cell tethering (Fig. 9).
A more complex scheme of inhibition by soluble LDV peptide was observed
on the spontaneous arrests of wt Jurkat cells, which developed during
cell rolling on VCAM-1, rather than immediately after cell tethering to
ligand. Although a portion of these spontaneous arrests were
insensitive to inhibition by the LDV-containing peptide, they could be
still blocked by sVCAM-1-Ig. An alkaline phosphatase-tagged VCAM-1-Ig
binds VLA-4 on Jurkat cells with an IC50 of 3 nM in
physiological medium (R. Lobb, unpublished observations), whereas the
LDV octapeptide used here binds VLA-4 with an IC50 of 100
µM (Fig. 4). This suggests the presence on Jurkat cells of
intermediate affinity states that are not inhibited by an
LDV-containing peptide but are inhibited by the more potent ligand,
VCAM-1-Ig. Taken together, these results suggest that the spontaneous
arrests developed by wt Jurkat rolling on VCAM-1 appear to be mediated
in part by high affinity and in part by intermediate affinity VLA-4.
Although a potent VLA-4 ligand, VCAM-1-Ig, did not affect the fractions
of wt Jurkat cells that established rolling or transient tethering to
VCAM-1 even at a concentration 20-fold higher than its IC50
(Fig. 9B). This is a further indication that rolling and
transient tethers are supported by extremely low affinity states of
VLA-4, which are not occupied even in the presence of saturating levels
of sVCAM-1. Similarly, in PBL, spontaneous arrests, but none of the
transient tethers or rolling adhesions, were inhibited by saturating
levels of ligand (Fig. 9). This finding suggests that high affinity
VLA-4 states constitutively expressed on circulating T lymphocytes are
mandatory for the ability of these lymphocytes to spontaneously arrest
on VCAM-1, but are dispensable for the rolling adhesions of these
lymphocytes on the vascular VLA-4 ligand.
A flowing T cell may form a primary tether to endothelial VCAM-1 through either its low affinity or its high affinity VLA-4 receptors. In the first case, low affinity interactions between VLA-4 and VCAM-1 would result in rolling adhesions until high affinity VLA-4, preexistent on the rolling cell, becomes available for binding immobilized VCAM-1 and instantaneously arrests the rolling cell. In the second case, an initial tether is mediated by high affinity VLA-4, and the tether bond is sufficiently strong to immediately arrest the tethered T cell under flow. Consistent with such a stochastic mechanism, prolonged periods of rolling were observed to increase the fractions of wt Jurkat or PBL that developed spontaneous arrests on VCAM-1. Interestingly, a general tyrosine phosphorylation inhibitor, genestein, known to interfere with VLA-4 outside-in signaling (53), did not reduce these fractions (data not shown). These results suggest that during rolling on VCAM-1, new high affinity states are not induced on the adherent cells, nor does VLA-4 avidity to the immobilized ligand increase through outside-in signaling. Nevertheless, a novel linkage between normal T cell activation and the ability of T cells, in their resting state, to maintain VLA-4 in the high affinity state has been established in this study. The 11.1 activation mutant characterized in the present study retained normal early TCR-mediated signaling and PKC expression (22). However, PMA activation of PKC, CD3 cross-linking, and augmentation of tyrosine phosphorylation by pervanadate pretreatment, which stimulated VLA-4 adhesiveness in wt cells, all failed to enhance VLA-4 adhesion in this mutant, although the mutant’s VLA-4 was functionally intact when activated with cations or mAbs. These findings locate the mutated function downstream to PKC and suggest that high VLA-4 affinity states on unstimulated wt T cells depend on PKC-dependent signaling pathways. The small GTPases, R-Ras and H-Ras, recently implicated in the control of the affinity of integrins in fibroblasts (54, 55), are candidate regulators of integrin affinity in T cells.
The pre-existent expression on T cells of high affinity VLA-4 may be of general significance, because VLA-4 adhesiveness is generally up-regulated by inside-out signaling pathways, which do not increase the intrinsic affinity of integrin for ligand (20, 32, 56). High affinity VLA-4 could support rapid lymphocyte arrest on inflamed vessels that express high levels of VCAM-1 (57) before or along with T cell activation by endothelium-displayed chemokines (14, 58). VCAM-1 is highly expressed on inflamed brain endothelium in models of experimental allergic encephalomyelitis (3), and in vivo blocking experiments indicate an exclusive role for VLA-4:VCAM-1 interactions in the recruitment of T lymphocytes to inflamed microvasculature of the central nervous system (57). In such settings and in other inflammatory sites that express high levels of VCAM-1, the application of soluble VLA-4 ligands or of reagents that elevate sVCAM-1 levels in serum (59) is expected to selectively block small subsets of high affinity VLA-4, leaving the majority of VLA-4 intact. These approaches may afford therapeutic advantages over VLA-4 blocking by mAbs, which interferes with normal leukocyte functions within noninflammed tissues (3).
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Acknowledgments |
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|
Footnotes |
|---|
2 Current address: Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., Ann Arbor, MI 48105.
3 Address correspondence and reprint requests to Dr. Ronen Alon, Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address:
4 Abbreviations used in this paper: VLA-4, very late Ag-4; wt, wild type; FN, fibronectin; LDV, leucine-asparatate-valine; HSA, human serum albumin; LIBS, ligand-induced binding site; 2-DOG, 2-deoxyglucose; DVL, aspartate-valine-leucine; sVCAM, soluble VCAM.
Received for publication June 1, 1998. Accepted for publication October 2, 1998.
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