© 1997 American Heart Association, Inc.
Articles |
Acetylated LDL Endocytosis by the Human Monocytic Mono Mac 6sr Cells Is Not Mediated by the Macrophage Type I and II Scavenger Receptors
From the Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universität, Pettenkoferstr 9, 80336 Munich, Germany.
Correspondence to N. Hrboticky, Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Pettenkoferstr 9, 80336 Munich, Germany.
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Abstract |
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Abstract We recently reported that the human monocytic Mono Mac 6sr cell line constitutively takes up and degrades acetylated (acLDL) and oxidized LDL through receptor-specific pathways. The present studies were undertaken to further characterize the acLDL binding site on a functional and molecular basis. The degradation of acLDL increased during differentiation of Mono Mac 6sr cells with lipopolysaccharide (10 ng/mL, 72 hours) and low concentrations of phorbol 12-myristate 13-acetate (PMA; 0.1 to 1.0 ng/mL, 72 hours). Higher doses of PMA (5 or 10 ng/mL), however, decreased acLDL degradation. Scatchard plots of acLDL binding in untreated and LPS-differentiated Mono Mac 6sr cells were nonlinear and suggested the presence of more than one binding site. Although the ligand specificity of the acLDL receptor in Mono Mac 6sr cells resembles that of the macrophage type I and type II scavenger receptors, we did not detect mRNA of either receptor type in untreated or differentiated Mono Mac 6sr cells by means of Northern blotting and reverse transcription polymerase chain reaction. Furthermore, ligand blotting with 125I-acLDL failed to detect the 220-kD types I and II scavenger receptor protein. Thus, Mono Mac 6sr cells express an acLDL receptor that is distinct from the type I and type II scavenger receptor found in human monocyte–derived macrophages but that, like the latter, is induced during monocytic differentiation.
Key Words: acLDL • Mono Mac 6 cells • scavenger receptor • monocyte differentiation • atherosclerosis
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Introduction |
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Macrophage scavenger receptors mediate the unregulated uptake of modified LDL and thus may promote the generation of foam cells in the arterial wall. In addition, these receptors play a role in macrophage adhesion,1 2 the binding and clearance of endotoxins,3 Gram-positive bacteria,4 and apoptotic cells.5
Matsumoto et al6 first purified and cloned two trimeric glycoproteins, called type I and type II scavenger receptors, that are predominant on tissue macrophages. Among human monocytic cell lines, THP-1 and KP-1 express type I and type II scavenger receptor activity. However, expression occurs only after differentiation with phorbol esters.7 8 9 In contrast, Mono Mac 6sr cells constitutively express receptor-specific uptake mechanisms for acLDL and oxLDL.10
There is now conclusive evidence that several other receptors for modified LDL exist that are structurally distinct from those identified by Matsumoto et al.6 Early ligand cross-competition studies indicated the presence of receptors specific for either acLDL or oxLDL, in addition to a receptor common for both ligands.11 12 More recently, the proteins Fc
RII-B2,13 CD36,14 and macrosialin, the mouse homologue of human CD68,5 15 16 were identified as receptors for oxLDL. The CD36-related protein SRBI has been shown to bind both modified and native LDL17 as well as HDL.18 In addition, a number of unknown proteins with binding properties for modified LDL but distinct from the type I and type II scavenger receptors have been described.19 20 21 22 23
Several factors have been reported to modulate the endocytosis of modified lipoproteins in different cell types.24 Unlike the receptor-mediated endocytosis of native LDL, the uptake of modified LDL in macrophages is not regulated by intracellular levels of cholesterol. Macrophage scavenger receptor expression, however, does correlate with cell maturation. For example, freshly isolated human monocytes express low scavenger receptor activity, which increases greatly during differentiation into macrophages.25 26
In the present study, we examined the acLDL binding and degradation in Mono Mac 6sr cells on a functional as well as molecular level. Differentiation of the cells with LPS and low concentrations of PMA led to an increase in the receptor-mediated endocytosis of acLDL. However, Northern blotting analysis and RT-PCR of RNA from Mono Mac 6sr cells failed to detect the types I and II macrophage scavenger receptors. Furthermore, no 220-kD protein band corresponding to the types I and II scavenger receptor was detected in ligand blots with 125I-acLDL. Our data suggest that Mono Mac 6sr cells constitutively express a receptor protein for acLDL other than the macrophage type I and type II scavenger receptors, which, like the latter, is upregulated during monocyte differentiation.
