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4
1 Governs Lymphocyte Migration1
*
Department of Vascular Biology, The Scripps Research Institute, La Jolla, CA 92037; and
Department of Immunology, Weizmann Institute of Science, Rehovot, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4
1 with
endothelial VCAM-1 controls the trafficking of lymphocytes from blood
into peripheral tissues. Cells actively regulate the affinity of
41 for VCAM-1 (activation). To
investigate the biological function of 41
activation, we isolated Jurkat T cell lines with defective
41 activation. Using these cells, we
found that 41-stimulated
L2-dependent cell migration was
dramatically reduced in cells with defects in
41 activation. These cells required 20
times more VCAM-1 to promote L2-dependent
cell migration. This defect was at the level of
41 affinity as an activating
41 Ab rescued
41-stimulated
L2-dependent migration. In contrast,
migration of 41 activation-defective
cells on VCAM-1 alone was enhanced at higher VCAM-1 densities. Thus,
41 activation determines a set point or
threshold at which VCAM-1 can regulate
L2-dependent as well as
41-dependent cell migration. Changes in
this set point may specify preferred anatomical sites of
integrin-dependent leukocyte emigration from the
bloodstream. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Changes in the function of leukocyte integrins may also contribute to
the control of leukocyte emigration from blood vessels. Two general
mechanisms have been described by which integrin-mediated adhesion is
regulated: 1) alterations in integrin affinity for extracellular
ligands (activation) and 2) affinity-independent mechanisms such as
changes in receptor mobility (6).
4 integrins play a major role in controlling
leukocyte emigration, but the role of activation in
4 integrin function has been questioned
(7). Recent studies established that
4 integrins undergo active affinity modulation
(8). Furthermore, NK cells express constitutively active
41, whereas the bulk
of resting T cells expresses inactive
41. Agonists can
activate 41 on
memory, but not naive T cells. Moreover, integrin affinity can control
multiple cellular responses in addition to cell adhesion. Thus, the
activation of integrin
41 is leukocyte type
specific, but its role in the control of leukocyte functions is
unclear.
4 integrins have potent signaling functions
that complement their capacity to mediate cell adhesion. For example,
4 integrins strongly promote cell migration
(9). Indeed, engagement of 4
integrins by trace quantities of VCAM-1 markedly stimulates
2 integrin-mediated cell adhesion and
migration (10, 11). In this sense, VCAM-1 is an agonist
for 41. The capacity
of agonists to initiate cellular responses is a function of the
affinity of their cellular receptors. The
41 ligand, VCAM-1, is
variably expressed at most vascular sites depending on the presence of
inflammatory responses (12, 13). Thus, affinity regulation
of this integrin might play a role in determining the threshold or
sensitivity of leukocytes to stimulation by VCAM-1.
41 affinity
modulation could potentially regulate a number of cellular functions
such as adhesion, migration, and signaling. In the present study, we
have assessed the biological role of activation of integrin
41 by deriving novel
cell lines that are incapable of activating
41. Using these cell
lines, we find that
41 affinity
determines the set point at which 1) VCAM-1 can stimulate
L2-dependent cell
migration on ICAM-1 and 2)
41-dependent
migration onVCAM-1 is regulated. In contrast, the activation of
41 had little impact
on static cell adhesion or adhesion in shear flow. The data suggest
that the affinity state of 4 integrins governs
the selection of preferred sites of integrin-dependent leukocyte
transmigration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Jurkat E6-1 T leukemic cell line was purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (BioWhittaker), 1% glutamine, 1% penicillin, and 1% streptomycin (Sigma, St. Louis, MO).
Reagents
The anti-human 1 mAb 8A2 was a
generous gift from N. Kovach and J. Harlan (University of Washington,
Seattle, WA). The anti-human 4, HP2/1;
anti-human 5, SAM1; and anti-human
1, K20 Abs were purchased from Immunotech
(Westbrook, ME). The anti-human 2 mAb
hybridoma cell line TS1/18 was obtained from American Type Culture
Collection and was used to generate ascities fluid. The cDNA encoding
the CS-1 region of fibronectin fused to GST was a gift from J. W.
