Cyclophilin A's Function To Monocytes/Macrophages In Rheumatoid Arthritis Via CD147 Signaling Pathway

Aim: Rheumatoid arthritis (RA) is a chronic destructive autoimmune disease characterized by the inflammation and progressive destruction of distal joints. Until now, the pathogenesis of RA has not been understood well and the therapy of RA just controls the symptom and development of RA, but doesn't cure it thoroughly. Multiple inflammatory cells and cytokines contribute to the development of RA. Among these cells, macrophage's rich array of secretory products, anatomic diversity and functional heterogeneity is unmatched by any other cell type. As a result of this remarkable versatility, the macrophage is able to influence every facet of the immune response and inflammation as well as playing a central role in the etiology and/or pathogenesis of a number of disease processes, especially in RA. Cyclophilin A (CypA), involved in numerous inflammatory events, is abundant in rheumatoid arthritis (RA) synovial fluid and is the major target of immunosuppressive drug cyclosporin A (CsA). It has multiple functions, such as peptidyle-prolyl-isomerase activity (PPIase) for protein folding and transducting, taking part in the immunologic derangement and contributes to inflammation. Its PPIase is detected in RA synovium, not in osteoarthritis (OA) synovium, and correlates well with the number of total cells in RA synovium. In RA synovial tissues, various cells, including macrophages and endothelial cells, are the sources of CypA. CypA can induce many typies of inflammatory cytokines to promote the destruction of cartilage and bone in RA. Although these findings showed the potential of CypA in RA pathogenesis, litte articles showed the receptor, signaling pathway of CypA and its function to macrophages in RA.CD147, the receptor of CyPA, also named extracellular matrix metalloproteinase inducer (EMMPRIN), belongs to immunoglobulin superfamily and is one of CypA's receptors. It is an important role in the embryonic development, organ formation, bony remodeling, wounds repairing in physiology, and tumor metastasis, arthritis and fibrosis in pathophysiology. Previous studies in our lab showed that membrane CD147 expression on monocytes/macrophages in RA synovium is much higher than that on monocytes/macrophages derived from RA patient's peripheral blood and that CD147 overexpression on synoviocytes in RA synovium enhances the production of MMP-2, -9 and the invasiveness of synoviocytes. But some cytokines which are involved in the development of RA via CD147 pathway signaling have not been reported yet.The potential role of interaction between CypA and CD147 has been shown in some diseases, such as AIDS, Severe Acute Respiratory Syndrome (SARS), and prostatic carcinoma. CypA can enhance the developments of these diseases via binding to membrane CD147 on host cells. But the CypA-CD147 pathway in RA pathogenesis has not been reported yet. So in this study, we want to identify whether CypA can upregulate the expression of matrix metalloproteinase 2 (MMP-2) and MMP-9 in monocytes/macrophages and the adhesion of monocytes/macrophages to Extracellular matrix (ECM); and RNAi, antibody or HAb18G/CD147 antagonistic peptide AP-9 against CD147 were used to confirm whether CD147 facilitates these functions.It is the first time that the interaction between CypA and CD147 will be studied for RA pathogenesis in our lab. The results of the study we believe may help us to gain a better understanding of the potential role of CypA-CD147 pathway in RA pathogenesis, and may give good ideas for the future RA therapy.Methods:1. CypA stimulation to MMP-2, -9 expression and invasion ability of RA monocytes/macrophagesThe monocytes/macrophages were isolated from RA peripheral blood and synovial fluid by using the Monocyte Negative Isolation Kits. Monocytes were differentiated into macrophages by recombinant human M-CSF. recombinant human cyclophilin A (CypA) stimulated monocytes/macrophages, MMP-2, -9 productions and invasion abilities were detected by gelatin zympgraphy and invasion assay respectively. At the same time, cyclosporin A (CsA) and HAb18G/CD147 antagonistic peptide AP-9 were used interrupting the interaction between CypA and CD147 to identify whether CD147 facilitates CypA's function.2. siRNA targeted against CD147siRNA fragments against CD147 were designed and transfect into human monocyte cell line THP-1 cells which were differentiated by PMA.. The suppressive efficiency of RNAi was detected by flow cytometry and quantitative real-time PCR (qRT-PCR).3. CypA stimulation to MMP-2, -9 expression and invasion ability of THP-1THP-1 cells were stimulated by CypA, the cells were run qRT-PCR for MMP-2, -9 mRNA levels. The supernatants were collected for detection of MMP-2, -9 protein activity by gelatin zymography. Invasion assay experiment was used to observer invasive ability of THP-1 cells to ECM under CypA stimulation. At the same time, RNAi, CsA and AP-9 were used interrupting the interaction between CypA and CD147 to identify whether CD147 facilitates CypA's function.4. CypA, CsA and AP-9 effects to CD147 expression on THP-1To identify whether CypA, CsA or AP-9 takes effect to CD147 expression which may influence MMPs expressions in RA, the experiments of qRT-PCR