a notch ligand, delta-like 1 functions as an adhesion molecule for
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of February 11, 2018.This information is current as
an Adhesion Molecule for Mast CellsA Notch Ligand, Delta-Like 1 Functions As
Yoshino and Shin-Ichi HayashiAkashi-Takamura, Sawako Moriwaki, Shumpei Niida, Miya Zúñiga-Pflücker, Kensuke Miyake, SachikoKajiki, Tomohisa Hirata, Hideo Yagita, Juan Carlos Akihiko Murata, Kazuki Okuyama, Seiji Sakano, Masahiro
ol.1000195http://www.jimmunol.org/content/early/2010/09/01/jimmun
published online 1 September 2010J Immunol
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The Journal of Immunology
A Notch Ligand, Delta-Like 1 Functions As an AdhesionMolecule for Mast Cells
Akihiko Murata,* Kazuki Okuyama,* Seiji Sakano,† Masahiro Kajiki,†
Tomohisa Hirata,† Hideo Yagita,‡ Juan Carlos Zuniga-Pflucker,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 mouseMCs 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: 000–000.
Mast cells (MCs) are derivatives of hematopoietic pro-genitor cells that play an essential role in normal hostdefense and various allergic disorders. MCs are widely
distributed throughout the body, especially in the skin, peritonealcavity, and gastrointestinal mucosa (1, 2). MCs usually accumulateat sites of chronic inflammation, and are thought to contributeto 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 andlocalization. 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 rheumatoidarthritis, chronic pancreatitis, and diabetic nephropathy (8–10).These conditions are frequently accompanied by accumulation ofMCs (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 fourNotch receptors (Notch1–Notch4) and twodistinct families ofNotchligands known as Delta-like (Dll) ligands (Dll1, Dll3, and Dll4) andJagged (Jag) ligands (Jag1 and Jag2) (14, 15). Binding of Notchligands to Notch receptors induces successive proteolytic cleavageof Notch receptors by a disintegrin and metalloproteases (ADAMs)at the extracellular domain (ECD) and subsequently by a g-secretasecomplex at the transmembrane domain. This results in the release ofNotch intracellular domain (ICD) and expression of Notch relatedgenes (14, 15). Overexpressed Notch ICD acts as a constitutivelyactive form of Notch receptors (16). A previous study suggests thatNotch signaling activates integrinb1 (17), indicating its involvementin cellular accumulation by enhancing cell adhesion. MCs mightinteract with increased Notch ligands in chronic inflammatory sites;however, the contribution ofNotch receptor–ligand interaction to theadhesion 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 culturedMCs by using the OP9 stromal cell line overexpressing a Notchligand, Dll1 (OP9-DL1), as a model for inflammatory sites wherethe expression of Notch ligands was upregulated. MCs adhered toOP9-DL1 more efficiently than to control OP9 cells. Surprisingly,the recognition was not due to Notch signaling in either MCs orstromal cells. Metabolically inactive MCs were still adhesive, and
*Division of Immunology, Department of Molecular and Cellular Biology, School ofLife 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 ofMedical Science, University of Tokyo, Minato-ku, ‖Laboratory of Genomics and Pro-teomics, National Center for Geriatrics and Gerontology, Obu, Japan; and xDepart-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 Technologyof 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 forthe Promotion of Science.
The sequences presented in this article have been submitted to Gene ExpressionOmnibus 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: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: ADAM, a disintegrin and metalloprotease; BM, bonemarrow; 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� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000195
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the recognition was blocked by addition of soluble DLL1, or mAbsagainst Dll1 or Notch2. Thus, our results show that Notch receptor(s) and Dll1 function as adhesion molecules for MCs.
Materials and MethodsMice
C57BL/6J mice were purchased from Japan CLEA (Tokyo, Japan). Ex-periments were approved and performed in accordance with the guidelinesof 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 lackedthe transmembrane and cytoplasmic domains were prepared as described(18, 19). Human IgG1 (Chemicon International, Temecula, CA) was used asa control for IgG1 Fc-fused Notch ligands. Sodium azide and DMSOwere purchased from Wako Pure Chemical Industries (Osaka, Japan). A g-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester (DAPT) was purchased from Peptide Institute (Osaka, Japan).
Cell lines
OP9 stromal cell lines (20) carryingDll1 andGFP genes (OP9-DL1), andGFPgene (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/mlstreptomycin (Meiji) (21).
