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. 2010 Sep 6;7(6):471–476. doi: 10.1038/cmi.2010.42

Contribution of functional KIR3DL1 to ankylosing spondylitis

Ivan V Zvyagin 1,5, Ilgar Z Mamedov 1,5, Olga V Britanova 1,5, Dmitriy B Staroverov 1, Evgeni L Nasonov 2, Anna G Bochkova 2, Anna V Chkalina 1, Alexei A Kotlobay 1, Dmitriy O Korostin 3,4, Denis V Rebrikov 3,4, Sergey Lukyanov 1, Yuri B Lebedev 1, Dmitriy M Chudakov 1
PMCID: PMC4002958  PMID: 20818412

Abstract

Increasing evidence points to a role for killer immunoglobulin-like receptors (KIRs) in the development of autoimmune diseases. In particular, a positive association of KIR3DS1 (activating receptor) and a negative association of KIR3DL1 (inhibitory receptor) alleles with ankylosing spondylitis (AS) have been reported by several groups. However, none of the studies analyzed these associations in the context of functionality of polymorphic KIR3DL1. To better understand how the KIR3DL1/3DS1 genes determine susceptibility to AS, we analyzed the frequencies of alleles and genotypes encoding functional (KIR3DL1*F) and non-functional (KIR3DL1*004) receptors. We genotyped 83 AS patients and 107 human leukocyte antigen (HLA)-B27-positive healthy controls from the Russian Caucasian population using a two-stage sequence-specific primer PCR, which distinguishes KIR3DS1, KIR3DL1*F and KIR3DL1*004 alleles. For the patients carrying two functional KIR3DL1 alleles, those alleles were additionally genotyped to identify KIR3DL1*005 and KIR3DL1*007 alleles, which are functional but are expressed at low levels. KIR3DL1 was negatively associated with AS at the expense of KIR3DL1*F but not of KIR3DL1*004. This finding indicates that the inhibitory KIR3DL1 receptor protects against the development of AS and is not simply a passive counterpart of the segregating KIR3DS1 allele encoding the activating receptor. However, analysis of genotype frequencies indicates that the presence of KIR3DS1 is a more important factor for AS susceptibility than the absence of KIR3DL1*F. The activation of either natural killer (NK) or T cells via the KIR3DS1 receptor can be one of the critical events in AS development, while the presence of the functional KIR3DL1 receptor has a protective effect. Nevertheless, even individuals with a genotype that carried two inhibitory KIR3DL1 alleles expressed at high levels could develop AS.

Keywords: ankylosing spondylitis, autoimmune disease, HLA-B27, KIR, KIR3DL1, KIR3DS1, KIR3DL1*004, NK cell, T cell

Introduction

The etiology of ankylosing spondylitis (AS) is thought to have a significant oligogenic component, with major input coming from the human leukocyte antigen (HLA)-B27.1, 2, 3 Indeed, more than 90% of patients with AS are born with the HLA-B27 gene, while only 5–15% of carriers are present in the total population.4, 5 Nevertheless, twin studies indicate that HLA-B27 contributes to only 16% of the total genetic risk,6 suggesting that AS has multiparameter heritability. Deciphering the heritable factors that contribute to AS is important in understanding the mechanisms of disease initiation and progression.

Several genetic polymorphisms have been reported to be associated with AS, including polymorphisms in the non-MHC genes IL23R and ERAP1.7, 8, 9, 10, 11, 12, 13 Another group of genes with polymorphisms that likely contribute to AS and other HLA-B27-associated diseases is the killer immunoglobulin-like receptor (KIR) genes.12, 14, 15, 16 These genes are located on chromosome region 19q13.4, an area that has been implicated in AS development by whole genome scans.12, 14, 15, 16. KIRs are expressed on the surface of natural killer (NK) cells and a subpopulation of T cells.15, 17, 18, 19, 20 On NK cells, KIRs play a key role in balancing the activating and inhibitory signals that determine the NK cell response, allowing NK cells to scan for the presence of MHC class I molecules and their antigenic load on target cells.15, 17, 18, 19 The role of KIRs expressed on a minor subpopulation of T cells is less clear. KIR–MHC class I interaction can inhibit cytolytic activity and cytokine production in T cells, but the level of KIR-mediated inhibition of T-cell effector functions depends on the strength of T-cell antigen receptor stimulation.20, 21, 22 Therefore, consistent with the role of KIRs on NK cells, KIRs probably regulate the activation and inhibition of specific T-cell subpopulation(s).