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Methods |
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Materials
LPS extracted from Escherichia coli serotype 055:B5 and PMA were obtained from Sigma Chemical Co. Human recombinant tumor necrosis factor-
(8.74x106 U/mg; LPS content <2.7 pg/mg) was generously provided by BASF. 125I (carrier free) was purchased from Amersham. Iodine monochloride was prepared as described in Reference 2727 . SP6 RNA polymerase, RNasin, and RNase-free DNase I were obtained from Promega. Cell culture media and ingredients as well as all other chemicals were purchased from Sigma unless otherwise specified.
Cell Culture
Mono Mac 6sr cells10 were maintained in RPMI 1640 medium containing 10% FCS, insulin (9 µg/mL), oxaloacetate (1 mmol/L), pyruvate (1 mmol/L), penicillin (200 U/mL), streptomycin (200 µg/mL), nonessential amino acids (1x), and L-glutamine (2 mmol/L). The medium was ultrafiltered through a Gambro 2000 column to eliminate LPS. THP-1 and J774 cells were obtained from American Type Tissue Culture Collection and were grown in RPMI 1640 and DMEM media, respectively, both supplemented with FCS (10%) and L-glutamine (2 mmol/L).
Preparation of Lipoproteins
LDL was isolated from plasma of normolipidemic fasting subjects in the density interval of 1.019 to 1.063 by sequential preparative ultracentrifugation.28 Acetylation and iodination of LDL were performed as in References 2929 and 3030 , respectively. Protein concentrations were determined,31 and lipoproteins were filter sterilized (0.22 µm), stored at 4°C, and used within a month.
Assays for Proteolytic Degradation and Cell Surface Binding of acLDL
For the determination of proteolytic acLDL degradation, Mono Mac 6sr cells (2x106 cells/mL) were incubated with 125I-acLDL at 37°C for 5 hours, with or without a 25-fold excess of unlabeled acLDL. Cells were then immediately put on ice, and all subsequent procedures were carried out at 4°C. Incubation medium was removed after centrifugation (800 rpmx5 minutes), and cells were washed three times each in PBS with or without 0.2% BSA. Proteolytic degradation of 125I-acLDL was measured as trichloroacetic acid–soluble, silver nitrate–soluble radioactivity released into the medium.32 Washed cell pellets were solubilized in 0.2N NaOH, and the protein content was measured.31
Cell surface acLDL binding was determined in Mono Mac 6sr cells (2x106/mL) incubated with 125I-acLDL, with or without a 50-fold excess of unlabeled acLDL at 4°C for 2 hours. Cells were washed and cell protein was determined as described above. Specific binding and degradation values were obtained by subtracting nonspecific values (with excess of unlabeled ligand) from total values (without unlabeled ligand). Cell-free lipoprotein degradation was minimal and was subtracted from total degradation.
