Scandinavian Journal of Gastroenterology. 2015; 50: 204–210
ORIGINAL ARTICLE
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Laval on 03/04/15 For personal use only.
Nitric oxide is a potential mediator of hepatic inflammation and fibrogenesis in autoimmune hepatitis YAVUZ BEYAZIT1, CUMALI EFE2, ALPASLAN TANOGLU3, TUGRUL PURNAK4, ABDURRAHIM SAYILIR5, ISMAIL TASKIRAN6, MURAT KEKILLI7, TURAN TURHAN8, ERSAN OZASLAN4 & STAFFAN WAHLIN9 1
Department of Gastroenterology, Canakkale State Hospital, Canakkale, Turkey, 2Department of Gastroenterology, Hacettepe University Medical Faculty, Ankara, Turkey, 3Department of Gastroenterology, Gulhane Military Medicine Academy, Istanbul, Turkey, 4Department of Gastroenterology, Numune education and Research hospital, Ankara, Turkey, 5Department of Gastroenterology, Giresun State Hospital, Giresun, Turkey, 6Department of Gastroenterology, Ordu State Hospital, Ordu, Turkey, 7Department of Gastroenterology, Hitit Univesity Hospital, Corum, ¸ Turkey, 8 Department of Biochemistry, Numune education and Research hospital, Ankara, Turkey, and 9Department of Gastroenterology and Hepatology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
Abstract Background. Despite advances in the understanding of the pathophysiological basis of autoimmune hepatitis (AIH), it is still difficult to delineate the mechanisms involved in progression from hepatic inflammation toward fibrosis. Our aim was to study serum concentrations of NO in AIH of different histological severity and possible effects of immunosuppressive therapy on NO production. Materials and methods. We studied serum NO metabolites (NOx) in 47 consecutive patients with AIH and in 28 age- and sex-matched controls. Results. Serum NOx concentrations were higher in AIH patients than in controls (10.3 (4.5–27.3 mmol/L) vs. 4.3 (1.6–14.3 mmol/L), p < 0.001). According to liver histology, median NOx concentrations were significantly higher in patients with severe interface hepatitis compared to patients with mild-moderate interface hepatitis (12.3 (4.5–27.3 mmol/L) vs. 9.3 (4.6–20.3 mmol/L), p = 0.029). Similarly, serum NOx concentrations were significantly higher in patients with advanced fibrosis than in those with early fibrosis (12.2 (4.6–27.3 mmol/L) vs. 9.3 (6.6–12.8 mmol/L), p = 0.018). NOx concentrations decreased in 16 AIH patients who were tested also after biochemical remission was achieved (12.6 (4.5–22.8 mmol/L) at baseline and 5.9 (2.8–10.5 mmol/L) after remission, p = 0.001). Conclusion. This study shows that serum NOx levels are associated with the histological severity of AIH. Hepatocyte inflammation and injury may activate hepatic stellate cells and kupffer cells, and the consequences may include release of NO, which ultimately promotes hepatic fibrosis. Immunosuppressive therapy inhibits this process and the production of NO.
Key Words: autoimmune hepatitis, fibrosis, nitric oxide, oxidative stress
Introduction Autoimmune hepatitis (AIH) is a chronic inflammatory hepatopathy that can lead end-stage liver failure and death [1,2]. The exact pathophysiology of the disease is not entirely understood. It is believed that some environmental factors trigger the inflammatory process in genetically susceptible individuals [3,4]. Chronic inflammation generally progresses into
hepatic fibrosis and cirrhosis if untreated. Currently, immunosuppression is the standard therapy for AIH and reverses or prevents hepatic fibrosis in 57–79% of patients [5]. However, complete treatment response is not achieved in all treated patients. A recent large population-based study showed that long-term mortality is higher in AIH than in the general population despite good initial response to immunosuppression [6].
Correspondence: Cumali Efe, MD, Department of Gastroenterology, Hacettepe University Medical Faculty, Ankara, Turkey. E-mail: [email protected]
(Received 29 July 2014; revised 30 September 2014; accepted 3 October 2014) ISSN 0036-5521 print/ISSN 1502-7708 online 2015 Informa Healthcare DOI: 10.3109/00365521.2014.974203
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Laval on 03/04/15 For personal use only.
