E-cadherin is a homophilic adhesion molecule that maintains homotypic intercellular adhesion between epithelial cells such as epidermal keratinocytes. E-cadherin is also expressed on resident murine epidermal γδ T cells, known as dendritic epidermal T cells (DETCs), but they express another receptor for E-cadherin, αE(CD103)β7 integrin, as well. In this study, we analyzed functional differences between E-cadherin–mediated homophilic binding and heterophilic binding of αEβ7 integrin to E-cadherin in heterotypic intercellular adhesion of DETCs to keratinocytes. E-cadherin, but not αEβ7 integrin, was downregulated on activation of DETCs in vivo and in vitro. Short-term (1-h) adhesion of DETCs to keratinocytes in vitro was primarily mediated by αEβ7 integrin, and blocking of the binding of αEβ7 integrin to E-cadherin inhibited the lysis of keratinocytes by DETCs. Stable binding of E-cadherin on DETCs to plate-bound recombinant E-cadherin was observed only after 24-h culture in vitro. Cytokine production and degranulation by DETCs in response to suboptimal TCR cross-linking and mitogen stimulation were augmented by coligation of αEβ7 integrin. In contrast, engagement of E-cadherin on DETCs with immobilized anti–E-cadherin Ab, plate-bound recombinant E-cadherin, and E-cadherin on keratinocytes inhibited DETC activation. Therefore, E-cadherin acts as an inhibitory receptor on DETCs, whereas αEβ7 integrin acts as a costimulatory receptor. Differential expression of E-cadherin and αEβ7 integrin on resting and activated DETCs, as well as their opposite functions in DETC activation, suggests that E-cadherin and αEβ7 integrin on DETCs regulate their activation threshold through binding to E-cadherin on keratinocytes.

E-cadherin is a calcium-dependent homophilic adhesion molecule that plays vital roles in embryonic development and maintenance of polarity and integrity of adult epithelial tissues (1, 2). Its cytoplasmic domain interacts with intracellular proteins including catenins, which connect E-cadherin to the actin cytoskeleton (1, 2). In the epidermis, E-cadherin constitutes the major adhesion molecule in intercellular adherens junctions between keratinocytes (2). Epidermal Langerhans cells also express E-cadherin, and the homophilic binding between E-cadherin molecules mediates heterotypic intercellular adhesion of Langerhans cells to keratinocytes in vitro (3). E-cadherin–mediated adhesion to keratinocytes may contribute to retention of Langerhans cells in the epidermis in vivo because E-cadherin is downregulated during maturation of Langerhans cells (3) that leads to their emigration from the epidermis.

Resident murine epidermal γδ T cells, known as dendritic epidermal T cells (DETCs), also express E-cadherin (4, 5). DETCs express a skin-specific invariant Vγ3 TCR (6) and are activated to exert various effector functions including immunoregulation (7, 8), tumor surveillance (9), and wound healing (10, 11) through TCR-mediated recognition of undefined self-Ags induced on neighboring stressed, damaged, or transformed keratinocytes (12, 13). Unlike Langerhans cells, blocking of the homophilic E-cadherin binding does not inhibit adhesion of DETCs to keratinocytes in vitro (4, 5). This is possibly because DETCs express another receptor for E-cadherin, αE(CD103)β7 integrin (14).

Besides DETCs, αEβ7 integrin is expressed on intestinal and other intraepithelial lymphocytes (IELs), intestinal lamina propria T cells (15), some effector CD8+ T cells (1618), and subsets of regulatory T cells (19), dendritic cells (20), and mast cells (21). Adhesion of intestinal IELs to epithelial cells in vitro is mediated by heterophilic binding of αEβ7 integrin to epithelial E-cadherin (2227). Because integrin αE-chain–deficient mice have reduced numbers of resident intestinal IELs (28), contribution of αEβ7 integrin to generation or maintenance of intestinal IELs in vivo through binding to epithelial E-cadherin has been suggested. CD8+ T cells stimulated with TGF-β acquire expression of αEβ7 integrin (1618, 29). Interactions of αEβ7 integrin with E-cadherin mediate adhesion of CD8+ T cells to epithelial cells in vitro (18) and are crucial for the lysis of E-cadherin–expressing tumor cells by CTLs (29). In addition, binding of αEβ7 integrin to E-cadherin may provide a costimulatory signal in intestinal IELs and CD8+ T cells because cross-linking of αEβ7 integrin leads to enhanced proliferation in response to suboptimal TCR stimulation (14, 25, 30).

Because DETCs are also reduced in integrin αE-chain–deficient mice (31), αEβ7 integrin has been implicated in retention of DETCs in the epidermis. In addition, a recent study showed that αEβ7 integrin influences cellular shape and motility of DETCs (32). However, roles of αEβ7 integrin in the adhesion of DETCs to keratinocytes, in the DETC-mediated lysis of keratinocytes, or in the costimulation of DETC activation have not been elucidated. Furthermore, it remains unknown whether homophilic E-cadherin binding and heterophilic binding of αEβ7 integrin to E-cadherin on keratinocytes have distinct functions in DETCs.

In this study, we analyzed functional differences between E-cadherin and αEβ7 integrin expressed on DETCs, both of which interact with E-cadherin on keratinocytes. In this article, we show that E-cadherin and αEβ7 integrin expression on DETCs are differently regulated depending on the activation state, and that they provide opposite signals for DETC activation through binding to E-cadherin on keratinocytes.

C57BL/6 mice were purchased from CLEA Japan (Tokyo, Japan). Female mice were used at 6–12 wk of age. All procedures have been approved by the Animal Care Committee of Kagoshima University following the institutional guidelines.

Epidermal cells were prepared as described previously (33) with a few modifications. In brief, the ear skin was treated for 40 min at 37°C with 1% trypsin (Invitrogen, Carlsbad, CA) in PBS containing 1 mM CaCl2. The epidermis was separated and collected in IMDM (Invitrogen) supplemented with 10% FCS (JRH Biosciences, Lenexa, KS) and 0.025% DNase I (Sigma-Aldrich, St. Louis, MO). Single-cell suspensions were obtained by mechanical agitation and sequential filtration through 70- and 30-μm nylon meshes.

