J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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The Journal of Lipid Research, Vol. 39, 482-494, March 1998
Copyright © 1998 by Lipid Research, Inc.

Original Article

Isolation of macrophage-like cell mutants resistant to the cytotoxic effect of oxidized low density lipoprotein

Hideki Hakamataa, Akira Miyazakia, Masakazu Sakaia, Hirofumi Matsudaa, Hiroshi Suzukib, Tatsuhiko Kodamac, and Seikoh Horiuchia
a Department of Biochemistry, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
b Chugai Pharmaceutical Co. Ltd, Shizuoka 412-0038
c Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-0041, Japan

Correspondence to: Seikoh Horiuchi.


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

A high concentration of oxidized low density lipoprotein (Ox-LDL) showed a cytotoxic effect on mouse macrophage-derived J774 cells. Mutant cells were selected from these cells that were resistant to the cytotoxic effect of Ox-LDL. One mutant form, named JO21b cells, was characterized in the present study. In spite of a marked resistance to the cytotoxic effect of Ox-LDL, JO21b cells were apparently as sensitive as the parent cells not only to toxic moieties of Ox-LDL, such as 7-ketocholesterol and lysophosphatidylcholine, but also to t-butyl hydroperoxide, an artificial lipid hydroperoxide analog. However, the cellular association of 125I-labeled Ox-LDL with, and subsequent endocytic degradation by JO21b cells was reduced by 70–80% compared with J774 cells. Similarly, accumulation of cholesteryl esters in JO21b cell by Ox-LDL was also reduced by 70%. Northern blot analyses of type I and type II macrophage scavenger receptors (type I and type II MSR) demonstrated that the mRNA levels of JO21b cells were lower than those of J774 cells. Moreover, peritoneal macrophages obtained from MSR-knockout mice showed a higher resistance to the cytotoxic effect of Ox-LDL than those from their wild-type littermates.

Our results suggest, therefore, that macrophage scavenger receptor-mediated endocytic uptake of oxidized low density lipoproteins (Ox-LDL) may play an enhancing role in Ox-LDL cytotoxicity to macrophages or macrophage-derived cells.—Hakamata, H., A. Miyazaki, M. Sakai, H. Matsuda, H. Suzuki, T. Kodama, and S. Horiuchi. Isolation of macrophage-like cell mutants resistant to the cytotoxic effect of oxidized low density lipoproteins. J. Lipid Res. 1998. 39: 482–494.

Supplementary key words: oxidized low density lipoprotein, macrophage, somatic cell mutant, cytotoxicity, macrophage scavenger receptors, atherosclerosis


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

The early stages of atherosclerotic lesions are characterized by the presence of lipid-laden macrophage foam cells (1). Macrophages are known to take up chemically modified low density lipoproteins (modified LDLs), such as acetylated LDL (acetyl-LDL) and oxidized LDL (Ox-LDL), by the macrophage scavenger receptors (MSR), leading to foam cell formation and massive intracellular accumulation of cholesteryl esters (CE) (2). MSR specific for these modified LDLs are preferentially expressed by macrophages or macrophage-derived cells (3) and at least three classes (class A, class B, and class C) have been recognized (4) (5). MSR class A receptors were the first to be cloned and divided into type I and type II MSR, representing the products of alternative splicing of a single gene (6) (7). Expression experiments using the MSR cDNA showed that both type I and type II MSR could recognize acetyl-LDL as well as Ox-LDL as effective ligands (8).

Among the modified LDLs, Ox-LDL is regarded as a major atherogenic lipoprotein in vivo, due to its presence in human and rabbit atherosclerotic plaques (9) (10). Ox-LDL has several biological properties such as (i) enhancement of adhesion of circulating monocytes to endothelial cells and subsequent migration into subendothelial space; (ii) induction of monocyte–macrophage differentiation; (iii) macrophage foam cell generation; (iv) induction of smooth muscle cell migration, proliferation, and foam cell generation; (v) cytotoxicity to vascular cells; (vi) modulation of expression of cytokines and adhesion molecules; and (vii) effects on the coagulation pathways (for reviews see (2), (11)). Of great importance is the cytotoxic effect of Ox-LDL on cells participating in the formation of atherosclerotic lesions, such as endothelial (12) (13) and smooth muscle cells (12) and macrophages (14) (15), as cellular components are lost from deep- to mid-intimal locations during the development of the lipid-rich necrotic core in atherosclerotic plaques (1). Among these cells, immunohistochemical observations suggest that those cells undergoing necrosis in human atherosclerotic plaque are mainly macrophages (16). In vitro experiments showed that Ox-LDL is able to induce the death of mouse peritoneal macrophages (14), P388D1 cells, a macrophage-like cell line (15), and human monocytes-macrophages (17). However, the cellular mechanism(s) of Ox-LDL-induced macrophage cytotoxicity are not fully known.

To investigate the cytotoxic mechanisms of Ox-LDL, a molecular and cell genetic approach was used to isolate mutant cells resistant to the cytotoxic effect of Ox-LDL from J774 cells, a cell line derived from mouse macrophages. In particular, one mutant cell called JO21b was characterized in the present study. Our results suggest that the endocytic uptake of Ox-LDL via the MSR plays an enhancing role in Ox-LDL-induced cytotoxicity in macrophages and macrophage-derived cells.


  MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES

Materials
Na125I (3.7 GBq/ml) and [{alpha}-32P]dCTP (111 TBq/mmol) were purchased from Amersham. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Dojindo Laboratories (Kumamoto, Japan). Cell culture media and reagents were obtained from Life Technologies. Newborn calf serum (NCS) was purchased from Hyclone Laboratories. All other chemicals were of the best grade from commercial sources.

