title: FPR2/ALX receptor expression and internalization are critical for lipoxin A4 and annexin-derived peptide-stimulated phagocytosis.
abstract: Lipoxins (LXs) are endogenously produced eicosanoids with well-described anti-inflammatory and proresolution activities, stimulating nonphlogistic phagocytosis of apoptotic cells by macrophages. LXA(4) and the glucocorticoid-derived annexin A1 peptide (Ac2-26) bind to a common G-protein-coupled receptor, termed FPR2/ALX. However, direct evidence of the involvement of FPR2/ALX in the anti-inflammatory and proresolution activity of LXA(4) is still to be investigated. Here we describe FPR2/ALX trafficking in response to LXA(4) and Ac2-26 stimulation. We have transfected cells with HA-tagged FPR2/ALX and studied receptor trafficking in unstimulated, LXA(4) (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 LXA(4) and Ac2-26 stimulated phagocytosis of apoptotic neutrophils. LXA(4) 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.
authors.affiliation: UCD Diabetes Research Centre, UCD School of Medicine and Medical Science, University College Dublin, Dublin, Ireland.
authors.forename: Paola
authors.initials: P
authors.lastname: Maderna
authors.forename: David C
authors.initials: DC
authors.lastname: Cottell
authors.forename: Tiina
authors.initials: T
authors.lastname: Toivonen
authors.forename: Neil
authors.initials: N
authors.lastname: Dufton
authors.forename: Jesmond
authors.initials: J
authors.lastname: Dalli
authors.forename: Mauro
authors.initials: M
authors.lastname: Perretti
authors.forename: Catherine
authors.initials: C
authors.lastname: Godson
mesh_terms: D000818:Animals; D017302:Annexins / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D044045:Lipoxins / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D010455:Peptides / Q000494:pharmacology*; D010587:Phagocytosis / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D044042:Receptors, Formyl Peptide / Q000235:genetics / Q000378:metabolism*; D044067:Receptors, Lipoxin / Q000235:genetics / Q000378:metabolism*
chemical_list: D017302:Annexins; C105101:FPR2 protein, human; D044045:Lipoxins; D010455:Peptides; D044042:Receptors, Formyl Peptide; D044067:Receptors, Lipoxin; C040527:lipoxin A4