• autoimmunity;
  • HCV;
  • peripheral blood B cells


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Hepatitis C virus (HCV) infection is associated with immune-mediated abnormalities and B-cell lymphoproliferation evolving to an overt lymphoma. Recently, CD81 was identified as an HCV receptor on B-lymphocytes, providing a mechanism by which B cells are infected and activated by the virus. In addition, expansion of CD5+ B lymphocytes was described to be associated with various non-HCV related autoimmune disorders. Therefore, we studied the possible role of peripheral B cells CD81 and CD5 over-expression in the development of HCV-related autoimmunity and their association with disease severity in chronic HCV infection. Peripheral B cells CD5 expression and mean fluorescence intensity (MFI) of CD81 were determined in 30 HCV-infected patients, 30 healthy controls and 15 patients with hepatitis B virus infection using fluorescence-activated cell scan (FACS). We have also investigated the association between peripheral CD5 and CD81 B-cell over-expression and markers of autoimmunity and disease severity in patients chronically infected by HCV. CD5+ B-cells were increased in chronic HCV infection (23·2 ± 7·2%) compared with those of healthy controls (15 ± 5·5%) (P < 0·0001) and chronic HBV infection (19 ± 3·7%) (P = 0·08). CD81 MFI was significantly higher in HCV-infected compared to HBV-infected patients and healthy controls. Both increased CD81 MFI and CD5+ B-cell expansion were associated with the production of rheumatoid factor and mixed cryoglobulins and positively correlated with HCV viral load and histological activity index. The overexpression of CD81 and the expansion of CD5+ peripheral B-cells in HCV-infected patients may possibly play a role in the development of HCV-associated autoimmunity and lymphoproliferation.


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Hepatitis C virus (HCV) infection is considered to be one of the most common important known causes of chronic liver disease worldwide affecting between 0·5% and 2% of western world populations [1]. In addition to its being hepatotrophic, HCV is also a lymphotrophic virus [2]. This peculiar lymphotropism may be responsible, at least in part, for the multiple immune-mediated extra hepatic manifestations of HCV infection, such as mixed cryoglobulinaemia [3–5], Sjögren-like syndrome [6], the presence of serum rheumatoid factor (RF), the production of autoantibodies [7–11] and B-cell non-Hodgkin lymphoma [12–14]. The pathogenetic link between HCV and the immune system in inducing both autoimmunity and lymphproliferation is unclear. The persistence of HCV in peripheral blood mononuclear cells, preferentially in B-cells [15], results in chronic stimulation of B-cells, leading to polyclonal and later to monoclonal proliferation of RF (IgMk)-producing cells, which may result eventually in malignant transformation and development of overt lymphoma [16–19]. The recent identification of CD81 protein as one of the HCV receptor candidates on B-lymphocytes [20] provides a mechanism by which B cells are infected with or activated by HCV and may raise a wide spectrum of interesting issues regarding the pathogenetic link between HCV infection, autoimmunity and lymphoproliferative disorders. On B cells, CD81 is a member of a signalling complex that includes CD19 and CD21 [21]. Cross-linking these complexes using either antibodies to CD81 or CD19 lowers the threshold for B-cell activation and proliferation. Similarly, binding of HCV particles to a CD81-containing complex might facilitate B-cell activation, possibly explaining, at least in part, the association between HCV, B-cell activation and lymphoproliferative diseases. In addition, it has been shown recently that peripheral blood CD5+ B cells subpopulation is expanded in patients with chronic HCV infection [22]. CD5+ B cells are rare in adults, but they are the predominant B cell population in the fetus, in which they appear to constitute a rather primitive but effective first line of defence against foreign antigens [23]. These cells are characterized by the production of low-affinity immunoglobulin M (IgM) with RF activity, arise early in ontogeny and are considered to represent the bridge linking the innate and acquired immune responses [24]. The production of circulating autoantibodies coupled with their increased frequency in rheumatoid arthritis and Sjögren’s syndrome has implicated them in the development of autoimmune diseases [25,26]. In addition, CD5+ B cells are also increased in Epstein–Barr virus and human immunodeficiency virus (HIV) infections associated with autoimmune manifestations as well as in patients with essential mixed cryoglobulinaemia [27–29]. Immunohistochemical techniques have identified CD5+ B cells in the hepatic lymphoid follicles of HCV-infected patients, where they produce both monoclonal and polyclonal IgM [30]. These CD5+ B-lymphoid aggregates decrease in number with advanced disease, suggesting that they may play a role in disease progression. Taken together, the aim of this study was therefore to determine the frequency of peripheral blood B cells over-expressing CD81 and CD5 in a cohort of chronically HCV-infected patients, and to investigate its possible relation to disease severity and autoimmune markers.


