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The Journal of Immunology, 2002, 168: 696-704.
Copyright © 2002 by The American Association of Immunologists

{alpha}4 Integrin Signaling Activates Phosphatidylinositol 3-Kinase and Stimulates T Cell Adhesion to Intercellular Adhesion Molecule-1 to a Similar Extent As CD3, but Induces a Distinct Rearrangement of the Actin Cytoskeleton1

Sharon J. Hyduk2 and Myron I. Cybulsky

Toronto General Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dynamic regulation of {beta}2 integrin-dependent adhesion is critical for a wide array of T cell functions. We previously showed that binding of high-affinity {alpha}4{beta}1 integrins to VCAM-1 strengthens {alpha}L{beta}2 integrin-mediated adhesion to ICAM-1. In this study, we compared {beta}2 integrin-mediated adhesion of T cells to ICAM-1 under two different functional contexts: {alpha}4 integrin signaling during emigration from blood into tissues and CD3 signaling during adhesion to APCs and target cells. Cross-linking either {alpha}4 integrin or CD3 on Jurkat T cells induced adhesion to ICAM-1 of comparable strength. Adhesion was dependent on phosphatidylinositol (PI) 3-kinase but not p44/42 mitogen-activated protein kinase (extracellular regulated kinase 1/2), because it was inhibited by wortmannin and LY294002 but not U0126. These data suggest that PI 3-kinase is a ubiquitous regulator of {beta}2 integrin-mediated adhesion. A distinct morphological change consisting of Jurkat cell spreading and extension of filopodia was induced by {alpha}4 integrin signaling. In contrast, CD3 induced radial rings of cortical actin polymerization. Inhibitors of PI 3-kinase and extracellular regulated kinase 1/2 did not affect {alpha}4 integrin-induced rearrangement of the actin cytoskeleton, but treatment with ionomycin, a Ca2+ ionophore, modulated cell morphology by reducing filopodia and promoting lamellipodia formation. Qualitatively similar morphological and adhesive changes to those observed with Jurkat cells were observed following {alpha}4 integrin or CD3 stimulation of human peripheral blood T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Integrins are a family of ubiquitously expressed {alpha}{beta} heterodimers with diverse ligands that include cell surface counterreceptors and components of the extracellular matrix. Over 20 {alpha}{beta} combinations have been identified and are expressed in a tissue-specific fashion on virtually all cell types. Integrins mediate adhesive interactions and activate signal transduction cascades that regulate diverse biological functions such as cell survival, differentiation, proliferation, and migration. Integrin adhesive capacity is not constitutive but is regulated by complex intracellular signal transduction pathways and conformational changes, as well as clustering and lateral associations with other cell surface proteins and cytoskeletal elements (1, 2).

The primary integrins on T lymphocytes include the {beta}2 integrins, which bind the ICAM family of adhesion receptors, and {alpha}4 integrins, which bind to VCAM-1 and fibronectin. {beta}2 integrins are essential for all aspects of leukocyte biology; they regulate T lymphocyte proliferation, provide costimulatory signals during Ag presentation, anchor target cells during cytotoxic T cell-mediated lysis, and participate in transendothelial migration. T cell activation and delayed-type hypersensitivity responses are severely compromised in {beta}2 integrin-null mice and in humans with leukocyte adhesion deficiency I (a deficiency in {beta}2 integrin expression or function) (3, 4).

{beta}2 integrins are "activated" in a highly regulated manner in response to stimulation by a wide array of cellular receptors. Intercellular adhesion between APCs and T cells or cytotoxic T cells and their targets is enhanced by signals from the TCR complex. Ligation of the TCR induces association of {beta}2 integrins with cytoskeletal proteins and adhesion to ICAM-1 (5, 6). Leukocyte emigration is a multistep process involving sequential leukocyte-endothelial adhesive interactions that include tethering, rolling, stable adhesion, spreading, and migration across the endothelium into extravascular tissues (7, 8). Intracellular signals generated during rolling interactions activate leukocyte integrins and lead to arrest, stable adhesion, and spreading. These signals may be generated by chemokine receptors (9) or selectins (10). {alpha}4{beta}1 integrins can also mediate leukocyte rolling interactions (11, 12, 13). We recently demonstrated that ligation of the {alpha}4 integrin also activates {beta}2 integrin-dependent adhesion of Jurkat cells and Mn2+-stimulated peripheral blood T cells (PB T cells)3 to ICAM-1, and similar observations have been obtained with monocytes (14, 15).

The process by which extracellular stimuli modulate integrin adhesive function has been termed inside-out signaling. Inside-out signals increase integrin ligand binding capacity in response to stimulation by a wide array of cellular receptors. Recent studies have begun to elucidate the intracellular signal transduction pathways that regulate {beta}2 integrin-mediated adhesion. To date, protein kinase C (5, 16), phosphatidylinositol (PI) 3-kinase (17, 18), ras-p44/42 mitogen-activated protein (MAP) kinase (extracellular regulated kinase (ERK)1/2) (18) calcium mobilization (19), and the rho family of small guanine nucleotide exchange proteins (20, 21) have been implicated in this process. Ligation of individual leukocyte receptors induces a complex cascade of molecular events, one of the consequences of which is activation of the {beta}2 integrins. Leukocyte adhesion is accompanied by polarization, shape change, and migration. Although a wide variety of receptors regulate {beta}2 integrin function and different signaling cascades are likely to be involved, common elements must exist.

