Human (THP-1) macrophages oxidize LDL by a thiol-dependent mechanism
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A Graham1, J L Wood, V J O'Leary
1Department of Biochemical Sciences Wellcome Research Laboratories, Beckenham, Kent, UK.
Abstract:
The oxidative modification of low-density lipoprotein by macrophages may be an important mechanism in the pathogenesis of atherosclerosis. The human monocytic leukaemia cell line THP-1, when stimulated with phorbol ester, shares many properties with human monocyte-derived macrophages. Oxidation of LDL by these cells was characterised by depletion of alpha-tocopherol, increases in thiobarbituric acid reactive substances and increases in electrophoretic mobility. The LDL particles were also converted to a form which increased accumulation of cholesteryl esters within macrophages. The oxidative mechanism appeared to be dependent upon the presence of thiols in the cellular medium. Oxidation of LDL by THP-1 macrophages, and production of thiols by these cells, were dependent upon the presence of L-cystine in the medium. Furthermore, cellular oxidation of LDL could be partially mimicked by the addition of cysteine to Hams F10 medium. Macrophage-independent oxidation of LDL, mediated by the addition of copper ions, was inhibited by cystine and cysteine in phosphate buffered saline, but not in Hams F10 medium. The glutathione content of THP-1 macrophages was also dependent upon the presence of cysteine or cystine in the medium, but inhibition of glutathione synthesis by buthionine sulfoximine did not prevent the production of thiols or the oxidation of LDL by THP-1 macrophages.
Insights
Oxidative modification of low-density lipoprotein (LDL) by macrophages is key in atherosclerosis. THP-1 cells mimic macrophages, showing LDL oxidation dependent on thiols and L-cystine.
Area of Science:
- Biochemistry
- Cell Biology
- Pathology
Background:
- Oxidative modification of low-density lipoprotein (LDL) is implicated in atherosclerosis pathogenesis.
- The human monocytic leukaemia cell line THP-1, when stimulated, exhibits properties similar to human monocyte-derived macrophages.
Purpose of the Study:
- To investigate the oxidative modification of LDL by THP-1 macrophages.
- To elucidate the role of thiols and L-cystine in this process.
Main Methods:
- Stimulation of THP-1 cells with phorbol ester.
- Characterization of oxidized LDL using alpha-tocopherol depletion, thiobarbituric acid reactive substances, and electrophoretic mobility.
- Assessment of cholesteryl ester accumulation in macrophages.
- Investigation of thiol and glutathione production.
- Mimicking LDL oxidation with cysteine and copper ions.
Main Results:
- THP-1 cells oxidized LDL, characterized by alpha-tocopherol depletion and increased thiobarbituric acid reactive substances.
- LDL oxidation and thiol production by THP-1 cells were dependent on L-cystine in the medium.
- Cellular LDL oxidation was partially mimicked by adding cysteine to the medium.
- Copper-mediated LDL oxidation was inhibited by cystine/cysteine in phosphate-buffered saline but not Hams F10 medium.
- Macrophage glutathione content was dependent on cysteine/cystine, but its synthesis was not required for thiol production or LDL oxidation.
Conclusions:
- THP-1 macrophages oxidize LDL through a thiol-dependent mechanism.
- L-cystine in the cellular medium is crucial for both LDL oxidation and thiol production by these cells.
- The findings suggest a specific role for thiols in macrophage-mediated LDL oxidation relevant to atherosclerosis.