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In vitro glycated low-density lipoprotein interaction with human monocyte-derived macrophages
0
INSTITUT
Res. Immunol. 1992, 143, 17-23
PASTEUR/ELSEVIER
Paris 1992
In vitro glycated low-density lipoprotein interaction human monocyte-derived macrophages
with
A. Gugliucci Creriche (‘), S. Dumont t2), J.C. Siffert c2), and A.J.C. Stahl (‘) “)Biochemistry
and 12)Immunopharmacology Laboratories, Facultt de Pharmacie, BP 24, 67401 Illkirch Cedex (France)
UniversitP Louis Pasteur,
SUMMARY Human low-density lipoprotein (LDL) was glycated in vitro (5 days, glucose 50 mmol/l), labelled with 1251, and its binding and uptake by human monocyte-derived macrophages studied. Glycation produced lower binding and lower uptake. Competition experiments using unlabelled LDL (control, glycated, and acetyl-LDL) showed that most glycated LDL was taken up by the apolipoprotein-Bl 00 : E receptor pathway. Results suggest that less of the glycated LDL may enter the cells via scavenger receptors, and very minute amount via non-saturable receptor-independent pathways. Key-words: Lipoprotein, Cell uptake, Competition.
Glycation,
Macrophage;
Introduction
Glycation (non-enzymatic glycosylation) of proteins is believed to be one of the primary initiating events in the pathogenesis of many of the chronic complications of diabetes mellitus (Brownlee et al., 1988; Vlassara et al., 1986). Glycation of apolipoprotein B from low-density lipoprotein (LDL) has been shown to occur both in vitro, (Gonen et al., 1981; Sasaki and Cottam, 1982 ; Schleicher et al., 1985 ; Witztum et al., 1982) and in vivo (Curtiss and Witztum, 1985; Witztum et al., 1984). Moreover, evidence for involvement of glycated LDL (glc-LDL) in the accelerated atherogenesis of long-term diabetes has appeared over the last few years. Reports exist showing impaired recognition of glcLDL by the ApoBlOO:E receptor (Gonen et al., 1981; Sasaki and Cottam, 1982; Schleicher et al., 1985). A recent publication (Lopes Virella et al., 1988) demonstrates that glc-LDL stimulates more choles-
LDL, Acetylation,
Receptor
binding,
teryl ester (CE) synthesis than does LDL (c-LDL) in human monocyte-derived macrophages (HMDMAC). The authors also suggest the existence of a putative ApoBlOO:E-receptor-independent mechanism for glcLDL uptake in HMDMAC. There is also evidence that different forms of modified LDL can enter the macrophage (MAC) through the scavenger receptor. Experiments with malonyldialdehyde (MDA)modified LDL have shown a threshold effect of derivatization which induces recognition by the scavenger receptor, while suppressing that of the ApoB100:E receptor (Haberland et al., 1982). To further characterize the interaction of glc-LDL with HMDMAC, we studied the binding and the uptake of the particles by HMDMAC. Materials
and Methods
Chemicals. - Medium : RPMI-1640 was purchased from GIBCO ; “‘1 was obtained from CEA
Submitted September 28, 1991, accepted November 21, 1991.
A. GUGLIUCCI
18
(Gif-sur-Yvette) : Iodogen was suppplied by Pierce; KC1 and EDTA were from Merck; Zymofren (aprotinin) from Specia. All other chemicals were purchased from Sigma. Plasma. - Pooled plasma from venipuncture of fasted subjects was obtained from the Laboratoire de Biochimie, Clinique Medicale “B” CHU Strasbourg. After blood collection on EDTA, aprotinin and NaN, were added as described by Fischer and Schumaker (1986). Cells. - Human monocytes were obtained from the CHU, Hautepierre, Strasbourg, Service d’OncoHtmatologie, Professeur Oberling. Pure human monocytes were prepared from the blood of normal human subjects by countercurrent centrifugal elutriation of leukapheresis specimens as described (Stevenson, 1985). The purity of the preparation was greater than 90 %, as checked by morphology, latex particle ingestion, and non-specific esterase staining. The cells were suspended in RPMI-1640 medium, supplemented with 10 % fcetal calf serum, to a final concentration of 2x IO6 cells/ml. Then, 2 x IO6 cells were pipetted in 35-mm tissue culture dishes and treated as by Lopes Virella et al. (1988). After one week of maturation to macrophages, serum was substituted for lipoprotein-depleted serum (LDS) for 24 h to allow upregulation of LDL receptors. LDL isolation Human LDL (d = 1019-1063 g/ml) was prepared from pooled EDTA plasma of fasted subjets by sequential flotation ultracentrifugation as previously described (Fischer and Schumaker 1986). Throughout the preparation procedures, lipoproteins (Lp) were kept in 1 mM EDTA, 1.5 mM 1 mM phenylmethylsulphonyl fluoride NaN,, (PMSF) and 0.1 mg/ml aprotinin. A single band was obtained in either SDS-PAGE (5 (70 acrylamide) or 1 070 agarose electrophoresis. Visualization of the bands was accomplished by staining with Coomassie brillant blue R250 and with Sudan black. After dialysis against PBS (10 mM sodium phosphate buffer, 0.15 M NaCI, pH 7.4), the LDL pre-
CRERICHE
ET AL.
paration was stored in 20 % sucrose at - 20°C until use (Fischer and Schumaker, 1986). For experiments, LDL was thawed, dialysed extensively, kept at 4°C and used within one week. Modification
of LDL
1) Glycation LDL (1 mg/ml) was incubated either in PBS containing 1 mM PMSF, 1.5 mM NaN,, and 1 mM EDTA for control LDL (c-LDL), or in the same solution supplemented with 50 mM glucose for glc-LDL. Incubation was at 37’C in screw-capped tubes for different periods up to five days. Absence of proteolysis was confirmed by SDS-PAGE as described above or by autoradiography of labelled samples. Relative mobility of LDL after glycation was measured by agarose electrophoresis (cc. 3 mA/cm, 3 h). Strips showed distinct, major bands of different anodie mobilities which depended on the length of incubation. Acetylation LDL was acetylated (ac-LDL) Basu et al. (1976). Radioiodination
as described by
of LDL
LDL (c-LDL, glc-LDL and ac-LDL) were labelled by the method of Fraker and Speck (1978) with iodogen and isolated by chromatography on a SephadexG25 column (10 x 300 mm). A specific activity of 100-400 cpm/ng was obtained. LDS LDS was prepared as by Lopes Virella (1988).
ac-LDL AGE ApoB BSA CE c-LDL EDTA glc-LDL
acetyl-LDL. advanced glycosylation endproduct. apolipoprotein B. bovine serum albumin. cholesteryl ester. control (non-glycated) LDL. ethylendiamine tetraacetate. glycated LDL.
