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Departments of Immunology and Gastroenterology, Hospital Universitario Central de Asturias
Department of Functional Biology, University of Oviedo, Oviedo, Spain
The aim of the present study was to investigate, in 152 Spanish patients infected with hepatitis C virus (HCV), the possibility that killer cell immunoglobulin-like receptors (KIRs) influence progression to hepatocellular carcinoma. KIRs are related to the activation and inhibition of natural killer cells and may play an important role in the innate response against infection with such viruses as HCV. We found that the human leukocyte antigenBw4I80 epitope and the KIR3DS1 gene were more frequent in HCV carriers than in patients with hepatocellular carcinoma. Moreover, these associations were not independent of each otherthe KIR3DS1/Bw4I80 genotype clearly was also more frequent in HCV carriers (odds ratio, 24.22).
NK cells provide defense against viral infections by producing cytokines and causing cytotoxicity, and this capacity is dependent on an equilibrium between the inhibitory and activating receptors [1]. The killer cell immunoglobulin-like receptor (KIR) genes encode a group of proteins that are expressed on NK cells and some T cells [2]. These genes are located on chromosome 19q13.4 in the leukocyte receptor complex. KIR proteins act as receptors that recognize major histocompatibility complex (MHC) class I molecules and are directly involved in the activation and inhibition of NK and possibly CD8+ T cells [3]. The difference between the inhibitory and activating receptors is mainly in the intracytoplasmatic tail. The inhibitory receptors have a long tail with an immunoreceptor tyrosine inhibitory motif, whereas the activating receptors have a short tail without this motif but with the capacity to interact with activating adaptor proteins such as DAP12 [4].
The KIR3DL1 and KIR3DS1 receptors segregate as alleles of the same locus. The KIR3DL1 inhibitory receptor binds to HLA-B with the serologically defined epitope Bw4. This interaction is more effective with those HLA-Bw4 alleles that have an isoleucine at position 80 (Bw4I80) than with those that have a threonine at this position (Bw4T80) [5]. The ligand for the KIR3DS1 activating receptor has not yet been defined, but KIR3DS1 shares 97% of the amino acid sequence in its extracellular domain with KIR3DL1, and these may share a similar set of ligands. Some recent studies have implicated the KIR3DS1 activating receptor in the rate of progression of HIV-1 infection [6] and in susceptibility to psoriatic arthritis [7].
HLA-Cw alleles are the natural ligands for KIR2D receptors. This interaction is characterized by the residue present at position 80 of the HLA-Cw molecule. An asparagine at this position defines the HLA-Cw group 1 alleles (HLA-C1), which bind to the KIR2DL2, 2DL3, and 2DS2 receptors. A lysine at this position defines the HLA-Cw group 2 alleles (HLA-C2), which bind to the KIR2DL1 and 2DS1 receptors [8].
Although the chronicity rate of acute hepatitis C virus (HCV) infection has not been well established, a considerable number of patients do develop hepatocellular carcinoma [9], the final stage of HCV infection. Mechanisms involved in the progression of HCV infection to hepatocellular carcinoma may be related to viral genotype, viral load, or diverse host factors. These host factorsespecially immunological and genetic onesseem to play an important role in the evolution of HCV infection. Recently, a study with a large number of patients implicated HLA and KIR inhibitory genes in the resolution of HCV infection [10]. In fact, it is well known that NK cells play an important role in the destruction of HCV-infected cells and that the loss of NK cell activity is correlated with the progression of HCV infection to hepatocellular carcinoma [11]. The aim of the present study was to analyze whether KIR genes (for 3DL1, 3DS1, 2DL2, 2DL3, and 2DS2) and their possible synergistic effect with HLA-B and HLA-C alleles influence the progression of HCV infection to hepatocellular carcinoma.
Patients, materials, and methods.
