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© The Rockefeller University Press, 0021-9525/1998//197 $5.00
The Journal of Cell Biology, Volume 140, Number 1, , 1998 197-210

Direct and Regulated Interaction of Integrin _Eβ₇ with E-Cadherin

Jonathan M.G. Higgins^*, Didier A. Mandlebrot^*,, Sunil K. Shaw^*, Gary J. Russell^*,, Elizabeth A. Murphy^*, Yih-Tai Chen^||, W. James Nelson^||, Christina M. Parker^*, and Michael B. Brenner^*

^* The Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy and Renal Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115; Combined Program in Pediatric Gastroenterology and Nutrition, and Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02115; and ^|| Department of Molecular and Cellular Physiology, Beckman Center, Stanford University School of Medicine, Stanford, California 94305

The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)–1, vascular cell adhesion molecule (VCAM)–1, or mucosal addressin cell adhesion molecule (MadCAM)–1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, _Eβ₇, has been proposed. Here, we demonstrate that a human E-cadherin–Fc fusion protein binds directly to soluble recombinant _Eβ₇, and to _Eβ₇ solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY' cells expressing the _Eβ₇ integrin adhere strongly to purified E-cadherin–Fc coated on plastic, and the adhesion can be inhibited by antibodies to _Eβ₇ or E-cadherin.

The binding of _Eβ₇ integrin to cadherins is selective since cell adhesion to P-cadherin–Fc through _Eβ₇ requires >100-fold more fusion protein than to E-cadherin–Fc. Although the structure of the _E-chain is unique among integrins, the avidity of _Eβ₇ for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of _Eβ₇ for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the _Eβ₇ integrin.

Abbreviations used in this paper: ICAM, intracellular adhesion molecule; iIEL, intestinal intraepithelial lymphocytes; MadCAM, mucosal addressin cell adhesion molecule; MFI, mean fluorescence intensity; PBL, peripheral blood lymphocytes; TCR, T cell receptor; VCAM, vascular cell adhesion molecule.

Address correspondence to Dr. M.B. Brenner, Brigham and Women's Hospital, Smith Building, Room 552, 75 Francis Street, Boston, MA 02115. Tel: (617) 525-1000. FAX: (617) 525-1010.

THE cadherins constitute a family of cell surface adhesion molecules that are involved in calcium- dependent homophilic cell to cell adhesion (Takeichi, 1990). The best studied human cadherins, E-, P-, N-, and VE-cadherin, have a restricted tissue distribution: E- and P-cadherin are expressed in epithelial tissues (Nose and Takeichi, 1986; Shimoyama et al., 1989a), N-cadherin is found mainly on neural cells (Hatta et al., 1987), and VE-cadherin is found on vascular endothelium (Lampugnani et al., 1992). Homophilic binding between cadherins on adjacent cells is vital for the maintenance of strong cell to cell adhesion in these tissues. For example, E-cadherin is required for the formation of adherens junctions between mature epithelial cells (Boller et al., 1985; Gumbiner et al., 1988) and is involved in Langerhans cell adhesion to keratinocytes (Tang et al., 1993), and VE-cadherin is needed for the maintenance of lateral association between endothelial cells (Lampugnani et al., 1992). During development cadherins are critically involved in the cell sorting required for tissue morphogenesis (Takeichi, 1995), and loss of E-cadherin function contributes to the metastasis of a variety of carcinomas (Ben-Ze'ev, 1997). The extracellular regions of mature mammalian cadherins are comprised five "CAD" modules of 110 amino acids. Crystallographic and biochemical studies indicate that cadherins probably form dimers on the cell surface (Shapiro et al., 1995; Nagar et al., 1996), and that interaction with dimeric cadherins on opposing cell surfaces can lead to the formation of "zipper-like" cell junctions (Shapiro et al., 1995; Tomschy et al., 1996).

The integrins are a second family of transmembrane adhesion molecules that are involved in both cell to cell and cell to matrix interactions. At least 15 chains associate with 8 β chains to form a large number of heterodimeric integrins that can be classified into several major subfamilies based on their shared use of a particular β chain (Hynes, 1992). Members of three such subfamilies, the β₁, β₂, and β₇ integrins, are commonly found on leukocytes. The expression of β₁ integrins is widespread (for example, ₅β₁, CD49e/CD29, is found on T cells, granulocytes, platelets, fibroblasts, endothelium, and epithelium), whereas the β₂ and β₇ integrins have a restricted pattern of expression. For example, _Lβ₂ (CD11a/CD18) is expressed on most lymphocytes and many myeloid cells but not on other cell types, and _Eβ₇ (CD103/unclustered) is found on >95% of intestinal intraepithelial lymphocytes (iIEL)¹ and on other mucosal T cells, macrophages, and mast cells, but on only 2% of peripheral blood lymphocytes (Cerf-Bensussan et al., 1987; Smith et al., 1994; Tiisala et al., 1995). The major ligands of the integrins fall into two categories: cell surface molecules that are members of the immunoglobulin superfamily (such as vascular cell adhesion molecule [VCAM]–1, intracellular adhesion molecule [ICAM]–1, 2, 3, and mucosal addressin cell adhesion molecule [MadCAM]–1) and a variety of large extracellular proteins (such as fibronectin, vitronectin, fibrinogen, and complement component iC3b; Hynes, 1992). A common feature of both groups is the presence of exposed acidic amino acids crucial for integrin binding (Bergelson and Hemler, 1995). Many integrins exist in states of low or high avidity for their ligands, and these states can be regulated from within the cell (Hynes, 1992). For example, the avidity of _Lβ₂ on resting T cells can be increased by stimulation through the T cell receptor (TCR) with anti–CD3 mAbs (Dustin and Springer, 1989; van Kooyk et al., 1989).

Recently, we reported that E-cadherin on human epithelial cells may be a ligand for the mucosal lymphocyte integrin, _Eβ₇, and a similar interaction has been suggested in the mouse (Cepek et al., 1994; Karecla et al., 1995). mAbs to E-cadherin or to _Eβ₇ block IEL adherence to epithelial cells, and transfection of cells with _Eβ₇ confers upon them the ability to adhere to cells transfected with E-cadherin (Cepek et al., 1994). E-cadherin has been defined extensively as a homophilic adhesion molecule, and its sequence is not related to known cell surface or extracellular integrin ligands. Thus, the concept that _Eβ₇ and E-cadherin are counter receptors is at variance with current knowledge of both integrin and cadherin interactions. Moreover, as we and others have pointed out, the indirect methods used in the studies to date are insufficient to conclusively demonstrate a direct physical interaction between these two adhesion receptors (Cepek et al., 1994; Erle, 1995; Karecla et al., 1995; Takeichi, 1995). For example, it is clear that antibodies to one integrin (e.g., _Vβ₃) can cause specific transdominant inhibition of the function of another integrin (e.g., ₅β₁; Blystone et al., 1995; Díaz-González et al., 1996) or could result in steric hindrance of an adjacent receptor. Furthermore, expression of an exogenous gene in a transfected cell can have profound effects on the surface expression of a variety of other proteins (Marks et al., 1996), and the transfection of constructs containing integrin β1 tails into fibroblasts alters the function of endogenous integrins (LaFlamme et al., 1994). Such effects can lead to erroneous conclusions about the interactions that are directly involved in adhesion.