Lipoproteins and their modifications
LDL (d 1.019–1.063 g/ml) was isolated by sequential ultracentrifugation of fresh human plasma and dialyzed against 0.15 M NaCl and 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.4). Acetyl-LDL was prepared as described previously (18). Ox-LDL was prepared as described previously (19) (20). Briefly, 0.1 mg/ml LDL was incubated for 18 h at 37°C with 5 µM CuSO4 in EDTA-free phosphate-buffered saline (PBS). The reaction was stopped by the addition of 1 mM EDTA and cooling on ice, followed by 50-fold concentration using ultrafiltration cell (Amicon, Danvers, MA) equipped with a PM-10 membrane. To remove CuSO4, Ox-LDL was subjected to extensive dialysis against 0.15 M NaCl and 1 mM EDTA (pH 7.4). Iodination of acetyl-LDL and Ox-LDL with 125I was performed according to McFarlane (21). The protein concentration was determined by BCA protein assay reagent (Pierce Chemical Co.) using BSA as a standard (22).

Cell culture
Unless otherwise specified, cell cultures were performed at 37°C in 5% CO2. J774 cells and their Ox-LDL-resistant cell lines were cultured in RPMI 1640 medium containing 10% newborn calf serum, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), 0.1 mg/ml streptomycin and 100 U/ml penicillin (medium A). Mouse peritoneal macrophages were cultured in RPMI 1640 medium containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), 0.1 mg/ml streptomycin, and 100 U/ml penicillin (medium B).

Isolation of Ox-LDL-resistant mutants from J774 cells
J774 cells (1 x 106) were plated into a 75-cm2 tissue culture flask (Falcon) in 20 ml of medium A and incubated at 37°C overnight. The medium was removed and the cells were treated with 400 µg/ml of ethylmethane sulfonate in 15 ml of medium A for 16 h to a survival rate of 10% of the initial cell number (23). The cells were washed three times with PBS and cultured for an additional 2 days in 20 ml of medium A without the mutagen. The harvested mutagenized cells (5 x 105) were seeded into 20 dishes (100 mm in diameter) and incubated for 2 weeks with 0.1 mg/ml of Ox-LDL in 10 ml of medium A. Each colony thus formed was transferred with the cloning cup to a culture dish containing 0.1 mg/ml of Ox-LDL in 10 ml of medium A, followed by incubation for 2 weeks (23). We obtained five colonies, subjected them to a further 3 months culture in the absence of Ox-LDL, and determined their Ox-LDL-resistant property by MTT assays described below. We finally obtained two Ox-LDL-resistant clones that were isolated independently from different dishes. One of them, JO21b clone, was investigated in the present study.

Cell viability
Cell viability was determined by the MTT method (20). Briefly, 50 µl of the suspension of JO21b cells or J774 cells (5 x 103 cells) in medium A was transferred to each well of 96-well tissue culture plates (7-mm diameter, Falcon). After incubation for 2 h at 37°C, 50 µl of the compound to be tested, such as lipoproteins (see Figure 1) and toxic components of Ox-LDL (see Figure 2), was added to each well, followed by further incubation for the indicated intervals (up to 7 days). At indicated times, 10 µl of 5 mg/ml of MTT was added to each well, followed by 4-h incubation at 37°C to reduce MTT to a blue formazan product. To each well was added 150 µl of 10% SDS in 0.01 N HCl, and the mixture was further incubated for 16 h at 37°C to dissolve the blue formazan product. The absorbance at 570 nm was then measured with each well in a multiwell spectrophotometer. Because MTT assay simply reflects the mitochondrial dehydrogenase activity of cells, the absorbance at 570 nm was used as an index of cell viability.



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  		Figure 1. Effects of Ox-LDL on the cell viability of JO21b cells (A) and J774 cells (B). The cells (5 x 103) were plated in microplates and incubated in 0.1 ml of medium A with 0.1 mg/ml Ox-LDL ({bullet}), acetyl-LDL ({circ}), LDL ({bigtriangleup}), or medium alone ({square}) for the indicated times. Four hours before the termination of the experiment, 10 µl of 5 mg/ml of MTT solution was added to each well, followed by the addition of 150 µl of 10% SDS in 0.01 M HCl. The mixture was further incubated for 16 h to dissolve the blue formazan product and absorbance was read with a multiwell spectrophotometer at 570 nm. Data are representative of three separate experiments with duplicate wells.



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  		Figure 2. Cytotoxic effects of 7-ketocholesterol, t-butyl hydroperoxide, and lysophosphatidylcholine on JO21b cells. JO21b cells (5 x 103) were incubated in 0.1 ml of medium A with 10 and 50 µg/ml of 7-ketocholesterol (A), 10 and 30 µM of t -butyl hydroperoxide (C) and 200 and 400 µM lysophosphatidylcholine (E) at the indicated time intervals. Under identical conditions, J774 cells were incubated with the same concentrations of 7-ketocholesterol (B), t -butyl hydroperoxide (D) and lysophosphatidylcholine (F). The cell viability was determined by the MTT assay as described in the legend of Figure 1. Data are representative of three separate experiments with duplicate wells.

