Killer immunoglobulin-like receptors (KIRs) are expressed on natural killer cells and some T-cell subsets and produce either activation or inhibitory signals upon binding with the appropriate human leucocyte antigen (HLA) ligand on target cells. Recent genetic association studies have implicated KIR genotype in the development of several inflammatory conditions. Ulcerative colitis (UC) is an inflammatory disorder of the colonic mucosa that results from an inappropriate activation of the immune system driven by host bacterial flora. We developed a polymerase chain reaction-sequence specific primer (SSP)-based assay to genotype 194 UC patients and 216 control individuals for 14 KIR genes, the HLA-Cw ligand epitopes of the KIR2D receptors and a polymorphism of the lectin-like-activating receptor NKG2D. Initial analysis found the phenotype frequency of KIR2DL2 and -2DS2 to be significantly increased in the UC cohort (P=0.030 and 0.038, respectively). Logistic regression analysis revealed a protective effect conferred by KIR2DL3 in the presence of its ligand HLA-Cw group 1 (P=0.019). These results suggest that KIR genotype and HLA ligand interaction may contribute to the genetic susceptibility of UC.
Ulcerative colitis (UC) is a common form of inflammatory bowel disease (IBD), affecting approximately one in 400 individuals in the UK population.1 UC is a complex polygenic condition, characterized by an inappropriate mucosal immune response within the gut to the commensal bacterial flora, leading to ulceration of the colonic mucosa.2 Genome-wide screening in UC has demonstrated replicated linkage to several chromosomal regions, including regions on chromosomes 123, 4 and 195, 6 termed IBD2 and IBD6, respectively. Within the IBD2 loci lies the lectin-like natural killer (NK) receptor gene NKG2D (KLRK1) located at 12p13.2–p12.3.7 NKG2D is expressed on NK cells, CD8 αβ T cells and γδ T cells.8 NKG2D produces activation signals via the adaptor molecule DAP109 when bound to its ligands. These include the major histocompatibility complex class I chain (MIC)-related genes MICA8 and MICB,10 and members of the UL16-binding proteins (ULBP)/retinoic acid early transcript 1 (RAET1) gene family.10, 11 Both the MIC genes (located on chromosome 6p21.312, 13) and ULBP/RAET1 genes (positioned at 6q2510) encode stress-inducible protein products, upregulated in malignant and virally infected cells and constitutively expressed within the epithelium of the gut.10, 11, 14, 15 The MIC-restricted Vδ1 subset of γδ T cells are the predominant lymphocyte present within intestinal epithelium. They require co-stimulation via NKG2D,8 suggesting that the interaction of NKG2D with its ligands may play a role in shaping the immune response within the gut. It has been proposed that this mechanism may also have an involvement in the development of inflammatory disorders of the gut. Investigations into the possible influence of MIC polymorphism in celiac disease, a gluten-sensitive enteropathy, suggest a causal role,16, 17, 18 although similar studies of IBD have produced conflicting results.19, 20, 21, 22 In contrast to the MIC genes, NKG2D displays limited polymorphism23 and has not previously been investigated in susceptibility to UC.
The killer immunoglobulin-like receptor (KIR) gene family is located within the IBD6 linkage region at chromosome 19q13.4. The family comprises 14 genes and two pseudogenes, although the number of loci varies on different KIR haplotypes. Only four loci are common to all KIR haplotypes (the so-called ‘framework’ genes KIR2DL4, -3DL1, -3DL3, and the pseudogene -3DP1), whereas the presence of remaining ‘non-framework’ loci can vary between haplotypes.24 KIR genes are also highly polymorphic, and their protein products display variegated expression on NK cells and some T-cell subsets.25, 26, 27, 28 KIR molecules interact with specific epitopes of human leucocyte antigen (HLA)-C and -B alleles, providing information on the HLA class I surface expression of target cells. Engagement of KIR with the appropriate HLA ligand induces either inhibitory or activating signalling depending on the presence of intracellular immunoregulatory tyrosine-based inhibitory motifs or immunoregulatory tyrosine-based activation motifs.