Given that the interaction of _Eβ₇ on intraepithelial lymphocytes with its receptor on epithelial cells is likely to be crucial for the normal development, function, and/or retention of lymphocytes in the epithelium (Cepek et al., 1993; Erle, 1995), we sought to determine if _Eβ₇ and E-cadherin are directly interacting counter receptors. In addition, we investigated whether this interaction is specific among cadherins and whether it can be regulated through alterations in _Eβ₇ avidity.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials
DNA manipulating enzymes were purchased from New England Biolabs Inc. (Beverly, MA). Oligonucleotides were obtained from Oligotech (Boston, MA). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). ICAM-1–Fc (the entire extracellular region of human ICAM-1 fused to the hinge and Fc portion of human IgG1) was a generous gift of Dr. Lloyd Klickstein (Brigham and Women's Hospital, Boston, MA). Purified human IgG1 was obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA).

mAbs
The mAb used (all mouse IgG against human antigens) were as follows: E4.6 (anti–E-cadherin, IgG1; Cepek et al., 1994), HECD-1 (anti–E-cadherin, IgG1; Shimoyama et al., 1989b), NCC-CAD299 (anti–P-cadherin, IgG1; Shimoyama et al., 1989b), HML-1 (anti-_Eβ₇, IgG2a; Cerf-Bensussan et al., 1987), BerACT-8 (anti-_Eβ₇, IgG1; Kruschwitz et al., 1991), E7-1 (anti-_Eβ₇, IgG2a; Russell et al., 1994), E7-2 (anti-_Eβ₇, IgG1; Russell et al., 1994), E7-3 (anti-_Eβ₇, IgG1; Russell et al., 1994), D6.21 (anti-_Lβ₂, IgG1; Cepek et al., 1993), TS1/22 (anti-_Lβ₂, IgG1; Sanchez-Madrid et al., 1982), TS1/18 (anti-β₂, IgG1; Sanchez-Madrid et al., 1983), ACT-1 (anti-₄β₇, IgG1; Lazarovits et al., 1984), B5G10 (anti-₄, IgG1; Hemler et al., 1987), 4B4 (anti-β₁, IgG1; Morimoto et al., 1985), RR1/1 (anti–ICAM-1, IgG1; Rothlein et al., 1986), SPVT3b (anti-CD3, IgG2a; Spits et al., 1983), OKT4 (anti-CD4, IgG2b; obtained from American Type Culture Collection [ATCC], Rockville, MD), OKT8 (anti-CD8, IgG2a; ATCC), W6/32 (anti-MHCI, IgG2a; Barnstable et al., 1978), TCR-1 (anti–TCR-, IgG1; Band et al., 1987), P3 (control IgG1; Kohler and Milstein, 1975), RPC5.4 (control IgG2a; Mohit and Fan, 1971).

Cells
Human iIEL were isolated as previously described (Roberts et al., 1993; Russell et al., 1996). The iIEL were stimulated with PHA-P (Difco Laboratories Inc., Detroit, MI) and irradiated feeder cells (80% PBMC and 20% JY lymphoblastoid cells) in 2 nM IL-2, 4% (vol/vol) heat-inactivated FBS (Hyclone Laboratories Inc., Logan, UT), 50 µM 2-mercaptoethanol, and Yssel's medium at 10% CO₂ (Russell et al., 1996). The iIEL lines 496 and 194 were maintained by periodic restimulation as described and were used in adhesion assays after 2–8 wk. At the time of assay, IEL496 was 100% CD3⁺, 90% CD8⁺, 10% CD4⁺, 95% _Lβ₂⁺ (MFI 400), 97% _Eβ₇⁺ (MFI 700), and IEL194 was 100% CD3⁺, 60% CD8⁺, 40% CD4⁺, 95% _Lβ₂⁺ (MFI 500), 80% _Eβ₇⁺ (MFI 600) by FACS^® analysis. Both lines maintained expression of _Eβ₇ without addition of exogenous TGF-β1.

A subline of the human B lymphoblastoid cell line, JY, that expresses the ₄β₇ integrin (JY'), was kindly provided by Dr. Martin Hemler (Dana-Farber Cancer Institute, Boston, MA; Chan et al., 1992) and was maintained in 10% (vol/vol) heat-inactivated FBS (Hyclone Laboratories Inc.), RPMI-1640 (GIBCO BRL, Gaithersburg, MD) at 37°C, and 5% CO₂. Human embryonic kidney HEK293 cells (obtained from ATCC) were maintained in 10% (vol/vol) heat-inactivated FBS (Hyclone Laboratories Inc.) and DME Medium (GIBCO BRL) at 37°C in 10% CO₂. COS-7 cells (obtained from ATCC) were grown in 10% (vol/vol) NuSerum (Collaborative Research, Inc., Waltham, MA), 10 mM Hepes, 2 mM l-glutamine, and DMEM (GIBCO BRL) at 10% CO₂. Human breast epithelial 16E6.A5 cells were maintained as described (Cepek et al., 1993).

Construction of E- and P-Cadherin–Fc Expression Vectors
E-cadherin–Fc.
A double-stranded DNA adapter containing a 5' Ngo MI cohesive end, the final five codons of the human E-cadherin extracellular region, and a 3' XhoI cohesive end was produced by annealing the complimentary oligonucleotides JON1 (5^'-CCGGCGTCTGTAGGAAGC-3^') and JON2 (5^'-TCGAGCTTCCTACAGACG-3^'). This adapter was then ligated to the 3'-end of an EcoRV–NgoMI fragment encoding the rest of the extracellular region of human E-cadherin derived from the plasmid pERF-1 (kindly provided by Dr. David Rimm, Yale University, New Haven, CT; Rimm and Morrow, 1994). After removal of excess adapters by centrifugation through a Centricon-100 filter (Amicon Corp., Danvers, MA), the resulting EcoRV–XhoI fragment was introduced inframe, upstream of coding for the hinge and Fc region of human IgG1 in a derivative of pCDM8 (pCDM8/Fc; Chen and Nelson, 1996) also cleaved with EcoRV and XhoI. The sequence of the junctional region is shown in Fig. 1 a. To confirm the integrity of the construct, the nucleotide sequence of the junctional region of the E-cadherin–Fc construct was determined by double-stranded sequencing using the Sequenase kit (United States Biochemical Corp., Cleveland, OH) according to the manufacturer's protocol. Finally, the E-cadherin–Fc cDNA was excised from pCDM8 using EcoRV and NotI and ligated into the expression vector pCEP4 (Invitrogen Corp., Carlsbad, CA) cleaved with PvuII and NotI.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Structure of soluble recombinant E-cadherin–Fc and truncated Eβ7. (a) Structure of the human E-cadherin–Fc fusion protein. The sequence of the extracellular juxtamembrane region of wild-type E-cadherin and the alterations resulting from fusion with the human Fc region are shown. Regions corresponding to the Fc portion are shown in bold. The corresponding sequences in P-cadherin, tgc cct gga ccc tgg aaa (encoding CPGPWK), become tgc cct gga ctc gag ctc (encoding CPGLEL) in the P-cadherin–Fc fusion protein. (b) Structure of soluble truncated Eβ7. In the E chain, the EF hand–like repeats are labeled I to VII, the extra "X" domain is shown in black, and the A domain in grey. In the β7 chain, the β integrin conserved region that may resemble an A domain (Lee et al., 1995) is shown in grey, and the cysteine-rich repeats are hatched. The transmembrane and cytoplasmic regions that were removed from each chain are shown as dotted lines. The change made in the cDNA sequence of each chain to introduce a stop codon immediately before the transmembrane region is shown in bold.

DNA for transfections was prepared using a Maxi-prep kit (QIAGEN Inc., Chatsworth, CA).

Construction of _E and β₇ Expression Vectors
Full-Length _E.
A full-length open reading frame encoding human _E was generated from cDNA clones described previously (Shaw et al., 1994). The final construct included the HindIII–NsiI fragment of clone 38, the NsiI–BsaBI fragment of clone 2-54, the BsaBI–AccI fragment of clone 1-39A, the AccI–BglII fragment of clone 2-54, and the BglII–XhoI fragment of clone 3-15. The full-length cDNA was introduced into the XhoI site of the expression vector pSR-neo (Takebe et al., 1988) to produce pSR-neo/E.

