Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
A regulated order of adhesion events directs
leukocytes from the vascular compartment into injured
tissues in response to inflammatory stimuli. We show
that on human T cells, the interaction of the 2 integrin
leucocyte function-associated antigen-1 (LFA-1) with
its ligand intercellular adhesion molecule-1 (ICAM-1)
will decrease adhesion mediated by
4
1 and, to a
lesser extent,
5
1. Similar inhibition is also seen when
T cells are exposed to mAb 24, which stabilizes LFA-1
in an active state after triggering integrin function
through divalent cation Mg2+, PdBu, or T cell receptor/
CD3 complex (TCR/CD3) cross-linking. Such cross
talk decreases
4
1 integrin-mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1
(VCAM-1). In contrast, ligand occupancy or prolonged
activation of
1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration
across fibronectin, unlike adhesion, is mediated solely
by
5
1, and is increased when the
4
1-mediated
component of fibronectin adhesion is decreased either
by cross talk or the use of
4-blocking mAb. The ability
of mAb 24 Fab
fragments to induce cross talk without
cross-linking LFA-1 suggests signal transduction
through the active integrin. These data provide the first
direct evidence for cross talk between LFA-1 and
1 integrins on T cells. Together, these findings imply that
activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the
subsequent migration on fibronectin. This sequence of
events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.
THE regulation of lymphocyte extravasation from the
circulation into sites of inflammation is critical in
coordinating an appropriate and effective immune
response. There is careful control not only of the particular tissues into which migration occurs, but also of the various subtypes of leukocytes involved (Butcher and Picker,
1996 There is increasing evidence that, on a given cell, one
subset of integrins may be negatively regulated by ligation
of another. Transfection of There is also indirect evidence of similar integrin regulation on T cells. For example, resting lymphocytes use both
LFA-1 and Preparation of T Lymphoblasts
Peripheral blood mononuclear cells were prepared from single donor leukocyte buffy coats by centrifugation through Lymphoprep® (Pharmacia
Diagnostics AB, Uppsala, Sweden). T cells were expanded from this population by culturing in RPMI 1640 plus 10% FCS (GIBCO BRL, Paisley,
UK) in the presence of phytohaemagglutinin (Murex Diagnostics, Dartford, UK) at 1 µg/ml for 72 h as previously described (Dransfield et al.,
1992a mAbs and Other Reagents
mAb 925.2 (CD11a; LFA-1 ICAM-1Fc was produced as a chimeric protein, consisting of the five
extracellular domains of ICAM-1 fused to the Fc fragment of human IgG1
(Berendt et al., 1992 Ligand-coated Beads
A modified protocol (Pyszniak et al., 1994 Cell Bead Attachment Assay
Multiwell Lab-Tek® Chamber Slides (Nunc, Inc., Naperville, IL) were left
uncoated as controls or coated with either ICAM-1Fc (10 µg/ml in PBS)
or rabbit anti-mouse Ig (1:100 dilution in PBS) overnight at 4°C. The next
day, mAb tissue culture supernatant was added to wells precoated with
anti-mouse Ig and left on ice for 1 h. Wells were washed twice with PBS,
and nonspecific binding sites were blocked with 0.1% denatured BSA for
2 h at room temperature. Cells (150 µl of 2 × 106/ml) in assay buffer (see
above) containing 3 mM Mg2+/2 mM EGTA were added to the wells and
allowed to settle on ice for 15 min. Freshly prepared ligand-coated beads
(see above) were added to the wells at 100:1 bead-to-cell ratio in 50 µl. After 30 min at 37°C, the unbound beads and cells were removed with four
washes in warmed assay buffer. Bound cells were fixed with 1% formaldehyde in PBS for 20 min at room temperature. Cells were then stained with
haematoxylin for 10 min. Beads and cells were counted per high power
field (×40 oil immersion objective; Carl Zeiss, Inc., Thornwood, NY), and
the number of beads per 100 cells was determined (attachment index).
