of June 13, 2013.
This information is current as
Potentiates Antigen Presenting Cell Function
Induces Tyrosine Phosphorylation and
Binding of DC-HIL to Dermatophytic Fungi
Akiyoshi, Ponciano D. Cruz, Jr. and Kiyoshi Ariizumi
Jin-Sung Chung, Tatsuo Yudate, Mizuki Tomihari, Hideo
2009; 183:5190-5198; Prepublished online 30
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The Journal of Immunology
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Binding of DC-HIL to Dermatophytic Fungi Induces Tyrosine
Phosphorylation and Potentiates Antigen Presenting Cell
Jin-Sung Chung,* Tatsuo Yudate,†Mizuki Tomihari,*‡Hideo Akiyoshi,*§Ponciano D. Cruz, Jr.,*
and Kiyoshi Ariizumi2*
APCs express receptors recognizing microbes and regulating immune responses by binding to corresponding ligands on immune
cells. Having discovered a novel inhibitory pathway triggered by ligation of DC-HIL on APC to a heparin/heparan sulfate-like
saccharide of syndecan-4 on activated T cells, we posited DC-HIL can recognize microbial pathogens in a similar manner. We
showed soluble recombinant DC-HIL to bind the dermatophytes Trichophyton rubrum and Microsporum audouinii, but not several
bacteria nor Candida albicans. Dermatophyte binding was inhibited completely by the addition of heparin. Because DC-HIL
contains an ITAM-like intracellular sequence, we questioned whether its binding to dermatophytes can induce tyrosine phos-
phorylation in dendritic cells (DC). Culturing DC with T. rubrum (but not with C. albicans pseudohyphae) induced phosphory-
lation of DC-HIL, but not when the tyrosine residue of the ITAM-like sequence was mutated to phenylalanine. To examine the
functional significance of such signaling on DC, we cross-linked DC-HIL with mAb (surrogate ligand), which not only induced
tyrosine phosphorylation but also up-regulated expression of 23 genes among 662 genes analyzed by gene-array, including genes
for profilin-1, myristoylated alanine rich protein kinase C substrate like-1, C/EBP, LOX-1, IL-1?, and TNF-?. This cross-linking
also up-regulated expression of the activation markers CD80/CD86 and heightened APC capacity of DC to activate syngeneic T
cells. Our findings support a dual role for DC-HIL: inhibition of adaptive immunity following ligation of syndecan-4 on activated
T cells and induction of innate immunity against dermatophytic fungi. The Journal of Immunology, 2009, 183: 5190–5198.
naive T cells. Subsets of DC include CD11c?/CD4?lymphoid DC,
CD11c?/CD8?myeloid DC, CD11c?/PDCA-1?plasmacytoid DC,
and I-A/I-E?epidermal Langerhans cells (LC) (1). In addition, im-
mature DC, which reside in tissues interfacing with the external en-
vironment, serve as sentinels that sense and distinguish among dif-
ferent microbes to be internalized and processed, leading to altered
DC gene expression profiles required for eliciting pathogen-specific
adaptive immunity (2). Thus, DC serve important roles in both innate
and adaptive immunity.
endritic cells (DC)3are the most potent APCs for initiating
and controlling adaptive immune responses through pre-
sentation of Ag in the context of costimulatory signals to
DC express several receptors that interact with corresponding
surface molecules on microbes, including pattern recognition re-
ceptors (PRRs) that bind directly to particular molecular compo-
nents of a given microbe (pathogen-associated molecular patterns)
(3). PRRs include: the leucine-rich repeat protein CD14 that binds
to LPS, lipoteichoic acid, and peptideglycan (4); scavenger recep-
tors (SR-A, CD36, and MARCO) that bind low density lipopro-
teins or lipid A on some bacteria (5); the adhesion molecule inte-
grin CR3 that binds to LPS, lipophosphoglycan, an acylpoly (1, 3)
galactoside (APG), and Candida albicans (6); TLRs, consisting of
nine members, that bind to zymosan, Staphylococcus aureus, LPS,
bacterial flagellin, CpG bacterial DNA (7, 8); and C-type lectin-
like receptors, such as dectin-1 (9), dectin-2 (10), and DC-SIGN
(11, 12), which bind to many pathogens via polysaccharide
moiety on their surface (13). Dectin-1 binds to ?-glucan poly-
saccharide expressed primarily on C. albicans yeast (14); dec-
tin-2 binds to hyphal (but not yeast) forms of C. albicans (15)
probably through high-mannose structures (16); and DC-SIGN
binds to mannose-type oligosaccharides (11). DC take advantage
of distinct ligand specificities of PRRs to distinguish among patho-
gens, thereby inducing pathogen-dependent up-regulated gene ex-
pression. In particular, DC-SIGN may be unique because it uses
adhesion properties to control DC migration and T cell activation
Previously, we discovered that DC-HIL is a highly glycosylated
type I transmembrane protein of 95 and 125 KDa containing an
extracellular Ig-like domain constitutively expressed at high levels
by DC and macrophages. We also found DC-HIL to deliver a
potent inhibitory signal to T cells, in an Ag-independent manner,
by binding a heparin/heparan-like saccharide of syndecan-4 (SD-4)
on activated T cells (19, 20). These findings led us to hypothesize
that DC-HIL binds microbial pathogens in a similar manner. We
*Department of Dermatology, The University of Texas Southwestern Medical Center
and; the Dermatology Section (Medical Service), Dallas Veterans Affairs Medical
Center; Dallas, Texas 75390;†Department of Dermatology, Kinki University School
of Medicine, Osaka-Sayama, Osaka, Japan;§Osaka Prefecture University, Graduate
School of Life and Environmental Science, Veterinary Medical Science, Sakai-shi,
Osaka, Japan; and‡Department of Clinical Veterinary Medicine, Obihiro University
of Agriculture and Veterinary Medicine, Obihiro, Japan
Received for publication April 29, 2009. Accepted for publication August 11, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the National Institutes of Health
(A164927-01) and from Galderma.
