Human leukocyte-associated immunoglobulin-like receptor (LAIR)-1 is expressed on many cells of the immune system and is predicted to mediate inhibitory functions based on the presence of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic domain. Although the role of LAIR-1 in the regulation of immune responses in vivo is unknown, LAIR-1 cross-linking by monoclonal antibody inhibits various immune cell functions in vitro. Here, we identify the coloncarcinoma-associated epithelial cellular adhesion molecule (Ep-CAM) as a ligand for LAIR-1 and LAIR-2, a related soluble LAIR-1 family member. Ep-CAM interacts with the LAIR molecules through its first epidermal growth factor domain; Ep-CAM–specific antibodies can abrogate the binding. Intraepithelial T lymphocytes express LAIR-1 and thus may interact with Ep-CAM present on human intestinal epithelium. We propose that LAIR-1–Ep-CAM interaction may contribute to mucosal tolerance and that LAIR-2 possibly modulates this function.
Inhibitory receptors, bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), play a pivotal role in balancing the immune response (for a review, see reference 1). Rather than relaying activation signals, these receptors suppress cellular functions. One or more ITIMs in the cytoplasmic tail are responsible for the inhibitory signal upon ligation of the receptors by their ligand. ITIMs are short amino acid motifs with the consensus sequence I/V/L/SxYxxL/V, which are able to recruit cytoplasmic protein tyrosine phosphatases upon phosphorylation of the central tyrosine residue. These phosphatases abrogate signaling through activating receptors, thereby preventing cellular immune functions such as cytotoxicity or proliferation. In recent years, many novel inhibitory receptors have been identified and recognized to be necessary for the immune system to prevent excessive activation or autoimmunity.
The leukocyte-associated Ig-like receptor (LAIR)-1 2 is a member of the Ig superfamily that is expressed on the majority of peripheral blood mononuclear cells, including NK cells, T cells, B cells, monocytes, and dendritic cells, as well as the majority of thymocytes. Cross-linking of LAIR-1 by mAb in vitro delivers a potent inhibitory signal that is capable of inhibiting cellular functions of NK cells, effector T cells, B cells, and dendritic cell precursors 2,3,4,5. In addition to LAIR-1, we identified LAIR-2, a putative secreted protein that is 84% homologous to LAIR-1 3.
LAIR-1 has two ITIM motifs and is structurally related to human killer cell Ig-like receptors (KIRs) and the immunoglobulin-like transcripts (ILTs/LIRs). The KIRs and some of the ILTs recognize MHC class I and are thought to play a role in the prevention of autoimmunity (for a review, see reference 6). Many of the ITIM-bearing inhibitory receptors are members of multigene families that contain genes encoding activating receptors. The activating isoforms are characterized by having short cytoplasmic domains and basic amino acids within their transmembrane regions. These receptors signal through their association with immunoreceptor tyrosine-based activating motif (ITAM)-bearing transmembrane adaptor molecules, such as FcεRIγ or DAP12 7,8.
Within the family of ITIM-bearing receptors, LAIR-1 is unique because it is extremely broadly expressed and does not recognize MHC class I 2. Furthermore, sequencing of the genomic region where the LAIR genes are located suggests that there are only two LAIR genes, neither of which is predicted to encode an activating receptor 9. To delineate the biological function of LAIR-1, identification of the natural ligand is imperative. We here report the identification of epithelial cellular adhesion molecule (Ep-CAM) as a binding partner for LAIR.
Materials And Methods
293T cells were provided by T. Kitamura (DNAX Research Institute). HT29 cells were obtained from American Type Culture Collection.
The mouse anti–LAIR-1 mAb DX26 was described previously 2. The 8A8 (IgG1) producing hybridoma was generated by fusing the Sp2/0 myeloma cell line with splenocytes from a BALB/c mouse immunized with purified LAIR-1 protein. 323/A3 is a mouse anti–human Ep-CAM mAb 10 and UBS54 is a human anti–human Ep-CAM mAb isolated from a phage library, as described previously 11.
Detection of LAIR-1 Ligand.
A chimeric protein composed of the leader sequence and the extracellular part of LAIR-1 (amino acids 1–162) fused to the Fc region of human IgG1 was inserted into the pCDNA3.1 vector. The protein, designated LAIR-1-hIg, was produced by transient expression in 293T cells and subsequent purification by affinity chromatography on protein A sepharose columns. Cell lines were screened for the presence of a putative LAIR-1 ligand by assaying for binding of the LAIR-1-hIg. 106 cells were incubated at room temperature (RT) for 30 min with 30 μl containing 5 μg LAIR-1-hIg, 1% BSA, 2% FCS, and 2% normal mouse serum. Upon washing, 10 μg/ml biotin-conjugated goat anti–human-IgG1 (Caltag Laboratories) was added for 15 min at RT, followed by washing and 15 min incubation with phycoerythrin-conjugated streptavidin. Cells were assayed on a FACSCalibur™ with the addition of propidium iodide to exclude dead cells. As control IgG, either 2% pooled human serum (HPS) or a mouse CTLA4-hIg protein was used, both giving similar results.
