Journal of Medical Virology 87:407–416 (2015)

Direct Role of Antibody-Secreting B Cells in the Severity of Chronic Hepatitis B Ayaid Khadem Zgair,1,2* Jenan A. Ghafil,1 and Razaq H E Al-Sayidi2 1 2

Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq Central Public Health Laboratory, Baghdad, Iraq

Chronic hepatitis B involves different immune cells. The direct role of antibody-secreting B cells in the severity of chronic hepatitis B unclear. In this study, the number of plaque forming cells [PFC-(IgG, IgM, anti-HBc IgG, and anti-HBc IgM)], liver function tests (LFT) [alkaline phosphatase (ALP), alanine aminotransferase (ALT), and total serum bilirubin (TSB)], the levels of IL-10 in sera and in lymphocyte cultures, the number of CD4þ and CD8þ cells were measured in the peripheral blood of patients and in the controls. In addition, the hepatocytotoxic effect of anti-HBc and anti-HBe in vitro was studied. The largest number of PFCs was observed in the peripheral blood of patients with chronic hepatitis B. This was concomitant with a decrease in CD4þ/CD8þ ratio versus this ratio in asymptomatic HBV carriers and in healthy volunteers (P < 0.05). An increase in immunoglobulin (IgG and IgM) levels, anti-HBc IgG, and anti-HBc IgM levels and LFTs in peripheral blood of patients with chronic hepatitis B was seen. Anti-HBc induced hepatocytotoxicity in vitro. The expression of mRNA and protein for IL-10 production was observed at a significant level in culture of lymphocytes isolated from patients with chronic hepatitis B. In addition, a high level of IL-10 was found only in the sera of patients with chronic hepatitis B. It is concluded that the antibody-secreting B cells and the antibodies, which are produced, play an important role in the severity of chronic hepatitis B, which was related negatively with CD4þ/CD8þ ratio and positively with IL-10 expression. J. Med. Virol. 87:407–416, 2015. # 2014 Wiley Periodicals, Inc.

KEY WORDS:

chronic hepatitis B; IL-10; liver function tests; plaque forming cells; T lymphocytes

C 2014 WILEY PERIODICALS, INC. 

INTRODUCTION Two billion people have been exposed to hepatitis B virus (HBV). Five million cases of acute hepatitis B occur annually and over 350 million people have a chronic infection [Lavanchy, 2005]. In total, hepatitis B results in 500,000-1.2 million deaths annually [Lavanchy, 2005]. This is a carcinogenic virus that causes 60–80% of the world’s hepatocellular carcinoma. The risk is 25–35 times higher among those with chronic HBV infection [Wands, 2004] causing 300,000– 500,000 deaths each year [Lavanchy, 2005]. The outbreak of HBV is similar to that seen with several other viruses including enteroviruses [Eissa, 2013; Eissa and Gupta, 2013]. HBV is transmitted by perinatal, percutaneous, and sexual exposure, as well as by close person-to-person contact presumably by open cuts and sores, especially among children in hyperendemic areas [Mast et al., 2005]. HBV can survive outside the body for prolonged periods [Petersen et al., 1976; Bond et al., 1981]. The risk of developing chronic HBV infection after acute exposure ranges from 90% in newborns of HBsAg-positive mothers to 25% to 30% in infants and children under 5 years and to less than 5% in adults [Beasley et al., 1983]. In addition, immunosuppressed persons are more likely to develop Abbreviations: PFC, plaque forming cell; HBc, Hepatitis B core; HBC, Hepatitis C virus; LFTs, liver function tests; HBeAg, Hepatitis e antigen; HBsAg, Hepatitis B surface antigen; HBVCH, Asymptomatic Hepatitis B virus healthy carrier; HCV, Hepatitis C virus; HDV, Hepatitis D virus; GBV-C, GB virus C; HIV, Human immunodeficiency virus; PBLs, peripheral blood lymphocytes; HBSS, Hank’s balance salt solution; SRBCs, Sheep red blood cells; ALP, Alkaline phosphatase; ALT, Aminotransferase; TSB, Total serum bilirubin. The authors have declared that there are no conflicts of interests.  Correspondence to: Prof. Ayaid Khadem Zgair, Department of Biology, College of Science, University of Baghdad, Baghdad, ALJadriya, Iraq, 10071. E-mail: [email protected] Accepted 1 August 2014 DOI 10.1002/jmv.24067 Published online 27 August 2014 in Wiley Online Library (wileyonlinelibrary.com).

