From the Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9069
Received for publication, September 19, 2000, and in revised form, November 14, 2000
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ABSTRACT |
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We isolated a novel molecule (DC-HIL) expressed
abundantly by the XS52 dendritic cell (DC) line and epidermal
Langerhans cells, but minimally by other cell lines. DC-HIL is a type I
transmembrane protein that contains a heparin-binding motif and an
integrin-recognition motif, RGD, in its extracellular domain (ECD). A
soluble fusion protein (DC-HIL-Fc) of the ECD and an immunoglobulin Fc
bound to the surface of an endothelial cell line (SVEC). This binding induced adhesion of SVEC to its immobilized form. Sulfated
polysaccharides (e.g. heparin and fucoidan) inhibited
binding of soluble DC-HIL-Fc and adhesion of SVEC. By contrast, an
integrin inhibitor (RGDS tetramer) had no effect on binding to SVEC,
but prevented adhesion of SVEC. This differential RGD requirement was
confirmed by the finding that DC-HIL-Fc mutant lacking the RGD motif
can bind to SVEC but is unable to induce adhesion of SVEC. Furthermore,
DC-HIL appears to recognize directly these sulfated polysaccharides. These results suggest that DC-HIL binds to SVEC by recognizing heparan
sulfate proteoglycans on endothelial cells, thereby inducing adhesion
of SVEC in an RGD-dependent manner. We propose that DC-HIL serves as a DC-associated, heparan sulfate
proteoglycan-dependent integrin ligand, which may be
involved in transendothelial migration of DC.
Dendritic cells (DCs1)
are a member of antigen-presenting cells (APC) family, which are
characterized morphologically by the extension of long, lamellar
dendrites (1). DC are distinguished from other APC (e.g.
macrophages and B cells) by an unsurpassed potency in presenting
antigens to naive T cells, thereby most efficiently initiating primary
T cell-mediated immune responses (1). DC are widely distributed but
comprise only a minuscule fraction (typically <5%) of the total cell
population of a given peripheral tissue. After capturing antigens,
tissue DC undergo maturation, losing endocytic capacity but acquiring
increased immunostimulatory capacity (2). During maturation, DC migrate from peripheral tissues to T cell areas of secondary lymphoid organs
where they activate T cells (2-4). Thus, DC migration is a critical
step in initiating antigen-specific immune responses.
Several adhesion molecules (e.g. cutaneous
lymphocyte-associated antigen (5), lymphocyte function-associated
antigen-1/CD11a (6), intercellular adhesion molecule-1 (ICAM-1)/CD54
(6), CD44 (7), E-cadherin (8), and Study on the unique properties of DC at molecular levels has been
hampered by the paucity of DC within tissues and the relative difficulty in maintaining their viability ex vivo. To
overcome this problem, we have previously established long-lived DC
lines, designated XS series, derived from the skin of newborn BALB/c mice (11). These XS cells retain many important features of LC,
including their morphology, surface phenotype (B7-2, major histocompatibility class II, CD11b, ICAM-1, CD49d/e, and E-cadherin), responsiveness to cytokines, expression of cytokines and their receptors, and potent APC function (11, 12). Having these cell lines,
our goal is to illuminate the molecular properties that distinguish
DC/LC from other APC, and our principal strategy has been to identify
and define genes and gene products expressed selectively by DC and LC.
We have previously isolated five novel molecules using a subtractive
cDNA cloning method in which the cDNA library prepared from the
XS52 DC line was subtracted with mRNA isolated from the J774
macrophage line. Two of these molecules, termed dectin-1 and dectin-2,
are expressed selectively by XS52 DC (13). They are type II
transmembrane glycoproteins that belong to the
Ca2+-dependent (C-type) lectin superfamily and
may serve as costimulatory molecules that are required for efficient
activation of T cells by DC. We now focus on the third gene, originally
designated as 2B4, which encodes a type I transmembrane protein
that can function as a ligand for integrins most likely through
recognition of heparan sulfate proteoglycans (HSPG) abundantly
expressed on endothelial cell surfaces (14). Based on its expression
pattern and function, we have renamed this molecule DC-associated,
HSPG-dependent integrin ligand (DC-HIL). Here we report its
structural, biochemical, and functional properties and discuss its
potential roles in DC-endothelial cell adhesion.
Animals--
Female BALB/c mice (6-10 weeks old) were purchased
from Harlan (Indianapolis, IN) and housed in the pathogen-free facility of the Animal Resource Center at The University of Texas Southwestern Medical Center. To study tissue distributions and Langerhans cell (LC)-specific expression of DC-HIL mRNA, skin and other tissues were excised from mice sacrificed by overdose of methoxyflurane inhalation.
Cell Lines--
XS52 is a long term DC line established from the
epidermis of BALB/c mouse (12). These cells were maintained and
expanded in complete RPMI 1640 supplemented with mouse recombinant
granulocyte/macrophage-colony stimulating factor (1 ng/ml) and NS47
fibroblast culture supernatant (10% v/v) as a source of
colony-stimulating factor (12). J774 and Raw macrophages, J558 myeloma
cells, BW5147 thymoma cells, HDK-1 (Th1) and D10 (Th2) T cells, SVEC
mouse vascular endothelial cells, and COS-1 cells were purchased from
the American Tissue Type Collection (ATCC, Rockville, MD). Pam 212 keratinocytes, NS47 fibroblasts, and 7-17 dendritic epidermal T cells
(DETC) were kindly supplied by Dr. Akira Takashima (UT Southwestern
Medical Center).
Epidermal Cell Isolation--
Epidermal cells were isolated from
abdominal skin of BALB/c mice using two sequential trypsin treatments
and enriched for LC by centrifugation over Histopaque (1.083, Sigma
Chemical Co., St. Louis, MO) as described previously (15-17). In some
experiments, LC were depleted from this cell population using anti-Ia
monoclonal antibody (mAb) plus complement treatment as described before
(16).
Isolation of DC-HIL cDNA Clone--
A DC-specific cDNA
library was constructed by subtracting the cDNA library prepared
from the XS52 DC with excess amounts of mRNAs isolated from the
J774 macrophage. Twelve thousand independent clones from this library
were screened by colony hybridization, slot blotting, and Northern
blotting for the genes expressed by the XS52 DC but only minimally or
not at all by J774 macrophages (15, 18). The original clone 2B4 was one
of the 50 clones selected in the above manner. The DNA sequences of
both sense and antisense strands were determined by Taq dye
deoxy termination cycle sequencing on a DNA sequencer (model 373A,
Applied Biosystems, Foster City, CA).