Cellular Cholesterol Accumulation
Mono Mac 6sr cells (2x105 cells/mL) were incubated in normal growth medium in the presence or absence of LPS (10 ng/mL) for 48 hours. Cells were then given fresh RPMI medium containing 10% delipidated FCS and incubated in the presence or absence of LPS and acLDL (200 µg/mL) for an additional 16 hours, as indicated in the figure legends. After incubation, cells were washed twice each with PBS with and without 0.1% BSA and extracted with hexane/isopropanol (3:2, vol/vol). Cellular cholesterol was determined in the total lipid extracts as in Reference 3333 , and protein content of extracted cell pellets was measured.34
RNA Isolation and Blotting
Total RNA isolation from Mono Mac 6sr and THP-1 cells, as well as RNA blotting, were done as described.35 Poly (A)+ RNA was purified by oligo(dT)-cellulose chromatography. RNA transcribed from DNAs subcloned in riboprobe vectors was used as probes: a 1585-bp EcoRI fragment from bovine type I scavenger receptor cDNA (a gift from M. Krieger, Massachusetts Institute of Technology) subcloned into the Bluescript SK vector and an 800-bp Pst fragment of rat GAPDH cDNA in SP65. The GAPDH probe was used to monitor the quality of the RNA and equal gel loading (internal reference). Riboprobes were prepared35 and hybridization was done as described,36 except that yeast RNA was omitted from the hybridization mix and washing was done at 65°C. The same blot was hybridized to both probes.
RT–Polymerase Chain Reaction
cDNA was produced from 250 ng total RNA by murine leukemia virus reverse transcriptase (Gibco-BRL) as described.37 Specific primers were synthesized (Dr Arnold, Genzentrum Martinsried, Munich, Germany): for ß-actin (length, 540 bp), forward primer was 5'-GTGGGGCGCCCCAGGCACCA-3' and reverse primer 5'-CTCCTTAATGTCACGCACGATTTC-3'; for scavenger receptors type I and II (366 bp), forward primer was 5'-CCAGGGACATGGGAATGCAA-3' and reverse primer 5'-CCAGTGGGACCTCGATCTCC-3'; and for the LDL receptor (258 bp), forward primer was 5'-CAATGTCTCACCAAGCTCTG-3' and reverse primer 5'-TCTGTCTCGAGGGGTAGCTG-3'. cDNA was amplified with Taq polymerase (Applied Biosystems) in a thermocycler 480 (Perkin Elmer). PCR products were separated on a 1.5% agarose gel. Further analysis of PCR products was performed by HPLC separation on a DEAE column (Applied Biosystems) with UV detection at 260 nm (Gilson 115 Variable Wavelength Detector, Abimed-Gilson).38
Membrane Solubilization and Ligand Blotting
Between 2x108 and 6x108 Mono Mac 6sr, THP-1, and J774 cells were suspended for 30 minutes in a hypotonic lysis buffer (10 mmol/L Tris-HCl, pH 7.4, containing EDTA 1 mmol/L, PMSF 0.6 mmol/L, apoprotinin 0.5 µg/mL, leupeptin 0.5 µg/mL, and pepstatin 0.7 µg/mL) and subsequently homogenized with 20 to 80 strokes in a Dounce homogenizer. After adjustment to 150 mmol/L NaCl, the homogenate was centrifuged at 800g for 10 minutes. The supernatant was centrifuged at 10 000g for 15 minutes, and the resulting supernatant was centrifuged at 100 000g for 1 hour to obtain a crude membrane pellet. Crude membrane proteins were solubilized with 40 mmol/L octyl glucoside, and insoluble material was sedimented by centrifugation at 100 000g for 1 hour. All procedures were carried out at 4°C.
Solubilized proteins were separated under nonreducing conditions on 5% to 12% gradient SDS-PAGE gels.39 Proteins were electrotransferred (50 V, 22 hours) to polyvinylidine difluoride membranes (BioRad) in a Towbin buffer (25 mmol/L Tris-HCl, 192 mmol/L glycine, pH 8.3) without methanol. Nonspecific binding was blocked by incubation at room temperature for 1.5 hours with 10 mmol/L Tris-HCl, 90 mmol/L NaCl, and 1 mmol/L EDTA, pH 8.0, containing 5% nonfat dry milk and 250 µg/mL native LDL. Membranes were then incubated with 125I-acLDL (20 µg/mL; specific activity, 468 cpm/ng) in the above buffer containing 1% nonfat dry milk and 250 µg/mL native LDL for 3 hours and subsequently washed 8 times (10 minutes each) in buffer containing 1% nonfat dry milk. Membranes were air dried and exposed to Kodak X-Omat AR film for 8 hours at -80°C.
