Macropinocytosis In Macrophages Research Articles

ATP2B4 is an essential gene for epidermal growth factor-induced macropinocytosis in A431 cells.

Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.

SARS-CoV-2 Spike Protein Stimulates Macropinocytosis in Murine and Human Macrophages via PKC-NADPH Oxidase Signaling.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While recent studies have demonstrated that SARS-CoV-2 may enter kidney and colon epithelial cells by inducing receptor-independent macropinocytosis, it remains unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated lung pneumonia, such as alveolar epithelial cells and macrophages. The goal of our study was to investigate the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in human alveolar epithelial cells and primary human and murine macrophages. Flow cytometry analysis of fluid-phase marker internalization demonstrated that SARS-CoV-2 spike protein subunits S1, the receptor-binding domain (RBD) of S1, and S2 stimulate macropinocytosis in both human and murine macrophages in an angiotensin-converting enzyme 2 (ACE2)-independent manner. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike-protein-induced fluid-phase marker internalization in macrophages both in vitro and in vivo. High-resolution scanning electron microscopy (SEM) imaging confirmed that spike protein subunits promote the formation of membrane ruffles on the dorsal surface of macrophages. Mechanistic studies demonstrated that SARS-CoV-2 spike protein stimulated macropinocytosis via NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS) generation. In addition, inhibition of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) in macrophages blocked SARS-CoV-2 spike-protein-induced macropinocytosis. To our knowledge, these results demonstrate for the first time that SARS-CoV-2 spike protein subunits stimulate macropinocytosis in macrophages. These results may contribute to a better understanding of SARS-CoV-2 infection and COVID-19 pathogenesis.

Abstract 15524: SARS-CoV-2 Spike Protein Stimulates Macropinocytosis in Murine and Human Macrophages

Coronavirus Disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The emergence of this virus has led to millions of deaths and the long-term cardiovascular and respiratory consequences of COVID-19 continue to pose a massive threat to public health. While recent studies have demonstrated that SARS-CoV-2 may enter kidney epithelial cells via angiotensin converting enzyme 2 (ACE2)-independent mechanisms by inducing receptor-independent macropinocytosis, it is currently unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated cardiovascular disease or pneumonia. Here, we investigated the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in alveolar epithelial cells and macrophages. Flow cytometry analysis of fluid-phase marker internalization demonstrated that SARS-CoV-2 spike protein subunits S1, the receptor binding domain (RBD) of S1, and S2 stimulate macropinocytosis in both human and murine macrophages, but not in human lung epithelial cells. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike protein-induced macropinocytosis in macrophages both in vitro and in vivo . High resolution scanning electron microscopy (SEM) imaging confirmed spike protein-induced plasma membrane activities characteristic of macropinocytosis. Mechanistic studies demonstrated that inhibition of protein kinase C and phosphoinositide 3-kinase in macrophages blocks SARS-CoV-2 spike protein-induced macropinocytosis. Further, pharmacological blockade of ACE2 did not inhibit macropinocytosis in SARS-CoV-2 spike protein-treated cells. To our knowledge, these results demonstrate for the first time that SARS-CoV-2 spike protein subunits stimulate macropinocytosis in macrophages. These results may contribute to a better understanding of SARS-CoV-2 infection and its cardiovascular and respiratory complications.

Anti-Atherogenic Actions of the Lab4b Consortium of Probiotics In Vitro

Probiotic bacteria have many protective effects against inflammatory disorders, though the mechanisms underlying their actions are poorly understood. The Lab4b consortium of probiotics contains four strains of lactic acid bacteria and bifidobacteria that are reflective of the gut of newborn babies and infants. The effect of Lab4b on atherosclerosis, an inflammatory disorder of the vasculature, has not yet been determined and was investigated on key processes associated with this disease in human monocytes/macrophages and vascular smooth muscle cells in vitro. The Lab4b conditioned medium (CM) attenuated chemokine-driven monocytic migration, monocyte/macrophage proliferation, uptake of modified LDL and macropinocytosis in macrophages together with the proliferation and platelet-derived growth factor-induced migration of vascular smooth muscle cells. The Lab4b CM also induced phagocytosis in macrophages and cholesterol efflux from macrophage-derived foam cells. The effect of Lab4b CM on macrophage foam cell formation was associated with a decrease in the expression of several key genes implicated in the uptake of modified LDL and induced expression of those involved in cholesterol efflux. These studies reveal, for the first time, several anti-atherogenic actions of Lab4b and strongly implicate further studies in mouse models of the disease in vivo and in clinical trials.

