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KIR genes and their human leukocyte antigen ligands in the progression to cirrhosis in patients with chronic hepatitis C
Human Immunology 72 (2011) 1074-1078
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KIR genes and their human leukocyte antigen ligands in the progression to cirrhosis in patients with chronic hepatitis C Amanda Vansan Marangon a,*, Giovanni Faria Silva b, Camila Fernanda Verdichio de Moraes c, Rejane Maria Tomasini Grotto c, Maria InËs de Moura Campos Pardini c, Dayse Sousa de Pauli d, Ana Maria Sell a, Jeane Eliete Laguila Visentainer a, Ricardo Alberto Moliterno a a
Immunogenetics Laboratory, Department of Basic Sciences, Maringà State University, UEM, Maringà, Paranà, Brazil Gastroenterology Division, Internal Medicine Department, Botucatu Medical School, Sâo Paulo State University, UNESP, Botucatu, Sâo Paulo, Brazil Molecular Biology Laboratory of Blood Transfusion Center, Botucatu Medical School, Sâo Paulo State University, UNESP, Botucatu, Sâo Paulo, Brazil d Londrina Clinical Hospital, Londrina State University, UEL, Londrina, Paranà, Brazil b c
A R T I C L E
I N F O
Article history: Received 5 January 2011 Accepted 25 August 2011 Available online 28 August 2011
Keywords: NK cells HLA KIR Hepatitis C Liver fibrosis
A B S T R A C T
Natural killer (NK) cells play pivotal roles in immune responses against infection with viruses, such as hepatitis C virus (HCV), and killer cell immunoglobulin-like receptors (KIRs) are related to the activation and inhibition of NK cells. The aim of this study was to investigate the possibility that KIR genes and their human leukocyte antigen (HLA) ligands influence progression to cirrhosis in patients infected with genotype 1 of HCV. A total of 145 Brazilian patients with confirmed chronic hepatitis C grouped from F0 to F4 according to fibrosis progression to cirrhosis were evaluated. Genotyping of KIR and HLA genes was performed by polymerase chain reaction with sequence-specific oligonucleotide probes. The HLA-C2 KIR ligand was more frequent in patients than in healthy controls (74.5% vs 64.3%, p ⫽ 0.04, odds ratio (OR) ⫽ 1.6, 95% confidence interval (CI) ⫽ 1.03–2.52). Moreover, the HLA-C1C2 genotype was more frequent in patients with advanced fibrosis or cirrhosis (F3–F4 group) than in patients in the F0 –F2 group (61.6% vs 44.7%, p ⫽ 0.06) and in the F4 group compared with the F0 –F3 group (65.7% vs 46.7%, p ⫽ 0.05, OR ⫽ 2.19, 95% CI ⫽ 1.01– 4.73). NK and KIR ligands may contribute to the development of liver damage in patients chronically infected by HCV. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Natural killer cells (NK) are crucial components of the innate response and provide defense mainly against viral infections and cancer. They also provide a link to priming of adaptive immunity. The function of NK cells is regulated by a fine balance of inhibitory and activating signals, which are mediated by a diverse array of cell-surface receptors including killer cell immunoglobulin-like receptors (KIR). These receptors can either inhibit or activate NK cells and the difference between them lies mainly in their intracytoplasmatic tail [1–3]. In humans, KIRs are encoded by a cluster of genes located in the leukocyte receptor complex on chromosome 19q13.4. At least 14 KIR genes have thus far been characterized, of which 8 are NK cell inhibitors (KIR2DL1, ⫺2DL2, ⫺2DL3, ⫺2DL4, ⫺2DL5, ⫺3DL1, ⫺3DL2, and ⫺3DL3), 6 are activating (KIR2DS1, -2DS2, -2DS3, -2DS4, -2DS5, and -3DS1), and 2 are pseudogenes (KIR2DP1 and -3DP1) [4,5]. Based on the genetic content and pattern of segregation at the population level, KIR haplotypes are divided into 2 groups, A and B, which differ in the number and kind of KIR genes. Common to both * Corresponding author. E-mail address: [email protected] (A.V. Marangon).
groups of haplotypes are the “framework genes”: KIR3DL2, -3DP1, -2DL4, and -3DL3 [4 – 6]. KIRs specifically recognize some human leukocyte class I antigens (HLA-I) present on target cells. HLA-Bw4 and distinct allotypes of HLA-C (C1 and C2 groups) are the predominant ligands for most KIRs [7]. The HLA-C alleles were classified as C1 or C2 KIR ligand groups, depending upon 2 amino acid positions encoded in exon 2. The HLA-C1 allotypes contain serine at position 77 and asparagine at position 80 and are ligands for the inhibitory receptors KIR2DL2 and KIR2DL3. The HLA-C2 allotypes contains asparagine and lysine at those positions and are ligands for the inhibitory receptor KIR2DL1 and for the activating receptor KIR2DS1 [8,9]. HLA-Bw4 allotypes are characterized by at least 5 different patterns of amino acid exchanges at positions 77 and 80 – 83 and serve as ligands for KIR3DL1. In addition, HLA-A3 and HLA-A11 are ligands of KIR3DL2 [10 –12]. Accumulated evidence suggests that NK cells play an important role not only in the host defense against pathogens in the liver but also in liver injury and repair [2,3]. Because of the important role of KIR genes in the control of NK cell activation, investigations have been conducted on different populations to evaluate the associations between KIR and liver diseases such as viral hepatitis [13–15].
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.08.017
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Most patients with hepatitis C virus (HCV) infection progress to chronic disease, which can lead to liver fibrosis and the subsequent occurrence of cirrhosis and hepatocellular carcinoma [16]. However, the mechanisms responsible for the onset and progression of hepatic lesions during chronic hepatitis C are not fully understood. Hence, the aim of the present study was to investigate the possible association of KIR genes and their HLA ligands with fibrosis progression to cirrhosis in patients with chronic hepatitis C.
certain amino acid in the adequate positions and classified into C1, C2, and Bw4 groups. The C1 group of alleles observed in patients were C*01, C*03, C*07, C*08, C*12, C*14, C*15:07, and C*16:01. The C2 group of alleles were C*02, C*04, C*05, C*06, C*07:07, C*12:05, C*15, C*16:02, C*17, and C*18. The group of alleles that belongs to the Bw4 group were B*13, B*27:05, B*27:09, B*37:01, B*38, B*44, B*47, B*49, B*51, B*52, B*53, and B*58. Only alleles observed in the patients were described.
2. Subjects and methods
2.6. KIR2DS4
2.1. Studied population
The 2 major full-length subtype and deleted forms of KIR2DS4 [20] were also analyzed by PCR with sequence-specific oligonucleotide probe protocols. The samples with doubtful results were confirmed by in-house PCR with sequence specific primers according to Martin [21] and optimized by Rudnick et al. [17].
