A Notch Ligand, Delta-Like 1 Functions As an Adhesion Molecule for Mast Cells

Abstract

Mast cells (MCs) accumulate in chronic inflammatory sites; however, it is not clear which adhesion molecules are involved in this process. Recently, the expression of Notch ligands was reported to be upregulated in inflammatory sites. Although Notch receptors are known as signaling molecules that can activate integrins, their contributions to the adhesion of MCs have not been studied. In this study, we demonstrated that mouse MCs efficiently adhered to stromal cells forced to express a Notch ligand, Delta-like 1 (Dll1). Surprisingly, the adhesion was a consequence of direct cell-cell interaction between MCs and Dll1-expressing stromal cells rather than activation of downstream effectors of Notch receptor(s)-Dll1. The adhesion of MCs to Dll1-expressing stromal cells remained even when the cell metabolism was arrested. The recognition was blocked only by inhibition of Notch receptor(s)-Dll1 interaction by addition of soluble DLL1, or mAbs against Dll1 or Notch2. Taken together, these results indicate that Notch receptor(s) and Dll1 directly promote the adhesion of MCs to stromal cells by acting as adhesion molecules. This appreciation that Notch receptor-ligand interactions have an adhesion function will provide an important clue to molecular basis of accumulation of MCs to inflammatory sites.

Full text

The Journal of Immunology
A Notch Ligand, Delta-Like 1 Functions As an Adhesion
Molecule for Mast Cells
Akihiko Murata,* Kazuki Okuyama,* Seiji Sakano,
†
Masahiro Kajiki,
†
Tomohisa Hirata,
†
Hideo Yagita,
‡
Juan Carlos Zu
´n
˜iga-Pflu
¨cker,
x
Kensuke Miyake,
{
Sachiko Akashi-Takamura,
{
Sawako Moriwaki,
‖
Shumpei Niida,
‖
Miya Yoshino,* and
Shin-Ichi Hayashi*
Mast cells (MCs) accumulate in chronic inflammatory sites; however, it is not clear which adhesion molecules are involved in this
process. Recently, the expression of Notch ligands was reported to be upregulated in inflammatory sites. Although Notch receptors
are known as signaling molecules that can activate integrins, their contributions to the adhesion of MCs have not been studied. In
this study, we demonstrated that mouse MCs efficiently adhered to stromal cells forced to express a Notch ligand, Delta-like 1 (Dll1).
Surprisingly, the adhesion was a consequence of direct cell–cell interaction between MCs and Dll1-expressing stromal cells rather
than activation of downstream effectors of Notch receptor(s)-Dll1. The adhesion of MCs to Dll1-expressing stromal cells remained
even when the cell metabolism was arrested. The recognition was blocked only by inhibition of Notch receptor(s)–Dll1 interaction
by addition of soluble DLL1, or mAbs against Dll1 or Notch2. Taken together, these results indicate that Notch receptor(s) and
Dll1 directly promote the adhesion of MCs to stromal cells by acting as adhesion molecules. This appreciation that Notch
receptor–ligand interactions have an adhesion function will provide an important clue to molecular basis of accumulation of
MCs to inflammatory sites. The Journal of Immunology, 2010, 185: 3905–3912.
Mast cells (MCs) are derivatives of hematopoietic pro-
genitor cells that play an essential role in normal host
defense and various allergic disorders. MCs are widely
distributed throughout the body, especially in the skin, peritoneal
cavity, and gastrointestinal mucosa (1, 2). MCs usually accumulate
at sites of chronic inflammation, and are thought to contribute
to pathogenesis by releasing a variety of inflammatory mediators
(3, 4).
Cell adhesion, which is mediated by specialized molecules, is
indispensable in the process of cellular recruitment, retention and
localization. Previous studies have reported that L-selectin, inte-
grins (a4b1 and a4b7) and Ig superfamily-cell adhesion molecules
(Icam1 and Vcam1) contribute to the accumulation of MCs in in-
flammatory sites (5–7). However, questions remain about molec-
ular mechanisms associated with accumulation of MCs.
Recently, the expression of Notch ligands was reported to be
upregulated in chronic inflammatory diseases such as rheumatoid
arthritis, chronic pancreatitis, and diabetic nephropathy (8–10).
These conditions are frequently accompanied by accumulation of
MCs (4, 11, 12). Notch receptors are widely expressed through he-
matopoietic cell lineages, including MCs, and are known as signal-
ing molecules that regulate a broad spectrum of cell fate decisions,
proliferation, and function (13–15). In mammals, there are four
Notch receptors (Notch1–Notch4)and two distinct families of Notch
ligands known as Delta-like (Dll) ligands (Dll1, Dll3, and Dll4) and
Jagged (Jag) ligands (Jag1 and Jag2) (14, 15). Binding of Notch
ligands to Notch receptors induces successive proteolytic cleavage
of Notch receptors by a disintegrin and metalloproteases (ADAMs)
at the extracellular domain (ECD) and subsequently by a g-secretase
complex at the transmembrane domain. This results in the release of
Notch intracellular domain (ICD) and expression of Notch related
genes (14, 15). Overexpressed Notch ICD acts as a constitutively
active form of Notch receptors (16). A previous study suggests that
Notch signaling activates integrin b1 (17), indicating its involvement
in cellular accumulation by enhancing cell adhesion. MCs might
interact with increased Notch ligands in chronic inflammatory sites;
however, the contribution of Notch receptor–ligand interaction to the
adhesion of MCs has not been studied.
