From the Department of Immunology, The Weizmann
Institute of Science, Rehovot 76100, Israel, § Biogen, Inc.,
Cambridge, Massachusetts 02142, ¶ Elan Pharmaceuticals, Inc.,
South San Francisco, California 94080, and
Department of
Pharmacology, The Bruce Rappaport Faculty of Medicine, Technion-Israel
Institute of Technology, Haifa 31096, Israel
Received for publication, June 7, 2000, and in revised form, November 28, 2000
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ABSTRACT |
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In circulating lymphocytes, the VLA-4 integrin
preexists in multiple affinity states that mediate spontaneous
tethering, rolling, and arrest on its endothelial ligand, vascular cell
adhesion molecule-1 (VCAM-1). The regulation and function of
VLA-4 affinity in lymphocytes has never been elucidated. We show here
that p56lck, the major Src kinase in T cells, is a key
regulator of high affinity VLA-4. This high affinity is essential for
the rapid development of firm adhesion of resting T cells to VCAM-1 and to their extracellular matrix ligand, fibronectin. Lck-regulated VLA-4
function does not require intact TCR nor several key components of the
TCR signaling pathway, including ZAP-70 and SLP-76. Furthermore, stimulation of p56lck by the phosphatase inhibitor,
pervanadate, triggers firm VLA-4-dependent adhesion to
VCAM-1. Although Lck is not required for chemokine receptor signaling
to mitogen-activated protein kinase, the presence of
Lck-regulated high affinity VLA-4 also facilitates firm adhesion triggered by the chemokine, SDF-1, at short-lived contacts.
Surprisingly, bond formation rates, ability to tether cells to VLA-4
ligand, and VLA-4 tether bond stability under shear flow are not
affected by VLA-4 affinity or Lck activity. Thus, the ability of high
affinity VLA-4 to arrest cells on VCAM-1 under flow arises from
instantaneous post-ligand strengthening rather than from increased
kinetic stability of individual VLA-4 bonds. These results suggest that
p56lck maintains high affinity VLA-4 on circulating
lymphocytes, which determines their ability to strengthen VLA-4
adhesion and rapidly respond to proadhesive chemokine signals at
endothelial sites.
Lymphocytes circulating throughout the blood and lymphatic tissues
respond to inflammatory cytokines and antigenic signals (1). These
cytokine and antigenic stimuli regulate both the expression and
function of vascular receptors on effector and memory lymphocytes,
which determine their preferential migration to extra lymphoid sites of
inflammation (2). To emigrate from the circulation into inflamed
tissues, lymphocytes must develop rapid firm adhesion to endothelial
ligands, displayed on the vascular endothelium within the target
tissue. This firm adhesion is mediated primarily by the binding of
lymphocyte integrins such as VLA-4 ( The p56lck (Lck) is a key Src tyrosine kinase of the TCR signaling
machinery of thymocytes and peripheral T lymphocytes (15-17). Lck can
physically associate with CD4, CD8, CD2, and CD28 coreceptors (18-20)
as well as with several receptors to major lymphocyte cytokines such as
IL-2 and IL-7 (21, 22) and can be membrane-anchored within
sphingolipid-rich microdomains, RAFTS (23, 24). Ligation of these
coreceptors and costimulatory molecules like L-selectin activate Lck
even in the absence of antigenic stimuli or TCR ligation (20, 25, 26).
Although cross-linking of TCR as well as of the aforementioned
costimulatory receptors can activate integrin-dependent adhesive processes (27-30), the majority of these processes require prolonged static contact with surface-ligands where the contribution of
integrin clustering, cytoskeletal remodeling, and cell spreading to the
generation of adhesive strength is large (31). Indeed, stimulation of
integrin function by TCR-signaling elements and associated
costimulatory molecules is not accompanied by induction of high
affinity states in the major lymphocyte integrins VLA-4, VLA-5, and
LFA-1 (31-33).
To test the involvement of the TCR machinery in modulating VLA-4
adhesion in T cells, we compared the affinity states and adhesive
properties of VLA-4 on Lck-deficient and -reconstituted Jurkat cells.
We demonstrate here that a high affinity VLA-4 subset continuously
regulated by Lck preexists on resting Jurkat cells as well as on
freshly isolated blood T cells. This subset is crucial for these
lymphocytes to firmly adhere to endothelial VCAM-1 and the
extracellular matrix ligand fibronectin (FN), both in the absence and
presence of chemokine-triggering signals. We show that this high
affinity subset is continuously regulated by Lck in resting cells,
independent of their TCR activation state and of key components of the
TCR signaling machinery. Lck-deficient cells are devoid of high
affinity VLA-4 subsets, display reduced adhesion strengthening to
VCAM-1 and FN, and exhibit impaired integrin responsiveness to T cell
chemokines compared with their Lck-reconstituted analogue cells. The
study also sheds new light on how integrin affinity to ligand
translates into tether adhesion strengthening events under dynamic
context of shear flow. Our work suggests that Lck activity in T cells
is a checkpoint in the generation of high affinity VLA-4, which may
determine the migratory patterns of T cells to specific sites of
inflammation expressing high levels of endothelial and extracellular
matrix VLA-4 ligands.
Reagents and Antibodies
The function-blocking HP1/2 mAb specific for the integrin
Cells
The Lck-deficient (JCAM1.6) and Lck-reconstituted (16),
TCR-negative and TCR-reconstituted (39), CD45-negative (40), SLP-76-deficient (J14-v-29), and SLP-76-reconstituted (J14-76-11) (41)
Jurkat lymphoblastoid cell lines were kindly provided by Dr. Arthur
Weiss (University of California, San Francisco, CA). The
ZAP-70-deficient (p116) and ZAP-70-reconstituted (p116.c39) (42) Jurkat
lines were a kind gift of Dr. Robert T. Abraham (Rochester,
Minnesota). Cells were maintained in RPMI 1640 supplemented with 10%
heat-inactivated FCS (Sigma), 2 mM L-glutamine
(Biological Industries, Beithemeek, Israel), 1 mM sodium
pyruvate (Biological Industries), 100 mM nonessential amino
acids (Biological Industries), 50 µM Western Blot Analysis
10 × 106 cells were solubilized in 100 µl of
lysis buffer (25 mM Tris, pH 7.5, 2 mM
vanadate, 0.5 mM EGTA, 10 mM NaF, 10 mM sodium pyrophosphate, 80 mM
Immunofluorescence Flow Cytometry
Cells were washed once with PBS, resuspended in FACS wash buffer
(PBS, 5% bovine serum albumin, and 0.05% sodium azide) and incubated
with primary antibodies (10 µg/ml) for 30 min at 4 °C. The samples
were then washed and incubated with secondary antibodies for 30 min at
4 °C. Cells were washed and analyzed immediately on a FACScan flow
cytometer (Becton Dickinson, Erembodegem, Belgium).
For surface staining of Binding of [3H]BIO-1211 to
[3H]BIO-1211, a radiolabeled LDV derivative was
prepared as described (44). For equilibrium binding measurements,
Jurkat cells were incubated for 40 min with increasing concentrations of [3H]BIO-1211 in Tris-buffered saline binding
medium (50 mM Tris HCl, 150 mM NaCl, 0.1%
bovine serum albumin, 2 mM glucose, 10 mM HEPES
pH 7.4) containing either Ca2+ and Mg2+ (1 mM each) or Mn2+ (2 mM). The cells
were pelleted by centrifugation, and bound ligand was quantitated by
scintillation counting as described (35). Background binding determined
in the absence of divalent cations was subtracted from total binding
counts. For kon measurements, cells were
incubated with 0.5-5 nM [3H]BIO-1211, and at
specific time points, cell samples were taken, diluted with 500-fold
excess of unlabeled BIO-1211 ligand, and immediately pelleted and
quantitated by scintillation counting. The kon
was determined by multiplying the observed binding rate constant by the
BIO-1211 concentration. For koff measurements, cells were incubated with 5 nM [3H]BIO-1211
for 40 min, diluted with a 500-fold excess of unlabeled BIO-1211 to
quench further binding of [3H]BIO-1211, and further
incubated for increasing periods up to 120 min. Cell samples were
pelleted at different time points, and radiolabeled ligand binding was
determined as above. Data were expressed as a percentage of the maximum
specific radioligand binding and were fit to an exponential curve by
nonlinear regression, yielding the first order
koff as described (35, 44).
