Liver fibrosis is the common sequel of chronic liver injury of variable origin (viral infection, metabolic diseases and toxin). Recent studies have identified hepatic stellate cells (HSCs) as the primary cell type mediating hepatic fibrosis 12. In normal liver, HSCs are the site of storage and metabolism of vitamin A 34. During hepatic fibrogenesis, HSCs proliferate and undergo a process of activation, developing a myofibroblast-like appearance. Activated HSCs appear to lose lipid droplet, increase rough endoplasmic reticulum, and express smooth muscle alpha actin (SMA) 567. Activated HSCs also increase the synthesis of extracellular matrix components that form the major part of the fibrotic liver 89.

Interferons were first discovered as anti-viral soluble protein and at present, interferon-α is the main medication for the treatment of viral hepatitis 1011. Interferons consist of type 1 interferon, which includes interferon-α, -β and -ω, and type II interferon, which is interferon γ. Several subtypes are observed in human interferon. Interferon-α has at least 14 subtypes, interferon-β has 3 subtypes and interferon-γ has 5 subtypes 1213. Biological activities of interferons are initiated by the interaction of interferons with cell surface type 1 and/or type II interferon receptors. This interaction brings together two receptor subunits. These two receptor subunits are not pre-associated on the cell surface but rather are induced to be associated in the presence of ligand 14. The formation of the heteromeric receptor results in the formation of a functionally active receptor that leads to the activation of cytoplasmic proteins and interferon signaling 15. IFN-α and IFN-β appear to utilize type 1 IFN receptor complex and activate similar intracellular signaling pathways 1617. IFN-γ seems to activate type II IFN receptor. Ligand-induced association of both types of IFN receptor results in the phosphorylation of the receptors by Janus kinases 181920, which subsequently results in the activation of STATs (signal transducers and activators of transcription) proteins by additional phosphorylation events 2122. Such events lead to the formation of IFN-inducible transcription factors that bind to IFN response elements presented in IFN-inducible gene 23.

Although it is generally considered that IFN-α and IFN-β utilize a common receptor complex, a number of observations suggest that differences may occur in the abilities of IFN-α and/or IFN-β to induce certain biological effects. These include the preferential induction of an IFN-specific gene 2425, different growth inhibitory effects 26, and erythropoietic effects 27. One possible explanation for the different signaling events between IFN-α and IFN-β would be the existence of IFN-β specific receptor associated phosphoprotein (BRAP), which appears to be tyrosine phosphorylated and to be associated with IFNAR1 (interferon alpha-receptor 1) 2829.

