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The Journal of Immunology, 1999, 163: 1592-1601.
Copyright © 1999 by The American Association of Immunologists

Lymphocyte Adhesion to Epithelia and Endothelia Mediated by the Lymphocyte Endothelial-Epithelial Cell Adhesion Molecule Glycoprotein1

Chi-Chang Shieh*,{ddagger}, Bhanu K. Sadasivan*, Gary J. Russell*,{dagger}, Michael P. Schön*, Christina M. Parker* and Michael B. Brenner2,*

* Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital, and {dagger} Combined Program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115; and {ddagger} Department of Pediatrics, National Cheng Kung University, Tainan, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Upon encountering the relevant vascular bed, lymphocytes attach to endothelial adhesion molecules, transmigrate out of circulation, and localize within tissues. Lymphocytes may then be retained at microanatomic sites, as in tissues, or they may continue to migrate to the lymphatics and recirculate in the blood. Lymphocytes also interact transiently, but with high avidity, with target cells or APC that are infected with microbes or have taken up exogenous foreign Ags. This array of adhesive capabilities is mediated by the selective expression of lymphocyte adhesion molecules. Here, we developed the 6F10 mAb, which recognizes a cell surface glycoprotein designated lymphocyte endothelial-epithelial cell adhesion molecule (LEEP-CAM), that is distinct in biochemical characteristics and distribution of expression from other molecules known to play a role in lymphocyte adhesion. LEEP-CAM is expressed on particular epithelia, including the suprabasal region of the epidermis, the basal layer of bronchial and breast epithelia, and throughout the tonsillar and vaginal epithelia. Yet, it is absent from intestinal and renal epithelia. Interestingly, it is expressed also on vascular endothelium, especially high endothelial venules (HEV) in lymphoid organs, such as tonsil and appendix. The anti-LEEP-CAM mAb specifically blocked T and B lymphocyte adhesion to monolayers of epithelial cells and to vascular endothelial cells in static cell-to-cell binding assays by ~40–60% when compared with control mAbs. These data suggest a role for this newly identified molecule in lymphocyte binding to endothelium, as well as adhesive interactions within selected epithelia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To localize to target organs, lymphocytes must circulate via the bloodstream and bind to regional endothelia to exit the vascular space into a specific tissue (1, 2, 3, 4). Homing to lymphoid organs is facilitated by high endothelial venules (HEV)3 that express specific homing receptors, known as addressins. Lymphocytes, in turn, express specific ligands for addressins. For example, naive peripheral T lymphocytes express L-selectin, which facilitates their extravasation in peripheral lymph nodes through an interaction with L-selectin ligands expressed on HEV, such as CD34 and GlyCAM-1, which constitute the peripheral lymph node addressin (PNAd) (5, 6). In contrast, memory T cells express additional adhesion molecules, such as the {alpha}4{beta}7 integrin, which recognizes mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expressed on HEV in Peyer’s patches and mesenteric lymph nodes. This {alpha}4{beta}7/MAdCAM-1 interaction is important in lymphocyte homing to mucosal sites (7, 8, 9). Similarly, the cutaneous lymphocyte Ag, expressed on a subset of lymphocytes, recognizes E-selectin on dermal vascular endothelium (10) and is thus thought to be involved in the homing of lymphocytes to the skin. Finally, activated lymphocyte trafficking in inflamed tissues is facilitated by other adhesion molecules, including ICAM-1 (11), VCAM-1 (12, 13), E-selectin (14), and P-selectin (15), which are up-regulated on inflamed endothelium. These endothelial molecules are recognized by the {alpha}L{beta}2 (LFA-1) and {alpha}4{beta}1 (VLA-4) integrins, which are expressed at increased levels on activated lymphocytes as well as by E- and P- selectin ligands. Combinations of these adhesion molecules confer specificity to leukocyte binding and extravasation in different organs constitutively or under inflammatory conditions. Once in tissues, lymphocytes must recognize target cells or professional APC, such as macrophages, to initiate immune responses. APC express adhesion molecules including the {alpha}L{beta}2 integrin, ICAM-1, 2, and 3, and LFA-3 (16, 17) that mediate adhesion with lymphocytes necessary for specific foreign Ag recognition and lymphocyte activation.

Lymphocyte adhesion within the epithelium also may be important in host defense. Except for those infectious agents that gain direct access to the body via trauma or arthropod vectors, most infectious microorganisms must interact with the mucosal or cutaneous epithelium to invade the host. Therefore, immune reactions in the epithelium are one of the first lines of defense against infections from the environment. The epithelium is also the origin of most adult cancers, such as of the breast, lung, colon, and uterine cervix (18). Lymphocytes in the epithelium may play important roles in both defending against infection and in tumor surveillance. Intraepithelial lymphocytes (IEL) represent a special subpopulation of lymphocytes, composed mainly of T cells that are resident in epithelial compartments. They occupy a unique anatomical site in direct contact with epithelial cells, enabling them to respond to infectious and malignant challenges within the epithelium. Due to the large surface area of epithelial organs, there are as many lymphocytes in the epithelium as in the organized peripheral lymphoid organs (19). Yet, little is known about the adhesive interactions between lymphocytes and epithelial cells. Recently, the first specific adhesion molecules mediating IEL adhesion to epithelium have been delineated. Intestinal IEL express the {alpha}E{beta}7 integrin (20, 21, 22, 23), which mediates specific adhesion to E-cadherin expressed on epithelial cells (24, 25). We have proposed that these molecules may mediate cell-to-cell interactions between T cells and epithelial cells that stabilize the retention of lymphocytes in the epithelium (24). Here, we describe a newly identified molecule that is expressed on selected epithelial cells and on endothelial cells. This molecule, designated lymphocyte endothelial-epithelial cell adhesion molecule (LEEP-CAM), is involved in the binding of lymphocytes to these tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