Nitric oxide levels in autoimmune hepatitis Oxidative stress has been shown to be important in the fibrogenesis of several chronic liver diseases, including chronic viral hepatitis, alcoholic liver disease, hepatoportal sclerosis, primary biliary cirrhosis (PBC), as well as in AIH [7,8]. Oxidative stress includes multiple pathways that start with apoptosis of hepatocytes. The released apoptotic bodies further increase serum levels of proinflammatory cytokines and this leads to fibrosis and finally cirrhosis [9,10]. Activated Kupffer cells contribute to this process by producing reactive oxygen species (ROS), nitric oxide (NO), cytokines, and chemokines [8,11]. NO has been shown to have a role in various clinical situations including vasodilatation, neurotransmission, tumoral development, and antimicrobial activity. NO is also a critical mediator in some organ-specific and non-organ-specific autoimmune disorders [12,13]. Accumulating evidence suggests that NO may promote fibrosis progression in choronic liver diseases [10]. Therefore, it is reasonable to investigate the possible role of NO in the pathogenesis and promotion of immune-mediated liver diseases. In this study, we aimed to evaluate oxidative stress in AIH patients by measuring serum NO levels. We hypothesize that circulating NO reflect Kupffer cell activity as well as oxidative facets of the disease process. We also investigated any correlations between the serum NO levels and histological features of AIH, including fibrosis stage and severity of interface hepatitis. Furthermore, we assessed the effects of immunosuppressive therapy on NO production.
205
aminotransferase and IgG levels while receiving treatment [15]. We included age- and sex-matched healthy subjects who did not have evidence of liver diseases or other chronic disorders. Neither patients nor controls were receiving any medications that might affect neutrophil function. The study was carried out in accordance with the guidelines of the Helsinki declaration. NO assay Fasting serum blood samples were obtained from each patient without using any anticoagulant and then centrifuged at 3000 rpm for about 10 min and stored at 80 C until the biochemical estimation of nitric oxide. As previously described [16] NO was determined as NO metabolites nitrite/nitrate (NOx) by the Griess reaction after conversion of nitrate into nitrite by nitrate reductase. A commercially available Nitric Oxide Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA) was used to assess NOx as an index of total NO production according to manufacturer’s instructions. Serological assessment
Materials and methods
Antinuclear antibodies (ANA), smooth muscle antibody (SMA), liver–kidney microsomal antibodies (LKM-1), and antibodies to soluble liver antigen were analyzed according to local laboratory standards. An indirect immunofluorescence titer of 1:40 or higher for ANA, SMA, and LKM-1 was considered to be positive.
Patient selection
Histological assessment
Serum from consecutive patients with AIH who were diagnosed at Ankara Numune Education and Research Hospital, Hitit University, and Canakkale ¸ State Hospital, Turkey, were used for the present study. AIH was diagnosed according to the simplified AIH criteria suggested by the International Autoimmune Hepatitis Group (IAIHG) [14]. Patients were excluded if they had other concomitant chronic liver disorders including HBV, HCV, PBC, primary sclerosing cholangitis, Wilson’s disease, hemochromatosis, or a history of excessive alcohol consumption. Patients with malignancies, diabetes mellitus, proliferative hematological disorders, or infectious diseases were also excluded. All patients were immunosuppression-naive at presentation. After collection of the first serum samples, the patients were treated with prednisone (30–60 mg/day) alone or in combination with azathioprine (50–100 mg/day). Biochemical remission was defined as the normalization of
The METAVIR score was used for staging liver fibrosis according to a five (0–4)-point scale [17], where F0 = no fibrosis, F1 = portal fibrosis without septa, F2 = few septa, F3 = numerous septa without cirrhosis, and F4 = cirrhosis. Patients with F1–2 were considered to have early-stage fibrosis and those with F3–4 to have advanced fibrosis. Presence of lymphocytic piecemeal necrosis was graded as mild (necrosis around 1–2 portal tracts), moderate (necrosis at the periphery of about half of the portal tracts), and severe (necrosis surrounding more than half of the circumference of almost all portal tracts). Typical and compatible histology for AIH was defined according to the IAIHG criteria [14]. Statistical analysis All data were analyzed using the SPSS version 18.0 (SPSS, Chicago, IL, USA), and p
ORIGINAL ARTICLE
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Laval on 03/04/15 For personal use only.