Short-term DETC lines were generated in each experiment by stimulating the epidermal cells (1–2 × 106/ml) for 7 d in 24-well plates coated with 10 μg/ml anti-TCR Cδ mAb (clone UC7-13D5; BD Biosciences, San Jose, CA). DETCs were harvested and restimulated for 24 h with 1 μg/ml Con A (Sigma-Aldrich), followed by expansion for an additional 14 d in IMDM supplemented with 10% FCS, 50 μM 2-ME, and 10 ng/ml mouse rIL-2 (R&D Systems, Minneapolis, MN). The resulting cell population that was >95% pure TCR Vγ3+ DETCs and had been ''rested'' for 14 d after the last stimulation was used as resting DETCs. Resting DETCs stimulated for 24 h with 10 μg/ml immobilized anti-TCR Cδ mAb (UC7-13D5) were used as activated DETCs. At the time of use, cells were harvested by incubation for 3 min with 1 mM EDTA in PBS.

The Pam 212 transformed keratinocyte cell line was grown in IMDM supplemented with 10% FCS. At the time of use, cells were harvested by pipetting after incubation for 5 min with 0.25% trypsin in PBS containing 1 mM CaCl2. The Pam 212 cells cultured in our laboratory express E-cadherin but not ICAM-1 even after 3-d culture in the presence of 50 ng/ml mouse rIFN-γ (R&D Systems) (Supplemental Fig. 1).

Cells were resuspended in PBS supplemented with 2% FCS and 0.1% NaN3. After preincubation with anti-CD16/CD32 mAb (2.4G2; BD Biosciences), cells were stained with the following mAbs: FITC-, PE-, or biotin-conjugated anti-TCR Vγ3 (clone 536; BD Biosciences or BioLegend, San Diego, CA), PE-conjugated anti-integrin αE-chain (2E7; eBioscience, San Diego, CA), PE-conjugated anti-integrin β7-chain (M293; BD Biosciences), biotin-conjugated anti-killer cell lectin-like receptor G1 (anti-KLRG1) (2F1; eBioscience), FITC-conjugated anti-integrin αL-chain (M17/4; eBioscience), FITC-conjugated anti-integrin αM-chain (M1/70; BD Biosciences), FITC-conjugated anti-integrin αX-chain (HL3; BD Biosciences), FITC-conjugated anti-integrin β2-chain (H18/2; eBioscience), PE-conjugated anti-integrin α4-chain (R1-2; eBioscience), PE-conjugated anti-integrin β1-chain (HMβ1-1; eBioscience), PE-conjugated anti-integrin αV-chain (RMV-7; eBioscience), PE-conjugated anti-integrin β3-chain (2C9.G3; eBioscience), FITC-conjugated anti-CD62L (MEL-14; eBioscience), PE-conjugated anti-CD25 (7D4; BD Biosciences), and FITC-, PE-, or biotin-conjugated isotype controls (BD Biosciences or eBioscience). Biotin-conjugated mAbs were visualized with Alexa Fluor 488-conjugated (Molecular Probes, Eugene, OR), PE-conjugated (Southern Biotechnology, Birmingham, AL), or PE-Cy5–conjugated (eBioscience) streptavidin. For double staining of TCR Vγ3 and E-cadherin, cells were preincubated with normal rabbit Igs (DAKO, Glostrup, Denmark) and stained with PE-conjugated anti-TCR Vγ3 (clone 536) and unlabeled anti–E-cadherin (ECCD-2; Takara Bio, Otsu, Japan) or isotype control mAbs, followed by FITC-conjugated rabbit anti-rat Igs Ab (DAKO) that was preincubated with unlabeled normal hamster IgG (eBioscience). For staining cells with anti–ICAM-1 or anti-integrin α5-chain mAb, cells were preincubated with normal goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and stained with unlabeled anti–ICAM-1 (3E2; BD Biosciences), anti-integrin α5-chain (HMα5-1; a gift from H. Yagita, Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan), or isotype control mAb, followed by PE-conjugated goat anti-hamster IgG (H+L) F(ab′)2 Ab (Caltag Laboratory, Burlingame, CA). After gating on forward and side scatters and propidium iodide, viable cells were analyzed on an EPICS XL flow cytometer with EXPO32 software (Beckman Coulter, Fullerton, CA), and data were analyzed using FlowJo software (Tree Star, San Carlos, CA).

Mice were anesthetized with 2,2,2,-tribromoethanol, and three sets of full-thickness wounds through the panniculus carnosus were created on the shaved back skin using a 3-mm punch tool (10). Mice were caged individually and wounds were left uncovered. The periwound skin and the control skin distant from the wounds were collected 24 h after wounding.

Epidermal sheets were prepared as described previously (34) using 0.5 M ammonium thiocyanate and fixed for 20 min in cold acetone. After preincubation for 15 min with 10% normal goat serum (Nichirei, Tokyo, Japan), the sheets were incubated overnight at 4°C with anti–E-cadherin (ECCD-2 or clone 36; BD Biosciences), anti–β-catenin (clone 14; BD Biosciences), or anti-integrin β7-chain (FIB27; BD Biosciences) mAb. After rinses in PBS, the sheets were incubated overnight at 4°C with Alexa Fluor 488-conjugated goat anti-rat IgG (H+L) (for ECCD-2 and FIB27) or goat anti-mouse IgG (H+L) F(ab′)2 (for clones 36 and 14) Ab (both from Molecular Probes). The sheets were then stained sequentially with 10% normal hamster serum (Jackson ImmunoResearch, West Grove, PA) for 15 min, biotin-conjugated anti-TCR Vγ3 mAb (clone 536) for 1 h at 37°C, and rhodamine-conjugated streptavidin (Southern Biotechnology) for 1 h at 37°C. The stained sheets were examined under a laser-scanning confocal microscopy (FV500; Olympus, Tokyo, Japan).

