FPR2/ALX receptor expression and internalization are critical for lipoxin A₄and 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.

Human Mesenchymal Stem (Stromal) Cells Promote the Resolution of Acute Lung Injury in Part through Lipoxin A4

“…The effects were associated with increased bacterial killing in part through enhanced phagocytosis activity of alveolar macrophages. LXA 4 is a potent stimulus for macrophage phagocytosis (32). Additionally, LXA 4 inhibits neutrophil transepithelial migration (33) and airway epithelial proinflammatory mediator expression in an ALX/FPR2-dependent manner (33,34).…”

Nonredundant protective properties of FPR2/ALX in polymicrobial murine sepsis

“…Analyses of cell behavior indicated, besides defective recruitment, a direct impairment of phagocyte functions in Fpr2/3 −/− neutrophils. Although not in these settings, studies have demonstrated the importance of AnxA1, LXA 4 , and more recently resolvin D 1 in promoting particle phagocytosis and efferocytosis by immune cells (17,33,34) together with ineffectiveness in cells lacking Fpr2/3 (35).…”

A vasculo-protective circuit centered on lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 operative in murine microcirculation

“…In sterile inflammation, this receptor is the main transducer of the pro-resolving actions of LXA 4 and AnxA1. Pharmacological delivery of either mediator engages Fpr2/3 to activate circuits of resolution, including attenuation of leukocyte trafficking, 18 promotion of apoptosis, 26 and efferocytosis, 27,28 favoring egress of leukocyte from the tissue. 29 Attenuation of postischemic leukocyte recruitment is crucial to prevent vascular and parenchymal damage as shown in many experimental settings in animals and also in human intestine, where removal of neutrophils for perfusates yielded consistent diminution of tissue injury.…”