Abstract
Dry eye is one of the most prevalent eye diseases and dry eye disease (DED) is associated with ocular surface inflammation. The interaction between killer cell immunoglobulin-like receptors (KIRs) and human leukocyte antigens (HLAs) regulates the activation of natural killer (NK) cells and certain T cell subsets in response to inflammation. The objective of this study was to explore whether KIR gene and HLA-C allele polymorphisms were associated with DED in a Chinese Han population. Polymerase chain reaction with sequence-specific primers method was used to genotype KIR genes and HLA-C alleles in 106 DED patients and 220 healthy controls. Framework genes KIR2DL4, KIR3DL2, KIR3DL3, and KIR3DP1 were present in all individuals. There were no significant differences in the frequencies of inhibitory KIR genes between the two groups. However, the frequency of KIR2DS2 was significantly higher in severe DED patients than that in healthy controls (p=0.031, odds ratio [OR]=1.828, 95% confidence interval [CI]=1.05-3.17). Significantly different distributions of HLA-C allele groups were not observed in severe DED patients and controls. The frequency of the combination of HLA-C1 allele group with KIR2DS2 was significantly higher in severe DED patients compared with controls (p=0.013, OR=2.083, 95% CI=1.16-3.74). These data suggested that this genotype combination was associated with susceptibility to severe DED and that NK cells might have a role in the pathogenesis of DED. The results led to an interesting future research question of whether or not KIR and HLA-C genotypes were involved in the predisposition to or pathogenesis of DED.
Introduction
D
To date, the pathogenesis of DED has not been fully understood, although it is widely recognized that DED is associated with ocular surface inflammation (Solomon et al., 2001; Luo et al., 2004). The clearance of inflammation is governed by a number of different factors, such as cytokine production, antigen presentation, and receptor recognition, which are determined by the host genetic background. One of the most important genetic factors known to be involved in immune response is human leukocyte antigen (HLA). The increased expression of HLA-DR in DED patient showed that inflammation may be a primary cause of ocular surface damage and those data supported the use of immunomodulatory drugs in the treatment of DED (Rolando et al., 2005).
HLA class I molecules are recognized by natural killer (NK) cells through killer immunoglobulin-like receptors (KIRs). Inhibitory KIR (iKIR) molecules bind to target cell HLA class I molecules and prevent the attack of NK cells on normal cells (Boyton and Altmann, 2007). When activating KIRs (aKIRs) bind to their ligands, activating signals are generated leading to the destruction of target cells (Campbell et al., 1996). KIR gene family clusters within the leukocyte receptor complex on chromosome 19q13.4, and 16 KIR genes have been characterized in humans: 7 inhibitory types (3DL1-3, 2DL1-3, and 2DL5), 6 activating types (3DS1 and 2DS1-5), 1 (2DL4) with both inhibitory and activating potential, and 2 pseudogenes (2DP1 and 3DP1) that do not encode functional KIR receptors. In general, two major groups of haplotypes of KIR genes may be distinguished: haplotype A with KIR2DS4 as the only aKIR gene and haplotype B characterized by the presence of at least one aKIR gene other than KIR2DS4.
Because of KIR specificity for HLA class I allotypes, and their extensive polymorphisms, there are accumulated research focuses on the association of KIR and HLA gene polymorphisms with different ocular diseases (Rolando et al., 2005; Goverdhan et al., 2008; Levinson et al., 2008; Lowe et al., 2009). For example, in patients with Vogt-Koyanagi-Harada (VKH) disease, putative aKIR-HLA combinations were more common in patients, and some iKIR-HLA combinations were more common in controls (Levinson et al., 2008). The genetic combination of KIR2DS2 in the presence of specific HLA-C ligand was associated with primary Sjögren's syndrome (SS) (Lowe et al., 2009). The HLA-Cw*0701 allele and KIR haplotype AA were associated with age-related macular degeneration (AMD) and this genotype combination suggested that NK cells had a role in the pathogenesis of AMD (Goverdhan et al., 2008).
Particularly relevant to NK recognition by KIRs are polymorphic HLA-C molecules. Based on the dimorphism in position 80 (epitope for KIR binding), all HLA-C alleles can be divided into two groups: the C1 group carrying asparagine, and the C2 group carrying lysine at this position. The following KIR-HLA-C interactions have been defined: 2DL1/2DS1+HLA-C2 and 2DL2/2DL3/2DS2+HLA-C1 (O'Connor et al., 2007). However, the HLA ligands for several KIR genes are not yet identified. Through interaction with KIRs, HLA-C molecules are able to modulate NK cell function.
It is implied that certain combinations of HLA-C and KIR gene variants may influence susceptibility to DED. To test this hypothesis we analyzed HLA-C and KIR genotypes, both individually and in combination, for association with DED.
Materials and Methods
Patients and controls
The study included 106 unrelated individuals with a diagnosis of DED and 220 healthy controls (Table 1).
