Signaling via Macrophage G2A Enhances Efferocytosis of Dying Neutrophils by Augmentation of Rac Activity

S Courtney Frasch,Ruby F Fernandez-Boyanapalli,Karin Zemski Berry,Christina C Leslie,Joseph V Bonventre,Robert C Murphy,Peter M Henson,Donna L Bratton

doi:10.1074/jbc.m110.181800

Abstract

Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PS(neg) apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.

Highlights

These short lived cells is imperative for resolution of inflammation and restoration of tissue function
Using exogenous lyso-PS supplied in liposomes to activate G2A, key signaling events and intermediaries downstream of G2A were identified and included macrophage calcium-dependent cytosolic PLA2 activation and prostaglandin E2 (PGE2) production leading to cyclic AMP-dependent protein kinase A (PKA) activation
Enhanced Efferocytosis of “Aged” Apoptotic Neutrophils by Macrophages Is Mediated by G2A Signaling—We had previously shown that neutrophils, activated either in vitro or in vivo, were ingested in an enhanced manner by a PS-dependent efferocytic mechanism

Summary

EXPERIMENTAL PROCEDURES

Materials—All lipids were purchased from Avanti Polar Lipids (Alabaster, AL) unless otherwise noted. The macrophages with target cells were co-cultured for 60 min at 37 °C in 10% CO2, washed three times with PBS, and stained with a modified Wright’s Giemsa stain (Fisher Scientific). For in vivo phagocytosis assays, 5 ϫ 106 fluorescent (Flash Red) 5-␮m carboxylate-modified beads in 1 ml of PBS or 1 ml of PBS containing 30 mol % lyso-PS liposomes (10 mg/kg of total lipid/mouse; Ref. 29) were injected intraperitoneally into WT or G2AϪ/Ϫ mice. Cells were co-cultured with the indicated target cells at a 4:1 targets/plated macrophage ratio in the absence or presence of 100 nmol of total lipid/5 ϫ 105 cells for the indicated times, and cell lysates were collected according to the manufacturer’s instructions. When significant analysis of variance was indicated, post hoc analysis using the Tukey-Kramer or Dunnett’s method for multiple comparisons or comparison with an internal control, respectively, was performed (JMP statistical program (SAS Institute, Cary, NC))

RESULTS

Geometric mean

DISCUSSION