Receptor-mediated Transcytosis of Lactoferrin through the
Blood-Brain Barrier*
Carine
Fillebeen
,
Laurence
Descamps§,
Marie-Pierre
Dehouck§,
Laurence
Fenart§,
Monique
Benaïssa
,
Geneviève
Spik
,
Roméo
Cecchelli§¶, and
Annick
Pierce
From the
Laboratoire de Chimie Biologique,
Université des Sciences et Technologies de Lille, Unité
Mixte de Recherche 111, CNRS, 59655 Villeneuve d'Ascq, the
§ Service d'Etude et de Recherche sur les
Lipoprotéines et l'Athérosclérose, INSERM,
Unité 325, Institut Pasteur, 59019 Lille, and the
¶ Université d'Artois, Faculté Jean Perrin,
62307 Lens, France
 |
ABSTRACT |
Lactoferrin (Lf) is an iron-binding protein
involved in host defense against infection and severe inflammation; it
accumulates in the brain during neurodegenerative disorders. Before
determining Lf function in brain tissue, we investigated its origin and
demonstrate here that it crosses the blood-brain barrier. An in
vitro model of the blood-brain barrier was used to examine the
mechanism of Lf transport to the brain. We report that differentiated
bovine brain capillary endothelial cells exhibited specific high
(Kd = 37.5 nM; n = 90,000/cell) and low (Kd = 2 µM;
n = 900,000 sites/cell) affinity binding sites. Only
the latter were present on nondifferentiated cells. The surface-bound
Lf was internalized only by the differentiated cell population leading
to the conclusion that Lf receptors were acquired during cell
differentiation. A specific unidirectional transport then occurred via
a receptor-mediated process with no apparent intraendothelial
degradation. We further report that iron may cross the bovine brain
capillary endothelial cells as a complex with Lf. Finally, we show that
the low density lipoprotein receptor-related protein might be involved
in this process because its specific antagonist, the
receptor-associated protein, inhibits 70% of Lf transport.
 |
INTRODUCTION |
Lactoferrin (Lf)1 (1) is
a mammalian cationic iron-binding glycoprotein belonging to the
transferrin (Tf) family. Despite some striking differences, mainly in
the glycan moiety, there are marked sequence and conformational
homologies among Lfs from different species, as well as similar general
functions (for review, see Ref. 2). Many physiological roles have been
ascribed to Lf, particularly in the host defense against infection and
severe inflammation (for review, see Ref. 3). This broad spectrum of
biological functions relies on the interaction of Lf with numerous cells. The binding of Lf to cells is independent of its degree of iron
saturation and is mediated mainly via interaction of the cluster of
basic amino acids at its NH2 terminus with sulfated molecules (4, 5). However, Lf is also targeted to specific cell
receptors, and only a few of these involved in its uptake have been
clearly identified. The 105-kDa Lf receptor characterized on activated
human T-cells (6) is expressed at the cell surface of platelets (7),
megacaryocytes (8), dopaminergic neurons, and mesencephalon
microvessels (9). Lf receptor internalizes Lf, which is subsequently
degraded (30-40%), whereas the remaining fraction is recycled (10).
In addition, the low density lipoprotein receptor-related protein (LRP)
displays a high affinity for Lf and is responsible for its clearance
(11-14). This is inhibited by RAP, the receptor-associated protein
known to be an antagonist for LRP (15). Transcytosis of Lf was
described for HT29 cells (16) and was a minor pathway, up-regulated
during iron deprivation (17).
Lf is produced by exocrine glands (1, 18) and is widely distributed in
the body fluids. It is stored in specific granules of neutrophilic
leukocytes (19) and is released during the inflammatory process. In
addition, the concentration of Lf present in cerebrospinal fluids is
enhanced in cases of acute cerebrovascular lesions (20, 21). Moreover,
as an iron-binding protein, Lf has been implicated in the pathogenesis
of brain lesions. Although practically absent from the normal human
cerebral cortex (22), Lf was found in brains associated with aging and,
more importantly, in increased amounts in the specific regions
adversely affected in neurodegenerative disorders (22-24). In the
mesencephalon, Lf is concentrated mainly in the dopaminergic neurons;
in the case of Parkinson's disease (25), the surviving neurons
accumulate higher concentrations of Lf. The origin and function of Lf
within either the normal or pathological brain are as yet uncharted.