Cell Attachment Assays
Flat-bottomed Immulon-1® 96-well plates (Dynatech Labs., Inc., Chantilly, VA) were precoated with 50 µl fibronectin (20 µg/ml), VCAM-1Fc
(7 µg/ml), or ICAM-1Fc (2.4 µg/ml) in PBS overnight at 4°C. The plates
were blocked with 2.5% BSA in PBS for 2 h at room temperature and
then washed four times in assay buffer (see above) at 4°C. T cells were
washed three times in assay buffer and labeled with 2.5 µM BCECF-AM
in the same buffer for 30 min at 37°C, followed by two further washes. T
cells (2 × 105 cells) were treated with 3 mM Mg2+/2 mM EGTA, 50 nM
phorbol-12,13-dibutyrate (PdBu), or CD3 mAb at indicated levels, as well
as inhibitors and mAbs in 100 µl assay buffer. Ca2+ and Mg2+ were included at 0.4 mM for experiments involving PdBu or T cell receptor/CD3
complex (TCR/CD3) cross-linking with mAb G19.4. Blocking mAbs were
titrated on T cells by FACS® analysis (Becton Dickinson, Mountain View,
CA) and used at saturating concentrations to block T cell function. For fibronectin- and VCAM-1-binding assays, all wells contained anti-LFA-1
mAb 38 at function-blocking concentrations of 10 µg/ml. This prevents
cells aggregating via LFA-1/ICAM-1 interactions, which would cause spuriously high binding to Transmigration Assays
Assays were performed in 20 mM Hepes, 140 mM NaCl, 2 mg/ml glucose,
pH 7.4, 0.25% BSA, 3 mM Mg2+/2 mM EGTA (transmigration buffer) using 6.5-mm-diam Transwell® plates (Costar Corp., Cambridge, MA). The
upper and lower surfaces of the inserts were coated with fibronectin at
concentrations ranging from 0 to 50 µg/ml in PBS overnight at 4°C. The
inserts were positioned in wells containing 600 µl transmigration buffer. Cells were then plated in the insert at a concentration of 5 × 105 cells in
100 µl transmigration buffer with appropriate mAbs. The mAbs were
used at the following final concentrations: mAb HP1/2 at 0.7 µg/ml, mAb
7.2 at 5 µg/ml, mAb SAM-1 at 5 µg/ml, mAb 24 at 5 µg/ml, and mAb 52U
at 5 µg/ml. The anti-LFA-1 mAb 38 was added to all inserts at function-blocking concentrations of 10 µg/ml to prevent a spurious decrease in migration due to cell aggregation when LFA-1 is activated. The plates were
then incubated for 6 h at 37°C. The bottom surface of the insert was then
scraped to release migrated but adherent cells into the bottom well, and
the migrated cells were counted in a hemocytometer. Nine grids (0.1 mm3
per grid) were counted per well, and readings were averaged from duplicate samples. All assays were performed in duplicate, and each experiment was repeated a minimum of four times.
In this study, we have investigated cross influences on
function between the Adhesion of T Cells to ICAM-1 Will Decrease Binding
of Fibronectin-coated Beads
To determine the effect of LFA-1 ligation on the function
of the
Adhesion of T Cells to ICAM-1 Decreases
Binding of Fibronectin- and VCAM-1-coated Beads
by Downregulating We then looked at the effects of LFA-1 ligand-binding on
each of the T cell fibronectin receptors,
Activation of the The inhibitory effect of LFA-1 on
The Effect of LFA-1 Activation on T Cells Is Mediated
Predominantly through Because T cells bind to fibronectin through both
Activation of We then reversed the situation to investigate the effect on
Inhibition of The effects of LFA-1-mediated cross talk on
Investigation of the Mechanism of LFA-1 Cross Talk
Treatment of T cell LFA-1 with Mg2+/EGTA directly induces a high affinity form of the integrin that is able to
bind soluble ICAM-1 (Stewart et al., 1996 We next tested the possibility that LFA-1 activation
might be influencing a cytoskeletal event. Although mAb
24 decreased the overall level of
This study was undertaken to examine the functional interaction on T cells between LFA-1 and the Previous studies have demonstrated the involvement of
In this study, we have demonstrated that, in contrast to
adhesion, the migration of activated T cells on fibronectin
is mediated by The mechanism for LFA-1 downregulation of It seemed possible that the cytoskeleton was a target of
LFA-1-mediated cross talk because both LFA-1 cross talk was evident after several different adhesion-inducing protocols, showing that the phenomenon
was not stimulus specific. The fact that cross talk was dependent on ICAM-1 or mAb 24 indicated that occupancy
of LFA-1 was a prerequisite. Although the signaling pathways activated upon engagement of the In summary, we describe inhibition of ). Under flow conditions, circulating lymphocytes can
attach and roll on vascular endothelium using the selectins and
4 integrins (Alon et al., 1995
; Berlin et al., 1995
; Luscinskas et al., 1995
). These adhesion receptors are able to
slow the transit of leukocytes and expose them to stimuli
causing activation-dependent firm adhesion. The integrins
4
1,
4
7, and leukocyte function-associated antigen-1
(LFA-1)1 have been implicated in activation-dependent
stable arrest of lymphocytes under flow (Bargatze et al.,
1995
). LFA-1, however, unlike the
4 integrins, cannot initiate adhesion under these conditions without L-selectin
and/or
4
7 first tethering the lymphocyte to the vessel
wall (Bargatze et al., 1995
). Subsequently, LFA-1 is the
principal integrin involved in transendothelial migration (van Epps et al., 1989
; Smith et al., 1989
; Oppenheimer-Marks et al., 1991
), although the stimulus that induces this
2 integrin-dependent movement across the endothelial
layer is unclear. Interaction with and migration across fibronectin and other extracellular matrix components are
then necessary for the successful completion of migration
into the tissues. Therefore, transmigration of any one T
cell is a multistep process dependent on the tight regulation of the sequential and often overlapping activities of
the expressed integrins (Bargatze et al., 1995
).
v
3 into K562 cells that endogenously express
5
1 provides a system in which ligation of
3 inhibits the phagocytic but not adhesive function of
5
1 (Blystone et al., 1994
). Similarly, ligation of
transfected
IIb
3 will inhibit the function of cotransfected
2
1 or endogenous
5
1 in CHO cells expressing
these integrins (Diáz-González et al, 1996). Such effects
are dependent on an intact
3 cytoplasmic tail, and are
considered to involve signal transduction (Blystone et al.,
1995
; Diáz-González et al., 1996
). In a further example,
ligand-binding by
4
1 on fibroblasts was able to suppress
the ability of
5
1 to induce metalloproteinase expression
(Huhtala et al., 1995
).
