Aims Crohn's disease is associated with reductions in bone mineral density and an increased risk of fracture. Measurement of bone turnover markers suggest that in children this is due to reduced bone formation. This study aimed to investigate the influence of resting and activated leucocytes and their secreted products on the growth of osteoblast-like Saos2 cells in vitro.
Methods Osteoblast-like Saos2 cells were cultured with resting or polyclonally activated populations of peripheral blood mononuclear cells (PBMCs) or isolated CD4+ T cells for 4 days. Cells were activated using anti-CD3/anti-CD28 monoclonal antibody-coated beads. The use of transwell inserts, which physically separate the different cell populations, determined the comparative influence of cell-cell contact and secreted factors on the observed effects.
Results The culture of Saos2 cells with resting PBMCs and CD4+ T cells increased their proliferation, whereas activated cells inhibited the proliferation of Saos2 cells. These effects were still present with the use of transwell inserts, although final cell numbers were overall slightly higher. Differences were not of statistical significance, but there was a clear dose-response effect. Supernatants from activated CD4+ T cells (50% v/v) significantly reduced the number of Saos2 cells at day 5 (492349 vs 224325; p=0.01). The effect of the PBMC supernatant at day 5 was only marginally greater than that of the CD4+ T cells (263824 vs 224325; not significant). All data are derived from three independent experiments.
Conclusions These results are consistent with a hypothesis that activated immune cells and/or their products are at least partially responsible for the reduced osteoblast activity that is seen in paediatric Crohn's disease. Furthermore, CD4+ T cells are mediators of inflammation in inflammatory bowel disease and these experiments demonstrate that these cells are primarily responsible for the effects seen.
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