Soluble Bivalent (VCAM-1)2-Ig Binding Assay
Cells (0.5 × 106) were incubated with
(VCAM-1)2-Ig (first two domain segments of human VCAM-1
fused to a single human Fc domain) for 30 min at room temperature in
H/H binding medium. Cells were then washed twice by suspension in H/H
binding medium and incubated with PE-donkey anti-human IgG (Jackson)
for 20 min at 4 °C (in binding medium) before washing with PBS, and
fluorescence was analyzed by FACS. The results were expressed as mean
fluorescence intensity units.
Preparation of Adhesive Substrates
Affinity-purified seven-domain human VCAM-1 (45) or human FN
(Life Technologies) was dissolved in PBS buffered with 20 mM bicarbonate, pH 8.5, and incubated on a polystyrene
plate (60 × 15-mm Petri dish; Becton Dickinson, Lincoln Park, NJ)
for 2 h at 37 °C or overnight at 4 °C. The plate was then
washed three times and blocked with human serum albumin (20 mg/ml PBS)
for 2 h at 4 °C. For SDF-1 coimmobilization on the adhesive
substrate, ligands were coated in the presence of normal or
heat-denatured chemokine (R&D Systems, Minneapolis, MN) (2 µg/ml) and
a carrier protein (human serum albumin, 2 µg/ml) and washed and
quenched as above.
Laminar Flow Adhesion Assays
Analysis of Cell Tethering, Rolling, and Arrest--
A
polystyrene plate on which purified ligand had been adsorbed was
assembled in a parallel plate laminar flow chamber (260-µm gap)
mounted on the stage of an inverted phase-contrast microscope (Diaphot
30, Nikon, Tokyo, Japan) as described previously (46, 47). Cells were
washed with cation-free H/H medium containing 5 mM EDTA and
stored in cation-free H/H medium at room temperature up to 1 h.
Cells were diluted with H/H binding medium and perfused through the
flow chamber at the desired shear stress generated with an automated
syringe pump (Harvard Apparatus, Natick, MA). All experiments were
carried out at 37 °C. Cell images were videotaped with a long
integration LIS-700 CCD video camera (Applitech, Holon, Israel) and a
Sony SLV E400 video recorder (Sony, Japan) and manually quantified by
analysis directly from the monitor screen. All adhesive interactions
between the cells flowing over ligand-coated substrates at low shear
stresses of 0.5-1.5 dyn/cm2 were tracked and quantified.
Tethering events were defined as adhesive interactions between cells
and the substrate. Four categories of cell tethers under flow were
defined as follows. Tethers were defined as transient if cells attached
briefly (<2 s) to the substrate in a VLA-4-dependent
manner, rolling if cells displayed rolling motions >5 s with a
velocity of at least 1 µm/s, and slow rolling if cells rolled >5 s
with a velocity of less than 1 µm/s, and arrests were defined as
cells that immediately arrested on the substrate, remaining adherent
and stationary for at least 20 s of continuous flow. Firm arrests
were defined as cells that arrested under flow and resisted detachment
when subjected to abruptly increased shear force of 2.5 dyn/cm2 for 5 s. The number of tethers for each was
divided by the flux of freely flowing cells in close proximity to the
substrate that moved through the same field during the analysis period.
Analysis of Transient Cell Tethers to Low Density
VCAM-1--
Transient tethers were determined as previously described
(47) at a resolution of 0.02 s in a digital still playback mode (Panasonic AG-7355, Osaka, Japan). Background tethering to
VCAM-1-coated fields blocked with the VCAM-1-blocking mAb, 4B9, was
less than 5% of overall tethers. All tethering events were set to
start at t = 0, and the natural log of the tethers that
remained bound after initiation of tethering derived from the duration
of all transient tethers and was plotted against tether duration to
yield a slope = Analysis of Cell Resistance to Detachment after Static Contact
with Ligand--
Cells were allowed to settle onto the substrate for
1-2 min at stasis. Flow was then initiated and increased stepwise
every 5 s. The number of cells that remained bound was expressed
relative to the number of cells originally settled on the substrate.
Prolonged adhesion assays were similarly performed by allowing cells to settle for 15 min on flow chamber-assembled substrates. Nonadherent cells were removed by a single 2-s wash of cells at a shear stress of
50 dyn/cm2.
Spontaneous VLA-4 Adhesiveness to VCAM-1 and Fibronectin and
Expression of the
Interestingly, Lck-reconstituted Jurkat cells displayed higher
constitutive staining of the Affinity of VLA-4 to Soluble Ligands Is Compromised in
Lck-deficient Cells--
We considered that the dramatically reduced
adhesiveness of VLA-4 on the Lck-deficient cells could be the result of
an altered intrinsic affinity of the integrin to ligand. VLA-4
recognizes an LDV-containing sequence on the CS-1 domain of FN (49)
that is homologous and isosteric with the tetrapeptide Gln-Ile-Asp-Ser (QIDS), the VLA-4 binding site in VCAM-1 (50). LDV octapeptides serve
as useful probes of VLA-4 affinity for both VCAM-1 and FN and can block
high affinity VLA-4 (14, 36). Since direct binding of monovalent VCAM-1
to cell surface VLA-4 cannot be monitored because of its extremely low
binding affinity in physiological medium (33), three approaches were
taken to compare VLA-4 affinity states in Lck-deficient and
Lck-reconstituted Jurkat cells: 1) LDV peptide saturation binding to
VLA-4 on Lck+ and Lck Lck-triggered High Affinity VLA-4 Is Essential for Firm Cell
Adhesion to VCAM-1--
To determine how the presence of high affinity
VLA-4 affects rapid adhesion developed by T cells on VCAM-1, we
utilized LDV peptides as selective blockers of high affinity VLA-4
states (14). Lck-expressing or -deficient Jurkat cells were allowed to
adhere briefly to VCAM-1 in the presence of control DVL or increasing concentrations of LDV octapeptide. The strong adhesion of
Lck-expressing cells (40% Lck+ versus 8%
Lck
We next compared VLA-4 tethering to VCAM-1 in shear flow in
Lck-deficient and -reconstituted Jurkat cells. The ability of VLA-4 to
tether cells to the integrin ligand did not depend on the presence of
Lck (Figs. 4A). However,
Lck-reconstituted Jurkat cells arrested on high density VCAM-1 within
fractions of seconds and developed firm shear-resistant adhesion much
more readily than Lck-deficient cells under shear flow (Fig.
4A). The high affinity VLA-4 subsets on Lck-expressing cells
were the exclusive mediators of firm arrest to VCAM-1, as evident by
the complete susceptibility of firm cellular arrests but not of weak
arrests, rolling, or transient tethering events to soluble LDV at 12 µM, a concentration selective for full occupancy of high
affinity VLA-4 (data not shown). Taken together these results suggest
that high affinity VLA-4 present exclusively in Lck-expressing cells is
critical for their ability to rapidly develop firm spontaneous adhesion
to VCAM-1 both under shear flow and during short static contact.