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CD81 mean fluorescence intensity (MFI) and CD5 over- expression on peripheral blood B cells was determined in 30 consecutive patients with chronic HCV infection attending the Liver Clinic at Bnai Zion Medical Center, Haifa, Israel. In addition two control groups were studies: group 1 consisted of 30 age- and sex-matched healthy individuals (recruited from Bnai Zion Medical Center staff, mean age ± s.d.: 45 ± 15, M/F: 17/13) and group 2 (disease-control group) consisted of 15 consecutive HIV-negative patients with hepatitis B virus (HBV)-related chronic liver disease (attending the Liver Clinic at Bnai Zion Medical Center). All HCV-infected patients were tested positive for anti-HCV antibody (ELISA II, Abbot Laboratories, North Chicago, IL, USA), had detectable HCV RNA (polymerase chain reaction assay, Amplicor; Roche Molecular Systems, Somerville, NJ, USA) and had histological evidence of chronic hepatitis with or without cirrhosis. Excluded were patients who had positive serological tests for HIV and/or hepatitis B surface antigen (HBsAg). All HBV-infected patients (control group 2) were HBsAg-positive and had clinical evidence of chronic liver disease. All HCV- positive and control group 2 patients underwent an ‘immunological screening’ including protein electrophoresis, rheumatoid factor (RF), cryoglobulin and a panel of autoantibodies including antinuclear (ANA), antismooth muscle (ASMA) and anticardiolipin antibodies. In HCV-positive patients, HCV genotype and viral load was determined and disease severity was assessed by serum level of alanine aminotransferase (ALT) and liver histology (inflammation and fibrosis). Patients gave written informed consent, and the study was approved by the Institutional Review Board of the Bnai Zion Medical Center.

Definition of immune-mediated phenomena

The definition of immune-mediated phenomena is the presence of Coombs positive autoimmune haemolytic anaemia (AIHA), immune thrombocytopenic purpura (ITP), autoimmune thyroiditis, vasculitis, Raynaud’s phenomena or lichen planus.

HCV quantification

HCV RNA concentration in serum was measured using the Amplicor Monitor Test Kit (Roche Diagnostic Systems, Basel, Switzerland). The test includes an RNA quantification standard of known copy number that is co-amplified with target and is used to calculate copy level of sample by colourimetric assay following hybridization to a specific probe.

HCV genotyping

HCV genotypes were assayed using a commercial assay (INNO-Lipa HCV II test; Innogenetics NV, Zwijndrecht, Belgium) based on amplification with biotinylated oligonucleotide primers for the 5′ untranslated region of HCV. This assay identifies six genotypes and major subtypes.

Histological analysis

Histological score was graded from 0 to 22, modified from Knodell et al.[31]. For the Knodell histological activity index (HAI), an inflammation score was obtained by combining scores of periportal injury (interface hepatitis and/or bridging necrosis) (0–10), parenchymal injury (0–4), portal inflammation (0–4) and fibrosis (0–4). Degree of inflammation and fibrosis was scored by an independent histopathologist unaware of the patients’ clinical status. The Knodell HAI was calculated only for the non-cirrhotic patients.

B cell separation

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficolle-Hypaque density gradient separation. B lymphocytes were positively selected with magnetic microbeads conjugated to antihuman CD22 (MAC System, Melteni Biotech) according to the manufacturer’s instructions. Following isolation, CD19+ cell population (also CD81+ cells) comprised 95% of the resulting population. The purified B cells were assayed for CD81 MFI and for the expression of CD5 on resting B cells.