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chronic HBV infection after acute infection [Bodsworth et al., 1989; Horvath and Raffanti, 1994]. The tests that are used for the screening of infection with HBV should include tests to detect hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs). Alternatively; hepatitis B core antibody (anti-HBc) can be used for initial screening. Anti-HBc IgM is an indicator of high activity of virus in a host, and anti-HBc IgG is an indicator of previous or ongoing exposure to HBV [Lok and McMahon, 2007]. In chronic viral hepatitis, an enrichment of CD8þ cytotoxic/suppressor T cell was observed in liver and peripheral blood [Lo¨hr et al., 1994]. Many investigators suggested the chronicity of HBV infection was caused by a deficient cellular immune function [Michalak et al., 1995; Khettry et al., 2000; Tu¨lek et al., 2000]. Thus, T-cell dysfunction might contribute towards viral persistence [You et al., 2008]. A significant decreasing trend in CD3þ and CD4þ cells and CD4þ/CD8þ ratio with increasing severity of hepatocyte damage with advancing age has been observed during HBV infection [You et al., 2008; Salih et al., 2013]. For a non-cytopathic virus like HBV to persist, it must either overwhelm or not induce an effective antiviral immune response, or it must be able to evade it. The HBV is not a cytopathic virus; hence the severity of this virus is probably due to immunopathological reaction against infected hepatocytes with HBV [Chisari and Ferrari, 1995]. Therefore, the major focus of current research has been to find out the reasons for the functional deterioration of the anti-viral T cell responses. Indeed, considerable progress has been made in identifying the potential targets for restoring T-cell function [Frebel et al., 2010]. However, the exact role of B cells in HBV infection needs to be defined as only few studies have addressed this subject. Oliviero et al. (2011) took a fresh look at the role of B cells in chronic HBV and HCV infection by assessing B-cell phenotype and function. These workers concluded that B lymphocytes play a crucial role in mediating immune response against HBV in a patient with chronic hepatitis B. Besides the ability of these cells in producing neutralizing antibodies that play an important role in eliminating HBV, they modulate the activity of CD4þ cells by up-regulating the negative co-stimulatory molecule PD-1 [Frommer et al., 2008]. In addition, B cells, at least in mice, are major sources of the anti-inflammatory cytokine interleukin-10 (IL10) [Madan et al., 2009] that has also been shown to promote viral persistence in vivo [Brooks et al., 2006]. Thus, B cells seem to feature at least two nonredundant mechanisms that inhibit viral elimination in vivo [Brooks et al., 2006; Madan et al., 2009]. Therefore, B cells may have an important role in the severity of chronic hepatitis B through inhibition or clearance of HBV or the hepatocytotoxic effect of antibodies produced by these cells against hepatocytes. Thereby, in the present study, the focus was on J. Med. Virol. DOI 10.1002/jmv

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the role of antibody-secreting cells [plaque forming cells (PFCs)] in the severity of chronic hepatitis B. MATERIALS AND METHODS Patients and Biochemical Tests This study was conducted following the approval of the local ethics committee of the ministry of health, Baghdad, Iraq. A written consent form was obtained from all the patients and healthy persons before withdrawing the blood. Sixty-four patients with HBV infection [43 men and 21 women, age 36.5  9.8 years (range: 25–59 years)] were included in the present study. Patients were classified into two groups. (1) Asymptomatic HBV healthy carriers (immune tolerance phase) [16 men and 10 women, age 36.1  8.1 years (range: 23– 39 years)]: positive for HBsAg and anti-HBc, negative for antibodies (Abs) to hepatitis C virus (HCV), delta virus (HDV), GB virus C (GBV-C), and human immunodeficiency virus (HIV)-1 and 2. High serum HBV DNA level and normal liver function tests [alkaline phosphatase (ALK) and alanine aminotransferase (ALT) and total serum bilirubin (TSB)] were reported. (2) Chronic hepatitis B patients (immune clearance phase) [27 men and 11 women, age 37.5  10.1 years (range: 20–59 years)]: positive for HBsAg and anti-HBc for more than six months, negative for anti-HCV, anti-HDV, anti-GBVC, and anti-HIV-1 and 2, and with persistently elevated LFT and serum HBV DNA. None of these patients had concurrent autoimmune liver diseases. The blood samples for patients with chronic hepatitis B and healthy carriers were collected before receiving antiviral therapy. The clinical and biochemical tests performed for these patients are listed in Table I. Thirty healthy blood donors were selected as healthy control. Healthy control volunteers included [18 men and 12 women, age 35.7  8.1years (range: 24–56 years)]: negative for HBsAg, HBeAg, anti-HBs, antiHBc, and normal levels of LFT (Table I). Detection of Virus Markers by ELISA HBsAg, HBeAg, anti-HBc IgM, anti-HBc IgG, antiHCV, anti-HDV IgM, and anti-HIV were detected in the sera of patients and healthy control groups by ELISA technique. HBsAg (Biotest, Puchong, Selangor, Malaysia.), HBeAg (BiocareTM Diagnostics, Xiangzhou, Zhuhai, China), anti-HBc (IgM and IgG) (BiocareTM Diagnostics, Xiangzhou, Zhuhai, China), anti-HCV (BiocareTM Diagnostics Xiangzhou, Zhuhai, China), anti-HDV IgM (Biokit, Barcelona, Spain), and antiHIV-1, -2 (Murex Diagnostic, Saluggia, Italy) kits were used according to manufacturer ’s instructions. Measurement of Essential Immunoglobulins Single radial immunodiffusion method was used to measure the concentration of essential immunoglobulins (IgM and IgG) in the sera of patients and control

