Roles Of CypA-CD147 Interactions On Actviated And Malignant Human B-cell Subsets In The Pathogenesis Of Autoimmune Diseases (Rheumatoid Arthritis) And Neoplasia (Acute Lymphoblastic Leukemia)

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by inflammation at synovium. The clinical feature of RA is manifested by proliferative and erosive synovitis similar to locally invasive tumor. 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.Previous studies 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. CD147 also named extracellular matrix metalloproteinase inducer (EMMPRIN), belongs to immunoglobulin superfamily and is one of Cyclophilin A (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. CypA, involved in numerous inflammatory events, is abundant in 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. The potential role of interaction between CypA and CD147 has been shown in some diseases, such as AIDS, SARS, and prostatic carcinoma.FACS analysis of peripheral blood from normal individuals revealed that small fractions of B-cells, T-cells, and monocytes expressed emmprin, whereas emmprin-expressing T-cells were much increased in number, and expressed this protein to a higher level, in ATLL patients. In vitro co-cultures of emmprin-positive HTLV-1-transformed lymphocytes (MT-2) and emmprinnegative human fibroblasts enhanced the production of pro-MMP-2 (gelatinase A) and active MMP-2, compared with cultures of either cell type alone. This stimulation was inhibited by an activity-blocking peptide against emmprin.In recent years a body of evidence has accumulated to show that chronic infl ammation can play an important role in the progression of some types of tumors from a premalignant state to full-blown disease. A link between cancer and infl ammation has long been suspected. Previous studies in our lab showed that CypA enhanced MMP-9 expression and adhesion of monocytes/macrophages by its direct binding to CD147. The expression of CD147 has been shown elevation on the surface of normal mononuclearcell, proliferative inflammatory cells and tumour cells successively. But CD147 expression on the actviated and malignant human B-cell subsets, and the pathogenesis of CypA-CD147 pathway in different activated B-cell stage has not been reported yet.So in this study, it is the first time that the interaction between CypA and CD147 will be studied for CD147 expression on B-cell development stage. The results of the study we believe may help us to gain a better understanding of the potential role of CypA-CD147 pathway in immflammation, precancerosis and neoplasia pathogenesis, and may give good ideas for the future biological tag and targeted therapy of autoimmune diseases and neoplasia therapy.Methods:1. Cyclophilin-mediated CD147 expression on B-cell from autoimmune diseases (RA)Samples of synovium and synovial fluid (SF) were obtained from 15 patients with active RA in Xijing Hospital. Streptavidin/peroxidase (SP) immunostainings were performed to detect the existence of B-cell germinal center (GC)-like reacrion and CD147 expression at the synovium derived from 15 patients with RA; FACS analyses were performed to quantify the levels of CD147 in synovial fluid B-cells (CD147-FITC,CD3-PrecP,CD19-APC) stimulated CypA, BAFF and LPS. At the same time, SF levels of RF and anti-CCP were quantified by rate nephelometry and ELISA, and corrected to synovial total IgM and IgG, respectively.2. CD147 expression on the malignant B-cell subsets in acute lymphoblastic leukemiaTo analyze CD147 expression by leukemic cell in boon marrow of acute lymphoblastic leukemia patients, especially by immature B-cell, were stained with a mixture of CD34/CD33 , CD10/CD19 , CD10/CD11b, and incubated with anti-CD147 monoclonal antibody were analyzed on properly compensated FACSCalibur. To study the relationship between CD147 and leukemia, we evaluated the expression of CD147 using this method on acute leukemia primary patient, post-treatment patients and contro and examined the availability of this procedure for evaluation of the quality of AL-remission.3. CypA stimulation to B lymphoma cell line invasion and malignant ability via CD147 pathway in vivoTo clarify the mechanism of in vivo invasion and metastases of Cyclophilin-CD147 interactions on B-cell, we examined the organ distribution of CD19+CD10+CD147high Raji cell (B lymphoma cell line) stimulated by CypA in severe combined immunodeficiency (SCID) mice using FACS and SP immunostainings.Results:1. Cyclophilin-mediated CD147 expression on B-cell from autoimmune diseases (RA)All the RA patients met the 1987 revised diagnostic criteria of the AmericanCollege of Rheumatology and have severe bone degradation. Expression of GC-like reacrion was detected at synovium from 13 of the 15 patients with RA, and the expression of CD147 of GC-like reacrion was detected in the 13 patients with RA. There was an apparently increased expression of CD147 in follicular synovitis the 7 patients with RA. Among the in folliculus lymphaticus of RA synovium expressing CD147, the lymphocytes share 30±8%, the folliculardendritic cells (FDCs) share 32±7%, the endothelial cells share 38±11%, and the plasma cells was negative, respectively; Mononuclearcells were isolated from synovial fluids of RA. The the mean expressional levels (MFI) of CD147 of CD19+ B-cell and corrected synovial fluid levels of RF and anti-CCP of RA patients were significantly higher after CypA, BAFFor LPS stimulation than those of control group(p<0.01), which was similar to result when stimulated by CypA, BAFFor LPS (p>0.05).2. CD147 expression on the malignant B-cell subsets in acute lymphoblastic leukemiaThe expression of CD147 on leukemic cells was evaluated by flow cytometry. The MFI of CD147 expression on leukemic cells (97.96±14.07,p< 0.01) was higher in the primary patient than in post-treatment patients (73.72±8.54) and normal control groups (66.61±15.59), with no significant difference between post-treatment patients and normal control (p >0.05). CD147 expression on lymphocyte, neutrophil, and erythroblast were also higher in the primary patient than in post-treatment patients and normal control groups (p< 0.01). The MFI of CD147 positive staining cells in leukemic B-cells (CD10/CD19) in primary B-ALL patient (105.96±5,p< 0.01) was higher than post-treatment patients (58.46±1.26) and normal control(35.76±8.49). At the same time, we found CD147 expression on leukemic cells (105.96±5) was higher than on mature B-cell (43.46±12.34,p< 0.01) in boon marrow of B-ALL. Relative to several differentiated B-ALL, the immaturer-B subtypes of ALL leukemia cells in the bone marrow of patients with the expression of CD147 higher, and with the increased percentage of immature cell, CD147 expression also increased. Its expression in the treatment of patients degraded to the normal expression, and there is certain degree of regularity of immune distribution. However, analysis of other acute leukemia leukemic cells and found no clear pattern.3. CypA stimulation to B lymphoma cell line invasion and migration ability via CD147 pathway in vivoOur next question was to address the correlation between CD147 expression and cyclophilin-mediated responses in malignant B-cell. Thus, we detected the capacity of CypA to induce malignant in Raji cell (B lymphoma cell line) by injected into SCID mice. For these studies, Raji cell expressed CD19+CD10+(about 85%), belong to immature B lymphocytes, and expressed CD147, MFI reached 337.56, which is CD19+CD10+CD147high expression, in line with further functional requirements of the experimental cells. The percentage of CD19+CD10+CD147+ cells in Raji cells stimulated by CypA in mice was significantly higher than in Raji cells alone immunized mice In mice immunized with the liver and spleen tissues observed (p <0.01), CD19+CD10+CD147+ cells expressed no significant difference in fluorescence in Raji cells stimulated by CypA and Raji cells alone after two groups of mice immunized. And CypA significantly increased MMP-9 expression, not MMP-2.Conclusions:Studies here showed that CD147 may be used for early diagnosis of ALL as biomarkers and for targeted therapy provide experimental basis. These findings may give some good ideas for the future biological tag and targeted therapy of autoimmune diseases and neoplasia therapy.