Hepatitis C virus (HCV) is a commonly encountered pathogen in medical practice. It is estimated that 2–3% of the world’s population is affected by HCV, with a prevalence of 170 million people (3% of the world’s population) and an incidence of 3–4 million per year 1. Chronic HCV infection is a progressive disease that may lead to liver cirrhosis and hepatocellular carcinoma. The hallmark of chronic hepatitis C is the presence of anti-HCV and HCV-RNA in the serum for more than 6 months after acute infection 2,3.
Occult HCV infection is a newly described form of HCV infection and is defined by the presence of HCV-RNA in liver cells with undetectable anti-HCV and serum viral RNA 4. The histological evaluation of the liver biopsies of patients with occult HCV infection documented different degrees of necroinflammatory activity and fibrosis (including cirrhosis), as reported for chronic hepatitis C 5. Patients with occult HCV infection present with a milder disease form compared with patients with chronic hepatitis C infection 6.
Several studies have suggested that the T-cell immunoregulatory cytokines have a key role in both HCV-related liver damage and the mechanism of viral persistence. Although some cytokines may exert a proinflammatory activity, such as interleukin-1 (IL-1), interferon-γ (IFN-γ), IL-8, tumour necrosis factor-α and IL-2, which can prime T cells towards a T helper type 1 (Th-1)-type immunity, others have a predominantly anti-inflammatory activity, as is the case for IL-4, IL-6 and IL-10, which are involved in Th2 immunity. In addition, some of these cytokines may have a fibrogenic (e.g. transforming growth factor) or an antifibrogenic (e.g. IFN-γ) role 7. Authors have suggested that a preferential shift towards either Th1 or Th2 response may influence the clinical outcome and disease progression 8–12.
The aim of this work was to study the Th1/Th2 cytokine levels in patients with occult HCV infection compared with those with chronic hepatitis C infection.
Patients and methods
This study was conducted on 27 consecutive untreated patients with proven occult HCV infection [HCV-RNA in liver specimens as detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and by in-situ hybridization, with negative anti-HCV antibodies and serum HCV-RNA]. The cytokine patterns and biochemical and virological characteristics of these patients were compared with those of a group of 28 untreated anti-HCV and serum HCV-RNA-positive patients with histopathologically proven chronic hepatitis C infection, equally matched in terms of age, sex and BMI during the same study period from June 2009 to June 2010.
We initially selected 108 patients attending the outpatient clinic of Tropical Medicine Unit, Mansoura University Hospital, with unexplained persistently abnormal liver function test results. Out of them, 81 patients were excluded for being negative for HCV-RNA in their liver biopsy specimens.
Exclusion criteria: All known causes of liver disease were excluded on the basis of analytical, clinical and epidemiological data, which included infection by hepatitis B virus (HBV) (i.e. patients were negative for hepatitis B surface antigen and for serum HBV-DNA), autoimmunity, metabolic and genetic disorders, HAV, HEV, CMV, HIV, NASH, alcohol intake and drug toxicity. This study was conducted following the guidelines of the Declaration of Helsinki, 1975.
Blood samples for separating peripheral blood mononuclear cells (PBMCs) were collected from all patients on the same day that the liver biopsy was performed.
From each patient 2 ml of venous blood was collected into clean, dry, plastic tubes and allowed to clot; the yielded serum was used for quantitative detection of IL-2, IL-4 and IFN-γ levels, which were measured using enzyme-linked immunosorbent assay kits (Bender MedSystems, Vienna, Austria).
The patients underwent an ultrasound-guided liver biopsy (using Tru-cut needles) for diagnostic purposes. The liver biopsy specimens were divided into two portions. One portion was fixed in 10% formalin and was paraffin embedded for routine histological diagnosis. Histological evaluation was performed by a pathologist who was blinded to the HCV-RNA status of the liver biopsy specimens. Necroinflammatory activity and fibrosis were scored according to the METAVIR score system 13,14. A minor fragment of the specimen (4–5 mm to result in an average weight of 15–20 mg) was immediately immersed in dry ice until extraction of RNA. The extracted RNA was immediately reverse transcribed and stored at −20°C until used for the detection of HCV-RNA by RT-PCR.