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TABLE I. Immunological and Clinical Features of Patients With Chronic Hepatitis B (CHB), Asymptomatic HBV-Healthy Carrier (HBV-HV), and Healthy Control

Number of patients Mean of age (year) Female Female (%) Males Male (%) Male/female ratio HBsAg positive case Anti-HBs positive case HBeAg positive case Anti-HBe positive case HBV DNA (Log10 IU/mL) Anti-HBc IgM positive case Anti-HBc IgG positive case ALP activity IU/l ALT activity IU/l TSB Con. mg/l

Chronic hepatitis B

HBV- HC

Healthy control

38 37.5  10.1 11 28.947368 27 71.052632 2.4545455 38 0 18 26 7.9  1.3 18 37 189.9  26.5 † 82.06  19.3 † 49.7  13.4 †

26 36.1  8.3 10 38.46 16 61.53 1.6 26 0 19 13 8.4  2.1 6 24 93.3  19.09 23.3  5.85 19.5  4.4

30 35.7  8.1 12 40 18 60 1.5 NA NA NA NA NA NA NA 88.18  21.7 19.5  6.3 16.6  6.3

 Significant †

difference from healthy control group. Significant differences from HC-HBV group. Student’s t-test for continuous variables; NA, not applicable; ALP, alkaline phosphatase; ALT, alanine aminotransferase; TSB, total serum bilirubin.

groups. The manufacturer’s instructions were followed (Imgenex, Bhubaneswar, India). Isolation of Peripheral Blood Lymphocytes (PBLs) Lymphocytes were separated from fresh heparinized venous blood by the modified method using Ficoll-Hypaque (Ficoll, Pharmacia Fine Chemicals Inc., Piscataway, NJ, Hypaque, Winthrop Laboratories, Sterling Drug Co., New York, USA) buoyant density gradient method of Boyum (1968). The viability of lymphocytes was determined by trypan blue exclusion test [Zgair, 2012]. The viable cells were adjusted to 106 cells/ml in Hank’s balance salt solution (HBSS) [Hassan and Zgair, 2013]. Preparation of Targeted Sheep Red Blood Cells The standard method of Golstein and Gomperts (1975) was followed to coat sheep red blood cells (SRBCs) with rabbit anti–(human)-IgG (Sigma–Aldrich), rabbit anti-(human)-IgM (Sigma–Aldrich) or HBcAg (gifted from Dr. Razaq Hadi Esa, CPHL, Baghdad, Iraq). The study was conducted following approval from the animal ethics committee of University of Baghdad. Indirect Plaque-Forming Cell (PFC) The number of PFCs was determined by using the modified method of Jerne plaque assay [Jerne and Nordin, 1963; Zgair, 2013a, Zgair, 2013b]. Nine test tubes were used in this experiment. Hundred micro liter of SRBCs (3%) coated with rabbit anti–(human)IgG was added to first and second test tubes. In third and fourth test tubes, 100 ml of SRBCs coated with rabbit anti–(human)-IgM was added. In fifth, sixth, and seventh test tubes, 100 ml of SRBCs coated with

HBcAg and 100 ml of uncoated SRBCs (3%) was added to 8th and 9th test tubes. Then, 100 ml of 5  106/ml [HBSS containing 0.5% w/v gelatin (BD DifcoTM, USA)] PBLs was added to first, third, fifth, sixth, and eighth test tubes. While, 100 ml of PBS (0.1 M, pH 7.2) was added to second, fourth, seventh, and ninth test tubes (technical control). Two milileter of 0.75% of agarose L [prepared in Eagle’s medium pH 7.2 (Flow Lab, Irvine, Scotland)] was added to each test tube. The test tubes containing the aliquots of mixture were maintained at 40˚C in a water bath. The mixture was spread quickly onto the warmed Petri dishes (39˚C) to form a thin layer. Plates were incubated for 2 hr at 37˚C. Two milileter of complement inactivated goat IgM anti-(human)IgG (1:500) (Sigma–Aldrich) was added to first, second, fifth, eighth, and ninth plates (corresponding to test tubes). All plates were washed three times with PBS (pH 7.2, 0.1M) after incubation for 1 hr at 37˚C. Two milileter of guinea-pig serum (1:10 v:v serum:PBS) was added to each plate and then the plates were incubated for 30 min at 37˚C. The plates were fixed by glutaraldehyde (0.25% PBS) and then PFCs were counted. Results were reported as PFCs/ million cells. The same method was conducted three times for each clinical and control sample. Flow Cytometric Quantitation of CD4þ and CD8þ Cells PBLs were stained for flow cytometric analysis, the following monoclonal antibodies were used in this study: CD4 (PerCP-Cy5.5, clone L200, BD Biosciences, Franklin Lakes, NJ, USA) and CD8 (BD Biosciences, Franklin Lakes, NJ, USA). Hundred micro litres of PBLs (106 cell/ml) were stained with antibodies for 20 min at room temperature. After washing the cells J. Med. Virol. DOI 10.1002/jmv