HMDMAC LDL LDS MAC MDA PBA PBS TBARS
= = = = = = = =
human monocyte-derived macrophage. low-density lipoprotein. lipoprotein-depleted serum. macrophage. malonyldialdehyde. (M-amino)phenylboronate agarose. phosphate-buffered saline. thiobarbiturie acid-reactive substance.
et al.
GLYCATED
LO W-DENSITY
Advanced glycosylation endproducts bovine serum albumin (BSA)
LIPOPROTEIN (AGE)
of
AGE-BSA was prepared by incubating BSA (5 mg/ml) in PBS with 100 mM glucose for 7 weeks (Lopes Virella et al., 1988). I) Thiobarbituric
INTERACTION 3) Glycation
WITH
MACROPHAGES
19
with “C-glucose
To assessthe degree of glycation attained in our incubation experiments, we also performed LDL glycation in the presence of traces of 14C-labelled glucose in a medium containing 10 or 50 mM glucose according to Witztum et al. (1982).
acid-reacting substances (TBARS)
The degree of oxidation of all LDL preparations was controlled by measuring the content of TBARS as described (Esterbauer and Chelseman, 1990), and expressed in nmol MDA equivalents/mg ApoB. 2) m-aminophenylboronate tography
agarose (PBA) chroma-
PBA affinity chromatography was carried out as previously described (Shakler et al., 1984).
4) Binding
and uptake experiments
As receptor activities have been shown to vary greatly, even with the same cell strain studied on different days, comparative experiments were always performed with the same batch of cells and LDS, and in the same week. In binding experiments, we incubated monolayers of 2 x lo6 cells/dish with radiolabelled Lp in RPM1 medium a 4°C for 2 h with gentle rocking (Innerarity et al., 1986).
80
60
Molar excess of competing ligands Fig. 1. Binding of rz51-c-LDL and rzsI-glc-LDL (7.5 ug/dish) to human monocyte-derived macrophages in the absence (100 % value) or in the presence of increasing amounts of non-labelled c-LDL, glc-LDL and ac-LDL. a) rzSI-c-LDL 100 Vo binding corresponds to 3.4 f 0.58 pM/mg cell protein. Each point represents mean of three experiments run in duplicate. b) iZ51-glc-LDL 100 % binding corresponds to 3.0 f 0.47 pM/mg cell protein. Each point represents mean of three experiments run in duplicate.
20
A. GUGLIUCCI
CRERICHE ET AL.
In uptake experiments, incubation was at 37°C in a 5% CO,-humidified incubator for 5 h. In both cases, the medium was discarded after incubation and dishes were thoroughly washed twice for 10 min with 50 mM TRIS-HCl at pH 7.4 containing 2% BSA, followed by three washings with the same buffer without BSA (Goldstein et al., 1983). Cells were lysed with three washings of 100 mM NaOH (0.5 ml/dish, 20 min each). Each washing was collected and radioactivity was counted. Cell protein was measured by the micromethod of Bradford (1976) using BSA in 100 mM NaOH to run a standard plot. Results LDL glycarion
“C-glucose incorporation was time-and dosedependent and reached a maximum of 4.0 mol/mol ApoB (assumed MW 550,000 indicated by Aggerbeck, 1990), in 5-day incubation and at a concentration of 50 mM (data not shown). With 10 mM glucose, the incorporation was 1.0 mol/mol ApoB. Both results are in accordance with previous publications (Gonen ef al., 1981; Schleicher ef al., 1985). Glc-LDL showed the same behaviour in SDSPAGE both with and without previous treatment with @mercaptoethanol. A single band for glc-LDL, with greater anodic mobility than c-LDL, was detected in agarose strips. The degree of glycation is therefore similar in the majority of the molecules in this preparation. Relative anodic mobility of glc-LDL (mobility of glc-LDL/mobility of c-LDL) as a function of time of incubation in 50 mM glucose shows a straight line (data not shown). Retention by PBA was 2.0 + 0.2 % for c-LDL and 25.0 + 1.5 070 for glc-LDL (mean + S.D., n = 3). Phenylboronate bound cis-diols present in ketoamine adducts of glycated proteins. TBARS contents of LDL preparations was 1.2 f 0.2 mM MDA equivalents/mg ApoB, and no significant differences between c-LDL and glc-LDL were observed. TBARS did not increase significantly during conservation of the preparations at -20°C up to three months. Binding and uptake
Competition of binding to macrophages between the labelled c-LDL and increasing molar concentrations of non-labelled c-LDL, ac-LDL and glc-LDL at 4°C is shown in figure la. 125-I-labelled c-LDL bound to the ApoBlOO:E receptor could be displac-
0
Molar excess of competing ligands Fig. 2. Uptake of “SI-c-LDL (15 t&dish) by human monocyte-derived macrophagesin the absence(100 % value) or in the presenceof increasingamountsof nonlabelledc-LDL, glc-LDL and ac-LDL. “‘1-LDL 100 % uptake correspondsto 35 f 4 pmol LDL/mg cell protein. Each point representsthe meanof three experimentsrun in duplicate.
ed by c-LDL, but also by glc-LDL, and far less efficiently by ac-LDL. Half-maximal binding at 4°C requires 30 Fg/l for c-LDL and 40 pg/ml for glcLDL. Glc-LDL is therefore recognized by the ApoBlOO:E receptor, though not to the same extent as is c-LDL, becausehigher concentrations are needed to displace c-LDL. Glycation of LDL reduces the affinity of LDL towards the ApoBlOO receptor, but does not completely hinder its binding. Mannan and AGE-BSA failed to displace c- and glc-LDL from their receptors (data not shown). When labelled glc-LDL is displaced from its binding to the macrophages by unlabelled ligands, there is some competition by ac-LDL, besides displacement by glc-LDL and c-LDL, showing that a certain proportion of glc-LDL is recognized by the scavenger receptors (fig. 1b). The uptake by macrophages of labelled c-LDL in the presence of increasing amounts of non-labelled c-LDL, glc-LDL and ac-LDL is shown in figure 2. Uptake of c-LDL by macrophages was most efficiently hindered by glc-LDL, and less so by ac-LDL.