Patients were recruited from the Ophthalmology Department of the 4th People's Hospital of Jinan. They were diagnosed by the symptoms, slit-lamp examination, and Schirmer's I test. In Schirmer's I test advised by the National Eye Institute workshop, a 35×5-mm-size filter paper strip is used to measure the amount of tears that are produced over a period of 5 min. More than 10-mm wetting of the filter paper is normal. Patients with moderate dry eyes have wetting values between 10 and 5 mm. Patients with severe dry eyes have wetting values of less than 5 mm. Of these patients, 47 were men and 59 were women, and aged 33-58 years, with an average age of 44 years. Meanwhile, 220 unrelated healthy subjects were recruited from the health examination center of this hospital as a control group who have wetting values of more than 10 mm in Schirmer's I test. This group consisted of 96 men and 124 women, aged 30-55 years, with an average age of 40 years. All subjects were Han people from the Shandong area. Valid informed consent was obtained from each participant and there is no conflict with ethical policy from local and national governments.
KIR and HLA-C genotyping with polymerase chain reaction with sequence-specific primers method
The KIR genotyping was performed using polymerase chain reaction with sequence-specific primers (PCR-SSPs) in all samples collected from recruited subjects for the following 16 KIR genes: 2DL1-5, 3DL1-3, 2DS1-5, 3DS1, 2DP1, and 3DP1. We used newly extracted DNA from frozen peripheral blood mononuclear cells in this study to avoid false-negative results of KIR genes in the PCR-SSP typing since most KIR-specific amplicons are longer than 1000 bp. DNA was extracted with an EZ Bead System-32 DNA workstation (Texas BioGene, Inc.) according to the manufacturer's instruction. The human growth hormone gene was served as a positive control for PCR. The primers were as follows: 5′CTTCCCAACCATTCCCTTA3′ and 5′CGGATTTCTGTTGTGTTTC3′. The PCR without DNA template was served as a negative control. The PCR sequence-specific polymorphism primers used for the detection of KIR genes were described previously (Uhrberg et al., 1997; Hsu et al., 2002b; Martin and Carrington, 2008) (Table 2). The primers (Lowe et al., 2009) were designed to detect the HLA-C ligand-specific groups (C1/C2 sense: 5′CCGGGGAGCCGCGCA3′; C1 antisense: 5′GCGCAGGTTCCGCAGGC3′; C2 antisense: 5′CGCGCAGTTTCCGCAGGT3′). All primers were synthesized and validated by BOYA. Bio Co., Ltd., Shanghai. In detail, 20-50 ng DNA was amplified in 10 μL volume containing 0.2 mM dNTP, 0.5 U Taq DNA polymerase (Promega Corporation), 0.4 μM primers (except for KIR2DS1, 0.8 μM), and 1×PCR buffer. PCR amplification was carried out in a 9700 thermal cycler (PerkinElmer) under the following conditions: initial denaturing at 94°C for 4 min, followed by 30 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 90 s, plus a final extension at 72°C for 10 min. Partial annealing temperatures were changed as follows: KIR2DS2 (63°C), KIR2DS3 (63°C), KIR2DS4 (61°C), and KIR2DS5 (63°C). The amplification products were analyzed in 1%-2% agarose gel with fluorescence dye and visualized by a Gene Genius Bio Imaging System (Syngene Ltd.).
Hsu et al. (2002b).
Uhrberg et al. (1997).
Martin and Carrington (2008).
KIR, killer cell immunoglobulin-like receptor.
Statistical analysis
The phenotypic frequencies were determined by direct counting of the individuals positive for a particular KIR and/or HLA-C specificity. KIR and HLA-C genotypic frequency differences were tested for significance by Fisher's exact probability test. p-Values less than 0.05 were regarded as statistically significant. The strength of association was estimated by calculating the odds ratio (OR). Statistical analysis was carried out using the SPSS15.0 (SPSS, Inc.) software package.
Results
KIR gene polymorphisms in DED patients and control subjects
In this study, all the tested KIR genes were present in different frequencies in DED patient and control groups, and their distributions in the control group were similar to those previously reported in Chinese Han populations (Jiao et al., 2008; Zhuang et al., 2012). Framework genes KIR2DL4, KIR3DL2, KIR3DL3, and KIR3DP1 were present in all individuals. There were no significant differences in the frequencies of KIR genes between the moderate DED patient and control groups (data not shown). However, significantly different distributions of KIR genes were found in severe DED patient and control groups (Table 3). The frequency of activating KIR2DS2 was increased significantly in the patients compared with controls (p=0.031, OR=1.828, 95% confidence interval [CI]=1.05-3.17). The other KIR genes and genotypes did not show any significantly different distributions in the two groups.
The PF of each KIR gene or genotype was calculated as the percentage of positive numbers among all subjects.
Fisher's exact test.
Statistical significance (p<0.05).
DED, dry eye disease; HLA, human leukocyte antigen; OR, odds ratio; 95% CI, 95% confidence interval; PF, phenotypic frequency.