In this study, we investigated the contribution of Notch ligands
to the adhesion of mouse bone marrow (BM)-derived cultured
MCs by using the OP9 stromal cell line overexpressing a Notch
ligand, Dll1 (OP9-DL1), as a model for inflammatory sites where
the expression of Notch ligands was upregulated. MCs adhered to
OP9-DL1 more efficiently than to control OP9 cells. Surprisingly,
the recognition was not due to Notch signaling in either MCs or
stromal cells. Metabolically inactive MCs were still adhesive, and
*Division of Immunology, Department of Molecular and Cellular Biology, School of
Life Science, Faculty of Medicine, Tottori University, Yonago;
†
Corporate R&D Lab-
oratories, Asahi Kasei Corp., Fuji;
‡
Department of Immunology, Juntendo Univer-
sity School of Medicine, Bunkyo-ku;
{
Division of Infectious Genetics, Institute of
Medical Science, University of Tokyo, Minato-ku,
‖
Laboratory of Genomics and Pro-
teomics, National Center for Geriatrics and Gerontology, Obu, Japan; and
x
Depart-
ment of Immunology, University of Toronto, Sunnybrook Research Institute, Toronto,
Canada
Received for publication January 20, 2010. Accepted for publication July 27, 2010.
This work was supported by grants-in-aid for Scientific Research (C [to S.I.H.] and B
[to S.N.]) from the Ministry of Education, Culture, Sports, Science, and Technology
of the Japanese government and a Canada Research Chair in Developmental Immu-
nology (to J.C.Z.-P.). A.M. and K.O. are Research Fellows of the Japan Society for
the Promotion of Science.
The sequences presented in this article have been submitted to Gene Expression
Omnibus under accession number GSE22659.
Address correspondence and reprint requests to Akihiko Murata, Division of Immu-
nology, Department of Molecular and Cellular Biology, School of Life Science,
Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503,
Japan. E-mail address: muratako@med.tottori-u.ac.jp
The online version of this article contains supplemental material.
Abbreviations used in this paper: ADAM, a disintegrin and metalloprotease; BM, bone
marrow; DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester;
Dll, delta-like; ECD, extracellular domain;ICD, intracellular domain; Jag, Jagged; Kitl,
Kit ligand; MC, mast cell; Tc, tetracycline.
Copyright Ó2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000195

the recognition was blocked by addition of soluble DLL1, or mAbs
against Dll1 or Notch2. Thus, our results show that Notch receptor
(s) and Dll1 function as adhesion molecules for MCs.
Materials and Methods
Mice
C57BL/6J mice were purchased from Japan CLEA (Tokyo, Japan). Ex-
periments were approved and performed in accordance with the guidelines
of the Animal Care and Use Committee of Tottori University.
Reagents
Human IgG1 Fc-fused human DLL1 (DLL1-Fc), DLL4 (DLL4-Fc), JAG1
(JAG1-Fc), and Flag (FL)-tagged human JAG2 (JAG2-FL), which lacked
the transmembrane and cytoplasmic domains were prepared as described
(18, 19). Human IgG1 (Chemicon International, Temecula, CA) was used as
a control for IgG1 Fc-fused Notch ligands. Sodium azide and DMSO
were purchased from Wako Pure Chemical Industries (Osaka, Japan). A g-
secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine
t-butyl ester (DAPT) was purchased from Peptide Institute (Osaka, Japan).
Cell lines
OP9 stromal cell lines(20) carrying Dll1 and GFP genes (OP9-DL1), and GFP
gene (OP9-control) were cultured in MEM a(aMEM; Life Technologies-
BRL, Grand Island, NY), supplemented with 20% FBS (JRH Biosciences,
Lenexa, KS), 50 mg/ml penicillin (Meiji Seika, Tokyo, Japan), and 50 U/ml
streptomycin (Meiji) (21).
ST2NIC cells, a BM-derived stromal cell line (ST2) transfected with
a constitutively active form of the Notch1 ICD gene, whose expression was
controlled by tetracycline (Tc; Sigma-Aldrich, St Louis, MO)-off system
and reflected by the expression of GFP, were maintained as described (22).
Cells were cultured with RPMI 1640 (Life Technologies-BRL), supple-
mented with 5% FBS, 50 mM 2-ME (Wako) and 1 mg/ml Tc.
Abs
For adhesion assays, rat antagonistic mAbs against mouse Kit (ACK2) (23)
and Il7ra(A7R34) (24); and hamster antagonistic mAbs against mouse
integrin a5 (HMa5-1) (25), Dll1 (HMD1-5) (26), Notch2 (HMN2-29)
(26), and Ctla4 (UC10-4F10-11) (27) were used.
For flow cytometric analyses, the above mAbs were conjugated with
biotin (Pierce, Rockford, IL). Biotin-conjugated hamster anti-mouse Cd3ε
(145-2C11) (BD Biosciences, San Jose, CA) or rat anti-mouse platelet-
derived growth factor receptor a(APA5) (28) mAbs were used as controls.
Stained cells were analyzed by a flow cytometer (EPICS XL; Coulter, Palo
Alto, CA).