In the present study, we compared {beta}2 integrin activation and alterations in T cell morphology by receptors that may be engaged during two very different functional contexts. {alpha}4 integrins participate in leukocyte emigration, whereas CD3 participates in responses to Ag and the lysis of target cells. Cross-linking of either {alpha}4 integrin or CD3 induced adhesion to ICAM-1-coated surfaces, which was dependent on PI 3-kinase and suggests that PI 3-kinase is a ubiquitous regulator of {beta}2 integrin-mediated adhesion. However, {alpha}4 integrin cross-linking induced extension of filopodia, whereas CD3 cross-linking induced cortical actin polymerization. Signaling via CD3 inhibited {alpha}4 integrin-induced spreading and extension of filopodia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

Recombinant human ICAM-1/Fc consisting of the five extracellular Ig domains of human ICAM-1 fused to the hinge region and Fc tail of human IgG was provided by Dr. D. Staunton (ICOS, Bothwell, WA) (22). The following Abs were used for cell cross-linking: anti-human {alpha}4 integrin (HP2/1; Serotec, Raleigh, NC), anti-human CD3 (OKT3; Ortho Biotech, Raritan, NJ) and F(ab')2 of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Rabbit polyclonal Abs against phospho Thr308 AKT (for immunohistochemistry) and phospho Ser473 AKT, and AKT (for Western blotting) were purchased from New England Biolabs (Mississauga, Canada). Mouse monoclonal anti-phospho Thr202/Tyr204 p44/42 MAP kinase (E10) and rabbit polyclonal anti-phospho Thr202/Tyr204 p44/42 MAP kinase were from New England Biolabs. Goat polyclonal anti-p44 MAP kinase (C16) was from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated anti-mouse IgG was purchased from Sigma-Aldrich (St. Louis, MO) and HRP-conjugated anti-rabbit IgG and anti-goat IgG were from Jackson ImmunoResearch Laboratories. Oregon green 488-conjugated goat anti-rabbit secondary Ab and rhodamine-phalloidin were obtained from Molecular Probes (Eugene, OR).

Other reagents included acrylamide, bis-acrylamide, Nonidet P-40, nonfat skim milk powder and polyvinylidene difluoride membrane from Bio-Rad (Mississauga, Canada), SDS, Tris, BSA, DMSO, PMSF, and wortmannin from Sigma-Aldrich, complete mini protease inhibitor tablets (Boehringer Mannheim, Laval, Canada), methanol (Fisher Scientific, Nepean, Canada), LY294002 and ionomycin (Calbiochem, La Jolla, CA), PD98059 and U0126 from New England Biolabs, SDF1{alpha} (Research Diagnostics, Flanders, NJ), and ECL Western blotting kit (Amersham Pharmacia Biotech, Baie d’Urfé, Canada).

Cell culture and T cell isolation

Jurkat E6-1, a human T cell line derived from an EBV-negative, non-Hodgkins lymphoblastic leukemia, was obtained from the American Type Culture Collection (Manassas, VA). Jurkat cells were routinely cultured in RPMI 1640 (Canadian Life Technologies, Burlington Canada) supplemented with 10% heat-inactivated FBS (Canadian Life Technologies) and 100 U/ml penicillin-streptomycin (Canadian Life Technologies).

Human PB T cells were isolated by density gradient centrifugation and Ab/magnetic particle depletion. Venous blood (30 ml) was obtained from healthy normal volunteers and collected in EDTA (3 mmol/l final concentration). Blood (diluted 1/1 with PBS) was layered over Histopaque 1077 (Sigma-Aldrich) and centrifuged at 300 x g to obtain mononuclear leukocytes. T cells were purified by negative depletion of monocytes, B lymphocytes, NK cells, and contaminating granulocytes according to the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA). Purified T cells were resuspended in RPMI 1640, 1% FBS and kept at 10°C. Just before experiments, cells were centrifuged and resuspended in the appropriate assay buffer.

Leukocyte detachment assay

ICAM-1/Fc was immobilized to 35-mm polystyrene tissue culture dishes as previously described (14). Briefly, 100 µg/ml goat anti-human IgG (Fc specific) F(ab')2 was passively adsorbed to the center of a dish for 1 h at room temperature in a humidified atmosphere. Dishes were washed with PBS and nonspecific binding sites were blocked with 5% FBS for 1 h at room temperature. The anti-Fc-coated area was incubated with a saturating concentration (20 µg/ml in PBS) of ICAM-1/Fc for 1 h at room temperature, which corresponds to a coating density of 1200 molecules/µm2 (14).

Cell detachment assays were performed using a parallel plate flow chamber purchased from Glycotech (Rockville, MD) as previously described (14). The ICAM-1/Fc-coated 35-mm tissue culture dish served as the bottom surface, and a silicone gasket formed a 0.254-mm (0.010-inch) high and 2.5-mm wide flow path. Cells (2 x 106/ml in HBSS (Canadian Life Technologies)) were injected via the outflow port into inverted flow chambers and allowed to settle onto the ICAM-1/Fc-coated surface for 10 min under static conditions once overturned. Shear stress was applied by pulling assay buffer through chambers with a Genie programmable syringe pump (Kent Scientific, Litchfield, CT). Cells were exposed to shear stress of 1, 4, and 10 dynes/cm2 at 30-s intervals. Cells were observed with a Diaphot 300 inverted phase contrast microscope (Nikon, Melville, NY) and recorded with a Sony DXC-151A color video camera and Sony SVT-S3100 time lapse video cassette recorder (both from Sony, Tokyo, Japan). Assay buffer was maintained at 37°C with a water bath and the flow chamber was maintained at 37°C using an infrared heat lamp, a thermocouple probe, and a temperature controller (CN76000; Omega Engineering, Laval, Canada). The number of cells before the introduction of shear stress and remaining attached after each shear stress interval was determined from videotape frames using Scion Image (www.scioncorp.com) and expressed as a percentage of input cells.