The inheritance of certain HLA and KIR alleles has an important impact on both protection against viral infections and predisposition to autoimmune diseases.15, 18, 23, 24 A number of studies has been conducted to identify possible associations between autoimmune conditions and KIR genes.25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 In particular, a number of works demonstrate the role of KIRs in spondyloarthropathies.16 Among the numerous KIR genes, the KIR3DL1/3DS1 locus is of particular interest with respect to AS because KIR3DL1 is the only KIR that recognizes HLA-B molecules of the Bw4 serotype, including HLA-B27.36 KIR3DL1 is an inhibitory receptor that interacts with HLA-Bw4 to suppress the cytolytic capacity of NK or T cells36, 37, 38 and likely plays a role in protecting against autoimmunity.15, 23 A counterpart of KIR3DL1 is the activating receptor, KIR3DS1. KIR3DL1 and KIR3DS1 have allelic relationships and are commonly considered alleles of the same KIR gene (IPD-KIR sequence database: http://www.ebi.ac.uk/ipd/kir/). Therefore, possible individual genotypes are 3DL1/3DL1, 3DL1/3DS1 or 3DS1/3DS1 (rare duplications of KIR3DL1/3DS1 genes have also been described39). In Caucasians, the frequency of KIR3DL1 is 79% and that of KIR3DS1 is 21%.40, 41

Inhibitory KIR3DL1 and activating KIR3DS1 differ by the motifs found in their intracytoplasmic tails, which contain either immunoreceptor tyrosine inhibitory motifs or motifs enabling interaction with activating adaptor proteins, such as DAP12.42 Conversely, the extracellular domains of KIR3DL1 and KIR3DS1 are strongly homologous. Therefore, although the particular ligand for KIR3DS1 has not yet been identified,43 KIR3DS1 is thought to interact with HLA-Bw4 carrying a specific load of exogenous peptides. Indeed, the interaction of KIR3DS1 with HLA-Bw4 has been shown to delay the progression of AIDS after HIV-1 infection.44, 45

Several recent works support the involvement of the KIR3DL1/3DS1 locus in AS development. The KIR3DS1 allele occurs more frequently and the KIR3DL1 allele less frequently in AS patients than in HLA-B27-positive controls from Caucasian populations in Spain,31 Portugal,31 China32 and Thailand.32 Another group also reported that KIR3DS1 is significantly more frequent in AS patients.33 In contrast, no increase in KIR3DS1 carriers among AS patients was noted in a study cohort from the United Kingdom.34 The contradictory data may be explained by the different inclusion criteria used in each study.

It is important to note that none of the studies analyzed associations of the polymorphic KIR3DL1 allele in the context of its functionality. Indeed, to perform its inhibitory function, KIR3DL1 must be expressed at the cell surface. At the same time, different KIR3DL1 alleles are expressed at least at three different levels: low (KIR3DL1*005, *006 and *007), high (KIR3DL1*001, *002, *003, *008, *009, *015 and *020), and not expressed/null (KIR3DL1*004).46, 47, 48, 49 The latter allele, KIR3DL1*004, carries the amino acid polymorphism Ser86Leu, which interrupts proper protein folding and leads to its intracellular retention.50 Because KIR3DL1*004 is absent from the cell surface, it cannot provide inhibitory signaling through recognition of an HLA-Bw4, such as HLA-B27. Thus, KIR3DL1*004 indirectly supports a more potent activation of immune cells. In agreement with this, a slower progression of AIDS has been noted in individuals with the HLA-Bw4/KIR3DL1*004 genotype.51 The functionality of KIR3DL1 may also be connected to the etiology of AS and other HLA-B27-associated diseases. In Caucasians, KIR3DL1*004 is rather frequent, comprising 16–18% of KIR3DL1/3DS1 alleles. Approximately 30% of Caucasians carry KIR3DL1*004 and thus its contribution to KIR3DL1 associations may be significant.40, 41