In situ synthesis of Lf occurs in brain, and Lf transcripts
were found in human (26) and mouse brain tissues (27). Moreover, we
have shown that Lf expression was up-regulated in mouse brain tissues
treated with the neurotoxic agent
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is used as a model
for Parkinson's disease (27). Nevertheless, this up-regulation was
slight and might not explain the large increase of Lf observed in the
case of neurodegenerative disorders (22, 25). Moreover, the presence of
Lf on the cerebral microvessels and its distribution in the vicinity of
the inflammatory foci led us to investigate whether Lf may cross the
blood-brain barrier (BBB).
The BBB is formed from specialized endothelial cells (ECs) that are
sealed together by continuous complex tight junctions to form a
polarized barrier that restricts the free exchange of most solutes
between plasma and the extracellular fluid of the brain. Furthermore,
even though brain capillary ECs contain no direct transendothelial
passageways such as fenestrations or channels, specific transport
mechanisms located in the cerebral ECs ensure that the central nervous
system receives an adequate supply of nutrients. Such receptors have
already been identified for proteins (28-30) and lipoproteins
(31).
We have used an in vitro model of the BBB which imitates the
in vivo situation by means of the co-culture of bovine brain capillary ECs (BBCECs) on one side of a porous filter and astrocytes plated at the bottom of six-well dish (32, 33) to provide direct
evidence that after binding to BBCECs, Lf crosses the endothelial monolayer from the apical to the abluminal surface. Lf transcytosis is
receptor-mediated, and our results indicate that LRP might be involved
in this transcellular transport across the brain endothelium.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
BBCECs isolated and characterized as described
previously (32, 33) were cloned, allowing us to obtain a pure EC
population uncontaminated by pericytes. The cells were cultured in the
presence of Dulbecco's modified Eagle's medium supplemented with 15%
(v/v) heat-inactivated calf serum (Hyclone Laboratories), 2 mM glutamine, 50 µg/ml gentamycin, and 1 ng/ml basic
fibroblast growth factor, added every other day. Nondifferentiated
cells were obtained by growing BBCECs in the absence of astrocytes.
Primary cultures of mixed astrocytes were prepared from newborn rat
cerebral cortex. After removing the meninges, the brain tissue was
forced gently through a nylon sieve (34). Astrocytes were plated on
six-well dishes (Nunclon; Nunc A/S) at a concentration of 1.2 × 105 cells/ml in 2 ml of Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum (Hyclone Laboratories).
The medium was changed twice a week. 3 weeks after the seeding was
completed, the purity of the astrocyte population was checked (33).
Stabilized cultures contained more than 95% astrocytes that were glial
fibrillary acidic protein-positive and were used for co-culture.
Establishing the in vitro model of BBB was performed using a
co-culture of BBCECs and astrocytes. Prior to cell culture, plate inserts (Millicell-CM 0.4 µm; 30-mm diameter; Millipore Corp.) were
coated on the upper side with rat tail collagen (35). They were then
set in the six-multiwell dishes containing the astrocytes prepared as
described above, and BBCECs were plated at a concentration of 4 × 105 cells/ml on the upper side of the filters in 1.5 ml of
co-culture medium. This BBCEC medium was changed every other day. Under
these conditions, differentiated BBCECs formed a confluent monolayer 7 days later (400,000 cells/cm2). Experiments were performed
5 days after confluence was reached. To be certain that differentiated
BBCECs are a legitimate model for the BBB in vitro, the
BBCEC phenotype was characterized and the tight junction network
vizualized. Cells were stained with a filamentous actin probe (28, 31),
and the localization to the plasma membrane of the tight
junction-associated protein ZO-1 was carried out as described (31).
Endothelial permeability was checked in each experiment and the
permeability coefficient (Pe, in cm/min)
calculated as described previously (33).
Preparation and Labeling of Bovine Lf (bLf)--
bLf was
purified from bovine colostrum by ion-exchange chromatography as
partially saturated bLf (36).
Fluorescent labeling of bLf on the glycan moiety was carried out by
coupling
5-((2-(carbhydrazino)methyl)-thio)acetyl)aminofluorescein (Hyf)
on aldehyde groups resulting from the mild periodate oxidation of
N-acetylneuraminic acid residues (7).
Iodination of bLf was performed using the IODO-GEN reagent (Pierce).
bLf (500 µg) was dissolved in phosphate-buffered saline and incubated
with 0.2 mCi of Na125I (carrier-free, 100 mCi/ml, Amersham
Pharmacia Biotech) for 15 min at 4 °C. Free iodine was removed on a
Sephadex G-50 column (PD-10, Amersham Pharmacia Biotech). The specific
activity range was 0.35-0.5 µCi/µg of protein; to avoid
radiolysis, the iodinated protein was stored at 4 °C and used the
same day.