Resting DETCs were labeled for 15 min at 37°C with 5 μM CFSE (Molecular Probes) in PBS, quenched for 30 min at 37°C with IMDM supplemented with 10% FCS, and resuspended in IMDM supplemented with 1% FCS. CFSE-labeled DETCs were preincubated for 15 min with indicated blocking mAbs or isotype control mAbs and seeded at 500 cells/mm2 on the confluent monolayer of Pam 212 keratinocytes precultured in chamber slides (Lab-Tek II; Nunc, Roskilde, Denmark). The following mAbs were used for blocking: 200 μg/ml anti–E-cadherin (ECCD-1; Takara Bio), 20 μg/ml anti–E-cadherin (ECCD-2), anti-integrin αE-chain (M290; BD Biosciences; and 2E7; eBioscience), β7-chain (FIB27), αL-chain (M17/4; eBioscience), β2-chain (H18/2; eBioscience), β1-chain (HMβ1-1; a gift from H. Yagita), and β3-chain (HMβ3-1; a gift from H. Yagita). DETCs were allowed to adhere to the monolayers for 1 h at 37°C, and nonadherent DETCs were removed by gentle washes with prewarmed IMDM containing 1% FCS. The number of adherent DETCs in a 0.17-mm2 area was counted using a calibrated ocular grid under a fluorescence microscopy (Optiphot; Nikon, Tokyo, Japan), and the density of adherent DETCs was recorded as the mean cell number per square millimeter of 10 random fields. The percentage of adherent DETCs was calculated as (adherent DETCs per mm2/500) × 100 (%).

Resting DETC effector cells were seeded at the indicated E:T ratios on Pam 212 target cells (1 × 104) precultured overnight in 96-well plates, and coincubated for 4 h in IMDM supplemented with 1% FCS. Effector cells were preincubated for 15 min with 20 μg/ml anti–E-cadherin (ECCD-2), anti-integrin αE-chain (M290), αL-chain (H17/4), β1-chain (HMβ1-1), β3-chain (HMβ3-1), or isotype control mAb before addition to the target cells, without washing out the mAb. The lactate dehydrogenase activity released in the supernatants was quantified by colorimetric assay using the Cytotoxicity Detection kit (Roche, Mannheim, Germany). Specific lysis was calculated as ([experimental release − effector cell control release − spontaneous release]/[maximum release − spontaneous release]) × 100 (%). The spontaneous release was <1% of the maximum release.

Chamber slides were coated overnight at 4°C with 1 μg/ml recombinant mouse E-cadherin (R&D Systems) in PBS containing 1 mM CaCl2. Resting DETCs were seeded on uncoated or E-cadherin–coated slides and cultured for 1 or 24 h. Adherent cells were fixed for 5 min in cold acetone and stained sequentially with 10% normal goat serum for 15 min; anti–E-cadherin (clone 36), anti–β-catenin (clone 14), or anti-integrin β7-chain (FIB27) mAb overnight at 4°C; and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) F(ab′)2 (for clones 36 and 14) or goat anti-rat IgG (H+L) (for FIB27) Ab for 1 h at 37°C. The slides were examined under confocal microscopy.

For stimulating DETCs with immobilized mAbs, 96-well plates were coated overnight at 4°C with indicated concentrations of anti-TCR Cδ mAb (UC7-13D5), and anti–E-cadherin (ECCD-2), anti-integrin αE-chain (M290), or isotype control mAb. For cytokine production assays, resting DETCs (1 × 105) were preincubated for 15 min with anti-CD16/CD32 mAb (2.4G2) and stimulated for 4 h with the immobilized mAbs in the presence of GolgiPlug (BD Biosciences) containing brefeldin A. Intracellular cytokine production was analyzed by flow cytometry as described previously (35) using the Cytofix/Cytoperm kit and PE-conjugated anti–IFN-γ (XMG1.2) or anti–TNF-α (MP6-XT22) mAb (all from BD Biosciences). For degranulation assays, resting DETCs preincubated with anti-CD16/CD32 mAb were stimulated for 4 h in the presence of GolgiStop (BD Biosciences) containing monensin and Alexa Fluor 488-conjugated anti-CD107a mAb (1D4B; eBioscience). After stimulation, viable cells were directly analyzed by flow cytometry.

For stimulating DETCs on the E-cadherin–coated plates, 96-well plates were coated overnight at 4°C with 1 μg/ml recombinant E-cadherin in PBS containing 1 mM CaCl2. Resting DETCs preincubated with anti-CD16/CD32 mAb were seeded on uncoated or E-cadherin–coated plates. Immediately after seeding or after culture for 24 h on the plates, DETCs were stimulated for 4 h with 1 μg/ml Con A in the presence of GolgiPlug for cytokine production assays or GolgiStop and Alexa Fluor 488-conjugated anti-CD107a mAb for degranulation assays.

For stimulating DETCs with fixed keratinocytes, Pam 212 keratinocytes (1 × 105) were cultured overnight in 96-well plates, and the keratinocyte monolayers were fixed for 15 min in methanol. Resting DETCs preincubated with anti-CD16/CD32 mAb were stimulated for 24 h with the fixed keratinocytes in the presence of 200 μg/ml anti–E-cadherin (ECCD-1) or isotype control mAb. GolgiPlug or GolgiStop and Alexa Fluor 488-conjugated anti-CD107a mAb were added during the last 4 h.

Differences between two groups were evaluated by t test. One-way ANOVA with post hoc multiple comparisons to a control (Dunnett’s procedure) was used to determine the significance of differences among three groups. Repeated-measures ANOVA was used to compare the cytotoxicity curves, with different E:T ratios serving as repeated measures. All reported p values are two-tailed, with a p value <0.05 considered significant. Statistical calculations were performed using JMP software (SAS Institute, Cary, NC).

Consistent with previous observations (4, 5, 14), both E-cadherin and αEβ7 integrin were expressed on freshly isolated DETCs (Fig. 1). Another receptor for E-cadherin, designated KLRG1, that has been identified as an inhibitory receptor expressed on subsets of NK cells and αβ T cells (36, 37) was not expressed on freshly isolated DETCs (Fig. 1). Besides αEβ7 integrin, low levels of αLβ2 integrin and integrin β1-chain, but not α4-chain, which also pairs with β7-chain, or αVβ3 integrin, were expressed on freshly isolated DETCs that exhibited the resting effector (CD62LlowCD25low) phenotype (Fig. 1).

