1 Molecular Virology and Hepatology Research, Division of Basic Medical Science, Faculty of Medicine, Health Sciences Centre, Memorial University, St John's, Newfoundland, Canada A1B 3V6
2 Liver Unit, Division of Gastroenterology, University of Calgary, Calgary, Alberta, Canada
3 Gastroenterology Unit, General Hospital, Faculty of Medicine, Memorial University, St John's, Newfoundland, Canada
4 Discipline of Laboratory Medicine, Faculty of Medicine, Memorial University, St John's, Newfoundland, Canada
Correspondence
Tomasz I. Michalak
timich{at}mun.ca
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ABSTRACT |
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INTRODUCTION |
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The liver is the main site of HCV replication. However, accumulated data indicate that the virus also replicates in the lymphatic system. This is reflected by a higher frequency of type II mixed cryoglobulinaemia (El-Serag et al., 2002) and non-Hodgkin's lymphoma (Musto, 2002
) in HCV-infected persons. The notion of lymphotropism being an intrinsic property of HCV has gained strong support from recent studies demonstrating the HCV genome and its replicative intermediate in peripheral blood mononuclear cells (PBMCs) from individuals with CHC and after spontaneous or therapeutically induced resolution of hepatitis C (Castillo et al., 2004
; Laskus et al., 2000
; Pham et al., 2004
; Radkowski et al., 2000
). In a previous study (Pham et al., 2004
), we provided evidence for long-term persistence of HCV RNA and its replicative strand in PBMCs and monocyte-derived dendritic cells after apparent complete recovery from hepatitis C. HCV RNA was also detected in PBMCs and liver tissue from patients with occult HCV infection accompanied by long-standing abnormal liver function tests (Castillo et al., 2004
). In addition, different types of lymphoid cells, including T (Shimizu et al., 1992
, 1998
) and B lymphocytes (Morsica et al., 1999
), as well as monocytes (Radkowski et al., 2004
), have been shown to support HCV propagation and the virus derived in vitro can be infectious (Kato et al., 1995
; Shimizu et al., 1998
; Sung et al., 2003
).
Considering that lymphotropism characterizes many viruses capable of long-term persistence (Grosjean et al., 1997; Oldstone, 1996
), the establishment of a method for reliable and sensitive detection of HCV in lymphoid cells may shed new light on the natural history of HCV infection and provide a tool for monitoring its replication. In earlier studies from this laboratory on occult infections with woodchuck hepatitis virus (WHV) (Coffin & Michalak, 1999
; Michalak, 2000
; Michalak et al., 1999
, 2004
) and HCV (Pham et al., 2004
), it was shown that treatment with certain non-specific mitogens led to greater expression of viral genomes in lymphoid cells, allowing identification of the residing virus in apparently negative cells.
With regard to occult HCV infection, HCV positive- and negative-strand RNAs were detected in the majority (81 and 75 %, respectively) of individuals with resolved hepatitis C when their PBMCs were cultured with phytohaemagglutinin (PHA) and interleukin-2 (IL2) (Pham et al., 2004). In the above study, the use of a highly sensitive RT-PCR nucleic acid hybridization (RT-PCR/NAH) assay [sensitivity of
10 virus genome equivalents (vge) ml1] undoubtedly contributed to enhanced detection of HCV RNA in PBMCs. The same assay revealed the virus genome in the sera of 88 % of the same individuals who were repeatedly HCV RNA non-reactive by a standard clinical assay (sensitivity of 102103 vge ml1). However, when taking the results from PBMCs and sera together, all individuals were HCV RNA positive (Pham et al., 2004
), raising the possibility that detection of the HCV genome in lymphoid cells could be further improved.
Based on the above reasoning, the aim of the current study was to design conditions for the most effective augmentation of HCV detection in lymphoid cells. We explored synergistic effects of mitogens stimulating T and B cells and the cytokines supporting their survival. The data reported here show that the detection of HCV positive- and negative-strand RNA, as well as HCV NS3 protein, in PBMCs can be markedly enhanced after ex vivo mitogen treatment. This approach offers a new tool to examine HCV lymphotropism and to monitor HCV infection.