Flow cytometry

Purified B cells were immunostained directly (one-step) with PE and FITC-conjugated monoclonal antibodies (MoAb) for CD81 and CD5 (Ancell, Bayport, MN, USA). Flow cytometry was carried out with a fluorescent-activated cell scan (FACS) operating with Cellquest software (Becton Dickinson, Mountain View, CA, USA). The total population of viable cells was gated according to their typical forward and right-angle light scatter. The fluorescence of cells treated with fluorescent isotype MoAb was evaluated in each experiment to determine the level of background fluorescence of negative cells. Data were acquired on the flow cytometer and CD5+ B-cells were expressed as a percentage of CD81+ cells. The percentage and mean fluorescence intensity (MFI) of stained cells were determined according only to the positive cells. MFI of CD81 was expressed as optical density (O.D.) units.

Statistical analysis

Continuous variables having a skewed distribution (i.e. percentage of CD5+ cells and ALT level) were transformed logarithmically before statistical analysis in order to stabilize their variances. However, the results are presented in their original, non- transformed form in order to simplify their clinical interpretation. Averages and 90% ranges (i.e. the 5th and 95th percentiles, the values bounding the central 90% of the data) are presented. Comparison between two groups was performed using Student’s t-test for independent groups. Association between categorical groups was tested using a χ2 test or Fisher’s exact test, as appropriate. Correlation between two continuous variables was evaluated using the Spearman’s Rank Correlation Coefficient. Two-tailed P-values of 0·05 or less were considered to be statistically significant.

Cryoglobulin detection and analysis

Venous blood samples were collected into prewarmed tubes after an overnight fast and allowed to clot at 37°C. After centrifugation, sera were incubated at 4°C for 3 days. The cryocrit was evaluated by centrifugation of the serum in haematocrit tubes at 4°C. The cryoprecipitates were further analysed and characterized by immunofixation electrophoresis (Immunofix Kit; Helena Laboratories, Beaumont, TX, USA).


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Table 1 summarizes the clinical characteristics of the HCV-infected patients and disease-control group. The two groups were comparable with respect to age, gender and disease severity, i.e. ALT levels, presence of cirrhosis and HAI score. However, the HCV-infected patients had significantly higher rate of RF positivity, detectable cryoglobulins and presence of autoantibodies. Genotype 1 was the most frequent genotype found among the HCV-positive patients: 22 of 30 (73%) (genotype 1b in 21 and 1a in 1), whereas the other eight patients had non-1 genotypes (3a in four, 2a in two, mixed in two). Cryoglobulin was detected in 11 of 30 (36%) HCV-infected patients (mean cryocrit: 4·5%, range: 1·1–28%). Analysis of the cryoglobulin in six of these 11 patients revealed type II mixed cryoglobulin with monoclonal IgMk in all.

Table 1.  Clinical characteristics of HCV-infected patients and disease- control group
 HCV-infected HBV-infected
Parameterpatients (n = 30)patients (n = 15)P
  1. HAI, Knodell histological activity index score; ALT, alanine aminotransferase; RF, rheumatoid factor; NS, non-significant. *Presence of one or more autoantibodies (i.e. antinuclear, antismooth muscle and/or anticardiolipin antibodies); **presence of Coombs positive autoimmune haemolytic anaemia (AIHA), immune thrombocytopenic purpura (ITP), autoimmune thryroiditis, vasculitis, Raynaud’s phenomena or lichen planus.

Age (mean ± s.d.), year46 ± 1447 ± 16n.s.
Sex, F/M18/128/7n.s.
Cirrhosis (%)5 (16)2 (13)n.s.
HAI (mean ± s.d.)9 ± 3 7·2 ± 2·7n.s.
ALT (IU/l)123 ± 9579 ± 25n.s.
HCV RNA titre, mean 5·2 ± 3 
(×105 copies/ml)
RF (%)11 (36)1 (7)0·03
Cryoglobulin (%)11 (36)1 (7)0·03
Autoantibodies (%)*18 (60)3 (20)0·017
Immune-mediated phenomena (%)**8 (27)2 (13)n.s.

