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An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization
Published on: June 12, 2013
Alpha-tocopherol supplementation of macrophages does not influence their ability to oxidize LDL
A Baoutina1, R T Dean, W Jessup
1Cell Biology Unit, The Heart Research Institute, Camperdown, New South Wales, Australia.
Abstract:
We have investigated the effect of alpha-tocopherol-loading of mouse peritoneal macrophages and human monocytes on their ability to oxidize human low density lipoprotein (LDL). Mouse peritoneal macrophages incorporated alpha-tocopherol (alpha-TOH) from culture medium supplemented with the vitamin in a time- and concentration-dependent manner. Subcellular fractionation by density gradient ultracentrifugation showed that the distribution of incorporated alpha-TOH within the cell was similar to that of free cholesterol. Most (approximately 88%) of alpha-TOH partitioned into the membrane fractions (plasma membrane approximately 41%, mitochondria and lysosomes approximately 26%, and endosomes plus endoplasmic reticulum approximately 21%). Cellular alpha-TOH was stable for at least 24 h in serum- or LDL-free media whether permissive (Ham's F-10) or non-permissive (Dulbecco's minimum essential medium, DMEM) for LDL oxidation. When incubated with LDL in DMEM, alpha-TOH-preloaded cells transferred small amounts of alpha-TOH (approximately 1 nmol/mg LDL protein after 9 h) to the lipoprotein. However, enrichment of the cells with alpha-TOH did not change the kinetics of oxidation of either normal or TOH-depleted LDL in Ham's F-10 medium compared with non-loaded cells, as assessed by alpha-TOH consumption, cholesteryl ester degradation, and cholesteryl ester hydroperoxide and 7-ketocholesterol accumulation. Nor did it alter superoxide release by the cells or their ability to reduce extracellular copper(II). Similar to mouse macrophages, enrichment of human monocytes with alpha-TOH did not change the kinetics of cell-mediated LDL oxidation. We conclude that elevated cellular levels of alpha-TOH in mouse peritoneal macrophages and in human monocytes do not affect their ability to oxidize LDL lipids in vitro. This suggests that either cell-mediated oxidation of LDL under the conditions of this study is not dependent on cell-derived radical species or that cellular alpha-TOH is unable to affect their formation.
Insights
Loading macrophages and monocytes with alpha-tocopherol (alpha-TOH) did not alter their ability to oxidize low-density lipoprotein (LDL). This suggests cellular alpha-TOH does not impact cell-mediated LDL oxidation in vitro.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Low-density lipoprotein (LDL) oxidation is implicated in atherosclerosis.
- Alpha-tocopherol (alpha-TOH), a form of vitamin E, is an antioxidant.
- The role of cellular alpha-TOH in modulating immune cell-mediated LDL oxidation is not fully understood.
Purpose of the Study:
- To investigate the impact of alpha-tocopherol loading on the LDL-oxidizing capacity of mouse peritoneal macrophages and human monocytes.
- To determine if increased intracellular alpha-TOH levels affect the kinetics of LDL oxidation by these cells.
Main Methods:
- Mouse peritoneal macrophages and human monocytes were loaded with alpha-tocopherol.
- Subcellular distribution of alpha-tocopherol was analyzed using density gradient ultracentrifugation.
- Cell-mediated LDL oxidation was assessed by measuring alpha-tocopherol consumption, cholesteryl ester degradation, and lipid peroxidation products.
- Superoxide release and copper reduction capacity of cells were also evaluated.
Main Results:
- Alpha-tocopherol incorporation into macrophages was time- and concentration-dependent, with most accumulating in membrane fractions.
- Alpha-tocopherol-loaded cells transferred minimal amounts of alpha-TOH to LDL.
- Enrichment with alpha-tocopherol did not alter the kinetics of LDL oxidation, cholesteryl ester degradation, or lipid peroxidation in either mouse macrophages or human monocytes.
- Cellular superoxide release and copper reduction were unaffected by alpha-tocopherol loading.
Conclusions:
- Elevated cellular alpha-tocopherol levels in macrophages and monocytes do not influence their in vitro capacity to oxidize LDL lipids.
- Cell-mediated LDL oxidation under these experimental conditions may not depend on cell-derived radical species, or cellular alpha-TOH may not effectively scavenge them.