Between September 2004 and January 2009 a total of 761 patients received a diagnosis of HCV infection at the Internal Medicine Department, Gastroenterology Division, Botucatu Medical School. Of these, 145 unrelated patients with infection only by the HCV genotype 1 were included in this study. A total of 616 patients were excluded based on the following criteria: human immunodeficiency virus–positive serology, hepatitis B virus coinfection, other hepatic diseases, presence of non-1 HCV genotype, and history of antiviral therapy before liver biopsy. In addition, 289 unrelated healthy individuals from Southern Brazil [17] were used as controls and compared with HCV patients. The studied protocol was approved by the Ethics Committee for Research Involving Human Beings of SÄo Paulo State University and all subjects provided written consent to participate in the study. 2.2. Liver biopsy Biopsies in all patients were carried out using the technique of Menghini or a Tru-Cut needle to determine the degree of fibrosis and progression of infection. The fragments were analyzed when at least 8 portal spaces were present and stained with hematoxylin and eosin, Masson trichrome, and reticulin stain. The hepatic biopsies were analyzed by a pathologist using the Metavir score: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis and few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis [18]. The patients were stratified into subgroups according to fibrosis stage: absence of fibrosis or mild fibrosis (F0 –F2, n ⫽ 85), advanced fibrosis or cirrhosis (F3–F4, n ⫽ 60), without cirrhosis (F0 –F3), and with cirrhosis (F4). 2.3. HCV genotyping The HCV genotype was defined by the reverse line probe assay (Innolipa v. 1.0, Innogenetics, Ghent, Belgium) according to the manufacturer’s instructions. Genotyping was preceded by the extraction of HCV viral RNA present in the patient’s plasma, followed by reverse-transcription polymerase chain reaction (PCR; Amplicor HCV test, v. 2.0, Roche Diagnostic Systems, Branchburg, NJ) according to the manufacturer’s instructions. 2.4. DNA extraction Genomic DNA was isolated from 250 L of peripheral blood using a NeoIsocolumn kit (One Lambda, Inc., San Diego, CA) or purelink (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. 2.5. KIR and HLA-I genotyping KIR (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, KIR2DP1, and KIR3DP1) and HLA-I (HLA-A, -B, and -C) genes were genotyped according to the manufacturer’s instructions by PCR with sequence-specific oligonucleotide probe protocols using commercial KIR and HLA genotyping test kits (One Lambda, Inc.) [19]. The sequences of all alleles were analyzed one by one in the HLA database (http://www.ebi.ac.uk/imgt/hla/) by the presence of a
2.7. Statistical analysis KIR and KIR-HLA ligand frequencies were obtained by direct counting. The 2 test with Yates’ correction or Fisher’s exact tests was applied to compare the distribution of the frequencies of alleles and genes between the groups. Odds ratios (OR) with 95% confidence intervals (CI) were also calculated. The analyses were carried out using SISA online statistics (http://www.quantitativeskills.com/sisa/). p values ⬍0.05 were considered statistically significant. Because the gametic phase of the alleles from the different loci is not known (segregation analysis into families was not carried out), haplotypes and the calculation of their frequency were determined by the likelihood-ratio test using the Arlequin statistical software program according to Excoffier et al. [22]. 3. Results 3.1. Patient characteristics Patient characteristics are summarized in Table 1. According to the Metavir score, 58.6% of patients presented no or mild fibrosis (4.8% were F0, 29.7% F1, and 24.1% F2) and 41.8% presented advanced fibrosis or cirrhosis (15.2% were F3 and 26.2% F4). The mean age of patients with advanced fibrosis or cirrhosis was higher than that of patients without fibrosis or mild fibrosis (48.6 ⫾ 9.8 years vs 39.8 ⫾ 9.3 years, respectively, p ⬍ 0.05). The same trend was seen when comparing the F4 group versus the F0 –F3 group (27.8 ⫾ 9.6 years vs 19.9 ⫾ 7.0 years, respectively, p ⬍ 0.05). With respect to possible risk factors for acquiring hepatitis C, the use of intravenous drugs was the main risk factor associated with the F0 –F2 group (25.8%) compared with only 2 patients (3.3%) in the F3–F4 group (p ⬍ 0.05%). The use of intravenous drugs was also the main risk factor associated with the F0 –F3 group compared with the F4 group (20.5% vs 5.3%, p ⬍0.05). 3.2. KIR genes and HLA ligand frequencies To evaluate the role of KIR genes and their HLA ligands in disease progression, patients were first stratified into individuals with absence of fibrosis or mild fibrosis (F0 –F2, n ⫽ 85) and individuals with advanced fibrosis or cirrhosis (F3–F4, n ⫽ 60) Next, patients with cirrhosis (F4 stage, n ⫽ 38) were separated and compared with patients without cirrhosis (F0 –F3 stage, n ⫽ 107). Furthermore, all patients were compared with unrelated healthy controls. The HLA epitopes (HLA-C1, -C2, -Bw4 and -A3/A11) were analyzed in all groups. The gene and genotype frequencies of the HLA ligands are presented in Table 2. HLA-C2 was more frequent in patients than in healthy controls (74.5% vs 64.3%, p ⫽ 0.04, OR ⫽ 1.6, 95% CI ⫽ 1.03–2.52). The heterozygote genotype HLA-C1C2 exhibited a trend of being more frequent in the F3–F4 group compared with the F0 –F2 group (61.6% vs 44.7%; p ⫽ 0.06, OR ⫽ 1.98, 95% CI ⫽ 1.02–3.9). The HLA-C1C2 genotype was also more frequent in patients with liver cirrhosis (F4) compared with patients without
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Table 1 Clinical and general features of patients with chronic hepatitis C attended in the Department of Internal Medicine, Gastroenterology Division, Botucatu Medical School, from September 2004 to January 2009 (n ⫽ 145) Characteristics
Patients (n ⫽ 145)
F0 –F2 (n ⫽ 85)
F3–F4 (n ⫽ 60)
F4 (n ⫽ 38)
p
Age (mean ⫾ SD)a Gender, n (%) Male Female Risk factor, n (%) Blood transfusion Intravenous druga Gluconergan Nasal drug Othersb Duration of infectionc (mean ⫾ SD) METAVIR stage,d n (%) F0 F1 F2 F3 F4
43.4 ⫾ 10.4
39.8 ⫾ 9.3
48.6 ⫾ 9.8
50.0 ⫾ 9.53
⬍0.05
112 (0.77) 33 (0.22)
67 (0.78) 18 (0.21)
45 (0.75) 15 (0.25)
17 (0.71) 11 (0.28)
36 (0.24) 24 (0.16) 22 (0.15) 5 (0.03) 58 (0.40) 21.8 ⫾ 8.3
17 (0.20) 22 (0.25) 10 (0.11) 2 (0.02) 34 (0.40) 19.0 ⫾ 6.9
19 (0.31) 2 (0.03) 12 (0.20) 3 (0.05) 24 (0.40) 25.7 ⫾ 8.7
10 (0.26) 2 (0.05) 6 (0.15) 3 (0.08) 17 (0.44) 27.8 ⫾ 9.6
0.15 ⬍0.05 0.25 0.64 0.97 0.10
7 (0.04) 43 (0.29) 35 (0.24) 22 (0.15) 38 (0.26)
7 (0.08) 43 (0.50) 35 (0.41) — —
— — — 22 (0.36) 38 (0.63)
— — — — 38 (1.00)
— — — — —
0.73 0.73
p ⬍ 0.05 when comparing F0 –F2 with F3–F4 and F0-F3 with F4. Tattoo, surgeries, occupational exposure, homosexuals or promiscuous sexual behavior, and unknown risk factors. c Duration of infection was calculated only for 90 patients (F0 ⫽ 4, F1 ⫽ 25, F2 ⫽ 24, F3 ⫽ 16, and F4 ⫽ 21). The duration of infection for 55 patients is unknown. d Metavir score: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis and few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis. a
b