GL YCA TED LO W-DENSITY
LIPOPROTEIN
INTERACTION
WITH
MACROPHAGES
21
Discussion
Interaction of modified LDL with arterial monocytes and macrophages has been proposed as one of the factors leading to their transformation in to foam cells, characteristic of the factors leading to their transformation into foam cells, characteristic of atherosclerotic lesions (Ross 1986; Steinberg and Olefsky, 1987). In this regard, much attention has recently been given to oxidative modifications occurring both in plasma and in situ (Brown and Goldstein, 1990; Halliwell and Gutteridge, 1990).
h
g
80
Glycation of LDL has been shown to occur in vitro and in vivo, and the effects of this modification upon its metabolism have been studied in fibroblasts, murine MAC and, more recently, in human MAC (Curtiss and Witztum, 1985; Gonen et al., 1981; Lopes Virella et al., 1988 ; Sasaki and Cottam, 1982; Schleicher et al., 1985; Witztum et al., 1982; Witztum et al., 1984). 10
20 Molar excess of competing ligands 0
Fig. 3. Uptake of ‘2SI-glc-LDL (15 vg/dish) by HMDMAC
in the absence (100 % value) or in the presence of increasing amounts of non-labelled c-LDL glc-LDL and ac-LDL. ‘251-glc-LDL 100 % uptake corresponds to 30 + 5 pmol LDL/mg cell protein. Each point represents the mean of three experiments run in duplicate.
Reverse experiments, measuring the uptake of glcLDL in the presence of increasing amounts of modified LDL, showed that glc-LDL uptake was partially restrained by c-LDL (fig. 3). Results indicate that glcLDL binds and is taken up by the ApoBlOO:E receptor and by scavenger receptors. In another set of experiments, we measured the uptake by macrophages of labelled glc-LDL in the presence of a large excess of both non-labelled c-LDL and ac-LDL (data not shown). Hence, when both ApoBlOO:E receptors and scavenger receptors were blocked, the macrophage was still able to take up small amounts of glc-LDL by a non-specific, nonsaturable pathway. Even the negatively charged polyvinylsulphate (2 mg/ml), which completely blocks the scavenger receptors, did not impair this uptake of glc-LDL. By measuring MDA in all LDL preparations, we checked that no strong oxidation occurred during chemical modifications of LDL. No significant differences were noted between values found in c-LDL, glc-LDL or ac-LDL preparations.
Recent reports suggest a link between glycation and oxidation ; glycation per se could lead to oxidation in a process referred to as autoxidative glycosylation (Hunt et al., 1990). To characterize the interaction of glc-LDL as compared to c-LDL, we studied binding and uptake of these lipoproteins by HMDMAC. Our binding studies clearly showed diminished binding of glc-LDL compared to c-LDL, as well as a lower affinity, in accordance with previous reports concerning fibroblasts and MAC (Gonen et al., 1981; Witztum et al., 1982). It has been argued (Lopes Virella et al., 1988) that glc-LDL stimulates greater cholesteryl ester synthesis in HMDMAC than does c-LDL, which suggests its uptake via pathways other than the classical ApoB1OO:E receptor. We performed competition studies for the binding and uptake of c-LDL, glc-LDL and ac-LDL. Our results confirm the lack of competition with mannan and AGE-BSA, which goes against the participation of the mannose-fucosereceptor and the AGE-receptor in this process. The lack of recognition of our glc-LDL by the latter receptor confirms the fact that glycation has only been pursued to the degree of Amadori rearrangement and has not reached the stage of AGE products. We performed glycation for 5 days (half life of ApoB) with 50 mM glucose in the absence of reducing agents in order to obtain protein modifications which could occur in a pathological situation such as in heavily decompensated diabetic subjects. Displacement curves (figs. 1 to 3) show that most of the binding and uptake of glc-LDL occur via the classical ApoBlOO:E receptor. However, there is a higher half-maximal binding concentration for glc-LDL than for c-LDL. When labelled glc-LDL is displaced by unlabelled ligands, there is a 50 % inhibition of uptake by ac-LDL at a lo-fold concentration. This
22
A. GUGLIUCCI
would indicate that at least a subfraction of glc-LDL is recognized by the scavenger receptors. This could be due to glc-LDL itself or to slight differential oxidation of the preparations involved. TBARS contents showed no significant differences between c-LDL and glc-LDL. Our results suggest that, though most glc-LDL is taken up by the ApoB1OO:E mechanism, glycation could transform LDL (or at least a subfraction) into a form recognized by scavenger receptors. Particles of LDL may exist which are both glycated and slightly oxidized. Indeed, reactive aldehydes produced in the oxidative cascade attack lysine residues as well as glycation. Since glucose itself can start the oxidative reaction, LDL particles from hyperglycaemic subjects might bear both modifications at the same time, which could certainly alter interactions with MAC. This warrants further research. Uptake experiments on labelled glc-LDL in the presence of a 20-fold molar excess of both unlabelled c-LDL and ac-LDL show that uptake of glc-LDL was not completely blocked. Hence, when both the ApoBlOO:E receptor and the scavenger receptors were blocked, MAC still could take up small amounts of glc-LDL, probably by non-specific, non-saturable pathways. The unusual increase in CE synthesis previously reported (Lopes Virella et al., 1988) in contrast with lower binding and uptake of glc-LDL shown in the present work, may have been due either to induction of CE synthesis by glc-LDL or to greater transfer or exchange of cholesterol from glc-LDL to the MAC. In any case, this situation is similar to that found for a modified LDL isolated as a small fraction from normal plasma (Avogaro et al., 1988). In conclusion, the results of our experiments suggest that the major part of glycated LDL binds to and is taken up by the macrophages, mainly through the ApoBlOO:E receptor pathway, but with apparent decreased efficiency compared to control nonglycated LDL. Along with this ApoBlOO:E receptor mechanism, glycation was able to convert nearly 20 % of LDL into a form recognized by the scavenger receptors. And finally, a small percentage of glcLDL enters the macrophage through a non-receptormediated pathway, probably by diffusion through the lipid bilayer or by endocytosis. Whether the glc-LDL molecules are sorted by the macrophage cells has not yet been determined. Detectable oxidation of the glcLDL preparation is not involved in such sorting, since assay for higher yields of malonyldialdehyde in glcLDL could not be found. At present, the following hypothesis can be made : slightly glycated LDL molecules are still able to be recognized by the ApoBlOO:E receptor of the macrophages, whereas more heavily glycated LDL mole-
CRERICHE ET AL.