Smith (Burnham Institute, La Jolla, CA). The expression and
purification of this fusion protein have been previously described
(14).
Construction and expression of VCAM-1- and ICAM-1-Ig fusion proteins
The cDNA for human VCAM-1 was a generous gift from T. Collins (Harvard University, Cambridge, MA). The coding sequence of the complete seven Ig domains of the extracellular region of VCAM-1 was PCR amplified and cloned into the NheI site of plasmid pB4Ig (a gift from R. Cobb, Tanabe Research Laboratories, San Diego, CA), which contains the human Fc coding sequence. The resulting VCAM-Ig fusion construct was excised with KpnI and cloned into pcDNA3.1- (Invitrogen, Carlsbad, CA). The resulting construct was transfected into Chinese hamster ovary (CHO)3 cells, and stable cell lines were isolated by selection in G418. A VCAM-Ig-expressing clonal cell line was isolated by limited dilution cloning and screening supernatant for VCAM-Ig production with a VCAM-1 ELISA (R&D Systems, Minneapolis, MN). Recombinant protein was purified from CHO cell supernatant using a protein A column. Similarly, an ICAM-Ig fusion construct, encoding the N-terminal two Ig-like domains of ICAM-1 (a generous gift from D. L. Simmons, CRF Laboratories, University of Oxford, Oxford, U.K.), was subcloned into pcDNA3.1- and transfected into CHO cells. ICAM-Ig fusion protein was isolated from the supernatants produced by a clonal cell line by protein A affinity chromatography.
Soluble VCAM-Ig-binding assay
Cells (5 x 105) were resuspended in a modified Tyrode’s buffer (150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 1 mg/ml glucose, and 1 mg/ml BSA) containing 1 mM CaCl2 and 1 mM MgCl2. The VCAM-Ig fusion protein was added at a final concentration of 100 nM and incubated for 30 min at room temperature. Cells were washed twice in Tyrode’s buffer and resuspended in the same buffer containing FITC-conjugated donkey anti-human IgG (Jackson ImmunoResearch, West Grove, PA) at a 1/100 dilution. After a 30-min incubation at 4°C, cells were washed twice, and bound Ab was detected using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed using CellQuest software.
Generation and isolation of Jurkat mutants
Jurkat cells were treated with ethyl methane sulfonate (EMS; 200
µg/ml) or ICR-191 (1.5 µg/ml; Sigma) for 24 h. After 5 days in
culture, soluble VCAM-1 (sVCAM-1)-binding assays were performed, and
low sVCAM-1-binding cells were isolated by cell sorting using a
FACStarPlus flow cytometer (BD Biosciences) into
96-well tissue culture-treated plates (Costar, Corning, Corning, NY).
Isolated clonal lines were sequentially reassayed for
4 expression with mAb HP2/1 and sVCAM-1
binding by flow cytometry. Jurkat lines with normal
4 expression and low sVCAM-1 binding were used
for further analysis.
Cell adhesion assays
Ninety-six-well Immulon 2HB plates (Dynex Technologies, Chantilly, VA) were incubated with indicated concentrations of VCAM-1 or the CS-1-containing fragment of fibronectin overnight at 4°C. Afterward, wells were blocked with 2% BSA in PBS for 30 min at room temperature. Cells in a modified Tyrode’s buffer were added to wells and allowed to adhere for 40 min at 37°C. Nonadherent cells were washed off with Tyrode’s buffer. Adherent cells were stained with 0.5% crystal violet stain in 20% methanol. The cell-incorporated crystal violet was solubilized with 10% acetic acid and measured in a microplate reader (Molecular Devices, Sunnyvale, CA) set at 560 nm.