cirrhosis (F0 –F3; 65.7% vs 46.7%, p ⫽ 0.05, OR ⫽ 2.19, 95% CI ⫽ 1.0 – 4.7). HLA-Bw4 molecules were divided into 2 groups based on whether isoleucine or threonine was present at position 80 (Bw480Ile and Bw4-80Thr, respectively). The Bw4-80Ile exhibited a trend of being less frequent in the group with liver cirrhosis (F4) compared with patients without cirrhosis (39.5% vs 56.1%, p ⫽ 0.09, OR ⫽ 0.51, 95% CI ⫽ 0.24 –1.0; Table 2). With respect to KIR genes, no association between the 16 KIR genes and susceptibility to HCV, progression to fibrosis, or cirrhosis was observed (Table 3). Nevertheless, a lower frequency of the homozygote genotype KIR2DL3/KIR2DL3 was observed in the F3–F4 group (45.4%) compared with the F0 –F2 group (62.0%; Table 4). The deleted variant of KIR2DS4 was the most frequent form in all groups, but the distribution of the KIR2DS4 forms (the full-length gene, the deletion variant, or both) was similar in all groups. In analysis of the interactions between activating and inhibitory KIR and HLA epitopes (KIR2DL1 and its ligand C2; KIR2DL2 with C1; KIR2DL3 with C1; KIR3DL1 with Bw4; KIR3DL1 with Bw4-80Ile; KIR3DL1 with Bw4-80Thr; KIR3DL2 with HLA-A3 and -A11; KIR2DS1 with C2; KIR2DS2 with C1; KIR3DS1 with Bw4; KIR3DS1 with Bw480Ile; and KIR3DS1 with Bw4-80Thr), none of the receptor–ligand
pairs was associated with susceptibility or progression of disease (Table 4). Furthermore, patients were identified as KIR AA, AB, or BB haplotype based on the multiple KIR genes they possessed. KIR BB haplotypes were characterized by the presence of one or more of the following genes: KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5, and KIR3DS1. Conversely, KIR AA haplotypes were characterized by both the absence of any 1 of the preceding genes and the presence of the genes KIR3DL3, KIR2DL3, KIR2DL1, KIR3DP1, KIR2DL4, KIR3DL1, KIR2DS4, and KIR3DL2. The AB haplotype was characterized by the presence of genes of the 2 groups [4,23,24]. The KIR AA haplotype was the most frequent haplotype in all groups (33.1% of all patients, 39.2% in the F3–F4 group, 33.3% in the F0 –F2 group, 34.2% in the F4 group, 32.7% in the F0 –F3 group, and 33.2% in unrelated healthy controls). No significant difference in the distribution of KIR haplotypes was observed. 4. Discussion NK cells represent a fundamental component of the innate immune system. The repertoire of NK cells is extensive in the liver [25] and cytolytic activity seems to have an inverse association with liver fibrosis stage, suggesting that the presence of cytolytic active
Table 2 Distribution of gene and genotype frequencies of KIR-HLA ligands in controls and in patients chronically infected by the HCV-1 genotype, classified according to their liver biopsies Metavir score HLA ligands
A3 and A11 Bw4 C1 C2b C1C1 C1C2c C2C2 Bw4-80Iled Bw4-80Thr
Metavir score groupsa F3–F4 n (f)
F0 –F2 n (f)
p
F4 n (f)
F0 –F3 n (f)
p
19 (0.31) 40 (0.66) 49 (0.81) 48 (0.80) 12 (0.20) 37 (0.61) 11 (0.18) 28 (0.46) 17 (0.28)
18 (0.21) 64 (0.85) 63 (0.74) 60 (0.70) 25 (0.29) 38 (0.44) 22 (0.25) 47 (0.55) 21 (0.24)
0.21 0.90 0.38 0.27 0.27 0.06 0.38 0.39 0.76
11 (0.29) 24 (0.63) 31 (0.81) 32 (0.84) 6 (0.15) 25 (0.65) 7 (0.18) 15 (0.39) 13 (0.34)
25 (0.23) 80 (0.74) 81 (0.75) 76 (0.71) 31 (0.29) 50 (0.46) 26 (0.24) 60 (0.56) 25 (0.23)
0.64 0.24 0.50 0.13 0.1 0.05 0.60 0.09 0.27
All patients
Controls
p
37 (0.25) 104 (0.71) 112 (0.77) 108 (0.74) 37 (0.25) 75 (0.51) 33 (0.22) 75 (0.51) 39 (0.26)
72 (0.24) 218 (0.75) 223 (0.77) 186 (0.64) 92 (0.31) 131 (0.45) 55 (0.19) 141 (0.48) 77 (0.26)
0.98 0.47 0.91 0.04 0.21 0.24 0.40 0.63 0.95
Metavir score: F3–F4, advanced fibrosis or cirrhosis (n ⫽ 60); F0 –F2, without fibrosis or mild fibrosis (n ⫽ 85); F4, with cirrhosis (n ⫽ 38); F0 –F3, without cirrhosis (n ⫽ 107). All patients (n ⫽ 145) versus controls (n ⫽ 289): odds ratios (OR) ⫽ 1.61; 95% confidence interval (CI) 1.03–2.52. c F3–F4 versus F0 –F2, OR ⫽ 1.98, 95% CI ⫽ 1.02–3.9; and F4 versus F0 –F3, OR ⫽ 2.19, 95% CI ⫽ 1.01– 4.7. d F4 versus F0 –F3: OR ⫽ 0.51, 95% CI ⫽ 0.24 –1.0. a
b
A.V. Marangon et al. / Human Immunology 72 (2011) 1074-1078
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Table 3 Distribution of KIR genes in controls and in patients chronically infected by the HCV-1 genotype, classified according to their liver biopsy Metavir score KIR genes
KIR2DL1 KIR2DL2 KIR2DL3 KIR2DL4 KIR2DL5 KIR2DP1 KIR2DS1 KIR2DS2 KIR2DS3 KIR2DS4 KIR2DS5 KIR3DL1 KIR3DL2 KIR3DL3 KIR3DP1 KIR3DS1
Metavir score groupsa F3–F4 n (f)
F0 –F2 n (f)
p
F4 n (f)
F0 –F3 n (f)
p
58 (0.96) 32 (0.53) 54 (0.90) 60 (1.00) 28 (0.46) 58 (0.96) 25 (0.41) 32 (0.53) 16 (0.26) 53 (0.88) 23 (0.38) 53 (0.88) 60 (1.00) 60 (1.00) 60 (1.00) 23 (0.38)
84 (0.98) 36 (0.42) 82 (0.96) 85 (1.00) 50 (0.59) 84 (0.98) 35 (0.41) 39 (0.46) 30 (0.35) 79 (0.93) 30 (0.35) 78 (0.92) 85 (1.00) 85 (1.00) 85 (1.00) 37 (0.44)
0.56 0.23 0.76 0.99 0.17 0.56 0.99 0.40 0.28 0.38 0.72 0.57 0.99 0.99 0.99 0.60
38 (1.00) 17 (0.45) 36 (0.95) 38 (1.00) 18 (0.47) 38 (1.00) 17 (0.45) 18 (0.47) 9 (0.24) 32 (0.84) 16 (0.42) 32 (0.84) 38 (1.00) 38 (1.00) 38 (1.00) 17 (0.45)
104 (0.97) 51 (0.48) 100 (0.93) 107 (1.00) 60 (0.56) 104 (0.97) 42 (0.39) 53 (0.50) 37 (0.35) 100 (0.93) 37 (0.35) 99 (0.93) 107 (1.00) 107 (1.00) 107 (1.00) 43 (0.40)
0.60 0.90 1.00 0.45 0.46 0.60 0.68 0.96 0.29 0.16 0.52 0.24 0.45 0.76 0.52 0.24
All patientsb
Controlsb
p
143 (0.99) 68 (0.47) 134 (0.92) 145 (1.00) 78 (0.54) 142 (0.98) 59 (0.41) 71 (0.49) 46 (0.32) 132 (0.91) 53 (0.37) 131 (0.90) 145 (1.00) 145 (1.00) 145 (1.00) 60 (0.41)
281 (0.97) 136 (0.47) 258 (0.89) 289 (1.00) 152 (0.53) 280 (0.97) 118 (0.41) 136 (0.47) 77 (0.27) 271 (0.94) 100 (0.35) 272 (0.94) 289 (1.00) 289 (1.00) 289 (1.00) 113 (0.39)
0.56 0.94 0.38 0.21 0.89 0.75 0.93 0.78 0.31 0.39 0.76 0.21 0.80 0.80 0.80 0.72
Metavir score: F3–F4, advanced fibrosis or cirrhosis (n ⫽ 60); F0 –F2, without fibrosis or mild fibrosis (n ⫽ 85); F4, with cirrhosis (n ⫽ 38); F0 –F3, without cirrhosis (n ⫽ 107). All patients (n ⫽ 145) versus controls (n ⫽ 289).
a
b
peripheral NK cells may be protective against liver disease progression [26]. NK cells likely play pivotal roles in immune responses against HCV infection and KIRs are the major group of receptors that modulate NK function. It has been observed that KIR genes and HLA–KIR interactions can influence disease susceptibility and various works suggest that NK activation enhances HCV clearance. An important association seems to involve the KIR2DL3 gene. Romero et al. [27] reported an association between the KIR2DL3/2DL3 genotype and its ligand HLA-C1C1 with spontaneous HCV clearance. Khakoo et al. [13] also suggested that the genotypic combination of KIR2DL3 with HLA-C1 was protective in terms of spontaneous clearance of the virus in individuals infected with low-dose viral inoculums. They hypothesized that this KIR–ligand pair could be protective and improve resistance to viral infection because it has a weak inhibitory function that could be easily overridden by signals through activating receptors upon appropriate ligand binding, resulting in lysis of the target. In the hierarchy of KIR2DL molecule–mediated inhibition, KIR2DL3/HLA-C1C1 appears to confer weaker inhibitory responses, whereas KIR2DL1 with C2C2 appears to have the highest inhibitory power [28].
Table 4 KIR genotypes and KIR-HLA ligand frequencies in patients chronically infected by the HCV-1 genotype KIR genotypes and KIR with HLA ligands
F3–F4a n (f)
F0 –F2a n (f)
p
KIR2DL2/2DL2 KIR2DL2/2DL3 KIR2DL3/2DL3b KIR2DL2 ⫹ HLA-C1 KIR2DL3 ⫹ HLA-C1 KIR2DL1 ⫹ HLA-C2 KIR3DL1 ⫹ HLA-Bw4 KIR3DL2 ⫹ HLA-A3 and ⫺ A11 KIR2DS1 ⫹ HLA-C2 KIR2DS2 ⫹ HLA-C1 KIR2DL2/2DL2 ⫹ HLA-C1 KIR2DL2⫹KIR2DL3 ⫹ HLA-C1 KIR2DL3/2DL3 ⫹ HLA-C1 KIR2DL2/2DL2 ⫹ HLA-C1C1 KIR2DL2⫹2DL3 ⫹ HLA-C1C1 KIR2DL3/2DL3 ⫹ HLA-C1C1
5 (0.15) 27 (0.45) 27 (0.45) 25 (0.41) 45 (0.75) 45 (0.75) 32 (0.53) 18 (0.30) 17 (0.28) 25 (0.41) 4 (0.12) 21 (0.35) 24 (0.43) 1 (0.03) 7 (0.11) 4 (0.06)
6 (0.16) 30 (0.35) 52 (0.62) 28 (0.32) 57 (0.67) 59 (0.69) 49 (0.57) 16 (0.18) 24 (0.28) 32 (0.37) 6 (0.16) 22 (0.25) 35 (0.41) 2 (0.05) 10 (0.11) 13 (0.15)
1.000 0.314 0.070 0.368 0.397 0.583 0.729 0.172 0.862 0.752 0.739 0.318 0.910 0.999 0.990 0.1911
a F3–F4, advanced fibrosis or cirrhosis (n ⫽ 60); F0 –F2,without fibrosis or mild fibrosis (n ⫽ 85) according to the Metavir score. b OR ⫽ 0.51, 95% CI ⫽ 0.25–1.02.