Northern Blotting and RT-PCR Analyses--
Northern blotting was
performed as described previously (15). Briefly, total RNAs (10 µg/lane) isolated from cell lines or mRNA (5 µg/lane) from
mouse organs were run on a vertical agarose gel, transferred onto a
nylon membrane, and hybridized with the 32P-labeled
cDNA probe for DC-HIL, glyceraldehyde-3-phosphate dehydrogenase, or
For RT-PCR, total RNAs were isolated from epidermal cells that were
treated with complement alone or complement plus anti-Ia Ab. After
conversion of the RNAs (1 µg) to cDNA forms by reverse transcriptase (Life Technologies, Inc., Rockville, MD), an aliquot (typically 5%) was used for PCR amplification as described before (19)
by using the following primer sets:
5'-CGACTCCGCCTCCACCTTCAACT-3' and 5'-CACAGCCCATCCACAGCCACAG-3' for
DC-HIL, 5'-TACAGGCTCCGAGATGAACAACAA-3' and
5'-TGGGGAAGGCATTAGAAACAGTC-3' for IL-1 Immunoblotting of DC-HIL Protein--
Anti-DC-HIL peptide Ab was
generated by immunizing rabbits with a synthetic 20-aa peptide of
C-GHEQYPNHMREHNQLRGWS (Alpha Diagnostics Intl., San Antonio, TX) (the
amino-terminal cysteine was attached for thiol coupling) corresponding
to aa 30-48 in the ECD of DC-HIL. After three rounds of immunization,
serum was collected and subjected to affinity purification of
peptide-specific Ab using the same 20-mer peptide as described before
(15).
Whole cell extracts were prepared from several different cell lines by
lysis with 0.3% Triton X-100 in Dulbecco's PBS( Sublocalization of DC-HIL--
XS52 DC were incubated in 0.25 M sucrose for 10 min at 4 °C. The pellet after
centrifugation was resuspended and homogenized in 10 mM
HEPES (pH 7.3) by 5-10 strokes with a 27-gauge needle on a 1-ml
syringe, and then centrifuged for 10 min at 1000 × g. The resulting supernatant was separated into cytosolic and membrane fractions by further centrifugation for 45 min at 50,000 × g at 4 °C. These samples were subjected to immunoblotting
for determining localization of DC-HIL proteins.
For experiments in which cell surface expression of DC-HIL was
assessed, XS52 DC and COS-1 cells transfected either with pCMV5-DC-HIL or a luciferase-expressing vector (pGL3-Control, Promega, Madison, WI)
were surface-biotinylated by incubation with 0.5 mg/ml
Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) for 60 min at 4 °C. After
washing, cell lysates were prepared using 0.3% Triton X-100 and
incubated with streptavidin-conjugated agarose beads (Sigma) by gently
rocking overnight at 4 °C. Following centrifugation, the supernatant
was collected as the streptavidin-unbound fraction, and the beads were
washed extensively with PBS and then eluted by boiling in Laemmli's
sample buffer (streptavidin-bound fraction). DC-HIL or luciferase in
each fraction was detected by immunoblotting with the respective
Ab.
N-Glycosidase Treatment--
The whole XS52 DC lysate was boiled
at 95 °C for 5 min with 1% SDS, and then incubated with or without
5 units/ml N-glycosidase (Roche Diagnostics) in the presence
of 1% Nonidet P-40 (SDS at final concentration of 0.2%) overnight at
37 °C. Samples were analyzed for molecular weights of DC-HIL by
SDS-PAGE followed by immunoblotting.
Immunocytochemistry--
Visualization of DC-HIL proteins in
XS52 DC was performed using anti-DC-HIL Ab and a peroxidase-based
immunostaining kit (LSAB, Dako, Carpenteria, CA) according to
manufacturer recommendations. Briefly, XS52 DC cultured on a coverslip
were air-dried and subjected to the following sequential treatment: 15 min fixation in 4% paraformaldehyde, 20 min permeabilization in 0.5%
saponin, 10 min blocking in goat IgG, and 60 min staining with
anti-DC-HIL Ab (10 µg/ml) in the presence of 0.5% saponin, and
followed by color development using HRP and 3-amino-9-ethylcarbazole
substrate. Subsequently, the specimens were counter-stained by
hematoxylin and mounted onto slides. Images were taken by slide films
using an Olympus BH-2 microscope; these were digitized and image
contrast was adjusted using Photoshop (Adobe, San Jose, CA).
Preparation of DC-HIL-Fc Fusion Protein--
DC-HIL-Fc protein,
consisting of, from the amino-terminus, the extracellular domain (ECD)
of DC-HIL and a Fc region of human IgG1
(hIgG1Fc), was produced in COS-1 cells. The DNA fragment encoding the ECD (aa 34-511) was PCR-amplified with primers containing HindIII and XbaI sites at the 5' and 3'
ends, respectively. Using these sites, the PCR product was
ligated in-frame to the coding sequence of the Fc region sequence (789 bp), which had been cloned into pSecTagB vector (Invitrogen, Carlsbad,
CA) at XbaI and ApaI sites in the 5' and 3'
ends (pSTB-DC-HIL-Fc). An expression vector for Dectin-1-Fc was
constructed by replacement of the ECD in pSTB-DC-HIL-Fc with the ECD of
Dectin-1 (aa 71-213) (15). We have reported previously that the
Dectin-1 recombinant protein tagged with the amino-terminal histidine
binds selectively to T cells (15), whereas the C-terminal Fc fusion
proteins lack the binding activity. The Fc-encoding expression vector
was constructed by insertion of the DNA fragment encoding
hIgG1Fc into the pSecTagA vector using XbaI and
ApaI sites. These expression vectors were introduced into
COS-1 cells by FuGene 6 (Roche Diagnostics). At 96 h after transfection, the culture supernatant was harvested and immunoglobulin fusion proteins were purified by protein A-agarose beads (Life Technologies, Inc.) according to manufacturer recommendations.