Flow cytometry analysis of B cells

The proportion of peripheral blood B cells co-expressing CD81 and CD5 antigens in patients with chronic HCV infection (n = 30) was significantly increased (mean ± s.d.) and the bounds of 90% range in parenthesis: 23·2 ± 7·2% (13·3–36·6) compared with that in normal controls (n = 30) (7·15 ± 5·5%, (10–30·3), P < 0·0001). Although CD5+ cells in HCV-infected patients were also expanded compared to patients with chronic HBV infection (n = 15) (5·19 ± 3·7% (15·3–25·6)) the difference did not reach statistical significance (P = 0·08) (Figs 1 and 2 ). In addition, MFI of CD81 expression was significantly higher in HCV-infected patients (n = 20) than in normal controls (n = 20) and HBV-infected patients (n = 15) (mean ± s.d.) and the bounds of 90% range in parenthesis: 205 ± 50 (94–275) O.D. vs. 148 ± 41 (78–220) O.D., P = 0·0004, and 166 ± 46 (70–235), P = 0·028, respectively) (Fig. 3). Interestingly, this increase of CD81 expression on B-cells from HCV-positive patients was significantly correlated with HCV RNA load (r = 0·52, P = 0·02) and HAI (r = 0·45, P < 0·05) and was associated with the presence of ANA (P = 0·01) and mixed cryoglobulins (P = 0·07).


Figure 1. A representative flow-cytometric profile of CD5+ B cells in normal controls, patients with chronic hepatitis B virus infection, and patients with chronic HCV infection. The y-axis represents CD5 staining and the x-axis represents CD81 staining.

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Figure 2. Expression of CD5 on B cells in normal controls, patients with chronic HBV infection and patients with chronic HCV infection. CD5+ B cells are expressed as a percentage of the total CD22/CD81+ population.

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Figure 3. CD81 mean fluorescence intensity (MFI) on peripheral B-cells from normal controls, patients with chronic HBV infection and patients with chronic HCV infection. The y-axis represents the CD81 MFI expressed by O.D. units. Mean MFI of isotype control.

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Whereas the expansion of CD5+ B-cell subpopulation in HCV-infected patients was not correlated with ALT levels (r = 0·04, P = 0·8), there was a positive correlation with histological activity index (r = 0·44, P = 0·03). In respect to the viral load, we found that the expansion of CD5+ B-cell was significantly associated with high HCV RNA levels (r = 0·54, P = 0·01) (Fig. 4).


Figure 4. Positive correlation between the titre of HCV RNA and the percentage of CD5+ peripheral B cells in HCV-infected individuals (r = 0·55, P = 0·006).

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In relation to the possible pathogenetic link between HCV infection and autoimmunity, the expansion of CD5+ B cells was found to be significantly associated with the production of RF (P = 0·02) and anticardiolipin antibodies (P = 0·04). However, although a trend for an association between the expansion of CD5+ cells and the presence of other autoantibodies (i.e. ANA and ASMA) and cryoglobulins was noted, it did not reach statistical significance (P = 0·06, P = 0·07, respectively).


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Chronic HCV infection is associated with a host of extrahepatic manifestations including autoimmune phenomena, benign clonal expansion of B-cells and B cell non-Hodgkin’s lymphoma, which suggest B cell activation and proliferation. However, the exact mechanism linking HCV infection with autoimmunity and lymphoproliferation is unknown. In this study, we demonstrated that peripheral B-cells from patients with chronic HCV infection overexpress both, CD5 and CD81 compared with chronic HBV infection and normal controls. This increase correlated with the levels of HCV RNA and histological severity, and was also found to be significantly associated with the presence of autoimmune markers.