Preparation of cultured MCs
Cultured MCs were generated as described (29). Cells from femora, adult
spleens, or E14.5 fetal livers were cultured in aMEM-supplemented with
10% FBS and mouse Il3 (a gift from Dr. Sudo, Toray Industries, Kana-
gawa, Japan) at 50 U/ml in a humidified atmosphere of 5% CO
2
in the air
at 37˚C. Nonadherent cells were replaced into fresh culture media every
5 d. After .6 wks, .97% of cells from BM and spleen in cultures were
MCs, as judged by their morphology and the surface expression of Kit on
the flow cytometry. MCs in fetal liver culture were enriched as Kit positive
cells with BD IMagnet system (BD Biosciences) and further cultured for
7 d with Il3. Adhesion assay were performed by using BM-derived cul-
tured MCs unless otherwise indicated.
Analysis of gene expression
For RT-PCR analyses, total RNA was prepared by using ISOGEN (Nippon
Gene, Toyama, Japan), and was reverse transcribed by using Reverse
TraAce (Toyobo, Osaka, Japan). The PCR conditions were as follows: 94˚C
(3 min) for primary; 94˚C (45 s), 60 or 55˚C (for Notch receptors and their
ligands or Kit ligand [Kitl], respectively) (1 min), 72˚C (1.5 min) for the
following 35 or 30 cycles (for Notch receptors and their ligands or Kitl,
respectively). The extension time in the last cycle was 3 min. Primers for
Kitl were as follows; 59-AAA TAG TGG ATG ACC TCG TG-39and 59-
ATT ACA AGC GAA ATG AGA GC-39. Other primers were as previously
described (22).
Gene expression was also analyzed by the “3D-Gene” mouse oligo chip
24k (Toray Industries, Kanagawa, Japan) (Supplemental Table I) (30).
Induction of adipocyte differentiation
The effective dose of DAPT was determined by adipocyte differentiation
assay. OP9 cells differentiate into adipocytes, and Notch signaling inhibits
adipocyte differentiation (31, 32). Forty-eight–well flat-bottomed culture
plates (Corning Costar, Corning, NY) were coated with 1 mg DLL1-Fc,
DLL4-Fc, JAG1-Fc, JAG2-FL, or human IgG1 in 100 ml PBS for 1 d at
4˚C. After washing, OP9-control (2 310
4
) in 200 ml of its culture media
were seeded with or without DAPT or the same volume of its solvent,
DMSO (0.1% v/v). After culturing for 5 d at 37˚C, cells were fixed with
3.7% formaldehyde (Wako), and stained with Oil Red O (Sigma-Aldrich)
solution. Stained cells were counted under a microscope (Fig. 3C,3D).
Cell adhesion assay
OP9-control and OP9-DL1 (2 310
4
) were seeded in 48-well plates and
cultured for 2 d at 37˚C to prepare confluent monolayers. After washing
wells with PBS, MCs (1.5 310
5
) suspended in 200 mlaMEM, supple-
mented with 10% FBS, were seeded into each well with or without mAbs
or reagents, and incubated for 1 h at 37˚C in a humidified atmosphere
of 5% CO
2
in the air, unless otherwise indicated. Nonadherent MCs were
recovered after vigorous agitation (low speed, scale 5) for 30 s with a Micro-
Mixer E-36 (Taitec Corporation, Saitama, Japan), and were counted with
a hemacytometer. Numbers of adherent MCs or ratios of nonadherent MCs
relative to ones initially added in a well were calculated.
For the assaywith fixed stromal cells, confluent monolayers of OP9-control
or OP9-DL1 were fixed with 4% paraformaldehyde (Wako) for 5 min at room
temperature. After washing cells, the adhesion assay was performed.
ST2NIC cells (4 310
4
) were seeded in 48-well plates with serial con-
centrations of Tc. After culturing for 3 d, the adhesion assay was per-
formed without Tc in culture.
Statistics
Data are presented as the mean 6SE of triplicate cultures unless otherwise
indicated. Statistical significance was established at p,0.05 by two-tailed
Student ttest.
Results
MCs adhered to OP9-DL1 more efficiently than to OP9-
control cells
We used OP9-DL1 as a model for tissues increased Dll1 density.
Semiquantitative RT-PCR analysis showed that OP9-DL1 expres-
sed more Dll1 than OP9-control, whereas Jag1 was expressed
equally by both stromal cells (Fig. 1A). Surface Dll1 expression on
OP9-DL1 was detected by flow cytometry (Fig. 1B).
To assess whether Dll1 contributes to the adhesion of MCs to
stromal cells, we incubated serial numbers of MCs on confluent
monolayers of OP9-control or OP9-DL1 for 1 h. We then counted
nonadherent MCs and calculated the numbers of adherent MCs
relative to ones initially added in a well. The survival of MCs was
not impaired during the adhesion assay (Supplemental Fig. 1). There
were significantly more adherent MCs on OP9-DL1 than on OP9-
control at each MC density (Fig. 1C). Numbers of adherent MCs on
OP9-control cells reached a maximum when we incubated 8 310
5
MCs. In contrast, numbers of adherent MCs on OP9-DL1 contin-
ued to increase with MC density.
