These data were presented in the Henry M. and Lillian Stratton Single Topic Conference (immunovirology and the liver), the American Association for the Study of Liver Diseases (AASLD), 11–13 June 2004, the Airlie Center, Warrenton, VA, USA. Abstract no. 22, p. 89 in conference book.
Interleukin (IL)-4, IL-10, IL-18 and IFN-γ Cytokines Pattern in Patients with Combined Hepatitis C Virus and Schistosoma mansoni Infections‡
Article first published online: 10 JAN 2005
Scandinavian Journal of Immunology
Volume 61, Issue 1, pages 87–91, January 2005
How to Cite
M. El-Kady, I., Lotfy, M., Badra, G., El-Masry, S. and Waked, I. (2005), Interleukin (IL)-4, IL-10, IL-18 and IFN-γ Cytokines Pattern in Patients with Combined Hepatitis C Virus and Schistosoma mansoni Infections. Scandinavian Journal of Immunology, 61: 87–91. doi: 10.1111/j.0300-9475.2005.01529.x
- Issue published online: 10 JAN 2005
- Article first published online: 10 JAN 2005
- Received 23 August 2004; Accepted in revised form 28 October 2004
Schistosoma mansoni infection is characterized by a strong T-helper type 2 (Th2) cell-associated immune response, but in the case of viral infection, it is associated with interferon-gamma (IFN-γ) increase and induction of Th1 immune response. Few data are available about the immune response of cases infected with combined hepatitis C virus (HCV) and schistosomiasis. Thus, the investigation of the cytokine pattern in patients coinfected with both HCV and Schistosoma mansoni was our rationale. This study included four patient groups: Group 1 included 20 patients infected with chronic HCV, Group 2 included 15 patients infected with schistosomiasis alone, Group 3 included 20 patients with chronic HCV and schistosomiasis and Group 4 included 15 healthy control individuals with matched age and sex. Serum levels of IFN-γ, interleukin (IL)-4, IL-10 and IL-18 were measured in all groups by enzyme-linked immunosorbent assay. The results showed that the patients infected with HCV had significantly higher serum levels of IFN-γ and IL-18 compared with the controls and with the patients with schistosomiasis and coinfection (P < 0.001). On the other hand, serum levels of IL-4 and IL-10 were significantly higher in patients with schistosomiasis and coinfection compared with the control group (P < 0.001 and 0.0001, respectively) and with the HCV patients (P < 0.05 and P < 0.001, respectively). A significant increase in serum levels of IL-4 and IL-10 was also found in HCV patients compared with the control (P < 0.05). Schistosomiasis appears to induce a Th2 cytokine profile, with increase in serum levels of IL-4 and IL-10, even in the presence of HCV coinfection. In conclusion, schistosomiasis may downregulate the stimulatory effect of HCV on Th1 cytokines and this may lead to the chronicity of HCV infection in coinfected patients.
Hepatitis C virus (HCV) infection and schistosomiasis are major public health problems in the Nile Delta of Egypt. Schistosomiasis is a chronic helminth disease that infects more than 200 million people worldwide . Infection with Schistosoma mansoni is endemic in Egypt, with a prevalence of 17.5–42.9%. Morbidity in humans infected with S. mansoni results primarily from the deposition of parasite ova in the portal areas that induces a T-cell-dependent granulomatous response, which progresses to irreversible fibrosis and severe portal hypertension in > 60% of cases . Schistosoma mansoni infection in mice is characterized by a strong T-helper type 2 (Th2) cell-associated immune response that is coupled with a defect in Th1-cell effect or function [4–6]. Schistosomiasis is most prevalent and severe in children and adults who are at maximal risk of suffering from the disease sequelae of splenic enlargement and shrunken liver . It has long been reported that populations where schistosomiasis is endemic are susceptible to other infections prevalent in that area .
HCV infection is characterized by a protracted clinical course with viral persistence, which leads to liver cirrhosis and hepatocellular carcinoma in some individuals [9–11]. Mechanisms underlying viral persistence and liver damage in patients with chronic HCV are not yet clarified, but a complex interplay of virological and immunological factors is implicated . HCV infection is hyperendemic in Egypt and its prevalence was found to be age related. HCV is responsible for more hepatic morbidity than schistosomiasis .