Khakoo et al. [13] also suggested that KIR2DL2 seems to counteract KIR2DL3/HLA-C1C1 protection once they determined that only individuals homozygous for KIR2DL3, and not KIR2DL2/ KIR2DL3 heterozygotes or KIR2DL2 homozygotes, were associated with HCV clearance. Nonetheless, another study reported an association of KIR2DL2 with viral clearance [29]. A recent study with intravenous drug users observed a significant association between the presence of the inhibitory receptors KIR2DL2 and/or KIR2DL3 and the HLA-C1 homozygous genotype together with the activating receptor KIR2DS4 in protection from HCV infection [30]. In the present study patients were carefully selected to form a homogeneous group in terms of HCV genotype and absence of viral coinfections. The data did not indicate any significant association between inhibitory or activating KIR genes and susceptibility to HCV infection, progression to fibrosis, or cirrhosis. Nonetheless, the frequency of the genotype KIR2DL3/KIR2DL3 was higher in the F0 –F2 group (62.0%) compared with the F3–F4 group (45.4%). Kakhoo et al. [13] attempted to explain the finding of protection of KIR2DL3/KIR2DL3 genotype based on the clonal expression of KIR on NK cells. Within an individual, a KIR gene is expressed on some, but not all NK cell clones [28]. In this way, homozygotes for KIR2DL3 will have more NK cells solely under the inhibitory control of KIR2DL3 than will KIR2DL2/KIR2DL3 heterozygotes [13]. Although the statistical power of the study was insufficient to confirm this association, the result might indicate that the KIR2DL3 homozygote genotype can be important not only for spontaneous HCV clearance, as previously observed, but also for protection against liver disease injury and is deserving of further investigation. With respect to the distribution of HLA-C ligands, HLA-C2 was more frequent in patients than in healthy controls, suggesting that the presence of HLA-C2 is associated with susceptibility to infection by the HCV-1 genotype. Because KIR2DL1 binds the C2 HLA ligand, it is possible that this very strong inhibitory interaction could account, in part, for the susceptibility to HCV-1 genotype infection, and the HLA ligand genotype could determine the relative effect of the KIR genotype. Accordingly, Khakoo et al. [13] reported a higher frequency of HLA-C2C2 genotype among patients with persistent infection and a higher frequency of HLA-C1C1 in individuals with resolved infection. Additionally, Lopez-Vazquez et al. [15] reported a positive association between HLA-C2 homozygosis and clinical evolution to hepatocellular carcinoma in patients with chronic HCV infection. According to the authors, the associations with HLA class I ligands may have an effect on the physiopathology of HCV because
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of the capacity of these molecules to present antigens that have different affinities for cytotoxic T lymphocytes, which may also be related to their ability to interact with KIR receptors. In this study, an association was observed only with the HLA-C2, but not with the HLA-C1 or HLA-C2C2 genotype. The heterozygote genotype HLA-C1C2 was more frequent in individuals with advanced fibrosis or cirrhosis compared with patients without fibrosis or mild fibrosis and was also more frequent in cirrhotic than in noncirrhotic patients. As previously discussed, HLA-C1C2 heterozygosis may have a worse effect on the physiopathology of HCV because of the ability of these molecules to interact with more inhibitory KIR receptors, leading to a higher NK hyporesponsiveness. In other words, HLA-C1C2 patients will probably have a higher proportion of NK cells that will be inhibited by KIR2DL1 [13]. With respect to the HLA-Bw4 ligands, we observed a trend toward a higher frequency of Bw4-80Ile in patients without liver cirrhosis. Recently, Lopez-Vazquez et al. [15] reported that the HLA-B Bw4-80Ile epitope and the KIR3DS1 gene had a protective effect against the development of hepatocellular carcinoma in patients infected with HCV, especially in those with minimal liver damage. In contrast, Paladino et al. [14] reported an increased frequency of 2 copies of KIR3DS1 and HLA-Bw4 in cirrhotic patients infected with HCV. 5. Conclusion A significant association with KIR genes to cirrhosis progression in patients with chronic HCV was not observed, although the KIR2DL3/KIR2DL3 genotype was more frequent in F0 –F2 patients, HLA-C2 was more frequent in patients than healthy controls, HLA B Bw480Ile was more frequent in noncirrhotic patients, and the HLA C1C2 genotype was more frequent in F3–F4 and F4 patients. NK cells and KIR genes may contribute to the development of liver damage in HCV-1 chronically infected patients, although further investigations in other homogeneous patient samples must be performed to confirm our data. Acknowledgments The authors thank all volunteers who took part in this study. This work was supported in part by Capes, CNPq, and The Brazil Health Ministry. References [1] Orange JS, Fassett MS, Koopman LA, Boyson JE, Strominger JL. Viral evasion of natural killer cells. Nat Immunol 2002;3:1006 –12. [2] Yamagiwa S, Kamimura H, Ichida T. Natural killer cell receptors and their ligands in liver diseases. Med Mol Morphol 2009;42:1– 8. [3] Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nat Rev Immunol 2001;1:41–9. [4] Uhrberg M, Valiante NM, Shum BP, Shilling HG, Lienert-Weidenbach K, Corliss B, et al. Human diversity in killer cell inhibitory receptor. Genes Immun 1997;7:753– 63. [5] Hsu KC, Liu XR, Selvakumar A, Mickelson E, O’Reilly RJ, Dupont B. Killer Ig-like receptor haplotype analysis by gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes, each with multiple subsets. J Immunol 2002;169:5118 –129. [6] Martin AM, Freitas EM, Witt CS, Christiansen FT. The genomic organization and evolution of the natural killer immunoglobulin-like receptor (KIR) gene cluster. Immunogenetics 2000;51:268 – 80.
[7] Winter CC, Gumperz JE, Parham P, Long EO, Wagtmann N. Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. J Immunol 1998;161:571–7. [8] Colonna M, Borsellino G, Falco M, Ferrara GB, Strominger JL. HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells. Proc Natl Acad Sci U S A 1993;90:12000 – 4. [9] Wagtmann N, Rajagopalan S, Winter CC, Peruzzi M, Long EO. Killer cell inhibitory receptors specific for HLA-C and HLA-B identified by direct binding and by functional transfer. Immunity 1995;3:801–9. [10] O’Connor GM, Guinan KJ, Cunningham RT, Middleton D, Parham P, Gardiner CM. Functional polymorphism of the KIR3DL1/S1 receptor on human NK cells. J Immunol 2007;178:235. [11] Martin MP, Gao XJ, Lee JH, Nelson GW, Detels R, Goedert JJ, et al. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 2002;31:429 –34. [12] Mu¨ller CA, Engler-Blum G, Gekeler V, Steiert I, Weiss E, Schmidt H. Genetic heterogeneity of the HLA-Bw4 and -Bw6 specificities lmmunogenetics 1989; 30: 200 –7. [13] Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, Astemborski J, et al. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 2004;305:872– 4. [14] Paladino N, Flores AC, Marcos CY, Fainboim H, Theiler G, Arruvito L, et al. Increased frequencies of activating natural killer receptors are associated with liver injury in individuals who do not eliminate hepatitis C virus. Tissue Antigens 2007;69 (suppl 1):109 –11. [15] Lopez-Vazquez A, Rodrigo L, MartÎnez-Borra J, PÊrez R, RodrÎguez M, FdezMorera JL, et al. Protective effect of the HLA-Bw4I80 epitope and the killer cell immunoglobulin-like Receptor 3DS1 gene against the development of hepatocellular carcinoma in patients with hepatitis C virus infection. J Infect Dis 2005;192:162–5. [16] Thomas DL, Seeff LB. Natural history of hepatitis C. Clin Liver Dis 2005;9: 383–98. [17] Rudnick CCC, Franceschi DSA, Marangon AV, Guelsin GAS, Sell AM, Visentainer JEL. Killer cell immunoglobulin-like receptor gene diversity in a Southern Brazilian population from the state of ParanÂ. Hum Immunol 2008;69:872– 6. [18] Bedossa P. Presentation of a grid for computer analysis for complication of hispathologic lesions in chronic viral hepatitis C. Ann Pathol 1993;13:260 –5. [19] Fulton RJ, Mc Dade RL, Smith PL, Kienker LJ, Kettman JR. Advanced multiplexed analysis with the FlowMetrix娃 system. Clin Chem 1997;43:1749 –56. [20] Maxwell LD, Wallace A, Middleton D, Curran MD. A common KIR2DS4 deletion variant in the human that predicts a soluble KIR molecule analogous to the KIR1D molecule observed in the rhesus monkey. Tissue Antigens 2002;60: 254 – 8. [21] Martin MP. Tipagem de genes KIR e sua aplicaÈÄo em estudos de populaÈÖes, associaÈÄo com doenÈas e transplantes. In: VIII Congresso da Sociedade Brasileira de Transplante De Medula ssea, 2004. Curitiba, ParanÂ, Brazil. [22] Excoffier LG, Laval G, Schneider S. Arlequin Ver. 3.0: An integrated software package for population genetics data analysis. Evol Bioinform Online 2005;1: 47–50. [23] Uhrberg M. The KIR gene family: life in the fast lane of evolution. Eur J Immunol 2005;35:10 –5. [24] Martin AM, Kulski JK, Gaudieri S, et al. Comparative genomic analysis, diversity and evolution of two KIR haplotypes A and B. Gene 2004;335:121–31. [25] Crispe IN, Mehal WZ. Strange brew: T cells in the liver. Immunol Today 1996; 17:522–5. [26] Morishima C, Paschal DM, Wang CC, Yoshihara CS, Wood BL, Yeo AET, et al. Decreased NK cell frequency in chronic hepatitis C does not affect ex vivo cytolytic killing. Hepatology 2006;43:573– 80. [27] Romero V, Azocar J, ZÛÒiga J, Clavijo OP, Terreros D, Gu X, et al. Interaction of NK inhibitory receptor genes with HLA-C and MHC class II alleles in hepatitis C virus infection outcome. Mol Immunol 2008;45:2429 –36. [28] Kulkarni S, Martin MP, Carrington M. The yin and yang of HLA and KIR in human disease. Semin Immunol 2008;20:343–52. [29] Montes-Cano MA, Caro-Oleas JL, Romero-GÔmez M, Diago M, Andrade R, carmona I, et al. HLA-C and KIR genes in hepatitis C virus infection. Hum Immunol 2005;66:1106 –9. [30] ZÛÒiga J, Romero V, Azocar J, Terreros D, Vargas-Rojas MI, Torres-GarcÎa D, et al. Protective KIR–HLA interactions for HCV infection in intravenous drug users. Mol Immunol 2009;46:2723–7.
