Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
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Abstract |
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The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues. Using lymphocyte function-associated antigen-1 (LFA-1) on T cells as a model, we have investigated adhesion to ligand intercellular adhesion molecule-1 induced by the Ca2+ mobilizers, ionomycin, 2,5-di-t-butylhydroquinone, and thapsigargin, and the well studied stimulators such as phorbol ester and cross-linking of the antigen-specific T cell receptor (TCR)- CD3 complex. We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca2+-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane. We propose that methods of leukocyte stimulation that cause Ca2+ fluxes induce LFA-1 adhesion by regulation of calpain activity. These findings suggest a mechanism whereby engagement of the TCR could promote adhesion strengthening at an early stage of interaction with an antigen-presenting cell.
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Introduction |
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LYMPHOCYTES have a dual function that requires they
must circulate in nonadherent form through blood
and lymph, but become adherent to allow transmigration across the vasculature or after contact with an antigen-presenting cell in a lymph node. Regulation of adhesion is achieved by controlling the activity of receptors such as the integrins on the cell surface. The major integrin on T cells is termed lymphocyte function-associated antigen-1 (LFA-1).1 This integrin is a heterodimeric transmembrane receptor composed of a unique subunit (
L;
CD11a) and a
2 subunit (CD18) that is common to a subset of leukocyte integrins. Leukocyte integrins such as
LFA-1 are not constitutively adhesive, but become firmly able to engage their ligands after stimuli received through
other cell membrane receptors such as the antigen-specific
T cell receptor (TCR) (Brown and Hogg, 1996
; Shaw and
Dustin, 1997
). How the signal transduction pathways involved in this "inside out" signaling alter the adhesive state
of integrin on the membrane, and whether these alterations are similar for all integrins, needs clarification. Integrin-mediated adhesion can occur through avidity changes
(Jakubowski et al., 1995
; Stewart et al., 1996
), but there is
also evidence that some naturally occurring agonists cause
an increase in the intrinsic affinity of the integrin (Faull
and Ginsberg, 1995
). Treatment with divalent cations Mn2+
or Mg2+/EGTA is an alternative method for inducing active integrin with increased affinity (Stewart and Hogg,
1996
). This form of integrin activation is considered to alter the ectodomain directly, bypassing the requirement for
intracellular signaling events (Kassner et al., 1994
; Stewart
et al., 1996
).
The cytoplasmic domains of integrins are essential for
control of their function. Mutation or deletion of specific
cytoplasmic sequences causes integrins to be constitutively
active and has also revealed links with the cytoskeleton
(Hughes et al., 1995; Peter and O'Toole, 1995
; Lu and
Springer, 1997
). Interaction with the cytoskeleton is regulated during the course of adhesion and may be involved
at more than one stage of the adhesion process. For example, LFA-1 is reported to associate with the cytoskeleton after TCR-CD3 cross-linking (Pardi et al., 1992
), but low
doses of the cytoskeletal disrupting agent cytochalasin facilitates adhesion by Mac-1 and LFA-1 (Elemer and Edgington, 1994
; Kucik et al., 1996
; Lub et al., 1997
).
Increases in cytoplasmic Ca2+ ([Ca2+]i) accompany triggering through the TCR and are also an important component
of other adhesion-inducing mechanisms. [Ca2+]i activates
enzymes and mediators such as protein kinase C (PKC), calcineurin, calmodulin, calreticulin, myosin light chain
kinase (MLCK), and calpain, many of which have been
implicated in integrin function. For example, Ca2+-activated calcineurin functions in the recycling of integrin v
3 on the moving neutrophil (Hendey et al., 1992
; Lawson and Maxfield, 1995
). Phorbol ester-induced T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is
Ca2+-dependent (Rothlein and Springer, 1986
; Stewart et
al., 1996
), and Ca2+ ionophores such as ionomycin can
cause integrin-mediated adhesion (Altieri et al., 1988
;
Hartfield et al., 1993
; van Kooyk et al., 1993
). Ca2+ fluxes
also activate actin-binding proteins causing actin dissociation and cytoskeletal rearrangements (Stossel, 1989
).