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METHODS |
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Previous investigations of occult infections with WHV (Coffin & Michalak, 1999; Michalak et al., 1999
, 2004
) and HCV (Pham et al., 2004
) showed that stimulation of lymphoid cells with mitogens could significantly enhance trace virus replication allowing detection of virus in cells that were supposedly non-reactive. To determine the most effective conditions augmenting HCV RNA in PBMCs, we assessed the effect of mitogens stimulating T and B cells and cytokines supporting their survival, either alone or in combination with each other. The following were tested: PHA (ICN Biomedicals), a T-cell proliferation inducing mitogen; pokeweed mitogen (PWM; ICN Biomedicals), a B- and T-cell proliferation-inducing lectin; IL2 (Roche Molecular Diagnostics), a T-cell-supportive cytokine; and IL4 (Roche Molecular Diagnostics), a B-cell-supportive cytokine (Table 2
). Thus, 5x106 PBMCs or T or B lymphocytes were cultured for 72 h in supplemented RPMI 1640 (Pham et al., 2004
) in the presence of mitogens and/or cytokines at the concentrations indicated in Table 2
.
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To assess the relative increase in HCV RNA expression in lymphoid cells after mitogen/cytokine treatments, PCR products (20 µl) of test samples and known amounts of HCV positive-strand sRNA were 10-fold serially diluted and immobilized by microfiltration on to a nylon membrane (Hybond; Amersham BioSciences). Blots were then hybridized to the rHCV UTRE2 probe (Pham et al., 2004).
In some instances, our in-house real-time RT-PCR assay using the LightCycler Fast Start Master Hybridization Probes (Roche) specific for HCV 5'-UTR (Pham et al., 2004) was used to quantify HCV RNA levels in sera and PBMCs of the individuals investigated. Ten-fold serial dilutions of rHCV UTRE2 were used for enumeration of viral load.
In cases where the cell number was sufficient and HCV RNA was detectable after stimulation with Combo 5, detection of HCV NS3 protein by flow cytometry was attempted. For this purpose, 5x105 PBMCs cultured with or without Combo 5 were permeabilized with 100 µl 0·1 % saponin in PBS, pH 7·4, containing 1 % BSA, 1 mM CaCl2, 1 mM MgS04, 0·05 % NaN3 and 10 mM HEPES for 1 h at room temperature. The cells were then exposed to mouse anti-HCV NS3 IgG1 monoclonal antibody (ViroGen) or to a relevant immunoglobulin isotype control for 1 h at 4 °C. Following washing with permeabilization buffer, cells were incubated with FITC-conjugated goat anti-mouse IgG and IgM antibody (Jackson Immunoresearch Laboratories), washed, fixed in 1 % paraformaldehyde and analysed using a FACSCalibur cytometer (BD Biosciences Pharmingen). The results were interpreted with the help of the CellQuest Pro software (BD Biosciences).
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RESULTS |
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Fig. 1 illustrates enhanced detection of the HCV genome in PBMCs from a patient with CHC (Case 1), which were virus-RNA reactive prior to treatment. Stimulation of the cells with PWM alone, PHA/IL2 or Combo 5 gave an increase in the HCV RNA level ranging from 1·4-fold (for PWM) to 3·5-fold (for Combo 5) compared with untreated PBMCs, as determined by densitometric analysis of Southern blot hybridization signals. Interestingly, in this case, treatment with PHA/IL2 led to slightly better enhancement of HCV RNA upregulation. The possibility that both T and B cells were infected and contributed to the augmented expression of virus genome was supported by the result from PBMCs treated with Combo 5 (Table 2
), which was the strongest enhancer of virus expression in this case (Fig. 1
). Identical treatment of healthy PBMCs did not lead to the appearance of a hybridization signal. PCR contamination controls were negative, further confirming the specificity of the augmentation detected (Fig. 1
).
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To determine whether augmentation of viral RNA was connected to enhanced synthesis of virus proteins, lymphoid cells from patients with CHC (Cases 4 and 5) were treated with Combo 5 and analysed for HCV RNA by RT-PCR/NAH and for the presence of NS3 protein by flow cytometry. As illustrated in Fig. 4(a), Combo 5 led to an approximately fourfold enhancement in the level of HCV RNA. Similarly, the production of NS3 protein was more evident in Combo 5-treated cells than in untreated cells, with an increase in the NS3 protein-positive cell population ranging from 3·9 to 5·3 % (Fig. 4b
). In mitogen-untreated cells, up to 1 % of the cells were NS3 reactive. The fact that cells from a healthy individual gave neither a specific signal by RT-PCR/NAH nor a shift after staining with anti-NS3 antibody validated the results obtained. Incubation of Combo 5-stimulated cells with an unrelated antibody of the same isotype (IgG1) was included to account for background binding.