Here, we investigated whether functional KIR3DL1 alleles, the non-functional allele KIR3DL1*004 and the activating allele KIR3DS1 were positively or negatively associated with AS in a Russian Caucasian cohort. Clarifying KIR3DL1/3DS1 associations by accounting for the functional activity of KIR3DL1 should provide a better understanding of the events occurring during activation of NK or T cells that may be critical to the initiation of AS.

Materials and methods

Patients and controls

AS patients were diagnosed at the Institute of Rheumatology, RAMS (Russian Academy of Medical Sciences), Moscow, Russia, in accordance with modified New York criteria (1984).52 Radiographs of the pelvis and lumbar spine were obtained from all patients. Inclusion criteria were as follows: all patients had a bilateral sacroiliitis of grade II or higher, and only HLA-B27-positive patients were selected for the study. The venous blood samples from the HLA-B27-positive controls were collected from a group of healthy, unrelated individuals living in the Moscow area. Venous blood was collected from adult patients (aged 21–63 years) without chronic disease during medical and/or diagnostic procedures. The presence of the HLA-B27 allele in controls and AS patients was confirmed by sequence specific primer PCR (SSP-PCR) and flow cytometry. Both patients and controls gave written informed consent prior to enrolling in the study. The study was conducted according to the Declaration of Helsinki.

Genotyping

Peripheral blood mononuclear cells were isolated from fresh whole peripheral blood samples using Ficoll–Urografin density gradient separation. Genomic DNA was prepared from peripheral blood mononuclear cells using the DNA extraction kit, Diatom DNA Prep (Isogen, Moscow, Russia). For genotyping, KIR3DL1 and KIR3DS1 were considered alleles of the same KIR gene. KIR3DL1/KIR3DS1 typing of DNA samples was performed using SSP-PCR as described previously.53 Results were confirmed by independent SSP-PCR using two pairs of primers specific for KIR3DL1 (KIR3DL1_DS1_For2 and KIR3DL1_Rev3) or KIR3DS1 (KIR3DL1_DS1_For2 and KIR3DS1_Rev3). To discriminate between KIR3DL1 allelic variants carrying Leu86 or Ser86, we used the KIR3DL1_DS1_For2–KIR3DL1_Rev3 PCR amplification product as a template for the two nested SSP-PCR reactions using primer pairs specific for KIR3DL1-Leu86 (KIR3DL1_For3 and KIR3DL1_Leu86Rev) or KIR3DL1-Ser86 (KIR3DL1_For3 and KIR3DL1_Ser86Rev). The same nested SSP-PCR approach was employed to identify the allelic variants KIR3DL1*005 and KIR3DL1*007 (see Supplementary Table 1 for primer sequences).

Quality guarantee measures

Genotyping results were reproduced at least twice for each of the individual genomes using one or both variations of the SSP-PCR assay. In addition, a subset of three healthy and three AS-affected individual genomes was analyzed by comprehensive accurate sequencing of KIR3D regions flanked by KIR3DL1_DS1_For2 and KIR3D_Rev2 (see Supplementary Table 1 for primer sequences). As a result, several variants of the KIR3DL1 allele, including KIR3DL1*004 and KIR3DS1 alleles, were identified. The sequencing results perfectly confirmed the genotyping by SSP-PCR for each of the analyzed individuals.

Statistical analysis

Allelic and genotypic frequencies were calculated by direct counting. Hardy–Weinberg equilibrium for KIR3DL1 and KIR3DS1 alleles was tested using Genome Data Analysis software. Associations were analyzed using the χ2 test with Yate's correction or Fisher's exact test. Values of P<0.05 were considered statistically significant. The odds ratio (OR) was calculated by the cross-product ratio, and the corresponding 95% confidence interval was determined.

