Lutheran blood group antigen as a receptor for alpha5 laminins in gingival epithelia.

BACKGROUND: Lutheran blood group glycoprotein (Lu) is a transmembrane receptor of the immunoglobulin superfamily. Lu serves as a receptor for alpha5 laminins (Lm). The Lm alpha5 chain is a constituent of Lms-511 and -521. Lm-511 is found in most human basement membranes (BMs) and also is detected in BM of gingival epithelia. Recent studies indicated that Lu mediates cell adhesion to Lms-511/521 independently or in concert with integrins. This study focused on the expression of Lu in gingival epithelia and on cultured immortalized gingival keratinocytes. The role of Lu and alpha(3) and beta(1) integrin subunits in the adhesion of oral epithelial cells to Lms-511/521 was also studied. METHODS: Immunofluorescence microscopy was used to study the expression of Lu in gingival tissues and in cultured gingival keratinocytes. Immunoprecipitation of radioactively metabolically labeled cells was used to detect Lu. Cell adhesion to Lm-511/521 preparation and to pure Lm-511 was studied in quantitative cell adhesion experiments. Morphological adhesion assays were carried out for visualization of the morphology and adhesion structure formation of the adhering cells. RESULTS: Immunofluorescence studies on gingiva showed complete coalignment of Lu on basal epithelial cells with the BM Lm alpha5 chain. A surface-confined, punctate immunoreaction for Lu was detected throughout cell surfaces on cultured gingival cells. Immunoprecipitation showed a broad polypeptide with molecular weight 85,000. In quantitative cell adhesion assays, the adhesion of cells to both Lm alpha5 preparations was diminished with monoclonal antibodies (MAbs) against integrin alpha(3) and even more effectively with MAbs against the beta(1) subunit. When the adhesion sites were blocked with soluble recombinant Lu (sol-Lu), the adhesion of gingival epithelial cells to Lms-511/521 was reduced significantly, and sol-Lu increased the inhibition with integrin alpha(3) antibody. Lm-511 did not induce lamellipodia or focal contacts in cultured gingival keratinocytes. CONCLUSIONS: Lu was in coalignment with Lm alpha5 chain in gingival epithelia. Lu also seemed to have a role in gingival epithelial cell adhesion together with integrin alpha(3)beta(1).

Junctional epithelium in rats is characterized by slow cell proliferation.

BACKGROUND: The integrity of junctional epithelium (JE) and a firm epithelial adhesion to the tooth surface are maintained by the balance between cell proliferation and cell death. Maintaining the JE structure is essential for the protection of periodontal connective tissues against oral microbes. In this study, the proliferative activity and the expression of caspase 3, a cysteine protease associated with cell death, were studied in rat JE and other epithelial structures during molar tooth development. METHODS: Fourteen rats aged 10 to 70 days were injected with bromodeoxyuridine (BrdU). Samples of first and second molars were selected for immunohistochemical staining. BrdU incorporation was studied in oral epithelium (OE) covering the erupting tooth, reduced enamel epithelium (REE), and gingival epithelium (GE), sulcular epithelium (SE), and JE. Samples were also subjected to immunohistochemical analysis for proliferating cell nuclear antigen (PCNA) and caspase 3. RESULTS: The basal cells of the GE were actively proliferating, but in the JE, only a few cells were positive for BrdU or PCNA immunostaining. Some outer REE cells were proliferating during tooth eruption. Caspase 3 expression was in specific areas of REE after completion of amelogenesis. CONCLUSIONS: Results showed slow proliferative activity in the rat JE. However, specific studies on cellular turnover and cell migration are needed to understand tissue homeostasis in this area.

Marginal periodontium as a potential reservoir of human papillomavirus in oral mucosa.

