Expression And Its Potential Role Of CD147 On Neutrophils In Rheumatoid Arthritis

ObjectivesRheumatoid arthritis (RA) is a chronic inflammatory disease characterized by pronounced synovial hyperplasia and bone destruction. Accumulating evidence suggests that the fibroblast-like synovial cell (FLS, also called synovial fibroblasts) is not only space-filling but also cartilage destruction-causing by producing a great amount of matrix metalloproteinases (MMPs). Although various cell types are present in the rheumatoid joint, neutrophils are the cells most abundant in number within RA synovial fluids and their activation products may serve as biomarkers of the disease activity. In our previous study, we found that CD147, rich on the surface of RA synovium cells, is involved in promoting the secretion and activation of MMPs, which, in turn increases the invasive potential of FLS. However, the exact mechanism underlying the up-regulated expression of MMPs during the cell-cell interaction remains unclear yet. We conducted this study in order to observe the expression of CD147 on neutrophils and testify its functions during the FLS-neutrophil interaction.MethodsCells were analyzed by a FACS Calibur flow cytometry and the positive cell count and the mean fluorescence intensity (MFI) of CD147 on the surface of the cells were both determined. The data were processed using Cell Quest software. Quantitative real-time PCR was performed to detect the mRNA expression of MMP-1, -2, -3, -9 and CD147. MxPro QPCR System Software was used to analyze relative quantification. The chemotaxis was assessed in a modified 48-well modified Boyden chamber using cyclophilin A (CyPA) as chemotactic reagent and anti-CD147 antibody was added to the upper cells to investigate their blockage effects. Gelatin zymography method was used to detect the secretion and activation of MMP-2 and MMP-9 in the culture medium. Quantities of MMP-1 and MMP-3 were determined using enzyme-linked immunosorbent assay (ELISA) kits. Cell invasion assay was performed using Boyden Chamber. The concentration of calcium in HL-60 cells were detected by confocol laser scanning microscopy. Cell adhesion ability was performed using adhesion assay. The expression of TRPM-7 in lipid raft was detected using western blot. The results presented were representative of a minimum of three experiments. Graphpad software was used for the above analyses and P<0.05 was considered significant.Results1.The percentage of positive staining cells of CD147 on RA FLS was higher than that on osteoarthritis (OA) FLS. The MFI of CD147 on differentiated HL-60 cells was higher than that on the undifferentiated HL-60 cells. The MFI of CD147 on RA synovial fluid neutrophils was higher than that on the RA peripheral blood neutrophils, while the MFI of CD147 on RA peripheral blood neutrophils was higher than that on healthy peripheral blood neutrophils.2.The expressions of MMP-1, -2, -3, -9 and CD147 mRNA were higher in RA FLS than those in OA FLS. The expressions of CD147, MMP-1, -2, -3 and -9 mRNA in RA FLS increased after co-culture with HL-60 cells. The expressions of CD147, MMP-1, -2, -3 and -9 mRNA in HL-60 cells were enhanced after HL-60 cells were differentiated by ATRA stimulation.3.The CyPA chemotactic index for differentiated HL-60 cells was higher than that for undifferentiated HL-60 cells. The CyPA chemotactic indexes decreased significantly in anti-CD147 antibody groups compared with those in nc-Ab groups.4.Gelatin zymography and ELISA results both showed that the secretion and activation of MMPs of RA FLS were higher than those of OA FLS. The increases were observed in the release of MMPs in differentiated HL-60 compared with that in undifferentiated HL-60; The secretion and activation of MMPs increased in the co-culture of RA FLS and differentiated HL-60 compared with those in the co-culture of RA FLS and undifferentiated HL-60. The values of MMPs activity of RA FLS co-cultured with HL-60 cells were higher than the values of co-cultured cells treated by anti-CD147 antibody, but no inhibitory effects were found in nc-Ab group.5.Gelatin zymography and ELISA both showed that MMP-1, -2, -3 and -9 secretion and activation increased in both the co-culture group of RA FLS with RA peripheral blood neutrophils and the co-culture group of RA FLS with healthy peripheral blood neutrophils, compared with culture group of RA FLS alone. Significant increases were observed in the release of MMPs in the co-culture of RA FLS and RA peripheral blood neutrophils compared with that of healthy peripheral blood neutrophils. The values of MMPs activity of RA FLS co-cultured with human neutrophils were higher than the values of co-cultured cells treated by anti-CD147 antibody, while no