Epithelial cell lines included the breast epithelial cell line 16E6.A5 (provided by Dr. V. Band, Tufts University, New England Medical Center, Boston, MA) derived by immortalization of the 76N normal human mammary epithelial cell line through transfection of the human papilloma virus E6 and E7 genes (26, 27). The clone was propagated in DFCI-1 medium that consists of {alpha}-MEM/HAM nutrient mixture F12 (1:1 v/v) (Life Technologies, Grand Island, NY) supplemented with 12.5 ng/ml epidermal growth factor, 10 nM triiodothyronine, 10 mM HEPES, 50 µM freshly dissolved ascorbic acid, 1 nM {beta}-estradiol, 1 µg/ml insulin, 2.8 µM hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, 10 µg/ml transferrin, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 15 nM sodium selenite (all from Sigma, St. Louis, MO), 1 ng/ml cholera toxin (Schwatz/Mann, New York, NY) and 1% FCS (HyClone Laboratories, Logan, UT). A431 (28) (epidermal squamous cell carcinoma cell line), 293T (29) (transformed embryonic kidney cell line), T84 (30) (colon carcinoma), THP1 (31) (monocytic cell line), U937 (32) (histiocytic lymphoma), HL60 (33) (premyelocytic leukemia), and JY (human B lymphoblastoid cell line) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 (Life Technologies) medium containing 5% FCS.

Endothelial cell lines included human umbilical vein endothelial cells (HUVEC) (34) maintained in culture under standard conditions on 1% gelatin-coated flasks with 199 medium (Life Technologies) supplemented with 20% FCS, 90 µg/ml heparin (Sigma), and 20 µg/ml endothelial growth supplement (Sigma). We used 3rd-7th passage HUVEC for the adhesion assays and biochemical analysis described in this study. CDC/EU.HMEC-1 (HMEC-1) (35) was derived from microvascular endothelial cells from human foreskin and was grown in endothelial basal medium (Clonetics, San Diego, CA) supplemented with 2 mM L-glutamine, 12.5 ng/ml epidermal growth factor, 2.8 µM hydrocortisone, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 5% FCS. ECV304, available from ATCC, is a spontaneously transformed endothelial cell line derived from an umbilical cord (36) and was grown in 199 medium (Life Technologies) with 10% FCS.

Human intestinal intraepithelial T lymphocyte (iIEL) cell line 390I was derived from T cells isolated from intestinal epithelium as previously described (21, 37). The iIEL line was maintained in Yssel’s medium (38) containing 2 nM human rIL-2 (gift of Ajinomoto, Kawasaki, Japan), 4% FCS (HyClone), and 50 µM 2-ME (2-ME, Sigma) at 10% CO2. Long-term culture of the 390I iIEL line was maintained by intermittent restimulation with PHA (1:2000; Difco, Detroit, MI) and irradiated feeder cells (80% PBMC and 20% JY B lymphoblastoid cells).

monoclonal Abs

The 6F10 mAb was produced by immunizing BALB/cJ mice with the human breast epithelial cell line 16E6.A5. Three i.p. injections and a final i.v. injection of 2 x 107 cells were given at 3-wk intervals. Three days after the i.v. immunization, splenocytes were isolated and fused with P3X63Ag8.653 myeloma cells in the presence of 50% PEG 1500, as described previously (39, 40, 41). Hybridomas were selected with aminopterin-containing medium, and hybridoma supernatants were screened for their ability to block iIEL adhesion to the 16E6.A5 epithelial cell monolayer on 96-well plate adhesion assays (see below). Selected hybridomas were subcloned three times by limiting dilution, and ascites containing the mAb were produced by i.p. injection of the hybridoma cells into pristane-treated BALB/cJ mice. The isotype of the 6F10 mAb is IgM {kappa}.

Previously described mAbs used were NS.4.1 (mouse anti-sheep RBC, IgM), Ber-ACT8 (mouse anti-human {alpha}E{beta}7, IgG1) (42), E4.6 (mouse anti-human E-cadherin, IgG1) (24), TS 1/22 (mouse anti-human LFA-1, IgG1) (43), 4B4 (mouse anti-human {beta}1, IgG1) (44), W6/32 (mouse anti-human MHC class I, IgG2a) (40), OKT3 (mouse anti-human CD3, IgG2a), and 187.1 (rat anti-mouse Ig {kappa} chain, IgG1), available from ATCC.

Cell preparations

Human PBMC were isolated from heparinized whole blood by density separation over Ficoll-Hypaque (Pharmacia Chemicals, Uppsala, Sweden). Monocytes were separated from PBMC by incubating the PBMC in plastic tissue culture flasks for 1 h. The adherent cells were collected as blood monocytes.