Immunolocalization of VLA-4 by electron microscopy indicated similar
distribution of the integrin on surface microvilli in both
Lck-deficient and -reconstituted cells (data not shown), suggesting
that VLA-4 topography is Lck-independent. To further show that the
adhesive defect in Lck-deficient cells is affinity and not integrin
topography-dependent, we substituted anti-
The ability of high affinity VLA-4 subsets to rapidly arrest
Lck-expressing cells on high density VCAM-1 under flow could reflect
stronger adhesive bonds. To test this possibility, we compared
transient VLA-4-mediated tethering of Lck-expressing or Lck-deficient
cells to low density VCAM-1 under shear flow. In this assay, the
contribution of integrin rearrangement during formation of transient
tethers to low density VCAM-1 is minimized since the majority of
tethers lasts <0.5 s (Fig. 4B), too rapid for diffusion-limited
integrin rearrangements (51). In agreement with the similar tethering
frequency mediated by VLA-4 in Lck-deficient and -reconstituted cells
on physiological high densities of VCAM-1 (Fig. 4A) and with
the similar kon measured for the binding of the
LDV derivative to VLA-4 on both cell types (Table I), the formation
rate of the quantal adhesive tethers mediated by high or low affinity
VLA-4 to low density VCAM-1 were found to be similar (Fig.
4B). Transient tethers dissociated from low VCAM-1 with a
single exponential corresponding to a single first order dissociation rate constant (koff) (Fig. 4B). This
dissociation rate constant varied with the shear stress but did not
increase upon further dilution of VCAM-1 (not shown), suggesting it
represented quantal adhesive units. Surprisingly, and in contrast to
the 2-fold lower koff measured for high affinity
VLA-4 bonds than for low affinity VLA-4 bonds (Table I) and to the
markedly higher binding of soluble VCAM-1 to high affinity VLA-4 on
Lck-reconstituted cells, no significant difference was noted in the
koff of VLA-4 tethers mediated by the high or
low affinity VLA-4 on Lck-reconstituted or Lck-deficient cells,
respectively. This suggests that shear flow conditions abolish
koff differences of tether bonds that are
evident in equilibrium binding measurements. Nevertheless, although not
contributing to stronger adhesive bonds formed between VLA-4 and very
low density VCAM-1 under shear flow, high affinity VLA-4 recognition of
VCAM-1 translated robustly within subseconds to adhesion strengthening events on high density ligand both under flow (Figs. 4A) and
in stasis (Fig. 3).
Regulation of VLA-4 Activity by Lck Occurs Independently of the TCR
Machinery--
It is well established that TCR stimulation can
modulate integrin adhesiveness (27, 28, 52). Lck plays a prominent and upstream role in TCR stimulation; Lck phosphorylates signaling motifs,
known as immunoreceptor tyrosine-based activation motifs, which
recruit the Syk family tyrosine kinase, ZAP-70, leading to
phosphorylation of a large number of signaling proteins, including the
adaptor protein, SLP-76. Therefore we asked whether the constitutive Lck-dependent VLA-4 adhesiveness implicates key components
of the TCR machinery. To test this hypothesis, we analyzed VLA-4 function in mutant Jurkat T cell lines lacking expression of the TCR
Lck-mediated Activation of VLA-4 Depends on the Kinase Activity of
Lck but Not ZAP-70--
Given our finding that Lck-mediated regulation
of VLA-4 occurs independently of its central role in TCR signaling, we
next asked whether the kinase activity of Lck is required for this function. We used the tyrosine phosphatase inhibitor, pervanadate (PV)
to augment Lck kinase activity (53-57). Treatment of cells with
pervanadate induced tyrosine phosphorylation of multiple cellular
proteins in an Lck-dependent manner, since it was blocked by the Src kinase inhibitor PP2 and was not observed in Lck-deficient cells (Fig. 6A). Pervanadate
treatment of Lck-expressing Jurkat cells resulted in increased
VLA-4 adhesion to VCAM-1 during rapid static contact but did not affect
VLA-4 adhesion of Lck-deficient cells to VCAM-1 (Fig. 6B).
In contrast, direct DAG-dependent PKC activation with
phorbol 12-myristate 13-acetate could bypass Lck in triggering VLA-4
adhesiveness (Fig. 6B). To test the specific role of Lck
kinase activity in regulating VLA-4 function, we tested the
contribution of ZAP-70 protein-tyrosine kinase in pervanadate-induced up-regulation of VLA-4-mediated adhesion. Notably, ZAP-70-deficient and
-reconstituted cells displayed similar pervanadate-induced increases in
VLA-4-mediated adhesion (Fig. 6C), suggesting that ZAP-70 is
not required, nor does it modulate pervanadate-induced Lck-mediated
up-regulation of VLA-4 adhesion. In addition, PV stimulation of VLA-4
activity in Lck-expressing T cells was inhibited by the selective
DAG-dependent PKC inhibitor GF 109203X (data not shown),
suggesting that Lck activation of VLA-4 is mediated by
DAG-dependent PKC activity. High affinity VLA-4 expressed
by resting Lck-expressing Jurkat cells could also be suppressed upon cell treatment with GF 109203X, further indicating that both
spontaneously maintained and PV-stimulated high affinity VLA-4 requires
intact PKC activity.
Jurkat cell integrin regulation by T cell activation signals resembles
that of effector VLA-4hi PBL (14). Consistent with a major
role of Lck activity in VLA-4 activation in Jurkat cells, short
pervanadate stimulation of freshly isolated human PBL triggered LDV
induction of VLA-4-associated LIBS, which was completely sensitive to
PP2 inhibition (Fig. 7A). Pervanadate also enhanced immediate VLA-4-dependent
lymphocyte arrests and strengthened lymphocyte rolling adhesions on
VCAM-1 under physiological shear flow (data not shown). Short exposure of T cells to IL-2 stimulates Lck activity independent of antigenic stimulation (21, 58, 59). Consistent with this finding, short
pretreatment of PBL with IL-2 enhanced firm VLA-4 adhesion to VCAM-1
under physiological flow relative to that of untreated PBL (Fig.
7B). Relatively low concentrations of LDV peptide, which failed to interfere with any adhesive interactions mediated by VLA-4 on
untreated cells, could completely inhibit the IL-2-augmented firm
VLA-4-mediated adhesion to VCAM-1 (Fig. 7B). Transient
VLA-4-mediated adhesions, which were not affected by the IL-2
stimulation, were also not susceptible to inhibition by the LDV
peptide. Whereas VLA-4 on Lck-deficient Jurkat showed no response to
IL-2 stimulation, Lck-reconstituted cells did develop firm adhesion to
VCAM-1 under flow upon short exposure to the cytokine (data not shown).
Thus, IL-2-triggered rapid VLA-4 adhesion strengthening to VCAM-1 is Lck-dependent and is mediated by LDV-susceptible high
affinity VLA-4 subsets on T cells.
The Lck-dependent High Affinity VLA-4 Subset
Facilitates Chemokine-stimulated T Cell Adhesion--
Chemokines can
rapidly trigger leukocyte adhesion to vascular integrin ligands both
under shear flow and during short static contact (6, 60). Chemokine
stimulation of integrin adhesion requires intact Gi protein
but is independent of DAG-dependent PKC (61). We wished to
examine whether the Lck-dependent high affinity VLA-4 on T
cells plays any regulatory role in the integrin response to chemokine
stimulation by SDF-1, a prototype T cell chemokine that strongly
triggers VLA-4 adhesiveness under physiological flow (62).
Lck-reconstituted and Lck-deficient Jurkat cells express comparably
high levels of the SDF-1 receptor CXCR4 (Fig. 8A), which was functional in
both cellular systems, triggering equivalent G-protein signaling
activity in an Lck-independent manner (Fig. 8B). Consistent
with this, Lck activity was not required for optimal SDF-1-induced
signaling activity of CXCR4 in normal T cells, since the Src kinase
inhibitor PP2 did not affect SDF-1-triggered mitogen-activated protein
kinase activation in Lck-reconstituted cells (Fig. 8B).
