Heightened turnover and failed maturation of monocyte-derived macrophages in murine chronic granulomatous disease.

Loss of NADPH oxidase activity leads to altered phagocyte responses and exaggerated inflammation in chronic granulomatous disease (CGD). We sought to assess the effects of Nox2 absence on monocyte-derived macrophages (MoMacs) in gp91phox-/y mice during zymosan-induced peritonitis. MoMacs from CGD and wild-type (WT) peritonea were characterized over time after zymosan injection. Although numbers lavaged from both genotypes were virtually identical, there were marked differences in maturation: newly recruited WT MoMacs rapidly enlarged and matured, losing Ly6C and gaining MHCII, CD206, and CD36, whereas CGD MoMacs remained small and were mostly Ly6C+MHCII-. RNA-sequencing analyses showed few intrinsic differences between genotypes in newly recruited MoMacs but significant differences with time. WT MoMacs displayed changes in metabolism, adhesion, and reparative functions, whereas CGD MoMacs remained inflammatory. PKH dye labeling revealed that although WT MoMacs were mostly recruited within the first 24 hours and remained in the peritoneum while maturing and enlarging, CGD monocytes streamed into the peritoneum for days, with many migrating to the diaphragm where they were found in fibrin(ogen) clots surrounding clusters of neutrophils in nascent pyogranulomata. Importantly, these observations seemed to be driven by milieu: adoptive transfer of CGD MoMacs into inflamed peritonea of WT mice resulted in immunophenotypic maturation and normal behavior, whereas altered maturation/behavior of WT MoMacs resulted from transfer into inflamed peritonea of CGD mice. In addition, Nox2-deficient MoMacs behaved similarly to their Nox2-sufficient counterparts within the largely WT milieu of mixed bone marrow chimeras. These data show persistent recruitment with fundamental failure of MoMac maturation in CGD.

Phagocytosis of microparticles by alveolar macrophages during acute lung injury requires MerTK.

Microparticles are a newly recognized class of mediators in the pathophysiology of lung inflammation and injury, but little is known about the factors that regulate their accumulation and clearance. The primary objective of our study was to determine whether alveolar macrophages engulf microparticles and to elucidate the mechanisms by which this occurs. Alveolar microparticles were quantified in bronchoalveolar fluid of mice with lung injury induced by LPS and hydrochloric acid. Microparticle numbers were greatest at the peak of inflammation and declined as inflammation resolved. Isolated, fluorescently labeled particles were placed in culture with macrophages to evaluate ingestion in the presence of endocytosis inhibitors. Ingestion was blocked with cytochalasin D and wortmannin, consistent with a phagocytic process. In separate experiments, mice were treated intratracheally with labeled microparticles, and their uptake was assessed though microscopy and flow cytometry. Resident alveolar macrophages, not recruited macrophages, were the primary cell-ingesting microparticles in the alveolus during lung injury. In vitro, microparticles promoted inflammatory signaling in LPS primed epithelial cells, signifying the importance of microparticle clearance in resolving lung injury. Microparticles were found to have phosphatidylserine exposed on their surfaces. Accordingly, we measured expression of phosphatidylserine receptors on macrophages and found high expression of MerTK and Axl in the resident macrophage population. Endocytosis of microparticles was markedly reduced in MerTK-deficient macrophages in vitro and in vivo. In conclusion, microparticles are released during acute lung injury and peak in number at the height of inflammation. Resident alveolar macrophages efficiently clear these microparticles through MerTK-mediated phagocytosis.

G2A Signaling Dampens Colitic Inflammation via Production of IFN-γ.

Proinflammatory consequences have been described for lysophosphatidylcholine, a lipid product of cellular injury, signaling via the G protein-coupled receptor G2A on myeloid and lymphoid inflammatory cells. This prompted the hypothesis that genetic deletion of G2A would limit intestinal inflammation in a mouse model of colitis induced by dextran sodium sulfate. Surprisingly, G2A(-/-) mice exhibited significantly worsened colitis compared with wild-type mice, as demonstrated by disease activity, colon shortening, histology, and elevated IL-6 and IL-5 in colon tissues. Investigation of inflammatory cells recruited to inflamed G2A(-/-) colons showed significantly more TNF-α(+) and Ly6C(hi)MHCII(-) proinflammatory monocytes and eosinophils than in wild-type colons. Both monocytes and eosinophils were pathogenic as their depletion abolished the excess inflammation in G2A(-/-) mice. G2A(-/-) mice also had less IFN-γ in inflamed colon tissues than wild-type mice. Fewer CD4(+) lymphocytes were recruited to inflamed G2A(-/-) colons, and fewer colonic lymphocytes produced IFN-γ upon ex vivo stimulation. Administration of IFN-γ to G2A(-/-) mice during dextran sodium sulfate exposure abolished the excess colitic inflammation and reduced colonic IL-5 and eosinophil numbers to levels seen in wild-type mice. Furthermore, IFN-γ reduced the numbers of TNF-α(+) monocyte and enhanced their maturation from Ly6C(hi)MHCII(-) to Ly6C(int)MHCII(+) Taken together, the data suggest that G2A signaling serves to dampen intestinal inflammation via the production of IFN-γ, which, in turn, enhances monocyte maturation to a less inflammatory program and ultimately reduces eosinophil-induced injury of colonic tissues.