Truncated _E.
PCR was used to introduce a stop codon and HindIII site immediately upstream of the transmembrane region of human _E cDNA (see Fig. 1 b). PCR with the primers 5^'-AAGAATGGCATTCAGTGAGC-3^' and 5^'-GGGAAGGTTGATGATAGGCTAAGAATGGTAC-3^' amplified a product that was subsequently cleaved with BglII and HindIII to generate a 180-bp fragment from the end of the _E extracellular region. This fragment was then introduced into the BglII site of the full-length _E cDNA to generate an open reading frame for the entire extracellular portion of human _E. After sequencing as described above, the truncated _E cDNA was introduced in either the sense or antisense direction into the HindIII site of the expression vector pAPRM8 (Wong and Farrell, 1991) to produce pAPRM8/tEs and pAPRM8/tEas.

Truncated β₇.
A full-length human β₇ cDNA was derived by ligation of the XhoI–EcoRI fragment from a partial β₇ cDNA (kindly provided by Dr. G.W. Krissansen, University of Auckland, New Zealand; Yuan et al., 1990) and the NotI–XhoI fragment of another partial β₇ cDNA cloned from an IEL cDNA library (Shaw et al., 1994). To introduce a stop codon and EcoRI site immediately upstream of the transmembrane region of human β₇ cDNA, PCR with the primers 5^'-TCGCTGCCAATGTGGAGTATG-3^' and 5^'-GGGGAATTCACAATGGCCTACGTGTGGTCTGC-3^' was carried out. The product obtained was subsequently cleaved with BsmI and EcoRI to generate a 400-bp fragment from the end of the β₇ extracellular region. This fragment was then introduced into the BsmI and EcoRI sites of pBluescript containing the β₇ cDNA to generate an open reading frame for the entire extracellular portion of human β₇ (see Fig. 1 b). After sequencing as described above, the truncated β₇ cDNA was excised from pBluescript with EcoRI and introduced in either the sense or antisense direction into the EcoRI site of the expression vector pAPRM8 to produce pAPRM8/tβ7s and pAPRM8/tβ7as.

Production of Cadherin–Fc Proteins
HEK293 cells (10⁶ cells per 75-cm² flask) were stably transfected with 25 µg plasmid DNA using the Mammalian transfection kit (Stratagene Inc.). After growth for 24 h in nonselective medium the cells were transferred to 96-well tissue culture plates and incubated in selective medium containing 300 µg/ml hygromycin B. After 15 d, supernatants from wells containing resistant colonies were assayed for fusion proteins by ELISA.

To produce the cadherin–Fc proteins, transfected cells were grown in triple-layer 500-cm² flasks (Nunc, Roskilde, Denmark) in 10% (vol/vol) Ultralow Ig FBS (GIBCO BRL), 300 µg/ml hygromycin B, and DMEM. After 5–10 d of culture, the medium was harvested and filtered through a 0.2-µm membrane. The E- and P-cadherin–Fc fusion proteins were then purified on separate, previously unused GammaBind G–Sepharose columns (Pharmacia Biotech Sverige, Uppsala, Sweden). The columns were washed with TBS and 1 mM CaCl₂, pH 7.4, and then eluted with 0.2 M glycine and 1 mM CaCl₂, pH 2.3. Fractions containing purified fusion proteins were dialyzed into TBS and 1 mM CaCl₂, pH 7.4 and then stored at –20°C. The purity of fusion protein was assessed by SDS-PAGE and Coomassie blue staining, and the concentration was determined by Bradford assay using BSA as a standard (Bio-Rad Labs., Hercules, CA).

Production of Soluble ³⁵S-labeled Recombinant _Eβ₇ Integrin
Soluble recombinant _Eβ₇ was produced by COS-7 cells after transient transfection using DEAE-dextran (Coligan et al., 1994, Unit 10-14) with the plasmids pAPRM8/tEs and pAPRM8/tβ7s. Control transfections were carried out with the antisense constructs pAPRM8/tEas and pAPRM8/tβ7as. After incubation for 48 h in complete medium, the cells were washed once with PBS and then 1 mCi ³⁵S-Express (Dupont-NEN, Boston, MA) in 6 ml 10% (vol/vol), dialyzed FBS, 5% DMEM, 85% methionine- and cysteine-free DMEM (GIBCO BRL), 10 mM Hepes, and 2 mM l-glutamine was added. After incubation at 37°C for 24 h, the medium, containing labeled secreted proteins, was filtered through a 0.2-µm membrane.

Generation of JY'-_E and JY'-Vector Cell Lines
To produce cells expressing cell surface _Eβ₇, JY' cells that express endogenous β₇ integrin chain were transfected by electroporation with 4 µg pSR-neo/E or with the pSR-neo vector alone as a control. Then transfected cells were selected by culture in 0.5 mg/ml G418. To generate the JY'-_E line, _Eβ₇-expressing transfectants were isolated by positive selection with the anti-_Eβ₇ mAb BerACT-8 on magnetic goat anti–mouse immunoglobulin dynabeads according to the manufacturer's recommendations (Dynal A.S., Oslo, Norway) and by flow cytometric sorting. FACS^® was carried out as previously described (Parker et al., 1990). The clone J6.7 was finally isolated by limiting dilution. The _Eβ₇ expressed on JY'-_E cells was indistinguishable from that on IEL when immunoprecipitated from ¹²⁵I surface-labeled cells with the anti–_Eβ₇ mAb, HML-1 (not shown).

Adhesion Assays
Unless otherwise stated, the wells of Linbro 96-well microtiter plates (ICN Flow Laboratories, Horsham, MA) were coated with human IgG1 Fc-containing proteins in 50 µl/well TBS and 1 mM CaCl₂, pH 7.4, for 18 h at 4°C. The wells were subsequently washed twice with 20 mM Hepes, 137 mM NaCl, and 3 mM KCl, pH 7.4 (HBS), with 1 mM CaCl₂, and was then blocked with 1% BSA (Calbiochem-Novabiochem Corp.), HBS, and 1 mM CaCl₂ for 2 h at room temperature. In assays in which the effects of divalent cations were assessed, coating and blocking was carried out in HBS and 10 mM EDTA. In assays in which adhesion to P- and E-cadherin–Fc was compared, the wells were coated with 1 µg/well goat anti–human IgG polyclonal antibody (Zymed, San Francisco, CA) in 100 µl TBS, pH 7.4, and blocked as described above before addition of Fc fusion proteins.

IEL or transfected JY' cells were labeled with BCECF-AM (Molecular Probes, Eugene, OR) as previously described (Cepek et al., 1993). During labeling of JY' cells, 10% (vol/vol) heat-inactivated normal human serum was included to block Fc receptors. Adhesion assays were carried out in 0.1% BSA and HBS with combinations of MnCl₂, MgCl₂, CaCl₂, or 1 mM EGTA, as indicated (see text). In antibody blocking experiments, cells or wells were preincubated with mAbs for 10 min at 4°C as described in the text. For cell activation experiments, cells were preincubated with antibodies or 50 ng/ml PMA at 4°C for 15 min. Adhesion assays were carried out as described previously (Cepek et al., 1993) with the following modifications. Labeled cells were brought into contact with the microtiter plate wells by centrifugation at 60 g for 2 min (IEL) or 1 min (JY'). After incubation at 37°C for 10 min, nonadherent cells were removed by washing with 1 mM MnCl₂, 1 mM MgCl₂, 1 mM CaCl₂, and HBS at 37°C unless the effect of divalent cations was being assessed, in which case HBS alone was used. Since in these assays the fluorescence of input cells was quenched to some degree by the presence of adhesion buffer, but the percent bound was determined after removing the buffer, some apparent readings of >100% are obtained.

Homophilic adhesion assays were carried out as described above with the following modifications. 16E6.A5 cells were released from culture dishes using 0.02% (wt/vol) trypsin, 2 mM CaCl₂, and HBS to minimize proteolysis of cadherins. After adding 2 vol of 0.04% (wt/vol) soy bean trypsin inhibitor, HBS, and washing twice with HBS, the cells were resuspended in 0.1% BSA, HBS, and 1 mM CaCl₂ and allowed to settle onto the microtiter plate wells for 10 min at 4°C. After incubation at 37°C for 30 min, and washing twice with HBS and 1 mM CaCl₂, the percentage of bound cells was determined using a fluorogenic assay of endogenous cellular phosphatase activity (Tolosa and Shaw, 1996).

