Effects of Lipid Interactions on Model Vesicle Engulfment by Alveolar Macrophages

The engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state ²H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.     

Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.     

Two-step engulfment of apoptotic cells

Apoptotic cells expose phosphatidylserine on their surface as an "eat me" signal, and macrophages respond by engulfing them. Although several molecules that specifically bind phosphatidylserine have been identified, the molecular mechanism that triggers engulfment remains elusive. Here, using a mouse pro-B cell line, Ba/F3, that grows in suspension, we reconstituted the engulfment of apoptotic cells. The parental Ba/F3 cells did not engulf apoptotic cells. Ba/F3 transformants expressing T cell immunoglobulin- and mucin-domain-containing molecule 4 (Tim4), a type I membrane protein that specifically binds phosphatidylserine, efficiently bound apoptotic cells in a phosphatidylserine-dependent manner but did not engulf them. However, Ba/F3 transformants expressing both Tim4 and the integrin α(v)β(3) complex bound to and engulfed apoptotic cells in the presence of milk fat globule epidermal growth factor factor VIII (MFG-E8), a secreted protein that can bind phosphatidylserine and integrin α(v)β(3). These results indicate that the engulfment of apoptotic cells proceeds in two steps: Tim4 tethers apoptotic cells, and the integrin α(v)β(3) complex mediates engulfment in coordination with MFG-E8. A similar two-step engulfment of apoptotic cells was observed with mouse resident peritoneal macrophages. Furthermore, the Tim4/integrin-mediated engulfment by the Ba/F3 cells was enhanced in cells expressing Rac1 and Rab5, suggesting that this system well reproduces the engulfment of apoptotic cells by macrophages.     

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI^−/− cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI^−/− versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI^−/− macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.     

Identification of TLT2 as an engulfment receptor for apoptotic cells

Clearance of apoptotic cells (efferocytosis) is critical to the homeostasis of the immune system by restraining inflammation and autoimmune response to intracellular Ags released from dying cells. TLRs-mediated innate immunity plays an important role in pathogen clearance and in regulation of the adaptive immune response. However, the regulation of efferocytosis by activation of TLRs has not been well characterized. In this study, we found that activation of TLR3 or TLR9, but not of TLR2, enhances engulfment of apoptotic cells by macrophages. We found that the activation of TLR3 upregulates the expression of triggering receptor expressed on myeloid cells (TREM)-like protein 2 (TLT2), a member of the TREM receptor family, on the surface of macrophages. Blocking TLT2 on the macrophage surface by either specific anti-TLT2 Ab or soluble TLT2 extracellular domain attenuated the enhanced ability of macrophages with TLR3 activation to engulf apoptotic cells. To the contrary, overexpression of TLT2 increased the phagocytosis of apoptotic cells. We found that TLT2 specifically binds to phosphatidylserine, a major "eat me" signal that is exposed on the surface of apoptotic cells. Furthermore, we found that TLT2 mediates phagocytosis of apoptotic cells in vivo. Thus, our studies identified TLT2 as an engulfment receptor for apoptotic cells. Our data also suggest a novel mechanism by which TREM receptors regulate inflammation and autoimmune response.     

ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.     

PTEN negatively regulates engulfment of apoptotic cells by modulating activation of Rac GTPase

Efficient clearance of apoptotic cells by phagocytes (efferocytosis) is critical for normal tissue homeostasis and regulation of the immune system. Apoptotic cells are recognized by a vast repertoire of receptors on macrophage that lead to transient formation of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] and subsequent cytoskeletal reorganization necessary for engulfment. Certain PI3K isoforms are required for engulfment of apoptotic cells, but relatively little is known about the role of lipid phosphatases in this process. In this study, we report that the activity of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatidylinositol 3-phosphatase, is elevated upon efferocytosis. Depletion of PTEN in macrophage results in elevated PtdIns(3,4,5)P(3) production and enhanced phagocytic ability both in vivo and in vitro, whereas overexpression of wild-type PTEN abrogates this process. Loss of PTEN in macrophage leads to activation of the pleckstrin homology domain-containing guanine-nucleotide exchange factor Vav1 and subsequent activation of Rac1 GTPase, resulting in increased amounts of F-actin upon engulfment of apoptotic cells. PTEN disruption also leads to increased production of anti-inflammatory cytokine IL-10 and decreased production of proinflammatory IL-6 and TNF-α upon engulfment of apoptotic cells. These data suggest that PTEN exerts control over efferocytosis potentially by regulating PtdIns(3,4,5)P(3) levels that modulate Rac GTPase and F-actin reorganization through Vav1 exchange factor and enhancing apoptotic cell-induced anti-inflammatory response.     

