Pharmacokinetics of mIFN-α injection into mice. (A) Serum concentrations of mIFN-α after s.c. injection of a single dose of 1,000 IU/g. The profile was obtained from two C57/BL6 mice receiving mIFN-α by s.c. injection. The peak of serum concentration was reached 1 h after the injection. After 8 h, the concentration of mIFN-α returned to pretreatment levels. The error bars represent the SEM. (B) Serum concentrations of mIFN-α after multiple injections. Two C57/BL6 mice were injected s.c. with 1,000 IU of mIFN-α/g as a priming dose and four times with 300 IU/g as a maintenance doses every 3 h. Sera were collected immediately before each injection and one h after each injection. The black arrows indicate time points of mIFN-α injections. The error bars represent the SEM.

Refractoriness of JAK-STAT signaling in the presence of continuously elevated mIFNα. (A) C57/BL6 mice were treated with human pegIFN-α-2b s.c. for 1 h at increasing doses (lanes 1 to 5) and for up to 6 h with 2,000 μg/kg (body weight) (lanes 7 to 11). Injection of mIFN-α served as positive control (lane 6). (B) Schematic diagram showing the time course of the multiple injection experiment. Mice were injected with 1,000 IU of mIFN-α/g as a priming dose, followed by four injections of 300 IU of mIFN-α/g as maintenance doses. The vertical bars in the diagram indicate the time points of animal sacrifice and liver resection. (C) The phosphorylation of STAT1, STAT2, and STAT3 was assessed by Western blotting with extracts of mouse liver after mIFN-α treatment. Blots were stripped and re blotted for total STAT1, STAT2, and STAT3 and stained with Blot-FastStain as a loading control. (D) DNA binding of ISGF3 in liver cells after continuous mIFN-α treatment. Liver nuclear extracts of C57/BL6 mice were analyzed in EMSAs using ISRE oligonucleotide probes. Extract from mouse liver 1 h after mIFN-α stimulation was used as a positive control (lane A), and shifts of the ISGF3 band were performed by using antibody specific for STAT1 (lane B) and STAT2 (lane C). (E) SOCS1 and SOCS3 mRNA expression level in the continuous presence of mIFN-α. Total RNA from mIFN-α-injected mice was prepared and subjected to Northern blot analysis for SOCS1 and SOCS3 mRNA expression. Equal loading of the gel was verified by ethidium bromide staining and comparing the intensities of the 28S rRNA and 18S rRNA bands. (F) SOCS1 protein expression was determined by Western blotting with whole-cell extracts of mouse liver after mIFN-α treatment.

USP18/UBP43-deficient mice remain responsive to IFN-α. (A) Quantitative RT-PCR analysis of USP18 mRNA expression in response to the first and second injections of mIFN-α. RNA was isolated from the livers of WT, IL-10^−/−, SOCS3^lox/lox Cre⁺, and STAT3^lox/lox Cre⁺ animals at time points 0, 1 h, and 9 h (1 h after the second injection). The data are plotted as the amount of USP18 mRNA relative to mRPL19 and are means (with the SEM) of two animals per time point for the KO mice and four animals per time point for the WT mice. (B) Activation of the JAK-STAT signaling pathway in response to the first and second injection of mIFN-α in WT and USP18/UBP43-deficient mice. Phosphorylation of STAT1, STAT2 (upper band), and STAT3 was assessed by Western blotting with extracts of mouse liver from WT and USP18/UBP43^−/− mice. Blots were stripped and reblotted for total STAT1, STAT2, and STAT3 and for actin as a loading control. (C) Bar diagram of phospho-STAT1 signals in WT and USP18/UBP43-deficient mice in response to the first and second injections of mIFN-α. Phospho-STAT1 and actin signals from three mice per time point were quantified by using an Odyssey infrared imaging system. The integrated intensities of phospho-STAT1 signals were divided by the actin values. The y axis displays arbitrary units. Shown are mean values and the 95% confidence intervals. (D) STAT activation in response to continuous presence of mIFN-α in WT and USP18/UBP43-deficient mice. Phosphorylation of STAT1 and STAT2 (upper band) was assessed by Western blotting with extracts of mouse liver from WT and USP18/UBP43^−/− mice. Blots were stripped and reblotted for total STAT1 and for actin as loading control. (E) Quantitative RT-PCR analysis of SOCS1 and PKR mRNA expression in response to the first and the second injections of mIFN-α in WT and in USP18/UBP43-deficient mice. Two mice were sacrificed per time point. Shown are mean values (with the SEM) of the ratio of SOCS1 and PKR mRNA over mRLP19 mRNA. (F) Quantitative RT-PCR analysis of SOCS1 mRNA expression in response to continuous stimulation with mIFN-α in WT mice and in USP18/UBP43-deficient mice.