FIGURE 1.

Freshly isolated DETCs express E-cadherin and αEβ7 integrin but not KLRG1. Freshly isolated epidermal cells were stained with indicated mAbs. Quadrant settings were determined by staining with isotype control mAbs. The percentages of cells for a given phenotype are shown. Representative profiles from five independent experiments are shown.

FIGURE 1.

Freshly isolated DETCs express E-cadherin and αEβ7 integrin but not KLRG1. Freshly isolated epidermal cells were stained with indicated mAbs. Quadrant settings were determined by staining with isotype control mAbs. The percentages of cells for a given phenotype are shown. Representative profiles from five independent experiments are shown.

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DETCs play a critical role in wound healing (10). When skin is wounded, DETCs in the periwound skin are rapidly activated through TCR-mediated recognition of unknown self-Ags induced on neighboring damaged keratinocytes and become round shaped (10). To determine whether the change in cellular shape of DETCs on activation in vivo is associated with alterations of E-cadherin or αEβ7 integrin expression, or both, we analyzed expression of these molecules on DETCs in the periwound skin by immunofluorescence staining of epidermal sheets. In contrast with the dendritic morphology of DETCs in the control skin, activated DETCs in the periwound skin became round 24 h after wounding (Fig. 2). E-cadherin expression on the rounded DETCs was downregulated as confirmed using two different mAbs that recognize extracellular (ECCD-2) and intracellular (clone 36) domains of E-cadherin (Fig. 2). Membrane staining of β-catenin was also reduced in the rounded DETCs (data not shown). Although αEβ7 integrin has a role for cellular shape of DETCs (32) and was reported to be downregulated on DETC activation (38), we could not find apparent downregulation of αEβ7 integrin on the rounded DETCs (Fig. 2). These results suggest that E-cadherin, but not αEβ7 integrin, is downregulated on activation of DETCs during wound healing. In this model, however, E-cadherin expression on the keratinocytes in the periwound skin also appeared to be diminished (Fig. 2). Therefore, it was unclear whether the downregulation of E-cadherin on rounded DETCs was directly associated with DETC activation or indirectly resulted from disruption of the homophilic E-cadherin binding caused by downregulation of E-cadherin on neighboring damaged keratinocytes.

FIGURE 2.

E-cadherin, but not αEβ7 integrin, is downregulated on activation of DETCs during wound healing. Full-thickness wound was created on the back skin. Epidermal sheets were prepared from the control skin distant from the wound and periwound skin 24 h after wounding, and double-stained with anti-TCR Vγ3 (red) and either anti–E-cadherin (clone ECCD-2 or 36) or anti-integrin β7-chain (green) mAbs. For each staining, five sheets were examined in three independent experiments, and representative fields are shown. Immunofluorescence stain, original magnification ×1100.

FIGURE 2.

E-cadherin, but not αEβ7 integrin, is downregulated on activation of DETCs during wound healing. Full-thickness wound was created on the back skin. Epidermal sheets were prepared from the control skin distant from the wound and periwound skin 24 h after wounding, and double-stained with anti-TCR Vγ3 (red) and either anti–E-cadherin (clone ECCD-2 or 36) or anti-integrin β7-chain (green) mAbs. For each staining, five sheets were examined in three independent experiments, and representative fields are shown. Immunofluorescence stain, original magnification ×1100.

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To determine whether downregulation of E-cadherin is intrinsic property of activated DETCs, we analyzed the expression levels of E-cadherin and αEβ7 integrin on short-term DETC lines before and after TCR stimulation in vitro. Expression of both E-cadherin and αEβ7 integrin, but not KLRG1, was also detected on resting DETC lines (Fig. 3A). Other integrins expressed on short-term DETC lines were similar to those on freshly isolated DETCs, except for the upregulation of αVβ3 integrin on resting DETCs (Fig. 3A). Although TGF-β upregulates αEβ7 integrin expression and modulates other integrins on resident intestinal IELs and peripheral T cells (1618, 22, 25, 29, 3941), TGF-β treatment did not affect expression of αEβ7 integrin or other integrins on short-term DETC lines (data not shown). After TCR stimulation, E-cadherin expression on DETCs was significantly downregulated (Fig. 3A, 3B). Downregulation of E-cadherin expression on activation of DETCs became detectable 10 min after TCR stimulation (data not shown), and the expression levels continued to decrease for 24 h. By contrast, activated DETCs showed no alteration in αEβ7 integrin expression (Fig. 3A, 3B). Therefore, DETCs downregulate E-cadherin, but not αEβ7 integrin, on activation, and the downregulation of E-cadherin is intrinsic property of activated DETCs.

FIGURE 3.

E-cadherin, but not αEβ7 integrin, is downregulated on activation of DETCs through TCR stimulation in vitro. A, Resting DETCs were stimulated for 24 h with 10 μg/ml immobilized anti-TCR mAb (activated DETCs). Resting and activated DETCs were stained with indicated mAbs (open histograms). Shaded histograms indicate cells stained with isotype control mAbs. Representative profiles from three independent experiments are shown. B, Relative geometric mean fluorescence intensity (MFI) was determined as geometric MFI of indicated mAb/geometric MFI of isotype control mAb. Data are expressed as the mean and SD of three experiments. Significant change in the relative geometric MFI on activation was evident only for E-cadherin (**p < 0.01).

FIGURE 3.

E-cadherin, but not αEβ7 integrin, is downregulated on activation of DETCs through TCR stimulation in vitro. A, Resting DETCs were stimulated for 24 h with 10 μg/ml immobilized anti-TCR mAb (activated DETCs). Resting and activated DETCs were stained with indicated mAbs (open histograms). Shaded histograms indicate cells stained with isotype control mAbs. Representative profiles from three independent experiments are shown. B, Relative geometric mean fluorescence intensity (MFI) was determined as geometric MFI of indicated mAb/geometric MFI of isotype control mAb. Data are expressed as the mean and SD of three experiments. Significant change in the relative geometric MFI on activation was evident only for E-cadherin (**p < 0.01).

