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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 9, 2001 as doi:10.1096/fj.00-0704fje. |
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Cell Biology Group, The Heart Research Institute, 145 Missenden Rd., Camperdown, Sydney, NSW, 2050, Australia
2Correspondence: Cell Biology Group, The Heart Research Institute, 145 Missenden Rd., Camperdown, N.S.W., 2050, Australia. E-mail: a.baoutina{at}hri.org.au
SPECIFIC AIMS
To better understand mechanisms by which arterial cells promote oxidation of low density lipoprotein (LDL), we investigated a role for the trans-plasma membrane electron transport (TPMET) system (which uses intracellular reductants to reduce extracellular acceptors, e.g., metal ions) of macrophages in cell-mediated LDL oxidation. We had previously shown that catalytic levels of extracellular transition metal ions are essential for cell-mediated oxidation of LDL; we studied how manipulation of TPMET activity of macrophages affects their ability to oxidize LDL in vitro.
PRINCIPAL FINDINGS
1. TPMET (ferricyanide-reductase) activity of macrophages and
monocytes
To study the TPMET activity of macrophages, we used ferricyanide
(FEC) as an extracellular electron acceptor and measured the formation
of its reduced form, ferrocyanide (FOC). Incubation of 1.2 x
106 mouse J774 macrophages or 1 x
106 human leukemia THP-1 monocytes or macrophages
with 100 µM FEC in 1 ml HBSS resulted in its time-dependent
reduction. By measuring the FEC reduction in the cell-conditioned
medium from cells, we showed that at least 45% of FEC-reductase
activity of J774 cells and more than 81% of that of THP-1 cells
required the continuous presence of cells and was mediated by their
TPMET system. The remaining FEC reduction is mediated by reductants,
possibly thiols, secreted from the cells. FEC-reductase activity of the
cells was not mediated by superoxide radical, as it was not affected by
the presence of added superoxide dismutase (SOD) (10 µg/ml).
2. Macrophage FEC-reductase activity is stimulated by loading cells
with ascorbate
To manipulate cellular TPMET, we induced an increase in the
intracellular reductant, ascorbate. J774 and THP-1 macrophages
pretreated with 100 µM dehydroascorbate (DHA) or ascorbate (Asc) in
HBSS for 1 h [further referred to as (+DHA)-cells or (+Asc)-
cells] had significantly enhanced FEC-reductase activity. The average
stimulation of FEC-reductase activity by (+DHA)-J774 macrophages was
7.7-fold and by (+Asc) cells, 4.6-fold. The enhanced reductase activity
of J774 macrophages was observed for at least 5 h after removing
DHA from the culture medium.
Next we demonstrated that this enhancement of macrophage TPMET was due to accumulation of Asc levels in the cells. Pretreatment of macrophages with 100 µM Asc or DHA for 1 h resulted in a significant increase in the cellular content of Asc: from undetectable levels in control cells to 3.9 and 11.6 nmol Asc per 1.2 x 106 cells (mean from three experiments), respectively. The intracellular level of the oxidized form of Asc, DHA, was very low (2.1% and 6.7% of total intracellular Asc in (+Asc)- and (+DHA)-cells, respectively). We observed a good correlation between intracellular Asc levels achieved by preincubation of macrophages with Asc or DHA and the consequent stimulation of cellular FEC-reductase in these cells.
3. Stimulation of macrophage FEC-reductase in Asc-loaded
cells is due to stimulation of their TPMET, but not to secretion of Asc
or other reductants from cells
To test the possibility that enhanced FEC reduction of
Asc-enriched cells was due to secretion of Asc and other reductants, we
studied the reduction of FEC in cell-conditioned media from control and
Asc-enriched macrophages and the effect of SOD on cell-mediated metal
ion reduction, measured secretion of Asc from cells, and studied the
effects of manipulation of cellular Asc recycling or of ascorbate
oxidase (AO) on FEC reduction.