Prior to 59Fe labeling, bLf was fully desaturated in
formiate buffer (0.2 M formic acid-sodium formiate;
containing 0.5 M NaH2PO4 and 25 mM EDTA) at pH 3.8 to give apobLf. 59Fe (18 nmol) (3-25 mCi/mg Fe, Amersham Pharmacia Biotech) was mixed with a
solution containing 0.4 mM nitriloacetic acid and 0.05 M NaOH for 5 min at room temperature, and then the pH was adjusted to 8.2 with NaOH. ApobLf (37 nmol) dissolved in 0.1 M Tris-bicarbonate buffer (pH 7.6) was added to the iron
solution and then incubated for 45 min at room temperature. Unbound
59Fe was captured mainly by adding 50 µl of Chelex 100 (Sigma) preequilibrated with 0.1 M Tris-bicarbonate buffer,
and the remaining free 59Fe was further removed on a PD-10
column. This method gave an average specific activity of 5 µCi/mg
protein and a yield of iron saturation equal to 99%.
For dual labeling of bLf, protein was first labeled with
59Fe and further iodinated with 0.05 mCi of
Na125I for 500 µg of 59Fe-bLf, giving a
specific activity of 0.1 µCi/µg protein for 125I and 4 µCi/mg protein for 59Fe.
Binding, Endocytosis, Internalization Kinetics, and
Transcytosis--
All of these studies were performed only with BBCECs
to avoid any interference from the astrocyte population. Moreover, to eliminate endogenous bLf, BBCECs were always incubated before each
experiment for 2 h in medium without serum. Because Tf has been
described as an effective blocking component, able to prevent high
levels of nonspecific Lf binding (37), all buffers contained 0.2% bTf
(Sigma). Each point was done in triplicate, and the data are
represented as the means ± S.E. Nonspecific controls were carried
out with a 100-fold excess of unlabeled protein.
Equilibrium binding was performed on differentiated and
nondifferentiated BBCECs in Ringer-HEPES-bTf for 2 h at 4 °C,
with a 125I-bLf concentration ranging from 0.2 to 200 µg/ml (2.5-2500 nM). The cells were washed carefully,
and cell-associated radioactivity was determined by removing the
membrane of the culture insert and counting it in a gamma counter. The
results were analyzed using the Enzfitter nonlinear regression data
analysis program (Elsevier-BIOSOFT, Cambridge, U. K.). Scatchard plots
and kinetic analyses were performed using the same software.
Determination of the luminal uptake of Hyf-bLf was performed before
cell fixation with 4% paraformaldehyde. The luminal compartment of the
differentiated BBCECs was exposed to Hyf-bLf (50 µg/ml; 625 nM) in Ringer-HEPES-bTf and left in contact with the cells for 45 min at 37 or 4 °C. Fluorescence microscopy experiments were
carried out as already described (28).
The time course of internalization of 125I-bLf by
differentiated BBCECs was measured using 125I-bLf (30 µg/ml; 375 nM) which was presented to the luminal surface for 1 h at 4 °C prior to the experiment. After washing off the unbound bLf, filters covered with cells were incubated in prewarmed medium at 37 °C. At various times, filters were removed, and all subsequent steps were performed at 4 °C. The medium compartments were collected, and cells were treated with 0.1% Pronase E (Merck) in
phosphate-buffered saline for 30 min at 4 °C. The surface-bound, internalized, released, and transported 125I-bLf was
counted in the Pronase-sensitive eluate, the cell extract, and the
trichloroacetic acid-precipitable fractions of incubation media of the
upper and lower compartments, respectively.
Transcytosis experiments were performed as follows. One insert covered
with BBCECs was set into a six-well dish with 2 ml of Ringer-HEPES-bTf
added to each well at 37 °C. 125I-bLf (30 µg/ml),
59Fe-bLf (50 µg/ml), and
125I-59Fe-double-labeled bLf (50 µg/ml) were
added to the upper side of the filter covered with cells. At various
times, the insert was transferred to another well to avoid a possible
reendocytosis of bLf by the abluminal side of the BBCECs. At the end of
each experiment, intact bLf was assessed using trichloroacetic acid precipitation of lower media, and protein degradation was assessed with
AgNO3 precipitation. For 125I-bLf studies, all
results were expressed as 125I-bLf equivalent flux
(ng/cm2), which represents trichloroacetic
acid-precipitable radioactivity recovered in the lower compartments.
The 59Fe equivalent flux (pg/cm2) corresponds
to the total iron radioactivity found in the lower compartment.
The influence of temperature on the transport of 125I-bLf
(30 µg/ml) was studied with the monolayers kept at 4 °C. In
parallel, the paracellular passage of sucrose (0.1 µCi of
[14C]sucrose) was assayed at 4 °C and 37 °C in the
same conditions.