Cloning of LAIR-1 Ligand.
The colorectal carcinoma cell line HT29 was found to highly express LAIR-1 ligand as assayed by LAIR-1-hIg binding. A cDNA library from this cell line was constructed into the pCDNA3.0 vector using oligo-dT–primed cDNA. cDNA cloning by transient transfection into 293T was performed as described 12 with modifications 13. Four independent cDNA clones were obtained and sequenced.
Generation of Ep-CAM Deletion Mutants.
Deletion mutants of the human Ep-CAM cDNA were constructed by using PCR. PCR fragments of the extracellular domain of Ep-CAM were cloned in frame into a pCDNA3.1 vector with an NH2-terminal Ep-CAM leader sequence, a COOH-terminal transmembrane region, and an intracellular domain of Ep-CAM, followed by a Myc epitope tag. All constructs were confirmed by nucleotide sequencing. After transfection into 293T cells, expression of the protein was checked by Western blotting using an anti-Myc mAb. Membrane expression and transfection efficiency was monitored by staining of methanol-fixed transfected cells with anti-Myc mAb.
Isolation and Staining of Intraepithelial Lymphocytes.
Intraepithelial lymphocytes (IELs) were isolated from the colon from donors that underwent partial colon resection because of malignancies. Unaffected parts of the colon were used to isolate IELs as described previously 14. Cells were stained immediately after isolation with anti-CD3 and anti–LAIR-1 Abs and analyzed by flow cytometry. All tissues were handled according to the guidelines of the institutional review board of the University Medical Center Utrecht on the use of human subjects in medical research.
Human ileum sections were snap frozen in liquid nitrogen and stored at −70°C. Frozen sections (6 μm) were cut, mounted on glass slides, dried at RT, and fixed in 3.7% formaldehyde in PBS at RT for 10 min. The sections were washed with PBS containing 1.5% glycine and incubated with biotin-conjugated UBS54 (anti–Ep-CAM) and a mixture of anti–LAIR-1 mAb DX26 and 8A8 for 1 h at RT. After washing, sections were incubated with tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti–mouse-Ig Abs and FITC-conjugated streptavidin for 1 h at RT. Samples were counterstained with 4′,6′-diamino-2-phenylindole (DAPI) and mounted in Vectashield anti-fade mounting medium (Vector Laboratories).
Results And Discussion
Ep-CAM Is a LAIR-1 Ligand.
To identify the natural ligand for LAIR-1 we constructed a fusion protein of the extracellular domain of LAIR-1 and the Fc portion of human IgG1 (LAIR-1-hIg). This protein was used as a staining reagent to screen cell lines. Human colon carcinoma cell lines bound this fusion protein but not a control Ig fusion protein and thus expressed a putative LAIR-1 ligand (LAIR-1L). The human colon carcinoma line HT29 displayed high LAIR-1L expression (Fig. 1 A). mRNA from HT29 cells was used to construct a cDNA expression library, and expression cloning was performed. We obtained four individual cDNAs that encoded a protein (LAIR-1L) that bound the LAIR-1-hIg (Fig. 1 B). Sequencing revealed that the cDNA of all four clones encoded full-length Ep-CAM (also known as EGP-2, GA733, CO17-1A, KSA, ESA, EGP40). Ep-CAM was indeed abundantly present on the surface of HT29 cells (Fig. 1 C). The identity of the protein encoded by the LAIR-1L cDNA was confirmed by staining of cells transfected with this cDNA with Ep-CAM–specific Abs (Fig. 1 D). Binding of the LAIR-1-hIg to Ep-CAM was abolished by prior incubation with the Ep-CAM–specific mAb 323/A3, demonstrating the specificity of the interaction (Fig. 2). The LAIR-1–specific Abs available (DX26 and 8A8) did not block the LAIR-1-hIg–Ep-CAM interaction (data not shown), indicating that these Abs do not recognize epitopes from LAIR-1 that are involved in Ep-CAM binding.
Ep-CAM Binds Both LAIR-1 and LAIR-2.
We previously described a gene highly related to LAIR-1, designated LAIR-2 2. LAIR-2 has 84% amino acid homology in the Ig domain with LAIR-1, and cDNA for two different splice variants were identified 3. LAIR-2 is not recognized by the LAIR-1–specific mAbs (data not shown). We constructed a fusion protein of LAIR-2 with the Fc portion of human IgG, similar to the LAIR-1-hIg protein. The protein was purified in the same way as LAIR-1-hIg and used to stain HT29 cells expressing natural Ep-CAM as well as 293T cells transfected with Ep-CAM cDNA. Like LAIR-1-hIg, LAIR-2-hIg was able to bind Ep-CAM on HT29 cells (Fig. 3 A), as well as Ep-CAM transfected 293T cells (Fig. 3 B).
Ep-CAM Binds to LAIR through Its First Epidermal Growth Factor–like Domain.