Contents lists available at SciVerse ScienceDirect
KIR genes and their human leukocyte antigen ligands in the progression to cirrhosis in patients with chronic hepatitis C Amanda Vansan Marangon a,*, Giovanni Faria Silva b, Camila Fernanda Verdichio de Moraes c, Rejane Maria Tomasini Grotto c, Maria InËs de Moura Campos Pardini c, Dayse Sousa de Pauli d, Ana Maria Sell a, Jeane Eliete Laguila Visentainer a, Ricardo Alberto Moliterno a a
Immunogenetics Laboratory, Department of Basic Sciences, Maringà State University, UEM, Maringà, Paranà, Brazil Gastroenterology Division, Internal Medicine Department, Botucatu Medical School, Sâo Paulo State University, UNESP, Botucatu, Sâo Paulo, Brazil Molecular Biology Laboratory of Blood Transfusion Center, Botucatu Medical School, Sâo Paulo State University, UNESP, Botucatu, Sâo Paulo, Brazil d Londrina Clinical Hospital, Londrina State University, UEL, Londrina, Paranà, Brazil b c
A R T I C L E
I N F O
Article history: Received 5 January 2011 Accepted 25 August 2011 Available online 28 August 2011
Keywords: NK cells HLA KIR Hepatitis C Liver fibrosis
A B S T R A C T
Natural killer (NK) cells play pivotal roles in immune responses against infection with viruses, such as hepatitis C virus (HCV), and killer cell immunoglobulin-like receptors (KIRs) are related to the activation and inhibition of NK cells. The aim of this study was to investigate the possibility that KIR genes and their human leukocyte antigen (HLA) ligands influence progression to cirrhosis in patients infected with genotype 1 of HCV. A total of 145 Brazilian patients with confirmed chronic hepatitis C grouped from F0 to F4 according to fibrosis progression to cirrhosis were evaluated. Genotyping of KIR and HLA genes was performed by polymerase chain reaction with sequence-specific oligonucleotide probes. The HLA-C2 KIR ligand was more frequent in patients than in healthy controls (74.5% vs 64.3%, p ⫽ 0.04, odds ratio (OR) ⫽ 1.6, 95% confidence interval (CI) ⫽ 1.03–2.52). Moreover, the HLA-C1C2 genotype was more frequent in patients with advanced fibrosis or cirrhosis (F3–F4 group) than in patients in the F0 –F2 group (61.6% vs 44.7%, p ⫽ 0.06) and in the F4 group compared with the F0 –F3 group (65.7% vs 46.7%, p ⫽ 0.05, OR ⫽ 2.19, 95% CI ⫽ 1.01– 4.73). NK and KIR ligands may contribute to the development of liver damage in patients chronically infected by HCV. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Natural killer cells (NK) are crucial components of the innate response and provide defense mainly against viral infections and cancer. They also provide a link to priming of adaptive immunity. The function of NK cells is regulated by a fine balance of inhibitory and activating signals, which are mediated by a diverse array of cell-surface receptors including killer cell immunoglobulin-like receptors (KIR). These receptors can either inhibit or activate NK cells and the difference between them lies mainly in their intracytoplasmatic tail [1–3]. In humans, KIRs are encoded by a cluster of genes located in the leukocyte receptor complex on chromosome 19q13.4. At least 14 KIR genes have thus far been characterized, of which 8 are NK cell inhibitors (KIR2DL1, ⫺2DL2, ⫺2DL3, ⫺2DL4, ⫺2DL5, ⫺3DL1, ⫺3DL2, and ⫺3DL3), 6 are activating (KIR2DS1, -2DS2, -2DS3, -2DS4, -2DS5, and -3DS1), and 2 are pseudogenes (KIR2DP1 and -3DP1) [4,5]. Based on the genetic content and pattern of segregation at the population level, KIR haplotypes are divided into 2 groups, A and B, which differ in the number and kind of KIR genes. Common to both * Corresponding author. E-mail address: [email protected] (A.V. Marangon).
groups of haplotypes are the “framework genes”: KIR3DL2, -3DP1, -2DL4, and -3DL3 [4 – 6]. KIRs specifically recognize some human leukocyte class I antigens (HLA-I) present on target cells. HLA-Bw4 and distinct allotypes of HLA-C (C1 and C2 groups) are the predominant ligands for most KIRs [7]. The HLA-C alleles were classified as C1 or C2 KIR ligand groups, depending upon 2 amino acid positions encoded in exon 2. The HLA-C1 allotypes contain serine at position 77 and asparagine at position 80 and are ligands for the inhibitory receptors KIR2DL2 and KIR2DL3. The HLA-C2 allotypes contains asparagine and lysine at those positions and are ligands for the inhibitory receptor KIR2DL1 and for the activating receptor KIR2DS1 [8,9]. HLA-Bw4 allotypes are characterized by at least 5 different patterns of amino acid exchanges at positions 77 and 80 – 83 and serve as ligands for KIR3DL1. In addition, HLA-A3 and HLA-A11 are ligands of KIR3DL2 [10 –12]. Accumulated evidence suggests that NK cells play an important role not only in the host defense against pathogens in the liver but also in liver injury and repair [2,3]. Because of the important role of KIR genes in the control of NK cell activation, investigations have been conducted on different populations to evaluate the associations between KIR and liver diseases such as viral hepatitis [13–15].
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.08.017
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Most patients with hepatitis C virus (HCV) infection progress to chronic disease, which can lead to liver fibrosis and the subsequent occurrence of cirrhosis and hepatocellular carcinoma [16]. However, the mechanisms responsible for the onset and progression of hepatic lesions during chronic hepatitis C are not fully understood. Hence, the aim of the present study was to investigate the possible association of KIR genes and their HLA ligands with fibrosis progression to cirrhosis in patients with chronic hepatitis C.
certain amino acid in the adequate positions and classified into C1, C2, and Bw4 groups. The C1 group of alleles observed in patients were C*01, C*03, C*07, C*08, C*12, C*14, C*15:07, and C*16:01. The C2 group of alleles were C*02, C*04, C*05, C*06, C*07:07, C*12:05, C*15, C*16:02, C*17, and C*18. The group of alleles that belongs to the Bw4 group were B*13, B*27:05, B*27:09, B*37:01, B*38, B*44, B*47, B*49, B*51, B*52, B*53, and B*58. Only alleles observed in the patients were described.
