No abstract

Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lysoPS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91 phox؊/؊ ). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91 phox؊/؊ mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.Neutrophils are often robustly recruited early in inflammation. Within hours of their activation in tissues, they are removed by phagocytes, an event required for resolution of inflammation and the return to normalcy of tissue function. It is known that neutrophils undergoing apoptosis drive the production of anti-inflammatory mediators such as transforming growth factor-␤ that actively suppress production of inflammatory cytokines, chemokines, eicosanoids, and nitric oxide (1, 2). Indeed, enhanced induction of neutrophil apoptosis in vivo is potently anti-inflammatory (3, 4). However, if recognition and clearance fail, activated and dying neutrophils ultimately disintegrate releasing injurious intracellular constituents (e.g. serine proteases) (5). Failure of timely cell clearance is associated with both autoimmunity and enhanced inflammation (6, 7).Phosphatidylserine (PS) 2 exposed in the plasma membrane outer leaflet of apoptotic cells has long been known as a key ligand important for their recognition and removal. Interaction with various PS receptors, including the recently identified TIM4 (8, 9), BAI1 (10), and stabilin 2 (11) or PS-recognizing bridge molecule-receptor combinations (e.g. MFG-E8 and ␣ v integrins or Gas6 and Mer (12)), have been demonstrated. In many, bu...

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 E 2 (PGE 2 ) via a calcium-dependent cytosolic phospholipase A 2 /cyclooxygenase-mediated mechanism. Subsequent signaling by PGE 2 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 PGE 2 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.

Respiratory syncytial virus (RSV) causes respiratory tract infections in young children, and signifi cant morbidity and mortality in the elderly, immunosuppressed, and immunocompromised patients and in patients with chronic lung diseases. Recently, we reported that the pulmonary surfactant phospholipid palmitoyl-oleoyl-phosphatidylglycerol (POPG) inhibited RSV infection in vitro and in vivo by blocking viral attachment to epithelial cells. Simultaneous application of POPG along with an RSV challenge to mice markedly attenuated infection and associated infl ammatory responses. Based on these fi ndings, we expanded our studies to determine whether POPG is effective for prophylaxis and postinfection treatment for RSV infection. In vitro application of POPG at concentrations of 0.2-1.0 mg/ml at 24 h after RSV infection of HEp-2 cells suppressed interleukin-8 production up to 80% and reduced viral plaque formation by 2-6 log units. In vivo, the turnover of POPG in mice is relatively rapid, making postinfection application impractical. Intranasal administration of POPG (0.8-3.0 mg), 45 min before RSV inoculation in mice reduced viral infection by 1 log unit, suppressed infl ammatory cell appearance in the lung, and suppressed virus-elicited interferon-␥ production. These fi ndings demonstrate that POPG is effective for short-term protection of mice against subsequent RSV infection and that it has potential for application in humans. Respiratory syncytial virus (RSV) infects nearly 90% of children under age 2. Immunity to the virus is incomplete, and reinfection of adults, especially the elderly, patients