Effect of annexin 1 on neutrophil-endothelial interactions in an in vitro flow chamber assay

Richard Hayhoe, Dianne Cooper and Mauro Perretti. William Harvey Research Institute, Bart’s and the London, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ.

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). The objectives of this study were to determine the effect of the AnxA1 on the dynamic interactions of neutrophils and the endothelium under flow conditions in vitro and to relate them to FPRL-1 activation.

For flow chamber assays, human umbilical vein endothelial cells (HUVEC) were stimulated with tumour necrosis factor (TNF)-α (10 ng/ml) for 4 h. Freshly isolated human neutrophils (gradient purification) were incubated for 10 min at 37ºC with or without AnxA1 prior to flow. In some experiments neutrophils were also incubated with a neutralising antibody against FPRL-1 (gift of Dr. D. Henderson, Astrazeneca). Neutrophils (1x106/ml) were then perfused over the HUVEC monolayers at a rate of 1 dyn/cm2 for 8 min, after which time 6 random fields were recorded for off-line analysis. Analysis of adhesion molecule expression was performed by flow cytometry; in brief, blood aliquots or purified neutrophils, isolated as for flow assays, were incubated with or without AnxA1 at 37°C for 30 min. Cells were stained with specific monoclonal antibodies against L-selectin, CD11b and PSGL-1 and fluorescence quantified. Experiments were repeated 3-5 times, and differences analysed by ANOVA and, if significant, by Dunnetts test.

Perfusion of untreated neutrophils over TNF-α -stimulated HUVEC resulted in a significant number of adherent and rolling neutrophils (30±4 and 240±37 cells per field, respectively, p≤0.05 vs. unstimulated HUVEC, n=5). Incubation of neutrophils with AnxA1 resulted in a marked reduction in firm adhesion when compared with untreated neutrophils (10±5 vs. 30±4 cells, p≤0.05, n=5), although total neutrophil capture and rolling remained unaffected. The effect of AnxA1 on firm adhesion was FPRL-1 dependent since it was abrogated by a neutralising anti-FPRL-1 antibody (12±4 cells for control antibody vs. 22±5 cells for anti-FPRL-1 and 24±9 cells in untreated controls). 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 role of AnxA1 is independent from alterations in adhesion molecule expression on the neutrophil, at least with respect to L-selectin, CD11b and PSGL-1. This study shows, for the first time, AnxA1 efficacy in the flow chamber assay, and the functional involvement of FPRL-1 in the anti-adhesive actions of this anti-inflammatory mediator.

This work was supported by the Research Advisory Board of Bart’s and the London Special Trustees (grant 03/PHD/07) and by the Arthritis Research Campaign (grant 15755).