Arherosclerosis, 92 (1992) 187-192 0 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved 0021-9150/92/%05.00
187
Printed and Published in Ireland
ATHERO 04758
The oxidative modification
of low density lipoprotein by human lymphocytes
David J. Lamb, Gary M. Wilkins and David S. Leake Department of Biochemistry and Physiology. School of Animal and Microbial Sciences, University of Reading, Whireknighrs. P.O. Box 228, Reading, Berkshire RG6 2AJ (U.K.)
(Received 24 July, 1991) (Revised, received 1 November, 1991) (Accepted 6 November, 1991)
Summary Oxidation of low density lipoprotein in atherosclerotic lesions may be involved in converting macrophages into lipid-laden foam cells. Lesions contain endothelial cells, smooth muscle cells, macrophages and lymphocytes. The first three types of cells have been shown previously to modify low density lipoprotein so as to increase its uptake by macrophages. We report here that lymphocytes from human blood are also capable of doing this. The modification process was an oxidative one because there was an increase in the thiobarbituric acid-reactive substances in media containing lymphocyte-modified low density lipoprotein and the modification could be inhibited by the antioxidants butylated hydroxytoluene and probucol. The lymphocyte-modified low density lipoprotein was taken up and degraded by macrophages by their scavenger receptor(s).
Key words: Antioxidants; Atherosclerosis; Low density lipoprotein; Lymphocyte; Macrophage: Oxidation
Introduction Uptake of low density lipoproteins (LDL) by arterial macrophages to generate foam cells in the arterial intima is believed to be an important step in the pathogenesis of atherosclerosis. Examination of human and animal atherosclerotic plaques by immunohistochemistry and other techniques Correspondence fo: Mr. D.J. Lamb, Department of Biochemistry and Physiology, School of Animal and Microbial Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading, Berkshire RG6 2AJ (U.K.)
indicates the presence of four major cell types: endothelial cells, smooth muscle cells, macrophages and lymphocytes [l-5]. It has been shown that incubating LDL with arterial endothelial cells [6], arterial smooth muscle cells [7] and mouse peritoneal macrophages [8,9] modifies LDL by an oxidative mechanism [lo], resulting in its more rapid uptake by cultured macrophages by means of their scavenger receptors. Native (unmodified) LDL is not recognised by the scavenger receptor(s) and is taken up only slowly by macrophages. Human blood monocytes [l l] and mononuclear
188 cells [ 121 have been reported to oxidise LDL, but not to increase its uptake by macrophages, and it has very recently been shown [ 131 that human monocyte-derived macrophages can oxidise LDL to increase its uptake by macrophages. We report here that human blood lymphocytes can modify LDL to greatly increase its uptake by macrophages. Materials and methods Isolation of lymphocytes and macrophages
Healthy human blood was defibrinated using glass beads in the presence of 15 mM EDTA and dextran (0.35 mg/ml) to sediment the erythrocytes over 4 h at room temperature. The white cell suspension was layered onto Histopaque 1077 (Sigma) and centrifuged at 400 x g for 30 min at room temperature. The mononuclear cell layer was recovered, washed with sterile Dulbecco’s phosphate-buffered saline (Gibco) then resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% (v/v) foetal calf serum and 50 pg/ml gentamicinlml. Monocytes were removed by adhesion over two incubations of 4-8 h in different culture dishes. Lymphocyte purity was assessed histologically using Wright’s stain (Sigma) which contains methylene blue and eosin which differentially stain cellular organelles thereby permitting identification of cell type. Monocyte contamination was shown to be no greater than 2% from triplicate counts of approximately 500 cells in several different experiments. Mouse resident peritoneal macrophages were isolated as described elsewhere [14]. Isolation and radioiodination of LDL
LDL (1.019-1.063 g/ml) was isolated from human blood by sequential density ultracentrifugation as described previously [ 151 and labelled with Na12’I using iodine monochloride [9]. Sterile 12’I-labelled LDL was stored in the dark at 4°C in a buffer containing 100 PM EDTA [9] to inhibit its autooxidation and discarded about a month after taking the blood. Modification of LDL