Statistical Analysis
Statistical analysis was performed with the nonpaired Student's t test.
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Effect of Cell Differentiation on acLDL Degradation by Mono Mac 6sr Cells
Regulation of acLDL degradation in Mono Mac 6sr cells during cellular maturation was studied by use of the bacterial endotoxin LPS and the phorbol ester PMA, two agents known to induce differentiation in this cell line.40 As shown in Fig 1A
, LPS increased 125I-acLDL degradation in a dose-dependent manner. After 72 hours of treatment, acLDL degradation increased by 76% (P<.05) with LPS concentrations as low as 0.1 ng/mL, whereas an increase of 239% (P<.01) was seen with 10 ng/mL LPS. In contrast to LPS, PMA treatment had a biphasic effect on acLDL degradation (Fig 1B). Although low concentrations of PMA (0.1 to 0.5 ng/mL) led to a significant rise in the degradation rate of acLDL (+130% with 0.5 ng/mL PMA; P<.01), higher PMA concentrations (5 to 10 ng/mL) inhibited acLDL degradation (-56% with 10 ng/mL PMA). DMSO, the carrier of PMA, had no effect on acLDL degradation at the concentration used (1 µL/mL) (data not shown).
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For purposes of comparison, the effect of LPS on acLDL receptor activity was examined in the monocytic THP-1 cell line, induced to express the type I and type II scavenger receptors with 100 ng/mL PMA for 48 hours.7 8 In contrast to Mono Mac 6sr cells, incubation of PMA-differentiated THP-1 cells with increasing concentrations of LPS for 48 hours suppressed acLDL degradation in a dose-dependent manner (Fig 2). This inhibitory effect of LPS was also observed when untreated THP-1 cells were simultaneously coincubated with PMA and LPS for 72 hours (data not shown). In both cell lines, cell viability, assessed by means of ethidium bromide/acridin orange fluorescence, was >90% at all PMA and LPS concentrations used.
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Cell Surface Binding of acLDL in Control and LPS-Differentiated Mono Mac 6sr Cells
acLDL binding (Fig 3A) and degradation (Fig 3C) were saturable in untreated Mono Mac 6sr cells and reached saturation at 
2.5 µg acLDL/mL. Average nonspecific binding and degradation were <20% of total binding and degradation (data not shown). Scatchard plots of cell surface binding data of untreated Mono Mac 6sr cells were concave and suggested the existence of two binding components (Fig 3B). Linear regression analysis was used to identify a high-affinity binding site with an estimated Bmax of 11.5 ng/mg protein and Kd of 0.22 µg/mL, or 4.34x10-10 mol/L, and an additional lower-affinity binding component (Bmax, 16.1 ng/mg; Kd, 1.71 µg/mL, or 3.33x10-9 mol/L).
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Parallel to its effect on acLDL degradation (Fig 3C
), the specific binding of acLDL was increased in LPS-differentiated cells (Fig 3A). LPS (10 µg/mL) increased the Bmax of both the high-affinity (11.5 versus 21.0 ng/mg protein, control versus LPS) and low-affinity (16.1 versus 60 ng/mg protein, control versus LPS) binding components (Fig 3B), indicating an increase in receptor number. LPS had no significant effect on the affinity of the high-affinity binding site (Kd LPS=0.21 µg/mL, or 4.09x10-10 mol/L) but increased the estimated Kd of the low-affinity component from 1.71 to 8.07 µg/mL (1.57x10-8 mol/L). Nonspecific binding was not affected by LPS treatment (data not shown).
