Human blood-brain barrier transferrin receptor

Human

Blood-Brain William

Barrier

M. Pardridge,

Transferrin

Jody Eisenberg,

Receptor

and Jing Yang

The kinetics of binding and endocytosis of ‘261-human holotransferrin by isolated human brain capillaries was examined using this system as a model of the human blood-brain barrier (BBB). Both binding and endocytosis of the peptide by human brain capillaries was temperature-dependent and the binding was saturated by holotransferrin, but not by insulin, somatostatin, or vasopressin. Scatchard analysis of the binding reaction revealed a dissociation constant of 448 + 110 ng/mL (5.6 f 1.4 nmol/L) and a maximal binding constant (If,) of 8.0 f 1.5 ng/mg protein. Thus, the affinity and capacity of the BBB transferrin receptor is within the same order of magnitude as the affinity and capacity of the BBB receptors for insulin, insulinlike growth factor-l, or insulinlike growth factor-II. The human brain capillary transferrin receptor was also detected with a mouse monoclonal antibody to the receptor using the avidinfbiotinlperoxidase technique. In conclusion, these studies characterize the human BBB transferrin receptor and support the hypothesis that this receptor acts as a transport system which mediates the transcytosis of transferrin-bound iron through the brain capillary endothelial cell in man. @ 7997 by Grune & Stratton, Inc.

T

is an 80 K glycoprotein that is the principal iron transport protein in the circulation.’ Transferrin is also a protein that is enriched in cerebrospinal fluid (CSF).’ The high concentration of transferrin in CSF is consistent with either de novo synthesis of the peptide, which has been shown to occur in brain,’ and/or transport of the peptide through the choroid plexus, ie, the blood-CSF barrier, or through the brain capillary wall, ie, the blood-brain barrier (BBB)! Since the brain needs iron to sustain intermediary metabolism and since iron circulates essentially only bound to transferrin, it is probable that brain iron originates from blood via receptor-mediated transport of circulating transferrin through the BBB. The initial evidence for this hypothesis came from the observation that a monoclonal antibody to the human transferrin receptor selectively binds human and rat brain capillaries, whereas the endothelia of other human tissues do not measurably bind the antibody to the transferrin receptor.’ The idea that circulating transferrin undergoes receptor-mediated transport through the BBB parallels recent studies supporting the hypothesis that receptor-mediated transport of other circulating peptides, such as insulin6 or insulinlike growth factors (IGFs),’ through the BBB occurs. Evidence for receptor-mediated endocytosis of insulin or insulin-like growth factors at the human BBB has been reported recently using isolated human brain capillaries as a model system for the human BBB.@ The present studies also employ isolated human brain capillaries to characterize the human brain capillary transferrin receptor.

From the Department of Medicine, UCLA School of Medicine, Los Angeles. Supported by NIH grant AM-25744. Presented at the Western Section American Federation of Clinical Research Meetings (Carmel, CA, February 1986) and the American Society of Clinical Investigation Annual Meeting (Washington, DC, May 1986). Address reprint requests to William M. Pardridge. MD, Department of Medicine, Division of Endocrinology, UCLA School of Medicine. Los Angeles, CA 90024. o I987 by Grune & Stratton, Inc. 0026-0495/87/3609/0013$03.00/0

a92

MATERIALS AND METHODS

RANSFERRIN

Autopsy

Data

Fresh brains (generally occipital lobes) were obtained from seven autopsies over a six-month period, and cortex was removed. The patient ages ranged from 29 to 71 years (4 women, 3 men) and final diagnoses included: cardiopulmonary arrest, motorcycle accident, suicide, breast cancer, and myocardial infarction. No patients had neurologic disease. The brains were obtained from the pathologist between 21 and 37 hours after death.

Microvessel

Isolation

Capillaries were isolated from human occipital cortex with a mechanical homogenization technique, as described previously.9 The final preparation consisted of pure microvessels free of adjoining brain material and photomicrographs of the human brain capillary have been published previously. ‘XI The final capillary pellet was suspended in 280 mmol/L sucrose, 0.02 mol/L Tris buffer (pH 7.4), and 2 mmol/L dithiothreitol, and was cryopreserved at -70°C in liquid nitrogen. Approximately 50% yield of the capillaries was recovered from the cryopreservation procedure, and the activity of the brain capillary insulin receptor6 or protein phosphorylation” has been shown to be unchanged in cryopreserved capillaries.