‘2’1-labelled LDL (100 pg protein/ml)
was in-
cubated at 37°C with lymphocytes (7.5 x lo6 cells/ml) or cell-free wells in 0.4 ml Ham’s F-10 medium (Flow Laboratories Ltd.) containing gentamicin (50 &ml) and 3 PM FeSO, in 16 mm multiwell dishes (Corning) under 5% CO2. After incubation (usually 18-24 h), the medium was centrifuged (1500 x g for 10 min at 4°C) to remove the cells. Stock solutions of the antioxidants butylated hydroxytoluene (Sigma) and probucol (a gift from Merrell Dow Pharmaceuticals Ltd., Staines) were prepared in ethanol such that the final concentration of ethanol in the modification media was 1% (v/v). Control cells were similarly adjusted to 1% ethanol. For the competition experiments, ‘251-labelled LDL was modified sterilely in F-10 medium containing 25 ccM CuSO, and gentamin (50 &ml) for 24 h at 37°C. Determination macrophages
of modified LDL
degradation by
Modified or control 1251-labelled LDL was diluted to 10 pg protein/ml in DMEM containing 10% (v/v) foetal calf serum and gentamicin (50 kg/ml). Diluted LDL was incubated for 20 h with mouse peritoneal macrophages (1 x lo6 peritoneal cells/well) or cell-free wells (22-mm multiwell dishes, Costar) at 1 ml per well. The rate of uptake was estimated by measuring the radioacnon-iodide tive trichloroacetic acid-soluble degradation products released into the medium as described previously [9]. Degradation products in cell-free wells were subtracted from those of the wells containing macrophages. The cells were washed and dissolved in 0.2 M NaOH as described elsewhere [9] and assayed for protein by a modified Lowry procedure [ 161. Thiobarbituric acid-reactive substances assay [17]
Samples of modified or control LDL (250 ~1 of 100 rg protein/ml) were taken and 3 ml of 0.335% (w/v) thiobarbituric acid in 10% (w/v) trichloroacetic acid was added to each sample and incubated at 100°C for 30 min. The absorbance was read at 535 nm. Standards of tetramethoxypropane (Sigma), up to 5 nmol per tube, were prepared in Ham’s F- 10.
189 Results
‘251-labelled LDL was modified by human blood lymphocytes so that it was taken up and degraded up to ten times or more faster by mouse peritoneal macrophages than 1251-labelled LDL incubated in cell-free wells rather than with lymphocytes. Increasing the number of lymphocytes per well increased the amount of LDL modification (Fig. 1). Human blood monocyte-derived macrophages were also able to modify LDL to increase its uptake by mouse peritoneal macrophages and were more active in doing so than the lymphocytes (data not shown), The modification of LDL by the lymphocyte preparations could not have been due to any contaminating macrophages, however, as a Wright’s stain showed the level of monocyte contamination of the lymphocyte preparations to be no greater than 2%, which could have accounted
I --
0
m
.
1
I
-
2
I
3
I
-
4
.
for less than 20% of the modification seen. In addition, lymphocytes prepared in culture medium that was obviously toxic to the macrophages isolated from the same sample of blood (possibly due to the batch of foetal calf serum used) still modified LDL well, whereas macrophages cultured separately died and did not modify the LDL. We therefore conclude that any contaminating macrophages in the lymphocyte preparation would also have been killed by the foetal calf serum and that the LDL modification observed would have been due to the lymphocytes themselves. Mouse peritoneal lymphocytes could also modify LDL to increase its uptake by macrophages, but were less effective in doing so than the human blood lymphocytes or mouse peritoneal macrophages (data not shown). Human blood lymphocytes were also less effective in modifying LDL than the mouse peritoneal macrophages (an example of this is shown in Fig. 3).
I
5
-I
LDL+lymph Number
of
cells
(mllllons)
Fig. 1. Modification of LDL by human lymphocytes. tz51labelled LDL was incubated for 24 h in Ham’s F-10 in the presence of increasing numbers of lymphocytes. The rate of degradation of the LDLs by mouse peritoneal macrophages was then determined. Each point is the mean f S.E.M. of three wells of cells.