Pioglitazone restores phagocyte mitochondrial oxidants and bactericidal capacity in chronic granulomatous disease.

BACKGROUND: Deficient production of reactive oxygen species (ROS) by the phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase in patients with chronic granulomatous disease (CGD) results in susceptibility to certain pathogens secondary to impaired oxidative killing and mobilization of other phagocyte defenses. Peroxisome proliferator-activated receptor (PPAR) γ agonists, including pioglitazone, approved for type 2 diabetes therapy alter cellular metabolism and can heighten ROS production. It was hypothesized that pioglitazone treatment of gp91(phox-/-) mice, a murine model of human CGD, would enhance phagocyte oxidant production and killing of Staphylococcus aureus, a significant pathogen in patients with this disorder. OBJECTIVES: We sought to determine whether pioglitazone treatment of gp91(phox-/-) mice enhanced phagocyte oxidant production and host defense. METHODS: Wild-type and gp91(phox-/-) mice were treated with the PPARγ agonist pioglitazone, and phagocyte ROS and killing of S aureus were investigated. RESULTS: As demonstrated by 3 different ROS-sensing probes, short-term treatment of gp91(phox-/-) mice with pioglitazone enhanced stimulated ROS production in neutrophils and monocytes from blood and neutrophils and inflammatory macrophages recruited to tissues. Mitochondria were identified as the source of ROS. Findings were replicated in human monocytes from patients with CGD after ex vivo pioglitazone treatment. Importantly, although mitochondrial (mt)ROS were deficient in gp91(phox-/-) phagocytes, their restoration with treatment significantly enabled killing of S aureus both ex vivo and in vivo. CONCLUSIONS: Together, the data support the hypothesis that signaling from the NADPH oxidase under normal circumstances governs phagocyte mtROS production and that such signaling is lacking in the absence of a functioning phagocyte oxidase. PPARγ agonism appears to bypass the need for the NADPH oxidase for enhanced mtROS production and partially restores host defense in CGD.

Neutrophils regulate tissue Neutrophilia in inflammation via the oxidant-modified lipid lysophosphatidylserine.

Resolution of neutrophilia characteristic of acute inflammation requires cessation of neutrophil recruitment and removal of tissue neutrophils. Based on in vitro studies, a role in these events was hypothesized for oxidant-generated lysophosphatidylserine (lyso-PS) on recruited neutrophils signaling via the G2A receptor on macrophages. Peritoneal exudate neutrophils harvested from wild type (WT) mice had 5-fold more lyso-PS (lyso-PS(high)) than those of gp91(phox)(-/-) (lyso-PS(low)) mice. Ex vivo engulfment of lyso-PS(high) neutrophils (95% viable) by WT peritoneal macrophages was quantitatively similar to UV-irradiated apoptotic blood neutrophils, although the signaling pathway for the former was uniquely dependent on macrophage G2A. In contrast, lyso-PS(low) neutrophils were poorly engulfed unless presented with exogenous lyso-PS. Enhanced clearance of lyso-PS(high) neutrophils was also seen in vivo following their adoptive transfer into inflamed peritonea of WT but not G2A(-/-) mice, further supporting a requirement for signaling via G2A. To investigate downstream effects of lyso-PS/G2A signaling, antibody blockade of G2A in WT mice reduced macrophage CD206 expression and efferocytosis during peritonitis. Conversely, adoptive transfer of lyso-PS(high) neutrophils early in inflammation in gp91(phox)(-/-) mice led to accelerated development of efferocytic(high) and CD206(high) macrophages. This macrophage reprogramming was associated with suppressed production of pro-inflammatory mediators and reduced neutrophilia. These effects were not seen if G2A was blocked or lyso-PS(low) neutrophils were transferred. Taken together, the results demonstrate that oxidant-generated lyso-PS made by viable tissue neutrophils is an endogenous anti-inflammatory mediator working in vivo to orchestrate the "early" and rapid clearance of recruited neutrophils as well as the reprogramming of "resolving" macrophages.