Results

Contribution of the KIR3DL1/3DS1 gene to AS development

To assess the possible association of the KIR3DL1 polymorphism with AS, we typed 83 HLA-B27-positive Russian Caucasian patients with AS and 107 HLA-B27-positive healthy controls.

As a first step, SSP-PCR analysis was used to distinguish KIR3DL1 and KIR3DS1 alleles, as has been performed in previous work.31, 32, 33, 34 KIR3DL1 and KIR3DS1 allele distribution was analyzed in both cohorts and showed no significant deviation from Hardy–Weinberg equilibrium (P>0.05). We found that the KIR3DL1 allele was underrepresented (P<0.01, OR=0.52) and the KIR3DS1 allele was overrepresented (P<0.01, OR=1.93) in the AS group compared to healthy controls (Table 1). Therefore, our data confirm that AS is associated with an increased frequency of KIR3DS1 and a decreased frequency of KIR3DL1, as has been reported for other study populations.31, 32, 33

Table 1. Allelic frequencies of KIR3DL1 and KIR3DS1 in Caucasian AS patients and healthy controls from Russian, Azorean, and Spanish populationsa.

Allele Frequency in AS patients % (number of alleles) Frequency in control individuals % (number of alleles) P value Russian AS versus Russian controls OR (95% CI) Russian AS versus Russian controls P value Combined Caucasian cohortsb OR (95% CI) Combined Caucasian cohortsb
Russian (2n=166) Spanish (2n=142) Azorean (2n=110) Russian (2n=214) Spanish (2n=210) Azorean (2n=114)          
3DL1 69.3% (115) 57% (81) 64.5% (71) 81.3% (174) 77.6% (163) 82.4% (94) <0.01 0.52 (0.32–0.84) <1×10−7 0.44 (0.33–0.60)
3DS1 30.7% (51) 42.9% (61) 35.4% (39) 18.7% (40) 22.3% (47) 17.5% (20) <0.01 1. 93 (1.20–3.11) <1×10−7 2.28 (1.70–3.05)
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a

Data for the Azorean and Spanish populations taken from Ref. 31.

b

Calculated for the combined cohorts of Russian, Spanish and Azorean AS patients and controls.

Abbreviations: AS, ankylosing spondylitis; CI, confidence interval; KIR, killer immunoglobulin-like receptor; OR, odds ratio.

We also confirmed that the frequency of the KIR3DL1/KIR3DL1 genotype was decreased (P=0.005, OR=0.42) and the frequency of the KIR3DL1/KIR3DS1 genotype was increased (P=0.01, OR=2.27) in AS patients compared to HLA-B27-positive controls. Analysis of combined Russian, Spanish and Azorean Caucasian populations also showed a statistically significant increase in the KIR3DS1/KIR3DS1 genotype frequency among AS patients (P=0.01, OR=2.35; Table 2). Thus, the findings in our study and previous work support an important role for the KIR3DL1/3DS1 gene in the etiology of AS.

Table 2. Genotypic frequencies of KIR3DL1 and KIR3DS1 in Caucasian AS patients and healthy controls in Russian, Azorean and Spanish populationsa.

Genotype Frequency in AS patients, % (no. of patients) Frequency in control individuals, % (no. of individuals) P value Russian AS versus Russian controls OR (95% CI) Russian AS versus Russian controls P value Combined Caucasian cohortsb OR (95% CI) Combined Caucasian cohortsb
Russian (n=83) Spanish (n=71) Azorean (n=55) Russian (n=107) Spanish (n=105) Azorean (n=57)          
3DL1/3DL1 48.2% (40) 35.2% (25) 36.3% (20) 69.2% (74) 61.9% (65) 68.4% (39) 0.005 0.42 (0.23–0.75) <1×10−7 0.35 (0.24–0.51)
3DL1/3DS1 42.2% (35) 43.6% (31) 56.1% (31) 24.3% (26) 31.4% (33) 28% (16) 0.01 2.27 (1.22–4.23) <1×10−4 2.24 (1.53–3.28)
3DS1/3DS1 9.6% (8) 21.1% (15) 7.2% (4) 6.5% (7) 6.6% (7) 3.5% (2) NS — 0.01 2.35 (1.23–4.48)
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a

Data for the Azorean and Spanish populations taken from Ref. 31.

b

Calculated for the combined cohorts of Russian, Spanish and Azorean AS patients and controls.