Liposome-mediated small RNA delivery to convert the macrophage polarity: A novel therapeutic approach to treat inflammatory uterine disease

Macrophages are present in all tissues for maintaining tissue homeostasis, and macrophage polarization plays a vital role in alleviating inflammation. Therefore, specific delivery of polarization modulators to macrophages in situ is critical for treating inflammatory diseases. We demonstrate that a size-controlled miRNA-encapsulated macrophage-targeting liposomes (miR/MT-Lip) specifically targets macrophages to promote M1-to-M2 polarization conversion, alleviating inflammation without cytotoxicity. miR/MT-Lip, approximately 1.2μm, showed excellent internalization through phagocytosis and/or macropinocytosis in macrophages. miR-10a/MT-Lip, but not scramble miR-Fluorescein amidite (FAM)/MT-Lip as control, effectively converted the polarization of lipopolysaccharide (LPS)-induced M1 macrophages to M2 invitro. When miR-10a/MT-Lip was intravenously delivered to mice insulted with LPS for inflammation, the proportion of M2 macrophages was significantly increased without disturbing the population of other immune cells. Furthermore, scramble miR-FAM/MT-Lip was mainly detected in macrophages, but not other immune cells. When our miR/MT-Lip was administered to mice with Asherman's syndrome that suffer from infertility because of sterile uterine inflammation, macrophage-specific targeting of miR-10a/MT-Lip facilitated M1-to-M2 conversion for angiogenesis in the impaired uterus, resulting in restoration of healthy uterine conditions. The results indicate that our MT-Lip encapsulating small RNAs has excellent potential to treat various inflammatory disorders by fine-tuning macrophage polarization invivo without any side effects.

Loss of GTPase activating protein neurofibromin stimulates paracrine cell communication via macropinocytosis

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) tumor suppressor gene, is a negative regulator of Ras signaling. Patients with mutations in NF1 have a strong predisposition for cardiovascular disease, which contributes to their early mortality. Nf1 heterozygous (Nf1+/−) bone marrow to wild type chimeras and mice with heterozygous recombination of Nf1 in myeloid cells recapitulate many of the vascular phenotypes observed in Nf1+/− mutants. Although these results suggest that macrophages play a central role in NF1 vasculopathy, the underlying mechanisms are currently unknown. In the present study, we employed macrophages isolated from either Nf1+/− or Lysm Cre+/Nf1f/f mice to test the hypothesis that loss of Nf1 stimulates macropinocytosis in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated that loss of Nf1 in macrophages stimulates macropinocytosis. We next utilized various cellular and molecular approaches, pharmacological inhibitors and genetically modified mice to identify the signaling mechanisms mediating macropinocytosis in Nf1-deficient macrophages. Our results indicate that loss of Nf1 stimulates PKCδ-mediated p47phox phosphorylation via RAS activation, leading to increased NADPH oxidase 2 activity, reactive oxygen species generation, membrane ruffling and macropinocytosis. Interestingly, we also found that Nf1-deficient macrophages internalize exosomes derived from angiotensin II-treated endothelial cells via macropinocytosis in vitro and in the peritoneal cavity in vivo. As a result of exosome internalization, Nf1-deficient macrophages polarized toward an inflammatory M1 phenotype and secreted increased levels of proinflammatory cytokines compared to controls. In conclusion, the findings of the present study demonstrate that loss of Nf1 stimulates paracrine endothelial to myeloid cell communication via macropinocytosis, leading to proinflammatory changes in recipient macrophages.

Constitutive and stimulated macropinocytosis in macrophages: roles in immunity and in the pathogenesis of atherosclerosis.