Signaling via macrophage G2A enhances efferocytosis of dying neutrophils by augmentation of Rac activity.

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.

SPLUNC1 promotes lung innate defense against Mycoplasma pneumoniae infection in mice.

Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is highly expressed in normal airways, but is dramatically decreased in allergic and cigarette smoke exposure settings. We have previously demonstrated SPLUNC1 in vitro antibacterial property against Mycoplasma pneumoniae (Mp). However, its in vivo biological functions remain unclear. The objectives of this study were to determine the in vivo functions of SPLUNC1 following bacterial (eg, Mp) infection, and to examine the underlying mechanisms. We generated SPLUNC1-deficient mice and utilized transgenic mice overexpressing human SPLUNC1 exclusively within the airway epithelium. These mice were infected with Mp and, twenty-four hours post infection, their host defense responses were compared to littermate controls. Mp levels and inflammatory cells increased in the lungs of SPLUNC1(-/-) mice as compared to wild type controls. SPLUNC1 deficiency was shown to contribute to impaired neutrophil activation. In contrast, mice overexpressing hSPLUNC1 exclusively in airway epithelial cells demonstrated lower Mp levels. Furthermore, neutrophil elastase activity was significantly increased in mice overexpressing hSPLUNC1. Lastly, we demonstrated that SPLUNC1 enhanced Mp-induced human neutrophil elastase (HNE) activity, and HNE directly inhibited the growth of Mp. Our findings demonstrate a critical in vivo role of SPLUNC1 in host defense against bacterial infection, and likely provide a novel therapeutic approach to restore impaired lung innate immune responses to bacteria in patients with chronic lung diseases.

PPARγ activation normalizes resolution of acute sterile inflammation in murine chronic granulomatous disease.

Absence of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase predisposes chronic granulomatous disease (CGD) patients to infection, and also to unexplained, exaggerated inflammation. The impaired recognition and removal (efferocytosis) of apoptotic neutrophils by CGD macrophages may contribute to this effect. We hypothesized that peroxisome proliferator-activated receptor γ (PPARγ) activation during CGD inflammation is deficient, leading to altered macrophage programming and decreased efferocytosis, and that PPARγ agonism would enhance resolution. using the gp91(phox-/-) murine model of X-linked CGD in a well-characterized model of sterile, zymosan-induced peritonitis, it was demonstrated that PPARγ expression and activation in CGD macrophages were significantly deficient at baseline, and acquisition was delayed over the course of inflammation relative to that of wild-type. Efferocytosis by macrophages reflected PPARγ activation during peritonitis and was impaired in CGD mice (versus wild-type), leading to accumulation of apoptotic neutrophils. Importantly, provision of the PPARγ agonist, pioglitazone, either prophylactically or during inflammation, significantly enhanced macrophage PPARγ-mediated programming and efferocytosis, reduced accumulation of apoptotic neutrophils, and normalized the course of peritonitis in CGD mice. As such, PPARγ may be a therapeutic target for CGD, and possibly other inflammatory conditions where aberrant macrophage programming and impaired efferocytosis delay resolution of inflammation.

Impaired phagocytosis of apoptotic cells by macrophages in chronic granulomatous disease is reversed by IFN-γ in a nitric oxide-dependent manner.

Immunodeficiency in chronic granulomatous disease (CGD) is well characterized. Less understood are exaggerated sterile inflammation and autoimmunity associated with CGD. Impaired recognition and clearance of apoptotic cells resulting in their disintegration may contribute to CGD inflammation. We hypothesized that priming of macrophages (Ms) with IFN-γ would enhance impaired engulfment of apoptotic cells in CGD. Diverse M populations from CGD (gp91(phox)(-/-)) and wild-type mice, as well as human Ms differentiated from monocytes and promyelocytic leukemia PLB-985 cells (with and without mutation of the gp91(phox)), demonstrated enhanced engulfment of apoptotic cells in response to IFN-γ priming. Priming with IFN-γ was also associated with increased uptake of Ig-opsonized targets, latex beads, and fluid phase markers, and it was accompanied by activation of the Rho GTPase Rac. Enhanced Rac activation and phagocytosis following IFN-γ priming were dependent on NO production via inducible NO synthase and activation of protein kinase G. Notably, endogenous production of TNF-α in response to IFN-γ priming was critically required for inducible NO synthase upregulation, NO production, Rac activation, and enhanced phagocytosis. Treatment of CGD mice with IFN-γ also enhanced uptake of apoptotic cells by M in vivo via the signaling pathway. Importantly, during acute sterile peritonitis, IFN-γ treatment reduced excess accumulation of apoptotic neutrophils and enhanced phagocytosis by CGD Ms. These data support the hypothesis that in addition to correcting immunodeficiency in CGD, IFN-γ priming of Ms restores clearance of apoptotic cells and may thereby contribute to resolution of exaggerated CGD inflammation.

