Figure 4.

S1 promotes leukocyte adhesion and NET formation on HMEC-1 under flow. (A) Quantification of leukocyte adhesion under flow conditions on HMEC-1 exposed for 24h to medium alone (CTR) or to subtoxic concentration of S1 (10 nM) in the presence of anti-ACE2 functional blocking Ab (ACE2, 2 μg/ml) or the corresponding Irr Ab (2 μg/ml). (B) Adhesion of leukocytes, incubated for one hour with control medium or with S1 (S1*, 10 nM) and perfused under flow conditions (1.5 dynes/cm²) on HMEC-1 exposed to medium alone (CTR) or with S1 (10 nM). (C) Representative images of leukocytes treated with medium alone (CTR) or S1 (S1*, 10 nM), which adhered to HMEC exposed for 24h to medium alone or to S1. In this setting, neutrophils were co-stained with histone H3 citrullinated (citHH3, green) and neutrophil elastase (NE, red). The release of neutrophil extracellular traps (NETs) was observed only when leukocytes were activated with S1 (10 nM S1+ 10 nM S1*, inset). All experiments were repeated at least 3 times. Data represent mean ± SEM and were analysed with Tukey’s multiple comparison test. ***p-value<0.001 vs CTR; °p-value<0.05, and °°°p-value<0.001 vs 10 nM S1; ^###p-value<0.001 vs 10 nM S1+Irr. Slides were counterstained with DAPI (blue). Scale bar 20 μm.