Cholesterol Accumulation by Macrophages Impairs Phagosome Maturation

Macrophages are key to the pathogenesis of atherosclerosis. They take up and store excessive amounts of cholesterol associated with modified low density lipoprotein, eventually becoming foam cells that display altered immune responsiveness. We studied the effects of cholesterol accumulation on phagosome formation and maturation, using lipid transport antagonists and cholesterol transport-deficient mutants. In macrophages treated with U18666A, a transport antagonist that prevents cholesterol exit from late endosomes/lysosomes, the early stages of maturation proceeded normally; phagosomes acquired Rab5, phosphatidylinositol 3-phosphate, and EEA1 and merged with LAMP-containing vesicles. However, fusion with lysosomes was impaired. Rab7, which is required for phagolysosome formation, was acquired by phagosomes but remained inactive. Maturation was also studied in fibroblasts from Niemann-Pick type C individuals that have defective cholesterol transport. Transfection of FcγIIA receptors was used to confer phagocytic capability to these fibroblasts. Niemann-Pick type C phagosomes failed to fuse with lysosomes, whereas wild type fibroblasts formed normal phagolysosomes. These findings indicate that cholesterol accumulation can have a detrimental effect on phagosome maturation by impairing the activation of Rab7, sequestering it and its effectors in cholesterol-enriched multilamellar compartments. Macrophages are key to the pathogenesis of atherosclerosis. They take up and store excessive amounts of cholesterol associated with modified low density lipoprotein, eventually becoming foam cells that display altered immune responsiveness. We studied the effects of cholesterol accumulation on phagosome formation and maturation, using lipid transport antagonists and cholesterol transport-deficient mutants. In macrophages treated with U18666A, a transport antagonist that prevents cholesterol exit from late endosomes/lysosomes, the early stages of maturation proceeded normally; phagosomes acquired Rab5, phosphatidylinositol 3-phosphate, and EEA1 and merged with LAMP-containing vesicles. However, fusion with lysosomes was impaired. Rab7, which is required for phagolysosome formation, was acquired by phagosomes but remained inactive. Maturation was also studied in fibroblasts from Niemann-Pick type C individuals that have defective cholesterol transport. Transfection of FcγIIA receptors was used to confer phagocytic capability to these fibroblasts. Niemann-Pick type C phagosomes failed to fuse with lysosomes, whereas wild type fibroblasts formed normal phagolysosomes. These findings indicate that cholesterol accumulation can have a detrimental effect on phagosome maturation by impairing the activation of Rab7, sequestering it and its effectors in cholesterol-enriched multilamellar compartments. Macrophages are key to the pathogenesis of atherosclerosis. They take up and store excessive amounts of cholesterol associated with oxidized low density lipoproteins through scavenger receptors, eventually becoming foam cells that display altered immune responsiveness. Circulating blood monocytes are attracted and activated by a variety of proinflammatory cytokines, including interleukin-1, -6, -8, -10, and -12 and tumor necrosis factor-α, produced by subendothelial cells in response to infiltrating lipoproteins (1Takahashi K. Takeya M. Sakashita N. Med. Electron Microsc. 2002; 35: 179-203Crossref PubMed Scopus (146) Google Scholar). In the arterial intima, monocytes differentiate into macrophages (2Bobryshev Y.V. Micron. 2006; 37: 208-222Crossref PubMed Scopus (275) Google Scholar), at least in part in response to macrophage colony-stimulating factor present in atherosclerotic lesions (3Clinton S.K. Underwood R. Hayes L. Sherman M.L. Kufe D.W. Libby P. Am. J. Pathol. 1992; 140: 301-316PubMed Google Scholar, 4Rosenfeld M.E. Yla-Herttuala S. Lipton B.A. Ord V.A. Witztum J.L. Steinberg D. Am. J. Pathol. 1992; 140: 291-300PubMed Google Scholar). These subendothelial macrophages proceed to ingest oxidized low density lipoprotein and other modified forms of low density lipoprotein by either endocytosis or phagocytosis, depending on the size of the particles. Scavenger receptors feature prominently in the uptake of modified LDL particles, but other receptors also contribute (1Takahashi K. Takeya M. Sakashita N. Med. Electron Microsc. 2002; 35: 179-203Crossref PubMed Scopus (146) Google Scholar, 5Vainio S. Ikonen E. Ann. Med. 2003; 35: 146-155Crossref PubMed Scopus (67) Google Scholar). A key event during atherogenesis is the conversion of blood-derived macrophages into foam cells, which are characterized by a massive accumulation of cholesterol esters in cytoplasmic lipid droplets (6Wustner D. Mondal M. Tabas I. Maxfield F.R. Traffic. 