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To determine the roles of E-cadherin–mediated homophilic binding and αEβ7 integrin-mediated heterophilic binding in DETC adhesion to keratinocytes, we tested inhibitory effects of a panel of mAbs including two different mAbs to E-cadherin, which selectively block homophilic binding between E-cadherin molecules (ECCD-1) and heterophilic binding of αEβ7 integrin to E-cadherin (ECCD-2) (27), on adhesion of DETCs to keratinocyte monolayers in vitro.

As reported previously (4, 5), blocking of the homophilic E-cadherin binding with anti–E-cadherin mAb ECCD-1 did not inhibit adhesion of DETCs to keratinocytes (Fig. 4). By contrast, blocking of the heterophilic binding of αEβ7 integrin to E-cadherin with anti–E-cadherin mAb ECCD-2, anti-integrin αE-chain mAbs, and anti-integrin β7-chain mAb inhibited DETC adhesion to keratinocytes (Fig. 4). Blocking mAbs to αLβ2 integrin had no effect (Fig. 4), and blocking mAbs to integrin β1- or β3-chains, or both, also failed to inhibit DETC adhesion to keratinocytes (data not shown). These results suggest that short-term adhesion of DETCs to keratinocytes in vitro primarily depends on heterophilic binding of αEβ7 integrin to E-cadherin but not on homophilic binding between E-cadherin molecules. Homophilic E-cadherin binding would not play a compensatory role for the DETC adhesion in this assay because anti–E-cadherin mAb ECCD-1 did not have an additional inhibitory effect on anti-integrin αE-chain mAb-mediated inhibition (Fig. 4).

FIGURE 4.

Short-term adhesion of DETCs to keratinocytes in vitro is mediated by heterophilic binding of αEβ7 integrin to E-cadherin but not by homophilic E-cadherin binding. CFSE-labeled resting DETCs were allowed to adhere for 1 h to the monolayers of Pam 212 keratinocytes in the presence of isotype control mAbs (filled bars) or indicated blocking mAbs (open bars). Adherent DETCs were counted under fluorescence microscopy, and data are expressed as mean adhesion and SD (%). Representative data from three independent experiments are shown. Significant inhibition of the adhesion as compared with control was evident with the anti–E-cadherin mAb ECCD-2 and mAbs to integrin αE- and β7-chains (*p < 0.05, **p < 0.01, ***p < 0.001). There was no significant difference between adhesion in the presence of anti-integrin αE-chain mAb (2E7) alone and anti–E-cadherin (ECCD-1) + anti-integrin αE-chain (2E7) mAbs (p = 0.766).

FIGURE 4.

Short-term adhesion of DETCs to keratinocytes in vitro is mediated by heterophilic binding of αEβ7 integrin to E-cadherin but not by homophilic E-cadherin binding. CFSE-labeled resting DETCs were allowed to adhere for 1 h to the monolayers of Pam 212 keratinocytes in the presence of isotype control mAbs (filled bars) or indicated blocking mAbs (open bars). Adherent DETCs were counted under fluorescence microscopy, and data are expressed as mean adhesion and SD (%). Representative data from three independent experiments are shown. Significant inhibition of the adhesion as compared with control was evident with the anti–E-cadherin mAb ECCD-2 and mAbs to integrin αE- and β7-chains (*p < 0.05, **p < 0.01, ***p < 0.001). There was no significant difference between adhesion in the presence of anti-integrin αE-chain mAb (2E7) alone and anti–E-cadherin (ECCD-1) + anti-integrin αE-chain (2E7) mAbs (p = 0.766).

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DETCs are potent CTLs against cutaneous tumor cells in vitro (42) and play an important role in cutaneous tumor surveillance in vivo (9). Although a crucial role for αEβ7 integrin in the killing of E-cadherin–expressing tumor cells by αEβ7 integrin-expressing CD8+ CTLs has been demonstrated (29), the role of αEβ7 integrin in the lysis of transformed keratinocytes by DETCs has not been determined.

DETCs could efficiently kill Pam 212 transformed keratinocytes, and the lysis was blocked with anti–E-cadherin mAb ECCD-2 and anti-integrin αE-chain mAb (Fig. 5). Anti-integrin αL-, β1-, or β3-chain mAb failed to inhibit the lysis (data not shown). Therefore, the lysis of Pam 212 keratinocytes by DETCs depends on αEβ7 integrin binding to E-cadherin. Anti–E-cadherin mAb ECCD-1, which blocks homophilic binding between E-cadherin molecules, could not be used in this assay because addition of this mAb disrupts homotypic intercellular adhesion between Pam 212 keratinocytes, leading to target cell death in the absence of DETC effector cells.

FIGURE 5.

Blocking of the interactions between αEβ7 integrin and E-cadherin inhibits the killing of keratinocytes by DETCs. Resting DETCs and Pam 212 keratinocytes were coincubated for 4 h at the indicated E:T ratios in the presence of isotype control or indicated blocking mAb. Data are expressed as mean specific lysis and SD (%). Representative data from three independent experiments are shown. Significant inhibition of the cytotoxicity as compared with control was evident with anti–E-cadherin and anti-integrin αE-chain mAbs (*p < 0.05, **p < 0.01).

FIGURE 5.

Blocking of the interactions between αEβ7 integrin and E-cadherin inhibits the killing of keratinocytes by DETCs. Resting DETCs and Pam 212 keratinocytes were coincubated for 4 h at the indicated E:T ratios in the presence of isotype control or indicated blocking mAb. Data are expressed as mean specific lysis and SD (%). Representative data from three independent experiments are shown. Significant inhibition of the cytotoxicity as compared with control was evident with anti–E-cadherin and anti-integrin αE-chain mAbs (*p < 0.05, **p < 0.01).

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The dependence of short-term adhesion of DETCs to keratinocytes in vitro on heterophilic binding of αEβ7 integrin to E-cadherin, but not on homophilic E-cadherin binding, indicates that formation of stable binding between E-cadherin molecules takes a longer time than binding of αEβ7 integrin to E-cadherin. To examine this possibility, we cultured DETCs on recombinant E-cadherin–coated slides instead of the keratinocyte monolayers because DETCs kill keratinocytes when coincubated for a prolonged period. We analyzed localization of E-cadherin, β-catenin, and αEβ7 integrin at the interface between adherent DETCs and E-cadherin–coated slide surface using confocal microscopy, rather than directly analyzing DETC adhesion, because DETCs can adhere to uncoated slides, as well as E-cadherin–coated slides.

