Characterization of LDL and VLDL Binding Sites on Human Basophils and Mast Cells
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.
- Received July 25, 1994.
- Accepted October 17, 1994.
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.
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.
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 5×40-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 (5×109) 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
In saturation experiments, the cells (5×105 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.
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 4.6×106 U/mg protein as determined by a myeloblast bioassay described by Griffin et al.30
Western Blot Analysis
For Western blotting experiments, cell pellets (5×107 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, β-galactosidase 116 kD, phosphorylase b 97 kD, bovine serum albumin 68 kD, ovalbumin 43 kD, carbonic anhydrase 29 kD, β-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 2.5×106/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.
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%.
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).
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.
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.
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⇓).
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.
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).
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 3⇓⇓a) 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 3⇓⇓b).
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).
Competition experiments with KU812 cells (Table 3⇑⇑a) as well as HMC-1 cells (Table 3⇑⇑b) 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 4⇓⇓a) and degradation (Table 4⇓⇓b) 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.
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).
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.
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.
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