Toll like receptors (TLRs) are “gate-keepers” of the mucosal immune response. The oral mucosa interacts with a high density of gram-positive and gram-negative bacteria. Recognition of bacterial structures by TLRs induces pro-inflammatory gene expression and prompts an appropriate acquired immune response. Objective: To determine the expression of TLRs in healthy and diseased gingival tissues and correlate this with innate immune response to lipopolysaccharide in vitro. Methods: Human gingiva was obtained after informed consent from chronic periodontitis (CP) (n=13) and healthy (n=11) subjects. TLR expression was analyzed by immunohistochemistry and quantitative real time RT-PCR (Qrt-PCR). Blood monocytes were used in vitro to analyze the effects of repeated stimulation with P. gingivalis (Pg) and E. coli LPS on TLR mRNA and protein expression. Inflammatory cytokine production was analyzed by Qrt-PCR, RT-PCR, and cytometric bead array. Results: Analysis of gingival tissue by immunohistochemistry revealed infiltration of basement membrane in healthy and CP with TLR2+ and TLR4+ cells in a pattern evocative of macrophages/dendritic cells. Qrt-PCR revealed that TLR2 and TLR4 are down-regulated (35-fold [p<0.05, Student's T test] and 10-fold, respectively) in CP compared to gingival health. Initial exposure of monocytes to either LPS up-regulated TLR2, TLR4, IL-1b mRNA/protein; moreover, the following cytokines were secreted: TNF-a, IL-1b, IL-6, IL-8, IL-10. Re-stimulation of monocytes resulted in much lower TLR2, TLR4 and IL-1b mRNA/protein and @10-fold reduction in TNF-a secretion, suggesting homo- and cross-tolerizing effects with either LPS. Less susceptible to tolerance by Pg LPS were IL-6, IL-10 and IL-8. Conclusion: These studies suggest that certain components of the innate oral mucosal immune system may become hyporesponsive during sustained exposure to bacterial structures such as LPS. It is likely that in chronic infections like periodontitis, tolerance is an adaptive measure to prevent inflammatory damage and to re-establish tissue homeostasis. Supported by NIH/NIDCR DE14328-03(to CWC)

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