To demonstrate whether Lf transendothelial transport was directional,
125I-bLf (30 µg/ml) was added to the lower compartment of
the wells, and the transcytosis experiment was conducted as above.
Effect of RAP on Lf Transcytosis--
The effect of RAP on the
transendothelial transport of 125I-bLf across BBCECs was
determined by adding 100 nM recombinant RAP to the upper
side of the filter covered with cells for 1 h at 37 °C before
experiments. Recombinant RAP was prepared in Escherichia coli as a fusion protein that contains the entire coding sequence of the 39-kDa protein fused to glutathione S-transferase
(38). All experiments were then performed with 125I-bLf as
described above.
Electrophoretic Characterization of bLf After
Transcytosis--
After 2 h of transcytosis, the apical and
basolateral compartment solutions were collected and analyzed by 10%
SDS-polyacrylamide gel electrophoresis. After electrophoresis, the gel
was dried and exposed for autoradiography 2 h at
80 °C (Kodak
X-Omat AR film).
 |
RESULTS |
Binding of bLf on BBCEC Monolayers--
Binding experiments were
performed both on differentiated and nondifferentiated BBCECs (cultured
in the absence of astrocytes) using radioiodinated ligand. Only the
binding data of differentiated cells are represented in Fig.
1. The binding was found to be
concentration-dependent, saturable, and inhibited by
approximately 85% in the presence of a 100-fold molar excess of
unlabeled bLf, suggesting that it was reversible and specific. The
binding data, analyzed by the method of Scatchard, were consistent with
a two-component binding curve. Fig. 1 shows the Scatchard analysis of
data from a single typical experiment (of five carried out). At a low
range of 125I-bLf concentrations, binding is determined
primarily by high affinity sites (Fig. 1A). At a high range
of 125I-bLf concentrations (Fig. 1B), low
affinity sites account for most of the binding. At intermediate
concentrations, binding is effected by both high and low affinity
sites. These data, summarized in Table I,
are in agreement with the presence of two binding sites on
differentiated BBCECs: a high affinity binding site with a
Kd of about 37.5 nM and 90,000 sites/cell and a low affinity binding site with a Kd
of about 1,900 nM and 890,000 sites/cell. Both sites were
specific for bLf. bLf binding to nondifferentiated BBCECs was also
investigated (Table I). Scatchard analysis revealed that the affinity
of bLf for these cells (Kd = 2, 100 nM)
and the number of bLf binding sites (920,000/cell) were comparable to
the low affinity binding site parameters found on the differentiated
cells. These results suggest that only differentiated BBCECs are able
to express high affinity binding sites for bLf.

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Fig. 1.
Scatchard analysis of the binding of
125I-bLf to differentiated BBCECs. bLf binding was
performed on BBCECs at 4 °C, and increased concentrations of
125I-labeled bLf were added at the luminal side of the
cells. Low (panel A) and high (panel
B) concentration ranges of bLf were used. Specific binding
was obtained after subtraction of the nonspecific binding in the
presence of a 100-fold excess of unlabeled bLf from the total counts.
The specific bound 125I-bLf was analyzed by the Scatchard
procedure. B/F, bound:free ratio.
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Table I
Dissociation constants (Kd) and numbers of bLf binding
sites/cell on BBCECs
Values are means ± S.E. for two separate experiments conducted in
triplicate.
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Endocytosis of bLf in BBCEC Monolayers--
To determine whether
bLf was internalized from the luminal surface of the BBCECs, Hyf-bLf
was first used as a probe. Staining visualized with a fluorescent
microscope is shown in Fig.
2A. Hyf-bLf was found as
small, individual vesicles throughout the differentiated cells. This
uptake was completely inhibited at 4 °C (Fig. 2B). The
endocytosis process was then monitored using radiolabeled bLf. Cells
were first incubated at 4 °C for 1 h until the binding site
occupancy reached a steady state. After washing off the unbound ligand,
cells were incubated at 37 °C and the distribution of surface-bound,
internalized, released, and transported radioactivities was counted at
various times. As shown in Fig. 3, the
Pronase-resistant fraction increased rapidly and reached a maximum of
18% of the initially bound 125I-bLf, 3 min after the start
of the incubation. The Pronase-resistant fraction decreased slowly
thereafter, as 125I-bLf started to appear in the lower
compartment a few minutes later, showing that transcytosis occurs.
During the experiment, the Pronase-sensitive fraction decreased, and
125I-bLf appeared in the upper compartment, suggesting a
marked dissociation from the cell surface or a release of
125I-bLf by the cells.

