chronic hepatitis b ( chb ) virus ( hbv ) infection is the principal cause of cirrhosis and hepatocellular carcinoma ( hcc ) . the pathogenesis of hbv - related chronic liver disease is not well understood . however , it is clear that the immune mechanisms associated with the antiviral response are responsible for chb outcome [ 24 ] . this concept stems from the observation that , in chronic hepatitis b , t - cells can potentially participate both in the immune clearance of hbv - infected cells and in the pathogenesis of hepatocellular injury . furthermore the numbers of b lymphocytes and plasma cells are significantly higher in patients with liver cirrhosis than of those with inactive chronic hepatitis [ 7 , 8 ] . enormous intrahepatic b - cells with massive production of igm and igg and infiltrating plasma cells into the hepatic lobules have also been shown in hbv - associated chronic active hepatitis . b - cells contribute to immune responses through the secretion of effector cytokines and it has been suggested that naive and memory b - cell subsets preferentially produce different effector cytokines [ 10 , 11 ] . nave b - cells undergo maturation by somatic hypermutation in immunoglobulin variable region of the b - cell receptor ( bcr ) genes following contact with a specific protein accessible on dendritic cells . then the high affinity antigen receptors which normally consist of two isotypes membranes igm and igd continue to mature to either ig - secreting plasma cells or memory b - cells . in comparison to antigen primary response , immunological memory presents the capacity to increase a faster and more vigorous humoral response subsequent to antigen re - exposure . although antibody associated mechanisms targeting hepatitis b core antigen ( hbcag ) was reported in earlier studies , few data exist on b lymphocytes population in the liver of patients with chb . cd20 is a b - cell specific surface antigen that is expressed in all stages of b - cell development except on either early pro - b - cells or plasma cells and plays an important role in b - cell activation and proliferation . to elucidate the role of intrahepatic b - cells in the pathogenesis of chronic hepatitis b , we investigated the expression of cd20 marker on b - cells in liver biopsy of these patients by immunohistochemistry . liver biopsy specimens from 57 patients with hbv - associated chronic liver disease without liver neoplasm attending the hepatitis clinic of shariati hospital , tehran university of medical sciences , during the years of 2008 to 2011 were studied . hbv infection was diagnosed by the positivity for hepatitis b surface antigen ( hbsag ) in the patients ' sera . all the patients were hbeag negative and had a history of familial hbv infection , without coinfection with human immunedeficiency virus ( hiv ) or other hepatitis viruses . the patients ' clinical data at the time of liver biopsy were acquired from their medical records . the presence of chb , stage of fibrosis , and histological activity were evaluated by modified histologic activity index ( hai ) scoring system   on the liver sections stained with hematoxylin - eosin and sirius red . commercially available primary monoclonal antibody against cd20 ( clone ucht1 , dako ) was used to stain 3.0  m sections of liver biopsies after deparaffinization by routine microwave antigen retrieval and horseradish peroxidase ( diaminobenzidine ) kit ( dako , carpinteria , ca ) , following manufacturer 's instructions . immunohistochemical scoring ranged from 1 to 5 score ( matched for 010% , 1025% , 2550% , 5075% , and > 75% cd20 positive cells ) with higher scores indicating a greater proportion of positive cells ( as determined from the positive control ) . for enumeration of cd20 positive b - cells , image acquisition was done with the + 10 , + 20 , and + 40 objectives , but stained cells were counted in the entire field of + 40 corresponding to a tissue area of 30  mm . positive stained cells appeared with no clear plasmacytoid morphology , but with granular cytoplasm and lobated nucleus of b - cells . correlations between variables were analyzed using the pearson correlation coefficient ( r ) or spearman rank correlation coefficient ( r ) , as appropriate . the baseline demographics are shown in table 1 . the mean  sd age of patients was 33  9 years and 40 ( 70% ) were male . the mean  sd of total hai score for fibrosis and necroinflammation of patients is shown in table 1 . nine patients ( 15.7% ) had significant fibrosis more than or equal to 3 . to analyze the impact of b - cell population in chb patients , the cd20 expression score , age , gender , log viral load , serum alt , and hai total score of necroinflammation and fibrosis were included in a multivariable - adjusted logistic regression model . there was a significant association between log hbv dna with both alt and hai total score ( r = 0.36 , p = 0.006 , and r = 0.3 , p = 0.02 ) , respectively . serum alt also showed positive correlation with total score of hai though which was not significant ( r = 0.25 , p = 0.06 ) . there were no lines of evidence of interactions and multicollinearity between variables age and sex with log hbv dna and alt . no significant correlation was observed between expression of cd20 b - cell marker expression with serum alt and hbv dna level . the liver was diffusely infiltrated by cd20 positive b - cells , distributed both as aggregates in the portal areas and as single cells within the lobules . the cd20 positive b - cells were expressed in all 57 liver biopsies with a submembranous and membranous staining pattern ( figure 1 ) . the expression of cd20 was significantly associated with hai total score ( r = 0.32 , p = 0.01 ) and stage of fibrosis ( r = 0.31 , p = 0.02 ) . fifteen cases had score 1 , thirty one had score 2 , ten had score 3 , and one had score 4 cd20 positive b - cells . in this study , the role of intrahepatic b - cells in the pathogenesis of chb infection has been investigated . the immunohistochemical findings of liver biopsies in this study indicate a major role for b - cells in the pathogenesis of chb which is driven by increased cd20 positive b - cells population . our evidence showed that cd20 positive b - cells clustered in the portal areas or as single cells within the lobule and correlated with necroinflammation and fibrosis which was consistent with previous studies . indeed our histological findings resembled the histological picture seen in patients with acute hepatitis b induced liver failure with a reflective b - cell response that could be responsible for huge expression of antibody to hbv core protein ( hbcab ) . our study indicates that b - cells are involved in inflammatory reactions for destruction of infected cells . thus , they play an important role in the immune response of chb infection . unlike hbsag and hbeag , the immunogenicity of hbcag is able to directly activate b - cells to produce specific antibodies in an extralymphatic situation and in the absence of t - cells . moreover , it has been shown that the activation of b - cells , in a t - cell independent pathway , produced immunoglobulins and cytokines in the liver that changed fibrotic responses [ 11 , 16 ] . our recent study showed the association of igd positive b - cells with the fibrosis stage in chronic hepatitis b . the intrahepatic lymphocytes are recognized as long - lived recirculating b - cells and are similar to splenic b2 cells that produce cytokines il-2 , il-4 , tnf , and il-6 with proinflammatory function and contribute to disease pathogenesis in an antibody - independent fashion , perhaps by modulating t cell responses [ 16 , 18 ] . earlier studies revealed that b - cells evidently contribute to the development of liver fibrosis by the production of profibrotic cytokine il-6 that induces the differentiation of hepatic stellate cells into myofibroblasts and raise collagen synthesis [ 16 , 18 , 19 ] . both mature b lymphocytes in liver that belong to b2 subset   and resting b - cells typically have cd20 receptor . therefore , we could suggest cd20 marker , as a representative marker of both naive and preswitch memory b - cells . evaluation of intrahepatic b - cells population is important to determine how b - cells would respond to hbv infection . in this setting , evidence from the current study indicates that exposure to viral antigens can increase the proportion of b - cells in the liver to carry an exhausted phenotype and therefore to dysfunction . our present data also support the property of liver as an immune system , which may potentially affect the pathogenesis of various liver diseases . the authors declare that there is no conflict of interests regarding the publication of this paper .