Binding and Endocytosis

Studies

Binding experiments were performed by thawing the cryopreserved microvessels followed by resuspension in Ringer-Hepes buffer (RHB), 141 mmol/L NaCl, 4 mmol/L KCI, 2.8 mmol/L CaCI,, 10 mmol/L Hepes (pH 7.4) containing 0.25 &i/mL ‘“‘I-transferrin and 2.5 &i/mL ‘H-inulin. The microvessels were incubated with various concentrations of unlabeled transferrin at either 4“C, 23OC, or 37°C for up to 180 minutes. Nonspecific binding was determined by incubating the microvessels in the presence of labeled transferrin and 40 Fg/mL unlabeled transferrin. The experimental procedure was exactly as described recently for the measurements of human brain capillary insulin receptor.6 The internalization (endocytosis) of ‘*‘I-transferrin was assessed with an acid wash technique described recently.’ After incubating the capillaries at various times with ‘*‘I-transferrin, 400 PL of the microvessel solution was transferred to 1.5 mL Beckman microfuge tubes and centrifuged at 10,000 for 45 seconds. The supernatant was aspirated and the microvessel pellet was resuspended in 450 *L of cold acid wash buffer (0.028 mol/L sodium acetate, 0.12 mol/L NaCI, 0.02 mol/L sodium barbital, pH 3.0), and was placed on ice for six minutes. Then 400 ML of this mixture was transferred to small

Metabolism, Vol36,

No 9 (September), 1987: pp 892-895

893

BLOOD-BRAIN BARRIER TRANSFERRIN RECEPTOR

Beckman microfuge tubes and these tubes were centrifuged at 10,000 g for 45 seconds. The supernatant was aspirated, the tubes were drained, and the pellets were cut into 0.5 mL 1 mol/L NaOH and counted for “‘1 and ‘H radioactivity as described previously.6 endocytosed The extent of metabolism, if any, of “‘1-transferrin over a 120-minute incubation was assessed by gel filtration of the microvessel extract obtained by suspending the capillary pellet in 3 mol/L acetic acid containing 1% Triton X-100 and 6 mol/L urea overnight at 4OC. The extract was centrifuged at 10,000 g for five minutes at 4OC and the supernatant was applied to a 1.6 x 63 cm column of Sephadex G-50 (fine). The column was eluted with 0.05 mol/L NaHPO, in 0.15 mol/L NaCl (pH 7.4) containing 0.1 g/100 mL bovine albumin and 1 mL fractions were counted for “‘1.

Miscellaneous Methods Human holotransferrin was iodinated to a specific activity of approximately 5 rCi/pg using ?-iodine and chloramine T.” The “‘1-transferrin was greater than 95% precipitable by trichloroacetic acid and was purified by Sephadex G-25 column chromatography on the morning prior to the experiment. The iodinated transferrin was discarded 1 week after iodination, and most experiments were performed within 24 hours of the iodination procedure. The detection of the human brain capillary transferrin receptor with an avidin/biotin/peroxidase assayI used 2 pg of a mouse monoclonal antibody to the human transferrin receptor as described previously.‘4 Mouse IgG,, (2 pg) was used as the control. The capillaries were adsorbed to a glass slide using a Cytospin centrifuge prior to fixation in 10% formaldehyde 4°C. ten minutes in Tris-buffered saline (pH 7.4).

Materials

Fig 2.