4
1 to bind endothelial cells, but when T cells
become activated, adhesion is mediated through LFA-1
with little or no contribution from
4
1 (van Kooyk et al.,
1993
). Additionally, in some leukemic T cell lines, functional
4
1 is found only when LFA-1 is either not expressed or inactive (van Kooyk et al., 1993
). Therefore, there appears to be a T cell integrin hierarchy in which
4
1 is inactive if LFA-1 is active. Here we provide direct
evidence of cross talk on T cells between
2 and
1 integrins. The avid state of the
2 integrin, LFA-1, can be maintained by the ligand intercellular adhesion molecule-1
(ICAM-1) or by activation mAbs. Such sustained activation of LFA-1 is shown to downregulate the adhesion
through
4
1 and, to a lesser extent, through
5
1 to the
ligands fibronectin and vascular cell adhesion molecule-1 (VCAM-1) (
4
1 alone). The result, phenotypically, is a
less adhesive, more migratory T cell. Therefore, we have
demonstrated another way in which integrin activities may
be regulated.
Materials and Methods
). Cells were then washed and maintained for 1-2 wk in medium
supplemented with 20 ng/ml recombinant IL-2 (Euro Cetus UK Ltd.,
Harefield, UK). The cells, which were used between days 10 and 14, were
a 99% CD3+ population, containing 65% CD8+ and 35% CD4+ cells. The
population was negative for the natural killer cell marker CD56.
subunit, nonblocking) was purchased from
Becton Dickinson (Oxford, UK). mAbs 38 (CD11a; LFA-1
subunit function-blocking), and 24 (CD11/CD18;
2 integrin activation reporter)
(Dransfield et al., 1992b
; Dransfield and Hogg, 1989
) and 52U (control antibody) were prepared in this laboratory, and purified from ascites or tissue culture supernatant by protein A-Sepharose chromatography (Ey et
al., 1978
). mAbs HP1/2 (CD49d;
4 subunit function-blocking) and TS2/
16 (CD29;
1 subunit-activating) were gifts from R. Lobb (Biogen, Inc.,
Cambridge, MA). mAb SAM-1 (CD49e;
5 subunit-blocking) was purchased from Eurogenetics (Hampton, UK). mAb 7.2 (CD49d;
4 subunit,
nonblocking) was a gift from J. Marshall (Imperial Cancer Research
Fund, London, UK). mAb UCHT2 (CD5) was a gift from P. Beverley
(University College, London, UK). mAb G19.4 was a gift from Bristol Myers-Squibb Pharmaceuticals (Princeton, NJ). Rabbit anti-mouse IgG was
purchased from Sigma Chemical Co. (Poole, UK).
). VCAM-1Fc, produced as a chimera consisting of
the two amino-terminal domains of human VCAM-1 fused to the Fc fragment of human IgG1 (Jakubowski et al., 1995
), was a gift from R. Lobb.
Fibronectin (0.1% solution from human plasma) was purchased from
Sigma Chemical Co., and cytochalasin D from GIBCO BRL. The fluorescent
cell label 2
,7
-bis-([carboxyethyl]-5[6
]-carboxyfluorescein) (BCECF-AM)
was purchased from Calbiochem Corp. (Nottingham, UK). All other
chemicals and reagents were purchased from Sigma Chemical Co.
) was developed in which 200 µl
(108) of 3.2-µm polystyrene beads (Sigma Chemical Co.) were washed twice in distilled water, followed by two further washes and resuspension in 0.1 M bicarbonate buffer, pH 9. Fibronectin, ICAM-1, VCAM-1, or
BSA as control were added to these beads to a final concentration of 10 µg/ml. The beads were rotated at room temperature for 1 h, washed once
in PBS, and blocked with 0.1% denatured BSA for 2 h at room temperature while being rotated. The beads were then washed twice in 20 mM
Hepes, 140 mM NaCl, 2 mg/ml glucose, pH 7.4 (assay buffer), containing 3 mM Mg2+/2 mM EGTA, for use in the adhesion experiments as described.
1 ligands through the piggy-back interaction of
nonadherent cells with truly adherent cells. Plates were incubated for 15 min on ice, followed by centrifugation at 40 g for 1 min, before 40-min incubation at 37°C. Nonadherent cells were removed by washing four times
in warmed assay buffer (150 µl/well). Adhesion was quantified by recording
emission at 530 nm, after excitation at 485 nm, using a Fluoroskan® II
(Labsystems, Inc., Basingstoke, UK), and by expressing the reading for
each well as a percentage of the total emission before incubation.
Results
1 integrins and LFA-1 on T cells.