cules would have sufficient positive charges masked in their ApoBlOO moiety to be recognized by the scavenger receptor. The existence of a separate specific receptor for glc-LDL could not be demonstrated, but the entry of some glc-LDL by the scavenger receptor pathway would favour the idea of altered catabolism of those molecules, leading to increased accumulation of cholesteryl esters in the macrophage.
References Aggerbeck, L.P. (1990),Transport du cholesterol,maladiescardiovasculaireset domainesstructuraux dans lesapolipoproteinesdu serum.Regard sur la Biochimie 51-56. Avogaro, P., Bittolo Bon, G. & Cazzolato, Q. (1988),Presence of a modified low density lipoprotein in humans.Arteriosclerosis, 8, 79-87. Basu, SK., Goldstein, J.L., Anderson, R.G. & Brown, M.S. (1976), Degradation of cationized LDL and regulationof cholesterolmetabolismin homozygous familial hypercholesterolemiafibroblasts.Proc. nut. Acud. Sci. (Wash.), 73, 3178-3181. Bradford, M.A. (1976).A rapid and sensitivemethod for the quantitation of microgramquantitiesof protein utilizing the principleof protein-dyebinding. Anulyf. Biochem,
72, 248-250.
Brown, MS. & Goldstein, J.L. (1990), Scavengingfor receptors.Nature (Lond.), 343, 508-509. Brownlee,M., Cerami,A. & Vlassara,H. (1988),Advanced glycosylation end productsin tissueand the biochemicalbasisof diabeticcomplications.New. Engl. J. Med., 318, 1315-1321. Curtiss,L.K. & Witztum, J.L. (1985),Plasmaapolipoproteins A,, A,,, B, C, and E are glucosylated in hyperglycemic diabetic subjects. Diubefes, 34, 452-461. Esterbauer,H. & Chelseman,K. (1990),Determinationof aldehydiclipid peroxidationproducts; malonaldehyde and 4-hydroxynonenal. Methods Enzymol., 186, 407-421. Fischer, W.R. & Schumaker,V.N. (1986), Isolation and characterization of apolipoprotein BlOO. Methods Enzymol., 128, 247-262. Fraker, P.J. &Speck, J.C. (1978),Protein and cell membrane iodinations with a sparingly solublechloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem.
biophys.
Res. Commun.,
80, 849-857.
Golstein,J.L., Basu,S.K. & Brown, M.S. (1983)Receptormediated endocytosis of LDL in cultured cells. Methods Enzymol., 98, 241-260. Gonen, B., Baenziger,J., Schonfeld, G., Jacobson,D. & Farner, P. (198l), Nonenzymatic glycosylation of LDL in vitro. Effects on cell-interactive properties. Diabetes,
30, 875-878.
Haberland, M.E., Fogelman,A.H.M. & Edmards, P.A. (1982), Specificity of receptor-mediatedrecognition of malondiadehydemodified LDL. Proc. nut/. Acud. Sci. (Wash.), 79, 1712-1716. Halliwell, B. & Gutteridge, J.M. (1990),Role of free radicals and catalytic metal ions in human disease:an overview. Methods Enzymol., 186, l-85.
GL YCA TED LO W-DENSITY
LIPOPROTEIN
Hunt, J.V., Smith, C.T. & Wolfe, S.P. (1990), Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose. Diabetes 39, 1420-1424. Innerarity, T.L., Pitas, R.E. & Mahley, R.W. (1986), Lipoprotein-receptor interactions. Methods Enzymol., 129, 542-561. Lopes Virella, M.T., Klein, R., Lyons, T.J., Stevenson, H.C. & Witztum, J.L. (1988), Glycosylation of LDL enhances cholesteryl ester synthesis in human monocyte-derived macrophages. Diabetes, 37, 550-557. Ross, R. (1986), The pathogenesis of atherosclerosis; An update. New. Engl. J. Med., 314, 488-500. Sasaki, J. & Cottam, G.L. (1982), Glycosylation of human LDL and its metabolism in human skin fibroblasts. Biochem. biophys. Res. Commun., 104, 997-1083. Schleicher, E., Olgemoller, B., Schon, J., Durst, T. & Wieland, O.H. (1985), Limited nonenzymatic glucosylation of LDL does not alter its catabolism in tissue culture. Biochem biophys Acta (Amst.), 846, 226-233.
INTERACTION
WITH
MACROPHAGES
23
Shaklei, N., Garlick, R.L. & Bunn, F. (1984) Nonenzymatic glycosylation of human serum albumin alters its conformation and function. J. biol. Chem., 259, 3812-3817. Steinberg, D. & Olefsky, J.M. (1987) in “Hypercholesterolemia and atherosclerosis” (p. 5-22). Churchill Livingstone, Edinburg. Stevenson, H.C. (1985), Separation of mononuclear leucocyte subsets by counter current centrifugal elutriation. Methods Enzymol., 162, 242-249. Vlassara, H., Brownlee, M. & Cerami, A. Non-enzymatic glycosylation: role in the pathogenesis of diabetic complications. C/in. Chem., 32, B 37-B 41. Witztum, J.L., Mahoney, E.M., Branks, M.J., Fisher, M., Elam, R. & Steinbery, D. (1982), Non-enzymatic glucosylation of LDL alters its biologic activity. Diabetes, 31, 283-291. Witztum, J.M., Steinbecher, U.P., Antew Kesaniemi, Y. & Fischer, M. (1984), Autoantibodies to glucosylated proteins in the plasma of patients with diabetes mellitus. Proc. null. Acad. Sci. (Wash.), 81, 3204-3208.
