The Study Of The Activity Of HLA-B27 Promoter And The Pathogenesy Of The Promoter-activating Factors In SpA

Objectives:The spondyloarthropathies (SpAs) are the second-commonest cause of inflammatory arthritis in humans, after rheumatoid arthritis. One genetic factor shared by all the varieties of SpA has already been identified, that is, HLA-B27. Although the presence of the HLA-B27 allele marks a strong predisposition for SpA, its role in disease pathogenesis is still unclear. Until now, there are three main hypotheses explaining the role of HLA-B27 in SpA: 1) Molecular mimicy: HLA-B27 or HLA-B27 bound peptides were thought to resemble bacterial peptides, and thus become the target of cross-reactive antibodies and/or CD8~+ cells. 2) Immunological recognition of aberrant forms of HLA-B27: the aberrant forms of HLA-B27 such as free heavy chains or HLA-B27 homodimers are specifically recognized by the receptors on NK, CD4~+ T cells or other immune cells. 3) Protein misfolding, ER stress, and inflammatory disease: HLA-B27 misfolding results in heavy chain retension and cause unfolded preotein response (UPR) activation that perhaps initiate or perpetuates a chronic inflammatory process. These hypotheses are based on the activities of the HLA-B27 protein, and hence would require the HLA-B27 gene to be transcribed.It is believed in addition to certain other genes and/or environmental factors, the expression degree of the HLA-B27 gene is also responsible for susceptibility to this disease. HLA-B27 positivity strongly influences spondyloarthropathy (SpA) susceptibility and phenotype, and experiments of HLA-B27 transgenic rats showed the gene level is probably linked to its arthritis-causing potential. Other people reported that the expression of the HLA-B27 molecule on the surface of PBMCs is higher in patients with AS than in HLA-B27-positive healthy volunteers. However, very little research has been conducted to investigate HLA-B27 gene regulation.Based on several observations suggesting that factors modulating HLA-B27 transcription may play a role in the pathogenesis of SpA, we designed this study to identify factors that might be responsible for the transcription of HLA-B27 in human monocytic cells and to assess whether any differences in the UPR can be detected following incubation with these promoter-activating factors.Methods:1. The IMGT/HLA Database was used to compare HLA-B2705 with other HLA B alleles and HLA A alleles at the 5'untranslated region to find out the similarities and differences especially at transcription binding sites.2. After that we used 25 specimens of synovial fluid of SpA patients to incubate the Hela B27 promoter transfectants for 48 hours, and TNF-αinhibitor was added into those positive specimens.3. Then the promoter region of HLA-B27 was transfected into U937 cell line to reveal the respond of B27 promoter in human monocyte cell line. The transfectant was challenged separately with 25 kinds of cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12-p70, IL-13, IL-15, IL-17, IL-18, TNF-α, M-CSF, GM-CSF, IP-10, MCP-1, RANTES, MIP-1α, IFN-α, IFN-β, IFN-γ, TGF-β1 and OSM) and 12 kinds of Toll Like Receptor ligands (MDP, PGN, Zyn, PIC, LPS, Flag, FSL, Lox, ssRNA, EcDNA, Pams and Decap KP). Promoter activity is measured by Flow Cytometry.4. Quantitive RT-PCR was used to study the mRNA expression of UPR related genes (GRP78, CHOP) in PBMC of SpA patients. Meanwhile, we constructed HLA-B27-EGFP, HLA-A2-EGFP and EGFP-alone stable transfected U937 cells and examined the FHC expression on them. Moreover, the UPR in B27 and A2 transfected U937 cells were studied by RT-PCR. HLA-B27 promoter-activating factors were used to incubate these cell lines.Resultes:1. There are just 6 B alleles including 3 B27 subtypes having the same sequence with HLA-B2705. X1/X2, S and CAAT Box binding site of B alleles have more variability than others. ISRE and kB1/2 are highly conservative in HLA B alleles.2. 5 out of 25 specimens of SF from SpA patients could significantly increase the activity of the HLA-B27 promoter. None of the RA specimens caused the same effect. The promoter activation caused by SF could be significantly inhibited by anti-TNF-αantibody3. The CBA and RT-PCR results showed that these cytokines, TNF-α,IL-10,IL-6 and IL-1, were significantly higher in the PBMC of AS patients than that of normal control.Out of TNF-α, only INF-α, -β, -γcan activate the B27 promoter in U937 cell line. And all the increase effects and the incubate time have positive correlation in 72 hours. The dose response relationship for HLA-B27 promoter activity in response to these cytokines was tested, and there were no significant differences in this cell line. When the peaks were compared, the most effective cytokine was INF-γ.4. Compared with the PBMC of normal control, the GRP78 and CHOP expression of SpA was significantly higher (P<0.05). The mRNA expression of GRP78, XBP-1 and CHOP were same in EGFP-U937, B27-U937 and A2-U937 cells. Then we then used IFN-γ, LPS, and TNF-αas stimulators to activate these U937 cells. The PCR results showed that after IFN stimulation for 8 h, CHOP and GRP78 expression were all increased compared with unstimulated cells, especially the GRP78 expression. But there were no significant differences between B27, A2-U937, and EGFP-U937 cells. LPS and TNF-αstimulation demonstrated the same results and we found that HLA-B27 does not modulate XBP-1 splicing following cytokine stimulation also.Conclusion:1. Our results confirmed that the cytokines including TNF-α,IL-10,IL-6 and IL-1βsecreted by PBMC play important roles in SpA. We found that Synovial fluids of spondyloarthropathy patients can significantly increased activity of HLA-B27 promoter and the increase was inhibited by TNF-αinhibitor. All these results indicate that cytokines play a very important role in pathogenesy of SpA.2. After compared HLA-B2705 with other HLA B alleles and HLA A alleles and screened cytokines and Toll Like Receptor ligands, we found out that only INF and TNF-αwhich associated with ISRE and kB1/2 binding sites can activate B27 promoter significantly. These results inferred that the HLA-B27 promoter activating factors could possibly stimulate the expression of HLA-B27 to induce the UPR in B27 expression cells.3. Quantitive RT-PCR was used to study the mRNA expression of UPR related genes (GRP78, CHOP) in PBMC of SpA patients. Compared with the PBMC of normal control, the GRP78 and CHOP expression of SpA was significantly higher. The result indicated there is UPR in SpA patients. Then we used these HLA-B27 promoter activating factors as stimulators to activate these transfected U937 cells. The PCR results showed that after stimulation, GRP78, CHOP and XBP-1 expression were all increased compared with unstimulated cells. But there were no significant differences between B27, A2-U937, and EGFP-U937 cells. These results were different with our early research. So, further studies on the response of the HLA-B27 promoter to cytokines in different kinds of cells, especially primary human monocytes/macrophages, combined with our findings will provide further understanding of the roles of HLA-B27 in the pathogenesis of SpA.