2Address correspondence and reprint requests to Dr. Kiyoshi Ariizumi, University of
Texas, 5323 Harry Hines Blvd., Dallas, TX 75390-9069. E-mail address:
3Abbreviations used in this paper: DC, dendritic cell; LC, Langerhans cell; PRR,
pattern recognition receptor; SD-4, syndecan-4; BM, bone marrow; HS, heparin/hepa-
ran sulfate; Marcksl-1, myristoylated alanine rich protein kinase C substrate like-1;
PD-1, programmed cell death-1.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
by guest on June 13, 2013
found DC-HIL to bind the dermatophytes Trichophyton rubrum
and Microsporum audouinii. Coculture of DC with T. rubrum in-
duces phosphorylation of the tyrosine residue in the ITAM-like
intracellular sequence of DC-HIL at a level markedly greater than
that induced by ligation to the T cell ligand SD-4. This phosphor-
ylation up-regulated expression of genes responsible for DC mat-
uration and augmentation of APC function. Thus, DC-HIL is a
PRR for dermatophytic fungi that can heighten APC properties,
while also negatively regulating T cell activation.
Materials and Methods
Female BALB/c and C57BL/6 (5- to 8-wk-old) mice were purchased from
Harlan Breeders, and OT-I and OT-II (5- to 8-wk-old) mice obtained from
Taconic Farms. Following National Institutes of Health guidelines, these
animals were housed and cared for in the pathogen-free facility of the
Institutional Animal Care Use Center of The University of Texas South-
western Medical Center.
Fc-fused recombinant protein
Fc-fused proteins (DC-HIL-Fc, SD-4-Fc, Dectin-2-Fc, and Fc alone) were
produced in COS-1 cells and purified as described previously (15, 19).
Rat mAb generated against CD4 (L3T4), CD8, I-A/I-E (2G9), CD11b (M1/
70), CD86 (GL1), CD317 (PDCA-1, eBio927), and hamster mAb against
CD11c (N418), CD80 (16A-10A1) were purchased from eBioscience. Sec-
ondary Abs were purchased from Jackson ImmunoResearch Laboratories.
UTX-103 rabbit anti-mouse DC-HIL mAb was generated as follows:
Rabbits were immunized four times with 0.5 mg of DC-HIL-hFc (fused
with human IgG-Fc) 3 wk apart. A week after the third immunization, sera
were collected and the titer of anti-mouse DC-HIL evaluated by ELISA
using DC-HIL-mFc (fused with mouse IgG-Fc) to eliminate anti-human
IgG Ab. Rabbit spleen cells were fused with a rabbit myeloma cell line
(Epitomics). One clone (UTX-103 mAb IgG1) was purified from the cul-
ture supernatant using protein A-agarose and entoxoin-free buffers. Com-
pared with our previous 1E4 rat anti-DC-HIL mAb (19), this mAb has
much higher affinity for native DC-HIL protein. Purified UTX-103 mAb
and control IgG preparations used for cross-linking experiments were
tested for endotoxin contamination using a LAL Chromagenic Endpoint
Assay (HyCult Biotechnology; Cedarlane Laboratories). Endotoxin levels
were ?0.5 EU/ml.
Preparation of APC
Epidermal cells were isolated from ear skin of BALB/c mice using trypsin
(Mediatech); LC were enriched by centrifugation over Histopaque (1.083,
Sigma-Aldrich) (21). Bone marrow (BM) DC were harvested from day 6
culture of BM cells with GM-CSF (22). Other DC subpopulations were
procured from the spleen cells of naive mice. Macrophages were harvested
from the peritoneal cavity of mice stimulated with thioglycolate (21).
Freshly isolated or cultured leukocytes were assayed for surface and in-
tracellular expression by flow cytometry. For surface expression, cells (1 ?
105) were incubated with UTX-103 rabbit anti-DC-HIL mAb or the iso-
typic control IgG1 (each 10 ?g/ml). After washing, cells were labeled with
fluorescent secondary Ab (PE-anti-rabbit IgG, 1 ?g/ml). For intracellular
staining, cells (1 ? 105) were fixed with 4% paraformaldehyde at room
temperature for 30 min. After washing with Dulbecco’s PBS, cells were
treated with permeabilization buffer (0.5% Saponin, 0.5% BSA in DPBS)
for 10 min, and then incubated with UTX-103 mAb (10 ?g/ml) in the
permeabilization buffer for 30 min, followed by incubation with the sec-
ondary Ab (1 ?g/ml). Fluorescence intensity of stained cells was analyzed
by FACSCalibur (BD Biosciences).