The binding of ‘*sl-

human transferrin to isolated human capillaries is selectively inhibited by 0.5 gmol/L human holotransferrin, but not by 0.5 pmol/L bovine insulin, arginine vasopressin. or somatostatin. lm), control: (01, 0.5 PmollL transferrin; ( ql, 0.5 gmol/L insulin: (El), 0.5 pmol/L somatostatin; (8). 0.5 pmol/L vasopressin.

peroxidase kit was purchased from Vector Labs (Burlingame, CA). Bovine albumin (Pentex Fraction V) was purchased from Miles Labs (Elkhart, IN). Human holotransferrin was obtained from U.S. Biochemical Company (Cleveland). Mouse monoclonal antibody to human transferrin receptor was obtained from Becton-Dickinson (Mountain View, CA), and the mouse IgGI, was obtained from Litton Bionetics, Inc (Kensington, MD). RESULTS ‘251-human transferrin readily bound to isolated human brain capillaries at 23% at a concentration of 0.6 nmol/L (Fig 1). The binding of the labeled transferrin to human brain capillaries was greatly depressed by the inclusion of 500 nmol/L unlabeled transferrin in the incubation medium, and this binding approximated that of 3H-inulin, an extracellular space marker (Fig 1). Conversely, 500 nmol/L concentrations of arginine vasopressin, somatostatin, or insulin did not inhibit transferrin binding (Fig 2). The inclusion of

‘251-iodine and ‘H-inulin were obtained from the New England Nuclear Corporation (Boston). Sephadex G-25 (medium), and G-50 (fine), and bovine insulin, and arginine vasopressin were obtained from Sigma Chemical Company (St Louis). Somatostatin was obtained from Peninsula Labs (Belmont, CA). The avidin/biotin/

0.05 r 0

0.04 25-

0.03 B F

Kg = 448 2 410 ng/ml Rg = 8.0 + 1.5 ng/mg,,

.o

\ 0.02 -

0

I = 0.90 P
O.Ol-

60

120 Minutes

I

180

Fig 1. Binding of “%human holotransferrin to isolated human brain capillaries at room temperature rises with time in the presence of low (0.6 nmol/LI concentrations of transferrin. The uptake of ‘261-transferrinin the presence of 606 nmol/L concantrations of unlabeled transferrin is low and approximates the uptake of ‘H-inulin.

0

0

I

I 4

I

I 8 Bound

I

, I 12 (ng /ml)

I 16

I 20

Fig 3. Scatchard plot of the binding of ‘2sl-human holotransferrin to isolated human brain capillaries in the presence of varying concentrations of unlabeled human transfarrin at 4°C. The dissociation constant (K,I and the maximal capacity &I were determined from the slope and intercept of the plot.

PARDRIDGE.

894

.“l/

‘1E 0

60

120

180

EISENBERG, AND YANG

and this process was slowed by incubation at 4°C. The uptake of transferrin into the space that was resistant to acid wash greatly exceeded that for an extracellular space marker such as ‘H-inulin (Fig 4), despite the fact that the molecular weight of inulin is much less than that of transferrin. The endocytosis of “‘1 represented uptake of unmetabolized ‘251-transferrin; when extracts of the capillary pellet were eluted from a 1.6 x 63 cm Sephadex G-50 column, all of the radioactivity eluted at a single peak just beyond the void volume (where transferrin standard elutes) and no other radioactive peaks (representing smaller metabolic products of “‘1-transferrin) eluted from the column. The mouse monoclonal antibody to the human transferrin receptor selectively bound to isolated brain capillaries compared to the mouse IgGza control, as shown by the diffuse speckled pattern in Fig 5. These studies were performed at 2 pg IgG/slide, and higher concentrations of monoclonal antibody resulted in a high background binding of the mouse IgGza isotype to the human brain capillary.

Minutes

DISCUSSION Fig 4. The total binding and the endocytosis of ‘261-human holotransferrin by isolated human brain capillaries is temperaturedependent, and both processes greatly exceed the endocytosis of ‘H-inulin. (0). the capillary associated transferrin that is resistant to a mild acid wash, and is believed to represent endocytosed peptide. (0). ‘261-transferrin (total): (W), ‘H-inulin (acid-resistant). Both the total binding and the endocytosis steps were slowed by incubations at 4°C.

various concentrations of unlabeled transferrin in the incubation medium resulted in a progressive decrease in the binding of ‘251-transferrin, and these data are shown in Fig 3 as a Scatchard analysis. The binding constants are within the same order of magnitude of insulin, IGF-I, or IGF-II, binding to human brain capillaries.‘5 The binding of ‘*‘I-transferrin to human brain capillaries was increased by incubation at 37OC and decreased at 4“C (Fig 4). At 37°C approximately 40% of the transferrin entered a brain capillary space that was resistant to acid wash (Fig 4). The entry of transferrin into a space that is resistant to acid wash is presumed to represent endocytosis,‘6