Cell migration assay
Cell migration was assayed in a modified Boyden chamber assay
system. Transwells (Costar; Corning) polycarbonate membranes containing
3-µm pores were incubated with VCAM-1 and/or ICAM-1 in 0.1 M
NaHCO3 (pH 8) overnight at 4°C. Membranes were
blocked with 2% BSA in PBS for 30 min at room temperature. A total of
2 x 105 cells in RPMI 1640 with 10% FCS
was added to the top chamber. Stromal cell-derived factor-1
(R&D Systems) at a final concentration of 15 ng/ml was added to the
bottom chamber. Cells were allowed to migrate for 4 h at 37°C.
Cells in the bottom chamber were enumerated with a hemocytometer.
VCAM-1 ELISA
The amount of recombinant VCAM-1 bound to the polycarbonate membranes of the transwells was measured by ELISA. Briefly, transwell membranes were coated as described above for cell migration assay, except in the case of the sVCAM-1 studies in which membranes were coated with ICAM-1 and blocked with BSA before addition of sVCAM-1. Afterward, the membranes were washed and anti-human VCAM-1 Ab, P8B1 (1/3000 diluted ascites) was added and incubated for 4 h at room temperature. After extensive washes, an HRP-conjugated goat anti-mouse IgG (BioSource International, Camarillo, CA) was incubated with the membranes for 2 h at room temperature. Bound Ab was detected with an 80 mM citrate phosphate buffer (pH 5) containing o-phenylenediamine and hydrogen peroxide using a Molecular Devices ELISA plate reader set at 490 nm.
Laminar flow assays
A polystyrene plate coated with sVCAM-1 (affinity-purified seven-domain human VCAM-1, a gift from R. Lobb, Biogen, Cambridge, MA) 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 (15). Jurkat cells were perfused at 106 cells/ml of binding medium (HBSS containing 2 mg/ml BSA and 10 mM HEPES, pH 7.4, supplemented with Ca2+ and Mg2+, each at 1 mM) at the desired shear stress generated with an automated syringe pump (Harvard Apparatus, Natick, MA). Cellular interactions on a field of view of 0.34 mm2 were visualized with a x10 objective and manually quantified by analysis of images directly from the monitor screen. The motion of each interacting cell was monitored for 10 s following its initial tethering, and three categories of tethers were defined: transient, if cells attached briefly (<2 s) to the substrate; rolling, if cells tethered and rolled on the substrate >5 s with a velocity >1 µm/s; arrest, if following rolling or immediately after tethering, cells came to a full arrest and remained stationary on the substrate for at least 20 s. The number of tethers for each category was divided by the flux of freely flowing cells. For calculations of cell flux, only the fraction of perfused cells that came into close proximity with the substrate, and therefore was potentially capable of interacting with the substrate, was considered.
Controlled flow detachment assays were performed on cells that were
settled at stasis on ligand-coated plates for 1 min and then were
subjected to wall shear stresses increased stepwise every 5 s (by
a programmed set of flow rates delivered by the syringe pump). 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
originally settled on the substrate in stasis. All assays were
performed at room temperature. To study peptide inhibition of
41-mediated tethering
events, cells were suspended in binding medium with 0.5 mM octapeptide
EILDVPST (containing the tripeptide very late Ag-4-binding motif
leucine-asparatate-valine, LDV) or its control analogue EIDVLPST for 5
min, and then perfused unwashed through the flow chamber over the
VCAM-1-coated substrate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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41 activation
To evaluate the functional importance
41 affinity
modulation, we generated a panel of variant Jurkat T cells with defects
in 41 activation
(Act. Defect. Jurkat). Jurkat cells were chemically mutagenized with
either EMS or ICR-191, and 4-expressing cells
were selected for reduced sVCAM-1 binding by flow cytometry. Clonal
lines were isolated that expressed 4, but
failed to bind sVCAM-1. The characterization of a representative mutant
line, JD6, is shown in Fig. 1
. In
contrast to wild-type Jurkat cells, the mutant cells showed marked
reduction in constitutive sVCAM-1 binding, but binding was
reconstituted in the presence of the exogenous
1 integrin-activating mAb, 8A2. Furthermore,
4, 5,
1, and 2 integrin
subunit expression was similar to that of wild-type Jurkat cells. These
results indicate that these mutant lines have a defect in
41 activation, which
is not due to a change in integrin expression nor a defect in the
integrin’s ligand binding site. Three independent cell lines were
isolated from three different mutagenesis experiments, two with EMS and
one with ICR-191. Similar results were obtained with all three mutant
lines in the experiments to be described.
