The Affinity of Integrin α₄β₁ Governs Lymphocyte Migration

Cells

The Jurkat E6-1 T leukemic cell line was purchased from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (BioWhittaker), 1% glutamine, 1% penicillin, and 1% streptomycin (Sigma, St. Louis, MO).

Reagents

The anti-human β₁ mAb 8A2 was a generous gift from N. Kovach and J. Harlan (University of Washington, Seattle, WA). The anti-human α₄, HP2/1; anti-human α₅, SAM1; and anti-human β₁, K20 Abs were purchased from Immunotech (Westbrook, ME). The anti-human β₂ mAb hybridoma cell line TS1/18 was obtained from American Type Culture Collection and was used to generate ascities fluid. The cDNA encoding the CS-1 region of fibronectin fused to GST was a gift from J. W. Smith (Burnham Institute, La Jolla, CA). The expression and purification of this fusion protein have been previously described (14).

Construction and expression of VCAM-1- and ICAM-1-Ig fusion proteins

The cDNA for human VCAM-1 was a generous gift from T. Collins (Harvard University, Cambridge, MA). The coding sequence of the complete seven Ig domains of the extracellular region of VCAM-1 was PCR amplified and cloned into the NheI site of plasmid pB4Ig (a gift from R. Cobb, Tanabe Research Laboratories, San Diego, CA), which contains the human Fc coding sequence. The resulting VCAM-Ig fusion construct was excised with KpnI and cloned into pcDNA3.1⁻ (Invitrogen, Carlsbad, CA). The resulting construct was transfected into Chinese hamster ovary (CHO)³ cells, and stable cell lines were isolated by selection in G418. A VCAM-Ig-expressing clonal cell line was isolated by limited dilution cloning and screening supernatant for VCAM-Ig production with a VCAM-1 ELISA (R&D Systems, Minneapolis, MN). Recombinant protein was purified from CHO cell supernatant using a protein A column. Similarly, an ICAM-Ig fusion construct, encoding the N-terminal two Ig-like domains of ICAM-1 (a generous gift from D. L. Simmons, CRF Laboratories, University of Oxford, Oxford, U.K.), was subcloned into pcDNA3.1⁻ and transfected into CHO cells. ICAM-Ig fusion protein was isolated from the supernatants produced by a clonal cell line by protein A affinity chromatography.

Soluble VCAM-Ig-binding assay

Cells (5 × 10⁵) were resuspended in a modified Tyrode’s buffer (150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO₃, 1 mg/ml glucose, and 1 mg/ml BSA) containing 1 mM CaCl₂ and 1 mM MgCl₂. The VCAM-Ig fusion protein was added at a final concentration of 100 nM and incubated for 30 min at room temperature. Cells were washed twice in Tyrode’s buffer and resuspended in the same buffer containing FITC-conjugated donkey anti-human IgG (Jackson ImmunoResearch, West Grove, PA) at a 1/100 dilution. After a 30-min incubation at 4°C, cells were washed twice, and bound Ab was detected using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and analyzed using CellQuest software.

Generation and isolation of Jurkat mutants

Jurkat cells were treated with ethyl methane sulfonate (EMS; 200 μg/ml) or ICR-191 (1.5 μg/ml; Sigma) for 24 h. After 5 days in culture, soluble VCAM-1 (sVCAM-1)-binding assays were performed, and low sVCAM-1-binding cells were isolated by cell sorting using a FACStar^Plus flow cytometer (BD Biosciences) into 96-well tissue culture-treated plates (Costar, Corning, Corning, NY). Isolated clonal lines were sequentially reassayed for α₄ expression with mAb HP2/1 and sVCAM-1 binding by flow cytometry. Jurkat lines with normal α₄ expression and low sVCAM-1 binding were used for further analysis.

Cell adhesion assays

Ninety-six-well Immulon 2HB plates (Dynex Technologies, Chantilly, VA) were incubated with indicated concentrations of VCAM-1 or the CS-1-containing fragment of fibronectin overnight at 4°C. Afterward, wells were blocked with 2% BSA in PBS for 30 min at room temperature. Cells in a modified Tyrode’s buffer were added to wells and allowed to adhere for 40 min at 37°C. Nonadherent cells were washed off with Tyrode’s buffer. Adherent cells were stained with 0.5% crystal violet stain in 20% methanol. The cell-incorporated crystal violet was solubilized with 10% acetic acid and measured in a microplate reader (Molecular Devices, Sunnyvale, CA) set at 560 nm.

