Quantitative Analysis of Apolipoprotein E Secretion by Human Monocyte-Derived Macrophages in Culture

Abstract

Apolipoprotein E (apo E) has an impact on lipid metabolism and its production by macrophages is considered to play a protective role against atherosclerosis. Apo A-I stimulates secretion of apo E from macrophages. We developed a new method to evaluate the ability of human monocyte-derived macrophages to secrete apo E, and the effects of factors such as apo A-I were examined. Monocytes separated from peripheral venous blood were cultured. The levels of apo E in macrophage-conditioned medium were quantified by immunoblotting with an anti-human apo E antiserum conjugated with alkaline phosphatase. The basal levels of apo E secretion and the response to exogenous apo A-I in macrophages from 10 healthy volunteers were measured. Sufficient accuracy and sensitivity were confirmed and coefficient of variation of the method was 18 +/- 11% (n = 10). It was confirmed that macrophage secreted apo E in a concentration-dependent manner in response to M-CSF and apo A-I. The average apo E concentration in the conditioned medium of macrophages from 10 healthy subjects was 30.9 +/- 14.7 ng/mg cell protein. After the addition of apo A-I, the average apo E concentration increased, by about 60%, to 49.4 +/- 29.7 ng/mg cell protein (p < 0.05). There was a positive correlation between the apo A-I-induced increase and plasma LDL cholesterol levels (r = +0.54, p < 0.05).