Macrophages respond to several stimuli by forming florid membrane ruffles that lead to fluid uptake by macropinocytosis. This type of induced macropinocytosis, executed by a variety of non-malignant and malignant cells, is initiated by transmembrane receptors and is involved in nutrient acquisition and mTOR signalling. However, macrophages also perform a unique type of constitutive ruffling and macropinocytosis that is dependent on the presence of extracellular calcium. Calcium-sensing receptors are responsible for this activity. This distinct form of macropinocytosis enables macrophages to continuously sample their microenvironment for antigenic molecules and for pathogen- and danger-associated molecular patterns, as part of their immune surveillance functions. Interestingly, even within the monocyte lineage, there are differences in macropinocytic ability that reflect the polarized functional roles of distinct macrophage subsets. This review discusses the shared and distinct features of both induced and constitutive macropinocytosis displayed by the macrophage lineage and their roles in physiology, immunity and pathophysiology. In particular, we analyse the role of macropinocytosis in the uptake of modified low-density lipoprotein (LDL) and its contribution to foam cell and atherosclerotic plaque formation. We propose a combined role of scavenger receptors and constitutive macropinocytosis in oxidized LDL uptake, a process we have termed 'receptor-assisted macropinocytosis'. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.

PKCδ stimulates macropinocytosis via activation of SSH1-cofilin pathway

Macropinocytosis is an actin-dependent endocytic mechanism mediating internalization of extracellular fluid and associated solutes into cells. The present study was designed to identify the specific protein kinase C (PKC) isoform(s) and downstream effectors regulating actin dynamics during macropinocytosis. We utilized various cellular and molecular biology techniques, pharmacological inhibitors and genetically modified mice to study the signaling mechanisms mediating macropinocytosis in macrophages. The qRT-PCR experiments identified PKCδ as the predominant PKC isoform in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated the functional role of PKCδ in phorbol ester- and hepatocyte growth factor (HGF)-induced macropinocytosis. Western blot analysis demonstrated that phorbol ester and HGF stimulate activation of slingshot phosphatase homolog 1 (SSH1) and induce cofilin Ser-3 dephosphorylation via PKCδ in macrophages. Silencing of SSH1 inhibited cofilin dephosphorylation and macropinocytosis stimulation. Interestingly, we also found that incubation of macrophages with BMS-5, a potent inhibitor of LIM kinase, does not stimulate macropinocytosis. In conclusion, the findings of the present study demonstrate a previously unidentified mechanism by which PKCδ via activation of SSH1 and cofilin dephosphorylation stimulates membrane ruffle formation and macropinocytosis. The results of the present study may contribute to a better understanding of the regulatory mechanisms during macrophage macropinocytosis.

Akt3 kinase suppresses pinocytosis of low-density lipoprotein by macrophages via a novel WNK/SGK1/Cdc42 protein pathway

Fluid-phase pinocytosis of LDL by macrophages is regarded as a novel promising target to reduce macrophage cholesterol accumulation in atherosclerotic lesions. The mechanisms of regulation of fluid-phase pinocytosis in macrophages and, specifically, the role of Akt kinases are poorly understood. We have found previously that increased lipoprotein uptake via the receptor-independent process in Akt3 kinase-deficient macrophages contributes to increased atherosclerosis in Akt3-/- mice. The mechanism by which Akt3 deficiency promotes lipoprotein uptake in macrophages is unknown. We now report that Akt3 constitutively suppresses macropinocytosis in macrophages through a novel WNK1/SGK1/Cdc42 pathway. Mechanistic studies have demonstrated that the lack of Akt3 expression in murine and human macrophages results in increased expression of with-no-lysine kinase 1 (WNK1), which, in turn, leads to increased activity of serum and glucocorticoid-inducible kinase 1 (SGK1). SGK1 promotes expression of the Rho family GTPase Cdc42, a positive regulator of actin assembly, cell polarization, and pinocytosis. Individual suppression of WNK1 expression, SGK1, or Cdc42 activity in Akt3-deficient macrophages rescued the phenotype. These results demonstrate that Akt3 is a specific negative regulator of macropinocytosis in macrophages.

Cytokine-induced macropinocytosis in macrophages is regulated by 14-3-3ζ through its interaction with serine-phosphorylated coronin 1.

