© 1995 American Heart Association, Inc.
Articles |
Characterization of LDL and VLDL Binding Sites on Human Basophils and Mast Cells
From the Department of Nuclear Medicine (I.V., S.-R.L., Q.Y., M.L.), Department of Internal Medicine I, Division of Hematology and Hemostaseology (W.R.S., P.V.), Department of Clinical Pharmacology (I.V.), Department of Physiology (E.K.), and Institute for Molecular Biology (J.N., W.S.), University of Vienna, and the Department of Radiochemistry (P.A.), Research Center Seibersdorf, Austria.
Correspondence to Irene Virgolini, MD, Department of Nuclear Medicine, University of Vienna, AKH, Währinger Gürtel 18-20, Vienna, Austria.
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Abstract |
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Abstract Recent data suggest that basophils and mast cells play a potential role in the processing and accumulation of plasma lipoproteins. This study investigated the interactions of 111In-low-density lipoprotein (LDL), 111In-acetyl-LDL, and 111In-very-low-density lipoprotein (VLDL) with purified primary human blood basophils, immortalized human basophils (KU812 cell line), and a human mast cell line, HMC-1. Binding sites for 111In-LDL resolved into curvilinear Scatchard plots indicating two classes of specific binding sites on primary basophils (Bmax1, 7404 sites/cell; Kd1, 1.9 nmol/L; Bmax2, 39 611 sites/cell; Kd2, 29 nmol/L), on KU812 cells (Bmax1, 8290±2690 sites/cell; Kd1, 2.4±0.6 nmol/L; Bmax2, 46 470 sites/cell; Kd2, 33.4±7.8 nmol/L), and on HMC-1 cells (Bmax1, 7840±360 sites/cell; Kd1, 1.8±0.8 nmol/L; Bmax2, 61 450±9900 sites/cell; Kd2, 28.4±9.4 nmol/L). On KU812 cells, binding of 111In-LDL was displaced by apolipoprotein (apo)-E–rich high-density lipoprotein (HDL) (IC50, 14±6 nmol/L), LDL (IC50, 29±11 nmol/L), VLDL (IC50, 55±21 nmol/L), HDL2 (IC50, 420±140 nmol/L), and heparin (IC50, 67±28 nmol/L), whereas no competition was produced by HDL, HDL3, or acetyl-LDL (IC50, >1 µmol/L). Western blot analysis using the monoclonal antibody C7 confirmed the presence of the LDL receptor on human basophils and HMC-1 cells. 111In-acetyl-LDL binding sites (scavenger receptor) could be detected neither on human basophils nor on HMC-1 cells. 111In-VLDL bound to a single class of high-affinity binding sites on primary basophils (Bmax, 4320 sites/cell; Kd, 10 nmol/L), KU812 cells (Bmax, 4020±840 sites/cell; Kd, 8±3 nmol/L), and HMC-1 cells (Bmax, 6143±1866 sites/cell; Kd, 4±2 nmol/L). 111In-VLDL binding was displaced by VLDL>LDL>apoE-rich HDL but not by heparin (IC50 >1 mmol/L). In the presence of prostaglandin E1, the number of 111In-LDL receptors increased by 150% (P<.05) in the high-affinity range and by 170% (P<.01) in the low-affinity range, whereas the number of 111In-VLDL binding sites remained unchanged. VLDL, LDL, HDL, and the subclasses HDL2 and HDL3 inhibited immunological histamine release by primary normal basophils (n=3) and mast cells (n=3). Our results provide evidence for the existence of LDL and VLDL binding sites on human basophils and HMC-1 mast cells. The exact biological and pathophysiological roles of these sites remain to be elucidated.
Key Words: lipoproteins • mast cells • histamine • arteriosclerosis
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Introduction |
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Alterations in lipid uptake and metabolism leading to accumulation of lipoproteins in the vessel wall are considered to be crucial steps in the process of arteriosclerosis. Uptake and processing of lipoproteins are mediated by specific cell surface membrane receptors. Circulating low-density lipoprotein (LDL) cholesterol is cleared from the plasma by "classic" LDL receptors that are specific for apolipoprotein (apo) B and apoE.1 2 3 These LDL receptors have been identified in various tissues and cells including hepatocytes,3 fibroblasts,4 5 lymphocytes,6 and mononuclear phagocytes.7 Macrophages, in addition, express scavenger receptors that mediate the uptake of modified LDL.8 This uptake is associated with foam cell formation in early atherogenesis.9 Recently, a very-low-density lipoprotein (VLDL) receptor has been identified and cloned from a rabbit heart cDNA library.10 This receptor mediates the uptake of apoE-containing lipoproteins in various extrahepatic organs including heart, lung, and adipose tissues.10
Tissue mast cells and circulating blood basophils are multifunctional effector cells of the immune system. They are involved in inflammatory reactions as well as in the regulation of vascular events and store and release vasoactive and immunomodulating compounds.11 12 Recent data suggest that human basophils and/or mast cells and their products may also play a role in lipid metabolism and atherogenesis.13 14 For example, heparin released from stimulated mast cells induces uptake of (modified) LDL by macrophages via scavenger receptor– mediated phagocytosis.15 16 17 The direct interactions between basophils and/or mast cells and the various lipoproteins have not been studied in detail. The aim of the present study was to characterize quantitatively the lipoprotein binding sites expressed on primary human basophils, the human basophil line KU812, and the human mast cell line HMC-1.