2005; 6: 396-412Crossref PubMed Scopus (83) Google Scholar). Lipid-laden foam cells are found within the subendothelial space of arteries in the fatty streaks of early lesions as well as in more advanced atherosclerotic plaques (7Lucas A.D. Greaves D.R. Expert Rev. Mol. Med. 2001; 3: 1-18Crossref PubMed Scopus (56) Google Scholar). Excessive uptake of oxidized low density lipoprotein is thought to be the primary cause of the accumulation of free and esterified cholesterol in foam cells. Normally, internalized lipids are delivered to lysosomes, where cholesterol esters are hydrolyzed. The released free cholesterol is then transported out of the lysosomes and, if present in excess, it is re-esterified in the endoplasmic reticulum for storage in lipid droplets (8Lange Y. Ye J. Chin J. J. Biol. Chem. 1997; 272: 17018-17022Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). During the early fatty streak phase of atherosclerotic disease, the inordinate amount of accumulated lipid generates the large cytoplasmic inclusions characteristic of foam cells. As the disease advances from a fatty streak to a fibrous plaque, lipid accumulates also in the lysosomes of macrophages. Lysosomes play a critical role in the innate immune function of macrophages by fusing with phagosomes. Insertion of lysosomal proton pumps is required for effective phagosomal acidification that, together with the delivery of microbicidal lysosomal contents, is key for the elimination of pathogens. Luminal acidification and protein degradation are also central to antigen presentation. These functions could in principle be altered by the excessive accumulation of cholesterol in the lysosomes of late stage foam cells. Remarkably little is known about the effects of excess lysosomal lipid accumulation on the immune function of macrophages. This paucity of information is most likely the result of technical limitations; it is virtually impossible to isolate and analyze primary late stage foam cells, and generation of foam cells in vitro by exposure to synthetically modified LDL is a time-consuming and expensive enterprise that has yielded varying results in different laboratories. In this report, we used pharmacological and genetic means to induce a pronounced and reliable accumulation of cholesterol in lysosomes and were able to study its consequences on the formation, acidification, and maturation of phagosomes. Reagents and Antibodies—Fetal bovine serum, Dulbecco's modified Eagle's medium, α-minimum Eagle's medium, Hepes-buffered solution RPMI 1640, and phosphate-buffered saline were from Wisent (St. Bruno, Canada). 3-β[2-(Diethylamino)-ethoxy]-androst-5-en-17-one (compound U18666A), o-phenylenediamine dihydrochloride, and human IgG were from Sigma. Sheep red blood cells (RBCs) 3The abbreviations and trivial name used are: RBC, red blood cell; NPC, Niemann-Pick type C; GFP, green fluorescent protein; RILP, Rab7-interacting lysosomal protein; U18666A, 3-β[2-(diethylamino)-ethoxy]-androst-5-en-17-one; PI(3)P, phosphatidylinositol 3-phosphate. and rabbit anti-sheep RBC antibody were from ICN-Cappel. LysoTracker Green and rhodamine-conjugated dextran (Mr 10,000) were from Molecular Probes, Inc. (Eugene, OR). Latex beads (3.87-μm diameter) were from Bangs Beads (Fishers, IN). Rat anti-mouse (ID4B) monoclonal antibodies to LAMP-1, rat anti-mouse monoclonal antibodies to LAMP-2 (ABL-93), mouse anti-human monoclonal antibodies to LAMP-1 (H4A3), and mouse anti-human monoclonal antibodies to LAMP-2 (H4B4) were from the Developmental Studies Hybridoma Bank, maintained by the University of Iowa and the Johns Hopkins University School of Medicine. Anti-EEA1 mouse monoclonal antibody was from CytoStore (Calgary, Canada). Antibodies against glyceraldehyde-3-phosphate dehydrogenase were purchased from Chemicon International. Alexa fluorochrome-conjugated antibodies were from Molecular Probes, whereas Cy2-, Cy3-, or Cy5-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Horseradish peroxidase-conjugated anti-mouse and anti-donkey antibodies were also from Jackson. Cell Culture and Transfection—RAW264.7 cells (called RAW cells hereafter) were obtained from the American Type Culture Collection (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium with 5! fetal bovine serum (v/v) at 37 °C in 5! CO2 under a humidified atmosphere. Where indicated, the cells were incubated overnight with the specified concentration of U18666A. Niemann-Pick type C (NPC) fibroblasts (cell lines 18684 and 20849) and control fibroblasts (cell lines 4212 and 4103) were a gift from Dr. John Callahan (Hospital for Sick Children, Toronto, Canada). The NPC and control fibroblasts were cultured in α-minimum Eagle's medium with 10! fetal bovine serum (v/v) and 1:100 antibiotic at 37 °C in 5! CO2 under a humidified atmosphere. Transfections were performed using either FuGene-6 or FuGene-HD (Roche Applied Science) according to the manufacturer's instructions, and the cells were used within 24–48 h of transfection. DNA Constructs—The