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twice with phosphate-buffered saline (PBS) containing 2% FBS, the cells were fixed with PBS containing 1% formaldehyde (Sigma, St. Louis, MO, USA). Results were obtained using a LSR II (BD Biosciences, USA) and analyzed using FlowJo software (TreeStar, Inc., Ashland, OR, USA) [Didier et al., 2012]. Isolation of Human Hepatocytes for Primary Culture The hepatocyte isolation procedure was described previously [Trevisan et al., 1982], with minor modification [Lau et al. 1991]. Briefly, the liver biopsies (1.5–3.5 cm long) obtained from patients (chronic hepatitis B and asymptomatic HBV-healthy carrier) and healthy volunteer (He was suffering from abnormal liver size, but after histopathological examination, no abnormal findings were observed) were immediately immersed in a HEPES-buffered RPMI 1640 medium (Himedia, Mumbai, India) containing 0.2% EDTA (Sigma–Aldrich), penicillin (200 U/ml), streptomycin (100 pg/ml), and 10% heat inactivated fetal calf serum (FCS; Sigma–Aldrich) adjusted to pH 7.4 with sterile 1 N NaOH. Under sterile conditions, the hepatocytes were digested with collagenase (Sigma–Aldrich) and dispase (Sigma–Aldrich) and a suspension was made by gentle teasing of the biopsies with a 21 G needle. The cell suspension was washed twice with oxygenated isolation medium without EDTA (culture medium). Cells were counted and resuspended at a concentration of 4  104 cells/ml with culture medium. Evaluation by phase-contrast microscopy showed that more than 95% of the isolated cells were hepatocytes with well-preserved morphology and cell-surface integrity. Contamination with non-parenchymal cells usually did not exceed 3% of the total cell number. The viability of the isolated hepatocytes assessed in parallel by trypan blue dye exclusion and by plastic adherence and eosin staining after 16-h culture was >80%. This result was comparable with the number of viable cells observed after short-term culture using similar assay in previous studies [Lau et al., 1991a; Lau et al., 1991b]. Preparation of Rabbit Anti-HBc and Rabbit Anti-HBe On days 1 and 7, rabbits were given a subcutaneous injection of 250 mg of either HBcAg (Virogen Corporation, Boston, USA) or HBeAg (GenScrip, USA) dissolved in 0.5 ml of complete Freund’s adjuvant (Sigma– Aldrich). A series of four intravenous injections containing 50, 100, 150, and 250 mg of antigens, respectively, in 0.5 ml of saline were given at three-day interval, starting 20 days after the first subcutaneous injection. Blood was collected from the marginal ear vein one week after the last injection. Sera were heated at 56˚C for 30 min to remove complement activity and stored in 0.5 ml aliquots at 20˚C. The levels of anti-HBc and anti-HBe in sera of immunized rabbit were determined J. Med. Virol. DOI 10.1002/jmv

using ELISA [Kryger et al., 1981]. The study was conducted following approval from the animal ethics committee of University of Baghdad. Cytotoxicity Assay Three sets of tissue culture tubes (Nunc, Denmark) (two tubes in each set) containing 0.2 ml of culture medium mixed with 0.1 ml of 4  104 hepatocytes suspension (isolated from liver of patients with chronic hepatitis B and an asymptomatic HBV carrier, and healthy control). 20 mI of rabbit anti-HBc (1:40), rabbit anti-HBe (1:40) and serum of unimmunized rabbit (1:40) as a control were added to each set. One tube of each pair that was treated with sera of immunized and unimmunized rabbit was supplemented with rabbit complement (Bangalore Geni, India) to a final dilution of 1:20, and a parallel tube with the same dilution of rabbit sera (anti-HBc, antiHBe, and control) was incubated with heat-inactivated rabbit serum (56˚C for 1 hr) that served as the inactivated complement control. Finally, in each set, there was activated complement medium (first tube) and inactivated complement medium (second tube). Complement dilution was determined in a pilot experiment in which hepatocytes isolated from liver biopsies from patients (chronic hepatitis B and HBVhealthy carrier) and control (healthy control) were incubated with two-fold serial dilutions of active and heat-inactivated rabbit complement and their cytotoxicity measured using both trypan blue dye exclusion and plastic adherence. The latter test was performed under conditions identical to those employed subsequently in the microcytotoxicity assay. The highest non-toxic concentration of active complement giving comparable rates of viable hepatocytes in both the assays was chosen for the cytotoxic experiments. After addition of active or inactivated complement serum, all samples were incubated for 30 min at 37˚C. Then, 1 ml of culture medium was added and cells were pelleted by centrifugation at 50 g for 5 min. Each pellet was resuspended in 200 ml of culture medium containing 15% heat-inactivated FCS, and hepatocytes seeded into wells of a microculture plate (Nunc, Inc., Naperville, IL, USA) at 10 ml (about 400 cells) per well. The plates were incubated in a humidified atmosphere of 95% O2 and 5% CO2 for 16 hr, inverted for 1 hr and gently washed with three changes of Hank’s balanced salt solution to remove the non-adherent, non-viable cells by trypan blue staining. The remaining hepatocytes were fixed in methanol for 7 min, stained with 1% eosin for 8–10 min and subsequently washed in distilled water four times. The number of hepatocytes in each well was counted. The quotient resulting from the division of the difference between mean number of cells in control and test wells was divided by mean number of cells in control (complement control) and this gave the percentage cytotoxicity [Michalak et al., 1995]. The experiment was repeated

B Cells in the Severity of Chronic Hepatitis B

three times independently, each time in triplicate. The data are represented as mean of percentage of cytotoxicity  the standard deviations.

