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February 2003, Volume 53, Issue 2

Original Article

Interleukin (IL) 1f3 and IL-1O Gene Polymorphism in Chronic Hepatitis C Patients with normal or elevated Alanine Aminotransferase Leyels

Z. Abbas  ( Gastroenterology Section, Sindh Institute of Urology and Transplantation, Karachi. )
T. Moatter  ( Civil Hospital and Department of Pathology, The Aga Khan University Hospital, Karachi. )

Abstract

Objective: To evaluate the frequency of cytokine gene single nucleotide polymorphisms (SNP) of IL-1ß and IL-1 at promoter sites -115 and -1082 respectively and their association with hepatocellular injury as suggested by alanine aminotransf erase (ALT) level.
Methods: Fifty six patients (31 males, 25 females) positive for HCV antibody were studied. Their mean age was 37.5±12.0 years (range 16-62). Fifty one were HCV RNA positive;14 had normal and 37 elevated ALT levels. Type 3 was the predominant genotype. For cytokine polymorphism DNA was extracted from the whole blood. After amplification with polymerase chain reaction, biallelic polymorphisms of IL-i ß at-115 and IL-lO at -1082 promotor site were determined using sequence specific oligonucleotide primers.
Results: IL-i ß-115 C allele was present in 49 (87.5%) and -115 T in 30 (53.6%) patients. Genotype CC was present in 26 (46%), TC 23 in (42 %), and TI in 07 (12%). IL-1 -1082 A allele was seen in 50 (89.3%) and -1082 G in 35 (62.5%). Genotype AG in 29 (52%), AA in 21(37%), and GG in 06 (11%). Though greater number of HCV RNA positive patients with normal ALT had IL-10-1082 AG genotype (10 out of 14), no statistically significant difference could be found in the genotype profile of the above promoter sites of lL-1f3 and lL-10 in patients with normal or elevated ALT.
Conclusion: IL-i ß-115 C and IL-1 -1082A are the more frequent alleles in our patients. Genotypes L-1ß -115 CC and TC, and IL-1 -1082AG and AA are common while -115 TI and -1082 GG are less common. Any effect of lL-1ß and IL-lO gene polymorphism on the degree of hepatocellular injury may not be apparent by the ALT levels (JPMA 53: 59;2003).

Introduction

Infection with the hepatitis C virus (HCV) is a leading cause of chronic liver disease world-wide. Pathogenesis of liver injury is not fully understood. There is a complex relationship between HCV and its host. The infection leads to viral persistence and chronic disease in a very high proportion of cases, despite broad humoral and cellular immunological responses to viral proteins. High rate of mutation leads to the generation of quasispecies, which continuously evolve in infected individuals. Recent data suggest that cytotoxic T cells and cytokines produced by both CD4+ (T helper) and cytotoxic T cells, may be responsible for much of the damage that occurs in the livers of infected patients.
Cytokines play a key role in the modulation of immune responses. Two distinct patterns of cytokines production may occur.’ Type I responses are characterized by production of IL­2, TNF-a and IFN-y, which prime and maintain antigen-specific cellular immunity23 and are important in defense against viruses. Type 2 responses are characterized by IL-4, IL-5, and IL- 10, which promote humoral immune responses. Interleukin (IL)I is a pro-inflammatory cytokine synthesized by mainly mononuclear cells. It stimulates T and B cell proliferation and cytokine production. Type 2 cells secrete IL-10, which is an anti-inflammatory cytokine and down regulates effective cell mediated mechanisms. The maximal capacity of cytokine production varies between individuals and may correlate with polymorphism in cytokine gene promoters. The objective of this study was to analyze the role of allelic or genotype variations of IL-1ß and IL-10 and its association with hepatocellular injury as suggested by ALT.

