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

No abstract

The pulmonary surfactant phospholipid, palmitoyloleoylphosphatidylglycerol (POPG), inhibits TLR2 and TLR4 signaling from the cell surface of macrophages. In this study we sought to develop structural analogs of POPG that vary in polar head group length, hydroxylation and alkyl branching, as novel compounds for suppressing the TLR inflammatory responses. These analogs were synthesized using a transphosphatidylation reaction. Analogs of POPG with C3 and C4 alkyl head group length (POP‐PROPANOL and POP‐BUTANOL) are less effective than POPG as TLR2 and TLR4 antagonists. However, adding a hydroxyl group at the alkyl chain 2, 3 or 4‐position (POP‐PROPANEDIOLS or POP‐BUTANEDIOLS) restored their inhibitory effects against TLR2 and TLR4 agonists. Interestingly, analogs with C1 and C2 alkyl head group length (POP‐METHANOL and POP‐ETHANOL) were effective as LPS antagonists. POP‐DIMETHYLPROPANEDIOL contains 2 methyl groups at the head group alkyl 2 position; and this analog is effective for inhibition of an LPS stimulus. However, it is a weak inhibitor of MALP‐2 induced AA release. Addition of amino group at the alkyl 2 position (POP‐SERINOL) completely abolished the potency of analogs. Collectively, these findings identify new compounds for antagonizing TLR2 and TLR4 activation and define structural properties for discriminating between the two receptor systems. NIH‐HL094629, COLORADO C2D2.

, we have identified the enzyme that catalyzes the -1 and -2 oxidation of LTB 4 in mouse myeloid cells as CYP4F18. As determined by mass spectrometry, this enzyme catalyzes the conversion of LTB 4 to 19-OH LTB 4 and to a lesser extent 18-OH LTB 4 . Inhibition of CYP4F18 resulted in a marked increase in calcium flux and a 220% increase in the chemotactic response of mouse PMN to LTB 4 . CYP4F18 expression was induced in bone marrow-derived dendritic cells by bacterial lipopolysaccharide, a ligand for TLR4, and by poly(I⅐C), a ligand for TLR3. However, when bone marrow-derived myeloid dendritic cells trafficked to popliteal lymph nodes from paw pads, the expression of CYP4F18 was down-regulated. The results identify CYP4F18 as a critical protein in the regulation of LTB 4 metabolism and functional responses in mouse PMN and identify it as the functional orthologue of human PMN CYP4F3A.How polymorphonuclear leukocytes (PMN), 2 macrophages, and dendritic cells (DC) control the initiation and amplification of innate and adaptive immune responses is a critical question, and the 5-lipoxygenase product of arachidonic acid metabolism leukotriene B 4 (LTB 4 ) is central to the amplification process. LTB 4 is equal to the most potent chemoattractant known for myeloid cells (1-6) and is synthesized from arachidonic acid by the action and interactions of 5-lipoxygenase, the five-lipoxygenase-activating protein, and leukotriene A 4 hydrolase (7). LTB 4 mediates its activity in these cells via the high affinity G proteincoupled receptor BLT1 (8 -10).LTB 4 has been implicated in the pathogenesis of multiple inflammatory diseases including inflammatory bowel disease (11-14), glomerulonephritis (15, 16), allograft rejection in kidney transplant models (17, 18), and cardiac allograft rejection (19). Studies with knock-out mice for the five-lipoxygenase-activating protein combined with LTB 4 receptor antagonists have supported a role for LTB 4 in murine collagen arthritis (20), in the EAE model of multiple sclerosis (21), and in mediating a primate model of asthma (22). In atherosclerosis, 5-lipoxygenase has been identified as a risk gene in a mouse model, and 5-lipoxygenase-rich cells have been identified in atheroscleotic plaques of mice and humans (23, 24). Furthermore, a protein closely related to CYP4F3A, presumably a mouse member of the CYP4F family, was strongly induced in foam cells in mice (23). Blockade of BLT1 has been associated with decreased progression of atherosclerosis in APOE1Ϫ/Ϫ mice (25,26). Understanding the molecular basis of LTB 4 signal termination is critical to elucidating how animals control the amplitude of inflammation in LTB 4 -dependent settings.There are two general cellular mechanisms that have the potential to terminate the responsiveness to LTB 4 and to all other chemoattractant molecules for G protein-coupled receptors. The first is the enzymatic metabolism of ligands. The second is receptor desensitization, which is based on G protein-coupled receptor kinases and ␤-arrestin (27, 28). The desensi...

The short palate, lung and nasal epithelial clone 1 (SPLUNC1) protein is a member of the palate, lung, and nasal epithelium clone (PLUNC) family, also known as bactericidal/permeability-increasing (BPI) fold-containing protein, family A, member 1 (BPIFA1). SPLUNC1 is an abundant protein in human airways, but its function remains poorly understood. The lipid ligands of SPLUNC1 as well as other PLUNC family members are largely unknown, although some reports provide evidence that lipopolysaccharide (LPS) could be a lipid ligand. Unlike previous hypotheses, we found significant structural differences between SPLUNC1 and BPI. Recombinant SPLUNC1 produced in HEK 293 cells harbored several molecular species of sphingomyelin and phosphatidylcholine as its ligands. Significantly, in vitro lipid-binding studies failed to demonstrate interactions between SPLUNC1 and LPS, lipoteichoic acid, or polymyxin B. Instead, one of the major and most important pulmonary surfactant phospholipids, dipalmitoylphosphatidylcholine (DPPC), bound to SPLUNC1 with high affinity and specificity. We found that SPLUNC1 could be the first protein receptor for DPPC. These discoveries provide insight into the specific determinants governing the interaction between SPLUNC1 and lipids and also shed light on novel functions that SPLUNC1 and other PLUNC family members perform in host defense.