Binding assays for microbes
Candida albicans (ATCC 10231) and the dermatophytes Trichophyton
rubrum (ATCC 14001) and Microsporum audouini (ATCC 10008) were
purchased from the American Type Culture Collection and grown in me-
dium recommended by the American Type Culture Collection. C. albicans
yeast was transformed into pseudohyphae by culturing freshly prepared
yeasts at 37°C for 90 min in HBSS containing 1.25 mM CaCl2, 1 mM
MgCl2, 10 mM HEPES (pH 7.7), and 10% heat-inactivated FCS (15).
Aliquots of pseudohyphae (4 ? 105cells) were washed with DPBS and
incubated with staining buffer (0.1% BSA, 2 mM CaCl2in DPBS) con-
taining 20 ?g/ml Fc proteins on ice for 1 h. After extensive washing with
buffer, cells were resuspended in 2.5 ?g/ml FITC-anti-human IgG Ab on
ice for 30 min. For immunstaining of dermatophyte fungi, single colonies
of fungi were grown on Sabouroud’s agar plates, harvested and suspended
in DPBS. After washing with DPBS and with water, small aliquots were
spotted on slide glass, air dried, and stained with Fc proteins as before.
Binding of Fc proteins to microbes was examined by confocal microscopy.
In inhibition experiments, 20 ?g/ml DC-HIL-Fc was preincubated with
indicated concentrations of heparin, chitin, D-galacto-D-mannan, ?-glu-
can, or mannan (all from Sigma-Aldrich) for 30 min on ice before binding
to T. rubrum. The fungi also were treated with 1,000 unit of N-glycosidase
(PNGase F, from New England Biolabs) at 37°C for 1 h before incubating
Either or both tyrosine residues (amino acids 523 and 529) in the intracellular
domain was replaced with phenylalanine using Quick Change Site-Directed
Mutagenesis and Pfu polymerase (Stratagene) performed according to the
manufacturer’s recommendations for the following oligonucleotide pairs:
Y523F 5?-GGTTACCATCTTGCTGTTCAAAAAACACAAGGCG-3? (5?
primer, where italic letters show mutations) and 5?-CGCCTTGTGTTTTTTG
AACAGCAAGATGGTAACC-3? (3? primer); Y529F 5?-AAAAACACAA
GGCGTTCAAGCCAATAGGAAACTG-3? and 5?-CAGTTTCCTATTGGC
AAAAAACACAAGGCGTTCAAGCCAATAG-3? and 5?-CTATTGGCTT
Immunoblotting and tyrosine phosphorylation assay
Whole cell extracts were prepared from untreated or activated BM-DC
(cultured for 2 days with 1 ?g/ml LPS) and assayed for protein concen-
tration (23). An aliquot (10 ?g/lane) was applied to 4–15% SDS-PAGE,
followed by immunoblotting using UTX-103 mAb and control IgG (each
1 ?g/ml) (23). To examine tyrosine phosphorylation of DC-HIL on DC,
BM-DC were treated with three different stimuli: BM-DC (3 ? 106
cells/ml complete medium) were cultured with indicated amounts (as dried
weight) of T. rubrum or C. albicans hyphae; BM-DC (5 ? 106cells in 500
?l of DPBS) were also incubated with UTX-103 mAb or control rabbit IgG
(10 ?g/ml) on ice for 30 min, followed by cross-linking with 100 ?g/ml
goat anti-rabbit IgG; and BM-DC (5 ? 106cells) were cultured in culture
dish precoated with SD-4-Fc or control Ig (20 ?g/ml). At indicated time
periods at 37°C, treated DC were lysed using 500 ?l of 2? lysis buffer
(15). DC-HIL protein was immunoprecipitated by incubation at 4°C for 3 h
with 2–5 ?g of UTX-103 mAb and overnight incubation with protein-A
agarose (50 ?l of 50% slurry). The immune-complexes were dissociated by
boiling and then analyzed for expression of phosphotyrosin by immuno-
blotting using biotinylated anti-phosphotyrosine (0.5 ?g/ml) (4G10, Up-
state Biotechnology) and HRP-streptavidin (1/10,000 dilution). The blotted
membranes were also stripped and reanalyzed using 1E4 rat anti-DC-HIL
mAb (1 ?g/ml) (23) and HRP-anti-rat IgG (1/10,000 dilution). To examine
tyrosine phosphorylation of DC-HIL mutants, COS-1 cells were seeded at
a density of 5 ? 105cells/dish and transfected with a plasmid vector en-
coding wild-type or tyrosine mutants of DC-HIL (2 ?g) using Fugene 6
(19). Two days after transfection, cells were cross-linked by UTX-103
mAb plus secondary Ab, and then immunoprecipitated as described above.