Site-directed Mutagenesis--
The RGD sequence of the
pSTB-DC-HIL-Fc was mutated to RAA using the QuikChange site-directed
mutagenesis kit according to the manufacturer recommendations
(Stratagene, La Jolla, CA). Sense and antisense oligonucleotides
(nucleotides 160-193) with the mutation (RGD to RAA) were synthesized
and high pressure liquid chromatography-purified. The sense strand,
5'-CCAGTGTGGAGGAGGGCAGCAGGCAGGTGGAAGG-3' (underlined sequence encodes RAA); and the antisense strand,
5'-CCTTCCACCTGCCTGCTGCCCTCCTCCAACACTGG-3'. The nucleotide
fragment coding entire ECD of DC-HIL was ligated into pBluescript II
KS( Cell Binding Assays of Soluble DC-HIL-Fc--
Binding properties
of soluble DC-HIL-Fc were examined by flow cytometric analyses:
Different cell lines were nonenzymatically harvested and incubated with
typically 10 µg/ml DC-HIL-Fc or control Fc fusion proteins,
Dectin-1-Fc or Fc, in binding buffer (Hanks' balanced salt solution
containing 1.3 mM CaCl2, 0.8 mM
MgSO4, 3% fetal calf serum, and 10 mM HEPES)
for 30 min on ice. After washing, the cells were labeled with 5 µg/ml
biotin-conjugated Fab fragment of goat anti-human Fc Binding Assays of DC-HIL-Fc to Heparin and Fucoidan--
The
96-well ELISA plates were coated with heparin-BSA or fucoidan overnight
at 4 °C. After extensive washing with PBS, 1 µg/ml DC-HIL-Fc or
control Fc fusion proteins were incubated for 60 min at room
temperature. After removing unbound fusion proteins, the wells were
incubated with biotin-conjugated Fab fragment of goat anti-human Fc Adhesion Assays of SVEC to Immobilized DC-HIL-Fc--
SVEC were
metabolically labeled with 3 µCi/ml [3H]thymidine (ICN,
Costa Mesa, CA) in 10% fetal calf serum-Dulbecco's modified Eagle's
medium overnight. After harvest with 0.5 mM EDTA and
subsequent washing with binding buffer, SVEC were counted for
radioactivity by a liquid scintillation counter (usually 2-4
cpm/cell). During labeling of SVEC, the 96-well ELISA plates were
coated with 100 µg/ml goat anti-human Fc Cloning of DC-HIL cDNA and Its Deduced Amino Acid
Structure--
We employed a subtractive cDNA cloning strategy to
isolate genes expressed by XS52 DC but not by J774 macrophage. Fifty
clones were selected and were searched for their identities in the
GenBankTM data base (updated in 1997). Five novel genes were
identified, one of which was designated 2B4 (the original clone name).
The clone 2B4 contained a cDNA insert of 2279 bp, including the
largest open reading frame (nucleotides 91-1815) with sequences that
matched Kozak consensus sequences (20). The open reading frame encoded
a putative polypeptide, termed DC-HIL, consisting of 574 amino acids
(aa) with features typical of type I integral membrane proteins (Fig.
1). DC-HIL consists of a leader sequence (aa 1-19), a long extracellular domain (ECD, aa 20-499), a
transmembrane domain (aa 500-523), and a cytoplasmic domain (aa
524-574). The ECD contains 11 potential N-glycosylation
sites (NX(S/T)) and several putative
O-glycosylation sites based on the stretch of proline-,
serine-, and threonine-rich region (21), and a proline-rich region (aa
320-352) that presumably forms a hinge, as seen in proteins like IgA
(22), which can mediate protein-protein interactions (23). Other
functional motifs identified were an RGD sequence (aa 64-66), an
integrin-binding sequence, and a KRFR sequence (aa 23-26) that matches
a heparin-binding motif composed of a stretch of basic residues
(BBXB, where B represents a basic residue) (24).
The cytoplasmic tail contained an immunoreceptor tyrosine-based activation motif (ITAM, YXXI, aa 529-532) and two lysosomal
targeting di-leucine motifs (LL, aa 548-549 and 566-567).
In a homology search, the aa sequence of DC-HIL had identity to mouse
nmb (GenBankTM accession number: AJ251685) with 99.4% (by the
Clustal method analyzed with the Lasergene Program, DNA Star,
Madison, WI) containing four substitutions of aa residues. The human
nmb (GenBankTM accession number: NM002510), a polypeptide that has
been reported to be expressed preferentially by lowly metastatic
melanoma cells (25), had homology of 71.1% to DC-HIL (Fig.
2). In addition, DC-HIL showed homology
with rat osteoactivin, for which the GenBankTM data annotates
the abundant expression in osteopetrotic bones (88.3%); QNR-71, which
is responsible for melanin production in quail neuroretina cells (26)
(48%); and pMEL17s melanoma markers (27) (24%). Less homology was
identified with lysosome-associated membrane protein (LAMP) family
members (11-13%), such as hLAMP-1 and -2 (28, 29), mCD68 (30), and hDC-LAMP (31). These structural characteristics led us to postulate that DC-HIL is a type I transmembrane protein that is heavily glycosylated, is involved in heparin binding, promotes
integrin-mediated cell adhesion, and is capable of transducing
tyrosine-based signaling.
Cell and Tissue Distributions of DC-HIL mRNA--
Northern
blot analysis showed DC-HIL mRNA (2.9 kb) to be expressed at
relatively high levels by XS52 DC, but only minimally by macrophages
J774 and Raw (Fig. 3A).
Importantly, DC-HIL mRNA was undetectable in other tested cell
lines, including Identification of DC-HIL Protein--
The DC-HIL protein was
characterized biochemically by immunoblotting with affinity-purified
rabbit Ab raised against a synthetic peptide corresponding to aa 30-48
of DC-HIL. This Ab detected under reducing conditions a broad band
migrating between 90 and 110 kDa in COS-1 cells that was transfected
with full-length cDNA for DC-HIL but not with the empty vector
(Fig. 4A). The same Ab recognized in XS52 DC lysates two bands of 95 and 125 kDa. Consistent with Northern blot data (Fig. 3A), DC-HIL protein was
expressed preferentially by XS52 DC, detectable in J774 macrophage and
B16 melanoma cells, but undetectable in J558 myeloma and BW5147 thymoma cells (Fig. 4B). The lower two bands visible in COS-1
transfectants, XS52 DC, and other cell lines were also detected by
control rabbit IgG (data not shown). The estimated molecular size of
the DC-HIL protein was considerably larger than that predicted from the
full-length amino acid sequence (67 kDa), a discrepancy probably due to
glycosylation, because DC-HIL contains 11 putative
N-glycosylation sites (Fig. 1). Indeed,
N-glycosidase treatment of XS52 DC lysate reduced significantly the molecular size of two bands (95 and 125 kDa) (Fig.
4C): A major band (76 kDa) was still larger than the
predicted molecular size, whereas a minor band (66 kDa) was almost
identical to the size. This heterogeneity in DC-HIL proteins may be
produced as a result of differential N- and/or
O-glycosylation, which may also explain the disparity in
molecular size between native DC-HIL in XS52 DC and its recombinant
form in COS-1 cells. DC-HIL is thus expressed preferentially by XS52 DC
as a heavily glycosylated protein.