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Fig. 2.
Hyf-Labeled bLf endocytosis in BBCECs.
BBCECs were incubated at 37 °C (panel A) or
4 °C (panel B) for 45 min with 50 µg/ml
Hyf-bLf. After washing, the cells were fixed and processed for
fluorescence microscopy as described under "Experimental
Procedures." Bar = 20 µm.
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Fig. 3.
Internalization kinetics of
125I-bLf in BBCECs. The cells were incubated for
1 h at 4 °C in the presence of bLf (30 µg/ml
125I-bLf, 3 mg/ml unlabeled bLf). After washing off the unbound
ligand, cells were incubated with 125I-bLf at 37 °C. At
the indicated times, filters were removed, and the amounts of
surface-bound, internalized, released, and transcytosed radioactivity
were counted. Ordinates represent the percentage of the
total radioactivity initially bound to BBCECs at 4 °C, which was
segregated within the cells ( ) and released into the lower
incubation medium ( ) or into the upper incubation medium ( ) or
associated with membranes ( ). The data are expressed as ng of
125I-bLf transported/cm2, which refers to the
surface area of the cells. Each point is a mean of three different
filters.
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Apical to Basolateral Transport of bLf Across BBCEC
Monolayers--
125I-bLf was added to the luminal chamber
of the culture, and its progressive transfer across the cell monolayer
was followed for 90 min. A range of different bLf concentrations
(10-100 µg/ml) was assayed, and no evidence of saturation was
detected at concentrations lower than 100 µg/ml (data not shown).
Fig. 4 shows the total, nonspecific and
calculated specific bLf flux values as a function of time with a
luminal concentration of 30 µg/ml bLf. Our results demonstrate that
differentiated BBCECs are not a barrier for the passage of bLf. The
transport of labeled bLf from the luminal to the abluminal compartment
was reduced severely by an excess of unlabeled bLf, suggesting that bLf
transport from the apical to the basal side of the cells was effected
by specific receptor-mediated transport. The rate of transcytosis was
evaluated, and about three to four bLf molecules were transported via
one high affinity binding site in 1 h, suggesting that one bLf
molecule crosses the BBCEC monolayer in 15-20 min. The possible
toxicity of a high concentration of bLf (3 mg/ml) for the integrity of
the BBCEC monolayer was assessed by calculating the permeability to
sucrose. No leakiness in the barrier function occurred
(Pe = (0.65 ± 0.02) × 10
3
and (0.67 ± 0.03) × 10
3 cm/min for the control and
in the presence of 3 mg/ml of bLf, respectively).

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Fig. 4.
Apical to basolateral transport of
125I-bLf across BBCEC monolayers. bLf (30 µg/ml
125I-bLf, 3 mg/ml unlabeled bLf) was added to the luminal
side of cells grown on porous filters coated with collagen. Intact
125I-bLf transport from the upper to the lower sides of the
filter represents radioactivity that was trichloroacetic
acid-precipitable in the lower compartments. Specific transport ( )
was calculated by subtracting the radioactivity obtained in the
presence of native bLf ( ) from that obtained in the absence of
native bLf ( ). The data are expressed as ng of 125I-bLf
transported/cm2, which refers to the surface area of the
cells. Each point is a mean of three different filters.
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The effect of temperature on bLf transport from the apical to basal
surfaces of BBCECs is shown in Fig. 5. A
decrease in the incubation temperature from 37 °C to 4 °C
slightly affected the paracellular passage of sucrose (Fig.
5A), whereas a dramatic decrease in the bLf transport
through the monolayer was observed (Fig. 5B), indicating
that bLf is directed to the abluminal compartment by a transcellular
route and that this transport system requires active mechanisms such as
receptor-mediated transcytosis.

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Fig. 5.
Effect of a reduction in temperature from
37 °C ( ) to 4 °C ( ) on the transport
of [14C]sucrose (panel A) and
125I-bLf (panel B) across BBCECs.
Results are expressed as a percentage of sucrose recovered in the lower
compartments. For bLf, data are expressed as an equivalent bLf flux
(ng/cm2), and values are means of triplicate inserts.
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Abluminal to luminal transport of bLf was assayed by added
125I-bLf to the lower chamber of the culture. No specific
transport of bLf was detected across the cell monolayer in these
conditions (data not shown), showing that the transport of bLf across
BBCECs is unidirectional.