The induction of macropinocytosis in macrophages during an inflammatory response is important for clearance of pathogenic microbes as well as the generation of appropriate immune responses. Recent data suggest that cytokine stimulation of macrophages induces macropinocytosis through phosphorylation of the protein coronin 1, thereby redistributing coronin 1 from the cell cortex to the cytoplasm followed by the activation of phosphoinositol-3 (PI-3) kinase. However, how coronin 1 phosphorylation regulates these processes remains unclear. We here define an essential role for 14-3-3ζ in cytokine-induced and coronin-1-dependent macropinocytosis in macrophages. We found that, upon stimulation, phosphorylated coronin 1 transiently associated with 14-3-3ζ and receptor of activated C kinase 1 (RACK1). Importantly, downregulation of 14-3-3ζ, but not RACK1, prevented relocation of coronin 1, as well as the induction of PI-3 kinase activity and thereby macropinocytosis upon cytokine stimulation. Together these data define an essential role for 14-3-3ζ in the regulation of macropinocytosis in macrophages upon cytokine stimulation through modulation of the localization of coronin 1.

Differential signaling during macropinocytosis in response to M-CSF and PMA in macrophages.

The cellular movements that construct a macropinosome have a corresponding sequence of chemical transitions in the cup-shaped region of plasma membrane that becomes the macropinosome. To determine the relative positions of type I phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) in this pathway, we analyzed macropinocytosis in macrophages stimulated by the growth factor macrophage-colony-stimulating factor (M-CSF) and by the diacylglycerol (DAG) analog phorbol 12-myristate 13-acetate (PMA). In cells stimulated with M-CSF, microscopic imaging of fluorescent probes for intracellular lipids indicated that the PI3K product phosphatidylinositol (3,4,5)-trisphosphate (PIP3) appeared in cups just prior to DAG. We then tested the hypothesis that PMA and DAG function after PI3K and prior to Ras and protein kinase C (PKC) during macropinosome formation in macrophages. Although the PI3K target Akt was activated by M-CSF, the Akt inhibitor MK-2206 did not inhibit macropinocytosis. The phospholipase C (PLC) inhibitor U73122 blocked macropinocytosis by M-CSF but not PMA. Macropinocytosis in response to M-CSF and PMA was inhibited by the Ras inhibitor farnesyl thiosalicylate (FTS), by the PKC inhibitor Calphostin C and by the broad specificity inhibitor rottlerin. These studies support a model in which M-CSF stimulates PI3K in macropinocytic cups, and the resulting increase in PIP3 activates PLC, which in turn generates DAG necessary for activation of PKC, Ras and the late stages of macropinosome closure.

Sorting nexin 5 (SNX5) selectively regulates dorsal ruffle-mediated macropinocytosis in primary macrophages

The regulation of macropinocytosis, a specialised endocytosis pathway, is important for immune cell function. However, it is not known whether the biogenesis of macropinosomes involves one or more distinct pathways. We previously identified sorting nexin 5 (SNX5) as a regulator of macropinocytosis in macrophages. Here, we show that bone-marrow-derived macrophages from SNX5-knockout mice had a 60-70% reduction in macropinocytic uptake of dextran or ovalbumin, whereas phagocytosis and retrograde transport from the plasma membrane to the Golgi was unaffected. In contrast, deficiency of SNX5 had no effect on macropinocytosis or antigen presentation by dendritic cells. Activation of macrophages with CSF-1 resulted in a localisation of SNX5 to actin-rich ruffles in a manner dependent on receptor tyrosine kinases. SNX5-deficient macrophages showed a dramatic reduction in ruffling on the dorsal surface following CSF-1 receptor activation, whereas peripheral ruffling and cell migration were unaffected. We demonstrate that SNX5 is acting upstream of actin polymerisation following CSF-1 receptor activation. Overall, our findings reveal the important contribution of dorsal ruffing to receptor-activated macropinocytosis in primary macrophages and show that SNX5 selectively regulates macropinosomes derived from the dorsal ruffles.

Differential regulation of macropinocytosis in macrophages by cytokines: Implications for foam cell formation and atherosclerosis

A key event during the formation of lipid-rich foam cells during the progression of atherosclerosis is the uptake of modified low-density lipoproteins (LDL) by macrophages in response to atherogenic mediators in the arterial intima. In addition to scavenger receptor-dependent uptake of LDL, macropinocytosis is known to facilitate the uptake of LDL through the constitutive and passive internalization of large quantities of extracellular solute. In this study we confirm the ability of macropinocytosis to facilitate the uptake of modified LDL by human macrophages and show its modulation by TGF-β, IFN-γ, IL-17A and IL-33. Furthermore we show that the TGF-β-mediated inhibition of macropinocytosis is a Smad-2/-3-independent process.