Cellular assays
JO21b and J774 cells (2 x 105) in 1 ml of medium A were seeded into each well of 24-well plates (16-mm diameter, Falcon) and incubated overnight. For the binding experiments, the monolayers thus formed were washed once with 1 ml of ice-cold PBS containing 0.3% BSA and twice with 1 ml of ice-cold PBS. Each well was incubated for 90 min on ice in 0.5 ml of medium A with the indicated concentrations of 125I-labeled Ox-LDL (83 cpm/ng protein) or 125I-labeled acetyl-LDL (117 cpm/ng protein). The cells were washed once with 1 ml of ice-cold PBS containing 0.3% BSA and twice with 1 ml of ice-cold PBS. The cells were dissolved with 1 ml of 0.1 M NaOH and the cell-bound radioactivity was determined as described previously (22). Nonspecific binding was measured by parallel incubation with an excess amount of the same unlabeled ligand. The specific binding was calculated by subtracting the nonspecific binding from the total binding.

To determine cellular association of 125I–labeled Ox-LDL or 125I-labeled acetyl-LDL and their endocytic degradation by macrophages, macrophage cell monolayers prepared as above were washed once with 1 ml of PBS containing 0.3% BSA and twice with 1 ml of PBS. Each well was incubated for 24 h at 37°C in 1 ml of medium A with various amounts of 125I-labeled Ox-LDL or 125I-labeled acetyl-LDL. After incubation, the culture medium was taken from each well and used for determination of endocytic degradation, while the cells in each well were washed three times with 1 ml of PBS, dissolved in 1 ml of 0.1 M NaOH, and the cell-associated radioactivity was determined as described previously (24). To determine endocytic degradation, to 0.75 ml of each culture medium was added 0.25 ml of ice-cold 40% TCA and 0.2 ml of 0.7 M AgNO3 and the mixture was incubated at room temperature for 30 min to precipitate free iodine. After centrifugation at 700 g for 10 min, 0.6 ml of the resulting supernatant was used for the determination of radioactivity using a gamma-counter (25).

Mass determination of cellular cholesterol contents
Macrophage cell monolayers prepared as above were incubated with various concentrations of Ox-LDL or acetyl-LDL. After incubation for 24 h, cellular lipids were extracted and both the free cholesterol mass and CE mass were quantified by a modification (22) of the enzymatic fluorometric methods (26). Briefly, cellular lipids extracted were dried under nitrogen flush and dissolved in 180 µl of isopropanol. Aliquots (30 µl) of the lipid extract was added to 0.4 ml of enzyme mixtures and incubated at 37°C for 1 h (for free cholesterol) or for 2 h (for total cholesterol), followed by addition of 0.81 ml of 0.5 M NaOH to terminate the reaction. Enzyme mixtures were identical to those reported previously (26) except that Carbowax-6000 was replaced by 0.01% Triton X-100 and enzyme concentrations used were 2 times higher (cholesterol oxidase; 0.16 U/ml and cholesteryl ester hydrolase; 0.16 U/ml). Fluorescence intensity was measured with an excitation at 320 nm and an emission at 407 nm. CE was calculated by subtracting free cholesterol from total cholesterol. Cells were dissolved in 0.1 M of NaOH to determine cell proteins using a BCA protein assay reagent.

Proteolytic activity of cell extracts
The proteolytic activity of macrophage cell extracts was determined according to the method described by Hoppe, O'Neil, and Hoff (27). Briefly, JO21b cells or J774 cells (2 x 106) were suspended in 1.0 ml of 250 mM sucrose, 1 mM EDTA, and 0.1% ethanol (pH 6.8) and ruptured by passing 10 times through a 25-gauge needle, by 10 cycles of freezing and thawing, and finally by 15-sec bursts of sonication. The cell homogenate was centrifuged at 5,000 g for 60 min at 4°C to remove particulate fractions. The supernatant solution (10 µg cell protein/ml) was incubated for 1 h at 37°C in 1.0 ml of 100 mM sodium acetate buffer (pH 4.0) with various concentrations (up to 40 µg/ml) of 125I-labeled acetyl-LDL as a protease substrate. After the reaction, the amount of degraded 125I-labeled acetyl-LDL was determined as TCA-soluble radioactivity as described above.

Northern blot analysis
Standard molecular biology techniques were used in this analysis (28). Poly (A)+ RNA prepared from J774 or JO21b cells using Fast Track® 2.0 Kit (Invitrogen) (2 µg/lane) was fractionated by electrophoresis through a denaturing formaldehyde 1% agarose gel and transferred to Hybond-N+ nylon membrane as described (29). Probes were generated according to Ashkenas et al. (30) from total RNA of J774 cells by RT-PCR (reverse transcriptase-polymerase chain reaction) (probes A through D; see Figure 8). The first strand cDNA was synthesized from 1.0 µg of J774 total RNA primed with oligo-dT using Moloney murine leukemia virus reverse transcriptase (BRL/GIBCO). Sense and antisense primer pairs for PCR were as follows. For probe A, 5'-CGC CGAGCGGCCGCGCTGTCTTCTTTACCAGC-3' and 5'- CGCCGGTCTAGATTATACTGATCTTGATCCGC-3'; for probe B, 5'-CCCCCTTAAGACAGTTCGAC-3' and 5'-GGTTTCATAATTGTAATTTC-3'; for probe C, 5'-ACT CCCTGAACATATTGGGG-3' and 5'-TAATAGGATTCT GCACAGTTA-3'; for probe D, 5'-ATTGATTAAGTAT TAGTTCT-3' and 5'-CGCCGGATCGATTTTAATAT ATTAGACTAC-3'. Specific primers for amplification of a 983 bp fragment of glyceraldehydephosphate dehydrogenase (GAPDH) (31) were included during the PCR process. The cycling conditions in the GeneAmp 9600 System consisted of 94°C denaturation for 2 min, followed by 30 cycles of annealing at 55°C for 60 sec, extension at 72°C for 90 sec, and denaturation at 93°C for 30 sec, with a final prolonged step of 75°C for 10 min. The amplification products were analyzed by 1% agarose gel electrophoresis. The amplification products extracted from the agarose gel were ligated into pGEM-T (Promega), and transfected into E. coli XL1-Blue. To obtain single-strand DNA (ssDNA), XL1-Blue cells containing this construct were infected with the VCSM13 helper phage. The purified ssDNAs were sequenced to verify that amplification products were consistent with the reported sequences of murine type I and type II MSR and GAPDH. For preparation of radioactive probes, the cDNA fragments were excised with Apa I and Sac I, and labeled with [{alpha}-32P]dCTP (29). Hybridization was performed with 50% formamide at 42°C as described previously (29). The final wash stringency was 0.2 x SSC, 0.1% SDS at a higher temperature (from 42 to 65°C) (29). The membrane was then exposed to an X-ray film (RX Fuji, Tokyo) for 24 h at room temperature. The intensity of MSR mRNA relative to that of GAPDH was calculated by densitometry (Quantity One, version 2.7, PDI) of the radiograph on a Macintosh computer.



