| Abstract|| |
Background and Aim: The aim of the study was to investigate the mast cell (MC) concentration in the liver tissue of patients diagnosed with chronic hepatitis C and to determine whether there was a correlation with clinical and pathological characteristics of patients. Materials and Methods: The study was conducted on liver biopsy samples from 60 patients with chronic hepatitis C. Clinical and laboratory data were obtained from follow-up records. Stained liver biopsies were examined. Immunohistochemical staining was performed by using an anti-c-kit antibody. Patients were divided into four groups (minimal, mild, moderate, and severe) based on the intensity of inflammation and their hepatic activity index scores and into two groups (no-mild, moderate-severe) according to fibrosis grade. Results: Among patients enrolled, 60.0% (n=36) were men with a mean age of 48.3±12.7 (range 18-64) years. The mean number of mast cells per portal area in the liver was 0.87±0.86 (0-4.0). No correlation was found between alanine aminotransferase (ALT) and hepatitis C virus (HCV). Ribonucleic acid ( RNA ) levels and the degree of inflammation of cases with number of mast cells and liver steatosis (P>0.05). As the degree of fibrosis increased in the liver so did the number of mast cells in portal areas (P=0.001). On the other hand, no correlation was found between the degree of fibrosis and the number of MCs in the sinusoids and steatosis (P<0.05). The increase in the number of MCs in the portal areas correlated with an increase in liver steatosis (t: 0.02, P=0.04). Conclusion: In light of these findings, it appears possible that MC accumulation in chronic HCV patients may be used as an indicator of fibrosis and possibly be considered in the follow-up of these patients.
Keywords: Fibrosis, hepatitis C virus, inflammation, mast cells
|How to cite this article:|
Koruk ST, Ozardali I, Dincoglu D, Bitiren M. Increased liver mast cells in patients with chronic hepatitis C. Indian J Pathol Microbiol 2011;54:736-40
|How to cite this URL:|
Koruk ST, Ozardali I, Dincoglu D, Bitiren M. Increased liver mast cells in patients with chronic hepatitis C. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Jun 19];54:736-40. Available from: http://www.ijpmonline.org/text.asp?2011/54/4/736/91510
| Introduction|| |
Mast cells (MCs) are regarded as multifunctional effector cells that differentiate from pluripotent hematopoietic cells. , They display special receptors for IgE and IgG subgroups on cell membranes. Hence, they play a role in hypersensitivity reactions and defense mechanisms against parasitic diseases.  However, it was previously shown that mast cells are also important for homeostasis, repair, and re-organization of tissues. 
Hepatitis C virus (HCV) can progress to liver cirrhosis and hepatocellular carcinoma (HCC) and is currently recognized as the primary cause of chronic liver disease worldwide.  Hepatic steatosis is a critical indicator of disease progression and fibrosis in chronic HCV patients.  It was previously shown that MCs are present in humans and laboratory animals under normal and pathological conditions. , Hepatic MCs are mainly observed in connective tissues around the bile ducts, hepatic artery and veins in the portal regions. , The liver of a healthy individual contains ≈1.2-3.9 MCs/mm 2.,
It was previously shown that in certain hepatic diseases, such as liver hemangiomas, hepatocellular carcinoma, autoimmune hepatitis, and primary biliary cirrhosis, the MC concentration is increased, and this causes the increase in collagen deposition observed in chronic hepatitis. ,,, Additionally, the significant correlation between MCs and an increase in micro-vascularisation concentration in liver neoplasia, emphasizes the fact that MCs may play a role in the progression of tumors by inducing angiogenesis.  Based on these facts, it was indicated that MCs, which induce the growth of fibroblasts and the synthesis of collagen, are increased in hepatic fibrosis and may play a role in extracellular matrix degradation. 
The aim of the study was to investigate the mast cell concentration in the liver tissue of patients diagnosed with chronic hepatitis C and to determine whether there was a correlation with clinical and pathological characteristics of patients.
| Materials and Methods|| |
The study was conducted on liver biopsy samples from 60 patients with chronic hepatitis C who were being treated and followed-up by the Department of Infectious Diseases and Clinical Microbiology between January 2009 and April 2010. Diagnosis and treatment were arranged in line with criteria.  Liver biopsies were obtained from patients who were positive for anti-HCV and HCV RNA, following individual written informed consent. Evaluations were performed in the Department of Pathology. Patients with autoimmune disease, a history of alcohol use, co-existing liver disease, diabetes mellitus, malignancy or who were receiving immunosuppressive treatment were excluded from the study. Clinical and laboratory data on patients were obtained from patient files.
Biopsy samples from the patients were stained with hematoxylin eosin, reticulin, and Masson's trichrome; the slides were examined to determine inflammation intensity, fibrosis, and the degree of steatosis. Samples with less than seven portal areas were not included in the study. The intensity of inflammation and the degree of fibrosis were determined according to the Ishak Modified Knodell system.  During investigation of inflammation, the total hepatic activity index (grade) was calculated by adding individual scores of periportal inflammation, piecemeal necrosis, confluent necrosis, and lobular necrosis. Cases were evaluated in terms of steatosis and were graded based on Brunt's scoring system.  According to this system, cases were divided into three groups; patients with fatty degeneration in 30% or less of hepatocytes were classified as having mild steatosis, patients with fatty degeneration in 30-60% of hepatocytes were classified as having moderate steatosis and patients with fatty degeneration in more than 60% of hepatocytes were classified as having severe steatosis. Cases with no fatty degeneration in hepatocytes were classified as negative for steatosis.