Supporting the dominant role for αEβ7 integrin in short-term adhesion of DETCs to keratinocytes, αEβ7 integrin, but not E-cadherin or β-catenin, was detected at the bottom surface of DETCs 1 h after seeding on the E-cadherin–coated and uncoated slides (Fig. 6). Although DETCs adhered equally well to uncoated and E-cadherin–coated slides 24 h after seeding, only DETCs cultured on the E-cadherin–coated slides extended cellular protrusions, which is mediated by interactions of αEβ7 integrin with E-cadherin (32). Accumulation of E-cadherin at the bottom surface of DETCs was observed only when cultured on the E-cadherin–coated slides for 24 h (Fig. 6). Because β-catenin colocalized with E-cadherin in areas of contact between DETCs and E-cadherin–coated slides only at this time point (Fig. 6), stable binding of cell-surface E-cadherin on DETCs to plate-bound E-cadherin occurred after 24-h, but not 1-h, culture in vitro.

FIGURE 6.

Stable binding of cell-surface E-cadherin on DETCs to plate-bound E-cadherin occurs after 24-h, but not 1-h, culture in vitro. Resting DETCs were cultured for 1 or 24 h on uncoated or E-cadherin–coated slides. Adherent DETCs were stained with anti–E-cadherin mAb specific for the intracellular domain (clone 36), anti–β-catenin mAb, or anti-integrin β7-chain mAb and examined under confocal microscopy. Focus planes are on the bottom of cells. Representative fields of three independent experiments are shown. Immunofluorescence stain, original magnification ×1200.

FIGURE 6.

Stable binding of cell-surface E-cadherin on DETCs to plate-bound E-cadherin occurs after 24-h, but not 1-h, culture in vitro. Resting DETCs were cultured for 1 or 24 h on uncoated or E-cadherin–coated slides. Adherent DETCs were stained with anti–E-cadherin mAb specific for the intracellular domain (clone 36), anti–β-catenin mAb, or anti-integrin β7-chain mAb and examined under confocal microscopy. Focus planes are on the bottom of cells. Representative fields of three independent experiments are shown. Immunofluorescence stain, original magnification ×1200.

Close modal

We next examined functions of E-cadherin and αEβ7 integrin in activation of DETCs. Cross-linking of E-cadherin or αEβ7 integrin on DETCs alone with immobilized mAb did not induce IFN-γ or TNF-α production or exocytosis of cytotoxic granules, which was identified by externalization of the CD107a protein (Fig. 7). Interestingly, cocross-linking of E-cadherin on DETCs with immobilized anti–E-cadherin mAb inhibited cytokine production and degranulation in response to TCR cross-linking (Fig. 7). By contrast, coligation of αEβ7 integrin augmented DETC activation in response to stimulation with a suboptimal concentration (0.1 μg/ml), but not an optimal concentration (10 μg/ml), of immobilized anti-TCR mAb (Fig. 7). These results suggest that E-cadherin on DETCs acts as an inhibitory receptor, whereas αEβ7 integrin acts as a costimulatory receptor.

FIGURE 7.

Engagement of E-cadherin on DETCs with immobilized mAb inhibits cytokine production and degranulation in response to TCR cross-linking, whereas coligation of αEβ7 integrin enhances DETC activation in response to suboptimal TCR stimulation. Plates were coated with indicated concentrations of anti-TCR mAb and isotype control, anti–E-cadherin, or anti-integrin αE-chain mAb. Resting DETCs were stimulated for 4 h on the plates, and intracellular IFN-γ and TNF-α production, as well as cell-surface CD107a expression, were analyzed by flow cytometry. Data are expressed as mean positive cells and SD (%). Representative data from three independent experiments are shown. Significant increase or decrease in the cytokine production or degranulation by cocross-linking of E-cadherin or αEβ7 integrin as compared with control is denoted with asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 7.

Engagement of E-cadherin on DETCs with immobilized mAb inhibits cytokine production and degranulation in response to TCR cross-linking, whereas coligation of αEβ7 integrin enhances DETC activation in response to suboptimal TCR stimulation. Plates were coated with indicated concentrations of anti-TCR mAb and isotype control, anti–E-cadherin, or anti-integrin αE-chain mAb. Resting DETCs were stimulated for 4 h on the plates, and intracellular IFN-γ and TNF-α production, as well as cell-surface CD107a expression, were analyzed by flow cytometry. Data are expressed as mean positive cells and SD (%). Representative data from three independent experiments are shown. Significant increase or decrease in the cytokine production or degranulation by cocross-linking of E-cadherin or αEβ7 integrin as compared with control is denoted with asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

Close modal

To determine whether E-cadherin and αEβ7 integrin on DETCs also provide inhibitory and costimulatory signals when bound to E-cadherin, mitogen-induced cytokine production and degranulation were analyzed in DETCs immediately after seeding or after 24-h culture on the E-cadherin–coated plates. DETCs stimulated for 4 h with Con A immediately after seeding on the E-cadherin–coated plates showed augmented cytokine production and degranulation as compared with DETCs stimulated on uncoated plates (Fig. 8A). Because only αEβ7 integrin but not E-cadherin on DETCs is engaged with plate-bound E-cadherin at this time point (Fig. 6), the augmented responses are attributable to the costimulatory signal through αEβ7 integrin.

FIGURE 8.

Engagement of αEβ7 integrin on DETCs with plate-bound E-cadherin enhances mitogen-induced cytokine production and degranulation, whereas stable binding of cell-surface E-cadherin on DETCs to plate-bound E-cadherin inhibits their activation. A, Resting DETCs were seeded on uncoated or E-cadherin–coated plates and immediately incubated for 4 h in the absence or presence of Con A. B, Resting DETCs were precultured on uncoated or E-cadherin–coated plates for 24 h. Cells were then incubated for 4 h in the absence or presence of Con A. Intracellular IFN-γ and TNF-α production, as well as cell-surface CD107a expression, were analyzed by flow cytometry, and data are expressed as mean positive cells and SD (%). Representative data from three independent experiments are shown. Significant differences in the cytokine production or degranulation between DETCs stimulated on uncoated and E-cadherin–coated plates are denoted with asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 8.