generation of the plasmids used for the expression of wild-type GFP-conjugated Rab5 (9Scott C.C. Cuellar-Mata P. Matsuo T. Davidson H.W. Grinstein S. J. Biol. Chem. 2002; 277: 12770-12776Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and Rab7, Rab7(Q67L) (10Bucci C. Thomsen P. Nicoziani P. McCarthy J. van Deurs B. Mol. Biol. Cell. 2000; 11: 467-480Crossref PubMed Scopus (810) Google Scholar), and EEA1 (11Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.M. Parton R.G. Stenmark H. J. 2000; PubMed Scopus Google Scholar) is in was purchased from The was by FcγIIA into at and A.D. Grinstein S. J. 2002; PubMed Scopus Google Scholar). beads and RBC were used as phagocytic with Latex beads were with human IgG for h at or overnight at Sheep RBC were with anti-sheep IgG at for h at or overnight at RAW cells grown on were to Dulbecco's modified Eagle's medium and were with of beads or In most was by of the cells the of beads or at for The were incubated with the beads or RBC for at 37 as that were internalized were by with beads were from beads by with Cy3-, or anti-human IgG The cells were then at 37 °C to maturation to proceed for the The phagocytic is as the of phagosomes macrophages. This was by and the beads were on as a result of secondary with antibodies against human IgG or rabbit In the of NPC was for by secondary antibody of beads and then maturation for the lysosomal RAW cells were incubated overnight in medium with dextran (Mr 10,000) in the or of U18666A. The medium was then and the cells were in medium in the or of for h to delivery of to lysosomal compartments. A was to NPC and control fibroblasts. and of LAMP-1 or LAMP-2 by was performed and the cells in in 5! serum, and then with rat anti-mouse LAMP-1 or rat anti-mouse LAMP-2 at for h at with phosphate-buffered were incubated with antibody for h at and then and on using A was used to LAMP-2 and of cholesterol by was performed cells in by with and then for h at °C with of the and density of the was performed using either or a with a was performed using grown on were with wild-type or and 24–48 were into that were on the stage of the of the was and then the of was The of the was to the of the The was used to and for during Electron were in in for of the and was at for at least a were then in in at for for h with in and then and in in were on and with and were using and were using a were in were by on were to for h with 5! bovine serum in phosphate-buffered saline (v/v) with and then for h at with the primary antibody in with phosphate-buffered were incubated with peroxidase-conjugated secondary antibody for h at and then by of cells were grown on and treated with or U18666A. with the the cells were in and to LAMP-1 or LAMP-2 by a peroxidase-conjugated The amount of associated with the cells was then using the o-phenylenediamine by at dehydrogenase was used as a control and was were performed at least in of or of were as A of was were if the was the phagosome with with that of the from or were as the was the phagosome with that of the or was the and as A was if the was from the of the phagosome or if its the phagosome was of the effects of oxidized low density lipoprotein on lysosomal cholesterol we treated cultured RAW macrophages with U18666A, a cholesterol transport This has to cholesterol from lysosomes L. PubMed Scopus Google Scholar, L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of low density cholesterol to the in the accumulation of free cholesterol in the of late The of this its and low As for other cells, macrophages incubated h with or a accumulation of by with A and with K. I. P. M. C. Parton R. J. van PubMed Scopus Google Scholar), the excess cholesterol was found in large late of cells a in the and density of and multilamellar that are of the late stages of the C and other lines of that the excess cholesterol accumulated in LAMP-1 and of lysosomes, in large in cells treated with and The of the LAMP-containing was in part to a accumulation of LAMP-1 and LAMP-2 in the cells, as by and by the under the of the lysosomes was this was using a with a fluorescent and We proceeded to study the effect of lysosomal cholesterol accumulation on the immune of macrophages. we the of control and macrophages a result of to by The phagocytic as the of cells, was in cells treated or with U18666A. The phagocytic was by and with and U18666A, the of was the phagocytic to excess cholesterol the of lysosomes to fuse with formed phagosomes. maturation fusion of with the formed phagosomes with early and by fusion with late and with lysosomes in a and T. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We the effects of cholesterol on the stages of the maturation Rab5, a of early P. M. J. Cell. Full Text PDF PubMed Scopus Google Scholar), is the on phagosomes C. L. J. A.D. Grinstein S. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). the be by with we macrophages with a of wild-type As in Rab5 was in the of control phagosomes within of of Rab5 was present on the of phagosomes formed by cells the the of of Rab5 was normal and cells and for cells to and U18666A, Rab5 is in the of EEA1 and to phagosomes. We these stages of the maturation was used to the EEA1 on the phagosomal and was with of As found for Rab5, most of the early phagosomes were for and a of was found for control and macrophages C and in and activation of the phosphatidylinositol results in the accumulation of phosphatidylinositol on the phagosomal L. Matsuo T. Davidson H.W. J.M. Grinstein S. J. Cell Biol. 2001; PubMed Scopus Google Scholar). The formation of this can be in cells using of a protein to a fluorescent protein that to the of with and We RAW cells with the and its by and As was on the phagosomal and The accumulation of for the of phagocytosis, virtually phagosomes were of the and the the of were in cells treated with and and these findings indicate that phagocytic is the early stages of phagosomal maturation are by the excessive accumulation of cholesterol in late the of and phagosomes and which as of the stages of The effects of cholesterol on these were and macrophages were to for and maturation was to proceed for a by virtually of the phagosomes in control and cells were for LAMP-1 and As for Rab5, we to of a fluorescent to study the of a and maturation that for LAMP-1, we found that the of the phagosomes in control and cells and were for either in The in the maturation is the fusion of the phagosome with and are present in late and lysosomes, the can be by with phase This a well of a to the lysosomal by a to the from of the of the We used this to lysosomes with fluorescent in to the effect of cholesterol accumulation on phagolysosome The cells were then to and the phagosomes were to maturation for As in cells a of the of phagolysosome of the control phagosomes were In most of the phagosomes in cells were of and that with cholesterol the fusion of lysosomes with phagosomes. The of late phagosomes is to the density of This acidification the accumulation of a fluorescent We of this feature of to analyze by the effects of cholesterol accumulation on As by at h virtually of the phagosomes of control cells were by In the phagosomes of cells failed to the However, the lysosomes that The of the lysosomes to a low that of the of of cholesterol has to function Mol. Biol. Cell. PubMed Scopus Google Scholar, J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. H. P. N. J. J. J. 2002; PubMed Scopus Google Scholar, M. M. M. T. Mol. Biol. Cell. PubMed Scopus Google Scholar). fusion phagosomes and lysosomes is thought to be by in its C. Grinstein S. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). This was found to be to the phagosomal its activation was In the of activation of can be from its the with a and in the whereas the is However, the of to on the lipid of the C. M. H. P. N. J. J. J. 2002; PubMed Scopus Google Scholar), which is likely to be altered in lysosomes and phagosomes. the of activation of on we with is a that with the of and is a reliable of its activation P. C. J. 2001; PubMed Scopus Google Scholar). As P. C. J. 2001; PubMed Scopus Google Scholar), was present together with in late endosomes/lysosomes, and its the of these A and In control cells, and also in phagosomes A and In phagosomes acquired Rab7, were of RILP, that most of the present on phagosomes was C and In to its Rab7-interacting other protein was that of the protein to normal phagosomes was by other out this we the (called of that with As in associated with normal the of In cells, phagosomes were virtually of that the is to effectors and was but was to that of in the cells, but these to the phagosomal a that also on late has to be on J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). that the of be on this we of Rab7, with by and As in A and was associated with the that to was also on and of in cholesterol-enriched is at least in to its of from the This was from the of where the of of the and was In this of can result from with the of of size were in control and cells, and the of were as in and As in the of as well as the of the that was the were in cells in the The of is to and on This is with the findings of and C. M. H. P. N. J. J. J. 2002; PubMed Scopus Google Scholar), that the of was in in cells. The of to with cholesterol-enriched together with its paucity on that the cholesterol of phagosomes be in cells. This was by the cells with As in and cholesterol was in the of virtually control phagosomes. In phagosomes in cells were of the that the of cholesterol was and these that is in its on is from is thought to by impairing cholesterol accumulation of the lipid in late L. PubMed Scopus Google Scholar, L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). this primary effect is likely for the in phagosome maturation, a of which is to cholesterol is We to analyze the role of