From the Medical Research Council Human Immunology
Unit, Institute of Molecular Medicine, John Radcliffe Hospital,
Headington, Oxford OX3 9DS, United Kingdom and the
§ Nuffield Department of Pathology, John Radcliffe Hospital,
Headington, Oxford OX3 9DS, United Kingdom
Received for publication, December 6, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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The glycosaminoglycan hyaluronan is a key
substrate for cell migration in tissues during inflammation, wound
healing, and neoplasia. Unlike other matrix components, hyaluronan (HA)
is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in
binding and uptake of hyaluronan within the lymphatics, one likely
candidate is the lymphatic endothelial hyaluronan receptor LYVE-1
recently described in our laboratory (Banerji, S., Ni, J., Wang, S.,
Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999)
J. Cell Biol. 144, 789-801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from
the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in
vitro and demonstrate using immunoelectron microscopy that it is
distributed equally among the luminal and abluminal surfaces of
lymphatic vessels in vivo. In addition, we show by means of
specific antisera that expression of mouse LYVE-1 remains restricted to
the lymphatics in homozygous knockout mice lacking a functional gene
for CD44, the closest homologue of LYVE-1 and the only other Link
superfamily HA receptor known to date. Together these results suggest a
role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function.
The extracellular matrix glycosaminoglycan hyaluronan
(HA)1 is a large polymer of
N-acetyl-D-glucosamine and
D-glucuronic acid (molecular mass
105-107 Da) which plays an important role in
maintaining tissue integrity as well as facilitating the migration of
cells during inflammation, wound repair, and embryonic development (1,
2). By comparison with other macromolecules of the extracellular
matrix, HA undergoes rapid turnover with a half-life of ~24 h (1).
Intriguingly, most degradation occurs not locally, but within distant
lymph nodes. During this process, tissue HA enters the afferent
lymphatic vessels and is transported with the lymph fluid to the
draining lymph nodes where ~90% of the glycosaminoglycan is degraded
by unknown mechanisms (3, 4). The remaining 10-15% of the HA exits
via the efferent lymphatics to the blood vasculature where it is
rapidly endocytosed by the liver sinusoid endothelial HA receptor (5),
a 300-kDa heterotrimeric complex of The majority of HA-binding proteins (8, 9) identified to date belong to
the Link protein superfamily, defined by the presence of a conserved
HA-binding domain known as the Link module (10, 11). This is a unit of
~100 amino acids that contains four conserved cysteine residues
interspersed with tracts of both hydrophobic and charged residues. The
three-dimensional structure of the Link module closely resembles that
of the C-type lectin fold, comprising two Recently we identified a novel Link superfamily HA receptor termed
LYVE-1 (LYmphatic Vessel
Endothelial HA receptor-1) from a homology search of the
human EST data base and showed its expression was exclusive to
lymphatic endothelial cells within normal adult tissues (35). These
initial studies revealed that the human LYVE-1 molecule binds HA with a
high degree of specificity and suggested a role for the receptor in
sequestering HA on the luminal surface of lymphatic vessels. To explore
such possibilities in an animal model (see also Refs. 36-40, 69-70)
we have isolated a murine LYVE-1 orthologue and here we describe its
detailed characterization together with sequence comparisons that
predict important similarities with the related CD44 molecule.
Intriguingly, we have found that mouse LYVE-1 mediates internalization
of HA and is located on both the luminal and abluminal faces of
lymphatic endothelial cells. The implications of these findings for the
function of LYVE-1 in vivo are discussed.
Cells, Antibodies, Plasmids, and Reagents--
The transformed
human primary embryonal kidney cell line 293T was obtained from the
Imperial Cancer Research Fund Cell Bank, Clare Hall, United Kingdom.
The eukaryotic expression vector pRcCMV was from InVitrogen, Groningen,
The Netherlands. The pCDM7Ig vector for soluble Ig fusion protein
expression was kindly provided by Dr. Alejandro Aruffo, Bristol-Myers
Squibb, Seattle, WA. Rat monoclonal antibody to mouse CD34 (RAM34) was
obtained from PharMingen. Texas RedTM-conjugated goat
anti-rabbit was purchased from Southern Biotechnologies. FITC-conjugated goat anti-rabbit Ig and phycoerythrin-conjugated goat
anti-rabbit Ig were obtained from Sigma. Alexa 488-conjugated goat
anti-rat was from Molecular Probes (Eugene, OR). Horseradish peroxidase-conjugated goat anti-rabbit and horseradish
peroxidase-conjugated goat anti-human IgG were from Pierce.
High molecular weight hyaluronan from rooster comb (catalog number
H-5388), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), and
Saponin were obtained from Sigma. Biotin-LC-hydrazide was obtained from
Pierce. Chondroitin 4-sulfate, chondroitin 6-sulfate, and heparan
sulfate were from Sigma. An intracellular adhesion molecule-2
fusion protein containing the extracellular domain of intracellular
adhesion molecule-2 fused to the Fc domain of human
IgG1 was kindly donated by Dr. S. Adams (Molecular
Parasitology Group, University of Oxford).
Tissue Sections from CD44 Cloning of Full-length Mouse LYVE-1--
The amino acid sequence
of human LYVE-1 was used to search for murine homologues within the
mouse EST data base using the NCBI BlastSearch tool via the TBlastN
program. The search yielded four overlapping ESTs, AI006667,
AI391129, AI226003, and AA8820234, which together encoded a contiguous
open reading frame of 957 base pairs bearing significant homology to
the human sequence. The coding sequence and flanking 5'- and
3'-untranslated regions were then amplified from mouse lung cDNA in
a two-stage PCR reaction using nested primers as follows. In the first
round of amplification (1 min 94 °C, 2 min 57 °C, 4 min
72 °C, 33 cycles), the primers 190F (GTCTCCCTTACTGCGGGTGG) and 1407R
(CTCTCTGGTCTGCTGTGAGCC) were used (nucleotides numbered as in Fig.