A

These studies provide evidence for a transferrin receptor on the isolated human brain capillary, which is used as a model system of the human BBB.6.‘0The acid wash studies in Fig 4 also provide evidence that the transferrin receptor mediates the endocytosis of unmetabolized transferrin into the endothelial cytoplasm. Presumably, the process that occurs in vivo is receptor-mediated endocytosis at the lumenal border of the brain capillary endothelial cell followed by receptor mediated exocytosis of transferrin at the antilumenal border of the brain capillary endothelial cell. This transcytosis process would allow for transport of circulating transferrin through the BBB and into brain interstitial space in man, similar to what is believed to occur for insulin and IGF.” This concept fits with other studies that have shown that the transferrin receptor is selectively enriched in the brain capillary compared to microvessels in other organs,5 although transferrin transcytosis is known to occur in endothelia of bone marrow microvessels.” Also, recent studies have shown that transferrin receptors are widely

:

Fig 5. Avidin / biotin / peroxidase reaction of isolated human brain capillaries with a mouse monoclonal antibody to the human transferrin receptor (a mouse IgG, subtype) (AL or with a nonspecific mouse IgO,, control (B). The peroxidase studies with the mouse monoclonel antibody to transferrin receptor generate a diffuse speckled pattern that is not present in the control slides.

BLOOD-BRAIN

BARRIER TRANSFERRIN

RECEPTOR

895

distributed throughout the rat brain in a pattern that it not related to iron distribution.” These results suggest a possible neuromodulatory role of transferrin similar to what has been shown for insulin.” Thus, circulating peptides such as insulin, IGFs, or transferrin may modulate brain functions subsequent to their receptor mediated transport through the BBB. The idea that a transcytosis system exists in the brain capillary is not completely testable using the isolated brain capillary model system. Owing to the homogenization procedure, the ATP level in isolated brain capillaries is near 0.” Since receptor-mediated exocytosis of peptides is ATPexocydependent, *‘J* it has not been possible to demonstrate tosis

of

peptides

with

isolated

bovine

brain

capillaries.’

showing exocytosis of ‘2SI-insulin from human brain capillaries were subsequently demonstrated to represent a nonspecific leakage of insulin from the postmortem human brain capillary. Both ‘*‘I-insulin or “‘1-transferrin nonspecifically leak out from human brain capillaries subsequent to endocytosis via a process that is independent of temperature (Pardridge WM, unpublished observation). However, recent studies using thaw-mount autoradiography Previous

studies

of rabbit brain after carotid infusion of “‘I-insulin provide evidence that insulin transport through the BBB into brain extravascular space is detectable,23 and this provides in vivo confirmation of the BBB peptide transcytosis hypothesis. If transferrin does undergo receptor-mediated transcytosis through the BBB, then the mechanism of this process is likely to be of considerable biologic interest. Generally, endocytosed transferrin enters into an acidic organelle called the endosome, which allows for dissociation of the iron from the transferrin followed by a return of the apotransferrin to the plasma membrane.24 However, in the case of transferrin transport through the endothelial cytoplasm, this process most likely occurs without iron dissociation from transferrin within the endothelial cytoplasm. Otherwise, net iron transport through the BBB would not occur. Since transferrin peroxidase conjugates have been prepared,*’ the use of such conjugates in future ultrastructural studies may illuminate the subcellular organelles involved in the movement of the transferrin-iron complex through the endothelial cytoplasm. ACKNOWLEDGMENT Dawn Brown skillfully

prepared

the manuscript.

REFERENCES 1, Seligman PA: Structure and function tor. Prog Hematol 13:131-147, 1983

of the transferrin

recep-

2. Walsh MJ, Limos L, Tourtellotte WW: Two-dimensional electrophoresis of cerebrospinal fluid and ventricular fluid proteins, identification of enriched and unique proteins, and comparison with serum. J Neurochem 43:1277-1285, 1984 3. Levin JJ, Tuil D, Uzan G, et al: Expression of the transferrin gene during development of non-hepatic tissues: High level of transferrin mRNA in fetal muscle and adult brain. Biochem Biophys Res Comm 122:2 12-2 I?, 1984 4. Pardridge WM: Neuropeptides Ann Rev Physiol45:73-82, 1983

and the blood-brain

barrier.

