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Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation

2016; Elsevier BV; Volume: 57; Issue: 6 Linguagem: Inglês

10.1194/jlr.m064089

1539-7262

Abigail S. Haka, Valéria C. Barbosa-Lorenzi, Hyuek Jong Lee, Domenick J. Falcone, Clifford A. Hudis, Andrew J. Dannenberg, Frederick R. Maxfield,

Immune cells in cancer

Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages. Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages. Macrophage interactions with adipocytes are important both in states of metabolic dysfunction and in healthy adipose tissue expansion and remodeling (1Hotamisligil G.S. 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Metab. 2013; 98: 1173-1181Crossref PubMed Scopus (91) Google Scholar). In the setting of obesity, it has been proposed that hypertrophic adipocytes release triglycerides and nonesterified fatty acids that the macrophage can then passively internalize using standard endocytic mechanisms (12Giordano A. Murano I. Mondini E. Perugini J. Smorlesi A. Severi I. Barazzoni R. Scherer P.E. Cinti S. Obese adipocytes show ultrastructural features of stressed cells and die of pyroptosis.J. Lipid Res. 2013; 54: 2423-2436Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). However, in this study, we show that, rather than endocytosis of released lipids, the macrophages themselves actively participate in lipid liberation from the adipocyte. Our laboratory and others have described a process in which large moieties or species tightly bound to the extracellular matrix are initially digested by macrophages in an extracellular acidic lytic compartment (13Zhang W.Y. Gaynor P.M. Kruth H.S. Aggregated low density lipoprotein induces and enters surface-connected compartments of human monocyte-macrophages. Uptake occurs independently of the low density lipoprotein receptor.J. Biol. Chem. 1997; 272: 31700-31706Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 14Grosheva I. Haka A.S. Qin C. Pierini L.M. Maxfield F.R. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1615-1621Crossref PubMed Scopus (33) Google Scholar, 15Haka A.S. Grosheva I. Chiang E. Buxbaum A.R. Baird B.A. Pierini L.M. Maxfield F.R. Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins.Mol. Biol. Cell. 2009; 20: 4932-4940Crossref PubMed Scopus (88) Google Scholar, 16Baron R. Neff L. Louvard D. Courtoy P.J. Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border.J. Cell Biol. 1985; 101: 2210-2222Crossref PubMed Scopus (595) Google Scholar, 17Stenbeck G. Formation and function of the ruffled border in osteoclasts.Semin. Cell Dev. Biol. 2002; 13: 285-292Crossref PubMed Scopus (97) Google Scholar, 18Jurdic P. Saltel F. Chabadel A. Destaing O. Podosome and sealing zone: specificity of the osteoclast model.Eur. J. Cell Biol. 2006; 85: 195-202Crossref PubMed Scopus (287) Google Scholar). We describe this process as exophagy. We have studied exophagy in the context of macrophage degradation of aggregated LDL, as occurs during atherogenesis (14Grosheva I. Haka A.S. Qin C. Pierini L.M. Maxfield F.R. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1615-1621Crossref PubMed Scopus (33) Google Scholar, 15Haka A.S. Grosheva I. Chiang E. Buxbaum A.R. Baird B.A. Pierini L.M. Maxfield F.R. Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins.Mol. Biol. Cell. 2009; 20: 4932-4940Crossref PubMed Scopus (88) Google Scholar). Exophagic catabolism of aggregated LDL results in uptake of cholesterol by the macrophage, leading to foam cell formation. While foam cell formation has been an area of extensive study in the atherosclerosis field, macrophage foam cell formation in CLSs has only been reported recently (11Shapiro H. Pecht T. Shaco-Levy R. Harman-Boehm I. Kirshtein B. Kuperman Y. Chen A. Bluher M. Shai I. Rudich A. Adipose tissue foam cells are present in human obesity.J. Clin. Endocrinol. Metab. 