Engagement of αEβ7 integrin on DETCs with plate-bound E-cadherin enhances mitogen-induced cytokine production and degranulation, whereas stable binding of cell-surface E-cadherin on DETCs to plate-bound E-cadherin inhibits their activation. A, Resting DETCs were seeded on uncoated or E-cadherin–coated plates and immediately incubated for 4 h in the absence or presence of Con A. B, Resting DETCs were precultured on uncoated or E-cadherin–coated plates for 24 h. Cells were then incubated for 4 h in the absence or presence of Con A. Intracellular IFN-γ and TNF-α production, as well as cell-surface CD107a expression, were analyzed by flow cytometry, and data are expressed as mean positive cells and SD (%). Representative data from three independent experiments are shown. Significant differences in the cytokine production or degranulation between DETCs stimulated on uncoated and E-cadherin–coated plates are denoted with asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

Close modal

In contrast, when DETCs were cultured for 24 h on the plates and then stimulated with Con A after the formation of stable binding between cell-surface E-cadherin on DETCs and plate-bound E-cadherin (Fig. 6), DETCs precultured on the E-cadherin–coated plates showed reduced responses as compared with DETCs precultured on uncoated plates (Fig. 8B). Thus, homophilic binding of E-cadherin on DETCs to plate-bound E-cadherin provides an inhibitory signal for mitogen-induced cytokine production and degranulation, and the inhibitory signal is dominant over the costimulatory signal through αEβ7 integrin in this assay condition.

It would be important to determine whether homophilic binding between E-cadherin molecules on DETCs and keratinocytes also provides an inhibitory signal in DETCs. When DETCs are coincubated with keratinocytes, however, lysis of keratinocytes by DETCs occurs within 4 h even in the presence of blocking mAbs that inhibit the binding of αEβ7 integrin to E-cadherin (Fig. 5), and the keratinocyte monolayers were completely destroyed within 18 h (data not shown). Because formation of stable binding between E-cadherin molecules on DETCs and keratinocytes would take a much longer time, we cultured DETCs on the fixed keratinocyte monolayers, which remained intact after 24-h coincubation (data not shown). Fixed keratinocytes stimulated cytokine production and degranulation by DETCs, though less efficiently than viable keratinocytes, when coincubated for 24 h (Fig. 9 and data not shown). Blocking of the homophilic binding between E-cadherin molecules with anti–E-cadherin mAb ECCD-1 augmented DETC activation in response to the fixed keratinocytes (Fig. 9). Therefore, E-cadherin on DETCs also acts as an inhibitory receptor through binding to E-cadherin on keratinocytes.

FIGURE 9.

Homophilic binding between E-cadherin molecules on DETCs and keratinocytes provides an inhibitory signal in DETCs. Resting DETCs were incubated for 24 h on uncoated plates or on the fixed monolayers of Pam 212 keratinocytes in the presence of isotype control or anti–E-cadherin (ECCD-1) mAb. Intracellular IFN-γ and TNF-α production, as well as cell-surface CD107a expression, were analyzed by flow cytometry, and data are expressed as mean positive cells and SD (%). Representative data from three independent experiments are shown. Significant augmentation of the cytokine production and degranulation in response to the fixed keratinocytes was evident with anti–E-cadherin mAb as compared with control (**p < 0.01, ***p < 0.001).

FIGURE 9.

Homophilic binding between E-cadherin molecules on DETCs and keratinocytes provides an inhibitory signal in DETCs. Resting DETCs were incubated for 24 h on uncoated plates or on the fixed monolayers of Pam 212 keratinocytes in the presence of isotype control or anti–E-cadherin (ECCD-1) mAb. Intracellular IFN-γ and TNF-α production, as well as cell-surface CD107a expression, were analyzed by flow cytometry, and data are expressed as mean positive cells and SD (%). Representative data from three independent experiments are shown. Significant augmentation of the cytokine production and degranulation in response to the fixed keratinocytes was evident with anti–E-cadherin mAb as compared with control (**p < 0.01, ***p < 0.001).

Close modal

We showed that E-cadherin, but not αEβ7 integrin, on activated and rounded DETCs in the periwound skin is downregulated in vivo. Because E-cadherin on short-term DETC lines is also downregulated on activation in vitro, E-cadherin expression on DETCs is intrinsically regulated depending on the activation state. It is possible, however, that the change in cellular shape of DETCs in the periwound skin is attributed to the downregulation of E-cadherin on keratinocytes rather than that on activated DETCs, because during wound healing, keratinocytes at wound margins undergo epithelial–mesenchymal transition and downregulate E-cadherin (43) as confirmed in this study. In addition to homophilic E-cadherin binding that controls cellular shape through connection to the actin cytoskeleton (2), heterophilic binding of αEβ7 integrin to E-cadherin also influences cellular shape (32). Therefore, the morphological change of DETCs during wound healing in vivo could result from disruption of E-cadherin–mediated homophilic binding and/or αEβ7 integrin-mediated heterophilic binding caused by downregulation of E-cadherin on DETCs, keratinocytes, or both.

We also showed that αEβ7 integrin expressed on DETCs mediates short-term adhesion and cytotoxicity to E-cadherin–expressing keratinocytes in vitro, and provides a costimulatory signal for cytokine production and degranulation in response to suboptimal TCR cross-linking and mitogen stimulation, similar to other intraepithelial T cells (14, 18, 2227, 29, 30). Therefore, αEβ7 integrin on DETCs may play critical roles in vivo for retention and effector functions of DETCs in the epidermis through binding to E-cadherin on keratinocytes. Although E-cadherin on DETCs is not involved in the short-term adhesion of DETCs to keratinocytes in vitro, our data suggest that this might be because of the delayed formation of stable homophilic binding between E-cadherin molecules on DETCs and keratinocytes as compared with heterophilic binding of αEβ7 integrin to E-cadherin. Alternatively, E-cadherin and αEβ7 integrin on DETCs could compete for binding to E-cadherin on keratinocytes, and high-affinity binding of αEβ7 integrin to E-cadherin might predominate over low-affinity binding between E-cadherin molecules in the short-term adhesion assay. This possibility is unlikely, however, because we found that homophilic E-cadherin binding cannot compensate for DETC adhesion to keratinocytes in vitro when interactions of αEβ7 integrin with E-cadherin are blocked. Stable homophilic E-cadherin binding may also contribute to retention of DETCs in the epidermis, but presumably in a different way from αEβ7 integrin-mediated rapid adhesion that regulates cell motility (32).