Nevertheless, whereas SDF-1 induced robust VLA-4 adhesion of
Lck-expressing cells interacting with either VCAM-1 or FN, it failed to
stimulate VLA-4-dependent adhesion in Lck-deficient cells
(Fig. 9A). To test whether the
defect in chemokine responsiveness of the Lck-deficient cells was
directly linked to impaired VLA-4 affinity state, we artificially
activated VLA-4 affinity on Lck-deficient cells by the
integrin-activating mAb TS2/16. Indeed, integrin affinity-stimulated
Lck-deficient Jurkat cells were rendered responsive to SDF-1
stimulation of VLA-4 adhesion (Fig. 9B). Lck-deficient cells
were also responsive to SDF-1 stimulation of VLA-4 adhesion to high
density VCAM-1 (data not shown), suggesting that the VLA-4 avidity and
contact strength rather than intrinsic affinity determine the ability
of VLA-4 to respond to chemokine. These experiments suggest for the
first time that proadhesive effects of chemokines on integrin function
are facilitated by integrin avidity to ligand and that preexistent high
affinity subsets can augment chemokine triggering of integrin
adhesion.
Integrin-mediated adhesion strengthening can result from changes
in intrinsic affinity to ligand as well as alterations in lateral
integrin clustering and post-ligand cell spreading, which generate high
avidity to multivalent ligand through low affinity integrin subsets
(31, 32, 63, 64). Leukocyte adhesion to vascular endothelium under
shear flow involves the establishment of rapid
integrin-dependent adhesion within subseconds to seconds of
leukocyte contact with the endothelium, suggesting that high affinity
recognition of ligand by integrin molecules is important rather than
integrin recruitment at the adhesive zone or cell spreading (31, 32).
In this study, we examined how integrin affinity contributes to cell
adhesion in different contexts of recognition of immobilized ligands,
i.e. ligand density, contact time, shear flow, and
proadhesive cytokines. Our study assigns a major role for high affinity
integrin recognition of ligand in regulating rapid development of firm
integrin-dependent adhesion at short-lived adhesive zones
under physiological flow and during short static contacts.
A subset of VLA-4, found by us to be constitutively regulated on T
cells by the Src kinase p56lck, exhibits high affinity to both
fibronectin-derived VLA-4 binding peptides and soluble VCAM-1. This
high affinity VLA-4 was found crucial for development of firm
VLA-4-mediated adhesions by T cells interacting with VCAM-1 and
fibronectin at short-lived contacts and under shear flow. Adhesive
interactions mediated by low affinity VLA-4 subsets were found to
support only weak adhesions to VCAM-1, which readily dissociated under
high shear forces (Figs. 1B, 3, and 4A).
Dissecting the relationship between ligand binding properties and
adhesiveness of distinct states of VLA-4, we found that both high
affinity and low affinity VLA-4 subsets (expressed by Lck-expressing and -deficient cells, respectively) bind a soluble monovalent fibronectin-derived VLA-4 peptide with similar
kon, which corresponded to similar rates of
tether formation supported by both VLA-4 subsets on immobilized VCAM-1
(Fig. 4). These results are consistent with our previous findings that
the formation rate of VLA-4 tethers to VCAM-1 does not depend on the
VLA-4 affinity state in Jurkat and PBL (14). High affinity VLA-4
recognition of VCAM-1 in Lck-expressing cells, but not in Lck-deficient
cells could, however, translate within subseconds of contact to rapid
adhesion strengthening on high density VCAM-1 under flow (Fig.
4A). Unexpectedly, high affinity VLA-4 dissociated from low
density VCAM-1 with similar rates to low affinity VLA-4 (Fig.
4B), suggesting that intrinsic tether bond lifetimes
mediated by either low or high affinity VLA-4 are similar. Similar
findings were observed with another Jurkat cell line, an activation
mutant defective in PKC signaling and high VLA-4 affinity that failed
to translate VLA-4 tethers to high density VCAM-1 into adhesion
strengthening events (14) but exhibited identical bond formation rates
and similar kinetic stability of tether bonds in the presence of shear
flow.2 This is a first
demonstration that equilibrium properties of integrin bonds do not
correlate to the kinetics of these bonds in the presence of tensile
shear forces. Nevertheless, despite its negligible contribution to
stability of individual VLA-4 tether bonds to low density VCAM-1 under
flow, high affinity VLA-4 has the capacity to instantaneously translate
its tethers to firm adhesion on high density VCAM-1 (Fig.
4A). When this high affinity subset is functionally
suppressed (by Lck deficiency) or blocked (by soluble LDV peptide), the
ability of VLA-4 tethers to develop rapid adhesion strengthening to
VCAM-1 under flow or upon static contact with ligand is completely
lost. The unique ability of high affinity VLA-4 to nucleate firm arrest
under flow could not be attributed to increased ability of high
affinity VLA-4 molecules to rearrange and cluster with ligand at
subsecond contact zones; both high and low affinity, VLA-4 on
Lck-reconstituted or Lck-deficient cells, respectively, shared a
comparable ability to tether to and cluster on immobilized VLA-4
binding mAbs (Fig. 1B and data not shown). We conclude that
high affinity occupancy of the integrin is obligatory to generate
instantaneous adhesion strengthening on multivalent ligand at subsecond
contact zones under shear flow and firm shear-resistant adhesion at
short static contacts.
Lymphocyte adherence to endothelium can be rapidly up-regulated upon
encounter with endothelium-displayed chemokines (6, 62, 65). We have
recently found that immobilized chemokine presented to adherent cells
in juxtaposition to VLA-4 or LFA-1 ligands can rapidly augment integrin
avidity to these ligands both at stasis and under shear flow (62, 65).
We now report that high affinity VLA-4 expressed on Lck-expressing
cells plays a crucial regulatory role in this process in that these
cells preferentially develop firm adhesion in response to surface-bound chemokines. Chemokine signaling is, however, Lck-independent, as SDF-1
induced rapid extracellular signal-regulated kinase 1/2 phosphorylation
in Lck-deficient cells as in Lck-reconstituted cells (Fig.
8B). Chemokine triggering of VLA-4 avidity results in
Gi-protein-dependent integrin clustering at the
adhesive contact zone and is not associated with alteration of integrin
affinity to ligand (62). Rather, the cells use preexistent affinity
states of VLA-4 to elicit various types of adhesive interactions with its ligands in response to adjacent chemokine signals. Thus, the ability of high affinity VLA-4 to nucleate high avidity interactions with ligand not only facilitates firm chemokine-independent VLA-4 adhesion to ligand but also augments the responsiveness of VLA-4 on
interacting cells to proadhesive chemokines presented to them at
adhesive contact sites. Indeed, above a threshold of integrin ligand
density, even low affinity VLA-4 on Lck-deficient cells could
efficiently respond to coimmobilized chemokine (data not shown). This
suggests that increased contact avidity to high density ligand can
compensate for the loss of high affinity VLA-4 in Lck-deficient cells
and rescue the poor responsiveness of VLA-4 on these cells to
immobilized chemokine. Further investigation is under way to test if
this novel role of high affinity integrin to function as a nucleation
element of both spontaneous and chemokine-triggered VLA-4 avidity to
ligand could apply to other vascular integrins such as LFA-1 and Mac-1.
Recent findings indicate that VLA-4 integrin ligation can increase
LFA-1 avidity to ICAM-1 at static contact (66). High affinity VLA-4 is
thought to be essential for this VLA-4 cross-talk with LFA-1,
suggestive of yet another function of high affinity VLA-4 subsets in
controlling vascular adhesion of circulating lymphocytes to inflamed
endothelium expressing both VCAM-1 and ICAM-1.