Preparation of liver cell lysates for total RNA isolation
The amount of tissue was determined by weighing the samples (all samples weighed 20 mg each), according to the recommendation of Norgen Biotek corporation (Ontario, Canada). The tissues were then slowly ground while still in dry ice. A volume of 600 μl of lysis solution was then added to the tissue samples and grinding was continued until the samples were homogenized. Homogenization was ensured by passing the lysate 5–10 times through a 25-G needle attached to a syringe. The lysate was then transferred to an RNase-free microfuge tube and spun down for 2 min to pellet out any cell debris. The supernatant was then transferred to another RNase-free microfuge tube, and an equal volume of 70% ethanol was added and vortexed for proper mixing.
Total RNA extraction and purification
A volume of 1200 μl of lysate with ethanol, obtained from the previous step, was applied to the assembled column and collection tube and centrifuged for 1 min. The flow-through was discarded and the spin column and collection tube were reassembled. This process was repeated until there was no lysate remaining. After this step, 400 μl of wash solution was added to the column and centrifuged for 1 min; the flow-through was discarded, and the process was repeated twice. RNA was then eluted by adding 50 μl of elution buffer to the column and centrifuged for a minute. The purified RNA was then immediately reverse transcribed into cDNA and stored at −20°C until used for PCR.
Peripheral blood mononuclear cell lysate for total RNA isolation
PBMCs were obtained using Ficoll-Hypaque density gradient of EDTA anticoagulated blood according to the manufacturer’s instructions (Lymphoflot, Biotest, Dreleich, Germany). Cells were washed three times with Mg2+-free and Ca2+-free PBS and resuspended to 1×106 cells/ml in PBS. Up to 100 μl of PBMC suspension in PBS was transferred to an RNase-free microfuge tube. To this was added 350 μl of lysis solution. Cells were then lysed by vortexing for 15 s (or until the mixture became clear). A volume of 200 μl of 95% ethanol was then added to the lysate and mixed by vortexing for 10 s. The peripheral RNA was reverse transcribed into cDNA and stored at 20°C until used for PCR. The lysate was used for RNA extraction following the same protocol for liver cell lysate.
Reverse transcription and nested PCR
The synthesis of cDNA and the two PCR cycles were performed using oligonucleotide primers from the highly conserved 5′ untranslated region of the genome; external primers P1 (sense, -GCGACACTCCACCATAGAT-; nucleotides 10–28) and P4 (antisense, -ACTCGCAAGCACCCTATCA-; nucleotides 303–285) for the first cycle of PCR and internal primers P2 (sense, -CTGTGAGCAACTACTGTCT-; nucleotides 36–55) and P3 (antisense, -CGGTGTACTCACCGGTTCC-; nucleotides 161–143) for the second cycle of PCR were used as described by Koziel in 1999 15.
Amplification of the cDNA was performed using 15 μl of the cDNA solution and 50 pmol of the outer primers (P1 and P4). Thirty cycles of DNA amplification were carried out, followed by an extension step for 10 min at 72°C. Each cycle of PCR was run under the following conditions: at 95°C for 45 s, at 50°C for 45 s and at 72°C for 45 s. The second PCR was carried out in the same way with 5 μl of the first PCR mixture and 50 pmol of each internal primer (P2 and P3). The amplified DNA was visualized by 2% agarose gel electrophoresis and ethidium bromide staining. The size of the second product generated by PCR was 126 bp. Further, several negative controls (no RNA) were included in each PCR step to ensure the specificity of the results.
Statistical analyses were performed using SPSS software, package release, version 15.0 (SPSS Inc., Chicago, Illinois, USA). Comparisons between the groups were made using the Student t-test for continuous variables and by either the χ 2-test or the Fisher exact test for categorical data. Correlations were determined by the Spearman rank correlation test. Epidemiological and clinical data of the patients (sex, age, BMI, estimated duration of abnormal liver function test results, previous blood transfusions, levels of aspartate transaminase, alanine transaminase, Gamma-glutamyl transpeptidase, cholesterol and triglycerides, and presence or absence of intrahepatic HCV-RNA) were included in a logistic regression analysis to identify the independent factors associated with necroinflammatory activity and fibrosis. A two-tailed P value of less than 0.05 was considered to denote statistical significance.