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  		Figure 3. The binding of 125I-labeled Ox-LDL to JO21b cells. JO21b cells (A) or J774 cells (B) as a control (2 x 105) were incubated for 90 min on ice with increasing amounts of 125I-labeled Ox-LDL with or without a 20-fold excess of unlabeled Ox-LDL. The cells were washed and the cell-bound 125I-labeled Ox-LDL was determined as described under Materials and Methods. The specific binding ({circ}) was calculated by subtracting the non-specific binding ({blacksquare}) from the total binding ({square}). The Scatchard plot of the specific binding is shown in the inset. Data are representative of three separate experiments with triplicate wells. Bars represent SD. SD values are small and are within the symbols.



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  		Figure 4. Cell-association of 125I-labeled Ox-LDL with and subsequent endocytic degradation by JO21b cells. JO21b cells or control J774 cells (2 x 105) were incubated for 24 h at 37°C with the indicated concentrations of 125I-labeled Ox-LDL. The cell-associated 125I-labeled Ox-LDL (A) and that degraded by the cells (B) were determined as described under Materials and Methods. Data are representative of three separate experiments with triplicate wells. SD values are small and within the symbols.



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  		Figure 5. The binding of 125I-labeled acetyl-LDL to JO21b cells (A) and control J774 cells (B) were incubated for 90 min on ice with increasing amounts of 125I-labeled acetyl-LDL with or without a 20-fold excess unlabeled acetyl-LDL. The cells were washed and the cell-bound radioactivity was determined as described under Materials and Methods. The specific binding ({circ}) was calculated by subtracting the non-specific binding ({blacksquare}) from the total binding ({square}). The Scatchard plot of the specific binding is shown in the inset. Data are representative of three separate experiments with triplicate wells. SD values are small and within the symbols.



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  		Figure 6. Cell-association of 125I-labeled acetyl-LDL with and subsequent endocytic degradation by JO21b cells. JO21b cells and control J774 cells (2 x 105) were incubated separately for 24 h at 37°C with the indicated concentrations of 125I-labeled acetyl-LDL. The amount of cell-associated 125I-labeled acetyl-LDL (A) and that degraded by these cells (B) was described under Materials and Methods. Data are representative of three separate experiments with triplicate wells. Bars represent SD. SD values are small and are within the symbols.



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  		Figure 7. The accumulation capacity of JO21b cells for cholesterol esters after exposure to Ox-LDL (A) and acetyl-LDL (B). JO21b cells or J774 cells (2 x 105) were plated and incubated in 1 ml of medium A with the indicated concentrations of Ox-LDL (A) and acetyl-LDL (B). The cells were harvested after 24 h incubation at 37°C, and the lipids were extracted and the mass of cellular cholesteryl esters (CE) were determined as described under Materials and Methods. Each value is the mean ± SD of quadruplicate experiments.



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  		Figure 8. Northern blot analysis of type I and type II MSR of JO21b cells. Schematic representation of mouse MSR mRNA (left panel). Open boxes: the common region of type I and type II forms (probe A), hatched box: the specific region for type I form (probe B), solid boxes: the specific region for all three type II forms (probe C), shaded boxes: the specific region for longer two type II forms (probe D). Complementary DNA probes were designated on the basis of the reported cDNA sequence and amplified by RT-PCR (probes A–D). Poly(A)+ RNA from J774 (P, parent) or JO21b (M, mutant) cells (2 µg/lane) was subjected to agarose electrophoresis, transferred to Hybond-N+ nylon membrane and hybridized with 32P-labeled probe (A–D, and GAPDH) as described under Materials and Methods (right panel). The mass of MSR mRNA compared with that of GAPDH was calculated by densitometry of the radiograph.

Cytotoxic effect of Ox-LDL on peritoneal macrophages from MSR-knockout mice
Cytotoxic effect of Ox-LDL on peritoneal macrophages obtained from MSR-knockout mice was examined by the MTT method (20). Peritoneal macrophages were collected with 8 ml of ice-cold PBS from MSR-knockout mice (32) and their wild-type littermates that were born to heterozygous mice (32). Cells were suspended in medium B. Fifty microliter of each cell suspension (5 x 105 cells) was transferred to each well of 96-well tissue culture plates (7-mm diameter, Falcon) and 50 µl of indicated concentrations of Ox-LDL in medium B was subsequently added to each well, followed by further incubation for 24 h or 48 h. Four hours before the termination of the experiment, 10 µl of 5 mg/ml of MTT was added to each well to reduce MTT to a blue formazan product. To each well was added 150 µl of 10% SDS in 0.01 N HCl, and the mixture was further incubated for 16 h at 37°C to dissolve the blue formazan product. The absorbance at 570 nm was then measured with each well in a multiwell spectrophotometer.