In this study, we have used three Ca2+ mobilizing agents, ionomycin, thapsigargin, and 2,5-di-t-butylhydroquinone (dBHQ) as tools to gain further understanding of the role of Ca2+ in the activation of adhesion by the integrin LFA-1. By inducing Ca2+ fluxing, the objective was to short circuit the early phases of signaling to dissect the events leading more immediately to adhesion. We show that these three reagents, as well as cross-linking the TCR- CD3 complex (XL-TCR-CD3) and phorbol ester treatment, induce LFA-1-mediated adhesion through a mechanism involving activation of calpain. The presented results suggest that Ca2+-mediated activation of calpain releases LFA-1 from cytoskeletal control allowing integrin clustering.
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Materials and Methods |
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Reagents
Ionomycin, thapsigargin, phorbol 12,13 dibutyrate (PdBu), and the membrane soluble calpain inhibitor calpeptin were obtained from Calbiochem/ Novabiochem Corp. (La Jolla, CA); dBHQ, and the calpain inhibitor, CBZ-LVG, were purchased from Sigma Chemical Co. (Dorset, UK). Jasplakinolide was obtained from Molecular Probes, Inc. (Eugene, OR). SK&F 96365 was purchased from BiomoL Feinchemikalien GmbH (Hamburg, Germany). With the exception of SK&F 96365, which was directly soluble in H2O, stock concentrations of inhibitors were prepared in DMSO and in every relevant experiment an equivolume of DMSO was added in the control sample. The above reagents had no effect on the viability of the T cells at the levels used in the reported experiments.
Flow Cytometry, mAbs, and Assessment of Soluble ICAM-1Fc Binding
A dimeric form of an ICAM-1Fc chimaeric protein consisting of the five
extracellular domains of ICAM-1 fused to the Fc fragment of human IgG1
was prepared as previously described (Berendt et al., 1992). Flow cytometry and measurement of the binding of soluble recombinant ICAM-1Fc to
T cells was carried out as previously described (Stewart et al., 1996
).
mAbs used in this study were the LFA-1
subunit (CD11a) mAbs 38 (Dransfield and Hogg, 1989
), F110.22 (Schmidt, 1989
), and G25.2 (Becton
and Dickinson Co., Mountain View, CA). The anti-CD3 mAbs G19.4 and
UCHT1 were obtained from Bristol Myers Squibb (Seattle, WA) and
P. Beverley (University College, London), respectively. The secondary
FITC-conjugated antibodies goat anti-mouse IgG Fc and goat anti-human
IgG Fc were obtained from Jackson ImmunoResearch Laboratories, Inc.
(West Grove, CA).
T Cell Adhesion to ICAM-1Fc
T lymphoblastoid cells were expanded from unstimulated peripheral
blood mononuclear cells by culture for 1-2 wk in RPMI-1640 medium
containing recombinant IL-2 (20 ng/ml; Cetus Corp., Berkeley, CA) with
details as previously described (Dransfield et al., 1992). Cells were used
between days 10 and 14. The method for quantifying T cell adhesion to
ICAM-1Fc protein has been previously described (Stewart et al., 1996
),
with the exception that the assay buffer used was RPMI 1640 unless controlled cation conditions were being analyzed. For these experiments T
cells were treated with 5 mM Mg2+/1 mM EGTA in Hepes/NaCl buffer.
The ICAM-1Fc protein was coated at 0.24 µg/well onto 96-well Immulon
1 plates (Dynatech, Chantilly, VA).
Confocal Microscopy
For immunofluorescence analysis by confocal microscopy, 13-mm-round glass coverslips were precoated with a 0.01% solution of poly-L-lysine (Sigma Chemical Co.) for 10 min at room temperature, washed twice in RPMI 1640, and then left to air dry. T cells were washed three times in RPMI 1640 buffer before addition onto coverslips (5 × 105 cells/coverslip), in the presence of stimulants and CD11a mAbs at 10 µg/ml. Coverslips were spun at 40 g, and then incubated for 30 min at 37°C. Unbound cells were removed by gentle washing in warm RPMI 1640 buffer. To prevent antibody-induced clusters, cells were fixed with 1% formaldehyde in PBS-A for 10 min at room temperature before a second incubation with 10 µg/ml FITC-conjugated goat anti-mouse IgG Fc (Jackson ImmunoResearch Laboratories) for 25 min at 4°C. Cells stimulated via CD3 triggering were incubated with CD3 mAbs UCHT1 or G19.4 for 30 min at 37°C and fixed as above.