Gene expression by cross-linked BM-DC was analyzed using oligonu-
cleotide probe-blotted gene arrays (Dendritic and APC and Autoim-
mune and Inflammation) according to the manufacturer’s recommenda-
tions (SuperArray Bioscience). In brief, BM-DC were harvested and
cultured in 96-well plate (1 ? 105cells/well) precoated with UTX-103
mAb or control IgG (20 ?g/ml). After 6 h of culture at 37°C, total RNA
was isolated from treated cells (1 ? 106) using ArrayGrade total RNA
isolation kit (SuperArray). Biotin-labeled cDNA probes were prepared
using the TrueLabeling-AMP 2.0 kit (SuperArray) and hybridized with
gene arrays; hybridization signals were then detected using chemilumi-
nescent detection kit (SuperArray). Image acquisition and analysis of
data were performed by ImageQuant 400 (Amersham Biosciences). Ex-
pression of some genes up-regulated by the cross-linking was reana-
lyzed by real-time PCR following the manufacturer’s recommendations
(LightCycler FastStart DNA MasterplusSYBR Green I, Roche). Primers
for oxidized low-density lipoprotein receptor (Olr-1, GenBank: AY057791)
include: 5?-CCCGGAAGCTGGACGAGA-3? (5? primer) and 5?-AGAACG
5191The Journal of Immunology
by guest on June 13, 2013
GGGAGGTGGTATGG-3? (3? primer); myristoylated alanine rich protein ki-
nase C substrate like-1 (Marcksl-1, GenBank: NM_009851): 5?-GCCCCCAG
CAGACCCCCATCAT-3? and 5?-CTCGCCCTGCTCCTGCTCTTCCTC-3?.
For production of TNF-? and IL-1?, BM-DC (1 ? 105cells) were incubated
similarly with immobilized UTX-103 mAb or control IgG for indicated time
periods. BM-DC (2 ? 105cells) were treated with 10 ?g/ml dried T. rubrum
or Candida hyphae at 37°C for 30 min, immediately after which cells were
and cytokines measured by ELISA kits (eBionscience).
BM-DC were cultured in 96-well plate precoated with UTX-103 mAb or
control IgG (20 ?g/ml) for 1 day. Treated DC were harvested and reseeded
on 96-well plate at a different cell density and pulsed for 6 h with MHC
class II-restricted OVA323–339and MHC class I-OVA257–264peptide (each
2 ?g/ml) synthesized by the Protein Chemistry Technology Center at The
University of Texas Southwestern Medical Center. After pulsing, DC were
cocultured with the constant number of CD4?or CD8?T cells (1 ? 105/
well) purified from spleen of unprimed OT-II or OT-I transgenic mice,
respectively, using T cell isolation kits (Miltenyi Biotec). Two days after
coculture, culture supernatant was recovered and IL-2 and IFN-? produc-
tion was assayed by ELISA kits (eBionscience).
Expression of DC-HIL by APC
Using a newly developed UTX-103 rabbit anti-DC-HIL mAb with
markedly higher affinity to native DC-HIL than our previously
generated 1E4 rat anti-DC-HIL mAb (19), we reexamined surface
expression of DC-HIL on different APC subsets by flow-cytomeric
analysis (Fig. 1). Epidermal LC were identified as I-A/I-E?epi-
dermal cells, almost all of which expressed DC-HIL constitutively
at high levels on the surface and intracellularly (Fig. 1A). By con-
trast, DC-HIL was not expressed by I-A/I-E?epidermal cells.
CD11c?BM-DC also expressed DC-HIL constitutively on their
surface. LPS stimulation up-regulated DC-HIL surface expression
by some (but not all) CD11c?DC (Fig. 1B). This up-regulation
was confirmed by immunoblotting of protein extracts from BM-DC
using UTX-103 mAb that immunostained two bands (95 and 125
KDa) (Fig. 1C). In spleen, there are at least three distinct DC subsets
(CD11c?/CD4?lymphoid DC, CD11c?/CD8?myeloid DC, and
CD11c?/PDCA-1?plasmacytoid DC (1), all of which also expressed
DC-HIL constitutively on the surface, albeit at lower levels (Fig. 1D).
DC-HIL expression by these DC subsets appeared invariant of in vivo
stimulation (data not shown). Finally, peritoneal macrophages from
mice treated with thioglycolliate also expressed surface and intracel-
lular DC-HIL at high levels (Fig. 1E). These results indicate that DC-
HIL is expressed by a wide variety of APC subsets.
DC-HIL binds to dermatophyte cell wall
Many C-type lectin receptors (e.g., mannose receptor, DC-SIGN,
dectin-1, and dectin-2) bind to saccharide ligands expressed by
microbes (13). Similarly, we showed DC-HIL to bind a heparin/
heparan sulfate (HS)-like saccharide of SD-4 on activated T cells
(20). We thus posited that DC-HIL may also recognize microbial
pathogens through a similar sugar moiety. To address this issue,
we performed binding assays using fluorescent-labeled DC-HIL-
Fc, dectin-2-Fc (as control), or Fc alone. Neither Staphylococcus
aureus, group A streptococci, Pseudomonas aeruginosa, nor Esch-
erichia coli bound to DC-HIL (data not shown). We then examined
binding to Candida albicans pseudohyphae consisting of round
yeast and filamentous hyphae (Fig. 2A). Using dectin-2-Fc as a
positive control because it is known to bind hyphal (but not yeast)
components (15) (Fig. 2A), we observed that neither DC-HIL-Fc
nor Fc alone bind to Candidal pseudohyphae. We then examined
binding to dermatophytes (Fig. 2, B and C). DC-HIL-Fc bound to
Trichophyton rubrum with high affinity and to Microsporum au-
douinii at lower affinity. Binding of DC-HIL to T. rubrum was
blocked completely by addition of heparin (2 ?g/ml) (Fig. 2D),
which we showed previously to inhibit DC-HIL binding to acti-
vated T cells (20). To sort the fungal ligands of DC-HIL, T.