LDL
lymph
medium
Fig. 2. Thiobarbituric acid-reactive substances in medium containing lymphocyte-modified LDL. LDL (100 pg protein/ml) was incubated for 24 h in Ham’s F-10 in the presence (LDL + lymph) or absence (LDL) of human lymphocytes and then assayed for thiobarbituric acid-reactive substances. Incubated F-10 (medium) and lymphocyte-conditioned medium (lymph) without LDL were also assayed. The mean f S.E.M. of three wells are shown.
190
lymph 0
Untreated
&j
Butylated
CO”
native
macro Concentration
Hydroxytoluene
Probucol
Fig. 3. Effect of butylated hydroxytoluene and probucol on modification of LDL by lymphocytes. 12sI-labelled LDL was incubated for 24 h with human lymphocytes (3 x IO6 per well) (lymph) or cell-free wells (con) with or without 10 pM butylated hydroxytoluene or 10 PM probucol. LDL was also incubated with mouse peritoneal macrophages 10~ (2 x IO6 peritoneal cells/well) (macro) for comparison. It should be noted that only about half the peritoneal cells would have been macrophages [ 131.The rate of degradation of all the LDLs (including non-incubated, native LDL) by mouse peritoneal macrophages was then determined. Each histobar is the mean f S.E.M. of three wells.
There was a large increase in the thiobarbituric acid reactive substances in media containing lymphocyte-modified LDL (Fig. 2). In addition, the modification of LDL by lymphocytes could be inhibited completely or almost completely by the chain-breaking antioxidant butylated hydroxytoluene or by probucol, an anti-atherosclerotic drug with antioxidant properties [18] (Fig. 3). The nature of the binding site on macrophages for human lymphocyte-modified LDL was investigated by competition experiments involving incubating with lymphocytemacrophages modified ‘251-labelled LDL in the presence of increasing concentrations of unlabelled ligands. Excess non-labelled copper-oxidised LDL competed effectively with lymphocyte-modified 1251-labelled LDL for degradation for macrophages, but nonlabeled native LDL gave little competition (Fig. 4).
of
competitor
(rg
/ ml)
Fig. 4. Competition between lymphocyte-modified labelled LDL and non-labelled hgands for degradation by macrophages. Mouse peritoneal macrophages were incubated for 20 h with lymphocyte-modified ‘251-labelled LDL (10 PM protein/ml) in the presence of increasing concentrations of nonlabelled copper-oxidised LDL ( . . ), native LDL (-), or polyinosinic acid (- - -) (obtained from Sigma) and its degradation measured. Each point is the mean f S.E.M. of three wells. The degradation of control 1251-labelledLDL was 1.04 + 0.11 pg protein/mg cell protein in 20 h.
Polyinosinic acid, a potent ligand for the scavenger receptor(s) [ 191, greatly decreased the degradation of lymphocyte-modified labelled LDL. Discussion We have shown that lymphocytes can oxidatively modify LDL to greatly increase its uptake by macrophages. The moditication of LDL by lymphocytes was by an oxidative process because there was a large increase in the thiobarbituric acid reactive substances in LDL-containing medium exposed by lymphocytes and the modification could be inhibited by antioxidants. Butylated hydroxytoluene, a chain-breaking antioxidant, and probucol, a lipid-lowering drug with antioxidant properties, inhibited completely or almost completely the modification of LDL by human lymphocytes. Similar effects have been reported with endothelial cell [ 10,171, macrophage [8,9] and human mononuclear cell [12] modification of LDL.
191 As the number of modifying lymphocytes was increased, there appeared to be a ‘lag’ before increased uptake by macrophages was observed (Fig. 1). This may be related to the ‘lag phase’ seen in the time course of LDL modification in other systems, due in part to the time taken for the depletion of endogenous antioxidants in the LDL, e.g., cr-tocopherol [20,21]; a small number of lymphocytes may not be able to exert a great enough oxidative stress to overcome this ‘lag phase’ in the time available. Lymphocyte-modified LDL was apparently taken up by the scavenger receptor(s) as copperoxidised LDL and polyinosinic acid, known ligands of the scavenger receptor(s), effectively inhibited its degradation by macrophages. Excess native LDL, however, only slightly decreased lymphocyte-modified LDL degradation. Lymphocytes are found in large numbers in atherosclerotic lesions [3-51, along with endothelial cells, smooth muscle cells and macrophages. The latter three cell types have been shown previously to modify LDL to increase its uptake by macrophages [6-10,131. It would now appear that all four major cell types within atherosclerotic lesions have the capacity to oxidise LDL and increase its uptake by macrophages contributing to their conversion into foam cells. Acknowledgements
We are grateful to the Research Endowment Fund of the University of Reading for a research studentship (D.J.L.) and the Wellcome Trust for financial support. We also thank Dr A.I. Tiffn for advice on isolating lymphocytes and Mrs Sylvia F. Dalton, Mrs Linda Bunn and Mrs Ellen Cook for carefully typing the manuscript.