2013; 98: 1173-1181Crossref PubMed Scopus (91) Google Scholar). Given the similarities between these two systems, we examined whether exophagy could be responsible for macrophage degradation of dead adipocytes. This would allow extracellular catabolism and subsequent uptake of pieces of the adipocyte, facilitating macrophage foam cell formation as a consequence of clearing dead adipocytes. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may overwhelm the metabolic capacity of the macrophage, as has been demonstrated in the setting of atherosclerosis (19Moore K.J. Tabas I. Macrophages in the pathogenesis of atherosclerosis.Cell. 2011; 145: 341-355Abstract Full Text Full Text PDF PubMed Scopus (1801) Google Scholar), leading to a maladaptive inflammatory response. This biology may have particular relevance during clearance of dramatically enlarged adipocytes, as occurs in the setting of obesity. Herein, we present evidence for CLS macrophage exophagic clearance of dead adipocytes in mouse WAT. To model this biology, an in vitro CLS cell culture model was developed that mirrors several features of in vivo CLS macrophage-adipocyte interactions. Using this model, we demonstrate that CLS macrophages form an extracellular acidic hydrolytic compartment containing lysosomal enzymes delivered via exocytosis. Initial catabolism of the dead adipocyte occurs in these extracellular compartments, allowing the macrophage to internalize pieces of the adipocyte and transform it into a foam cell. We show that macrophage foam cell formation is specific to interaction with dead or dying adipocytes and is blocked when exophagy is inhibited. C57BL/6J wild-type male and female mice were purchased from Jackson Laboratory (Bar Harbor, ME). At 6 weeks of age, all male mice were randomized to receive either a low fat diet (LFD) or high fat diet (HFD) for 12 weeks. The LFD (12450Bi) and HFD (D12492i) contain 10 kcal% fat and 60 kcal% fat, respectively (Research Diets, New Brunswick, NJ) and are commonly used in studies of obesity (20Hong J. Stubbins R.E. Smith R.R. Harvey A.E. Nunez N.P. Differential susceptibility to obesity between male, female and ovariectomized female mice.Nutr. J. 2009; 8: 11Crossref PubMed Scopus (190) Google Scholar). Male mice were euthanized and epididymal fat was removed and fixed with 1% formalin for immunofluorescence analysis. At 5 weeks of age, ovariectomized C57BL/6J female mice received a HFD for 10 weeks. Following euthanization, mammary fat was removed and stained for immunofluorescence or paraffin blocks were prepared for hematoxylin and eosin (H&E) staining. For each patient, paraffin blocks were prepared. Samples were examined with H&E staining. Primary murine adipocytes were isolated from epididymal fat as described previously (21Motoshima H. Wu X. Sinha M.K. Hardy V.E. Rosato E.L. Barbot D.J. Rosato F.E. Goldstein B.J. Differential regulation of adiponectin secretion from cultured human omental and subcutaneous adipocytes: effects of insulin and rosiglitazone.J. Clin. Endocrinol. Metab. 2002; 87: 5662-5667Crossref PubMed Scopus (361) Google Scholar). Briefly, epididymal fat in DMEM/F-12 medium (Invitrogen, Carlsbad, CA) containing 1.0% BSA were chopped with surgical scissors and then digested with 0.2% collagenase type 2 (Worthington, Lakewood, NJ) for 25 min at 37°C. After passing the mixture through a 200 μm mesh filter to remove undigested fragments, the filtrate was centrifuged at 100 g for 10 min at 4°C. Separated adipocytes were collected with a disposable transfer pipette, washed with DMEM/F-12 medium, and attached to poly-D-lysine-coated coverslip dishes for microscopy. Coverslip dishes were functionalized with 6.15 mg/ml Bis(NHS)PEO5 (Pierce, Rockford, IL) in PBS (pH 9.0) for 1 h so that primary adipocytes would adhere to the dish rather than float. Dishes were then washed with HBSS, inverted, and placed on top of a cuvette containing primary adipocytes. Primary adipocytes were incubated with inverted functionalized coverslip dishes in HBSS (pH 7.8) for 1 h. To quench any unreacted N-hydroxysuccinimidyl ester (NHS) dishes were incubated with DMEM containing 50 mg/ml fatty acid-free BSA for 1 h. 3T3 L1 fibroblasts were cultured in DMEM supplemented with 10% calf serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. Cells were differentiated into adipocytes as described previously and used 7–10 days after differentiation (22Frost S.C. Lane M.D. Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes.J. Biol. Chem. 1985; 260: 2646-2652Abstract Full Text PDF PubMed Google Scholar). To induce apoptosis, adipocytes were either incubated with 25 nM TNF-α for 24 h (23Lin J. Page K.A. Della-Fera M.A. Baile C.A. Evaluation of adipocyte apoptosis by laser scanning cytometry.Int. J. Obes. Relat. Metab. Disord. 2004; 28: 1535-1540Crossref PubMed Scopus (15) Google Scholar) or coverslip bottom dishes were exposed to 365 nm UV radiation using a 2UV Transilluminator (UVP, Upland, CA) for 1 h. To induce pyroptosis, adipocytes were incubated with 10 ng/ml lipopolysaccharide for 4 h followed by a 2 h incubation with 10 μM nigericin (24Sagulenko V. Thygesen S.J. Sester D.P. Idris A. Cridland J.A. Vajjhala P.R. Roberts T.L. Schroder K. Vince J.E. Hill J.M. et al.AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC.Cell Death Differ. 2013; 20: 1149-1160Crossref PubMed Scopus (342) Google Scholar). Adipocyte death was confirmed with propidium iodide staining, performed according to the manufacturer's protocol (Clontech, Moutainview, CA). J774a.1 and RAW264.7 macrophage-like cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM supplemented with 10% FBS, 50 units/ml penicillin, and 50 μg/ml streptomycin in a humidified atmosphere (5% CO2) at 37°C. Bone marrow-derived macrophages (BMMs) isolated from C57BL/6 mice were differentiated for 7 days by culture in the same medium supplemented with 20% L-929 cell-conditioned medium. Human monocytes (Life Line Cell Technology, Frederick, MD) were differentiated into macrophages in vitro by incubation in RPMI containing 10% heat-inactivated FBS and 10 ng/ml macrophage colony stimulating factor (R&D Systems, Minneapolis, MN) for 7 days. For all live cell imaging experiments, medium was changed to DMEM containing 25 mM HEPES without phenol red or sodium bicarbonate. Adipocytes were labeled using succinimidyl esters of AlexaFluor (Alexa)546 and Alexa488 (Invitrogen), FITC, biotin (Sigma-Aldrich, St. Louis, MO), or CypHer 5E (GE Healthcare, Chalfont St. Giles, UK). Alexa546-biocytin, Alexa488-cholera toxin B (CtB), and LipidTOX-Red were purchased from Invitrogen. Streptavidin, bafilomycin A1, and protease inhibitor cocktail (P1860), containing aprotinin, bestatin, E-64, leupeptin, and pepstatin A, were purchased from Sigma-Aldrich. Whole-mounted epididymal fat from male mice on a LFD or HFD was incubated in 5% FBS for 1 h at room temperature for blocking after fixation with 1% formalin at 4°C for 12 h. To analyze macrophage plasma membrane lysosome-associated membrane protein-1 (LAMP-1) expression levels, murine adipose tissues were incubated with primary antibodies at 4°C for 12 h. After three washes with PBS, samples were incubated with secondary antibodies at room temperature for 4 h. For the detection of macrophage lysosomal LAMP-1 expression levels, adipose tissue was treated with 0.3% Triton for permeabilization. The primary antibodies used in this experiment were LAMP-1 (1:2,000, ab24170; Abcam, Cambridge, MA), F4/80 (1:2,000, MCA497R; AbD Serotec, Raleigh, NC), and calnexin (1:2,000, ab192439; Abcam). Anti-rabbit-cy3 (1:2,000), anti-rat-FITC (1:2,000), and anti-goat-cy5 (1:2,000) (Jackson ImmunoResearch, West Grove, PA) were used as secondary antibodies. Tissues