Abbreviations: AS, ankylosing spondylitis; CI, confidence interval; KIR, killer immunoglobulin-like receptor; NS, not significant; OR, odds ratio.

However, it remained unclear which allele provided the most prominent input. Is the predisposition to AS attributable to the absence of KIR3DL1 (which protects against autoimmunity by interacting with HLA-B27) or the presence of KIR3DS1 (which promotes stimulation of NK and/or T cells), or are both conditions required? The genotyping studies conducted above could not answer this question, as KIR3DL1 and KIR3DS1 are segregating alleles.

Protective role of the functional KIR3DL1 allele

Although most KIR genes seem to be expressed stochastically and independently of one another, their expression is tightly regulated.54 In particular, it has been demonstrated that in KIR3DS1/KIR3DL1 heterozygous donors, most NK cells express either KIR3DS1 only or KIR3DL1 only.55 Selective expression of either inhibitory or activating KIRs has also been recently reported for a minor subset of CD4+ T cells.56 Therefore, the association of the KIR3DS1 allele with AS could be explained by a statistically decreased frequency of NK or T cells expressing KIR3DL1 in KIR3DS1-positive individuals, rather than by a direct functional activation of KIR3DS1-expressing immune cells, especially as KIR3DS1 has not yet been shown to interact with HLA-Bw4.43 To investigate this possibility, we performed second-stage analysis using nested SSP-PCR, which allowed us to distinguish KIR3DL1 alleles known to be functionally expressed on the cell surface (including KIR3DL1*001–003, *005–009, *015 and *020; designated KIR3DL1*F) from the non-functional allele KIR3DL1*004. The aggregate frequency of rare KIR3DL1 alleles other than KIR3DL1*F and *004 is below 1% and therefore their input was ignored.

Because the KIR3DL1*004 receptor plays neither a stimulatory nor an inhibitory role, we expected that its allelic frequency would depend on the pressure of the functional alleles that play a dominant role. Indeed, if expression of activating KIR3DS1 plays a dominant role in disease development (direct association), then its allelic frequency should be increased at the expense of both KIR3DL1*F and KIR3DL1*004 independently of the functionality of these alleles. Alternatively, if the absence of the protective KIR3DL1*F allele plays a dominant role, then the frequency of this allele should be decreased in AS patients in favor of both KIR3DS1 and KIR3DL1*004 independently of their ability to transfer an activating signal (KIR3DS1) or inability to transfer any signal (KIR3DL1*004).

Remarkably, results from the analysis of the allelic frequencies of KIR3DL1*F, KIR3DL1*004 and KIR3DS1 in AS patients supported neither of the two hypotheses. As expected, AS patients had a lower frequency of KIR3DL1*F (P=0.005, OR=0.54) and a higher frequency of KIR3DS1 (P<0.01, OR=1.94) alleles compared to healthy controls. However, the frequency of the 3DL1*004 allele between AS patients and healthy controls was approximately equal (Table 3). The frequency of KIR3DL1*004 carriers was also equal between patients with AS and healthy controls (Table 4).

Table 3. Allelic frequencies of KIR3DL1*F, KIR3DL1*004 and KIR3DS1 in AS patients and healthy controls in Russian Caucasian population.

Allele Frequency, % (no. of alleles) P value OR (95% CI)
AS (2n=166) Control (2n=214)      
3DL1*F 48.2% (80) 63.1% (135) 0.005 0.54 (0.36–0.82)
3DL1*004 21.1% (35) 18.2% (39) NS —
3DS1 30.7% (51) 18.7% (40) <0.01 1.94 (1.21–3.12)
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Abbreviations: AS, ankylosing spondylitis; CI, confidence interval; KIR, killer immunoglobulin-like receptor; NS, not significant; OR, odds ratio.