Cells were mounted for confocal microscopy which was carried out using a MRC-600 Confocal Laser Scanning System (Bio-Rad Laboratories Ltd, Hertfordshire, UK). The regions of the confocal microscopy images with the highest fluorescence intensity (i.e., with the pixel color intensity in the 150-255 range) were highlighted in red using the segmentation utility of the IP Lab Spectrum Version 3.1 software (Signal Analytics Co., Vienna, VA).
This software was also used to quantify the levels of fluorescence on the images. Background fluorescence was estimated by measuring the signal strength in areas visibly devoid of specific staining but in close proximity to cells. The average of these values was then subtracted from the original image to give a corrected image. The areas of interest (e.g., free membrane or membrane at cell-cell contact points) on the corrected images were selected and the average signal strength calculated automatically by the computer software. A statistical assessment of the differences between the treatment groups and the untreated resting T cells was performed using the one-way ANOVA test.
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Results |
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Agents That Mobilize Intracellular Ca2+ Induce LFA-1 Adhesion
To gain insight into the role of Ca2+ in LFA-1 adhesion, we
have analyzed the effect on leukocyte adhesion to ICAM-1
of the Ca2+ ionophore ionomycin and two other Ca2+ mobilizers, thapsigargin, and dBHQ. The latter two compounds act by inhibiting the ATPase pumps on Ca2+ storage organelles, which maintain homeostasis by pumping
Ca2+ from the cytosol into these organelles and the endoplasmic reticulum (Thomas and Hanley, 1994). Inhibition
of pump action causes emptying of intracellular Ca2+ stores
and a resultant Ca2+ influx across the plasma membrane
via capacitative entry (Thastrup et al., 1990
). The three
agents showed a dose-dependent stimulation of T cell
LFA-1-mediated adhesion to immobilized ICAM-1 (Fig. 1). The peak adhesion with ionomycin, thapsigargin, and
dBHQ occurred at 0.7, ~5, and ~50 µM, respectively. The
higher optimal concentration of dBHQ may be due to its
comparatively poor ability to penetrate the plasma membrane (Thomas and Hanley, 1994
). The reason for the
phase of decline in the bell-shaped response curves was
not due to loss of cell viability, but was not further pursued. Therefore, mobilization of [Ca2+]i by three distinct
agents can cause LFA-1-mediated T cell adhesion, highlighting a role for [Ca2+]i in the LFA-1 activation pathway.
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LFA-1-mediated Adhesion after Ca2+ Mobilization Requires Extracellular Ca2+
The tested pharmacological agents raise [Ca2+]i levels not
only by inducing release from intracellular stores, but also
by capacitative fluxing in through the plasma membrane
(Putney and Bird, 1993; Breittmayer et al., 1994
; Wenzel-Seifert et al., 1996
). To define the source of Ca2+ that
facilitates T cell adhesion, we made use of the imidazole compound SK&F 96365, which blocks Ca2+ channels on leukocytes that open as a result of depletion of Ca2+ stores
(Wenzel-Seifert et al., 1996
). Adhesion stimulated by ionomycin, thapsigargin, through the TCR-CD3 complex,
and by PdBu was inhibited by SK&F 96365 (Fig. 2). As expected, T cell adhesion to ICAM-1 induced by Mg2+/EGTA
was not altered by the Ca2+ channel blocker (Stewart et
al., 1996
). Further evidence that extracellular Ca2+ has an
intracellular function came from the use of the intracellular Ca2+ chelator BAPTA-AM, which at 20-30 µM reduced LFA-1 adhesion by 50-80% for all inducers in this
study with the exception of Mg2+/EGTA (data not shown).