CD4+ and CD8+ lymphocytes were purified from PBMC by magnetic cell sorting (Miltenyi Biotech, Hamburg, Germany). Briefly, 107 cells suspended in 80 µl PBS/5% FCS were incubated in 20 µl anti-CD4/CD8 mAb coupled to magnetic Biobeads (Miltenyi Biotech) for 20 min on ice. After washing once, the cells were passed through a column with strong magnetic field. After extensive washing, the column was removed from the magnetic field and the bound cells were eluted with 5 column volumes of PBS/5% FCS. The eluted cells were then subjected to flow cytometry analysis with the corresponding mAb. Purity of the cells obtained was routinely >90%. PHA blast T cells were generated by culturing PBMC or purified CD4+ or CD8+ T cells and irradiated feeder cells in the presence of 1:2000 PHA and rIL-2 for 2 wk. B cells were prepared from surgically obtained human tonsillar tissues kindly provided by Cheryl Greene of Massachusetts Eye and Ear Hospital (Boston, MA). Tonsillar lymphocytes were isolated by gently teasing tonsils in Levkowitz media (Life Technologies) over a fine wire mesh followed by density separation over Ficoll-Hypaque. B cells were then positively selected by magnetic cell sorting using CD19-magnetic beads (Miltenyi Biotech), as described above. Purity of the preparation was assessed by direct immunofluorescence using CD20-FITC and CD3-FITC Abs (Becton Dickinson, Mountain View, CA). Preparations typically contained 95–98% pure B cells. B cells were activated by culturing 2 x 106 B cells in 1:5000 v/v dilution of Staphylococcus aureus Cowan I strain (SAC; Pansorbin, Calbiochem, La Jolla, CA) and 500 nM human rIL-2 for 5 days at 37°C.

Adhesion assays

Static cell-to-cell adhesion assays were performed with modifications of previously described methods (20). Briefly, monolayers of 104 adherent cells were grown to confluence in 96-well flat-bottom tissue culture plates. Suspension cells were labeled with 25 µg of 2', 7'-bis-(2-carboxyethyl)-5 (and-6) carboxyfluorescein (BCECF-AM; Molecular Probes, Eugene, OR), dissolved in 5 µl of DMSO, and added to complete media for 30 min at 37°C. After washing with PBS, 40,000 labeled suspension cells were resuspended in 100 µl of adhesion medium (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2) with or without blocking Abs, added to each well of adherent cells and incubated at 37°C for 50 min. In Ab-blocking experiments, the adherent cells were incubated with 50 µl hybridoma culture supernatant, 1/250 dilution of ascites or 10 µg/ml of purified mAb for 30 min at 37°C before adding the suspension cells. mAb NS4.1 (noncell binding IgM) and W6/32 (cell binding IgG) were used as indicated in adhesion assays as nonblocking controls. Unbound cells were then washed from the plates with adhesion media by flicking (three to five washes), and the bound cells were detected as fluorescence units using fluorescence plate reader (IDEXX, Portland, ME). The fluorescence intensity and cell number form a linear relationship within the fluorescence detection range of the fluorescence plate reader. At least four replicates were performed in each experiment. Student’s t test was used to analyze the data obtained in adhesion assays.

Flow cytometry

Flow cytometry analysis was performed as previously described (45) using the FACSort flow cytometer (Becton Dickinson). Primary and secondary mAbs were used at saturating concentrations. Isotype-matched negative controls were used and W6/32 mAb (mouse anti-human MHC class I) was used as positive control. The mean fluorescence intensity (MFI) in negative controls was consistently <10 fluorescence units.

Immunohistochemistry

Tissue samples were mounted in OCT compound (Ames, Elkart, IN), snap frozen in liquid nitrogen, and stored at -70°C. Frozen tissue sections 4 µm thick were fixed in acetone for 5 min, air dried, and stained by an indirect immunoperoxidase method (46) using avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) and 3-amino-9-ethylcarbazole (Aldrich, Milwaukee, WI) as the chromogen. OKT3 mAb reactive with the human CD3{epsilon} molecule was used as a positive mAb in the immunohistochemical staining.

Immunoprecipitation

Epithelial and endothelial cells were surface radiolabeled with Na125I (DuPont-New England Nuclear, Boston, MA) as previously described (47). The cells were solubilized in lysis buffer containing TBS (pH 7.6) with 1% Triton X-100 (TX100), 0.5% sodium deoxycholate (DOC), 8 mM iodoacetamide, and 1 mM PMSF (Sigma) for 1 h. After centrifugation to remove insoluble debris, the lysates were precleared with 200 µl of 10% SAC (Pansorbin, Calbiochem, La Jolla, CA). Lysates from 3–5 x 106 cells were used in each immunoprecipitation. The lysates either were directly incubated with 100 µl of 10% (v/v) Ab-coupled Sepharose 4B beads (Pharmacia, Piscataway, NJ) or with 0.5 µl of ascites of the relevant Ab and 125 µl of culture supernatant from 187.1 hybridoma (rat anti-mouse {kappa} chain) followed by 100 µl of 10% (v/v) protein A-Sepharose (Pharmacia). The immunoprecipitates were washed five times with TBS containing 1% TX100, 0.5% DOC, and 0.2% SDS, eluted with sample buffer containing 10% glycerol, 3% SDS, and 5% 2-ME by boiling for 3 min, and analyzed by SDS-PAGE as described previously (48). For N-glycanase digestion, washed immunoprecipitates were resuspended in 50 µl of 30 mM Tris buffer (pH 7.6) containing 0.1% SDS and 0.1 M 2-ME. After boiling for 5 min, the samples were allowed to cool, following which, 5 µl of 10% TX100 and 0.3 Genzyme unit of N-glycanase (Genzyme, Cambridge, MA) were added. The reaction was allowed to proceed overnight at 37°C. The samples were then boiled in sample buffer and analyzed by SDS-PAGE. Signals were visualized using Kodak X-OMAT scientific imaging films.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a mAb inhibiting lymphocyte adhesion to epithelial cells