Lck maintains basal levels of constitutive activity in resting T cells
(59), regulating a steady state level of tyrosine phosphorylation on a
large number of lymphocyte proteins. Consistent with tight regulation
of VLA-4 function by p56lck, short inhibition of Jurkat cell or
blood T lymphocyte phosphatases by pervanadate directly activates
p56lck (53-57) concomitantly with activation of VLA-4
adhesion. Recently, the two major T cell Src kinases Fyn and Lck were
shown to have partially overlapping but distinct functions in Jurkat
cells (67). The complete lack of effect of PV on VLA-4 activity in
Lck-deficient Jurkat cells demonstrates that Lck is an indispensible
regulator of high affinity VLA-4 in these T cells. Our results suggest
a minor if any compensatory role for other Src kinases, such as Fyn, at
the level of which they are expressed and active in Jurkat cells (16,
67). Of course, our results do not rule out the possibility that other
Src kinases may play some role in regulating VLA-4 affinity in other
cell types. Additionally, the full effect of PV in triggering VLA-4
activity in ZAP-70-deficient cells, impaired in their activation of
PLC The dramatic differences in VLA-4-dependent adhesion to
VCAM-1 caused by small changes in binding affinity on subset of VLA-4 molecules shown by us suggests that VLA-4 affinity in circulating T
cells plays a crucial Lck-regulated function in versatile rapid adhesion strengthening processes mediated by this integrin.
p56lck activity is subjected to complex intracellular
regulation by both antigen-dependent TCR ligation as well
as by antigen-independent exposure to inflammatory cytokines, shown to
induce integrin activation epitopes that correlate with high affinity
states on effector lymphocytes (77). Indeed, short T cell exposure to
IL-2 is sufficient to enhance rapid T cell adhesion to VCAM-1 under
shear flow mediated by high affinity VLA-4 subsets (Fig. 7). Prolonged
activation of p56lck on effector T cells by IL-2 (22) is
therefore likely to up-regulate both VLA-4 affinity and rapid adhesion
strengthening on ligand at dynamic contexts. TCR activation of VLA-4
adhesion by antigen (27-29) and costimulatory elements physically
associated with Lck (19, 20, 30, 78) likely involve Lck activation as
well. Lck may thus be a key translator of both cytokine and antigenic information transmitted to effector T cells at lymphoid tissues before
they exit these sites and return to the circulation.
Lck-dependent acquisition of firm T cell adhesiveness to
VCAM-1, a key homing receptor of effector cells at sites of
inflammation, may serve as a checkpoint in the ability of these
lymphocytes to emigrate from blood vessels at inflamed extra-lymphoid
target organs.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
1), LFA-1, and
4
7 to their respective endothelial
ligands expressed or induced on vessel walls of target sites (3, 4). To
induce cell arrest on their endothelial ligands, lymphocyte integrins
must rapidly stabilize their short-lived contacts with endothelial
ligands without modulation of integrin surface expression. Stabilization of lymphocyte contacts could be critical for their subsequent response to endothelium-displayed chemoattractants (chemokines) (5-7), which further up-regulate integrin avidity as well
as lymphocyte spreading on the endothelium, critical steps in
lymphocyte extravasation to the surrounding tissue.
4 integrins are rather unique among other leukocyte
integrins in that they maintain basal heterogeneous affinity states, which enable binding to their endothelial ligands spontaneously in the
absence of cellular activation (3, 8-13). Previously we and others
have demonstrated that low affinity VLA-4 subsets mediate weak
tethering and rolling interactions of VLA-4-expressing lymphocytes on
vascular cell adhesion molecule
(VCAM-1)1 under shear flow
(3, 9, 14). We further demonstrated that a small subset of high
affinity VLA-4 expressed by a portion of circulating peripheral blood T
lymphocytes (PBL) or resting T leukemia Jurkat cells supports tethers
followed by firm arrest on high density VCAM-1, even in the absence of
stimulatory chemokines (14). The mode of regulation of this high
affinity subset on effector T cells by intracellular signaling pathways
has not been elucidated to date.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 subunit (34), anti-CXCR4-mAb 12G5 (Pharmingen, San
Diego, CA), the function-blocking anti-VCAM-1 mAb 4B9, the anti-VLA-5
mAb (Serotec, Oxford, UK), the nonblocking 5BG10 mAb specific for the
integrin
4 subunit (35), the
1
integrin-activating TS2/16 mAb (35) as well as the
1
integrin subunit-specific mAbs 15/7 (36), 9EG7 (37), and Huts4 and 21 (38) were all used as purified Ig. PE-conjugated goat anti-mouse IgG
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA),
PE-conjugated donkey anti-human IgG (Jackson), and fluorescein
isothiocyanate-conjugated mouse anti-rat IgG (Jackson) were used as
secondary antibodies. EILDVPST, an eight-residue peptide containing the
tripeptide integrin binding motif (leucine-aspartate-valine (LDV)),
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). Bovine serum
albumin fraction V, Ca2+ and Mg2+-free Hanks'
buffered saline solution, and Ficoll-Hypaque 1077 were obtained
from Sigma. Human serum albumin (fraction V), phorbol 12-myristate
13-acetate, Src kinase inhibitor PP2, phosphatidylinositol 3-kinase inhibitor Wortmannin, and PKC inhibitor GF 109203X
(bisindolylmaleimide I) were purchased from Calbiochem.
Pervanadate was prepared by mixing 20 mM sodium
orthovanadate (Aldrich) and equimolar concentrations of hydrogen
peroxide (Merck) for 15 min at room temperature. The reaction was
terminated by adding 200 µg/ml catalase (Sigma) and placed on ice
until use.
-mercaptoethanol
(Merck), 100 IU/ml penicillin (Life Technologies, Inc.), and 100 µg/ml streptomycin (Life Technologies, Inc.). The Lck-expressing
transfectants were grown in the same medium supplemented with 250 µg/ml hygromycin (Calbiochem-Novabiochem). Human PBL (obtained from
healthy donors) were isolated from citrate-anticoagulated buffy coats
by dextran sedimentation and density gradient centrifugation separation
as previously described (43). The mononuclear cells were further
purified on nylon wool, and monocytes were removed by plastic
adsorption. The resulting PBL were >90% CD3+ T lymphocytes.
-glycerol phosphate, 25 mM NaCl, 10 mM MgCl2, 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotonin, 20 µg/ml
leupeptin) and, 10-µl lysates were separated by SDS-polyacrylamide
gel electrophoresis in reducing buffer. Proteins were
electrotransferred to nitrocellulose membranes and reacted with either
monoclonal anti-Lck (Santa Cruz Biotechnology, Santa Cruz, CA),
anti-phosphotyrosine (PY-20, Transduction Laboratories, Lexington, KY),
or anti-phosphospecific extracellular signal-regulated kinase 1/2 (a
kind gift from Dr. Rony Seger, Weizmann Institute) followed by
peroxidase-labeled goat anti-mouse (Jackson) or reacted with polyclonal
anti-extracellular signal-regulated kinase 1/2 (provided by R. Seger)
followed by peroxidase-labeled protein A (ICN Biomedicals, Inc. Aurora,
Ohio). Blots were developed using enhanced chemiluminescence (ECL, Sigma).
1 integrin activation epitopes
and for monitoring the induction of ligand-induced binding site (LIBS) epitopes by soluble peptide ligand, intact or treated cells were incubated for 5 min at 24 °C in H/H binding medium (Hanks' buffered saline solution containing 2 mg/ml bovine serum albumin and 10 mM Hepes, pH 7.4 supplemented with 1 mM
CaCl2 and 1 mM MgCl2) alone or in
the presence of LDV- or DVL-containing octapeptides. The LIBS-specific
anti-
1 mAbs, 15/7, 9EG7, or HUTS, were then added at 10 µg/ml to the cell suspension for an additional 5 min at 24 °C, and
cells were then incubated on ice for an additional 30-min period.
