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Abstract

Background:

This article is devoted to the inquiry of three diseases of the liver: alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), and autoimmune hepatitis (AIH). The aim of the study was to assess the changes in populations of circulating lymphocytes expressing antiapoptotic bcl-2 molecule and proapoptotic Fas (cluster of differentiation 95(CD95)) receptor in patients with ALD, AIH, and PBC.

Materials and Methods:

The study population consisted of 110 patients with ALD (n = 50), PBC (n = 30), and AIH (n = 30) and age-matched healthy volunteers (n = 25). Peripheral blood lymphocytes were isolated, stained with monoclonal antibodies against CD4, CD8, and CD19 antigen; intracellular bcl-2; and surface Fas receptor (CD95) antigens, and estimated using the flow cytometric method.

Results:

Bcl-2 expression was the highest in CD4+ and CD19+ T lymphocytes in ALD; however, only the differences in median/mean fluorescence intensity values of CD4+bcl-2+ lymphocytes between ALD and PBC group and CD19+bcl-2+ between ALD and PBC groups were statistically significant, indicating the different role of B cells in pathology of ALD and PBC. In contrast to that, statistically significant higher percentage of CD4+, CD8+, and CD19+ bearing Fas receptor in all groups of patients with liver diseases in comparison with the control subjects were estimated. The highest expression of Fas in CD4+ lymphocytes in ALD and in CD8+ cells of PBC and AIH groups were detected.

Conclusion:

Low expression of bcl-2 molecule and high expression of Fas in peripheral blood lymphocytes indicate significant dysregulation of apoptotic mechanisms not only in the liver but also in peripheral blood lymphocytes in all examined groups, especially in ALD group.

Keywords

Alcoholic liver disease autoimmune hepatitis primary biliary cirrhosis FasR bcl-2 blood lymphocytes

Introduction

Apoptosis is a physiological cell death essential to the normal development and homeostatic maintenance of the liver. Stimulation of apoptosis may ameliorate the course of autoimmune disease and cancer by eliminating autoreactive lymphocytes and tumor cells, respectively. On the other hand, suppression of apoptosis is supposed to aggravate autoimmune diseases and cancers.¹

There are two overlapping signaling pathways leading to apoptosis, termed as intrinsic and extrinsic pathway. The intrinsic pathway is characterized by mitochondria dysfunction. The damage of the mitochondrial inner membrane may be caused by various stimuli, leading to the release of proapoptotic factors such as cytochrome c and apoptosis-inducing factor. These factors cause nuclear changes. Cytochrome c complexes with Apaf-1 to activate procaspase 9, which in turn activates caspases 3, 6, and 7. This pathway is regulated by the relative proportion of antiapoptotic (e.g. bcl-2 and bcl-xl) and proapoptotic (e.g. bax and bad) bcl-2 family members. The extrinsic pathway is mediated by cell surface receptors, including Fas and tumor necrosis factor α (TNF-α) receptors. Upon activation by FasL or TNF-α, respectively, both complexes bind adapter proteins and procaspase 8. This initiates cleavage of procaspase 8 into its active form. Cytotoxic lymphocytes express FasL and after binding to Fas receptor release granules containing granzyme B and perforin, which in turn activates procaspases in target cells.^2,3

Autoimmune hepatitis (AIH) is an autoimmune liver disease characterized by chronic active inflammation of the liver. A variety of immunological disorders are observed in AIH patients, including high titers of antinuclear antibodies and hypergammaglobulinemia. Although it is suggested that T cell-mediated immune responses are associated with hepatocellular damage, details of the mechanism remain unclear. The results indicate that activated cluster of differentiation 4 (CD4) cells play a critical role in the damage of hepatocytes.^4
–6

Primary biliary cirrhosis (PBC) is characterized histologically by the progressive loss of intrahepatic small bile ducts and serologically by the presence of antimitochondrial (AMA) antibodies. It is believed that enhanced apoptosis, mediated by FasR/FasL interaction, may contribute to the bile duct injury. The high intensity of FasL expressing mononuclear cells and strong expression of Fas in epithelial cells of the injured bile ducts were observed in the liver of PBC patients.⁷