As one method of activating these leukocyte integrins, we
treated T cells with 3 mM Mg2+/2 mM EGTA (Dransfield
et al., 1992a
). For the
2 integrin, LFA-1, the advantage
of such treatment is that it directly alters the integrin
ectodomain, bypassing the requirement for an intracellular stimulus (Stewart et al., 1996
). This form of LFA-1 is
considered to be of high affinity because it is able to bind
soluble ICAM-1 (Stewart et al., 1996
). There have been
both positive and negative reports of the ability of Mg2+/
EGTA to induce fibronectin receptor-mediated adhesion
(Shimizu and Mobley, 1993
; Luque et al., 1996
). In this
study, we show that T cells do bind fibronectin, immobilized either on plates or on beads, in an Mg2+-dependent
manner. To examine further the generality of cross-influences between these integrins, we also investigated T cells
stimulated with phorbol ester or by TCR/CD3 cross-linking. Both of these stimuli act from within the cell to activate integrins, so called inside-out signaling, and may be
considered more representative of the in vivo situation.
1 integrins on the same T cell, we developed a
ligand-coated bead-binding system. T cells were adhered
to immobilized ICAM-1 via LFA-1, or to a control substrate, and their ability to bind beads coated with ligand
for the
1 integrins,
4
1 and
5
1, was then investigated.
Fibronectin-coated beads were bound by T cells adherent
to the control substrate, anti-CD5 mAb, immobilized on
plastic (Fig. 1 A), and the specificity of adhesion was demonstrated by blocking bead-binding with a combination of
4 and
5 mAbs (Fig. 1 B). However, when T cells were
adherent to ICAM-1 as substrate, they bound fewer fibronectin-coated beads (Fig. 1 C). When binding of the fibronectin-coated beads was quantified, there was a decreased level of fibronectin bead-binding when T cells
were adherent to ICAM-1 (Fig. 2 A) (inhibition: 65.0 ± 23.4% = mean ± SD; n = 6). This result demonstrated
that, on human T cells, the interaction of LFA-1 with its
ligand ICAM-1 could downregulate the function of the
1
integrins. In contrast, T cells adhered to anti-LFA-1 mAb
bound beads at a similar level as T cells adherent to control mAb. This indicated that the LFA-1 inhibitory effect
could not be mimicked by cross-linking LFA-1 with immobilized CD11a mAb 38 (Fig. 2 A). Conversely, there was
no difference between the ability of T cells adherent to
anti-CD5 mAb, fibronectin, or ICAM-1 to bind ICAM-1-
coated beads (Fig. 2 B), indicating that adhesion to immobilized fibronectin did not alter the extent of ICAM-1 bead binding by LFA-1. This is the first evidence that
LFA-1 could dominate the activity of the fibronectin-binding receptors and that the reverse situation did not hold.
Fig. 1.
Fibronectin bead-binding is reduced when T cells adhere to ICAM-1. Cultured human T cells treated with 3 mM
Mg2+/2 mM EGTA and adhered to plastic coated with anti-CD5
(mAb UCHT2) (A and B) and ICAM-1 (C) were incubated with
fibronectin-coated beads. Bead-binding was blocked using a combination of the 4 and
5 function-blocking mAbs HP1/2 (0.5 µg/
ml) and SAM-1 (0.5 µg/ml) (B). Bar, 20 µm.
[View Larger Version of this Image (91K GIF file)]
Fig. 2.
Inhibition of fibronectin bead-binding when T cells adhere to ICAM-1. (A) Cultured human T cells treated with 3 mM
Mg2+/2 mM EGTA and adhered to plastic coated with anti-CD5
(mAb UCHT2), anti-LFA-1 (mAb 38), or ICAM-1 and incubated with fibronectin-coated beads (dark bars). Bead attachment was blocked with a combination of 4 and
5 function-
blocking mAbs HP1/2 and SAM-1 as previously (open bars). (B)
Cultured human T cells treated with 3 mM Mg2+/2 mM EGTA
and adhered to plastic coated with anti-CD5 (mAb UCHT2), fibronectin, or ICAM-1 were incubated either with ICAM-1-coated beads (stippled bars) or with BSA-coated control beads (open
bars). The bead-binding assays were performed as described in
Materials and Methods, and data are expressed as binding index
(beads bound/100 cells). Data represent the mean of six high-power fields ± SEM. One representative experiment of three is
shown.
[View Larger Version of this Image (32K GIF file)]
4
1 Activity
4
1 and
5
1.
Both of these integrins can be involved in T cell binding to
fibronectin, and, if one is blocked, the other can partially
compensate, as previously described (Wayner et al., 1989
).
Fig. 3 A shows the binding of fibronectin-coated beads by
T cells adherent to anti-CD5 mAb. This binding can be
partially blocked with either an
4-blocking mAb, an
5-blocking mAb, or completely with a combination of both blocking mAbs, showing that T cells bind these beads
through a mixture of
4
1 and
5
1 integrins. However,
when T cells are adherent to ICAM-1, the binding index
for fibronectin-coated beads is lower, and is reduced only
by an
5-blocking mAb (Fig. 3 B). Therefore, binding in
this situation is mediated mainly through
5
1 with little
contribution from
4
1, demonstrating that the binding
activity of
4
1 has been compromised. The binding by T
cells of VCAM-1-coated beads, which is mediated exclusively by
4
1, reveals a similar downregulation when T
cells are adherent to ICAM-1 as compared to anti-LFA-1
mAb (Fig. 4).
