Monocyte recruitment during inflammation: a novel role for the annexin A1/Fpr2 pathway

Simon McArthur1, Chris Reutlingsperger2, Rod Flower1, Mauro Perretti1. 1Queen Mary, University of London, London, UK, 2Maastricht University, Maastricht, The Netherlands

The efficient and timely resolution of inflammation is critical for the restoration of normal tissue function and for the prevention of chronic disease. Resolution is not a passive decline in inflammation, but is an active process initiated by inflammatory mediators themselves, of which the protein annexin A1 (AnxA1) has received much interest1, 2. Both this protein, and its principal receptor, the GPCR formyl peptide receptor 2 (Fpr2) are expressed at high levels in monocytes3, but the role it plays in their behaviour has been relatively unexplored. Using a combined in vivo and in vitro approach and exploiting a mouse model lacking Fpr2/34, we studied how AnxA1 affects monocyte recruitment during inflammation.

Adult male C57Bl/6 and Fpr2/3-/- mice (n=4) were injected i.p. with 0.5mg zymosan, animals were killed by a schedule 1 method and peritoneal lavages were taken 0h, 4h, 24h, 48h and 72h post-injection. Immigrant cell populations were characterised by flow cytometry (monocytes: CD115+, F4/80Low, CD11b+; macrophages CD115+ F4/80High, CD11b+; neutrophils CD115-, F4/80-, CD11b+). Ex vivo analysis of human monocyte chemotaxis to AnxA1 (10pM, 30pM, 100pM, 300pM or 1nM) was performed in 96-well format Boyden chambers (n=3 independent donors), sometimes following 10 minute pre-treatment of cells with either the Fpr2 antagonist WRW4 (10µM), the p38α MAP kinase inhibitor SB203580 (20nM, 200nM or 2µM) or the Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (6nM, 60nM or 600nM). Data was analysed by one- or two-way ANOVA as appropriate, followed by Tukey’s HSD post hoc test, with p<0.05 being considered significant.

Mice lacking Fpr2/3 showed a clear deficiency in monocyte recruitment to the peritoneal cavity, which was most apparent at 48h post-zymosan administration (wild-type 3.3±0.3x106, Fpr2/3-/- 1.5±0.3x106, p<0.05) and was due primarily to a lack of CCR2+, inflammatory monocytes. Ex vivo analysis of primary human monocytes revealed a dose-dependent chemoattractant effect of AnxA1, an activity mediated via Fpr2 (chemotaxis attenuated ˜45% by 10µM WRW4, p<0.01) and dependent upon activation of p38α MAP kinase (chemotaxis attenuated ˜85% by 200nM SB203580, p<0.01). Intriguingly, this chemoattractant action of AnxA1 was significantly reduced by administration of the iPLA2 inhibitor bromoenol lactone (chemotaxis attenuated ˜50% by 60nM bromoenol lactone, p<0.001), suggestive of a possible role for phospholipid metabolism in AnxA1-driven chemotaxis, given the well-known inhibitory activity of AnxA1 upon cytosolic phospholipase A25.

Together, these data allow us to propose a significant new role for AnxA1 in the recruitment of monocytes to sites of inflammation, aiding in the removal of apoptotic neutrophils and promoting inflammatory resolution.

This work was funded by the Wellcome Trust (programme grant 086867/Z/08/Z)

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