Both KIR and their HLA ligands display considerable genetic diversity and segregate independently from each other during meiosis, resulting in variation in possible KIR and HLA ligand combinations between individuals. Analysis of KIR/HLA ligand combination is increasingly of interest in the genetic study of inflammatory disorders, resulting in the discovery of KIR associations with psoriasis vulgaris,29, 30 psoriatic arthritis,31 scleroderma,32 rheumatoid arthritis33 and type I diabetes.34, 35 In addition, celiac disease has been linked to 19q13.4,36 where the KIR cluster is located, although genotype analysis of a limited number of KIR genes (KIR2DL1, -2DL2, -2DL3 and -2DL5) failed to detect an association.37
The aim of this study was to investigate the role of the functional and positional candidate genes encoding NKG2D, the KIRs and the HLA ligands of KIR in determining susceptibility to UC.
To assess the genetic contribution of the NK receptor gene NKG2D in UC, we genotyped for the Ala72 and Thr72 substitution in both UC patients and controls. The NKG2D single-nucleotide polymorphism (SNP) frequency did not differ between the two cohorts, with frequencies of 0.84 and 0.16 for Ala72 and Thr72, respectively. NKG2D genotype frequencies fell within Hardy–Weinberg distribution in both cohorts.
We also genotyped both cohorts for the presence or absence of all 14 KIR genes and their HLA-Cw ligand epitopes. The phenotype frequencies of non-framework KIR loci and HLA-Cw epitopes are displayed in Table 1. The framework genes KIR2DL4, -3DL2 and -3DL3 were present in all individuals, and presumably on both haplotypes in each individual. The genotype frequencies of HLA-Cw epitopes fell within Hardy–Weinberg distribution in both cohorts. Statistical analysis using Fisher's exact test showed that phenotype frequencies of KIR2DL2 and -2DS2 were significantly increased in the UC patients (Table 1 and Figure 1). None of the other loci differed significantly, although KIR2DL3 displayed a 7% decrease in frequency in the UC cohort (P=0.06).
We next performed logistic regression analysis of KIR phenotype (both in combination with, and independent of, their corresponding HLA-Cw ligands) to assess the impact of KIR gene and HLA-Cw ligand interaction on UC susceptibility, a known risk factor in several other inflammatory diseases.29, 30, 31, 32, 33, 35, 38 Analysis revealed KIR2DL3 in the presence of HLA-C1 as the dominant association, significantly reduced in UC patients (P=0.019, odds ratio (OR)=0.577, 95% confidence interval (CI)=0.364–0.915), and the loss of significance with KIR2DL2 and -2DS2 (Figure 1). KIR2DL2 and -2DS2 in the presence of their shared ligand HLA-C1 also failed to reach significance (-2DL2/HLA-C1: OR=1.311, 95% CI=0.881–1.950; -2DS2/HLA-C1: OR=1.286, 95% CI=0.866–1.911). Analysis of KIR2DL2, -2DL3 and -2DS2 ligand interactions stratified by HLA-C1 or -C2 homozygosity did not produce significant results (data not shown).
Previous disease association studies have implicated a role of activating KIR in the development of several inflammatory disorders.29, 30, 31, 32, 33, 34, 35 Our initial statistical analysis indicated a possible involvement of the activating KIR2DS2 and the inhibitory KIR2DL2 in the development of UC. Both genes displayed a high level of linkage disequilibrium with each other (previously reported in Hsu et al.39), occurring independently in only three of the samples genotyped in this study. However, logistic regression analysis failed to find an association of UC with KIR2DS2 or -2DL2 in combination with their shared ligand HLA-C1.
KIR2DS2 and -2DL2 are both present on the centromeric section of ‘B’ KIR haplotypes, whereas KIR2DL3 is located on the opposing position within the ‘A’ haloptype.24 An increase in KIR2DS2/-2DL2 gene frequency will result in a reciprocal decrease in the frequency of KIR2DL3. Although the decrease in phenotype frequency was not significant, it did result in a significant decrease in KIR2DL3 homozygosity (i.e. KIR2DL3 in the absence of KIR2DL2) in the UC cohort (P=0.03, Fisher's exact test). In addition, logistical regression revealed an association with KIR2DL3 when in combination with HLA-Cw1. This result suggests that the interaction between KIR2DL3 and its ligand exerts a mild protective effect in UC.