Therefore, adhesion stimulated in several ways by "inside
out" signaling is dependent upon an extracellular source
of Ca2+.
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Measurements of Soluble ICAM-1 Binding
To investigate the mechanism of Ca2+ action, the characteristics of LFA-1-mediated adhesion caused by the Ca2+
mobilizers was examined. One question of interest was
whether the Ca2+-mediated signaling increased the ability
of LFA-1 to bind soluble ICAM-1 (sICAM-1). In a previous study, sICAM-1 binding distinguished high affinity
Mg2+-stimulated LFA-1 from low affinity LFA-1 on phorbol ester-stimulated and XL-TCR-CD3 T cells (Stewart et
al., 1996). None of the Ca2+ mobilizers showed any induction of sICAM-1 binding even at sICAM-1 levels of 1 mg/ml
(4.5 µM), in contrast to the enhanced ability of Mg2+-stimulated T cells to bind sICAM-1, which served as a positive control (Fig. 3). It can be concluded that the Ca2+ mobilizers resemble the phorbol ester or TCR-CD3 model of adhesion by failing to promote an increase in the ability of
LFA-1 to bind to soluble ICAM-1.
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Distribution of LFA-1 on T Cells after Exposure to Ca2+-mobilizing Agents
As the Ca2+ mobilizers did not cause a detectable increase in the affinity of LFA-1, we used confocal microscopy to investigate whether the membrane distribution of LFA-1 was altered in a manner facilitating adhesion. To analyze LFA-1 distribution, we highlighted the confocal microscopy images such that the membrane regions with the highest LFA-1 fluorescence (i.e., in the pixel intensity range 150-250, where 250 is the highest possible value) are depicted in red and all others in white (Fig. 4). Unstimulated cells (Fig. 4 a) and Mg2+/EGTA-stimulated cells (Fig. 4 b) have very little high intensity LFA-1 fluorescence (red) in comparison to that of thapsigargin-stimulated cells (Fig. 4 c). These observations were confirmed by quantifying (see Materials and Methods) the levels of fluorescence for each sample on regions of the membrane where there was no contact between cells (Table I, Free membrane). Statistical analysis (one way ANOVA) of these measurements confirmed that the level of fluorescence on the thapsigargin-stimulated cells, but not on the Mg2+/EGTA-stimulated T cells, was significantly increased compared to the level on the resting T cells (Table I, Significance levels). This increase in the intensity of LFA-1 fluorescence upon thapsigargin stimulation was detected with three distinct CD11a mAbs, 38, F110.22, and G25.2 (data not shown), and therefore does not represent the exposure of a particular epitope. These results suggested that thapsigargin might stimulate an increase in the cell surface expression of LFA-1. When measured by flow cytometry, however, results revealed that the level of LFA-1 expression after thapsigargin stimulation did not differ from LFA-1 expression on the resting or Mg2+/EGTA-treated cells (Table I, Flow cytometry). This suggested that the increase in signal strength observed by confocal microscopy upon thapsigargin stimulation reflects increased clustering that creates a higher LFA-1 fluorescence intensity. Similar results were obtained with the other Ca2+-mobilizing agents, with PdBu, and by XL-TCR-CD3 (data not shown).
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It was observed that some of the brightest LFA-1 fluorescence was found where cells were in contact with each other, and we therefore quantified the level of fluorescence in these regions (Table I, Contact zone) to determine whether this high fluorescence reflected a redistribution of LFA-1 to cell-cell contacts or was merely the additive value of the two cell membrane measurements. As the ratio of the average fluorescence intensity at cell contact areas to that of free membrane was ~2 (Table I, Contact/Free), we concluded that there is no large scale redistribution of LFA-1 to points of cell contact upon thapsigargin treatment. In summary, the confocal results revealed that upon thapsigargin stimulation LFA-1 becomes clustered but does not change its distribution to regions of the cell membrane in contact with other cells. Similar results were obtained with the other Ca2+-mobilizing agents as well as PdBu (data not shown), and by cross-linking the TCR/CD3 (see Fig. 7) suggesting that local LFA-1 clustering may be a general feature of several T cell adhesion- activating protocols.
