| BackgroundRheumatoid arthritis (rheumatoid arthritis, RA) is an autoimmune disease with chronic synovitis as the main pathology, often leading to joint destruction and disability. Pathogenesis of RA is not yet very clear, which includes genetics, environmental factors and autoimmune disorders and so on. Above all, genetic factors play the most important role, which HLA-DRB1 alleles coding the five amino acid sequence of QKRAA, QRRAA or RRRAA are associated with this disease. This five amino acid sequence locate in the 70-74 bits of β chain of the third hypervariable region coded by these HLA-DRB1 alleles, which is called the shared epitope (SE). Shared epitope hypothesis was raised by PETER K in 1987. Shared epitope generally refers to the five highly conserved amino acid sequence locating in the P chain third high 70-74-bit variable region coded by some HLA-DRB1 alleles, which is high expressed in rheumatoid arthritis patients. There are three homologous amino acid sequence variants:1. QKRAA mainly coded by HLA-DRB 1*0401 allele.2. QRRAA, mainly coded by HLA-DRB 1*0404, HLA-DRB1*0101 and HLA-DRB1*0405 alleles.3. RRRAA, coded by HLA-DRB1*1001 allele. Wherein QKRAA is mainly common in Caucasians and RRRAA is very rare in all populations. Studies also showed that HLA-DRB1 SE is not only associated with the susceptibility of RA but also associated with the severity of RA. In addition, DRB1 alleles coding SE have the alleles dose effect, which the homozygotes (2 alleles coding SE) have a higher severity of RA compared with the heterozygous (1 alleles coding SE) and the heterozygous have a higher severity of RA compared with those without alleles coding SE in RA patients. However, these studies are based on genetic factors. The role and the mechanism of HLA-DRB1 shared epitope in RA are still not clear yet.In addition to rheumatoid arthritis, several studies inicated that HLA-DRB1 shared epitope is also associated with other diseases, including polymyalgia rheumatica, giant cell arteritis, type I diabetes, psoriatic arthritis, lupus, autoimmune hepatitis, and the early onset of chronic lymphocytic leukemia. In addition, HLA-DRB1 SE is associated with spontaneous arthritis in canine. In the HLA-DRB 1*0401 transgenic mice, SE is related to the susceptibility of spontaneous diabetes, collagen-induced arthritis and the severity of autoimmune encephalomyelitis. However, the relationship between SE and ankylosing spondylitis (AS), which is also HLA-DRB 1 relevant, have not been reported yet.Therefore, this study aims to investigate the immune response of HLA-DRB 1 in rheumatoid arthritis and the association of HLA-DRB 1 shared epitope and ankylosing spondylitis.Part I:Immune response of CD3+CD4+T cells to HLA-DRB1 shared epitope in rheumatoid arthritis patientsObjectiveTo observe immune response of CD3+CD4+T cells to shared epitope in rheumatoid arthritis patients.Method1. Peripheral blood of rheumatoid arthritis patients and healthy people were collected, including 18 cases of rheumatoid arthritis patients,17 cases of healthy people.2. QIAGEN DNA extraction kit was used to extract DNA from peripheral blood.3. SSP-PCR was used to DNA samples for HLA-DRB1 genotyping. First, low-resolution typing for HLA-DRB1*01, HLA-DRB1*04 and HLA-DRB1*10 was performed. And then the high-resolution typing for HLA-DRB1*01, HLA-DRB1*04 was performed. Genotypes were determined to the PCR results.4. Peripheral blood mononuclear cells (PBMC) were collected by using Ficoll (density gradient centrifugation).5. PBMC were divided into five groups and stimulus were added to stimulate cells for six hours. The first group served as positive controls, Jiarufobo ester (PMA) and ionomycin were added. In the second group, HLA-DRB1*0401 peptide (KDLLEQKRAAVDTYC) was added. In the third group, HLA-DRB1*0405 peptide (KDLLEQRRAAVDTYC) was added. In the fourth group, HLD-DRB1*0402 peptide (KDILEDERAAVDTYC) was added. In the fifth group, medium was added serving as the control group.6. PBMC were dye-labeled by fluorescent and CD3+CD4+ cells were selected. Intracellular cytokines IFN-γ and IL-17 were dye-labeled. Results were analyzed by flow cytometry and were presented by the frequency of IFN-γ, IL-17 positive cells.7. All datas presented by mean±standard deviation and were analyzed by using SPSS 19.0. Kruskal-Wallis Test was used to compare multiple sets of