Constitutive Receptor-independent Low Density Lipoprotein Uptake and Cholesterol Accumulation by Macrophages Differentiated from Human Monocytes with Macrophage-Colony-stimulating Factor (M-CSF)

Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352–2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors. Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352–2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors. Macrophage cholesterol accumulation is considered a critical process in the development of atherosclerotic plaques, the cause of most heart attacks and strokes. LDL, 2The abbreviations used are: LDL, low density lipoprotein; HDL, high density lipoprotein; FBS, fetal bovine serum; DPBS, Dulbecco's phosphate-buffered saline; BSA, bovine serum albumin; IL-10, interleukin-10; PMA, phorbol 12-myristate 13-acetate; M-CSF, macrophage-colony-stimulating factor; FITC, fluorescein isothiocyanate.2The abbreviations used are: LDL, low density lipoprotein; HDL, high density lipoprotein; FBS, fetal bovine serum; DPBS, Dulbecco's phosphate-buffered saline; BSA, bovine serum albumin; IL-10, interleukin-10; PMA, phorbol 12-myristate 13-acetate; M-CSF, macrophage-colony-stimulating factor; FITC, fluorescein isothiocyanate. the main carrier of blood cholesterol, enters the blood vessel wall and by some mechanism, monocyte-derived macrophages take up the LDL in the vessel wall. Macrophage cholesterol accumulation converts the macrophages into so-called foam cells and stimulates the macrophages to secrete proteases and tissue factor that contribute to plaque rupture and thrombosis, respectively (1Galis Z.S. Sukhova G.K. Kranzhofer R. Clark S. Libby P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 402-406Crossref PubMed Scopus (515) Google Scholar, 2Lesnik P. Rouis M. Skarlatos S. Kruth H.S. Chapman M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10370-10374Crossref PubMed Scopus (73) Google Scholar, 3Rouis M. Nigon F. Lafuma C. Hornebeck W. Chapman M.J. Arteriosclerosis. 1990; 10: 246-255Crossref PubMed Google Scholar). Because incubation of macrophages with native LDL did not produce substantial macrophage cholesterol accumulation in past studies, it was proposed that LDL modifications that promote recognition and uptake of large amounts of the modified LDL by macrophage scavenger receptors is required to generate macrophage foam cells (4Brown M.S. Goldstein J.L. Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar). LDL oxidation has been the most commonly studied LDL modification that stimulates LDL uptake (5Steinberg D. J. Biol. Chem. 1997; 272: 20963-20966Abstract Full Text Full Text PDF PubMed Scopus (1453) Google Scholar). Recently, we have shown that treatment of human monocyte-derived macrophages with protein kinase C activators such as PMA stimulates fluid-phase macropinocytosis of native (unmodified) LDL (6Kruth H.S. Huang W. Ishii I. Zhang W.Y. J. Biol. Chem. 2002; 277: 34573-34580Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 7Kruth H.S. Jones N.L. Huang W. Zhao B. Ishii I. Chang J. Combs C.A. Malide D. Zhang W.Y. J. Biol. Chem. 2005; 280: 2352-2360Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). This results in uptake of LDL in amounts directly proportional to its concentration in the medium. As a result, incubation of human monocyte-derived macrophages with LDL at concentrations similar to those that exist within the normal vessel wall (0.7–2.7 mg/ml) (8Smith E.B. Ashall C. Biochim. Biophys. Acta. 1983; 754: 249-257Crossref PubMed Scopus (48) Google Scholar, 9Smith E.B. Eur. Heart J. 1990; 11: 72-81Crossref PubMed Google Scholar, 10Hoff H.F. Gaubatz J.W. Gotto Jr., A.M. Biochem. Biophys. Res. Commun. 1978; 85: 1424-1430Crossref PubMed Scopus (32) Google Scholar) leads to macrophage foam cell formation. Human monocytes can differentiate into macrophage phenotypes with different morphology and function depending on the culture conditions and differentiation factors included in the culture medium (11Akagawa K.S. Int. J. Hematol. 