Effects Of Neutrophil On The Matrix Metalloproteinase Production And The Invasiveness Of FLS In RA

Objective: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by chronic hyperplastic synovitis and progressive joint and cartilage destruction. As we known that progressive degradation extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is an essential step for joint destruction in RA. However, the precise pathogenesis of MMP production at the site of joint destruction remains unknown. Fibroblast-like synoviocytes (FLS) are known as the most active synoviocytes and are close to the articular cartilage in a position to invade the cartilage. Although IL-1 and tumor necrosis factor alpha are reported by some studies as key regulators of matrix degradation in RA, other molecules, such as CD147, are also thought to have played some regulatory role in RA pathogenesis. CD147, also called extracellular matrix metalloproteinase inducer, was initially identified on the surface of human cancer cells and has been proven to stimulate the adjacent stromal cells to produce several MMPs. Previous studies have confirmed that CD147 was expressed on many kinds of inflammatory cells from peripheral blood (PB) and synovial fluid (SF) in RA. As we known, Neutrophil is a main component of synovial fluid in RA, which played an important role in cartilage erosion in RA. This study will investigate the effect of neutrophil via CD147 on MMP production and the invasive potential of RA FLS in vitro. This study deploys the model of HL-60 cell differentiation. And HL-60 cells are differentiated into mature neutrophil by an inducer in vitro. On the basis of confirming high expression of CD147 on HL-60 cells and neutrophils from PB /SF in RA, this study will investigate the effect of CD147 on neutrophil to the MMPs production and the invasive potential of FLS in RA further, and antagonist peptide against EMMPRIN/CD147 (AP-9) and anti-CD147 monoclone antibody (HAb 18) are added to the coculture. Studying these problems may not only facilitate us understanding the potential role of neutrophil in joint destruction in RA, but also provide us a better understanding of the possible mechanism and regulation of CD147 on MMPs production and the invasive potential, which will lay a foundation for the development of innovative therapies to RA.Methods:1. HL-60 cells were differentiated into mature neutrophil after by an inducer named alltransretinoic acid (ATRA) for 3~5 days. Cell morphous was observed by microscope, and the degree of cell differentiation was detected through NBT reduction reaction. FLS were isolated from synovial tissues from the patients of rheumatoid arthritis (RA) and ostarthritis (OA) during synovectomy and confirmed by detection of CD14, CD90 using flow cytometry(FCM) and morphologic study.2. CD147 expression on the differentiated/undifferentiated HL-60 cells and RA FLS were detected by FCM.3. The release and activity of MMP-2 and MMP-9 were detected in the supernatants of HL-60 cells cocultured with RA FLS using Gelatin zymography. The invasive potential of RA FLS was detected by invasion assay. To further investigate the effect of CD147 on neutrophil roles in the MMPs production by FLS and the invasive potential of FLS in RA, antagonist peptide against EMMPRIN/CD147 (AP-9) and anti-CD147 monoclone antibody (HAb 18) were added to the coculture.4. Samples of peripheral blood (PB) and synovial fluid (SF) were obtained from 12 patients with active RA in Xijing Hospital. And Samples of peripheral blood were taken from 8 healthy human donor volunteers and 8 patients of other rheumatic disease as control. The expression of CD147 on CD66b+ cells were detected by Flow cytometry. The release and acitivity of MMP-2 and MMP-9 were detected in the supernatants of neutrophil cocultured with RA FLS using Gelatin zymography. The invasive potential of RA FLS was detected by invasion assay. The antagonist peptide against EMMPRIN/CD147 (AP-9) was also added to the coculture.Results:1. The diversity of cell morphology and NBT reduction reaction both showed that HL-60 cells were differentiated into full-grown neutrophils by ATRA. Flow cytometry confirmed the isolated FLS of RA by negative staining for CD14 and positive for CD90.2. High expression of CD147 on undifferentiated/differentiated HL-60 cells were both detected by FCM. CD147 expression on differentiated HL-60 cells was higher than undifferentiated HL-60 cells, and CD147 expression on both of them were higher than that on RA FLS of the fifth generation (P<0.05).3. The Pro-MMP-2 and MMP-2 secretion in RA FLS were higher than those in OA FLS (P<0.05). The production of Pro-MMP-2, MMP-2, Pro-MMP-9 and MMP-9 were significantly increased when RA FLS were cocultured with the undifferentiated or differentiated HL-60 cells (P<0.05). And the increase of ProMMP-2 and MMP-2 in differentiated HL-60 cells cocultured with RA FLS were higher than those in undifferentiated HL-60 cells with RA FLS (P<0.05). The increase was inhibited by AP-9 significantly (P<0.05). Invasion assay showed that the invasive potential of RA FLS was higher than that of OA FLS (P<0.05), and undifferentiated or differentiated HL-60 cells increased the invasive potential of RA FLS (P<0.05), which could be blocked by AP-9 or HAb 18.4. FCM analysis showed that the neutrophils from SF and PB both expressed high levels of CD147 expression. Compared with the control, the mean fluorescence intensity (MFI) of CD147 expression on neutrophil from peripheral blood and synovial fluids in RA patients was significantly higher(P<0.05), and the MFI of CD147 expression on neutrophil from synovial fluid in RA patients was higher than that on neutrophil from peripheral blood(P<0.05). The in vitro studies showed that the co-culture of neutrophil from PB or SF with RA FLS led to higher levels of MMP-2, 9 in coculture supernatants. And the invasion assay revealed that a significant increase in the number of cells invading through the Matrigel layer in the coculture of the neutrophils from SF or PB with RA FLS. While the higher levels of MMP-2, 9 and the invasive potential of RA FLS could be suppressed by CD147 antagonistic peptide (AP-9).Conclusions:1. The experimental system of FLS separation and culture and HL-60 differentiation were established successfully. 2. The expression of CD147 on HL-60 cells was significantly higher than that on RA FLS, and its expression on differentiated HL-60 cells was higher than that on undifferentiated HL-60 cells.3. It was found that undifferentiated and differentiated HL-60 could enhance the MMP-2, 9 production and invasive potential of RA FLS in the coculture system, while CD147 antagonistic peptide(AP-9) and antibody can block this effect. These results suggest that suppression of neutrophils-CD147-MMPs pathyway may be a potential alternative therapeutic target for the treatment of joint destruction in RA.4. We found high expression of CD147 on neutrophils from PB or SF in RA. And the level of MMP-2, 9 production by FLS and it's invasive potential can be greatly enhanced by cocultured with these neutrophils, while CD147 antagonistic peptide(AP-9) had inhibitory effects on MMP production and the invasion of RA FLS. These findings indicate that CD147 expressed on neutrophil may be responsible for the elevated MMP secretion, and play an important role in the degradation of bone in RA.5. These experimental systems we established have provide us a good foundation for further research on the roles of neutrophil in the pathogenesis of RA, and also give us some good ideas for RA therapy in future.