Patients were divided into four groups based on the intensity of inflammation and their hepatic activity index scores; cases with scores of 1-3 were regarded as minimal, 4-8 were accepted as mild, 9-12 were regarded as moderate and 13-18 were regarded as severe. In terms of degrees of fibrosis, each case was initially evaluated according to the Ishak system.  Upon evaluation, cases were classified into two groups: the first group was composed of cases with scores between 0 and 2 with no fibrosis or mild fibrosis, and the second group had scores of 3-6 with moderate-severe fibrosis.
For immunohistochemical examination, paraffin blocks of patient samples were taken out of archive, cross-sectioned at 4 μm and placed on slides. Cross-sections were de-paraffinized with xylene and rehydrated with an alcohol series. For the antigen retrieval, cross-sections were kept in a microwave oven in a solution of 1 mmol/L EDTA (pH:8.0) for 20 min. Cross-sections were kept in 3% hydrogen peroxide for 5 min to inactivate endogen peroxidase. As a primary antibody, CD117 (Dako, polyclonal rabbit-antihuman, code no: A4502) was added and then the sample was incubated for 80 min. Following addition of the primary antibody, biotinized anti-mouse and anti-rabbit IgG were applied to the cross-sections for 45 min, followed by incubation with streptavidin peroxidase, and then the cross-sections were stained with 3,3´-diaminobenzidine tetrahydrochloride. Hematoxylin was used for cross-staining.
Prepared samples were examined under a light microscope for CD117 (C-kit)-stained mast cells [Figure 1]. This evaluation was performed according to the recommendations of Wataru et al. Based on this approach, mast cells in hepatic lobuli and portal areas were individually counted and evaluated. Mast cells in the hepatic lobuli in zones 2 and 3 were counted and evaluated in at least 10 locations under magnification (×200), and the number of mast cells in each area was calculated. Mast cells in portal areas were evaluated in at least 7 portal regions under magnification of the microscope (×200), and the number of mast cells per portal area was calculated.
|Figure 1: C-kit (CD117) stained mast cell in the portal area (CD117, ×1000)|
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Statistical analysis was performed with the SPSS 11.5 program. Descriptive statistics and Kendall's Tau correlation test were used for analysis of the data. A P-value <0.05 was considered significant.
| Results|| |
Among patients enrolled, 60.0% (n=36) were men with a mean age of 48.3±12.7 (range 18-64) years. The mean alanine aminotransferase (ALT) value of patients was 70.0±57.2 (range 27-260) U/L, and the mean HCV RNA level was 3.9×10 6±4.9x10 6 (range 2.9×10 4 -3.8x10 7 ) IU/mL. Of patients, 46 (76.6%) had genotype 1 and 14 (23.4%) had genotype 2. Classification of inflammation and fibrosis and mean number of mast cells in liver biopsy samples of patients are shown in [Table 1].
|Table 1: Classification of inflammation and fibrosis and mean number of mast cells in liver biopsy samples of patients|
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The mean number of mast cells per portal area in the liver was 0.87±0.86 (0-4.0). The mean number of MCs in the ten magnification areas of sinusoids (number of MCs in 2.2 mm 2 ) was 0.46±0.68 (range 0-2) [Figure 2], [Figure 3], [Figure 4] and [Figure 5].
|Figure 2: Image of a mild fibrosis and moderate inflammation of a liver biopsy section (H and E, ×100)|
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|Figure 3: C-kit stained mast cell of the case in Figure 2 in hepatic lobulus (CD117, ×400)|
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|Figure 4: Image of a moderate inflamation with moderate steatosis of a liver biopsy section (H and E, ×100)|
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|Figure 5: C-kit stained mast cell of the case in Figure 4 in the portal area (CD117, ×400)|
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The gender and age of the patients, levels of ALT and HCV RNA, the degree of inflammation and fibrosis in liver biopsies, MCs in portal areas and sinusoids, and the correlation with steatosis are shown in [Table 2]. No correlation was found between ALT and HCV RNA levels and the degree of inflammation of cases with number of mast cells and liver steatosis (P>0.05 for all). As the degree of fibrosis increased in the liver so did the number of mast cells in portal areas, and this difference was statistically significant (P=0.001) [Table 1]. On the other hand, no correlation was found between the degree of fibrosis and the number of MCs in the sinusoids and steatosis. Additionally, it was determined that the increase in the number of MCs in the portal areas correlated with an increase in liver steatosis, and this difference was statistically significant (t: 0.02, P=0.04).