1A) with 1 µl of mouse lung cDNA (41) in a reaction
mixture (50 µl) containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 1 mM dNTPs and 2.5 units of Pyrococcus furiosus
(Pfu) DNA polymerase. In the second round of amplification, 4 µl of
the first round product was used as template in a similar reaction
using the primers 229F Hind (CGCGAAGCTTGGGATCTGCACAATGCTCCAG) and 1231R Not
(AGAGAAAAGGGCGGCCGCTTGCCTCGTGTGCACTTTCTCC). The
restriction sites in each case are underlined. After digestion with
HindIII and NotI, the PCR product was ligated
into HindIII/NotI cut pRcCMV. The cloned
construct was then sequenced on both strands to confirm its integrity.
Northern Blot Analysis--
A Northern blot containing a variety
of normal mouse tissue RNAs (2 µg of poly(A)+
mRNA/lane) was purchased from CLONTECH and
hybridized in ExpressHyb solution (CLONTECH) with a
probe encompassing the extracellular domain of mouse LYVE-1 (694 base
pairs) labeled with [ Cloning of a Soluble Mouse LYVE-1 Fusion Construct and
Purification of the Fc Fusion Protein--
The extracellular domain of
mouse LYVE-1 including the cleavable N-terminal leader (residues
1-228, MLQHTS ... FKNEAAG in Fig. 1B) was amplified
from mouse stomach cDNA (41) by nested PCR. Reactions (1 µl of
cDNA template) were performed as described above for full-length
LYVE-1 using the primers 190F (GTCTCCCTTACTGCGGGTGG) and 994R
(CAGCCAGCACAGCGGCAGC; 1 min 94 °C, 1 min 59 °C, 3 min 72 °C,
33 cycles) followed by the primers 229F Hind
(CGCGAAGCTTGGGATCTGCACAATGCTCCAG) and 923R Bam
(GGTCGGGATCCCCAGCTGCTTCGTTCTTGAATG; 1 min 94 °C, 1 min
57 °C, 3 min 72 °C, 33 cycles) using 2.5 units of Pfu polymerase (nucleotides numbered as in Fig. 1A). After digestion with
HindIII and BamHI the PCR product was ligated
into HindIII/BamHI cut pCDM7Ig vector yielding a
construct encoding the first 228 amino acids of mouse LYVE-1 fused to
the Fc region of human IgG1. The cloned construct was
sequenced on both strands to confirm integrity.
For expression and purification of the Fc fusion protein, the construct
was transfected into 293T cells using calcium phosphate and
transfectants grown in UltraCHO medium (Bio-Whittaker) for 3 days prior
to harvesting the culture supernatant. After adjustment of the pH by
the addition of 0.05 volumes of 2 M Tris-HCl buffer, pH
8.0, the fusion protein was purified by affinity chromatography on a
column (1-ml bed volume) of protein A-Sepharose (Sigma) eluted with 0.1 M glycine-HCl buffer, pH 3.0. Fractions containing Fc fusion protein were neutralized by the addition of 0.05 volume of 2 M Tris-HCl buffer, pH 8.0, and the purity confirmed by
SDS-polyacrylamide gel electrophoresis.
Biotinylated HA and FITC-conjugated HA--
High molecular
weight HA was biotinylated by a modification of the method of Yu and
Toole (42) exactly as described previously (35). Conjugation of HA with
FITC was carried out using the method of De Belder and Wik (43).
HA Binding Assays--
Binding of mouse LYVE-1 fusion protein to
immobilized HA was tested in 96-well ELISA plates (Nunc Maxisorp) as
described previously (35). Plates were coated by overnight incubation
with 1 mg/ml HA in coating buffer (15 mM sodium carbonate
and 34 mM sodium bicarbonate, pH 9.3). Wells were blocked
for 2 h in PBS, 1% (w/v) bovine serum albumin, 0.05%
(v/v) Tween 20, and subsequently incubated with purified mouse LYVE-1
Fc fusion protein (62.5-1000 ng/ml) in PBS, 0.05% Tween 20 for 1 h at room temperature. Human LYVE-1 Fc (35) and intracellular adhesion
molecule-2 Fc fusion proteins were used as positive and negative
controls, respectively. After washing (3 times with PBS, once with PBS,
0.05% Tween 20), bound fusion protein was detected with horseradish
peroxidase-conjugated goat anti-human IgG (1:4000; Pierce) followed by
o-phenylenediamine substrate (Sigma). Subsequently,
absorbance at 490 nm was measured in a Bio-Rad microplate reader.
Competition experiments with free glycosaminoglycans including HA,
chondroitin 4-sulfate, chondroitin 6-sulfate, and heparan sulfate were
performed by preincubating the mouse LYVE-1 Fc fusion protein (10 µg/ml) with the appropriate glycosaminoglycan (3.13-100 µg/ml) for
30 min in PBS, 0.05% Tween 20. The mixtures were subsequently added to
96-well HA-coated plates and bound fusion protein was detected as
described above.
For binding of LYVE-1 to soluble HA, 96-well plates were first coated
overnight with either mouse or human LYVE-1 fusion protein or a control
syndecan-2 Fc fusion protein (62.5-1000 ng/ml in coating buffer)
followed by blocking and washing as described above. The wells were
then incubated with biotinylated HA (5 µg/ml) in PBS, 0.05% Tween 20 (with or without a 20-fold molar excesss of unlabeled HA as a control
for specificity) and bound biotinylated-HA detected using horseradish
peroxidase-conjugated avidin (1:500; DAKO) with
o-phenylenediamine as substrate. Binding was measured as the
absorbance at 490 nm.
For binding of HA to LYVE-1-transfected cells (see below), these were
incubated (20 min, 25 °C) in PBS, pH 7.5, containing FITC-conjugated
HA (25 µg/ml), 0.1% azide, and 5% fetal calf serum followed by
washing (×3) in PBS alone. Cells were then fixed in 2% formaldehyde,
mounted with fluorescent mounting medium, and viewed under a Zeiss
Axioskop microscope equipped with epifluorescent illumination.