Poly(ADP-ribosyl)ation of high mobility group box 1 (HMGB1) protein enhances inhibition of efferocytosis

Phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is a critical process in the resolution of inflammation. High mobility group box 1 (HMGB1) protein was first described as a nuclear nonhistone DNA-binding protein, but is now known to be secreted by activated cells during inflammatory processes, where it participates in diminishing efferocytosis. Although HMGB1 is known to undergo modification when secreted, the effect of such modifications on the inhibitory actions of HMGB1 during efferocytosis have not been reported. In the present studies, we found that HMGB1 secreted by Toll-like receptor 4 (TLR4) stimulated cells is highly poly(ADP-ribosyl)ated (PARylated). Gene deletion of poly(ADP)-ribose polymerase (PARP)-1 or pharmacological inhibition of PARP-1 decreased the release of HMGB1 from the nucleus to the extracellular milieu after TLR4 engagement. Preincubation of macrophages or apoptotic cells with HMGB1 diminished efferocytosis through mechanisms involving binding of HMGB1 to phosphatidylserine on apoptotic cells and to the receptor for advanced glycation end products (RAGE) on macrophages. Preincubation of either macrophages or apoptotic cells with PARylated HMGB1 inhibited efferocytosis to a greater degree than exposure to unmodified HMGB1, and PARylated HMGB1 demonstrated higher affinity for phosphatidylserine and RAGE than unmodified HMGB1. PARylated HMGB1 had a greater inhibitory effect on Ras-related C3 botulinum toxin substrate 1 (Rac-1) activation in macrophages during the uptake of apoptotic cells than unmodified HMGB1. The present results, showing that PARylation of HMGB1 enhances its ability to inhibit efferocytosis, provide a novel mechanism by which PARP-1 may promote inflammation.     

Tim4- and MerTK-Mediated Engulfment of Apoptotic Cells by Mouse Resident Peritoneal Macrophages

Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.     

Cell surface-expressed phosphatidylserine as therapeutic target to enhance phagocytosis of apoptotic cells

Impaired efferocytosis has been shown to be associated with, and even to contribute to progression of, chronic inflammatory diseases such as atherosclerosis. Enhancing efferocytosis has been proposed as strategy to treat diseases involving inflammation. Here we present the strategy to increase ‘eat me'' signals on the surface of apoptotic cells by targeting cell surface-expressed phosphatidylserine (PS) with a variant of annexin A5 (Arg-Gly-Asp–annexin A5, RGD–anxA5) that has gained the function to interact with α_vβ₃ receptors of the phagocyte. We describe design and characterization of RGD–anxA5 and show that introduction of RGD transforms anxA5 from an inhibitor into a stimulator of efferocytosis. RGD–anxA5 enhances engulfment of apoptotic cells by phorbol-12-myristate-13-acetate-stimulated THP-1 (human acute monocytic leukemia cell line) cells in vitro and resident peritoneal mouse macrophages in vivo. In addition, RGD–anxA5 augments secretion of interleukin-10 during efferocytosis in vivo, thereby possibly adding to an anti-inflammatory environment. We conclude that targeting cell surface-expressed PS is an attractive strategy for treatment of inflammatory diseases and that the rationally designed RGD–anxA5 is a promising therapeutic agent.     

Human lysosomal beta-glucosidase: kinetic characterization of the catalytic, aglycon, and hydrophobic binding sites

