Association Of IKIR And HLA-Cw Gene Polymorphisms With Susceptibility To Inflammatory Bowel Disease

Inflammatory bowel disease(IBD) includes ulcerative colitis(UC) and Crohn's disease(CD) which are characterized by chronic illness of unknown etiology,it is thought that environmental stimuli including inflammation trigger inappropriate immune response in a genetically susceptible host with some genetic defect.A variety of epidemiological and clinical data suggest that genetic factors are intimately involved in the pathogenesis of IBD including familial aggregation pattern of disease with a much higher disease frequency in first degree relatives of affected individuals compared with the general population.Twin with a much higher rate of disease concordance observed in monozygotic than in dizygotic twins and wide variations in the incidence and prevalence of IBD among different populations.The whole genome scans studies and candidate gene studies have supported that IBD are likely to share some susceptibility genes.Linkage analysis allows scanning of the whole genome. Eleven of these total genome scans have been undertaken in IBD,resulting in a number of susceptibility regions on chromosomes 1,3,4,5,6,7,10,12,14,16,19 and X.According to their initial date of reporting and independent confirmations,the regions on chromosomes 16q,12,6,14,5,19,1,16p and 10 have been renamed IBD1 to IBD9,respectively.UC and CD are considered to be complex polygenic diaeases.The killer immunoglobulin-like receptor(KIR) gene family is located with in the IBD6 linkage region at chromosome 19q13.4.KIRs are expressed on natural killer cells and subsets ofγδ⁺T cells and memory and effectorαβ⁺T cells(usually CD8⁺T cells and some CD4⁺T cells),and can be divided on functional grounds into inhibitory and activating receptors.KIR possess a variety of inhibitory and activating receptors that upon recognizing and binding to their HLA classâ… ligands on target cells,and regulate activation and inhibition of NK cell and some T cell.Inhibitory receptors with specificity for HLA classâ… appear to be important mediators of self-tolerance for NK cells.At present,clinical studies had shown that KIR gene associated with varied diseases,including tumor immune,virus infection,Maternal-fetal immune tolerance, bone marrow transplantation,autoimmune disease,and so on.An emerging body of evidence is accumulating to suggest that KIRs contribute to the pathogenesis of diverse kinds of autoimmune diseases such as Rheumatoid arthritis(RA),systemic lupus crythematosus(SLE),Ankylosing spondylitis(AS),Psoriatic arthritis(PsA), and Multiple sclerosis(MS).A much higher risk for RA,AS,PsA,and MS was observed in IBD patients,IBD may share an underlying pathogenesis with other autoimmune diseases.HLA-Cw belongs to classical HLA classâ… ,in resent studies, HLA-Cw was shown to bind KIR on NK cells,which could modulate NK cells and some T cells.Combinations of KIR and HLA classâ… ligand variants that reduce NK cell inhibition have been shown to increase susceptibility to autoimmune diseases.At present,the studies of KIR gene are very few in IBD.The aim of this study was to explore whether iKIR-HLA-Cw combinations are associated with IBD susceptibility by PCR-SSP.HLA-Cw^*0602 gene were cloned,expressed on K562 cells to interact with NK cells from peripheral blood.So that we could observe how KIR2DL1-HLA -Cw^*0602 combination influence NK cytotoxicity and IFN-γsecretion from PBMCs,and explore how KIR2DL1-HLA-Cw^*0602 would influence the development of IBD.To provide the theory foundation for clinical therapy in IBD.Objectives1.To investigate the gene polymorphism of iKIR in patients with IBD and explore whether the iKIR gene polymorphisms are associated with IBD.2.To investigate the distribution of iKIR-HLA-Cw combinations in patients with IBD,and to explore whether iKIR/HLA-Cw combinations are associated with IBD susceptibility.3.To investigate influences of iKIR-HLA-Cw combinations on NK cytotoxicity and on IFN-γsecretion from PMBC in IBD patients.To explore how KIR2DL1-HLA-Cw^*0602 