Expansion of innate CD5pos B cells expressing high levels of CD81 in hepatitis C virus infected liver
Introduction
Hepatitis C virus (HCV) is a major cause of chronic liver disease with over 170 million people infected world-wide [1], [2]. Almost one third of chronically infected individuals will develop cirrhosis and, of these, up to 2% per year will develop hepatocellular carcinoma [3], [4]. Currently, end-stage liver disease secondary to HCV infection results in 8000–10 000 deaths per year and is a leading indication for liver transplantation in the United States [1], [5]. HCV is predominantly hepatotropic with convincing immunohistochemical and in situ hybridisation data to show that hepatocytes are a primary site of infection [6], [7]. However, HCV can also infect and replicate within peripheral blood mononuclear cells (PBMCs), which may be important for viral dissemination and sequestration [8], [9], [10], [11], [12], [13], [14], [15], [16].
Immunological studies of HCV infection classically focus on T lymphocytes. However, the association of HCV infection with mixed cryoglobulinaemia, the presence of HCV antigen in the cryoprecipitate, clonal B cell proliferation in peripheral blood, bone marrow and liver and the high prevalence of non-Hodgkin's lymphoma suggest a major role for B cells in the pathogenesis of HCV infection [17], [18], [19], [20], [21], [22], [23], [24], [25]. Moreover, B cells are more frequently infected than T cells, monocytes and neutrophils and limiting dilution studies indicate higher HCV titres in B-lymphocytes than T cells or monocytes [26], [27]. We, and others, have reported an expansion of peripheral blood innate (CD5pos) B cells, the human equivalent of murine B-1 cells, in HCV infection [28], [29]. B-1 cells have restricted receptor gene segment usage [30], are a primary source of autoantibodies [31], can be activated by T-independent antigens [32] and are thus thought to comprise part of the functional and evolutionary bridge between the innate and adaptive immune systems. In this respect, B-1 cells are considered the B cell equivalent of γδ T cells [33]. CD5pos B cells have been shown to produce the monoclonal WA cross reactive idiotype rheumatoid factor in HCV infection [34], [35], [36] and may be responsible for the production of other auto-antibodies associated with HCV infection. Significant populations of innate-type T Lymphocytes including NT (CD3posCD56pos) and γδ-TCRpos T-cells have been detected in normal human liver [37], however, little is known about the equivalent populations of hepatic innate B cells (B-1/CD5pos). A primary aim of this study was to examine CD5pos B cell populations in normal and HCV infected liver.
CD81, a widely expressed tetraspanin molecule involved in the regulation of T cell homeostasis, is expressed on hepatocytes and lymphocytes including B-lymphocytes. Recently, CD81 has been shown to bind HCV RNA in association with the E2 region of HCV envelope protein [38]. The role for CD81 as a viral co-receptor remains unclear although CD81 is capable of binding recombinant E2. It has been demonstrated that combination Interferon-α and Ribavirin anti-viral therapy down-regulates CD81 expression both in vitro and in vivo [39], suggesting a role for CD81 in HCV pathogenesis. In addition, a potential role for CD81:HCV interactions is suggested through inhibition of NK cell activity [40], [41]. In this study, we explore differential surface expression of CD81 on hepatic and peripheral blood CD5pos B cells and CD5neg B cells. We also investigate the presence of HCV RNA and quantitate the intra-cellular viral load in purified peripheral B cell subpopulations.
Section snippets
Subjects
Six groups of patients and sero-negative normal controls were analysed. Subjects have been divided based on sample type (liver/peripheral blood), viremia (PCR+/−) and severity of HCV-associated liver disease (cirrhosis) to facilitate comparison of possible compounding variables between these groups (Table 1). All subjects gave written informed consent to participate in this study, which was approved by the Ethics Committee of St. Vincent's University Hospital. The first group included 34
Expression of CD5 by peripheral blood and hepatic B cells
Small proportions of B cells found in normal liver tissue express the CD5 molecule (Mean±standard error of the mean, SEM, 8.16±1.98%, % of total CD19pos B cells, n=9). However, this population was significantly expanded in livers of individuals with HCV cirrhosis (20.32±4.79%, n=11, P<0.05). There is an even greater expansion of these cells in the livers of non-cirrhotic HCV-infected individuals (38.95±5.64%; n=10, P<0.002, Fig. 2a). Interestingly, there was no overall expansion in the total B
Discussion
Innate lymphocytes recognise conserved structures that signal viral infection. Failure to eliminate HCV may result from a deficiency of such innate lymphocytes. Indeed, depletion of NK cells and CD56+ T cells have been reported to occur in the livers of chronically HCV-infected individuals [48], [49]. In this study we focus on another innate lymphocyte population, the CD5pos B cells, in chronic HCV infection. The most frequent and well-characterised extrahepatic manifestations of chronic HCV
Acknowledgements
The authors wish to thank the patients who participated in this study. We are also grateful to Ann Farrell, Siobhan Cronin, Kate Frazer and Deirdre O'Sullivan for their assistance in collecting samples. The assistance of the transplant co-ordinators and surgeons at the National Liver Transplant Unit is also gratefully acknowledged. This work was made possible through funding from the Health Research Board Ireland.
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Contributed equally to the direction of this study.