INSTITUT
Res. Immunol. 1992, 143, 17-23
PASTEUR/ELSEVIER
Paris 1992
In vitro glycated low-density lipoprotein interaction human monocyte-derived macrophages
with
A. Gugliucci Creriche (‘), S. Dumont t2), J.C. Siffert c2), and A.J.C. Stahl (‘) “)Biochemistry
and 12)Immunopharmacology Laboratories, Facultt de Pharmacie, BP 24, 67401 Illkirch Cedex (France)
UniversitP Louis Pasteur,
SUMMARY Human low-density lipoprotein (LDL) was glycated in vitro (5 days, glucose 50 mmol/l), labelled with 1251, and its binding and uptake by human monocyte-derived macrophages studied. Glycation produced lower binding and lower uptake. Competition experiments using unlabelled LDL (control, glycated, and acetyl-LDL) showed that most glycated LDL was taken up by the apolipoprotein-Bl 00 : E receptor pathway. Results suggest that less of the glycated LDL may enter the cells via scavenger receptors, and very minute amount via non-saturable receptor-independent pathways. Key-words: Lipoprotein, Cell uptake, Competition.
Glycation,
Macrophage;
Introduction
Glycation (non-enzymatic glycosylation) of proteins is believed to be one of the primary initiating events in the pathogenesis of many of the chronic complications of diabetes mellitus (Brownlee et al., 1988; Vlassara et al., 1986). Glycation of apolipoprotein B from low-density lipoprotein (LDL) has been shown to occur both in vitro, (Gonen et al., 1981; Sasaki and Cottam, 1982 ; Schleicher et al., 1985 ; Witztum et al., 1982) and in vivo (Curtiss and Witztum, 1985; Witztum et al., 1984). Moreover, evidence for involvement of glycated LDL (glc-LDL) in the accelerated atherogenesis of long-term diabetes has appeared over the last few years. Reports exist showing impaired recognition of glcLDL by the ApoBlOO:E receptor (Gonen et al., 1981; Sasaki and Cottam, 1982; Schleicher et al., 1985). A recent publication (Lopes Virella et al., 1988) demonstrates that glc-LDL stimulates more choles-
LDL, Acetylation,
Receptor
binding,
teryl ester (CE) synthesis than does LDL (c-LDL) in human monocyte-derived macrophages (HMDMAC). The authors also suggest the existence of a putative ApoBlOO:E-receptor-independent mechanism for glcLDL uptake in HMDMAC. There is also evidence that different forms of modified LDL can enter the macrophage (MAC) through the scavenger receptor. Experiments with malonyldialdehyde (MDA)modified LDL have shown a threshold effect of derivatization which induces recognition by the scavenger receptor, while suppressing that of the ApoB100:E receptor (Haberland et al., 1982). To further characterize the interaction of glc-LDL with HMDMAC, we studied the binding and the uptake of the particles by HMDMAC. Materials
and Methods
Chemicals. - Medium : RPMI-1640 was purchased from GIBCO ; “‘1 was obtained from CEA
Submitted September 28, 1991, accepted November 21, 1991.
A. GUGLIUCCI
18
(Gif-sur-Yvette) : Iodogen was suppplied by Pierce; KC1 and EDTA were from Merck; Zymofren (aprotinin) from Specia. All other chemicals were purchased from Sigma. Plasma. - Pooled plasma from venipuncture of fasted subjects was obtained from the Laboratoire de Biochimie, Clinique Medicale “B” CHU Strasbourg. After blood collection on EDTA, aprotinin and NaN, were added as described by Fischer and Schumaker (1986). Cells. - Human monocytes were obtained from the CHU, Hautepierre, Strasbourg, Service d’OncoHtmatologie, Professeur Oberling. Pure human monocytes were prepared from the blood of normal human subjects by countercurrent centrifugal elutriation of leukapheresis specimens as described (Stevenson, 1985). The purity of the preparation was greater than 90 %, as checked by morphology, latex particle ingestion, and non-specific esterase staining. The cells were suspended in RPMI-1640 medium, supplemented with 10 % fcetal calf serum, to a final concentration of 2x IO6 cells/ml. Then, 2 x IO6 cells were pipetted in 35-mm tissue culture dishes and treated as by Lopes Virella et al. (1988). After one week of maturation to macrophages, serum was substituted for lipoprotein-depleted serum (LDS) for 24 h to allow upregulation of LDL receptors. LDL isolation Human LDL (d = 1019-1063 g/ml) was prepared from pooled EDTA plasma of fasted subjets by sequential flotation ultracentrifugation as previously described (Fischer and Schumaker 1986). Throughout the preparation procedures, lipoproteins (Lp) were kept in 1 mM EDTA, 1.5 mM 1 mM phenylmethylsulphonyl fluoride NaN,, (PMSF) and 0.1 mg/ml aprotinin. A single band was obtained in either SDS-PAGE (5 (70 acrylamide) or 1 070 agarose electrophoresis. Visualization of the bands was accomplished by staining with Coomassie brillant blue R250 and with Sudan black. After dialysis against PBS (10 mM sodium phosphate buffer, 0.15 M NaCI, pH 7.4), the LDL pre-
CRERICHE
ET AL.
paration was stored in 20 % sucrose at - 20°C until use (Fischer and Schumaker, 1986). For experiments, LDL was thawed, dialysed extensively, kept at 4°C and used within one week. Modification
of LDL
1) Glycation LDL (1 mg/ml) was incubated either in PBS containing 1 mM PMSF, 1.5 mM NaN,, and 1 mM EDTA for control LDL (c-LDL), or in the same solution supplemented with 50 mM glucose for glc-LDL. Incubation was at 37’C in screw-capped tubes for different periods up to five days. Absence of proteolysis was confirmed by SDS-PAGE as described above or by autoradiography of labelled samples. Relative mobility of LDL after glycation was measured by agarose electrophoresis (cc. 3 mA/cm, 3 h). Strips showed distinct, major bands of different anodie mobilities which depended on the length of incubation. Acetylation LDL was acetylated (ac-LDL) Basu et al. (1976). Radioiodination
as described by
of LDL
LDL (c-LDL, glc-LDL and ac-LDL) were labelled by the method of Fraker and Speck (1978) with iodogen and isolated by chromatography on a SephadexG25 column (10 x 300 mm). A specific activity of 100-400 cpm/ng was obtained. LDS LDS was prepared as by Lopes Virella (1988).
ac-LDL AGE ApoB BSA CE c-LDL EDTA glc-LDL
acetyl-LDL. advanced glycosylation endproduct. apolipoprotein B. bovine serum albumin. cholesteryl ester. control (non-glycated) LDL. ethylendiamine tetraacetate. glycated LDL.