Unbound mAbs were washed out at 4 °C by multiple washing followed by
staining with PE-conjugated goat-anti mouse Ig. Induction of the 15/7
or 9EG7 epitope was determined and expressed as mean fluorescence
intensity units, with the preimmune mIgG control staining subtracted.
To compare ligand-induced 15/7 staining among differently treated PBL,
15/7 staining in the presence of 10 µg/ml control DVL peptide was
subtracted from the 15/7 staining in the presence of equimolar LDV peptide.
4
1-expressing Jurkat Cells
koff.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 Activation Epitope Is Impaired in
Lck-deficient T Cells--
The adhesive properties of VLA-4 in a
mutant Jurkat line that lack functional Lck were compared with those in
the same mutant reconstituted with functional Lck (Ref. 16 and Fig.
1A). Both Lck-deficient
and -reconstituted T cells expressed comparable levels of the
4 and
1 subunits of the VLA-4
heterodimer, the major integrin receptor on these cells for the
endothelial ligand VCAM-1 and the extracellular matrix ligand FN, along
with moderate but similar levels of another
1 integrin,
the VLA-5 integrin (Fig. 1A). Notably, the Lck-reconstituted
cells had restored basal levels of constitutive tyrosine
phosphorylation strongly suppressed in the Lck depleted line (Fig.
1A). We therefore considered that Lck-regulated tyrosine
phosphorylation events might regulate integrin-mediated adhesion of
lymphocytes. To compare the adhesiveness of VLA-4 toward its
endothelial ligand, VCAM-1, and toward its extracellular matrix ligand,
FN, in both Lck-deficient and Lck-reconstituted cells, we tested the
ability of both cell types to develop shear resistant adhesion after a
brief static contact with the purified ligands, analyzed in parallel
plate flow chamber assays. VLA-4-dependent adhesion to
VCAM-1 or FN of Lck-expressing cells was strikingly greater than that
exhibited by Lck-deficient cells (Fig. 1B). Lck-deficient
cells remained defective in VLA-4-dependent adhesion to
VCAM-1 or FN even after 15 min of contact with ligand (data not shown).
VLA-4 on Lck-deficient cells was, however, structurally intact with
respect to acquisition of activated phenotype, since external
activation of VLA-4 with the nonphysiological affinity-stimulating mAb
TS2/16 restored firm VLA-4 adhesiveness to both VCAM-1 and FN (data not
shown). The strength of
4 integrin-dependent
adhesion of both Lck-expressing and Lck-deficient Jurkat cells to
surface-bound
4-specific mAb was, however,
indistinguishable (Fig. 1B), consistent with similar
clustering capability of
4 integrins on both cell types.
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Fig. 1.
Lck increases
1 integrin-dependent T cell
adhesion and induces
1 activation
epitope without altering the expression of the VLA-4 and VLA-5
integrins. A, absence of Lck and reduced levels of
constitutive tyrosine phosphorylation of multiple substrates in
Lck-deficient Jurkat cells. Top left panel, immunoblot
analysis of Lck in Lck-deficient cells, Lck-deficient cells
reconstituted with intact Lck cDNA, and parental Jurkat cell line.
Middle panel, phosphotyrosine immunoblots of total cell
lysates of Lck-deficient or -reconstituted Jurkat cells. Equal amounts
of total proteins of each cell type were separated on reducing 7.5%
SDS-polyacrylamide gel electrophoresis and immunoblotted with the
phosphotyrosine-specific mAb, PY-20. Right panel, FACS
staining of VLA-4 and VLA-5 integrins on LCK-deficient (thin
line) and -reconstituted (thick line) Jurkat
cells using the
4 and
5 integrin
subunit-specific mAbs followed by secondary fluorescein
isothiocyanate-labeled-anti-mouse Ig. B, spontaneous
adhesion of Lck-deficient (open circles) or
Lck-reconstituted (closed squares) cells to VCAM-1 (medium
density; 0.2 µg/ml) or fibronectin (high density; 5 µg/ml) or to
the
4 integrin-specific mAb HP1/2 (coated at 0.2 µg/ml
on the substrate) at stasis. Strength of adhesion was analyzed by
measuring the resistance to detachment by controlled shear forces of
compared Jurkat populations settled for 2 min on ligand-coated
substrates assembled as the lower plate of a flow chamber. Cells were
then subjected to an incremented shear stress assay. The number of
cells remaining bound at the end of each 5-s interval of shear increase
was expressed relative to the number of cells originally settled on the
substrate. Antibody blocking with the anti-
4 HP1/2 mAb
completely eliminated T cell adhesion to VCAM-1 (Fig. 1B,
left panel) and reduced adhesion to FN by >90% (data not
shown) in these short term static assays. All measurements were
performed at 37 °C in adhesion binding medium. The mean ± range of two independent experiments is depicted. Results are
representative of six and three independent experiments, respectively.
C, FACS staining of the
1 activation epitope
15/7 in Lck-deficient (thin line) and-reconstituted
(thick line) Jurkat cells.
1 subunit activation
epitope 15/7 (36, 48) than Lck-deficient cells in the absence of
soluble integrin ligand (Fig. 1C) or divalent cations (data
not shown), suggesting a higher activation state of
1
integrins in these cells than in the Lck-deficient ones.
Lck-reconstituted cells also expressed higher levels of other
1 integrin activation epitopes including 9EG7 (37),
HUTS4, and HUTS21 (38) than the Lck-deficient cells, despite similar
levels of total
1 integrins on both cells (data not
shown). Collectively, these results suggest that constitutive Lck
activity in resting T cells is essential for
1 integrin
activation and strong VLA-4 adhesiveness developed during short static
contacts with the physiological integrin ligands VCAM-1 and FN.
Jurkat populations,
monitored by the peptide induction of LIBS on the
1
integrin subunit of VLA-4; 2) direct binding of a radiolabeled high
affinity LDV peptide derivative, BIO-1211 (44) to VLA-4 on Lck mutant
and -reconstituted Jurkat cells; and 3) direct binding of bivalent
VCAM-Ig monitored by secondary antibody staining on both cell types. An
LDV-containing octapeptide induced the LIBS epitope 15/7 on entire
populations of both Lck-deficient and -reconstituted Jurkat cells in a
dose-dependent manner (Fig.
2A and data not shown).
However, only Lck-reconstituted cells exhibited saturation binding of
LDV, mediated by two classes of VLA-4 subsets; the first high affinity
subset saturated at 5 µM peptide (EC50 of <1
µM), and the second moderate affinity subset (second
plateau) saturated at 100 µM (EC50 of 25 µM) (Fig. 2A). Consistent with the presence of
high affinity VLA-4 subsets in Lck-expressing cells, significantly
lower Kd values were measured in direct binding
measurements of [3H]BIO-1211 to VLA-4 on Lck-expressing
cells both in physiological medium as well as in medium containing the
integrin-activating cation Mn2+ (Table
I). Notably, the difference in VLA-4
affinity between the Lck-expressing and Lck-deficient cells toward
BIO-1211 was much smaller than the difference in apparent VLA-4
affinity (i.e. EC50) between Lck-expressing and
Lck-deficient cells toward the LDV octapeptide. This reflects the
106-fold higher binding affinity of BIO-1211
versus the LDV octapeptide to VLA-4 (35, 44). Interestingly,
the lower Kd values (higher affinity) of BIO-1211
bound to VLA-4 on Lck-expressing cells were exclusively due to 2-fold
slower dissociation rates of the ligand from high affinity VLA-4, with
no change in the Kon values between the higher
and lower affinity VLA-4 states. Ligand dissociated more slowly from
VLA-4 on Lck-expressing cells than from VLA-4 on Lck-deficient cells
even in the presence of Mn2+, even though this cation
prolonged VLA-4 bond lifetime by 100-fold relative to physiological
cations (Table I). The increased affinity of ligand recognition by
VLA-4 in Lck-expressing cells is thus due to an inherently slower
dissociation rate of ligand from VLA-4 in these cells. Consistent with
a markedly stronger binding of soluble ligands to VLA-4 on
Lck-reconstituted cells, Lck-reconstituted cells exhibited saturated
binding of bivalent VCAM-1, with an EC50 of 1.4 nM, whereas the Lck-deficient cells bound the same ligand
with an EC50 of >10 nM (Fig. 2B and
data not shown).