Statistical Analysis
Data were evaluated by Student's t -test. Differences were considered significant when the P value was less than 0.05.


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

Cytotoxic effects of Ox-LDL on JO21b cells
JO21b cells, a mutant cell line resistant to the cytotoxic effect of Ox-LDL, were selected as described under Materials and Methods. In the first step, we compared the effects of Ox-LDL on the cell viability of JO21b cells with those on parent cells (J774 cells) using the MTT assay. As shown in Figure 1B, 0.1 mg/ml of Ox-LDL had a negligible effect on cell viability until day 3, but became cytotoxic to J774 cells after day 4, indicating that J774 cells were sensitive to the cytotoxic effect of Ox-LDL. At the same concentration, however, Ox-LDL had no effect at all on the cell viability of JO21b cells, indicating resistance of JO21b cells to the cytotoxic effect of Ox-LDL (Figure 1A). A similar concentration of acetyl-LDL exhibited no cytotoxic effect on JO21b cells as well as J774 cells (Figure 1A and Figure B), rather, it had a weak stimulating effect on the viability of J774 cells. Similarly, the cell viability of these cells was not influenced by unmodified LDL, suggesting that the cytotoxic effect of Ox-LDL on J774 cells was highly selective.

Cytotoxic effects of 7-ketocholesterol, t-butyl hydroperoxide and lysophosphatidylcholine on JO21b cells
Results of previous studies have suggested that the toxicity of Ox-LDL does not reside in its protein moiety, but rather in its lipid moiety (12). The potential cytotoxic compounds suggested so far include oxysterol (33) (34) (35) (36), lipid hydroperoxide (37) (38) (39), and lysophosphatidylcholine (40). To examine the involvement of these compounds in Ox-LDL-induced cytotoxicity, we determined the cytotoxic effects of 7-ketocholesterol, tertiary-butyl hydroperoxide (a lipid hydroperoxide analog), and palmitoyl-lysophosphatidylcholine, on JO21b and J774 cells ( Figure 2). At 10 µg/ml, 7-ketocholesterol reduced the viability of JO21b cells at day 2 and that of J774 cells at day 1. The effect was more marked, causing death of all cells, at a concentration of 50 µg/ml (Figure 2A and Figure B), indicating that JO21b cells were as sensitive as J774 cells to the cytotoxic effect of 7-ketocholesterol. Similar experiments using 10 µM of t -butyl hydroperoxide, an artificial lipid hydroperoxide analog, did not influence the viability of J774 or JO21b cells, whereas a higher amount of the compound (30 µM) severely retarded an increase in the viability of J774 cells (Figure 2D) and induced death of JO21b cells (Figure 2C). These results suggest that the parent cells were slightly more resistant to t -butyl hydroperoxide. Lysophosphatidylcholine at 400 µM caused death of all JO21b and J774 cells (Figure 2E and Figure F), indicating that both cells were equally sensitive to the cytotoxic effects of lysophosphatidylcholine. Considered together, these results indicate that the sensitivity of JO21b cells to these cytotoxic compounds is essentially indistinguishable from that of the parent cells. Thus, these experiments failed to explain the mechanisms of resistance of JO21b cells to the cytotoxic effect of Ox-LDL. Accordingly, in the next series of experiments we examined the combined effects of these compounds on JO21b and J774 cells. Compared with the parent cells, however, JO21b cells were not specifically resistant to two or more compounds when combined together (data not shown), indicating again that the resistance of JO21b cells to Ox-LDL was not explained by these compounds.

Interaction of JO21b cells with Ox-LDL
The interaction of Ox-LDL with JO21b cells was determined by cellular binding, cell-association, and intracellular degradation. Figure 3 shows the binding of 125I-labeled Ox-LDL to JO21b and J774 cells at 0°C. Total binding was effectively replaced by >60% by an excess unlabeled ligand. The specific binding, calculated by subtracting nonspecific binding from the total binding, exhibited a saturation pattern in both JO21b and J774 cells. However, the total and specific bindings of 125I-labeled Ox-LDL to JO21b cells (Figure 3A) were reduced by 50% compared with those to J774 cells (Figure 3B). The Scatchard analysis of this specific binding of Ox-LDL to JO21b cells disclosed a binding site with an apparent dissociation constant (Kd) of 2.6 µg/ml and maximal surface binding (Bmax) of 335 ng/mg cell protein, whereas the same analysis of the specific binding to J774 cells showed a binding site with an apparent Kd of 3.4 µg/ml and Bmax of 616 ng/mg cell protein. The nonspecific binding to JO21b cells was also reduced by 50% as compared with that to J774 cells. Figure 4 shows the results of cellular association and degradation experiments. The total degradation of 125I–labeled Ox-LDL by J774 cells in 24 h increased in a dose-dependent manner (Figure 4B). The amount of Ox-LDL degraded by the mutant cells also increased dose-dependently (0.7 µg/mg cell protein/24 h at 100 µg/ml of Ox-LDL). However, the latter was only 10% of that degraded by J774 cells (6.3 µg/mg cell protein/24 h at 100 µg/ml of Ox-LDL) (Figure 4B). Similarly, the total cellular association of 125I-labeled Ox-LDL with JO21b cells was less than 20% of that with J774 cells (Figure 4A). These results strongly suggested that the cellular binding of Ox-LDL and its subsequent endocytic degradation by JO21b cells were significantly less than those by parent cells.