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ware. A value of P < 0.05 was considered to be statistically significant. RESULTS

Peripheral Blood Lymphocytes Culture The isolated peripheral blood lymphocytes were adjusted to 106 viable cells/ml and cultured under standard cell culture condition (37˚C, 5% CO2) in RPMI 1640 medium with 2 mM l-glutamine adjusted to contain 1.5 g/l sodium bicarbonate, 4.5 g/l glucose and 10 mM HEPES (complete media) and supplemented with 10% heat inactivated fetal bovine serum (FBS; Sigma–Aldrich). The culture medium, chemicals, and FBS were endotoxin-free. 0.1 ml of samples was collected from cell cultures at different time intervals (0, 2, 4, 8, 24 hr) for mRNA IL-10 and IL-10 protein quantification. Preparation of Total RNA and Real Time RT-PCR The culture samples were mixed with a Trizol reagent (InvitrogenTM, Applied Bioscience, USA) using an Ultra Turrax homogenizer and stored at 80˚C. The total RNA was then isolated using an RNA isolation kit (Ultraspec-II; Biotecx, Houston, TX, USA). The IL-10 mRNA levels were measured using a real time-polymerase chain reaction (RT-PCR) [Chuang et al., 2012]. The purified total RNA was used as the template in the RT-PCR. The cDNA was synthesized using moloney murine leukemia virus reverse transcriptase (Promega, USA) according to the manufacturer’s instruction. The PCR primers for IL-10 and G3PDH were described previously [Bujalska et al., 2007; Vouldoukis et al., 2011] and the reactions were performed as described previously [GarciaRodriguez et al., 2012]. The relative expression of IL-10 with G3PDH as the reference gene was determined using the 2-~~CT (Livak) method.

Levels of Anti-HBc IgG and Anti-HBc IgM Figure 1 shows the level of anti-HBc in the sera of patients with chronic hepatitis B (CHB) as well as asymptomatic HBV-healthy carriers (HBV-HC). These results proved that significant increase of anti-HBc level was seen in the sera of patients with chronic hepatitis B as compared to the level of anti-HBc in the sera of asymptomatic HBV-healthy carriers. Significant elevation was observed in case of anti-HBc IgM (P < 0.001) (Fig. 1a), while moderate increase (P < 0.05) was observed in case of anti-HBc IgG (Fig. 1b). Number of CD4þ and CD8þ Cells in Peripheral Blood Flow cytometery was used to estimate the number of T helper cells (CD4þ cells) and suppressor/cytotoxic T lymphocytes (CD8þ cells) in the peripheral blood of patients with chronic hepatitis B and asymptomatic HBV carriers. On the basis of the results, the number of CD4þ and CD8þ cells in healthy control group was determined (Fig. 2). Significant decrease (P < 0.01) in the number of CD4þ cells was observed in peripheral blood of patients with chronic hepatitis B as compared to number of CD4þ cells in peripheral blood of asymptomatic HBV-healthy carrier and healthy control groups (Fig. 2a). A significant increase in the number of CD8þ T lymphocyte in peripheral blood of patients with chronic hepatitis B was observed as compared to other studied groups (Fig. 2b). On this basis, significant decrease in CD4þ/CD8þ ratio was observed in the

a 1.6

Equal volumes were collected from PBL cultures at different time intervals (0, 2, 4, 8, 24 hr). The samples were centrifuged at 600 g for 5 min to pellet cells. Supernatants were then collected and stored at 20˚C until use for ELISA. 10 ml of blood was collected from patients and healthy volunteers for cytokine assay. The concentrations of IL-10 were measured using the human IL-10 ELISA kit (BD Biosciences, USA). Statistical Analysis All values have been used to give a mean value and the standard deviation (s.d.) calculated. The correlation coefficient test [Pearson correlation (r)] was used to check the relationship between two groups. The differences were analyzed by using Student’s t-test employing original version 8.0 soft-

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Fig. 1. Levels of anti-HBc in terms of optical density (OD) at 450 nm. a, anti-HBc IgM; b, anti-HBc IgG in sera of patients with chronic hepatitis B and asymptomatic HBV-healthy carriers. Asterisk indicate a significant differences between asymptomatic HBV-healthy carrier (HBV-HC) and chronic hepatitis B (CHB) ( P < 0.05;  P < 0.001). Upper and lower lines represent the quantization range of OD.