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Methods |
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Lipoprotein Isolation and Characterization
Lipoproteins were isolated from healthy normolipemic blood donors. None of the volunteers had received any medication for at least 3 weeks before blood donation. Blood (60 mL) was collected through siliconized needles into heparin-coated vials after an overnight fast. Lipoproteins were prepared from the fresh plasma by isopycnic ultracentrifugation in a Beckman ultracentrifuge (Typ L5-75, Rotor 40.3 Ti) using potassium bromide for density adjustment as described.18 19 The following lipoproteins were isolated: VLDL (<1.006 g/mL), LDL (1.019 to 1.063 g/mL), high-density lipoprotein (HDL) (1.063 to 1.21 g/mL), HDL2 (1.063 to 1.125 g/mL), and HDL3 (1.125 to 1.21 g/mL). In selected experiments, HDL3 was fractionated into apoE-depleted HDL and apoE-enriched HDL by heparin-agarose affinity chromatography using 0.05 mol/L NaCl and 25 mmol/L MnCl2 to elute the apoE-free lipoproteins and 0.6 mol/L NaCl to elute the apoE-enriched lipoproteins, as described.20 Acetyl-LDL was prepared by treatment of LDL with acetic anhydride,20 and its formation was monitored by its enhanced mobility on electrophoresis in agarose gel at pH 8.6. The apoprotein composition of each of the lipoprotein classes was assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The apoE content of each HDL subclass was determined by radioimmunoassay (RIA) and amounted to 5.2% for apoE-enriched HDL and to <0.05% for apoE-depleted HDL.
Lipoproteins were dialyzed against phosphate-buffered saline (PBS), pH 7.4, containing 0.1 mg/mL EDTA and stored at 4°C for not longer than 1 week. The total protein content of the lipoproteins was analyzed by the method of Lowry et al.21 Lipoproteins were concentrated by ultrafiltration using Centrisart UF membranes (Sartorius).
Lipoprotein-deficient serum (LPDS) was prepared from fetal bovine serum by a single centrifugation at 100 000g for 48 hours at 10°C after density adjustment to 1.215 with solid KBr and dialyzed for 48 hours at 4°C against three changes of 50 vol each of 0.15 mol/L NaCl.
Radiolabeling
For each series of experiments, lipoproteins of one normolipemic
subject were used for labeling with 111In, as described
previously.22 Briefly, to a 0.5-mol/L
NaHCO3-containing microvial equipped with a magnetic
stirrer, the lipoprotein and cyclic diethylene-triaminepentaacetic acid
anhydride (DTPA, Sigma) were added. The reaction mixture was slowly
stirred for 1 hour and applied to a 5x40-mm Sephadex G50F column
(Pharmacia) equilibrated in metal-free acetate-buffered saline (ABS),
pH 5.5. The column was eluted with ABS and the protein fraction
collected into another microvial. To this, 100 µCi
111In-Cl3 was added with gentle mixing. After 1
hour at room temperature, the reaction mixture was applied onto a
second ABS-equilibrated Sephadex G50F column. The
111In-labeled protein fraction was collected and mixed with
1 mol/L DTPA in PBS to give 1 mL of final product solution. The amount
of free iodine was tested by trichloroacetic acid (TCA) precipitation
(10% TCA final concentration) and was not more than 3% of the total
incorporated radioactivity. The amount of radioactivity localized in
the lipid moiety was 5% to 10%, as estimated by extraction with
chloroform/methanol according to Folch et al.23
Radiochemical purity of lipoproteins was determined by (1) thin-layer chromatography (TLC) using Silica Gel Merck (SG) plates and an eluant composed of methanol:10% ammonium formate:0.5 mol/L citric acid (20:20:10; vol/vol/vol) and by (2) cellulose acetate electrophoresis with 0.05 mol/L barbital buffer, pH 8.6, containing 1 mmol/L EDTA and 1% human serum albumin at 300 V for 20 minutes.
Purification of Primary Human Basophils and Cell Culture
Primary basophils were purified to homogeneity from one chronic
myeloid leukemia (CML) patient after informed consent was given.
Basophils were purified by negative selection technique using
monoclonal antibodies (mAbs) as described previously.24 25
The mAbs used for basophil isolation were VIM13 (CD14), VIBC5 (CD24),
VIT3 (CD3), VIMD5 (CD15), VIEG4 (antiglycophorin A), VIT6 (CD1), and
VID1 (anti–HLA-DR) (Institute of Immunology, University of Vienna).
The mAbs Leu1 (CD5), Leu7 (CD57), and Leu9 (CD7) were purchased from
Becton Dickinson, BMA 022 (anti–HLA-DR) and BMA 0110 (CD2) from
Behring, and mAb E-124-2-8 (anti-IgE) from Immunotech. The mAb CLB-Ery3
(anti–blood group H) was a generous gift from Dr P.A.T. Tetteroo
(Amsterdam, The Netherlands). Mononuclear cells (MNCs) were isolated
from peripheral venous blood by gradient density centrifugation with
Ficoll (1.077 g/mL). MNCs (5x109) were incubated in RPMI
1640 medium containing 1 mg mAb VIMD5 at 4°C for 45 minutes. After
washing, cells were exposed to 50 mL of rabbit complement (Behring AG)
at 37°C for 90 minutes. Washed cells were then exposed to a mixture
of mAbs (VIT3, VIBC5, VIM13, Leu1, Leu7, Leu9, VIEG4, BMAO1110, BMAO22,
CLB-Ery3, and VIM-D5; 25 µg/108 cells for each mAb) at
4°C for 45 minutes and then to rabbit complement for another 90
minutes (37°C). After washing, cells were again layered over Ficoll
and examined for the percentage of basophils by Giemsa staining.