Surface {alpha}4 integrin or CD3 were ligated by incubation with mAb (HP2/1 or OKT3) for 30 min on ice followed by cross-linking with F(ab')2 of goat anti-mouse IgG for 10 min at 37°C before introduction into the flow chamber. For inhibitor experiments, leukocytes were preincubated with wortmannin (100 nM), LY294002 (20 µM), PD98059 (50 µM), U0126 (10 µM), or DMSO (vehicle control) for 30 min at 37°C before cross-linking.

SDS-PAGE and Western blotting

For Western blot analysis lysates were collected from unstimulated, {alpha}4 integrin, or CD3 cross-linked cells. Lysis buffer contained 50 mM Tris, 150 mM NaCl, 1 mM sodium orthovanadate, 0.25% sodium deoxycholate, 1% Nonidet P-40, PMSF, and protease inhibitors (complete mini protease inhibitor mixture). Unstimulated, anti-{alpha}4 integrin, or anti-CD3-treated cells were incubated in HBSS for 2–10 min at 37°C with cross-linking (anti-mouse) Ab. Cells were washed three times with ice-cold PBS and incubated in lysis buffer for 15 min on ice. Lysates were collected by centrifugation at 12,000 rpm for 15 min at 4°C. Total lysates (20 µg) were separated on 8% SDS-PAGE gels and transferred overnight to polyvinylidene difluoride membrane.

Membranes were probed with primary Abs and developed using HRP-conjugated secondary Abs and the ECL system as per the manufacturer’s protocol. Individual blots were probed initially for phospho Ser473 AKT and then stripped and reprobed for total AKT, phospho ERK1/2 (E10), or total ERK1.

Confocal microscopy

Nunclon eight-well glass chamber slides (Canadian Life Technologies) were coated with various combinations of ICAM-1 (10 µg/ml), VCAM-1 (2.5 µg/ml), anti-{alpha}4 integrin (HP2/1; 2.5 µg/ml) or anti-CD3 (OKT3; 2.5 µg/ml) for 1 h at room temperature. Slides were stored overnight at 4°C and wells were blocked with 5% FBS for 2 h before use. A total of 200,000 cells were added to each well in HBSS and allowed to adhere at 37°C for 1–10 min. Nonadherent cells were removed by washing with warm HBSS and adherent cells were rapidly fixed with 2% paraformaldehyde for 15 min at room temperature. Cells were subsequently permeabilized with 0.2% Triton X-100 in PBS for 5 min and blocked with 2% BSA for 1 h at room temperature before incubation with primary Abs (4°C, overnight). Polyclonal rabbit anti-phospho Thr308 AKT was used to detect activated AKT, and polyclonal rabbit anti-phospho Thr202/Tyr204 ERK1/2 was used to detect activated ERK1/2. Cells were washed and incubated with Oregon green 488-conjugated goat anti-rabbit secondary Ab and rhodamine-phalloidin for 45 min at room temperature. For studies of cell shape change and actin rearrangement, cells were prepared as described above with omission of the BSA block, primary, and secondary Ab incubations.

Slides were mounted in Vectashield anti-fade mounting medium (Vector Laboratories, Burlingame, CA) and visualized with a Bio-Rad MRC-1024ES confocal microscope equipped with a krypton/argon laser and a x60 oil immersion objective with a numerical aperture of 1.4 (Nikon). Images were collected at a zoom factor of 2.00. Oregon green (488) was excited at a wavelength of 488 nm and detected with a band-pass filter of 506–538 nm. Rhodamine was excited at 568 nm and fluorescence was detected at 589–621 nm.

Quantification of cell cytoskeletal rearrangement

Cell spreading and morphological changes were observed using immunoconfocal micrographs of rhodamine-phalloidin stained cells adherent to coated surfaces (see above). A "morphological index" was calculated using the following formula, as previously described (23):

(1)

(2)
The area in contact with the coverslip and perimeter measurements of randomly sampled cells from at least three individual experiments were obtained using scion image. Cell perimeters were determined as the linear measurement of peripheral phalloidin staining. The morphological index measures how much a cell perimeter deviates from a circle (index = 1). Therefore, using this formula, cells with greater perimeters (such as those with extensive membrane projections) will have a higher morphological index.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
{alpha}4 integrin signaling stimulates {beta}2 integrin-mediated adhesion of Jurkat cells to ICAM-1 to a similar extent as CD3 but induces a distinct rearrangement of the actin cytoskeleton

We showed that binding of high-affinity {alpha}4{beta}1 integrins to VCAM-1 strengthens {alpha}L{beta}2 integrin-mediated adhesion to ICAM-1 (14) and now compare this to CD3-induced adhesion. Cross-linking {alpha}4 integrin or CD3 strengthened the adhesion of Jurkat T cells to recombinant purified ICAM-1 to a similar extent (Fig. 1Goa). The number of cells that remained adherent following application of 4 or 10 dynes/cm2 fluid shear stress increased 2- to 3-fold. This interaction with ICAM-1 is mediated by {alpha}L{beta}2 integrin (14). Cross-linking {alpha}4 integrin led to cell spreading and polarization on ICAM-1 (Fig. 1Goc), whereas cells in which CD3 was cross-linked became more adherent but did not undergo significant shape change (Fig. 1God) as compared with untreated adherent cells (Fig. 1Gob). Cell shape changes were observed under static conditions and were not significantly altered by the application of fluid flow.