Reduced adaptive immune response, increased susceptibility to infections, and cancer development observed in individuals exposed to alcohol are probably related to the status of ethanol (EtOH) intake and the existing liver injury. Acute exposure to alcohol has been linked to reduced antigen presentation by monocytic cells and lower secretion of inflammatory cytokines. In turn, chronic alcoholism has been considered to result in T cell activation, higher secretion of monocyte-derived inflammatory cytokines, and increased expression of the co-stimulatory molecules on macrophages. Alcoholic liver disease (ALD) is a term that encompasses hepatic lesions due to alcohol overconsumption, including liver steatosis, alcoholic hepatitis, and chronic hepatitis with hepatic fibrosis or cirrhosis.^8

–13

Apoptosis plays also a prominent role in the pathogenesis of liver diseases. Induction of extrinsic apoptotic pathway by cytotoxic lymphocytes predominates in autoimmune liver disease and in viral hepatitis. Biliary cell apoptosis is also regulated by bcl-2 family members. Both the extrinsic and intrinsic pathways are activated in alcohol-related liver disease.¹

The description of individual changes in the apoptosis in each of the mentioned diseases has already been reported by several authors. However, the comparison of expression on peripheral blood cells of antiapoptotic marker such as bcl-2 molecule and proapoptotic ones such as Fas receptor in the three diseases is not yet carried out. Moreover, the expression of those molecules on B cells from patients with ALD, PBC, and AIH were poorly estimated in spite of the important role of those cells in the production of autoantibodies.

The aim of this study was to assess the changes in populations of circulating CD4+, CD8+, and CD19+ lymphocytes (% of cells bearing bcl-2 and Fas) and the level of expression, measured as median/mean fluorescence intensity (MFI) of bcl-2 and Fas molecules in peripheral blood lymphocytes in patients with liver diseases, including ALD, AIH, and PBC.

Materials and methods

Study population

The study population comprised of 110 patients with liver disorders hospitalized at the Gastroenterology Department of the Medical University of Lublin, Poland. The enrolled individuals were newly diagnosed with ALD (n = 50), AIH (n = 30), or PBC (n = 30). In parallel, 25 age-matched healthy volunteers with no history of chronic alcohol use, liver disease, or any autoimmune processes were recruited (control subjects).

Diagnosis of ALD was based on the history of chronic alcohol consumption and ascites, hepatic encephalopathy or variceal bleeding, the presence of symptoms and signs of chronic liver disease, as well as on gastroscopic and/or ultrasonographic findings. None of the patients had acute alcoholic hepatitis or decompensated cirrhosis at the time of entering the study. Alcohol use habits of the studied participants were determined using a health questionnaire.¹⁴ The average alcohol consumption in ALD group was required to be ≥90 g of EtOH day⁻¹.

Patients recruited to AIH and PBC groups had clinical and biochemical features suggestive of the respective diseases. Briefly, patients with AIH had high levels of serum aminotransferases and immunoglobulin G (IgG) and were positive for antinuclear antibodies. The final diagnosis, however, was confirmed by histopathology. Thus, the inclusion criteria to the AIH group combined clinical, laboratory, and histologic components and were consistent with international guidelines and scoring system for AIH diagnosis codified and updated by International Autoimmune Hepatitis Group.^15,16

All subjects enrolled into PBC group had high serum levels of alkaline phosphatase and IgM and were characterized by the presence of serum AMA or anti-M2 antibodies. Moreover, the histology of liver biopsy specimens showed chronic nonsuppurative destructive cholangitis.¹⁷ Patients recruited to the AIH, PBC, and control groups were required to satisfy the criterion of not excessive alcohol intake (i.e. the average alcohol consumption <15 g of EtOH day⁻¹).

None of the study participants had hepatitis A, B, or C virus or HIV infection or had been taking any systemic medication known to be hepatotoxic or to affect evaluated immunological parameters (i.e. steroids or immunosuppressants). Moreover, patients with respective liver disorders were specific treatment naive and had no other comorbid liver conditions. Other exclusion criteria included any ongoing infection, malnutrition, and blood transfusions.

The study protocol was approved by the Human Subjects Ethics Committee at the Medical University of Lublin, Poland, in accordance with the ethical standards of the revised Helsinki Declaration. Following detailed explanation of the nature and purpose of the study, written informed consent was obtained from each participant.