To assess the relationship between incubation time and adhesion
efficiency, we incubated 1.5 310
5
MCs on OP9-control or OP9-
DL1 for 15–240 min. The percentage of nonadherent MCs on
OP9-DL1 was significantly lower than that on OP9-control at each
time point (Fig. 1D). One hour was needed to reach a plateau of
adhesion of MCs to OP9-control. In contrast, MCs stably adhered
to OP9-DL1 within 15 min. These results suggest that Dll1 on
stromal cells contributes to the efficient adhesion of MCs. Similar
results were obtained from MCs induced from adult spleen or fetal
liver (Fig. 4D).
The adhesion of MCs to OP9-DL1 was not inhibited by
treatment with antagonistic Abs against Kit and integrin a5
A receptor protein tyrosine kinase, Kit, is one of the important
adhesion molecules for MCs (33). MCs highly express cell surface
3906 CELL ADHESION BY A NOTCH LIGAND

Kit molecules (Fig. 2A). Semiquantitative RT-PCR showed that
both OP9-control and OP9-DL1 expressed comparable amounts of
the Kitl, which consisted of membrane bound and soluble forms
(Fig. 2B).
To investigate the contribution of Kit to the adhesion of MCs to
stromal cells, we treated the cocultures with anti-Kit antagonistic
mAb. Treatment with anti-Kit mAb was significantly effective on
the percentage of nonadherent MCs to OP9-control (32.7 64.1%
[control], 64.9 62.5% [anti-Kit] [p,0.005]) but not to OP9-DL1
(8.4 60.6% [control], 13.6 62.3% [anti-Kit] [p= 0.09]) (Fig. 2C).
Notch signaling is reported to activate a5b1 integrin (17), which
binds to fibronectin and is activated by Kit signaling (34). MCs
expressed integrin a5 (Fig. 2A). To assess the contribution of this
molecule to the adhesion of MCs, we treated the cocultures with
anti-Kit and anti-integrin a5 antagonistic mAbs (25). This mani-
pulation had little influence on the adhesion of MCs to OP9-DL1
(Fig. 2C). These results indicate that Kit plays a critical role in
the adhesion of MCs to OP9-control but not to OP9-DL1. The
a5b1 integrin that is reported to be activated by Notch signaling
did not contribute to the efficient adhesion of MCs to OP9-DL1.
Notch signaling in stromal cells did not account for the
efficient adhesion of MCs to OP9-DL1
RT-PCR analysis showed that MCs expressed Notch1 and Notch2,
and both OP9-control and OP9-DL1 comparably expressed Notch1,
Notch2, and Notch3 (Fig. 3A, Supplemental Table I), suggesting
that the efficient adhesion of MCs to OP9-DL1 resulted from ad-
ditional adhesion molecules on MCs or OP9-DL1 by Notch sig-
naling. There are at least three possible explanations for the effi-
cient adhesion: 1) OP9-DL1 cells expressed additional adhesion
molecule(s) after interaction with MCs by reciprocal signaling via
Notch between cells; 2) OP9-DL1 cells expressed additional ad-
hesion molecule(s) by Notch signaling after interaction with each
other; and 3) MCs expressed additional adhesion molecule(s) by
signal transduction including Notch signaling after interaction with
OP9-DL1.
To assess the first possibility that OP9-DL1 expressed additional
adhesion molecule(s) by reciprocal signaling between MCs, we
incubated MCs with fixed stromal cells. Fixed stromal cells would
be unable to express additional cell surface molecules after in-
teraction with MCs, though they would still provide cell surface
molecules expressed before the adhesion assay. MCs also adhered
to fixed OP9-DL1 more efficiently than to fixed OP9-control cells
(Fig. 3B), indicating that the additional adhesion molecule(s) on
OP9-DL1 after interaction with MCs did not account for the ef-
ficient adhesion of MCs to OP9-DL1.
To assess the second possibility that OP9-DL1 expressed ad-
ditional adhesion molecule(s) following Notch signaling, we used
ag-secretase inhibitor, DAPT, which can block Notch cleavage at
the transmembrane site and thus impairs Notch signaling (35). The
effective dose of DAPT was determined by adipocyte differenti-
ation assay (see Materials and Methods). Adipocyte differentia-
tion of OP9-control was significantly inhibited by stimulation with
immobilized Notch ligands but not control human IgG1 (Fig. 3C).
Inhibition of adipocyte differentiation by immobilized DLL1-Fc
was blocked by treatment with 10 mM but not 1 mM DAPT or
DMSO during the culture (Fig. 3D). OP9-DL1 cells also had re-
duced tendency to differentiate into adipocytes and treatment with
10 mM DAPT was also effective on this inhibition (Supplemental
Fig. 2). Therefore, we selected the 10 mM concentration of DAPT
to inhibit Notch signaling.
If the efficient adhesion of MCs to OP9-DL1 resulted from the
Notch signaling in stromal cells, pretreatment of stromal cells with
DAPTwould inhibit the adhesion of MCs. To this end, we pretreated
OP9-control or OP9-DL1 with DAPTor DMSO for 2 to 3 d and then
performed the adhesion assay. These reagents had no effect on the
adhesion of MCs to either type of stromal cells (Fig. 3E).