Cell migration assay

Cell migration was assayed in a modified Boyden chamber assay system. Transwells (Costar; Corning) polycarbonate membranes containing 3-μm pores were incubated with VCAM-1 and/or ICAM-1 in 0.1 M NaHCO₃ (pH 8) overnight at 4°C. Membranes were blocked with 2% BSA in PBS for 30 min at room temperature. A total of 2 × 10⁵ cells in RPMI 1640 with 10% FCS was added to the top chamber. Stromal cell-derived factor-1α (R&D Systems) at a final concentration of 15 ng/ml was added to the bottom chamber. Cells were allowed to migrate for 4 h at 37°C. Cells in the bottom chamber were enumerated with a hemocytometer.

VCAM-1 ELISA

The amount of recombinant VCAM-1 bound to the polycarbonate membranes of the transwells was measured by ELISA. Briefly, transwell membranes were coated as described above for cell migration assay, except in the case of the sVCAM-1 studies in which membranes were coated with ICAM-1 and blocked with BSA before addition of sVCAM-1. Afterward, the membranes were washed and anti-human VCAM-1 Ab, P8B1 (1/3000 diluted ascites) was added and incubated for 4 h at room temperature. After extensive washes, an HRP-conjugated goat anti-mouse IgG (BioSource International, Camarillo, CA) was incubated with the membranes for 2 h at room temperature. Bound Ab was detected with an 80 mM citrate phosphate buffer (pH 5) containing o-phenylenediamine and hydrogen peroxide using a Molecular Devices ELISA plate reader set at 490 nm.

Laminar flow assays

A polystyrene plate coated with sVCAM-1 (affinity-purified seven-domain human VCAM-1, a gift from R. Lobb, Biogen, Cambridge, MA) was assembled in a parallel plate laminar flow chamber (260-μm gap) and mounted on the stage of an inverted phase-contrast microscope (Diaphot 300; Nikon, Tokyo, Japan), as previously described (15). Jurkat cells were perfused at 10⁶ cells/ml of binding medium (HBSS containing 2 mg/ml BSA and 10 mM HEPES, pH 7.4, supplemented with Ca²⁺ and Mg²⁺, each at 1 mM) at the desired shear stress generated with an automated syringe pump (Harvard Apparatus, Natick, MA). Cellular interactions on a field of view of 0.34 mm² were visualized with a ×10 objective and manually quantified by analysis of images directly from the monitor screen. The motion of each interacting cell was monitored for 10 s following its initial tethering, and three categories of tethers were defined: transient, if cells attached briefly (<2 s) to the substrate; rolling, if cells tethered and rolled on the substrate >5 s with a velocity >1 μm/s; arrest, if following rolling or immediately after tethering, cells came to a full arrest and remained stationary on the substrate for at least 20 s. The number of tethers for each category was divided by the flux of freely flowing cells. For calculations of cell flux, only the fraction of perfused cells that came into close proximity with the substrate, and therefore was potentially capable of interacting with the substrate, was considered.

Controlled flow detachment assays were performed on cells that were settled at stasis on ligand-coated plates for 1 min and then were subjected to wall shear stresses increased stepwise every 5 s (by a programmed set of flow rates delivered by the syringe pump). At the end of each 5-s interval of the increase in shear stress, the number of cells that remained bound was expressed relative to the number of cells originally settled on the substrate in stasis. All assays were performed at room temperature. To study peptide inhibition of α₄β₁-mediated tethering events, cells were suspended in binding medium with 0.5 mM octapeptide EILDVPST (containing the tripeptide very late Ag-4-binding motif leucine-asparatate-valine, LDV) or its control analogue EIDVLPST for 5 min, and then perfused unwashed through the flow chamber over the VCAM-1-coated substrate.

Isolation of Jurkat cell lines with defective integrin α₄β₁ activation

To evaluate the functional importance α₄β₁ affinity modulation, we generated a panel of variant Jurkat T cells with defects in α₄β₁ activation (Act. Defect. Jurkat). Jurkat cells were chemically mutagenized with either EMS or ICR-191, and α₄-expressing cells were selected for reduced sVCAM-1 binding by flow cytometry. Clonal lines were isolated that expressed α₄, but failed to bind sVCAM-1. The characterization of a representative mutant line, JD6, is shown in Fig. 1⇓. In contrast to wild-type Jurkat cells, the mutant cells showed marked reduction in constitutive sVCAM-1 binding, but binding was reconstituted in the presence of the exogenous β₁ integrin-activating mAb, 8A2. Furthermore, α₄, α₅, β₁, and β₂ integrin subunit expression was similar to that of wild-type Jurkat cells. These results indicate that these mutant lines have a defect in α₄β₁ activation, which is not due to a change in integrin expression nor a defect in the integrin’s ligand binding site. Three independent cell lines were isolated from three different mutagenesis experiments, two with EMS and one with ICR-191. Similar results were obtained with all three mutant lines in the experiments to be described.