BACKGROUND: Although human papillomaviruses (HPVs) are associated with a number of proliferative epithelial lesions including squamous cell malignancies, they can also be detected in the normal oral mucosa in 10% to 20% of the adult population. However, the point of entry and the site of replication of HPV in the oral cavity are not known. Since the gingival pocket is the only site in the oral mucosa where basal cells, known to be targets of HPV at other mucosal sites, are normally exposed to the environment, we hypothesized that this could be the site of latent HPV. METHODS: Gingival biopsies taken from 38 individuals with clinically diagnosed periodontal disease were examined. The presence of HPV DNA was studied by using nested PCR (polymerase chain reaction with MY09/MY11 and GP05+/GP06+ primers targeting the L1 region of HPV), followed by subsequent hybridization with a cocktail of 12 high-risk HPV oligoprobes and in situ hybridization (ISH) with probes for HPV screening and the HPV subtype 16. RESULTS: In the present study, high-risk HPV types were detected in 26% (8/31) of the gingival biopsies with PCR. By using in situ hybridization, the viral DNA was localized to the coronal part of the junctional epithelium in the gingival pocket. CONCLUSIONS: The results suggest that the periodontal pocket might serve as a reservoir of HPVs in oral mucosa. While having important implications in understanding the HPV transmission, this observation does not rule out the possibility that HPV may be involved in the initiation of periodontal disease.

Function of laminins and laminin-binding integrins in gingival epithelial cell adhesion.

BACKGROUND: In human gingiva, epithelial cells attach to their adjacent tissues by means of specialized molecular adhesion complexes and a basement membrane. Little is known about the synthesis of adhesion proteins by gingival keratinocytes; we, therefore, studied how cultured immortalized gingival epithelial cells produce laminins and express laminin-binding integrins. We presumed that different laminins and integrins would be involved in the adhesion of gingival epithelial cells. METHODS: We cultured gingival keratinocytes and studied their production of laminins and expression of integrins using immunofluorescence microscopy, immunoprecipitation, and immunoblotting methods and by quantitative cell adhesion experiments. We also studied how gingival tissue expresses these adhesion proteins in vivo by using immunofluorescence microscopy. RESULTS: In immunofluorescence microscopy, the cells were seen to organize chains of laminin-5 (alpha3beta03gamma2) to extracellular patches, whereas the alpha5 chain of laminin-10 (alpha5betalgamma1) could only be seen intracellularly. Of the laminin-binding integrin subunits, integrin a6 subunit was organized to dotted arrays, typical of prehemidesmosomal adhesions, whereas integrin alpha3 subunit was located at cell-cell junctions, in prehemidesmosomal structures, and at some locations also in small focal-contact like patches. Integrin beta1 subunit was found at cell-cell junctions and in focal contacts. Immunoprecipitation experiments showed that the cells synthesize and secrete chains of laminin-5 and laminin-10. In quantitative cell adhesion experiments, the cells adhered efficiently to these laminins by using cooperatively integrin alpha3beta1 and alpha6beta1 integrin complexes. None of the other known laminin-binding integrin subunits appeared to be significantly involved in cell adhesion to these laminin isoforms. CONCLUSIONS: Our results provide new information on gingival epithelial cell adhesion and extracellular matrix production and may thus aid in the understanding of periodontal physiology.

An organotypic in vitro model that mimics the dento-epithelial junction.

BACKGROUND: The dento-epithelial junction forms the primary periodontal defense structure against oral microbes. The cells of the junctional epithelium (JE) attach both to a basement membrane (BM) facing the connective tissue and to a hard dental tissue by structurally similar but molecularly distinct mechanisms. Here we describe a new organotypic cell culture model for the dento-epithelial junction comprising not only epithelial and mesenchymally derived components, but also a tooth surface equivalent. METHODS: Rat palatal keratinocytes were seeded on fibroblast-collagen gels. A tooth slice was placed on top of the epithelial cells and the multilayer cultures were grown at the air-liquid interface. Formation of the epithelial structures, BM components, and the epithelial attachment to the tooth surface were studied by immunofluorescence and light and electron microscopy. The findings were compared to the structure of the dento-epithelial junction in vivo. RESULTS: A well-differentiated stratified epithelium was formed. Under the tooth slice the epithelium remained thin and non-differentiated. Attachment of the epithelial cells to the tooth surface was mediated by hemidesmosomes (HDs) as in vivo. Laminin-5 (Ln-5) was present in the extracellular matrix (ECM) between the tooth and the epithelium as well as in the BM structure between the epithelium and the fibroblast-collagen matrix. Instead, Ln-10/11 was present only at the mesenchymal tissue side as is known to be the case in vivo. CONCLUSIONS: The organotypic model presented expresses the characteristic structural and molecular features of the dento-epithelial junction and may be applied for studying physiological and pathological processes in the epithelial attachment.