Surface Labeling of iIEL with ¹²⁵I
Cultured iIEL (4 x 10⁷) were isolated by centrifugation on Ficoll-Paque (Pharmacia Biotech Sverige) and subjected to cell surface labeling with 2 mCi Na¹²⁵I (Dupont-NEN) using the lactoperoxidase method (Coligan et al., 1994, Unit 8-11). The labeled cells were then lysed in 0.5% Triton X-100, TBS, and 4 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride for 4 h at 4°C. Insoluble material was removed by centrifugation at 12,000 g for 20 min.

Immunoadsorption
Batches of ¹²⁵I-labeled IEL lysate or ³⁵S-labeled transfected COS-7 medium were supplemented divalent cations (see text) and precleared twice with 0.4% (vol/vol) normal rabbit serum, 1.5% (vol/vol) protein A–Sepharose (Pharmacia), and 1.5% (wt/vol) Pansorbin (Calbiochem-Novabiochem Corp.) for a total of 24 h at 4°C. Subsequently, aliquots were incubated with mouse mAbs or human IgG1 Fc-containing proteins for 3 h at 4°C. Then, 10 µl protein A–Sepharose resin was added to each tube, and the incubation at 4°C was continued for a further 3 h. For immunoprecipitations using mouse IgG1 mAbs, protein A–Sepharose precoated with rabbit anti–mouse immunoglobulin polyclonal antibody (Cappel, West Chester, PA) was used. Then the immobilized complexes were washed six times with TBS containing the same concentration of divalent cations used during the adsorption steps. For immunoadsorption from IEL lysates, 0.1% Triton X-100 was also present during washing. Proteins bound to the resin were eluted by boiling in 3% (wt/vol) SDS, 10% (vol/vol) glycerol, 50% (wt/vol) urea, and 60 mM Tris, pH 7, before analysis by SDS-PAGE.

SDS-PAGE
SDS-PAGE on 7.5% (wt/vol) polyacrylamide gels (Protogel; National Diagnostics, Atlanta, GA) was carried out as described (Coligan et al., 1994, Unit 8-4). Samples were reduced by the inclusion of 25 mM dithiothreitol. Radiolabeled proteins were visualized by autoradiography using Biomax MR and MS film (Kodak, Rochester, NY) and quantitated using phosphorimaging and the ImageQuant package (Molecular Dynamics Inc., Sunnyvale, CA).

Statistical Analysis
P values testing the hypothesis that two populations had equal means were calculated using a two-tailed Welch t test (which assumes the populations follow a normal distribution, but not that they have equal variance). Also, a nonparametric two-tailed Mann-Whitney test (which does not assume normal distributions) was used to test whether the population medians were equal. Calculations were carried out using the Instat package (Graphpad Software, San Diego, CA).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Production of Human E- and P-cadherin–Fc Fusion Proteins
Constructs encoding the extracellular portion of either human E-cadherin or P-cadherin were linked in frame to a construct encoding the Fc region of human IgG1 (including the hinge, C_H2, and C_H3 domains). Transfection of HEK293 cells and selection with hygromycin B led to the generation of stable lines expressing soluble E-cadherin– Fc or P-cadherin–Fc fusion proteins. The cadherin–Fc fusion proteins were expected to be dimeric due to the presence of disulfide bonds in the Fc region (Fig. 1 a), possibly similar to cadherin dimers on the cell surface (Shapiro et al., 1995; Nagar et al., 1996).

After purification on protein G–Sepharose, SDS-PAGE in reducing conditions revealed the presence of proteins of the expected size for both E- and P-cadherin–Fc monomers (120 kD, Fig. 2). Minor species of 135 or 130 kD were also present in preparations of the E- and P-cadherin fusion proteins, respectively. Since cadherins are synthesized as proproteins and the sizes of these bands match those predicted for the immature forms, it is likely that a small proportion of partially processed procadherin is present in each case. In nonreducing conditions both fusion proteins migrate at 240 kD (Fig. 2) as expected for dimeric fusion proteins linked through disulfide bonds in the hinge of the Fc region. A small proportion of monomeric cadherin fusion protein is also present in each case. In an ELISA, E-cadherin–Fc but not P-cadherin–Fc is recognized by the antihuman E-cadherin mAb E4.6, and P-cadherin–Fc but not E-cadherin–Fc is recognized by the antihuman P-cadherin mAb NCC-CAD299, further confirming the identity of the two recombinant products (not shown).

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 2 SDS-PAGE of purified recombinant P- and E-cadherin–Fc fusion proteins. Approximately 2 µg of P-cadherin–Fc and 1.5 µg of E-cadherin–Fc protein were subjected to 7.5% SDS-PAGE in reducing and nonreducing conditions and Coomassie blue staining.

View larger version (48K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Adhesion of cultured intestinal IEL and epithelial cells to E- and P-cadherin–Fc. (a) Dose response of IEL adhesion to E- and P-cadherin–Fc. Serial dilutions of purified E-cadherin– Fc, P-cadherin–Fc, and human IgG1 were immobilized on microtiter plate wells coated with polyclonal goat anti– human IgG antibody and blocked with BSA. The adhesion of cultured IEL was determined in the presence of 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, 0.1% BSA, HBS, pH 7.4, as described in Materials and Methods. Adhesion was determined in two independent experiments, each in triplicate, and the results are expressed as the mean percent bound ± 1 SD (n = 6). (b) Inhibition of adhesion of IEL to E-cadherin–Fc and ICAM-1–Fc by mAbs. Microtiter plate wells were coated directly with 0.06 µg/well of human E-cadherin–Fc or 0.6 µg/well ICAM-1–Fc protein, and blocked with BSA. The adhesion of cultured IEL was determined in the presence of 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, 0.1% BSA, HBS, pH 7.4, and various mAbs. The purified mAbs E7-2 (anti-Eβ7), D6.21 (anti-Lβ2), 4B4 (anti-β1), ACT-1 (anti-4β7), E4.6 (anti–E-cadherin), and RR1/1 (anti–ICAM-1) were used at 10 µg/ml. Ascites fluid containing HML-1 (anti-Eβ7), TS1/22 (anti-Lβ2), and TS1/18 (anti-β2) were used at a dilution of 1:50. All antibodies were preincubated with IEL for 10 min on ice before addition to the plate except for RR1/1 and E4.6, which were preincubated in the microtiter plate. All mAbs were mouse IgG1 except HML-1 (mouse IgG2a). The results are expressed as the mean percent bound + 1 SD (n = 5). (c) Dose response of 16E6.A5 cell adhesion to E- and P-cadherin–Fc. Microtiter plates were coated as described for a. Adhesion was determined as described in Materials and Methods and the results are expressed as the mean percent bound ± 1 SD (n = 3). The adhesion of 16E6.A5 cells to E-cadherin–Fc is inhibited by >95% with the anti–E-cadherin mAb HECD-1, but not with the anti–P-cadherin mAb NCC-CAD299. In contrast, the adhesion of 16E6.A5 cells to P-cadherin–Fc is inhibited by 80% with the anti–P-cadherin mAb NCC-CAD299, but not with the anti– E-cadherin mAb HECD-1 (data not shown).