FIGURE 1. MCs adhered to OP9-DL1 more efficiently than to OP9-
control. A, Serial dilutions of cDNAs were subjected to PCR amplification
specific for the Dll1,Jag1, or hypoxanthine guanine phosphoribosyl trans-
ferase 1 (Hprt1). B, The expression of Dll1 on stromal cells was analyzed by
flow cytometry using anti-Dll1 mAb (open) or anti-Cd3εmAb (filled) as
a control. Cand D, On OP9-control or OP9-DL1, (C) serial numbers of MCs
were incubated for 1 h, and (D) MCs (1.5 310
5
) were incubated for in-
dicated times. Nonadherent MCs were counted and the numbers of adherent
MCs (C) or the percentages of nonadherent MCs (D) in total MCs were
calculated. Data indicate mean 6SE of triplicate cultures and were statis-
tically significant between OP9-control and OP9-DL1 at each point (p,
0.05). Data are representatives of at least two independent experiments.
FIGURE 2. The adhesion of MCs to OP9-DL1 was not inhibited by
treatment with antagonistic mAbs against Kit and integrin a5. A, The
expression of Kit and integrin a5 on MCs was analyzed by flow cytometry
(open). Anti–platelet-derived growth factor receptor a(left) or anti-Cd3ε
(right) mAbs were used as controls (filled). B, Serial dilutions of cDNAs
were subjected to PCR amplification. Upper and lower bands in Kitl
products correspond to soluble form (contain exon 6, upper arrow) and
membrane-bound form (not contain exon 6, lower arrow) of Kitl tran-
scripts, respectively. C, The adhesion assay was performed with indicated
combinations of anti-Il7ra, anti-Kit, anti-Ctla4 and/or anti-integrin a5
antagonistic mAbs (5 mg/ml each). Anti-Il7raand anti-Ctla4 mAbs were
used as controls for anti-Kit and anti-integrin a5 mAbs, respectively.
Percentages of nonadherent MCs in total MCs were indicated. Significant
differences compared with the responses on OP9-control were indicated by
an asterisk (p,0.05).
The Journal of Immunology 3907

If the efficient adhesion of MCs to OP9-DL1 arose from the
Notch signaling in stromal cells, stimulation of Notch signaling
in OP9-control might promote the adhesion of MCs. To this end,
we incubated MCs on OP9-control stimulated with immobilized
DLL1-Fc or human IgG1 for 2 d. These manipulations also had no
effect on the adhesion of MCs (Fig. 3F). We assessed possible
differences in expression of molecules associated with Notch sig-
naling and cell adhesion between OP9-control and OP9-DL1 by
cDNA microarray analysis. No significant difference except Dll1
was observed (Supplemental Table I). Flow cytometric analysis
also showed that both stromal cells comparably expressed Vcam1,
integrin aV, a5, and b1, and Cd44 (Supplemental Fig. 3).
To further investigate the importance of Notch signaling in
stromal cells, we used ST2NIC cells carrying Notch1 ICD, a con-
stitutive active form of Notch1 regulated under the Tc-off sys-
tem (22). In the ST2NIC cells, the expression of Notch1 ICD was
induced in the absence of Tc (Fig. 3G). We incubated MCs with
ST2NIC cells precultured with serial doses of Tc for 3 d, and ob-
served that the ratio of nonadherent MCs on ST2NIC cells was
increased with the expression of Notch1 ICD (Fig. 3H, Supple-
mental Fig. 4). Taken together, these results indicate that Notch
signaling in stromal cells did not account for the efficient adhesion
of MCs to OP9-DL1.
Notch signaling in MCs did not account for the efficient
adhesion of MCs to OP9-DL1
To assess the third possibility that MCs expressed additional ad-
hesion molecules by Notch signaling after interaction with OP9-
DL1, we treated the cocultures with DAPT or DMSO during the
adhesion assay. These reagents had no effect on the adhesion of
MCs to either OP9-control or OP9-DL1 (Fig. 4A).
To further assess the contribution of signal-transduction in MCs
on their adhesion, we suppressed all possible expression of addi-
tional adhesion molecules during the adhesion assay. Cocultures
were treated with sodium azide, which inhibits the function of
ATPase and thus impairs ATP-dependent cell metabolism (36, 37).
Treatment with sodium azide significantly inhibited the adhesion
of MCs to OP9-control but not to OP9-DL1 (Fig. 4B). Moreover,
simultaneous treatment with sodium azide and anti-Kit mAb com-
pletely inhibited the adhesion of MCs to OP9-control. Surpris-
ingly, even in this condition, MCs were still adhered to OP9-DL1
cells and the percentage of nonadherent MCs was only 33.3 6
3.9%.
In addition, we performed the adhesion assay on ice to arrest
almost all signal-transduction and cell metabolism (37). It is known
that Kit molecules on MCs are immediately internalized after
binding to soluble Kit ligand (38). Because the expression of Kit
on MCs is subject to a rapid turnover even when cells are at rest
(38), their expression on MCs is rapidly recovered after treatment
with trypsin. In cultures on ice, soluble Kit ligand-induced Kit
internalization and recovery of Kit expression after treatment with
trypsin on MCs were inhibited (data not shown). This indicates
that turnover of cell surface protein is inhibited on ice. Conducting
the adhesion assay on ice completely inhibited the adhesion of
MCs to OP9-control, but more than 60% of MCs still adhered to
OP9-DL1 (Fig. 4C). Similar results were obtained from MCs in-
duced from adult spleen and fetal liver (Fig. 4D).
