Expression of receptor activator of nuclear factor‐κB ligand by B cells in response to oral bacteria

Abstract

Introduction: We investigated receptor activator of nuclear factor‐κB ligand (RANKL) expression by B lymphocytes during early and late aspects of the immune response to Aggregatibacter actinomycetemcomitans, a gram‐negative, anaerobic bacterium associated with aggressive periodontal disease. Methods: Expression of messenger RNA transcripts (tumor necrosis factor‐α, Toll‐like receptors 4 and 9, interleukins 4 and 10, and RANKL) involved in early (1‐day) and late (10‐day) responses in cultured rat splenocytes was examined by reverse transcription–polymerase chain reaction (RT‐PCR). The immune cell distribution (T, B, and natural killer cells and macrophages) in cultured rat splenocytes and RANKL expression in B cells were determined by flow cytometric analyses. B‐cell capacity for induction of osteoclast differentiation was evaluated by coculture with RAW 264.7 cells followed by a tartrate‐resistant acid phosphatase (TRAP) activity assay. Results: The expression levels of interleukins 4 and 10 in cultured cells were not changed in the presence of A. actinomycetemcomitans until cultured for 3 days, and peaked after 7 days. After culture for 10 days, the percentages of B and T cells, the overall RANKL messenger RNA transcripts, and the percentage of RANKL‐expressing immunoglobulin G‐positive cells were significantly increased in the presence of A. actinomycetemcomitans. These increases were considerably greater in cells isolated from A. actinomycetemcomitans‐immunized animals than from non‐immunized animals. RAW 264.7 cells demonstrated significantly increased TRAP activity when cocultured with B cells from A. actinomycetemcomitans‐immunized animals. The addition of human osteoprotegerin‐Fc to the culture significantly diminished such increases. Conclusion: This study suggests that B‐lymphocyte involvement in the immune response to A. actinomycetemcomitans through upregulation of RANKL expression potentially contribute to bone resorption in periodontal disease.