Primary rat hepatic stellate cells were isolated and cultured for 9 days before the cells were detached and sub-cultured for the study of interferon regulation of proliferation and activation. Figure 1 shows the morphology of sub-cultured HSCs at days 3 (Figure 1A) and 12 (Figure 1B) under phase contract microscope. The fluorescent images of SMA expression in rat HSCs were presented in Figure 1C and 1D for sub-cultured 3-day and 12-day HSCs respectively. At day 12, all HSCs displayed myofibroblast phenotype and expressed SMA. The expression of SMA and desmin proteins at different times (1, 3, 6, 9, and 12 days) of the sub-cultured HSCs was shown in Figure 2. SMA gradually increased from day 1 to day 12 and reached a maximum level at day 12 while desmin abundance did not elevate as much as observed on the SMA. The result indicated that HSCs fully differentiated into myofibroblast-like phenotype after 12 days sub-culture in vitro. We then examined the effect of IFN-α, IFN-β and IFN-γ on cell proliferation and DNA synthesis in sub-cultured HSCs. Figure 3 showed the effect of IFN-α, IFN-β and IFN-γ on proliferation of subcultured HSCs by WST-1 cell proliferation assay. As shown in Figure 3A, IFN-α did not affect sub-cultured HSC proliferation and it also did not affect Bromo-2'-deoxy-urindne (BrdU) incorporation in HSCs (Figure 3D). In contrast, both IFN-β and IFN-γ significantly inhibited subcultured HSC proliferation and BrdU incorporation. IFN-β inhibition of HSC proliferation was observed at day 6 (32% decrease relative to control) and still could be observed at day 9 (18% decrease relative to control) (Figure 3B). IFN-γ also inhibited HSC proliferation at day 9 (40% decrease relative to control) (Figure 3C). Both IFN-β and IFN-γ significantly inhibited BrdU incorporation in HSCs (about 10 % decrease relative to control for both IFN-β and IFN-γ) (Figure 3D). Moreover, both IFN-β and IFN-γ reduced the cell number after 7 days treatment (500 U/ml IFB-β = 4.5 × 10⁵ ± 0.10 × 10⁵ cells/ml vs. control = 5.2 × 10⁵ ± 0.12 × 10⁵ cells/ml, p < 0.01; 500 U/ml IFN-γ = 4.8 × 10⁵ ± 0.1 × 10⁵ cells/ml vs. control = 6.0 × 10⁵ ± 0.15 × 10⁵ cells/ml, p < 0.01) The inhibitory effect of IFN-β on HSC proliferation was not dose dependent (Figure 4) while the effect of INF-γ on HSC proliferation seemed to correlate with the dose. However, the statistically significant difference of inhibition was only observed at the highest concentration of INF-γ employed.

We then examined the effect of IFNs on the expression of SMA, which is a phenotypic marker of activated HSCs. Western blot analysis indicated that both IFN-β and IFN-γ decreased SMA expression in sub-cultured HSCs when they were exposed to the cytokines for 3 or 6 days (Figure 5A), but IFN-α did not affect SMA abundance after the same length of treatment. However, when sub-cultured HSCs were exposed to the cytokines for 9 days, both IFN-α and IFN-β reduced SMA level in a small attitude while IFN-γ did not alter the abundance of SMA. Analyses of the blots by densitometric scanning revealed that IFN-β and IFN-γ reduced the SMA level by 76% ± 3% and 73% ± 7% respectively after HSCs were incubated with 500 U/ml IFN-β and IFN-γ for six days (Figure 5B). In addition, we also observed that water alone did not affect the expression of SMA in 6-day rat HSCs (Figure 6A) as well as transforming growth factor beta1, bone morphogenetic protein 2 and bone morphogenetic protein 4 promoted the expression of SMA in rat HSCs (Figure 6B).

The present study investigated the effect of IFN-α, IFN-β, and IFN-γ on the proliferation and SMA expression of rat hepatic stellate cells cultured on uncoated plastic dish. Rat hepatic stellate cells have been documented to exhibit proliferation and morphological change in experimental hepatic fibrosis 30 or in human liver specimens obtained from patients with fibrotic liver disease 3132. IFN-α is an effective drug for the treatment of patients with hepatitis B virus or C virus infection. The antiviral efficiency of IFN-α is almost the same as IFN-γ; however, IFN-α has fewer side effects than IFN-γ. In the other aspect, IFN-γ has anti-fibrogenic effect as it is documented that IFN-γ inhibits collagen synthesis in several cell types 33343536. Moreover, IFN-γ delays phenotypic trans-differentiation of rat hepatic stellate cells in vitro 37. Furthermore, IFN-γ reduces SMA expression in human HSCs, arterial smooth muscle cells and dermal myofibroblasts 3839. Our results with IFN-γ were consistent with these reports. We also observed that IFN-γ reduced SMA expression in cultured rat HSCs. Several clinical studies have suggested that IFN-α has anti-fibrogenic activity. Most of the studies were conducted in patients with chronic hepatitis C. This suggests that IFN-α may reduce the histological fibrosis index of the Knodell score in responders, which may be consequent to the antiviral properties of the drug 101140. Furthermore, some studies point to a direct anti-fibrogenic effect of IFN-α, independent of its antiviral property 41. In these studies, IFN-α decreased collagen concentration and SMA index in not only responders but also non-responders or relapsers. An in vitro study confirmed these results by documenting that IFN-α-2c inhibits human hepatic stellate cell proliferation and collagen product at the concentration (10000 U/ml) higher than a single therapeutic dose 42 (peak plasma concentrations range between 40 U/ml to 150 U/ml 4344). In our current study, we did not observe that rat IFN-α inhibited rat hepatic stellate cell proliferation and SMA expression at the concentrations from 100 to 1000 U/ml. In contrast to IFN-α, we showed that rat IFN-β significantly inhibited rat hepatic stellate cell proliferation and SMA expression at the concentration of 500 U/ml. Although the concentration was higher than the single therapeutic dose of IFN-α used in clinical treatment, it is within the range of drug accumulation after repeated administration, which is by the factor of 2 to 5 of the single administration 45. Moreover, the difference between this study and others regarding the effect of IFN-α on HSCs could also be due to the different species employed in studies. In this study, rats were employed while in the other study 42 human HSCs were used. In addition, more than 14 subtypes of human IFN-α have been identified and each subtype of IFN-α has different binding affinity to type 1 IFN receptor 46. At present, only one subtype of rat IFN-α has been identified.

