Soluble IL-15 triggers a differential cell signal in RCC and RPTEC.
A) Scatchard's plot analysis: Effects of anti-IL-15Rβ and γc mAbs on IL-15 binding to RCC. For the IL-15 binding experiments, RCC7 cells were incubated with increasing concentrations of radioiodinated rIL-15 in presence or not of the following neutralizing mAbs: anti-IL-2Rβ and IL-2Rγ. The nonspecific cell binding was determined in the presence of radioiodinated rhIL-15 and a 100-fold excess of unlabeled rhIL-15. Cell-bound (B) and unbound (free, F) fractions were measured, and the specific bound fraction was calculated by subtracting the nonspecific binding from the cell-bound fraction. On the ordinate is plotted the ratio of the specific bound fraction (expressed in sites per cell) over the total concentration (bound plus free) of radioiodinated rIL-15 (expressed in pM). On the abscissa bound fraction (expressed in sites per cell). The high affinity specific IL-15 binding (Kd = 375 pM, 413 IL-15 binding sites per cell), which was completely abrogated by neutralizing antibody against the IL-2Rβ (inset) but not the γc chain, suggested the presence on RCC of an IL-15Rα/IL-2Rβ complex. B) Detection of IL-15Rαβ complex by immunoprecipitation (IP) with anti-IL-15Rα (M161) or mouse IgG protein G-Sepharose-conjugate on total lysate (TL) of RCC7. Immunoprecipitated complexes were blotted either with anti-IL-2Rβ (sc-1046) and anti-IL-15Rα (sc-9172). C) Stimulation for 10 and 40 min with physiologic (10 pg/mL) and supra-physiologic (10 ng/mL) concentrations of rhIL-15 induces the phosphorylation of MAPK ERK1/2 and IκBα in RPTEC and RCC7, whereas STAT5 activation was only observed in RPTEC. Histograms represent densitometry comparison of each factor normalized to β-actin in 3 different RCC (RCC5, RCC7, RCC8) and 3 RPTEC batches. * P<0.05 versus control, Mann-Whitney test. One experiment representative of a total of three is shown.