ST2NIC cells, a BM-derived stromal cell line (ST2) transfected witha constitutively active form of the Notch1 ICD gene, whose expression wascontrolled by tetracycline (Tc; Sigma-Aldrich, St Louis, MO)-off systemand 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 mouseintegrin 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 withbiotin (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, PaloAlto, CA).
Preparation of cultured MCs
Cultured MCs were generated as described (29). Cells from femora, adultspleens, or E14.5 fetal livers were cultured in aMEM-supplemented with10% FBS and mouse Il3 (a gift from Dr. Sudo, Toray Industries, Kana-gawa, Japan) at 50 U/ml in a humidified atmosphere of 5% CO2 in the airat 37˚C. Nonadherent cells were replaced into fresh culture media every5 d. After .6 wks, .97% of cells from BM and spleen in cultures wereMCs, as judged by their morphology and the surface expression of Kit onthe flow cytometry. MCs in fetal liver culture were enriched as Kit positivecells with BD IMagnet system (BD Biosciences) and further cultured for7 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 RNAwas prepared by using ISOGEN (NipponGene, Toyama, Japan), and was reverse transcribed by using ReverseTraAce (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 theirligands or Kit ligand [Kitl], respectively) (1 min), 72˚C (1.5 min) for thefollowing 35 or 30 cycles (for Notch receptors and their ligands or Kitl,respectively). The extension time in the last cycle was 3 min. Primers forKitl were as follows; 59-AAA TAG TGG ATG ACC TCG TG-39 and 59-ATTACA AGC GAA ATG AGA GC-39. Other primers were as previouslydescribed (22).
Gene expression was also analyzed by the “3D-Gene” mouse oligo chip24k (Toray Industries, Kanagawa, Japan) (Supplemental Table I) (30).
Induction of adipocyte differentiation
The effective dose of DAPT was determined by adipocyte differentiationassay. OP9 cells differentiate into adipocytes, and Notch signaling inhibitsadipocyte differentiation (31, 32). Forty-eight–well flat-bottomed cultureplates (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 at4˚C. After washing, OP9-control (2 3 104) in 200 ml of its culture mediawere 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 with3.7% 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 3 104) were seeded in 48-well plates andcultured for 2 d at 37˚C to prepare confluent monolayers. After washingwells with PBS, MCs (1.5 3 105) suspended in 200 ml aMEM, supple-mented with 10% FBS, were seeded into each well with or without mAbsor reagents, and incubated for 1 h at 37˚C in a humidified atmosphereof 5% CO2 in the air, unless otherwise indicated. Nonadherent MCs wererecovered after vigorous agitation (low speed, scale 5) for 30 s with aMicro-Mixer E-36 (Taitec Corporation, Saitama, Japan), and were counted witha hemacytometer. Numbers of adherent MCs or ratios of nonadherent MCsrelative to ones initially added in a well were calculated.
For theassaywithfixed stromal cells, confluentmonolayersofOP9-controlor OP9-DL1were fixedwith 4% paraformaldehyde (Wako) for 5min at roomtemperature. After washing cells, the adhesion assay was performed.
ST2NIC cells (4 3 104) 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 mean6 SE of triplicate cultures unless otherwiseindicated. Statistical significance was established at p, 0.05 by two-tailedStudent t test.
ResultsMCs 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 expressedequally by both stromal cells (Fig. 1A). Surface Dll1 expression onOP9-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 confluentmonolayers of OP9-control or OP9-DL1 for 1 h. We then countednonadherent MCs and calculated the numbers of adherent MCsrelative to ones initially added in a well. The survival of MCs wasnot impaired during the adhesion assay (Supplemental Fig. 1). Therewere significantly more adherent MCs on OP9-DL1 than on OP9-control at eachMC density (Fig. 1C). Numbers of adherent MCs onOP9-control cells reached a maximum when we incubated 83 105
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 3 105 MCs on OP9-control or OP9-DL1 for 15–240 min. The percentage of nonadherent MCs onOP9-DL1 was significantly lower than that on OP9-control at eachtime point (Fig. 1D). One hour was needed to reach a plateau ofadhesion of MCs to OP9-control. In contrast, MCs stably adheredto OP9-DL1 within 15 min. These results suggest that Dll1 onstromal cells contributes to the efficient adhesion of MCs. Similarresults were obtained from MCs induced from adult spleen or fetalliver (Fig. 4D).