KIR are expressed on some T-cell subsets, including a CD4+ CD28null subset which has a suspected role in rheumatoid arthritis.40 The expression of KIR on the T-cell effector subsets in IBD has yet to be assessed. KIRs are predominantly expressed on NK cells, lymphocytes which have been found to produce both protective and detrimental effects in autoimmune conditions (reviewed by Johansson et al.41), although the potential role of NK cells in the development of IBD has been largely overlooked by investigators. However, several studies have reported reduced cytotoxicity of peripheral blood NK cells from UC patients.42, 43, 44 Giacomelli et al.43 suggested that this NK inhibition was due to circulating soluble factors, after observing reduced NK activity in control peripheral blood samples upon addition of IBD patient's blood serum. This negative modulation of NK activity may abrogate a possible regulatory mechanism in gut inflammation. NK cells display variegated expression of both activating and inhibitory receptors, their effector responses governed by the resulting balance of signalling. As most NK cells are believed to express at least one inhibitory receptor to self,45, 46 an individual possessing both KIR2DL3 and HLA-C1 is likely to have NK cell subsets which rely on this interaction as the main source of inhibitory signalling. The affinity of KIR2DL3 to its ligand is the weakest known of all inhibitory KIR;47 therefore, KIR2DL3-positive NK cells may theoretically have a greater potential for activation, leading to possible protective effects in UC.
Our results suggest a possible influence of KIR genotype in the development of UC, although confirmation is required owing to the relatively small sample size and the moderate levels of significance obtained. We found the protective effect of KIR2DL3 in combination with its ligand HLA-C1 provided the strongest association, although a possible influence of KIR2DL2/-2DS2 in UC susceptibility cannot be ruled out. Further analysis using larger cohorts may identify the precise genetic source of association.
Materials and methods
The UC patient cohort consisted of 194 unrelated, Caucasoid individuals recruited at the IBD clinic at the John Radcliffe Hospital, Oxford. Diagnosis was based upon standard clinical, radiological and histological criteria.48
The control group consisted of either the partners of patients suffering from IBD (n=124) or were recruited in general practice well person clinics in Oxfordshire (n=92). No control subject had either a personal or family history of IBD. The control and patient groups were matched for age, ethnicity and gender.
In order to produce polymerase chain reaction (PCR)-SSP reactions able to detect and discriminate each of the known KIR genes, primers were designed using sequence alignments comprising all KIR allelic variants present in the immunopolymorphism database (IPD) KIR sequence database (http://www.ebi.ac.uk/ipd/kir/). All reactions were designed to produce an amplicon of between 140 and 290 bp in size in order to achieve robust amplification. Individual KIR genes are detectable by single reactions, although reactions for KIR2DL2, -2DL3 and –2DS1 contain more than one primer pair to allow amplification of all known alleles at those loci. KIR reactions were validated using 18 Epstein–Barr virus-transformed lymphoblastoid cell lines from the 10th International Histocompatibility Workshop previously typed by Vilches et al.49 KIR PCR-SSP genotyping of two of these samples are shown in Figure 2a.
Reactions were also designed for the detection of the HLA-C class I ligands of KIR: epitopes C1 (defined by the presence of Asn80 in the α1 domain of the HLA-C molecule, recognized by KIR2DL2, -2DL3 and -2DS247) and C2 (Lys80, recognized by KIR2DL1 and -2DS147). The reaction to detect HLA-Cw C1 epitope will also co-amplify the rare HLA-Bw alleles *0713, *6702, *5401, *5402 and *5507. Consequently, reactions were included to detect the presence of these alleles to improve overall specificity of the reaction set. HLA primer sequences were designed using the latest alignments from the IMGT/HLA Sequence Database (http://www.ebi.ac.uk/imgt/hla/allele.html). Reactions were validated using the same 18 International Histocompatibility Workshop lymphoblastoid cell lines used in the validation of the KIR PCR-SSP assays. An example of HLA-KIR ligand typing is shown in Figure 2b.
