Figure 6.
sIL-6R produced by monocytes sensitizes neuroblastoma cells to IL-6-mediated STAT3 activation. A, CHLA-255 cells were treated with IL-6 at indicated concentrations in the absence (top panel) or presence (lower panel) of sIL-6R (25ng/mL) for 30 minutes. The cell lysates were examined for expression of pSTAT3 and STAT3 by Western blot. B, CHLA-255 cells were treated with IL-6 alone (10 ng/ml) and in the presence of increased concentrations of sIL-6R (0.1 to 25 ng/mL) for 30 minutes. The cell lysates were then examined for expression of pSTAT3 and STAT3 by Western blot. The bar diagram represents the ratio pSTAT3/STAT3 obtained by scanning of the blot. C, CHLA-255 (4.5×105) and human BMMSC or monocytes (4.5×105) were cultured either alone or together in Transwell plates for 48 hours. The culture medium was collected and the concentrations of IL-6 and sIL-6R were determined by ELISA. The data represent the mean concentration (±SD) of duplicate (upper) and triplicate (lower) samples. D, CHLA-255 cells were co-cultured with monocytes in Transwell plates in the absence or presence of an anti-IL-6R antibody (tocilizumab). After 48 hours cell lysates were examined for pSTAT3 and STAT3 by Western blot. E. Monocytes isolated from the peripheral blood of patients with neuroblastoma and CHLA-255 cells were cultured alone or in contact (ratio tumor cells:monocytes 4:1) for 24 hours. Cells were then harvested and examined by flow cytometry for the presence of nuclear pSTAT3 and expression of cell surface CD56 and CD14 to separate tumor cells from monocytes. The data represent the average percent of pSTAT3 positive cells (±SD) from 3 separate samples.