The anti‐adhesive effects of annexin 1 on neutrophil‐endothelial interactions under flow

Annexin 1 (AnxA1) is an anti‐inflammatory protein with established activity in a range of in vivo and in vitro experimental models. Recently, some of these inhibitory effects have been associated with activation of a specific G‐protein coupled receptor, termed FPR like‐1 (FPRL‐1). Freshly isolated human neutrophils (1x10 6 /ml) were incubated (10 min; 37°C) with or without AnxA1 prior to perfusion over TNF activated (10 ng/ml; 4h) human umbilical vein endothelial cells (HUVEC) at 1 dyn/cm 2 for 8 min, then 6 random fields were recorded for analysis. In some experiments neutrophils were also incubated with a specific FPRL‐1 antagonist WRWWWW or with a selective anti‐FPRL‐1 mAb. Analysis of adhesion molecule expression was performed by staining neutrophils, incubated with or without AnxA1, with specific monoclonal antibodies against L‐selectin, CD11b and PSGL‐1 and measuring fluorescence by flow cytometry. Incubation of neutrophils with AnxA1 resulted in a marked reduction in firm adhesion, but not rolling, onto HUVEC monolayers when compared with untreated neutrophils. This effect was FPRL‐1 dependent since abrogated by WRWWWW and anti‐FPRL‐1 mAb. AnxA1 did not modify neutrophil adhesion molecule expression. These results indicate that AnxA1 interferes with neutrophil recruitment onto the endothelium under flow conditions in vitro , causing a significant reduction in adherent cells. This effect of AnxA1 does not result from alteration in the adhesion molecules investigated, however there is evidence for the functional involvement of FPRL‐1. Supported by the Research Advisory Board of Bart’s and the London Special Trustees (grant 03/PHD/07).