Reto Asmis

Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages

Abstractu2002Macrophages are key players in many aspects of human physiology and disease. It has been hypothesized that in a given microenvironment monocytes differentiate into specific subpopulations with distinct functions. In order to study the role of macrophage heterogeneity in atherogenesis, we established a novel isolation and culture technique for human monocyte-derived macrophages. The present technique does not select for monocyte subpopulations prior to the onset of differentiation. Monocytes were cultured for 2 weeks in the presence of autologous lymphocytes before being plated quantitatively. They differentiated into mature macrophages in terms of morphology, lipid composition, and biological activity. Based on phagocytic activity as well as on the expression of CD14, CD36, and the low-density lipoprotein (LDL) receptor, we have identified macrophage subpopulations that may play distinct roles in atherogenesis. While virtually all adherence-purified monocytes expressed CD14, CD36, and the LDL-R, we characterized three subpopulations of macrophages based on the expression of these antigens: CD36+CD14–LDL-R– (58±12%), CD36+CD14+LDL-R+(18±5%), the remaining cells being CD36–CD14–LDL-R–. The first two subsets decreased in size during further differentiation (51±12% and 8±3%, respectively). Our culture technique may also serve as a good model for studying the implications of macrophage heterogeneity in diseases other than atherosclerosis.

Large variations in human foam cell formation in individuals: a fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids

The transformation of monocyte-derived macrophages into lipid-laden foam cells constitutes a characteristic and crucial event in the development of the earliest atherosclerotic lesions. We investigated whether the propensity to form foam cells varies among individuals. We developed a fully autologous foam cell assay based on a recently developed novel culture technique for human monocyte-derived macrophages (Wintergerst ES, Jelk J, Asmis, R. Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages. A novel cell culture system to study macrophage differentiation and heterogeneity, Histochem. Cell Biol. 1998;110:231-241). Thin layer chromatography and laser densitometry were used to determine cholesterol, triglyceride and cholesteryl ester levels in human macrophages. Aggregated LDL obtained by vortexing was found to be a reproducible stimulus of foam cell formation in human macrophages. In our hands, Cu(2+)-oxidized LDL also induced cholesteryl ester accumulation, but only when vortexed. We found that foam cell formation in an individual varied by less than 25% over a 10-month period. In contrast, we observed a sevenfold difference in foam cell formation among eight male volunteers. The transfer of foam cells into culture medium with freshly thawed autologous serum resulted in a 75% regression within 1 week, independent of the amount of cellular cholesteryl esters accumulated. Foam cell formation correlated neither to serum nor to cellular cholesterol and triglyceride levels. The propensity to form foam cells could therefore represent a novel indicator of individual risk of atherogenesis.

Vitamin E Supplementation of Human Macrophages Prevents Neither Foam Cell Formation Nor Increased Susceptibility of Foam Cells to Lysis by Oxidized LDL

Several studies in macrophage cell lines, rodent macrophages, and animal models of atherosclerosis suggest that vitamin E may prevent the formation of foam cells. We tested this hypothesis in a recently developed, fully autologous in vitro model of human foam cell formation. During maturation, macrophages continuously increased their &agr;-tocopherol/total cholesterol ratio, demonstrating that these cells accumulate &agr;-tocopherol at an even higher rate than cholesterol. In the presence of unsupplemented serum, we observed no correlation between serum vitamin E levels and the increase in the cellular &agr;-tocopherol/total cholesterol ratio. In contrast, under supplemented conditions, a 3.1-fold increase in the mean serum &agr;-tocopherol/total cholesterol ratio resulted in a corresponding mean 3.5-fold increase in the cellular &agr;-tocopherol/total cholesterol ratio. Vitamin E loading had no effect on the lipid composition of macrophages and did not affect their growth. Foam cell formation was stimulated in mature unsupplemented and vitamin E–loaded macrophages for 1 week with 50 &mgr;g autologous aggregated low density lipoprotein (LDL) in the presence of unsupplemented and vitamin E–loaded serum, respectively. We observed no effect of vitamin E supplementation on the formation of foam cells. However, foam cell formation resulted in a 36% and 44% reduction in the cellular &agr;-tocopherol/total cholesterol ratio in unsupplemented and vitamin E–supplemented foam cells, respectively. The loss of vitamin E was accelerated with increasing concentrations of aggregated LDL and was accompanied by an increase in the susceptibility of these foam cells to succumb to the cell lytic effects of oxidized LDL (OxLDL). However, vitamin E supplementation did not protect macrophages or foam cells from OxLDL-mediated cell lysis, suggesting that vitamin E loss in foam cells is not the cause of their increased susceptibility to cell lysis. Our results suggest that the beneficial effects of vitamin E on cardiovascular disease observed in humans are due neither to a reduction in the propensity of macrophages to form foam cells nor to an increased resistance of these cells to cytolytic OxLDL.

Concurrent quantification of cellular cholesterol, cholesteryl esters and triglycerides in small biological samples Reevaluation of thin layer chromatography using laser densitometry

Absolute specificity and high accuracy is required for the quantitation of cholesterol, cholesteryl esters and triglycerides in small biological samples, particularly in a limited number of cells. Both can be achieved through thin-layer chromatography and molybdatophosphoric acid staining, while the shortcomings of traditional spot detection are overcome by laser densitometry. The major advantage of the proposed technique is the concurrent assay of nanogram quantities of cholesterol, cholesteryl esters and triglycerides. Our assay is at least ten-fold more sensitive than common thin-layer chromatography-based techniques and at least four-fold more sensitive than common enzymatic methods. The present low-cost assay is highly reproducible and may be particularly suitable for the routine lipid analysis of a 10% aliquot of relatively small tissue and cell samples, equivalent, for instance, to > or = 10(4) human monocytes.