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 [31
]. 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 [33
]. 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 [38
]. 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 [10
]. 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 [43
]). 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 [48
]. 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 [24
]. Two different research groups demonstrated this tyrosine-phosphorylated protein to be IFNAR2.2 [28
]. 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.