rubrum fungi were pretreated with N-glycosidase that removes
saccharide residues from glycoproteins (Fig. 2E), or DC-HIL-Fc
was preincubated with fungal saccharides, including chitin, galac-
tomannan, ?-glucan, and mannan before binding assays (Fig. 2, F
and G). N-glycosidase treatment inhibited binding of DC-HIL-Fc
to T. rubrum almost completely. None of the saccharide inhibitors
showed complete inhibition as was observed with heparin: Chitin
and galactomannan were moderate inhibitors and others (?-glucan
and mannan) weak inhibitors. These results indicate that DC-HIL
can bind dermatophytes, suggesting that the fungal ligands of DC-
HIL may be saccharides structurally related to HS, chitin, and/or
Ligation of DC-HIL leads to tyrosine phosphorylation of its
Because DC-HIL has an ITAM-like signaling motif (YxxI), we
questioned whether binding of DC-HIL to T. rubrum transduces
tyrosine phosphorylation of this protein in DC (Fig. 3A). BM-DC
were cocultured with varying doses of C. albicans hyphae or T.
rubrum (as dried weight), and tyrosine phosphorylation on DC-
HIL was assayed by immunoprecipitation and blotting using anti-
p-tyrosine Ab. Tyrosine phosphorylation of DC-HIL was induced
in DC following coculture with T. rubrum, but not with C. albicans
hyphae even at the highest dose tested, consistent with selective
binding by DC-HIL. Such phosphorylation was also detected in
BM-DC after cross-linking of DC-HIL with UTX-103 mAb (but
not with control IgG) (Fig. 3B). However, the level of phosphor-
ylation induced by the mAb was considerably less than by T.
rubrum. We then questioned whether the T cell ligand SD-4 can
induce tyrosine phosphorylation (Fig. 3C). Treatment of BM-DC
with immobilized SD-4-Fc (but not control Ig) induced phosphor-
ylation, albeit at a weaker level compared with the two previous
DC-HIL contains two tyrosine residues in its intracellular do-
main: at aa 523 proximal to the transmembrane domain and aa 529
in the YxxI sequence corresponding to the ITAM-like motif (note
that a typical ITAM has two tandem-repeats of YxxI/L) (24). To
determine which tyrosine residue is responsible for phosphoryla-
tion, point-mutation analysis was performed (Fig. 3D), in which
either or both Tyr 523 or 529 was (or were) mutated to phenylal-
anine (designated Y523F, Y529F, or Y523F/Y529F). Mutants and
wild-type DC-HIL were transfected separately into COS-1 cells
and assayed for tyrosine phosphorylation (Fig. 3E). Surface ex-
pression of mutant DC-HIL on COS-1 cells was similar to those of
wild-type DC-HIL (data not shown). Wild-type DC-HIL was ty-
rosine phosphorylated as early as 10 min in COS-1 cells cross-
linked with UTX-103 mAb (but not with control IgG). DC-HIL
bearing Y523F mutation was phosphorylated at a similar level,
whereas Y529F mutant and doubly mutated DC-HIL failed to
undergo phosphorylation (Fig. 3E), thereby identifying the
former (tyrosine on aa 529 in the YxxL motif) as the relevant
Cross-linking of DC-HIL with UTX-103 mAb up-regulates
particular genes in DC
Because phosphorylated YxxL (even just one unit) can induce
gene expression (25), we examined changes in DC gene expres-
sion profile following stimulation of DC-HIL (Fig. 4). For this
study, we chose UTX-103 mAb as a surrogate ligand for DC-
HIL because cell wall extracts from T. rubrum are toxic to
BM-DC (?2 h coculture with extract kills most DC). BM-DC
5192DC-HIL IS A PATTERN RECOGNITION RECEPTOR
by guest on June 13, 2013
control IgG) and marker Ab. Surface or intracellular expression of DC-HIL was analyzed by flow cytometry. A, Epidermal LC were identified by I-A/I-E
expression in epidermal cell suspension. Frequency (%) of positive cells is shown on dot-blots. B and C, BM-DC were left untreated or activated with LPS and
by high expression of CD11b from peritoneal cells of mice stimulated with thioglycolate. A second experiment showed similar results.
DC-HIL expression by DC and macrophages. Different APC subpopulations were labeled fluorescently with UTX-103 anti-DC-HIL mAb (or
5193 The Journal of Immunology
by guest on June 13, 2013
were cross-linked with UTX-103 mAb or control IgG, followed
6 h later by isolation of mRNA, and then gene expression anal-
ysis using two different Oligo GEArray Dendritic and APC and
Autoimmune and Inflammatory microassays, on which 260 and
440 gene-specific olignucletide probes were blotted, respec-
tively. (These were a total of 662 genes because the two arrays
have overlapping gene probes.) Gene expression in DC treated
with UTX-103 mAb was expressed relative to that of the house
keeping gene GAPDH (Fig. 4, A and C) and compared with that
of DC treated with control IgG (evaluated by fold difference).