Cholesterol Accumulation in Mono Mac 6sr Cells
As shown in Fig 4, the incubation of Mono Mac 6sr with acLDL (200 µg/mL) for 16 hours resulted in significant increases in intracellular cholesterol levels. This intracellular cholesterol accumulation was further increased in cells differentiated with LPS (10 ng/mL, 48 hours) before incubation with acLDL. LPS treatment alone had no effect on cellular cholesterol levels (data not shown).
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, Values significantly different from untreated cells and cells incubated with 200 µg acLDL, respectively.
RT-PCR and Northern Blot Analysis
To characterize the acLDL binding sites in Mono Mac 6sr cells at the mRNA level, RT-PCR was performed on total cell RNA. Mono Mac 6sr RNA failed to yield a PCR signal specific for type I and type II scavenger receptors (Fig 5). Treatment of the cells with LPS did not induce type I and type II scavenger receptor expression, whereas exposure of Mono Mac 6sr cells to PMA gave rise to trace amounts of scavenger receptor–specific RNA, detectable only by the very sensitive HPLC method (Fig 5B). RNA degradation did not occur, because RNA of all samples was positive for ß-actin and the LDL receptor. In contrast, THP-1 cells treated with PMA (100 ng/mL PMA for 24 hours) gave rise to a 366-bp PCR product detectable by both electrophoresis (Fig 5A) and HPLC (Fig 5B). In untreated THP-1 cells, this band was barely detectable.
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The absence of the type I and type II scavenger receptor mRNA in Mono Mac 6sr cells was confirmed in three separate Northern blotting experiments using a cDNA probe able to detect both types of the scavenger receptor. As illustrated in Fig 6, no specific mRNA bands were seen in this cell line. In contrast, the expected scavenger receptor mRNA bands41 42 were observed in PMA-treated THP-1 cells. To increase sensitivity, poly(A)+ RNA from untreated Mono Mac 6sr cells was assayed by Northern hybridizations. No signal corresponding to type I and type II scavenger receptor mRNA could be detected (data not shown).
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To eliminate the possibility that scavenger receptor mRNA was present earlier than at the time of isolation (72 hours after passage), total RNA from untreated cells was isolated at 1, 3, 5, 8, and 24 hours after passage. No mRNA specific for type I and type II scavenger receptors was detected at any of the tested time points (data not shown). Furthermore, scavenger receptor mRNA was not induced in cells treated with 10 ng/mL LPS (Fig 6), which was maximally effective in enhancing 125I-acLDL binding and degradation.
Ligand Blotting With 125I-acLDL
In agreement with our results at the mRNA level, ligand blotting with 125I-acLDL failed to detect protein bands corresponding to the type I and type II scavenger receptors in plasma membrane proteins isolated from Mono Mac 6sr cells. As shown in Fig 7, 125I-acLDL showed ligand binding to a 210- to 240-kD protein band in membrane proteins isolated from the PMA-differentiated human THP-1 cells (lane 3) and from the mouse macrophage J774 cells (lane 4). This band, presumably corresponding to the type I and type II scavenger receptors, was absent in untreated THP-1 cells (lane 2) and could be blocked in PMA-treated THP-1 and J774 cells by preincubation of strips with 1 mg/mL fucoidan, a known ligand for the type I and type II scavenger receptors (data not shown). Furthermore, this band was not blocked by native LDL, which was present in the blocking buffer. Plasma membranes from J774 cells showed additional ligand binding to a band at 95 kD (lane 4), which was not inhibitable with fucoidan (data not shown). This latter band may correspond to the recently identified mouse macrophage membrane protein, which recognizes oxLDL and phosphatidylserine-rich liposomes15 16 but can also bind acLDL in ligand blots.15 In contrast, we detected no 125I-acLDL ligand binding to plasma membrane proteins of untreated (data not shown) or LPS-differentiated Mono Mac 6sr cells (lane 1) under blotting conditions that proved positive for the detection of the type I and type II scavenger receptor in PMA-differentiated THP-1 cells and in the J774 cells. Ligand binding of native 125I-LDL to a 130-kD protein, corresponding to the classic LDL receptor, was possible in identical preparations of Mono Mac 6sr plasma membranes (data not shown).