2. Subjects and methods
2.6. KIR2DS4
2.1. Studied population
The 2 major full-length subtype and deleted forms of KIR2DS4 [20] were also analyzed by PCR with sequence-specific oligonucleotide probe protocols. The samples with doubtful results were confirmed by in-house PCR with sequence specific primers according to Martin [21] and optimized by Rudnick et al. [17].
Between September 2004 and January 2009 a total of 761 patients received a diagnosis of HCV infection at the Internal Medicine Department, Gastroenterology Division, Botucatu Medical School. Of these, 145 unrelated patients with infection only by the HCV genotype 1 were included in this study. A total of 616 patients were excluded based on the following criteria: human immunodeficiency virus–positive serology, hepatitis B virus coinfection, other hepatic diseases, presence of non-1 HCV genotype, and history of antiviral therapy before liver biopsy. In addition, 289 unrelated healthy individuals from Southern Brazil [17] were used as controls and compared with HCV patients. The studied protocol was approved by the Ethics Committee for Research Involving Human Beings of SÄo Paulo State University and all subjects provided written consent to participate in the study. 2.2. Liver biopsy Biopsies in all patients were carried out using the technique of Menghini or a Tru-Cut needle to determine the degree of fibrosis and progression of infection. The fragments were analyzed when at least 8 portal spaces were present and stained with hematoxylin and eosin, Masson trichrome, and reticulin stain. The hepatic biopsies were analyzed by a pathologist using the Metavir score: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis and few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis [18]. The patients were stratified into subgroups according to fibrosis stage: absence of fibrosis or mild fibrosis (F0 –F2, n ⫽ 85), advanced fibrosis or cirrhosis (F3–F4, n ⫽ 60), without cirrhosis (F0 –F3), and with cirrhosis (F4). 2.3. HCV genotyping The HCV genotype was defined by the reverse line probe assay (Innolipa v. 1.0, Innogenetics, Ghent, Belgium) according to the manufacturer’s instructions. Genotyping was preceded by the extraction of HCV viral RNA present in the patient’s plasma, followed by reverse-transcription polymerase chain reaction (PCR; Amplicor HCV test, v. 2.0, Roche Diagnostic Systems, Branchburg, NJ) according to the manufacturer’s instructions. 2.4. DNA extraction Genomic DNA was isolated from 250 L of peripheral blood using a NeoIsocolumn kit (One Lambda, Inc., San Diego, CA) or purelink (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. 2.5. KIR and HLA-I genotyping KIR (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, KIR2DP1, and KIR3DP1) and HLA-I (HLA-A, -B, and -C) genes were genotyped according to the manufacturer’s instructions by PCR with sequence-specific oligonucleotide probe protocols using commercial KIR and HLA genotyping test kits (One Lambda, Inc.) [19]. The sequences of all alleles were analyzed one by one in the HLA database (http://www.ebi.ac.uk/imgt/hla/) by the presence of a
2.7. Statistical analysis KIR and KIR-HLA ligand frequencies were obtained by direct counting. The 2 test with Yates’ correction or Fisher’s exact tests was applied to compare the distribution of the frequencies of alleles and genes between the groups. Odds ratios (OR) with 95% confidence intervals (CI) were also calculated. The analyses were carried out using SISA online statistics (http://www.quantitativeskills.com/sisa/). p values ⬍0.05 were considered statistically significant. Because the gametic phase of the alleles from the different loci is not known (segregation analysis into families was not carried out), haplotypes and the calculation of their frequency were determined by the likelihood-ratio test using the Arlequin statistical software program according to Excoffier et al. [22]. 3. Results 3.1. Patient characteristics Patient characteristics are summarized in Table 1. According to the Metavir score, 58.6% of patients presented no or mild fibrosis (4.8% were F0, 29.7% F1, and 24.1% F2) and 41.8% presented advanced fibrosis or cirrhosis (15.2% were F3 and 26.2% F4). The mean age of patients with advanced fibrosis or cirrhosis was higher than that of patients without fibrosis or mild fibrosis (48.6 ⫾ 9.8 years vs 39.8 ⫾ 9.3 years, respectively, p ⬍ 0.05). The same trend was seen when comparing the F4 group versus the F0 –F3 group (27.8 ⫾ 9.6 years vs 19.9 ⫾ 7.0 years, respectively, p ⬍ 0.05). With respect to possible risk factors for acquiring hepatitis C, the use of intravenous drugs was the main risk factor associated with the F0 –F2 group (25.8%) compared with only 2 patients (3.3%) in the F3–F4 group (p ⬍ 0.05%). The use of intravenous drugs was also the main risk factor associated with the F0 –F3 group compared with the F4 group (20.5% vs 5.3%, p ⬍0.05). 3.2. KIR genes and HLA ligand frequencies To evaluate the role of KIR genes and their HLA ligands in disease progression, patients were first stratified into individuals with absence of fibrosis or mild fibrosis (F0 –F2, n ⫽ 85) and individuals with advanced fibrosis or cirrhosis (F3–F4, n ⫽ 60) Next, patients with cirrhosis (F4 stage, n ⫽ 38) were separated and compared with patients without cirrhosis (F0 –F3 stage, n ⫽ 107). Furthermore, all patients were compared with unrelated healthy controls. The HLA epitopes (HLA-C1, -C2, -Bw4 and -A3/A11) were analyzed in all groups. The gene and genotype frequencies of the HLA ligands are presented in Table 2. HLA-C2 was more frequent in patients than in healthy controls (74.5% vs 64.3%, p ⫽ 0.04, OR ⫽ 1.6, 95% CI ⫽ 1.03–2.52). The heterozygote genotype HLA-C1C2 exhibited a trend of being more frequent in the F3–F4 group compared with the F0 –F2 group (61.6% vs 44.7%; p ⫽ 0.06, OR ⫽ 1.98, 95% CI ⫽ 1.02–3.9). The HLA-C1C2 genotype was also more frequent in patients with liver cirrhosis (F4) compared with patients without
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Table 1 Clinical and general features of patients with chronic hepatitis C attended in the Department of Internal Medicine, Gastroenterology Division, Botucatu Medical School, from September 2004 to January 2009 (n ⫽ 145) Characteristics
Patients (n ⫽ 145)
F0 –F2 (n ⫽ 85)
F3–F4 (n ⫽ 60)
F4 (n ⫽ 38)
p
Age (mean ⫾ SD)a Gender, n (%) Male Female Risk factor, n (%) Blood transfusion Intravenous druga Gluconergan Nasal drug Othersb Duration of infectionc (mean ⫾ SD) METAVIR stage,d n (%) F0 F1 F2 F3 F4
43.4 ⫾ 10.4
39.8 ⫾ 9.3
48.6 ⫾ 9.8
50.0 ⫾ 9.53
⬍0.05
112 (0.77) 33 (0.22)
67 (0.78) 18 (0.21)
45 (0.75) 15 (0.25)
17 (0.71) 11 (0.28)
36 (0.24) 24 (0.16) 22 (0.15) 5 (0.03) 58 (0.40) 21.8 ⫾ 8.3
17 (0.20) 22 (0.25) 10 (0.11) 2 (0.02) 34 (0.40) 19.0 ⫾ 6.9
19 (0.31) 2 (0.03) 12 (0.20) 3 (0.05) 24 (0.40) 25.7 ⫾ 8.7
10 (0.26) 2 (0.05) 6 (0.15) 3 (0.08) 17 (0.44) 27.8 ⫾ 9.6
0.15 ⬍0.05 0.25 0.64 0.97 0.10
7 (0.04) 43 (0.29) 35 (0.24) 22 (0.15) 38 (0.26)
7 (0.08) 43 (0.50) 35 (0.41) — —
— — — 22 (0.36) 38 (0.63)
— — — — 38 (1.00)
— — — — —
0.73 0.73
p ⬍ 0.05 when comparing F0 –F2 with F3–F4 and F0-F3 with F4. Tattoo, surgeries, occupational exposure, homosexuals or promiscuous sexual behavior, and unknown risk factors. c Duration of infection was calculated only for 90 patients (F0 ⫽ 4, F1 ⫽ 25, F2 ⫽ 24, F3 ⫽ 16, and F4 ⫽ 21). The duration of infection for 55 patients is unknown. d Metavir score: F0, no fibrosis; F1, portal fibrosis without septa; F2, portal fibrosis and few septa; F3, numerous septa without cirrhosis; and F4, cirrhosis. a