inhibitory effects were found in nc-Ab group.6.MMPs secretion and activation in the co-culture of RA FLS with RA synovial fluid neutrophils were all higher than those in the culture of RA FLS alone or in the co-culture with RA peripheral blood neutrophils. Anti-CD147 antibody was found to have inhibitory effects on MMPs release and activation of co-cultured RA FLS and neutrophils. However, no inhibitory effects were found in nc-Ab group.7.A higher number of RA FLS were found to have invaded through transwell chambers compared with that of OA FLS and a higher number of RA FLS co-cultured with differentiated HL-60 were found to have invaded compared with that of RA FLS co-cultured with undifferentiated HL-60 both of which were higher than the number in the culture of RA FLS alone. RA FLS co-cultured with RA peripheral blood neutrophils were found to have a higher number invading through the transwell chambers compared with that of RA FLS co-cultured with healthy peripheral blood neutrophils. RA FLS co-cultured with RA synovial fluid neutrophils were found to have a higher number invading through the transwell chambers compared with that of RA FLS co-cultured with RA peripheral blood neutrophils. The invasion block assay showed that the number of cells decreased after the treatment with anti-CD147 antibody in RA FLS co-cultured with neutrophils, while no inhibitory effects were found in nc-Ab group.8.Calcium detection and cell adhesion assay showed that concentrationof calcium and cell adhesion ability were enhanced after stimulation of CyPA. Anti-CD147 antibody was found to have some inhibitory effects. The results of western blot showed that this may be due to the expression and function of TRPM-7 in lipid raft fractions.Discussion1.In this study, we found that expression of CD147 on differentiated HL-60 cells were higher than that on undifferentiated HL-60 cells. Gelatin zymography, ELISA and invasion assay all showed that the values of MMPs were significantly enhanced when HL-60 cells were stimulated into a maturation stage. The higher expression levels of CD147 observed in the differentiated process may explain the increased MMPs production, imbalanced activity between MMPs and tissue inhibitors of MMPs caused by enhanced expression of CD147 may eventually leads to RA joint destruction.2.We had RA FLS co-cultured with HL-60 cells to explore the effect of cell-cell interaction on the production of MMPs and the invasiveness of RA synoviocytes. Gelatin zymography and ELISA both showed that the co-culture of HL-60 and RA FLS resulted in higher levels of MMPs than the culture of HL-60 or FLS alone. Cell invasion assay confirmed these results, which partly prove that CD147-upregulated neutrophils may act as an amplifier of the pathogenetic cascade by interacting with FLS.3.We also had neutrophils from peripheral blood or synovial fluid of RA co-cultured with RA FLS to further confirm the effect of neutrophil-FLS interactions in rheumatic joints as the environments of co-cultured FLS and neutrophils from RA are more like the real environments in vivo compared with those of co-cultured RA FLS and HL-60. The results from the co-culture of neutrophils from peripheral blood or synovial fluid of RA co-cultured with RA FLS were consistent with the results from the co-culture of RA FLS with HL-60, indicating that the over expression of CD147 in vivo induces elevated levels of MMPs, which in turn increase the invasive potential of RA FLS.4.We found that the addition of anti-CD147 monoclonal antibody has some inhibitory effects not only on MMPs production but also on the invasive potential of RA FLS, suggesting that the inhibition of CD147 can decrease the MMPs production and the invasive potential of RA FLS5 . The results of chemotaxis assay have testified that CyPA have a chemotactic effect on neutrophils. Moreover, we have also found that this chemotaxis can be significantly suppressed by anti-CD147 antibody. These findings suggest that CyPA does interact with CD147 and it is highly possible that CyPA may act as a ligand of CD147 to induce the accumulation of inflammatory cells which highly express CD147.6.CD147 may enhance the concentration of calcium and cell adhesion ability induced by CyPA through TRPM-7; Moreover, we have also found that this can be significantly suppressed by anti-CD147 antibody.7.In conclusion, Our study demonstrates that the increased expression of CD147 on neutrophils in RA may be responsible for CyPA-mediated neutrophil migration into the joints, elevated MMPs secretion and cell invasion of synoviocytes, all of which may contribute to the cartilage invasion and bone destruction of RA. The inhibition of CD147 may be a promising target for novel therapeutic strategies in RA.