The amount of FEC reduced during 1 h in cell-conditioned medium from (+Asc)- and (+DHA)-J774 cells was at the most 0.3 µM higher than the amount of FEC reduced in the cell-conditioned medium from control cells. Under the same conditions, the additional formation of FOC by (+Asc)- and (+DHA)-cells compared with control cells was 13.6 and 19.2 µM, respectively. This suggests that secreted reductants are not responsible for the enhanced FEC-reducing activity of these cells. The enhanced FEC reduction by Asc-enriched cells was not affected by SOD, indicating that the process is independent of superoxide.
Culturing Asc-enriched J774 macrophages in HBSS resulted in the loss of
intracellular Asc and its appearance in the medium. After 1 h
incubation, 0.49 and 0.53 µM Asc were secreted from Asc- or
DHA-pretreated cells, respectively; more that 85% of it was present in
its reduced form. The presence of FEC in the culture medium did not
increase Asc release from Asc-enriched macrophages. We estimated that
the accumulation of FOC due to Asc release from (+Asc)- or (+DHA)-J774
cells could be 
1.1 µM. On the other hand, these cells
reduced on average 14.4 and 26.4 µM FOC more than control cells. The
results indicate that at the most, 6.8 and 4.1% of enhanced
FEC-reductase activity of Asc-enriched cells could be attributed to the
Asc released by (+Asc)- or (+DHA)-cells, whereas the rest could be
explained by the stimulation of cellular TPMET system.
Additional support against a role for secreted Asc was obtained in experiments with AO. Addition of AO (4 units/ml) did not affect generation of FOC by Asc- or DHA-preincubated cells or control cells.
These results thus indicate that Asc secreted from Asc-enriched macrophages could be only a minor contributor to the total FEC-reductase activity of these cells. However, this could become significant if cells continuously recycle extracellular Asc. To test whether Asc recycling contributed to FEC reduction by Asc-enriched cells, we used inhibitors of DHA uptake by the GLUT-1 transporter. We demonstrated that DHA uptake was inhibited by 76 and 32% in the presence of cytochalasin B and 2-deoxy-glucose, respectively, but this had no effect on FEC reduction by Asc-enriched macrophages.
We conclude that the enhanced FEC reduction by Asc-loaded macrophages is due to stimulation of their TPMET activity rather than to increased secretion of reductants from these cells.
4. Macrophage-mediated LDL oxidation is stimulated by Asc
enrichment of the cells and this stimulation is not due to secretion of
Asc
To investigate the contribution of TPMET to
macrophage-mediated LDL oxidation, we examined whether stimulation
of their TPMET activity by Asc enrichment affected the ability of the
cells to oxidize LDL. We and others have previously shown that low
concentrations of transition metals are essential for cell-mediated LDL
oxidation, and propose that by maintaining extracellular metal ions in
their reduced form, TPMET could stimulate this lipid peroxidation.
Enrichment of J774 macrophages with Asc increased the rate of
cell-mediated LDL oxidation in Ham’s F-10 (contains 3 µM of Fe(III)
and 0.01 µM of Cu(II)), as was seen by the more rapid kinetics of
consumption of lipoprotein tocopherol (TOH), free cholesterol (FC),
cholesteryl esters [cholesteryl arachidonate (CCA) and cholesteryl
linoleate (CL)], accumulation of the cholesterol oxidation product
7-ketocholesterol (7-KC), as well as formation and decomposition of
cholesteryl ester hydroperoxide and hydroxide (CEO(O)H)
(Fig. 1
).
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To link the increased ability of Asc-loaded macrophages to oxidize LDL with their enhanced TPMET activity, it was necessary to eliminate a possible role of Asc secreted by the cells. First, we showed that addition of Asc in the amounts secreted by cells to LDL oxidizing system (either in the presence or absence of cells) profoundly inhibited oxidation of LDL. DHA (0.2–1 µM) had little or no inhibitory effect on LDL oxidation. Second, we showed that AO has no significant effect on LDL oxidation by control (+DHA)- and (+Asc)-macrophages. These findings indicate that enhanced LDL oxidation by Asc-enriched macrophages is not due to secretion of Asc, but rather to activation of TPMET.
