Analysis of fluorescent low density lipoprotein uptake by lymphocytes, paradoxical increase in the elderly

Traill, Karine N.; Jürgens, Günther; Böck, Günther; Huber, Lukas A.; Schönitzer, D; Widhalm, Kurt; Winter, Ute; Wick, Georg

doi:10.1016/0047-6374(87)90023-6

Cited by 29 publications

(22 citation statements)

References 43 publications

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“…On the other hand, two investiga tions support our findings that rates of 125I-LDL degradation by derepressed blood lym phocytes are not dependent on the age-related rise in plasma cholesterol. Nevertheless, the following points deserve close scrutiny: (1) in the study by Cortese et al [43] the subjects were under age 55, and their plasma cholester ol concentration varied over a much wider range; (2) Bilheimer et al [20] studied control subjects and normolipidemic relatives of fa milial hypcrcholesterolemics, but included only 1 older case, even though this investiga tion has often been referred to by others as evidence that the cell metabolism of l25I-LDL does not vary with age [14,16,17], More recently, Stulnig et al [41] confirmed a preGcroniology 1997;43:232-241 vious study by Traill et al [44] on freshly col lected peripheral blood lymphocytes. The re sults showed greater activity of LDL receptors as age advanced and that these cells displayed higher levels of mRNA for the LDL receptors, without, however, significantly increasing the niRNA content of 3-hydroxy-3-mcthylglutaryl coenzyme A reductase, as would be ex pected.…”

Section: Discussionmentioning

confidence: 55%

The Age-Related Rise of Plasma Cholesterol in Humans Is Not Caused by a Cell Removal Defect of LDL Particles: ‘In vitro’ Studies in Peripheral Mononuclear Blood Cells

Neves

Carvalho²,

Passarelli³

et al. 1997

Gerontology

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In healthy subjects with a normal body mass index, total plasma cholesterol, low-density lipoprotein (LDL) cholesterol, and apoB lipoprotein levels are higher in older individuals (n = 34) than in younger subjects (n = 43). The two groups studied ranged in age from 60 to 93 years and from 17 to 30 years, respectively. The cholesterol synthesis rates of peripheral mononuclear blood cells from ¹⁴C-acetate, total and unesterified cholesterol concentrations in freshly isolated cells, and the rates of uptake of pooled donor LDL, labeled with ¹²⁵I- or ¹⁴C-cholesteryl oleoyl ether, by cells derepressed in a lipoprotein-free medium were similar in both experimental groups. Thus, the rise of LDL cholesterol with age would seem to be likely secondary to the higher rate of very-low-density lipoprotein production, as shown by other investigators.

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Section: Discussionmentioning

confidence: 55%

The Age-Related Rise of Plasma Cholesterol in Humans Is Not Caused by a Cell Removal Defect of LDL Particles: ‘In vitro’ Studies in Peripheral Mononuclear Blood Cells

Neves

Carvalho²,

Passarelli³

et al. 1997

Gerontology

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“…proportional to the lymphocyte's cholesterol require ment [1][2][3][4], Thus, LDL receptor expression is very low on resting T lymphocytes freshly isolated from the pe ripheral blood, higher on resting T lymphocytes 'starved' of cholesterol by incubation for 1-4 days in serum-free medium, and highest on actively dividing T cell lines [2]. Expression of high-affinity receptors on freshly isolated resting cells from young healthy subjects correlates negatively with serum cholesterol levels [5][6][7] and positively with polyunsaturated fatty acid levels [8]; this may not be the case, however, in el derly subjects whose freshly isolated T cells paradoxi cally display an elevated receptor activity despite ele vated serum cholesterol levels [9]. In fact, the role of high-affinity LDL receptors in delivering cholesterol to circulating lymphocytes, which only show a low degree of membrane turnover, is questionable [4], Studies with cells from LDL receptor-deficient patients suffering from homozygous familial hyper cholesterolemia (FH) have shown that endogenous cholesterol synthesis is elevated in vitro to maintain cholesterol levels, but this does not occur in vivo be cause cholesterol can be obtained via plasma LDL through mechanisms other than the classical LDL re ceptor pathway [3].…”