HMDMAC LDL LDS MAC MDA PBA PBS TBARS
= = = = = = = =
human monocyte-derived macrophage. low-density lipoprotein. lipoprotein-depleted serum. macrophage. malonyldialdehyde. (M-amino)phenylboronate agarose. phosphate-buffered saline. thiobarbiturie acid-reactive substance.
et al.
GLYCATED
LO W-DENSITY
Advanced glycosylation endproducts bovine serum albumin (BSA)
LIPOPROTEIN (AGE)
of
AGE-BSA was prepared by incubating BSA (5 mg/ml) in PBS with 100 mM glucose for 7 weeks (Lopes Virella et al., 1988). I) Thiobarbituric
INTERACTION 3) Glycation
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19
with “C-glucose
To assessthe degree of glycation attained in our incubation experiments, we also performed LDL glycation in the presence of traces of 14C-labelled glucose in a medium containing 10 or 50 mM glucose according to Witztum et al. (1982).
acid-reacting substances (TBARS)
The degree of oxidation of all LDL preparations was controlled by measuring the content of TBARS as described (Esterbauer and Chelseman, 1990), and expressed in nmol MDA equivalents/mg ApoB. 2) m-aminophenylboronate tography
agarose (PBA) chroma-
PBA affinity chromatography was carried out as previously described (Shakler et al., 1984).
4) Binding
and uptake experiments
As receptor activities have been shown to vary greatly, even with the same cell strain studied on different days, comparative experiments were always performed with the same batch of cells and LDS, and in the same week. In binding experiments, we incubated monolayers of 2 x lo6 cells/dish with radiolabelled Lp in RPM1 medium a 4°C for 2 h with gentle rocking (Innerarity et al., 1986).
80
60
Molar excess of competing ligands Fig. 1. Binding of rz51-c-LDL and rzsI-glc-LDL (7.5 ug/dish) to human monocyte-derived macrophages in the absence (100 % value) or in the presence of increasing amounts of non-labelled c-LDL, glc-LDL and ac-LDL. a) rzSI-c-LDL 100 Vo binding corresponds to 3.4 f 0.58 pM/mg cell protein. Each point represents mean of three experiments run in duplicate. b) iZ51-glc-LDL 100 % binding corresponds to 3.0 f 0.47 pM/mg cell protein. Each point represents mean of three experiments run in duplicate.
20
A. GUGLIUCCI
CRERICHE ET AL.
In uptake experiments, incubation was at 37°C in a 5% CO,-humidified incubator for 5 h. In both cases, the medium was discarded after incubation and dishes were thoroughly washed twice for 10 min with 50 mM TRIS-HCl at pH 7.4 containing 2% BSA, followed by three washings with the same buffer without BSA (Goldstein et al., 1983). Cells were lysed with three washings of 100 mM NaOH (0.5 ml/dish, 20 min each). Each washing was collected and radioactivity was counted. Cell protein was measured by the micromethod of Bradford (1976) using BSA in 100 mM NaOH to run a standard plot. Results LDL glycarion
“C-glucose incorporation was time-and dosedependent and reached a maximum of 4.0 mol/mol ApoB (assumed MW 550,000 indicated by Aggerbeck, 1990), in 5-day incubation and at a concentration of 50 mM (data not shown). With 10 mM glucose, the incorporation was 1.0 mol/mol ApoB. Both results are in accordance with previous publications (Gonen ef al., 1981; Schleicher ef al., 1985). Glc-LDL showed the same behaviour in SDSPAGE both with and without previous treatment with @mercaptoethanol. A single band for glc-LDL, with greater anodic mobility than c-LDL, was detected in agarose strips. The degree of glycation is therefore similar in the majority of the molecules in this preparation. Relative anodic mobility of glc-LDL (mobility of glc-LDL/mobility of c-LDL) as a function of time of incubation in 50 mM glucose shows a straight line (data not shown). Retention by PBA was 2.0 + 0.2 % for c-LDL and 25.0 + 1.5 070 for glc-LDL (mean + S.D., n = 3). Phenylboronate bound cis-diols present in ketoamine adducts of glycated proteins. TBARS contents of LDL preparations was 1.2 f 0.2 mM MDA equivalents/mg ApoB, and no significant differences between c-LDL and glc-LDL were observed. TBARS did not increase significantly during conservation of the preparations at -20°C up to three months. Binding and uptake
Competition of binding to macrophages between the labelled c-LDL and increasing molar concentrations of non-labelled c-LDL, ac-LDL and glc-LDL at 4°C is shown in figure la. 125-I-labelled c-LDL bound to the ApoBlOO:E receptor could be displac-
0
Molar excess of competing ligands Fig. 2. Uptake of “SI-c-LDL (15 t&dish) by human monocyte-derived macrophagesin the absence(100 % value) or in the presenceof increasingamountsof nonlabelledc-LDL, glc-LDL and ac-LDL. “‘1-LDL 100 % uptake correspondsto 35 f 4 pmol LDL/mg cell protein. Each point representsthe meanof three experimentsrun in duplicate.
ed by c-LDL, but also by glc-LDL, and far less efficiently by ac-LDL. Half-maximal binding at 4°C requires 30 Fg/l for c-LDL and 40 pg/ml for glcLDL. Glc-LDL is therefore recognized by the ApoBlOO:E receptor, though not to the same extent as is c-LDL, becausehigher concentrations are needed to displace c-LDL. Glycation of LDL reduces the affinity of LDL towards the ApoBlOO receptor, but does not completely hinder its binding. Mannan and AGE-BSA failed to displace c- and glc-LDL from their receptors (data not shown). When labelled glc-LDL is displaced from its binding to the macrophages by unlabelled ligands, there is some competition by ac-LDL, besides displacement by glc-LDL and c-LDL, showing that a certain proportion of glc-LDL is recognized by the scavenger receptors (fig. 1b). The uptake by macrophages of labelled c-LDL in the presence of increasing amounts of non-labelled c-LDL, glc-LDL and ac-LDL is shown in figure 2. Uptake of c-LDL by macrophages was most efficiently hindered by glc-LDL, and less so by ac-LDL.