were next stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) at room temperature for 10 min. For the detection of F-actin, epididymal fat from a male mouse on a HFD was treated with 0.3% Triton for 1 h at room temperature and then incubated with Alexa488-phalloidin (1:200, Invitrogen) at 4°C for 12 h. Images were acquired with a Zeiss LSM510 laser scanning confocal microscope using a 40× 0.8 numerical aperture (NA) objective. Data were analyzed with MetaMorph image analysis software, Molecular Devices Corporation (Downingtown, PA). Images were convolved with a 5 × 5 pixel Gaussian filter. For LAMP-1 analysis, a binary mask was created using the FITC signal intensity, to select F4/80 positive cells, and multiplied by an inverted binary mask created using the Cy5 signal, to select calnexin-negative nonpermeabilized cells. The resultant mask was then applied to the Cy3 channel to isolate LAMP-1 signals from nonpermeabilized macrophages (F4/80 positive and calnexin negative). The surface LAMP-1 signal in each image was divided by the number of cells in that field, as determined by DAPI staining, to generate an average value for plasma membrane LAMP-1 per cell. For permeabilized tissues, a binary mask was created using the FITC signal intensity to select F4/80 positive cells. This mask was applied to the Cy3 channel to isolate LAMP-1 signals from macrophages. The LAMP-1 signal was divided by the number of cells in each image, as determined by DAPI staining, to calculate a value for lysosomal LAMP-1 per macrophage. Plasma membrane labeling with Alexa488-CtB (15Haka A.S. Grosheva I. Chiang E. Buxbaum A.R. Baird B.A. Pierini L.M. Maxfield F.R. Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins.Mol. Biol. Cell. 2009; 20: 4932-4940Crossref PubMed Scopus (88) Google Scholar) and F-actin labeling with Alexa488-phalloidin (14Grosheva I. Haka A.S. Qin C. Pierini L.M. Maxfield F.R. Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1615-1621Crossref PubMed Scopus (33) Google Scholar) were performed as described previously. Plasma membrane labeling with Alex488-CtB was either followed by fixation in 3% paraformaldehyde (PFA) for 15 min (to allow the CtB on its lipid receptor to redistribute into the sealed zone between the adipocyte and macrophage following fixation) or 3% PFA and 0.5% glutaraldehyde for 30 min (to avidly cross-link plasma membrane proteins preventing postfixation diffusion of the CtB on its lipid receptor). Apoptotic adipocytes were labeled with various fluorophores or biotin conjugated to succinimidyl esters by incubation of the cells with 0.02 mg/ml of the conjugated fluorophore in DMEM for 1 h. Streptavidin labeling of adipocytes was accomplished by incubating biotin-labeled cells with 0.5 mg/ml streptavidin in DMEM for 1 h. To determine surface LAMP-1 expression in the CLS cell culture model, J774 cells were incubated with apoptotic adipocytes or live adipocytes, fixed without permeabilization in 1% PFA for 5 min, and incubated in PBS with 10% goat serum for 1 h. Next, cells were labeled with an antibody against LAMP-1 (Abcam) at 1:125 dilution in the presence of 3% goat serum for 60 min, washed, and incubated with anti-rabbit-Alexa488 at 1:250 dilution for 30 min. An irrelevant antibody was used as a control for LAMP-1 immunostaining. Surface LAMP-1 imaging was carried out with the pinholes open (to improve signal collection) resulting in an axial resolution of 14 μm. For imaging, cells were plated on poly-D-lysine-coated glass coverslip bottom dishes. Images were acquired with a Zeiss LSM510 laser scanning confocal microscope using either a 40× 0.8 NA plan Apochromat objective or a 63× 1.4 NA plan Apochromat objective. Lysosome labeling of macrophages plated in a tri-partitioned Petri dish was accomplished via an 18 h pulse with 2.2 mg/ml biotin-fluorescein-dextran. Cells were chased for 4 h in DMEM