Effect of RAP on the Transendothelial Transport of
125I-bLf Across BBCECs--
To determine whether LRP might
be involved in the receptor-mediated transport of bLf, transcytosis
experiments were performed in the presence of RAP. RAP is known to
interact with LRP and block the binding of any kind of LRP ligand. The
fusion molecule with glutathione S-transferase was used
because it has been shown that LRP does not bind glutathione
S-transferase (15, 39).The integrity of the monolayer was
evaluated, and no increase in the permeability of sucrose in the
presence of recombinant RAP was detected (Pe = (0.52 ± 0.02) × 10
3 and (0.55 ± 0.03) × 10
3 cm/min for the control and in the presence of 100 nM RAP, respectively). As shown in Fig.
6, recombinant RAP decreases the rate of
the passage of bLf through the BBCEC monolayer. A decrease of 70% of
the initial transcytosis was observed, suggesting the involvement of
LRP in this intracellular traffic.

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Fig. 6.
Effect of RAP on the transport of
125I-bLf across BBCEC monolayers. After incubation for
1 h at 37 °C with ( ) or without ( ) RAP (4 µg/ml), bLf
(30 µg/ml 125I-bLf, 3 mg/ml unlabeled bLf) was added to
the luminal side of the cells. The data are expressed as ng of
125I-bLf transported/cm2. Each point is a mean
of three different filters.
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Characterization of bLf After Transcytosis--
To determine
whether bLf undergoes degradation within BBCECs during transcytosis,
125I-bLf recovery was followed by counting the
trichloroacetic acid precipitate, and 125I-bLf degradation
was followed by measuring the AgNO3 precipitate in the
upper and lower compartments. Controls were carried out with filters
coated with collagen and without cells as above. The results showed
that 125I-bLf degradation was no more than 2 ± 0.5%
either in the upper or in the lower compartment. Moreover, Fig.
7 shows that 125I-bLf was
recovered as an intact protein after its transport through the BBCEC
monolayers. These results suggest that bLf was transported across
BBCECs via a specialized pathway that does not lead to degradation.

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Fig. 7.
Electrophoretic characterization of
125I-bLf after transcytosis across BBCEC monolayers.
bLf (30 µg/ml 125I-bLf) was added to the luminal side of
cells for 2 h at 37 °C. Lane 1, medium of the
luminal compartment after transcytosis experiment; lane 2,
medium of abluminal compartment after transcytosis experiment;
lane 3, 125I-bLf before its addition to the
luminal compartment. The arrow indicates the dye
front.
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Transendothelial Iron Transport Studies Using 59Fe-bLf
and 125I-59Fe-Double-labeled bLf--
To
determine whether iron-bound Lf might reach brain tissues, iron
transport was studied across BBCECs. Transcytosis experiments were
repeated with 59Fe-bLf. Fig.
8 represents specific transcytosis of
59Fe through BBCECs. This transport was reduced by an
excess of unlabeled bLf, suggesting that 59Fe crosses the
monolayer associated with bLf. To elucidate this point, experiments
were then carried out with 125I-59Fe-double
labeled bLf, and the data are summarized in Table
II. 59Fe-bLf equivalent flux
was calculated from 59Fe flux because one molecule of bLf
binds two molecules of iron. The 59Fe-bLf equivalent flux
(0.044 pmol cm
2 h
1), which is equal to
one-half of the 59Fe flux (0.088 pmol cm
2
h
1), was very close to that of the 125I-bLf
equivalent flux observed (0.046 pmol cm
2
h
1). These results suggest that iron found in the lower
compartment crossed the BBCEC monolayers bound to bLf.

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Fig. 8.
Apical to basolateral transport of
59Fe-labeled bLf on BBCEC monolayers. bLf (50 µg/ml
59Fe-bLf, 3 mg/ml unlabeled bLf) was added to the luminal
side of the cells. Specific transport ( ) was calculated by
subtracting the radioactivity obtained in the presence of unlabeled bLf
( ) from that obtained with only labeled bLf ( ). All values are
means of triplicate inserts ± S.E. (bars;
n = 3).
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Table II
125I-59Fe-double-labeled Lf transport across BBCEC
monolayers
Values are means ± S.E. for three different experiments.
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DISCUSSION |
The purpose of the present study was to determine whether Lf was
able to cross the BBB, which would explain its presence in brain
tissues. We have demonstrated that Lf binding to BBCECs was followed by
transcytosis of the protein from the luminal to the abluminal surface
of brain capillary ECs in the absence of any demonstrable degradation.
Using RAP as an antagonist of LRP, we have been able to show that this
receptor might be involved in the intracellular traffic of Lf.