4
5
6
7
8
Henriksen, T., Mahoney, E.M. and Steinberg. D.. Enhanced macrophage degradation of biologically modified LDL, Arteriosclerosis, 3 (1983) 149. Parthasarathy. S., Printz, D.J., Boyd. D., Joy, L. and
9
Steinberg, D., Macrophage oxidation of low density lipoprotein generates a modified form recognised by the scavenger receptor, Arteriosclerosis, 6 (1986) 505. Leake, D.S. and Rankin, S.M., The oxidative modifica-
10
II
Jonasson,
L., Helm.
J., Skalli.
O.,
Bondjers,
G. and
tion of low density lipoproteins by macrophages. Biochem. J.. 270 (1990) 741. Steinbrecher, U.P., Parthasarathy, S.. Leake. D.S.. Witzturn, J.L. and Steinberg, D.. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids, Proc. Natl. Acad. Sci. USA, 81 (1984) 3883. Cathcart, M.K., Morel, D.W. and Chisolm, III, G.M., Superoxide dismutase (SOD) and catalse (CAT) inhibit the oxidation of low density lipoprotein (LDL) by monocyte derived macrophages. J. Cell. Biochem., 599 (1985) 153.
12
Hiramatsu, K., Rosen, H., Heinecke, J.W., Wolfbauer, G. and Chait, A., Superoxide initiates oxidation of low density lipoprotein by human monocytes. Arteriosclerosis, 7 (1987) 55.
13
Jialal, I. and Grundy, SM., Preservation of the endogenous antioxidants in low density lipoprotein by ascorbate but not probucol during oxidative modification. J. Clin. Invest., 87 (1991) 597.
14
Cohn, Z.A. and Benson, B., The differentiation of mononuclear phagocytes. Morphology, cytochemistry and biochemistry, J. Exp. Med., 121 (1965) 153. Rankin, S.M.. Knowles, M.E. and Leake, D.S.. Macrophages possess both neutral and acidic protease activities towards low density lipoprotein. Atherosclerosis, 79 (1989) 71. Schacterle, G.R. and Pollak, R.L.. A simple method for
I5
Aqel, N.M., Ball, R.Y., Waldmann, H. and Mitchinson. M.J., Identification of macrophages and smooth muscle cells in human atherosclerosis using monoclonal antibodies, J. Pathol., 146 (1985) 197.
hypercholesterolesis in the non-human primate. I. Fatty streak formation, Arteriosclerosis, 10 (1990) 164. Henriksen, T.. Mahoney, E.M. and Steinberg, D., Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells: recognition of receptors for acetylated low density lipoprotein, Proc. Natl. Acad. Sci. USA, 78 (1981) 6499.
References Fowler, S., Shio, H. and Haley, N.J., Characterisation of lipid-laden aortic cells from cholesterol-fed rabbits. IV. Investigation of macrophage-like properties of aortic cell population, Lab. Invest., 41 (1979) 372.