would influence the development of IBD,and To provide the theory foundation for clinical therapy in IBD.Methods1.iKIR genotypingThe samples were recruited from the Second Affiliated Hospital of Zhengzhou University,the First Affiliated Hospital of Henan University of Traditional Chinese Medicine,the Central Hospital of Xinxiang,Suzhou Municipal Hospital during October 2006 to January 2008.The study included 100 patients with UC,73 patients with CD.IBD was diagnosed and classified according to standard clinical,endoscopic or radiological and histological criteria.All patients met the diagnosis criteria of IBD in Jinan meeting(2007),excluding other autoimmune diseases,virus infection and tumor.106 age,ethnical and gender-matched healthy controls was randomly selected. Venous blood was gathered and anticoagulated with EDTA,Genomic DNA was extracted from whole blood using a Relax Gene Blood DNA system(TIANGEN, China).The iKIR genotyping was performed by means of sequence specific primer polymerase chain reaction(PCR-SSP) in all the recruited subjects for the following iKIRs:KIR2DL1,KIR2DL2,KIR2DL3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2 and KIR3DL3.The primers were synthesized by Sangon Co,Shanghai.Based on the KIR typing result,iKIR expressing frequencies was summed and compared in IBD and controls.2.HLA-Cw genotyping and analysis of iKIR and HLA-Cw combinationHLA-Cw genotyping were performed by the HLA-C LOCUS SSP UNITRAY^? typing kits(Invitrogen,USA).PCR-SSP sets was used for typing according to the manufacturer's instructions.HLA-Cw alleles were assigned using the UniMatch Program provided by Dynal(Invitrogen,USA).Based on the iKIR and HLA typing result,Distribution of different combination of iKIR and its HLA ligand was compared in IBD and controls.3.Construction of mammalian cell expression vector of human leukocyte antigen-Cw gene and expression on K562 cellsTotal cell RNA was extracted from peripheral blood cells in a healthy people, and the HLA-Cw0602^*cDNA was amplified by RT-PCR.the cDNA fragments were directly bind with pMD-19 T vector,transformation,the recombinant pMD-19 T vector and HLA-Cw^*0602 was identified by sequencing,the plasmid of pcDNA3.1-HLA -Cw^*0602 was transfected into K562 cells by lipofectamine 2000 and positive clones were selected by G418.The expressions of HLA-Cw^*0602 on K562 were analysed by RT-PCR and FACS.4.Influences of HLA-Cw antigens on cytotoxicity of NK cells and on IFN-γsecretion from PBMC in IBD patients.â‘ The expressions of KIR2DL1 on PBMC were analysed by FACS in IBD patients and healthy controls.â‘¡NK cells from IBD patients and healthy controls with KIR2DL1 positive were randomly selected to detect their cytotoxicity on K562 cells that were transfected with HLA-Cw^*0602 and empty pcDNA3.1 plasmid respectively by the LDH method.â‘¢NK cells from IBD patients and healthy controls with KIR2DL1 positive were randomly selected to detect the influences of K562-HLA-Cw^*0602 cells on IFN-γsecretion from PBMC by the ELISA method.Results1.iKIR genotypingiKIR typing results in 100 cases of patients with UC showed that,KIR gene phenotype frequency was KIR2DL1(0.710),KIR2DL2(0.150),KIR2DL3(0.620), KIR2DL4(1.000),KIR2DL5(0.230),KIR3DL1(1.000),KIR3DL2(1.000),KIR3DL3 (1.000),respectively;iKIR typing results in 73 cases of patients with CD showed that, KIR gene phenotype frequency was KIR2DL1(0.740),KIR2DL2(0.164),KIR2DL3 (0.767),KIR2DL4(1.000),KIR2DL5(0.192),KIR3DL1(0.973),KIR3DL2(1.000), KIR3DL3(1.000),respectively.The framework genes KIR2DL4,KIR3DL2 and KIR3DL3 were present in all individuals.The KIR2DL1 and KIR2DL3 gene phenotype frequencies in UC patients were 0.710 and 0.620 respectively,both significantly lower than those in healthy controls(P=0.001).The KIR2DL1 gene phenotype frequency in CD patients was 0.731,significantly lower than that in healthy controls(P=0.007),and none of the other iKIR differed significantly.2.HLA-Cw genotyping and analysis of iKIR and HLA-Cw combinationThe HLA-Cw genotype were analyzed in patients with IBD and healthy controls