Purified basophils were cultured in RPMI 1640 medium containing 10%
fetal calf serum (FCS), glutamine, and antibiotics at 37°C in a
humidified CO2 atmosphere as described.25 26
CML basophils were kept in culture (10% FCS, 37°, 5%
CO2) for at least 24 hours before being analyzed.
The basophil (precursor) cell line KU812 was established from a patient suffering from CML27 and kindly provided by Dr K. Kishi, Niigata University, Japan. The mast cell line HMC-1 was kindly provided by Dr J.H. Butterfield (Mayo Clinic, Rochester, Minn).28 KU812 cells were cultured in RPMI 1640 medium and HMC-1 cells were cultured in IMDM medium. For long-term culture, cells were maintained in 10% FCS at 37°C and 5% CO2. For the assessment of lipoprotein receptors, basophils and mast cells were grown in medium in the presence of 10% FCS or 10% LPDS for 24 hours. Cells were harvested by centrifugation and washed in assay buffer containing 50 mmol/L Tris HCl and 5 mmol/L MgCl2. For control studies, human fibroblasts were cultured as described29 and preincubated in medium supplemented with 10% LPDS for 24 hours.
Binding of Lipoproteins to Basophils and Mast Cells
To investigate ligand binding to basophils and mast cells,
direct binding experiments were carried out essentially as reported
earlier.18 19 22 All incubations were done in duplicate.
In initial experiments, the time course of association and dissociation
as well as the temperature dependency of lipoprotein binding were
studied as described.18 19
Saturation Experiments
In saturation experiments, the cells (5x105
cells in each tube) were incubated with increasing concentrations of
111In-lipoprotein (0.1 to 70 µg protein/mL) in the
absence (total binding) and the presence of the same unlabeled
lipoprotein (100 µg protein/mL, nonspecific binding). Specific
binding was determined as the difference of total and nonspecific
binding. In typical experiments, nonspecific binding (determined in the
presence of an excess of unlabeled LDL) amounted to less than 10% of
total binding in the high-affinity ligand range.
Competition Experiments
In competition experiments, the cells were incubated at
room temperature for 45 minutes with 5 µg protein/mL
111In-LDL (925 cpm/ng protein) in the absence (total
binding) and the presence of increasing concentrations (0.1 to 500 µg
protein/mL) of unlabeled lipoproteins (VLDL, LDL, HDL,
HDL2, HDL3, apoE-rich HDL,
apoE-depleted HDL). To evaluate the specificity of
111In-lipoprotein binding onto basophils and mast cells,
several unrelated proteins (albumin, fibrinogen, myoglobin, ovalbumin,
soybean trypsin inhibitor, heparin, and transferrin) were also tested
for their capacity to displace bound 111In-lipoprotein
using a 25- to 50-fold molar excess of each unlabeled competitor
protein.
After incubation, the tubes were rapidly centrifuged (1500g, 10 minutes, 4°C) to separate free from membrane-bound radioligand. After twice washing, the pellet was counted in a gamma counter for 1 minute. In the absence of cells, the application of 30 µg protein of 111In-lipoprotein resulted in the recovery of less than 1 µg protein of 111In-lipoprotein in the tip of the tube after centrifugation (<4%). This amount was identical for incubations of total and nonspecific binding.
To determine the response of the basophil lipoprotein receptors, cells were preincubated for 30 minutes with either prostaglandin (PG) E1 (Advanced Magnetics) at a concentration of 10-7 to 10-5 mol/L or with recombinant human interleukin-3 (rhIL-3; 1 to 1000 U/mL) in RPMI 1640 medium at 37°C. Thereafter, cells were washed in 50 mmol/L Tris HCl buffer, pH 7.5, and analyzed for lipoprotein receptor expression. RhIL-3, expressed in Escherichia coli, was provided by the Genetics Institute (Cambridge, Mass). Purified rhIL-3 had a specific activity of 4.6x106 U/mg protein as determined by a myeloblast bioassay described by Griffin et al.30
Western Blot Analysis
For Western blotting experiments, cell pellets
(5x107 cells) were solubilized by addition of 200 mL of
solubilization buffer (200 mmol/L Tris/maleate, 2 mmol/L
CaCl2, 0.5 mmol/L PMSF, 2.5 mmol/L leupeptide, 1.4%
Triton X-100, pH 6.5). The cell suspension was kept on ice for 15
minutes, the extracts were centrifuged at 30 000g (Beckman
TLX-Optima) for 40 minutes at 4°C, and the pellets were discarded.
The supernatants were subjected to one-dimensional SDS-PAGE. Gradient
gel electrophoresis was carried out according to Laemmli31
containing 4.5% to 18% polyacrylamide using a Minisystem (BioRad) at
200 V for 1 hour. Gels were calibrated with the following molecular
standards: myosin 200 kD, ß-galactosidase 116 kD, phosphorylase b 97
kD, bovine serum albumin 68 kD, ovalbumin 43 kD, carbonic anhydrase 29
kD, ß-lactoglobulin 18 kD, and lysozyme 14 kD. Electrophoretic
transfer of the separated proteins to nitrocellulose was performed as
described previously.32 Western blotting was carried out
using PBS supplemented with 5% nonfat dry milk for blocking. The
monoclonal antibody C732 against the human LDL receptor
was used at a concentration of 6 µg/mL incubation buffer. Bands were
visualized by chemiluminescence (ECL-system, Amersham) according to the
manufacturer's instructions, and membranes were exposed to
ECL-Hyperfilm (Amersham) for 1 to 10 minutes.