Interaction of JO21b cells with acetyl-LDL
Although Ox-LDL is a ligand for type I and type II MSR, acetyl-LDL is known as a representative ligand for these MSR. We therefore determined the cellular binding of 125I-labeled acetyl-LDL to JO21b cells at 0°C. As was the case with Ox-LDL, the specific binding of acetyl-LDL to both J774 and JO21b cells, calculated by subtracting nonspecific binding from the total binding, exhibited a saturation pattern. However, the amount of 125I-labeled acetyl-LDL specifically bound to JO21b cells at each ligand concentration ( Figure 5A) was less than 30% of that obtained with the parent cells (Figure 5B). The Scatchard analysis of this specific binding of acetyl-LDL to JO21b cells disclosed a binding site with an apparent Kd of 2.1 µg/ml and Bmax of 480 ng/mg cell protein, whereas the same analysis of the specific binding to J774 cells showed a binding site with an apparent Kd of 4.2 µg/ml and Bmax of 2,035 ng/mg cell protein. The endocytic activity of JO21b cells for acetyl-LDL was determined under conditions identical to those of Ox-LDL (Figure 4). The cellular association of 125I-labeled acetyl-LDL in JO21b cells was 60% of that in parent cells ( Figure 6A). Similarly, the subsequent endocytic degradation of 125I-labeled acetyl-LDL by these cells was only 70% of that with J774 cells (Figure 6B). Thus, the endocytic capacity of these mutant cells for acetyl-LDL is also reduced in the same way as that for Ox-LDL, suggesting a significantly reduced MSR function in JO21b cells.

CE-accumulation capacity of JO21b cells upon incubation with Ox-LDL or acetyl-LDL
Results of the above experiments (Figure 3, Figure 4, Figure 5, and Figure 6) suggested that the processes of Ox-LDL and acetyl-LDL binding to, and subsequent endocytic degradation by, JO21b cells were significantly weaker than J774 cells. As the endocytic degradation of these modified LDLs by macrophages is associated with intracellular accumulation of cholesteryl esters and foam cell formation, the mass of cholesteryl esters accumulating in JO21b cells was measured and compared with that in parent cells. Upon incubation with Ox-LDL, CE-accumulation increased dose-dependently in JO21b cells in a manner similar to J774 cells. However, the amount of accumulated CE in mutant cells was less than 20% of that in J774 cells ( Figure 7A). Furthermore, acetyl-LDL-induced CE-accumulation in JO21b cells was also less than one-third of that in J774 cells (Figure 7B). As the loading of these cells with Ox-LDL and acetyl-LDL did not increase the cellular free cholesterol, the increase in total cholesterol was exclusively explained by intracellular accumulation of CE (data not shown).

These results are consistent with reduced endocytic activity of mutant cells for the ligands of MSR such as Ox-LDL and acetyl-LDL (Figure 4 and Figure 6). One cannot exclude, however, the possibility that the resistance of JO21b cells to Ox-LDL might be in part due to altered levels of lysosomal protease activity. However, determination of the lysosomal protease activity indicated that the activity in JO21b cells was not different from that in parent cells (data not shown), indicating that lysosomal protease activity did not contribute to the resistance of JO21b cells to Ox-LDL.

Northern blot analysis of type I and type II MSR
Our results so far suggested a possible reduction of MSR activity in JO21b cells. Northern blot analysis was performed to determine whether type I and type II MSR expression was reduced at the mRNA level using J774 cells as a control. Previous studies indicated that two isoforms (type I and type II) of MSR are formed by alternative splicing of transcripts derived from a single gene (41). Because type I has a single transcript, whereas type II has three transcripts (30), we designated four probes (A, B, C, and D) to detect these different species of MSR mRNA ( Figure 8); probe A can recognize all four species of transcripts, probe B can detect only type I transcript, probe C recognizes all three species of type II transcript, and probe D recognizes longer two type II transcripts. As shown in Figure 8, [32P]-labeled probe A cross-hybridized with poly(A)+ RNAs of JO21b and J774 cells, with strong three signals at 4.0, 2.5, and 1.5 kb. Probe B cross-hybridized with poly(A)+ RNAs, with a signal at 1.5 kb. Three signals at 4.0, 2.5, and 1.5 kb were detected by probe C, whereas two signals at 4.0 and 2.5 kb were detected by probe D. Parallel experiments showed that [32P]-labeled GAPDH probe cross-hybridized with poly(A)+ RNAs of JO21b and J774 cells, with a single signal at 1.0 kb. The densitometry units for MSR bands were divided by the corresponding units of GAPDH. The ratio of type I MSR mRNA level detected by probe B to the GAPDH level in JB23 cells (type I MSR mRNA mass/GAPDH mRNA mass = 0.27) was 2.5-fold larger than the corresponding ratio in J774 cells (type I MSR mRNA mass/GAPDH mRNA mass = 0.11). However, from the result using probe A which hydridized both type I and type II MSR mRNA, total MSR mRNA (type I and type II) was reduced by 70% in JO21b cells (MSR mRNA mass/GAPDH mRNA mass = 0.49) compared to J774 cells (MSR mRNA mass/GAPDH mRNA mass = 1.66). These results suggested the presence of a reduced activity of MSR in JO21b cells at the mRNA level.