and flow cytometry were used to detect the changing levels of CD147 mRNA and protein.5. CypA stimulation to NF-κB activity via MAPK pathway in the THP-1NF-κB is the most important factor for regulating MMP-9 expression in many types of cells. Under CypA stimulation, the p50 protein level in cytoplasm or nucleus was detected by immunofluorescence. CsA or AP-9 was used to interrupt the interaction between CypA and CD147, in order to identify whether CD147 facilitates CypA's function. CD147 is a regulator of MMP production by MAPK pathway, which includes extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38. The inhibitors of MAPK pathway were used to identify whether CypA activates NF-κB activity via MAPK pathway and which pathway is involved.6. CypA mediated Ca2+ mobilization and adhesion of THP-1 to ECMUnder CypA stimulation, Calcium mobilization in the THP-1 cells was detected by laser scanning confocal fluorescence microscope, the adhesion of THP-1 cells to ECM was also investigated. CsA or AP-9 was used to interrupt the interaction between CypA and CD147, in order to identify whether CD147 facilitates CypA's function.Results:1. CypA stimulation to MMP-2 and -9 expression and invasion ability of RA monocytes/macrophagesHuman peripheral blood monocytes were successfully differentiated into macrophages. After differentiation, pro-MMP-9 and MMP-9 production were significantly increased in macrophages of normal human and RA patients peripheral blood, while pro-MMP-2 production was not increased (P>0.05). Under CypA stimulation, the secretion and activation of MMP-9 were increased in macrophages, while it was a little increased in monocytes. No significant changes with CypA stimulation were observed when the cells were respectively treated with CsA or AP-9. For pro-MMP-2, its secretion and activation had no significant changes in monocytes or macrophages after CypA stimulation. A higher number of macrophages (cells/filter) which differentiated from monocytes were found to have invaded through transwell chambers than that of monocytes, and the invading number cells of monocytes/macrophages from RA SF was the highest in those cells. The number of invading macrophages in CypA's stimulation group was much bigger than that in negative control group, while the number of monocytes was a little increased after CypA stimulation. CypA also increased the number of the invading monocytes/macrophages from RA SF. With or without CypA stimulation, the number of invading cells were both decreased when the monocytes or macrophages were pretreated with CsA or AP-9 respectively.2. Suppression of CD147 expression by RNAiThe undifferentiated THP-1 cells were successfully differentiated. siRNA I and siRNA II were both strongly suppress CD147 expression and the suppressive efficiency of siRNA I was better than that of siRNA II. Negative control siRNA had no effect to CD147 expression. siRNA I was selected for the following experiment.3. CypA stimulation to MMP-2, -9 expression and invasion ability of THP-1CypA significantly increased MMP-9 expression, not MMP-2, in the THP-1 cells by enhancing the NF-κB activity. CsA, siRNA or AP-9 respectively dramatically decreased MMP-2 and MMP-9 expression, both in the absence or presence of CypA. Invasion assay showed that CypA enhanced invasive ability of the undifferentiated or differentiated THP-1 cells to ECM, while CsA or AP-9 downregulated their invasive abilities.4. CypA, CsA and AP-9 effects to CD147 expression on THP-1The results of qRT-PCR and flow cytometry showed that CypA had no effect to CD147 expression, while CsA inhibited CD147 expression. AP-9 could strongly blockade membrane CD147.5. CypA stimulation to NF-κB activity via MAPK pathway in the THP-1The results of immunofluorescence showed that CypA induced nuclear translocation of NF-κB. AP-9 or CsA blockaded CypA-induced the nuclear translocation of NF-κB. Gelatin zymography showed that P38 MAPK inhibitor, SB203580, had no effect on the MMP-9 secretion in the THP-1 cells, while ERK1/2 and JNK inhibitors (U0126 and SP600125) significantly decreased MMP-9 secretion, both in the absence or presence of CypA. Similar results were observed in NF-κB activity by immunofluorescence.6. Ca2+ mobilization in the THP-1 cells Adhesion of THP-1 cell to Matrigel Matrix with CypA stimulationLaser scanning confocal fluorescence microscope showed that CypA increased Ca2+ mobilization in the THP-1 cells. Anti-CD147 antibody HAb18 strongly decreased cytosolic Ca2+ concentration. No cytosolic Ca2+ mobilization response was observed when CypA was preincubated in the presence of CsA or cells were preincubated with HAb18. A significant increase in both theTHP-1 cell adhesion was observed with CypA. In contrast, CsA or anti-CD147 antibody HAb18 dramatically reduced THP-1 cell adhesion, both in the absence or presence of CypA.Conclusions:Studies here showed that CypA enhanced MMP-9 expression and adhesion of monocytes/macrophages by its direct binding to CD147. These findings suggest that in RA, the abundant CypA might enhance the adhesion of monocytes/macrophages to cartilage and upregulate MMP-9 production in monocytes/macrophages to destruct cartilage which may contribute to the development of RA. These findings may give some good ideas for the therapy of RA in the future.