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Discussion |
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We recently reported that the human monocytic Mono Mac 6sr cell line constitutively exhibits receptor-specific uptake mechanisms for acLDL and oxLDL.10 The present study indicates that endocytosis of acLDL in these cells is mediated by a receptor protein other than the macrophage type I and II scavenger receptors.6
Ligand blotting of plasma membrane proteins prepared from Mono Mac 6sr cells with 125I-acLDL did not yield the 210- to 240-kD protein band corresponding to the type I and type II macrophage scavenger receptors under blotting conditions positive in the mouse macrophage J774 cells and the PMA-differentiated human THP-1 cells. The reason behind the lack of a protein band binding 125I-acLDL in the ligand blot of Mono Mac 6sr cells under the blotting conditions used remains to be elucidated. However, it suggests the presence of a receptor protein with binding properties different from those of the type I and type II macrophage scavenger receptors. Furthermore, we failed to detect mRNA specific for both types of the macrophage scavenger receptors in this cell line. Trace amounts of type I and type II scavenger receptor mRNA were observed in Mono Mac 6sr cells treated with PMA. However, our data suggest that this very weak induction was not responsible for the acLDL endocytosis in Mono Mac 6sr cells. First, both 0.5 and 100 ng/mL PMA equally induced type I and type II scavenger receptor-specific mRNA, but only the lower PMA concentration led to an increased 125I-acLDL degradation. Second, LPS did not induce scavenger receptor mRNA but increased acLDL binding and degradation to a larger extent than PMA. Furthermore, small amounts of type I and type II scavenger receptor mRNA could also be measured in untreated THP-1 cells, which showed no significant uptake of 125I-acLDL.
The ligand specificity of the acLDL binding sites in the Mono Mac 6sr cell line10 does not correspond to other previously described alternative scavenger receptors shown to bind acLDL. Unlike SR-BI, a CD36-related receptor for acLDL,17 acLDL uptake in Mono Mac 6sr cells was inhibited by polyanions such as fucoidan and polyinosinic acid but not by native LDL.10 The not yet fully characterized acLDL receptor protein expressed in Chinese hamster ovary cells cultured in the presence of the cholesterol synthesis inhibitor simvastatin and acLDL is also not blocked by polyanions.21 Furthermore, the 94- to 97-kD receptor protein recently purified from mouse peritoneal macrophages binds oxLDL but not acLDL with high affinity.15 16
A classification of scavenger receptors has recently been proposed.24 In this scheme, the acLDL endocytosis in Mono Mac 6sr cells appears to be mediated by class A receptor(s), because thus far, it exhibits a ligand pattern identical to the type I and type II scavenger receptors.10 Recently, a novel bacteria-binding receptor that also recognizes acLDL and is structurally related to type I and type II scavenger receptors has been cloned.22 Endothelial cells also express an acLDL receptor distinct from type I and type II scavenger receptors but with a similar ligand specificity and a common estimated molecular mass of 220 kD.20 23 The presence of a novel class A scavenger receptor protein in the Mono Mac 6sr cell line is therefore conceivable.
