Interferon-alpha reduced inflammatory effects of filgrastim (G-CSF) in the liver of Syrian mice

Introduction: granulocyte colony-stimulating factor (G-CSF) has been widely used for the treatment of chemotherapy-induced neutropenia. One of the major sideeffects of interferon-α (IFN-α) therapy is neutropenia. Previous studies have confirmed the beneficial effects of co-administration of G-CSF and IFN-α on neutropenia in patients infected with hepatitis C. In this study for the first time, the effects of co-administration of type I IFNs and G-CSF on liver and liver enzymes investigated. Methods: forty-two mice (male, eight weeks) were randomly divided into six groups of seven: distilled water, G-CSF (200μg/kg), IFN-α (200μg/kg), IFN-β (200μg/kg), IFN-α+G-CSF and IFN-β+G-CSF. After 28 days, blood was taken from the heart of each mouse and histological changes in the liver and liver enzymes including aspartate transaminase (AST) and alanine transaminase (ALT), as well as bilirubin, were measured. Results: Surprisingly, in most cases, the G-CSF and type one IFNs alone or simultaneously reduced the levels of AST and bilirubin. The levels of ALT induced by IFN-α, the addition of G-CSF to IFN-α reduced the level of this liver enzyme. G-CSF induced cell infiltrations into the liver tissue, addition of IFN-α but not IFN-β to G-CSF obviously reduced the cell infiltration into the liver. Conclusion: Since the changes in liver enzymes and bilirubin were not at harmful levels, and the administration of IFN-α to G-CSF reduced the cell infiltrations into the liver, our results suggested that co-administration of type I IFNs and G-CSF had no harmful effects on liver histology and functions. iD D ow nl oa de d fr om p pj . p hy ph a. ir at 1 0: 21 + 03 30 o n M on da y N ov em be r 4t h 20 19 29 | Physiol Pharmacol 23 (2019) 28-35 Namjou et al. multiple reports indicated an increased expression of these factors in inflammatory diseases (Nakamura et al., 2000). G-CSF has been introduced in several studies as an inflammatory factor that plays a role in the inflammatory response (Eyles et al., 2008). Pharmaceutical forms of this factor are filgrastim and lenograstim, which are widely used in cases of neutropenia following chemotherapy and bone marrow transplants and improve heart function after acute myocardial infarction. Filgrastim drugs may have several side effects such as allergic reactions, enlarged spleen, arrhythmia and vascular inflammation. Other known side effects of this drug are hepatomegaly (enlarged liver), transient hypotension, urinary disorders (such as hematuria), osteoporosis, thrombocytopenia, anemia, decreased transiently in blood glucose and increased uric acid (Dale et al., 2003; Renwick et al., 2009). Interferons are produced by the host cells as a defensive response to pathogens such as viruses and tumors. Type I interferons are widely used in the treatment of leukemia, viral hepatitis and multiple sclerosis. Previous studies have indicated the antiinflammatory properties of type I interferons (IFN-α and IFN-β). Type I IFNs down-regulate the formation of proinflammatory cytokines such as IL-1b and TNFα in fibroblast-like synoviocytes (Palmer et al., 2004). IFN-β inhibits the proliferation and the migration of Tcells (Stüve et al., 1996). The type one IFNs also induce the production of immunosuppressive cytokine of IL-10 that inhibits the pro-inflammatory gene (Benveniste and Qin, 2007). The liver plays a pivotal role in drug degradation as well as in the metabolism of carbohydrates, fats and protein. Changes in liver enzymes can indicate liver damage or changed bile flow. Transaminases are among of the most important enzymes of the liver that is interested in laboratory professionals. Two important transaminase enzymes often measured in clinical laboratories are aspartate aminotransferase (AST) and alanine aminotransferase (ALT). One of the substances secreted by the liver is bilirubin and like other secretions such as alkaline phosphatase, cholesterol and products of cholesterol (bile acids) are excreted by the liver through the bile. Changes in liver enzymes along with other clinical investigations can be used to diagnose diseases (Giannini et al., 2005). AST is currently used to diagnose liver disease. The level of AST has greatly increased in liver parenchymal diseases such as viral hepatitis, toxic hepatitis and infectious mononucleosis. AST is also increased in obstructive liver disease (obstruction of the liver, both inside and outside). During the process of cirrhosis, AST levels could be higher than average (Sookoian and Pirola, 2015). The levels of liver enzymes such as AST and ALT can also indicate damage to the liver parenchymal cells in the inflammatory process. It has been suggested that the levels of AST and ALT in the serum can be used as markers for inflammation (except in cirrhosis) of the liver (Gaia et al., 2006). Neutropenia is a major side effect of IFN-α therapy which could be due to the suppression of G-CSF production in the presence of IFN-α (Tajuddin et al., 2010). Previous studies have confirmed the beneficial effects of G-CSF administration on neutropenia in patients with hepatitis C infections (Sharvadze et al., 2007; Koskinas et al., 2009). Since the co-treatment of G-CSF and IFNs could be beneficial, and due to the wide therapeutic use of these cytokines, in this paper for the first time we investigated the simultaneous effects of G-CSF and IFN-α or IFN-β on liver and liver enzymes. Materials and methods Animals and treatments Forty-two male Syrian mice (eight-weeks) were obtained from the animal house at the School of Pharmacy, Mashhad University of Medical Sciences, Iran. The whole process of the study was carried out in compliance with the guidelines for the care and use of Laboratory Animals published by the National Institutes of Health (NIH Publications No.8023, revised 1978) and was approved by the ethical committee of The Ferdowsi University of Mashhad. The mice had free access to food and water and were maintained on a 12h dark/light cycle in a room with controlled temperature (26-27°C) and humidity (60±10%). Recombinant human G-CSF and type one IFNs were purchased from Pooyesh Darou (Tehran, Iran) and were reconstituted in distilled water. Mice were randomly divided into six groups which received intraperitoneal injection (n=7 in each group): group one (control): distilled water, group two: G-CSF (200μg/kg), group three: IFN-α (200μg/kg), group four: IFN-β (200μg/kg), group five: same dosages of D ow nl oa de d fr om p pj . p hy ph a. ir at 1 0: 21 + 03 30 o n M on da y N ov em be r 4t h 20 19 Filgrastim, type I interferons and liver Physiol Pharmacol 23 (2019) 28-35 | 30 G-CSF + IFN-α and group six: same dosages of GCSF + IFN-β. After 28 days, the mice were anesthetized with ketamine and then, one ml of blood was taken from their heart with a syringe. After collecting blood, serum was separated from blood cells using a centrifuge for 15 minutes at 4,000 rpm (Kakusan, Japan) and samples were sent to the medical laboratory of Veterinary Medicine of Ferdowsi University to measure serum levels of liver enzymes (AST and ALT) and bilirubin. In the next step to assess liver histology, small sample tissues were isolated and were maintained in 10% formaldehyde solution to fix it. After standard tissue processing, the samples were stained with hematoxylin-eosin. Statistical analysis Differences among groups were assessed by twoway ANOVA with Tukey’s post hoc analysis to identify statistical differences among the treatments when P was less than 0.05.