MCs were spherical and looked refractile by phase-contrast mi-
croscopy. Once tightly adhered to stromal cells, MCs spread on
stromal cells and looked dark (Fig. 4E,upper). Interestingly, all of
MCs still bound to OP9-DL1 in the presence of sodium azide and
anti-Kit mAb or on ice were spherical and refractile (Fig. 4E,lower).
This appearance may correspond to the tethering phase of cell ad-
hesion (39).
These results suggest that the efficient adhesion of MCs to OP9-
DL1 did not result from adhesion molecule(s) additionally ex-
pressed on MCs by any signal transduction after interaction with
OP9-DL1. This means that adhesion molecule(s) that strongly
support the adhesion of MCs must exist on the surface of OP9-DL1
stromal cells.
Notch receptor(s) and Dll1 themselves functioned as adhesion
molecules for MCs
The previous findings begged the question of whether Dll1 could
itself function as an adhesion molecule for MCs. To examine this,
FIGURE 3. Notch signaling in stromal cells did not account for the efficient adhesion of MCs to OP9-DL1. A, Serial dilutions of cDNAs were subjected
to PCR amplification specific for Notch receptors and Hprt1.B, The adhesion assay was performed on fixed stromal cells. Cand D, The effective dose of
DAPT was determined by adipocyte differentiation assay. Adipocytes were induced from OP9-control cells in the presence of (C) immobilized Notch
ligands or human IgG1 as a control, and (D) immobilized DLL1-Fc or human IgG1 with or without DAPT (1 or 10 mM) or the same volume of DMSO
(0.1% v/v). Eand F, The adhesion assay was performed (E) on stromal cells precultured with or without DMSO or 10 mM DAPT for 2 d, and (F) on OP9-
control stimulated with immobilized DLL1-Fc or human IgG1 for 2 d. Gand H, After culturing for 3 d with indicated concentration of Tc, (G) the ex-
pression of Notch1 ICD in ST2NIC was assessed by flow cytometry, and (H) the adhesion assay was performed on each ST2NIC cells. B,E,F, and H,
Percentages of nonadherent MCs were indicated. Significant differences compared with the responses with human IgG1 (C,D) or on OP9-control (B,E)
were indicated by an asterisk (p,0.05).
3908 CELL ADHESION BY A NOTCH LIGAND

we added soluble DLL1-Fc to the coculture as an antagonist. The
adhesion of MCs to OP9-DL1 was inhibited by soluble DLL1-Fc
but not human IgG1 in a dose-dependent manner (Fig. 5A). Ad-
dition of soluble DLL1-Fc at 30 mg/ml significantly inhibited the
adhesion of MCs to OP9-DL1, and the percentage of nonadherent
MCs on OP9-DL1 became comparable to that on OP9-control
(25.1 61.4% on OP9-control, 28.0 61.0% on OP9-DL1 [p=
0.16]) (Fig. 5B, third columns). Moreover, almost all of the adhe-
sion of MCs remained on OP9-DL1 held on ice was inhibited in the
presence of soluble DLL1-Fc (Fig. 5C). Addition of anti-Dll1 mAb
also significantly inhibited the adhesion of MCs to OP9-DL1 on ice
(Fig. 5E). These findings indicate that Dll1 on OP9-DL1 functions
as an adhesion molecule, directing the remarkable adhesion of
MCs to OP9-DL1.
We also assessed whether the Dll1-associated adhesion was in-
hibited by other soluble Notch ligands. The adhesion of MCs to
OP9-DL1 was also inhibited by soluble DLL4-Fc (Fig. 5B). In-
terestingly, soluble JAG ligands did not inhibit the adhesion of
MCs to OP9-DL1. None of soluble Notch ligands tested here in-
hibited the adhesion of MCs to OP9-control (Fig. 5B).
Dll1 was reported to interact with Notch1 and Notch2 (40, 41),
and MCs expressed both receptors (Fig. 5D, Supplemental Fig.
5A) (42). To assess whether Notch receptors are counter-receptors
for Dll1, we treated the coculture with anti-Notch2 mAb (26). The
FIGURE 4. Notch signaling in MCs did not account for the efficient adhesion of MCs to OP9-DL1. A–C, The adhesion assay was performed (A) with or
without 10 mM DAPT or the same volume of DMSO (0.1% v/v), (B) in the presence of 5 mg/ml anti-Il7ra(control) or anti-Kit mAbs with or without 50
mM sodium azide, and (C) at 37˚C or on ice without 5% CO
2
in the air. D, The adhesion assays were performed at 37˚C or on ice with MCs induced from
adult spleen (left) or fetal liver (right). Significant differences compared with the responses on OP9-control were indicated by an asterisk (p,0.05). E,
Photomicrographs (original magnification 3200) of adherent MCs in the graph (C) were shown (insets, higher magnification of an adherent MC). Scale
bars, 50 mm.