Hansson, G.K., Regional accumulation of T-cells, macrophages and smooth muscle cells in human atherosclerotic plaque, Arteriosclerosis, 6 (1986) 13 I. Hansson, G.K., Holm, J. and Jonasson, L., Detection of activated T-lymphocytes in the human atherosclerotic plaque, Am. J. Pathol., 135 (1989) 169. Masuda, J. and Ross, R., Atherogenesis during low level
16
17
18
the quantitative assay of small amounts of protein in biological material, Anal. Biochem. 51 (1973) 654. Bernheim, F., Bernheim, M.L.C. and Wilbur. K.M., The reaction between thiobarbitric acid and the oxidation of products of certain lipids, J. Biol. Chem., 174 (1948) 257. Parthasarathy, S., Young, S.G.. Witzum. J.L., Pittman, R.C. and Steinberg, D.. Probucol inhibits oxidative
192 modification of low density lipoprotein, J. Clin. Invest., 77 (1986) 641. 19 Brown, MS., Basu, S.K., Falck, J.R., Ho, Y.K. and Goldstein, J.L., The scavenger cell pathway for lipoprotein degradation: specitity of the binding site that mediates the uptake of negatively-charged LDL by macrophages, J. Supramol. Struct., 13 (1980) 67. 20 Esterbauer, H., Jiirgens, G., Quehenberger, 0. and
21
Keller, E., Autooxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes, J. Lipid Res., 28 (1987) 495. Jessup, W., Rankin, S.M., De Whalley, C.V., Ho&, J.R.S., Scott, J. and Leake, D.S., ol-Tocopherol consumption during low density lipoprotein oxidation, Biochem. J., 265 (1990) 399.
187
Printed and Published in Ireland
ATHERO 04758
The oxidative modification
of low density lipoprotein by human lymphocytes
David J. Lamb, Gary M. Wilkins and David S. Leake Department of Biochemistry and Physiology. School of Animal and Microbial Sciences, University of Reading, Whireknighrs. P.O. Box 228, Reading, Berkshire RG6 2AJ (U.K.)
(Received 24 July, 1991) (Revised, received 1 November, 1991) (Accepted 6 November, 1991)
Summary Oxidation of low density lipoprotein in atherosclerotic lesions may be involved in converting macrophages into lipid-laden foam cells. Lesions contain endothelial cells, smooth muscle cells, macrophages and lymphocytes. The first three types of cells have been shown previously to modify low density lipoprotein so as to increase its uptake by macrophages. We report here that lymphocytes from human blood are also capable of doing this. The modification process was an oxidative one because there was an increase in the thiobarbituric acid-reactive substances in media containing lymphocyte-modified low density lipoprotein and the modification could be inhibited by the antioxidants butylated hydroxytoluene and probucol. The lymphocyte-modified low density lipoprotein was taken up and degraded by macrophages by their scavenger receptor(s).
Key words: Antioxidants; Atherosclerosis; Low density lipoprotein; Lymphocyte; Macrophage: Oxidation
Introduction Uptake of low density lipoproteins (LDL) by arterial macrophages to generate foam cells in the arterial intima is believed to be an important step in the pathogenesis of atherosclerosis. Examination of human and animal atherosclerotic plaques by immunohistochemistry and other techniques Correspondence fo: Mr. D.J. Lamb, Department of Biochemistry and Physiology, School of Animal and Microbial Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading, Berkshire RG6 2AJ (U.K.)