CONCLUSIONS
Oxidation of LDL in the arterial intima is believed to play a key role in atherogenesis. A wealth of in vitro studies has demonstrated that macrophages, one of the most important cell types in the developing lesions, promote LDL oxidation; however, the exact mechanisms responsible for this process remain unclear. The results of our study make a substantial contribution to the elucidation of the mechanisms responsible for cell-mediated LDL oxidation in vitro by directly implicating the macrophage TPMET system.
We demonstrated that macrophages, both mouse and human, possess TPMET activity as measured by cell-mediated reduction of FEC. We showed that a large portion of FEC reduction by macrophages was due to the TPMET; some of this macrophage reductase activity could be attributed to reductants, probably thiols, secreted by cells.
The FEC-reductase activities of macrophages could be significantly enhanced by their enrichment with Asc. A direct correlation between intracellular concentration of Asc and the rate of FEC reduction was observed. Several lines of evidence demonstrate that stimulation of FEC reduction by macrophage enrichment with reduced Asc could not be explained by direct reduction of external FEC by Asc or other reductants secreted by cells. First is the low and equal reducing capacities of cell-conditioned media from control and Asc-enriched macrophages and the absence of the effect of SOD on FEC-reductase activity of Asc-enriched cells. Second, the fact that disruption of cellular Asc recycling process, using the inhibitors of DHA uptake, did not affect FEC-reductase activities of Asc-enriched macrophages strongly supports the conclusion that continuous recycling of secreted Asc does not contribute to FEC-reductase activity of these cells. Third, the finding that Asc in the amounts secreted from (+Asc)- or (+DHA)-cells accounts for less than 7% of FOC generated in the presence of these cells and the absence of an effect of AO on FEC reduction by Asc-enriched macrophages further support the idea that secreted Asc is only a minor contributor to the total FEC-reductase activity of these cells.
Thus, the results indicate that intracellular Asc directly stimulates TPMET of macrophages. Enhancement of TPMET by intracellular Asc has been previously reported for erythrocytes, HL-60 and endothelial cells, and the proposed mechanism involves the role of Asc as the electron donor for the TPMET system in these cells. Our results are consistent with this hypothesis.
If TPMET contributes to macrophage-mediated LDL oxidation, then the
stimulation of TPMET by intracellular Asc should also accelerate
cell-mediated LDL oxidation. This was indeed the observed effect (Fig. 1)
. Dissociation between effects of Asc loading on cell-mediated
oxidation of LDL and Asc secretion was established through inability of
added Asc to stimulate the former process and the lack of effect of AO
on it. Thus, stimulation of cellular TPMET activity in Asc-enriched
cells is the most likely mechanism for the accelerated LDL oxidation by
these cells. We propose that the cells preloaded with Asc and
possessing enhanced TPMET activity mediate more efficient reduction of
extracellular transition metal ions than control cells. The reduced
transition metal ions then facilitate lipid hydroperoxide decomposition
via a Fenton-type reaction and chain lipid peroxidation in the
lipoprotein (Fig. 2
).
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The presented results make a significant contribution to the elucidation of the mechanisms responsible for cell-mediated LDL oxidation in vitro. The reported presence and colocalization of macrophages, transition metals, and oxidized LDL in atherosclerotic plaque without depletion of tissue ascorbate make this mechanism plausible for the accumulation of oxidatively modified LDL in vivo and thus may be an important target for further antiatherogenic therapies.
Our results also broaden the current view on the cellular functions of the TPMET system and add new weight to the possibility that in some circumstances, particularly pathological ones, ascorbate may play a potentially pro-oxidant and perhaps proatherogenic role in vivo.
In conclusion, the results here show that enhancing TPMET activity of macrophages increases their ability to oxidize LDL. These findings strongly support our hypothesis that TPMET plays a significant role in macrophage-mediated LDL oxidation via reduction of extracellular transition metals ions.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0704fje ; to cite this
article, use FASEB J. (May 9, 2001) 10.1096/fj.00-0704fje 
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), Asc-enriched cells
(resulting from preincubation of J774 cells in HBSS containing 100 µM
DHA for 1 h) (•), or cell-free wells (