Heterotropic modulation of selectin affinity by allosteric antibodies affects leukocyte rolling

Abstract

Selectins are a family of adhesion receptors designed for efficient leukocyte tethering to the endothelium under shear. As a key property to resist premature bond disruption, selectin adhesiveness is enhanced by tensile forces that promote the conversion of a bent into an extended conformation of the N-terminal lectin and epidermal growth factor-like domains. Conformation-specific Abs have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins. In this study, we show that the anti-human L-selectin mAbs DREG-55 and LAM1-5 but not DREG-56, DREG-200, or LAM1-1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber, and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin Abs and introduce the new concept of heterotropic modulation of receptor function.

Figure 1

Anti-human L-selectin antibodies of the DREG and LAM family differentially regulate L-selectin dependent rolling on PSGL-1. A) The rolling velocity of human PBL (dashed line) and L-selectin (CD62L)-coated beads (solid line) was investigated in a parallel plate flow chamber on immobilized PSGL-1-F_c. PBL rolled on 30 μg/ml PSGL-1 at 2 dyn/cm² and beads on 20 μg/ml PSGL-1 at 3 dyn/cm². Anti-L-selectin mAb DREG-55 or isotype anti-human IgG₁ (10 μg/ml) were added prior to the experiment. Mean velocities were 112.8 vs. 13.3 μm/s for PBL and 36.4 vs. 4.8 μm/s for beads (untreated vs. DREG-55 mAb). Mean ± S.D is shown of n = 3-4. B) Jurkat T cells rolling on PSGL-1-F_c at 2 dyn/cm². ‘No interaction’ indicates full inhibitory activity with cell displacement equal to the hydrodynamic flow. Mean ± S.D. of 4 experiments. C) Cumulative frequency histogram of the rolling velocity showing concentration-dependent effects of DREG-55 mAb. Mean velocities are 13.1, 19.5, 39.5 and 111.5 μm/s at 10, 1, 0.1 and 0 μg/ml DREG-55 mAb concentration, respectively. 32 – 79 analyzed cells per condition, representative of 2 independent experiments. D) Jurkat cells rolling on 30 μg/ml PSGL-1 were subjected to increasing shear forces in a flow chamber. Cells were either left untreated (open circle) or incubated with 10 μg/ml DREG-55 mAb (solid circle) prior to the experiment. The number of interacting cells was determined for each shear level at 1, 1.5, 3, 6 and 10 dyn/cm² and expressed relatively to 1 dyn/cm². Mean ± S.D. of 3 experiments. FOV = field of view. E) Rolling velocity of Jurkat cells on 20 μg/ml immobilized PSGL-1 at 2 dyn/cm² with or without addition of LAM1.1 or LAM1.5. Mean ± S.D. of 3-4 experiments, more than 100 cells analyzed per condition.

Figure 2

Microkinetic analysis of DREG-55 mAb induced slow rolling. A) The impact of the PSGL-1 ligand density on Jurkat cell rolling was determined with and without a saturating amount of DREG-55 mAb. Mean rolling velocities are 117.7 ± 5.8 (30 μg/ml coated ligand), 161.5 ± 17.2 (10 μg/ml) and 225.6 ± 16.8 μm/s (6 μg/ml) and decrease to 25.3 ± 3.7 (30 μg/ml), 26.7 ± 3.7 (10 μg/ml) and 29.9 ± 2.7 μm/s (6 μg/ml) upon 10 μg/ml DREG-55 treatment. Data show mean ± S.D. of n = 3 with at least 30 cells analyzed per experiment. B) and D) High-resolution velocity profile of Jurkat cells rolling on low density PSGL-1 recorded at 100 fps. A representative two second time course of an individual untreated (B) and DREG-55 antibody treated (D) Jurkat cell rolling at 0.96 dyn/cm² on 6 μg/ml immobilized PSGL-1 is shown. C) Comparative k_off values were calculated from B and D as described in methods.

Figure 3

DREG-55 mAb-induced slow rolling is ligand specific. Flow chamber assays were performed with Jurkat cells at 2 dyn/cm² using different immobilized L-selectin ligands. Numbers on top of the bars indicate the mean rolling velocity. ‘No interaction’ indicates full inhibitory activity of DREG-55 mAb. DREG-200 antibody blocked all interactions (not shown). n= 3-4, mean ± S.D.

Figure 4

DREG-55 mAb increases force-free affinity of L-selectin. Jurkat cells were incubated with human PSGL-1-F_c or E-selectin-F_c in the presence of DREG-55 or -200 mAb and the fluorescence of the secondary anti-human IgG-FITC antibody was measured by flow cytometry. In untreated samples, only soluable ligand and secondary antibody but no DREG antibody was added. MFI is expressed relative to DREG-200 mAb samples. Mean ± S.D. of 3 to 4 experiments.

Figure 5

PMN string formation via L-selectin-PSGL-1 interactions is enhanced by DREG-55 mAb. Flow chambers were coated with 20 μg/ml E-selectin and human PMN string formation was analyzed at 1 dyn/cm² as described in materials and methods. A) Typical readouts for untreated, DREG-55 and -200 mAb-treated PMNs under flow are shown. The arrow indicates the direction of the flow. B) Determination of the number of strings. 5 FOVs were averaged per experiment. Mean, n = 3 – 4. C) Histogram showing the average lengths of all strings in the untreated (n = 60) and DREG-55 treated (n = 89) condition.

Figure 6

Mapping of DREG antibody epitopes onto the N-terminal lectin domain. A) Sequence alignment of the N-terminal portion of human selectins. The four switch regions in the lectin domain that undergo conformational changes upon selectin extension (19) are underlined and labeled S1-S4. Residues involved in PSGL-1 binding are indicated in orange (PSGL-1 polypeptide) and magenta (PSGL-1 glycan). Residues involved in calcium binding are marked by cyan circles. Residues whose mutation abolishes mAb DREG-55 and -200 binding (30) are indicated in blue and green, respectively. B) Cartoon drawings of P-selectin crystal structures in the bent (left panel) and extended (right panel) conformation (17). The lectin domain is at the top and the EGF-like domain at the bottom. The pivot at the interdomain hinge is indicated by a filled black triangle and switch regions S2 and S3 are labeled. The calcium ion is shown as a cyan sphere. The PSGL-1 ligand is shown in orange (polypeptide) and magenta (glycan). The two sulfated tyrosines are shown in atomic detail. Arg-85, which reorients dramatically upon PSGL-1 binding, is shown in red. Residues whose mutation abolishes DREG-55 and -200 mAb binding are indicated by blue and green Cα spheres, respectively. The putative DREG-55 mAb epitope in the extended conformation is indicated by a blue oval.