GL YCA TED LO W-DENSITY
LIPOPROTEIN
INTERACTION
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21
Discussion
Interaction of modified LDL with arterial monocytes and macrophages has been proposed as one of the factors leading to their transformation in to foam cells, characteristic of the factors leading to their transformation into foam cells, characteristic of atherosclerotic lesions (Ross 1986; Steinberg and Olefsky, 1987). In this regard, much attention has recently been given to oxidative modifications occurring both in plasma and in situ (Brown and Goldstein, 1990; Halliwell and Gutteridge, 1990).
h
g
80
Glycation of LDL has been shown to occur in vitro and in vivo, and the effects of this modification upon its metabolism have been studied in fibroblasts, murine MAC and, more recently, in human MAC (Curtiss and Witztum, 1985; Gonen et al., 1981; Lopes Virella et al., 1988 ; Sasaki and Cottam, 1982; Schleicher et al., 1985; Witztum et al., 1982; Witztum et al., 1984). 10
20 Molar excess of competing ligands 0
Fig. 3. Uptake of ‘2SI-glc-LDL (15 vg/dish) by HMDMAC
in the absence (100 % value) or in the presence of increasing amounts of non-labelled c-LDL glc-LDL and ac-LDL. ‘251-glc-LDL 100 % uptake corresponds to 30 + 5 pmol LDL/mg cell protein. Each point represents the mean of three experiments run in duplicate.
Reverse experiments, measuring the uptake of glcLDL in the presence of increasing amounts of modified LDL, showed that glc-LDL uptake was partially restrained by c-LDL (fig. 3). Results indicate that glcLDL binds and is taken up by the ApoBlOO:E receptor and by scavenger receptors. In another set of experiments, we measured the uptake by macrophages of labelled glc-LDL in the presence of a large excess of both non-labelled c-LDL and ac-LDL (data not shown). Hence, when both ApoBlOO:E receptors and scavenger receptors were blocked, the macrophage was still able to take up small amounts of glc-LDL by a non-specific, nonsaturable pathway. Even the negatively charged polyvinylsulphate (2 mg/ml), which completely blocks the scavenger receptors, did not impair this uptake of glc-LDL. By measuring MDA in all LDL preparations, we checked that no strong oxidation occurred during chemical modifications of LDL. No significant differences were noted between values found in c-LDL, glc-LDL or ac-LDL preparations.
Recent reports suggest a link between glycation and oxidation ; glycation per se could lead to oxidation in a process referred to as autoxidative glycosylation (Hunt et al., 1990). To characterize the interaction of glc-LDL as compared to c-LDL, we studied binding and uptake of these lipoproteins by HMDMAC. Our binding studies clearly showed diminished binding of glc-LDL compared to c-LDL, as well as a lower affinity, in accordance with previous reports concerning fibroblasts and MAC (Gonen et al., 1981; Witztum et al., 1982). It has been argued (Lopes Virella et al., 1988) that glc-LDL stimulates greater cholesteryl ester synthesis in HMDMAC than does c-LDL, which suggests its uptake via pathways other than the classical ApoB1OO:E receptor. We performed competition studies for the binding and uptake of c-LDL, glc-LDL and ac-LDL. Our results confirm the lack of competition with mannan and AGE-BSA, which goes against the participation of the mannose-fucosereceptor and the AGE-receptor in this process. The lack of recognition of our glc-LDL by the latter receptor confirms the fact that glycation has only been pursued to the degree of Amadori rearrangement and has not reached the stage of AGE products. We performed glycation for 5 days (half life of ApoB) with 50 mM glucose in the absence of reducing agents in order to obtain protein modifications which could occur in a pathological situation such as in heavily decompensated diabetic subjects. Displacement curves (figs. 1 to 3) show that most of the binding and uptake of glc-LDL occur via the classical ApoBlOO:E receptor. However, there is a higher half-maximal binding concentration for glc-LDL than for c-LDL. When labelled glc-LDL is displaced by unlabelled ligands, there is a 50 % inhibition of uptake by ac-LDL at a lo-fold concentration. This
22
A. GUGLIUCCI
would indicate that at least a subfraction of glc-LDL is recognized by the scavenger receptors. This could be due to glc-LDL itself or to slight differential oxidation of the preparations involved. TBARS contents showed no significant differences between c-LDL and glc-LDL. Our results suggest that, though most glc-LDL is taken up by the ApoB1OO:E mechanism, glycation could transform LDL (or at least a subfraction) into a form recognized by scavenger receptors. Particles of LDL may exist which are both glycated and slightly oxidized. Indeed, reactive aldehydes produced in the oxidative cascade attack lysine residues as well as glycation. Since glucose itself can start the oxidative reaction, LDL particles from hyperglycaemic subjects might bear both modifications at the same time, which could certainly alter interactions with MAC. This warrants further research. Uptake experiments on labelled glc-LDL in the presence of a 20-fold molar excess of both unlabelled c-LDL and ac-LDL show that uptake of glc-LDL was not completely blocked. Hence, when both the ApoBlOO:E receptor and the scavenger receptors were blocked, MAC still could take up small amounts of glc-LDL, probably by non-specific, non-saturable pathways. The unusual increase in CE synthesis previously reported (Lopes Virella et al., 1988) in contrast with lower binding and uptake of glc-LDL shown in the present work, may have been due either to induction of CE synthesis by glc-LDL or to greater transfer or exchange of cholesterol from glc-LDL to the MAC. In any case, this situation is similar to that found for a modified LDL isolated as a small fraction from normal plasma (Avogaro et al., 1988). In conclusion, the results of our experiments suggest that the major part of glycated LDL binds to and is taken up by the macrophages, mainly through the ApoBlOO:E receptor pathway, but with apparent decreased efficiency compared to control nonglycated LDL. Along with this ApoBlOO:E receptor mechanism, glycation was able to convert nearly 20 % of LDL into a form recognized by the scavenger receptors. And finally, a small percentage of glcLDL enters the macrophage through a non-receptormediated pathway, probably by diffusion through the lipid bilayer or by endocytosis. Whether the glc-LDL molecules are sorted by the macrophage cells has not yet been determined. Detectable oxidation of the glcLDL preparation is not involved in such sorting, since assay for higher yields of malonyldialdehyde in glcLDL could not be found. At present, the following hypothesis can be made : slightly glycated LDL molecules are still able to be recognized by the ApoBlOO:E receptor of the macrophages, whereas more heavily glycated LDL mole-
CRERICHE ET AL.
