Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters alpha 4beta 1- and alpha 5beta 1-mediated Function

Joanna C. Porter, and Nancy Hogg

Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Abstract
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
Abbreviations used in this paper
References


Abstract

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 beta 2 integrin leucocyte function-associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by alpha 4beta 1 and, to a lesser extent, alpha 5beta 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 alpha 4beta 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 beta 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 alpha 5beta 1, and is increased when the alpha 4beta 1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of alpha 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 beta 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). Under flow conditions, circulating lymphocytes can attach and roll on vascular endothelium using the selectins and alpha 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 alpha 4beta 1, alpha 4beta 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 alpha 4 integrins, cannot initiate adhesion under these conditions without L-selectin and/or alpha 4beta 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 beta 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).

There is increasing evidence that, on a given cell, one subset of integrins may be negatively regulated by ligation of another. Transfection of alpha vbeta 3 into K562 cells that endogenously express alpha 5beta 1 provides a system in which ligation of beta 3 inhibits the phagocytic but not adhesive function of alpha 5beta 1 (Blystone et al., 1994). Similarly, ligation of transfected alpha IIbbeta 3 will inhibit the function of cotransfected alpha 2beta 1 or endogenous alpha 5beta 1 in CHO cells expressing these integrins (Diáz-González et al, 1996). Such effects are dependent on an intact beta 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 alpha 4beta 1 on fibroblasts was able to suppress the ability of alpha 5beta 1 to induce metalloproteinase expression (Huhtala et al., 1995).

There is also indirect evidence of similar integrin regulation on T cells. For example, resting lymphocytes use both LFA-1 and alpha 4beta 1 to bind endothelial cells, but when T cells become activated, adhesion is mediated through LFA-1 with little or no contribution from alpha 4beta 1 (van Kooyk et al., 1993). Additionally, in some leukemic T cell lines, functional alpha 4beta 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 alpha 4beta 1 is inactive if LFA-1 is active. Here we provide direct evidence of cross talk on T cells between beta 2 and beta 1 integrins. The avid state of the beta 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 alpha 4beta 1 and, to a lesser extent, through alpha 5beta 1 to the ligands fibronectin and vascular cell adhesion molecule-1 (VCAM-1) (alpha 4beta 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

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). 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.

mAbs and Other Reagents

mAb 925.2 (CD11a; LFA-1alpha subunit, nonblocking) was purchased from Becton Dickinson (Oxford, UK). mAbs 38 (CD11a; LFA-1 alpha  subunit function-blocking), and 24 (CD11/CD18; beta 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; alpha 4 subunit function-blocking) and TS2/ 16 (CD29; beta 1 subunit-activating) were gifts from R. Lobb (Biogen, Inc., Cambridge, MA). mAb SAM-1 (CD49e; alpha 5 subunit-blocking) was purchased from Eurogenetics (Hampton, UK). mAb 7.2 (CD49d; alpha 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).

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). 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.

Ligand-coated Beads

A modified protocol (Pyszniak et al., 1994) 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.

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 beta 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.

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.


Results

In this study, we have investigated cross influences on function between the beta 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 beta 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.

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 beta 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 beta 1 integrins, alpha 4beta 1 and alpha 5beta 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 alpha 4 and alpha 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 beta 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 alpha 4 and alpha 5 function-blocking mAbs HP1/2 (0.5 µg/ ml) and SAM-1 (0.5 µg/ml) (B). Bar, 20 µm.
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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 alpha 4 and alpha 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.
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Adhesion of T Cells to ICAM-1 Decreases Binding of Fibronectin- and VCAM-1-coated Beads by Downregulating alpha 4beta 1 Activity

We then looked at the effects of LFA-1 ligand-binding on each of the T cell fibronectin receptors, alpha 4beta 1 and alpha 5beta 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 alpha 4-blocking mAb, an alpha 5-blocking mAb, or completely with a combination of both blocking mAbs, showing that T cells bind these beads through a mixture of alpha 4beta 1 and alpha 5beta 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 alpha 5-blocking mAb (Fig. 3 B). Therefore, binding in this situation is mediated mainly through alpha 5beta 1 with little contribution from alpha 4beta 1, demonstrating that the binding activity of alpha 4beta 1 has been compromised. The binding by T cells of VCAM-1-coated beads, which is mediated exclusively by alpha 4beta 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 alpha 4beta 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 alpha 4 and alpha 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.
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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 alpha 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.
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Activation of the beta 2 Integrin LFA-1 on T Cells Inhibits Their beta 1-Mediated Binding to Fibronectin

