The p - VLDL receptor pathway of murine P 388 D 1 macrophages

Very low density lipoproteins Sf 100-400 (VLDL1) from hypertriglyceridemic (HTG) subjects and chylomicrons cause receptor-mediated lipid engorgement in unstimulated macrophages in vitro via the @-VLDL receptor pathway. We now report that the murine macrophage P388D1 cell line possesses the characteristics of the 8-VLDL receptor pathway observed previously in freshly isolated resident murine peritoneal macrophages or human monocyte-macrophages. HTG-VLDL, isolated from the plasma of subjects with hypertriglyceridemia types 3, 4, and 5 interact with P388D1 macrophages in a high-affinity, curvilinear manner. @-VLDL, HTG-VLDL1, chylomicrons, and thrombin-treated HTG-VLDL1 (which do not bind to the LDL receptor) compete efficiently and similarly for the uptake and degradation of HTG-VLDL,. LDL and acetyl LDL do not compete, indicating that uptake of HTG-VLDL1 is via neither the LDL receptor nor the acetyl LDL receptor. Binding of thrombin-treated HTG-VLDLI to the @-VLDL receptor indicates that the thrombin-accessible apoE, which is absolutely required for interaction of HTG-VLDL Sf > 60 with the LDL receptor, is not required for binding to the /3-VLDL receptor. The uptake and degradation of 1251-labeled HTG-VLDLI is suppressed up to 80-90% by preincubation of the cells with sterols, acetyl LDL, or /3-VLDL, indicating that this process is not via the irrepressible chylomicron remnant (apoE) receptor. Chylomicrons, HTG-VLDL1, and thrombin-treated HTG-VLDLI but not normal VLDL', /3-VLDL, LDL, or acetyl LDLproduce massive triglyceride accumulation (10-20-fold mass increases in 4 hr) in P388D1 macrophages. Triglyceride loading is not enhanced by heparin, indicating that lipoprotein lipase is not involved under these conditions. The P388D1 cell line cultured as described herein represents an ideal model system for studying interactions of lipoproteins with the @-VLDL receptor. -Gianturco, S. H., S. A. Brown, D. P. Via, and W. A. Bradley. The 8-VLDL receptor pathway of murine P388D1 macrophages. J. Lipid f i x 1986. 27: 412-420. Supplementary key words chylomicrons hypertriglyceridemic VLDL apoE LDL acetyl LDL receptors Very low density lipoproteins (VLDL) from hypertriglyceridemic (HTG) subjects and chylomicrons are the only native human plasma lipoproteins known to rapidly convert macrophages into foam cells in vitro via a specific receptor pathway (1). The 0-VLDL receptor, the responsible receptor (2), is genetically distinct from the LDL receptor (3, 4). It is also present in other cells of reticuloendothelial origin, such as endothelial cells (4) and arterial foam cells (5), but not in non-macrophage cells (for a recent review, see ref. 6). This alternate pathway for disposal of large triglyceride-rich lipoproteins may produce the foam cells that occur in some forms of hypertriglyceridemia (7). This pathway was first identified in resident murine peritoneal macrophages by its interaction with the dietinduced abnormal cholesteryl ester-rich 0-migrating VLDL from cholesterol-fed dogs (8-VLDL) (2). Uptake of 8-VLDL triggers a sequence of events analogous to that which follows uptake of low density lipoproteins (LDL) via the LDL receptor. Internalized particles are hydrolyzed in lysosomes, and excess, released cholesterol is reesterified by acyl-coenzyme A:cholesteryl acyl transferase (ACAT), with oleate as the preferred fatty acid, and stored as cytoplasmic cholesteryl ester droplets. 0-VLDL from other cholesterol-fed animals produce saturable cholesterol reesterification in macrophages from murine, rabbit, monkey, and human sources via this receptor pathway (8). By contrast, VLDL, LDL, and HDL from normal animals and humans fail to cause cholesteryl ester accumulation in unstimulated macrophages of any source (6). We have demonstrated that HE-VLDL bind with high affinity to the 0-VLDL receptor of fresh resident murine peritoneal macrophages (1). After uptake and degradation of HE-VLDL, the cells acquire morphologic characteristics of foam cells. The major lipid that accumulates in macrophages exposed to HE-VLDL or chylomicrons is triglyceride, and not cholesteryl ester. Analogous to the incorporation of exogenous oleate into cellular cholesteryl ester after uptake of 0-VLDL, oleate incorporation into cellular triglyceride was stimulated by incubation with HE-VLDL or chylomicrons. Thus the lipid that accumulates in macrophages after receptor-mediated uptake of a lipoprotein reflects the predominant lipid transAbbreviations: HTG-VLDLI, hypertriglyceridemic very low density lipoproteins; VLDL', Sf 100-400; VLDL?, Sf60-100; VLDLs, Sf 20-60; LDL, low density lipoproteins; apoE, apolipoprotein E; EDTA, ethylenediaminetetraacetic acid; SDS, sodium dodecylsulfate. 'To whom correspondence should be addressed at The Methodist Hospital, A-601, 6565 Fannin, Houston, TX 77030. 