Transient Transfection of 293T Cells and Assays for
LYVE-1-mediated HA Internalization--
Human 293T cells in 12-well
plates were transfected with full-length mouse or in some cases human
LYVE-1 (35) in pRcCMV (2 µg/well) using calcium phosphate
precipitation. For flow cytometric analysis of ligand internalization,
triplicate wells were incubated with FITC-HA (1-10 µg/ml) alone or
in the presence of a 500-fold molar excess of unconjugated HA (control)
for 0-5 h at 37 °C. After appropriate time intervals, cells were
washed briefly with ice-cold PBS and detached by suspension in ice-cold
PBS, 5 mM EDTA with gentle pipetting. One-half of each
detached cell sample was then fixed directly in 4% (w/v)
paraformaldehyde in PBS (to determine total HA accumulation) and the
other digested with papain (0.5 mg/ml) for 45 min at 37 °C to remove
cell surface LYVE-1·HA complexes followed by fixation (to determine
internalized HA). The efficiency of LYVE-1 cleavage under the latter
conditions was assessed by staining cells before and after papain
treatment with polyclonal anti-mouse LYVE-1 (1/500) followed by
phycoerythrin-conjugated goat anti-rabbit Ig. In each case fluorescence
was quantitated by flow cytometry using a Becton-Dickinson FacScan.
For analysis of HA internalization by immunofluorescence microscopy,
cells were prepared as described for flow cytometry except that cell
surface LYVE-1 was stained using Texas Red-conjugated goat anti-rabbit
Ig rather than a phycoerythrin conjugate. Slides were then viewed using
a Zeiss Axiophot microscope equipped with epifluorescent illumination.
For analysis of LYVE-1/HA internalization by confocal microscopy,
transfectants were incubated (5 h, 37 °C) with FITC-HA (10 µg/ml)
and antibodies to either mouse or human LYVE-1 (diluted 1/500). Cells
were then fixed (10 min, room temperature) in 4% (w/v)
paraformaldehyde and permeabilized with 0.25% (w/v) saponin, in PBS,
pH 7.5, containing 1% bovine serum albumin and 1% fetal calf serum
(30 min, room temperature) prior to the addition of Texas
RedTM anti-Ig conjugate. Slides were viewed on a Bio-Rad
Radiance 2000 laser scanning confocal microscope equipped with argon
and green helium/neon lasers.
Generation of Mouse LYVE-1 Antisera in Rabbits--
Rabbits were
immunized by subcutaneous injection with purified mouse LYVE-1 fusion
protein (100 µg) in complete Freund's adjuvant followed by two
further injections in Freund's incomplete adjuvant at 14-day
intervals. Antiserum was tested in ELISAs by assessing reactivity with
immobilized mouse LYVE-1 fusion protein or CD44Fc fusion protein as a
negative control. Sera were then subjected to affinity chromatography
on human IgG-agarose to deplete antibodies directed against the Fc
portion of the immunogen.
Generation of Mouse Lymphangiomas--
Female Balb/c mice were
injected (× 2) intraperitoneally with 0.1 ml of a 1:1 emulsion of
Freund's incomplete adjuvant in PBS, pH 7.5, at 2-week intervals as
described in Ref. 44. Two weeks after the second injection, the mice
were sacrificed by cervical dislocation and lymphangiomas which
developed on the abdominal surfaces of the diaphragm and in the liver
resected for fixation and staining as described below.
Preparation of Tissues and Cells for
Immunoperoxidase/Immunofluorescent Antibody Staining--
Tissues were
removed from female Balb/c mice or C57B16 homozygous
CD44
For double-immunofluorescent staining, tissues (Balb/c mice) were snap
frozen in liquid N2, cut into thin sections using a cryotome and fixed in acetone (room temperature, 10 min) prior to
incubation with polyclonal anti-mouse LYVE-1 and rat anti-mouse CD34
(10 µg/ml). Sections were then treated with a mixture of Texas
RedTM-conjugated goat anti-rabbit Ig (1:50) and Alexa
488-conjugated goat anti-rat Ig (1:200). Slides were fixed in 2%
formaldehyde, mounted with fluorescent mounting medium (Vectashield),
and viewed under a Zeiss fluorescent microscope. For fluorescent
staining of LYVE-1-transfected 293T cells, these were incubated with
rabbit anti-mouse LYVE-1 (1/400) in PBS, 5% fetal calf serum, 0.1%
azide for 30 min prior to washing in PBS and re-incubation with
FITC-conjugated goat anti-rabbit Ig (1/100).
Immunoelectron Microscopy--
For immunoelectron microscopy,
sections of formaldehyde-fixed mouse small intestine (see above) were
washed (3 times) in 0.1 M phosphate buffer and cut into
2-mm cubes. These were then incubated (room temperature, overnight)
with LYVE-1 polyclonal serum (1/100 dilution), washed, and stained with
either immunogold or horseradish peroxidase-conjugated anti-rabbit Ig.
Samples were post-fixed in osmium tetroxide in 0.1 M
phosphate buffer, dehydrated, and embedded in Spurr's epoxy resin.
Thin sections were cut and examined in a JEOL 1200EX electron microscope.
Cloning of Murine LYVE-1 and Comparison with Human LYVE-1 and
CD44--
We originally identified the human LYVE-1 cDNA by
searching the EST data base for homologues with the amino acid sequence of the Link HA-binding domain of the human CD44 molecule. Here we used
the human LYVE-1 amino acid sequence to identify the mouse orthologue
by BlastSearching the mouse EST data base with the program TBlastN. The
search yielded four overlapping ESTs, AI006667, -391129, and -226003, and AA8820234 that formed a contiguous sequence of 1516 base pairs. The
cDNA which was subsequently cloned from mouse lung using nested PCR
(Fig. 1A) contains a large
open reading frame of 318 amino acids (four residues shorter than the
human receptor) starting with a Kozak consensus ATG initiation codon at
position 241 and terminating with a TAG at position 1195. In brief, the
deduced amino acid sequence encompasses: 1) an N-terminal 23 residue
hydrophobic leader sequence. 2) A 211-residue hydrophilic segment
corresponding to the extracellular domain containing seven cysteines,
four of which (Cys60, Cys84,
Cys105, and Cys127) are predicted to form the
conserved disulfide bridges that stabilize the link module (see below),
two N-glycosylation sites (Asn52 and
Asn129), and a serine/threonine-rich tract (residues
144-188) likely to be heavily O-glycanated. 3) A 21-residue
hydrophobic transmembrane anchor. 4) A 63-residue cytoplasmic tail.
Alignment of the murine and human LYVE-1 sequences (Fig. 1B)
revealed an overall similarity between the two orthologues of 74%
(69% identity). Within the extracellular domain, the regions corresponding to the HA-binding Link homology unit (residues 44-89) are 79% similar, while the downstream, serine/threonine-rich
membrane-proximal domains are only 48% similar. Nevertheless eight of
the 13 potential O-glycanation sites in this region are
completely conserved, as are both the N-glycanation motifs
bracketing the Link module, suggesting that glycosylation, which is
known to regulate HA-binding in CD44, may also be important for LYVE-1 function.