To determine which domain of Ep-CAM was responsible for binding to the LAIR molecules, deletion constructs of Ep-CAM were designed. Ep-CAM contains two epidermal growth factor (EGF)-like repeats in its extracellular domain and has a short cytoplasmic domain 15. Various PCR fragments containing the different domains of the extracellular part of Ep-CAM were produced (Fig. 4 A). These were ligated into a vector containing the leader sequence of Ep-CAM at the NH2 terminus and the transmembrane region and cytoplasmic tail at the COOH terminus. All constructs were Myc-tagged at the COOH terminus. Upon transfection in 293T, protein expression was compared by anti-Myc Western blotting and transfection efficiency was assessed by staining of methanol-fixed cells with anti-Myc Abs. Microscopic analysis of the stained cells indicated membrane expression of the proteins encoded by the various constructs and protein expression and transfection efficiency was comparable for all constructs (data not shown). The only construct, apart from full-length Ep-CAM, that retained binding of LAIR-1-hIg, was a construct containing the first EGF-like repeat from Ep-CAM (Fig. 4 B). Similar results were obtained for LAIR-2-hIg (data not shown). We conclude that Ep-CAM binds to the LAIR molecules through its first EGF-like repeat.
This observation is in accordance with the finding that the anti–Ep-CAM mAb 323/A3 was able to block Ep-CAM–LAIR interaction. The epitope for this mAb maps to the first EGF-like domain of Ep-CAM 15.
Intraepithelial Lymphocytes Express LAIR-1.
Ep-CAM is a 38-kD transmembrane glycoprotein that is expressed on the basolateral surface of the majority of human simple epithelia 16,17. Therefore, we investigated whether LAIR-1–expressing cells in the intestine would be able to interact with Ep-CAM on these epithelial cells. Isolated intraepithelial cells expressed LAIR-1 as analyzed by flow cytometry (Fig. 5 A). Moreover, tissue sections from human colon stained with anti–LAIR-1 and anti–Ep-CAM Abs demonstrated LAIR-1–expressing intestinal intraepithelial lymphocytes in close proximity to Ep-CAM–expressing epithelial cells (Fig. 5 B). This suggests that these cells might be subject to downregulation through Ep-CAM–LAIR-1 interactions, which might provide a mechanism to regulate mucosal immunity in the intestine.
The physiological role proposed for Ep-CAM so far is in intercellular adhesion 18. It has been shown to function as a Ca2+-independent homophilic intercellular adhesion molecule 19. Ep-CAM resembles known adhesion molecules, although the adhesion mediated by Ep-CAM is relatively weak and its role in maintaining tissue integrity is uncertain 15. Ep-CAM is also abundantly and homogeneously expressed on human carcinomas of different origins. Recently, Ep-CAM has been noted as a promising target for immunotherapy 20. The advantage for tumor cells to express Ep-CAM and the mechanisms of action of anti–Ep-CAM therapy have not yet been elucidated. It is tempting to speculate that Ep-CAM expression might equip tumor cells with a tool to suppress antitumor responses through its interaction with LAIR-1. Preliminary studies have failed to demonstrate diminished NK cell lysis against Ep-CAM–bearing target cells. Further studies are required to address this issue.
We demonstrate that the widely expressed immune inhibitory receptor LAIR-1 has an abundantly expressed ligand, Ep-CAM. Therefore, tight regulation of this interaction is probably needed, which might be provided at different levels. First, LAIR-1 expression can be regulated on cells in different stages of differentiation or upon activation, as we previously demonstrated for B cells on which LAIR-1 expression is tightly regulated 4. In addition, we now demonstrate that LAIR-2 can bind the same ligand as LAIR-1, suggesting regulation of LAIR-1 function by competition for the same ligand.
Ep-CAM molecules have been identified in several species, including mice 21. The murine NS-1 cell line expresses mouse Ep-CAM 22, but did not bind LAIR-1-hIg, indicating that human LAIR-1 does not interact with mouse Ep-CAM (data not shown). As yet, no structural homologues of LAIR molecules have been identified in rodents. The human LAIR genes are located on chromosome 19q13.4, in close proximity to the KIR and ILT genes. These gene families are not conserved in evolution, such that counterparts of these human receptors have not been identified in mice 23. However, KIRs have functional homologues in the mouse, the structurally unrelated Ly49 family of molecules. It will be interesting to pursue the possibility that mouse Ep-CAM has a cellular receptor of unrelated sequence, yet with similar function to human LAIR.
In summary, our data reveal a novel interaction between two previously known proteins. We propose that Ep-CAM, through interaction with the inhibitory receptor LAIR-1, plays a role in the control of mucosal immune responses, possibly preventing excessive inflammatory responses in regions, like the intestine, with high antigen exposure. To further establish the biological importance of the interaction of LAIR with Ep-CAM, experimental evidence that Ep-CAM binding can stimulate the inhibitory function of LAIR-1 is needed.
We thank Drs. Wim de Lau, Marc van de Wetering, Johan van Es, Jeroen Kuipers, and Gerwin Huls for technical advice and helpful discussions and Sandra Zurawski (DNAX) for the LAIR-1-hIg construct.
Linde Meyaard is supported by a fellowship from the Royal Netherlands Academy of Arts and Sciences. Lewis L. Lanier is supported by National Institutes of Health grant CA89294-01.