The combined viral hepatitis and schistosomiasis incidence was found to be increasing . Schistosomiasis and HCV coinfection is common in Egypt [15–18] and other developing countries . Patients coinfected with HCV and schistosomiasis exhibit a unique clinical, virological and histological pattern manifested by viral persistence with high HCV-RNA titres, as well as higher necroinflammatory and fibrosis scores in their liver biopsy samples [17, 18]. Dual infections of schistosomiasis and viral infections display significant influences on host immune reactions including cytokine shift pattern alteration, cytotoxic T-lymphocyte response and other impaired immunologic functions with diminished capacity to clear the virus [20–22]. Interleukin (IL)-18, a recently identified proinflammatory cytokine, has been implicated in a variety of pathological conditions such as rheumatoid arthritis, insulin-dependent diabetes mellitus and inflammatory liver injury . IL-18 functions primarily to promote a Th1 response through induction of IFN-γ. It is produced by Kupffer's cells, intestinal epithelial cells and activated macrophages and acts synergistically with IL-12 to induce IFN-γ production by T lymphocytes . However, scarce studies are available that investigated the influence of S. mansoni on the HCV-specific immune response. Therefore, determination of different cytokine profile in cases infected with combined HCV and S. mansoni was our rationale in the present study.
Patients and methods
Patients. The history of our patients was completely reviewed, evaluated and recorded. The clinical examination for assessment of the severity of the liver disease and its complications such as jaundice, ascites and encephalopathy was carried out. All the required laboratory, radiological and pathological investigations were performed.
Sera obtained from all chronic schistosomal and HCV-infected patients were screened for the presence of anti-HCV antibodies. ETI-AB-HCV K-3 kit was used; an enzyme immunoassay that utilizes third generation of enzyme-linked immunosorbent assay (ELISA; Sorin Biomedica, Saluggia, Italy). Highly antigenic determinants of both structural (recombinant C22 and C33) and nonstructural (synthetic NS4 and NS5) regions of HCV genome were coated onto microtitre plate to capture specific antibodies present in serum samples. Samples reactive for anti-HCV were further verified for RNA positivity to identify those who have viraemia. Confirmation of S. mansoni infection was carried out through detection of S. mansoni ova in stool as determined by quantitative examination of repeated stool samples  or rectal snip and a serological test (indirect haemagglutination assay; Femouz Laboratories, Asniéres, France).
This study included four patient groups (Table 1): Group 1 included 20 patients infected with chronic HCV only, Group 2 included 15 patients infected with schistosomiasis alone, Group 3 included 20 patients with chronic HCV and schistosomiasis coinfection and Group 4 included 15 control healthy individuals with matched age and sex and have no evidence of liver diseases as determined by the above-mentioned investigations.
|Group 1*||Group 2†||Group 3‡||Group 4§|
|Age (years)||45.6 ± 3.7||40.5 ± 2.4||45.3 ± 4.1||43.7 ± 3.4|
|ALT (IU/ml)||53.6 ± 26.3||24.6 ± 3.7||71.4 ± 23.4||34.6 ± 6.7|
|AST (IU/ml)||52.4 ± 22.5||26.6 ± 7.5||68.9 ± 27.3||36.6 ± 8.5|
Reverse-transcription polymerase chain reaction (RT-PCR). RT-PCR technique was performed to confirm the obtained results with ELISA and to identify patients with viraemia by detection of HCV-RNA. In brief, HCV-RNA was extracted using phenol/isopropanol method as described by Chanczynski and Sacchi . The synthesis and extension of complementary DNA was performed according to the method of Sullivan and Gerber  using deoxynucleotide triphosphate bases, primers and RT enzyme (Promega, Madison, WI, USA); the reaction was run in a programmable DNA thermal cycler (Perkin Elmer 4020, Perkin Elmer, Foster City, CA, USA) for 35 cycles: denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min and primer extension at 72 °C for 1 min. Detection of the amplified specific DNA sequences was performed using DNA enzyme immunoassay kit (Sorin Biomedica). The kit utilizes biotinylated single-stranded nucleic acid probes specific for HCV genome coated onto a microtitre plate. The manufacturer's instructions were followed.
Cytokine measurements. Serum cytokines were quantitated by commercially available ELISA for IFN-γ (Biosource International, Camarillo, CA, USA) and for IL-4, IL-10 and IL-18 (Diaclone Research, Besancon Cedex, France) according to the manufacturer's instructions.
Statistical analysis. Results were expressed as mean ± standard deviation and were analysed by using nonparametric Mann–Whitney U-test and/or Kurskal–Wallis test, as appropriate. A P-value of ≤ 0.05 was considered significant. All statistical procedures were performed by using spss software, version 11 for windows.