Antibodies to _Eβ₇ and E-Cadherin Block IEL Adhesion to E-Cadherin–Fc
To determine if lymphocyte _Eβ₇ was responsible for adhesion of IEL to E-cadherin–Fc, we attempted to block the interaction with mAbs to human lymphocyte surface integrins. The binding of IEL to E-cadherin–Fc coated directly on plastic was completely blocked by anti–_Eβ₇ mAbs HML-1, BerACT8, E7-1, and E7-2 and by the anti–E-cadherin mAb E4.6, while adhesion to ICAM-1–Fc was not affected (Fig. 3 b and data not shown). In contrast, the anti-β₂ integrin mAb, TS1/18, the anti–_Lβ₂ mAbs, TS1/22 and D6.21, and the anti–ICAM-1 mAb RR1/1 all prevented IEL adhesion to ICAM-1–Fc, but not to E-cadherin–Fc. The anti-β1 integrin mAb, 4B4, blocked adhesion of IEL to human fibronectin (not shown) but not to E-cadherin–Fc or ICAM-1–Fc. A blocking mAb to ₄β₇ (ACT-1) also did not inhibit IEL adhesion to E-cadherin– Fc or ICAM-1–Fc (Fig. 3 b). Adhesion of IEL to P-cadherin–Fc was also completely blocked by the anti–_Eβ₇ mAb, E7-2, but was unaffected by the anti–_Lβ₂ mAb, D6.21 (data not shown). Thus, adhesion of IEL to E- or P-cadherin–Fc involves _Eβ₇, and adhesion to ICAM-1–Fc involves _Lβ₂.

JY Cells Transfected with _E Adhere to E-Cadherin–Fc
To confirm that expression of exogenous _Eβ₇ would confer upon a cell the ability to adhere to E-cadherin–Fc, adhesion assays were performed with JY' cells transfected with a full-length human _E-encoding cDNA construct. JY' cells, or JY' cells transfected with vector only (JY'-vector), expressed the ₄β₇ integrin, very little β₁ integrin, but no _Eβ₇ by FACS^®. JY' transfected with _E (JY'-_E) had levels of ₄ and β₁ integrins similar to untransfected or JY'-vector cells, but now also expressed _Eβ₇ (99% _Eβ₇⁺, MFI 80). Both JY'-vector and JY'-_E transfectants also expressed similar levels of _Lβ₂ (Fig. 4 a).

View larger version (72K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Analysis of JY' cells transfected with E cDNA. (a) Flow cytometric analysis of the cell surface expression of integrins on JY' cells transfected with pSR-neo vector alone (JY'-vector) or with pSR-neo/E (JY'-E). The staining with control mAb P3 is shown unshaded. Staining with E7-2 (anti-Eβ7), 4B4 (anti-β1), B5G10 (anti-4), ACT-1 (anti-4β7), and TS1/22 (anti-Lβ2) is shown shaded. All mAbs were mouse IgG1. (b) Adhesion of transfected JY' cells to E-cadherin–Fc. Microtiter plate wells were coated directly with serial dilutions of E-cadherin–Fc or human IgG1, and blocked with BSA. The adhesion of JY'-vector and JY'-E cells was determined in the presence of 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2, 0.1% BSA, HBS, pH 7.4, as described in Materials and Methods. The results are expressed as the mean percent bound ± 1 SD (n = 3). (c) Inhibition of adhesion of JY'-E cells to E-cadherin–Fc by mAbs. Microtiter plate wells were coated directly with 0.63 µg/well of E-cadherin–Fc or human IgG1, and blocked with BSA. The adhesion of transfected JY' cells in the presence of various mAbs was determined as described for IEL in Fig. 3 b. The purified mAbs E7-2, and BerACT8 (anti-Eβ7), ACT-1 (anti-4β7), 4B4 (anti-β1), D6.21 (anti-Lβ2), E4.6 (anti–E-cadherin), and RR1/1 (anti–ICAM-1) were used at 10 µg/ml. Ascites fluid containing TS1/18 (anti-β2) was used at a dilution of 1:100. All mAbs were mouse IgG1. The results are expressed as the mean percent bound + 1 SD (n = 4). Adhesion of JY'-vector and JY'-E cells to human IgG1 in this experiment was <3%.

The adhesion of JY'-_E cells to E-cadherin–Fc was blocked from 40% cells bound to <5% (the level seen for JY'-vector cells) by mAbs to _Eβ₇ (E7-2 and BerACT8) and E-cadherin–Fc (E4.6), but not by blocking mAbs to _Lβ₂, ₄β₇, β₁, or β₂ integrins or to ICAM-1 (Fig. 4 c). In contrast, the adhesion of JY'-_E cells to ICAM-1–Fc was blocked by antibodies to _Lβ₂ (TS1/22 and D6.21), β₂ integrins (TS1/18), and ICAM-1 (RR1/1), but not by antibodies to _Eβ₇, ₄β₇, or β₁ integrins (data not shown). These mAb-blocking experiments further confirm that the cell adhesion measured is mediated by _Eβ₇ binding to E-cadherin–Fc, or by _Lβ₂ binding to ICAM-1–Fc.

E-Cadherin–Fc Binds Directly to ¹²⁵I-labeled _Eβ₇ from a Lysate of IEL
To demonstrate that E-cadherin molecules interact directly with _Eβ₇ molecules, the proteins that bound to E-cadherin–Fc from a ¹²⁵I-labeled IEL lysate in the presence of 1 mM MnCl₂, 1 mM MgCl₂, and 1 mM CaCl₂ were analyzed. Since the fusion protein contains a human IgG1 Fc region that binds to protein A, a standard immunoadsorption procedure was used. Proteins of the expected size for _Eβ₇ (175, 135, and 110 kD) and _Lβ₂ (160 and 100 kD) were visible on SDS-PAGE in nonreducing conditions after immunoprecipitation with anti–_Eβ₇ and anti–_Lβ₂ mAbs, respectively (Fig. 5). Remarkably, E-cadherin–Fc bound to radiolabeled species identical in size and relative intensity to those seen with the anti–_Eβ₇ mAb (Fig. 5, compare lanes 1 and 3), but distinct from those seen with the anti–_Lβ₂ mAb (Fig. 5, compare lanes 3 and 8). In contrast, no radiolabeled species were detected binding to the P-cadherin–Fc or IgG1 proteins. After immunoadsorption with the ICAM-1–Fc, proteins of the expected size for _Lβ₂ could be visualized only on overexposed phosphorimages (not shown). While the same number of cell equivalents were used in each of the human IgG1 containing protein-binding experiments, 40-fold fewer cell equivalents were required to immunoadsorb an equal amount of radiolabeled _Eβ₇ with the anti–_Eβ₇ mAb compared with E-cadherin–Fc (see legend to Fig. 5). This is consistent with the expected lower affinity of a cell adhesion receptor interaction compared with that of an antibody–antigen interaction.

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Binding of Eβ7 from an IEL lysate to E-cadherin–Fc. Cultured intestinal IEL were surface labeled with 125I and solubilized as described in the text. Batches of lysate were subjected to immunoadsorption with mAbs or human IgG1 Fc containing proteins in the presence of 1 mM MnCl2, 1 mM MgCl2, 1 mM CaCl2. Samples were resolved by 7.5% SDS-PAGE in nonreducing conditions, and visualized by autoradiography. The precipitations with E7-1 and RPC5.4 mAbs (both mouse IgG2a) represent the material obtained using 0.1 µl ascites from 7 x 104 cell equivalents, for TS1/22 and P3 mAbs (both mouse IgG1, with rabbit anti–mouse IgG) using 0.5 µl ascites from 7 x 105 cell equivalents, and for E-cadherin–Fc, P-cadherin–Fc, ICAM-1–Fc and human IgG1 using 5 µg fusion protein from 3 x 106 cell equivalents. For the preclearing experiments, batches of 1.5 x 106 cell equivalents were preabsorbed with the stated antibodies and protein A–Sepharose, before immunoadsorption with 2.5 µg E-cadherin–Fc.