In this study, 27 occult HCV patients and 28 patients with HCV infection were included after giving a well-informed written consent.
Table 1 shows the characteristics of patients with occult HCV infection and those with chronic hepatitis C infection; no significant differences regarding age, sex and BMI were observed. Significant increase was noted in classic HCV infection compared with occult HCV infection regarding bilirubin (P<0.001), alanine transaminase (P=0.009), aspartate transaminase (P=0.013) and alfa-fetoprotein (P<0.001) levels, whereas serum albumin was found to be significantly higher in occult HCV cases than in those with chronic HCV infection (P<0.001).
Table 2 shows the histological characteristics of both studied groups. Hepatic necroinflammation (P<0.001), fibrosis (P<0.001) and cirrhosis (P=0.03) were significantly observed in patients with chronic HCV infection compared with those with occult HCV.
Table 3 shows the Th1/Th2 cytokine patterns of the studied patient groups. The levels of Th1 cytokines, IL-2 and IFN-γ, were statistically significantly increased in patients with chronic HCV infection (P<0.001) compared with those with occult HCV infection. The Th2 cytokine IL-4 was significantly increased in patients with occult HCV infection (P<0.001) compared with those with chronic HCV infection. These results indicate that Th1 cytokines are predominant in patients with chronic HCV infection than in those with occult HCV infection, whereas Th2 cytokine IL-4 is predominant in patients with occult HCV infection than in those with chronic HCV infection.
Epidemiological studies of HCV infection have suggested that the Nile-Delta region of Egypt has the highest prevalence rates of HCV infection in the world, with seroprevalence rates of 20–30% in villagers above the age of 30 years. Women had a similar or a slightly lower prevalence of antibodies to HCV than did men 16.
In this work, the prevalence of occult HCV infection was 25% (27/108) among patients with unexplained abnormal liver function test results. This finding should draw the attention of clinicians to HCV as an underestimated hidden liver viral pathogen and may recategorize the aetiologic diagnostic possibilities of unexplained persistent liver abnormalities. Large epidemiolocal studies are needed to clarify the approximate prevalence of occult HCV infection among Egyptians.
There is suggestive evidence that T-cell immunoregulatory cytokines may have a key role in the aetiopathogenesis of occult HCV infection. The activated CD4+ T cells can be divided into two subsets on the basis of their cytokine secretion profiles 17–19. The Th1 subset produces IFN and ILK2 and participates in cell-mediated immune responses 18, whereas the Th2 subset produces IL-4 and IL-10 and mediates humoral immune responses 20. The Th1/Th2 cytokine balance is likely to be important in determining the rate of HCV chronicity and the pattern and extent of HCV-induced liver injury 21,22.
In this study, there was a significant increase in Th1 cytokines IL-2 and IFN-γ in patients with chronic HCV infection (193.6±15.4 and 64.8±6.76 pg/ml, respectively) compared with those with occult HCV infection (102.1±6.7 and 28.6±4.2, respectively) (P<0.001), as shown in Table 3.
This significantly greater shift in cytokine pattern towards Th1 pattern in chronic HCV infection compared with occult HCV infection may suggest that the clinical and histological differences observed between the studied groups are a consequence of the hosts’ immunological system responses and their derived cytokines, as the cytokine shift towards Th1 is associated with greater progression of the disease. These data are supported by previous findings denoting that IFN-γ expression was clearly augmented in the serum of chronic hepatitis C patients 23–25, its increase being correlated with an increase in the severity of disease 26.