Blood sampling, cell preparation, and phenotyping

Blood samples were taken by venipuncture after an overnight fast. Specimens were collected into sterile, heparinized tubes and immediately processed. The mononuclear cells were isolated by density gradient centrifugation on Lymphoprep (Nycomed, Norway) at 400g at room temperature for 20 min. Interphase cells were removed and washed twice in phosphate-buffered saline without calcium and magnesium ions containing 0.5% bovine serum albumin and 2 mM ethylenediaminetetraacetic acid. The cells were then counted in a hemocytometer and resuspended in the mentioned buffer. After surface staining with phycoerythrin (PE)-conjugated monoclonal antibody against CD4, CD8, and CD19 (Miltenyi-Biotec, Germany), 100 µl of FACS permeabilizing solution were added (10 min, room temperature, in the dark), and cells were stained with anti-bcl-2 antibodies (fluorescein isothiocyanate (FITC), Miltenyi-Biotec, Germany). Subsequently, the double-color immunofluorescence staining was performed using the following monoclonal antibodies: mouse anti-human CD19-CyChrome (Pharmingen, San Diego, California, USA), CD95 (Fas)-FITC, anti CD4-PE, and anti BD8-PE (Becton Dickinson, Franklin Lakes, New Jersey, USA). Mouse anti-human IgG_2a isotype control was used for staining. The staining procedure was conducted according to the manufacturers’ protocols. The cells (aliquots of ≤1 × 10⁷ cells) were briefly incubated with optimum amounts of monoclonal antibodies in the dark at 4°C for 10 min, then washed in the buffer, and centrifuged at 300g and 4°C for 10 min. To minimize nonspecific Fc receptor (FcR)–monoclonal antibody binding, the immunostaining was performed in the presence of FcR blocking reagent (20 µl × 10⁻⁷ cells) or fewer cells resuspended in 80 µl of the buffer (Miltenyi-Biotec, Germany).

Flow cytometric analysis

The immunolabeled cells were collected (300,000 events) using a FACSCalibur flow cytometer equipped with 488-nm argon laser (Becton Dickinson) and analyzed with Cell-Quest Software. Cell debris and dead cells were excluded from the analysis based on scatter signals. The number of CD4+, CD8+, and CD19+ were expressed as the percentage of the mononuclear cells. MFI was also measured. Since there was no significant difference in the absolute value of peripheral blood leukocytes between the groups of patients examined, MFI was used to compare the results.

Statistical analysis

Data were analyzed using STATISTICA Data Analysis Software, version 8.0 (StatSoft, Inc. Poland). Continuous variables were expressed as mean ± standard deviation (SD), median, and range. For variable distribution normality, Shapiro–Wilk’s test was applied. Given the non-normal distribution of the involved variables, the differences between the groups (>2) were assessed with Kruskall–Wallis test. Nonlinear correlation of coefficient between continuous variables was calculated using Spearman’s rank test. For all analyses, a probability value of p < 0.05 was considered as statistically significant.

Results

Clinical data

The data on age, sex, and clinical characteristics of the study population with regard to the etiology of liver disorder are detailed in Table 1. As for the laboratory parameters, there were no significant differences in the aspartate transaminase, γ-glutamyl transferase, albumin, and bilirubin serum levels between the selected patients. The serum level of alanine transaminase was markedly lower in patients with ALD compared to AIH group (p = 0.001), and serum levels of alkaline phosphatase in patients with PBC were significantly higher than those in patients with AIH (p = 0.002). All liver function tests of control subjects were within normal ranges and differed significantly from those observed in patients with liver disease, regardless of its etiology (p < 0.05).

Table 1.