datas, NemenyiTest was used to compare two sets of datas. P values less than 0.05 are determined as statistically significant.Result1. Genotyping results showed that a total of nine cases were SE positive in rheumatoid arthritis patients, including two cases of HLA-DRB 1*0401 and 7 cases of HLA-DRB 1*0405. The rest 9 cases were SE negative.3 cases were SE positive in healthy people and all were HLA-DRB 1*0405. The rest 14 cases were SE negative.2. In rheumatoid arthritis patients, stimulating by SE postive HLA-DRB 1*0401 peptide (KDLLEQKRAAVDTYC), the frequency of CD3+CD4+IFN-γ+T cells was 0.06±0.04%, the frequency of CD3+CD4+IL-17+T cells was 0.16±0.16% in HLA-DRB1*0401 patients. The frequency of CD3+CD4+IFN-γ+T cells was 0.11±0.04%, the frequency of CD3+CD4+IL-17+T lymphocytes was 0.26±0.11% in HLA-DRB 1*0405 patients. The frequency of CD3+CD4+IFN-γ+T cells was 0.04±0.02%, the frequency of CD3+CD4+IL-17+T cells was 0.20±0.07% in SE negative patients.Stimulating by SE postive HLA-DRB 1*0405 peptide (KDLLEQRRAAVDTYC), the frequency of CD3+CD4+IFN-γ+T cells was 0.05±0.05%, the frequency of CD3+CD4+IL-17+T cells was 0.70±0.10% in HLA-DRB1*0401 patients. The frequency of CD3+CD4+IFN-γ+T cells was 0.43±0.09%, the frequency of CD3+CD4+IL-17+T lymphocytes was 0.40±0.11% in HLA-DRB1*0405 patients. The frequency of CD3+CD4+IFN-γ+T cells was 0.03±0.01%, the frequency of CD3+CD4+IL-17+T cells was 0.27±0.09% in SE negative patients.Stimulating by SE negative HLA-DRB 1*0402 peptide (KDILEDERAAVDTYC), the frequency of CD3+CD4+IFN-γ+T cells was 0.02±0.02%, the frequency of CD3+CD4+IL-17+T cells was 0.18±0.07% in HLA-DRB 1*0401 patients. The frequency of CD3+CD4+IFN-γ+T cells was 0.05±0.03%, the frequency of CD3+CD4+IL-17+T lymphocytes was 0.07±0.03% in HLA-DRB 1*0405 patients. The frequency of CD3+CD4+IFN-γ+T cells was 0.02±0.01%, the frequency of CD3+CD4+IL-17+T cells was 0.09±0.02% in SE negative patients.3. In healthy people,Stimulating by SE postive HLA-DRB 1*0401 peptide (KDLLEQKRAAVDTYC), the frequency of CD3+CD4+IFN-γ+T cells was 0.02±0.02%, the frequency of CD3+CD4+IL-17+T cells was 0.10±0.05% in HLA-DRB1*0405 people. The frequency of CD3+CD4+IFN-y+T cells was 0.07±0.03%, the frequency of CD3+CD4+IL-17+T cells was 0.10±0.04% in SE negative people.Stimulating by SE postive HLA-DRB 1*0405 peptide (KDLLEQRRAAVDTYC), the frequency of CD3+CD4+IFN-γ+T cells was 0.09±0.03%, the frequency of CD3+CD4+IL-17+T cells was 0.19±0.08% in HLA-DRB 1*0405 people. The frequency of CD3+CD4+IFN-γ+T cells was 0.07±0.02%, the frequency of CD3+CD4+IL-17+T cells was 0.15±0.06% in SE negative people.Stimulating by SE negative HLA-DRB 1*0402 peptide (KDILEDERAAVDTYC), the frequency of CD3+CD4+IFN-y+T cells was 0.02±0.01%, the frequency of CD3+CD4+IL-17+T cells was 0.12±0.10% in HLA-DRB 1*0405 people. The frequency of CD3+CD4+IFN-y+T cells was 0.05±0.03%, the frequency of CD3+CD4+IL-17+T cells was 0.18±0.08% in SE negative people.4. In SE+RA patients, the frequency of CD3+CD4+INF-γ+T cells was significant differenct (P=0.015<0.05) in three groups. The frequency of CD3+CD4+INF-γ+T cells stimulated by the genotype-corresponding peptides was significantly higer than the other two groups (P= 0.035<0.05 and P=0.003<0.05). While the frequency of CD3+CD4+IL-17+T cells had no significant differences in the three groups (P=0.053> 0.05). In SE-RA patients both the frequency of CD3+CD4+INF-γ+T cells and CD3+CD4+IL-17+T cells had no significant differences in the three groups (P=0.354>0.05 and P=0.348> 0.05).In healthy people, the frequency of CD3+CD4+INF-γ+T cells and CD3+CD4+IL-17+T cells of SE+and SE-people had no significant differences in Ihe three groups (P=0.095>0.05 and P=0.651>0.055 P=0.623>0.05 and P=0.932>0.05).Conclusion1. In rheumatoid arthritis, HLA-DRB1 shared epitope may stimulate CD3+CD4+T cells of shared epitope-positive patients to produce more IFN-y, resulting in the susceptibility of rheumatoid arthritis and exacerbating disease severity.2. In rheumatoid arthritis, HLA-DRB1 shared epitope can not stimulate the CD3+CD4+T cells to produce more IL-17.Part II:Immune response of CD11c+CD8-dendritic cells to HLA-DRB1 shared epitope in rheumatoid arthritis patientsObjectiveTo observe immune response of CDllc+CD8-dendritic cells (DCs) to shared epitope in rheumatoid arthritis patients.Method1. Peripheral blood of 12 cases of rheumatoid arthritis patients