Uptake and Degradation Studies
KU812 or HMC-1 cells (105 cells/well) were
preincubated (24 hours) in the medium supplemented with 5% LPDS. After
addition of increasing concentrations (1 to 100 µg protein/mL) of
111In-LDL or 111In-VLDL, the cells were further
incubated at 4° and 37°C, respectively, for up to 8 hours. Cells
were then washed three times with ice-cold PBS and solubilized in 1N
NaOH. Cell-associated radioactivity was measured in a gamma counter and
is expressed as nanograms of (lipoprotein) protein bound per milligram
of total cell protein. The extent of proteolytic degradation of
111In-LDL/111In-VLDL was measured by assaying
the amount of the 123I-labeled TCA-soluble (final
concentration, 10% vol/vol) radioactivity formed by the cells and
excreted into the culture medium. Nonspecific association and
degradation was determined in the presence of an excess of unlabeled
lipoprotein (100 µg protein of LDL and VLDL, respectively).
Histamine Release Assay
Histamine release from blood basophils of nonallergic
individuals (n=3) was determined as described
previously.33 Briefly, peripheral blood cells were
fractionated by incubation in 1.1% dextran 70 and 0.008 mmol/L EDTA
for 90 minutes at 22°C. Cells of the granulocyte-rich upper layer
were then centrifuged (200g, 4°C, 8 minutes) and washed
twice in PIPES buffer (25 mmol/L PIPES, 110 mmol/L NaCl, and 5 mmol/L
KCl, pH 7.35). Granulocytes were resuspended in PIPES buffer containing
2.0 mmol/L CaCl2. Cells were adjusted to a final
concentration of 2.5x106/mL and incubated
with various concentrations of lipoprotein (VLDL, LDL, HDL,
HDL2, HDL3) in 96 multiwell
plates. Cells then were incubated with these lipoproteins for 45
minutes at 37°C and thereafter exposed to various concentrations of
anti-IgE mAB E-124-2-8 (37°C) for another 20 minutes. Thereafter,
cells were centrifuged (200g, 4°C) and the cell-free
supernatants recovered.
Histamine release was also determined in human uterine mast cells (n=3). Tissue was obtained from patients suffering from uterus myomatosis after informed consent was given. Mast cells were isolated as described previously.24 Histamine release was carried out essentially as described for blood basophils.
Histamine was measured by a commercially available RIA (Immunotech).33 Total histamine in cell suspensions was quantified after cell lysis in distilled water. Extracellular histamine was measured in cell-free supernatants after centrifugation at 4°C.
Analysis
Binding data were calculated according to
Scatchard.34 Values are presented as mean±SD if not
otherwise indicated. Statistical analysis was done by standard
statistical tests including Student's t test, ANOVA, and
simple linear regression analysis at a confidence level of
95%.
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Results |
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Initial Binding Studies With Human Basophils and Mast Cells
In initial binding studies, the interaction of 111In-lipoproteins with human basophils or HMC-1 mast cells was assessed as a function of time and temperature. Labeled lipoproteins bound to the washed (intact) cells at 4°C, and the time course of the binding reaction gave a rapid increase of binding for approximately 5 minutes and reached an apparent equilibrium at 15 minutes. In the same experiment, after a 30-minute incubation time, displacement of 111In-lipoprotein by a 25- to 50-fold excess of unlabeled lipoprotein was achieved, indicating that 90% of the bound 111In-lipoproteins were notporated under these conditions. In all experiments, lipoprotein binding was measured at 4°C, with a 30-minute incubation time to ensure constant conditions.
Evaluation of Lipoprotein Receptor Numbers and Binding
Affinities
The capacity to saturate the human basophil and mast cell binding
sites for a variety of lipoproteins was assessed by incubating the
cells with increasing concentrations of 111In-lipoproteins
in the absence or presence of the same unlabeled lipoprotein. Specific
binding was defined by subtraction of the binding observed in the
presence from that observed in the absence of excess unlabeled
lipoprotein.
Binding of 111In-LDL to Human Basophils and Human Mast
Cells
Specific binding of 111In-LDL to primary CML basophils
(Fig 1
) was saturable and indicated a curvilinear
Scatchard plot representing two binding classes, one high-affinity
binding class capable of binding 615 ng protein
111In-LDL/108 cells (ie, 7404 sites per cell;
Kd, 1.9 nmol/L) and one low-affinity
binding class capable of binding 3290 ng protein
111In-LDL/108 cells (ie, 39 611 sites per
cell; Kd, 29 nmol/L).
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) was calculated by subtracting the
amount of 111In-LDL bound in the presence of an excess of
unlabeled LDL (100 µg protein/mL; nonspecific binding) from that
bound in its absence (
, total binding). Scatchard analysis
indicated two classes of saturable binding sites: a high-affinity
component that bound 615 ng protein of
111In-LDL/108 basophils (ie, 7404 sites per
cell) and a low-affinity binding component capable of binding 3290 ng
protein of 111In-LDL/108 basophils (ie, 39 611
sites per cell) specifically. The corresponding dissociation constants
were 0.95 (ie, 1.9 nmol/L) and 14.5 (ie, 29 nmol/L) µg protein/mL.
Two binding classes for 111In-LDL were also found on KU812
basophils as well as on HMC-1 mast cells (Table 1).
Specific binding of 111In-LDL to KU812 cells represented
two binding classes, one high-affinity binding class capable of binding
689±224 ng protein of 111In-LDL/108 cells
(Kd, 2.4±0.6 nmol/L) and one
low-affinity binding class capable of binding 3862±765 ng protein of
111In-LDL/108 cells
(Kd, 33.4±7.8 nmol/L). On HMC-1 cells,
similar Kd values and numbers of binding sites
were found (Table 1). The high-affinity receptors bound 651±302 ng
111In-LDL/108 cells, and the low-affinity
receptors bound 5107±823 ng 111In-LDL/108
cells. The corresponding Kd values were 1.8±0.8
nmol/L and 28.4±9.4 nmol/L, respectively.