E-Cadherin–Fc Binds to Soluble Recombinant _Eβ₇
To produce a soluble form of _Eβ₇, stop codons were introduced immediately upstream of the transmembrane coding regions in the cDNAs encoding the human _E and β₇ proteins (see Fig. 1 b, and Materials and Methods). Supernatant containing ³⁵S-labeled soluble _Eβ₇ was produced by transient transfection of COS-7 cells followed by metabolic labeling. Proteins in the supernatant were then subjected to immunoprecipitation with a panel of anti– _Eβ₇ antibodies that recognize at least three distinct _E epitopes (Russell et al., 1994). In the medium from cells transfected with plasmids encoding truncated _E and β₇ in the sense orientation, all the anti–_Eβ₇ mAbs tested (E7-1, E7-2, E7-3, HML-1, and BerACT8), but not a control mAb (TCR-1), precipitated two major bands of 140 and 105 kD on reducing SDS-PAGE. On nonreducing SDS-PAGE, two bands of 170 and 100 kD were observed (Fig. 6 and data not shown). These sizes correspond to those expected for the truncated _E and β₇ chains, respectively. No such proteins were precipitated from the medium of COS-7 cells transfected with constructs containing truncated _E and β₇ cDNAs in the antisense orientation. These results confirm the secretion of a soluble form of _Eβ₇ that retains all of the epitopes of cell surface _Eβ₇ that were tested.

View larger version (59K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Direct binding of soluble recombinant Eβ7 to E-cadherin–Fc. COS-7 cells transiently transfected with either E and β7 constructs in the sense or in the antisense orientation were metabolically labeled with 35S amino acids as described in the text. The medium was then made 1 mM with respect to MnCl2 and subjected to immunoadsorption with antibodies or Fc-fusion proteins. Samples were resolved on 7.5% SDS-PAGE in nonreducing conditions, and visualized by autoradiography. The precipitations with E7-1 and RPC5.4 mAbs (both mouse IgG2a) represent the material obtained using 0.25 ml medium and 0.5 µl ascites, and for E-cadherin–Fc, P-cadherin–Fc, and ICAM-1–Fc, using 1.0 ml medium and 5 µg fusion protein. Aggregated material is present in the both control and test E-cadherin–Fc adsorptions.

The Avidity of Cell Surface _Eβ₇ Is Regulated
Although the avidity of many integrins is regulated from within the cell, similar regulation of _Eβ₇ has not previously been reported. Furthermore, the structure of the _E chain is unique among integrins due to the presence of an extra domain in the extracellular region, membrane distal of the A domain, that is cleaved in the mature protein (the "X" domain, see Fig. 1 b; Shaw et al., 1994). This raises the possibility that regulation of the avidity of _Eβ₇ due to conformational changes could be different from other integrins. Therefore, studies were performed to investigate the regulation of _Eβ₇ avidity on JY'-_E cells and IEL, and to compare it to the well studied _Lβ₂ integrin.

JY' cells transfected with _E adhered poorly to E-cadherin–Fc in the presence of 1 mM MgCl₂ and 1 mM CaCl₂ in the absence of manganese. The addition of 1 mM manganese caused a 12-fold or greater rise in the adhesion of JY'-_E cells to E-cadherin–Fc. PMA stimulation of JY'-_E also caused a sevenfold increase in binding to E-cadherin–Fc (Fig. 7 a). Adhesion of JY'-_E cells to ICAM-1–Fc was also enhanced by manganese or PMA, suggesting that both _Eβ₇ and _Lβ₂ can be activated by similar means. Relative to the effect of manganese, PMA was better able to stimulate adhesion of JY'-_E to ICAM-1–Fc than to E-cadherin–Fc. The reason for this difference is unclear, but valid comparisons are difficult since _Lβ₂ is expressed at a higher level than _Eβ₇ on JY'-_E cells (Fig. 3 a).

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Regulation of JY'-E cell and IEL adhesion to E-cadherin–Fc and ICAM-1–Fc. (a) The adhesion of JY'-E cells to 0.6 µg/well directly coated E-cadherin–Fc and ICAM-1–Fc under various conditions. EDTA (10 mM) was present during coating and blocking of the microtiter plates, and was washed out with HBS before adding cells. The adhesion buffer was 0.1% BSA/ HBS, pH 7.4, containing divalent cations and 50 ng/ml PMA or 1 mM EGTA where appropriate. The results are expressed as mean percent adhesion + 1 SD (n = 6 for adhesion to E-cadherin–Fc, n = 3 for adhesion to ICAM-1–Fc). The percent adhesion to human IgG1 under the each condition was subtracted from the results (<5% in each case). (b) The adhesion of IEL to 0.1 µg/well E-cadherin–Fc under various conditions. The adhesion buffer was 0.05 mM MgCl2, 1 mM CaCl2, 0.1% BSA, HBS, pH 7.4, unless otherwise stated. Ascites containing antibodies (all mouse IgG2a) to MHC I (W6/32), CD8 (OKT8), nonbinding control (RPC5.4), and CD3 (SPVT3b) were used at a dilution of 1:100 and cross-linked with 10 µg/ml rabbit anti–mouse IgG polyclonal antibody. Where appropriate, 1 mM MnCl2 or 50 ng/ml PMA was also included. The results are expressed as mean percent adhesion + 1 SD (n = 6, except for experiments with OKT8 and RPC5.4 where n = 3). In both a and b, P values calculated by Welch's alternate t test compared with the Mg/Ca alone are as follows: ***P < 0.0001, **P < 0.001, *P < 0.01. In addition, for adhesion to E-cadherin–Fc, P values by the Mann-Whitney test compared with Mg/Ca alone are: ***P = 0.002, **P < 0.005 (see Materials and Methods).

It is known that calcium is required for the rigidification of the structure of E-cadherin (Nagar et al., 1996; Pokutta et al., 1994) and for E-cadherin–mediated homotypic cell to cell adhesion (Hyafil et al., 1981; Yoshida and Takeichi, 1982). However, in the presence of magnesium, even when calcium has been depleted with EGTA, both JY'-_E cells (Fig. 7 a) and IEL (not shown) adhere to E-cadherin–Fc. Thus, the heterophilic interaction of E-cadherin appears to be independent of calcium, at least in this system. It is unlikely that this is due to restriction of conformational changes within plate-bound E-cadherin–Fc, since similar results were found whether the fusion protein was immobilized directly on plastic or through its Fc region on antiimmunoglobulin antibodies (not shown). In contrast, the adhesion of E-cadherin–expressing 16E6.A5 epithelial cells to E-cadherin–Fc is dependent on calcium (not shown). Also, since mouse E-cadherin does not have appreciable affinity for magnesium (Hyafil et al., 1981), and magnesium cannot support homophilic adhesion to E-cadherin–Fc (not shown), it is unlikely that magnesium substitutes for calcium in the cadherin structure. The results also suggest that calcium is not required for the function of _Eβ₇ integrin. This is also the case for _Lβ₂ binding to ICAM-1 (Fig. 7 a; Shimizu and Mobley, 1993; Stewart et al., 1996).

In summary, _Eβ₇, like _Lβ₂, can exist in both high and low avidity states on the cell surface. In common with _Lβ₂–mediated adhesion to ICAM-1, treatment of cells expressing _Eβ₇ in a low avidity state with manganese, with PMA, with anti–CD3 antibodies, or by removing calcium and elevating magnesium, leads to increased adhesion to E-cadherin–Fc.

The Direct Binding of E-Cadherin–Fc to Solubilized _Eβ₇ Is Modulated by Divalent Cations
We wished to determine if the effects of divalent cations on cell adhesion to E-cadherin–Fc were due to a direct influence on the binding of _Eβ₇ molecules to E-cadherin– Fc molecules. The binding of E-cadherin–Fc to ¹²⁵I-labeled _Eβ₇ in an IEL lysate was analyzed by immunoadsorption as described in Fig. 5, but in the presence of various concentrations of divalent cations (Fig. 8). No binding of E-cadherin–Fc to _Eβ₇ was detected in the presence of 5 mM EDTA, confirming the cation dependency of the interaction (Fig. 8 b). Binding of E-cadherin–Fc to _Eβ₇ was clear in the presence of 1 mM MgCl₂ and 1 mM CaCl₂, but was increased eightfold by the addition of 1 mM MnCl₂ (Fig. 8 b). Approximately equal quantities of _Eβ₇ were immunoadsorbed by the anti–_Eβ₇ mAb E7-1 in all conditions, confirming that the differences seen in binding to the E-cadherin–Fc fusion protein were not due to dissociation or degradation of the _Eβ₇ chains (Fig. 8 a). Furthermore, since cadherins are known to be protease sensitive in the absence of calcium, we confirmed that an equal quantity of E-cadherin–Fc protein was present in each condition by Coomassie Blue staining of the immunoadsorbed material (Fig. 8 b, ii).