Genes up-regulated more than 2-fold greater than control are
listed (Fig. 4, B and D), including profilin-I (an actin-binding
protein involved in turnover and restructuring of the actin cy-
toskeleton) (26), Marcksl-1 (myristoylated alanine-rich C-ki-
nase substrate involved in regulating cell shape, motility, se-
cretion, transmembrane transport, and cycling) (27), CCAAT/
enhancer binding protein (C/EBP or Cebpb) involved in LC
commitment (28), and a lectin-like receptor for oxidatively
modified low-density lipoprotein (LOX-1 or Orl-1) (29, 30).
Our analyses showed 23 genes to be up-regulated following
cross-linking with UTX-103 mAb, corresponding to 3% of 662
genes tested. Not one gene examined was down-regulated ?2-
fold. Because endotoxin content in preparations of UTX-103
mAb and control IgG were similar (?0.5 EU/ml), we think it
can be excluded as a cause of the changes.
To verify our results, we examined mRNA expression of
Marcksl-1 and Orl-1, using real-time PCR. Both genes were chosen
because they were highly up-regulated in each microarray (Fig. 5, A
examined in this manner because we were unable to find relevant
primers capable of producing dose-dependent amplification. After
BM-DC were cross-linked with UTX-103 mAb or control IgG,
mRNA expression was analyzed and expression levels calculated as
fold-increases. Consistent with results of microarray-gene expression
analysis, Orl-1 and Marcksl-1 genes were up-regulated (12 and 4.5-
fold, respectively, greater than controls) 6 h after cross-linking. Their
up-regulation was transient because a return to baseline expression
levels was noted 2 days after stimulation.
dermatophyte fungi. Candida albicans
pseudohyphae (A), Trichophyton rubrum
(B and D), and Microsporum audouinii
(C) were incubated with Fc alone, DC-
HIL-Fc, or Dectin-2-Fc (20 ?g/ml) and
fluorescently labeled with FITC-anti-
human IgG Ab. D, DC-HIL-Fc was al-
lowed to bind T. rubrum in the presence
of heparin (2 ?g/ml). E, T. rubrum fungi
were pretreated with N-glycosidase be-
fore binding assay. F and G, DC-HIL-Fc
was pretreated with indicated concen-
trations of chitin (F) or galactomannan
(G) before incubation with T. rubrum.
Phase contrast (Phase) and fluorescent
(FITC) images were taken by visual
and confocal microscopy, respec-
tively. The scale bar is 10 (A) or 100
?m (B). Data shown are representa-
tive of three separate experiments.
Binding of DC-HIL to
5194 DC-HIL IS A PATTERN RECOGNITION RECEPTOR
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We also examined protein expression of TNF-? and IL-1? by
ELISA (Figs. 5C and D). Without stimulation, BM-DC did not
secrete detectable levels of either cytokine, and control IgG treat-
ment induced very low levels of expression. By contrast, treatment
with UTX-103 mAb led to markedly elevated levels of TNF-? and
IL-1? secretion that lasted at least 2 days. We then questioned
with/without 0.1 or 1 mg/ml (dried weight) C. albicans pseudohyphae or T. rubrum, 15 min after of which whole cell extracts were prepared. DC-HIL
protein was then immunoprecipitated, followed by immunoblotting using anti-p-tyrosine or anti-DC-HIL mAb (phosphorylation assay). B and C, At
indicated time point after cross-linking of DC-HIL on BM-DC with UTX-103 mAb (B) or culturing BM-DC with immobilized SD-4-Fc (C) or control IgG
(Ctrl), phosphorylation levels on DC-HIL in treated cells was examined as before. D, Amino acid structures of DC-HIL with no mutations (WT), a mutation
of Y523F or Y529F, or double mutations (DM) are aligned and schematically shown, consisting of, from the N terminus, the extracellular domain (ECD),
transmembrane (TM), and intracellular domain (ICD) in which location of tyrosine residues at a 523 and 529 is indicted by a triangle and circle,
respectively. Closed and open symbols represent no mutation and replacement with phenylalanine, respectively. At the right, results of tyrosine phos-
phorylation assays are shown: ? and – indicate significant phosphorylation levels detected and no phosphorylation. E, Tyrosine phosphorylation assays
using WT and mutants. Two days after transfection of COS-1 cells with WT or mutant DC-HIL gene, cells were treated similarly with UTX-103 mAb or
control IgG for 10 min and the level of tyrosine phosphorylation assayed. A second experiment showed similar results.
Ligation of DC-HIL induces tyrosine phosphorylation of an ITAM-like motif in the intracellular domain. A, BM-DC were cocultured
or control IgG (Ctrl IgG), cDNA probe was prepared from RNA of treated BM-DC and hybridized to oligo-microarrays Dendritic and APC (A) and
Autoimmune and Inflammatory (C). Hybridization images (A and C) on the array are shown. Gene location on the array matrix is shown by alphabetic and
numerical numbers according to designation by Super Array Inc (Refer to Gene Table at http://www.sabiosciences.com/genetable). B and D, After
computer-assisted expression analysis, genes whose expression levels were more than 2-fold greater than in control IgG-treated DC are listed with the
matrix location code (shown in the parentheses), among of which genes marked with an asterisk are further analyzed using real-time PCR or ELISA (Fig.
5). These data are registered as the accession number of GSE17699 at Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/).