FIGURE 5. Notch receptor(s) and Dll1 themselves functioned as adhesion molecules for MCs. A, The adhesion assay was performed on OP9-DL1 with
a serial concentration of DLL1-Fc or human IgG1 as a control. Bars indicate percentages of nonadherent MCs of one-well culture. Dotted line indicates the
mean of percentage of nonadherent MCs cultured with PBS in triplicate cultures. Band C, The adhesion assay was performed (B) at 37˚C with human IgG1
or indicated soluble Notch ligands (30 mg/ml each), and (C) at 37˚C or on ice with human IgG1 or DLL1-Fc (30 mg/ml each). The bars of nonadherent MCs
on OP9-control on ice in Care the same data of culturing with PBS on ice. D, The expression of Notch2 on MCs was analyzed by flow cytometry using
anti-Notch2 mAb (open) and anti-Cd3εmAb (filled) as a control. E, The adhesion assay was performed on ice with indicated combinations of anti-Ctla4
(110 mg/ml), anti-Dll1 (10 mg/ml) and/or anti-Notch2 (100 mg/ml) mAbs. Anti-Ctla4 mAb was used as a control. Percentages of nonadherent MCs were
indicated. Significant differences compared with the responses on OP9-control (B,C) or on OP9-DL1 with anti-Ctla4 mAb (E) were indicated by an asterisk
(p,0.05).
The Journal of Immunology 3909

adhesion of MCs to OP9-DL1 on ice was significantly inhibited by
anti-Notch2 mAb at a concentration of 100 mg/ml in nine of 10
experiments (Fig. 5E). The effect of simultaneous addition of anti-
Dll1 and anti-Notch2 mAbs was comparable to that of single ap-
plications of them (Fig. 5E). These results indicate that at least
Notch2 on MCs functions as an adhesion receptor and permits
binding to Dll1.
Taken together, Notch receptor(s) and Dll1 themselves function
as adhesion molecules and contribute to the efficient adhesion of
MCs to stromal cells.
Discussion
Our findings describe a new mechanism through which Notch
family members can mediate cell–cell communication. A Notch
ligand, Dll1 effectively functions as an adhesion ligand, binding
MCs to stromal cells. This study was initiated because of reports
that Notch ligand levels are elevated in inflammatory sites where
MCs accumulate, and our observations provide an important in-
sight into molecular basis of cell accumulation.
The adhesion to OP9-DL1 occurred even when cell metabolism
was arrested and did not require induced expression of additional
molecules. Importantly, it was inhibited by addition of soluble DLL
ligands. Several studies have suggested that soluble Notch ligands
can activate Notch signaling in some conditions (43–45); however,
it is not the case in this study because Notch signaling was irrel-
evant to the adhesion of MCs to OP9-DL1.
Members of the Notch family are potential counter-receptors
for Dll1. Addition of anti-Notch2 mAb significantly inhibited the
adhesion of metabolically inactive MCs to OP9-DL1, indicating
that Notch2–Dll1 interactions have an adhesion function. Remain-
ing MC adhesion to OP9–DL1 in the presence of anti-Notch2
mAb was significantly inhibited by further addition of recombi-
nant mouse Notch1-Fc as an antagonist (Supplemental Fig. 5), im-
plying that Notch1–Dll1 interaction might also contribute to the
adhesion of MCs.
We assessed whether the Jag ligand could also contribute to the
adhesion of MCs using JAG2-expressing fibroblasts as a substitute
for OP9-DL1, and observed that MCs also adhered to them more
efficiently than to control fibroblasts (Supplemental Fig. 6). This
efficient adhesion of MCs was not inhibited by addition of anti-Kit
mAb, but inhibited by culturing on ice (Supplemental Fig. 6). These
observations support that Jag2 also promotes the adhesion of MCs,
although it might not directly function as an adhesion ligand.
In studies of adhesion molecules, the affinity characterized by
aK
d
is an important index of adhesion force. We previously ob-
served that
125
I-labeled IgG1 Fc-tagged soluble DLL1 bound to
CCRF-CEM T cell line expressing Notch1,Notch2, and Notch3
with an apparent K
d
of 9.23 nM (Supplemental Fig. 7). The K
d
of
soluble Jag1 for Ba/F3 cells was reported to be 0.4 nM (46). Pre-
vious studies showed the K
d
of other adhesion receptor–ligand pairs
involved in cell–cell interactions; soluble P-selectin for neutrophils
was ∼70 nM (47); soluble Icam1 for Lfa1 on activated T cells was
400 nM (48); soluble VCAM1 for a4b1 integrin on U937 cells
was 33 nM (49). These results indicate that the affinity of Notch
receptor–ligand interactions is relatively high compared with those
of other adhesion receptor–ligand pairs. Analysis of purified or
recombinant receptor–ligand protein binding also showed compa-
rable results (Supplemental Table II). This high affinity might allow
Notch receptor–Dll1 interactions to function in cell adhesion.
Interestingly, soluble JAG ligands did not inhibit the adhesion
of MCs to OP9-DL1 contrary to soluble DLL ligands at the same
concentration. This result suggests that the affinity of Dll ligands to
Notch receptors on MCs is higher than that of Jag ligands. The
glycosylation of Notch receptors by fringes is known to influence
the Notch receptor–ligand binding specificity. Fringes are fucose-
specific b-1,3-N-acetylglucosaminyltransferases localized to the
Golgi, which elongate O-linked fucose residues on Notch ECD
(50, 51). Several reports have suggested that fringe modification
enhances the affinity of Dll but not Jag ligands to Notch receptors
(40, 41, 52, 53). Because MCs expressed three homologs, Lunatic,
Manic, and Radical fringes (RT-PCR analysis, data not shown),
Notch receptors on MCs might be potentiated to bind soluble Dll
ligands selectively.