2002; 76: 27-34Crossref PubMed Scopus (144) Google Scholar). In the studies discussed above, we differentiated human monocytes into macrophages in the presence of human serum, producing the macrophage phenotype that resembles a “fried egg.” In the present study, we show that differentiation of human monocytes into macrophages in the presence of fetal bovine serum with added M-CSF produces a macrophage phenotype having an elongated shape and showing high levels of constitutive macropinocytosis not dependent on activation of the macrophage with PMA. The constitutive macropinocytosis in these macrophages mediated fluid-phase uptake of LDL causing high levels of macrophage cholesterol accumulation. Thus, our findings demonstrate a macrophage model system showing constitutive cholesterol accumulation without the need for either LDL modification or LDL uptake mediated by receptors. Culture of Human Monocyte-derived Macrophages—Human monocytes were purified with counterflow centrifugal elutriation of mononuclear cells obtained by monocytopheresis of normal human donors. M-CSF monocyte-derived macrophage cultures were begun with the elutriated human monocytes suspended in RPMI 1640 medium (MediaTech) with 10% fetal bovine serum (FBS) (Invitrogen) seeded in 6-well plates (MatTek and Corning) at a density of 4 × 105 monocytes/cm2. After 2-h incubation in a cell culture incubator with 5% CO2/95% air at 37 °C, the attached monocytes were rinsed three times with RPMI 1640 and then incubated with RPMI 1640 containing 10% FBS, 50 ng/ml M-CSF (PeproTech), and 25 ng/ml interleukin-10 (IL-10) (PeproTech) (12Hashimoto S. Yamada M. Motoyoshi K. Akagawa K.S. Blood. 1997; 89: 315-321Crossref PubMed Google Scholar). The medium was exchanged with fresh medium on day 6, and monocyte-derived macrophages were used for experiments on day 8. Elutriated human monocytes differentiated in human serum were cultured in 12-well plates as described previously (6Kruth H.S. Huang W. Ishii I. Zhang W.Y. J. Biol. Chem. 2002; 277: 34573-34580Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). For experiments, monocyte-derived macrophage cultures (about 0.25 mg of protein/well and 0.15 mg of protein/well for human serum and FBS+M-CSF+IL-10 cultured macrophages, respectively) were first rinsed three times with serum-free RPMI 1640. Cultures were incubated in RPMI 1640 without serum with the indicated concentration of LDL and the reagents specified in each experiment. For monocytes differentiated in FBS+M-CSF+IL-10, experimental incubations with these monocyte-derived macrophages were carried out with M-CSF and IL-10 added to RPMI 1640 medium. Preparation of Lipoproteins for Use in Experiments—Before use, human lipoproteins, LDL, oxidized LDL, and HDL (Intracel) were dialyzed against 1 liter of 0.15 m sodium chloride and 0.3 mm EDTA (pH 7.4) for 12 h at 4 °C, then against RPMI 1640 medium (2 changes, 1 liter/each change) for 24 h. LDL was incubated 24 h at 37 °C with 5 μm CuSO4 to prepare oxidized LDL as described previously (13Mori M. Itabe H. Higashi Y. Fujimoto Y. Shiomi M. Yoshizumi M. Ouchi Y. Takano T. J. Lipid Res. 2001; 42: 1771-1781Abstract Full Text Full Text PDF PubMed Google Scholar, 14Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3883-3887Crossref PubMed Scopus (1415) Google Scholar). Human 125I-LDL (Biomedical Technologies) was dialyzed against 0.15m sodium chloride and 0.3 mm EDTA (pH 7.4) over 36 h at 4 °C (three changes, 1 liter/each change). All dialysis was carried out with Pierce Slide-A-Lyzer cassettes (10,000 molecular weight cut-off). After dialysis, lipoproteins were sterilized by passage through a 0.45-μm (pore size) low protein-binding filter (Gelman Acrodisc). 125I-LDL specific activity was adjusted to 2.25 × 10–5 μCi/ng by adding unlabeled LDL. LDL concentration is expressed in terms of protein. Assays of 125I-LDL Cell Association and Degradation—Macrophage cell association and degradation of 125I-LDL were determined according to the methods of Goldstein et al. (15Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1282) Google Scholar). Lipoprotein degradation was quantified by measurement of trichloroacetic acid-soluble organic iodide radioactivity in supernatants of culture media samples that were centrifuged at 15,000 × g for 10 min. Values obtained in the absence of cells were <5% of those