Age and sex distribution and clinical characteristics of the study population.^a

	Reference range	ALD (n = 50)	AIH (n = 30)	PBC (n = 30)	Controls (n = 25)
Mean age (years)		51.60 ± 10.10	47.63 ± 16.43	53.40 ± 8.83	43.39 ± 8.35
Gender (female:male)		13:37	27:3	30:0	13:12
White blood cells (10³ mm⁻³)	(4.00–10.00)	7.45 ± 2.52	7.43 ± 1.91	6.40 ± 2.52	6.86 ± 1.66
Serum levels of:
Alanine transaminase (U L⁻¹)	(10.00–40.00)	141,52 ± 47.56	253,26 ± 144.86	101.80 ± 28.89	17.84 ± 4.43
Aspartate transaminase (U L⁻¹)	(0.00–34.00)	136.18 ± 65.19	278.50 ± 291.62	103.63 ± 38.76	16.44 ± 3.77
γ-Glutamyl transferase (U L⁻¹)	(2.00–30.00)	325.02 ± 218.38	158.83 ± 109.33	322.63 ± 151.60	30.48 ± 8.13
Alkaline phosphatase (U L⁻¹)	(25.00–100.00)	153.91 ± 101.19	136.96 ± 111.88	260.92 ± 201.53	83.06 ± 13.99
Bilirubin (mg dL⁻¹)	(0.30–1.20)	7.68 ± 10.17	3.75 ± 2.80	6.75 ± 9.87	0.55 ± 0.28
Albumin (g dL⁻¹)	(3.50–5.00)	3.26 ± 1.82	3.76 ± 0.99	2.01 ±1.23	4.54 ± 0.44
Immunoglobulin M (mg dL⁻¹)	(50.00–300.00)	Not done	271.34 ± 84.68	603.40 ± 242.38	not done
Immunoglobulin G (mg dL⁻¹)	(652.00–1600.00)	Not done	2081.55 ± 644.80	1357.64 ± 506.52	not done
Prothrombin time (s)	(9.40–14.10)	15.95 ± 3.92	13.76 ± 2.93	14.4 ± 5.03	11.56 ± 0.52
Average alcohol intake (g of EtOH day⁻¹)		≥90	<15	<15	<15
Markers of current infection with hepatitis A, B, or C viruses		Absent	Absent	Absent	Absent
Recent exposure to hepatotoxic medications		Absent	Absent	Absent	Absent

ALD: alcoholic liver disease; AIH: autoimmune hepatitis; PBC: primary biliary cirrhosis; EtOH: ethanol.

^aData are presented as mean values ± standard deviation.

Bcl-2 expression in blood lymphocytes of ALD, PBC, and AIH patient groups

No significant differences in intracellular bcl-2 expression in different lymphocyte populations including CD4+, CD8+, and CD19+ cells were found when comparing patients with liver diseases to the healthy controls (Figure 1). Statistically significant difference was only seen between percent of CD8+bcl-2+ in ALD and PBC patients and MFI of CD4+bcl-2+ cells in ALD patients in comparison with PBC patients. When comparing MFI of blood CD19+ B cells in three examined groups, a higher MFI in bcl-2 staining in ALD statistically significant and in PBC was observed in comparison with the control. Only B cells from AIH patients expressed similar staining for bcl-2 as control subjects, while CD19+ cells in ALD patients expressed more bcl-2 molecule than controls. When bcl-2 expressing cells were characterized in our study, it was detected that mainly CD4+ cells of patients with ALD, PBC, and AIH strongly express bcl-2 molecule. MFI of CD4+bcl-2+ cells was the highest in comparison with other cell populations. One exception was the strong expression of bcl-2 molecule in CD19+ cells of ALD patients. However, the MFI of all cell populations in patients with ALD was the highest in comparison to PBC and AIH patients.

Figure 1.

Percentages and MFI of bcl-2+ lymphocyte subsets in ALD, PBC, and AIH group in comparison with the control. **p ≤ 0.01: statistically significant between percentage of cells; ^## p ≤ 0.01: statistically significant between MFI. MFI: median/mean fluorescence intensity; ALD: alcoholic liver disease; PBC: primary biliary cirrhosis; AIH: autoimmune hepatitis.

Fas expression in blood lymphocytes in ALD, PBC, and AIH patient groups

Bcl-2 expression in all subpopulations of lymphocytes and the percent of blood lymphocytes expressing Fas receptor on the cell surface were significantly higher in ALD, PBC, and AIH patients in comparison with the controls (Figure 2). Comparison of Fas-expressing lymphocytes among the different groups revealed that percent of CD4+ and CD19+ cells with Fas expression was the highest in ALD group, while the expression in CD8+ cells was the lowest in this group. In AIH group, CD4+ and CD19+ cells expressed higher percent than that observed in PBC group. However, these differences were not statistically significant.

Figure 2.