The adhesion of MCs to OP9-DL1 was not inhibited bytreatment with antagonistic Abs against Kit and integrin a5
A receptor protein tyrosine kinase, Kit, is one of the importantadhesion molecules for MCs (33). MCs highly express cell surface
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Kit molecules (Fig. 2A). Semiquantitative RT-PCR showed thatboth OP9-control and OP9-DL1 expressed comparable amounts ofthe 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 antagonisticmAb. Treatment with anti-Kit mAb was significantly effective onthe percentage of nonadherent MCs to OP9-control (32.7 6 4.1%[control], 64.96 2.5% [anti-Kit] [p, 0.005]) but not to OP9-DL1(8.46 0.6% [control], 13.66 2.3% [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). MCsexpressed integrin a5 (Fig. 2A). To assess the contribution of thismolecule to the adhesion of MCs, we treated the cocultures withanti-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 inthe adhesion of MCs to OP9-control but not to OP9-DL1. Thea5b1 integrin that is reported to be activated by Notch signalingdid not contribute to the efficient adhesion of MCs to OP9-DL1.
Notch signaling in stromal cells did not account for theefficient adhesion of MCs to OP9-DL1
RT-PCR analysis showed that MCs expressed Notch1 and Notch2,and both OP9-control and OP9-DL1 comparably expressedNotch1,Notch2, and Notch3 (Fig. 3A, Supplemental Table I), suggestingthat 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 adhesionmolecule(s) after interaction with MCs by reciprocal signaling viaNotch 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) bysignal transduction including Notch signaling after interaction withOP9-DL1.To assess the first possibility that OP9-DL1 expressed additional
adhesion molecule(s) by reciprocal signaling between MCs, weincubated MCs with fixed stromal cells. Fixed stromal cells wouldbe unable to express additional cell surface molecules after in-teraction with MCs, though they would still provide cell surfacemolecules expressed before the adhesion assay. MCs also adheredto fixed OP9-DL1 more efficiently than to fixed OP9-control cells(Fig. 3B), indicating that the additional adhesion molecule(s) onOP9-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 useda g-secretase inhibitor, DAPT, which can block Notch cleavage atthe transmembrane site and thus impairs Notch signaling (35). Theeffective 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 withimmobilized Notch ligands but not control human IgG1 (Fig. 3C).Inhibition of adipocyte differentiation by immobilized DLL1-Fcwas blocked by treatment with 10 mM but not 1 mM DAPT orDMSO during the culture (Fig. 3D). OP9-DL1 cells also had re-duced tendency to differentiate into adipocytes and treatment with10 mM DAPT was also effective on this inhibition (SupplementalFig. 2). Therefore, we selected the 10 mM concentration of DAPTto inhibit Notch signaling.If the efficient adhesion of MCs to OP9-DL1 resulted from the
Notch signaling in stromal cells, pretreatment of stromal cells withDAPTwould inhibit the adhesion ofMCs. To this end, we pretreatedOP9-control or OP9-DL1with DAPTor DMSO for 2 to 3 d and thenperformed the adhesion assay. These reagents had no effect on theadhesion 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-Cd3ε mAb (filled) as
a control.C andD, On OP9-control or OP9-DL1, (C) serial numbers ofMCs
were incubated for 1 h, and (D) MCs (1.5 3 105) 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 6 SE 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-Il7ra and 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).
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If the efficient adhesion of MCs to OP9-DL1 arose from theNotch signaling in stromal cells, stimulation of Notch signalingin OP9-control might promote the adhesion of MCs. To this end,we incubated MCs on OP9-control stimulated with immobilizedDLL1-Fc or human IgG1 for 2 d. These manipulations also had noeffect on the adhesion of MCs (Fig. 3F). We assessed possibledifferences in expression of molecules associated with Notch sig-naling and cell adhesion between OP9-control and OP9-DL1 bycDNA microarray analysis. No significant difference except Dll1was observed (Supplemental Table I). Flow cytometric analysisalso 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 wasinduced in the absence of Tc (Fig. 3G). We incubated MCs withST2NIC cells precultured with serial doses of Tc for 3 d, and ob-served that the ratio of nonadherent MCs on ST2NIC cells wasincreased with the expression of Notch1 ICD (Fig. 3H, Supple-mental Fig. 4). Taken together, these results indicate that Notchsignaling in stromal cells did not account for the efficient adhesionof MCs to OP9-DL1.