Gene expression analysis of BM-DC stimulated by cross-linking of DC-HIL. Six hours after cross-linking with UTX-103 mAb (?DC-HIL)
5195 The Journal of Immunology
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whether T. rubrum (or Candida hyphae) stimulates DC to produce
these proinflammatory cytokines (Fig. 5, E and F). BM-DC were
stimulated with/without dried T. rubrum or Candida hyphae and
measured for secretion of these cytokines. DC treated with T.
rubrum produced TNF-? and IL-1? cytokines at a level similar or
higher than by DC stimulated with cross-linking of DC-HIL. T.
rubrum was a stronger stimulator than Candida.
Because IL-1? induces DC maturation and acquisition of
strong immunostimulatory capacity (31), we next examined the
effect of cross-linking of DC-HIL on DC expression of activa-
tion/maturation markers. BM-DC were cross-linked with UTX-
103 mAb and then cultured for 2 days. Surface expression of
CD80 and CD86 was examined by flow cytometry (Fig. 6, A
and B). Treatment with UTX-103 mAb markedly increased ex-
pression of CD80 (47 vs 356 mean fluorescent intensity, MFI)
and CD86 (18 vs 49). We then examined T cell-stimulatory
capacity by UTX-103 mAb-treated DC (Fig. 6, C–E). After
cross-linking and pulsing DC with MHC class I- or II-restricted
OVA peptide, increasing numbers of these cells were cocul-
tured with a constant number of CD4?or CD8?T cells isolated
from OT-II or OT-I transgenic mice, respectively. Activation of
T cells was measured by IL-2 production for CD4?T cells (Fig.
6C) and IL-2 and IFN-? for CD8?T cells (Fig. 6, D and E).
UTX-103 mAb-treated DC stimulated OT-II CD4?T cells to
produce IL-2, greater than by control IgG-treated DC at each
dose tested, with highest response of 8-fold increase. UTX-103
mAb also stimulated DC to exhibit a greater capacity to activate
OT-I CD8?T cells, but to a lesser degree than for OT-II CD4?
T cells: ?2-fold increase in IL-2 (Fig. 6D) and 50% increase in
IFN-? (Fig. 6E). Altogether, cross-linking of DC-HIL up-reg-
ulated DC gene expression, resulting in DC maturation and aug-
mented T cell-stimulatory capacity.
linking of DC-HIL on BM-DC with mAb or control IgG, RNA or culture supernatant were isolated/recovered, and then mRNA for Orl-1 and Marcksl-1
genes or protein expression of TNF-? and IL-1? was measured by real-time PCR (A and B) or ELISA (C and D, shown as mean ? SD, n ? 3), respectively.
mRNA expression levels are normalized by expression of GAPDH and expressed as fold-increase over values in control IgG-treated DC. E and F, BM-DC
also were treated with/without dried T. rubrum or Candida pseudohyphae (10 ?g/ml) and cultured for 2 days. Secretion of TNF-? (E) or IL-1? (F) was
measured by ELISA. Statistical significance of presented data is p ? 0.001 (Student’s t test) compared with controls. Data shown are representative of at
least two independent experiments.
Expression of up-regulated genes by DC following stimulation with anti-DC-HIL mAb or T. rubrum. At varying time points after cross-
control IgG (IgG-treated), BM-DC were harvested and examined by flow cytometry for expression of CD80 (A) and CD86 (B) activation markers. Staining
of DC treated with UTX-103 mAb or control IgG are shown by histograms with solid or dashed lines, respectively. Expression levels on untreated DC (just
before stimulation) are also shown. Histogram filled in gray represents staining of untreated DC with isotypic control IgG (for marker Ab). Mean fluorescent
intensity for CD80 (or for CD86) was 47 (or 18) for control IgG-treated and 356 (or 49) for UTX-103 mAb-treated DC. C–E, These treated DC were also
assayed for APC capacity: Increasing numbers of DC were pulsed with OVA peptides and cocultured with a constant number (1 ? 105cells/well) of splenic
CD4?T cells from OT-II transgenic mice (C) or CD8?T cells from OT-I mice (D and E). Activation of T cells was measured by production levels of
IL-2 (C and D) and IFN-? (E) (mean ? SD, n ? 3). Statistical analysis (Student’s t test) for all data (C–E) shows p ? 0.0001 compared with values by
IgG-treated DC. These results are representative of at least two independent experiments.
DC-HIL-stimulated DC exhibit augmented T ell-stimulatory capacity. Two days after cross-linking with UTX-103 mAb (UTX-treated) or
5196DC-HIL IS A PATTERN RECOGNITION RECEPTOR
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While infecting skin, hair, and nails, dermatophytic fungi obtain
nutrients from keratinized material (32). Although these fungi usu-
ally do not invade living tissue, their metabolic by-products cause
inflammation, which is especially severe in immunosuppressed pa-
tients (33). An early defense mechanism against dermatophytic
infection may be mediated by epidermal LC (skin-resident DC)
that reside close to the usual primary site of infection. Having
shown that DC-HIL acts as a PRR for dermatophytes and that
epidermal LC express DC-HIL constitutively at high levels (hu-
man LC also express DC-HIL at high levels; Ref. 34), we posit that
DC-HIL exerts antifungal immunity via innate immune recogni-
tion and potentiation of LC function. Concurrently, DC-HIL may
suppress cutaneous inflammation by attenuating activity of T cells
that home to skin. Thus, the net effect of these positive and neg-
ative regulations exerted by DC-HIL may determine, at least in
part, the outcome of antidermatophyte immunity.