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FIGURE 1.

Characterization of mutant Jurkat T cell lines. Top panels, Histograms of sVCAM-1 binding, as measured by flow cytometry. Solid lines are sVCAM-1 binding in a modified Tyrode’s buffer containing 1 mM CaCl₂ and 1 mM MgCl₂. Filled histograms are the binding with the addition of anti-α₄ Ab, HP2/1. Dotted lines are binding with the addition of activating β₁ Ab, 8A2. Quantification of sVCAM-1 binding was assessed as an activation index (AI) defined as 100 × (F_o − F_r)/(F_max − F_r), in which F_o is mean fluorescence intensity of sVCAM-1 binding, F_r is fluorescence intensity in the presence of mAb HP2/1, and F_max is the fluorescence intensity in the presence of mAb 8A2. Subsequent panels are flow cytometric analysis of integrin expression. Binding of specific mAb for α₄ (HP2/1), β₁ (8A2), α₅ (SAM1), and β₂ (TS 1/18) was detected with a FITC-conjugated goat anti-mouse IgG (filled histograms). Open histograms are the binding of purified mouse IgG. Geometric mean fluorescence intensity (MFI) is shown in each panel. Results are representative of three separate experiments with similar results.

The rescue of sVCAM-1 binding in the mutant lines by mAb 8A2 indicates that the intrinsic ligand-binding capacity of the α₄β₁ is retained and the defect impacts the activation process. Defective activation could be due to mutations in either subunit of integrin α₄β₁ or a signaling molecule that is required for α₄β₁ activation. To distinguish between these two possibilities, mutant lines were transfected with wild-type α₄ and β₁ integrin subunits. Failure to rescue sVCAM-1 binding by introduction of wild-type α₄β₁ indicates that cells have a defect in signaling machinery needed to activate α₄β₁. The mutant lines were transiently transfected with green fluorescent protein (as a markers of transfection) and cDNAs encoding either α₄, β₁, or a combination of subunits, and sVCAM-1-binding assays performed by flow cytometry. In all three lines examined, transfection of exogenous α₄β₁ failed to rescue sVCAM-1 binding (data not shown). This result indicates that the defect in integrin activation is not ascribable to integrin mutations, but rather is due to a defect in cellular mechanisms required for activation of α₄β₁.

The affinity state of α₄β₁ controls cell migration

α₄β₁ integrins can regulate adhesion and migration mediated by β₂ integrins (10, 11, 16). Under these conditions, VCAM-1 acts as agonist that regulates β₂ integrin functions. The regulation of integrin function by soluble agonist, such as chemokines, is a function of the affinity of the agonist receptor. By analogy, we reasoned that the capacity of VCAM-1 to stimulate β₂ integrins would be a function of the affinity state of α₄β₁ (Fig. 2⇓A). To examine α₄β₁-stimulated β₂-dependent migration, Jurkat cells were allowed to migrate on a substrate coated with 200 μg/ml ICAM-1, a ligand for β₂ integrins. We examined the effect of addition of trace quantities of VCAM-1 (0.1–10 μg/ml). Under these conditions, the filters were coated with ∼250 molecules/μm² of ICAM-1, and the addition of small quantities of VCAM-1 did not significantly displace ICAM-1 (Fig. 2⇓B).

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FIGURE 2.

α₄β₁ integrin interaction with VCAM-1 stimulates β₂ integrin-dependent Jurkat T cell migration. A, Agonist modulation of integrin function. In heterologous modulation, a cell surface receptor (e.g., chemokine receptor) activates a signaling pathway, which modulates integrin function. In homologous modulation, an integrin (integrin 1), engages ligand and activates a signaling pathway that modulates the function of a second integrin (integrin 2). B, Quantification of ICAM-1 coating on polycarbonate membranes of transwell migration chambers. ICAM-1 was radiolabeled with ¹²⁵I. Membranes (0.33 cm²) were incubated with ¹²⁵I-labeled ICAM-1 mixed with the indicated amounts of VCAM-1. Specific activity of ¹²⁵I-labeled ICAM-1 was used to calculate ICAM-1 coating expressed as molecules per square micrometer. C, Migration of wild-type Jurkat T cells. Transwell membranes were coated with 200 μg/ml ICAM-1 and indicated concentrations of VCAM-1. Jurkat cells were added to the top chamber either untreated or with the addition of anti-β₂ Ab (TS 1/18) (20 μg/ml) or anti-VCAM-1 (P8B1) (20 μg/ml). SDF-1α (15 ng/ml) was added to the bottom chamber, and cells were allowed to migrate for 4 h at 37°C. Cells in the bottom chamber were enumerated, and migration was expressed as a percent change relative to no VCAM-1 addition. Results are the mean ± SEM of three separate experiments. D, sVCAM-1 stimulates β₂-dependent migration of Jurkat T cells. Transwell membranes were coated with ICAM-1 (200 μg/ml). Jurkat cells were pretreated for 5 min with the indicated concentrations of sVCAM-1 and then added to the top chamber of the transwell. SDF-1α (15 ng/ml) was added to the bottom chamber, and cells were allowed to migrate for 4 h at 37°C. Cells in the bottom chamber were enumerated, and migration was expressed as a percent change relative to no VCAM-1 addition. VCAM-1 bound to the transwell membrane was measured by ELISA, as described in Materials and Methods, and expressed as OD units read at 490 nm. For reference, an OD of 0.46 U was observed when the membranes were directly coated with VCAM-1 at a concentration of 1 μg/ml. Results shown are representative of three separate experiments.