Methods

Fifty six patients (31 males, 25 females) of chronic hepatitis C were included in this study. Their mean age was 37.5± 12.0 years (range 16-62). HCV RNA was determined by reverse transcriptase polymerase chain reaction (Roche Molecular Systems, Nutley, NJ) in all cases. This assay had a sensitivity of detecting HCV RNA _100 copies/mi. Fifty-one patients were HCV RNA positive and type 3 was the predominant genotype. The participants had not received interferon therapy. They did not have any co-infection with either the human immunodeficiency virus or hepatitis B and no other associated forms of chronic liver disease. For cytokine polymorphism DNA was extracted from the whole blood by proteinase K digestion of fresh peripheral mononuclear cells, followed by phenolic extraction and ethanol precipitation. After amplification with polymerase chain reactions, the distribution of cytokine gene polymorphisms of interleukin (IL) 1 ß and JL-10 was examined in these patients. Single nucleotide polymorphisms (SNPs) were detennined using sequence specific oligonucleotide primers as described previously.4-6 Biallelic polymorphism was assessed for the IL­10 promotor at position -1082 (A versus G) and for IL-1ß pomotor at position-I 15 (C versus T).

Results

IL-1ß-l15 C allele was present in 49(87.5%) and -115 T in 30 (53.6%) patients. Genotype CC was present in 26 (46%), TC in 23 (42%), and TI’ in 07 (12%). IL-10 -1082A allele was seen in 50 (89.3%) and -1082 0 in 35 (62.5%). Genotype AG in 29 (52%), AA in 21(37%) and GG in 06 (11%).
Five patients had normal ALT and undetectable HCV RNA. They had probably recovered from the infection. In these patients, IL-1ß genotype TC was present in three, CC in one and TT in one while IL- 10 genotype AA was present in four and AG in one case. Out of fifty-one HCV RNA positive patients, there were 14 (27.5%) patients who had their ALT levels normal while 37 (72.5%) patients had elevated ALT levels. Allelic and genotype distribution of IL-1ß and IL-10 in these two groups of patients is shown in Table.

There was no statistical difference in the distribution. However, it was noted that IL-10 genotype AG was more prevalent in HCV RNA positive patients with normal ALT (10/14 patients).