Nitric oxide is a potential mediator of hepatic inflammation and fibrogenesis in autoimmune hepatitis YAVUZ BEYAZIT1, CUMALI EFE2, ALPASLAN TANOGLU3, TUGRUL PURNAK4, ABDURRAHIM SAYILIR5, ISMAIL TASKIRAN6, MURAT KEKILLI7, TURAN TURHAN8, ERSAN OZASLAN4 & STAFFAN WAHLIN9 1
Department of Gastroenterology, Canakkale State Hospital, Canakkale, Turkey, 2Department of Gastroenterology, Hacettepe University Medical Faculty, Ankara, Turkey, 3Department of Gastroenterology, Gulhane Military Medicine Academy, Istanbul, Turkey, 4Department of Gastroenterology, Numune education and Research hospital, Ankara, Turkey, 5Department of Gastroenterology, Giresun State Hospital, Giresun, Turkey, 6Department of Gastroenterology, Ordu State Hospital, Ordu, Turkey, 7Department of Gastroenterology, Hitit Univesity Hospital, Corum, ¸ Turkey, 8 Department of Biochemistry, Numune education and Research hospital, Ankara, Turkey, and 9Department of Gastroenterology and Hepatology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
Abstract Background. Despite advances in the understanding of the pathophysiological basis of autoimmune hepatitis (AIH), it is still difficult to delineate the mechanisms involved in progression from hepatic inflammation toward fibrosis. Our aim was to study serum concentrations of NO in AIH of different histological severity and possible effects of immunosuppressive therapy on NO production. Materials and methods. We studied serum NO metabolites (NOx) in 47 consecutive patients with AIH and in 28 age- and sex-matched controls. Results. Serum NOx concentrations were higher in AIH patients than in controls (10.3 (4.5–27.3 mmol/L) vs. 4.3 (1.6–14.3 mmol/L), p < 0.001). According to liver histology, median NOx concentrations were significantly higher in patients with severe interface hepatitis compared to patients with mild-moderate interface hepatitis (12.3 (4.5–27.3 mmol/L) vs. 9.3 (4.6–20.3 mmol/L), p = 0.029). Similarly, serum NOx concentrations were significantly higher in patients with advanced fibrosis than in those with early fibrosis (12.2 (4.6–27.3 mmol/L) vs. 9.3 (6.6–12.8 mmol/L), p = 0.018). NOx concentrations decreased in 16 AIH patients who were tested also after biochemical remission was achieved (12.6 (4.5–22.8 mmol/L) at baseline and 5.9 (2.8–10.5 mmol/L) after remission, p = 0.001). Conclusion. This study shows that serum NOx levels are associated with the histological severity of AIH. Hepatocyte inflammation and injury may activate hepatic stellate cells and kupffer cells, and the consequences may include release of NO, which ultimately promotes hepatic fibrosis. Immunosuppressive therapy inhibits this process and the production of NO.
Key Words: autoimmune hepatitis, fibrosis, nitric oxide, oxidative stress
Introduction Autoimmune hepatitis (AIH) is a chronic inflammatory hepatopathy that can lead end-stage liver failure and death [1,2]. The exact pathophysiology of the disease is not entirely understood. It is believed that some environmental factors trigger the inflammatory process in genetically susceptible individuals [3,4]. Chronic inflammation generally progresses into
hepatic fibrosis and cirrhosis if untreated. Currently, immunosuppression is the standard therapy for AIH and reverses or prevents hepatic fibrosis in 57–79% of patients [5]. However, complete treatment response is not achieved in all treated patients. A recent large population-based study showed that long-term mortality is higher in AIH than in the general population despite good initial response to immunosuppression [6].
Correspondence: Cumali Efe, MD, Department of Gastroenterology, Hacettepe University Medical Faculty, Ankara, Turkey. E-mail: [email protected]
(Received 29 July 2014; revised 30 September 2014; accepted 3 October 2014) ISSN 0036-5521 print/ISSN 1502-7708 online 2015 Informa Healthcare DOI: 10.3109/00365521.2014.974203
Scand J Gastroenterol Downloaded from informahealthcare.com by University of Laval on 03/04/15 For personal use only.