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Western Blot Analysis
To provide a molecular substrate for lipoprotein binding sites, we
used C7 monoclonal antibody directed against the human LDL receptor
expressed on fibroblasts.32 As shown in Fig 2, in the absence of plasma lipoproteins, HMC-1 mast
cells as well as KU812 basophils expressed the classic LDL receptor
with a molecular weight of 130 kD.
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Binding of Acetylated 111In-LDL to Human Basophils and
Mast Cells
Receptors for acetylated LDL have been detected on monocytes and
macrophages.8 9 In this study we tested whether acetylated
LDL (111In–acetyl-LDL) would bind to primary blood
basophils, KU812 cells, or HMC-1 cells. However, we were unable to
detect specific or saturable binding sites for acetyl-LDL on basophils
or HMC-1 mast cells (Fig 3).
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) no specific or saturable binding could be
detected.
Binding of 111In-VLDL to Basophils and Mast Cells
111In-VLDL bound to primary CML basophils in a
specific and saturable manner. The binding isotherm (Fig 4) resolved into a straight line on the Scatchard plot,
indicating a single class of binding sites for 111In-VLDL.
These sites had a binding capacity (Bmax) of 720 ng
111In-VLDL/108 CML basophils (ie, 4320
sites/cell); the corresponding Kd was 10
nmol/L.
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A single class of 111In-VLDL binding sites was also
observed on KU812 basophils as well as on HMC-1 mast cells (Table 2). The Bmax for KU812 cells amounted to
670±140 ng protein/108 cells
(Kd, 8±3 nmol/L) and for HMC-1 cells to
1020±310 ng protein/108 cells
(Kd, 4±2 nmol/L).
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Ligand Specificity of 111In-LDL Binding Sites
On primary human basophils 111In-LDL could be
displaced (Fig 5) by LDL (IC50, 25
nmol/L), VLDL (IC50, 46 nmol/L), and apoE-rich HDL
(IC50, 450 nmol/L) but not by modified LDL, HDL, or
apoE-depleted HDL. The competition produced by unlabeled LDL for
111In-LDL sites expressed on KU812 cells (Table 3A) amounted to 29±11
nmol/L (IC50). Whereas VLDL (IC50,
55±21 nmol/L) was a strong competitor at LDL binding sites, HDL was
about 20 times less active in replacing bound 111In-LDL
from the membrane. The HDL2 subfraction
(IC50, 420±140 nmol/L) was a somewhat better
competitor as compared with the HDL3 subfraction
(IC50 >1 mmol/L). ApoE-rich HDL was the best competitor at
111In-LDL sites, producing an IC50 value of
14±6 nmol/L. The competition produced by various lipoproteins for
binding of 111In-LDL onto HMC-1 cells indicated a similar
rank order of potency for lipoprotein binding (Table 3B).
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), 62 nmol/L for
very-low-density lipoprotein (
), >1 µmol/L for HDL (
).
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Ligand Specificity for 111In-VLDL Binding Sites
On primary CML basophils, 111In-VLDL binding to
basophils was inhibited by more than 90% at a 20-fold excess of
unlabeled VLDL (Fig 6). The IC50 value for
unlabeled VLDL amounted to 21 µg protein/mL (21 nmol/L). LDL
recognized 111In-VLDL sites with an IC50 of 25
nmol/L. Modified LDL, HDL, and the apoE-depleted HDL subfraction did
not recognize 111In-VLDL binding sites, whereas
apoE-enriched HDL produced an IC50 value of 400 µg
protein/mL (400 nmol/L).
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Competition experiments with KU812 cells (Table 3
A)
as well as HMC-1 cells (Table 3B) confirmed that
111In-VLDL binding sites were recognized by unlabeled LDL
(IC50, 45±12 and 26±8 nmol/L, respectively) but
not by modified LDL, HDL, HDL2, or
HDL3. A significant (P<.001) difference,
however, was found for apoE-rich HDL compared with apoE-depleted HDL
subfractions for both KU812 cells and HMC-1 cells. ApoE-rich HDL bound
to VLDL receptors with IC50 values of about 400 nmol/L,
indicating a 10-fold lower affinity than that observed for VLDL binding
for 111In-VLDL binding sites.
To validate specificity of lipoprotein binding to cell membranes, the capacity of unrelated proteins to inhibit basophil interaction with 111In-LDL and 111In-VLDL was assessed. Except for heparin, all other unrelated proteins investigated (albumin, fibrinogen, myoglobin, ovalbumin, soybean trypsin inhibitor, and transferrin) were unable to inhibit binding of 111In-LDL or 111In-VLDL onto KU812 cells even at a 50-fold molar excess relative to the final concentration of 111In-labeled lipoproteins. Interestingly, heparin considerably reduced 111In-LDL binding (IC50, 67±28 nmol/L), whereas it had only minimal effect on 111In-VLDL binding to basophils and mast cells.
Effect of PGE1 on Expression of
111In-LDL Binding Sites
As reported previously for the classic LDL receptor expressed on
hepatocytes,35 PGE1 dose-dependently enhanced
binding of 111In-LDL onto KU812 cells (Table 1), whereas no
effect on binding of 111In-VLDL to KU812 cells was observed
(Table 2). In the same set of experiments, the IC50 values
also decreased, which indicated that PGE1 might render
basophils more sensitive for LDL binding. No significant effect of
rhIL-3 on the binding of 111In-LDL and
111In-VLDL onto KU812 basophils was observed (Tables 1 and 2).