Discussion

Cytokines play an important role in the defense against viral infections, both indirectly, through determination of the predominant pattern of host response, and directly, through inhibition of viral replication. However, in the context of an inflammatory response against a virus, cytokines may also lead to liver damage. Approximately 80 to 90% of acutely infected individuals develop persistent infection after acute disease; about one-half of them have elevated transaminases indicative of ongoing liver inflammation.7 Chronic HCV infection is marked by the presence of abundant amounts of cytokines. HCV may be inherently resistant to the effects of cytokines produced by HCV-specific cytotoxic T lymphocytes. Thus, the continuing inflammation results in liver damage in the absence of complete virologic recovery.8 In fact, the expression of these cytokines correlates with the amount of inflammation. The presence of an ongoing cellular immune response probably also contributes to the process of hepatic fibrosis. Kupifer cells can be activated by the production of cytokines such as TNF-a, which in turn produce TGF-ß.9
Most liver-infiltrating T cells in chronic hepatitis C are type 1 cells. Studies to date in liver tissue show that intrahepatic mRNA for type 1-like cytokines, such as IL-1ß, IL-2, IL-6, IL­8, TNF-cz, and IFN-y are upregulated in chronic HCV.10 The level of expression of type 1 cytokines, such as IL-2 and IFN­y, is correlated with the degree of histologic injury as well as the likelihood of nonresponsiveness to IFN-atherapy.11
Sustained response to interferon and ribavirin, defined as undetectable HCV RNA at 6 months after discontinuation of therapy, is achievable in 30% to 60% of treated patients.12,13 Predictors of response include viral factors such as viral genotypes, viral load and early disappearance of HCV RNA after initiation of therapy while the host factors include gender, age and degree of In recent years, increasing attention has been drawn to the role of host variation in cytokine levels in inflammatory and immune responses. Interleukin 10 (IL-10) is a potent anti-inflammatory Th2 cytokine that down-regulates the expression of major histocompatibility complex (MHC) class I and class II molecules, as well as the production of Thi cytokines.15-20 IL-10 levels differ widely between individuals, possibly because of polymorphisms in the promoter region of the IL-10 gene.21.22 Specifically, three single nucleotide polymorphisms (SNPs) in the promoter (at positions -1082, -819, and -592 relative to the transcription start site) form three SNP combinations (ATA, ACC, 0CC) associated with differential IL- 10 expression.22-24 It is reasonable to assume that patients of hepatitis C who produce high levels of IL-10 will have less hepatocellular injury and less ability to control infection.
There might be an association of cytokine gene polymorphism and susceptibility to hepatitis C infection. Hohler et al25 reported such an association with polymorphism at the TNF-a promoter. Patients with low secretion of IL-l0 should have better capability to eliminate the hepatitis infection. Perhaps low IL-l0 production can skew the immune system into the Thi type of response, facilitating the clearance of viral load. -1082A allele is associated with reduced IL-10 production in vitro.23 Individuals who are homozygous for IL-10 A at position -1082 have significantly lower levels of plasma IL-10 levels. Interleukin 10 polymorphisms may also modulate the response to interferon therapy. It has been shown that hepatitis C patients, genotyped as high IL-lO producers, have poor response to IFN-a therapy.24 These patients may benefit from additional treatment strategies designed to enhance a T-helper type 1 (Thi) response. Such polymorphisms may also predict sustained viral response to antiviral therapy.26 Liver transplant recipients, who are genotyped as having low production profile of IL- 10, are more prone to rejection and less likely to have hepatitis C recurrence.27
We, in this study, determined SNP only at position -1082 in case of 1L-10 and position -115 in IL-1ß and analyzed the frequency of its distribution and any correlation with the ALT level. Allelic variation of IL-10 at position -1082 influences plasma IL-10 levels as discussed above. The IL-10 gene polymorphism at -511 has also been shown to play a role in modulating the disease process e.g. development of coronary arterial disease in patients with chlamydia pneumoniae infection28 and gastric mucosal atrophy.29 In our series no significant difference in the genotype profile of IL-1ß
-115 and IL-lO -1082 was detected between patients with normal or elevated ALT. This may be due to the small sample size or because ALT level is not a good parameter to judge the ongoing hepatocellular injury. There is a need of a larger well-controlled study to suggest any role of cytokine gene polymorphism and hepatocellular injury in hepatitis C by assessing histological activity. Cytokine gene polymorphism of IL-1 TNFa, TGF ß IL-10 and INFyat different promoter sites should be considered in such a study.