Percentages and MFI of Fas+ (CD95+) lymphocyte subsets in ALD, PBC, and AIH group in comparison to control. **p ≤ 0.01: statistically significant at between percentage of cells. ^## p ≤ 0.01: statistically significant between MFI. MFI: median/mean fluorescence intensity; ALD: alcoholic liver disease; PBC: primary biliary cirrhosis; AIH: autoimmune hepatitis.

Correlations between laboratory markers and changes in immunological parameters of blood cells in ALD, PBC, and AIH patients are presented in Table 2.

Table 2.

Correlations between laboratory markers and changes in immunological parameters of blood cells in ALD, PBC, and AIH patients.

Markers	R	p
CD4+CD95+% and body weight	−0.4065	0.2570
CD4+bcl-2+MFI and bilirubin	0.4240	0.0310^a
CD19+bcl-2+MFI and bilirubin	0.3789	0.0389^a
CD19+bcl2+MFI and AST	0.4990	0.0120^a
CD4+bcl-2+MFI and CD8+bcl-2+MFI	0.6729	0.00004^a
CD4+bcl-2+MFI and CD19+bcl-2+MFI	0.4571	0.0110^a
CD8+bcl-2+MFI and CD19+bcl-2+MFI	0.5604	0.00127^a
CD19+CD95+% and body weight	−0.3889	0.0336^a
CD19+CD95+MFI and GGTP	−0.3957	0.0303^a
CD8+CD95+% and AST	0.4419	0.0144^a
CD4+CD95+% and GGTP	0.3809	0.0378^a
CD4+CD95+MFI and CD8+CD95+MFI	0.7262	0.00000^a
CD19+CD95+MFI and CD4+CD95+MFI	0.3847	0.0057^a
CD19+CD95+MFI and CD8+CD95+MFI	0.3172	0.0247^a

R: Spearman’s correlation test; ALD: alcoholic liver disease; AIH: autoimmune hepatitis; PBC: primary biliary cirrhosis; CD: cluster of differentiation; GGTP: gamma-glutamyl transferase; AST: aspartate transaminase.

^a p ≤ 0.05: statistically significant.

Discussion

The present study demonstrated that MFI of CD19+ B cells was higher in ALD and PBC, but AIH patients expressed similar staining as control subjects. Our results are in contrast to those observed by Yachida et al. who diagnosed high MFI of bcl-2 expression in mononuclear cells from peripheral blood of AIH patients.⁵ Moreover, Yachida et al. indicated that mainly CD4+ Th-1–like cells express this molecule. A possible explanation of different results obtained in our study is that our experiment encompassed nearly a two times larger group of patients. It is also important that very high SD was observed in our experiment as well as in Yachida et al.’s experiments.⁵ Another explanation of such discrepancies is the accumulation of cells expressing antiapoptotic molecule in the area of liver tissue, which can depend on the stage of disease. Such observation was described by Kurokohchi et al. in patients with AIH.⁴ In the portal area of liver specimen, they observed mainly the accumulation of CD4+ cells expressing bcl-2 molecule. Moreover, our results are in agreement with those obtained by Ogawa et al. who did not observe significant differences in bcl-2 expression in control and AIH patients, despite the increase in the number of Fas-expressing T cells.⁶

In an in vitro experiment on mixed lymphocyte reaction model and regulation of apoptosis of T cells, authors detected that during the reaction time, bcl-2 expression significantly increased but after that exhausted lymphocytes decreased bcl-2 expression and undergo apoptotic death. It can be speculated that expression of bcl-2 molecule and susceptibility of cells to apoptosis not only depends on the localization of lymphocytes in peripheral blood or in inflammed liver but can be also due to the stage of liver disease.¹⁸ It should be noted that bcl-2 expression was found to decrease significantly in mononuclear cells from blood of patients with advanced stage of chronic kidney diseases, at the same time, soluble Fas (sFas) increased significantly.¹⁹ It is well documented that culmination of immune response involves death of the majority of activated antigen-specific lymphocytes via two separate types of cell death. Either extrinsic or intrinsic apoptotic pathway would dominate depending on several factors such as lymphocyte localization, type and stage of the disease, and other factors as yet poorly described. The association between apoptosis and bcl-2 expression in the liver of patients with alcoholic steatohepatitis is well documented. Expression of bcl-2 correlates with a degree of portal and lobular inflammation.¹⁰ O’Flaherty et al. suggested that such a high expression of antiapoptotic molecule is probably an adaptive response to alcohol-related stress. In rats used as experimental model of ALD, bcl-2 protein presentation was observed in bile duct epithelial and in inflammatory cells infiltrating liver, and it was connected with liver injury and lipid peroxidation.⁹ Even in patients without liver failure, EtOH induced dysfunction not only of hepatocytes but also of blood leukocytes, especially of B cells by promoting their apoptosis.⁸ In our study, very high expression of bcl-2 in B cells can be considered an adaptive response of those cells. The results obtained concur with the available data from Lian et al., who proved high expression of bcl-2 in cells producing autoantibodies in nearly 70% of patients with ALD.¹³ Moreover, antibodies against liver-specific autoantigens such as P450 2E1 have frequently been detected in ALD. These antibodies are usually correlated to the extension of lymphocyte infiltration and hepatocyte apoptosis.