We have identified a novel role for E-cadherin as an inhibitory receptor on DETCs. Although, to our knowledge, this is the first demonstration of a role for E-cadherin as an inhibitory receptor on lymphocytes, inhibitory functions of E-cadherin in epithelial cell growth have been established. E-cadherin is a tumor-suppressor protein, and its loss of expression or function is associated with uncontrolled cell growth (1). Conversely, restoration of E-cadherin into epithelial cancer cells leads to an inhibition of proliferation. In normal epithelial cells, homophilic E-cadherin binding directly transduces a growth inhibitory signal (44) and has been postulated to be responsible for the phenomenon of contact inhibition. In addition to the inhibitory role for E-cadherin in epithelial cells, ligation of E-cadherin on Langerhans cells has been shown to inhibit their maturation and chemokine production (45, 46). Besides DETCs, murine intestinal IELs (47, 48) and a subset of mast cells (49) express E-cadherin, as well as αEβ7 integrin. Although in humans, E-cadherin expression has not been detected on intestinal IELs (26) or other lymphocytes, some human T cell neoplasms express N-cadherin (5054). Whether cadherins have inhibitory functions in these cells remains to be determined in future studies.

Signal transduction pathways that mediate inhibitory signals from E-cadherin have remained elusive. E-cadherin is an important regulator of the Wnt/β-catenin signaling pathway through binding of β-catenin, which is a key signal transducer in this pathway (1). The Wnt pathway does not appear to be involved in the E-cadherin–mediated inhibition of DETC activation, however, because cross-linking of E-cadherin on DETCs with immobilized anti–E-cadherin mAb, which induces an inhibitory signal, does not recruit β-catenin in areas of E-cadherin accumulation (unpublished data). The inhibition would not be mediated through segregation of TCR, other activating/costimulatory receptors, or intracellular signaling molecules away from the contact site by homophilic E-cadherin binding, because soluble mitogen-induced DETC activation can be inhibited by engagement of E-cadherin only on the bottom surface of cells with plate-bound E-cadherin. A number of signaling pathways other than the Wnt pathway can also be triggered by homophilic E-cadherin binding (1). Further studies are necessary to explore the signaling pathways that mediate the inhibitory signal through E-cadherin on DETCs.

Differential expression of E-cadherin on resting and activated DETCs and opposite signals provided in DETCs by engagement of E-cadherin and αEβ7 integrin, both of which bind to E-cadherin on keratinocytes, suggest that activation threshold of DETCs is fine-tuned by E-cadherin expression on DETCs or keratinocytes, or both, in vivo. DETC activation is regulated by the balance between positive and negative signals provided through various activating/costimulatory receptors including TCR, 2B4 (55, 56), NKG2D (9, 33, 57), and junctional adhesion molecule-like protein (JAML) (58), and inhibitory receptors including Ly49E, CD94-NKG2A (59), and CD200R (60). In normal skin, E-cadherin and αEβ7 integrin may also be involved in the regulation of activation threshold of DETCs through binding to E-cadherin on keratinocytes. Selective downregulation of an inhibitory receptor E-cadherin, but not a costimulatory receptor αEβ7 integrin, on activation of DETCs would augment activating signals and lead to exerting effecter functions. Because E-cadherin expression on keratinocytes is downregulated during wound healing (43), spongiotic dermatitis (61, 62), and tumor progression of transformed keratinocytes (63), these environmental insults and cellular stress may also alter the activation threshold of DETCs.

Notably, both E-cadherin and αEβ7 integrin are expressed on fetal thymic precursors of DETCs, and E-cadherin is also expressed on fetal thymic stromal cells (14, 64). DETC precursors undergo positive selection through TCR-mediated signals in the fetal thymus to maturate and migrate to the skin (65). Therefore, inhibitory and costimulatory signals provided through E-cadherin and αEβ7 integrin might also influence the thymic selection process of DETC precursors. In this context, analysis of thymic development of DETC precursors in integrin αE-chain–deficient mice will be needed to determine whether impaired development of DETC precursors is involved in the reduction of DETCs in this strain (31).

In summary, we have demonstrated that E-cadherin, but not αEβ7 integrin, is downregulated on activation of DETCs. Although both E-cadherin and αEβ7 integrin may contribute to retention of DETCs in the epidermis through binding to E-cadherin expressed on keratinocytes, their binding kinetics is different. Importantly, we have shown that E-cadherin on DETCs acts as an inhibitory receptor, whereas αEβ7 integrin costimulates DETC activation. Another adhesion molecule, junctional adhesion molecule-like protein (JAML) expressed on DETCs, has been identified to be an important costimulatory receptor for DETC activation through interactions with a tight junction protein, coxsackie and adenovirus receptor expressed on keratinocytes (58). Therefore, interactions between adhesion molecules expressed on DETCs and keratinocytes are not only involved in adhesion but also in regulating DETC activation by acting as costimulatory or inhibitory receptors. Together, these novel findings imply potential functions of other adhesion molecules in regulation of activation thresholds of DETCs, as well as other intraepithelial T cells.

We thank Tomoko Fukushige, Kanayo Gunshin, Narumi Sagara, and Nobue Uto for technical assistance, Hideo Yagita for providing mAbs, Michael P. Schön for helpful information on blocking mAbs, and Ayano Takeuchi for advice on stimulation assays using fixed keratinocytes.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • DETC

    dendritic epidermal T cell

  •  
  • IEL

    intraepithelial lymphocyte

  •  
  • KLRG1

    killer cell lectin-like receptor G1

  •  
  • MFI

    mean fluorescence intensity.

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The authors have no financial conflicts of interest.