FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexin‐derived peptide‐stimulated phagocytosis

Lipoxins (LXs) are endogenously produced eicosanoids with well‐described anti‐inflammatory and proresolution activities, stimulating nonphlogistic phagocytosis of apoptotic cells by macrophages. LXA4 and the glucocorticoid‐derived annexin A1 peptide (Ac2–26) bind to a common G‐proteincoupled receptor, termed FPR2/ALX. However, direct evidence of the involvement of FPR2/ALX in the anti‐inflammatory and proresolution activity of LXA4 is still to be investigated. Here we describe FPR2/ALX trafficking in response to LXA4 and Ac2–26 stimulation. We have transfected cells with HA‐tagged FPR2/ALX and studied receptor trafficking in unstimulated, LXA4 (1–10 nM)‐ and Ac2–26 (30 µM)‐treated cells using multiple approaches that include immunofluorescent confocal microscopy, immunogold labeling of cryosections, and ELISA and investigated receptor trafficking in agonist‐stimulated phagocytosis. We conclude that PKC‐dependent internalization of FPR2/ALX is required for phagocytosis. Using bone marrow‐derived macrophages (BMDMs) from mice in which the FPR2/ALX ortholog Fpr2 had been deleted, we observed the nonredundant function for this receptor in LXA4 and Ac2–26 stimulated phagocytosis of apoptotic neutrophils. LXA4 stimulated phagocytosis 1.7‐fold above basal (P< 0.001) by BMDMs from wild‐type mice, whereas no effect was found on BMDMs from Fpr2‐/‐ mice. Similarly, Ac2–26 stimulates phagocytosis by BMDMs from wild‐type mice 1.5‐fold above basal (P<0.05). However, Ac2–26 failed to stimulate phagocytosis by BMDMs isolated from Fpr2‐/‐ mice relative to vehicle. These data reveal novel and complex mechanisms of the FPR2/ALX receptor trafficking and functionality in the resolution of inflammation.—Maderna, P., Cottell, D. C., Toivonen, T., Dufton, N., Dalli, J., Perretti, M., Godson, C. FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexinderived peptide‐stimulated phagocytosis. FASEB J. 24, 4240–4249 (2010). www.fasebj.org

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