We have used an in vitro BBB model consisting of a
co-culture of brain capillary EC and astrocytes. When cultured on
permeable supports, these cells form a well polarized monolayer that
mimics the in vivo situation (28, 31, 33) and displays
remarkable phenotypic similarities to the in vivo BBB with
respect to the expression of specific markers and characteristics such
as tight junctions, electrical resistance, low paracellular
permeability, specific enzymatic activities, and carrier-mediated
transport (40, 41).
The binding parameters of Lf have already been published for numerous
cells. The BBCEC constants are within the same range as those reported
(4, 6) because the apical surface of differentiated BBCEC cells
exhibited 9 × 104 high specificity binding sites/cell
for bLf with a dissociation constant of about 37.5 nM.
These high affinity binding sites represent one-tenth of the total
number of binding sites and were not present on the nondifferentiated
BBCECs, demonstrating that Lf receptors were acquired during differentiation.
Our data are equally consistent with the existence of low affinity
binding sites on both types of BBCEC cells with a Kd of 2 µM and approximately 9 × 105
binding sites/cell. As already shown, Lf binds to various specific ligands at the cell surface. Its clearance from the circulation involves at least two classes of binding sites, a large number of low
affinity binding sites and a smaller number of high affinity binding
sites including LRP (39). Two classes of binding sites have also been
described for Lf on human lymphoblastic Jurkat cells because treatment
with chlorate, which inhibits the sulfation of the carbohydrate
residues on intact cells (42), resulted in a 40% decrease of the total
number of binding sites for native Lf, suggesting that part of the
sites consist of cell-associated sulfate groups (4). The low affinity
binding components present in large numbers on BBCECs may also be
sulfated molecules such as glucosaminoglycans or proteoglycans, which
are known to trap biologically active molecules and retain them until
they are targeted to their specific receptors (43).
In this study we have shown that after binding to the cell surface, Lf
enters the differentiated BBCECs. This uptake is a very dynamic,
receptor-mediated process followed by transcytosis. Furthermore the
transcytosis process itself seems highly regulated in that it is both
receptor-mediated and independent of a high concentration of Lf.
Accumulation of Lf occurs at the abluminal side of the BBCECs, and the
passage of Lf through the BBB therefore potentially corresponds to a
source of brain Lf. Lf was also found at the luminal side of the cells,
and this mechanism might be the result of either cell surface
dissociation or recycling. Because of the large numbers of low affinity
binding sites, Lf bound to these sites could dissociate rapidly on
equilibrium with extensive washes.
We next investigated the possibility that Lf might be internalized via
LRP, which is a member of the LDL receptor family involved in the
internalization and subsequently degradation of remnant chylomicron
particles. LRP is a multiligand receptor also responsible for the
cellular internalization of many ligands such as Lf,
2-macroglobulin, Pseudomonas endotoxin, and
both proteinases and proteinase-inhibitor complexes (for review, see
Refs. 44 and 45). The functional involvement of LRP in bLf uptake by
BBCECs was tested by inhibition experiments in the presence of RAP, the
LRP universal antagonist (15). RAP is an endoplasmic reticulum resident
protein (46) that functions intracellularly as a molecular chaperone
for LRP and maintains LRP in an inactive, non-ligand binding state
along the secretory pathway (47). We demonstrated that Lf transcytosis was mediated by LRP because 70% of the Lf traffic was inhibited by
RAP. The distribution of LRP on ECs was reported previously (48, 49).
It has also been studied in the central nervous system and was found
associated with neurons (50, 51), weakly on some glial cells, and
discontinously along the membranes of the capillaries (51). LRP was
also associated with neurodegenerative disorders such as Alzheimer's
disease (52) and was found in increased concentrations in some neurons.
LRP might therefore be responsible for Lf accumulation in some specific
brain areas.
Lf endocytosis (10, 13) and transcytosis (16, 17) have been reported
previously, and partial or complete degradation of Lf always occurs. Lf
released during neutrophil activation has a rapid turnover, and its
clearance by the liver keeps its level in the plasma very low. Lf
internalized by Jurkat cells accumulates in the endosomal compartment,
and then both intact and degraded Lf is released, suggesting that part
of the Lf is recycled (10). Lf is also endocytosed by the intestinal
epithelial cells HT-29. In this case, transcytosis of 10% of Lf
occurred, whereas 90% of the Lf followed a major degradative pathway
(16). In the present study, we report that Lf is taken up and
transported through the BBCECs without any degradation. The
nondegradation of Lf during the transcytosis indicates that the
transcytotic pathway in BBCECs is different from the classical Lf
receptor pathways described above. The existence of a receptor-mediated process that bypasses lysosomes seems to be a feature of ECs; the
absence of degradation of proteins such as insulin (29), albumin (30),
Tf (28), and lipoproteins such as LDL (31) through ECs was reported
previously. Microscopy studies also confirmed that no accumulation of
LDL was observed in lysosomes (31). The precise transcellular pathway
for the passage of blood-borne molecules across the BBB is not yet
elucidated. One of the characteristics of brain capillaries ECs is the
paucity of clathrin-coated pits, and previous studies strongly support
the involvement of caveolae in endocytosis (53, 54). These structures
seemed responsible for the uptake and transcellular transport of
albumin (55) and human Tf (28). They have recently been implicated in
the traffic of LDL through the brain microvascular endothelia leading
to LDL accumulation in early endosomes and in multivesicular body
structures (31). To determine whether Lf transport through BBCECs is
caveolae-dependent, electron microscopy investigations are
currently under way.