b
cirrhosis (F0 –F3; 65.7% vs 46.7%, p ⫽ 0.05, OR ⫽ 2.19, 95% CI ⫽ 1.0 – 4.7). HLA-Bw4 molecules were divided into 2 groups based on whether isoleucine or threonine was present at position 80 (Bw480Ile and Bw4-80Thr, respectively). The Bw4-80Ile exhibited a trend of being less frequent in the group with liver cirrhosis (F4) compared with patients without cirrhosis (39.5% vs 56.1%, p ⫽ 0.09, OR ⫽ 0.51, 95% CI ⫽ 0.24 –1.0; Table 2). With respect to KIR genes, no association between the 16 KIR genes and susceptibility to HCV, progression to fibrosis, or cirrhosis was observed (Table 3). Nevertheless, a lower frequency of the homozygote genotype KIR2DL3/KIR2DL3 was observed in the F3–F4 group (45.4%) compared with the F0 –F2 group (62.0%; Table 4). The deleted variant of KIR2DS4 was the most frequent form in all groups, but the distribution of the KIR2DS4 forms (the full-length gene, the deletion variant, or both) was similar in all groups. In analysis of the interactions between activating and inhibitory KIR and HLA epitopes (KIR2DL1 and its ligand C2; KIR2DL2 with C1; KIR2DL3 with C1; KIR3DL1 with Bw4; KIR3DL1 with Bw4-80Ile; KIR3DL1 with Bw4-80Thr; KIR3DL2 with HLA-A3 and -A11; KIR2DS1 with C2; KIR2DS2 with C1; KIR3DS1 with Bw4; KIR3DS1 with Bw480Ile; and KIR3DS1 with Bw4-80Thr), none of the receptor–ligand
pairs was associated with susceptibility or progression of disease (Table 4). Furthermore, patients were identified as KIR AA, AB, or BB haplotype based on the multiple KIR genes they possessed. KIR BB haplotypes were characterized by the presence of one or more of the following genes: KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5, and KIR3DS1. Conversely, KIR AA haplotypes were characterized by both the absence of any 1 of the preceding genes and the presence of the genes KIR3DL3, KIR2DL3, KIR2DL1, KIR3DP1, KIR2DL4, KIR3DL1, KIR2DS4, and KIR3DL2. The AB haplotype was characterized by the presence of genes of the 2 groups [4,23,24]. The KIR AA haplotype was the most frequent haplotype in all groups (33.1% of all patients, 39.2% in the F3–F4 group, 33.3% in the F0 –F2 group, 34.2% in the F4 group, 32.7% in the F0 –F3 group, and 33.2% in unrelated healthy controls). No significant difference in the distribution of KIR haplotypes was observed. 4. Discussion NK cells represent a fundamental component of the innate immune system. The repertoire of NK cells is extensive in the liver [25] and cytolytic activity seems to have an inverse association with liver fibrosis stage, suggesting that the presence of cytolytic active
Table 2 Distribution of gene and genotype frequencies of KIR-HLA ligands in controls and in patients chronically infected by the HCV-1 genotype, classified according to their liver biopsies Metavir score HLA ligands
A3 and A11 Bw4 C1 C2b C1C1 C1C2c C2C2 Bw4-80Iled Bw4-80Thr
Metavir score groupsa F3–F4 n (f)
F0 –F2 n (f)
p
F4 n (f)
F0 –F3 n (f)
p
19 (0.31) 40 (0.66) 49 (0.81) 48 (0.80) 12 (0.20) 37 (0.61) 11 (0.18) 28 (0.46) 17 (0.28)
18 (0.21) 64 (0.85) 63 (0.74) 60 (0.70) 25 (0.29) 38 (0.44) 22 (0.25) 47 (0.55) 21 (0.24)
0.21 0.90 0.38 0.27 0.27 0.06 0.38 0.39 0.76
11 (0.29) 24 (0.63) 31 (0.81) 32 (0.84) 6 (0.15) 25 (0.65) 7 (0.18) 15 (0.39) 13 (0.34)
25 (0.23) 80 (0.74) 81 (0.75) 76 (0.71) 31 (0.29) 50 (0.46) 26 (0.24) 60 (0.56) 25 (0.23)
0.64 0.24 0.50 0.13 0.1 0.05 0.60 0.09 0.27
All patients
Controls
p
37 (0.25) 104 (0.71) 112 (0.77) 108 (0.74) 37 (0.25) 75 (0.51) 33 (0.22) 75 (0.51) 39 (0.26)
72 (0.24) 218 (0.75) 223 (0.77) 186 (0.64) 92 (0.31) 131 (0.45) 55 (0.19) 141 (0.48) 77 (0.26)
0.98 0.47 0.91 0.04 0.21 0.24 0.40 0.63 0.95
Metavir score: F3–F4, advanced fibrosis or cirrhosis (n ⫽ 60); F0 –F2, without fibrosis or mild fibrosis (n ⫽ 85); F4, with cirrhosis (n ⫽ 38); F0 –F3, without cirrhosis (n ⫽ 107). All patients (n ⫽ 145) versus controls (n ⫽ 289): odds ratios (OR) ⫽ 1.61; 95% confidence interval (CI) 1.03–2.52. c F3–F4 versus F0 –F2, OR ⫽ 1.98, 95% CI ⫽ 1.02–3.9; and F4 versus F0 –F3, OR ⫽ 2.19, 95% CI ⫽ 1.01– 4.7. d F4 versus F0 –F3: OR ⫽ 0.51, 95% CI ⫽ 0.24 –1.0. a
b
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Table 3 Distribution of KIR genes in controls and in patients chronically infected by the HCV-1 genotype, classified according to their liver biopsy Metavir score KIR genes
KIR2DL1 KIR2DL2 KIR2DL3 KIR2DL4 KIR2DL5 KIR2DP1 KIR2DS1 KIR2DS2 KIR2DS3 KIR2DS4 KIR2DS5 KIR3DL1 KIR3DL2 KIR3DL3 KIR3DP1 KIR3DS1
Metavir score groupsa F3–F4 n (f)
F0 –F2 n (f)
p
F4 n (f)
F0 –F3 n (f)
p
58 (0.96) 32 (0.53) 54 (0.90) 60 (1.00) 28 (0.46) 58 (0.96) 25 (0.41) 32 (0.53) 16 (0.26) 53 (0.88) 23 (0.38) 53 (0.88) 60 (1.00) 60 (1.00) 60 (1.00) 23 (0.38)
84 (0.98) 36 (0.42) 82 (0.96) 85 (1.00) 50 (0.59) 84 (0.98) 35 (0.41) 39 (0.46) 30 (0.35) 79 (0.93) 30 (0.35) 78 (0.92) 85 (1.00) 85 (1.00) 85 (1.00) 37 (0.44)
0.56 0.23 0.76 0.99 0.17 0.56 0.99 0.40 0.28 0.38 0.72 0.57 0.99 0.99 0.99 0.60
38 (1.00) 17 (0.45) 36 (0.95) 38 (1.00) 18 (0.47) 38 (1.00) 17 (0.45) 18 (0.47) 9 (0.24) 32 (0.84) 16 (0.42) 32 (0.84) 38 (1.00) 38 (1.00) 38 (1.00) 17 (0.45)
104 (0.97) 51 (0.48) 100 (0.93) 107 (1.00) 60 (0.56) 104 (0.97) 42 (0.39) 53 (0.50) 37 (0.35) 100 (0.93) 37 (0.35) 99 (0.93) 107 (1.00) 107 (1.00) 107 (1.00) 43 (0.40)
0.60 0.90 1.00 0.45 0.46 0.60 0.68 0.96 0.29 0.16 0.52 0.24 0.45 0.76 0.52 0.24
All patientsb
Controlsb
p
143 (0.99) 68 (0.47) 134 (0.92) 145 (1.00) 78 (0.54) 142 (0.98) 59 (0.41) 71 (0.49) 46 (0.32) 132 (0.91) 53 (0.37) 131 (0.90) 145 (1.00) 145 (1.00) 145 (1.00) 60 (0.41)
281 (0.97) 136 (0.47) 258 (0.89) 289 (1.00) 152 (0.53) 280 (0.97) 118 (0.41) 136 (0.47) 77 (0.27) 271 (0.94) 100 (0.35) 272 (0.94) 289 (1.00) 289 (1.00) 289 (1.00) 113 (0.39)
0.56 0.94 0.38 0.21 0.89 0.75 0.93 0.78 0.31 0.39 0.76 0.21 0.80 0.80 0.80 0.72
Metavir score: F3–F4, advanced fibrosis or cirrhosis (n ⫽ 60); F0 –F2, without fibrosis or mild fibrosis (n ⫽ 85); F4, with cirrhosis (n ⫽ 38); F0 –F3, without cirrhosis (n ⫽ 107). All patients (n ⫽ 145) versus controls (n ⫽ 289).
a
b
peripheral NK cells may be protective against liver disease progression [26]. NK cells likely play pivotal roles in immune responses against HCV infection and KIRs are the major group of receptors that modulate NK function. It has been observed that KIR genes and HLA–KIR interactions can influence disease susceptibility and various works suggest that NK activation enhances HCV clearance. An important association seems to involve the KIR2DL3 gene. Romero et al. [27] reported an association between the KIR2DL3/2DL3 genotype and its ligand HLA-C1C1 with spontaneous HCV clearance. Khakoo et al. [13] also suggested that the genotypic combination of KIR2DL3 with HLA-C1 was protective in terms of spontaneous clearance of the virus in individuals infected with low-dose viral inoculums. They hypothesized that this KIR–ligand pair could be protective and improve resistance to viral infection because it has a weak inhibitory function that could be easily overridden by signals through activating receptors upon appropriate ligand binding, resulting in lysis of the target. In the hierarchy of KIR2DL molecule–mediated inhibition, KIR2DL3/HLA-C1C1 appears to confer weaker inhibitory responses, whereas KIR2DL1 with C2C2 appears to have the highest inhibitory power [28].