SD-4 is a transmembrane protein heavily laden with HS chains
consisting of alternating disaccharide residues (glucuronic acid
and iduronic acid with glucosamine). It is expressed constitutively
by B cells but not by naive T cells (its expression can be induced
by activation; Ref. 20). Despite its expression profile, DC-HIL
binds to activated T cells, but not to B cells (our unpublished data).
Because the binding is abrogated by heparin or by heparinase treat-
ment of activated T cells, DC-HIL is likely to recognize the struc-
ture of HS chain expressed on T cells. Our results suggest that
nonself DC-HIL ligands are expressed by T. rubrum and M. au-
douinii. Unlike TLR ligands, expression of these ligands may be
restricted to only some fungi because DC-HIL does not bind bac-
teria and C. albicans. The cell wall of dermatophytes is made up
primarily of chitin, mannan, and galactomannan, none of which
structurally resemble HS (35). In fact, chitin and galactomannan
did not strongly inhibit DC-HIL binding as heparin did, and
mannan was a weak inhibitor. We thus postulate that the puta-
tive ligand(s) on dermatophytes are saccharides structurally re-
sembling HS on T cells.
Our findings indicate that the membrane-proximal YxxI se-
quence of DC-HIL is the functional tyrosine-based signal motif. It
is not a typical ITAM because two ITAM units have been shown
to be required for signal transduction (36). ITAM was identified
originally by mutation analysis of Ag receptors containing multi-
ple activation motifs comprising two YxxL/I sequences with de-
fined spacing between them (24). ITAM is phosphorylated by Src
family kinases and the resultant phosphorylated ITAMs are rec-
ognized by two Src homology 2 domains of Syk kinases, that
enable transduction of signals (37). Thus, the space between two
ITAMs is important for recognition by Syk kinases. However, only
one ITAM unit was demonstrated to be sufficient to initiate Syk-
mediated signal transduction (e.g., dectin-1, which has one YxxL
motif; Ref. 9, 14, 25). In fact, ligation of dectin-1 by ?-glucan (or
zymosan) can induce expression of a number of genes necessary
for innate immunity against yeast pathogens, cooperatively with
TLR (8, 38). Like dectin-1, we speculate that the YxxI motif in
DC-HIL is capable of inducing Syk-mediated signaling responsi-
ble for potentiating DC function.
Several ligand/receptor pairs controlling T cell activation are
involved in reciprocal signaling between APC and T cells. Al-
though the impact of these pairs has been well studied for T cells,
relatively less is known about effects on APC. Programmed cell
death-1 ligands (PD-L1 and PD-L2/B7-DC) on APC negatively
regulate T cell activation by interacting with PD-1 on T cells (39,
40). Because PD-L1 and PD-L2 have short cytoplasmic tails lack-
ing known motifs for signal transduction, these ligands are thought
to be incapable of transducing signals following binding to PD-1.
PD-L2 possesses a four amino acid-long intracellular domain and
recent studied have shown that cross-linking PD-L2 directly po-
tentiates DC function by enhancing DC presentation of Ag-loaded
MHC molecules, promoting DC survival and increasing secretion
of IL-12 (41, 42). Possibly, PD-L2 associates with a coreceptor
(that contains intracellular signaling motifs) through two charged
amino acids in its transmembrane domain. To our knowledge, PD-
L1-induced signaling has not been formally reported. Herpesvirus
entry mediator on APC is a ligand for coregulatory receptors
BTLA, CD160, and LIGHT on T cells. Herpesvirus entry mediator
is a member of the TNF receptor family that can recruit several
members of the TNFR-associated factor family, enabling activa-
tion of NF-?B and Jun N-terminal kinase, eventually leading to
augmented immune responses (43). These coinhibitory ligands in-
cluding DC-HIL deliver negative signals to T cells when engaged
to their corresponding T cell receptors, and conversely they can
transduce positive signals within APC.
Such reciprocal signaling may confer balance in the activation
of APC and T cells. Immature DC such as epidermal LC consti-
tutively express coinhibitory ligands at levels higher than costimu-
latory ligands (44). This is true for DC-HIL, which is expressed
highly on epidermal LC. Such DC are less potent activators of
naive T cells than mature DC (45), but may down-regulate the
status of recently activated T cells because they express high levels
of coinhibitory receptors. The DC-HIL ligand SD-4 also is ex-
pressed on activated (but not resting) T cells. Shortly after engag-
ing with activated T cells, signals induced by coinhibitory ligands
may drive DC to undergo maturation, enabling them to become
highly potent APC in activating naive T cells. Infection by T.
rubrum may release fungal products containing the DC-HIL li-
gands that modulate this DC-HIL/SD-4 pathway.
In sum, our results document direct binding of DC-HIL to the cell
wall of dermatophytes to transduce a signal potentiating DC function,
indicating that DC-HIL is a PRR for these fungi. Thus, DC-HIL can
regulate immune responses in a dual manner: previously we showed
it to be an inhibitor of adaptive immunity following ligation of SD-4
on activated T cells, and now we present evidence that it can induce
innate immunity against dermatophytes.
We thank Irene Dougherty for technical expertise and Susan Milberger for
The authors have no financial conflict of interest.
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