cules would have sufficient positive charges masked in their ApoBlOO moiety to be recognized by the scavenger receptor. The existence of a separate specific receptor for glc-LDL could not be demonstrated, but the entry of some glc-LDL by the scavenger receptor pathway would favour the idea of altered catabolism of those molecules, leading to increased accumulation of cholesteryl esters in the macrophage.
References Aggerbeck, L.P. (1990),Transport du cholesterol,maladiescardiovasculaireset domainesstructuraux dans lesapolipoproteinesdu serum.Regard sur la Biochimie 51-56. Avogaro, P., Bittolo Bon, G. & Cazzolato, Q. (1988),Presence of a modified low density lipoprotein in humans.Arteriosclerosis, 8, 79-87. Basu, SK., Goldstein, J.L., Anderson, R.G. & Brown, M.S. (1976), Degradation of cationized LDL and regulationof cholesterolmetabolismin homozygous familial hypercholesterolemiafibroblasts.Proc. nut. Acud. Sci. (Wash.), 73, 3178-3181. Bradford, M.A. (1976).A rapid and sensitivemethod for the quantitation of microgramquantitiesof protein utilizing the principleof protein-dyebinding. Anulyf. Biochem,
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Brown, MS. & Goldstein, J.L. (1990), Scavengingfor receptors.Nature (Lond.), 343, 508-509. Brownlee,M., Cerami,A. & Vlassara,H. (1988),Advanced glycosylation end productsin tissueand the biochemicalbasisof diabeticcomplications.New. Engl. J. Med., 318, 1315-1321. Curtiss,L.K. & Witztum, J.L. (1985),Plasmaapolipoproteins A,, A,,, B, C, and E are glucosylated in hyperglycemic diabetic subjects. Diubefes, 34, 452-461. Esterbauer,H. & Chelseman,K. (1990),Determinationof aldehydiclipid peroxidationproducts; malonaldehyde and 4-hydroxynonenal. Methods Enzymol., 186, 407-421. Fischer, W.R. & Schumaker,V.N. (1986), Isolation and characterization of apolipoprotein BlOO. Methods Enzymol., 128, 247-262. Fraker, P.J. &Speck, J.C. (1978),Protein and cell membrane iodinations with a sparingly solublechloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem.
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Haberland, M.E., Fogelman,A.H.M. & Edmards, P.A. (1982), Specificity of receptor-mediatedrecognition of malondiadehydemodified LDL. Proc. nut/. Acud. Sci. (Wash.), 79, 1712-1716. Halliwell, B. & Gutteridge, J.M. (1990),Role of free radicals and catalytic metal ions in human disease:an overview. Methods Enzymol., 186, l-85.
GL YCA TED LO W-DENSITY
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Hunt, J.V., Smith, C.T. & Wolfe, S.P. (1990), Autoxidative glycosylation and possible involvement of peroxides and free radicals in LDL modification by glucose. Diabetes 39, 1420-1424. Innerarity, T.L., Pitas, R.E. & Mahley, R.W. (1986), Lipoprotein-receptor interactions. Methods Enzymol., 129, 542-561. Lopes Virella, M.T., Klein, R., Lyons, T.J., Stevenson, H.C. & Witztum, J.L. (1988), Glycosylation of LDL enhances cholesteryl ester synthesis in human monocyte-derived macrophages. Diabetes, 37, 550-557. Ross, R. (1986), The pathogenesis of atherosclerosis; An update. New. Engl. J. Med., 314, 488-500. Sasaki, J. & Cottam, G.L. (1982), Glycosylation of human LDL and its metabolism in human skin fibroblasts. Biochem. biophys. Res. Commun., 104, 997-1083. Schleicher, E., Olgemoller, B., Schon, J., Durst, T. & Wieland, O.H. (1985), Limited nonenzymatic glucosylation of LDL does not alter its catabolism in tissue culture. Biochem biophys Acta (Amst.), 846, 226-233.
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Shaklei, N., Garlick, R.L. & Bunn, F. (1984) Nonenzymatic glycosylation of human serum albumin alters its conformation and function. J. biol. Chem., 259, 3812-3817. Steinberg, D. & Olefsky, J.M. (1987) in “Hypercholesterolemia and atherosclerosis” (p. 5-22). Churchill Livingstone, Edinburg. Stevenson, H.C. (1985), Separation of mononuclear leucocyte subsets by counter current centrifugal elutriation. Methods Enzymol., 162, 242-249. Vlassara, H., Brownlee, M. & Cerami, A. Non-enzymatic glycosylation: role in the pathogenesis of diabetic complications. C/in. Chem., 32, B 37-B 41. Witztum, J.L., Mahoney, E.M., Branks, M.J., Fisher, M., Elam, R. & Steinbery, D. (1982), Non-enzymatic glucosylation of LDL alters its biologic activity. Diabetes, 31, 283-291. Witztum, J.M., Steinbecher, U.P., Antew Kesaniemi, Y. & Fischer, M. (1984), Autoantibodies to glucosylated proteins in the plasma of patients with diabetes mellitus. Proc. null. Acad. Sci. (Wash.), 81, 3204-3208.