Three binding sites on highly purified lysosomal beta-glucosidase from human placenta were identified by studies of the effects of interactions of various enzyme modifiers. The negatively charged lipids, taurocholate and phosphatidylserine, were shown to be noncompetitive, nonessential activators of 4-methylumbelliferyl-beta-D-glucoside hydrolysis. Similar results were observed using the natural substrate, glucosyl ceramide, and low concentrations of taurocholate (less than 1.8 mM) or phosphatidylserine (0.5 mM). However, higher concentrations resulted in a complex partial inhibition of glucosyl ceramide hydrolysis. Increasing concentrations of phosphatidylserine obviated the effects of taurocholate, suggesting that these compounds compete for a common binding site on the enzyme. Glucosyl sphingosine and its N-hexyl derivative were potent noncompetitive inhibitors of the enzyme activity using either substrate. Taurocholate (or phosphatidylserine) and glucosyl sphingosine were shown to be mutually exclusive, indicating competition for a common binding site. In contrast, octyl- and dodecyl-beta-glucosides were linear-mixed-type inhibitors of glucosyl ceramide or 4-methylumbelliferyl-beta-D-glucoside hydrolysis, indicating at least two binding sites on the enzyme. Inhibition by these alkyl beta-glucosides was observed only in the presence of taurocholate or phosphatidylserine. The competitive component [Ki (slope)] for the two alkyl beta-glucosides decreased with increasing alkyl chain length, and was unaffected by increasing taurocholate or phosphatidylserine concentration. The noncompetitive component [Ki (intercept)] was nearly identical for both alkyl beta-glucosides and was decreased by increasing taurocholate or phosphatidylserine concentration. These results indicated that the negatively charged lipids and alkyl beta-glucosides were not mutually exclusive, but interacted with different binding sites on the enzyme. Gluconolactone was shown to protect the enzyme from inhibition by the catalytic site-directed covalent inhibitor, conduritol B indicating an interaction at a common binding site. In the presence of substrate, taurocholate facilitated the inhibition of gluconolactone or conduritol B epoxide. These studies indicated that lysosomal beta-glucosidase had at least three binding sites: (i) a catalytic site which cleaves the beta-glucosidic moiety, (ii) an aglycon site which binds the acyl or alkyl moieties of substrates and some inhibitors, and (iii) a hydrophobic site which interacts with negatively charged lipids and facilitates enzyme catalysis.     

Participation of the receptor for advanced glycation end products in efferocytosis

Clearance of apoptotic cells by macrophages and other phagocytic cells, called efferocytosis, is a central process in the resolution of inflammation. Although the receptor for advanced glycation end products (RAGE) has been shown to participate in a variety of acute and chronic inflammatory processes in the lungs and other organs, a role for RAGE in efferocytosis has not been reported. In the present studies, we examined the potential involvement of RAGE in efferocytosis. Macrophages from transgenic RAGE(-/-) mice showed a decreased ability to engulf apoptotic neutrophils and thymocytes. Pretreatment of RAGE(+/+) macrophages with advanced glycation end products, which competitively bind to RAGE, or Abs against RAGE diminished phagocytosis of apoptotic cells. Overexpression of RAGE in human embryonic kidney 293 cells resulted in an increased ability to engulf apoptotic cells. Furthermore, we found that incubation with soluble RAGE enhances phagocytosis of apoptotic cells by both RAGE(+/+) and RAGE(-/-) macrophages. Direct binding of RAGE to phosphatidylserine (PS), an "eat me" signal highly expressed on apoptotic cells, was shown by using solid-phase ELISA. The ability of RAGE to bind to PS on apoptotic cells was confirmed in an adhesion assay. Decreased uptake of apoptotic neutrophils by macrophages was found under in vivo conditions in the lungs and peritoneal cavity of RAGE(-/-) mice. These results demonstrate a novel role for RAGE in which it is able to enhance efferocytosis through binding to PS on apoptotic cells.     

Tethering of apoptotic cells to phagocytes through binding of CD47 to Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1

Apoptotic cells are swiftly phagocytosed by macrophages and immature dendritic cells. In this study, we found that one mouse macrophage cell line (BAM3) engulfed apoptotic thymocytes, but not a lymphoma cell line (WR19L). mAbs that inhibited the phagocytosis of apoptotic thymocytes by BAM3 were identified. Purification of the Ag revealed that it was Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1 (SHPS-1). CD47, the ligand for SHPS-1, was expressed in mouse thymocytes, but not in WR19L. When WR19L was transformed with CD47, the transformants, after induction of apoptosis, could be phagocytosed by BAM3. The WR19L transformants expressing CD47 were more efficiently engulfed in vivo by splenic dendritic cells than the parental WR19L. Masking of the phosphatidylserine exposed on apoptotic thymocytes inhibited the engulfment, whereas the anti-SHPS-1 mAb inhibited not only the engulfment, but also the binding of apoptotic cells to phagocytes. These results indicate that macrophages require CD47 and phosphatidylserine on apoptotic cells for engulfment, and suggest that the interaction between CD47 and SHPS-1 works as a tethering step in the phagocytosis.     

Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts

Removal of apoptotic cells during tissue remodeling or resolution of inflammation is critical to the restoration of normal tissue structure and function. During apoptosis, early surface changes occur, which trigger recognition and removal by macrophages and other phagocytes. Loss of phospholipid asymmetry results in exposure of phosphatidylserine (PS), one of the surface markers recognized by macrophages. However, a number of receptors have been reported to mediate macrophage recognition of apoptotic cells, not all of which bind to phosphatidylserine. We therefore examined the role of membrane phospholipid symmetrization and PS externalization in uptake of apoptotic cells by mouse macrophages and human HT-1080 fibrosarcoma cells by exposing them to cells that had undergone apoptosis without loss of phospholipid asymmetry. Neither mouse macrophages nor HT-1080 cells recognized or engulfed apoptotic targets that failed to express PS, in comparison to PS-expressing apoptotic cells. If, however, their outer leaflets were repleted with the l-, but not the d-, stereoisomer of sn-1,2-PS by liposome transfer, engulfment by both phagocytes was restored. These observations directly demonstrate that loss of phospholipid asymmetry and PS expression is required for phagocyte engulfment of apoptotic cells and imply a critical, if not obligatory, role for PS recognition in the uptake process.     

Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.     

MFG-E8 and HMGB1 Are Involved in the Mechanism Underlying Alcohol-Induced Impairment of Macrophage Efferocytosis

Efferocytosis is a unique phagocytic process for macrophages to remove apoptotic cells in inflammatory loci. This event is maintained by milk fat globule-EGF factor 8 (MFG-E8), but attenuated by high mobility group box 1 (HMGB1). Alcohol abuse causes injury and inflammation in multiple tissues. It alters efferocytosis, but precise molecular mechanisms for this effect remain largely unknown. Here, we showed that acute exposure of macrophages to alcohol (25 mmol/L) inhibited MFG-E8 gene expression and impaired efferocytosis. The effect was mimicked by hydrogen peroxide. Moreover, N-acetylcysteine (NAC), a potent antioxidant, blocked acute alcohol effect on inhibition of macrophage MFG-E8 gene expression and efferocytosis. In addition, recombinant MFG-E8 rescued the activity of alcohol-treated macrophages in efferocytosis. Together, the data suggest that acute alcohol exposure impairs macrophage efferocytosis via inhibition of MFG-E8 gene expression through a reactive oxygen species dependent mechanism. Alcohol has been found to suppress or exacerbate immune cell activities depending on the length of alcohol exposure. Thus, we further examined the role of chronic alcohol exposure on macrophage efferocytosis. Interestingly, treatment of macrophages with alcohol for seven days in vitro enhanced MFG-E8 gene expression and efferocytosis. However, chronic feeding of mice with alcohol caused increase in HMGB1 levels in serum. Furthermore, HMGB1 diminished efferocytosis by macrophages that were treated chronically with alcohol, suggesting that HMGB1 might attenuate the direct effect of chronic alcohol on macrophage efferocytosis in vivo. Therefore, we speculated that the balance between MFG-E8 and HMGB1 levels determines pathophysiological effects of chronic alcohol exposure on macrophage efferocytosis in vivo.     

Calcium-Lipid Interactions Observed with Isotope-Edited Infrared Spectroscopy

Calcium ions bind to lipid membranes containing anionic lipids; however, characterizing the specific ion-lipid interactions in multicomponent membranes has remained challenging because it requires nonperturbative lipid-specific probes. Here, using a combination of isotope-edited infrared spectroscopy and molecular dynamics simulations, we characterize the effects of a physiologically relevant (2 mM) Ca²⁺ concentration on zwitterionic phosphatidylcholine and anionic phosphatidylserine lipids in mixed lipid membranes. We show that Ca²⁺ alters hydrogen bonding between water and lipid headgroups by forming a coordination complex involving the lipid headgroups and water. These interactions distort interfacial water orientations and prevent hydrogen bonding with lipid ester carbonyls. We demonstrate, experimentally, that these effects are more pronounced for the anionic phosphatidylserine lipids than for zwitterionic phosphatidylcholine lipids in the same membrane.     

Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells

During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways.The removal of apoptotic cells, known as efferocytosis, is a series of arranged events from the recruitment of phagocytes to sites where apoptotic cells are generated to the digestion of apoptotic cells by phagocytes.^{1, 2, 3} One of the key steps during efferocytosis is the recognition of dying cells by phagocytes. Phagocytes can detect apoptotic cells by the direct or indirect association of multiple receptors on phagocytes with ligands on apoptotic cells.^{4, 5, 6, 7, 8, 9} Some receptors on the surface of phagocytic cells not only bind to apoptotic cells but also transduce apoptotic cell recognition signals into phagocytes in order to mediate the ingestion of apoptotic cells. For instance, brain-specific angiogenesis inhibitor 1 (BAI1) and stabilin-2, which are phosphatidylserine (PtdSer) receptors, recognize PtdSer on apoptotic cells and relay signals to the Elmo-Dock-Rac module and Gulp, respectively, via their cytoplasmic tails.^{8, 10, 11} By contrast, it has been suggested that other receptors, called tethering receptors, merely tether apoptotic cells to phagocytes without mediating downstream signal transduction, following which the internalization of apoptotic cells is mediated by the association of these receptors with co-receptors or other engulfment receptors located nearby.^{12, 13, 14, 15, 16} However, it is unclear whether co-receptors for tethering receptors exist in tethering receptor-mediated phagocytosis of apoptotic cells, and, if they do, whether they are indispensable for this process.One intriguing characteristic of tethering receptors is that they have cytoplasmic tails lacking any signaling motifs or are anchored via glycophosphatidylinositol (GPI) to the outer leaflet of the plasma membrane. For example, Tim-4, a PtdSer receptor with a short cytoplasmic tail that promotes the engulfment of apoptotic cells by the binding of its IgV domain to PtdSer on apoptotic cells, lacks signaling motifs in its cytoplasmic tail. It has been known that neither the cytoplasmic tail nor the transmembrane region of Tim-4 is essential for Tim-4-mediated engulfment of apoptotic cells. Accordingly, it functions as a tethering receptor to secure apoptotic cells on phagocytes.^{9, 14} CD14 is located at the exofacial leaflet of the plasma membrane through its GPI anchor, which rules out the possibility that it mediates direct signal transduction into phagocytes after binding to apoptotic cells. Consequently, it is also considered to be a tethering receptor.¹⁵Phospholipids such as PtdSer and phosphatidylcholine (PtdCho) are unequally distributed between the inner and outer leaflets of the plasma membrane in the normal state. For instance, uncharged phospholipids such as PtdCho and sphingomyelin are primarily located in the outer leaflet, whereas positively or negatively charged phospholipids (such as phosphatidylethanolamine or PtdSer, respectively) are restricted to the inner leaflet facing the cytosol.^{17, 18, 19} However, this asymmetric distribution of phospholipids in the plasma membrane is disrupted during apoptosis. In the plasma membrane of apoptotic cells, PtdSer is exposed to the outer leaflet of the plasma membrane by the activity of scramblases and flippases.^{18, 20, 21} Thus, exposed PtdSer is a hallmark of apoptotic cells and is the best characterized ligand on apoptotic cells for efferocytosis. PtdSer on the surface of apoptotic cells can be recognized by various PtdSer-sensing membrane proteins on phagocytes, collectively called PtdSer receptors, including tethering receptors.Besides PtdSer receptors, many PtdSer-binding proteins have been identified. These proteins are involved in various biological processes such as blood coagulation, synaptic vesicle fusion, membrane scaffolding, and signal transduction.²² One of the best known proteins is annexin A5, which has been extensively studied as a PtdSer-binding protein. Annexin A5 belongs to the family of annexins, which are characterized by their Ca²⁺-dependent ability to bind to negatively charged phospholipids and share structural properties. Annexins are considered to be cytosolic proteins because they lack a 5′ leader sequence; however, some annexins, including annexin A5, are also found on the cell surface and in the circulation. This and related properties imply that annexins participate in diverse biological events from membrane dynamics to cell differentiation and migration.^{23, 24, 25} However, the physiological significance of this family is poorly understood. Among annexins, annexin A5 binds to PtdSer with high affinity. Because of this property, annexin A5 has been harnessed as a molecular probe to distinguish apoptotic cells from live cells both in vivo and in vitro for decades.^{25, 26}In this study, annexin A5 was expressed on the cell surface through a GPI anchor to delineate whether a tethering receptor without its co-receptor can promote efferocytosis. GPI-anchored annexin A5 (Anxa5-GPI) should not interact with any plasma membrane or extracellular protein, at least those involved in the engulfment of apoptotic cells. Thus, it is possible to exclude the effects of co-receptors on Anxa5-GPI-mediated phagocytosis of apoptotic cells. The expression of Anxa5-GPI in phagocytes promoted not only the binding but also the internalization of apoptotic cells. By contrast, phagocytosis of carboxylate beads and Escherichia coli was not affected by the expression of Anxa5-GPI in phagocytes. Anxa5-GPI-induced efferocytosis was not only partially dependent on a specific engulfment pathway but also relied on the generally known cytoskeletal engulfment machinery. Our observations suggest that co-receptors are dispensable for tethering receptor-mediated efferocytosis. In addition, tethering receptors could enhance efferocytosis through diverse engulfment machinery located nearby.     