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Fig. 2.
Induction of
1 integrin-specific LIBS epitope by
soluble monovalent VLA-4 ligand and the binding of soluble VCAM-1-Ig
are saturable at lower ligand concentrations in Lck-reconstituted
Jurkat cells than in Lck-deficient cells. A,
dose-dependent induction of the 15/7 epitope by increasing
concentrations of LDV containing octapeptide, but not by control DVL
octapeptide, on Lck-deficient (Lck-defic.) and
-reconstituted (Lck-reconst.) Jurkat cells. The 15/7 epitope
staining, as detected with PE-anti-mouse IgG and analyzed by FACScan is
expressed in mean fluorescence intensity units (M.F.I.). One
experiment is representative of three. B, bivalent
(VCAM-1)2-Ig binding to Lck-deficient and -reconstituted
Jurkat cells. Cells were incubated with increasing amounts of
(VCAM-1)2-Ig, washed, incubated with PE-anti human IgG, and
analyzed by FACScan. Background binding, determined in the presence of
5 mM EDTA, was less than 10% of the total binding and was
subtracted from that binding. One experiment is representative of
three.
Binding of 3H-BIO1211 to LCK-deficient and reconstituted Jurkat
cells
) was highly sensitive to the presence of LDV peptide,
indicating the involvement of high affinity VLA-4 molecules. By
contrast, the low level of VLA-4 adhesiveness to VCAM-1 developed by
Lck-deficient cells was completely insensitive to inhibition by LDV
peptide, indicating that these cells lack high affinity VLA-4 (Fig.
3). Consistent with these results, a
soluble VCAM-Ig fusion protein at a concentration saturating the high
affinity subset (10 nM) completely blocked all
VLA-4-dependent adhesion of Lck-expressing to VCAM-1-coated
substrates but did not affect the weak adhesion developed by
Lck-deficient cells on identical substrates (data not shown). This
demonstrates that high VLA-4 affinity maintained by Lck activity plays
an indispensable role in early VLA-4-mediated adhesion-strengthening
events of Jurkat cells adhered to VCAM-1 at stasis.
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Fig. 3.
Dose-dependent occupancy of high
affinity VLA-4 subsets by soluble LDV blocks adhesion to VCAM-1
developed by Lck-reconstituted (B) but not by Lck-deficient
(A) cells. Spontaneous adhesion of Lck-deficient or
Lck-reconstituted cells to VCAM-1 (coated at 0.2 µg/ml) is shown in
the presence of increasing concentrations of LDV octapeptide. Relative
extent of adherent cells from original input cells and their strength
of adhesion were analyzed by measuring the resistance to detachment of
cells preincubated with the indicated concentrations of peptides for 2 min and settled (unwashed) for an additional 2 min on the VCAM-1-coated
substrate. Cells were then subjected to an incremented shear stress
assay as described in Fig. 1. Extent and strength of cell adhesion in
the presence of the DVL control octapeptide was similar to that
measured in the absence of any peptide. The difference between adhesion
developed by Lck-reconstituted cells adhered to VCAM-1 in the presence
of 6 µM DVL versus 6 µM LDV
octapeptides was highly significant, p < 0.01. A
representative experiment of three is shown.
4 integrin-specific antibodies for VLA-4 ligands as the adhesive substrate. Lck-deficient cells showed no defect in their ability to
tether and arrest on a surface-immobilized anti-
4
integrin mAb HP1/2 under shear flow, consistent with the similar
adhesion to these mAbs after short static contact (Fig. 1B
and data not shown).
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Fig. 4.
Lck-regulated high affinity VLA-4 supports
tethers to VCAM-1 resulting in rapid adhesion strengthening under
flow. A, tethering, rolling, and spontaneous arrest of
Lck-deficient and -reconstituted Jurkat cells interacting with VCAM-1
under shear flow. The motions of all tethered cells were analyzed on
high density VCAM-1, coated at 0.5 µg/ml, and grouped into four
categories of tethers as described under "Experimental Procedures."
The fraction of each category is presented in each stacked
bar. One representative experiment of four is shown. B,
transient tethering of Lck-deficient and -reconstituted cells to low
density VCAM-1 (coated at 0.02 µg/ml) at low shear stress of 0.5 dyn/cm2. Kinetics of formation and dissociation of
VLA-4-mediated tethers, formed by equal number of Lck-deficient or
-reconstituted Jurkat cells perfused over the substrate for 1 min. The
natural log of tethers with duration lower or equal to the indicated
time as described under "Experimental Procedures." is shown.
Residual number of tethers longer than 1 s were not fitted to the
first order dissociation curve. In A and B, more
than 95% of all tethers were blocked by T cell pretreatment with the
anti- 4 integrin mAb, HP1/2 (not shown). One experiment
is representative of three.
chain, ZAP-70, or SLP-76. Each mutant was compared with a paired
reconstituted clone stably transfected with the wild-type cDNA and
expressing comparable levels of
4 integrin subunit of VLA-4 (Fig. 5, A-C). Analysis
of the ability of each pair to develop shear resistant adhesion to
VCAM-1 showed that the absence of the TCR, ZAP-70, or SLP-76 did not
impair VLA-4 adhesive function nor did it effect the activation state
of the integrin, as detected by the
1 epitope-specific
mAb 15/7 (Fig. 5, A-C). This is in direct contrast to the
highly impaired level of mAb 15/7 activation epitope and markedly
reduced VLA-4-mediated adhesion in Lck-deficient cells (Fig. 1,
B and C). Together, these results rule out a role for the TCR and other key TCR components in the constitutive
Lck-dependent up-regulation of VLA-4 function.
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Fig. 5.
Lck-regulated VLA-4 activity in Jurkat cells
is independent of the TCR, ZAP-70, or SLP-76. TCR, ZAP-70, and
SLP-76-deficient or -reconstituted Jurkat pairs are depicted in
panels A, B, and C, respectively.
Left panels (A-C), spontaneous rapid adhesion to
VCAM-1 (medium density; 0.15 µg/ml) at stasis. Strength of adhesion
was analyzed by measuring the resistance to detachment by controlled
shear forces of compared deficient or reconstituted Jurkat populations
settled for 2 min on VCAM-1-coated substrates as described in Fig. 1.
Right panels (A-C), FACS staining of VLA-4
integrin and 1 activation epitope on deficient
(thin line) and reconstituted (thick line)
Jurkat cells using the
4 subunit-specific mAb and 15/7
mAb, respectively, followed by secondary PE labeled-anti-mouse
Ig.
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Fig. 6.
Lck stimulation by pervanadate augments VLA-4
adhesion to VCAM-1. A, immunoblots with the
anti-phosphotyrosine specific mAb, PY-20, showing enhanced tyrosine
phosphorylation in total Jurkat lysates induced by short treatment with
the phosphatase inhibitor PV. The indicated cells were left intact or
pretreated for 30 min with the Src kinase inhibitor PP2 (40 µM) before a 2-min treatment with PV (100 µM, see "Experimental Procedures"). B,
extent of cell adherence and resistance to detachment by shear flow
developed by differently treated Lck-deficient or -reconstituted Jurkat
cells settled on medium density VCAM-1 (coated at 0.1 µg/ml).