cholesterol in phagolysosome NPC is lipid storage associated with in and function is the by and to be in late transport of cholesterol and cause accumulation of cholesterol that that in cells. the has used to the NPC in K. I. P. M. C. Parton R. J. van PubMed Scopus Google Scholar, C. M. H. P. N. J. J. J. 2002; PubMed Scopus Google Scholar, E. M.E. PubMed Scopus Google Scholar). secondary effects of U18666A, we studied phagosome maturation also in NPC cells. primary are to and are from NPC we used fibroblasts. to fibroblasts from NPC used in this study accumulated cholesterol in A and lysosomal C and and amount of LAMP-1 and amounts of LAMP-1 and LAMP-2 were by and and are In to study phagosome maturation in the NPC fibroblasts were with the human phagocytic expression of this to confer phagocytic to a variety of and phagolysosome that is from that in Y. P. A.D. Grinstein S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). As in A and normal and NPC fibroblasts receptors and internalized particles. phagosomes of control and NPC fibroblasts acquired LAMP-1 during the of However, the control fibroblasts merged with lysosomes A and which were by with dextran as under whereas the NPC phagosomes failed to fuse In with the results obtained with we found that NPC control failed to the results were obtained using different NPC lines and control These obtained with that the maturation results from cholesterol accumulation and is by or effects of the fusion in NPC fibroblasts. and NPC fibroblasts were with and lysosomes were with fluorescent dextran using the in The cells were then to beads for to induce phagocytosis, and internalized beads as the phagosomes were to for are of control and NPC fibroblasts are as C and control and NPC fibroblasts were to beads in A and and then with of the of phagosomes that acquired are means of at least phagosomes for The control and NPC fibroblasts was The to whereas to phagosomes. the the altered immune of foam cells, we macrophages with cholesterol using and also studied primary fibroblasts with a genetic to which were in a role for cholesterol as a critical of phagosome we found that of free cholesterol the of lysosomes to fuse with phagosomes. The early stages of the maturation are Rab5 and are acquired and with normal This that the of maturation results from with a stage and is by to the In LAMP-1 was also acquired by phagosomes of cells. LAMP-1 was thought to be a of lysosomes but is known to be present also in late and, to a in other of the and In the of cells, LAMP-1 found on phagosomes from late or other from lysosomes, which to is that the fusion of of LAMP-1 with phagosomes from using of phosphatidylinositol L. Matsuo T. Davidson H.W. J.M. Grinstein S. J. Cell Biol. 2001; PubMed Scopus Google Scholar, J.M. J. S. J. Cell Biol. 2001; PubMed Scopus Google Scholar). the present in these to be critical of the phagolysosome fusion phagosome maturation is at early stage in cells from LAMP-1 and LAMP-2 C.C. P. Grinstein S. J. PubMed Scopus Google Scholar). was also to phagosomes in normal and cells. However, a was least a the associated with normal phagosomes was and of with was on phagosomes from cells. was in these cells, in with cholesterol-enriched that, C. M. H. P. N. J. J. J. 2002; PubMed Scopus Google Scholar), cholesterol Mol. Biol. Cell. PubMed Scopus Google Scholar), M. M. M. T. Mol. Biol. Cell. PubMed Scopus Google Scholar), Rab5, and J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) are also in cholesterol-enriched where are from by This the that most of the is in these becoming to fusion with the we found that the phagosomes of cells were of the of the lipid in the cells. of cholesterol have the of the phagosomes to or to to defective However, the these is required for phagosome maturation C. Grinstein S. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar), it is that of to prevents is well that in and cells, late a multilamellar amounts of in of critical other in be the cause of phagolysosome the normal of lipids from to other of is a key of known as that are critical for normal E. C. J. PubMed Scopus Google Scholar, H. T. K. M. T. J. 2002; PubMed Scopus Google Scholar, 2005; PubMed Scopus Google Scholar). However, a by K. I. P. M. C. Parton R. J. van PubMed Scopus Google Scholar) found in the of of cells to U18666A, in lipid We that the cause of the in and NPC cells is the accumulation of In accumulation of cholesterol in can cause in the of phagosomes. fusion with lysosomes, excessive cholesterol is to with the microbicidal role of phagosomes. In this is for immune function in macrophages with of lipids in in from is the accumulation of cholesterol in lysosomes of late stage foam cells and of macrophages incubated in vitro with modified low density In these defective phagolysosome formation can be This in the of for the well of with atherosclerotic with

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