Table 4 KIR genotypes and KIR-HLA ligand frequencies in patients chronically infected by the HCV-1 genotype KIR genotypes and KIR with HLA ligands
F3–F4a n (f)
F0 –F2a n (f)
p
KIR2DL2/2DL2 KIR2DL2/2DL3 KIR2DL3/2DL3b KIR2DL2 ⫹ HLA-C1 KIR2DL3 ⫹ HLA-C1 KIR2DL1 ⫹ HLA-C2 KIR3DL1 ⫹ HLA-Bw4 KIR3DL2 ⫹ HLA-A3 and ⫺ A11 KIR2DS1 ⫹ HLA-C2 KIR2DS2 ⫹ HLA-C1 KIR2DL2/2DL2 ⫹ HLA-C1 KIR2DL2⫹KIR2DL3 ⫹ HLA-C1 KIR2DL3/2DL3 ⫹ HLA-C1 KIR2DL2/2DL2 ⫹ HLA-C1C1 KIR2DL2⫹2DL3 ⫹ HLA-C1C1 KIR2DL3/2DL3 ⫹ HLA-C1C1
5 (0.15) 27 (0.45) 27 (0.45) 25 (0.41) 45 (0.75) 45 (0.75) 32 (0.53) 18 (0.30) 17 (0.28) 25 (0.41) 4 (0.12) 21 (0.35) 24 (0.43) 1 (0.03) 7 (0.11) 4 (0.06)
6 (0.16) 30 (0.35) 52 (0.62) 28 (0.32) 57 (0.67) 59 (0.69) 49 (0.57) 16 (0.18) 24 (0.28) 32 (0.37) 6 (0.16) 22 (0.25) 35 (0.41) 2 (0.05) 10 (0.11) 13 (0.15)
1.000 0.314 0.070 0.368 0.397 0.583 0.729 0.172 0.862 0.752 0.739 0.318 0.910 0.999 0.990 0.1911
a F3–F4, advanced fibrosis or cirrhosis (n ⫽ 60); F0 –F2,without fibrosis or mild fibrosis (n ⫽ 85) according to the Metavir score. b OR ⫽ 0.51, 95% CI ⫽ 0.25–1.02.
Khakoo et al. [13] also suggested that KIR2DL2 seems to counteract KIR2DL3/HLA-C1C1 protection once they determined that only individuals homozygous for KIR2DL3, and not KIR2DL2/ KIR2DL3 heterozygotes or KIR2DL2 homozygotes, were associated with HCV clearance. Nonetheless, another study reported an association of KIR2DL2 with viral clearance [29]. A recent study with intravenous drug users observed a significant association between the presence of the inhibitory receptors KIR2DL2 and/or KIR2DL3 and the HLA-C1 homozygous genotype together with the activating receptor KIR2DS4 in protection from HCV infection [30]. In the present study patients were carefully selected to form a homogeneous group in terms of HCV genotype and absence of viral coinfections. The data did not indicate any significant association between inhibitory or activating KIR genes and susceptibility to HCV infection, progression to fibrosis, or cirrhosis. Nonetheless, the frequency of the genotype KIR2DL3/KIR2DL3 was higher in the F0 –F2 group (62.0%) compared with the F3–F4 group (45.4%). Kakhoo et al. [13] attempted to explain the finding of protection of KIR2DL3/KIR2DL3 genotype based on the clonal expression of KIR on NK cells. Within an individual, a KIR gene is expressed on some, but not all NK cell clones [28]. In this way, homozygotes for KIR2DL3 will have more NK cells solely under the inhibitory control of KIR2DL3 than will KIR2DL2/KIR2DL3 heterozygotes [13]. Although the statistical power of the study was insufficient to confirm this association, the result might indicate that the KIR2DL3 homozygote genotype can be important not only for spontaneous HCV clearance, as previously observed, but also for protection against liver disease injury and is deserving of further investigation. With respect to the distribution of HLA-C ligands, HLA-C2 was more frequent in patients than in healthy controls, suggesting that the presence of HLA-C2 is associated with susceptibility to infection by the HCV-1 genotype. Because KIR2DL1 binds the C2 HLA ligand, it is possible that this very strong inhibitory interaction could account, in part, for the susceptibility to HCV-1 genotype infection, and the HLA ligand genotype could determine the relative effect of the KIR genotype. Accordingly, Khakoo et al. [13] reported a higher frequency of HLA-C2C2 genotype among patients with persistent infection and a higher frequency of HLA-C1C1 in individuals with resolved infection. Additionally, Lopez-Vazquez et al. [15] reported a positive association between HLA-C2 homozygosis and clinical evolution to hepatocellular carcinoma in patients with chronic HCV infection. According to the authors, the associations with HLA class I ligands may have an effect on the physiopathology of HCV because
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of the capacity of these molecules to present antigens that have different affinities for cytotoxic T lymphocytes, which may also be related to their ability to interact with KIR receptors. In this study, an association was observed only with the HLA-C2, but not with the HLA-C1 or HLA-C2C2 genotype. The heterozygote genotype HLA-C1C2 was more frequent in individuals with advanced fibrosis or cirrhosis compared with patients without fibrosis or mild fibrosis and was also more frequent in cirrhotic than in noncirrhotic patients. As previously discussed, HLA-C1C2 heterozygosis may have a worse effect on the physiopathology of HCV because of the ability of these molecules to interact with more inhibitory KIR receptors, leading to a higher NK hyporesponsiveness. In other words, HLA-C1C2 patients will probably have a higher proportion of NK cells that will be inhibited by KIR2DL1 [13]. With respect to the HLA-Bw4 ligands, we observed a trend toward a higher frequency of Bw4-80Ile in patients without liver cirrhosis. Recently, Lopez-Vazquez et al. [15] reported that the HLA-B Bw4-80Ile epitope and the KIR3DS1 gene had a protective effect against the development of hepatocellular carcinoma in patients infected with HCV, especially in those with minimal liver damage. In contrast, Paladino et al. [14] reported an increased frequency of 2 copies of KIR3DS1 and HLA-Bw4 in cirrhotic patients infected with HCV. 5. Conclusion A significant association with KIR genes to cirrhosis progression in patients with chronic HCV was not observed, although the KIR2DL3/KIR2DL3 genotype was more frequent in F0 –F2 patients, HLA-C2 was more frequent in patients than healthy controls, HLA B Bw480Ile was more frequent in noncirrhotic patients, and the HLA C1C2 genotype was more frequent in F3–F4 and F4 patients. NK cells and KIR genes may contribute to the development of liver damage in HCV-1 chronically infected patients, although further investigations in other homogeneous patient samples must be performed to confirm our data. Acknowledgments The authors thank all volunteers who took part in this study. This work was supported in part by Capes, CNPq, and The Brazil Health Ministry. References [1] Orange JS, Fassett MS, Koopman LA, Boyson JE, Strominger JL. Viral evasion of natural killer cells. Nat Immunol 2002;3:1006 –12. [2] Yamagiwa S, Kamimura H, Ichida T. Natural killer cell receptors and their ligands in liver diseases. Med Mol Morphol 2009;42:1– 8. [3] Cerwenka A, Lanier LL. Natural killer cells, viruses and cancer. Nat Rev Immunol 2001;1:41–9. [4] Uhrberg M, Valiante NM, Shum BP, Shilling HG, Lienert-Weidenbach K, Corliss B, et al. Human diversity in killer cell inhibitory receptor. Genes Immun 1997;7:753– 63. [5] Hsu KC, Liu XR, Selvakumar A, Mickelson E, O’Reilly RJ, Dupont B. Killer Ig-like receptor haplotype analysis by gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes, each with multiple subsets. J Immunol 2002;169:5118 –129. [6] Martin AM, Freitas EM, Witt CS, Christiansen FT. The genomic organization and evolution of the natural killer immunoglobulin-like receptor (KIR) gene cluster. Immunogenetics 2000;51:268 – 80.
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