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  		FIGURE 1. Cross-linking {alpha}4 integrin or CD3 stimulates quantitatively similar but qualitatively different adhesion of Jurkat cells to ICAM-1. a, Jurkat cell {alpha}4 integrin or CD3 were cross-linked and cells were allowed to adhere under static conditions for 10 min before the application of 4 or 10 dynes/cm2 of shear fluid flow. The mean ± SEM of the percentage of cells remaining adherent are plotted. Significant differences from control adhesion at 4 (*) and 10 dynes/cm2 (#) were determined using ANOVA and Fisher PLSD post hoc test (p < 0.05). b–d, Phase contrast micrographs of unstimulated (b), {alpha}4 integrin cross-linked (c), and CD3 cross-linked (d) Jurkat cells adherent to ICAM-1 following the application of shear stress of 10 dynes/cm2. Scale bar represents 10 µm.

 
{alpha}4 integrin and CD3 induce distinct rearrangement of the actin cytoskeleton revealed by immunohistochemical staining of F-actin with rhodamine-phalloidin. Unstimulated cells (which adhere weakly to ICAM-1) show a fine band of F-actin at the cell periphery (data not shown). A dense band of cortical F-actin was observed in cells adherent to coimmobilized ICAM-1 and anti-CD3. Adhesion to ICAM-1 coimmobilized with VCAM-1 or anti-{alpha}4 integrin induced cell spreading and extension of numerous fine filopodia without extensive actin polymerization (Fig. 2Go). Analogous shape changes were observed in cells adherent to VCAM-1, anti-{alpha}4, or anti-CD3 immobilized in the absence of ICAM-1. The extent of cell process extension was quantified using a morphological index (23). Adherence to either anti-{alpha}4 integrin or VCAM-1 induced comparable morphological changes, which were distinct from those observed upon adherence to anti-CD3 (Fig. 2Go).



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  		FIGURE 2. {alpha}4 integrin signaling induces filopodia extension, whereas CD3 induces cortical actin polymerization. Immunoconfocal micrographs of F-actin distribution at the adherent surface of Jurkat cells on VCAM-1 (a and g)-, anti-{alpha}4 integrin (b and h)-, or anti-CD3 (c and i)-coated surfaces, in the presence (g–i) or absence (a–c) of ICAM-1. d–f, Perimeter tracings and perimeter (P) and area (A) measurements of the cells illustrated in a–c, respectively. j, A morphological index of adherent cells was determined from area and perimeter measurements of rhodamine-phalloidin-stained cells from three independent experiments (mean ± SEM).

 
Adherence of Jurkat cells to a combination of anti-CD3 and anti-{alpha}4 integrin resulted in a cell shape and cytoskeletal actin polymerization that was virtually indistinguishable from that observed on a surface coated with anti-CD3 alone (Fig. 3Go). The majority of cells exhibited a dense band of cortical actin, with only occasional cells extending filopodia. This suggests that signals generated by ligation of CD3 promote cortical actin polymerization and inhibit the formation of filopodia.



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  		FIGURE 3. CD3 signaling inhibits {alpha}4 integrin-induced filopodia extension. Immunoconfocal micrographs of F-actin distribution at the adherent surface of Jurkat cells on surfaces coated with anti-CD3 (a), anti-{alpha}4 integrin (b), or anti-CD3 and anti-{alpha}4 integrin (c). Scale bar represents 10 µm. d, Morphological index determined from rhodamine-phalloidin-stained cells from three independent experiments (mean ± SEM).

 
PI 3-kinase is activated by {alpha}4 integrin and mediates adhesion to ICAM-1

Recent studies have begun to elucidate signaling molecules that regulate integrin adhesiveness. PI 3-kinase has been implicated in the regulation of lymphocyte adhesion mediated by {beta}2 integrins (17, 18). We used AKT (protein kinase B), a downstream effector of PI 3-kinase that promotes cell survival, as a marker for PI-3 kinase activity. AKT phosphorylation was examined 2 and 10 min following cross-linking of Ab-bound {alpha}4 integrin or CD3. Fig. 4Goa illustrates a similar level of activation of AKT at both 2- and 10-min time points following the ligation of {alpha}4 integrin or CD3. In contrast, AKT phosphorylation was not detected following treatment with cross-linking Ab (XL) alone. Immunoconfocal microscopy was used to examine the cellular distribution of AKT following adhesion of Jurkat cells to surface-immobilized anti-CD3 or anti-{alpha}4 integrin. Phospho-AKT staining was detected at the adherent surface as early as 1 min, was maximal after 2 min (Fig. 4Go, f and i), and in some cells remained positive at 10 min. Phospho-AKT was localized primarily at the plasma membrane, probably through binding of its plekstrin homology domain. In more apical optical sections through adherent cells, membrane-localized phospho-AKT was not detected (Fig. 4Gog). Membrane-localized staining was absent when a nonimmune IgG was used in place of the primary Ab (Fig. 4Goh). High-intensity staining was observed in the nucleus of all cells examined (adherent or nonadherent).