were collected.2. QIAGEN DNA extraction kit was used to extract DNA from peripheral blood.3. SSP-PCR was used to DNA samples for HLA-DRB1 genotyping. First, low-resolution typing for HLA-DRB1*O1, HLA-DRB1*04 and HLA-DRB1*10 was performed. And then the high-resolution typing for HLA-DRB1*01, HLA-DRB1*04 was performed. Genotypes were determined to the PCR results.4. Peripheral blood mononuclear cells (PBMC) were collected by using Ficoll (density gradient centrifugation).5. Dendritic cells (DCs) were cultured by adherent PBMC. GM-CSF and IL-4 were added to the medium in day 1,3,5. DCs were collected in day 6.6. DCs surface markers CDllc and CD83 were dye-labeled by fluorescent and analyzed by flow cytometry.7. DCs were divided into five groups and stimulus were added to stimulate cells for six hours. The first group served as positive controls, TNF-a was added. In the second group, HLA-DRB1*0401 peptide (KDLLEQKRAAVDTYC) was added. In the third group, HLA-DRB 1*0405 peptide (KDLLEQRRAAVDTYC) was added. In the fourth group, HLD-DRB 1*0402 peptide (KDILEDERAAVDTYC) was added. In the fifth group, medium was added serving as the control group.8. DCs were dye-labeled by fluorescent and CD11c+CD8-cells were selected. Intracellular cytokine IL-6 was dye-labeled. Results were analyzed by flow cytometry and were presented by the frequency of IL-6 positive cells.9. All datas presented by mean±standard deviation and were analyzed by using SPSS 19.0. Kruskal-Wallis Test was used to compare multiple sets of datas. P values less than 0.05 are determined as statistically significant.Result1. Genotyping results showed that a total of 6 cases were SE positive in rheumatoid arthritis patients, including 2 cases of HLA-DRB 1*0401 and 4 cases of HLA-DRB1*0405. The rest 6 cases were SE negative.2. The frequency of CD11c+DCs was 89.94±2.82% and CD83+DCs was 6.46± 0.98%. With microscopic morphology, DCs collected in day 6 were immature DCs.3. Stimulating by SE postive HLA-DRB 1*0401 peptide (KDLLEQKRAAVDTYC), the frequency of CDllc+CD8-IL-6+DCs was 0% in HLA-DRB1*0401 patients, the frequency of CDllc+CD8-IL-6+DCs was 1.66±0.32% in HLA-DRB 1*0405 patients, the frequency of CDllc+CD8-IL-6+DCs was 0.83±0.46% in SE negative patients.Stimulating by SE postive HLA-DRB 1*0405 peptide (KDLLEQRRAAVDTYC), the frequency of CDllc+CD8-IL-6+DCs was 0.04±0.04% in HLA-DRB1*0401 patients, the frequency of CDllc+CD8-IL-6+DCs was 0.11±0.07% in HLA-DRB 1*0405 patients, the frequency of CDllc+CD8-IL-6+DCs was 0.52±0.18% in SE negative patients.Stimulating by SE postive HLA-DRB 1*0402 peptide (KDILEDERAAVDTYC), the frequency of CD11c+CD8-IL-6 DCs was 0.03±0.03% in HLA-DRB1*0401 patients, the frequency of CDllc+CD8-IL-6+DCs was 0.11±0.11%in HLA-DRB 1*0405 patients, the frequency of CD11c+CD8-IL-6+DCs was 0.26±0.17% in SE negative patients.In rheumatoid arthritis patients, the frequency of CDllc+CD8-IL-6+DCs of SE+and SE-patients had no significant differences in the three groups (P=0.304>0.05 and P=0.354> 0.05).ConclusionIn rheumatoid arthritis, HLA-DRB1 shared epitope can not stimulate CD11c+CD8-dendritic cells to produce more IL-6.Part Ⅲ:Association of HLA-DRB1 shared epitope and ankylosing spondylitisObjectiveTo explore the association of shared epitope and ankylosing spondylitis.Method1. DNA of 182 cases of ankylosing spondylitis patients were collected. Dates of 404 cases of healthy people were from the laboratory of Department of Rheumatology and Immunology, Changzheng Hospital, Second Military Medical University.2. SSP-PCR was used to DNA samples for HLA-DRB1 genotyping. First, low-resolution typing for HLA-DRB1*01, HLA-DRB1*O4 and HLA-DRB1*10 was performed. And then the high-resolution typing for HLA-DRB1*01, HLA-DRB1*04 was performed. Genotypes were determined to the PCR results.3. All datas presented by mean±standard deviation and were analyzed by using SPSS 19.0 and Chi-square test was used. P values less than 0.05 are determined as statistically significant.Result1. In ankylosing spondylitis patients,37 cases were SE positive with a ratio of 20.33%. In healthy people,91 cases were SE positive wih a ratio of 22.52%.2. It’s no significant differences between ankylosing spondylitis patients and healty people (P=0.56>0.05).ConclusionHLA-DRB1 shared epitope is not association with ankylosing spondylitis. |
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