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Toll-like Receptors, NOD1, and NOD2 in Oral Epithelial Cells

¹ Division of Oral Diagnosis, Department of Oral Medicine and Surgery, and
² Department of Microbiology and Immunology, Tohoku University Graduate School of Dentistry, Sendai, Japan;
³ Department of Chemistry, Graduate School of Science, Osaka University, Japan; and
⁴ Division of Control of Oral Infection, Hokkaido University Graduate School of Dentistry, Sapporo, Japan

View larger version (95K): [in a new window]   Figure 1. Expression of TLR2, TLR4, NOD1, and NOD2 in human gingival epithelial tissues. Cryosections of healthy gingival tissues (a,c,e,g) and inflamed gingival tissues (b,d,f,h) were stained brown with anti-NOD1 antibody (a,b), anti-NOD2 antibody (c,d), anti-TLR2 antibody (e,f), and anti-TLR4 antibody (g,h). The sections were counterstained blue with hematoxylin (a–h). Scale bars: 100 µm.

View larger version (162K): [in a new window]   Figure 2. Up-regulated expression of TLR2 and TLR4 in inflamed gingival epithelial tissues. Cryosections of healthy gingival tissues (a,c) and inflamed gingival tissues (b,d) were stained brown with anti-TLR2 antibody (a,b) and anti-TLR4 antibody (c,d). The sections were counterstained blue with hematoxylin (a–d). Scale bars: 20 µm.

View larger version (45K): [in a new window]   Figure 3. Expression of TLR2, TLR4, NOD1, and NOD2 on human oral epithelial cells. (a) Human oral epithelial HSC-2, HO-1-u-1, KB cells, and primary gingival epithelial cells were cultured until confluent at 37°C. After incubation, the total RNA was extracted, and the mRNA expression of TLR2, TLR4, NOD1, and NOD2 was analyzed by PCR. (b) Human oral epithelial cells were cultured until confluent at 37°C. The expression of TLR2, TLR4, NOD1, and NOD2 was assessed by flow cytometry. Thin lines represent the isotype Ab control. The results presented are representative of 4 different experiments demonstrating similar results. (c) Human oral epithelial HSC-2 cells were cultured until confluent at 37°C. After fixation, cells were treated with anti-TLR2 antibody or anti-TLR4 antibody and then visualized with Alexa Fluor 488 (green). Nuclei were visualized by being stained with propidium iodide (red). For NOD staining, cells were treated with anti-NOD1 antibody (brown) or anti-NOD2 antibody (brown). The results are representative of 3 different experiments with similar results. Scale bars: 20 µm.

View larger version (20K): [in a new window]   Figure 4. Induction of ß-defensin 2 triggered by TLR2, TLR4, NOD1, or NOD2 ligand in human oral epithelial cells. (a) Human oral epithelial HSC-2 cells were incubated for 8 hrs in the presence or absence of Pam3CSSNA (100 pg/mL), lipid A (100 ng/mL), muramyldipeptide (100 µg/mL), or iE-diaminopimelic acid (100 µg/mL). After incubation, total RNA was extracted, and the mRNA expression of ß-defensin 2 was analyzed by real-time PCR. (b) Human oral epithelial HSC-2 cells were incubated for 24 hrs in the presence or absence of Pam3CSSNA (100 pg/mL), lipid A (100 ng/mL), muramyldipeptide (100 µg/mL), or iE-diaminopimelic acid (100 µg/mL). After fixation, cells were treated with anti-ß-defensin 2 antiboby and then visualized with Alexa Fluor 488 (green). Nuclei were visualized by being stained with 4',6-diamino-2-phenylindole (blue). Scale bars: 20 µ m. (c) Human oral epithelial HSC-2 cells were incubated for 24 hrs in the presence or absence of Pam3CSSNA (100 pg/mL), lipid A (100 ng/mL), muramyldipeptide (100 µg/mL), or iE-diaminopimelic acid (100 µ g/mL). The expression of ß-defensin 2 was assessed by flow cytometry. Thin lines represent the isotype antibody control. The results presented are representative of 4 different experiments demonstrating similar results.