Abstract 912: Spleen Tyrosine Kinase Regulates TLR4 dependent Macropinocytosis And Lipid Accumulation In Macrophages

Toll-like receptors play a key role in the signaling pathways of innate immune response and have been recently shown to contribute to vascular inflammation and atherosclerosis. Oxidation of LDL is considered a leading mechanism of atherogenesis, and in our previous studies, we demonstrated that minimally modified LDL (mmLDL) induced TLR4-dependent chemokine secretion as well as robust actin polymerization and spreading of macrophages. We noticed that these mmLDL-induced cytoskeletal rearrangements led to extensive vacuolization, characteristic of macropinocytosis. In this study, we examined the mechanisms of TLR4-induced actin polymerization and macropinocytosis. It has been reported that spleen tyrosine kinase (Syk) regulates actin cytoskeleton and that it may interact with TLR4. We found in the immunoprecipitation experiments that mmLDL induced Syk association with TLR4 as well as Syk phosphorylation. Next we developed a J774 macrophage cell line, which stably expressed Syk shRNA and had the Syk expression reduced by 70% or more. Using this Syk-deficient macrophage cell line and specific Syk inhibitors, we demonstrated that Syk is necessary for mmLDL-induced activation of Ras and phosphorylation of Raf-MEK1-ERK1/2. The ERK1/2 activation resulted in phosphorylation of paxillin and an increase in the formation of N-WASP/Arp2 complex, which initiates actin polymerization. Taken together, these results suggest that Syk is a critical kinase in the mmLDL macrophage activation and that the mmLDL-induced cytoskeleton signaling. In addition, we demonstrated that mmLDL induced macrophage uptake of native LDL and of fluorescent 10 KDa dextran, and that this effect was Syk-dependent. These data suggest a novel pathway for the mmLDL-induced macropinocytosis in macrophages, which may constitute a mechanism for macrophage lipid accumulation and foam cell formation, a characteristic step in the development of atherosclerosis.

Survival of Mycobacteria in Macrophages Is Mediated by Coronin 1-Dependent Activation of Calcineurin

Pathogenic mycobacteria survive within macrophages by avoiding lysosomal delivery, instead residing in mycobacterial phagosomes. Upon infection, the leukocyte-specific protein coronin 1 is actively recruited to mycobacterial phagosomes, where it blocks lysosomal delivery by an unknown mechanism. Analysis of macrophages from coronin 1-deficient mice showed that coronin 1 is dispensable for F-actin-dependent processes such as phagocytosis, motility, and membrane ruffling. However, upon mycobacterial infection, coronin 1 was required for activation of the Ca(2+)-dependent phosphatase calcineurin, thereby blocking lysosomal delivery of mycobacteria. In the absence of coronin 1, calcineurin activity did not occur, resulting in lysosomal delivery and killing of mycobacteria. Furthermore, blocking calcineurin activation with cyclosporin A or FK506 led to lysosomal delivery and intracellular mycobacterial killing. These results demonstrate a role for coronin 1 in activating Ca(2+) dependent signaling processes in macrophages and reveal a function for calcineurin in the regulation of phagosome-lysosome fusion upon mycobacterial infection.

Macropinocytosis Is the Endocytic Pathway That Mediates Macrophage Foam Cell Formation with Native Low Density Lipoprotein

Previously, we reported that fluid-phase endocytosis of native LDL by PMA-activated human monocytederived macrophages converted these macrophages into cholesterol-enriched foam cells (Kruth, H. S., Huang, W., Ishii, I., and Zhang, W. Y. (2002) J. Biol. Chem. 277, 34573-34580). Uptake of fluid by cells can occur either by micropinocytosis within vesicles (<0.1 microm diameter) or by macropinocytosis within vacuoles ( approximately 0.5-5.0 microm) named macropinosomes. The current investigation has identified macropinocytosis as the pathway for fluid-phase LDL endocytosis and determined signaling and cytoskeletal components involved in this LDL endocytosis. The phosphatidylinositol 3-kinase inhibitor, LY294002, which inhibits macropinocytosis but does not inhibit micropinocytosis, completely blocked PMA-activated macrophage uptake of fluid and LDL. Also, nystatin and filipin, inhibitors of micropinocytosis from lipid-raft plasma membrane domains, both failed to inhibit PMA-stimulated macrophage cholesterol accumulation. Time-lapse video phase-contrast microscopy and time-lapse digital confocal-fluorescence microscopy with fluorescent DiI-LDL showed that PMA-activated macrophages took up LDL in the fluid phase by macropinocytosis. Macropinocytosis of LDL depended on Rho GTPase signaling, actin, and microtubules. Bafilomycin A1, the vacuolar H+-ATPase inhibitor, inhibited degradation of LDL and caused accumulation of undegraded LDL within macropinosomes and multivesicular body endosomes. LDL in multivesicular body endosomes was concentrated >40-fold over its concentration in the culture medium consistent with macropinosome shrinkage by maturation into multivesicular body endosomes. Macropinocytosis of LDL taken up in the fluid phase without receptor-mediated binding of LDL is a novel endocytic pathway that generates macrophage foam cells. Macropinocytosis in macrophages and possibly other vascular cells is a new pathway to target for modulating foam cell formation in atherosclerosis.