Fig. 3.
Fibronectin bead-binding mediated by 4
1 is differentially inhibited when T cells are adherent to ICAM-1. Cultured human T cells treated with 3 mM Mg2+/2 mM EGTA were adhered to plastic coated with anti-CD5 (mAb UCHT2) (A) or ICAM-1 (B) and incubated with fibronectin-coated beads. Bead attachment was assessed in the presence or absence of the
4 and
5 function-blocking
mAb HP1/2 and SAM-1 either alone or in combination, as previously. The data are expressed as binding index (beads bound/100 cells),
and are the mean of six high-power fields ± SEM. One representative experiment of three is shown.
[View Larger Version of this Image (15K GIF file)]
Fig. 4.
Inhibition of
VCAM-1 bead-binding when
T cells adhere to ICAM-1.
Cultured human T cells
treated with 3 mM Mg2+/2
mM EGTA were adhered to
plastic coated with anti-CD5
(mAb UCHT2), anti-LFA-1
(mAb 38), or ICAM-1 and
incubated with VCAM-1-
coated beads (hatched bars).
Bead attachment was prevented by the 4-blocking mAb HP1/2 (open bars).
Data are expressed as binding index (beads bound/100
cells) and represent the mean
of six high-power fields ± SEM. One representative experiment of three is shown.
[View Larger Version of this Image (45K GIF file)]
2 Integrin LFA-1 on T Cells Inhibits
Their
1-Mediated Binding to Fibronectin
1-mediated ligand-binding could not be demonstrated by cross-linking LFA-1
with an anti-LFA-1 mAb, but required binding to ligand
ICAM-1. This suggested that high affinity LFA-1 rather
than receptor cross-linking was necessary for cross talk.
This led to the development of an assay in which T cells
were first stimulated with Mg2+/EGTA, TCR/CD3 cross-linking, or PdBu and then exposed to mAb 24, which holds
LFA-1 in an active conformation as if occupied by ligand
(Dransfield et al., 1992b
). mAb 24 caused increased T cell
binding to ICAM-1 after titration of Mg2+ (Fig. 5 A), CD3
mAb G19.4 (Fig. 5 B), and PdBu (not shown). In contrast,
mAb 24 caused inhibition of T cell binding to fibronectin after TCR/CD3 cross-linking (Fig. 6 A) or Mg2+/EGTA
(data not shown). Monovalent Fab
fragments of mAb 24 produced the same degree of inhibition as bivalent mAb
24 (data not shown). This demonstrated that activation or
ligand occupancy of LFA-1 in the absence of clustering is
sufficient to alter fibronectin-mediated adhesion. mAb
KIM185 (CD18;
2-activating) behaved similarly to mAb
24, depressing binding of T cells to fibronectin while enhancing adhesion to ICAM-1 (data not shown).
Fig. 5.
Prolonged activation of LFA-1 by mAb 24 increases T cell adhesion to ICAM-1 induced either by Mg2+ and 2 mM EGTA (A)
or by TCR/CD3 cross-linking through CD3 mAb G19.4 in the presence of Ca2+ and Mg2+ at 0.4 mM (B). The LFA-1 activation antibody, mAb 24 () or mAb 52U (IgG1 isotype control) (
) were used at 20 µg/ml. Specificity of adhesion was shown by block of ICAM-1
binding with mAb 38 (LFA-1-function blocking, 10 µg/ml) (
). Data represent means of triplicates ± SD. One representative experiment of three is shown.
[View Larger Version of this Image (15K GIF file)]
Fig. 6.
Prolonged activation of LFA-1 blocks 4
1- and to a lesser extent
5
1-mediated binding to fibronectin. Adhesion to fibronectin was induced by TCR/CD3 cross-linking through CD3 mAb G19.4 in the presence of Ca2+ and Mg2+ at 0.4 mM. (A) Adhesion
mediated by
4
1 and
5
1 together. (B)
5
1-mediated adhesion after the
4
1 component had been blocked with mAb HP1/2 (0.5 µg/ml). (C)
4
1-mediated adhesion after the
5
1 component had been blocked with mAb SAM-1 (0.5 µg/ml). Adhesion was assessed
in the presence of the LFA-1 activation antibody, mAb 24 (open symbols), or the isotype-matched control antibody, mAb 52U (closed
symbols), used at 20 µg/ml. Data represent means of triplicates ± SD. One representative experiment of three is shown. The specificity
of the adhesion is shown by the block achieved using
4- and
5-blocking mAbs as indicated.