values obtained with cells. Cell-associated 125I-LDL was determined by rinsing macrophages three times with Dulbecco's phosphate-buffered saline (DPBS) plus Ca2+, Mg2+, and 0.2% bovine serum albumin (BSA), followed by 3 times with DPBS plus Ca2+ and Mg2+ all at 4 °C. Then, macrophages were dissolved overnight in 0.1 n NaOH at 37 °C. Aliquots of cell samples were assayed for 125I radioactivity with a γ counter. Values were subtracted for 125I radioactivity determined for wells incubated with 125I-LDL but without macrophages. These values were <1% of the cell-associated 125I-LDL. Determination of Fluid-phase Endocytosis—125I-BSA (MP Biomedicals) cell association and degradation by macrophages was determined similar to 125I-LDL described above and served as a means to determine fluid-phase uptake by the macrophages. Before incubations, 125I-BSA specific activity was adjusted to 2.25 × 10–5 μCi/ng by adding unlabeled BSA. Macrophages were incubated 24 h with varying concentrations of 125I-BSA (31, 62, 125, and 250 μg/ml) to show non-saturable uptake of BSA. Macrophages were incubated with 250 μg/ml 125I-BSA in incubations parallel to 125I-LDL when fluid-phase uptake was to be determined. The amount of fluid that macrophages ingested was calculated by dividing the total amount of 125I-BSA taken up by the macrophages (i.e. cell-associated plus degraded 125I-BSA) by the concentration of 125I-BSA added to the medium (i.e. 250 μg/ml). Assay of Cholesterol and Protein Contents of Macrophages—After incubations, macrophages were rinsed three times each with DPBS plus Mg2+, Ca2+, and 0.2% BSA and then DPBS plus Mg2+ and Ca2+. Macrophages were harvested from wells by scraping into 1 ml distilled water and then processed as described previously (16Kruth H.S. Skarlatos S.I. Lilly K. Chang J. Ifrim I. J. Cell Biol. 1995; 129: 133-145Crossref PubMed Scopus (74) Google Scholar). Lipids were extracted from an aliquot of cell suspension the J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The cholesterol of macrophages was determined according to the of et al. W. M. Kruth H.S. J. J. Lipid Res. 1978; Full Text PDF PubMed Google Scholar). Macrophage protein was determined on aliquot of cell suspension by the of et al. J. Biol. Chem. Full Text PDF PubMed Google Scholar) BSA as a was carried out by macrophages in a with a on an 5% CO2/95% air and 37 °C were in an from the macrophages. were a and were and at (i.e. the from the is of of LDL, macrophages were seeded in 6-well culture After incubation with LDL, macrophages were in and for as described previously (16Kruth H.S. Skarlatos S.I. Lilly K. Chang J. Ifrim I. J. Cell Biol. 1995; 129: 133-145Crossref PubMed Scopus (74) Google Scholar) that of BSA was used to LDL was by with 10 μg/ml LDL and then with a of 10 For the LDL was with the concentration of purified This showed of Cell a incubation with LDL or μg/ml oxidized LDL, macrophages were rinsed with serum-free culture then the macrophages were incubated at with 1 ml of 50 mm (pH containing mm BSA, ng/ml and 1 μg/ml cell The cells were rinsed with The and iodide to the macrophages were by at and at for at and at for for each of three culture wells were by and and macrophage was of and macrophages were in each for LDL, and oxidized are as the means of the The means were determined from three culture wells for each are not shown the is the of means were the was considered first determined human monocytes differentiated with M-CSF and IL-10 to as M-CSF LDL from monocytes differentiated in human M-CSF-differentiated macrophages showed an high of 125I-LDL uptake times with human serum differentiated macrophages of the 125I-LDL that was taken up by the M-CSF macrophages was of the 125I-LDL was not to of the as incubation of M-CSF macrophage media with 125I-LDL did not cause degradation of 125I-LDL. 125I-LDL degradation was when the was added to incubations of macrophages with 125I-LDL. Macrophage uptake of 125I-LDL was over a incubation of 125I-LDL uptake by M-CSF-differentiated macrophages. were differentiated in containing M-CSF and IL-10, then the differentiated monocyte-derived macrophages were incubated in serum-free M-CSF, and IL-10 for the