Absence of alphavbeta6 integrin is linked to initiation and progression of periodontal disease

Abstract

Integrin alphavbeta6 is generally not expressed in adult epithelia but is induced in wound healing, cancer, and certain fibrotic disorders. Despite this generalized absence, we observed that alphavbeta6 integrin is constitutively expressed in the healthy junctional epithelium linking the gingiva to tooth enamel. Moreover, expression of alphavbeta6 integrin was down-regulated in human periodontal disease, a common medical condition causing tooth loss and also contributing to the development of cardiovascular diseases by increasing the total systemic inflammatory burden. Remarkably, integrin beta6 knockout mice developed classic signs of spontaneous, chronic periodontal disease with characteristic inflammation, epithelial down-growth, pocket formation, and bone loss around the teeth. Integrin alphavbeta6 acts as a major activator of transforming growth factor-beta1 (TGF-beta1), a key anti-inflammatory regulator in the immune system. Co-expression of TGF-beta1 and alphavbeta6 integrin was observed in the healthy junctional epithelium. Moreover, an antibody that blocks alphavbeta6 integrin-mediated activation of TGF-beta1 initiated inflammatory periodontal disease in a rat model of gingival inflammation. Thus, alphavbeta6 integrin is constitutively expressed in the epithelium sealing the gingiva to the tooth and plays a central role in protection against inflammatory periodontal disease through activation of TGF-beta1.

Figure 1

JE expresses αvβ6 integrin and TGF-β1. A: Immunolocalization of CK19 (a), laminin-5 (b), tenascin-C (c), αvβ6 integrin (d), EDA-fibronectin (e), and TGF-β1 (f) in JE from fully erupted human teeth. B: Immunolocalization of αvβ6 integrin in rat (a) and mouse (b) gingiva from molar teeth. Insets show magnified area indicated by a box in each panel. CT, connective tissue; SE, sulcular epithelium; OE, oral epithelium. Scale bars = 100 μm.

Figure 2

The expression of αvβ6 integrin is restored in healing JE. JE was removed surgically, and samples of the healing tissue were collected immediately after tissue removal (D–F) and after 1 (G–I), 3 (J–L), or 7 (M–O) days of healing. Normal tissue (A–C) is shown as a control. H&E staining of healing tissue (A, D, G, J, M); immunolocalization of CK19 (B, E, H, K, N) and αvβ6 integrin (C, F, I, L, O). Arrowheads point to the wound edge. Higher magnifications of the migrating epithelial front and restored JE are shown in the insets. CT, connective tissue; GT, granulation tissue; OE, oral epithelium; E, epithelium; FC, fibrin clot. Scale bar = 200 μm.

Figure 3

The expression of integrin αvβ6 is down-regulated in periodontal disease. Tissue was collected from four patients. Healthy tissue was collected as a control. Tissue sections from individuals with periodontal disease (B–E, G–J, L–O) or from healthy patients (A, F, K) were immunostained for CK19 (A–E) and αvβ6 integrin (F–J). 4,6-diamidino-2-phenylindole staining (K–O) was performed to highlight the nuclei. CT, connective tissue; PE, pocket epithelium. Scale bar = 200 μm.

Figure 4

P. gingivalis, LAP, and anti-TGF-β1 reduce αvβ6 integrin expression in cultured gingival keratinocytes. A: Gingival keratinocytes were treated with cytokines (TGF-β1, IL-1β, prostaglandin E2, TNF-α, IL-6), bacterial LPS (P. gingivalis, T. denticola, F. nucleatum, T. forsythensis), or P. gingivalis whole cell lysate. Total RNA was extracted, and the expression of β6 integrin subunit was analyzed by real-time PCR using β-actin as a control. PCR reactions were performed in triplicates, and the experiments were repeated four to seven times. B: The cells were exposed to cell lysate of P. gingivalis, TGF-β1, or a combination of both. Total cell surface expression of αvβ6 integrin was measured by flow cytometry. The experiment was repeated three times. C: The cells were treated with LAP or function-blocking anti-TGF-β1 antibody. Relative expression of β6 integrin was analyzed as in A. The experiment was repeated three times. Relative β6 integrin expression is expressed as mean ± SD of parallel experiments in all figures. Only differences that were deemed statistically significant are indicated: *P < 0.05; **P < 0.01, ***P < 0.001 (analysis of variance and Tukey’s post test or Student’s t-test). Results of the effect of anti-TGF-β1 antibody treatment are from a single experiment, and SD was calculated from three parallel samples.