[View Larger Version of this Image (22K GIF file)]
4
1
4
1 and
5
1, we analyzed the effects of LFA-1 activation individually on these integrins using function-blocking mAbs and
either TCR/CD3 cross-linking (Fig. 6, B and C) or Mg2+/
EGTA (data not shown) to stimulate adhesion. Prolonged
activation of LFA-1 with mAb 24 had only a small effect
on total fibronectin-binding (Fig. 6 A) and on
5
1-mediated adhesion (Fig. 6 B), but had a much greater effect on
4
1-mediated adhesion (Fig. 6 C). Similar levels of
4
1
inhibition by mAb 24 were seen when the integrins were
activated with PdBu or Mg2+/EGTA (Fig. 7). In addition,
there was no effect of the nonfunction-altering anti-LFA-1
mAb G25.5, which again emphasized the requirement for LFA-1 activation (Fig. 7). Under equivalent activating
conditions, mAb 24 and the
2 integrin-activating mAb
KIM185 decreased
4
1-mediated adhesion to VCAM-1
to the same extent as to fibronectin (data not shown). Together, these results reinforced the findings that
4
1
function is particularly sensitive to the state of LFA-1 activation.
Fig. 7.
Prolonged activation of LFA-1 blocks 4
1-mediated fibronectin-binding after various stimuli.
Adhesion to fibronectin was induced by TCR/CD3 cross-linking using mAb G19.4 (2.5 µM) with 0.4 mM Ca2+ and
Mg2+ (white bars); PdBu 50 nM with 0.4 mM Ca2+ and
Mg2+ (dark bars); or 3 mM
Mg2+/2 mM EGTA (cross-hatched bars). Integrin
5
1
was blocked with SAM-1 (0.5 µg/ml), allowing
4
1 adhesion to be investigated in isolation. Adhesion was assessed
in the presence of mAb G25.2 (an LFA-1 nonactivating and
nonblocking mAb), mAb 24, or mAb 52U (the IgG1 isotype-matched control antibody), each used at 20 µg/ml. Data represent means of
triplicates ± SD. One representative experiment of three
is shown. The specificity of
the adhesion is shown with an
4-blocking mAb.
[View Larger Version of this Image (29K GIF file)]
1 Integrins on T Cells Has No Effect on
LFA-1 Binding to ICAM-1
2 integrin-mediated adhesion of maintaining
1 integrins
in an active state, using the
1 integrin-stimulating mAb
TS2/16. This mAb increased binding to fibronectin after
the three activating treatments (Fig. 8 A), but had no effect on
2-mediated binding to ICAM-1 (Fig. 8 B), confirming that the
1 integrins were unable to influence the
ligand-binding activity of LFA-1.
Fig. 8.
2 integrin activation decreases
1-mediated
binding of T cells, but the reverse is not true. T cell adhesion to fibronectin (A) and
ICAM-1 (B) was induced by
TCR/CD3 cross-linking using mAb G19.4 (2.5 µg/ml)
with 0.4 mM Ca2+ and Mg2+,
PdBu 50 nM with 0.4 mM
Ca2+ and Mg2+, or 3 mM
Mg2+/2 mM EGTA, and the
adhesion assay was performed in the presence or absence of the
1-activating
mAb TS2/16 (10 µg/ml) or
the isotype-matched control,
mAb 52U (10 µg/ml). Specificity of adhesion was shown by blocking of fibronectin-binding with a combination
of
4- and
5-blocking
mAbs, and ICAM-1-binding
with the LFA-1-blocking antibody, mAb 38. Adherent
cells are expressed as percentage of total cells added,
and data represent means of
triplicates ± SD. One representative experiment of three
is shown.
[View Larger Version of this Image (34K GIF file)]
4
1 with Blocking mAbs or by LFA-1
Activation Increases
5
1-Mediated Migration
4
1- and
5
1-mediated T cell migration on fibronectin were then
investigated. Using the Transwell® system, we established
that T cells undergo random migration on fibronectin using
5
1 exclusively, and that this migration was enhanced
by an
4-blocking mAb HP1/2, but not affected by an
4-
nonblocking mAb 7.2 (Fig. 9 A). In addition, migration at
different concentrations of fibronectin remained solely dependent on
5
1 (Fig. 9 B), and could be enhanced either
with
4-blocking mAb HP1/2 (Fig. 9 C), or by maintaining
LFA-1 activation with mAb 24 (Fig. 9 D). Such increased
migration was blocked with an
5-blocking mAb. Therefore, by decreasing the
4
1 contribution to fibronectin
adhesion with either an
4-blocking mAb or an LFA-1- activation mAb, the ability of
5
1 to mediate migration
was increased.
Fig. 9.
Migration of T cells
on fibronectin is mediated
by 5
1 and promoted by
blocking
4
1 function. (A)
Migration of T cells on fibronectin (10 µg/ml) is mediated by
5
1 and enhanced
when
4
1-mediated adhesion to fibronectin is blocked
by the function-blocking
4 antibody mAb HP1/2. The
non-function-blocking
4 antibody mAb 7.2 had no effect.
(B) Migration of T cells
across membranes coated with fibronectin at various
concentrations (0-50 µg/ml)
is dependent on
5
1. (C)
Addition of the
4-blocking
mAb HP1/2 increases migration above control mAb 52U.
This increased migration can
be blocked with the
5-blocking mAb SAM-1 (5 µg/
ml). (D) Addition of the LFA-1-activation mAb 24 increases migration above
control mAb 52U. This increased migration can be
blocked with the
5-blocking mAb SAM-1. The assay was
performed as described in
Materials and Methods, and
data are expressed as the total number of migrated cells.