Notch signaling in MCs did not account for the efficientadhesion 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 theadhesion assay. These reagents had no effect on the adhesion ofMCs to either OP9-control or OP9-DL1 (Fig. 4A).
To further assess the contribution of signal-transduction in MCson their adhesion, we suppressed all possible expression of addi-tional adhesion molecules during the adhesion assay. Cocultureswere treated with sodium azide, which inhibits the function ofATPase 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-DL1cells and the percentage of nonadherent MCs was only 33.3 63.9%.In addition, we performed the adhesion assay on ice to arrest
almost all signal-transduction and cell metabolism (37). It is knownthat Kit molecules on MCs are immediately internalized afterbinding to soluble Kit ligand (38). Because the expression of Kiton MCs is subject to a rapid turnover even when cells are at rest(38), their expression on MCs is rapidly recovered after treatmentwith trypsin. In cultures on ice, soluble Kit ligand-induced Kitinternalization and recovery of Kit expression after treatment withtrypsin on MCs were inhibited (data not shown). This indicatesthat turnover of cell surface protein is inhibited on ice. Conductingthe adhesion assay on ice completely inhibited the adhesion ofMCs to OP9-control, but more than 60% of MCs still adhered toOP9-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 onstromal cells and looked dark (Fig. 4E, upper). Interestingly, all ofMCs still bound to OP9-DL1 in the presence of sodium azide andanti-Kit mAb or on icewere 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 withOP9-DL1. This means that adhesion molecule(s) that stronglysupport the adhesion of MCs must exist on the surface of OP9-DL1stromal cells.
Notch receptor(s) and Dll1 themselves functioned as adhesionmolecules for MCs
The previous findings begged the question of whether Dll1 coulditself 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. C and 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). E and 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. G and 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).
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we added soluble DLL1-Fc to the coculture as an antagonist. Theadhesion of MCs to OP9-DL1 was inhibited by soluble DLL1-Fcbut not human IgG1 in a dose-dependent manner (Fig. 5A). Ad-dition of soluble DLL1-Fc at 30 mg/ml significantly inhibited theadhesion of MCs to OP9-DL1, and the percentage of nonadherentMCs on OP9-DL1 became comparable to that on OP9-control(25.1 6 1.4% on OP9-control, 28.0 6 1.0% 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 thepresence of soluble DLL1-Fc (Fig. 5C). Addition of anti-Dll1 mAbalso 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 ofMCs to OP9-DL1.We also assessed whether the Dll1-associated adhesion was in-
hibited by other soluble Notch ligands. The adhesion of MCs toOP9-DL1 was also inhibited by soluble DLL4-Fc (Fig. 5B). In-terestingly, soluble JAG ligands did not inhibit the adhesion ofMCs 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-receptorsfor 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% CO2 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. B and 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 C are 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-Cd3ε mAb (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).
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adhesion of MCs to OP9-DL1 on ice was significantly inhibited byanti-Notch2 mAb at a concentration of 100 mg/ml in nine of 10experiments (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 leastNotch2 on MCs functions as an adhesion receptor and permitsbinding to Dll1.Taken together, Notch receptor(s) and Dll1 themselves function
as adhesion molecules and contribute to the efficient adhesion ofMCs to stromal cells.