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Effect of Jasplakinolide on T Cell Adhesion to ICAM-1 and on LFA-1 Distribution
The implication of the above confocal experiments was
that the Ca2+ mobilizers promoted LFA-1 clustering. This
clustering might occur as a result of breakdown of cytoskeletal tethering (Elemer and Edgington, 1994; Kucik et al.,
1996
; Lub et al., 1997
) or, conversely, might follow from
the formation of new cytoskeletal connections (Pardi et al.,
1992
). To address these alternative possibilities, we investigated the effects on LFA-1 adhesion and membrane distribution of jasplakinolide, a compound that stabilizes pre-existing actin filaments, promotes actin polymerization, and
prevents actin depolymerization (Bubb et al., 1994
). Jasplakinolide caused dose-dependent inhibition of LFA-1-mediated
adhesion to ICAM-1 with complete inhibition between 1 to
2 µM (data not shown). Therefore, the general inhibitory effects of jasplakinolide on agonists that indirectly stimulate LFA-1-mediated adhesion is consistent with a requirement for disassembly of the actin cytoskeleton.
We next used confocal microscopy to ask whether jasplakinolide affected the distribution of LFA-1 after pretreatment of thapsigargin-stimulated T cells. As seen in Fig. 5, jasplakinolide diminished the fluorescence levels of LFA-1 detected by confocal microscopy (i.e., there is less fluorescence highlighted [red] in Fig. 5 b than in a). Identical levels of LFA-1 expression were detected by flow cytometry before and after jasplakinolide treatment (data not shown), indicating that the inhibitor interfered with LFA-1 clustering rather than causing receptor loss. Similar results were obtained after T cell stimulation with phorbol ester and through CD3-TCR (data not shown). Therefore, clustering of LFA-1 on the membrane is dependent on the disassembly of the actin fibers, suggesting that adhesion through LFA-1 clustering is promoted by active release from restraints imposed by the cytoskeleton.
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Analysis of the Intracellular Molecules Controlling LFA-1-mediated Adhesion: Involvement of Calpain in Ca2+-induced Effects
In an attempt to understand the molecular mechanism by
which Ca2+ induces LFA-1 clustering, we investigated the
effect of inhibitors and mAbs specific for several major
Ca2+-using enzymes that have a link with adhesion. After
stimulation with ionomycin or thapsigargin, we failed to find
a role for PKC, calcineurin, or calmodulin using the specific inhibitors Ro 31-8220 (1-5 µM), FK506 (0-7 ng/ml),
and trifluoperazine (0.5-10 µM), respectively. Similarly,
immunoprecipitation and blotting with specific antibodies
failed to show an association of the 208-kD MLCK (Gallagher et al., 1995) or calreticulin (Coppolino et al., 1995
)
with stimulated LFA-1 (data not shown).
Another enzyme activated by Ca2+ is calpain, a multifunctional protease that is located in the cytosol (Sorimachi et al., 1994). We monitored LFA-1 adhesion stimulated
by thapsigargin, PdBu, XL-TCR-CD3, and Mg2+/EGTA
after preincubation with the membrane permeable calpain inhibitor, calpeptin (Tsujinaka et al., 1988
; Kwak et al.,
1993
). This agent caused maximal inhibition of T cell adhesion at 100 µg/ml (280 µM) after stimulation by thapsigargin, PdBu, and XL-TCR-CD3, but had no effect on
Mg2+-induced adhesion (Fig. 6). A further calpain inhibitor, CBZ-LVG, also blocked adhesion at similar concentrations (data not shown). There was no alteration in cell
viability at concentrations at which these inhibitors were
maximally active. We next found that the levels of clustered LFA-1 detected after treatment with thapsigargin (Fig. 7 a), XL-CD3 (Fig. 7 b), and PdBu (data not shown)
were diminished by calpeptin treatment (i.e., there is less
fluorescence highlighted [red] in Fig. 7 c than in a and in
Fig. 7 d than in b) to levels similar to those on resting T
cells (Table I, Free membrane).