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Direct binding of Eβ7 to E-cadherin–Fc is modulated by divalent cations. (a) Immunoadsorption of Eβ7 by an anti– Eβ7 mAb is unaffected by divalent cations. Batches of 125I-labeled IEL lysate were subjected to immunoadsorption with the E7-1 mAb in the presence of divalent cations or EDTA as shown. Samples were resolved by 7.5% SDS-PAGE in nonreducing conditions, and Eβ7 was visualized by autoradiography. (b) Immunoadsorption of Eβ7 by E-cadherin–Fc is modulated by divalent cations. (i) In duplicate, batches of 125I-labeled IEL lysate were subjected to immunoadsorption with E-cadherin–Fc in the conditions shown in a. (ii) The presence of an equal quantity of E-cadherin–Fc in the immunoadsorbed material was confirmed by 7.5% SDS-PAGE in reducing conditions and Coomassie blue staining. (iii) Quantitation of Eβ7 bound to E-cadherin–Fc in each condition. Phosphorimage quantitation of Eβ7 was based upon the 175- and 135-kD bands corresponding to the E-chain (Shaw et al., 1994). The averages of duplicates for each band were combined. The precipitations with E7-1 represent the material obtained using 0.05-µl ascites from 6 x 104 cell equivalents and for E-cadherin–Fc using 5 µg fusion protein from 2 x 106 cell equivalents.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Here, we demonstrate that an E-cadherin fusion protein binds directly both to _Eβ₇ from solubilized intraepithelial lymphocytes, and to a soluble recombinant form of _Eβ₇. Furthermore, purified E-cadherin fusion protein can serve as a potent substrate for cell adhesion through the _Eβ₇ integrin. Thus, although E-cadherin is classically thought to be a homophilic adhesion molecule and is unlike known integrin ligands, it is a direct counter receptor for the _Eβ₇ integrin.

We found that _Eβ₇ exhibits selectivity in binding to cadherins. The adhesion of IEL cells to P-cadherin–Fc requires >100-fold more fusion protein than adhesion to E-cadherin–Fc. In both cases the adhesion is dependent on _Eβ₇, since it is abolished by antibodies to _Eβ₇. Thus, although human epithelial cells express both E- and P-cadherin, it is likely that E-cadherin is the primary epithelial receptor for _Eβ₇ in vivo. The idea that _Eβ₇ is selective in binding cadherins is consistent with the proposed role of _Eβ₇ in tissue-specific leukocyte adhesion, and with the finding that IEL do not adhere to all cadherin-expressing cells. For example, IEL do not adhere to endothelial cells that possess VE-cadherin (Cepek et al., 1994).

Regulation of _Eβ₇ avidity for its counter receptor has not previously been reported. An increase in IEL adhesion to epithelial cells in the presence of manganese has been observed (Cepek et al., 1993; Karecla et al., 1995). However, it is not clear that this change is due to regulation of _Eβ₇ binding to E-cadherin since other receptors, including _Lβ₂ and ICAM-1, are involved in this complex cell to cell interaction (Cepek et al., 1993; Roberts et al., 1993). Here, the adhesion of cells expressing both _Eβ₇ and _Lβ₂ to purified E-cadherin–Fc or ICAM-1–Fc allowed us to compare the regulation of _Eβ₇ avidity with that of _Lβ₂ in a system in which only a single integrin counter receptor was available in each case. The _Lβ₂ integrin on freshly isolated PBL has low avidity for immobilized ICAM-1 in the presence of 1 mM MgCl₂ and 1 mM CaCl₂. However, in the presence of manganese ions PBL adhere strongly to purified ICAM-1 (Dransfield et al., 1992). Changes in the concentrations and presence of divalent cations are thought to have a direct effect on the conformation of the integrin at the cell surface, leading to increased ligand binding through an increase in the affinity of the integrin and/or changes in clustering of the integrin in the membrane. In addition, signaling from inside the cell can lead to increased integrin avidity. For example, PMA stimulation of resting PBL leads to increased adhesion to ICAM-1 through _Lβ₂. This type of regulation may involve changes in integrin clustering on the cell surface due to alterations in integrin association with the cytoskeleton (Lub et al., 1995, 1997; Stewart et al., 1996). In contrast to resting PBL, _Lβ₂ on a proportion of IL-2/PHA-activated PBL has high avidity for ICAM-1 (Lub et al., 1997). Furthermore, the avidity state of integrins transfected into different cell types can vary. For example, _Lβ₂ expressed on K562 cells is in a constitutively inactive state, while _Lβ₂ expressed on L cells is constitutively active (Lub et al., 1995).

We find that in 1 mM MgCl₂ and 1 mM CaCl₂, _E-transfected JY' cells exhibit poor adhesion to both E-cadherin–Fc and ICAM-1–Fc. Thus, on these cells, both _Eβ₇ and _Lβ₂ exist in a low avidity state. However, these cells can be induced to adhere to both integrin ligands by the addition of 1 mM MnCl₂. Since parallel changes in the direct binding of solubilized _Eβ₇ to E-cadherin–Fc were also observed, it is likely that this difference in cell adhesiveness is due to a direct effect of manganese on the conformation of the _Eβ₇ integrin. Thus, changes in extracellular cations can regulate the affinity of _Eβ₇ for its ligand. In contrast, cultured IEL, which are maintained in IL-2 with periodic stimulation with PHA and feeder cells, adhere avidly to both E-cadherin–Fc and ICAM-1–Fc in 1 mM MgCl₂ and 1 mM CaCl₂ even in the absence of manganese. Both _Eβ₇ and _Lβ₂ on IEL appear to be maintained in a constitutively active state, like _Lβ₂ on a proportion of IL-2/PHA-stimulated peripheral blood T cells.

We also show that signaling from inside the cell is able to increase the avidity of _Eβ₇. The addition of the phorbol ester, PMA, which acts intracellularly to upregulate protein kinase C, leads to increased adhesion of _E-transfected JY' cells to E-cadherin–Fc or ICAM-1–Fc. In the presence of a suboptimal concentration of magnesium, PMA also enhanced IEL adhesion to E-cadherin–Fc. The physiological triggers for such changes in lymphocyte integrin avidity in vivo remain unclear. In vitro, chemokines such as MCP-1, MIP-1β, and RANTES can activate lymphocyte integrins (Campbell et al., 1996; Carr et al., 1996; Lloyd et al., 1996), and cross-linking of components of the T cell receptor can boost the adhesion of resting PBL to ICAM-1 through _Lβ₂ (Dustin and Springer, 1989; van Kooyk et al., 1989) or to fibronectin and laminin through β1-integrins (Shimizu et al., 1990). Here, we report that antibody cross-linking of cell surface CD3 is similarly able to increase IEL adhesion to E-cadherin–Fc, while cross-linking of MHC class I or CD8 receptors had no such effect. Thus, recognition by an IEL of an antigen presented by an epithelial cell or a professional antigen-presenting cell expressing E-cadherin (such as a Langerhans cell; Tang et al., 1993) could trigger increased adhesion to that cell through an upregulation of _Eβ₇ avidity. This interaction may also provide costimulation to the T cell, since anti–_Eβ₇ antibodies, in common with anti–_Lβ₂ antibodies, are known to increase T cell proliferation in the presence of suboptimal anti-CD3 (Russell et al., 1994; Sarnacki et al., 1992; Wacholtz et al., 1989), and ICAM-1 on an antigen-presenting cell can costimulate through _Lβ₂ (Dubey et al., 1995). Such events may be important in arresting lymphocytes within the epithelium when a specific antigen is recognized. In addition, since IEL contact more than one cell when resident within the epithelium, localized upregulation of _Eβ₇ avidity within an IEL could aid in polarizing lymphocyte interactions toward the relevant antigen-presenting cell.