Figure 5

The JE of β6 integrin knockout mice shows epithelial invasion into the connective tissue, migration along the root surface, and increased inflammation. A: H&E staining of the JE of 6-month-old WT (a) and Itgb6^−/− mice (β6^−/−, b). D, dentin; asterisk, enamel. Arrows point to invading epithelial cells, and arrowheads mark infiltrated inflammatory cells. B: H&E staining (a, b, d, e) and pentachrome staining (c, f) of the JE of 12-month-old WT (a–c) and Itgb6^−/− mice (d–f). Green color in the Movat pentachrome-stained tissues indicates collagen. g and h: Enlarged image of the H&E-stained pocket epithelium and the pentachrome staining of a bone lesion in the Itgb6^−/− mice, respectively. Small arrows in g point to infiltrated inflammatory cells. PE, pocket epithelium; D, dentin; B, alveolar bone; AB, abscess-like lesion surrounded by epithelial cells; BV, blood vessel; OC, osteoclast. Scale bars = 100 μm.

Figure 6

Twelve-month-old β6-knockout mice show significantly increased epithelial migration, higher levels of inflammation, and elevated expression of active MMPs. A: Quantitative assessment of the distance of epithelial migration in stained paraffin sections representing multiple teeth from WT (n = 6) and Itgb6^−/− mice (β6^−/−, n = 6). B: The relative number of inflammatory cells adjacent to the JE in WT (n = 6) and Itgb6^−/− mice (n = 6) with apical migration. C: MMPs in the gingival extracts from WT (n = 4) and Itgb6^−/− mice (n = 5) were analyzed by gelatin zymography. The quantified band intensities are shown in D. Results are expressed as mean ± SD in all figures. Only differences that were deemed statistically significant are indicated: *P < 0.05; **P < 0.01 (Student’s t-test or Mann-Whitney U-test).

Figure 7

β6 integrin-knockout mice develop severe alveolar bone loss. A: The jaws of 3-month-old (a, d), 6-month-old (b, e), and 12-month-old (c, f) WT (a–c) and Itgb6^−/− mice (β6^−/−, d–f) were defleshed and stained to show the area of exposed root surface. Arrows mark the CEJ. B: The exposed area between the CEJ and the crest of the alveolar bone of the first and second molars was quantified. Mean values of 48 teeth from 3- and 6-month-old mice and 125 teeth from 12-month-old mice ± SD are presented. Statistically significant differences are indicated: *P < 0.05; **P < 0.01 (Student’s t-test). C: Examples of severe bone loss extending between the roots of molar teeth in 12-month-old Itgb6^−/− mice (c, d). The jaw of a WT mouse is shown in a for comparison.

Figure 8

β6 integrin-knockout mice show extensive bone resorption between the teeth. A: Radiographs (a, b) and scanning electron microscopy images (c–f) of WT (a, c, e) and Itgb6^−/− (β6^−/−; b, d, f) mouse half-mandibles from 12-month-old animals. The arrow in b points to an area of decreased bone density, indicating bone loss. In c and d, an interproximal resorption is noted between the first and second molars. M1, first molar; M2, second molar; M3, third molar. B: Quantitative assessment of the degree of bone loss measured from radiographs. Data presented are mean ± SD from 11 WT and 11 Itgb6^−/− half-mandibles, providing 110 sites for measurement. The difference between WT and Itgb6^−/− is statistically significant (**P < 0.01; Mann-Whitney U-test). Scale bars = 500 μm.

Figure 9

Blocking of αvβ6 integrin-mediated TGF-β1 activation increases apical epithelial migration and inflammation. A: H&E staining of rat gingiva treated daily with H₂O (a), LPS (b), LPS and a nonblocking anti-αvβ6 antibody (7.6B3, c) or LPS and an antibody that blocks αvβ6 integrin-mediated TGF-β1 activation (6.3G9, d) for 8 weeks. Arrowheads and arrows show the distance of epithelial migration. CT, connective tissue; PE, pocket epithelium. B: The epithelial migration was measured from 15 H&E-stained tissue sections from three rats in each treatment group. The results are presented as a mean area of migrated epithelium ± SD. C: The inflammatory infiltrate adjacent to the epithelium was quantified in H&E-stained sections using a combination index based on both the area occupied by inflammatory cells and the inflammatory cell density. The results are presented as mean score for inflammation ± SD. Only differences that were deemed statistically significant are indicated: *P < 0.05; **P < 0.01, ***P < 0.001 (analysis of variance and Tukey’s post test). Scale bar = 200 μm.