Spontaneous or agonist-stimulated VLA-4-dependent adhesion
is depicted. Cells were left intact or pretreated for 2 min with either
phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) or PV (100 µM) before being settled for 2 min on the VCAM-1
substrate. Cells were then subjected to an incremented shear stress
assay as described in Fig. 1. No adhesion to VCAM-1 of either type of
Jurkat cells was observed if cells were pretreated with the
anti- 4 subunit mAb HP1/2 (not shown). The mean ± range of two independent experiments is depicted. Results are
representative of four independent experiments. C, extent of
cell adherence developed by resting or agonist-treated ZAP-70-deficient
or -reconstituted Jurkat cells settled on medium density VCAM-1 (coated
at 0.1 µg/ml). All treatments and assays are as described in
B. The mean ± range of two independent experiments is
depicted. Results are representative of three independent
experiments.
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Fig. 7.
Effect of short term IL-2 and pervanadate
treatment of PBL on VLA-4 affinity to ligand and high affinity
VLA-4-mediated firm adhesion under shear flow. A,
effect of pervanadate pretreatment on VLA-4 binding to soluble LDV.
Binding was monitored by LDV-dependent induction of LIBS
epitope 15/7. The 15/7 staining of intact or treated PBL in the
presence of 12 µm of LDV octapeptide, expressed in mean fluorescence
intensity units (M.F.I.), was corrected for background
staining in the presence of control DVL peptide. This background
staining was identical for intact and PV-treated PBL. To block PV
stimulation of Src activity, PBL were pretreated for 30 min with the
Src kinase inhibitor PP2 (40 µM) before incubation with
PV. B, effects of LDV peptide on intact or IL-2-treated PBL
rolling and arrest on VCAM-1 (2 µg/ml) under flow. The motions of all
tethered PBL were analyzed and grouped into the indicated categories of
tethers, and the frequencies of the different tether categories are
depicted in the stacked bars, as in Fig. 4. Cells were left
intact or pretreated for 15 min with 100 units/ml IL-2 at
37 °C in binding medium before incubation with or without LDV
peptide (240 µM) (a subsaturating concentration for high
affinity VLA-4 on PBL (14)). DVL-containing peptide had no inhibitory
effect on PBL tethers to VCAM-1 under identical experimental conditions
(not shown). The experiments described in A and B
are representative of four independent experiments with multiple
donors.
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Fig. 8.
CXCR4 expression and SDF-1 signaling are
Lck-independent. A, FACS staining of the SDF-1 receptor
CXCR4 on Lck-deficient (thin line) and -reconstituted
(thick line) cells with the mAb 12G5 or isotype-matched
control mAb. B, SDF-1 stimulation of extracellular
signal-regulated kinase 1/2 phosphorylation in Lck-deficient and
Lck-reconstituted cells. 1 × 106 cells (intact or
pretreated with the Src kinase inhibitor PP2 (40 µM) for
30 min) were stimulated with PV (100 nM, 2 min), phorbol
12-myristate 13-acetate (PMA, 100 ng/ml, 2 min), or SDF-1
(100 nM, 30 s) at 37 °C. Cells were lysed
immediately after stimulation, and lysates were separated on reducing
10% SDS-polyacrylamide gel electrophoresis and immunoblotted with
anti-phosphospecific extracellular signal-regulated kinase 1/2
(upper panel). Immunoblots were stripped and reprobed with
anti-extracellular signal-regulated kinase (lower
panel).
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Fig. 9.
VLA-4 affinity enhances SDF-1 triggering of
VLA-4-mediated adhesion to VCAM-1 and FN. A, resistance
to detachment by shear flow developed by Lck-deficient or
-reconstituted Jurkat cells settled for 2 min on low density VCAM-1
(0.05 µg/ml) or fibronectin (2 µg/ml) alone or in the presence of
coimmobilized SDF-1 (2 µg/ml). Note that at low density integrin
ligand in the absence of chemokine, both types of Jurkat cells failed
to develop detectable adhesion. No adhesion of either cell type to the
VCAM-1 or FN coimmobilized with chemokine was observed in the presence
of the 4 integrin-blocking mAb, HP1/2, or the integrin
inhibitor EDTA, respectively (data not shown). The mean ± range
of data collected from two representative fields of view are depicted.
One experiment is representative of three. B, resistance to
detachment by shear flow developed by Lck-deficient Jurkat cells
settled on VCAM-1 alone or coimmobilized with SDF-1 after artificial
activation of VLA-4 affinity with the
1
integrin-activating mAb TS2/16. Cells were preincubated in binding
medium alone or with 0.5 µg/ml mAb for 5 min and allowed to settle
for 2 min on low density VCAM-1 (0.05 µg/ml) before being subject to
detachment. TS2/16-stimulated adhesion to VCAM-1 in the absence of
presence of chemokine was integrin-specific as it was completely
blocked in the presence of EDTA. One experiment is representative of
three.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 in response to PV (42), suggests that Lck activation of VLA-4
function does not require and is not modulated by ZAP-70 or PLC
1
activities. Although we link Lck-regulated tyrosine phosphorylation to
VLA-4 affinity, tyrosine phosphorylation of integrin tail cytoplasmic
domains per se is unlikely to be implicated in Lck-regulated
VLA-4 affinity. Integrin tyrosine phosphorylation does not result in
altered binding to soluble ligand (68, 69) but, rather, has been
implicated in physical association with the cytoskeleton (70).
Furthermore, activation of VLA-4 function through p56lck
required intact activities of DAG-dependent PKC (data not
shown), implicating serine/threonine phosphorylation events in
Lck-regulated VLA-4 function. It has been shown that Lck localizes
within discrete lipid microdomains, RAFTS, (24) which are enriched in
heterotrimeric G proteins and TCR signaling elements (71). As VLA-4
colocalizes to specific RAFTS (72), these domains may serve as
platforms for Lck to sequester and activate affinity modulators of
VLA-4. Candidate Lck substrates for induction of high VLA-4 affinity subsets are members of the Ras family, shown to modulate
1 integrin affinity to ligand in fibroblasts (73, 74)
and T cells (75). Rap1, a Ras-family GTPase, has been recently shown to
regulate integrin adhesiveness in T cells, but it does not appear to
control constitutive VLA-4 avidity to ligand (76).
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. A. Weiss for providing us with the Lck-deficient (JCAM1.6) and Lck-reconstituted as well as TCR- and SLP-76-depleted and -reconstituted Jurkat cell lines. We also thank Dr. B. Abraham for providing us with ZAP-70-deficient and -reconstituted Jurkat cells. We thank Dr. R. Seger for providing anti-extracellular signal-regulated kinase 1/2 antibodies. We also wish to thank G. Cinamon for assistance in PBL isolation and Dr. S. Schwarzbaum for editorial assistance. Special thanks to Drs. A. Peled and R. Seger for helpful discussions.
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FOOTNOTES |
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* This work was supported in parts by the Israel Science Foundation and the Minnerva Foundation, Germany.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** Incumbent of The Tauro Career Development Chair in Biomedical Research and a recipient of the Jakubskind-Cymerman award. To whom correspondence should be addressed. Tel.: 972-8-934-2482; Fax: 972-8-934-4141; E-mail: ronalon@wicc.weizmann.ac.il.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M004939200
2 C. Chen and R. Alon, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: VCAM-1, vascular cell adhesion molecule-1; PBL, peripheral blood T lymphocytes; DVL, aspartate-leucine-valine; FN, fibronectin; LDV, leucine-aspartate-valine; SDF-1, stromal-derived factor-1; TCR, T cell receptor; PE, phosphatidylethanolamine; DAG, diacylglycerol; IL, interleukin; mAb, monoclonal antibody; PKC, protein kinase C; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; LIBS, ligand-induced binding site; PV, pervanadate.
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