The threeway alignment of mouse LYVE-1, human LYVE-1, and human CD44
(Fig. 1B) highlights a number of potentially important similarities between the HA-binding domains of the two receptors, both
within the Link module and in the downstream
membrane-proximal/transmembrane domains. For example, the three
residues Lys46, Tyr87, and
Asn109 in human LYVE-1 Link previously shown to correspond
to the known CD44 HA-binding residues Lys38,
Tyr79, and Asn100 (45, 46) are fully conserved
in mouse LYVE-1 Link. This interspecies conservation further
strengthens the prediction that these amino acids bind HA in the LYVE-1
link domain, a prediction supported by preliminary results from
site-directed mutagenesis experiments (data not shown).
Downstream of the Link module, both mouse and human LYVE-1 contain
tracts of basic residues (RKTKK and RRKK, respectively) conserved in
identical locations. Indeed a virtually identical tract (RKRK) is also
conserved in a bovine LYVE-1 orthologue identified in a further search
of the EST data base (not shown). This feature may have functional
significance in light of the fact that a tract of basic residues in the
membrane-proximal domain of CD44 has been shown to contribute to HA
binding (45). We also note that an adjacent cysteine residue which is
absent in CD44 is conserved in both mouse and human LYVE-1
(Cys197 and Cys201, respectively).
This may in turn be functionally important as this cysteine is
predicted to be unpaired and thus to form a free thiol that could form
an intermolecular disulfide bond leading to LYVE-1 dimerization. In
addition, the transmembrane anchors of mouse and human LYVE-1 both have
a conserved cysteine residue (Cys253 and
Cys257) in the same position as Cys286 in the
CD44 molecule that is implicated in covalent dimerization and HA
binding (47, 48). Together these sequence comparisons raise the
interesting possibility that LYVE-1, like CD44, may possess a regulated
HA-binding domain that extends beyond the immediate link module
(46).
Binding to Hyaluronan and Other Glycosaminoglycans--
To assess
the functionality of the murine receptor we first transfected 293T
human fibroblasts with full-length cDNA in pRcCMV and measured
binding of FITC-HA to the cell surface. As shown by the data in Fig.
2D, the majority of LYVE-1
transfectants but none of the control mock-transfected cells bound
FITC-HA. Surface expression of the receptor in these experiments was
confirmed clearly by immunofluorescent antibody staining with mouse
LYVE-1-specific polyclonal sera (see below). To investigate HA binding
in more detail we expressed the extracellular domain of murine LYVE-1 as a soluble Fc fusion protein and measured binding to ligand immobilized in 96-well microtiter plates. The results (Fig. 2, A and B) confirm the mouse molecule binds both
immobilized high molecular weight HA and soluble biotinylated HA in a
concentration-dependent fashion. Furthermore, binding to
immobilized HA was inhibited only by free hyaluronan and not by the
glycosaminoglycans chondroitin 4-sulfate, chondroitin 6-sulfate, or
heparan sulfate (Fig. 2C). These latter results indicate
that the murine LYVE-1 receptor has a specificity for hyaluronan that
is similar to that of the human orthologue but is distinct from the
closely related CD44 molecule which binds both HA and chondroitin
sulfates.
LYVE-1 Mediates HA Internalization--
The capacity of LYVE-1 to
function as a receptor for HA internalization was assessed in
experiments where LYVE-1-transfected 293T cells were incubated with
FITC-HA and the accumulated ligand assayed by flow cytometry.
Internalized ligand was distinguished from surface bound ligand in
these assays by measuring FITC-HA fluorescence both before and after
treatment of cells with papain to cleave exposed LYVE-1·HA complexes.
As shown by the progress curve in Fig.
3A, LYVE-1-transfected 293T
cells bound and internalized FITC-HA rapidly. The rate of total
accumulation (the sum of both cell surface and internalized components)
was logarithmic, reaching a plateau within 1-2 h, while the rate of
internalization was linear, reaching a plateau after 4 h. Indeed
some 50% of the accumulated HA was internalized by LYVE-1-transfected
cells within this time interval. As binding of FITC-HA was blocked
( Tissue-specific Expression of Murine LYVE-1 mRNA--
As an
initial test for the tissue specificity of mouse LYVE-1 expression we
compared transcript levels in different tissues by Northern blotting.
As shown by the blot in Fig. 4,
hybridization to a single 2.6-kilobase transcript was seen only in
lung, liver, and heart, while no mRNA was detected in either
spleen, muscle, kidney, testis, or brain, even after extended exposure
(data not shown). This contrasts markedly with the human orthologue
where the mRNA is readily detected in each of these tissues and is
particularly abundant in spleen (35). These results suggest there are
significant differences either in the pattern of LYVE-1 gene expression
or in LYVE-1 mRNA stability in humans and mice.
Immunohistochemical Detection of Murine LYVE-1 in Normal and
Neoplastic Lymphatics--
In order to immunolocalize the LYVE-1
polypeptide in murine tissues we generated a polyclonal serum by
immunizing rabbits with soluble murine LYVE-1 Ig Fc fusion protein. The
resulting antiserum was highly specific for the murine receptor even at high dilution (>1/5000) as assessed by an ELISA and failed to bind
either the human LYVE-1 or human CD44 fusion proteins (Fig. 5). Correspondingly, antibodies to the
human LYVE-1 molecule failed to recognize the mouse orthologue (data
not shown). The specificity of the LYVE-1 antiserum was additionally
confirmed by immunofluorescent staining of 293T cells transfected with
full-length LYVE-1 which detected the receptor in an intense
ring-staining pattern at the cell surface (Fig. 2D).
Furthermore, the antiserum blocked binding of LYVE-1 Fc fusion protein
to immobilized HA, indicating the presence of antibodies directed
against the ligand-binding domain (data not shown).
Immunoperoxidase staining of a range of paraffin-embedded normal C57Bl6
mouse tissues including large intestine, lung, and cardiac smooth
muscle (Fig. 6), and stomach, skin, and
skeletal muscle (not shown) using LYVE-1 polyclonal serum revealed
strong staining of vessels. These were identified as lymphatics on the basis of their characteristic flattened, irregular morphology, empty
lumena (lacking erythrocytes), and the absence of a basement membrane.