Alterations in iron metabolism occur in neurodegenerative diseases
which lead to excessive iron deposits (56-58). The causes and
consequences of such deposits in the brain are unknown, as is the
nature of the responsible iron complex. Conflicting results have been
reported on the implication of the proteins responsible for maintaining
brain iron homeostasis, but no significant variation in the levels of
expression of Tf, transferrin receptor, and ferritin have been found in
pathological brain tissues (59, 60), whereas changes in the
distribution, the amount (22-25), and the level of expression (27) of
Lf were observed. In the case of inflammatory responses, high plasma
levels of Lf are available, and larger amounts of Lf may cross the BBB
and accumulate in the inflammatory foci. This process might explain why
a strong labeling of Lf was observed on cerebral microvessels located
in the vicinity of the neurodegenerative lesions (22). On the other
hand, in response to local tissue injury or inflammation, additional
transport pathways for large molecules may be opened and existing
pathways modified or made less restrictive. It was shown recently that
the intracellular traffic kinetics of Tf and LDL are disturbed in
BBCECs in the presence of tumor necrosis factor
(40).
The most striking characteristic of Lf is its high affinity for iron.
In this study, we have been able to show that Lf crosses the BBB as
both the iron-saturated and native forms. Under physiological conditions, Lf functions as a major specialized iron scavenger and acts
as an antioxidant (61) rather than an iron donor. Sequestration of free
iron by Lf may inhibit the iron-catalyzed formation of hydroxyl
radicals, and the presence of Lf at sites where oxidative stress occurs
may limit cell damage. Nevertheless, iron-saturated Lf may act as a
prooxidant agent and contribute to cell injury (62). The precise role
of Lf in the brain is not yet known, but we can hypothesize that in
response to oxidative stress in brain tissue, Lf could have a
beneficial effect in neurodegenerative disorders by capturing the iron
in higher concentrations in some specific brain regions and act in this
way as a natural scavenger of reactive oxidative species.
Alternatively, its in situ synthesis at abnormal levels, its
release from necrosing neurons, or its possible increased uptake and
transcytosis by BBB ECs during the inflammatory process may exacerbate
and amplify the lesions, leading to a cytotoxic effect resulting in an
increase in neuronal death. Our current approach toward testing these
conflicting hypotheses is to investigate Lf intracellular traffic in
BBCECs in the presence of inflammatory mediators.
 |
ACKNOWLEDGEMENTS |
We are indebted to Prof. Manfred
Hüttinger from the University of Vienna (Institute of Medical
Chemistry, Waehringerstrasse 10, A-1090 Vienna, Austria) who provided
us with the recombinant RAP. We are grateful to Dr. R. J. Pierce for
reviewing the manuscript.
 |
FOOTNOTES |
*
This investigation was supported in part by CNRS Unité
Mixte de Recherche 111 (Relations Structure-Fonction des Constituants Membranaires), by the Université des Sciences et Technologies de
Lille I, by INSERM Unité 325 (Service d'Etude et de Recherche sur les Lipoprotéines et l'Athérosclérose), by the
Institut Pasteur de Lille, and by grants from the Conseil
Régional Nord-Pas de Calais (Axe régional: maladies
neurodégénératives et vieillissement).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratoire de
Chimie Biologique, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France. Tel.: 33-3-2033-7238; Fax: 33-3-2043-6555; Email: annick.pierce{at}univ-lille1.fr.
 |
ABBREVIATIONS |
The abbreviations used are:
Lf, lactoferrin;
bLf, bovine lactoferrin;
Tf, transferrin;
bTf, bovine transferrin;
LRP, low density lipoprotein receptor-related protein;
RAP, receptor-associated protein;
BBB, blood-brain barrier;
EC, endothelial
cell;
BBCEC, bovine brain capillary endothelial cell;
Pe, permeability coefficient;
LDL, low density
lipoprotein.
 |
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