Table 4. Frequency of KIR3DL1*F, KIR3DL1*004 and KIR3DS1 carriers in AS patients and healthy controls in the Russian Caucasian population.

Alleles Frequency, % (no. of individuals) P value OR (95% CI)
AS (n=83) Control (n=107)      
3DL1*F 75.9% (63) 83.2% (89) NS —
3DL1*004 36.1% (30) 31.8% (34) NS —
3DS1 51.8% (43) 30.8% (33) 0.03 1.72 (1.08–2.92)
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Abbreviations: AS, ankylosing spondylitis; CI, confidence interval; KIR, killer immunoglobulin-like receptor; NS, not significant; OR, odds ratio.

Thus, neither allelic nor carrier frequency of KIR3DL1*004 correlated with frequency of the KIR3DL1*F or KIR3DS1 allele. Both allelic and carrier frequency of KIR3DL1*004 demonstrated remarkable similarity between patients and controls. This result indicates that the input of the activating KIR3DS1 allele is not the only factor in AS initiation, and that the inhibitory KIR3DL1*F allele plays an antagonistic, protective role in disease development.

Discussion

Typing of KIR3DL1*F/3DL1*004/3DS1 allowed us to distinguish among six functionally distinct genotypes: 3DL1*F/3DL1*F, 3DL1*F/3DL1*004, 3DL1*004/3DL1*004, 3DL1*F/3DS1, 3DL1*004/3DS1 and 3DS1/3DS1 (Table 5). Although a statistically reliable interpretation of distribution of these genotypes requires a much higher number of patients in the study, the analysis still allows for preliminary conclusions. Among these genotypes, 3DL1*F/3DL1*F was the only genotype assuring that each immune cell expressing the KIR3DL1/3DS1 locus would present the inhibitory KIR3DL1 receptor at its surface. In the healthy population, this genotype had the highest frequency, constituting approximately 37–40% of individuals (Ref. 41 and Table 5). Both genotypes 3DL1*F/3DL1*004 and 3DL1*F/3DS1 accounted for immune cells that may have no functional inhibitory KIR3DL1 receptor on their surface despite upregulated expression of the gene. Finally, immune cells of the individuals with the 3DL1*004/3DL1*004, 3DL1*004/3DS1 or 3DS1/3DS1 genotypes would not express the KIR3DL1 receptor at their surface.

Table 5. Genotypic frequencies of KIR3DL1*F, KIR3DL1*004 and KIR3DS1 in AS patients and healthy controls in the Russian Caucasian population.

Genotype Frequency, % (no. of individuals) P value OR (95% CI)
AS (n=83) Control (n=107)      
3DL1*F/3DL1*F 20.5% (17) 43.0% (46) <0.002 0.34 (0.18–0.66)
3DL1*F/3DL1*004 21.7% (18) 21.5% (23) NS —
3DL1*004/3DL1*004 6.0% (5) 4.7% (5) NS —
3DL1*F/3DS1 33.7% (28) 18.7% (20) 0.03 2.22 (1.14–4.31)
3DL1*004/3DS1 8.4% (7) 5.6% (6) NS —
3DS1/3DS1 9.6% (8) 6.5% (7) NS —
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Abbreviations: AS, ankylosing spondylitis; CI, confidence interval; KIR, killer immunoglobulin-like receptor; NS, not significant; OR, odds ratio.

Hypothesizing that the protective role of the KIR3DL1*F receptor drives the association of the locus with AS, we would expect to see the following changes in genotypic frequencies in AS patients:

  1. a decrease in the percentage of 3DL1*F/3DL*F genotypes;

  2. a decrease, albeit to a lesser extent, in the percentage of 3DL1*F/3DL1*004 and 3DL1*F/3DS1 genotypes;

  3. a uniform increase in the percentage of 3DL1*004/3DL1*004, 3DL1*004/3DS1 and 3DS1/3DS1 genotypes.