The LYVE-1-stained vessels were particularly abundant in tissues such
as the lamina propria of large intestinal mucosa that have an extensive
lymphatic network (Fig. 6, panel A) and in experimental
lymphangiomas induced by intraperitoneal injection of Freund's
adjuvant (Fig. 6, panel D). Moreover the LYVE-1-stained lymphatics could be clearly distinguished from blood capillaries by
double immunofluorescence staining with LYVE-1 and the vascular endothelial marker CD34 (Fig. 7,
panels A-C) which revealed two discrete populations of
single positive vessels (green CD34+ve and red
LYVE-1+ve). Together these results confirm mouse LYVE-1,
like its human orthologue is largely restricted to lymphatic vessel
endothelium.
LYVE-1 Expression in Homozygous CD44
Importantly, no CD44 expression was detected in the lymphatics of
wild-type mice or in any tissues in the CD44 Murine LYVE-1 Is Located on the Luminal and Abluminal Faces of
Lymphatic Endothelial Cells--
To distinguish between the
possibilities that LYVE-1 functions as a receptor for the uptake of HA
from the lymph or as a receptor for HA in the tissues immediately
underlying the lymphatics, we used immunoelectron microscopy to
determine whether LYVE-1 is exposed to the luminal or the basolateral
face of lymphatic endothelial cells.
Intriguingly, the analyses (Fig. 8)
revealed expression of LYVE-1 (detected both with immunogold and
horseradish peroxidase conjugates) on both faces of lymphatic
endothelium. Individual endothelial cells were visible as thin
elongated cells containing many intracellular vesicles, that were
devoid of basement membrane and formed characteristic overlapping
cell:cell junctions. The bipolar distribution of LYVE-1 was
consistently observed in multiple different sections, further
supporting the possibility that the receptor is engaged in transport of
HA into the vessel lumen.
In this article we have described the cloning, expression, and
functional characterization of the mouse lymphatic endothelial HA
receptor, LYVE-1, and shown that it mediates both binding and internalization of HA from the surrounding medium. In addition, we have
shown that mouse LYVE-1, similar to its human orthologue, is expressed
almost exclusively on endothelial cells in lymphatic vessels and
capillaries in normal tissues and in a primary lymphatic endothelial
tumor and have demonstrated, using immunoelectromicroscopy, that the
receptor is present on both the luminal and abluminal endothelial
surfaces. We have detailed the degree of sequence homology between the
HA receptors CD44 and LYVE-1 from different species and have predicted
on the basis of key conserved structural features that LYVE-1, in
common with CD44, contains an extended HA-binding domain that is
potentially subject to regulatory control. Finally, we have presented
evidence that the pattern of LYVE-1 expression is not significantly
altered in homozygous CD44 In a previous article (35) we showed that human LYVE-1 functions as a
specific high-affinity receptor for HA in lymphatic vessel endothelium.
In this present article we have shown using the mouse orthologue that
LYVE-1 also mediates the endocytosis of HA. Using flow cytometry to
quantitate the accumulation of FITC-HA we showed LYVE-1-transfected
293T fibroblasts bind and internalize ligand relatively rapidly, with
~50% of total HA partitioning into a protease-resistant
intracellular compartment within 4 h (see also below). Moreover,
analysis of FITC-HA-loaded cells using both standard fluorescence and
laser scanning confocal microscopy clearly demonstrated that HA and
LYVE-1 co-localize within numerous small vesicles beneath the plasma
membrane. The co-localization of ligand and receptor, coupled with the
observations that HA binding/internalization is inhibited by LYVE-1
polyclonal sera2 and that
untransfected 293T cells fail to internalize FITC-HA, all point to
uptake by means of receptor-mediated endocytosis rather than fluid
phase pinocytosis.
Classical receptor-mediated endocytosis, during which receptor-ligand
complexes become concentrated within membrane pits coated with the
geodesic lattice protein clathrin, is exemplified by transferrin, low
density lipoprotein, and immunoglobulin Fc receptors and receptor
tyrosine kinases such as epidermal growth factor receptor and
fibroblast growth factor receptor (49-51). In common with other
receptors that utilize the coated pit pathway these all have
cytoplasmic tails that contain either tyrosine-based motifs
(YXX An alternative possibility is that LYVE-1 mediates the uptake of HA
via caveolae. These are small invaginations of the plasma membrane lined with the 22-kDa membrane-anchored protein caveolin (57)
that mediate endocytosis and transcellular transport by a mechanism
that is distinct from the coated pit pathway (58). It is interesting to
note that caveolin-mediated transport is particularly apparent in
endothelial cells and that the LYVE-1 cytoplasmic tail contains the
hydrophobic motif YXXXFXF, that approximates the
consensus found within proteins that bind to caveolin (59). Further
experiments to investigate the possible association between LYVE-1 and
caveolae are currently in progress.
The capacity to mediate HA-internalization suggests a physiological
role for LYVE-1 in lymphatic HA turnover. The majority of HA turnover
in tissues such as the skin and digestive tract is known to occur in
draining lymph nodes. This process is highly efficient and studies have
shown that some 80% or more of the HA entering afferent lymphatics is
degraded during passage through the nodes, the remaining 10-20% being
rapidly cleared by the liver and hydrolyzed to the level of small
saccharides (1, 3, 4). However, recent evidence points to the
oligomeric HA receptor "HARE" (HA receptor for endocytosis, see
Ref. 60) rather than LYVE-1 as the major receptor for HA-uptake in
lymph node.