Cytotoxic effect of Ox-LDL on peritoneal macrophages from MSR-knockout mice
In order to directly demonstrate the correlation of reduced MSR activity to reduced sensitivity to the cytotoxic effect of Ox-LDL, we attempted to transfect type II MSR cDNA or type I MSR cDNA into JO21b cells. However, several trials eventually failed. Thus, we compared the sensitivity of peritoneal macrophages obtained from MSR-knockout mice with that of their wild-type littermates by MTT methods (20). As shown in Table 1, incubation with 0.2 mg/ml of Ox-LDL for 48 h caused total cell death of both MSR-wild-type macrophages and MSR-knockout macrophages (Table 1). However, at 0.1 mg/ml of Ox-LDL, a decrease in cell viability of wild-type macrophages (36.9%) was significantly larger than that of knockout macrophages (1.3%) (Table 1). Similar results were obtained upon 24-h incubation with Ox-LDL (data not shown). These results clearly indicate that peritoneal macrophages from MSR-knockout mice are more resistant to the cytotoxic effect of Ox-LDL than those from MSR-wild-type mice, suggesting that MSR plays an enhancing role in the cytotoxic effect of Ox-LDL toward macrophages.


 
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  		Table 1. Cytotoxic effect of Ox-LDL on peritoneal macrophages obtained from MSR-knockout mice


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

The present study provides several lines of experimental evidence that MSR plays an important role in Ox-LDL-induced macrophage cell death. First, we have isolated mutant cells resistant to the cytotoxic effect of Ox-LDL from J774 cells (a murine macrophage-like cell line), and one mutant form, JO21b cells, showed a marked reduction in MSR activity and a concomitant reduction in their ability to take up Ox-LDL. Second, macrophages from MSR-knockout mice (32), whose activity for endocytic uptake of Ox-LDL was less than 50% of that of wild-type macrophages (32), showed significantly higher resistance to the cytotoxic effect of 0.1 or 0.12 mg/ml of Ox-LDL than those from their wild-type littermates (Table 1). However, 0.2 mg/ml of Ox-LDL became cytotoxic to macrophages from both MSR-knockout mice and their littermates (Table 1), suggesting that MSR-mediated uptake of Ox-LDL enhances macrophage cell death, rather than being essential for cell death. It is therefore highly likely that MSR plays an enhancing role in the cytotoxic effect of Ox-LDL toward macrophages or macrophage-derived cells.

In the current study, JO21b cells were characterized by their reduced expression of MSR. However, it is not clear whether single or multiple gene alternations occur in JO21b cells. Subsequent observations seem to support the latter possibility. First, the specific binding of Ox-LDL to JO21b cells was reduced by 50% compared to that to J774 cells (Figure 3A), while the endocytic degradation of Ox-LDL by JO21b cells was reduced by >80% compared to that by J774 cells (Figure 4B). These results suggest that an additional gene alteration(s) in JO21b cells may independently affect the endocytic process of Ox-LDL. Alternatively, it is also possible that there are cell surface binding sites for Ox-LDL that do not mediate endocytosis. Second, as the nonspecific binding of Ox-LDL to JO21b cells was also reduced by 50% compared with that to J774 cells (Figure 3), another gene alteration(s) could reduce the expression of cellular machinery involved in the nonspecific binding of Ox-LDL. This reduction may cause a decrease in the nonspecific transfer of toxic components of Ox-LDL and cause JO21b cells to be resistant to Ox-LDL. However, all these possibilities are speculative and have no molecular basis at the present. Further studies are therefore needed to identify a cDNA(s) that corrects the mutant phenotype of JO21b cells.

Ox-LDL is reported to be toxic not only to macrophages (14) (15) (17), but also to other cells, such as endothelial, smooth muscle, and fibroblast cells (for a review, see (42)). Previous studies showed that the cytotoxic effects of Ox-LDL were not cell specific (43), suggesting that the essential toxic component(s) of Ox-LDL is delivered to the cell interior via a non-specific exchange reaction between lipoproteins and plasma membranes. However, Schmitt et al. (44) and Nègre-Salvayre et al. (45) showed that the cytotoxic effects of ultraviolet-oxidized LDL on cultural bovine aortic endothelial cells and EB virus-transformed B lymphocytes were related to the amount of oxidized neutral lipids taken up specifically by these cells through the apoB/E receptor pathway (the LDL receptor pathway), suggesting that receptor-mediated endocytosis of Ox-LDL is involved in the cytotoxic effect of Ox-LDL. This notion was greatly supported by the present conclusion that MSR plays an enhancing role in Ox-LDL-induced cell death of macrophages. As extensive oxidative modification of LDL by Cu2+ results in a loss of its ligand activity for the apoB/E receptor (2) and Ox-LDL thus formed is specifically taken up by macrophages via MSR which leads to foam cell formation (46), our experimental system using macrophages and Cu2+-oxidized LDL likely reflects the events in the early stage of atherosclerotic lesions. Thus, the present study may explain that MSR-mediated uptake of Ox-LDL enhances the Ox-LDL-induced cell death of macrophages in a macrophage-specific manner during atherosclerotic lesion formation (16).