To identify adhesion molecules involved in lymphocyte binding to epithelial cells, we immunized BALB/cJ mice with the 16E6.A5 cell line derived from human breast epithelium and produced mAbs. The hybridoma supernatants were screened to identify those that blocked the binding of in vitro cultured T cells to 16E6.A5 epithelial cell monolayers in static cell-to-cell adhesion assays. One mAb, designated 6F10, stained the immunizing epithelial cell line and blocked the adhesion between T cells and epithelial cells effectively and was selected for further study. The 6F10 mAb ascites reproducibly blocked the binding of T cells to epithelial cell monolayers by ~60%, similar to the degree of blocking observed with anti-E-cadherin mAb, E4.6 (Fig. 1Goa). Previously, we have shown that T cell adhesion to epithelial cells can be mediated by the T cell integrin, {alpha}E{beta}7, and epithelial cell E-cadherin (24). To determine whether the 6F10 Ag was involved in {alpha}E{beta}7/E-cadherin adhesion, studies were performed using PHA blast T cells that lack significant levels of {alpha}E{beta}7 expression. As expected, when short-term PHA-stimulated T cell lines were examined, adhesion to epithelial cell monolayers was not blocked by mAb E4.6 against E-cadherin. Nevertheless, the 6F10 mAb still significantly blocked PHA blast T cell adhesion to epithelial cells by 50%, in comparison to negative control mAb NS.4.1 or W6/32 (Fig. 1Gob). Thus, 6F10 Ag-dependent adhesion between T cells and epithelial cells is distinct from that mediated through the {alpha}E{beta}7 integrin-E cadherin interaction.



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  		FIGURE 1. The 6F10 mAb blocks the binding between lymphocytes and epithelial cells. Adhesion assays were performed with 16E6.A5 epithelial cells as an adherent monolayer and either iIEL (a) or PHA blasts (b) as BCECF fluorescent-labeled suspension cells. NS.4.1 (isotype-matched nonbinding Ab), W6/32 (mouse anti-human MHC class I) were used as negative controls. E4.6 mAb, which binds to E-cadherin and inhibits the adhesion between the {alpha}E{beta}7 integrin and E-cadherin was used for comparison. Fluorescence units reflecting suspension cell binding to 16E6.A5 adherent cells are shown with error bars representing SDs. Experiments were performed with six replicates and repeated three times with similar results. One representative experiment is shown.

 
Tissue distribution of the 6F10 Ab staining

Flow cytometric analysis and immunoperoxidase tissue staining were used to determine the cellular distribution of the 6F10 Ag expression. First, a panel of cultured human cell lines was analyzed by flow cytometry. As shown in Table IGo, several epithelial-derived cell lines, including 16E6.A5 (breast origin), A431 (epidermal squamous cell carcinoma), and primary cultures of keratinocytes, were stained brightly with MFI of 448, 445, and 751, respectively. Other epithelial cell lines were stained weakly (T84) or were negative (293T). Cells of endothelial origin, including HUVEC (endothelial cell primary culture), ECV304, a spontaneously transformed HUVEC cell line, and HMEC-1, a transformed microvascular endothelial cell line, all stained with the 6F10 mAb with MFIs of 641, 165, and 69, respectively. Thus, several cell lines of endothelial or epithelial origin expressed the 6F10 Ag. In addition, platelets (MFI 161) and freshly isolated blood monocytes (MFI 308) were stained with the 6F10 mAb. Interestingly, these freshly isolated blood monocytes lost expression of the 6F10 Ag during 3 days of in vitro culture. All other cell lines of myelomonocytic or lymphocytic lineages lacked reactivity with the 6F10 mAb (Table IGo).


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  		Table I. FACS analysis of cell lines stained with 6F10 Ab

 
To evaluate the 6F10 Ag expression in vivo, immunoperoxidase staining of human tissue sections was performed. In this analysis, the 6F10 mAb stained the basal layer (B) of breast ductal epithelium (Fig. 2Goa), the suprabasal layer (Sb) of stratified epithelium in skin (Fig. 2Gob), the basal and suprabasal layers of tonsillar epithelium (Fig. 2Goc), the basal cells (B) of bronchiolar epithelium (Fig. 2God), and the vaginal and endometrial epithelia of the uterine cervix (data not shown). However, epithelial tissue expression of the 6F10 Ag was selective since the columnar epithelium (Ep) of intestine (Fig. 2Go, e and f) and the cuboidal epithelium of renal tubules were negative (data not shown). Endothelial expression was also noted with prominent staining of HEV endothelium in lymphoid tissues, such as appendix, tonsil (Fig. 2Go, g and h), mesenteric lymph node, and peripheral lymph node (data not shown). This staining was intense on the lumenal side of the HEV, where endothelial cell-lymphocyte interactions occur (Fig. 2Goh, arrow). Scattered cells with a dendritic appearance typical of tissue macrophages (M) or dendritic cells were stained in the lamina propria (Lp) just under the epithelium of the appendix and the large intestine (Fig. 2Go, e and f).