Between March 1994 and May 2001, a total of 602 patients received a diagnosis of HCV infection at our center. From those, we selected a group of 152 unrelated patients, all of whom were positive for both anti-HCV antibodies and serum HCV RNA and met the criteria (defined below) either for being an HCV carrier or for having liver cirrhosis or hepatocellular carcinoma. All patients were Spanish and white and lived in the same geographic area. The patients selected for the present study were diagnosed between 1994 and 1998 and have been followed since HCV infection was diagnosed. The protocol was approved by the ethics committee of our hospital, and all patients provided written, informed consent before enrollment.
The HCV-infected patients were divided into 3 groups, according to the clinical features that developed during follow-up. The first group, HCV carriers, consisted of 51 HCV-infected patients with minimal liver disease and persistently normal levels of alanine transaminase; this group had a sex distribution of 1.55 female patients to each male patient. The second group, patients with liver cirrhosis (defined by clinical and biological criteria and confirmed by liver biopsy), consisted of 47 patients and had a nearly equal sex distribution (24 male patients and 23 female patients). The third group, patients with hepatocellular carcinoma (defined as 1 or more tumoral nodules in a cirrhotic liver), consisted of 54 patients and had a sex distribution that was inversed relative to that of the HCV carriers, with 1.38 male patients to each female patient. All diagnoses of hepatocellular carcinoma were confirmed by methods of diagnosis by image (helicoidal computed tomography and echography) and by anatomopathological criteria. The 3 groups were similar in age; other demographic and clinical features are shown in table 1. One hundred and sixteen unrelated blood donors, all of whom belonged to the same ethnic group as that of the patients and were negative for HCV infection, served as healthy control subjects.
HCV genotypes were determined by polymerase chain reaction (PCR). The HLA-Cw and HLA-B alleles and the Bw4 and Bw6 epitopes were typed by PCR with sequence-specific oligoprobes (SSO), using RELI-SSO typing kits (Dynal). KIR genotyping was performed by PCR with gene-specific primer pairs, in accordance with the method described elsewhere [12].
Allelic frequencies were calculated by direct counting, and the significances of associations were determined either by the 2 test with Yates's correction or by Fisher's exact test. Odds ratios (ORs) were calculated by the cross-product ratio. Exact 95% confidence intervals (CIs) were determined. Multiple-variable logistic regression analysis was conducted by use of SPSS software (version 10; SPSS). P < .05 was considered to be significant.
Results.
To begin, we analyzed the distribution of the Bw4 and Bw6 epitopes among the 3 groups of patients; no significant association was found. Nevertheless, the frequency of the Bw4I80 allele was notably increased in the HCV carriers, compared with that in the patients with hepatocellular carcinoma (49.01% vs. 12.96%), and the difference reached statistical significance (P = .0003; OR, 6.45) (table 2). The frequency of the Bw4I80 allele was also increased in the patients with liver cirrhosis, compared with that in the patients with hepatocellular carcinoma (corrected P = .02; OR, 4.16) (table 2). However, the Bw4T80 allele had the same distribution among the different groups studied. Multiple-variable logistic regression analysis of Bw4I80, Bw4T80, and Bw6 showed the persistence of the protective effect of Bw4I80 (P = .002).
No association between the frequency of the KIR3DL1 gene and progression to hepatocellular carcinoma was found. This gene was present in 95% of the HCV carriers, 95% of the patients with liver cirrhosis, and 98% of the patients with hepatocellular carcinoma. Nevertheless, differences in the distribution of the KIR3DS1 gene among these groups were found. This gene was significantly more frequent in the HCV carriers than in the patients with hepatocellular carcinoma (50.98% vs. 25.92%; P = .008; OR, 2.97) (table 2). It was also more frequent in the patients with liver cirrhosis than in the healthy control subjects (44.68% vs. 25.92%), but this difference did not reach statistical significance.