IL-2 is considered to be a Th1-type cytokine and is involved in enhancing the proliferation and activation of most T lymphocytes, NK cells and B lymphocytes. Liver sinusoidal and inflammatory cells have been reported to be sources of IL-2, and no consensus exists on the predictive value of this cytokine. However, it is apparent that the expression of IL-2 is associated with a more advanced stage of the disease 27–29. Napoli and colleagues demonstrated that as liver injury worsens there is an increase in intrahepatic Th1-like cytokine mRNA levels. In particular, there was a positive correlation between IFN-γ and IL-2 mRNA expression and the severity of both the inflammatory and fibrotic components in the portal tracts 8.
Our data showed a significant increase in serum IL-4 in patients with occult HCV infection (120.3±9.2 pg/ml) in comparison with those with chronic HCV infection (68.9±12.15 pg/ml) (P<0.001). IL-4 has been shown to regulate a wide spectrum of functions of B cells, monocytes/macrophages and other nonhaematopoietic cells 30. An increased level of Th2 cytokine IL-4 may be responsible for the decrease in IFN-γ and IL-2 production among occult HCV-infected patients in the present study. This finding is concordant with another study stating that Th2 cytokines regulate the antibody secretion by B cells and have suppressor functions 31. In addition, the increased levels of Th2 cytokines IL-4 and IL-10 may be responsible for the decrease in IFN-γ production 8.
Patients with occult HCV infection have abnormal liver function tests, and 35% of them have histological damage, including liver cirrhosis 4. However, patients with occult HCV infection present a milder disease form than those with chronic hepatitis C infection 6. The small number of infected hepatocytes found in patients with occult HCV infection may be related to less-severe liver damage. Furthermore, the immune response of those patients may be better fine-tuned than that of patients with chronic hepatitis C, leading to a more effective control of the infection 6. There is suggestive evidence that T-cell immunoregulatory cytokines may play a key role in the persistence of HCV infection and may influence the extent of liver damage 15,29,32–34.
Despite a less-aggressive course of occult HCV and absence of HCV-RNA in peripheral blood, the body’s immune system could not eradicate the infection. This could be because of the predominant Th2 cytokine profile in occult HCV that may be responsible for persistence of infection in these patients, agreeing with reports from other authors 6,28. It has been proposed that the inability to terminate HCV infection may also result from the inappropriate release of some T-cell-derived and monocyte–macrophage-derived cytokines, such as IL-4 and IL-10 35,36. These cytokines may inhibit cell-mediated antiviral responses by interfering with T-cell activation and function 37–40. These data probably express the conditions in our study, in which an undetectable amount of serum HCV-RNA in case of occult HCV infection can explain the low level of Th1 cytokines in this study among patients with occult infection compared with those with chronic HCV infection. The existence of a relationship between chronic HCV replication and Th1 predominance was further supported by the findings of a marked decrease in the Th1 response in patients in whom the HCV-RNA titre dropped to undetectable levels following in-vivo IFN-γ parenteral manipulations 41,42.
Our data revealed a high prevalence of occult HCV infection (25%) in patients who presented with unexplained persistently abnormal liver function test results. These patients exhibited a distinct immunoregulatory cytokine profile, with a predominant Th2 cytokine response favouring viral persistence in liver tissue despite its absence from peripheral blood. The lack of Th1 cytokine response in occult HCV infection could explain the less-aggressive course of this disease entity compared with chronic HCV infection.
The authors thank Professor Sawsan M. Abd Al-Monem, Internal Medicine Department, for her effort and contribution.
Conflicts of interest
There are no conflicts of interest.