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The calpain inhibitors used in this study are not entirely specific for calpain but also inhibit lysosomal cathepsins and proteasome activity. To rule out the involvement of the proteasome, we investigated the effect of the highly specific proteasome inhibitor lactacystin on T cell adhesion to ICAM-1. Although antigen presentation was inhibited by 20 µM lactacystin (Correa, I., and J. Trowsdale, personal communication) T cell adhesion was unaffected by concentrations as high as 200 µM (data not shown). Furthermore, T cells lack lysosomal cathepsins that are also susceptible to the calpain inhibitors. In summary, these results indicate that a calpain-like enzyme is pivotal in causing the clustering of LFA-1 and by this means adhesion to ICAM-1 is facilitated.
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Discussion |
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In this study, we have examined a role for Ca2+ in the induction of integrin-mediated adhesion, using the leukocyte integrin LFA-1 on T cells as the model. Three agents that cause Ca2+ mobilization trigger LFA-1-mediated adhesion to ICAM-1 to the same extent as other inside out inducers of adhesion such as XL-TCR-CD3 and phorbol ester. Furthermore, all three forms of stimulation cause LFA-1 clustering on T cells. The following points can be made about the Ca2+-dependent mechanism by which adhesion is brought about: (a) LFA-1 clusters in the cell membrane thereby strengthen adhesion by increasing the integrin avidity; (b) the adhesion is dependent upon Ca2+ influx; (c) the overall affinity of LFA-1 for ligand does not alter as no increase in the ability to bind soluble ICAM-1 can be detected; (d) both adhesion and LFA-1 redistribution are prevented by inhibiting cytoskeletal disassembly, implying a role for actin reorganization; (e) calpain has a central role in the clustering of LFA-1; (f) the above features are not shared by Mg2+/EGTA-treated LFA-1, which binds ICAM-1 with high affinity and mediates adhesion without dependence on the intracellular events described here.
Stimulation of T cells by the Ca2+ mobilizers, through
the TCR-CD3 complex and by phorbol ester, results in an
increase in integrin avidity by clustering of LFA-1 in the
membrane. These results are in agreement with previous
reports of integrin clustering after phorbol ester stimulation
(Burn et al., 1988; Kupfer and Singer, 1989
). Both phorbol
esters and the other Ca2+-dependent adhesion inducers
used in this study cause LFA-1 clustering over the entire
cell membrane. In a more physiological setting, when the
TCR is triggered upon T cell-APC contact Ca2+ fluxes occur locally (Zweifach and Lewis, 1993
; Hall et al., 1993
;
Donnadieu et al., 1994
; Negulescu et al., 1996
). The subsequent effect on LFA-1 would be confined to the immediate points of cell contact. Support for this prediction comes
from the observation that LFA-1 segregates away from the
"TCR clusters" after TCR signaling, and therefore is no
longer uniformly distributed (Dustin et al., 1996
). Whether
clustering involves direct lateral association between integrin subunits or is driven by ligand (Singer, 1992
) requires
further investigation. The expression of the epitopes of
both nonfunction blocking (G25.2) and function blocking
(38, F110.22) anti-LFA-1
-subunit mAb epitopes is comparable when detected by confocal microscopy, implying
that the clustering in this study is probably not driven by
ligand (data not shown). The work of van Kooyk et al.
(1994)
suggests that Ca2+ may also have an extracellular
role in integrin multimerization. Finally, a recent analysis
of
4 integrin made use of the confocal microscope to investigate integrin clustering in the absence of an alteration
in
4 integrin cell membrane expression (Yauch et al., 1997
)
in a similar manner to the approach described in our study.