Subcellular Localization of DC-HIL--
The subcellular
localization was determined by a biochemical method. As shown in Fig.
5A, the 95- and 125-kDa
immunoreactivities were detected exclusively in the membrane fraction,
documenting that DC-HIL is a membrane-integrated polypeptide. To
further separate the membrane fractions, whole cell lysates of
surface-biotinylated XS52 DC were incubated with
streptavidin-conjugated agarose beads and separated into unbound
(cytoplasm) and bound (plasma membrane) fractions. Immunoblotting with
anti-DC-HIL Ab revealed that a majority of DC-HIL proteins was
recovered in the streptavidin-unbound fraction (intracellular
fraction), with less amounts in the plasma membrane fraction. Similar
results were obtained using COS-1 cells transfected with full-length
cDNA for DC-HIL (Fig. 5C). On the other hand,
recombinant luciferase, used as an intracellular marker when expressed
transiently in COS-1 cells, was unrecoverable in the streptavidin-bound
fraction (Fig. 5C), validating the efficacy of our
system.
The intracellular localization was further examined by
immunocytochemistry. When XS52 DC were membrane-permeabilized with saponin, most DC-HIL accumulated in a single, large perinuclear vesicle
and also in small vesicles scattered toward the periphery (Fig.
6). This reactivity was significantly
reduced when cells were stained in the absence of saponin (data not
shown). Together, these results suggest that DC-HIL mostly localize in
the cytoplasm, but is present at lower levels on the surface of XS52
DC.
Binding of Soluble DC-HIL-Fc to Cell Lines--
To study the
function of DC-HIL on DC, we prepared a soluble Fc fusion protein,
DC-HIL-Fc. DC-HIL-Fc was produced in COS-1 cells as a disulfide-linked
homodimer (data not shown), which was highly purified by affinity
chromatography with protein A-agarose, and detected as a single band by
SDS-PAGE and subsequent CBB staining (Fig.
7A). By using this recombinant
protein, together with control proteins consisting of Dectin-1 ECD and
hIgG1Fc (Dectin-1-Fc) or hIgG1Fc (Fc) alone, we
were able to test binding of DC-HIL-Fc to the cell surface by flow
cytometric analysis. Among the cell lines tested, only mouse vascular
endothelial cells (SVEC), mouse melanoma cells (Queens), and dermal
fibroblasts (NS47) showed significant binding (Fig. 7B). No
binding was detected with the control Fc fusion proteins Dectin-1-Fc or
Fc. We focused on characterizing the interaction of DC-HIL and SVEC,
because this system may provide a useful model for understanding
transendothelial migration of DC.
Because the ECD of DC-HIL contained both RGD and heparin-binding motifs
(Fig. 1), we determined which motif is responsible for the binding of
soluble DC-HIL-Fc. Inhibition studies showed that synthetic
RGD-containing peptide (1 mM RGDS) fails to inhibit binding
on SVEC (Fig. 7C), whereas binding was reduced significantly by 1 µg/ml heparin (Fig. 7C). Further experiments
demonstrated that other highly sulfated polysaccharides such as dextran
sulfate (500 kDa) and fucoidan were also inhibitory in a
dose-dependent manner, whereas the less sulfated
polysaccharide, chondroitin sulfate A, and a nonsulfated polysaccharide
mannan were much less or not inhibitory at all (Fig. 7D).
These results suggest that HSPG, but not RGD, is involved in the
binding of DC-HIL ECD to SVEC. We then tested whether DC-HIL binds
directly to heparin or fucoidan. DC-HIL-Fc showed
dose-dependent binding to wells coated with either
heparin-BSA or fucoidan (Fig.
8A). Binding to heparin was
inhibited by heparin, fucoidan, and dextran sulfate, but less so by
chondroitin sulfate A or mannan (Fig. 8B). On the other
hand, binding to fucoidan was not inhibited by heparin (Fig. 8C). These data suggest that a class of binding sites on
DC-HIL is shared by heparin, fucoidan, and dextran sulfate, and a
second class not recognized by heparin. Thus, DC-HIL binds to HSPG
probably via multiple sites, including a heparin-binding motif.
Recognition of HSPG on EC by DC-HIL was supported by the findings that
just preincubation of SVEC with these inhibitors did not affect binding of DC-HIL-Fc (Fig. 8D), and that pretreatment with chlorate,
a reversible inhibitor of glycosaminoglycan sulfation, significantly reduced binding (Fig. 8E). It is therefore highly likely
that DC-HIL binds to SVEC through recognition of HSPG on the surface of
SVEC.
Adhesion of SVEC to Immobilized DC-HIL-Fc--
The presence of the
RGD motif in the ECD of DC-HIL led us to test the possibility that
binding of DC-HIL-Fc to SVEC leads to cell adhesion. We employed an
in vitro adhesion assay in which cells were allowed to
adhere to the recombinant proteins-immobilized plates. Typically
50-60% of input SVEC adhered to plates coated with DC-HIL-Fc in a
dose-dependent (Fig.
9A) and
time-dependent manner (Fig. 9B), but they
adhered to Dectin-1-Fc or Fc-coated plates only negligibly (data not
shown). Maximal adhesion of SVEC was observed at 45- to 60-min
incubation and at 37 °C temperature. Importantly, an RGDS peptide
but not control peptide RPKP inhibited adhesion of SVEC in a
dose-dependent manner, with maximum inhibition (90%) at
0.5 mM (Fig. 9C). Moreover, chelation of
divalent cations by EDTA, which interferes with the integrin subunit
assembly (34, 35), also inhibited adhesion
dose-dependently. These data strongly suggest that SVEC
adhere to immobilized DC-HIL-Fc through recognition of its RGD sequence
by some members of integrin family, which are expressed on EC and play
pivotal roles in the adhesion to leukocytes (36, 37). Of interest was
that the adhesion of SVEC was also inhibited by most of the sulfated
polysaccharides that could inhibit soluble DC-HIL-Fc binding on SVEC
(Fig. 7D). On the other hand, adhesion of SVEC to
vitronectin, a well known extracellular matrix component containing RGD
sequence as well as heparin-binding motifs (38, 39), was blocked
completely by RGDS peptide but not affected by any of these sulfated
polysaccharides (data not shown). Thus DC-HIL-mediated adhesion is more
dependent on HSPG recognition than vitronectin.