DiscussionOur findings describe a new mechanism through which Notchfamily members can mediate cell–cell communication. A Notchligand, Dll1 effectively functions as an adhesion ligand, bindingMCs to stromal cells. This study was initiated because of reportsthat Notch ligand levels are elevated in inflammatory sites whereMCs 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 additionalmolecules. Importantly, it was inhibited by addition of soluble DLLligands. Several studies have suggested that soluble Notch ligandscan 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 theadhesion of metabolically inactive MCs to OP9-DL1, indicatingthat Notch2–Dll1 interactions have an adhesion function. Remain-ing MC adhesion to OP9–DL1 in the presence of anti-Notch2mAb 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 theadhesion of MCs.We assessed whether the Jag ligand could also contribute to the
adhesion of MCs using JAG2-expressing fibroblasts as a substitutefor OP9-DL1, and observed that MCs also adhered to them moreefficiently than to control fibroblasts (Supplemental Fig. 6). Thisefficient adhesion of MCs was not inhibited by addition of anti-KitmAb, but inhibited by culturing on ice (Supplemental Fig. 6). Theseobservations 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
a Kd is an important index of adhesion force. We previously ob-served that 125I-labeled IgG1 Fc-tagged soluble DLL1 bound toCCRF-CEM T cell line expressing Notch1, Notch2, and Notch3with an apparent Kd of 9.23 nM (Supplemental Fig. 7). The Kd ofsoluble Jag1 for Ba/F3 cells was reported to be 0.4 nM (46). Pre-vious studies showed theKd of other adhesion receptor–ligand pairsinvolved in cell–cell interactions; soluble P-selectin for neutrophilswas ∼70 nM (47); soluble Icam1 for Lfa1 on activated T cells was400 nM (48); soluble VCAM1 for a4b1 integrin on U937 cellswas 33 nM (49). These results indicate that the affinity of Notchreceptor–ligand interactions is relatively high compared with thoseof other adhesion receptor–ligand pairs. Analysis of purified orrecombinant receptor–ligand protein binding also showed compa-rable results (Supplemental Table II). This high affinity might allowNotch 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 sameconcentration. This result suggests that the affinity of Dll ligands toNotch receptors on MCs is higher than that of Jag ligands. Theglycosylation 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 theGolgi, which elongate O-linked fucose residues on Notch ECD(50, 51). Several reports have suggested that fringe modificationenhances 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 Dllligands selectively.The expression of Notch receptors is widely detected through
hematopoietic cell lineages (14, 15). We observed that T cells andB cells in lymph nodes of naive C57BL/6mice also adhered to OP9-DL1 more efficiently than to OP9-control, and their adhesion toOP9-DL1 still remained in cultures on ice (A. Murata, unpublisheddata). Unlike MCs, however, their recognitions were not inhibitedby addition of soluble DLL1 (30 mg/ml) or anti-Notch2 mAb (100mg/ml) (A.Murata, unpublished data). These results suggest that themechanism that Dll1 promotes cell adhesion might be differentbetween MCs and lymphocytes.Despite their expression of both Notch receptors and Dll1, OP9-
DL1 cells do not tend to form cell aggregation or adhere tightlyeach other. Notch ligands are known to form cis interactions withNotch receptors expressed in the same cell (54, 55). The cis in-teraction is shown to inhibit the trans-activation of Notch receptorsby Notch ligands expressed on adjacent cells (55–57). It mightmean that the cis interactions inhibit the trans interactions of Notchreceptors and Dll1, resulting in abrogation of the self-adhesion ofOP9-DL1.The adhesion function of Notch receptors requires intact Notch
ECD, whereas the signaling function requires proteolytic cleavageof Notch ECD by ADAMs (14). This is interesting because thesefunctions of Notch receptor–ligand interaction are exclusive andirreversible. ADAMs might not only initiate Notch signaling butalso diminish cell adhesion mediated by Notch receptor–ligandinteractions. To determine how the function of ADAMs is regu-lated will provide an insight of how these two functions of Notchreceptor–ligand interactions are controlled.Homologs of Notch receptors and their ligands have been id-
entified in a variety of multicellular organism (58). It is noteworthythat Drosophila Notch–Delta interaction is reported to have a highadhesion force (59). When Drosophila Schneider (S2) cells ex-pressing Notch are mixed with S2 cells expressing Delta, huge cellaggregates 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 hamsterovary cell line, can adhere to that expressing mouse Dll1 (62).These results are not only consistent with our finding that Dll1functions as an adhesion molecule but also suggest that its adhesionfunction 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 ligandsmight be involved in recruitment and retention of MCs in tissuesas adhesion molecules. Recently, Notch2 signaling is reported toinduce 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 onendothelial cells in inflammatory sites (64, 67, 68), suggesting thatthey might contribute to the process of cellular extravasation. In
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fact, MCs were tethered to OP9-DL1 in cultures held on ice onlybyNotch receptor(s)–Dll1 interactions. In addition, Notch signalingis 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 processof 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 tomalignancy. This appreciation that in addition to signaling and ac-tivation of transcription, cell adhesion is involved provides a newperspective on these issues. Determining the relationship betweenadhesion and signaling functions of Notch receptors and their li-gands should dissect these important processes.
AcknowledgmentsWe 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.
DisclosuresS.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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