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DISCUSSION |
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Due to limitations in the clinical material available, evaluation of both HCV positive- and negative-strand RNA and NS3 protein were only rarely possible in the same PBMC sample. Nevertheless, it should be emphasized that the treatment conditions established in this study, i.e. culture of PBMCs with Combo 5 for 72 h, consistently resulted in greater detection of at least two of the three viral parameters mentioned above, indicating that our findings were not random observations.
The ability of PHA and PWM to regulate the expression and replication of the viral genome positively is not unique to HCV, as similar observations have been made for other viral pathogens, including measles virus (MV) (Hyypia et al., 1985; Lucas et al., 1978
), human herpesvirus 6 (Frenkel et al., 1990
), cytomegalovirus (Braun & Reiser, 1986
), human immunodeficiency virus type 1 (HIV-1) (Gowda et al., 1989
) and feline immunodeficiency virus (FIV) (Joshi et al., 2004
). In the case of MV and FIV, it has been shown that activation of latently infected lymphocytes by PHA or concanavalin A (ConA) gave a 515-fold increase in the number of virus-producing cells relative to that seen in unstimulated cells (Joshi et al., 2004
; Lucas et al., 1978
). Furthermore, infectious MV was released only by infected lymphoid cells treated with PHA, and the treated cells were able to establish a productive infection with features comparable to those of the lytic infection. It was postulated that lymphocytes not challenged by mitogen could persistently harbour the virus without being recognized as targets by immune effector mechanisms (Lucas et al., 1978
). The fact that mitogen stimulation can enhance productive virus replication suggests that, for example, a coincidental activation of lymphocytes may play a role in the reactivation of latent infection or augment pathogenicity of the ongoing infection. In the case of HCV, virus reactivation in PBMCs has been reported following liver transplantation (Radkowski et al., 1998
) or as a result of immunosuppression due to chemotherapy (Melisko et al., 2004
) or HIV infection (Laskus et al., 1998
).
Although molecular mechanisms underlying the ability of certain mitogen/cytokine combinations to upregulate HCV replication are not clear and will require separate studies, it is unlikely that the augmented HCV RNA expression was an attribute of cell proliferation, since we did not see a correlation between the rate of lymphoid-cell proliferation and the degree to which HCV genome detection was enhanced (data not shown). This discrepancy could be somehow similar to that recently described for different CD4+ T-lymphocyte subpopulations latently infected with FIV (Joshi et al., 2004). Specifically, it was found that although CD4+CD25 T cells could proliferate efficiently in response to either IL2 or ConA, virus reactivation was only seen in cells treated with ConA. However, the reverse was true for CD4+CD25+ T cells, in which virus replication, but not cell proliferation, was seen following stimulation with ConA.
The enhanced detection of HCV positive- and negative-strand RNA in PBMCs after apparent complete resolution of HCV infection observed in this study confirms our earlier findings (Pham et al., 2004), which demonstrated HCV persistence in circulating lymphoid cells during long-term follow-up of individuals with spontaneous resolution of acute hepatitis C or SVR following antiviral treatment of CHC. Furthermore, the current data clearly showed that treatment with mitogens inducing both T and B lymphocytes (i.e. Combo 5) resulted in a substantially greater detection of the virus genome in lymphoid cells in both study groups over that observed after PHA/IL2 treatment. Taking into consideration the fact that the amounts of HCV RNA and viral proteins are very low in hepatic tissue, even in active CHC (Bartenschlager & Lohmann, 2000
), examination of PBMCs after their ex vivo treatment using conditions established in this study might be advantageous for identifying HCV occult infection. As such, it could also be used for monitoring HCV replication during the entire course of infection, including the pre-acute and acute phases, and potentially applied to the assessment of the efficacy of sterilizing antiviral therapy. Further investigations are required to validate fully the applicability of such evaluations for clinical purposes.
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ACKNOWLEDGEMENTS |
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Received 20 September 2004;
accepted 19 November 2004.
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