Heparin inhibited binding of soluble DC-HIL-Fc to SVEC, and blocked
adhesion of SVEC to immobilized DC-HIL-Fc. However, RGDS had no effect
on binding of soluble protein to SVEC, but prevented cell adhesion to
immobilized DC-HIL-Fc. Therefore, we investigated whether DC-HIL-Fc
interaction with sulfated polysaccharides was needed prior to
integrin-mediated adhesion. Notably, BW5147 thymoma cells, which lacked
the ability to capture DC-HIL-Fc (Fig. 7B) did not adhere to
immobilized DC-HIL-Fc (Fig. 10). On the
other hand, BW5147 cells showed constitutive adhesion to vitronectin, which was comparable to those of SVEC and Queens (Fig. 10), indicating a good correlation between cell binding and cell adhesive capacities of
DC-HIL. Moreover, SVEC once adhered to immobilized DC-HIL-Fc were no
longer removable by incubation with sulfated polysaccharides (data not
shown). These data suggest that RGD-dependent cell adhesion is preceded by interaction of this molecule with HSPG.
Binding and Adhesion Profiles of RGD-deficient DC-HIL-Fc
Mutant--
To more definitively demonstrate involvement of RGD
sequence in adhesion of SVEC to immobilized DC-HIL-Fc, we generated
RGD-deficient DC-HIL-Fc, termed RAA mutant, by altering RGD to RAA
using site-directed mutagenesis. This strategy has successfully
documented the critical role of RGD in the interaction of several
different molecules with integrins (40-42). RAA mutant DC-HIL-Fc was
produced in COS-1 cells and purified as highly as the wild-type was
(Fig. 11A). When tested for
its binding ability to SVEC, the RAA mutant showed an almost identical
binding profile to the wild-type, including the inhibition by heparin
(Fig. 11B). In sharp contrast, when tested for the adhesive
activity, the mutant was unable to induce adhesion of SVEC (Fig.
11C). Thus, these results clearly demonstrate that the
RGD-integrin interactions are indispensable for the adhesion of SVEC to
immobilized DC-HIL-Fc but not for the binding of soluble DC-HIL-Fc to
SVEC.
In searching for DC-specific genes that provide unique properties
of DC, a number of DC-specific or -associated, novel molecules have
been identified, e.g. DC-SIGN (43), Langerin (44), and DC-LAMP (31). In fact, functional studies on these molecules demonstrated that they are involved in establishing distinct properties of DC. In addition to dectin-1 and dectin-2, we added to the current list of DC-associated molecules a new member, termed DC-HIL, that may
contribute to unique mechanisms for transendothelial migration of
DC.
DC-HIL showed significant homologies with
melanoma/melanosome-associated membrane proteins and lower degree with
members of the LAMP family (Fig. 2). In addition, its structural and
biochemical features are also in accordance with these membrane protein
families, which include: 1) a proline-rich hinge region containing
numerous O-glycans, 2) highly N-glycosylated
forms, 3) lysosomal targeting motifs, 4) a tyrosine-based signaling
motif in a cytoplasmic tail, 5) high accumulation in a perinuclear
region (Fig. 6) (28, 29), and 6) relatively low expression on the cell
surface (Fig. 5) (45-47). Because DC do not have melanosomes, we
speculate that DC-HIL may be a newly identified member of the LAMP
family and may play roles in the intracellular events.
In this study we focused on characterizing the function of the ECD
rather than the intracellular domain of DC-HIL. The ECD contains an RGD
motif and a heparin-binding motif, both of which are frequently found
in adhesion molecules, leading us to hypothesize a role as an adhesion
molecule. Using the recombinant fusion protein DC-HIL-Fc, we found two
independent functions. The soluble form of DC-HIL-Fc binds to limited
cell types, including EC, and the immobilized form of DC-HIL-Fc
promotes adhesion of SVEC. In further studies, we demonstrated that: 1)
binding of DC-HIL to cells is inhibited by heparin and fucoidan; 2)
DC-HIL recognizes heparin and fucoidan; 3) SVEC adhesion to immobilized
forms absolutely requires RGD recognition; and 4) the adhesion is
induced by the recognition of HSPG. Based on these findings, we propose
dual roles of DC-HIL: first, recognition of HSPG, which functions to specify the target cells; second, a ligand for integrins, which mediates an RGD-dependent cell adhesion only when DC-HIL
binds to cells. Although it is unclear at present how the interaction of DC-HIL and HSPG on EC induces an RGD-dependent cell
adhesion, two possible molecular models are considered: First, the
interaction activates integrins expressed on SVEC, resulting in
recognition of the RGD. Second, the interaction induces conformational
changes of DC-HIL, thereby exposing the cryptic RGD sequence, as
suggested for von Willebrand factor and thrombospondin (48, 49).
Further studies will be required for determining which model accounts for the DC-HIL-induced cell adhesion.
This working model shares some features with the two-step adhesion
cascade for neutrophil/EC interactions, in which the primary adhesion
involves three members of selectin family and their oligosaccharide ligands. Subsequently, secondary adhesion involves Heparan sulfate, as products of the enzymatic degradation of HSPG, is
rapidly released from cell surface and extracellular matrices under
conditions of inflammation and tissue damages (53, 54). Recently, this
heparan sulfate has been demonstrated to induce phenotypic as well as
functional maturation of DC characterized by enhanced surface
expression of major histocompatibility class II, CD80 (B7-1), and CD86
(B7-2), lowered rate of antigen uptake, and increased allostimulatory
capacity (55). Although it is not defined how heparan sulfate induces
maturation signals to DC, direct binding of heparan sulfate to its
putative receptors on DC is thought to be one of the most possible
mechanisms. Because a toll-like receptor, which is expressed on DC,
binds to peptidoglycan, including lipopolysaccharide and heparan
sulfate (56, 57), this receptor is proposed to be a candidate
responsible for inducing maturation signals. It is thus tempting to
propose that DC-HIL is a second candidate for such a receptor, which
may transduce maturation signals to DC via the residues ITAM on its
cytoplasmic tail.
We were unable to block adhesion of XS52 DC to SVEC by pretreatment of
SVEC with DC-HIL-Fc even at 75 µg/ml (data not shown). Because our
anti-DC-HIL antibody does not recognize proteins expressed on the cell
surface of XS52 DC (probably the epitope for the Ab is cryptic in the
molecule), we have not examined blocking of SVEC adhesion by Ab.