1. Antonelli A, Ferri C, Galeazzi M, Giannitti C, Manno D, Mieli-Vergani G, et al. HCV infection: pathogenesis, clinical manifestations, and therapy. Clin Exp Rheumatol. 2008;26(Suppl 48):S39–S47
2. Chen SL, Morgan TR. The natural history of hepatitis C virus
infection. Int J Med Sci. 2006;3:47–52
3. Seeff LB. Natural history of chronic hepatitis C. Hepatology. 2002;36(Suppl. 1):S35–S46
4. Castillo I, Pardo M, Bartolomé J, Ortiz-Movilla N, Rodríguez-Iñigo E, de Lucas S, et al. Occult hepatitis C virus
infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown. J Infect Dis. 2004;189:7–14
5. Yano M, Kumada H, Kage M, Ikeda K, Shimamatsu K, Inoue O, et al. The long-term pathological evolution of chronic hepatitis C. Hepatology. 1996;23:1334–1340
6. Pardo M, López-Alcorocho JM, Rodríguez-Iñigo E, Castillo I, Carreño V. Comparative study between occult hepatitis C virus
infection and chronic hepatitis C. J Viral Hepat. 2007;14:36–40
7. Tilg H, Kaser A, Moschen AR. How to modulate inflammatory cytokines
in liver diseases. Liver Int. 2006;26:1029–1039
8. Napoli J, Bishop GA, McGuinness PH, Painter DM, McCaughan GW. Progressive liver injury in chronic hepatitis C infection correlates with increased intra-hepatic expression of Th1-associated cytokines
. Hepatology. 1996;24:759–765
9. Tsai SL, Liaw YF, Chen MH, Huang CY, Kuo GC. Detection of type 2-like helper cells in hepatitis C virus
infection: implications for hepatitis C virus
chronicity. Hepatology. 1997;25:449–458
10. Sarih M, Bouchrit N, Bebslimane A. Different cytokine profiles of peripheral blood mononuclear cells from patients with persistent and self-limited hepatitis C virus
infection. Immunol Lett. 2000;74:117–120
11. Sobue S, Nomura T, Ishikawa T, Ito S, Saso K, Ohara H, et al. Th1/Th2 cytokine profiles and their relationship to clinical features in patients with chronic hepatitis C virus
infection. J Gastroenterol. 2001;36:544–551
12. Yamaki K, Uchida H, Li X, Yanagisawa R, Takano H, Hayashi H, et al. Effect of varying types of anti-arthritic drugs on Th1 and Th2 immune responses in mice. Int J Immunopathol Pharmacol. 2005;18:133–144
13. Bedossa P. Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. METAVIR Cooperative Study Group. Hepatology. 1994;20:15–20
14. Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology. 1996;24:289–293
15. Koziel MJ. Cytokines
in viral hepatitis. Seminar Liver Dis. 1999;2:157–168
16. Frank C, Mohamed MK, Strickland GT, Lavanchy D, Arthur RR, Magder LS, et al. The role of parenteral antischistosomal therapy in the spread of hepatitis C virus
in Egypt. Lancet. 2000;355:887–891
17. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 1986;136:2348–2357
18. Gioia C, Horejsh D, Agrati C, Martini F, Capobianchi MR, Ippolito G, Poccia F. T-Cell response profiling to biological threat agents including the SARS coronavirus. Int J Immunopathol Pharmacol. 2005;18:525–530
19. Lanzilli G, Falchetti R, Tricarico M, Ungheri D, Fuggetta MP. In vitro effects of an immunostimulating bacterial lysate on human lymphocyte function. Int J Immunopathol Pharmacol. 2005;18:245–254
20. Clerici M, Shearer GM. The Th1-Th2 hypothesis of HIV infection: new insights. Immunol Today. 1994;15:575–581
21. Vecchiet J, Dalessandro M, Travasi F, Falasca K, Di Iorio A, Schiavone C, et al. Interleukin-4 and interferon-gamma production during HIV-1 infection and changes induced by anti-retroviral therapy. Int J Immunopathol Pharmacol. 2003;16:157–166
22. Perrella A, Borgia G, Borrelli F, Di Sirio S, Gnarini M, Grattacaso S, et al. TNF-alpha serum level elevations in chronic hepatitis C patients with diabetes mellitus. Int J Immunopathol Pharmacol. 2005;18:189–193
