Interferons (IFNs) are widely expressed cytokines with potent antiviral and growth-inhibitory effects. These cytokines are the first line of defence against viral infections and have important roles in immunosurveillance of malignant cells. The IFN family includes two main classes of related cytokines: type I IFNs and type II IFN (1). Unlike the many different types of type I IFNs there is only one type II IFN, Interferon gamma (IFNG). After binding of IFNG to the two-parted IFNG cell membrane receptor janus thyrosine kinase 1 (JAK1) is rapidly associated to the receptor and reveals after phosphorylation a docking site for the latent cytoplasmatic signal transducer and activator of transcription 1 (STAT1). Upon phosphorylation, STAT1 homodimerizes, translocates in the nucleus and regulates IFNG dependent transcription by binding to IFNG activated sequences (GAS) in IFNG inducible genes (2).
In many clinical trials and in-vitro-studies IFNG has been reported as a possible agent to affect fibroproliferative diseases in an antifibrotic manner, e.g. IFNG has been demonstrated to inhibit collagen synthesis in human fibroblasts (3), to modulate galactosaminoglycans produced of human skin fibroblasts (4) and to block induction of myofibroblasts (5).
Fibrosis is characterized by transdifferentiation of quiescent fibroblasts to myofibroblasts that results in overexpression and deposition of extracellular matrix proteins that subsequently leads to organ impairment or dysfunction(6–11). On the molecular level, profibrotic changes and regulatory mechanisms in fibroblasts are induced and controlled by members of the transforming growth factor beta (TGFB) family that transduce their signals via receptor regulated Smads (R-SMADs), common mediator Smads (Co-SMADs), and inhibitory Smads (I-SMADs) following specific receptor activation(12, 13). During signaling phosphorylated R-SMADs form oligomeric complexes with the common mediator SMAD4 (14, 15). These complexes translocate into the nucleus and regulate transcription of target genes(15).
Dupuytren’s disease (DD) is a fibroproliferative disorder of the hand, characterized by formation of nodules and cords that appear in the palmar and digital fascia, leading to disfigurement and functional impairment of the hand. Recently, DD was characterized as a useful model of fibrosis, since it displays the entire temporal and histological architecture of cells, cytokines and extracellular matrix involved in fibroproliferative processes(16). Like other fibroproliferative disorders, TGFB1 is a pivotal factor during pathogenesis of palmar fibrosis, where inhibitory SMAD7 acts to oppose signal(17) transducing R- and Co-SMADs by forming stable associations with activated type I receptors, thereby preventing phosphorylation of R-SMADs, thus acting as a negative feedback regulator(18).
Since this model provides a reasonable explanation for induction and progression of fibroproliferative phenomena, it does not explain clinical findings in patients suffering from DD, where sudden arrest of the disease through all stages can be observed.
During an immunohistochemical study we surprisingly found high levels of intra- and extracellular INFG. Since other groups have demonstrated that INFG leads to SMAD7 upregulation and thereby blocking TGFB1 signaling our study addressed the question whether endogenous expression of INFG is influenced by TGFB1, thus indicating a bidirectional crosstalk of signaling pathways.
Cells – Tissues from Dupuytren’s disease (DF) and Control Fibroblasts (CF) derived from normal tendon pulleys were obtained, isolated and cultured as described previously (21). All individuals suffered from contractures of the fingers rated as second or third degree deformities according to the Tubiana score (22).
Immunohistochemistry/-fluorescence – DF and CF were incubated on 8-well-Chamber Slides (Nunc) and fixed in ice-cold ethanol at the end of each experiment. Subsequently, cells were rehydrated in PBS, blocked with fetal calf serum (Biochrom) and incubated with primary antibodies at 4 °C diluted in antibody diluent (DAKO) overnight. The next day, slides were washed 3 times with PBS and incubated for 1 hour with secondary antibodies conjugated to Biotin. After another 3 washing steps with PBS slides were incubated with StreptABComplex/HRP (DAKO) for 30 minutes and stained with AEC + Substrat-Chromogen (DAKO) for 5 minutes. Mayers-Hämalaun (Merck) was used for nucleus-staining. Slides were mounted with Faramount Mounting Medium (DAKO) for microscopy. For Immunofluorescence specimen were rehydrated in PBS, blocked with donkey-serum (Jackson ImmunoResearch) and incubated with primary antibodies diluted in antibody diluent overnight at 4 °C. The following day, slides were washed 3 times with PBS and incubated with multi-labeling secondary antibodies conjugated to either cy2 or cy3 or cy5 (Jackson ImmunoResearch) at room temperature for 1 hour avoiding light expression. After secondary labeling, slides were washed 3 times in PBS and rinsed in DAPI (Sigma-Aldrich) for nucleus staining and mounted in fluorescent mounting medium (DAKO). Imaging of stained slides was performed using an Axioplan2 microscope (Zeiss). All slides were treated identically and scanned using the same settings in each experiment. A list of used antibodies is provided in Supplemental Table 1.