Uptake and Degradation Studies
Previous studies have demonstrated that binding of LDL to
its receptor leads to internalization and degradation of the
lipoprotein.5 6 8 The significance of the interaction
between LDL and VLDL, in terms of cellular metabolism of LDL/VLDL by
KU812 and HMC-1 cells, was tested by measuring the uptake (Table 4A) and degradation
(Table 4B) of the lipoproteins. The cellular content of
111In-LDL and 111In-VLDL rose and reached a
steady state plateau at about 2 hours and remained constant over 8
hours, while the rate of degradation of labeled lipoproteins continued
to increase in a linear fashion. By 8 hours, approximately 5 times as
much lipoprotein had been degraded as was present in the cells in
the steady state. In the presence of unlabeled LDL and VLDL, the uptake
of 111In-LDL and 111In-VLDL and the rates of
degradation were reduced proportionally.
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Effects of Lipoproteins on Histamine Release by Human Basophils and
Mast Cells
To confirm functional activity of lipoprotein basophil/mast
cell interactions, histamine release experiments were performed on
blood basophils and tissue mast cells. The lipoproteins investigated
(LDL, VLDL, HDL, HDL2, and HDL3)
had no effect on spontaneous histamine release (less than 10% of total
histamine) over the dose range tested (1 to 100 mg protein/mL).
However, remarkable inhibition (60% to 70% of anti–IgE-induced
histamine release) was found in three basophil donors as well as in
human mast cells (Table 5). Fig 7 shows
the inhibitory effect of the lipoproteins on human basophil
releasability; Fig 8 shows a dose-dependent inhibition
of histamine secretion in basophils induced by LDL and VLDL. Inhibitory
effects were observed between 0.1 and 100 µg protein per mL.
According to previous findings,36 PGE1 reduced
the capacity of the basophils to release histamine upon induction with
anti-IgE, whereas rhIL-3 was found to upregulate the capacity of the
basophils to release histamine and stem cell factor (SCF) was found to
upregulate IgE-dependent histamine release from human mast cells (not
shown).
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). Basophils were obtained
from a nonallergic donor and exposed to various concentrations of
lipoproteins as indicated. Histamine release is expressed as
percentages of total histamine. Abbreviations as in Fig 5 |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Uptake and metabolism of plasma lipoproteins are mediated through specific cellular receptors. The purpose of this study was to characterize lipoprotein receptors expressed on human basophils and human mast cells. On primary human blood basophils, on the human basophil cell line KU812 as well as on the human mast cell line HMC-1 we were able to detect LDL and VLDL binding sites recognizing both apoB and apoE. In contrast, the scavenger receptor, likewise expressed on monocyte-macrophages, could not be detected on human basophils or mast cells.
The LDL receptor (apoB, E-receptor) is a major site of cholesterol uptake and is ubiquitously distributed in various tissues and cells. In the present study, we were able to identify the LDL receptor on basophil granulocytes and HMC-1 mast cells. 111In-LDL bound to cell surface membrane receptors with two different binding affinities, suggesting a high- and low-affinity binding class. These LDL binding sites were also recognized by VLDL and apoE-rich HDL but not by acetylated LDL or apoE-depleted HDL. Together, our data suggest the presence of a classic apoB, E-receptor likewise expressed on hepatocytes or fibroblasts.1 2 3 4 5 6 7 32 These binding sites facilitate the uptake and degradation of LDL. The presence of an apoB, E-receptor in mast cells (HMC-1) and basophils (KU812) was also confirmed by using the specific monoclonal antibody C7.32 The reason for the discrepancies between binding and Western blot experiments concerning the number of LDL receptors on KU812 versus HMC-1 cells cannot be explained readily. One explanation would be that both cells express equal amounts of surface LDL binding sites but differ in expression of cytoplasmic C7 domains. Alternatively, KU812 cells express surface LDL receptors that lack C7 epitopes.
Recent data suggest the existence of specific VLDL binding sites in extrahepatic tissues.10 In this study, 111In-VLDL bound to human basophils and mast cells and binding of VLDL to the LDL receptor were demonstrable. Although a molecular substrate for a specific VLDL receptor could not be substantiated, several data point to the presence of a unique VLDL binding site on human basophils and HMC-1 cells. First, in contrast to the LDL binding sites, Scatchard transformation of VLDL binding onto human basophils and mast cells resolved into a single class of high-affinity binding sites. Furthermore, heparin was able to inhibit LDL binding to basophils and/or mast cells but did not influence binding of VLDL. Studies are under way to clarify whether indeed human basophils and mast cells express specific VLDL receptors.
Mast cells and macrophages share a number of antigens and a common hemopoietic progenitor. In this study, we tested whether the mast cell line HMC-1 or human basophils would express or lack the scavenger receptor previously reported to be specific for monocytes and/or macrophages.8 9 However, we were unable to detect significant amounts of acetyl-LDL binding sites on basophils or mast cells. We also found that unlike mononuclear phagocytes, mast cells and basophils lack LRP/CD91 (manuscript submitted). These findings favor the concept that mast cells and macrophages represent two different cell lineages within the hemopoietic system.37
Previous studies have shown that human mast cells, like monocyte-macrophages, are present in the intima of the vessel wall.13 A significant functional role for mast cells (and basophils) in lipoprotein metabolism and during the process of atherogenesis has recently been suggested. For example, mast cell proteases were found to degrade LDL.16 Other studies suggest that heparin (a mast cell–specific compound) induces LDL uptake by macrophages via the scavenger receptor.17 Moreover, histamine (produced by mast cells and basophils) was described as a potential mediator of coronary atherosclerosis,38 of platelet aggregation,39 and of transendothelial lipid transport.40 In this study, IgE-mediated secretion of histamine from mast cells and basophils was inhibited by addition of lipoproteins. As histamine has been reported to be atherogenic,38 the deactivating effect of lipoproteins on human basophils and mast cells could represent a feedback mechanism in inflammation-related atherogenesis.