The inhibitory effect of LFA-1 on beta 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; beta 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 (open circle ) or mAb 52U (IgG1 isotype control) (bullet ) 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) (black-triangle). Data represent means of triplicates ± SD. One representative experiment of three is shown.
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Fig. 6. Prolonged activation of LFA-1 blocks alpha 4beta 1- and to a lesser extent alpha 5beta 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 alpha 4beta 1 and alpha 5beta 1 together. (B) alpha 5beta 1-mediated adhesion after the alpha 4beta 1 component had been blocked with mAb HP1/2 (0.5 µg/ml). (C) alpha 4beta 1-mediated adhesion after the alpha 5beta 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 alpha 4- and alpha 5-blocking mAbs as indicated.
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The Effect of LFA-1 Activation on T Cells Is Mediated Predominantly through alpha 4beta 1

Because T cells bind to fibronectin through both alpha 4beta 1 and alpha 5beta 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 alpha 5beta 1-mediated adhesion (Fig. 6 B), but had a much greater effect on alpha 4beta 1-mediated adhesion (Fig. 6 C). Similar levels of alpha 4beta 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 beta 2 integrin-activating mAb KIM185 decreased alpha 4beta 1-mediated adhesion to VCAM-1 to the same extent as to fibronectin (data not shown). Together, these results reinforced the findings that alpha 4beta 1 function is particularly sensitive to the state of LFA-1 activation.


Fig. 7. Prolonged activation of LFA-1 blocks alpha 4beta 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 alpha 5beta 1 was blocked with SAM-1 (0.5 µg/ml), allowing alpha 4beta 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 alpha 4-blocking mAb.
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Activation of beta 1 Integrins on T Cells Has No Effect on LFA-1 Binding to ICAM-1

We then reversed the situation to investigate the effect on beta 2 integrin-mediated adhesion of maintaining beta 1 integrins in an active state, using the beta 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 beta 2-mediated binding to ICAM-1 (Fig. 8 B), confirming that the beta 1 integrins were unable to influence the ligand-binding activity of LFA-1.


Fig. 8. beta 2 integrin activation decreases beta 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 beta 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 alpha 4- and alpha 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.
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Inhibition of alpha 4beta 1 with Blocking mAbs or by LFA-1 Activation Increases alpha 5beta 1-Mediated Migration

The effects of LFA-1-mediated cross talk on alpha 4beta 1- and alpha 5beta 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 alpha 5beta 1 exclusively, and that this migration was enhanced by an alpha 4-blocking mAb HP1/2, but not affected by an alpha 4- nonblocking mAb 7.2 (Fig. 9 A). In addition, migration at different concentrations of fibronectin remained solely dependent on alpha 5beta 1 (Fig. 9 B), and could be enhanced either with alpha 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 alpha 5-blocking mAb. Therefore, by decreasing the alpha 4beta 1 contribution to fibronectin adhesion with either an alpha 4-blocking mAb or an LFA-1- activation mAb, the ability of alpha 5beta 1 to mediate migration was increased.


Fig. 9. Migration of T cells on fibronectin is mediated by alpha 5beta 1 and promoted by blocking alpha 4beta 1 function. (A) Migration of T cells on fibronectin (10 µg/ml) is mediated by alpha 5beta 1 and enhanced when alpha 4beta 1-mediated adhesion to fibronectin is blocked by the function-blocking alpha 4 antibody mAb HP1/2. The non-function-blocking alpha 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 alpha 5beta 1. (C) Addition of the alpha 4-blocking mAb HP1/2 increases migration above control mAb 52U. This increased migration can be blocked with the alpha 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 alpha 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.
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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). Therefore, we looked at the ability of beta 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 (alpha 4beta 1 and alpha 5beta 1)- and VCAM-1 (alpha 4beta 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 beta 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.