Phosphoinositide-3-kinase-independent contractile activities associated with Fcgamma-receptor-mediated phagocytosis and macropinocytosis in macrophages.

Previous studies have shown that Fcgamma receptor (FcR)-mediated phagocytosis and macropinocytosis in macrophages consist of two dissociable activities: a phosphoinositide 3-kinase (PI3K)-independent extension of phagocytic cups and a PI3K-dependent contractile mechanism that closes phagosomes and ruffles into intracellular organelles. Here, we identify an additional contractile activity that persists in the presence of the PI3K inhibitor wortmannin. ML-7, an inhibitor of myosin-light-chain kinase (MLCK), inhibited FcR-mediated phagocytosis, macropinocytosis and cell movements associated with ruffling. Scanning electron microscopy demonstrated a striking difference in morphology between phagocytic cups in the different inhibitors: whereas phagocytic cups of control cells and wortmannin-treated cells conformed closely to particles and appeared to have constricted them, the phagocytic cups in cells treated with ML-7 were more open. Video microscopy of macrophages expressing green-fluorescent-protein (GFP)-actin fusions revealed that bound IgG-opsonized erythrocytes were squeezed during phagosome formation and closure. In ML-7, GFP-actin-rich protrusions extended outward but failed to squeeze particles. Moreover, in contrast to the effects of PI3K inhibitors, ML-7 markedly reduced ruffle movement, and perturbed circular ruffle formation. These PI3K-independent myosin-II-based contractile activities that squeeze phagocytic cups and curve ruffles therefore represent a third component activity of the actin cytoskeleton during phagocytosis and macropinocytosis.

Hypoglossal-Facial Anastomosis

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) tumor suppressor gene, is a negative regulator of Ras signaling. Patients with mutations in NF1 have a strong predisposition for cardiovascular disease, which contributes to their early mortality. Nf1 heterozygous (Nf1+/−) bone marrow to wild type chimeras and mice with heterozygous recombination of Nf1 in myeloid cells recapitulate many of the vascular phenotypes observed in Nf1+/− mutants. Although these results suggest that macrophages play a central role in NF1 vasculopathy, the underlying mechanisms are currently unknown. In the present study, we employed macrophages isolated from either Nf1+/− or Lysm Cre+/Nf1f/f mice to test the hypothesis that loss of Nf1 stimulates macropinocytosis in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated that loss of Nf1 in macrophages stimulates macropinocytosis. We next utilized various cellular and molecular approaches, pharmacological inhibitors and genetically modified mice to identify the signaling mechanisms mediating macropinocytosis in Nf1-deficient macrophages. Our results indicate that loss of Nf1 stimulates PKCδ-mediated p47phox phosphorylation via RAS activation, leading to increased NADPH oxidase 2 activity, reactive oxygen species generation, membrane ruffling and macropinocytosis. Interestingly, we also found that Nf1-deficient macrophages internalize exosomes derived from angiotensin II-treated endothelial cells via macropinocytosis in vitro and in the peritoneal cavity in vivo. As a result of exosome internalization, Nf1-deficient macrophages polarized toward an inflammatory M1 phenotype and secreted increased levels of proinflammatory cytokines compared to controls. In conclusion, the findings of the present study demonstrate that loss of Nf1 stimulates paracrine endothelial to myeloid cell communication via macropinocytosis, leading to proinflammatory changes in recipient macrophages.