Data represent the mean of
two readings from each well. All assays were performed in
duplicate, with bars indicating the range of readings. Experiments are representative
of four similar experiments.
[View Larger Version of this Image (31K GIF file)]
). Therefore, we
looked at the ability of
1 integrins to adopt a high affinity
state. However, treatment with Mg2+/EGTA yielded no
detectable binding of soluble fibronectin or VCAM-1,
even when these ligands were used at concentrations up to
1.2 mg/ml. In contrast, 0.5 mM Mn2+ was able to induce fibronectin (
4
1 and
5
1)- and VCAM-1 (
4
1)-binding to both T cells and Jurkat cells (data not shown), as has
been reported by others (Jakubowski et al., 1995
; Gomez
et al., 1997
). Similarly, while 0.5 mM Mn2+ was able to induce expression of the
1 activation reporter epitopes recognized by mAb 15/7 (Yednock et al., 1995
) or mAb
HUTS-21 (Luque et al., 1996
), no expression of these
epitopes was observed with Mg2+/EGTA treatment (data
not shown). These findings imply that the fibronectin-binding integrins are in a low affinity state after all three
methods of stimulation, and that LFA-1 cross talk causes inhibition of postreceptor occupancy events, rather than
direct modulation of receptor affinity.
4
1-mediated adhesion
to fibronectin, there was no change in the sensitivity of
binding to cytochalasin D (Fig. 10). Therefore, LFA-1
cross talk affects an event in cell adhesion after receptor
occupancy but before changes in the actin cytoskeleton,
and is independent of both.
Fig. 10.
Inhibition of 4
1-mediated adhesion by mAb 24 does not alter the sensitivity of adherent T cells to cytochalasin
D. Integrin
5
1 was blocked with SAM-1, as previously, allowing
4
1 adhesion to be investigated in isolation. Adhesion was
stimulated with 3 mM Mg2+/2 mM EGTA in the presence of the
LFA-1-activation antibody, mAb 24 (
), or the isotype-matched
control antibody, mAb 52U (
), as previously. Cytochalasin D
was used at 0-10 µg/ml (0-20 µM). Specificity of the adhesion
was shown by blocking with mAb HP1/2 (anti-
4) (
). Data represent means of triplicates ± SD. One representative experiment
of four is shown.
[View Larger Version of this Image (14K GIF file)]
Discussion
1 integrin fibronectin receptors
4
1 and
5
1. The main findings are
that (a) the occupation of T cell LFA-1 by its ligand
ICAM-1 decreases the binding of
4
1 to ligands fibronectin and VCAM-1; (b) this inhibitory cross talk also
results from the prolonged activation of LFA-1 induced by the activation reporter mAb 24 in combination with several T cell adhesion-inducing protocols; (c) the adhesive
activity of
5
1 is affected to a lesser extent; (d) while active LFA-1 downregulates the avidity of
4
1, the reverse
does not occur, as neither
1 integrin-activating mAb
TS2/16 nor
1-mediated binding to fibronectin affected
the avidity of LFA-1; and (e) downregulation of
4
1 activity increases the efficiency of
5
1-mediated migration
on fibronectin. Therefore, we have demonstrated differential regulation of two integrin subclasses and a hierarchy of
integrin usage in which the
2 integrin LFA-1 will suppress the function of
1 integrins, particularly
4
1.
4
1 in leukocyte adhesion to but not migration across endothelium, and of LFA-1 as the chief integrin in transendothelial migration (van Epps et al., 1989
; Oppenheimer-Marks et al., 1991
; Moser et al., 1992
). Furthermore, in
vitro experiments during flow have emphasized the requirement that an integrin hierarchy allow coordinated migration of lymphocytes across the endothelium into the
tissues (Butcher and Picker, 1996
). Our finding that active
LFA-1 is able to decrease the ligand-binding activity of
4
1 has direct implications for the sequential activity of
these integrins in such an adhesion cascade; LFA-1 may
function optimally in the absence of
4
1 adhesion, allowing the T cell to deadhere from the apical surface of the endothelium and transmigrate. Our findings also argue
against a redundancy among integrin-ligand pairs in leukocyte transmigration, and imply specific roles for each integrin.
5
1 with no contribution from
4
1. In
addition, suppressing
4
1 activity on T cells either by
mAb 24 or
4 function-blocking mAbs enhanced the level
of
5
1 migration, particularly at low fibronectin levels.
This may reflect the compensatory increase in
5
1 adhesion, with its migratory potential, when binding through the nonmigratory
4
1 is blocked. Another possibility is
that the enhanced migration by
5
1 is due to removal of
a restraint imposed by
4
1. The importance of strength
of adhesion in regulating cell migration is well documented (Huttenlocher et al., 1996
; Palecek et al., 1997
),
suggesting that firm adhesion by both
4
1 and
5
1 may
make conditions suboptimal for migration. Alternatively,
4
1 may be involved in a more specific inhibition of
5
1
function, as has been described in the control of metalloproteinase expression in fibroblasts (Huhtala et al., 1995
).
The promotion of migratory behavior by
5
1 through
loss of
4
1-binding activity is in keeping with its more
prominent role within the tissues after successful negotiation of T cells across the endothelium (Miyake et al., 1992
). Therefore, a hierarchy of integrin activity may feature at this later stage of the adhesion cascade, with LFA-1
providing a link between
4
1 and
5
1.