Impaired apoptotic cell clearance in CGD due to altered macrophage programming is reversed by phosphatidylserine-dependent production of IL-4.

Chronic granulomatous disease (CGD) is characterized by overexuberant inflammation and autoimmunity that are attributed to deficient anti-inflammatory signaling. Although regulation of these processes is complex, phosphatidylserine (PS)-dependent recognition and removal of apoptotic cells (efferocytosis) by phagocytes are potently anti-inflammatory. Since macrophage phenotype also plays a beneficial role in resolution of inflammation, we hypothesized that impaired efferocytosis in CGD due to macrophage skewing contributes to enhanced inflammation. Here we demonstrate that efferocytosis by macrophages from CGD (gp91(phox)(-/-)) mice was suppressed ex vivo and in vivo. Alternative activation with interleukin 4 (IL-4) normalized CGD macrophage efferocytosis, whereas classical activation by lipopolysaccharide (LPS) plus interferon gamma (IFNgamma) had no effect. Importantly, neutralization of IL-4 in wild-type macrophages reduced macrophage efferocytosis, demonstrating a central role for IL-4. This effect was shown to involve 12/15 lipoxygenase and activation of peroxisome-proliferator activated receptor gamma (PPARgamma). Finally, injection of PS (whose exposure is lacking on CGD apoptotic neutrophils) in vivo restored IL-4-dependent macrophage reprogramming and efferocytosis via a similar mechanism. Taken together, these findings support the hypothesis that impaired PS exposure on dying cells results in defective macrophage programming, with consequent efferocytic impairment and has important implications in understanding the underlying cause of enhanced inflammation in CGD.

Role of gp91phox in hepatic macrophage programming and alcoholic liver disease.

Hepatic macrophages (MΦs) are important in the development and progression of alcoholic liver disease (ALD). This study investigates the role of gp91phox (nicotinamide adenine dinucleotide phosphate oxidase 2) in the severity of ALD and specifically in regulating hepatic MΦ efferocytic capability and the subsequent reprogramming associated with resolution of inflammation. After 4 weeks of ethanol feeding, more severe ALD developed in gp91phox-/- mice than in wild-type (WT) C57Bl/6J mice, evidenced by increased liver injury and inflammation. This phenomenon was not sex dependent, and thus the majority of experiments were performed with female mice. While total hepatic MΦ numbers did not differ between genotypes, hepatic infiltrating MΦs (IMs) were slightly more numerous in gp91phox-/- mice, and both IMs and resident Kupffer cells displayed enhanced proinflammatory and reduced tissue-restorative programming compared with these cells from WT mice. The ratio of proinflammatory IMs with higher expression of Ly6C (Ly6Chi) to anti-inflammatory IMs with lower expression of Ly6C (Ly6Clow) was significantly higher in gp91phox-/- mice compared to WT mice. Greater numbers of apoptotic cells accumulated in the liver of gp91phox-/- mice compared to WT mice, and receptors for binding and engulfing apoptotic cells were expressed at much lower levels on both Kupffer cells and IMs of gp91phox-/- mice. Interactions with apoptotic cells (binding and engulfment) in vitro were significantly fewer for gp91phox-/- MΦs than for WT MΦs, resulting in diminished expression of tissue restorative mediators by hepatic MΦs of gp91phox-/- mice. Conclusion: gp91phox plays a critical role in the differentiation of proinflammatory hepatic MΦs to a tissue-restorative phenotype, likely through programming for efferocytosis, and thereby lessens the severity of ALD. These findings enhance our understanding of the tissue environmental cues that regulate MΦ phenotypes. This knowledge could help in designing MΦ-targeting strategies to prevent and treat ALD. (Hepatology Communications 2017;1:765-779).