PCR-SSP reactions were also designed to detect a non-synonymous polymorphism within the transmembrane region of NKG2D, Ala72Thr, which results from guanine to adenine substitution at nucleotide 214 of the coding sequence.23 An example of NKG2D PCR-SSP typing is shown in Figure 2c. The results of the NKG2D reactions were confirmed by the direct sequencing of genomic DNA from two representative samples for each possible genotype detected (i.e. 214 G/G, G/A and A/A) using primers located either side of this SNP (sense primer: IndexTermGCTGCTTCATCGCTGTAGC and antisense: IndexTermCGGTCAAGGGAATTTGAACTTC).
All primers were synthesized by Sigma Genosys (Haverhill, UK); PCR-SSP primer sequences are listed in Table 2.
PCR reactions were composed of 5 μl 2 × Biomix (containing Biotaq DNA polymerase) (Bioline, UK), between 0.01 and 0.1 μg of DNA, primer combinations (final reaction concentrations of each primer are given in Table 2), and brought to a total volume of 10 μl with 0.2 μm filtered H2O (Sigma). The Final 1 × concentrations of the Biomix components are as follows: 1 mM deoxynucleoside triphosphates, 16mM (NH4)2SO4, 62.5 mM Tris-HCl (pH 8.8 at 25°C), 0.01% Tween 20 and 2 mM MgCl2. Reaction mixtures were dispensed under 10 μl of mineral oil (Sigma, UK) in 96-well PCR plates (Costar, High Wycombe, UK). DNA samples were amplified on either MJ Research (Reno, NV, USA) Research Dyad DNA Engines or MJ Research PTC-200 or PTC-225 thermal cyclers. Cycling parameters were as follows: 1 min at 96°C followed by five cycles of 96°C for 20 s; 70°C for 45 s and 72°C for 25 s, followed by 21 cycles of 96°C for 25 s; 65°C for 50 s and 72°C for 30 s, followed by four cycles of 96°C for 30 s and 55°C for 60 s and 72°C for 90 s. Following the PCR, 5 μl of loading buffer, comprising 0.25% Orange G, 30% v/v glycerol and 0.5 × Tris-Borate-ethylenediaminetetraacetic acid (EDTA) (TBE) buffer (89 mM of Tris base, 89 mM of boric acid and 2 mM of EDTA (pH 8.0)), was added to each reaction mix. PCR products were electrophoresed in 2.0% agarose gels containing 0.5 μg/ml ethidium bromide, for 25–30 min at 15 V/cm in 0.5 × TBE buffer and visualized with UV illumination.
PCR was carried out using 50 μl volume reactions. All concentrations and conditions are identical to those used in the standard PCR-SSP protocol. The resulting PCR products were gel purified using QIAquick gel extraction kit (Qiagen, Hilden, Germany). Cycle sequencing was performed using BigDye Terminator v 3.1 methodology (Applied Biosystems, Foster City, CA, USA) and an Applied Biosystems 3730 × l DNA Analyser.
A comparison of the phenotype distribution of KIR genes, HLA-Cw epitopes and NKG2D polymorphism between the two cohorts was performed using Fisher's exact test employing a 2 × 2 contingency table (SPSS Inc., Chicago, IL, USA). The influence of KIR genes both in combination with, and independent from their HLA-C ligands, was assessed jointly using stepwise binary logistical regression analysis (SPSS 12.0.1).
The HLA-Cw and NKG2D genotype and allele frequencies in both populations were tested for conformity to Hardy–Weinberg equilibrium using a 2 × 2 χ2 test comparing expected and the observed values.
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NTY and DCJ are funded by the Leukaemia Research Fund; JT is funded by the Medical Research Council and the Wellcome Trust.
About this article
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