4
1 was
explored in several ways. We first established that there
was no alteration in expression of either
4
1 or
5
1
during the experimental period (data not shown). Furthermore, confocal microscopy using mAbs specific for
4
1,
5
1, and the
1-activation reporter mAb 15/7 indicated
that avid LFA-1 did not cause
1 integrin redistribution on the T cell membrane (data not shown). In addition, although stimulation of T cells with Mg2+/EGTA induces
high affinity LFA-1 (Stewart et al., 1996
), none of the
three stimulating protocols induced high affinity
4
1 or
5
1. This implied that cross talk was not affecting high
affinity
1 integrins. Together, these findings suggested
that the
1 integrins had not undergone a detectable alteration in affinity nor been redistributed or shed from the
cell surface, and that LFA-1 cross talk was targeting events
after ligand-binding. This result is in keeping with other
studies in which cross talk is ultimately dependent on the
presence of the
subunit cytoplasmic tail and steps subsequent to modulation of integrin affinity (Blystone et al.,
1994
; Diáz-González et al., 1996
).
4
1- and
5
1-mediated adhesion were more sensitive to changes in actin
than was adhesion through LFA-1 (data not shown). However, for
1 integrin-mediated adhesion, the similarity in
cytochalasin D sensitivity of mAb 24-treated and untreated cells and the synergism between suboptimal doses of cytochalasin D and mAb 24 in the inhibition of
4
1-mediated binding to fibronectin (data not shown) supported the evidence that inhibition occurs upstream of cytoskeletal changes. These results implied that cross talk
affects an event in cell adhesion occurring after receptor
occupancy but before actin-mediated cytoskeletal changes,
and independent of both. In addition, protein kinase A,
associated with LFA-1 signaling and deadhesion (Rovere
et al., 1996
), and protein kinase C, implicated in some previous cross talk studies (Blystone et al., 1994
; Pacifici et al.,
1994
), were not involved in this phenomenon (data not
shown).
2 integrins are
not well understood, certain observations suggested that
cross talk did activate specific intracellular signaling pathways. Cross talk was not observed using the Jurkat T cell
line, which is known to have a defect in LFA-1 signaling
(Mobley et al., 1994
). In addition, cross talk was induced
by mAb 24 Fab
fragments but not by immobilized anti-
LFA-1 mAb, emphasizing the requirement for a mechanism beyond LFA-1 clustering. For
5
1 on human fibroblasts, although clustering by mAbs of integrin on beads
induced phosphorylation and accumulation of p125 focal
adhesion kinase and tensin, ligand occupancy recruited
further cytoskeletal proteins to the signaling complex
(Miyamoto et al., 1995a
,b). One speculation is that the targets of LFA-1 cross talk may be the proteins providing the link between integrins and actin. However, several observations suggested that cross talk does not represent a simple sequestering of such intracellular proteins. First, integrin activity operates in one direction only, so prolonged
activation of the
1 integrins using mAb TS2/16 does not
alter LFA-1 binding to ICAM-1. Second, LFA-1 predominantly affects the activity of
4
1, despite a sixfold abundance of
4
1 over
5
1 (data not shown). Future work
will address the role of potential integrator molecules in
the cross talk phenomenon.
4
1-binding activity in T cells as a consequence of LFA-1 activation. A
speculation is that deadhesion of
4
1 from the apical surface of the endothelium is required for LFA-1-mediated
migration across endothelium to proceed. Another observation is that T cell migration on fibronectin is mediated
by
5
1, and that this migration is enhanced by interfering with
4
1 adhesion. LFA-1 might provide a link between
4
1 and
5
1 by uncoupling the former in order to enhance migration by the latter. While the actual mechanism
by which cross talk is achieved is unclear, our findings implicate a downstream signaling event brought about by
maintaining LFA-1 in a highly avid state.
Received for publication 3 April 1997 and in revised form 24 June 1997.
Address all correspondence to J.C. Porter, Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Tel.: 44-171-269-3569; Fax: 44-171-269-3093; e-mail: porterj{at}europa.lif.icnet.ukWe gratefully acknowledge Dr. Roy Lobb for supplies of VCAM-1Fc and
mAbs, Dr. Fumio Takei (Vancouver, Canada) for invaluable assistance
with the bead-binding assays, Dr. Martyn Robinson, Dr. Carlos Cabañas,
Dr. John Marshall, Dr. Peter Beverley, and Dr. Ted Yednock for mAbs,
Alison McDowall for preparation of mAb 24 Fab fragments, and all our
colleagues in the Leucocyte Adhesion Laboratory for their helpful comments and critical reading of the manuscript.
This work was supported by the Imperial Cancer Research Fund, London, UK. J.C. Porter is a Medical Research Council (UK) Clinical Training Fellow.
ICAM-1, intercellular adhesion molecule-1; LFA-1, lymphocyte function-associated antigen-1; PdBu, phorbol-12,13-dibutyrate; TCR/CD3, T cell receptor/CD3 complex; VCAM-1, vascular cell adhesion molecule-1.
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