Preparation of whole cell lysates and immunoblot analysis – Total lysates from DF and CF were prepared by solubilization in RIPA lysis buffer (Santa Cruz) according to manufacturer’s instructions. The amount of protein in lysates was estimated by BCA protein quantification assay (Pierce). Twenty micrograms per lane of protein were loaded onto NuPAGE Novex Bis-Tris or NuPAGE Novex Tris-Acetate Gels (Invitrogen) for electrophoresis in a Xcell SureLock Electrophoresis cell (Invitrogen). Separated proteins were transferred to polyvinyldifluorid membranes (Roth) using an Xcell II Blot Module (Invitrogen). Primary antibody and horseradish-peroxidase-secondary antibody labeling was performed using guidelines proposed by blot module manufacturer followed by incubation with western blotting luminol Reagent (Santa Cruz). Chemoluminescence signal was detected by using a Lumi-Imager LAS 1000 (FujiFilm). Quantitation of bands in immunoblot results was performed by using Syngene GeneTools 3.08 software (Synoptics Ltd). A list of used antibodies is provided in Supplemental Table 1.
Qualitative and quantitative RT-PCR – Total RNA was purified from DF and CF monolayer cell cultures with an RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. During purification RNA was treated with DNase1 (Qiagen) to avoid contamination with genomic DNA. To generate cDNA 2 micrograms of total RNA were reverse transcribed using Omniscript RT Kit (Qiagen) according to manufacturer’s instructions. Qualitative PCR was performed using Hot Star Taq Plus DNA Polymerase Kit (Qiagen) following manufacturer’s guidelines. Real time quantitative PCR was conducted using an iQ iCycler Real-Time PCR Detection System (BioRad) with SYBR green fluorophore (ABgene). Quantification was performed by using the ΔΔCT method. All used primers were QuantiTect Primer Assay primers (Qiagen). PCR conditions are provided in Supplemental Table 2.
siRNA – siRNA for JAK1 and STAT1 was synthesized by Qiagen (Qiagen) and was transfected by HiPerfect Transfection reagent (Qiagen) according to manufacturer’s instructions.
Adenoviral construction and purification – Smad7 expressing adenovirus has been described previously (23). Human IFNγ coding adenovirus was constructed using the Transpose-Ad Adenoviral Vector System (Qbiogene). Briefly, human IFNγ was excised from pORF hINFγ (Invitrogen) using SgrAI and NheI and ligated into transfervector pCR259. Subsequently, pCR259 was transposed in Transpose-Ad 294 vector. Transpose-Ad 294 vector was linearized using PacI and transfected in HEK 293 cells (Biochrom) using Superfect Transfection Kit (Qiagen). Adenoviruses were amplified using AdenoX Virus Purification Kit (Clontech) corresponding to Manufacturer’s instructions. Infections of DF and CF were performed according to other reports (24). Routine infections were accomplished at 50 m.o.i. with single virus clones of the same virus stock preparation. Infection efficiency was proven by adenoviral constructs expressing β-Gal and, each experiment, about 90% of the cells were infected. Staining of infected cells was performed using an X-Gal staining kit (Roche).
TGFB1 responsive reporter assay – For TGFB1 responsive reporter assay DF and CF were infected with (CAGA)9-MLP-Luc (25) as described previously (21). Cell lysis and luciferase assays were performed using Steady-Glo® Luciferase Assay System (Promega). Luciferase activity was measured in a CENTRO LB 960 Luminometer (Berthold Technologies).
Human subjects – Written informed consent to perform research on surgically exzised human tissues was obtained from all patients. The research protocol applied during the experiments was approved by the Ethics Committee of the Medical Faculty of the University of Erlangen.
Statistical analysis – Results were given mean ± standard deviation. Statistical analysis was performed by using GraphPad Prism 4.02. According to data 1- or 2-way ANOVA with Bonferroni post hoc tests was applied. P-values < 0.05 were considered significant.