Increased Expression of High-Affinity Low-Density Lipoprotein Receptors on Human T-Blasts

Abstract

Like all cells, lymphocytes need cholesterol for proper function, a requirement met by a finely tuned homeostasis between intracellular synthesis and uptake from the environment via low-density lipoproteins (LDL). We used flow cytometry to analyze the receptor activity of resting cells and T blasts incubated/activated in serum-free culture medium, or in medium supplemented with 25–5,000 μg/ml LDL. Dioctadecyl-indocarbocyanine has proved to be a useful fluorescent probe for investigating the LDL receptor activity of lymphocytes. The results show the receptor activity of day-3 resting T cells to be reduced more than 50% by 50 μg LDL/ml, whereas 100-fold higher concentrations are necessary to achieve the same level of reduction in day-3 PHA blasts. The LDL receptor activities of individual blood donors’ resting T cells, in vitro cholesterol-deprived resting T cells, and activated T blasts, were compared using two analytical techniques: spectrofluorometric analysis of detergent-solubilized cell suspensions and flow cytometric analysis of single living cells. Receptor affinity was determined by Scatchard analysis of spectrofluorometric binding curves, and by Line-weaver-Burke plots of flow cytometric data. Both methods yielded essentially identical dissociation constants (K_d) for cholesterol-deprived resting T cells and mitogen-activated T blasts, which fell in the expected range for the high-affinity LDL receptor (4.1–8.9 nM). In addition, spectrofluorometric analysis, but not flow cytometry, permitted quantification of LDL uptake. The low receptor activity of freshly isolated T cells, however, could only be reliably measured by flow cytometry; this highly sensitive technique not only afforded specific binding curves, but also determination of K_d of the same high affinity (7.5 nM) as that of T cells with upregulated LDL receptor activity.

17β-Estradiol Increases Ca2+ Influx and Down Regulates Interleukin-2 Receptor in Mouse Thymocytes

“…In other experiments, where it was necessary to show the extent of 17␤-estradiol inhibitory effect on T lymphocyte proliferation, PHA stimulation was adopted by treating thymocytes for 16 h with mitogen at a concentration of 2.5 g/1.0 ϫ 10 6 cells together with varying doses of estradiol. After incubation the expression of IL-2 receptor was monitored by spectrofluorometry, a technique which has been shown to give highly reproducible results and is comparable to flow cytometry for the expression of receptors on lymphocytes (20).…”

Improved detection of familial hypercholesterolemia by determining low density lipoprotein receptor expression in mitogen-induced proliferating lymphocytes

“…It has been known for sometime that mitogen stimulation of lymphocytes increased the LDL-R expression (26,28,49,50). However, what exactly caused the up-regulation is not clear.…”