Macrophages transfer cholesterol to lymphocytes in culture.

Abstract

Macrophages have been recognized to present an intense cholesterol metabolism, recycling the entire plasma membrane once every 30 minutes [I] and having an enormous ability to produce and export cholesterol, this being modulated by prostaglandins [2, 31. On the other hand, lymphocytes treated with cholesterol have been shown enrichment in the amount of the sterol in plasma membrane, markedly suppressing mitogenicinduced differentiation to blast cells [4]. This information led us to investigate whether cholesterol released from rat macrophages would be transferred to rat lymphocytes in culture. Mesenteric lymph node lymphocytes and peritoneal resident macrophages were obtained from male Wistar rats killed by cervical dislocation. Inflammatory macrophages were obtained from thioglycollate-injected animals [5]. Mesenteric lymph node lymphocytes obtained from these animals are called here as thioglycollate-stimulated lymphocytes. Cells were then cultured at 37°C in a 5% CO, atmosphere in air in Eagle's essential medium supplemented with antibiotics and lO?? foetal calf serum (FCS). Macrophages were left to attach on polystyrene dish and after 2 h non-attached cells were removed. Lymphocytes (> 98% pure) were seeded and pre-cultivated for 2 h in order to eliminate any attachable cells. To label macrophage intracellular cholesterol pool, resident and thioglycollate-stimulated cells (1 O'/well) were cultivated at 37°C in a 5% C02 atmosphere in air for 18 hours with pre-purified (thin-layer chromatography, TLC) 0.1 pCi/ml [4-C] cholesterol (57.1 mCimmol, NEN, Du Pont Co., USA) dissolved in ethanol (0.05% final concentration, by volume) in medium supplemented with lipid-free bovine serum albumin (BSA) prepared as described [6]. Preliminary experiments established that, under the above conditions, there is a maximal incorporation of [4-'4C]-cholesterol into cells. After labeling, supernatants were discharged and macrophage monolayers were washed three times with MEM (remaining radioactivity equalling background counts). A lymphocyte suspension (-2 x 10 cells) was then added to macrophage monolayers and cells were cocultured for 12, 24 or 48 h. Lymphocytes were obtained from the same macrophage-donor animals. At the end of culture periods, lymphocytes were harvested by using a micropipette. In order to eliminate any macrophage eventually detached during co-cultures, lymphocytes were incubated for 30 min at 37°C under agitation with iron powder (mesh 400, -10 mg/107 lymphocytes in 2 ml of medium). The suspension was then layered over a 1.077 g/ml Lymphoprep solution (Nycomed Pharma AS, Oslo, Norway). The turbid interface between the medium and Lymphoprep solution (> 99% lymphocytes) was collected and the bottom of the tube (containing macrophages with phagocytosed iron particles) discharged. Lymphocytes were then transferred to 2nd Eppendorf microcentrihge tubes, total lipids extracted [2] and total cholesterol contents (cholesterol and cholesteryl esters) determined [7]. An appreciable transference of radioactivity to the lymphocyte free cholesterol pool was found when these cells were co-cultivated with [4-14C]-cholesterol pre-labeled resident macrophages (Table 1). This process gradually increased with the time. Thioglycollate-stimulated macrophages presented a much lower transference of cholesterol to lymphocytes (by up to 91%), Table 1 Cholesterol incorporated into lynphocvtes after cocultivation with r4-14C1-cholesterol labeled macrophages. The results are presented as the mean f S.E.M. of 6 experiments and expressed as nm0V10'~ lymphocytedlO1O macrophages.