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 PLA 2 (cPLA 2α) activation and PGE 2 production leading to cyclic AMP (cAMP)-dependent protein kinase A (PKA) activation. Similar to the findings of our earlier studies of activated neutrophils, lyso-PS-positive apoptotic neutrophils signaled via macrophage G2A for enhanced engulfment. Here, we show that the modified phosphatidylserine lyso-PS was generated during neutrophil apoptosis only under conditions where the NADPH oxidase was activated. In this investigation, we sought to define the signaling pathway downstream of G2A resulting in enhanced engulfment. We demonstrated further that cell-associated lyso-PS signaled to macrophages via the G-protein-coupled receptor G2A for enhanced PS-dependent removal of activated neutrophils. Surprisingly, substantial amounts of lyso-phosphatidylserine (lyso-PS) species, rather than oxidized PS species, were generated in an NADPH oxidase-dependent manner during neutrophil activation both in vitro and in vivo ( 15). Given this, we had previously hypothesized that activation of the NADPH oxidase would enhance PS oxidation and contribute significantly to the removal of neutrophils. CD36) ( 11– 14), adding to the array of possible receptors utilized by macrophages to recognize different PS species and structures. More recently, oxidized PS has also been shown to facilitate recognition of apoptotic cells through scavenger receptors ( e.g. The consequences of PS-dependent interactions are actively anti-inflammatory, resulting in the production of mediators, such as TGFβ and prostaglandin E 2 (PGE 2) ( 9, 10). The exofacially exposed phosphatidylserine (PS) 2 head group is the best described ligand and is recognized by an increasing number of bridge molecules and receptors on macrophages, including MFG-E8, Gas6, BAI1, Tim4, and Stabilin 2 ( 5– 8). Relatively little is known of the ligands presented by apoptosing neutrophils, or any apoptosing cell for that matter, that signal for their recognition and engulfment. serine proteases and cationic proteins), and contribute to ongoing inflammation, tissue destruction, and, in some cases, autoimmunity ( 2– 4). Where defects in efferocytosis have been identified, dying neutrophils ultimately disintegrate, release phlogistic cargo ( e.g. Removal of activated and dying neutrophils is most often orchestrated by professional phagocytes, such as macrophages, in a process that has been called efferocytosis (“to carry to the grave”) ( 1). Although robust neutrophil recruitment is critical for host defense, removal of these short lived cells is imperative for resolution of inflammation and restoration of tissue function. 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.Ī hallmark of acute inflammation is the recruitment of large numbers of neutrophils from the vasculature into tissues in response to microbial infection and/or injury. 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. These data were surprising in light of previous reports demonstrating that signaling by PGE 2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. 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. Subsequent signaling by PGE 2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. 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 E 2 (PGE 2) via a calcium-dependent cytosolic phospholipase A 2/cyclooxygenase-mediated mechanism. Here, we investigated the signaling pathway downstream of G2A. 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. 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.
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