References

1.Mosniann TR, Sad S. The expanding universe of T-cell subsets: Thi. Th2 and more. Immunology Today 1996:17:138-46.
2.Biron CA. Cytokines in the generation of immune responses to, and resolution of virus infection. Curr Opin Immunol 1994;6: 530-8.
3.Tough DF, Borrow P. Spent J. Induction of bystander T cell proliferation by viruses and type I interferon in vivo. Science 1996;272: 1947-50.
4.Kato S. Onda M, Yamada S. et al. Association of the interleukin-1 beta genetic polymorphism and gastric cancer risk in Japanese. J Gastroenterol 2001:36:696-9.
5.di Giovine FS. Takhsh E, Blakemore Al. et al. Single base polymorphism at -511 in the human interleukin-l beta gene (ILl beta). Hum Mol Genet 1992:1450,
6.Perrey C. Turner Si. Pravica V.et al. ARMS-PCR methodologies to determine IL­10, TNF-alpha, TNF-beta and TGF-beta I gene polymorphisms. Transpl Immunol I 999;7: 127-8.
7.Alter MJ, Mast EE, Moyer LA, et al. Hepatitis C. Infect Dis Clin North Am 1998;1 2:13-26.
8.Koziel MJ. The role of immune responses in the pathogenesis of hepatitis C virus ­infection. J Viral Hepatitis 1997;4:3 141.
9.Roulot D, Durand H, Coste T, et al. Quantitative analysis of transforming growth ­factor beta I messenger RNA in the liver of patients with chronic hepatitis C: ­absence of correlation between high levels and seventy of disease. Hepatology ­1995:21:298-304.
10.Llorent L, Richaud.Patin Y, Alcocer-Castillejos N. et al. Cytokinc gene expression in cirrhotic and non-cirrhotic human liver. J Hepatol 1996;24:555-563.
11.Fukuda R, Ishimura N, Ishihara S, et al. Intrahepatic expression of pro-inflammatory cytokine mRNAs and interferon efficacy in chronic hepatitis C. Liver 1996;1 6:390.9.
12.Poynard T. Marcellin P. Lee S. et al. Randomized trial of interferon 2b plus ribavirin for 48 weeks or for 24 weeks versus interferon 2b plus placebo for 48 ­weeks for treatment of chronic infection with hepatitis C virus. Lancet 1 998;352: 1426-32.
13.McHutchison JG, Gordon SC, Schiff E, et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engi J Med 1998;339:485-92.
14.Brouwer JT Hansen, BE, Niesters HG, et al. Early prediction of response in interferon monotherapy and in interferon-ribavinn combination therapy for chronic hepatitis C: HCV RNA at 4 weeks versus ALT. J Hepatol I 999:30:192-8.
15.Fiorentino DF, Zlotnik A, Vieira P. et al. IL- 10 acts on the antigen-presenting cell to inhibit cytokine production by ThI cells. J Immunol 1991;l46:3444-51.
16.Yue FY Dummer R, Geertsen R, et al. Interleukin- 10 is a growth factor for human melanoma cells and down-regulates HLA class I. HLA class II and ICAM-l molecules. IntJ cancer 1997:71:630-37.
17.Tsuruma T, Yagihashi A, Torigoe T, et al. Interleukin- 10 reduces natural killer sensitivity and downregulates MHC class I expression on H-ras transformed cells. Cell Immunol 1998;184:121-8.
18.Zeller JC, Panoskaltsis-Mortan A, Murphy WJ, et al. Induction of CD4 T cell alloantigen specific hyporesponsiveness by ILl 0 and TGF-.ß J Immunol 1999;163:3684-91.
19.de Waal Malefyt R, Haanen J, Spits H, et al. Interleukin 10 (IL10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via down regulation of class II major histocompatibility complex expression. J Exp Med 1991:174:915-24.
20.de Waal Malefyt R, Abrams J, Bennett B, et al. lnterleukin 10 (IL-JO) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-JO produced by monocytes. J Exp Med 1991:174:1209-20.
21.Eskdale J, Gallagher G, Verweij CL. et al. Interleukin 10 secretion in relation to human IL-10 locus haplotypes. Proc NatI Acad Sci USA I 998;95:9465-70.
22.Eskdale J, Keijsers V. Huizinga T, et aJ. Microsatellite alleles and single nucleotide polymorphisms (SNP) combine to form four major haplotype families at the human interleukin-10 (IL-JO) locus. Genes and Immunity 1999;1:151-5.
23.Turner D, Williams D, Sankaran D, et al. An investigation of polymorphism in the interleukin- JO gene promoter. Eur J Immunogenet 1997:24:1-8.
24.Edwards-Smith C, Jonsson JR, Purdie D. et al. Interleukin-10 promoter polymorphism predicts initial response of chronic hepatitis C to interferon alpha. Hepatology 1999:30:526-30.
25.Hohler T, Kruger A. Gerken G. et al. Tumor necrosis factor alpha promoter polymorphism at position-238 is associated with chronic active hepatitis C infection. J Med Virol 1998:54:173.
26.Yee U, Tang J, Gibson AW, et al. Interleukin 10 polymorphisms as predictors of sustained response in antiviral therapy for chronic hepatitis C infection. Hepatology 2001 ;33 708-12.
27.Tambur AR, Ortegel JW, Ben-Ari Z, et ab. Role of cytokine gene polymorphism in hepatitis C recurrence and allograft rejection among liver transplant recipIents. Transplantation 2001:71:1475-80.
28.Momiyama Y, Hirano R, Taniguchi H, et al. Effects of interleukin-l gene polymorphisms on the development of coronary artery disease associated with Chlamydia pneumoniae infection. JAm CoIl Cardiol 2001:38:712-17.
29.Kato S. Onda M, Yaniada S. et al. Association of the interleukin-l beta genetic polymorphism and gastric cancer risk in Japanese. J Gastroenterol 2001,36:696-9.

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