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41 is retained and
the defect impacts the activation process. Defective activation could
be due to mutations in either subunit of integrin
41 or a signaling
molecule that is required for
41 activation. To
distinguish between these two possibilities, mutant lines were
transfected with wild-type 4 and
1 integrin subunits. Failure to rescue sVCAM-1
binding by introduction of wild-type
41 indicates that
cells have a defect in signaling machinery needed to activate
41. The mutant lines
were transiently transfected with green fluorescent protein (as
a markers of transfection) and cDNAs encoding either
4, 1, or a
combination of subunits, and sVCAM-1-binding assays performed by flow
cytometry. In all three lines examined, transfection of exogenous
41 failed to rescue
sVCAM-1 binding (data not shown). This result indicates that the
defect in integrin activation is not ascribable to integrin mutations,
but rather is due to a defect in cellular mechanisms required for
activation of 41.
The affinity state of 41 controls
cell migration
41 integrins can
regulate adhesion and migration mediated by 2
integrins (10, 11, 16). Under these conditions, VCAM-1
acts as agonist that regulates 2 integrin
functions. The regulation of integrin function by soluble agonist, such
as chemokines, is a function of the affinity of the agonist receptor.
By analogy, we reasoned that the capacity of VCAM-1 to stimulate
2 integrins would be a function of the
affinity state of 41
(Fig. 2A). To examine
41-stimulated
2-dependent migration, Jurkat cells were
allowed to migrate on a substrate coated with 200 µg/ml ICAM-1, a
ligand for 2 integrins. We examined the effect
of addition of trace quantities of VCAM-1 (0.1–10 µg/ml). Under
these conditions, the filters were coated with 
250
molecules/µm2 of ICAM-1, and the addition of
small quantities of VCAM-1 did not significantly displace ICAM-1 (Fig. 2B).
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C). The stimulated migration was completely blocked with an
anti-2 Ab, TS1/18, indicating that the
migration was 2 dependent. Furthermore, the
stimulated migration was blocked with an anti-VCAM-1 Ab, P8B1 (Fig. 2C), as well as an anti-4 Ab,
HP2/1 (data not shown). As an alternative means of assessing VCAM-1
trans activation of 2-dependent
cell migration, we used sVCAM-1 to stimulate migration across modified
Boyden chambers coated with ICAM-1 alone. As shown in Fig. 2D, the addition of sVCAM-1 to Jurkat cells stimulated
2-dependent cell migration. ELISA confirmed
that negligible amounts of the sVCAM-1 became absorbed to the membranes
under these conditions (Fig. 2D). These results indicate
that the VCAM-1 acted as an agonist, stimulating
2-dependent migration by binding to
41.
To investigate the role of
41 affinity
modulation in 4-stimulated
2-dependent migration, the migration of
activation-defective JD6 cells on a mixed substrate of ICAM-1 and
VCAM-1 was examined. In contrast to wild-type Jurkat cells, the
stimulated migration of activation-defective cells required a more than
20-fold higher concentration of VCAM-1 (Fig. 3A). Since these mutant lines
were derived by chemical mutagenesis, it is possible that cellular
changes other than those affecting
41 affinity could
account for the decreased sensitivity of
2-dependent migration to VCAM-1 stimulation.
To test this possibility, we used an activating
1 Ab, 8A2, to reconstitute high-affinity
41 on the mutant
lines. In the presence of 8A2, VCAM-1-stimulated
2-dependent migration was identical in the
mutant and wild-type Jurkat T cell lines (Fig. 3B). This
rescue in function was not observed when a nonactivating
anti-1 Ab, K20, was used (data not shown).
Thus, the capacity of differing quantities of VCAM-1 to stimulate
2 integrin-dependent events is a function of
the affinity state of
41.