X-ray crystallography and NMR studies have recently revealed that cadherin modules adopt a tertiary structure rather like immunoglobulin domains (Overduin et al., 1995; Shapiro et al., 1995; Nagar et al., 1996). Thus E-cadherin has a structure resembling that of the known cellular integrin ligands and can now be placed within the family of immunoglobulin-like, integrin-binding proteins. E-cadherin may share another feature of well-defined integrin ligands; the presence of a solvent-exposed acidic residue vital for integrin binding (Bergelson and Hemler, 1995). Mutation of an aspartate or glutamate residue in the CD-loop region in domain 1 of the immunoglobulin superfamily integrin ligands VCAM-1 (Osborn et al., 1994; Renz et al., 1994; Vonderheide et al., 1994; Jones et al., 1995; Wang et al., 1995), ICAM-1, (Staunton et al., 1990; Holness et al., 1995), and MadCAM-1 (Briskin et al., 1996; Viney et al., 1996) abolishes integrin binding. The tenth immunoglobulin-like FN III repeat of fibronectin also has an acidic residue within the RGD sequence on the FG-loop that is involved in integrin binding (Main et al., 1992). In mouse E-cadherin, the BC loop of domain 1 contains a glutamate residue required for adhesion of _Eβ₇-expressing lymphocytes to E-cadherin–transfected L cells (Karecla et al., 1996). Since this residue is conserved in human E-cadherin and we demonstrate that E-cadherin is a direct counter receptor for _Eβ₇, it is likely that this amino acid contributes directly to the _Eβ₇-binding site on E-cadherin, in a manner similar to that proposed for other integrin ligands. It is interesting to note that while three different families of immunoglobulin-like structures with exposed acidic amino acids serve as integrin ligands, the face of the module involved appears to be different in each case. It is also intriguing that both E-cadherin and ICAM-1 are likely to be dimeric on the cell surface (Miller et al., 1995; Reilly et al., 1995; Nagar et al., 1996), and that both are ligands of integrins that contain an A domain in the chain (_Eβ₇ and _Lβ₂, respectively). Since it has been proposed that integrin β-chains also contain a ligand-binding A domain (Lee et al., 1995; Puzon-McLaughlin and Takada, 1996), it is possible that _Eβ₇ and _Lβ₂ actually possess two A domains each. As suggested for _Lβ₂ binding to ICAM-1 (Miller et al., 1995), it is tempting to speculate that the binding of dimeric E-cadherin by _Eβ₇ could involve both these domains.

Although E-cadherin is a calcium-binding molecule, we find that E-cadherin–Fc binding to _Eβ₇ is independent of calcium. It seems that calcium is not directly involved in maintenance of the _Eβ₇ binding site on E-cadherin. Studies with recombinant soluble forms of mouse E-cadherin and Xenopus C-cadherin suggest that homophilic cadherin interactions do require the presence of calcium (Brieher et al., 1996; Tomschy et al., 1996). Interestingly, in their NMR studies of the first module of mouse E-cadherin, Overduin et al. (1995) find that the addition of calcium leads to a large shift in the orientation of histidine-79 within the HAV motif implicated in homophilic E-cadherin interaction (Blaschuk et al., 1990; Shapiro et al., 1995). In contrast, no such shift is found in any of the residues in or around the BC-loop implicated in _Eβ₇ interaction (Overduin et al., 1995). Thus it is possible that changes in extracellular calcium levels (for review see Maurer et al., 1996) would differentially regulate E-cadherin binding to _Eβ₇ versus other E-cadherin molecules. However, since calcium is required to rigidify and extend E-cadherin (Pokutta et al., 1994; Nagar et al., 1996) and to protect it from proteolysis (Hyafil et al., 1981; Yoshida and Takeichi, 1982), the lack of calcium dependence for _Eβ₇-mediated adhesion to the E-cadherin–Fc fusion protein may not hold true on the cell surface.

Although the role of _Eβ₇ binding to E-cadherin in vivo has yet to be established, the direct, specific, and regulated binding of _Eβ₇-expressing cells and of _Eβ₇ itself to the E-cadherin fusion protein very strongly suggests a physiological function for this interaction. Although there is an increase in the number of β₇-integrin positive iIEL in chimeric mice with a defect in intestinal E-cadherin expression (Hermiston and Gordon, 1995), this is perhaps not surprising since the observed disruption of the epithelial layer and accompanying infectious and inflammatory response is likely to result in the attraction of many leukocytes into the intestine. In contrast, recently developed _E knockout mice have reduced numbers of intraepithelial IEL (Parker, C.M., unpublished results).

The role in vivo of the low but detectable _Eβ₇-mediated adhesion of IEL to human P-cadherin is more difficult to assess. The relative abundance of E- and P-cadherin on epithelial cells depends upon the state of cell differentiation (Hirai et al., 1989a,b), but this may not be the sole determinant of _Eβ₇-mediated interactions. On a single cell type, cadherins can display differential association with the cytoskeleton and thus exist in distinct pools on the cell surface (Salomon et al., 1992), and E- and P-cadherin are found in separate complexes on A431 cells (Johnson et al., 1993). Cell adhesion through homophilic cadherin–cadherin interactions requires their association with the cytoskeleton through intracellular catenins (Nagafuchi and Takeichi, 1988). However, while E-cadherin lacking its cytoplasmic tail is unable to associate with the catenins and cannot support strong homophilic adhesion, it is able to support adhesion of lymphocytes expressing _Eβ₇ (Karecla et al., 1996). So, since different pools of cadherins may differ in their availability for interaction with different counter receptors, it is difficult to predict whether or not a high local concentration of a distinct population of P-cadherin on the cell surface could contribute to _Eβ₇-mediated adhesion.

Like human, mouse, canine, and Xenopus E-cadherin, human P-cadherin possesses an acidic residue at the tip of the BC loop in the first cadherin module (Shimoyama et al., 1989b). Thus human E- and P-cadherin may share a similar mode of binding to _Eβ₇. In contrast, mouse P-cadherin does not possess an acidic residue at this position (Nose et al., 1987), and it has been reported that murine lymphocytes expressing _Eβ₇ do not adhere to L cells transfected with mouse P-cadherin (Karecla et al., 1996). Although this cell to cell adhesion assay is likely to be less sensitive than the cell to fusion protein assay used here, these findings further complicate the question of the potential importance of _Eβ₇ binding to P-cadherin. It is possible that in some respects the function of P-cadherin may differ in the two species, and this may be reflected in the different expression pattern of P-cadherin in human and mouse (Shimoyama et al., 1989a,b).

Recently, a second direct heterophilic noncadherin ligand of a cadherin has been identified. The Listeria surface protein internalin binds to E-cadherin and invasion of cells expressing E-cadherin by Listeria in vitro is inhibited by anti–E-cadherin antibodies (Mengaud et al., 1996). Together with studies suggesting that different cadherins can bind to each other (for example N- and R-cadherin) (Takeichi, 1995), it is now clear that the biology of cadherin function is not limited to homophilic interactions, and is more complex than previously imagined.

	Acknowledgments

 
This work was supported by a Wellcome Trust International Prize Travelling Research Fellowship (J.M.G. Higgins), and grants from the National Institutes of Health (NIH; M.B. Brenner), NIH grant AI01212 (D.A. Mandlebrot), the Crohn's and Colitis Foundation of America and NIH grant DK43351 (G.J. Russell), an NIH R29 First Award GM49342 and a Cancer Research Institute Investigator Award (C.M. Parker), NIH grant GM35527 (W.J. Nelson), and a postdoctoral fellowship from the National Kidney Foundation (Y.-T. Chen).

Submitted: 8 August 1997

Revised: 10 November 1997

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