indicated times with μg/ml 125I-LDL. 125I-LDL uptake was determined as the of cell-associated and degraded Macrophage uptake of LDL to macrophage accumulation of of M-CSF macrophages with concentrations of LDL up to 4 did not show of cholesterol accumulation This in a of macrophage cholesterol to a that of cell protein with of the cholesterol macrophages were incubated 24 h with HDL and LDL at similar total cholesterol concentrations (i.e. to for HDL and mg of for the macrophages and of of cell showing that cholesterol accumulation was not specific for LDL. were differentiated in the presence of M-CSF plus IL-10 of IL-10 has been shown to produce a macrophage culture by morphology and to the and differentiation of human monocytes with M-CSF (12Hashimoto S. Yamada M. Motoyoshi K. Akagawa K.S. Blood. 1997; 89: 315-321Crossref PubMed Google Scholar). Monocyte-derived macrophages differentiated with M-CSF showed cholesterol accumulation incubation with LDL. these macrophages cholesterol macrophages differentiated from monocytes with M-CSF and in without M-CSF did not as previously (11Akagawa K.S. Int. J. Hematol. 2002; 76: 27-34Crossref PubMed Scopus (144) Google Scholar). Macrophage uptake of 125I-LDL was not by of that of LDL to the LDL U. Goldstein J.L. Brown M.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) of LDL its to the LDL I. A.M. J. M. J. Lipid Res. Full Text PDF PubMed Google Scholar). that LDL uptake was not mediated by the LDL was shown by the that of LDL did not cholesterol accumulation not of on 125I-LDL uptake by 125I-LDL of of cell (2 (2 in a the mechanism of macrophage LDL uptake and cholesterol we examined the concentration of 125I-LDL uptake by the M-CSF macrophages. that uptake of 125I-LDL was not showing of with 125I-LDL concentrations up to as high as 250 μg/ml Thus, 125I-LDL uptake was directly proportional to its concentration in the medium. These results that the mechanism of LDL uptake was fluid-phase we determined the amount of macrophage fluid uptake to macrophage uptake of LDL in the fluid account for 125I-LDL uptake by macrophages. is uptake of BSA by macrophages, and we used this to macrophage fluid uptake as was previously for J.L. Brown M.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). we that uptake of 125I-BSA was and did not show with This was the as M-CSF macrophage uptake of 125I-BSA was up to the concentration 250 μg/ml not of M-CSF macrophages was incubated with 250 μg/ml of and of macrophages was incubated with μg/ml 125I-LDL for 24 h. The total uptake (i.e. cell-associated and of the fluid-phase was determined to be cell h of the BSA taken up was The fluid uptake that have produced this amount of 125I-BSA uptake was cell h. This amount of macrophage fluid uptake have 250 LDL cell h. The 125I-LDL uptake was of LDL of cell a not different from the Thus, macrophage fluid endocytosis account for the amount of 125I-LDL that these macrophages with the that the 125I-LDL uptake be for by fluid-phase endocytosis was the that a of LDL or HDL did not 125I-LDL uptake by macrophages Fluid-phase endocytosis can occur by either in or macropinocytosis in large Because is and macropinocytosis is we examined the effect of an that with on macrophage LDL uptake and cholesterol accumulation. by macrophage 125I-LDL uptake and cholesterol accumulation that macrophage LDL uptake and cholesterol accumulation were mediated by fluid-phase macropinocytosis of LDL. of M-CSF macrophages showed that were in these macrophages, and as a result, the macrophages and in the that LDL was within the by with the macrophages incubated with LDL for and then for LDL. Macrophages incubated with LDL showed of LDL the macrophages incubated without LDL showed of LDL uptake in M-CSF-differentiated macrophage were differentiated in containing M-CSF and is a of the monocyte-derived macrophages and the macrophage of these macrophages showed that the were within the macrophages in the LDL within a of macrophages incubated with LDL for min. C the absence of LDL in of macrophages incubated without we that monocytes differentiated to macrophages in human serum showed macropinocytosis of LDL that was protein kinase and be with the