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B). The dose-response curve
was biphasic, with maximal migration occurring at an ICAM-1-coating
concentration of 10 µg/ml. Furthermore, the migration of wild-type
Jurkat cells on VCAM-1 in the absence of ICAM-1 was biphasic, with
maximal migration occurring at a VCAM-1-coating concentration of 10
µg/ml (Fig. 3A). However, the migration of the mutant
lines on VCAM-1 was augmented relative to wild-type cells, and maximal
migration occurred at a VCAM-1-coating concentration of 40 µg/ml.
Thus, the mutant cells do not have a general defect in
integrin-dependent cell migration. Furthermore, the
41-dependent
migration on different densities of VCAM-1 is a function of
41 affinity state.
These effects of 41
activation on cell migration were similarly observed in the two other
mutant Jurkat lines (Table I).
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41 activation has
little effect on cell adhesion under static or flow conditions
We next examined the adhesive properties of the
4 activation-defective Jurkat lines. Under
static conditions, adhesion of these lines to a wide concentration
range of coating concentrations of VCAM-1 or the CS-1 fragment of
fibronectin was not markedly different from wild-type Jurkat cells
(Fig. 5). For both cell lines, the
adhesion was 4 dependent, as it was blocked
with anti-4 Ab, HP2/1. Thus,
41 activation plays
little role in regulating static Jurkat cell adhesion.
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41 supports
dynamic and reversible tethering and rolling of cells in flowing blood
(17). At a shear flow of 1 dyne/cm2,
however, there was no significant difference in the number of
activation-defective Jurkat cells rolling and subsequently arresting on
VCAM-1 as compared with wild-type Jurkat cells (Fig. 6A). Furthermore, when
increasing shear force was applied, there was no difference in the
detachment of mutant and wild-type cells from VCAM-1 (Fig. 6B). Thus, the activation-defective cells exhibit similar
adhesion strengthening as wild-type cells. Chen and coworkers
(15) previously implicated the importance of high-affinity
41 in adhesion
strengthening of cells under flow conditions. This was partially based
on the ability of the LDV-containing peptide to inhibit adhesion
strengthening. Consequently, we questioned whether soluble LDV peptide
would inhibit adhesion strengthening of the activation-defective Jurkat
variants. As shown in Fig. 6C, adhesion of the
activation-defective and wild-type Jurkat cells on VCAM-1 was equally
inhibited by LDV peptide. This suggests that while the
activation-defective Jurkat cells have a reduced affinity for sVCAM-1,
their binding affinity for soluble LDV peptide is unaffected.
Collectively, these results suggest that loss of
41 activation in
these Jurkat variants has little effect on cell adhesion to VCAM-1
under static or flow conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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41 for VCAM-1
(activation) (8). In the present study, we have assessed
the biological role of
41 activation by
deriving Jurkat T cell lines that lack high-affinity VCAM-1 binding.
Using these cell lines, we observed that loss of
41 activation
dramatically reduces the sensitivity of cells to VCAM-1-stimulated
L2-dependent cell
migration. In contrast, defective
41 activation
enhanced migration on purified VCAM-1 substrates, alone, and had little
impact on static cell adhesion to VCAM-1 or on adhesion in shear flow.
These results lead us to propose that the activation of integrin
41 is involved in the
specification of preferred anatomical sites of leukocyte transmigration
from the vasculature.
To study the functional role of
41 affinity
modulation, we used chemical mutagenesis and flow cytometry to isolate
Jurkat lines with defects in
41 activation. Using
a VCAM-Ig fusion protein as a soluble ligand for
41, we previously
established that 20% of
41 expressed on
Jurkat cells is capable of binding sVCAM-1 with high affinity
(EC50 50 nM) (8), whereas <1%
of the 41 was
activated on the defective Jurkat cells. The defect in sVCAM-1 binding
in these mutant lines was not due to a reduction in integrin
expression, as the mutant lines expressed wild-type levels of
41. Furthermore, the
loss of sVCAM-1 binding was not due to an intrinsic defect in the
ligand binding site as sVCAM-1 binding could be rescued with exogenous
integrin activators such as Mn2+ or activating
Abs. The defect was not ascribable to an integrin mutation, because
overexpression of wild-type 4 and/or
1 subunits failed to rescue sVCAM-1 binding.