|Table 2: Correlation between ALT and HCV RNA levels, degree of inflammation and fibrosis in liver biopsies of patients with MCs in portal areas and sinusoids and steatosis|
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| Discussion|| |
MCs are found in critical organs of the human body, mainly in body surfaces that contact the surrounding environment like the skin, respiratory system, and gastrointestinal system. Mast cells initiate defense mechanisms when foreign materials attack the host.  Mast cells are connective tissue cells that contain chemicals like histamine, heparin, neutral proteases, eosinophilic chemotactic factor, tumor necrosis factor α (TNF-α), and many interleukins (IL-3, IL-4, IL-5, IL-6, IL-9, IL-10) in their cytoplasmic granules and have a role in many physiological and physiopathological processes in the body. , Studies indicate that MCs play an unexpected role in liver disease.  It was previously shown that in humans and in animal models, fibrosis in the liver is increased due to liver damage induced by viral infections, chemical toxins, and autoimmune reactions. The number of MCs in the liver increased in response to fibrotic response, which developed as a result of chronic inflammation. ,
Hepatic steatosis, also known as lipid accumulation in the liver, is a frequent histological finding in chronic hepatitis C (CHC). Hepatocellular steatosis is associated with HCV and is caused by the cytopathic effects of HCV genotype 3, while in other cases, metabolic risk factors are emphasized.  In a study conducted by Minakari et al., infection with HCV genotype 3 and high viral load in these patients are associated with significant steatosis. However, it is not clear to what extent the virus itself and/or host factors influence the development of steatosis. , In the present study, all of the patients had genotype 1 and there were no genotype 3 patients. Also, no correlation was found between HCV RNA levels and the degree of inflammation of cases with number of mast cells and liver steatosis. Adipose tissue is an important resource for certain soluble tissue factors like tissue cytokines, hormones, and TNF-α. All of these factors play a primary role in the accumulation of MCs and inflammatory cells in various organs and in providing an increase of liver steatosis.  Nearly 10% of liver MCs were found in the peri-sinusoidal region, but this figure is increased by MC hyperplasia during development of liver fibrosis. ,
In this study, which evaluated liver biopsies of chronic HCV patients it was determined that: (a) there is no correlation between age and gender of patients and the concentration or number of MCs. (b) No correlation was found between MC concentration and serum ALT levels. In chronic HCV infection, aminotransferase levels are normal in about one third of patients. In some of these patients with normal aminotransferase levels, progression to histological damage was observed.  Therefore, fluctuating ALT levels may not directly affect MC accumulation. (c) No significant correlation was found between the degree of inflammation and the concentration of MCs in both the sinusoidal and portal areas. Similar to this study, in patients with HCC that was due to chronic HCV infection, no correlation was found between MC concentration and stages or grades of patients. In the same study, although a causal relationship was not established between HCC and MCs, it was indicated that they might have induced formation of new capillaries.  (d) It was determined that as the degree of fibrosis in the liver tissue is increased, MCs in the portal area increased. Similarly, MC accumulation in the portal areas was found to correlate with an increase in the degree of steatosis. Hepatic steatosis is a frequent pathological finding in liver biopsies of patients with chronic HCV-related diseases. In vitro and in vivo studies suggest that the HCV core protein may induce lipid accumulation in transfected cells and transgenic mice. , It was also shown that HCV has the capacity to induce steatosis directly, by interfering with lipid metabolism that follows increased viral replication after entry of HCV into hepatocytes.  In a study conducted by Leandro et al., it was confirmed that steatosis is significantly and independently associated with fibrosis in CHC. Upon evaluation of these studies, it is confirmed that fibrosis is increased in HCV infection. In the present study, we observed an increase in fibrosis and steatosis in portal areas with an increase and accumulation of MCs. In a related study performed by Franceschini et al., it was determined that the concentration of MCs is increased in patients with HCV infection and steatosis, compared to HCV patients without steatosis. Persico et al. indicated that in HCV patients, hepatic steatosis is a very critical co-factor for liver disease, due to its ability to interact with the antiviral response and its ability to trigger HCC development.
Several risk factors for liver disease progression are known for both chronic hepatitis C (CHC) and non-alcoholic fatty liver disease (NAFLD). These factors are, high BMI, type 2 diabetes mellitus, older age and alcohol consumption and these can also affect the presence and the gravity of hepatic steatosis.  In our study, patients with autoimmune disease, a history of alcohol use, co- existing liver disease, diabetes mellitus, malignancy, and patients who were receiving immunosuppressive treatment were excluded from the study. Unfortunately the BMIs of patients were not assessed in the study. This is a limitation of our study. However, we took into consideration other factors that lead to steatosis, indicated above and did not include these patients in our study.
In conclusion, the number of studies that found a correlation between mast cell concentration and HCV is limited. In light of these findings, it appears possible that MC accumulation in chronic HCV patients may be used as an indicator of fibrosis and possibly be considered in the follow-up of these patients. Nevertheless, the sample size is too small and further large-scale studies are needed to verify this conclusion.
| Acknowledgments|| |
We would like to acknowledge Dr. Ibrahim Koruk at Harran University, Faculty of Medicine for his valuable contribution to statistical analysis of the results.
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Suda Tekin Koruk
Harran University, Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, Yenisehir Campus, 63100, Sanliurfa
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]