TLR4 Expression by Human Pulp Stem Cells and Fibroblasts

The receptor complex CD14/TLR4-MD2 recognizes bacterial lipopolysaccharides (LPS) and Toll-Like receptor 4 (TLR4) initiates the intracellular signaling in immune cells. Previously we demonstrated that LPS induced VEGF expression in odontoblast-like cells (MDPC-23) and macrophages was mediated at least in part by TLR4 signaling. Recently we found VEGF up-regulation by human pulp stem cells (DPSC) and fibroblasts (HDPF) after exposure to E. coli and P. endodontalis LPS. However, TLR4 expression levels in human dental pulp cells are still unknown. The purpose: of this study was to evaluate the expression of TLR4 in human dental pulp cells by immunocytochemistry and Western Blot. Methods: 3 x 104 HDPF and DPSC and 6 x 104 MDPC-23 and macrophages were seeded in 8 well chamber slides and left to attach overnight. Cells were then treated with 0 or 20 µg/mL of E. coliLPS for 24 hours and then fixed. Immunocytochemistry was performed to identify TLR4 or IgG as a control. Binding was visualized with an AEC chromogen kit and a hematoxylin counterstain. Western Blot assays were also used to analyze TLR4 expression. 40 x 104 HDPF and DPSC cells were seeded and treated with either 0 or 20 µg/mL of E. coli LPS. Protein was then collected and run in a 10% SDS polyacrylamide gel. Membranes were exposed to TLR4 or GAPDH antibodies. Macrophages, MDPC-23 and THP-1 cells were used as controls. Four independent experiments were performed for each assay. Results: We observed low levels of TLR4 expression in both HDPF and DPSC in comparison with controls by immunocytochemistry. There was no difference between treated and untreated cells as seen by Western Blots. Conclusions: We conclude that HDPF and DPSC express TLR4 and treatment with E. coli LPS did not increased TLR4 expression.

Supported by CRSE department, School of Dentistry, University of Michigan.