Our experiments showed that in ALD group, percent of CD4+ and CD19+ cells with Fas expression was the highest, while in CD8+ cells was the lowest. These findings are consistent with the results of other authors.¹¹ Ishimaru detected the increased ratio of CD4+ cells positive for Fas and the decrease in ratio of CD8+Fas-positive cells by measuring T cell subsets in peripheral blood of patients with alcoholic hepatitis.¹¹ Moreover, we observed accelerated apoptosis of lymphocytes from blood of patients with alcoholic cirrhosis and high serum level of sFas.¹² It should be mentioned, however, that in this experiment, our group of ALD patients were diagnosed as having mainly alcoholic steatosis, therefore the differences between T cell subsets positive and negative for Fas were not so striking. Considering the AIH group of patients, our results confirmed those obtained by Ogawa et al. who proved that the surface expression of Fas was significantly higher in the subsets of both CD4+ and CD8+ T lymphocytes compared with normal subjects.⁶ The authors mentioned that in normal immune responses, the activated lymphocytes associated with strong Fas expression have been deleted by an apoptotic mechanism that downregulates an excessive immune response.⁶ The expanded Fas+ T cells most likely reflect a continuous antigen-specific or nonspecific activation and abnormalities in the peripheral deletion of activated lymphocytes. In our study, in PBC group lower percent of CD4+Fas+expressing cells was detected in comparison with AIH group. PBC and AIH are two distinct autoimmune liver diseases. AIH is characterized by the presence of antinuclear and anti-liver–kidney microsome antibodies and immune-mediated destruction of hepatocytes. Fox et al. noted that both FasL and granzyme B levels were increased in AIH, while in PBC only the levels of granzyme B were elevated.⁷ In PBC, patients express AMA antibodies and initially develop immune-mediated interlobular bile duct damage. The mode of apoptosis in both diseases is mediated by surrounding cytotoxic lymphocytes. Iwata et al. showed that Fas expression was upregulated in damaged bile ducts of PBC.²⁰ Moreover, FasL and Fas were increased on surrounding cytotoxic lymphocytes in PBC. In other autoimmune diseases, such as lupus erythematosus, an increase in the number of CD95-bearing T cells was also observed.²¹ FasL on peripheral lymphocytes, as well as Fas, play a crucial role in inducing apoptosis involved in negative selection of autoreactive T cells during differentiation and maturation. FasL/Fas system was proved to be involved not only in prevention but also in aggravation of autoimmune diseases. While the main function of cytotoxic lymphocytes is immune surveillance for virus-infected and tumor cells, this system often causes tissue destruction in tissue-specific autoimmune diseases such autoimmune liver diseases. Strong expression of cell-associated Fas in both PBC and AIH groups indicates that this mechanism is not only connected to immune cells infiltrating liver but also present in peripheral blood. Positive correlation between CD8+Fas+ and AST or CD4+Fas+ and gamma-glutamyl transferase (GGTP) confirm the role of these cells in the liver injury.

Conclusion

Low expression of bcl-2 molecule and high expression of Fas in peripheral blood lymphocytes indicate significant dysregulation of the apoptotic mechanisms present not only in the liver but also in peripheral blood lymphocytes in all examined groups, especially in the ALD group. Positive correlations between CD4+ and CD19+ expressing bcl-2 and bilirubin and also CD8+Fas+ and AST, or CD4+ and CD19+ positive for Fas and GGTP suggest the role of these cells in the pathology of liver diseases.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Bai

Odin

. Apoptosis and the liver: relation to autoimmunity and related conditions. Autoimmun Rev 2003; 2: 36–42.