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  		FIGURE 4. Cross-linking {alpha}4 integrin or CD3 induces activation of PI 3-kinase. AKT phosphorylation is dependent on upstream PI-3 kinase activity and was used as an indirect measure of PI 3-kinase activity. a, Western blots of Jurkat cell lysates were probed for phospho-Ser473 AKT followed by stripping and reprobing for total AKT. Cross-linking either CD3 or {alpha}4 integrin in solution induced phosphorylation of AKT. XL alone had no effect. A representative of four separate experiments is shown. b–i, Immunoconfocal micrographs of F-actin (b–e) and phospho-Thr308 AKT (f–i) or nonimmune IgG control (h) in cells adherent to immobilized anti-CD3 or anti-{alpha}4 for 2 min. Phospho-Thr308 AKT was localized to the cell membrane on the adherent surface of the cell (f and i) but not in a region 1 µm apical to the adherent surface (g). Scale bar represents 10 µm.

 
Inhibitors of PI 3-kinase (wortmannin and LY294002) attenuated {alpha}4 integrin- and CD3-induced adhesion of Jurkat cells to ICAM-1 in detachment assays (Fig. 5Go). Basal adhesion was also reduced by wortmannin and LY294002, but this effect was not statistically significant. Wortmannin abrogated {alpha}4 integrin- and CD3-induced phosphorylation of AKT (Fig. 5Goc) and partially inhibited activation of ERK1/2 in response to CD3 and {alpha}4 integrin ligation (data not shown).



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  		FIGURE 5. Inhibitors of PI 3-kinase attenuate adhesion to ICAM-1. The percentage of cells remaining adherent following application of 10 dynes/cm2 shear fluid flow are shown. Pretreatment with the PI 3-kinase inhibitors wortmannin (a) or LY294002 (b) inhibited {alpha}4 integrin- and CD3-induced adhesion of Jurkat cells to ICAM-1. Mean ± SEM (n = 4). Significant differences from control-DMSO-treated adhesion (*) and the equivalent DMSO-treated group (#) were determined by ANOVA and Fisher PLSD post hoc test (p < 0.05). c, Western blots of Jurkat cell lysates were probed for phospho-Ser473 AKT followed by stripping and reprobing for total AKT. Wortmannin pretreatment completely abolished {alpha}4 integrin- and CD3-induced phosphorylation of AKT. A representative of three independent experiments is shown.

 
ERK1/2 is activated to a lesser extent by {alpha}4 integrin as compared with CD3 and does not mediate {beta}2 integrin adhesion

Because ERK1/2 has also been implicated in {beta}2 integrin-mediated adhesion of T cells to ICAM-1, we investigated its role in {alpha}4 integrin-induced adhesion to ICAM-1 (18). Cross-linking of either {alpha}4 integrin or CD3 induced the activation of ERK1/2. ERK1/2 phosphorylation (phospho Thr202/Tyr204) was examined 2 and 10 min following ligation of {alpha}4 integrin or CD3 with XL. CD3 cross-linking induced a strong activation of ERK1/2 at 2 min, which was reduced 10 min following ligation. Cross-linked {alpha}4 integrin stimulated a much weaker phosphorylation of ERK1/2 at 2 min, which was back to control levels at 10 min (Fig. 6Goa). This {alpha}4 integrin-induced ERK1/2 phosphorylation was significantly weaker than CD3-induced phosphorylation in all experiments. Treatment with the XL alone for 2 (data not shown) or 10 min (Fig. 6Goa) did not stimulate ERK activation. Using immunoconfocal microscopy, phospho-ERK1/2-specific staining was maximally detected in cells adherent to surface-immobilized anti-CD3 for 2 min (Fig. 6Goe). Staining for phosphorylated ERK1/2 was also detected following the adherence of Jurkat cells to anti-{alpha}4 integrin (Fig. 6Gof) but was less intense than that observed in cells adherent to anti-CD3.



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  		FIGURE 6. Cross-linking {alpha}4 integrin or CD3 induces activation of p44/42 MAP kinase. a, Western blots of Jurkat cell lysates were probed for phospho-Thr202/Tyr204 p44/42 MAP kinase (ERK 1/2) followed by stripping and reprobing for total ERK1/2. Cross-linking of CD3 induced a dramatic phosphorylation of ERK1/2 at 2 min which was reduced at 10 min. {alpha}4 integrin-induced phosphorylation of ERK1/2 was much weaker than CD3-induced phosphorylation at 2 min and was undetectable at 10 min. XL alone had no effect. A representative of four separate experiments is shown. b–g, Immunoconfocal micrographs of F-actin (b–d) and phospho ERK1/2 (e and f) or nonimmune IgG control (g) in cells adherent to immobilized anti-{alpha}4 or anti-CD3 for 2 min. Scale bar represents 10 µm.