Trace quantities of VCAM-1 stimulated Jurkat migration on ICAM-1 (Fig. 2⇑C). The stimulated migration was completely blocked with an anti-β₂ Ab, TS1/18, indicating that the migration was β₂ dependent. Furthermore, the stimulated migration was blocked with an anti-VCAM-1 Ab, P8B1 (Fig. 2⇑C), as well as an anti-α₄ Ab, HP2/1 (data not shown). As an alternative means of assessing VCAM-1 trans activation of β₂-dependent cell migration, we used sVCAM-1 to stimulate migration across modified Boyden chambers coated with ICAM-1 alone. As shown in Fig. 2⇑D, the addition of sVCAM-1 to Jurkat cells stimulated β₂-dependent cell migration. ELISA confirmed that negligible amounts of the sVCAM-1 became absorbed to the membranes under these conditions (Fig. 2⇑D). These results indicate that the VCAM-1 acted as an agonist, stimulating β₂-dependent migration by binding to α₄β₁.

To investigate the role of α₄β₁ affinity modulation in α₄-stimulated β₂-dependent migration, the migration of activation-defective JD6 cells on a mixed substrate of ICAM-1 and VCAM-1 was examined. In contrast to wild-type Jurkat cells, the stimulated migration of activation-defective cells required a more than 20-fold higher concentration of VCAM-1 (Fig. 3⇓A). Since these mutant lines were derived by chemical mutagenesis, it is possible that cellular changes other than those affecting α₄β₁ affinity could account for the decreased sensitivity of β₂-dependent migration to VCAM-1 stimulation. To test this possibility, we used an activating β₁ Ab, 8A2, to reconstitute high-affinity α₄β₁ on the mutant lines. In the presence of 8A2, VCAM-1-stimulated β₂-dependent migration was identical in the mutant and wild-type Jurkat T cell lines (Fig. 3⇓B). This rescue in function was not observed when a nonactivating anti-β₁ Ab, K20, was used (data not shown). Thus, the capacity of differing quantities of VCAM-1 to stimulate β₂ integrin-dependent events is a function of the affinity state of α₄β₁.

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FIGURE 3.

Loss of α₄β₁ integrin activation causes decreased sensitivity to VCAM-1-stimulated β₂ integrin-dependent migration. A, Migration of wild-type and α₄β₁ activation-defective Jurkat T cell variants. Transwell membranes were coated with 200 μg/ml ICAM-1 and indicated concentrations of VCAM-1. Migration assays were performed as described in Fig. 2⇑ legend. B, Activating β₁ Ab rescues activation-defective Jurkat T cell migration. Migration assays were performed as in A, but cells were either untreated or treated with mAb 8A2 (0.1 μg/ml). Results are means ± SEM of three separate experiments.

We next questioned whether the mutant lines might have a general migration defect. In the absence of VCAM-1, the migration of wild-type and activation-defective Jurkat cells on ICAM-1 was similar (Fig. 4⇓B). The dose-response curve was biphasic, with maximal migration occurring at an ICAM-1-coating concentration of 10 μg/ml. Furthermore, the migration of wild-type Jurkat cells on VCAM-1 in the absence of ICAM-1 was biphasic, with maximal migration occurring at a VCAM-1-coating concentration of ∼10 μg/ml (Fig. 3⇑A). However, the migration of the mutant lines on VCAM-1 was augmented relative to wild-type cells, and maximal migration occurred at a VCAM-1-coating concentration of ∼40 μg/ml. Thus, the mutant cells do not have a general defect in integrin-dependent cell migration. Furthermore, the α₄β₁-dependent migration on different densities of VCAM-1 is a function of α₄β₁ affinity state. These effects of α₄β₁ activation on cell migration were similarly observed in the two other mutant Jurkat lines (Table I⇓).

