Transition from Rolling to Firm Adhesion Can Be Mimicked by Extension of Integrin α_Lβ₂ in an Intermediate Affinity State^*

Cell Lines, Antibodies, and Small Molecule Inhibitors

Overlap extension PCR was used to generate cysteine substitution mutations in intact α_L, as described previously (3). K562 cells were stably transfected with K160C/F299C-α_L, K160C/T300C-α_L, L161C/F299C-α_L, or L161C/T300C-α_L in pcDNA3.1(hygro) together with wild-type β₂ and then selected in 200 μg/ml hygromycin. The 1% highest expressing cells were sorted by immunofluorescent flow cytometry into 96-well plates at ~1 cell/well. Wells with an outgrowth of a single colony were retested by immunofluorescence, and clones with similar expression to wild-type and high affinity transfectants were expanded and frozen in liquid N₂. Clones were maintained in medium containing 200 μg/ml hygromycin. For each mutant, all results shown are for the same clone, and only one clone was tested. Similar α_Lβ₂ surface expression as wild-type was selected and routinely verified by immunofluorescence flow cytometry. K562 cells stably transfected with wild-type α_Lβ₂ or K287C/K294C-α_L/β₂ (high affinity) in pEF(puro) were as described previously (3). K562 transfectants were maintained in RPMI 1640 medium containing 10% fetal bovine serum, penicillin/streptomycin, and 3 μg/ml puromycin (wild-type α_Lβ₂ and high affinity α_Lβ₂) or 200 μg/ml hygromycin for the rest of the mutants. A non-binding mouse IgG1 (X63) as control, an anti-human α_L I domain mAb (TS1/22), an α_L β -propeller domain mAb (TS2/4), and an anti-β₂ I-like domain mAb (TS1/18) were used to determine surface expression of the transfectants by immunofluorescent flow cytometry or to specifically block α_Lβ₂-ICAM-1 interactions (11, 12). CBR LFA-1/2 was employed to induce activation of α_Lβ₂ (11). Activation-dependent mAbs KIM127 (13) and m24 (4, 14) were kindly provided by Dr. M. Robinson (Celltech, Slough, UK) and Dr. N. Hogg (Imperial Cancer Research Fund, London, UK), respectively. XVA143 (15) was obtained from Dr. Paul Gillespie (Hoffman-La Roche, Nutley, NJ). BIRT377 (16) was from Dr. Terence Kelly (Boehringer Ingelheim Pharmaceuticals, Inc., Ridgeway, CT).

Soluble ICAM-1 Binding Assay

K562 transfectants were washed with HBS (20 mM HEPES, 150 mM NaCl, pH 7.3) containing 5 mM EDTA and resuspended in HBS buffer containing the indicated cation concentrations. To induce activation, wild-type α_Lβ₂ cells were pre-incubated with 10 μg/ml CBR LFA-1/2 for 15 min. In some experiments, cells were pre-incubated with 10 μg/ml αI blocking mAb TS1/22 or isotype mouse IgG control X63 before the addition of ICAM-1.

Binding of dimeric soluble ICAM-1 was assayed as follows. A chimera containing the five Ig domains of human ICAM-1 fused to the Fc portion of IgA (ICAM-1-Fcα) (17) was added to the cells at 50 μg/ml and incubated at 37 °C for 30 min. To test the effect of XVA143 and BIRT377 on soluble ICAM-1-Fcα binding to the K562 transfectants, cells were pre-incubated with 0.001% Me₂SO, 1 μM XVA143, or 10 μM BIRT377 for 10 min at 22 °C. The cells were washed and incubated with a 1:100 dilution of goat anti-human IgA-FITC (Zymed Laboratories Inc.) for 30 min at room temperature, washed, and analyzed by flow cytometry.

In another series of experiments, multimeric ICAM-1 complexes were prepared by mixing ICAM-1-Fcα with affinity-purified goat anti-human IgA antibody labeled with FITC (1:10 w/w) and incubated at 22 °C for 30 min. The mixture (4 μl) was then added to 50 μl of transfected cells to yield final concentrations of 50 μg/ml anti-human IgA-FITC and 5 μg/ml ICAM-1-Fcα and incubated for 30 min at 22 °C. The cells were washed and subjected to immunofluorescence flow cytometry.

Cell Adhesion to Immobilized ICAM-1 under Shear Flow

Cell adhesion in a parallel wall flow chamber was quantified as described previously (9). A chimera containing the five immunoglobulin domains of human ICAM-1 fused to the Fc portion of IgG (ICAM-1-Fcγ) was a kind gift from Dr. Lloyd Klickstein (Brigham and Women’s Hospital, Boston, MA). When indicated, Me₂SO (0.001%) as control or XVA143 (1 μM) was added to the cells immediately before infusion into the flow chamber.