 
U0126, an inhibitor of MAP/ERK kinase (MEK)1/2, completely inhibited ERK1/2 phosphorylation in response to both CD3 and {alpha}4 integrin ligation but had no effect on adhesion (Fig. 7Go). In contrast, PD98059, an inhibitor of MEK1, only partially reduced CD3-induced phosphorylation of ERK1/2 and did not significantly affect CD3-induced adhesion. PD98059 completely prevented {alpha}4 integrin-induced phosphorylation of ERK1/2 and attenuated {alpha}4 integrin-stimulated adhesion to ICAM-1.



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  		FIGURE 7. Involvement of MAP kinases in adhesion to ICAM-1. The percentage of cells remaining adherent following application of 10 dynes/cm2 shear fluid flow is shown. U0126 (a) did not inhibit either {alpha}4 integrin or CD3-induced adhesion, whereas pretreatment with PD98059 (b) inhibited {alpha}4 integrin but not CD3-induced adhesion. Mean ± SEM (n = 4). Significant differences from control-DMSO-treated adhesion (*) and the equivalent DMSO-treated group (#) were determined by ANOVA and Fisher PLSD post hoc test (p < 0.05). c, Western blots of Jurkat cell lysates were probed for phospho-Thr202/Tyr204 ERK1/2 followed by stripping and reprobing for total ERK1/2. U0126 pretreatment completely abolished {alpha}4 integrin- and CD3-induced phosphorylation of ERK1/2. PD98059 completely inhibited {alpha}4 integrin-induced but only partially inhibited CD3-induced phosphorylation. A representative of two individual experiments is shown.

 
Pretreatment with PD98059, but not wortmannin or U0126, prevented the {alpha}4 integrin-induced filopodia extension. In contrast, the cortical ring of polymerized actin observed in Jurkat cells adherent to anti-CD3 was unaffected by any of the inhibitors (Fig. 8Go).



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  		FIGURE 8. {alpha}4 integrin-induced filopodia extension is inhibited by PD98059 and ionomycin. a–j, Immunoconfocal micrographs of F-actin distribution at the adherent surface of Jurkat cells on anti-{alpha}4 integrin (a–e)- or anti-CD3 (f–j)-coated surfaces. Cells were pretreated with DMSO (a and f), 100 nM wortmannin (b and g), 50 µM PD98059 (c and h), or 10 µM U0126 (d and i) for 30 min or 500 nM ionomycin (e and j) for 5 min at 37°C before adhesion to the Ab-coated plates. Scale bar represents 10 µm. k, Morphological index of cells adherent to anti-{alpha}4 integrin- or anti-CD3-coated plates was determined from rhodamine-phalloidin-stained cells from three individual experiments (mean ± SEM). Significant differences from the corresponding DMSO-treated group (*) were determined using ANOVA and Scheffe post hoc test (p < 0.05).

 
Ligation of {alpha}4 integrins and CD3/TCR induces a biphasic calcium flux. In both Jurkat cells and PB T cells, TCR-induced calcium mobilization is 2- to 3-fold greater than that triggered by {alpha}4 integrin (24). Ionomycin, a calcium ionophore, was used to determine whether the differential morphological changes were due to differences in calcium mobilization. Pretreatment of Jurkat cells with ionomycin reduced {alpha}4 integrin-induced filopodia formation and promoted peripheral, lamellipodial-like extensions. These changes resulted in a reduction in the morphological index (Fig. 8Go). In contrast, ionomycin did not alter CD3-induced cortical actin polymerization.

Morphological and adhesive responses of purified human PB T cells to {alpha}4 integrin or CD3 cross-linking

Unlike Jurkat cells, PB T cells are a largely heterogenous population of cells and respond to stimuli as such. Ligation of CD3 induced spreading and cortical actin polymerization in a minority of PB T cells; however, when costimulatory molecules such as {alpha}4 or {beta}2 integrins were coligated with CD3, the majority of PB T cells spread in a similar fashion to Jurkat cells (Fig. 9Goc). {alpha}4 integrins on Jurkat T cells are in a high affinity state, as are T cell blasts (25), whereas on circulating PB T cells they are at low affinity. Consequently, few unstimulated PB T cells spread in response to {alpha}4 integrin ligation, and most cells remain rounded (Fig. 9Goa). However, costimulation with SDF1{alpha}, a chemokine that transiently increases {alpha}4 integrin affinity (26), led to extension of filopodia and spreading in occasional T cells, which was morphologically similar to that observed in Jurkat cells. In detachment assays, cross-linking of high-affinity {alpha}4 integrin induced adhesion to ICAM-1; however, adhesion was much stronger following cross-linking of CD3.



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  		FIGURE 9. Stimulation of adhesion and morphological changes in PB T cells following cross-linking of {alpha}4 integrin or CD3. a–c, Immunoconfocal micrographs of PB T cells showing F-actin distribution at the adherent surface of slides coated with anti-{alpha}4 (a), anti-{alpha}4 plus SDF1{alpha} (b), or anti-CD3 plus anti-{beta}2 (c). Scale bar represents 10 µm. d, {alpha}4 integrins or CD3 were cross-linked in solution on Mn2+-treated human PB T cells, and cells were allowed to adhere to ICAM-1 for 10 min under static conditions before the application of 10 dynes/cm2 shear fluid flow. A representative of three individual experiments is shown, mean ± SEM of six individual fields. *, Significant differences from the control group, determined by ANOVA and Fisher’s PLSD post hoc test (p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
{beta}2 integrins are essential for T lymphocyte function and participate in diverse processes including proliferation, activation, target cell lysis, and emigration into tissues. Integrins are not constitutively active but become functionally competent following activation of the appropriate signaling cascades. Although diverse cell surface receptors may mediate integrin activation in each situation, it is probable that parallel signal transduction pathways are involved. The most characterized pathway for the activation of T cell adhesion to ICAM-1 via {beta}2 integrins is induced by ligation of the TCR. However, engagement of the TCR is unlikely to participate during emigration from blood into tissues. In this process, chemokine receptors (27), selectins (10), and {alpha}4 integrins (14, 15) may be involved in regulating {beta}2 integrin-mediated adhesion. In this study, we demonstrate that cross-linking of either {alpha}4 integrin or CD3 activate similar signaling pathways and stimulate {beta}2 integrin-mediated adhesion but mediate distinct cytoskeletal rearrangement.