Nitric oxide levels in autoimmune hepatitis Oxidative stress has been shown to be important in the fibrogenesis of several chronic liver diseases, including chronic viral hepatitis, alcoholic liver disease, hepatoportal sclerosis, primary biliary cirrhosis (PBC), as well as in AIH [7,8]. Oxidative stress includes multiple pathways that start with apoptosis of hepatocytes. The released apoptotic bodies further increase serum levels of proinflammatory cytokines and this leads to fibrosis and finally cirrhosis [9,10]. Activated Kupffer cells contribute to this process by producing reactive oxygen species (ROS), nitric oxide (NO), cytokines, and chemokines [8,11]. NO has been shown to have a role in various clinical situations including vasodilatation, neurotransmission, tumoral development, and antimicrobial activity. NO is also a critical mediator in some organ-specific and non-organ-specific autoimmune disorders [12,13]. Accumulating evidence suggests that NO may promote fibrosis progression in choronic liver diseases [10]. Therefore, it is reasonable to investigate the possible role of NO in the pathogenesis and promotion of immune-mediated liver diseases. In this study, we aimed to evaluate oxidative stress in AIH patients by measuring serum NO levels. We hypothesize that circulating NO reflect Kupffer cell activity as well as oxidative facets of the disease process. We also investigated any correlations between the serum NO levels and histological features of AIH, including fibrosis stage and severity of interface hepatitis. Furthermore, we assessed the effects of immunosuppressive therapy on NO production.
205
aminotransferase and IgG levels while receiving treatment [15]. We included age- and sex-matched healthy subjects who did not have evidence of liver diseases or other chronic disorders. Neither patients nor controls were receiving any medications that might affect neutrophil function. The study was carried out in accordance with the guidelines of the Helsinki declaration. NO assay Fasting serum blood samples were obtained from each patient without using any anticoagulant and then centrifuged at 3000 rpm for about 10 min and stored at 80 C until the biochemical estimation of nitric oxide. As previously described [16] NO was determined as NO metabolites nitrite/nitrate (NOx) by the Griess reaction after conversion of nitrate into nitrite by nitrate reductase. A commercially available Nitric Oxide Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA) was used to assess NOx as an index of total NO production according to manufacturer’s instructions. Serological assessment
Materials and methods
Antinuclear antibodies (ANA), smooth muscle antibody (SMA), liver–kidney microsomal antibodies (LKM-1), and antibodies to soluble liver antigen were analyzed according to local laboratory standards. An indirect immunofluorescence titer of 1:40 or higher for ANA, SMA, and LKM-1 was considered to be positive.
Patient selection
Histological assessment
Serum from consecutive patients with AIH who were diagnosed at Ankara Numune Education and Research Hospital, Hitit University, and Canakkale ¸ State Hospital, Turkey, were used for the present study. AIH was diagnosed according to the simplified AIH criteria suggested by the International Autoimmune Hepatitis Group (IAIHG) [14]. Patients were excluded if they had other concomitant chronic liver disorders including HBV, HCV, PBC, primary sclerosing cholangitis, Wilson’s disease, hemochromatosis, or a history of excessive alcohol consumption. Patients with malignancies, diabetes mellitus, proliferative hematological disorders, or infectious diseases were also excluded. All patients were immunosuppression-naive at presentation. After collection of the first serum samples, the patients were treated with prednisone (30–60 mg/day) alone or in combination with azathioprine (50–100 mg/day). Biochemical remission was defined as the normalization of
The METAVIR score was used for staging liver fibrosis according to a five (0–4)-point scale [17], where F0 = no fibrosis, F1 = portal fibrosis without septa, F2 = few septa, F3 = numerous septa without cirrhosis, and F4 = cirrhosis. Patients with F1–2 were considered to have early-stage fibrosis and those with F3–4 to have advanced fibrosis. Presence of lymphocytic piecemeal necrosis was graded as mild (necrosis around 1–2 portal tracts), moderate (necrosis at the periphery of about half of the portal tracts), and severe (necrosis surrounding more than half of the circumference of almost all portal tracts). Typical and compatible histology for AIH was defined according to the IAIHG criteria [14]. Statistical analysis All data were analyzed using the SPSS version 18.0 (SPSS, Chicago, IL, USA), and p