and subsequently incubated with apoptotic streptavidin-labeled adipocytes for 90 min. Labeling of extracellular streptavidin-conjugated adipocytes was accomplished with a 30 s pulse of 200 μM Alexa546-biocytin at the end of the 90 min incubation. Cells were incubated with 200 μM biotin for 10 min in order to bind any unoccupied streptavidin sites prior to cell permeabilization. Cells were then fixed with 3% PFA for 15 min, washed, and permeabilized with 1% Triton for 10 min. Images were acquired with a Zeiss LSM 510 laser scanning confocal microscope using a 40× 0.8 NA objective. Macrophages were incubated for 60 min with CypHer 5E (a pH sensitive fluorophore) and Alexa488 (a pH insensitive fluorophore) dual-labeled apoptotic adipocytes. The pH value within each pixel was assessed quantitatively by comparison with ratio images obtained in calibration buffers of varying pH, as described previously (15Haka A.S. Grosheva I. Chiang E. Buxbaum A.R. Baird B.A. Pierini L.M. Maxfield F.R. Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins.Mol. Biol. Cell. 2009; 20: 4932-4940Crossref PubMed Scopus (88) Google Scholar). Nuclear regions were excluded from the calibration curve, as the two dyes accumulated at different rates within the nucleus. Cell temperature was maintained at 37°C with a heated stage. All data were analyzed with MetaMorph image analysis software, Molecular Devices Corporation. A binary mask was created using the Alexa488 signal intensity and applied to both channels to remove background noise. Images were convolved with a 7 × 7 pixel Gaussian filter, and ratio images were generated. J774 cells were incubated with Alexa546-labeled TNF-α-induced apoptotic primary murine adipocytes for 90 min in the presence or absence of 2 μM bafilomycin A1. The macrophage plasma membrane was labeled with Alexa488-CtB, as described above, and samples were fixed with 1% PFA for 15 min. The Alexa546 signal in each macrophage touching an adipocyte was quantified. We note that the coculture system often contains small amounts of cellular debris from the apoptotic adipocytes. Macrophages may internalize this debris using endocytic mechanisms other than exophagy. To minimize the effects of adiopcyte cellular debris on our results, Alexa546 signal was only quantified in those macrophages in contact with an adipocyte. The cells in each image were identified using Alexa488-CtB cell surface staining. The fluorescence power was then calculated as the sum of all the pixel intensities within the cell boundaries. As a control, J774 cells were incubated with Fluoresbrite YG latex beads (Polysciences, Inc., Warrington, PA) for 90 min in the presence or absence of 2 μM bafilomycin A1. RAW264.7 cells were incubated with apoptotic 3T3 L1 adipocytes or primary murine adipocytes for 90 min. For transmission electron microscopy (EM), the cells were fixed with a modified Karnovsky's solution containing 2.5% glutaraldehyde, 4% PFA, and 0.02% picric acid, postfixed with 1% osmium tetroxide, 1.5% potassium ferricyanide, treated with uranyl acetate, dehydrated through a graded ethanol series, and embedded in LX112 resin. En face serial sections were cut at 70 nm thickness and picked up on formvar-coated 4-slot copper grids. Sections were further contrasted with uranyl acetate and lead citrate. Images were acquired at Weill Cornell Medical College on a JEOL JEM 100CX-II electron microscope operating at 80 kV. For 3D focused-ion beam scanning EM (FIB-SEM), the resin block was attached to a scanning EM stub with double-sided carbon tape and painted with colloidal silver. It was then coated with a thin layer of Au/Pd in an Ar environment (25 mA current, 60 mtorr, 5 min). The sample was loaded into an FEI Helios Nanolab 650 FIB-SEM for ion-abrasion tomography. An area of interest was found and coated with 1 μm Pt using the gas injection system of the