HLA-C allele groups in severe DED patients and control subjects
Based on known KIR ligand specificity to a dimorphism on HLA-C allotypes, HLA-C alleles were initially grouped into C1 and C2 allele groups. The frequencies of two HLA-C groups in our controls were similar to that reported in the Chinese Han population (Jiao et al., 2008). Since no significant differences of KIR gene distributions were observed in moderate DED patients and controls, we just compared the difference of HLA-C groups' distributions between the severe DED patients and controls (Table 4). The HLA-C alleles did not show any significantly different distributions in the two groups, although the frequency of HLA-C1 alleles was lower in severe DED patients compared with controls.
The PF of each HLA-C allele group was calculated as the percentage of positive numbers among all subjects.
Fisher's exact test.
Combination of HLA-C allele groups with their KIR ligands in severe DED patients and control subjects
The frequencies of KIR-HLA gene pairs in patients with severe DED were compared with controls (Table 5). A significant association with DED was observed for the HLA-C1 allele group in combination with its known KIR ligand, KIR2DS2 (p=0.013, OR=2.083, 95% CI=1.16-3.74). The remaining HLA-C allele groups in combinations with their KIR ligands did not show any significant association with DED.
The PF of each combination of HLA-C and KIR gene was calculated as the percentage of positive numbers among all specimens.
Fisher's exact test.
Statistical significance (p<0.05).
Discussion
NK cells have been increasingly reported to be an important effector in autoimmune diseases (Kulkarni et al., 2008). KIR genes encode for glycoprotein receptors that bind polymorphic HLA class I ligands and regulate the activation of NK cells and a subset of T cells. KIR genes exhibit allelic, haplotypic, and gene content variability (Hsu et al., 2002a). This, in conjunction with receptor-ligand interaction, raised the possibility that this family included candidate genes that confer susceptibility to chronic inflammatory diseases. However, nothing is known in this regard in DED, the most common eye pathology, which is characterized by sustained inflammation on the ocular surface. In the current study, our data indicated that the distributions of iKIRs did not show any significant differences in severe DED patient and control groups. However, the frequency of activating KIR2DS2 was significantly higher in the patients than that in controls (p<0.05). This result suggested that KIR2DS2 was associated with susceptibility to DED. Recent investigations have showed that the KIR2DS2 gene significantly increased the risk of patients with rheumatoid arthritis (Yen et al., 2001), scleroderma (Momot et al., 2004), psoriatic arthritis (Nelson et al., 2004), idiopathic bronchiectasis (Boyton et al., 2006), and type I diabetes mellitus (van der Slik et al., 2003; Middleton et al., 2006). In view of these findings, it was suggested that individuals with KIR2DS2 might be susceptible to certain autoimmune and inflammatory diseases. The ligands for the activating KIR2DS2 are unknown. The activating receptor KIR2DS2 shares sequence similarity in its extracellular domains with its corresponding inhibitory counterparts (KIR2DL2) and is thought to share HLA ligand-binding specificities as well. KIR2DS2 may bind weakly to HLA-C1 (the ligand of KIR2DL2), though this has not been conclusively established (Stewart et al., 2005). In this study, the distribution of HLA-C1 allele group and KIR2DS2 genotype was significantly different in severe DED patients and controls, adding further support for the hypothesis that this receptor-ligand pair was involved in the ocular surface damage occurring in DED patients. Similar to previous reports in VKH disease (Levinson et al., 2008), the aKIR-HLA combinations were more common in patients and the data suggested that the signals transduced by the aKIRs upon their binding to putative HLA class I ligands may overcome HLA class I-dependent inhibition and trigger NK reactivity, leading to the autoimmune condition in VKH. More interestingly, a study in SS (Lowe et al., 2009), the combination of KIR2DS2 and the corresponding HLA-C1 ligand in the complete absence of KIR2DL2 was associated with risk to primary SS, although no significant difference of KIR2DS2 distribution was observed in the SS patients and controls. Combining with our data, these findings support previously proposed models of KIR-mediated autoimmunity (Nelson et al., 2004).
Disease-promoting roles of NK cells in autoimmunity include release of IFN-γ, actual killing of target tissue cells, and activation of dendritic cells in lymph nodes (Johansson et al., 2005). A similar IFN-γ-dependent and disease-promoting effect of NK cells was made in a study of murine experimental autoimmune encephalomyelitis (Shi et al., 2000). Interestingly, El Annan et al. (2009) found that significantly higher levels of IFN-γ were expressed in a DED mice model. It was reported that NK cells secreted IFN-γ promoted induction of DED via direct target tissue damage, and subsequently influenced on the immune responses in secondary lymphoid tissue (Chen et al., 2011). The activation of NK cells producing IFN-γ is modulated by several different receptor-ligand combinations, including most important KIR and HLA interaction. Therefore, we speculate that IFN-γ productions in response to DED maybe have association with combination of KIR2DS2 with HLA-C1, but this needs to be confirmed further. Here, we consider two hypotheses by which KIR2DS2 and HLA-C1 might affect NK cells producing IFN-γ in responses to DED. First, signals produced from the interaction of KIR2DS2 and HLA-C1 might modulate the degree of activation of NK cells. Second, binding of KIR2DS2 by HLA-C1 during NK cell ontogeny might lead to greater education of NK cells.
Footnotes
Author Disclosure Statement
No competing financial interests exist.