V-bottom Cell Adhesion Assay

Cell adhesion to a V-bottom-well plate was assayed as described previously (18). Briefly, V-bottom 96-well plates (Corning) were coated with affinity-purified human tonsil ICAM-1 or with bovine serum albumin at 4 °C overnight and then blocked with 2% bovine serum albumin for 1 h at 37 °C. Cells were labeled for 15 min at 37 °C with 2′,7′-bis-(carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (Molecular Probes), washed, resuspended in L-15 medium/2.5% fetal calf serum (3 × 10⁴ cells in 50 μl), and when indicated, incubated for 15 min with 1 mM MnCl₂. The cells were added to the plates and immediately centrifuged at 200 × g for 15 min at room temperature.

Flat Bottom Cell Adhesion Assay

Binding of the transfectants to immobilized ICAM-1 was as described previously (12). Briefly, cells and plates (96-well flat bottom plates; Corning) were prepared as described above. In some experiments, wild-type α_Lβ₂ transfectants were pre-incubated with 10 μg/ml CBR LFA-1/2, Me₂SO (0.001%), BIRT377 (10 μM), or XVA143 (1 μM) for 15 min. Cells were added to the plates in 100 μl of L15/2.5% fetal calf serum with the indicated agents. After incubation at 37 °C for 30 min, unbound cells were washed off using a plate washer and fluorescence read.

Statistical Analysis

Data were analyzed when indicated using Student’s unpaired t test. Values are reported as mean ± S.E. Statistical significance was defined as p < 0.05.

Characterization of Intermediate Affinity α_Lβ₂ Mutants and Adhesion to ICAM-1

Measurements of the solution affinity for ICAM-1 of different double cysteine mutants of the α_L I domain (1, 2) show that they can be classified as low affinity (wild-type and K160C/T300C), intermediate affinity (L161C/T300C, K160C/F299, L161C/F299C), and high affinity (K287C/K294C) (Table 1). To correlate the solution affinity with adhesive behavior, the mutations were introduced into the full-length α_L subunit. K562 cells were stably transfected with the double cysteine α_L I domain substitution mutants together with wild-type β₂. In preliminary experiments, two to three clonal cell lines expressing each mutant were tested in shear flow assays. No significant differences in rolling versus firm adhesion behavior was seen among clones of the same mutant. In all subsequent experiments, one clone for each mutant with the most similar expression to the wild-type α_Lβ₂, as revealed by staining with an anti-β₂ β-propeller mAb TS2/4 (Fig. 1) and an anti-β₂ I-like domain mAb TS1/18 (data not shown), was selected. The previously described high affinity (K287C/K294C)- α_L/β₂ mutant (3) was expressed on transfectants 50–70% as well as the others.

Transfectants were tested for their ability to bind to immobilized human ICAM-1 in a flat bottom or a V-well assay. As previously described, adhesion of K562 transfectants expressing wild-type α_Lβ₂ is hardly detectable in flat bottom adhesion assays when cells are incubated at room temperature (22 °C) (data not shown) (19), and <10% of the cells adhere at 37 °C (Fig. 2A) (4). Activation of wild-type α_Lβ₂ with Mn²⁺ (4, 19) or an activating antibody, CBR LFA-1/2, greatly increases adhesion to levels seen with high affinity (K287C/K294C)- α_L/β₂ (Fig. 2A) (4, 19).

As expected, adhesion to immobilized ICAM-1 of K562 cells expressing the low affinity K160C/T300C-α_L/β₂ mutant was as low as that of wild-type α_Lβ₂ (Fig. 2A). Moreover, none of the cells transfected with intermediate affinity α_Lβ₂ mutants showed significantly higher binding to immobilized ICAM-1 in this assay (Fig. 2A). This contrasted with the higher affinity for ICAM-1 of the same mutations in isolated I domains as measured with surface plasmon resonance (Table 1).

We next explored the ability of the K562 transfectants expressing α_Lβ₂ mutants to bind to immobilized ICAM-1 under less stringent conditions than those of the flat bottom assay. In the V-well assay, cells are spun down at 200 × g and are never subjected to washing. Non-adherent cells roll or slide to the bottom of the V under centrifugal force, whereas adherent cells remain evenly distributed on the V-well bottom. The V-well assay detects lower affinity or avidity states that cannot be detected by conventional flat bottom assay or soluble ligand binding (6, 18). In the V-well assay, ~40–50% of the high affinity α_Lβ₂ transfectants or Mn²⁺-treated wild-type α_Lβ₂ transfectants bound to the ICAM-1-coated wells, whereas the wild-type and low affinity K160C/T300C-α_L/β₂ transfectants were significantly less adherent (Fig. 2B). Despite the less stringent conditions of this assay, among the intermediate affinity mutants, significant adhesion (p < .05) was seen only with L161C/F299C (Fig. 2B), which has the highest affinity for ICAM-1 of the three intermediate affinity mutants (Table 1).