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Fig. 2. Flow cytometrical technique to count the number of T helper lymphocyte (CD4þ cells) and cytotoxic/suppressor T lymphocyte (CD8þ cells) in peripheral blood of patients (chronic hepatitis B, asymptomatic HBV-healthy carriers) and healthy control groups. a, Number of CD4þ cells in patients and

control groups; b, number of CD8þ cells in patients and control groups; c, CD4þ/CD8þ ratio in patients and control groups. Each value represents the mean  s.d. a, compared to asymptomatic HBV-healthy carrier; b, compared to control group. Asterisks indicate a significant difference from studied groups (P < 0.05).

peripheral blood of patients with chronic hepatitis B as compared to asymptomatic HBV carriers and the healthy control groups (Fig. 2c).

Figure 3. The significant elevation of PFC-IgG, PFCIgM, PFC-anti-HBc IgM, and PFC-anti-HBc IgG in peripheral blood of patients with chronic hepatitis B was noticed as compared to number of PFCs in asymptomatic HBV-healthy carrier and healthy control volunteers (P < 0.05). The present study proved that the blood of patient with chronic hepatitis B had significantly higher number of antibody-secreting cells that produced IgG, IgM, anti-HBc IgM, and HBc-IgG spontaneously without any external stimulation.

Plaque Forming Cells in Peripheral Blood Number of plaque forming cells in peripheral blood of patients (chronic hepatitis B and asymptomatic HBV carriers) and healthy controls is shown in

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Fig. 3. Plaque forming cell (PFC) in peripheral blood of patients with chronic hepatitis B, asymptomatic HBV-healthy carriers and healthy control group (control). a, number of PFCIgM/106; b, number of PFC-IgG/106; c, number of PFC-anti-HBc IgG/106; d, number of PFC-anti-HBc IgM/106. Each value represents the mean  s.d. a, compared to asymptomatic HBVhealthy carrier; b, compared to control group. Asterisks indicate a significant difference from studied groups (P < 0.05).

J. Med. Virol. DOI 10.1002/jmv

In the current study, the relationship between the number of PFC and the levels of antibody in peripheral blood of patients with chronic hepatitis B, asymptomatic HBV-healthy carrier and healthy volunteers was evaluated. The total concentration of essential immunoglobulins (IgG and IgM) is presented. The correlation was calculated in terms of the Pearson correlation (r) value. The positive relationship was observed between the number of PFC-(IgG, IgM, anti-HBc IgG, and anti-HBc IgM) and levels of total IgG, IgM, anti-HBc IgG, and anti-HBc IgM in peripheral blood of patients with chronic hepatitis B (r > þ0.5; P < 0.05) (Supplement file 1 a, b, c, and d). However, no relationship was found between PFCs and levels of antibody in peripheral blood of the asymptomatic HBV carrier group (r < 0.5, P > 0.05) (Supplement file. 1. e, f, g, and h). In the healthy control group, the relationship was calculated between PFC-(IgG and IgM) and IgG and IgM levels only as in this group, the PFC-anti-HBc and antiHBc antibodies were not detected in peripheral blood. In control group, no correlation was found between PFC-(IgG and IgM) and IgG and IgM levels (r < 0.5,

B Cells in the Severity of Chronic Hepatitis B

The Correlation Between PFC and Severity of Chronic Hepatitis B and Asymptomatic HBV Carriers (LFTs) To evaluate whether the hyperactivity of antibodysecreting cells affects the severity of chronic hepatitis B, the relationship between the severity of disease in terms of LFTs levels, PFC numbers, and antibodies in peripheral blood of patients (chronic hepatitis B and asymptomatic HBV-healthy carrier) and controls was studied. The significant relationship (P < 0.05) between the numbers of PFC-(IgG, IgM, anti-HBc IgG, and HBc IgM) and LFTs (ALK, ALT, and TSB) (r > þ0.5) was seen. Similar trend was observed between the levels of antibodies (IgG, IgM, anti-HBc IgG, and anti-HBc IgM) and the levels of LFTs (r> þ0.5, P < 0.05). However, when the same parameters were studied in the peripheral blood of control group, no significant relationship was found (r < þ0.5, P > 0.05) (Supplemented file 2). Cases of low severity hepatitis disease (asymptomatic HBV carriers) were included in this study to prove the role of PFC activity in the severity of any previous infection with HBV. No relationships were found among all the studied parameters (P > 0.05) (Supplemented file 3). The current study proved that in case of asymptomatic HBV-healthy carriers, there is no effect of antibody-secreting cells (PFC number) or antibodies produced by them on the level of LFTs. Hence, the study strongly confirms the involvement of antibodysecreting cells in the severity of chronic hepatitis B. Cytotoxic Effect of Anti-HBc and Anti-HBe on Human Hepatocytes In Vitro The present study showed that the sera collected from rabbits immunized with HBcAg- and HBeAginduced hepatocytotoxicity in presence of complement. This was found only in the case of hepatocytes isolated from patients with chronic hepatitis B and asymptomatic HBV carriers but not in hepatocytes isolated from healthy controls. The maximum hepatocytotoxicity was found in the hepatocytes that were isolated from patients with chronic hepatitis B and exposed to anti-HBc. Anti-HBc also induced significant hepatocytotoxicity in hepatocytes isolated from asymptomatic HBV-healthy carriers as compared to hepatocytotoxicity seen in cells isolated from healthy controls and those exposed to anti-HBc. Furthermore, the current study showed that anti-HBe also induced