5. Jefferies WA, Brandon MR, Hunt SV, et al: Transferrin receptor on endothelium of brain capillaries. Nature 3 12: 162-163, 1984 6. Pardridge WM. Eisenberg J, Yang J: Human blood-brain barrier insulin receptor. J Neurochem 44:1771-1778, 1985 7. Frank HJL, Pardridge WM, Morris WL, et al: Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels. Diabetes 35:654-661, 1986 8. Duffy KR, Pardridge WM, Rosenfeld RG: Human bloodbrain barrier insulin-like growth factor receptor. Clin Res 35: I49A, 1987 9. Pardridge WM. Eisenberg J, Yamada T: Rapid sequestration and degradation of somatostatin analogues by isolated brain microvessels. J Neurochem 44:1178-l 184,1985 10. Choi TB, Pardridge WM: Phenylalanine transport at the human blood-brain barrier. Studies in isolated human brain capillaries. J Biol Chem 26 1:6536-6541, 1986 11. Pardridge WM, protein phosphorylation 45:1141-l 147, 1985

Yang J, Eisenberg J: Blood-brain barrier and dephosphorylation. J Neurochem

12. Larrick JW, Cresswell P: Transferrin receptors on human B and T lymphoblastoid cell lines. Biochim Biophys Acta 583:483-490, 1979 13. Hsu S, Raine L, Fanger H: A comparative peroxidase method and an avidin-biotin complex

study of the method for

studying polypeptide hormone with radioimmunoassay antibodies. Am J Clin Pathol 75:734-738, 1981 14. Pardridge WM. Yang J, Eisenberg J, et al: Antibodies to blood-brain barrier bind selectively to brain capillary endothelial lateral membranes and to a 46K protein. J Cereb Blood Flow Metab 6:203-211, 1986 15. Pardridge WM: Receptor-mediated transport through the blood-brain barrier. Endocr Rev 7:314-330, 1986 16. Haigler HT, Maxfield FR, Willingham MC, et al: Dansylcadaverine inhibits interalization of ‘*51-epidermal growth factor in BALB 3T3 cells. J Biol Chem 255:1239-1241, 1980 17. Soda R, Tavassoli M: Transendothelial transport (transcytosis) of iron-transferrin complex in bone marrow. J Ultrastruct Res 88:18-29, 1984 18. Hill JM, RUB MR, Weber RJ, et al: Transferrin receptors in rat brain: Neuropeptide-like pattern and relationship IO iron distribution. Proc Nat1 Acad Sci USA 82:4553-4557, 1985 19. Kessler JA, Spray DC, Saez JC, et al: Determination of synaptic phenotype: Insulin and CAMP independently initiate development of electrotonic coupling between cultured sympathetic neurons. Proc Nat] Acad Sci USA 81:6235-6239, 1984 20. Lasbennes F, Gayet J: Capacity for energy metabolism in microvessels isolated from rat brain. Neurochem Res 9: I - 10, 1984 21. Pan BT, Johnston R: Selective externalization of the transferrin receptor by sheep reticulocytes in vitro. J Biol Chem 259~9776-9782, 1984 22. Clarke BL, Weigel PH: Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. J Biol Chem 260:128-133, 1985 23. Duffy KR, Pardridge WM: Blood-brain barrier transcytosis of insulin in developing rabbits. Brain Res (in press) 24. Dickson RB, Hanover JA, Willingham MC, et al: Prelysosoma1 divergence of transferrin and epidermal growth factor during receptor-mediated endocytosis. J Cell Biol 94:207-212. 1982 25. Willingham MC, Hanover JA, Dickson RB, et al: Morphologic characterization of the pathway of transferrin endocytosis and recycling in human KB cells. Proc Nat1 Acad Sci USA 8 1: 175- 179, 1984