Time course of induction of haptoglobin mRNA expression by IL-6 and Hyper-IL-6. Haptoglobin mRNA expression was analyzed in NMRI mice injected i.p. with 40 μg of IL-6/mouse (A) or with 4 μg of Hyper-IL-6/mouse (B). Mice were sacrificed at the time points indicated. RNA was prepared from the liver and subjected to Northern blot analysis. Filters were hybridized with a 32P-labeled 0.9-kb HinfI restriction fragment of human haptoglobin cDNA. The lower panels in A and B represent photographs of the ethidium bromide-stained RNA gel to demonstrate equal loading of RNA.
Dose response of induction of haptoglobin mRNA expression by IL-6 and Hyper-IL-6. Haptoglobin and GAPDH mRNA expressions were analyzed in NMRI mice injected i.p. with various dosages of IL-6 (A) or Hyper-IL-6 (B) as indicated. Mice were sacrificed 4 h after injection. RNA was prepared from the liver and subjected to Northern blot analysis. Filters were hybridized with a 32P-labeled 0.9-kb HinfI restriction fragment of human haptoglobin cDNA and with a 32P-labeled 1.3-kb HifI restriction fragment of GAPDH.
Plasma levels of IL-6 and Hyper-IL-6. A, Time-course study of IL-6 and Hyper-IL-6 plasma levels measured with a human IL-6 ELISA in NMRI mice injected with 40 μg of IL-6/mouse (white bars) or with 4 μg of Hyper-IL-6/mouse (black bars) at the time points indicated. B and C, Dose-response study of IL-6 and Hyper-IL-6 serum levels measured with a human IL-6 ELISA in NMRI mice injected with various dosages of IL-6 (B) and Hyper-IL-6 (C). Mice were sacrificed 4 h after injection. The mice represented in the figure are the same animals as those described in Figures 1 and 2.
Stimulation of HepG2 cells with IL-6 and Hyper-IL-6. Time course (A and B) and dose-response studies (C) of induction of haptoglobin and GAPDH mRNA expression in human hepatoma (HepG2) cells by IL-6 and Hyper-IL-6. For the time-course experiment, cells were treated for the time periods indicated with 10 ng/ml of IL-6 (A) or Hyper-IL-6 (B), respectively. Total RNA was prepared and subjected to Northern blot analysis for haptoglobin and GAPDH expression. For the dose-response experiment (C), cells were treated with increasing amounts of IL-6 (white bars) or Hyper-IL-6 (black bars), respectively. Concentrations of IL-6 and Hyper-IL-6 are given in nanograms per milliliter. Stimulation of HepG2 cells was measured as the secretion of the acute phase protein haptoglobin analyzed by ELISA as recently described (22). Total RNA was prepared and subjected to Northern blot analysis for haptoglobin and GAPDH mRNA expression.
Hyper-IL-6 stimulation is resistant to washing. HepG2 cells were treated with 10 ng/ml of Hyper-IL-6. Twenty-four hours after stimulation, cells were either left untreated with the original medium containing the respective cytokine (lanes 2 and 3) or were subjected to intensive washing at 37°C with PBS followed by incubation with cytokine-free medium (lanes 4 and 5). Forty-eight and seventy-two hours poststimulation, RNA was prepared from the cells and subjected to Northern blot analysis for human haptoglobin and GAPDH mRNA expression. Filters were hybridized with a 32P-labeled 0.9-kb HinfI restriction fragment of human haptoglobin cDNA and with a 32P-labeled 1.3-kb HifI restriction fragment of GAPDH.
Activity and stability in vivo of radiolabeled cytokines on BAF/3-gp130 cells. Murine B9 cells were used to compare the bioactivity of radiolabeled [125I]IL-6 with that of unlabeled IL-6 (A). BAF/3 cells stably transfected with the human gp130 cDNA (27) were used to compare the bioactivity of radiolabeled [125I]Hyper-IL-6 with that of unlabeled Hyper-IL-6 (B). The proliferation of cells treated with IL-6/sIL-6R or Hyper-IL-6 at the concentrations indicated was measured after 72 h using the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide proliferation assay. Data are given as the mean proliferation (±SD) of three independent experiments. To investigate the stability of the designer protein, radiolabeled [125I]Hyper-IL-6 (10.000 cpm/well; 100 μl/well) was incubated with HepG2 cells in a 96-well plate in DMEM containing 10% FCS at 37°C (C). At the time points indicated in the figure, supernatant was harvested from the HepG2 cells. Ten microliters of cell supernatant, respectively, were loaded onto a 12.5% SDS gel and subjected to autoradiography. The arrow marks the 55-kDa Hyper-IL-6 protein. C, Five hundred counts per minute of radiolabeled [125I]Hyper-IL-6.
Competition of binding of [125I]IL-6 and [125I]Hyper-IL-6 to HepG2 cells. Cells were washed twice with ice-cold binding medium (DMEM containing 0.2% (w/v) BSA). Binding was initiated by the addition of [125I]IL-6 (A) or [125I]Hyper-IL-6 (B) at the concentration of 100 pM for 4 h at 4°C in the presence of increasing amounts of unlabeled IL-6 or Hyper-IL-6, respectively, as indicated. After 4 h, medium was removed, and the cells were washed three times with PBS containing 1 mM MgCl2, 0.1 mM CaCl2, and 0.2% BSA. Cells were subsequently lysed in 1 ml of 1 M NaOH, and radioactivity was measured in a gamma counter. Data are expressed as a percentage of the values for control incubations containing only labeled ligand. Values are the mean (±SD) of three independent experiments.
Internalization of surface-bound [125I]IL-6 (A) and [125I]Hyper-IL-6 (B) in HepG2 cells. Cells were incubated with 100 pM for 2 h at 4°C. Internalization was induced by a temperature shift to 37°C for the indicated times. Surface-bound protein (squares) was determined after subjecting the cells to 0.5 M NaCl/HCl, pH 1, for 3 min. Internalized protein (triangles) was measured in the gamma counter after lysis of the cells in 1 M NaOH. Data are expressed as a percentage of initial binding of protein at 4°C. Values are the mean (±SD) of three independent experiments.