HECA-452 is a non-function blocking antibody for isolated sialyl Lewis x adhesion to endothelial expressed E-selectin under flow conditions

Abstract

E-selectin, expressed on inflamed endothelium, and sialyl Lewis x (sLe(x)), present on the surface of leukocytes, play a key role in leukocyte-endothelial interactions during leukocyte recruitment to sites of inflammation. HECA-452 is a monoclonal antibody (mAb) that recognizes sLe(x) and is routinely used by investigators from diverse fields who seek to unravel the mechanisms of leukocyte adhesion. The data regarding the ability of HECA-452 to inhibit carbohydrate-mediated leukocyte adhesion to E-selectin remains conflicted, in part due to the presence of a variety of potential E-selectin reactive moieties on leukocytes. Recognizing this, we utilized a complementary approach to gain insight into HECA-452 adhesion assays. Specifically, we used sLe(x) microspheres to investigate the hypothesis that HECA-452 is a non-function blocking mAb for isolated sLe(x) mediated adhesion to endothelial expressed E-selectin. Flow cytometric analysis revealed that HECA-452 recognizes and binds to the sLe(x) microspheres. Perfusion of the sLe(x) microspheres over human umbilical vein endothelial cells (HUVEC) at 1.5 dyn/cm² revealed that the microspheres attach to 4h interleukin (IL)-1β activated HUVEC specifically via E-selectin. Pretreatment of the sLe(x) microspheres with HECA-452 did not influence sLe(x) microsphere initial tethering and accumulation on IL-1β activated HUVEC. Neuraminidase and fucosidase treatments of sLe(x) microspheres revealed that sialic acid and fucose are required for E-selectin binding, whereas HECA-452 recognition of sLe(x) does not depend on the fucose moiety to the extent required for E-selectin recognition. This latter finding suggests there are potential subtle differences between the sLe(x) antigens for E-selectin and HECA-452. Combined, the data indicate that HECA-452 is a non-inhibitor of sLe(x)-mediated adhesion to endothelial expressed E-selectin.

Figure 1

Analysis of sLe^x microspheres by flow cytometry. The sLe^x microspheres were first treated with HECA-452 and then a FITC labeled goat anti-rat IgM antibody. The histograms represent the fluorescence distribution of the population of microspheres analyzed. The log fluorescence correlates with the levels of HECA-452 epitopes bound to the sLe^x microspheres. The coating concentrations used to prepare the sLe^x microspheres are shown on the right side. The data presented represent n≥3 experiments.

Figure 2

SLe^x microsphere initial tethering to 4 h. IL-1β activated HUVEC occurs primarily through E-selectin. Initial tethering of sLe^x microspheres to unactivated HUVEC, 4 h. IL-1β activated HUVEC and 4 h. IL-1β activated HUVEC pretreated with an anti-E-selectin mAb (HAE-1f) was determined. Shear stress = 1.5 dynes/cm²; *p ≤ 0.05 compared with the center bar.

Figure 3

HECA-452 does not block sLe^x microsphere initial tethering and accumulation on IL-1β activated HUVEC and does not significantly increase the rolling velocity. (A) SLe^x microspheres were treated with various concentrations of HECA-452, as indicated, and then analyzed via flow cytometry. The Mean Channel Fluorescence (MCF) for the population of sLe^x microspheres is plotted vs. the concentration of HECA-452. (B, C and D) SLe^x microspheres were untreated or treated with 100 μg/ml HECA-452, as indicated, prior to the adhesion assay and perfused over 4 h. IL-1β activated HUVEC at a shear stress of 1.5 dynes/cm². The initial tethering during the first 2.5 minutes of flow (B), the number of sLe^x microspheres present after the 2.5 minutes of flow (C), and the rolling velocity (D) was determined. A coating concentration of 0.125 μg/mL sLe^x was used for preparing the sLe^x microspheres; shear stress = 1.5 dynes/cm²; Results shown are the average of n=3.

Figure 4

HECA-452 does not inhibit sLe^x microsphere, generated with a relatively low concentration of sLe^x, tethering and accumulation on IL-1β activated HUVEC and does not significantly increase the rolling velocity. (A) sLe^x microspheres generated with different concentrations of sLe^x [(A, B, C) 0.0625 μg/mL and (D) 0.03125 μg/mL] were either untreated or treated with HECA-452, as indicated, and perfused over 4 h. IL-1β activated HUVEC at a shear stress of 1.5 dynes/cm². (A and D) The initial tethering during the first 2.5 minutes of flow. (B) The number of 0.0625 μg/mL sLe^x microspheres present after 2.5 minutes of flow. Note that accumulation data is not given for the sLe^x microspheres prepared with 0.03125 μg/mL sLe^x since there were an insignificant number of sLe^x microspheres present for both untreated and mAb treated conditions. (C) The rolling velocity of the 0.0625 μg/mL sLe^x microspheres. 0.03125 μg/mL sLe^x microspheres pretreated with HECA-452 did not exhibit an increase in the rolling velocity compared to untreated microspheres (data not shown). Data are the average of n ≥ 3 separate experiments.

Figure 5

Neuraminidase treatment of sLe^x microspheres eliminated both HECA-452 recognition of sLe^x microspheres and adhesion to E-selectin. (A) Levels of HECA-452 reactive epitopes on sLe^x microspheres, with and without neuraminidase treatment, were analyzed by flow cytometry. Top panels - microspheres prepared with no sLe^x and not treated with neuraminidase; Middle panels-microspheres prepared with 0.125 μg/ml multivalent sLe^x and not treated with neuraminidase; Bottom panels-microspheres prepared with sLe^x and treated with neuraminidase (0.1 U/ml). (B) SLe^x microspheres, with and without neuraminidase treatment 30 min prior to the adhesion assay, were perfused over 4 h. IL-1β activated HUVEC at a shear stress of 1.5 dynes/cm². N indicates neuraminidase treatment; *p≤0.05; n≥2.

Figure 6

Fucosidase treatment of sLe^x microspheres had a slight inhibitory effect on HECA-452 recognition of sLe^x microspheres but a relatively robust inhibitory effect on sLe^x microsphere adhesion to E-selectin. (A) Levels of HECA-452 reactive epitopes on sLe^x microspheres, with and without fucosidase treatment, were analyzed by flow cytometry. Top panels - microspheres prepared with no sLe^x and not treated with fucosidase; Middle panels - microspheres prepared with 0.125 μg/ml sLe^x and not treated with fucosidase; Bottom panels - microspheres prepared with sLe^x and treated with fucosidase (0.1 U/ml). (B) SLe^x microspheres, treated with and without fucosidase 24 hr prior to the adhesion assay, were perfused over 4 hr. IL-1β activated HUVEC at a shear stress of 1.5 dynes/cm². F indicates fucosidase treatment; *p≤0.05; n ≥4.

Figure 7

HECA-452 does not block neutrophil accumulation on 4 hr. IL-1β activated HUVEC. Freshly isolated human neutrophils were untreated, treated with rat IgM or treated with 100 μg/ml HECA-452, as indicated, prior to the adhesion assay and perfused over 4 h. IL-1β activated HUVEC for 2.5 minutes. The number of adherent neutrophils was determined after the perfusion period and normalized to the area of the field of view. Shear stress = 1.5 dynes/cm²; Results shown are the average of n=3.