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FIGURE 4.

Migration of wild-type and activation-defective Jurkat variants on ICAM-1 or VCAM-1. Transwell membranes were coated with the indicated concentrations of either ICAM-1 (left panel) or VCAM-1 (right panel). Wild-type (□) and α₄β₁ activation-defective (⋄) Jurkat T cell variants were allowed to migrate for 4 h at 37°C using SDF-1α (15 ng/ml) as a chemoattractant. Cells migrating to the bottom chamber were enumerated with a hemocytometer, and migration was expressed as a percentage of input cells. Results are mean ± SEM of three separate experiments.

Disruption of α₄β₁ activation has little effect on cell adhesion under static or flow conditions

We next examined the adhesive properties of the α₄ activation-defective Jurkat lines. Under static conditions, adhesion of these lines to a wide concentration range of coating concentrations of VCAM-1 or the CS-1 fragment of fibronectin was not markedly different from wild-type Jurkat cells (Fig. 5⇓). For both cell lines, the adhesion was α₄ dependent, as it was blocked with anti-α₄ Ab, HP2/1. Thus, α₄β₁ activation plays little role in regulating static Jurkat cell adhesion.

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FIGURE 5.

Static adhesion of wild-type and α₄β₁ activation-defective Jurkat variants. Plates were coated with the indicated concentrations of either VCAM-1 (left panel) or CS-1 fragment of fibronectin (right panel). Wild-type Jurkat (□) or α₄β₁ activation-defective Jurkat variants (⋄) alone or with the addition of anti-α₄ Ab (HP2/1) (20 μg/ml) were allowed to adhere for 40 min at 37°C. Adhesion was quantified by crystal violet staining and expressed as a percentage of input cells. Results are mean ± SEM of three separate experiments.

Integrin α₄β₁ supports dynamic and reversible tethering and rolling of cells in flowing blood (17). At a shear flow of 1 dyne/cm², however, there was no significant difference in the number of activation-defective Jurkat cells rolling and subsequently arresting on VCAM-1 as compared with wild-type Jurkat cells (Fig. 6⇓A). Furthermore, when increasing shear force was applied, there was no difference in the detachment of mutant and wild-type cells from VCAM-1 (Fig. 6⇓B). Thus, the activation-defective cells exhibit similar adhesion strengthening as wild-type cells. Chen and coworkers (15) previously implicated the importance of high-affinity α₄β₁ in adhesion strengthening of cells under flow conditions. This was partially based on the ability of the LDV-containing peptide to inhibit adhesion strengthening. Consequently, we questioned whether soluble LDV peptide would inhibit adhesion strengthening of the activation-defective Jurkat variants. As shown in Fig. 6⇓C, adhesion of the activation-defective and wild-type Jurkat cells on VCAM-1 was equally inhibited by LDV peptide. This suggests that while the activation-defective Jurkat cells have a reduced affinity for sVCAM-1, their binding affinity for soluble LDV peptide is unaffected. Collectively, these results suggest that loss of α₄β₁ activation in these Jurkat variants has little effect on cell adhesion to VCAM-1 under static or flow conditions.

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FIGURE 6.

Adhesion of wild-type and α₄β₁ activation-defective Jurkat variants under flow conditions. A, Cells were analyzed at a shear stress of 1 dyne/cm² on a VCAM-1 (soluble monovalent seven-domain VCAM-1) coated at 1 or 0.2 μg/ml. Cells were monitored for 10 s after the initial tethering event and were grouped into cells either transiently tethered, tethered and rolling, or arresting immediately after tethering to the adhesive substrates (see Materials and Methods). The fraction of cells (of the cell flux coming in close contact with the substrate) is presented in frequency units in a stacked bar graph. B, Resistance to detachment by shear flow. Cells were settled for 1 min on VCAM-1 at a coating of 0.1 μg/ml under static conditions. Shear stress was increased stepwise every 5 s. The number of cells that remained bound was expressed relative to the original number settled on the VCAM-1-coated substrate. C, Effect of LDV octapeptide and DVL octapeptide control on α₄β₁-mediated tethering of Jurkat and activation-defective Jurkat variant to low-density VCAM-1. Tethering frequencies were determined at a shear stress of 1 dyne/cm² in binding medium containing 0.5 mM of either peptide. Tethering categories (transient or arrest) are depicted, respectively, in the filled and open bars. Data depicted are representative of three independent flow experiments.