indicates the presence of four major cell types: endothelial cells, smooth muscle cells, macrophages and lymphocytes [l-5]. It has been shown that incubating LDL with arterial endothelial cells [6], arterial smooth muscle cells [7] and mouse peritoneal macrophages [8,9] modifies LDL by an oxidative mechanism [lo], resulting in its more rapid uptake by cultured macrophages by means of their scavenger receptors. Native (unmodified) LDL is not recognised by the scavenger receptor(s) and is taken up only slowly by macrophages. Human blood monocytes [l l] and mononuclear
188 cells [ 121 have been reported to oxidise LDL, but not to increase its uptake by macrophages, and it has very recently been shown [ 131 that human monocyte-derived macrophages can oxidise LDL to increase its uptake by macrophages. We report here that human blood lymphocytes can modify LDL to greatly increase its uptake by macrophages. Materials and methods Isolation of lymphocytes and macrophages
Healthy human blood was defibrinated using glass beads in the presence of 15 mM EDTA and dextran (0.35 mg/ml) to sediment the erythrocytes over 4 h at room temperature. The white cell suspension was layered onto Histopaque 1077 (Sigma) and centrifuged at 400 x g for 30 min at room temperature. The mononuclear cell layer was recovered, washed with sterile Dulbecco’s phosphate-buffered saline (Gibco) then resuspended in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% (v/v) foetal calf serum and 50 pg/ml gentamicinlml. Monocytes were removed by adhesion over two incubations of 4-8 h in different culture dishes. Lymphocyte purity was assessed histologically using Wright’s stain (Sigma) which contains methylene blue and eosin which differentially stain cellular organelles thereby permitting identification of cell type. Monocyte contamination was shown to be no greater than 2% from triplicate counts of approximately 500 cells in several different experiments. Mouse resident peritoneal macrophages were isolated as described elsewhere [14]. Isolation and radioiodination of LDL
LDL (1.019-1.063 g/ml) was isolated from human blood by sequential density ultracentrifugation as described previously [ 151 and labelled with Na12’I using iodine monochloride [9]. Sterile 12’I-labelled LDL was stored in the dark at 4°C in a buffer containing 100 PM EDTA [9] to inhibit its autooxidation and discarded about a month after taking the blood. Modification of LDL
‘2’1-labelled LDL (100 pg protein/ml)
was in-
cubated at 37°C with lymphocytes (7.5 x lo6 cells/ml) or cell-free wells in 0.4 ml Ham’s F-10 medium (Flow Laboratories Ltd.) containing gentamicin (50 &ml) and 3 PM FeSO, in 16 mm multiwell dishes (Corning) under 5% CO2. After incubation (usually 18-24 h), the medium was centrifuged (1500 x g for 10 min at 4°C) to remove the cells. Stock solutions of the antioxidants butylated hydroxytoluene (Sigma) and probucol (a gift from Merrell Dow Pharmaceuticals Ltd., Staines) were prepared in ethanol such that the final concentration of ethanol in the modification media was 1% (v/v). Control cells were similarly adjusted to 1% ethanol. For the competition experiments, ‘251-labelled LDL was modified sterilely in F-10 medium containing 25 ccM CuSO, and gentamin (50 &ml) for 24 h at 37°C. Determination macrophages
of modified LDL
degradation by
Modified or control 1251-labelled LDL was diluted to 10 pg protein/ml in DMEM containing 10% (v/v) foetal calf serum and gentamicin (50 kg/ml). Diluted LDL was incubated for 20 h with mouse peritoneal macrophages (1 x lo6 peritoneal cells/well) or cell-free wells (22-mm multiwell dishes, Costar) at 1 ml per well. The rate of uptake was estimated by measuring the radioacnon-iodide tive trichloroacetic acid-soluble degradation products released into the medium as described previously [9]. Degradation products in cell-free wells were subtracted from those of the wells containing macrophages. The cells were washed and dissolved in 0.2 M NaOH as described elsewhere [9] and assayed for protein by a modified Lowry procedure [ 161. Thiobarbituric acid-reactive substances assay [17]
Samples of modified or control LDL (250 ~1 of 100 rg protein/ml) were taken and 3 ml of 0.335% (w/v) thiobarbituric acid in 10% (w/v) trichloroacetic acid was added to each sample and incubated at 100°C for 30 min. The absorbance was read at 535 nm. Standards of tetramethoxypropane (Sigma), up to 5 nmol per tube, were prepared in Ham’s F- 10.
189 Results
‘251-labelled LDL was modified by human blood lymphocytes so that it was taken up and degraded up to ten times or more faster by mouse peritoneal macrophages than 1251-labelled LDL incubated in cell-free wells rather than with lymphocytes. Increasing the number of lymphocytes per well increased the amount of LDL modification (Fig. 1). Human blood monocyte-derived macrophages were also able to modify LDL to increase its uptake by mouse peritoneal macrophages and were more active in doing so than the lymphocytes (data not shown), The modification of LDL by the lymphocyte preparations could not have been due to any contaminating macrophages, however, as a Wright’s stain showed the level of monocyte contamination of the lymphocyte preparations to be no greater than 2%, which could have accounted
I --
0
m
.