Yonehara

. Death receptor Fas and autoimmune disease: from the original generation to therapeutic application of agonistic anti-Fas monoclonal antibody. Cytokine Growth Factor Rev 2002; 13: 393–402.

Precone

Stornaiuolo

Amato

. Different changes in mitochondrial apoptotic pathway in lymphocytes and granulocytes in cirrhotic patients with sepsis. Liver Int 2013; 33: 834–842.

Kurokohchi

Arima

Masaki

. Analysis of CD28 and bcl-2 expression on peripheral blood and liver-infiltrating mononuclear cells in patients with autoimmune hepatitis. J Clin Immunol 2006; 26: 323–330.

Yachida

Kurokohchi

Arima

. Increased bcl-2 expression in lymphocytes and its association with hepatocellular damage in patients with autoimmune hepatitis. Clin Exp Immunol 1999; 116: 140–145.

Ogawa

Sakaguchi

Takaki

. Increase in CD95 (Fas/APO-1)-positive CD4+ and CD8+ T cells in peripheral blood derived from patients with autoimmune hepatitis or chronic hepatitis C with autoimmune phenomena. J Gastroenterol Hepatol 2000; 15: 69–75.

Fox

Furtwaengler

Nepomuceno

. Apoptotic pathways in primary biliary cirrhosis and autoimmune hepatitis. Liver 2001; 21: 272–279.

Gheorghiu

Bâră

Păsărică

. Ethanol-induced dysfunction of hepatocytes and leukocytes in patients without liver failure. Roum Arch Microbiol Immunol 2004; 63: 5–33.

Yacoub

Fogt

Griniuviene

. Apoptosis and bcl-2 protein expression in experimental alcoholic liver disease in the rat. Alcohol Clin Exp Res 1995; 19: 854–859.

10.

Ramalho

Cortez-Pinto

Castro

. Apoptosis and Bcl-2 expression in the livers of patients with steatohepatitis. Eur J Gastroenterol Hepatol 2006; 18: 21–29.

11.

Ishimaru

. Measurement of circulating T cell subsets positive for Fas antigen (CD95) in patients with alcoholic hepatitis. Nihon Arukoru Yakubutsu Igakkai Zasshi 1999; 34: 161–172.

12.

Szuster-Ciesielska

Daniluk

Bojarska-Junak

Apoptosis of blood mononuclear cells in alcoholic liver cirrhosis. The influence of in vitro ethanol treatment and zinc supplementation. Toxicology 2005; 212: 124–134.

13.

Lian

Hua

Sheng

. Prevalence and significance of autoantibodies in patients with alcoholic liver disease. J Dig Dis 2013; 14: 396–401.

14.

Ewing

JA.

Detecting alcoholism. The CAGE questionnaire. JAMA 1984; 252: 1905–1907.

15.

Czaja

. Performance parameters of the diagnostic scoring systems for autoimmune hepatitis. Hepatology 2008; 48: 1540–1548.

16.

Czaja

Freese

American Association for the Study of Liver Disease. Diagnosis and treatment of autoimmune hepatitis. Hepatology 2002; 36: 479–497.

17.

Prince

Chetwynd

Craig

. Asymptomatic primary biliary cirrhosis: clinical features, prognosis, and symptom progression in a large population based cohort. Gut 2004; 53: 865–870.

18.

O’Flaherty

Wong

Pettit

. Regulation of T-cell apoptosis: a mixed lymphocyte reaction model. Immunology 2000; 100: 289–299.

19.

Dounousi

Koliousi

Papagianni

. Mononuclear leukocyte apoptosis and inflammatory markers in patients with chronic kidney disease. Am J Nephrol 2012; 36: 531–536.

20.

Iwata

Harada

Hiramatsu

. Fas ligand expressing mononuclear cells around intrahepatic bile ducts co-express CD68 in primary biliary cirrhosis. Liver 2000; 20: 129–135.

21.

Amasaki

Kobayashi

Takeda

. Up-regulated expression of Fas antigen (CD95) by peripheral naive and memory T cell subsets in patients with systemic lupus erythematosus (SLE): a possible mechanism for lymphopenia. Clin Exp Immunol 1995; 99: 245–250.