Originally thought to be a liver-specific endothelial HA receptor (LEC
HAR, Refs. 5, 6, and 61), HARE is also expressed abundantly in spleen
and lymph node medullary sinuses (60). In common with HARE-mediated
uptake of HA in rat liver cells (62), the uptake and degradation of HA
in lymph node (63) can be blocked by heparin and chondroitin sulfate,
yet as shown in this present article neither glycosaminoglycan blocks
binding/uptake of HA by LYVE-1. Moreover HARE, which recycles rapidly
via clathrin-coated pits, can transport HA at a rate (250 molecules/s/cell) similar to that reported for professional scavengers
such as the asialoglycoprotein receptor and the macrophage mannose
receptor (62). Although it is difficult to make a quantitative
comparison of endocytic rates between the HARE receptor and LYVE-1, we
estimate using previously published data (62) that the rate of
LYVE-1-mediated HA uptake is considerably lower (50% of total HA
internalized/4 h, 37 °C) than that of HARE (90% of total HA
internalized/1.5 h, 37 °C). Indeed LYVE-1 appears to mediate HA
internalization at a rate that is closer to that reported for its
closest homologue CD44 in primary chondrocytes (14% of total HA/8 h;
20% of total HA/8 h) and chondrosarcomas (55, 56). Based on these
considerations we consider it unlikely that LYVE-1 plays a significant
role in the rapid uptake and degradation of HA within lymph nodes and suggest HARE rather than LYVE-1 is the primary receptor for lymphatic HA degradation.
What then is the likely role of LYVE-1 in the lymphatic system? Rather
than facilitating the degradation of HA in lymph node, we suggest
LYVE-1 may be involved in its transport across lymphatic endothelium,
specifically the movement of tissue HA from interstitium to lymph. This
hypothesis is supported by our finding (using immunoelectron microscopy) that LYVE-1 is present on both the luminal and abluminal faces of lymphatic capillaries, a disposition that suggests shuttling across the endothelium or "transcytosis." Prominent examples of receptors involved in transcytosis are the Lastly, it is possible that LYVE-1 regulates the entry of leukocytes or
tumor cells into the lumen of afferent lymphatic capillaries. For
example, in the skin, resident CD44+ve epidermal Langerhans
cells are known to migrate to draining lymph nodes in response to
proinflammatory cytokines and HA breakdown products produced during
tissue inflammation (32). The initial entry of these cells to the lumen
of lymphatic capillaries could conceivably be facilitated by
interaction with LYVE-1·HA complexes on the abluminal face of the
endothelium or in overlapping cell junctions. A similar mechanism could
regulate the entry of metastasizing tumor cells, many examples of which
disseminate to regional lymph nodes in human cancers. Such hypotheses
are amenable to testing by experiments in animal models using LYVE-1
antibodies (see e.g. Refs. 36-40) or soluble LYVE-1 Fc
fusion protein as adhesion blocking reagents. These studies and the
construction of a LYVE-1 knockout mouse will ultimately determine the
true physiological function of LYVE-1 in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
subunits that
clears not only HA but also chondroitin and heparan sulfate from the
circulation (6). While it is clear that HA can rapidly permeate the
lymphatics in skin and other tissues (7), the mechanisms responsible
for its transport across lymphatic endothelium, and the receptors
involved in its uptake and transport within lymphatic vessels are all unknown.
sheets flanked by two
short
helices and stabilized by two disulfide linkages enclosing a
central hydrophobic core (12, 13). Members of the Link superfamily
include versican (14), the cartilage structural proteins aggrecan and
link protein (15), the brain proteoglycans neurocan (16) and brevican
(17), the inflammation-associated TSG-6 protein (18), and the integral membrane glycoprotein CD44; until recently the only known cell surface
(Link superfamily) HA receptor (19). Expressed on a variety of cell
types, the CD44 molecule engages in multivalent HA binding that is
tightly regulated by glycosylation, so-called "inside-out"
signaling and receptor clustering/oligomerization (20-24). Although
inactive on normal circulating leukocytes, CD44 can be induced to bind
HA in response to inflammatory cytokines (25-27). Binding to HA is
thought to direct the extravasation of leukocytes in inflamed tissues,
where CD44 engages HA induced on or below the surface of vascular
capillary endothelial cells (28-31) and to mediate dendritic cell
migration in inflamed skin (32). Yet the unique involvement of CD44 in
the aforementioned processes has been called into question by the
demonstration that homozygous CD44
/
mice contain no
obvious defects in either the vascular or lymphatic systems (33, 34).
These factors have highlighted the likelihood that novel HA receptors
must occur within the immune system and elsewhere and have prompted a
search for such candidates within the human genome.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Knockout
Mice--
Homozygous CD44
/
knockout mice (33) bred on the
C57Bl/6 background and wild-type C57Bl/6 controls were obtained from
the MRC Center for Inflammation Research, University of Edinburgh, UK.
Mice were sacrificed by cervical dislocation and the organs dissected
and formalin-fixed by Dr. Ian Dransfield, (University of Edinburgh,
United Kingdom). These were prepared for microscopy as described below.
-32P]dCTP (Amersham Pharmacia
Biotech) by random hexamer-primed labeling (Merck). Blots were washed
according to the manufacturer's instructions in 2 × SSC, 0.05%
SDS at room temperature for 30 min and in 0.1 × SSC, 0.1% SDS at
50 °C for 40 min followed by autoradiography. After stripping in
0.5% SDS (100 °C), blots were re-probed with human
-actin
cDNA (supplied by the manufacturer).
/
knockout mice, fixed in PBS, 4%
paraformaldehyde, and embedded in paraffin wax. Prior to staining,
sections were dewaxed and rehydrated by successive incubation in
CitroclearTM (2 × 5 min) 100% industrial methylated
spirit (2 × 5 min), 50% industrial methylated spirit (5 min),
and water (5 min). Antigen retrieval was performed by microwave
treatment (95-100 °C, 10 min) in 0.1 M Tris, 2 mM EDTA, pH 9.0. Sections were then blocked by incubation
in PBS, 5% fetal calf serum for 5 min and treated with a peroxidase
quenching agent (DAKO) for 5 min prior to incubation with rabbit
polyclonal anti-mouse LYVE-1 (1:400) for 45 min. After washing with
PBS, slides were incubated with anti-rabbit Ig peroxidase conjugate
(Envision kit, DAKO) for a further 45 min and developed with
diaminobenzidine (DAKO) before counterstaining with hematoxylin. All
incubations were performed at room temperature.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and deduced amino acid sequence of
murine LYVE-1 and comparison with the human orthologue.
Panel A shows nucleotide (nucleotides 1-1440) and deduced
amino acid sequence from the 1516-base pair mouse LYVE-1 cDNA
identified by BlastSearching the mouse EST data base and subsequently
cloned from mouse lung cDNA (see "Experimental Procedures").
