Introduction: Bisphosphonates (BPPs) directly affect osteoclasts in undergoing apoptosis and impeding bone loss, and therefore, they are some of the most commonly prescribed drugs for osteoporosis. It is well known that osteoclasts and dendritic cells (DCs) share a common precursor, which can be interpreted as BPPs similarly having a potential influence on DCs. DCs play critical roles in innate immune responses as antigen presenting cells, and thus, they are the main players that cause rejection after organ transplantation. In this study, we focused on the effects of BPPs on DCs and determined the immune-modulating function of BPPs by examining the molecular changes of DCs after BPP treatments.
Materials and Methods: A mice immature DC cell line, DC2.4 cells were plated at 1*10⁶ cells/a 100mm culture dish and cultured for 24 or 48 hours with 20 or 40μM of either pamidronate, risedronate, or zoledronate, which were three different kinds of BPPs. An immortalized human proximal tubule epithelial cell line, HK2, and an immortalized human endothelial cell line, EA.hy926, were also treated and cultured with BPPs as control groups to examine the cytotoxic effect of BPPs on other cell types. Cytotoxicity on each cell type was measured using both MTT and LDH assays. The mRNA expression levels of various molecules of BPPs-treated DC2.4 were also qualified using real-time PCR.
Results: The viability of DC2.4 cells was significantly decreased by 60 percent of the control level when treated with 40μM of pamidronate for 48 hours. When treated with the same dose of risedronate and zoledronate for the same period of time, the viability decreased by 50 and 60 percent when compared to the control, respectively. The cell viabilities of both HK2 and EA.hy926, however, were maintained at over 80 percent to those of the control under all conditions. The mRNA expression of DC-derived cytokines IL-6 and IL-1β of DC2.4 significantly increased more than 20- and 80- folds, respectively, when treated with 40μM of zoledronate for 48 hours. However, the gene expression of apoptosis related molecules, such as Bcl-2, BclxL, or BAX, had no significant changes.
Discussion: Based on the results of the cytotoxicity assay, BPPs might influence DCs, or DC-like cells, more effectively and specifically than it does to other cell types. The gene expression data indicate that the BPP-mediated cytotoxic events of DC2.4 might result from changes in cytokine production, not from apoptosis mechanism.
Conclusion: Considering the selective cytotoxic effects of BPPs on DCs, we suggest that BPPs might be used as an immune modulatory agent by regulating the population of DCs. Further study will be conducted in co-culture systems focusing on functional and numerical changes in DC surface markers to identify the molecular links between BPPs and DCs.