Scavenger receptor activity in human monocyte–derived macrophages is known to increase with cell maturation or differentiation.25 26 We thus investigated whether the acLDL receptor in the Mono Mac 6sr cells is similarly upregulated during monocyte maturation. LPS and PMA have been shown to induce distinct patterns of differentiation in Mono Mac 6 cells.40 We found LPS (0.1 to 10 ng/mL) to stimulate acLDL degradation and binding in a dose-dependent manner. In contrast to Mono Mac 6sr cells, LPS suppressed scavenger receptor activity in PMA-treated THP-1 macrophages. LPS also suppresses macrophage scavenger receptor expression in human monocyte–derived macrophages,43 Chinese hamster ovary cells transfected with type I and type II scavenger receptors,44 and mouse peritoneal macrophages.45
In contrast to LPS, PMA had a biphasic effect on acLDL degradation in Mono Mac 6sr cells. Low PMA concentrations (0.1 to 1 ng/mL) increased and higher PMA concentrations (5 to 10 ng/mL) decreased acLDL degradation. The mechanism of this biphasic effect is not known. In the premonocytic THP-1 cell line, PMA in concentrations of 10 to 400 ng/mL are required to induce type I and type II scavenger receptor expression,8 presumably via a protein kinase C–mediated stimulation of yet unidentified transcription factors.46 Conversely, PMA in similarly high doses can reduce the binding and uptake of acLDL in mouse peritoneal macrophages45 47 by posttranscriptional mechanisms that induce changes in intracellular and/or cell surface receptor distribution.47 The biphasic effect of PMA on acLDL degradation in our cells may thus reflect a sum of pretranslational and posttranslational PMA effects. Our results in LPS- and PMA-differentiated Mono Mac 6sr cells led us to conclude that, like the scavenger receptor activity in human monocyte–derived macrophages, the increase in acLDL receptor activity in these cells correlates with cell differentiation. The upregulation of the acLDL endocytosis in Mono Mac 6sr cells by the bacterial endotoxin LPS further suggests that the acLDL receptor expressed in this cell line is influenced by mediators of the inflammatory response. In support of this, the cytokine tumor necrosis factor- (100 U/mL, 72 hours) also increased acLDL degradation by 58% (data not shown). However, the present data do not allow us to speculate as to whether the atypical effects of LPS and PMA in Mono Mac 6sr cells are cell specific or receptor protein specific.
Scatchard plots derived from the acLDL binding data in Mono Mac 6sr cells were nonlinear. Although some studies of acLDL binding to the macrophage scavenger receptor show linear Scatchard plots,47 48 nonlinear plots have also been reported, especially when binding is measured over a broad ligand concentration range.44 49 50 Nonlinear Scatchard plots usually suggest the presence of multiple, nonidentical binding sites on a single protein or the existence of two different binding proteins. However, such nonlinear plots are also consistent with the presence of multiple identical sites exhibiting negative cooperativity or with a lattice of closely spaced identical sites at which the initial binding of large ligands sterically hinders subsequent binding. The latter lattice model was recently used to describe the kinetics of LDL binding to the LDL receptor.51 At this point, we cannot say which one of the above interpretations best explains the acLDL binding kinetics in our cells. LPS differentiation resulted in an increase in the estimated receptor number of both the high- and low-affinity binding components. Whether the LPS-related increase in the estimated affinity of the low-affinity component was due to changes in protein conformation or other mechanisms cannot be answered by the present data.
The physiological relevance of the acLDL receptor in the Mono Mac 6sr cells is unknown. The specific binding of acLDL and oxLDL suggests its involvement in the clearance of modified lipoproteins. In support of this, cellular cholesterol accumulation was observed after incubation of Mono Mac 6sr cells with acLDL. Furthermore, cross-competition with the polysaccharide fucoidan and the polyribonucleotide polyinosinic acid indicates the presence of a multiligand receptor capable of diverse biological functions. In conclusion, this work points to the presence of a novel acLDL receptor protein in the monocytic Mono Mac 6sr cell line, which, in contrast to other newly identified acLDL receptors, is expressed constitutively on a cell of human origin. Intensive studies are under way to further characterize this protein at the molecular level and to elucidate its physiological and pathophysiological function.
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Acknowledgments |
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This work was supported by BMFT grant 07ERG03 and the August Lenz Stiftung, Germany. Dr Hrboticky was partially supported by the Alexander von Humboldt Stiftung and the University of Munich HSPII grant program. We thank Monica Laliberte and Birgit Vetter for expert technical assistance and Dr Monty Krieger (Massachusetts Institute of Technology) for the gift of bovine type I scavenger receptor cDNA.
Received May 31, 1996; accepted September 3, 1996.
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