Binding of Soluble Dimeric and Multimeric ICAM-1-Fcα to Intermediate Affinity α_Lβ₂ Mutants

For comparison to the cell adhesion assay results, we examined binding of soluble ligands using dimeric or multimeric soluble ICAM-1-Fcα and fluorescent flow cytometry. In contrast to the cell adhesion assays, soluble ligand binding by α_Lβ₂ transfectants (Fig. 3) correlated well with previous affinity measurements on purified I domains (Table 1). The panels in Fig. 3, A and B, are arranged in the same order as the affinities in Table 1. The rank order of these affinities in Table 1 is entirely concordant with the rank order of binding of dimeric and multimeric ICAM-1-Fcα in Fig. 3. Activation of wild-type α_Lβ₂ with CBR LFA-1/2 mAb induced binding of ICAM-1-Fcα similar to the K287C/K294C mutant. Blocking by TS1/22 abrogated binding of dimeric and multimeric ICAM-1-Fcα to all of the transfectants studied, supporting the specificity of the binding.

Binding of Intermediate Affinity Mutants to Immobilized ICAM-1 in the Presence of Shear Forces

Both the isolated wild-type α_L I domain and α_Lβ₂ on the cell surface can mediate weak adhesion and rolling when low shear forces are applied (20–22), despite the lack of binding in standard adhesion assays (Figs. 2 and 3) (2, 3). Previously, we have shown (22) that wild-type α_Lβ₂ mediates mostly weak, rolling interactions under shear forces and that activated α_Lβ₂ or the high affinity mutant supports primarily firm adhesion that is resistant to high shear forces, as confirmed here (Fig. 4).

Examination of the low and intermediate affinity mutants showed that adhesion in shear flow was correlated with affinity (Fig. 4). The low affinity mutant, which exhibits a 3-fold affinity increase over wild-type, exhibited more rolling cells than wild-type (Fig. 4). Furthermore, the three intermediate affinity mutants all showed a mixture of rolling and firmly adherent cells under basal conditions, in contrast to rolling by the low affinity mutant and firm adhesion by the high affinity mutant (Fig. 4).

Differential Effect of the α/β I Allosteric Antagonist XVA143 on Low, Intermediate, and High Affinity Mutants

The small molecule α_Lβ₂ antagonist XVA143 binds in a metal-dependent fashion to the β₂ I-like domain and induces extension of the integrin legs, as shown by activation epitope exposure. XVA143 blocks activation of the α_L I domain by the β₂ I domain and therefore inhibits ligand binding by wild-type α_Lβ₂ but not high affinity mutant α_Lβ₂ (8), or as shown below, intermediate affinity I domain mutants. Binding of XVA143 and extension of α_Lβ₂ is reflected by the exposure of activation-dependent epitopes in the β₂ I-like and integrin epidermal growth factor-like 2 domains detected by the antibodies m24 and KIM127, respectively (8, 9). The mutations used here to introduce disulfide bonds into the α_L I domain did not interfere with exposure by XVA143 of the m24 activation epitope in the β₂ I domain or the KIM127 activation epitope in the integrin epidermal growth factor-like 2 domain in the β₂ leg (Fig. 1). Thus, binding of this small molecule and extension of α_Lβ₂ are not impaired by the α_L I domain cysteine substitutions.

XVA143 had an effect on the intermediate affinity mutants, unlike that seen on other classes of mutants (Fig. 5). For all three intermediate affinity mutants, XVA143 promoted a marked increase in the total number of firmly adherent cells that were bound and remained attached under shear forces (Fig. 5). As previously reported (9), XVA143 enhanced rolling by wild-type α_Lβ₂ and had no effect on the high affinity mutant. These results suggest that, when α_Lβ₂ has intermediate affinity for ligand, extension is a major factor influencing the strength of the adhesion. This may be similar to the case with wild-type α_Lβ₂, where extension greatly promotes rolling (Fig. 5) (9). However, in contrast to intermediate affinity mutants, extension of the wild-type or low affinity mutant was not sufficient to promote firm adhesion (Fig. 5).

Following up on these findings, we tested the hypothesis that XVA143 could induce adhesion by the intermediate affinity mutants in the flat bottom assay described above. Indeed, incubation with XVA143 induced a significant increase in adhesion mediated by the intermediate affinity α_Lβ₂ mutants but had no effect on the low affinity mutant K160C/T300C-α_L/β₂ (Fig. 6). In keeping with our previous findings (9), XVA143 abrogated binding mediated by resting and activated wild-type α_Lβ₂, but as expected for an allosteric inhibitor, had no effect on adhesion mediated by the high affinity K287C/K294C-α_L/β₂-transfected cells (Fig. 6).

As a contrast, we tested the effect of the small molecule BIRT377, which belongs to the α I allosteric class of inhibitors that stabilize the closed conformation of the α_L I domain and do not induce extension of α_Lβ₂ (8, 16). BIRT377 did not increase binding by the intermediate affinity α_Lβ₂ transfectants and, if anything, slightly decreased their binding (Fig. 6). As expected, BIRT377 completely blocks adhesion mediated by the resting or activated wild-type α_Lβ₂ and had no effect on the high affinity K287C/K294C-α_L/β₂ mutant (Fig. 6).