The predicted N-terminal leader and C-terminal transmembrane anchor are
underlined and two motifs for potential
N-glycosylation are boxed. Panel B shows an
alignment of the amino acid sequences for mouse LYVE-1, human LYVE-1,
and human CD44 generated with the GCG programs Pileup and PrettyPlot
with similar residues highlighted in yellow. The solid
blue line indicates the consensus Link module. Key cysteine
residues are highlighted and indicated by colored circles.
These are as follows; the four highly conserved structural cysteines of
the Link module (red); the two conserved flanking cysteines
essential for folding and function of CD44 Link (green); a
conserved cysteine within the transmembrane anchor implicated for
receptor dimerization and HA-binding in CD44 (violet) and a
seventh free cysteine unique to LYVE-1 (blue). Conserved
residues within the LYVE-1 Link module equivalent to those implicated
in CD44 HA-binding are highlighted with orange arrowheads.
In addition, a conserved tract of basic residues downstream of the Link
module in mouse and human LYVE-1 and a similar tract in CD44 that forms
an extension to the HA-binding domain are highlighted with pink
arrowheads.
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Fig. 2.
Characteristics of murine LYVE-1 binding to
immobilized and soluble HA and competition with free
glycosaminoglycans. Soluble mouse LYVE-1 Fc fusion proteins and
(full-length) LYVE-1-transfected 293T cells were assayed for their
capacity to bind HA using an ELISA or fluorescence microscopy (see
"Experimental Procedures"). In panel A, mouse LYVE-1 Fc,
human LYVE-1 Fc, and Syndecan 2 Fc (negative control) immobilized in
microtiter plates were incubated with biotinylated HA and the extent of
binding determined with streptavidin peroxidase. Binding of mouse
LYVE-1 Fc in the presence of a 20-fold molar excess of unlabeled HA was
also included as a control for ligand specificity. In panel
B, mouse LYVE-1 Fc, human LYVE-1 Fc, and ICAM-2 Fc
(negative control) were incubated with HA immobilized in microtiter
plates and binding was determined with peroxidase-conjugated anti-human
Fc antibody. In panel C, the binding of mouse LYVE-1 Fc to
immobilized HA was assayed in the presence or absence of free HA or the
glycosaminoglycans chondroitin 4-SO4 (C4S),
chondroitin 6-SO4 (C6S), or heparan
SO4 (HS). Data in each case are the mean ± S.E. of three replicate determinations. In panel D, 293T
cells transfected with either control vector or with mouse LYVE-1
cDNA were incubated with either rabbit anti-mouse LYVE-1/FITC
anti-rabbit IgG, or with FITC-labeled HA and viewed with a fluorescence
microscope.
80%) by a 500-fold excess of unlabeled HA and no uptake was
observed with untransfected 293T cells (Fig. 3A), the
internalization of FITC-HA most likely represents LYVE-1
receptor-mediated endocytosis rather than simple pinocytosis. Further
quantitation of the FITC-HA internalized by cells in these experiments
showed that protease treatment sufficient to cleave 90% of cell
surface LYVE-1 released only 56% of the accumulated FITC-HA (Fig.
3B). The intracellular localization of the residual
papain-resistant HA was confirmed by immunofluorescent microscopy which
revealed FITC-HA within numerous small vesicles beneath the plasma
membrane (Fig. 3C). Furthermore, these vesicles were also
shown to contain internalized LYVE-1 when FITC-HA-loaded cells were
permeabilized with saponin for fluorescent antibody staining and
confocal microscopy (Fig. 3D, top panels). Similar uptake of
FITC-HA was also observed in experiments with human LYVE-1-transfected
293T cells (Fig. 3D, bottom panels, and data not shown).
These results provide clear evidence that LYVE-1 promotes both binding
and internalization of HA through receptor-mediated endocytosis.
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Fig. 3.
LYVE-1 mediates HA internalization.
Human 293T cells were transiently transfected with full-length murine
or human LYVE-1 cDNA to assess the capacity of the receptor to
internalize FITC-labeled HA. Measurement of internalized HA was made by
flow cytometry after removal of surface bound LYVE-1/HA with papain as
described under "Experimental Procedures." Panel A shows
the time course of FITC-HA total accumulation ( ) and internalization
(
) by murine LYVE-1-transfected and control vector-transfected cells
(
) at 37 °C. The level of FITC-HA total accumulation in the
presence of a 500-fold molar excess of unconjugated HA (
) is shown
as a control for binding specificity. Panels B and
C show a comparison of the levels of FITC-HA accumulation
and cell surface LYVE-1 in 5-h incubated mouse LYVE-1-transfected 293T
both before and after papain treatment, assessed, respectively, by flow
cytometry and fluorescence microscopy. In panel C,
internalized FITC-HA is visible within intracytoplasmic vesicles below
the plasma membrane. Panel D shows the localization of
FITC-HA (green) and Texas RedTM-labeled LYVE-1
in 5-h incubated mouse LYVE-1-transfected 293T (top) and
human LYVE-1-transfected 293T (bottom) analyzed by laser
scanning confocal microscopy. Intracellular vesicles containing
endocytosed LYVE-1 and FITC-HA appear yellow.
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Fig. 4.
Northern blot hybridization of murine LYVE-1
cDNA. RNA blots containing 2 µg of poly(A)+ RNA
per lane from each of the tissues shown were hybridized to a
32P-labeled mouse LYVE-1 extracellular domain DNA probe
(upper panel), or actin probe (lower panel),
and washed at high stringency prior to autoradiography (see
"Experimental Procedures"). The sizes (kilobases) and migration
positions of RNA calibration markers are shown at the left
of the figure.
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Fig. 5.
Specificity of an antiserum to murine
LYVE-1. The specificity of a rabbit polyclonal antiserum generated
against soluble mouse LYVE-1 Fc was assessed in a microtiter plase
binding assay. Wells were coated with mouse LYVE-1 Fc ( ,
), human
LYVE-1Fc (
), or CD44 Fc (
) were incubated with appropriately
diluted immune (
,
,
) or control preimmune serum (
), and
binding detected with peroxidase-conjugated anti-rabbit Ig as described
under "Experimental Procedures." Data are the mean ± S. D. of triplicate determinations.
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Fig. 6.