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  		FIGURE 2. Immunoperoxidase tissue staining with mAb 6F10. Human tissue sections including: breast ducts (a), skin (b), tonsil epithelium (c), bronchiole (d), appendix (e), and colon (f), and HEV in appendix (g) and tonsil (h) were stained with the 6F10 mAb, visualized by immunoperoxidase methods, and counterstained with hematoxylin. Note staining of the basal or suprabasal regions in various epithelia and in the HEV. Arrow in h indicates staining on the lumenal surface of HEV. The magnification was x400 (a–g) and x600 (h) in the original photograph. Digital images are shown. B, basal layer; D, dermis; Ep, epithelial cell; L, lumen; Lp, lamina propria; M, macrophage; Sb, suprabasal layer; Sc, stratum corneum

 
Because of the prominent staining on the suprabasal layers of epidermis, the 6F10 mAb staining in psoriasis, an autoimmune disorder of skin involving T cells, was evaluated (49, 50). In human psoriatic skin, the suprabasal layers of the thickened epidermis were prominently stained by the 6F10 mAb (Fig. 3Gob) with a distribution similar to that observed in normal human skin (Fig. 3Goa). While dermal blood vessel (Bv) endothelium in the normal skin was stained weakly, some HEV-like blood vessels were stained intensively in the dermis of psoriatic skin.



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  		FIGURE 3. Expression of the 6F10 Ag on normal and psoriatic skin. Samples from normal (a) and psoriatic (b) human skin were stained with the 6F10 mAb and visualized by immunoperoxidase methods and counterstained with hematoxylin. Dotted lines indicate the epithelial basement membrane. Note staining of the thickened epithelium and prominent blood vessels in the psoriatic lesion. The magnification was x200 in the original photograph. Bv, blood vessel.

 
The 6F10 mAb inhibits the adhesion of lymphocytes to endothelial cells

The 6F10 mAb was identified based on its ability to block T cell adhesion to epithelial cells. Since the 6F10 Ag also was expressed on endothelia (Table IGo and Fig. 2Go), adhesion assays between iIEL and monolayers of HUVEC were performed. The binding of lymphocytes and HUVEC is known to be mediated by several adhesion molecule-counterreceptor interactions, including LFA-1 ({alpha}L{beta}2)-ICAM-1 and -2, and VLA-4 ({alpha}4{beta}1)-VCAM-1. With these other adhesive interactions intact, the 6F10 mAb inhibited T cell-HUVEC adhesion by only 20%, compared with the level of adhesion seen using control mAb against MHC class I (Fig. 4Goa). The inhibition became more evident when the lymphocytes were preincubated with anti-LFA-1 mAb, TS1/22 (Fig. 4Gob) and was readily observed when the lymphocytes had been preincubated in the presence of both anti-LFA-1 mAb, TS1/22, and anti-{beta}1 integrin mAb, 4B4, with >50% inhibition of binding by the 6F10 mAb compared with control mAb (Fig. 4Goc). As expected, the mAb E4.6 against E-cadherin had no significant effects in these experiments, even in the presence of other anti-integrin Abs, as E-cadherin is not expressed by HUVEC. Thus, 6F10 Ag binding contributes to lymphocyte adhesion to endothelial as well as to epithelial cell substrates.



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  		FIGURE 4. The 6F10 mAb inhibits the binding of iIEL to endothelial cells. HUVEC were grown to confluence as a monolayer in 96-well plates and fluorescence-labeled iIEL were used as suspension cells in the adhesion assays. The assays were performed with adhesion buffer without mAb (a), buffer containing anti-LFA-1 mAb (TS1/22) (b), or buffer containing both anti-LFA-1 mAb (TS1/22) and anti-{beta}1 integrin mAb (4B4) (c). Fluorescence units reflecting suspension cell binding to adherent HUVEC in the presence of control and specific blocking mAb W6/32, E4.6, and 6F10 mAb are shown as means and SDs under conditions using the three buffers described in a–c. Experiments were performed with six replicates and repeated three times with similar results. One representative experiment is shown.

 
Divalent cation requirement for 6F10 mediated adhesion

We characterized the divalent cation requirements for the 6F10 Ag-mediated lymphocyte-epithelial cell adhesion and found that the 6F10 Ag-mediated binding is not dependent on Ca2+ or Mn2+. The 6F10 mAb blocked the binding of iIEL T cells to epithelial cell monolayers by ~60% when compared with control mAb in the presence of 1 mM Ca2+, Mg2+, and Mn2+ (Fig. 5Goa). Monoclonal Ab E4.6 against E-cadherin also blocked the binding of {alpha}E{beta}7+ iIEL T cells to 16E6.A5 cell monolayers to levels that were similar to that noted for the newly developed 6F10 mAb (Fig. 5Goa).



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  		FIGURE 5. 6F10-mediated adhesion is independent of the presence of Ca2+ and Mn2+. 3901 iIEL cell line was used as the suspension cells and the breast epithelial cell, 16E6.A5, monolayers were used as the adherent cells in a static cell to cell adhesion assay. In experiment a, normal medium (TBS containing 1 mM each of Ca2+, Mg2+, and Mn2+) was used in the incubation and washing steps. In experiment b, medium containing 1 mM Mg2+ and 25 mM EGTA was used. NS.4.1 (isotype-matched nonbinding Ab), W6/32 (mouse anti-human MHC class I Ab), and E4.6 (anti-E-cadherin Ab) were used as control Abs. Fluorescence units reflecting suspension cell binding to 16E6.A5 adherent cells are shown with error bars representing SDs. Each bar represents a mean of six replicates. The experiment was repeated twice with similar results.