These results indicate that an element(s) of the intrinsic signaling
pathway required for
41 activation in
these cells was disrupted. These
41
activation-defective cells provide a tool to study the role of
41 affinity
modulation on T cell function.
41 affinity
modulation is involved in regulating 4
signaling to 2 integrins.
4 integrins can function as signaling
receptors as well as adhesion receptors. 4
integrin engagement influences biochemical pathways that affect cell
function such as metalloproteinase gene expression (18).
4 integrin signaling can also affect the
function of other integrin family members, a process termed
trans activation/suppression or cross-talk (19, 20). 41
signaling stimulates 2-dependent cell adhesion
(10, 11) and, as confirmed in the present work,
2-dependent cell migration. Coimmobilization
of VCAM-1, as a ligand for
41, and ICAM-1, as a
ligand for 2 integrins, stimulated Jurkat cell
migration. The stimulated cell migration was mediated by
2 integrins, as the increased migration was
blocked with an anti-2 Ab. The stimulated
migration was also blocked with an anti-VCAM-1 Ab, indicating that
engagement of 41 with
ligand was required for the stimulated migration. Consequently,
41 functions as a
VCAM-1 receptor to stimulate 2
integrin-dependent cell migration. In contrast to wild-type Jurkat
cells, the activation-defective Jurkat cells required 20-fold more
VCAM-1 to stimulate 2-dependent migration. The
defect in 41
signaling to 2 integrins in these Jurkat
variants was at the level of VCAM-1 binding, because the rescue of
high-affinity 41 by
an activating 1 Ab also rescued the
41-stimulated
2-dependent cell migration. Thus,
41 affinity, in
conjunction with VCAM-1 ligand density, regulates the migratory
function of 2 integrins.
41 activation plays a
minor role in the control of Jurkat cell adhesion under static and flow
conditions. The wild-type and activation-defective Jurkat lines
manifested similar adhesion to VCAM-1 and the CS-1 fragment of
fibronectin under static conditions. Thus, the high-affinity
41 on Jurkat cells do
not appear to play a major role in regulating static cell adhesion.
This finding is not unique to
41, as integrin
51 activation also
plays only a minor role in static cell adhesion (21).
Furthermore, wild-type and
41
activation-defective Jurkat cells showed similar tethering/rolling and
adhesion strengthening on VCAM-1 under flowing conditions. These
results are in agreement with Yauch et al. (22), who
reported that 41
affinity modulation plays a minor role in adhesion. However, one study
found that Jurkat variants with defective
4-dependent static adhesion also had a
defect in adhesion strengthening under flow (15). In that
same study, a small LDV-containing peptide was found to inhibit
adhesion strengthening of cells on VCAM-1. This led to the proposal
that high-affinity 41
was required for adhesion strengthening. It was surprising to find no
difference in adhesion strengthening between our
41
activation-defective and wild-type Jurkat cells. Furthermore, the
soluble LDV peptide inhibited equally well in these two cell types. It
is possible that our Jurkat variants may have lost high affinity for
VCAM-1, but retained a high-affinity recognition of LDV peptide.
Another possibility is that our sVCAM-1-binding mutants may have a
specific defect in recognition of Ig domain 4 of VCAM-1. VCAM-1
contains two 41
binding sites, Ig domains 1 and 4. It may be that soluble binding of
VCAM-1 requires recognition of both domains 1 and 4. A loss of domain 4
recognition could interfere with sVCAM-1 binding and cell signaling
without interfering with tethering under flow, which has been found to
be dependent on the VCAM-1 Ig domain 1. We are currently testing such a
possibility with the use of specific mutants and Abs to VCAM-1 Ig
domains 1 and 4.