412 Journal of Lipid Research Volume 27, 1986 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom ported by that lipoprotein (1, 6). VLDL, LDL, and HDL from normal humans, in contrast to H E V L D L and pVLDL, failed to stimulate oleate incorporation into either cellular cholesteryl ester or triglyceride (1). The ligands for the P-VLDL receptor remain to be defined. Studies in murine peritoneal macrophages in several laboratories indicate that neither apoE alone nor apoB-100 alone is sufficient for binding (2, 9). Definitive studies for the identification of binding determinants are complex and require large numbers of macrophages, preferably with few or no LDL receptors. Because of the potential importance of this receptor in several clinicopathological phenomena (foam cell accumulation, atherosclerosis, hypertriglyceridemia), we sought a long-term macrophage line for use as a model system to facilitate studies of the P-VLDL receptor pathway. The line P388D1 was an ideal source for the study and isolation of the acetyl LDL receptor (10, 11). These cells contain very low levels of the LDL receptor without induction (lo), they do not express the chylomicron remnant receptor as do human monocyte-macrophages (12) as reported here, and, finally, they do not secrete apoE (13); they seemed, therefore, potentially well suited for studying the 0-VLDL receptor pathway. Because both 8-VLDL and HTG-VLDL can also bind to the LDL receptor, the presence of high levels of LDL receptors on human monocyte-macrophages and on many long-term macrophage lines introduces ambiguity into the interpretation of direct and competitive uptake and degradation studies designed to identify binding determinants for the P-VLDL receptor. We now report that the characteristics of the 0-VLDL receptor pathway in the murine macrophage line P388D I are like those previously described in freshly isolated, unstimulated murine peritoneal macrophages. Moreover, since these cells do not appear to secrete lipoprotein lipase under our culture conditions, cellular triglyceride accumulation can be used as an intracellular end point to monitor receptor-mediated uptake of triglyceride-rich particles. MATERIALS AND METHODS Cells and cell culture The murine macrophage cell line P388D1 was obtained from the Salk Institute Cell Repository and was cultured in RPMI-1640 (high glucose) supplemented with 1% glutamine, 10% fetal bovine serum (Gibco), 100 pg/ml penicillin, and 100 unitdm1 streptomycin (10). Cells were maintained in 100-mm dishes in a humidified incubator (5% COP) at 37°C. For experimental purposes, approximately 3.7 x lo5 cellddish were seeded into dishes (60 x 15 mm) in 2 ml of complete medium. Cultures were refed 24 hr later with complete medium, and on the second day the medium was removed. After washing twice with saline, the cells were incubated in 2 ml of serum-free RPMI-1640 containing the desired concentration of lipoprotein, as indicated in the figures and tables. For binding studies, lipoproteins were iodinated by a modification of the iodine monochloride method of McFarland (14). Free iodine was removed by gel filtration and extensive dialysis. Samples were filtered (0.45 pm Millex) immediately before use; specific activities ranged from 35 to 200 cpm per ng protein. Less than 10% of the label was extractable into organic solvent. Duplicate dishes of cells and empty dishes were incubated with labeled lipoproteins alone and in the presence of indicated quantities of unlabeled lipoprotein at 4OC or 37OC for up to 4 hr as indicated in the figure legends. After 6 hr at 37OC, the H E VLDLl particles denature in the absence of albumin or serum and show greatly increased nonspecific binding to the control dishes that contain no cells. Since inclusion of albumin, fetal calf serum, or lipoprotein-deficient serum to stabilize VLDL diminishes cellular triglyceride accumulation and introduces soluble, transferable apoproteins that affect the binding and uptake of added VLDL, all experiments were conducted at 2 to 4 hr of incubation in the absence of serum components other than lipoproteins. Total cell-associated radioactivity (representing both surface-bound and internalized) was determined after the cells were washed five times with chilled buffer containing 2 mg of albumidml and once with albumin-free buffer (15). The amount of non-iodide, non-lipid, trichloroacetic acid-soluble radioactivity in the medium was used as a measure of iodinated lipoprotein degradation (15). There was no evidence of deiodination of '251-labeled monotyrosine following the cellular degradation of radiolabeled apoproteins. Each value was corrected by subtracting the amount 'bound" or degraded in control dishes that contained no cells. Specific binding, uptake, and degradation curves were calculated by subtracting the curve generated by plotting the amount of '251-labeled lipoprotein processed by cells in the presence of excess unlabeled homologous lipoprotein (these plots were linear) from the curve representing the amount bound in the absence of unlabeled lipoproteins (curvilinear), as in Fig. 1. Triglyceride mass was determined using an enzymatic kit (Boehringer Mannheim, cat. no. 701912). To determine cellular triglyceride mass, duplicate 60-mm dishes containing cells (and no cells, for blanks) were incubated with the indicated quantities of lipoproteins for 4 hr at 37°C and washed extensively with albumin-containing buffer as in the binding studies. The lipids were then extracted with hexane-isopropanol 3:2 (v/v). The solvent was evaporated under nitrogen, and lipids were resolubilized in 0.5 ml of the kit reagent containing Triton X-100 (10 pllml reagent). Cholesterol and cholesteryl ester masses were determined using an enzymatic method (16). Cells for oil-red 0 staining were grown, incubated with lipoproteins, and extensively washed as described for Gianturco et al. P-VLDL receptor pathway in murine P388D1 macrophages 413 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom binding studies. The cells were fixed with 60% isopropyl alcohol and stained with oil-red 0 and hematoxylin as described previously (1). Before examination by light microscopy, cover slips were placed on the stained cells using a 50% solution of polyvinylpyrrolidone as an aqueous mounting medium.