by PCR-SSP.20 HLA-Cw genotype were detected,iKIR and HLA typing results in further studied by iKIR and HLA-Cw combination.KIR2DL2/2DL3-HLA-C1 is dominant in IBD patients and controls.The KIR2DL1-HLA-C2 combination (2DL1⁺-HLA-C2⁺) in UC patients and CD patients were 0.380 and 0.411 respectively,both significantly lower than that in healthy controls(P=0.005 and P= 0.030,respectively).The phenotype frequencies of KIR2DL2/KIR2DL3-HLA-C1 combination in IBD patients did not significantly differ from those in healthy controls. KIR-HLA disparity occurred in this study.Frequency of expressing KIR2DL1 or KIR2DL2/2DL3 without corresponding HLA ligand was 0.330,0.060 in UC patients. Frequency of expressing HLA-C1,HLA-C2 without corresponding iKIR was 0.200, 0.100 in UC patients.Frequency of expressing KIR2DL1 or KIR2DL2/2DL3 without corresponding HLA ligand was 0.329,0.137 in CD patients.Frequency of expressing HLA-C1,HLA-C2 without corresponding iKIR was 0.151,0.110 in CD patients. There isn't significantly different in IBD patients and healthy controls.3.Construction of mammalian cell expression vecor of human leukocyte antigen-Cw gene and expression on K562 cellsthe sequences of the insert was identical to the published sequences encoding HLA-Cw^*0602 gene.The recombinant mammalian cell expression vectors of pcDNA3.1-HLA-Cw^*0602 was constructed,Transfected K562 cell clone was obtained,which expressed HLA-Cw^*0602 in high level and constitutively.4.Influences of HLA-Cw antigens on cytotoxicity of NK cells and on IFN-γ secretion from PBMC in IBD patients.â‘ KIR2DL1 mean expression was(31.78±10.42)%on PBMC of healthy controls,KIR2DL1 mean expression showed significantly higher in UC [(45.90±16.52)%]and CD[(44.39±13.30)%]than that in healty controls(P=0.035å’ŒP=0.033).â‘¡NK from IBD patients and healthy controls showed significantly decreased cytotoxicity on K562-HLA-Cw^*0602 cells than that on empy pcDNA3.1 transfected K562 cells(P=0.000),NK cytotoxicity significantly increased(P=0.000)when KIR2DL1 were blocked with a specific monoclonal antibody.Comparing to healty controls,NK cells from UC and CD patients showed decreased cytotoxicity on K562-HLA-Cw^*0602 cells(P=0.000).â‘¢In IBD patients and healthy controls,Comparing to empy pcDNA3.1 transfected K562 cells,IFN-γsignificantly decreased from PBMC on K562-HLA-Cw^*0602 cells(P=0.000).Comparing to healty controls,IFN-γsignificantly increased from PBMC on empy pcDNA3.1 transfected K562 cells in UC and CD patients(P=0.000).Conclusion1.The KIR2DL1 and KIR2DL3 gene phenotype frequencies were decreased in UC patients,suggesting that decreased gene phenotype frequencies of KIR2DL1 and KIR2DL3 are associated with the susceptibility of UC.The KIR2DL1 gene phenotype frequency was significantly decreased in CD group than in healthy control group, suggesting that decreased gene phenotype frequencies of KIR2DL1 are associated with the susceptibility of CD.2.The KIR2DL1-HLA-C2 combinations in IBD patients significantly decreased than that in healthy controls.Reduced combinations of KIR2DL1-HLA-C2 in patients with UC and CD probably make NK cell and some killer T cell lack sufficently inhibitory signal,attack self cell,and finally result in autoimmune disease. Our results showed that IBD(UC and CD) susceptibility is mainly associated with a decrease in KIR2DL1-HLA-C2 combinations. 3.KIR-HLA disparity occurred in this study.4.KIR2DL1 mean expression showed significantly higher in UC and CD,which showed a pathological condition of IBD self.5.HLA-Cw^*0602 on K562 could inhibit NK cytotoxicity significantly though binding KIR2DL1.Comparing to healty controls,NK from UC and CD patients showed decreased cytotoxicity on K562-HLA-Cw^*0602 cells,which may be associated with higher KIR2DL1 expression on PBMC in IBD.6.HLA-Cw^*0602 on K562 could inhibit IFN-γsecretion from PBMC though binding KIR2DL1.Comparing to healty controls,IFN-γsignificantly increased from PBMC on empy pcDNA3.1 transfected K562 cells in UC and CD patients,which showed that IFN-γwas associated with the development of UC and CD.