1
I
-
2
I
3
I
-
4
.
for less than 20% of the modification seen. In addition, lymphocytes prepared in culture medium that was obviously toxic to the macrophages isolated from the same sample of blood (possibly due to the batch of foetal calf serum used) still modified LDL well, whereas macrophages cultured separately died and did not modify the LDL. We therefore conclude that any contaminating macrophages in the lymphocyte preparation would also have been killed by the foetal calf serum and that the LDL modification observed would have been due to the lymphocytes themselves. Mouse peritoneal lymphocytes could also modify LDL to increase its uptake by macrophages, but were less effective in doing so than the human blood lymphocytes or mouse peritoneal macrophages (data not shown). Human blood lymphocytes were also less effective in modifying LDL than the mouse peritoneal macrophages (an example of this is shown in Fig. 3).
I
5
-I
LDL+lymph Number
of
cells
(mllllons)
Fig. 1. Modification of LDL by human lymphocytes. tz51labelled LDL was incubated for 24 h in Ham’s F-10 in the presence of increasing numbers of lymphocytes. The rate of degradation of the LDLs by mouse peritoneal macrophages was then determined. Each point is the mean f S.E.M. of three wells of cells.
LDL
lymph
medium
Fig. 2. Thiobarbituric acid-reactive substances in medium containing lymphocyte-modified LDL. LDL (100 pg protein/ml) was incubated for 24 h in Ham’s F-10 in the presence (LDL + lymph) or absence (LDL) of human lymphocytes and then assayed for thiobarbituric acid-reactive substances. Incubated F-10 (medium) and lymphocyte-conditioned medium (lymph) without LDL were also assayed. The mean f S.E.M. of three wells are shown.
190
lymph 0
Untreated
&j
Butylated
CO”
native
macro Concentration
Hydroxytoluene
Probucol
Fig. 3. Effect of butylated hydroxytoluene and probucol on modification of LDL by lymphocytes. 12sI-labelled LDL was incubated for 24 h with human lymphocytes (3 x IO6 per well) (lymph) or cell-free wells (con) with or without 10 pM butylated hydroxytoluene or 10 PM probucol. LDL was also incubated with mouse peritoneal macrophages 10~ (2 x IO6 peritoneal cells/well) (macro) for comparison. It should be noted that only about half the peritoneal cells would have been macrophages [ 131.The rate of degradation of all the LDLs (including non-incubated, native LDL) by mouse peritoneal macrophages was then determined. Each histobar is the mean f S.E.M. of three wells.
There was a large increase in the thiobarbituric acid reactive substances in media containing lymphocyte-modified LDL (Fig. 2). In addition, the modification of LDL by lymphocytes could be inhibited completely or almost completely by the chain-breaking antioxidant butylated hydroxytoluene or by probucol, an anti-atherosclerotic drug with antioxidant properties [18] (Fig. 3). The nature of the binding site on macrophages for human lymphocyte-modified LDL was investigated by competition experiments involving incubating with lymphocytemacrophages modified ‘251-labelled LDL in the presence of increasing concentrations of unlabelled ligands. Excess non-labelled copper-oxidised LDL competed effectively with lymphocyte-modified 1251-labelled LDL for degradation for macrophages, but nonlabeled native LDL gave little competition (Fig. 4).
of
competitor
(rg
/ ml)
Fig. 4. Competition between lymphocyte-modified labelled LDL and non-labelled hgands for degradation by macrophages. Mouse peritoneal macrophages were incubated for 20 h with lymphocyte-modified ‘251-labelled LDL (10 PM protein/ml) in the presence of increasing concentrations of nonlabelled copper-oxidised LDL ( . . ), native LDL (-), or polyinosinic acid (- - -) (obtained from Sigma) and its degradation measured. Each point is the mean f S.E.M. of three wells. The degradation of control 1251-labelledLDL was 1.04 + 0.11 pg protein/mg cell protein in 20 h.
Polyinosinic acid, a potent ligand for the scavenger receptor(s) [ 191, greatly decreased the degradation of lymphocyte-modified labelled LDL. Discussion We have shown that lymphocytes can oxidatively modify LDL to greatly increase its uptake by macrophages. The moditication of LDL by lymphocytes was by an oxidative process because there was a large increase in the thiobarbituric acid reactive substances in LDL-containing medium exposed by lymphocytes and the modification could be inhibited by antioxidants. Butylated hydroxytoluene, a chain-breaking antioxidant, and probucol, a lipid-lowering drug with antioxidant properties, inhibited completely or almost completely the modification of LDL by human lymphocytes. Similar effects have been reported with endothelial cell [ 10,171, macrophage [8,9] and human mononuclear cell [12] modification of LDL.