Many signaling pathways have been implicated in the regulation of {beta}2 integrin-mediated adhesion to ICAM-1 including protein kinase C (5, 16), calcium mobilization (19), PI 3-kinase (17, 18, 28) (29), ras, and ERK1/2 (18). In monocytes, a urokinase receptor-dependent pathway regulated the {alpha}4 integrin-mediated activation of {beta}2 integrins (15). Other pathways involved in {beta}2 integrin adhesion are also activated by {alpha}4 (or {beta}1) integrins and may play additional roles in this signaling cascade (24, 30). In this paper, we investigated the role of PI 3-kinase and ERK1/2.

A recently identified cytoplasmic protein, cytohesin-1, is a candidate proximal effector of {beta}2 integrin function. Cytohesin-1 regulates {beta}2 integrin-mediated adhesion in both monocytes and T cells in a PI 3-kinase-dependent manner (17, 29, 31). Physical interaction of cytohesin-1 with the {beta}2 integrin cytoplasmic domain and regulation of cell spreading have been implicated in this process (17, 23). We used AKT phosphorylation as an indicator of PI 3-kinase activity. Phosphorylation of AKT in PB T cells may play an important role in their protection from apoptosis (32). Cross-linking the integrin {beta}1 subunit or adherence of cells to {beta}1 integrin ligands such as fibronectin leads to activation of AKT (33, 34). Our data suggest that {alpha}4 integrin can also use this pathway. The {alpha}4 integrin- and CD3-induced phosphorylation of AKT was entirely dependent on PI 3-kinase activity because it was abrogated following pretreatment with wortmannin, consistent with previous reports (32, 33). Activation of AKT requires PI 3-kinase-dependent translocation to the cell membrane where phosphorylation occurs. Following activation, AKT detaches from the cell membrane and translocates to the nucleus (35). At early time points following adhesion of Jurkat cells to plates coated with anti-CD3 or anti-{alpha}4, we observed phospho-AKT at the cell membrane; at later time points (>10 min), fewer cells had membrane-localized AKT. In contrast, phospho-AKT was observed in the nucleus of all cells, including nonadherent cells, which suggests that a basal level of AKT activation exists in T cells.

Adhesion of {alpha}4 integrin- and CD3-cross-linked cells to ICAM-1 was inhibited to a comparable extent by two PI 3-kinase inhibitors, wortmannin and LY294002. This confirms other reports of PI 3-kinase-dependent, TCR-induced adhesion to ICAM-1 (17, 18). In addition, PI 3-kinase activation is important for LPS- and IL-2-mediated alterations in {beta}2 integrin adhesion (28, 29). Therefore, PI 3-kinase may be an important and ubiquitous regulator of stimulated adhesion via {beta}2 integrins. It may also play a role in basal adhesion because we consistently observed decreased adhesion of inhibitor-treated unstimulated cells. Our data suggest that other pathways are also involved, because complete inhibition of PI 3-kinase only partially inhibited adhesion.