Successful blocking may require antibodies reactive with the DC-HIL
proteins on cell surface. It thus remains uncertain to which extent
DC-HIL contributes to the DC/EC adhesion under physiological
conditions. Nevertheless, DC-HIL is potentially involved in endothelial
adhesion of DC, and its preferential expression of the protein in DC
suggests that it plays critical roles in the unique mechanisms for DC migration.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 integrins (9))
regulate the migration of DC through the entire life cycle. For
example, blood-circulating precursors of Langerhans cells (LC), which
are DC that reside in epidermis, attach to the blood vessel wall prior to initiating transendothelial migration into the epidermis where they
develop into APC with phenotypes distinct from other members of the DC
family. The LC homing and anchoring in the epidermis are probably
controlled by lymphocyte-associated antigen (5) and E-cadherin (8),
respectively. Conversely, after antigenic stimulation, LC dissociate
from surrounding keratinocytes by down-regulating E-cadherin expression
(8, 10), then adhere via
6 integrins to the basement
membrane (9) with subsequent passage into the dermis, where they
re-enter the afferent lymphatics or blood vessels. This endothelial
migration is essential for completion of the DC life cycle. Despite
this importance, very little is known about the molecular mechanisms
underlying the adhesion of DC to endothelial cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin.
, or
5'-GTGGGCCGCTCTAGGCACCAA-3'and 5'-CTCTTTGATGTCACGCACGATTTC-3' for
-actin. Following 30-cycle amplification, PCR products were
separated on 1.5% agarose gel, transferred onto a membrane under
alkaline conditions, and hybridized with 32P-labeled
cDNA probe for DC-HIL, IL-1
, or
-actin.
) for 15 min,
followed by centrifugation for 20 min at 10,000 × g.
The full-length cDNA for DC-HIL was enzymatically excised from
clone 2B4 and inserted into a mammalian expression vector pCMV5 (a gift from Dr. D. Russel, UT Southwestern Medical Center). COS-1 cells were
transfected with the resulting vector (pCMV5-DC-HIL) or an empty vector
using FuGene 6 (Roche Diagnostics, Indianapolis, IN), cultured for 3 days, and the whole cell lysate was prepared as described above. The
samples were separated by 4-15% SDS-PAGE, transferred onto
polyvinylidene fluoride membrane (Millipore, Bedford, MA), and then
blotted with 1 µg/ml purified rabbit anti-DC-HIL or control rabbit
IgG. After washing, the membrane was further blotted with horseradish
peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson
ImmunoResearch Laboratory, West Grove, PA) and developed with ECL
system (Amersham Pharmacia Biotech, Piscataway, NY).
) vector (Stratagene) and used as a template DNA for the following
PCR. Using the above oligonucleotides, the entire plasmid vector with
the designed mutation was PCR-amplified with the cycling protocol; 18 cycles of 1-min annealing at 52 °C and 18-min extension at 68 °C.
The resulting PCR products were treated with DpnI
restriction enzyme to digest selectively the template DNA and then
transformed into Escherichia coli. The nucleotide sequence
of full-length mutated ECD was confirmed by DNA sequencing. The DNA
fragment for wild-type ECD in pSTB-DC-HIL-Fc was replaced with the
mutated ECD and produced in COS-1 cells, followed by affinity
chromatography with protein A-agarose.
(Jackson
ImmunoResearch Laboratory) for 10 min, followed by fluorescein
isothiocyanate-conjugated streptavidin at 1:100 dilution (Jackson
ImmunoResearch Laboratory) for 10 min. Binding of soluble DC-HIL-Fc to
cells was examined by FACSCalibur (Becton Dickinson, San Jose, CA). In
some experiments, the binding was performed in the presence of
inhibitors (heparin, fucoidan, dextran sulfate (500 kDa),
chondroitin sulfate A, mannan, RGDS and RPKP tetramer peptides, and
EDTA; all purchased from Sigma).
and then incubated with HRP-conjugated streptavidin at 1:100 dilution
for 10 min. The 3,3',5,5'-tetramethylbenzidine (TMB) substrate reagent
(PharMingen) was added to the wells, and the enzymatic reaction was
allowed to progress at room temperature for 60 min. After the reaction
was stopped by addition of 1 M phosphoric acid, the
absorbance at 450 nm was measured by an ELISA plate reader. The
background binding to BSA-coated wells or noncoated wells was
negligible (A450 nm < 0.03). Each
value represents the mean of three separate reactions.
IgG overnight at 4 °C.
After washing, DC-HIL-Fc or control Fc fusion proteins (10 µg/ml)
were added to the wells and incubated for 1 h at 4 °C for
immobilization, and then washed with binding buffer. As an additional
control, vitronectin (5 µg/ml) was also immobilized. The labeled SVEC
(1 × 104 cells) were incubated for 60 min, in most
experiments, at 37 °C in the Fc fusion protein-coated wells. After
extensive washing, SVEC adhered to the wells were lysed with 1 N NaOH and counted for the radioactivity of 3H
(cpm). Adhesion activity was shown by the cpm/total input cpm; each
value represents the mean of three independent wells. Like SVEC, other
cell lines that were used for the DC-HIL binding assays were also
tested for the adhesion.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Deduced amino acid sequence and putative
domain organization of DC-HIL. Deduced amino acid sequence
of DC-HIL is shown and segmented, from the amino terminus, into a
putative leader sequence (underlined), an extracellular
domain, a transmembrane domain (dashed line), and a
cytoplasmic domain. Putative functional motifs found are a
heparin-binding motif (BBXB, shown by the # symbol), RGD
motifs (boxed), putative N-glycosylation sites
(arrowheads), a proline-rich region (dotted
line), an immunoreceptor tyrosine-based activation motif (ITAM,
YXXI, double underlined), and lysosomal targeting
motifs LL (asterisks).
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Fig. 2.
Amino acid sequence comparison of DC-HIL with
melanoma/melanosome- and lysosome-associated proteins.
A, DC-HIL, rat osteoactivin (GenBankTM accession number:
AF184983), human nmb (NM002510), and Q (quail) NR-71 (X94144)
were aligned by the Clustal method in the Lasergene program (DNA star).
Amino acid residues that are conserved between more than two molecules
are shaded. B, phylogenic relationships between
DC-HIL and melanoma/melanosome or lysosome-associated membrane proteins
are indicated in a tree.
T cells (7-17 DETC), two
T cells (HDK-1
and D10), B cell hybridoma clones (5C5), keratinocytes (Pam 212), and
dermal fibroblasts (NS01). As noted in Fig. 3B, DC-HIL
mRNA was detected in greatest quantities in bone marrow and adipose
tissues, in modest levels in thymus, and at low levels in skin.