Immunoperoxidase staining of LYVE-1 in normal
and homozygous CD44 /
mouse tissues. LVYE-1
was immunolocalized in a range of wild-type CD44+ and
homozygous CD44
/
mouse tissues and in adjuvant-induced
lymphangioma by immunoperoxidase staining with a rabbit polyclonal
LYVE-1 antiserum. The tissue sections shown are large intestinal mucosa
(panel A), normal lung (panel B), cardiac smooth
muscle (panel C), and adjuvant-induced lymphangioma
(panel D) from wild-type (CD44+) C57/Bl6 and
Balb/c mice, respectively. The tissue sections in panels E
and F are large intestinal mucosa and lung from homozygous
CD44
/
C57/Bl6 mice. Individual lymphatic vessels
(intense brown staining) are in each case indicated with black
arrows and bronchiole/arterioles (no staining) are indicated with
red arrows. Some alveolar lining cells in lung (panel
B) were also stained. Nuclei appear in blue.
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Fig. 7.
Immunofluorescent localization of LYVE-1 in
lymphatic but not vascular endothelium. Tissue sections from
normal wild-type mice were double-stained with antibodies to LYVE-1 and
the vascular endothelial marker CD34 prior to detection with Texas
RedTM or Alexa L88-labeled anti-Ig, respectively, as
described under "Experimental Procedures." Tissues shown are small
intestine (panel A), liver (panel B), and cardiac
smooth muscle (panel C). In A,
LYVE-1+ve lacteal lymphatic vessels (red) within
individual villi are surrounded by a necklace of LYVE-1 ve
blood capillaries (green). In panels B and
C, LYVE-1+ve lymphatic vessels (red)
are located at the perimeter of large CD34+ve blood vessels
(green).
/
Knockout
Mice--
Recent reports demonstrating apparently normal HA metabolism in
CD44
/
knockout mice suggest the possibility that
additional compensatory HA receptors may be up-regulated in these
animals. To test the hypothesis that LYVE-1 might fulfill such a
compensatory role we compared its expression pattern in adult tissues
prepared from wild-type and CD44
/
animals by means of
immunohistochemical staining. The results revealed equally intense
staining of lymphatic vessels in both C57/Bl6 wild-type and C57/Bl6
CD44
/
mouse tissues (Fig. 6 compare panels A
and B with panels E and F). Moreover
no gross differences in the level or distribution of LYVE-1 staining or
in the numbers of lymphatic vessels were apparent when a comprehensive
panel of different tissues was compared between the two mouse
populations (not shown).
/
mice as
assessed by immunostaining with the monoclonal antibody IM-7 (not
shown). Although our analyses do not exclude the possibility of more
subtle alterations in LYVE-1 expression in CD44
/
tissues or of alterations occurring during embryogenesis, the results
generally indicate that LYVE-1 is unlikely to compensate for the loss
of CD44 expression and that additional as yet unidentified HA-receptors
are likely to be involved.
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Fig. 8.
Immunoelectron microscopy of LYVE-1 in
lymphatic endothelium. Mouse intestinal tissue was stained with an
antibody to mouse LYVE-1 followed by either horseradish peroxidase
(panel A) or immunogold-conjugated anti-Ig (panels
B and C) as described under "Experimental
Procedures." Panels A and B show sections
through lymphatic vessels where the LYVE-1 antibody has decorated both
the luminal and abluminal surfaces of the endothelial cells
(arrows). Panel C shows an adjacent area of the
same section as panel B depicting part of a fenestrated
blood capillary (L, lumen) that does not stain for
LYVE-1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
knockout mice, implying
that additional as yet unidentified HA receptors are present within the genome.
, or NPXY, where X represents
any amino acid and
represents a bulky hydrophobic residue) or
dihydrophobic repeats (e.g. LL, LV) which promote the
formation of coated vesicles through binding the AP2
clathrin-adapter/clathrin protein complex (51, 52). However, no
such sequences are present within the cytoplasmic tail of either mouse
LYVE-1 or human LYVE-1. Furthermore, in preliminary studies, incubation
of LYVE-1-transfected 293T cells in hyperosmolar conditions (0.4 M buffered sucrose) similar to those previously shown to
inhibit clathrin-mediated endocytosis in fibroblasts (53) did not
significantly affect LYVE-1 mediated HA
uptake.3 These properties are
similar to CD44, which mediates uptake of HA by fibroblasts,
macrophages, and chondrocytes by a mechanism that is not understood but
which involves neither coated pit formation nor pinocytosis
(54-56).4 Hence the
possibility exists that both LYVE-1 and CD44 mediate endocytosis
via novel pathways.
1-acidic
glycoprotein receptor on vascular endothelium (64) and the polymeric Ig
receptor on mucosal epithelium (65). Recent observations that HA
applied to the skin can rapidly enter the dermal lymphatics (7) are also consistent with the occurrence of a pathway for rapid
transendothelial transport of this glycosaminoglycan in
vivo. Efficient macromolecular transport is of course one of the
key roles of the lymphatic endothelium (66) and the process has been
studied in detail by means of tracer labeling experiments with isolated
perfused renal lymphatics (67). Moreover, ultrastructural analyses of
lymphatic endothelia have consistently revealed an abundance of
intracytoplasmic vesicles, and clusters of these have been suggested to
form stable channels that facilitate trans-endothelial transport (68).
Regardless of whether HA permeates the lymphatic endothelium by means
of channels or by conventional vesicular transfer, it is quite likely that LYVE-1 is directly involved in the process. Clearly, experiments with primary LYVE-1+ve lymphatic endothelial cells will
help clarify these issues.
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FOOTNOTES |
---|
* This work was supported in part by the United Kingdom Medical Research Council and by project Grants 99-250 and 00-311 from the Association for International Cancer Research (to D. J.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ311501.
¶ Supported by a Wellcome Trust grant for equipment.
To whom correspondence should be addressed: Medical Research
Council Human Immunology Unit, Institute for Molecular Medicine, John
Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom. Tel.:
44-1865-222313; Fax: 44-1865-222502; E-mail: djackson@
enterprise.molbiol.ox.ac.uk.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M011004200
2 R. Prevo and D. G. Jackson, unpublished observation.
3 R. Prevo and D. G. Jackson, unpublished data.
4 R. Tammi, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: HA, hyaluronan; LYVE-1, lymphatic vessel endothelial HA receptor-1; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; HARE, HA receptor for endocytosis.
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