 
Static adhesion assays between 16E6.A5 epithelial cells and iIEL were also performed in medium without Ca2+ and Mn2+. To ensure the integrity of epithelial cell monolayers, 1 mM of Mg2+ was added to the adhesion medium along with 25 mM of EGTA, which has a 105-fold greater affinity for Ca2+ than for Mg2+. In the absence of Ca2+ and Mn2+ in the adhesion medium, the blocking effects of the anti-E-cadherin mAb E4.6 decreased from 55 to 0% (Fig. 5Go, a and b; E4.6 compared with W6/32), as expected based on the requirements for activation of integrin {alpha}E{beta}7 by Mn2+ and E-cadherin for calcium in adhesion. In contrast, blocking by the 6F10 mAb was not significantly affected by the removal of Ca2+ and Mn2+. Blocking was 60 and 50% in the presence and absence of Ca2+ and Mn2+ (Fig. 5Go, a and b; 6F10 compared with W6/32). Thus, the adhesion mediated by the 6F10 Ag, in contrast to the adhesion mediated by {alpha}E{beta}7-E-cadherin, was not dependent on the presence of Ca2+ and Mn2+. Assays to determine the role of Mg2+ in adhesion were not conclusive, since the monolayer of epithelial cells that served as the adhesion substrate was not adequately maintained in the absence of Mg2+.

Leukocyte subpopulations that express the 6F10 counterreceptors

The counterreceptor for the 6F10 Ag has not yet been determined. To identify the cells that can bind to epithelial cells through 6F10 Ag recognition, several cell types were tested as suspension cells in adhesion assays using 16E6.A5 epithelial cell monolayers as adherent cells. The cells tested included iIEL, PBL, PHA-stimulated T cell blasts (PHA blasts) and their CD4+ or CD8+ subsets, freshly isolated and activated B cells, and polymorphonuclear cells (PMN).

iIEL and PHA blast T cells bind 16E6.A5 cells in a 6F10-dependent manner (Fig. 1Go). To determine whether freshly isolated PBL also were capable of binding 16E6.A5 epithelial cells in a 6F10-dependent manner, monocyte-depleted PBMC were used as the suspension cells in the adhesion assays. In comparison with the paired experiment with iIEL in which 60% of the binding could be blocked by the 6F10 mAb (data not shown), fresh PBL binding could be blocked by only ~10% with the 6F10 mAb (Fig. 6Goa; 6F10 and W6/32, p > 0.05). CD4+ and CD8+ subpopulations of freshly isolated PBL were also tested for 6F10 Ag-mediated binding in adhesion assays. Both CD4+ and CD8+ PBL showed minimal 6F10 mAb blockable adhesion (data not shown), indicating that neither the whole population of fresh PBLs nor the CD4+/CD8+ subpopulations of PBL had significant 6F10 mAb blockable binding to epithelial cells. Similarly, freshly isolated PMN also showed no blockable adhesion to the epithelial cells (Fig. 6Gob) when compared with the 60% blocking in a paired experiment with iIEL as the suspension cells (data not shown).



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  		FIGURE 6. Expression of 6F10 counterreceptor on leukocyte subpopulations. Static adhesion assays between 16E6.A5 cells and PBL (a), PMN (b), CD4+ PHA blast T cells (c), CD8+ PHA blast T cells (d), freshly isolated tonsillar B cells (e), and activated tonsillar B cells (f) were performed to test the blocking effect of the 6F10 mAb. Each bar represents the mean of six replicates in the adhesion assay and each error bar represents one SD. The result of one experiment is shown. The experiments were repeated at least three times with similar results.

 
Since PHA-activated PBL adhesion to epithelial cells was 6F10 Ag-dependent (Fig. 1Go), we examined potential differences in the CD4+ or CD8+ subpopulations of PHA blast T cells in adhesion to epithelial cells. Both the CD4+ and CD8+ PHA-stimulated lymphoblasts bound epithelial cells similar to the mixed population of PHA blasts and could be blocked with the 6F10 mAb by ~50% when compared with blocking with control mAb (Fig. 6Go, c and d). Thus, the 6F10 Ag-mediated binding contributes comparably to CD4+ and CD8+ populations of PHA blasts in binding to epithelial cells.

B cells also were tested for their ability to bind 16E6.A5 epithelial cells. Although slight decreases in the binding of freshly isolated B cells to 16E6.A5 epithelial cells were seen in the presence of the blocking 6F10 mAb, these differences were not significant when compared with mAb NS.4.1, the isotype-matched control, or mAb w6/32, the cell-binding control (Fig. 6Goe). However, B cells activated with the B cell-specific mitogen, formalin-treated SAC, bound 16E6.A5 cells in a 6F10-dependent manner (Fig. 6Gof), such that the binding could be blocked with the 6F10 mAb by 40% when compared with blocking with control mAbs. Similarly, binding of B lymphoblastoid cell lines to 16E6.A5 cells was also blocked by the 6F10 mAb (data not shown).

Thus, PHA lymphoblasts, activated B cells, as well as iIEL cell lines bind epithelial cells in a 6F10-dependent fashion that is independent of adhesion mediated through the {alpha}E{beta}7 integrin-E-cadherin interaction. The suspension cells (iIEL, PBL, PHA blasts, B cells, PMN) tested in these adhesion assays did not express the 6F10 Ag themselves as seen by flow cytometric analysis (Table IGo) and, therefore, presumably express a heterophilic counterreceptor for the 6F10 Ag.

The 6F10 mAb immunoprecipitates an N-glycanase-sensitive molecule distinct from other known cell adhesion molecules

After cell surface labeling with 125I, 16E6.A5 epithelial cells, or HUVEC, were solubilized in TBS containing 1% TX100 and 0.5% DOC, immunoprecipitated with the 6F10 mAb, and resolved in SDS-PAGE (Fig. 7Goa). The immunoprecipitated radiolabeled species resolved as a major broad band having a mean relative mobility of 105 kDa from epithelial cells (Fig. 7Goa, lane 3, bracket A) and 100 kDa and 145 kDa from endothelial cells (Fig. 7Goa, lane 6, brackets B and C). After treatment with N-glycanase, the radiolabeled species from epithelial cells (105 kDa; Fig. 7Gob, lane 1, bracket D) decreased in apparent m.w. to ~65 kDa (Fig. 7Gob, lane 2, bracket E) with several more weakly labeled species, the smallest of which was 55 kDa (Fig. 7Gob, lane 2, arrow head). The apparent m.w. of immunoprecipitates were not changed after O-glycanase digestion (data not shown).