Section: Introductionmentioning

confidence: 72%

“…Aliquots werp frozen, and thawed on the day of assay [9]; these suspensions are re ferred to as 'freshly isolated resting PBL'.…”

Section: Isolation O F Human Peripheral Blood Lymphocytesmentioning

confidence: 99%

“…Anti-coagulant-treated blood was diluted 1:2 in RPMI (Seromed, Berlin, FRG) and peripheral blood lymphocytes (PBL) were isolated by density gradient centrifugation over Lympho-Paque (density 1.086 g /l, Nyegaard & Co., Oslo, Norway) as described elsewhere [9,14], Monocytes were depleted by plastic adherence in tissue culture flasks [9,14] when lipoprotein uptake was to be mea sured; the resulting lymphocyte suspensions contained more than 90% T cells, as determined by fluorescence staining. Aliquots werp frozen, and thawed on the day of assay [9]; these suspensions are re ferred to as 'freshly isolated resting PBL'.…”

Section: Isolation O F Human Peripheral Blood Lymphocytesmentioning

confidence: 99%

“…Flow cytometric analysis with dioctadecylindocarbocyanine-labeled LDL [11] offers many advantages over conventional radiometric stud ies, such as measuring LDL uptake in individual living cells, sorting and functional analysis of the labeled cells, phenotypic double-labeling studies with monoclonal antibodies, and the requirement of only low cell numbers even for elaborate binding studies involving very low receptor activity, and thus weak fluorescence intensity [9,12,13]. Recently, we re ported that double reciprocal transformation (Lineweaver-Burke plots) of the specific binding curves obtained with a fluorescence-activated cell sorter (FACS) allows for a simple and accurate assessment of the dissociation constant [14].…”

Section: Introductionmentioning

confidence: 99%

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

Huber

Böck²,

Jürgens³

et al. 1990

Int Arch Allergy Immunol

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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.

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Flow cytometric assessment of LDL receptor activity in peripheral blood mononuclear cells compared to gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia

et al. 1999

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Background: Studies indicate that human peripheral blood mononuclear cells mirror low‐density lipoprotein (LDL) receptor activity of other cells in the body. To measure LDL receptor activity in patients with heterozygous familial hypercholesterolemia (FH), we prepared peripheral blood mononuclear cells from individuals with molecularly verified LDL receptor defective (Trp66‐Gly mutation, n = 18) or receptor negative (Trp23‐stop mutation, n = 17) heterozygous FH and from healthy individuals (n = 24). Methods: The cells were stimulated to express maximum LDL receptor by preincubation in lipoprotein‐free medium. They were then incubated at 4° or 37°C with fluorescently conjugated LDL (DiI‐LDL). T‐lymphocytes and monocytes were identified by fluorescently conjugated monoclonal antibodies. DiI‐LDL bound (at 4°C) or internalized (at 37°C) by the cells was measured using flow cytometry. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of our functional assay with the DNA diagnosis. Results: The diagnostic accuracy did not allow our assay to be used for diagnosis of individual cases of heterozygous FH. Conclusions: We suggest that our two‐color fluorescence flow cytometry assay can be used to characterize functionally gene mutations causing LDL receptor dysfunction in patients with heterozygous FH. Cytometry 36:52–59, 1999. © 1999 Wiley‐Liss, Inc.

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Analysis of fluorescent low density lipoprotein uptake by lymphocytes, paradoxical increase in the elderly

Cited by 29 publications

References 43 publications

The Age-Related Rise of Plasma Cholesterol in Humans Is Not Caused by a Cell Removal Defect of LDL Particles: ‘In vitro’ Studies in Peripheral Mononuclear Blood Cells

The Age-Related Rise of Plasma Cholesterol in Humans Is Not Caused by a Cell Removal Defect of LDL Particles: ‘In vitro’ Studies in Peripheral Mononuclear Blood Cells

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

Flow cytometric assessment of LDL receptor activity in peripheral blood mononuclear cells compared to gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia

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