Conversely, hypothesizing that the activating KIR3DS1 receptor drives the association of the locus with AS, we would expect the following alterations in frequencies in AS patients:

  1. an increase in the percentage of 3DS1/3DS1 genotypes;

  2. a smaller increase in the percentage of 3DL1*004/3DS1 and 3DL1*F/3DS1 genotypes;

  3. a uniform decrease in the percentage of 3DL1*F/3DL*F, 3DL1*F/3DL1*004 and 3DL1*004/3DL1*004 genotypes.

Our data generally support the latter hypothesis. Indeed, the frequency of the 3DL1*F/3DS1 genotype was greater in AS patients than in healthy controls (P=0.03, OR=2.22; Table 5), arguing against the first hypothesis. In general, the frequency of genotypes carrying the KIR3DS1 allele was increased in AS patients (P=0.03, OR=1.72; Table 4) independently of the presence or absence of the functional inhibitory KIR3DL1*F allele (Table 5). Therefore, we conclude that the input of the activating KIR3DS1 allele is probably the strongest in the hypothetical checkpoint that precedes initiation of AS.

At the same time, the functional role of the KIR3DL1 receptor demonstrated by analysis of the allelic and carrier frequencies (Tables 3 and 4) was also notable and presumably affects genotype frequencies. Indeed, in AS patients, the decrease in the frequency of the ‘always functional' genotype, 3DL1*F/3DL1*F, was prominent and statistically significant (P<0.002, OR=0.34), while the frequency of the 3DL1*F/3DL1*004 genotype was approximately equal between patients and controls (Table 5).

In the context of the proposed protective role of the functional KIR3DL1 receptor, a plausible explanation for disease development in 3DL1*F/3DL1*F AS patients could be the presence of either the KIR3DL1*005 or the KIR3DL1*007 allele, which are known to be expressed at low levels.46, 47, 48, 49 Another allele expressed at low levels, KIR3DL1*006, is not characteristic of the Caucasian population.40, 41 To verify if the low expression level alleles are present in 3DL1*F/3DL1*F AS patients, we additionally typed them for the presence of KIR3DL1*005 and KIR3DL1*007 alleles. Notably, the typing revealed that 9 of 17 3DL1*F/3DL1*F patients carried low expression alleles (5× 3DL1*F/3DL1*005, 3× 3DL1*005/3DL1*005 and 1× 3DL1*F/3DL1*007). However, the remaining eight patients had two functional KIR3DL1 alleles, both expressed at a high level. Therefore, even those 3DL1*F/3DL1*F genotypes that carried both KIR3DL1 alleles expressed at a high level still did not guarantee protection against AS.

In summary, analysis of KIR3DL1*F, KIR3DL1*004 and KIR3DS1 allelic and genotypic frequencies in AS patients showed that both inhibitory KIR3DL1*F receptors and the activating receptor, KIR3DS1, functionally contribute to the probability of disease development. Nevertheless, input of the KIR3DS1 receptor is probably more important, as it followed from the analysis of genotypic frequencies. The protective function of KIR3DL1*F likely becomes realized when KIR3DS1 is absent from the surface of the immune cell. Therefore, the interaction between KIR3DS1 and HLA-B27 is probably a key event that should be investigated to uncover the etiology of AS and other HLA-B27-associated diseases. Our findings should be confirmed by the analysis of an expanded cohort of AS patients, which should provide a more profound understanding of the role of KIR receptors in autoimmunity. In general, the analysis of linked alterations in frequencies of functional and non-functional alleles should provide a deeper understanding of how the balance of activating and inhibitory receptors functionally determines susceptibility to autoimmune diseases.

Acknowledgments

This work was supported by the State Support of the Leading Scientific Schools NS-2395.2008.4, Molecular and Cell Biology Program RAS, Rosnauka 02.512.12.2053, Rosobrazovanie P 256 and Basic Research for Medicine RAS.

Footnotes

Note: Supplementary information is available on the Cellular & Molecular Immunology website(http://www.nature.com/cmi/).

Supplementary Information

Supplementary information
Click here for additional data file. (28.4KB, pdf)

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