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  		FIGURE 7. The 6F10 mAb recognizes an N-glycanase-sensitive protein. Immunoprecipitation using the 6F10 mAb was conducted from 125I surface-labeled cell lines, resolved by SDS-PAGE and visualized by autoradiography. a, The mAb 6F10 immunoprecipitation from cell lysates of epithelial cells (16E6.A5 breast epithelial cell line) and endothelial cells (HUVEC). Lanes 1–3, Immunoprecipitates with NS.4.1 mAb (isotype-matched control Ab), W6/32 mAb (mouse anti-human MHC class I), and the 6F10 mAb, respectively, from epithelial cells. Lanes 4–6, Immunoprecipitates from endothelial cells using the same panel of Abs. b, The 6F10 immunoprecipitate from epithelial cells after N-glycanase digestion. Lanes 1 and 2, Immunoprecipitates with the 6F10 mAb. Lanes 3 and 4, W6/32 mAb. Lanes 2 and 4, N-glycanase-digested immunoprecipitates with the 6F10 and W6/32 mAbs, respectively. a, Resolved in 5–15% gradient SDS-PAGE; b, Resolved on 7.5% SDS-PAGE. Both panels were resolved under reducing conditions. N-ase: N-glycanase.

 
Based on these biochemical studies, the 6F10 Ag appears to be a glycoprotein containing ~40 kDa of asparagine (N)-linked additions. These biochemical features and the prominent expression on selected epithelia and endothelia distinguishes the 6F10 Ag from other known cell adhesion molecules to which lymphocytes bind. Based on its prominent expression on both endothelia and epithelia and its role in lymphocyte adhesion to these cell types, the 6F10 Ag was designated LEEP-CAM.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have developed the 6F10 mAb against an adhesion molecule called LEEP-CAM, that is expressed prominently on selected epithelial tissues, HEV, endothelial cells, fresh monocytes, and platelets. The pattern of expression of LEEP-CAM and its biochemical features distinguish it from other cell adhesion molecules known to mediate lymphocyte adhesion. Immunoprecipitation and SDS-PAGE analysis of radiolabeled cells revealed that LEEP-CAM consists of 90–115 and 145 kDa species containing N-glycanase-sensitive asparagine (N)-linked glycans (Fig. 7Go, a and b) but no O-glycanase-sensitive modification. These N-linked glycans might function in the peptide folding, intracellular targeting or as an important epitope recognized in the adhesion function of LEEP-CAM. In repeated experiments, weaker bands of 90, 160, and 180 kDa were also visualized. The broad range of mobilities of LEEP-CAM polypeptides noted using endothelial cells and epithelial cells in the immunoprecipitation experiments could be due to variable posttranslational carbohydrate modification, multiple peptide backbones, or alternative splicing of a single gene product. Preliminary data from peptide mapping suggested that predominant radiolabeled species from epithelial and endothelial cells shared many of the same peptides (data not shown). However, the nature of the additional species has not been determined.

Both O- and N-linked glycans have been found to play key roles in the leukocyte adhesion of other molecules (51, 52) and are useful in identifying molecules important in lymphocyte binding to tissue-specific endothelial receptors. Given the glycosylation of LEEP-CAM and its prominent expression on the HEV, we considered its possible relationship to known mucosal addressins. The PNAd is important for the tethering and rolling of leukocytes on the endothelium through the calcium-dependent adhesive interaction mediated by L-selectin, a C-type lectin present on resting T cells and neutrophils (53, 54, 55). MECA 79 (5), a rat IgM Ab that recognizes PNAd in mice, immunoprecipitates a series of molecules with different polypeptide backbones, including CD34 (56) and GlyCAM-1 (57), with m.w. ranging from 40 to 200 kDa. LEEP-CAM is distinct from the PNAd, since PNAd molecules are mainly O-glycosylated, while LEEP-CAM is N-glycosylated and is resistant to O-glycoprotease digestion (data not shown). In addition, the anti-LEEP-CAM mAb blocked the binding of L-selectin-negative cell lines to epithelial cells (Fig. 1Goa), demonstrating that LEEP-CAM must be interacting with a molecule other than L-selectin to mediate lymphocyte-epithelial cell adhesion. Like LEEP-CAM, CD44 is expressed on many cell types, including epithelial cells and endothelial cells, and is expressed with many apparent m.w., due to extensive alternative splicing and variable glycosylation, including proteoglycan forms, which are decorated with glycosaminoglycans (58, 59). However, LEEP-CAM is resistant to heparitinase and chondroitinase ABC digestion and cannot be labeled with 35S sulfate, distinguishing it from known proteoglycans (data not shown). CD44 and LEEP-CAM immunoprecipitation from the same cell sources and side-by-side SDS-PAGE analysis did not reveal comigrating species (data not shown). LEEP-CAM was also distinct biochemically from MAdCAM-1, based on each displaying distinct Mr on SDS-PAGE analysis (9, 60). Finally, cutaneous lymphocyte Ag, a counterreceptor of E-selectin, is a lymphocyte cell-surface glycoprotein mediating tissue-specific trafficking of skin homing lymphocytes (10, 61, 62). However, E-selectin is known to be expressed only on endothelial cells (63) and thus must be distinct from LEEP-CAM, which is also prominently expressed on various epithelia (Fig. 2Go). Thus, although glycosylation makes up a large component of LEEP-CAM, it appears distinct from other glycosylated cell adhesion molecules expressed on activated endothelium or on HEV. While adhesion to HEV was not examined directly here, we hypothesize that like MAdCAM-1 and PNAd molecules, LEEP-CAM may play a role in lymphocyte homing by binding to HEV and flat endothelium at sites of inflammation as noted in psoriasis and tonsil samples.