Calpain is implicated in the clustering and adhesive activity of LFA-1 by the ability of selective inhibitors to diminish these processes. Calpains are Ca2+-dependent neutral cysteine proteases that are activated by local Ca2+ fluxes and
are widely expressed in mammalian cells (Suzuki et al., 1987;
Saido et al., 1994
; Sorimachi et al., 1994
). Calpain is highly
expressed in T cells and is increased at both mRNA and protein levels by phorbol ester, calcium ionophore, and
anti-CD3 treatment, all agents that can induce LFA-1 adhesion (Deshpande et al., 1995
). In platelets Ca2+ ionophore will directly activate calpain, but when platelets are
stimulated by a physiological agonist, such as thrombin,
calpain activation is then dependent upon interaction of
integrin
IIb
3 with ligand (Fox et al., 1993
). However,
for T cells, the evidence suggests that the Ca2+ flux is not
dependent on ligand binding by LFA-1, but, as we argue
here, is directly responsible for LFA-1 clustering, with ligand binding a secondary event. Firstly, anti-CD3 stimulation of T cells causes activation of calpain proteolytic activity (Selliah et al., 1996
). Secondly, the Ca2+ flux in T cells
stimulated in this manner is not affected by function
blocking CD11a and CD18 mAbs (Monard, 1991
). Finally, there has been no indication that LFA-1 is physically associated with Ca2+ channel activity as has been suggested for
IIb
3 (Rybak et al., 1988
; Fujimoto et al., 1991
). Therefore, the evidence suggests that calpain activity is regulated differently in these two cell types, but this does not
preclude further action of calpain after the ICAM-1 binding phase of T cells.
There are a number of possibilities to explain how calpain might be acting. Calpain may be activated to cleave a
key protein, physically releasing LFA-1 from its cytoskeletal restraint and allowing movement in the membrane as
observed by single particle tracking (Kucik et al., 1996)
and resonance energy transfer studies (Poo et al., 1994
).
Proteins that have been identified as calpain targets include talin (Inomata et al., 1996
), filamin (Collier and Wang,
1982
), and
-actinin (Selliah et al., 1996
). In resting PBMC, both filamin and
-actinin are associated with
CD18 (Pavalko and LaRoche, 1993
; Sharma et al., 1995
),
and in T cells calpain cleaves
-actinin after TCR-CD3 triggering (Selliah et al., 1996
) with kinetics similar to that of
LFA-1-mediated adhesion (Dransfield et al., 1992
; Dustin
and Springer, 1989
). Another possibility is that calpain
cleaves a signaling protein. Potential candidates include
focal adhesion kinase (Cooray et al., 1996
) and phosphotyrosine phosphatase kinase 1B (Frangioni et al., 1993
). It
will be of interest to test the sensitivity to calpain of LFA-1-binding proteins such as cytohesin-1 (Kolanus et al.,
1996
). There is evidence that calpain can cleave the
3
subunit (Du et al., 1995
). Whether the
2 subunit can also
be cleaved by calpain remains to be investigated, and it is
uncertain whether a
2 integrin cleaved at the sites homologous to the
3 calpain sensitive sites would be able to attach to the cytoskeleton or to bind ligand.
The cytoskeleton is intimately involved in the adhesive
process. Use of jasplakinolide, an agent that inhibits actin
disassembly (Bubb et al., 1994) prevents LFA-1 clustering
and T cell adhesion. Alternatively, low levels of cytochalasin D promote integrin-mediated adhesion (Elemer and
Edgington, 1994
; Kucik et al., 1996
; Lub et al., 1997
; Yauch
et al., 1997
) and allow movement of the integrin in the membrane (Kucik et al., 1996
). Put together, these findings indicate that in the nonactive state, LFA-1 is tethered to the cytoskeleton and release from this constraint allows motility leading to LFA-1 clustering and adhesion to ICAM-1.
LFA-1 has also been reported to associate with cytoskeletal elements after activation (Pardi et al., 1992
), and it is
possible that clustered integrin, either before or after contact with ligand, might renew interaction with the cytoskeleton. The role of calpain in cytoskeletal rearrangements
remains unresolved. Cytoskeletal reorganization may be
necessary for the exposure of the proteolytic target of calpain or, conversely, calpain activation may lead to alterations in the cytoskeleton itself. Phorbol ester and other
stimulants also cause coincident PKC and calpain translocation to the membrane (Pontremoli et al., 1989
; Hong et al.,
1995
). In platelets activating agonists are reported to cause
calpain to move from a generalized distribution in cells to
a peripheral location (Fox et al., 1993
), and this could be
brought about by cytoskeletal reorganization.