191 As the number of modifying lymphocytes was increased, there appeared to be a ‘lag’ before increased uptake by macrophages was observed (Fig. 1). This may be related to the ‘lag phase’ seen in the time course of LDL modification in other systems, due in part to the time taken for the depletion of endogenous antioxidants in the LDL, e.g., cr-tocopherol [20,21]; a small number of lymphocytes may not be able to exert a great enough oxidative stress to overcome this ‘lag phase’ in the time available. Lymphocyte-modified LDL was apparently taken up by the scavenger receptor(s) as copperoxidised LDL and polyinosinic acid, known ligands of the scavenger receptor(s), effectively inhibited its degradation by macrophages. Excess native LDL, however, only slightly decreased lymphocyte-modified LDL degradation. Lymphocytes are found in large numbers in atherosclerotic lesions [3-51, along with endothelial cells, smooth muscle cells and macrophages. The latter three cell types have been shown previously to modify LDL to increase its uptake by macrophages [6-10,131. It would now appear that all four major cell types within atherosclerotic lesions have the capacity to oxidise LDL and increase its uptake by macrophages contributing to their conversion into foam cells. Acknowledgements
We are grateful to the Research Endowment Fund of the University of Reading for a research studentship (D.J.L.) and the Wellcome Trust for financial support. We also thank Dr A.I. Tiffn for advice on isolating lymphocytes and Mrs Sylvia F. Dalton, Mrs Linda Bunn and Mrs Ellen Cook for carefully typing the manuscript.
4
5
6
7
8
Henriksen, T., Mahoney, E.M. and Steinberg. D.. Enhanced macrophage degradation of biologically modified LDL, Arteriosclerosis, 3 (1983) 149. Parthasarathy. S., Printz, D.J., Boyd. D., Joy, L. and
9
Steinberg, D., Macrophage oxidation of low density lipoprotein generates a modified form recognised by the scavenger receptor, Arteriosclerosis, 6 (1986) 505. Leake, D.S. and Rankin, S.M., The oxidative modifica-
10
II
Jonasson,
L., Helm.
J., Skalli.
O.,
Bondjers,
G. and
tion of low density lipoproteins by macrophages. Biochem. J.. 270 (1990) 741. Steinbrecher, U.P., Parthasarathy, S.. Leake. D.S.. Witzturn, J.L. and Steinberg, D.. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids, Proc. Natl. Acad. Sci. USA, 81 (1984) 3883. Cathcart, M.K., Morel, D.W. and Chisolm, III, G.M., Superoxide dismutase (SOD) and catalse (CAT) inhibit the oxidation of low density lipoprotein (LDL) by monocyte derived macrophages. J. Cell. Biochem., 599 (1985) 153.
12
Hiramatsu, K., Rosen, H., Heinecke, J.W., Wolfbauer, G. and Chait, A., Superoxide initiates oxidation of low density lipoprotein by human monocytes. Arteriosclerosis, 7 (1987) 55.
13
Jialal, I. and Grundy, SM., Preservation of the endogenous antioxidants in low density lipoprotein by ascorbate but not probucol during oxidative modification. J. Clin. Invest., 87 (1991) 597.
14
Cohn, Z.A. and Benson, B., The differentiation of mononuclear phagocytes. Morphology, cytochemistry and biochemistry, J. Exp. Med., 121 (1965) 153. Rankin, S.M.. Knowles, M.E. and Leake, D.S.. Macrophages possess both neutral and acidic protease activities towards low density lipoprotein. Atherosclerosis, 79 (1989) 71. Schacterle, G.R. and Pollak, R.L.. A simple method for
I5
Aqel, N.M., Ball, R.Y., Waldmann, H. and Mitchinson. M.J., Identification of macrophages and smooth muscle cells in human atherosclerosis using monoclonal antibodies, J. Pathol., 146 (1985) 197.
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