significant hepatocytotoxicity in hepatocytes isolated from patients with chronic hepatitis B and asymptomatic HBV carriers as compared to those isolated from healthy controls and exposed to anti-HBe (Fig. 4). The current study proved the ability of antiHBc to induce hepatocytotoxicity in hepatocytes isolated from chronic hepatitis B patients higher than the ability of anti-HBe to induce the hepatocytotoxicity in vitro. IL-10 mRNA and Protein Levels in Cultured PBLs The level of IL-10 expression by lymphocytes obtained from peripheral blood of patients with chronic hepatitis B, asymptomatic HBV carriers and controls, and cultured under standard conditions are presented in Figure 5. The results showed significant increase (P < 0.05) in IL-10 mRNA expression by PBLs isolated from cases of chronic hepatitis B at 0 hr and 2 hr post incubation as compared to its expression by PBLs isolated from asymptomatic HBV carriers and control groups (Fig. 5a). Figure 5b shows the level of IL-10 in the suspension of PBLs cultures isolated from peripheral blood of patients with chronic hepatitis B, asymptomatic HBV carriers, and healthy controls. Significant increase in IL-10 concentration started as early as 2 hr post incubation in case of culture of PBLs obtained from patients with chronic hepatitis B as compared to controls. While, no significant change in IL-10 concentrations was seen in the suspension of PBLs isolated from patients

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#α

70

% cytotoxicity

P < 0.05) (Supplement file 1. i, and j). Significant elevation of IgG and IgM was observed in the peripheral blood of patients with chronic hepatitis B when compared with IgG and IgM levels in peripheral blood of asymptomatic HBV-healthy carrier and healthy control groups (P < 0.05) (Supplement file 1. k). The present study showed that the levels of IgG, IgM, anti-HBc (IgG and IgM) are associated with number of corresponding PFCs in the peripheral blood for patients with chronic hepatitis B.

413

60 50 40 30 20

*

* *

10 0 Anti-HBc

Anti-HBe

NS

Applied antibodies Fig. 4. Hepatocytotoxic effect of rabbit anti-HBc and rabbit anti-HBe in vitro on hepatocytes isolated from patients with chronic hepatitis B, asymptomatic HBV-healthy carrier and healthy volunteer. Columns represent mean of percentage of cytotoxicity  s.d. for each group of patients and healthy volunteers (control).  P < 0.05 versus % cytotoxicity of hepatocytes isolated from healthy control and exposed to anti-HBc or anti-HBe; #P < 0.05 versus % cytotoxicity of hepatocytes isolated from asymptomatic HBV-healthy carrier and exposed to antiHBc or anti-HBe; aP < 0.05 versus % cytotoxicity of hepatocytes isolated from chronic hepatitis B and exposed to anti-HBe.

J. Med. Virol. DOI 10.1002/jmv

414

Zgair et al.

Time

2h

* *

*

* 0h

2h

4h

Time

8h

24h

5

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4 3 2 1 0

HBV-HC

0h

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CHB

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8 7 6 5 4 3 2 1 0

c IL-10 pg/ml

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IL-10 pg/ml

8 7 6 5 4 3 2 1 0

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b

Relative expression of IL-10 mRNA

a

Fig. 5. IL-10 mRNA expression, a, and IL-10 protein levels; b, by human peripheral lymphocytes obtained from patients with chronic hepatitis B, asymptomatic HBV-healthy carrier and healthy control, and cultured in standard conditions. c, IL-10 concentrations in sera of patients with chronic hepatitis B, asymptomatic HBV-healthy carrier and healthy con-

trol. Total RNA was extracted and subjected to real time polymerase chain reaction (RT-PCR) as described in materials and methods. Data are presented as means of data collected from experiments of ten test and control.  P < 0.05 versus (control and asymptomatic HBV-healthy carrier groups).

with asymptomatic HBV-healthy carrier as compared to control. The current study proved the ability of lymphocytes obtained from patients with chronic hepatitis B to produce IL-10 without being exposed to external stimulators. While, the lymphocytes isolated from patient with asymptomatic HBV-healthy carrier lacked this ability.