Here we have provided evidence that the protease calpain plays a role in the regulation of LFA-1 adhesion induced by Ca2+ flux. This process involves an increase in
ligand-binding avidity of the T cell through clustering of
LFA-1 but no detectable increase in LFA-1 affinity. In many
aspects this route to LFA-1 adhesion differs from that of
Mg2+/EGTA treatment, which requires neither Ca2+ nor
calpain but activates from the "outside" causing an affinity increase in LFA-1, indicating direct integrin conformational change (Stewart et al., 1996). Thus, there are two alternative pathways to LFA-1-mediated adhesion with different mechanisms. Migrating cells need adhesive contacts
that can be rapidly made and easily broken. We suggest
here that such contacts may be regulated through proteolysis and cytoskeletal control. On the other hand, once a
leukocyte has arrived at an inflammatory site, migration is halted and stable cell-cell contacts form, which may then
allow the bi-directional signaling necessary for other aspects of T cell function (Shaw and Dustin, 1997
). High affinity adhesion would be an efficient means of facilitating
these interactions and therefore it is of interest that the
Mg2+ concentration of wound fluids is in favorable balance with Ca2+ and considerably higher than in normal
plasma (Grzesiak and Pierschbacher, 1995
). There is also
evidence that this higher affinity LFA-1 may succeed the
primary low affinity interaction of LFA-1 as a result of
conformational change brought about by contact with ligand (Cabañas and Hogg, 1993
).
In summary, our findings show that the Ca2+-regulated
activation of calpain causes the clustering of LFA-1, possibly by cleaving a key cytoskeletal component. In vivo this
Ca2+-induced clustering would be speculated to be confined to local sites of receptor triggering. Recent studies
have outlined the phases of contact between T cells with
antigen-presenting cells and highlight the importance of
local Ca2+ fluxes in converting the motile "scanning" T cell
into an immobile cell, stably engaged with its partner
(Donnadieu et al., 1994; Negulescu et al., 1996
). Our observations lead to the prediction that any receptor trigger
which stimulates a Ca2+ flux in T cells would induce LFA-1
adhesion. It will be interesting to discover whether this
mechanism is unique to control of LFA-1 adhesion or part
of the adhesion mechanism for integrins found on other
cells.
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Footnotes |
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Received for publication 23 September 1997 and in revised form 25 November 1997.
Address all correspondence to Nancy Hogg, Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Tel.: 44 171 269 3255. Fax: 44 171 269 3093. E-mail: hogg{at}icrf.icnet.ukWe gratefully acknowledge the help of Imperial Cancer Research Fund colleagues Rainer Pepperkok, Peter Jordan, Alex Stokes, and Andrew Edwards (Digital Imaging Microscopy Department) and Henry Potts (Medical Statistics Group) for performing the statistical analysis. We thank K.K. Wang (Parke Davis, Ann Arbor, MI) for discussion about calpain and A. Koffer (University College of London, London) and G. Nash (University of Birmingham, Birmingham, UK) for discussion about jasplakinolide. We thank T. Plesner (University of Copenhagen, Copenhagen, Denmark) for LFA-1 mAb F110.22, F. Pavalko (Indiana School of Medicine, Indianapolis, IN) for sharing unpublished data and for the kind gift of embMLCK-specific mAb, S. Dedhar (Terry Fox Institute, Vancouver, British Columbia, Canada) for antisera specific for calreticulin, and N. Clipstone (Stanford, Palo Alto, CA) for FK506. We thank colleagues Matthew Robinson, Birgit Leitinger, Joanna Porter, and Rebecca Newton for their helpful comments about the manuscript.
This work was supported by the Imperial Cancer Research Fund.
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Abbreviations used in this paper |
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dBHQ, 2,5-di-t-butylhydroquinone; ICAM-1, intercellular adhesion molecule-1; LFA-1, lymphocyte function-associate antigen-1; MLCK, myosin light chain kinase; Pdbu, phorbol 12,13 dibutyrate; PKC, protein kinase C; sICAM-1, soluble intercellular adhesion molecule-1; TCR, T cell receptor.
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