We next tested the possibility that LFA-1 activation might be influencing a cytoskeletal event. Although mAb 24 decreased the overall level of alpha 4beta 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 alpha 4beta 1-mediated adhesion by mAb 24 does not alter the sensitivity of adherent T cells to cytochalasin D. Integrin alpha 5beta 1 was blocked with SAM-1, as previously, allowing alpha 4beta 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 (open circle ), or the isotype-matched control antibody, mAb 52U (bullet ), 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-alpha 4) (black-square). Data represent means of triplicates ± SD. One representative experiment of four is shown.
[View Larger Version of this Image (14K GIF file)]


Discussion

This study was undertaken to examine the functional interaction on T cells between LFA-1 and the beta 1 integrin fibronectin receptors alpha 4beta 1 and alpha 5beta 1. The main findings are that (a) the occupation of T cell LFA-1 by its ligand ICAM-1 decreases the binding of alpha 4beta 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 alpha 5beta 1 is affected to a lesser extent; (d) while active LFA-1 downregulates the avidity of alpha 4beta 1, the reverse does not occur, as neither beta 1 integrin-activating mAb TS2/16 nor beta 1-mediated binding to fibronectin affected the avidity of LFA-1; and (e) downregulation of alpha 4beta 1 activity increases the efficiency of alpha 5beta 1-mediated migration on fibronectin. Therefore, we have demonstrated differential regulation of two integrin subclasses and a hierarchy of integrin usage in which the beta 2 integrin LFA-1 will suppress the function of beta 1 integrins, particularly alpha 4beta 1.

Previous studies have demonstrated the involvement of alpha 4beta 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 alpha 4beta 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 alpha 4beta 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.

In this study, we have demonstrated that, in contrast to adhesion, the migration of activated T cells on fibronectin is mediated by alpha 5beta 1 with no contribution from alpha 4beta 1. In addition, suppressing alpha 4beta 1 activity on T cells either by mAb 24 or alpha 4 function-blocking mAbs enhanced the level of alpha 5beta 1 migration, particularly at low fibronectin levels. This may reflect the compensatory increase in alpha 5beta 1 adhesion, with its migratory potential, when binding through the nonmigratory alpha 4beta 1 is blocked. Another possibility is that the enhanced migration by alpha 5beta 1 is due to removal of a restraint imposed by alpha 4beta 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 alpha 4beta 1 and alpha 5beta 1 may make conditions suboptimal for migration. Alternatively, alpha 4beta 1 may be involved in a more specific inhibition of alpha 5beta 1 function, as has been described in the control of metalloproteinase expression in fibroblasts (Huhtala et al., 1995). The promotion of migratory behavior by alpha 5beta 1 through loss of alpha 4beta 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 alpha 4beta 1 and alpha 5beta 1.

The mechanism for LFA-1 downregulation of alpha 4beta 1 was explored in several ways. We first established that there was no alteration in expression of either alpha 4beta 1 or alpha 5beta 1 during the experimental period (data not shown). Furthermore, confocal microscopy using mAbs specific for alpha 4beta 1, alpha 5beta 1, and the beta 1-activation reporter mAb 15/7 indicated that avid LFA-1 did not cause beta 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 alpha 4beta 1 or alpha 5beta 1. This implied that cross talk was not affecting high affinity beta 1 integrins. Together, these findings suggested that the beta 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 beta  subunit cytoplasmic tail and steps subsequent to modulation of integrin affinity (Blystone et al., 1994; Diáz-González et al., 1996).

It seemed possible that the cytoskeleton was a target of LFA-1-mediated cross talk because both alpha 4beta 1- and alpha 5beta 1-mediated adhesion were more sensitive to changes in actin than was adhesion through LFA-1 (data not shown). However, for beta 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 alpha 4beta 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).

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 beta 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 alpha 5beta 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 beta 1 integrins using mAb TS2/16 does not alter LFA-1 binding to ICAM-1. Second, LFA-1 predominantly affects the activity of alpha 4beta 1, despite a sixfold abundance of alpha 4beta 1 over alpha 5beta 1 (data not shown). Future work will address the role of potential integrator molecules in the cross talk phenomenon.

In summary, we describe inhibition of alpha 4beta 1-binding activity in T cells as a consequence of LFA-1 activation. A speculation is that deadhesion of alpha 4beta 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 alpha 5beta 1, and that this migration is enhanced by interfering with alpha 4beta 1 adhesion. LFA-1 might provide a link between alpha 4beta 1 and alpha 5beta 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.


Footnotes

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.uk

We 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.


Abbreviations used in this paper

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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