The expression of Notch receptors is widely detected through
hematopoietic cell lineages (14, 15). We observed that T cells and
B cells in lymph nodes of naive C57BL/6 mice also adhered to OP9-
DL1 more efficiently than to OP9-control, and their adhesion to
OP9-DL1 still remained in cultures on ice (A. Murata, unpublished
data). Unlike MCs, however, their recognitions were not inhibited
by addition of soluble DLL1 (30 mg/ml) or anti-Notch2 mAb (100
mg/ml) (A. Murata, unpublished data). These results suggest that the
mechanism that Dll1 promotes cell adhesion might be different
between MCs and lymphocytes.
Despite their expression of both Notch receptors and Dll1, OP9-
DL1 cells do not tend to form cell aggregation or adhere tightly
each other. Notch ligands are known to form cis interactions with
Notch receptors expressed in the same cell (54, 55). The cis in-
teraction is shown to inhibit the trans-activation of Notch receptors
by Notch ligands expressed on adjacent cells (55–57). It might
mean that the cis interactions inhibit the trans interactions of Notch
receptors and Dll1, resulting in abrogation of the self-adhesion of
OP9-DL1.
The adhesion function of Notch receptors requires intact Notch
ECD, whereas the signaling function requires proteolytic cleavage
of Notch ECD by ADAMs (14). This is interesting because these
functions of Notch receptor–ligand interaction are exclusive and
irreversible. ADAMs might not only initiate Notch signaling but
also diminish cell adhesion mediated by Notch receptor–ligand
interactions. To determine how the function of ADAMs is regu-
lated will provide an insight of how these two functions of Notch
receptor–ligand interactions are controlled.
Homologs of Notch receptors and their ligands have been id-
entified in a variety of multicellular organism (58). It is noteworthy
that Drosophila Notch–Delta interaction is reported to have a high
adhesion force (59). When Drosophila Schneider (S2) cells ex-
pressing Notch are mixed with S2 cells expressing Delta, huge cell
aggregates are formed (60). Overexpressed zebrafish Delta in cul-
tured human keratinocytes also promotes cell cohesiveness (61).
Moreover, Ba/F3 cells, which hardly adhere to the Chinese hamster
ovary cell line, can adhere to that expressing mouse Dll1 (62).
These results are not only consistent with our finding that Dll1
functions as an adhesion molecule but also suggest that its adhesion
function might be evolutionally conserved.
It is still vague for what kinds of physiological situation Notch
receptor–ligand interactions play important roles through MCs.
Our observations suggest that Notch receptors and their ligands
might be involved in recruitment and retention of MCs in tissues
as adhesion molecules. Recently, Notch2 signaling is reported to
induce differentiation of MCs from BM progenitor cells in vitro,
but MCs were not depleted in Notch2 conditional knockout mice
(63). The expression level of Notch ligands is reported to be up-
regulated at sites of chronic inflammatory diseases (8–10, 64–66),
where are frequently accompanied by the accumulation of MCs (4,
11, 12). These results suggest that Notch receptor–ligand inter-
actions might be important for accumulation of MCs in inflam-
matory but not normal condition. Notch ligands are upregulated on
endothelial cells in inflammatory sites (64, 67, 68), suggesting that
they might contribute to the process of cellular extravasation. In
3910 CELL ADHESION BY A NOTCH LIGAND

fact, MCs were tethered to OP9-DL1 in cultures held on ice only
by Notch receptor(s)–Dll1 interactions. In addition, Notch signaling
is reported to confer APC function on MCs by inducing the expres-
sion of MHC class II and OX40 ligand (42). In view of these evi-
dences, Notch receptor–ligand interactions could affect the process
of inflammation through modulating cell adhesion, differentiation,
and effector functions of MCs.
Notch receptor–ligand interactions are critical to a wide range
of biological processes that range from normal development to
malignancy. This appreciation that in addition to signaling and ac-
tivation of transcription, cell adhesion is involved provides a new
perspective on these issues. Determining the relationship between
adhesion and signaling functions of Notch receptors and their li-
gands should dissect these important processes.
Acknowledgments
We thank Dr. Paul W. Kincade (Oklahoma Medical Research Foundation,
Oklahoma) for helpful suggestions and critical reading of the manuscript.
We also thank Drs. Tetsuo Sudo and Hideo Akiyama (Toray Industries,
Kanagawa, Japan) for reagents and cDNA microarray, Yousuke Takahama
(Tokushima University, Tokushima, Japan) and Toshiyuki Yamane (Mie
University, Mie, Japan) for stromal cells, Noriko Kato (Olympus, Tokyo,
Japan) for fluorescence correlation spectroscopy assay, Motokazu Tsuneto
and Fritz Melchers (Max Planck Institute for Infection Biology, Berlin, Ger-
many), Shiro Ono (Osaka Ohtani University, Osaka, Japan), Kazuo Yamada,
and Haruaki Ninomiya (Tottori University) for helpful discussion, and
Toshie Shinohara for technical assistance.
Disclosures
S.S., M.K., and T.H. are employed by Asahi Kasei Corp. The other authors
have no financial conflicts of interest.
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3912 CELL ADHESION BY A NOTCH LIGAND