Analysis of mixed cryoglobulins from patients infected with HCV shows that hepatitis C virion is bound to monoclonal IgM bearing the WA cross-idiotype (XId) [4]. In addition, data on the molecular composition of WA monoclonal RF (mRF), and the characterization of monoclonal B cells in the liver of patients with mixed cryoglobulinaemia type II [30] suggest that a specific population of B cells may be involved in the host response to HCV infection. These are B cells that proliferate with little or no somatic mutations of the immunoglobulin genes, are self- replicating, are stimulated by self-antigens in a T cell-independent manner and bear the CD5 marker [32]. Thus, it has been suggested that HCV infection, may induce antigen-driven benign proliferation of selected B cells producing IgM mRF. The HCV-induced RF response is biased to produce IgM RF encoded by a restricted set of immunoglobulin V genes, predominantly the VH/VL gene pair 51p1/kv325 [33,34]. Additionally, the recent report on somatic hypermutation and preferential expression of the VH 51p1/VL kv325 immunoglobulin gene combination in HCV-associated immunocytomas [16] supports the role of chronic antigenic stimulation in inducing B-cell clonality in HCV-infected patients. B cell proliferation in HCV-infected patients is probably enhanced by HCV-specific properties, including the ability of HCV proteins to bind to CD81 on B cell surface, and to influence intracellular regulatory functions following viral entry into B cells. In respect of our finding of the positive correlation between CD81 overexpression and HCV viral load, one may speculate that viral factors enhance CD81 up-regulation, possibly leading to the enhanced binding of HCV proteins to this receptor. In support of this hypothesis is the recent finding that lower levels of cell surface-associated CD81 are associated with genotype HCV-3 and the initial decline of HCV RNA after initiation of antiviral therapy [35]. The binding of HCV proteins to CD81-containing complex may lower the threshold for B-cell activation and proliferation, and thus may facilitate B-cell activation and proliferation. Our finding of CD5+ B-cell subpopulation expansion in HCV-infected individuals and its association with the presence of RF, cryoglobulins and anticardiolipin may support this hypothesis and reflect facilitated activation of CD5+ B cells which is induced by HCV binding to CD81 antigen. These findings, along with the observation that levels of CD5+ cells correlate with serum RF activity in patients with mixed cryoglobulinaemia (MC) [29], may indicate that CD5+ cells are involved in the production of mRF, cryoglobulins and possibly other autoantibodies in patients with chronic HCV infection.

Interestingly, the proportion of CD5+ B cells population in our HCV-infected patients was correlated positively with the levels of HCV-RNA. It might be reasonable to speculate that an increased number of HCV particles in peripheral blood may enter, infect and activate a larger number of peripheral B cells expressing CD5 and CD81, as occurs when a higher level of HCV viraemia is associated with more advanced stage of liver disease [36]. In support of this hypothesis are recent data indicating that HCV infection and clonal expansion of B cells within the liver preferentially involve RF-producing cells [37]. Furthermore, in selected cases of type II MC patients, B cell subsets expressing IgM RF are the prevalent cell type targeted by HCV [38].

In respect to the association between the expansion of CD5+ B cells and the production of autoantibodies, we have found such an association with anticardiolipin antibodies (IgM isotype in all) and RF but not with other autoantibodies such as ANA and ASMA. Our findings are in agreement with the recent data showing that antismooth muscle antibody, a serological marker of autoimmunity, was not linked to clinical findings of autoimmune diseases in HCV-infected individuals, whereas RF and cryoglobulins were frequently present in patients who had autoimmune phenomena [39]. Although an increase of CD5+ B cells in patients with Epstein–Barr virus and HIV infections was found to be associated with autoimmune manifestations [27,28], the lack of an association between increase of CD5+ cells and the presence of ANA and ASMA in our study may indicate a different stimulatory and pathogenetic mechanism for the production of these antibodies in HCV-infected subjects.

In contrast to the findings of Curry et al.[22] who showed a negative correlation between CD5+ B cells and HAI, increased CD5+ percentage in our HCV-infected patients was associated with a higher HAI. The reason for this difference may be due to the inclusion of a larger number of patients with advanced disease (i.e. five with cirrhosis and four with significant fibrosis) in our study, whereas in Curry’s study most of the patients had a mild to moderate disease [22]. The role of CD5+ B cells in progression of liver disease is not clear and remains speculative. However, the recent observation that blood levels of cryoglobulins and RF correlate with the amount of fibrosis in patients with HCV [40] may suggest that these cells play some role in liver injury. B cells are not usually associated with cytokine production; however, CD5+ B cells are known to produce the immunoregulatory cytokine, interleukin-10 (IL-10) [41]. The production of IL-10, which is thought to have an autocrine loop, may result in further expansion of this B-cell subpopulation [42].

In conclusion, peripheral B-cells from patients chronically infected with HCV show expansion of CD5+ subpopulation and CD81 overexpression. The correlation of CD5+ B-cells expansion and CD81 overexpression with HCV viral load, and their association with the presence of autoimmune markers, may be relevant to the development of immunoregulatory disturbances, lymphoproliferation and progression of liver disease associated with chronic HCV infection. The potential effect of antiviral therapy in down-regulating CD81 and CD5 should be a subject for further studies.


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