Comparison of human eosinophil and neutrophil adhesion to endothelial cells under nonstatic conditions: Role of L-selectin

To simulate adhesion that occurs under conditions of flow, we investigated the attachment of eosinophils to endothelium under rotational conditions. Tissue-culture plates containing monolayers of HUVEC were placed on a horizontal rotator (80 revolutions per minute (rpm)), and equal numbers of purified human eosinophils or neutrophils were added to separate wells at 4°C. Binding of eosinophils and neutrophils to unstimulated endothelial cells was 15 ± 3 and 31 ± 11 cells/four high power fields (HPF), respectively. After preincubation of HUVEC with IL-1β (1 ng/ml, 4 h, 37°C), adhesion increased to 56 ± 4 and 290 ± 26 cells/four HPF, respectively (p < 0.0002 for both, n = 8-14). Eosinophils with reduced levels of L-selectin (blood eosinophils activated in vitro or eosinophils obtained from bronchoalveolar lavage (BAL) performed after segmental lung allergen challenge of allergic subjects) demonstrated reduced binding under rotating conditions. Several L-selectin Abs inhibited adhesion of eosinophils and neutrophils (e.g., LAM1-3: 43 ± 14% vs 63 ± 3% inhibition; LAM1-6: 73 ± 5% vs 36 ± 6% inhibition, respectively, n ≥ 6). Interestingly, one additional L-selectin Ab, LAM1-11, inhibited eosinophil but not neutrophil adhesion (51 ± 2% vs 1 ± 7% inhibition, respectively, n ≥ 5). We conclude that eosinophils, like neutrophils, use L-selectin to bind to activated endothelial cells under conditions of flow, although mAb LAM1-11 can selectively inhibit eosinophil attachment to stimulated endothelial cells in vitro, suggesting different functional epitopes on L-selectin among eosinophils and neutrophils.