Investigation of TLR4 and CD14 on human periodontal ligament fibroblasts

Objectives: The aim of this study was to investigate Toll like receptor 4(TLR4) and CD14 expression on cultured human periodontal ligament fibroblasts. Methods: Healthy human premolars extracted due to orthodontics therapy were collected, periodontal ligaments were scraped and cut into 1mmx1mm explants. Then they were placed on the culture dish and cultured with Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (Hyclone USA) in 37°C, 5%CO₂. Human periodontal ligament fibroblasts were digested with 0.25% typsin when they reached 80% confluence. 3rd passage cells were fixed with 4% paraformaldehyde and prepared for immunofluoresence detection. And 1x10⁶ cells were collected and incubated with 10µl antibody for 30 minutes in darkness and tested with flow cytometry. Antibodies used were PE conjugate mouse anti-human CD14 (Chemicon), mouse anti-human TLR4 and FITC conjugate mouse anti-human TLR4 (Serotec). Secondary antibody was rabbit anti-mouse FITC conjugate IgG. Isotype antibodies were mouse anti-human FITC and PE conjugate monoclonal IgG2a. Results: It was found that there were TLR4 and CD14 expressed on the surface of periodontal ligament fibroblasts by immunofluoresence and FACS. The expression of periodontal ligament fibroblasts for TLR4 and CD14 was 19.5±2.3% and 47±4.8% respectively. Conclusions: Periodontal ligament fibroblasts are heterogenous cell population. Both TLR4 and CD14 are positive on the surface of some human periodontal ligament fibroblasts. These molecules maybe take part in immune response of periodontal tissues.