Although mRNA was not detectable by Northern analysis in lymph node
and spleen, RT-PCR demonstrated its expression in lymph node (data not
shown). Tissue distribution of DC-HIL mRNA does not well correlate
with that of DC, suggesting that the expression may be restricted to
certain subpopulations of DC. The expression of DC-HIL mRNA in skin
was consistent with its establishment from skin-derived XS52 DC (12),
which resemble skin resident DC, i.e. LC in many features
(11, 12). We then assessed by RT-PCR the source of DC-HIL mRNA
expression in epidermis (Fig. 3C). DC-HIL mRNA was
detected in epidermal cell suspensions freshly prepared from BALB/c
mice. Importantly, depletion of LC (Ia+) by treatment with anti-Ia mAb
plus complement abrogated most of the signal for DC-HIL. The absence of
PCR signals for IL-1
, which is known to be specific for LC in mouse
epidermis (32, 33), shows complete depletion of LC. This corroborates our observations that DC-HIL mRNA was detected by Northern blot in
the LC-like XS52 cells, but was totally absent from cells derived from
other epidermal populations (i.e. Pam 212 keratinocytes and the 7-17 epidermal
T cells) (Fig. 3A). On the other
hand, the incomplete abrogation of DC-HIL mRNA by the depletion
suggests a different minor source for this signal in epidermis,
possibly melanocytes, because DC-HIL mRNA was detected in several
murine melanoma lines by RT-PCR (data not shown). Collectively, our
data suggest that DC-HIL mRNA is expressed constitutively and
preferentially by XS52 DC and epidermal LC.
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Fig. 3.
Cellular and tissue distributions of DC-HIL
mRNA. Total RNAs were isolated from XS52 DC, J774, and Raw
macrophages, 7-17 DETC, HDK-1 Th1 cells, D10 Th2 cells, 5C5 B cell
hybridoma, Pam 212 keratinocytes, and NS01 dermal fibroblasts
(A), and mRNA isolated from the indicated tissues in
adult BALB/c mice (B). The total RNAs and mRNAs were
examined in Northern blot for expression of mRNA for DC-HIL,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (A), or
-actin (B). C, epidermal cells freshly
isolated from skin tissues of adult BALB/c mice were examined for
DC-HIL mRNA expression by RT-PCR using the primer (see
"Experimental Procedures") designed to amplify nucleotides
1029-1404. Some epidermal cells were treated with anti-Ia mAb plus
complement to deplete LC. The extent of depletion was assessed by
measuring IL-1
mRNA, which is known to be expressed exclusively
by LC in the mouse epidermis.
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Fig. 4.
Expression of DC-HIL protein. Whole cell
extracts were prepared from XS52 DC and COS-1 cells transfected with
cDNA encoding the full-length DC-HIL (A) or from the
indicated cell lines (B) in lysis buffer containing 0.3%
Triton X-100. These samples were analyzed for DC-HIL protein expression
by immunoblotting with affinity-purified rabbit Ab raised against
synthetic peptide of DC-HIL. Control rabbit Ab detected two bands shown
by asterisks. C, whole cell extracts from XS52 DC
were treated with 5 units/ml N-glycosidase overnight at
37 °C in the presence of 0.1% SDS and 0.5% Triton X-100, and then
subjected to immunoblotting of DC-HIL.
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Fig. 5.
Subcellular localization of DC-HIL.
The subcellular localization was examined by biochemical method.
A, crude homogenate prepared from XS52 DC was centrifuged at
50,000 × g for 45 min, and the supernatant (cytosol
fraction) and the pellet (membrane fraction) were separately recovered
and tested for the amounts of DC-HIL by immunoblotting. An aliquot
(equivalent to 1 × 105 cells) in each fraction was
loaded on SDS-PAGE. B, surface proteins on XS52 DC were
labeled with biotin at 4 °C, followed by detergent extraction of
cells and precipitation by streptavidin-conjugated agarose beads. After
centrifugation, the supernatant (streptavidin-unbound fraction, shown
by "U") and the eluate from the beads
(streptavidin-bound fraction, "B") were immunoblotted
for DC-HIL. The sample proteins loaded for streptavidin-unbound and
bound fractions were equivalent to 1 and 3 × 105
cells, respectively. C, the efficacy of the surface
biotinylation method was confirmed by immunoblotting for recombinant
DC-HIL (left) and luciferase (Luc, used as an
intracellular control protein, right) in COS-1 cells
transfected with the corresponding cDNAs. The sample proteins
loaded for streptavidin-unbound (U) and bound fractions
(B) were the same as those in B. The data shown
are representative of three independent experiments.
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Fig. 6.
Immunostaining of DC-HIL in XS52 DC.
XS52 DC were fixed with 4% paraformaldehyde and stained with
anti-DC-HIL Ab in the presence of 0.5% saponin. Stained XS cells shown
in the inset at magnification × 40, one of which was
enlarged to show that DC-HIL proteins (shown in red)
accumulated in a single large vesicle adjacent to the
hematoxylin-stained nuclei (in blue) as well as in small
vesicles scattered toward the periphery.
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Fig. 7.
Soluble DC-HIL-Fc binds to the surface of
particular cell types. Binding properties of soluble DC-HIL-Fc to
the cell surface was examined by flow cytometric analyses.
A, production of recombinant proteins. Recombinant DC-HIL-Fc
as well as control proteins, Dectin-1-Fc and Fc alone, were produced in
COS-1 cells and affinity purified by protein A-agarose. Their purity
and molecular weights were examined by SDS-PAGE (2-4 µg of
protein/lane) under reducing conditions and CBB staining. B,
binding assays. The indicated cell lines were incubated with 10 µg/ml
soluble DC-HIL-Fc (closed histograms) or Dectin-1-Fc
(open histograms) for 30 min at 4 °C and then labeled
with biotin-conjugated anti-human Fc Ab and fluorescein
isothiocyanate-conjugated streptavidin, followed by flow cytometric
analysis. Raw macrophages were pretreated with 10 µg/ml anti-Fc Ab to
block Fc-mediated binding. C, inhibition of binding. SVEC
were incubated with soluble DC-HIL-Fc in the absence (open
histograms) or the presence of 1 mM RGDS tetramer or 1 µg/ml heparin (closed histograms). Dectin-1-Fc
(dotted histograms) served as controls. D,
dose-dependent inhibition. Different concentrations of the
indicated sulfated or nonsulfated polysaccharides were added to the
binding reaction of soluble DC-HIL-Fc to SVEC. Inhibitory capacities
are expressed as the percentage of mean fluorescence relative to that
obtained in the absence of inhibitors. The data shown for binding
assays are representative of three independent experiments.
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Fig. 8.