A notable feature of LEEP-CAM was its prominent but selective expression on epithelia. In contrast to the many well-defined examples of leukocyte adhesion to endothelium, relatively few studies have addressed the adhesion of immune cells in the epithelium. Of the human epithelial adhesion molecules known to mediate adhesion to lymphocytes, only E-cadherin displays highly restricted expression at this location. Previously, we described the binding of E-cadherin by {alpha}E{beta}7 integrin expressed on iIEL at wet mucosal sites (24). Of the known cellular adhesion molecules in the epidermis, most are expressed only at the basal layers (e.g., ICAM-1 (66) and {beta}1 integrins (67)). In contrast, LEEP-CAM is expressed specifically in the suprabasal layer of the epidermis. LEEP-CAM expression also was found on psoriatic epidermis and in psoriatic vascular endothelium (Fig. 3Go). Thus, it is possible that LEEP-CAM contributes to the extravasation and epidermotropism of T lymphocytes during the pathogenesis of psoriasis (49, 68). Prominent staining of the basal cells in the mammary duct also was observed. This finding corresponds with the origin of the papilloma virus E6/E7 gene-transformed breast epithelial cell line 16E6.A5, against which the 6F10 Ab was made (64). The Ab also stained the entire tonsillar epithelium, which is often infiltrated by lymphocytes and which overlies the tonsillar lymphoid tissues (65).

Lymphocytes home to specific tissues through binding to a distinct combination of adhesion molecules on the vascular endothelium, following which they extravasate into the target tissue (4). However, it is less well understood how the circulating lymphocytes destined for various epithelia migrate to and bind their target epithelia. The finding that LEEP-CAM is present on both HEV and selected epithelia suggests a potential mechanism linking lymphocyte exit from the circulation and epithelial adhesion. For example, LEEP-CAM expression on HEV or flat endothelium could contribute to the extravasation of the lymphocytes into tissues in conditions like psoriasis. After extravasation, the presence of LEEP-CAM on the epithelium could have two functions. First, LEEP-CAM might anchor or retain the lymphocytes once they enter the epithelium. Second, the binding mediated by the LEEP-CAM might activate or induce the expression of other adhesion molecules, such as {alpha}E{beta}7 or {alpha}L{beta}2 integrins (43), which might further mediate lymphocyte epithelial adhesion. Thus, the LEEP-CAM/lymphocyte interaction may support immune responses in the epithelium or stabilize the localization of lymphocytes within the epithelium, such as the IEL, which may be important for immune surveillance against epithelial infections, tumors, or injury. Because LEEP-CAM is selectively absent from intestinal and renal epithelia, the molecular mechanism involving LEEP-CAM may facilitate the trafficking of lymphocytes into selected epithelia, such as skin, tonsil, bronchiole, vagina, and breast. Future studies will aim to define the molecular structure of LEEP-CAM and its counterreceptor. As a newly identified molecule mediating lymphocyte adhesion to endothelia and epithelia, LEEP-CAM should provide new insights into how lymphocytes traffic to and within organs to mediate host defense.


   Acknowledgments
 
We thank Cheryl Greene, Rachael Clark, and Dr. Michael Detmar for providing tissue samples; Dr. Martin Hemler, Dr. Edmond Yunis, Dr. Jonathan Higgins, and Karen Taraszka for critical reading of the manuscript; and Dr. Sam T. Hwang for helpful discussions.


   Footnotes
 
1 This work was supported by grants from National Institutes of Health (to M.B.B. and C.M.P. (First Award GM 49342)); a Pilot Feasibility Grant from The Center for the Study of Inflammatory Bowel Disease (DK 43351) (to G.J.R.); a Pilot Feasibility Grant from Harvard Skin Disease Research Center at Brigham and Women’s Hospital and an Investigator Award from the Cancer Research Institute (to C.M.P.); grants from the Crohn’s and Colitis Foundation of America (to G.J.R. and C.M.P.); a grant from the Deutsche Forschungsgemeinschaft (to M.P.S.); and a postdoctoral fellowship from the Arthritis Foundation (to B.K.S.). Back

2 Address correspondence and reprint requests to Dr. Michael B. Brenner, Smith Building Room 522, One Jimmy Fund Way, Boston, MA 02115. E-mail address: Back

3 Abbreviations used in this paper: HEV, high endothelial venules; PNAd, peripheral lymph node addressin; MAdCAM, mucosal addressin cell adhesion molecule; IEL, intraepithelial lymphocyte; LEEP-CAM, lymphocyte endothelial-epithelial cell adhesion molecule; HUVEC, human umbilical vein endothelial cell; iIEL, intestinal IEL; SAC, Staphylococcus aureus Cowan I strain; MFI, mean fluorescence intensity; PMN, polymorphonuclear cell. Back

Received for publication September 11, 1998. Accepted for publication May 12, 1999.


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