Expression and synthesis of lipoprotein receptors are controlled by various factors. In this study, cholesterol depletion was associated with increased expression of lipoprotein receptors on basophils and mast cells. An additional increase of LDL binding sites was noted when cells were exposed to PGE1. This is consistent with findings obtained for hepatocytes35 and fibroblasts2 3 and with the observation that basophils express functionally active PGE1 binding sites.36
So far, the critical cells involved in atherogenesis were thought to be endothelial cells, monocyte-macrophages, smooth muscle cells, and platelets. Recent data suggest that basophils and mast cells produce mediators potentially involved in the process of atherogenesis. Moreover, studies by Kokkonen and Kovanen15 16 suggest that mast cells may actively participate in foam cell formation. We provide evidence that plasma lipoproteins can interact with human blood basophils and mast cells and may trigger their functions via cellular binding sites. These findings support the novel concept that basophils and mast cells may play a more important role in atherogenesis than has so far been assumed.
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Acknowledgments |
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This study was supported in part by the Jubiläumsfonds of the Austrian National Bank (projects No. 3778 and No. 4560), by the Foundation of the Mayor of the City of Vienna, and by the Fonds "zur Förderung der wissenschaftlichen Forschung in Österreich" (grants No. P-9359 and No. P-10427). We wish to thank Eva Spanblöchl for skillful technical assistance.
Received July 25, 1994; accepted October 17, 1994.
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Brown MS, Goldstein JL. Receptor-mediated control of cholesterol metabolism: study of human mutants has disclosed how cells regulate a substance that is both vital and lethal. Science.. 1986;232:34-47.
2.
Goldstein JL, Brown MS. Regulation of low-density lipoprotein
receptors: implications for pathogenesis and therapy of
hypercholesterolemia and atherosclerosis.
Circulation. 1987;76:504-507.
3.
Kovanen PT, Brown MS, Basu SK. Saturation and
suppression of hepatic lipoprotein receptors: a mechanism for
hypercholesterolemia of cholesterol fed rabbits. Proc Natl Acad
Sci U S A.. 1981;78:1396-1400.
4. Oram JF, Brinton EA, Bierman EL. Regulation of high density lipoprotein receptor activity in cultured human skin fibroblasts and human arterial smooth muscle cells. J Clin Invest.. 1983;72:1611-1622.
5.
Basu SK, Goldstein JL, Anderson GW, Brown SM. Degradation of
cationized low density lipoprotein and regulation of cholesterol
metabolism in homozygous familial hypercholesterolemia fibroblasts.
Proc Natl Acad Sci U S A.. 1976;73:3178-3182.
6. Ho YK, Brown MS, Bilheimer DW, Goldstein JL. Regulation of low density lipoprotein-receptor activity in freshly isolated human lymphocytes. J Biol Chem.. 1986;58:1465-1471.
7. Goldstein L, Brown MS. The low-density lipoprotein pathway and its relation to atherosclerosis. Ann Rev Biochem.. 1987;46:897-930.
8.
Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on
macrophages that mediates uptake and degradation of acetylated low
density lipoprotein, producing massive cholesterol deposition.
Proc Natl Acad Sci U S A.. 1979;76:333-337.
9. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med.. 1989;320:915-924. [Medline] [Order article via Infotrieve]
10.
Takahashi S, Kawarabayasi Y, Nakai T, Sakai J, Yamamoto T.
Rabbit very low density lipoprotein receptor: a low density lipoprotein
receptor-like protein with distinct ligand specificity. Proc Natl
Acad Sci U S A.. 1992;89:9252-9256.
11. Galli SJ. Biology of disease: new insights into "the riddle of the mast cells": microenvironmental regulation of mast cell development and phenotypic heterogeneity. Lab Invest.. 1990;62:5-33. [Medline] [Order article via Infotrieve]
12. Serafin WE, Austin KF. Mediators of immediate hypersensitivity reactions. N Engl J Med.. 1987;317:30-34. [Medline] [Order article via Infotrieve]
13. Tas J, Greenen LHM. Microspectrophotometric detection of heparin in mast cells and basophilic granulocytes stained metachromatically with toluidine blue O. Histochem J.. 1975;7:231-248. [Medline] [Order article via Infotrieve]
14. Metcalfe DD, Lewis RA, Silbert JE, Rosenberg RD, Wasserman SI, Austen FK. Isolation and characterization of heparin from human lung. J Clin Invest.. 1979;64:1537-1543.
15.
Kokkonen JO, Kovanen PT. Low density lipoprotein degradation
by rat mast cells: demonstration of extracellular proteolysis caused by
mast cell granules. J Biol Chem.. 1985;260:14756-14763.
16.
Kokkonen JO, Kovanen PT. Proteolytic enzymes of mast cell
granules degrade low density lipoproteins and promote their
granule-mediated uptake by macrophages. J Biol Chem.. 1989;264:10749-10755.