The effect of IFNs on rat HSC proliferation in this study was low and marginal especially at early time points. Since rat HSCs were cultured in 10% serum condition, proliferation of rat HSCs has been stimulated by the other factors in serum. In other studies relating to HSC proliferation, serum-reducing condition has been employed which utilized 0.1%-1% serum 47. Dramatic differences of cell proliferation and activation of HSCs were observed in this culture condition. Recently we also observed that when rat HSCs were cultured in 5% serum condition or serum-free condition for a short period, IFNs and other cytokines could dramatically affect cell proliferation and expression of SMA in rat HSCs (data not shown).

Our results also showed the first time that IFN-α and IFN-β induced different biological effects on rat hepatic stellate cells. The different biological effects of IFN-α and IFN-β have been documented in human glioma cells 26 and in the selected Tyk2-deficient cell lines 4849. In these cells, growth inhibition and gene expression are responsive to IFN-β but not to IFN-α. The mechanism of the differences between IFN-α and IFN-β biological effects is still unknown. However, it is known that type 1 IFNs bind to un-associated type 1 IFN receptor and assemble two chains of IFNAR1 and IFNAR2. The assembled type 1 IFN receptor will be phosphorylated by associated kinases, which would lead to intracellular signaling events. One difference between IFN-α and IFN-β at the receptor level is that there is a phosphoprotein selectively involved in IFN-β signaling 2425. Two different research groups demonstrated this tyrosine-phosphorylated protein to be IFNAR2.2 2829. By employing specific antibody against IFNAR2.2, they documented that IFNAR2.2 is present in Daudi cells as a cell surface protein approximately 90–100 kDa, which is tyrosine-phosphorylated and associated with IFNAR1 upon stimulation of cells with IFN-β but not IFN-α. Their studies suggest that there are some differences in receptor interaction between IFN-α and IFN-β in HSCs. However, it is still unclear why this phosphoprotein is not related with IFN-α and what IFN-β specific responses are associated with the IFN-β induced phosphoprotein. Our results suggested that HSCs might serve as the cell type to investigate the different responses of IFN-α and IFN-β.

Rat interferon-α and interferon-β have different biological effects on rat hepatic stellate cells and different signaling events might exist between interferon-α and interferon-β in hepatic stellate cells.

Dr. Shen H carried out the isolation of hepatic stellate cells, Western blot analysis and cell proliferation assay. Mrs. Zhang MN participated in the cell imaging. Dr. Minuk GY participated in the design of the study. Dr. Gong Y conceived of the study and participated in its design and coordination.

All authors read and approved the final manuscript.