The obtained data of both patient groups and controls are shown in Figs 1–4. Liver biopsy in HCV and coinfected patients was ranged from mild hepatitis with mild piecemeal necrosis to moderate chronic active hepatitis. All HCV patients were positive for HCV-RNA as detected by the RT-PCR, and the controls were confirmed for their negativity in that test. Patients infected with HCV had significantly higher serum levels of IFN-γ and IL-18 compared with controls and patients with schistosomiasis and coinfection (P < 0.001) (Figs 1 and 2). On the other hand, a highly significant increase in serum level of IL-4 was found in the coinfected patients and in patients infected with schistosomiasis alone compared with the controls (P < 0.001) or with HCV patients (P < 0.05) (Fig. 3). A very highly significant increase in serum level of IL-10 was found in the coinfected patients and in patients infected with schistosomiasis alone compared with the controls (P < 0.0001) or with the HCV group (P < 0.001) (Fig. 4). A significant increase in serum levels of IL-4 and IL-10 was also found in HCV patients compared with the control (P < 0.05). No correlation was found between the liver enzymes and the investigated cytokines.
Many studies reported that patients coinfected with HCV and schistosomiasis have a more severe clinical course, higher HCV-RNA titres, higher incidence of cirrhosis, hepatocellular carcinoma, poor response to IFN therapy and higher mortality rates due to liver-related causes compared with patients infected with HCV or schistosomiasis alone [17, 19, 22, 28, 29].
The present results showed that the Th1 response is dominant in HCV-infected patients, but Th2 response is dominant in schistosomiasis infection alone and in coinfected patients. The Th2 cytokine dominance in schistosomiasis infection only and in coinfected patients was in agreement with previous reports in humans [6, 21, 30] and in murine models .
In coinfected patients, HCV infection was probably acquired after the establishment of S. mansoni infection, possibly via parenteral antischistosomal therapy and/or blood transfusions. Thus, in the first encounter of HCV with the host immune system, it was confronted with a prevailing Th2 pattern. This Th2 profile, induced by S. mansoni infection, has probably antagonized and downregulated the antiviral activities of Th1 cytokines. This may result in increased viral replication and more aggressive progression to fibrosis . In our study, the Th1 cytokine pattern was demonstrated to be dominant in HCV-infected patients, as reported in previous report .
Imbalance towards a Th2 profile at the time of acquisition of HCV infection or during the acute stage of the disease may favour the faster progression to chronicity in coinfected patients [17–19, 29]. This finding is further reported by Tsai et al. , as their analysis of the cytokine profile of bulk cultures, as well as of CD4+ T-cell clones from patients infected with HCV, has revealed that viral clearance is more likely to occur in patients displaying a Th1, rather than Th2, cytokine profile.
Coinfected patients had significantly high HCV-RNA titres and an inverse relationship between virus load and CD4+ T-cell responses. This may be explained by the lack of viral control due to defective HCV-specific CD4+ T-cell response or abrogation of the HCV-specific Th1 response which is antagonized by the Schistosoma mansoni-induced Th2 response. The high initial virus load in these coinfected patients may cause immune exhaustion thereby aggravating HCV infection . Actor et al. showed that mice infected with vaccinia virus alone rapidly cleared the virus, whereas viral clearance in animals coinfected with S. mansoni was delayed in the liver by as much as 3 weeks and in the spleen and lungs by several days. These previous observations in the mouse model support our findings, which suggest that helminth infection may influence immune responses to concurrent viral infections. Schistosoma mansoni causes liver pathology through an immune-mediated mechanism rather than through direct hepatic injury .
Schistosoma mansoni ova are trapped in the liver, evoking a highly skewed Th2 immune response profile with granuloma formation that progresses to periportal fibrosis. However, the ultrastructure and function of hepatocytes are minimally affected. Our study as well as previous reports [17–19, 29] showed that patients infected with schistosomiasis alone had very low inflammatory scores with no piecemeal necrosis. The high fibrosis scores of patients with concomitant infection may be immunologically mediated with predominance or upregulation of the collagen-inducing cytokine IL-4 and the downregulation of the collagen-suppressing cytokine IFN-γ as was detected in our study and in other studies of murine [35, 36] and clinical schistosomiasis .
Our results showed a significant increase in IL-18 level in hepatitis C infection and this may be due to the dominance of Th1 response and this in agreement with McGuinness et al. . In addition, a significant increase in IL-10 level in schistosomal and coinfected patients may be due to the dominance of Th2 response and this in agreement with Kamal et al. .
In conclusion, S. mansoni infection seems to induce a Th2-related cytokines, with increase in serum levels of IL-4 and IL-10, even in the presence of HCV coinfection. In addition, schistosomiasis may downregulate the stimulatory effect of HCV on Th1 cytokines and this may lead to the chronicity of HCV infection and may play a role in unresponsiveness to interferon therapy in coinfected patients. Further investigations are required to clarify this hot point.
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