Signaling through {alpha}4 integrin or CD3 induced distinct morphological changes and rearrangement of the actin cytoskeleton in Jurkat cells. Cross-linking of {alpha}4 integrin and adherence to ICAM-1 (or adhesion to VCAM-1 or anti-{alpha}4 integrin) stimulated cell spreading and the extension of numerous fine filopodia. Identical shape changes were observed in the absence of ICAM-1, suggesting that the shape changes were receptor induced and not a consequence of postreceptor binding events regulated by the {beta}2 integrin. Adhesion of fibroblasts to {beta}1 integrin ligands initiates the formation of filopodia or lamellopodia that are required for locomotion (36). Filopodia are also observed during random locomotory activity of activated T cells on integrin substrates (37). During emigration, spreading may render an adherent cell less susceptible to detachment by shear forces of flowing blood. Newly formed filopodia and pseudopodia mediate adhesive interactions and provide the protrusive forces necessary for leukocyte motility (38). {alpha}4 integrin is involved in multiple steps of emigration and may have a key role in mediating individual adhesive and signaling functions. In contrast, CD3-induced adhesion may be more relevant for interactions with individual APCs or target cells where close apposition is required, but not extensive spreading. CD3-cross-linked cells exhibited cortical actin polymerization, radial lamellipodial extensions, and no extension of filopodia. Bunnell et al. (39) recently reported similar morphological changes in Jurkat cells triggered by TCR engagement. Real-time analysis of the spreading contact area revealed lamellipodial contacts that dynamically remodeled to form a dense, actin-rich ring. {beta}2 integrins are colocalized with CD3-induced subcortical actin, and reorganization of actin may drive the accumulation of actin-linked receptors to the target-T cell interface (5, 40, 41). {beta}2 integrins are regulated by interactions with the cytoskeleton. Integrins associate with the cytoskeleton through linker proteins such as {alpha}-actinin, vinculin, and talin (1). Integrin release from the cytoskeleton promotes diffusion in the plasma membrane and formation of integrin clusters, which confer strengthened adhesion upon reestablishment of cytoskeletal connections (19, 42). A role for redistribution of {beta}2 integrins in the formation of the "immunological synapse" during Ag presentation is well established (43, 44). Redistribution of {beta}2 integrins to the filopodia in {alpha}4 integrin-stimulated cells may similarly play an important role in mediating leukocyte adhesion and/or migration. In the presence of ionomycin, fewer {alpha}4 integrin-induced filopodia were observed, and radial lamellipodial projections comparable to those observed on anti-CD3 were present. This suggests that calcium flux may contribute to the differential spreading patterns. This is supported by data illustrating the inhibition of TCR-induced actin rearrangement following incubation with BAPTA, an intracellular calcium chelator (39). A greater CD3-induced calcium flux may result in greater calcium-dependent myosin activation and contraction, leading to radial actin polymerization and suppression of filopodia. Indeed, when both CD3 and {alpha}4 integrin were ligated together, the cytoskeletal changes in response to signals from CD3 dominated those from {alpha}4 integrin (Fig. 3Go). These two signals may be activated during the interactions of T cells with dendritic cells or target cells and emphasizes the importance of the tight collar of actin in maintaining and regulating T cell-target interactions (44).

ERK1/2 is an important regulator of T cell function. Activation of the ras, MEK, MAP kinase pathway by ligation of the TCR complex is critically important for the proliferation, differentiation, and activation of T cells (45). We observed a rapid phosphorylation of ERK1/2 following the cross-linking of CD3 in Jurkat T cells. Activation of ERK1/2 was also observed following {alpha}4 integrin ligation, but the response was much weaker than that seen with CD3 ligation. Cross-linking {alpha}4{beta}1 integrin on monocytic cells also leads to the activation of ERK1/2 (46, 47).

PD98059 blocked filopodia extension and inhibited the adhesion to ICAM-1 induced by ligation of {alpha}4 integrin. In a previous study, TCR-activated adhesion of murine thymocytes and resting CD4+ T cells to ICAM-1 was inhibited by PD98059; however, maximal inhibition was achieved in the presence of PI 3-kinase inhibitors (18). We also observed a greater inhibition of {alpha}4 integrin-induced adhesion when both inhibitors were used in combination (data not shown). The lack of significant inhibition of CD3-induced adhesion by PD98059 in our studies may relate to our use of the Jurkat T cell line, which resembles a mature T cell rather than an immature resting cell. U0126, another MEK inhibitor which completely inhibited ERK1/2 phosphorylation by both CD3 and {alpha}4 integrin ligation, did not inhibit adhesion, cell spreading, or cytoskeletal rearrangements induced by either stimulus. Because U0126 is a more efficient inhibitor of MEK, these conflicting results may indicate nonspecific effects of the PD98059 inhibitor, as recently suggested by several other investigators (48, 49).

PI 3-kinase inhibition did not influence the cytoskeletal changes induced by {alpha}4 integrin. A role for ras activation of PI 3-kinase has been demonstrated in chemokine-induced actin polymerization and adhesion to ICAM-1 (50). Similarly, Cytohesin-1 is reported to regulate cell spreading on ICAM-1 (23). Our data suggest that PI 3-kinase does not play a role in {alpha}4 integrin-induced morphological changes.

ERK activation in response to ligation of both {alpha}4 integrin and CD3 has important consequences that are not related to cell adhesion or cytoskeletal rearrangements. PD98059, but not U0126, blocked {alpha}4 integrin-induced spreading and filopodia extension and reduced the strength of adhesion to ICAM-1. As discussed previously, this effect of PD98059 is likely related not to its inhibition of MEK1, but to nonspecific inhibition of another pathway. In any case, these data suggest that cell spreading and filopodia extension induced by {alpha}4 integrin signaling strengthens {alpha}L{beta}2 integrin-mediated adhesion.


   Acknowledgments
 
We are grateful to Dr. Donald Staunton (ICOS) for providing chimeric ICAM-1/Fc and VCAM-1/Fc.


   Footnotes
 
1 This work was supported by an Astra Zeneca-Medical Research Council of Canada-Pharmaceutical Manufacturers of Canada Research Fellowship (PFE-36617, to S.J.H.) and Canadian Institutes of Health Research (MT-14151, to M.I.C.). M.I.C. was a recipient of an Established Investigatorship from the American Heart Association. Back

2 Address correspondence and reprint requests to Dr. Sharon J. Hyduk, Toronto General Research Institute, 200 Elizabeth Street, CCRW 1-828, Toronto, Ontario M5G 2C4, Canada. E-mail address: sharon.hyduk{at}utoronto.ca Back

3 Abbreviations used in this paper: PB T cell, peripheral blood T cell; PI, phosphatidylinositol; MAP, mitogen-activated protein; ERK, extracellular regulated kinase; XL, cross-linking Ab; MEK, MAP/ERK kinase. Back

Received for publication March 30, 2001. Accepted for publication November 14, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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