DC-HIL-Fc recognizes heparin and
fucoidan. DC-HIL-Fc was assessed for binding capacity to heparin
and fucoidan. A, dose-dependent binding. Varying
concentrations of DC-HIL-Fc were incubated for 60 min at room
temperature in the ELISA plates precoated with 100 µg/ml heparin-BSA
or fucoidan overnight. After extensive washing, the plates were
sequentially incubated with biotin-conjugated anti-human Fc and with
HRP-conjugated streptavidin. After adding TMB substrate, the amounts of
DC-HIL-Fc bound to the plates were measured by absorbance at 450 nm.
B and C, inhibition of binding. Binding of
DC-HIL-Fc (1 µg/ml) to immobilized heparin (B) or fucoidan
(C) was performed in the presence of heparin (H),
fucoidan (F), dextran sulfate (500 kDa) (D),
chondroitin sulfate A (C), or mannan (M) at the
increasing concentrations. D, blocking of DC-HIL-Fc-binding
to SVEC by pretreatment with heparin. SVEC were treated with
(closed histogram) or without (open histogram) 1 µg/ml heparin, washed, and then examined for binding of DC-HIL-Fc by
flow cytometric analysis. E, pretreatment with sodium
chlorate. SVEC were treated with (closed histogram) or
without (open histogram) 30 mM sodium chlorate
for 24 h prior to the binding assays. Dotted histograms
show the binding of Dectin-1-Fc.
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Fig. 9.
SVEC adhere to immobilized DC-HIL-Fc.
A, dose-dependent adhesion.
[3H]Thymidine-pulsed SVEC (2 × 104
cells/well) were cultured for 60 min at 37 °C in 96-well plates
precoated with different concentrations of DC-HIL-Fc or control fusion
proteins. After washing, the adherent cells were lysed and measured for
the radioactivity, and shown as percent cpm relative to total input
cpm. B, time course of SVEC adhesion. In the same adhesion
assays, the adhesive activity was measured at the various time points
after incubation of SVEC in the wells precoated with 10 µg/ml
DC-HIL-Fc. C, blocking of SVEC adhesion by polysaccharides.
SVEC were incubated for 60 min in the wells precoated with 10 µg/ml
DC-HIL-Fc in the presence of sulfated or nonsulfated polysaccharides at
the 10-fold increasing concentrations. Inhibitory activity is shown by
percentage of 3H cpm obtained in the absence of inhibitors.
D, blocking with inhibitors for integrin-mediated adhesion.
Increasing doses of inhibitors (an RGDS tetramer, an RPKP control
tetramer, and EDTA) were added to the same adhesion assays. Note that
adhesion of SVEC to control fusion proteins typically showed as little
as 1%. Data shown are representative of five independent
experiments.
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Fig. 10.
Adhesive capacity of cell lines to
immobilized DC-HIL-Fc correlates with their binding capacity.
Soluble DC-HIL-Fc binds to SVEC and Queens, but not to BW5147, J558,
and Raw. These cell lines were examined for adhesive capacity to
immobilized DC-HIL-Fc (10 µg/ml) or vitronectin (5 µg/ml), as
performed in Fig. 9. The data shown are representative of two
independent experiments.
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Fig. 11.
Binding and adhesive properties of
RGD-defective DC-HIL-Fc mutant. A, generation of
RGD-defective DC-HIL-Fc, termed RAA mutant. The nucleotide sequence
AGGGGAGAC, encoding RGD in DC-HIL-Fc, was genetically altered to the
nucleotide sequence AGGGCAGCA, which encodes RAA, by using
site-directed mutagenesis (upper part of
A). RAA mutant DC-HIL-Fc was produced and purified in
the same way as the wild-type. Both recombinant proteins were compared
for their electrophoretic mobility and tested for the purity on
SDS-PAGE/CBB staining (lower part). B, binding
capacity of soluble RAA mutant to SVEC. Wild-type or RAA mutant
DC-HIL-Fc (each at 10 µg/ml) was incubated with SVEC in the absence
(closed histograms) or presence (open histograms)
of 1 µg/ml heparin, and binding activity was measured by flow
cytometric analysis. Fc was used as control fusion protein
(dotted histograms). C, adhesive capacity of RAA
mutant. According to the same procedures as used in Fig. 9, adhesive
capacity of RAA mutant was compared with that of wild-type DC-HIL-Fc
and a Fc control protein. The data shown are representative of three
independent experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 (CD18)
integrins on neutrophils and immunoglobulin gene superfamily
counter-receptors on EC, primarily ICAM-1 (37). However,
DC-HIL-mediated adhesion differs from this two-step model in that
DC-HIL serves also as one of the counter-receptors for integrins. In
this respect, DC-HIL is distinguishable from other adhesion molecules
on the cell surface: e.g. Thy-1, Ly-5, and NCAM are all
capable of interacting with heparin (50-52), whereas they do not
function as ligands for integrins. This feature of DC-HIL may exemplify
that DC display not only common but also unique mechanisms for their
transendothelial migration.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Akira Takashima for providing 7-17 DETC, NS47 fibroblast, and Pam 212 keratinocyte lines and Drs. Ponciano D. Cruz Jr. and Dorothy Yuan for critical reading of this manuscript. We are also grateful to Alok Das for excellent technical assistance and Susan Milberger for secretarial assistance in preparation of this manuscript.
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FOOTNOTES |
---|
* This work was supported by Research Grant RO1-AR44189 from the National Institutes of Health.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) AF184983.
To whom correspondence should be addressed: Dept. of Dermatology,
University of Texas Southwestern Medical Center, 5323 Harry Hines
Blvd., Dallas, TX 75390-9069. Tel.: 214-648-3419; Fax: 214-648-3472; E-mail: kariiz@mednet.swmed.edu.
Published, JBC Papers in Press, December 12, 2000, DOI 10.1074/jbc.M008539200
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ABBREVIATIONS |
---|
The abbreviations used are: DC, dendritic cells; EC, endothelial cells; Ab, antibody; mAb, monoclonal Ab; APC, antigen-presenting cells; CBB, Coomassie Brilliant Blue; ECD, extracellular domain; HSPG, heparan sulfate proteoglycans; HRP, horseradish peroxidase; IL, interleukin; LC, Langerhans cells; TMB, 3,3',5,5-tetramethylbenzodine; ICAM-1, intercellular adhesion molecule-1; SVEC, surface of an endothelial cell line; DETC, 7-17 dendritic epidermal T cells; PBS, phosphate-buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); kb, kilobase(s); ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; LAMP, lysosoma-associated membrane protein.
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