protein kinase C did not M-CSF macrophage uptake of 125I-LDL μg/ml) a This showed that the constitutive macropinocytosis of LDL by M-CSF macrophages was not protein kinase not we examined macrophage cholesterol accumulation mediated by macropinocytosis was incubation of macrophages for 24 h with LDL macrophages with and cholesterol accumulation of and of cell cholesterol accumulation did not or of the macrophages. of macrophages showed with incubation with LDL. This was not different from the of macrophages, was macrophages showed with iodide cell In incubation with oxidized LDL, most macrophages with and some macrophages with iodide with the effect of oxidized LDL on cells human macrophages C. M.J. 1995; PubMed Scopus Google Scholar, Arteriosclerosis. 1983; PubMed Google Scholar). In this we have a plausible model for macrophage foam cell formation that does not depend on LDL modification or macrophage receptors. Differentiation of human monocytes in the presence of and M-CSF produces a macrophage phenotype that high levels of constitutive Because of the constitutive macropinocytosis in these M-CSF-differentiated monocyte-derived macrophages, the macrophages can take up large amounts of LDL and high levels of LDL uptake was directly proportional to its concentration with LDL uptake in the fluid-phase of Because fluid-phase endocytosis is lipoprotein be taken up by this and produce macrophage cholesterol accumulation. we that incubation of macrophages with HDL produced cholesterol accumulation. The amount of cholesterol accumulation that with HDL was with a similar amount of LDL cholesterol, HDL is to cholesterol from macrophages. In to LDL, it is that HDL can contribute to macrophage cholesterol accumulation in the vessel wall. This is LDL concentration in the vessel wall can 1 mg the vessel wall concentration of HDL is of the LDL concentration (8Smith E.B. Ashall C. Biochim. Biophys. Acta. 1983; 754: 249-257Crossref PubMed Scopus (48) Google Scholar, 9Smith E.B. Eur. Heart J. 1990; 11: 72-81Crossref PubMed Google Scholar, 10Hoff H.F. Gaubatz J.W. Gotto Jr., A.M. Biochem. Biophys. Res. Commun. 1978; 85: 1424-1430Crossref PubMed Scopus (32) Google Scholar, H.F. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, E.B. and Scholar). studies showed non-saturable LDL uptake by cultured human that was to fluid-phase endocytosis in these cells J.L. Brown M.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.S. Goldstein J.L. PubMed Scopus Google Scholar). Because are not to out the fluid-phase endocytosis in those cells was most mediated by LDL taken up by fluid-phase endocytosis in does not cause cholesterol accumulation the cholesterol by LDL is by the M.S. Goldstein J.L. PubMed Scopus Google Scholar). the we have shown that LDL macrophages by fluid-phase endocytosis mediated by macropinocytosis does cause cholesterol accumulation. to be determined this in cholesterol is of a in of cholesterol by macropinocytosis and or of a in and macrophage of cholesterol to these cells. Macrophage macropinocytosis fluid as a LDL at a some times that shown by from 5 in J.L. Brown M.S. J. Biol. Chem. Full Text PDF PubMed Google and this be factor to macrophage accumulation of cholesterol by fluid-phase uptake of LDL not in cultured cells but in and it to LDL and tissue cholesterol M. J. Lipid Res. Full Text PDF PubMed Google Scholar, J. S. J. Lipid Res. Full Text PDF PubMed Google Scholar). The system in macrophages are a cell in of LDL J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Thus, it is that macrophage uptake of LDL that we have in to of LDL in In we have shown that when monocytes are differentiated into macrophages human serum, not show constitutive these macrophages show macropinocytosis of LDL causing foam cell formation when protein kinase C is in these macrophages (6Kruth H.S. Huang W. Ishii I. Zhang W.Y. J. Biol. Chem. 2002; 277: 34573-34580Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 7Kruth H.S. Jones N.L. Huang W. Zhao B. Ishii I. Chang J. Combs C.A. Malide D. Zhang W.Y. J. Biol. Chem. 2005; 280: 2352-2360Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). 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