microscope. A trench was dug in front of the area using the ion beam. Imaging of the cross-sectional face was done at an electron beam viewing angle of −30 degrees. The FEI Slice and View G2 programs were used to collect serial images using the following parameters: working distance, 2.4 mm; landing energy, 2 keV; current, 200 pA; horizontal field width, 41 μm; dwell time, 1 μs; 4× integration; 4096 pixels across (10 nm/pixel); 20 nm slice width; 1,500 images. Images were stacked into a single file, shrunk by a factor of 2 in X and Y then aligned using IMOD tools. The aligned stack was imported into Imaris (Bitplane, Oxford Instruments, Concord, MA) for segmentation and analysis. Sealed compartments contained no gap in electron density between the macrophage and apoptotic adipocytes (resolution 10 nm). J774 cells were incubated with primary murine apoptotic adipocytes for 24 h in the presence or absence of a protease inhibitor cocktail used at a 1:800 dilution. Samples were fixed with 1% PFA for 15 min and then labeled with LipidTOX-Red according to the manufacturer's protocol. The LipidTOX signal in each macrophage touching an adipocyte was calculated as the sum of all the pixel intensities within the cell boundaries. As a control, J774 cells were incubated with Fluoresbrite YG latex beads (Polysciences, Inc.) for 90 min in the presence or absence of protease inhibitor cocktail used at a 1:800 dilution. To examine foam cell formation in the presence of live or dead adipocytes, J774 cells were incubated with untreated 3T3 L1 adipocytes or TNF-α-induced apoptotic 3T3 L1 adipocytes for 24 h. The macrophage plasma membrane was labeled with Alexa488-CtB as described above. Samples were fixed with 1% PFA for 15 min, labeled with LipidTOX-Red according to the manufacturer's protocol, and the LipidTOX intensity per macrophage touching an adipocyte quantified. Statistical analysis was performed using Matlab R2012A (Mathworks, Natick, MA). Data acquired on different days were compared by normalizing the data from each day to the median control value for that day. Macrophage response to apoptotic adipocytes is heterogeneous, with some cells responding vigorously and others not at all. This results in a nonnormal distribution of the macrophage response. Thus, for such comparisons of two groups, a Wilcoxon rank sum test was used. In experiments where the data was normally distributed, a Student's t-test was performed. The animal protocol was approved by the Institutional Animal Care and Use Committee at Weill Cornell Medical College (New York, NY). Studies involving human tissue were approved by the Institutional Review Boards of Memorial Sloan-Kettering Cancer Center and Weill Cornell Medical College. Women undergoing mastectomy at Memorial Sloan-Kettering Cancer Center were consented under a standard tissue acquisition protocol. To see whether exophagy occurs during macrophage interactions with dead adipocytes, we first examined the amount of lysosome exocytosis in CLSs and resident macrophages in an established mouse model of obesity. As a marker of lysosome exocytosis, we quantified macrophage plasma membrane LAMP-1 levels. LAMP-1 on the surface of cells is normally at extremely low levels (25Huynh K.K. Eskelinen E.L. Scott C.C. Malevanets A. Saftig P. Grinstein S. LAMP proteins are required for fusion of lysosomes with phagosomes.EMBO J. 2007; 26: 313-324Crossref PubMed Scopus (468) Google Scholar, 26Harter C. Mellman I. Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane.J. Cell Biol. 1992; 117: 311-325Crossref PubMed Scopus (208) Google Scholar), and surface LAMP-1 expression has been shown to be a marker of fusion of lysosomes with the plasma membrane (27Colvin R.A. Means T.K. Diefenbach T.J. Moita L.F. Friday R.P. Sever S. Campanella G.S. Abrazinski T. Manice L.A. Moita C. et al.Synaptotagmin-mediated vesicle fusion regula