IFN-α induces refractoriness of JAK-STAT signaling in the mouse liver. (A) C57/BL6 mice were treated with two injections of mIFN-α (1,000 IU/ml) or PBS, according to the time course indicated in panel D. IFN-α-induced tyrosine phosphorylation of STAT1 and STAT3 was assessed by immunoblotting of liver whole-cell extracts. Blots were stripped and reprobed for total STAT1 and STAT3 and for actin as a loading control. (B) DNA binding of STAT1 and STAT3 homo- and heterodimers upon stimulation with mIFN-α. Nuclear extracts of liver cells obtained from two-dose mIFN-α injected mice were analyzed in EMSAs with the SIE-m67 oligonucleotide probe. Nuclear extract from mouse liver 1 h after mIFN-α stimulation was used as a positive control (lane A). Antisera specific to STAT1 (lane B) and STAT3 (lane C) were used to shift the STAT1 homodimers, STAT3 homodimers, and STAT1-STAT3 heterodimers (all marked with arrows). (C) DNA binding of ISGF3 in liver cells after two-dose mIFN-α treatment. Liver nuclear extracts of C57/BL6 mice were analyzed in EMSAs using ISRE oligonucleotide probes. Nuclear extract from mouse liver 1 h after mIFN-α stimulation was used as a positive control (lane A), and shifts of the ISGF3 band were performed by using antiserum specific for STAT1 (lane B) and STAT2 (lane C). (D) Schematic diagram showing the time course of the experiment with two injections of mIFN-α. The interval between the injections is 8 h and the vertical bars in the diagram indicate time points of animal sacrifice and liver resection.

IL-10, STAT3, SOCS3, and SOCS1 are not responsible for refractoriness of IFN-α-induced JAK-STAT signaling. (A) In the left panel, injection of a single s.c. dose of mIFN-α (1,000 IU/g) induces a transient increase in IL-10 serum concentrations. The level of mouse IL-10 as determined by ELISA is shown. In the right panel, repeated injections of 300 IU of mIFN-α/g every 4 h after an initial priming dose of 1,000 IU/g induce constantly high mouse IL-10 serum concentrations. During mIFN-α maintenance doses, the concentration of IL-10 stays at levels of ∼40 pg/ml (black triangles). As a negative control, the level of serum IL-10 was determined in a mouse multiply injected with PBS (see the graph with black squares). (B) STAT1 activation in response to two injections of mIFN-α in IL-10-deficient mice. The upper panel shows a schematic representation of the experimental design with two mIFN-α injections performed 8 h apart. Animals were sacrificed at time points 0 (control) and 1 h and 9 h (1 h after the second injection). Immunoblotting for pSTAT1/STAT1, pSTAT3/STAT3, and actin is shown for C57BL/6 WT mice and for IL-10^−/− animals. (C) The upper panel shows results for STAT activation in response to two injections of mIFN-α in mice with liver-specific KO of STAT3 or SOCS3. Immunoblotting for pSTAT1/STAT1, pSTAT3/STAT3, and actin is shown for WT C57BL/6 mice and for SOCS3^lox/lox Cre⁺ and STAT3^lox/lox Cre⁺ animals. The lower panel presents the results of quantitative RT-PCR analysis of SOCS3 mRNA expression in the livers of WT, SOCS3^lox/lox Cre⁺, and STAT3^lox/lox Cre⁺ animals at time points 0, 1 h, and 9 h (1 h after the second injection). The data are plotted as the amount of SOCS3 mRNA relative to mRPL19 mRNA and are means (with the SEM) of two animals per time point for the KO mice and four animals per time point for the WT mice. (D) STAT activation in response to two injections of mIFN-α in mice deficient for SOCS1. Immunoblotting for pSTAT1/STAT1 and actin is shown for IFN-γ^−/− mice and for IFN-γ^−/− SOCS1^−/− mice.