17. Lindstedt KA, Kokkonen JO, Kovanen PT. Soluble heparin proteoglycans released from stimulated mast cells induce uptake of low density lipoproteins by macrophages via scavenger receptor-mediated phagocytosis. J Lipid Res.. 1992;33:65-75. [Abstract]
18.
Virgolini I, Li, SR, Yang Q, Koller E, Banyai M, Angelberger
P, Sinzinger H. Binding of 111In-labeled low density
lipoprotein to platelets of normolipemic volunteers and patients with
heterozygous familial hypercholesterolemia (FH).
Arteriosclerosis.. 1993;13:536-547.
19.
Virgolini I, Li SR, Yang Q, Koller E, Banyai M, Angelberger P,
Sinzinger H. Binding of 111In-labeled high density
lipoprotein to platelets of normolipemic volunteers and patients with
heterozygous familial hypercholesterolemia (FH).
Arteriosclerosis.. 1992;12:849-861.
20. Roll SC, Weisgraber KH, Mahley RW. Isolation and characterization of apolipoprotein E. Methods Enzymol.. 1986;128:273-279. [Medline] [Order article via Infotrieve]
21.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
measurement with the Folin phenol reagent. J Biol Chem.. 1951;193:265-275.
22.
Virgolini I, Angelberger P, Li SR, Koller F, Koller E, Pidlich
J, Lupattelli G, Sinzinger H. 111In-labeled low density
lipoprotein (LDL) binds with higher affinity to the human liver as
compared to 123I-labeled LDL. J Nucl Med.. 1992;32:2132-2138.
23.
Folch J, Lees M, Stanley GHS. A simple method for the
isolation and purification of total lipides from animal tissues.
J Biol Chem.. 1957;226:497-509.
24. Valent P, Majdic O, Maurer D, Bodger M, Muhm M, Bettelheim P. Further characterization of surface membrane structures expressed on human basophils and mast cells. Int Arch Allergy Appl Immunol.. 1990;91:198-203. [Medline] [Order article via Infotrieve]
25. Valent P, Besemer J, Kishi K, Kaltenbrunner R, Kuhn B, Maurer D, Lechner K, Bettelheim P. IL-3 promotes basophilic differentiation of KU812 cells through high affinity binding sites. J Immunol.. 1990;145:1885-1889. [Abstract]
26. Stain C, Stockinger H, Scharf M, Jäger U, Gössinger H, Lechner K, Bettelheim P. Human blood basophils display a unique phenotype including activation linked membrane structures. Blood. 1987;70: 1872-1879.
27. Kishi K. A new leukemic cell line with Philadelphia chromosome characterized as basophil precursors. Leuk Res.. 1985;9:381-387. [Medline] [Order article via Infotrieve]
28. Butterfield JH, Weiler D, Dewald G, Gleich GJ. Establishment of an immature mast cell line from a patient with mast cell leukemia. Leuk Res.. 1988;12:345-355. [Medline] [Order article via Infotrieve]
29.
Hayashi K, Nimpf J, Schneider WJ. Chicken oocytes and
fibroblasts express different apolipoprotein-B-specific receptors.
J Biol Chem.. 1989;264:3131-3139.
30.
Griffin JD, Sullivan R, Beveridge RP, Garcon P, Schlossmann
SF. Induction of proliferation of purified human myeloid progenitor
cells: a rapid assay for granulocyte colony stimulating factors.
Blood.. 1984;63:904-911.
31. Laemmli UK. Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature.. 1970;227:680-685. [Medline] [Order article via Infotrieve]
32.
Beisiegel U, Schneider WJ, Goldstein JL, Anderson RGW, Brown
MS. Monoclonal antibodies to the LDL receptors as probes for study of
receptor-mediated endocytosis and the genetics of familial
hypercholesterolemia. J Biol Chem.. 1981;256:11923-11931.
33.
Valent P, Besemer J, Muhm M, Lechner K, Bettelheim P.
Interleukin 3 activates human blood basophils via high-affinity binding
sites. Proc Natl Acad Sci U S A.. 1989;86:5542-5546.
34. Scatchard G. The attractions of proteins for small molecules and ions. Ann N Y Acad Sci.. 1949;51:660-672.
35. Virgolini I, Li SR, Lupattelli G, Pidlich J, Banyai M, Angelberger P, Sinzinger H. Effect of prostaglandin E1 on low density lipoprotein apo-B, E-receptor binding. Prostaglandins.. 1991;42:81-93. [Medline] [Order article via Infotrieve]
36.
Virgolini I, Li SR, Sillaber C, Majdic O, Sinzinger H, Lechner
K, Bettelheim P, Valent P. Characterization of prostaglandin (PG)
binding sites expressed on human basophils. J Biol Chem.. 1992;267:12700-12708.
37. Agis H, Willheim M, Sperr WR, Wilfing A, Krömer E, Kabrna E, Spanblöchl E, Strobl H, Geissler K, Spittler A, et al. Monocytes do not make mast cells when cultured in the presence of SCF. J Immunol.. 1993;151:4221-4227. [Abstract]
38. Forman MB, Oates JA, Robertson D, Robertson RM, Roberts LJ, Virmani R. N Engl J Med.. 1985;313:1138-1141. [Medline] [Order article via Infotrieve]
39.
Kalsner S, Richards R. Coronary arteries of cardiac patients
are hyperreactive and contain stores of amines: a mechanism for
coronary spasm. Science.. 1984;223:1435-1437.
40. Gonen B, O'Donnel P, Ost TJ, Quinn TJ, Schulman ES. Very low density lipoprotein (VLDL) triggers the release of histamine from human basophils. Biochim Biophys Acta.. 1987;917:418-424.[Medline] [Order article via Infotrieve]
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