disease in terms of LFTs. This study also highlights the elevation of PFC concomitant with decrease in number of CD4þ cells and increase in number of CD8þ cells in peripheral blood of patients with chronic hepatitis B as compared to their numbers in the peripheral blood of asymptomatic HBV carriers and healthy volunteers. The direct role of antibodysecreting cells in the hepatocytotoxicity of cells in patients with chronic hepatitis B and asymptomatic HBV carriers was seen and this effect was mediated by antibodies (anti-HBc). In the present study, the asymptomatic HBV carriers were included in addition to healthy controls as this group carried HBV but without any clinical symptoms and to strengthen the relationship between the activity of PFCs and severity of chronic hepatitis B. As in asymptomatic HBV carriers, no relationship was observed between the activity of PFCs and pathogenicity of HBV. HBcAg stimulated production of IL-10 and negatively regulated HBcAg-specific T-helper cell responses in chronic hepatitis B patients. This may represent one evasion strategy for HBV to subvert specific antiviral responses in human. T helper cells have been shown to have an important role in host defense against viral infections [Li et al., 2010]. However, IL-10 helps B-lymphocytes to produce antibody spontaneously [Llorente et al., 1995]. This observation therefore supports the findings of the present study that proved the enhanced antibody production was seen following IL-10 production. The role of B lymphocytes in persistence of HBV in patients with chronic hepatitis B was demonstrated in the study of Oliviero et al. (2011). Herkel and Carambia (2011) showed the possible role of IL-10 in inducing the immunity of patients with chronic hepatitis B. In the present study, ad significant expression of IL-10 by lymphocytes isolated was

IL-10 Measurement in Sera of Patients The sera were collected from patients with chronic hepatitis B, asymptomatic HBV-healthy carrier, and healthy volunteers to measure the levels of IL-10. Figure 5c shows that the significant increase in IL-10 concentrations was found in sera of patients with chronic hepatitis B as compared to levels of IL-10 in sera of asymptomatic HBV carriers and healthy control groups (P < 0.005). DISCUSSION Chronic hepatitis B is responsible for the death of millions of people all over the world. Many studies have shown that cellular immune response against HBV virus is important in controlling the infection with this virus [Lavanchy, 2005; You et al., 2008]. T lymphocytes subgroups such as CD4þ and CD8þ cells are known to respond to this virus and directly affect on the severity of chronic hepatitis B. The literature dealing with the role of B-lymphocytes in the severity of chronic hepatitis B is very scanty. In the present study, we demonstrate the direct role of antibodysecreting cell (PFC) in the severity of chronic hepatitis B that was mediated by either total Igs (IgG and IgM) or anti-HBc (IgG and IgM). The current study provides evidence about the direct relationship between the number of PFC-(IgG, IgM, anti-HBc IgG, and anti-HBc IgM) obtained from peripheral blood of patients with chronic hepatitis B and severity of this J. Med. Virol. DOI 10.1002/jmv

B Cells in the Severity of Chronic Hepatitis B

found in patients with chronic hepatitis B and no such expression was evident by lymphocytes obtained from asymptomatic HBV carriers. Thus, this study suggests that B-lymphocytes in chronic hepatitis B reduce the activity of specific CD4þ cells by secreting IL-10. In addition, the involvement of B cells in the severity of chronic hepatitis B was due to the hepatotoxic effect of antibodies especially anti-HBc (IgG and IgM). Recent study reports significant high activity of B-lymphocytes associated with chronic hepatitis B [Oliviero et al., 2011]. The present study explains the role of B cells in allowing the persistence of HBV in chronic hepatitis B patients and the cytotoxic effect of antibodies in hepatotoxicity. The high activity of antibody-secreting cells was concomitant with low number of T-helper cell (CD4þ cell). That is why; the antibody-secreting cells had dual effect on the severity of chronic hepatitis B. Firstly, it played a crucial role in the persistence of HBV in patients with chronic hepatitis B. Secondly, it led to increase the severity of chronic hepatitis B through hepatocytotoxic effect of antibodies (anti-HBc) produced by these cells. The results of this study suggest that the chronicity of HBV infection is dependent on the ability of virus to decrease the activity of T-helper cells and that concomitant with high activity of antibodysecreting cells. That is why; the antibody-secreting cells, which are independent of T helper, play an important role in the severity of chronic hepatitis B. Thus, a decrease in activity of antibody-secreting cells along with an increase in the activity of T-helper cells might contribute in reducing the severity of HBV in patients with chronic hepatitis B. Work on these lines is in progress in our laboratory. In conclusion, the antibody-secreting cells (PFCs) play an important role in severity of chronic hepatitis B mediated by polyclonal antibodies (IgG and IgM) and specific antibodies (anti-HBc IgG and anti-HBc IgM). In addition, the activity of PFCs studied was not related to CD4þ cells therefore, the virus may activate B lymphocytes directly. Also, B cells reduced the activity of T-helper cells in patients with chronic hepatitis B by producing IL-10. The cytotoxic effect of antibodies produced by activated antibody-secreting cells was proved in present study. REFERENCES Beasley RP, Hwang LY, Lee GC, Lan CC, Roan CH, Huang FY, Chen CL. 1983. Prevention of perinatally transmitted hepatitis B virus infections with hepatitis B virus infections with hepatitis B immune globulin and hepatitis B vaccine. Lancet 2:1099– 1102. Bodsworth N, Donovan B, Nightingale BN. 1989. The effect of concurrent human immunodeficiency virus infection on chronic hepatitis B: A study of 150 homosexual men. J Infect Dis 160:577–582. Bond WW, Favero MS, Petersen NJ, Gravelle CR, Ebert JW, Maynard JE. 1981. Survival of hepatitis B virus after drying and storage for one week. Lancet 1:550–551. Boyum A. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Invest 21:77–89.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.