Integrin activation is one mechanism for regulating integrin function
(23). The biologic role of integrin affinity modulation is
integrin and cell type specific (24). Activation of
IIb3 is critical for
platelet aggregation, but it has little impact on platelet adhesion to
fibrinogen (25). Similarly, activation of integrin
51 appears to play a
minor role in static cell adhesion, but is important in fibronectin
matrix assembly (26, 27). The results reported in this
work show little effect of
41 activation on
static adhesion. However, they define a new function for integrin
activation: regulation of the sensitivity or threshold of leukocytes to
stimulation by immobilized integrin ligands, such as VCAM-1.
Leukocyte subpopulations show tissue-specific trafficking patterns that
govern immune responses (2, 3). These tropisms are
dependent, in part, on adhesion molecule expression on leukocytes and
vascular endothelium (5). Integrin abundance on leukocytes
and counter ligand density on vascular endothelial cells is one
proposed means to regulate leukocyte trafficking (28). For
example, a subset of memory T cells, expressing high levels of
41, migrates
preferentially to nongastrointestinal, extralymphoid inflammatory
sites, in which VCAM-1 is expressed (28, 29, 30). Our results
suggest that 41
activation may play an important role in determining leukocyte
responses to different levels of VCAM-1. The affinity of
41 acts as a set
point for the cells’ response to VCAM-1 and subsequent stimulation of
2-dependent migration. Primary circulating
leukocytes express varying levels of high-affinity
41 (8),
and VCAM-1 expression changes several-fold on blood vessels during the
course of inflammation (13, 31). Consequently, the
activation of 41 may
specify preferential recruitment of leukocyte subsets to sites of
differing VCAM-1 densities.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark H. Ginsberg, Department of Vascular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, VB-2, La Jolla, CA 92037. E-mail address: ginsberg{at}scripps.edu
3 Abbreviations used in this paper: CHO, Chinese hamster ovary; EMS, ethyl methane sulfonate; SDF, stromal cell-derived factor; sVCAM, soluble VCAM.
Received for publication January 24, 2001. Accepted for publication June 18, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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41 integrins is cell-type specific and activation dependent and is disrupted during apoptosis in T cells. Blood 95:602.4 cytoplasmic domain. Mol. Biol. Cell 6:661.[Abstract]
41 integrin/VCAM-1 integration activates L2 integrin-mediated adhesion to ICAM-1 in human T cells. J. Immunol. 164:746.2 integrin-dependent leukocyte adhesion involving the urokinase receptor. Blood 96:506.41. Cell Adhes. Commun. 3:487.[Medline]
4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80:413.[Medline]
41- and 51-mediated function. J. Cell Biol. 138:1437.4 and VLA-1 discriminates multiple subsets of CD4+CD45RO+ "memory" T cells. J. Immunol. 149:4082.[Abstract]
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D. G. Woodside, R. M. Kram, J. S. Mitchell, T. Belsom, M. J. Billard, B. W. McIntyre, and P. Vanderslice Contrasting Roles for Domain 4 of VCAM-1 in the Regulation of Cell Adhesion and Soluble VCAM-1 Binding to Integrin {alpha}4beta1. J. Immunol., April 15, 2006; 176(8): 5041 - 5049. [Abstract] [Full Text] [PDF]
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M. H. Qureshi, J. Cook-Mills, D. E. Doherty, and B. A. Garvy TNF-{alpha}-Dependent ICAM-1- and VCAM-1-Mediated Inflammatory Responses Are Delayed in Neonatal Mice Infected with Pneumocystis carinii J. Immunol., November 1, 2003; 171(9): 4700 - 4707. [Abstract] [Full Text] [PDF]
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J. Han, D. M. Rose, D. G. Woodside, L. E. Goldfinger, and M. H. Ginsberg Integrin {alpha}4{beta}1-dependent T Cell Migration Requires Both Phosphorylation and Dephosphorylation of the {alpha}4 Cytoplasmic Domain to Regulate the Reversible Binding of Paxillin J. Biol. Chem., September 12, 2003; 278(37): 34845 - 34853. [Abstract] [Full Text] [PDF]
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) and
) Jurkat T
cell variants were allowed to migrate for 4 h at 37°C using
SDF-1
