Increased expression of high-affinity low-density lipoprotein receptors on human T-blasts.
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 micrograms/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 microgram 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 (Kd) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Huber
L A
LA
Institute for General and Experimental Pathology, University of Innsbruck Medical School, Austria.
Böck
G
G
Jürgens
G
G
Traill
K N
KN
Schönitzer
D
D
Wick
G
G
D000328:Adult; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D008213:Lymphocyte Activation; D008297:Male; D011973:Receptors, LDL / Q000032:analysis*; D013601:T-Lymphocytes / Q000737:chemistry*
D008077:Lipoproteins, LDL; D011973:Receptors, LDL