23. Tilg H, Wilmer A, Vogel W, Herold M, Nölchen B, Judmaier G, Huber C. Serun levels of cytokynes in chronic liver diseases. Gastroenterology. 1992;103:264–274
24. Cacciarelli TV, Martinez OM, Gish RG, Villanueva JC, Krams SM. Immunoregulatory cytokines
in chronic hepatitis C virus
infection: pre- and post-treatment with interferon alfa. Hepatology. 1996;24:6–9
25. Kamal SM, Graham CS, He Q, Bianchi L, Tawil AA, Rasenack JW, et al. Kinetics of intrahepatic hepatitis C virus
(HCV)-specific CD4+ T cell responser in HCV and Schistosoma mansoni coinfection: relation to progression of liver fibrosis. J Infect Dis. 2004;189:1140–1150
26. González-Peralta RP, Fang JW, Davis GL, Gish R, Tsukiyama-Kohara K, Kohara M, et al. Optimization for the detection of hepatitis C virus
antigens in the liver. J Hepatol. 1994;20:143–147
27. Makris M, Preston FE, Ralph S. Increased soluble IL-2 receptor levels in HCV-infected haemophiliacs: a possible indicator of liver disease severity. Br J Haematol. 1994;87:419–421
28. Bozkaya H, Bozdayi AM, Aslan N, Türkay C, Sarioglu M, Cetinkaya H, et al. Circulating IL-2 and IL-10 in chronic active hepatitis C with respect to the response to IFN treatment. Infection. 2000;28:309–313
29. Gramenzi A, Andreone P, Loggi E, Foschi FG, Cursaro C, Margotti M, et al. Cytokine profile of peripheral blood mononuclear cells from patients with different outcomes of hepatitis C virus
infection. J Viral Hepat. 2005;12:525–530
30. Paul WE. Interleukin4: a prototypic immunoregulatory lymphokine. Blood. 1991;77:1859–1870
31. Bahri A, Abdullah C, Hikmet A. Serum profile of T helper 1 and T helper 2 cytokines
in patients with chronic hepatitis C virus
infection. Turk J Gastroenterol. 2003;14:7–11
32. Kempuraj D, Donelan J, Frydas S, Iezzi T, Conti F, Boucher W, et al. Interleukin-28 and 29 (IL-28 and IL-29): new cytokines
with anti-viral activities. Int J Immunopathol Pharmacol. 2004;17:103–106
33. Petrarca C, Frydas S, Donelan J, Boucher W, Papadopoulou N, Cao J, et al. Interleukin 27 (IL-27): a novel pleiotropic cytokine involved in T cell differentiation and T cell response modulation. Int J Immunopathol Pharmacol. 2005;18:191–194
34. Huang SH, Frydas S, Conti P, Kempuraj D, Barbacane RC, Grilli A, et al. Interleukin-17: a revisited study. Int J Immunopathol Pharmacol. 2004;17:1–4
35. Salgame P, Abrams JS, Clayberger C, Goldstein H, Convit J, Modlin RL, Bloom BR. Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science. 1991;254:279–282
36. Mosmann TR, Subash S. The expanding universe of T cell subsets: Th1, Th2 and more. Immunol Today. 1996;17:138–146
37. Sher A, Gazzinelli RT, Oswald IP, Clerici M, Kullberg M, Pearce EJ, et al. Role of T-cell derived cytokines
in the down regulation of immune responses in parasitic and retroviral infection. Immunol Rev. 1992;127:183–204
38. Scott P, Kaufmann SH. The role of T-cell subsets and cytokines
in the regulation of infection. Immunol Today. 1991;12:346–348
39. Ada GL, Blanden RV. Immunity and cytokine regulation in viral infection. Res Immunol. 1994;145:625–629
40. Del Prete G, Maggi E, Romagnani S. Th1 and Th2 cells: functional properties, mechanisms of regulation, and role in diseases. Laboratory Invest. 1994;70:299–306
41. Brinkmann V, Geiger T, Alkan S, Heusser CH. Interferon α increases the frequency of interferon gamma-producing CD4+ T cells. J Exp Med. 1993;178:1655–1662
42. Parronchi P, De Carli M, Manetti R, Simonelli C, Sampognaro S, Piccinni MP, et al. IL-4 and IFN-gamma exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones. J Immunol. 1992;149:2977–2983