Induction of TGF-ß1 synthesis by macrophages in response to apoptotic cells requires activation of the scavenger receptor CD36.

BACKGROUND/OBJECTIVE: Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-ß (TGF-ß) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, using RAWTßRII cells in which a truncated dominant negative TGF-ß receptor II was stably transfected in order to avoid auto-feedback induction of TGF-ß, we show that TGF-ß1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-ß1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-ß1 expression. CONCLUSION/SIGNIFICANCE: Since CD36 has been previously implicated in activation of secreted latent TGF-ß, the present study indicates its role in the multiple steps to generation of this important biological mediator.

Emerging roles for lysophosphatidylserine in resolution of inflammation.

Despite overlapping structural aspects with other phospholipids, lysophosphatidylserine (lysoPS), the monoacyl derivative of phosphatidylserine (diacylPS), appears to exert unique signaling characteristics important in both the early stages of initiating acute inflammation and in the orchestration of its resolution. LysoPS has long been known as a signaling phospholipid in mast cell biology, markedly enhancing stimulated histamine release and eicosanoid production. More recently, there has been a resurgence of interest in lysoPS as new roles in the promotion of phagocytosis of apoptotic cells, so-called efferocytosis, and resolution of inflammation have been identified. With regard to the latter, lysoPS generated in/on activated or aged apoptotic neutrophils enhances their clearance by macrophages via signaling through the macrophage G-protein coupled receptor G2A. In macrophages, this early acting pathway results in PKA-dependent augmentation of Rac1 activity via increased production of PGE2 and cAMP. As such, macrophages stimulated with lysoPS demonstrate significantly increased efferocytic capacity necessary to clear large numbers of recruited neutrophils typical of acute inflammation. Given that clearance of these cells is critical for restoration of tissue function, lysoPS, as a pro-resolving lipid mediator, is hypothesized to play a key role in promoting timely resolution of inflammation. This article will review our current knowledge of lysoPS biology including receptor signaling and mechanisms of generation as well as summarize the more recent evidence of its expanding roles in inflammation.

Modulation of macrophage efferocytosis in inflammation.

A critical function of macrophages within the inflammatory milieu is the removal of dying cells by a specialized phagocytic process called efferocytosis ("to carry to the grave"). Through specific receptor engagement and induction of downstream signaling, efferocytosing macrophages promote resolution of inflammation by (i) efficiently engulfing dying cells, thus avoiding cellular disruption and release of inflammatory contents, and (ii) producing anti-inflammatory mediators such as IL-10 and TGF-ß that dampen pro-inflammatory responses. Evidence suggests that plasticity in macrophage programming, in response to changing environmental cues, modulates efferocytic capability. Essential to programming for enhanced efferocytosis is activation of the nuclear receptors PPARγ, PPARδ, LXR, and possibly RXRα. Additionally, a number of signals in the inflammatory milieu, including those from dying cells themselves, can influence efferocytic efficacy either by acting as immediate inhibitors/enhancers or by altering macrophage programming for longer-term effects. Importantly, sustained inflammatory programming of macrophages can lead to defective apoptotic cell clearance and is associated with development of autoimmunity and other chronic inflammatory disorders. This review summarizes the current knowledge of the multiple factors that modulate macrophage efferocytic ability and highlights emerging therapeutic targets with significant potential for limiting chronic inflammation.

NADPH oxidase-dependent generation of lysophosphatidylserine enhances clearance of activated and dying neutrophils via G2A.

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 (lyso-PS), 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 (gp91phox-/-). 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 gp91phox-/- 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.

Lysophospholipids of different classes mobilize neutrophil secretory vesicles and induce redundant signaling through G2A.

Lysophosphatidylcholine has been shown to enhance neutrophil functions through a mechanism involving the G protein-coupled receptor G2A. Recent data support an indirect effect of lysophosphatidylcholine on G2A rather than direct ligand binding. These observations prompted the hypothesis that other lysophospholipids (lyso-PLs) may also signal for human neutrophil activation through G2A. To this end, 1-oleoyl-2-hydroxy-sn-glycero-3-[phospho-L-choline], but also C18:1/OH lyso-PLs bearing the phosphoserine and phosphoethanolamine head groups, presented on albumin, were shown to signal for calcium flux in a self- and cross-desensitizing manner, implicating a single receptor. Blocking Abs to G2A inhibited calcium signaling by all three lyso-PLs. Furthermore, inhibition by both pertussis toxin and U-73122 established signaling via the Galphai/phospholipase C pathway for calcium mobilization. Altered plasma membrane localization of G2A has been hypothesized to facilitate signaling. Accordingly, an increase in detectable G2A was demonstrated by 1 min after lyso-PL stimulation and was followed by visible patching of the receptor. Western blotting showed that G2A resides in the plasma membrane/secretory vesicle fraction and not in neutrophil primary, secondary, or tertiary granules. Enhanced detection of G2A induced by lyso-PLs was paralleled by enhanced detection of CD45, confirming mobilization of the labile secretory vesicle pool. Together, these data show that lyso-PLs bearing various head groups redundantly mobilize G2A latent within secretory vesicles and result in G2A receptor/Galphai/phospholipase C signaling for calcium flux in neutrophils.

Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of LRP on the phagocyte.

Apoptotic-cell removal is critical for development, tissue homeostasis, and resolution of inflammation. Although many candidate systems exist, only phosphatidylserine has been identified as a general recognition ligand on apoptotic cells. We demonstrate here that calreticulin acts as a second general recognition ligand by binding and activating LDL-receptor-related protein (LRP) on the engulfing cell. Since surface calreticulin is also found on viable cells, a mechanism preventing inadvertent uptake was sought. Disruption of interactions between CD47 (integrin-associated protein) on the target cell and SIRPalpha (SHPS-1), a heavily glycosylated transmembrane protein on the engulfing cell, permitted uptake of viable cells in a calreticulin/LRP-dependent manner. On apoptotic cells, CD47 was altered and/or lost and no longer activated SIRPalpha. These changes on the apoptotic cell create an environment where "don't eat me" signals are rendered inactive and "eat me" signals, including calreticulin and phosphatidylserine, congregate together and signal for removal.

Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neutrophils.

Movement of phosphatidylserine (PS) to the plasma membrane outer leaflet is a nearly universal marker of apoptosis and occurs during activation of many cells. Neutrophils stimulated with the chemotactic peptide formylated Met-Leu-Phe (fMLP) demonstrated transient PS exposure. Stimulated outward movement of PS was accompanied by enhanced inward movement of several phosphorylcholine lipid probes and was associated with enhanced FM 1-43 staining indicative of phospholipid packing changes. Unlike apoptosis, inward movement of exogenously added fluorescent PS did not decline, and DNA was not cleaved during fMLP stimulation. Movement of phospholipids occurred within minutes following stimulation, was independent of endocytosis/pinocytosis, and was consistent with bidirectional, transbilayer phospholipid flip-flop. While the role of phospholipid scramblase 1 (PLSCR1) is controversial in flip-flop, we sought evidence for its role in enhanced phospholipid movements during fMLP stimulation. Using antibodies to the carboxyl-terminal domain of PLSCR1, its presence in the plasma membranes of non-permeabilized neutrophils was confirmed by flow cytometry. Additionally subcellular fractionation demonstrated that PLSCR1 was also located in secretory vesicles and tertiary and secondary granules. Activation of neutrophils with fMLP, however, did not significantly alter surface labeling suggesting that stimulated phospholipid flip-flop does not require additional mobilization of PLSCR1 to the plasma membrane. As expected for palmitoylated proteins, PLSCR1 was enriched in detergent-insoluble membranes and co-localized with raft markers at the neutrophil uropod after stimulation. Of note, PS exposure, phospholipid uptake, and FM 1-43 staining also localized to the uropod following stimulation demonstrating that both PLSCR1 and phospholipid flip-flop characterize this specialized domain of polarized neutrophils.

Phosphorylation of Bax Ser184 by Akt regulates its activity and apoptosis in neutrophils.

Although important for apoptosis, the mechanism of Bax regulation is poorly understood. This study demonstrates that phosphorylation of Ser(184) regulates Bax activity. The phosphorylation required phosphatidylinositol 3-kinase/Akt activation and appeared to be mediated by Akt itself. In the serine-phosphorylated form, Bax was detected in the cytoplasm, could not be immunoprecipitated with the activation-specific antibody 6A7, and promoted heterodimerization with Mcl-1, Bcl-x(L), and A1. Apoptotic neutrophils possessed reduced levels of serine-phosphorylated Bax correlating with an increase in activated Bax as well as an increase in the amount of Bax found translocated to the mitochondria. We suggest that Bax is regulated by phosphorylation of Ser(184) in an Akt-dependent manner and that phosphorylation inhibits Bax effects on the mitochondria by maintaining the protein in the cytoplasm, heterodimerized with antiapoptotic Bcl-2 family members.