Sialoside-binding macrophage lectins in phagocytosis of apoptotic bodies

Elimination of apoptotic bodies is one of the important functions of macrophages. The aim of this work was to study the role of macrophage lectins in this process. Macrophage lectins were probed with neoglycoconjugates Glyc-PAA-fluo where carbohydrate is linked to fluorescein-labeled polyacrylamide (MW 30 kD). It was shown that neoglycoconjugates containing a Neu5Ac2-3Gal fragment bound to macrophages isolated from blood of healthy donors. Besides, carbohydrate chains containing the same fragment were revealed on apoptotic bodies. Phagocytosis of apoptotic bodies by macrophages was inhibited with sialooligosaccharide ligands of siglec-5 and MAbs to siglec-5. Thus, siglec-5 expressed on macrophages could participate in phagocytosis of apoptotic bodies. In addition, the role of siglecs in engulfment of apoptotic bodies by tumor-associated macrophages was studied. The phagocytic potency of macrophages isolated from blood of breast cancer patients was lower than engulfment ability of macrophages obtained from healthy donors and depended on tumor degree. Staining of macrophages obtained from blood of tumor patients with sialylated Glyc-PAA-fluo probes was more intense than that of macrophages from healthy donors; phagocytosis of apoptotic bodies by tumor associated macrophages was inhibited by carbohydrates that are known to be ligands for siglecs.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 406–415.Original Russian Text Copyright © 2005 by Rapoport, Sapotko, Pazynina, Bojenko, Bovin.     

Interleukin-6 protects human macrophages from cellular cholesterol accumulation and attenuates the proinflammatory response

Cholesterol-laden monocyte-derived macrophages are phagocytic cells characteristic of early and advanced atherosclerotic lesions. Interleukin-6 (IL-6) is a macrophage secretory product that is abundantly expressed in atherosclerotic plaques but whose precise role in atherogenesis is unclear. The capacity of macrophages to clear apoptotic cells, through the efferocytosis mechanism, as well as to reduce cellular cholesterol accumulation contributes to prevent plaque progression and instability. By virtue of its capacity to promote cellular cholesterol efflux from phagocyte-macrophages, ABCA1 was reported to reduce atherosclerosis. We demonstrated that lipid loading in human macrophages was accompanied by a strong increase of IL-6 secretion. Interestingly, IL-6 markedly induced ABCA1 expression and enhanced ABCA1-mediated cholesterol efflux from human macrophages to apoAI. Stimulation of ABCA1-mediated cholesterol efflux by IL-6 was, however, abolished by selective inhibition of the Jak-2/Stat3 signaling pathway. In addition, we observed that the expression of molecules described to promote efferocytosis, i.e. c-mer proto-oncogene-tyrosine kinase, thrombospondin-1, and transglutaminase 2, was significantly induced in human macrophages upon treatment with IL-6. Consistent with these findings, IL-6 enhanced the capacity of human macrophages to phagocytose apoptotic cells; moreover, we observed that IL-6 stimulates the ABCA1-mediated efflux of cholesterol derived from the ingestion of free cholesterol-loaded apoptotic macrophages. Finally, the treatment of human macrophages with IL-6 led to the establishment of an anti-inflammatory cytokine profile, characterized by an increased secretion of IL-4 and IL-10 together with a decrease of that of IL-1β. Taken together, our results indicate that IL-6 favors the elimination of excess cholesterol in human macrophages and phagocytes by stimulation of ABCA1-mediated cellular free cholesterol efflux and attenuates the macrophage proinflammatory phenotype. Thus, high amounts of IL-6 secreted by lipid laden human macrophages may constitute a protective response from macrophages to prevent accumulation of cytotoxic-free cholesterol. Such a cellular recycling of free cholesterol may contribute to reduce both foam cell formation and the accumulation of apoptotic bodies as well as intraplaque inflammation in atherosclerotic lesions.