We further analyzed whether a synergistic effect existed between the KIR3DS1 gene and the Bw4I80 epitope in the development of hepatocellular carcinoma. We found these 2 factors to be combined (KIR3DS1/Bw4I80 genotype) in 16 of the HCV carriers and in only 1 of the patients with hepatocellular carcinoma (31.37% vs. 1.85%; corrected P = .0003; OR, 24.22). The frequency of this genotype was also increased in the patients with cirrhosis, compared with that in the patients with hepatocellular carcinoma, but the difference was not significant. We also analyzed whether the above-mentioned associations were independent of each other. The frequencies of the Bw4I80 ligand in the 3 groups of patients were similar (in the HCV carriers, 17.64%; in the patients with liver cirrhosis, 23.40%; and in the patients with hepatocellular carcinoma, 11.11%), when we considered only those patients who were negative for KIR3DS1. Similarly, when we analyzed the distribution of KIR3DS1 in those patients who did not carry Bw4I80, the frequencies were similar in the 3 groups (in the HCV carriers, 19.38%; in the patients with liver cirrhosis, 21.27%; and in the patients with hepatocellular carcinoma, 24.07%).
The analysis of the distribution of HLA-Cw showed that more HCV carriers had 2 copies of HLA-C1 alleles than did patients with hepatocellular carcinoma (P = .013; OR, 3.11), whereas more patients with hepatocellular carcinoma had 2 copies of HLA-C2 alleles than did the HCV carriers (P = .004; OR, 4.99) (table 2). There were no statistically significant associations between the frequencies of the KIR2Ds genes in the different groups studied. When we finally analyzed whether a possible synergistic effect existed among the different KIR2D receptors and their ligands, no association with the different progressive forms of HCV infection was found.
Discussion.
Previous studies have demonstrated several associations between various HLA class I alleles and the clinical progression of HCV infection [13]. In fact, we recently demonstrated an association between the HLA-B*18 allele and the development of hepatocellular carcinoma in HCV-infected patients [14]. These associations could be due to the capacity of these molecules to present HCV peptides with different affinities to cytotoxic T lymphocytes (CTLs) and, additionally, to their capacity to interact with KIRs.
The present study has shown that the frequencies of both the Bw4I80 ligand and the KIR3DS1 gene were increased in patients who had not developed hepatocellular carcinoma, especially in those with minimal liver damage. Although these genetic factors might seem to be separately associated, we have demonstrated that the individual associations were not independentwe have been able to show that the observed protective effect these genetic factors had against the development of hepatocellular carcinoma was related to the KIR3DS1/Bw4I80 genotype. This has a certain similarity to the effect observed in the progression of HIV-1 infection reported previously [6] and is in accordance with the weak protective effect this genotype has against the development of chronic HCV infection found by Khakoo et al [10].
No associations were found between KIR2D/HLA-C genotypes and the progression of HCV infection. This could be due, in some cases, to the relatively small groups used in the present study. This was the case with the KIR2DS2/HLA-C1 genotype, which was present in 47% of the HCV carriers but in only 27% of the patients with hepatocellular carcinoma.
As mentioned above, KIR genes encode a group of receptors that seem to be implicated in the activation and inhibition of the cytotoxic capacity of NK cells. These proteins recognize HLA class I moleculesespecially HLA-C and HLA-Band this leads to inhibition when these molecules interact with L receptors and to activation when they interact with S receptors. It is well known that some viruses are able to down-regulate the expression of HLA class I molecules, as a mechanism for evasion of the immune system's CTL response. Host defenses can circumvent this viral adaptation through the capacity of NK cells to detect the absence of HLA class I molecules on the surface of infected cells. This mechanism provides a strong stimulus for the activation of NK cells. The impairment of NK cell activity has been implicated in the maintenance of HCV infection and in the progression of this infection to hepatocellular carcinoma [13, 15].
The data obtained in the present study suggest that HLA-B molecules and KIRs play roles in the natural history of HCV infection. Although the receptor-ligand interaction between HLA-B alleles that carry the Bw4I80 epitope and KIR3DS1 has not been demonstrated, it is possible to suggest a model in which the interaction between these molecules leads to better NK and/or CD8+ T cell activation, which could provide a more adequate elimination of HCV-infected cells. Further genetical and functional studies of this interaction could provide a new perspective on the pathogenic mechanisms involved in HCV infection and may offer new approaches to its treatment.
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