Recent cDNA expression and protein purification experiments have identified other Ox-LDL receptors, including Fc{gamma}RII-B2 (47), SR-BI (4), macrosialin (48), CD36 (49) and LOX-1 (50). Interestingly, Nozaki et al. (51) showed that the specific endocytic degradation of 40 µg/ml of 125I-labeled Ox-LDL by monocyte-derived macrophages from CD36-deficient subjects was 75% of that by the corresponding cells from normal subjects, suggesting an important role for CD36 in endocytosis of Ox-LDL by macrophages or macrophage-derived cells. However, our previous experiments showed that the endocytic degradation of 125I-labeled Ox-LDL by peritoneal macrophages obtained from the MSR-knockout mice was reduced by more than 50% compared with those of their wild-type littermates (32) (52). Although the involvement of other Ox-LDL receptors in the endocytic uptake of Ox-LDL in J774 cells is not well known, from all these results it can be expected that type I and type II MSR play a major role in the endocytic uptake of Ox-LDL in J774 cells as well as mouse peritoneal macrophages.

It was reported that oxysterol (33) (34) (35) (36), lipid hydroperoxide (37) (38) (39), and lysophosphatidylcholine (40) are cytotoxic components of Ox-LDL. In fact, all these compounds were cytotoxic to J774 cells as well as JO21b cells (Figure 2). However, we could not identify a toxic component(s) of Ox-LDL specifically involved in Ox-LDL-induced death of J744 cells. A recent study identified 7ß-hydroperoxycholesterol as a potent toxic component of Ox-LDL in human carotid atherosclerotic lesions (34). Moreover, Colles, Irwin, and Chisolm (35) reported that 7ß-hydroperoxycholesterol was predominantly responsible for Ox-LDL-induced cytotoxicity to fibroblasts, smooth muscle cells, and endothelial cells. Further studies are necessary to compare the cytotoxic effects of this compound on J744 and JO21b cells.

Two types of cell death, apoptosis and necrosis, have been so far described (53) (54). However, the mechanisms of macrophage cell death induced by Ox-LDL are not fully understood. In a previous study, Reid, Hardwick, and Mitchinson (14) showed that exposure of murine macrophage-like cells (P388D1 cells) to Ox-LDL led to DNA fragmentation, a process often used as a biochemical marker of apoptosis. We tested whether apoptosis might represent a mechanism of Ox-LDL-induced J774 cell death in accordance with Reid's procedure (14). However, repeated experiments failed to detect DNA fragmentation in J774 cells (data not shown). Several recent reports have demonstrated morphological apoptosis without DNA fragmentation (55) (56) (57). However, our electron microscopic observations failed to detect morphological features typical of apoptosis, such as condensation of nuclear chromatin, compact cytoplasmic organelles, and blebbing of the cell surface (data not shown). The exact reason for the apoptotic effect of Ox-LDL on P388D1 cells (14) but not J774 cells is not completely understood. The different response may reflect differences in cell type or methodology, or both. When all these observations are combined it seems that J774 cells mainly undergo necrosis rather than apoptosis when they are exposed to a high concentration of Ox-LDL.

In contrast to Ox-LDL-induced macrophage cell death, we reported previously that Ox-LDL can paradoxically induce macrophage cell growth in mice (58), humans (59), and rats (60). In this process, endocytic uptake of Ox-LDL containing lysophosphatidylcholine by MSR plays an important and efficient role in the mitogenic effect of Ox-LDL (20) (52). Under these conditions, incubation of mouse peritoneal macrophages with Ox-LDL at concentrations less than 0.1 mg/ml led to the growth of these cells (20) (52) (58), whereas incubation with Ox-LDL at concentrations higher than 0.1 mg/ml resulted in cell death in a manner similar to that observed with J774 cells (Figure 1A). Therefore, it seems likely that the mechanisms of Ox-LDL-induced macrophage cell growth are, in essence, related or similar to those of Ox-LDL-induced death of the same cells. This notion is supported by the fact that MSR-mediated endocytic uptake of Ox-LDL plays an enhancing role in both phenomena. Therefore, both the mitogenic and cytotoxic effects of Ox-LDL on macrophages should be pursued in parallel.


  ACKNOWLEDGMENTS

We are grateful to Dr. Michihiko Kuwano of the Department of Biochemistry, Kyushu University School of Medicine, for his helpful advice on the somatic cell genetics.

We are also grateful to Dr. Takashi Mori from the Second Department of Pathology, Kumamoto University School of Medicine (present address: Department of Internal Medicine, Miyazaki Prefectural Nobeoka Hospital) for his help with electron microscopy. We are also grateful to Drs. Yi Ding, Yu-Ichiro Sakamoto, Takeshi Biwa, Takashi Kawano, Yuko Hasunuma, and Takashi Kawasaki for their endeavor throughout this study. This work was supported by Grants-in-Aid for Scientific Research (No, 08044304, 09877200, and 09770789) from the Ministry of Education, Science and Culture of Japan.

Manuscript received August 27, 1997; and in revised form November 10, 1997.

Abbreviations: LDL, low density lipoprotein; acetyl-LDL, acetylated LDL; Ox-LDL, oxidized LDL; CE, cholesteryl esters; MSR, macrophage scavenger receptors; MTT, 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide; BSA, bovine serum albumin; TCA, trichloroacetic acid; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehydephosphate dehydrogenase; ssDNA, single-strand DNA


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T. Biwa, H. Hakamata, M. Sakai, A. Miyazaki, H. Suzuki, T. Kodama, M. Shichiri, and S. Horiuchi
Induction of Murine Macrophage Growth by Oxidized Low Density Lipoprotein Is Mediated by Granulocyte Macrophage Colony-stimulating Factor
J. Biol. Chem., October 23, 1998; 273(43): 28305 - 28313.
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