Haemopoietic cells influence stromal cell gene expression during human osteoclast development

Abstract onlyWe have used a culture system of human peripheral blood mononuclear cells (PBMC)as a source of osteoclast (OC) precursors and murine stromal cells to define the cytokine environment in which human OC form, and to determine the separate contributions of the stromal and haemopoietic elements. We designed a panel of reverse transcription-polymerase chain reaction (RT-PCR) primers that specifically amplify the respective murine or human mRNA species that correspond to cytokines and their receptors previously shown to promote or inhibit OC formation. Murine ST-2 cells and human PBMC were cocultured for up to 21 days in the presence of 1,25(OH) 2vitD3, dexamethasone and human macrophage-colony stimulating factor (M-CSF). OC formation was monitored by the appearance of cells that were positive for tartrate resistant acid phosphatase and able to form resorption lacunae on slices of dentine. We found that the ST-2 cells in these cultures expressed mRNA encoding a repertoire of many of the reported osteoclastogenic factors, as well as the recently described OC differentiation factor (ODF/RANKL). The stromal cells also expressed mRNA encoding osteoprotegerin (OPG), a potent inhibitor of OC formation. We found that agonists and antagonists of OC formation were expressed by both the stromal cells and the PBMC. RANK, the receptor for ODF/RANKL, was expressed only by the PBMC as were IL-1R2 and c-FMS. We identified three features of the cytokine environment that may be a characteristic of normal OC formation. Firstly, the ratio of mouse ODF:OPG mRNA was found to increase during the cocultures, consistent with a key role for ODF in the promotion by stromal cells of OC formation. Secondly, we found that mRNA encoding IL-1 and IL-17, as well as IL-6 and sIL-6R, were coordinately expressed by the PBMC. Thirdly, analysis of the culture medium showed that the PBMC secreted IL-1, IL-6 and TNF-alpha protein only in coculture with ST-2 cells during the first few days of osteoclast development. Similarly, prostaglandin E2, shown to synergise with ODF during OC development, was secreted only in cocultures. Together, these data show OC develop in a complex cytokine environment and suggest that haemopoietic cells provide signals to stromal cells during OC development. Work is in progress to extend these studies to human PBMC interacting with normal human osteoblasts.G.J. Atkins, D R Haynes, A.C.W. Zannettino, M Capone, T Crotti and D.M. Findla