Platelets Activation And Regulation Of Monocyte Subsets In Rheumatoid Arthritis

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.Multiple inflammatory cells and cytokines contribute to the development of RA.Blood platelets not only play critical roles in homeostasis but also in immune response and inflammation.In humans, increased platelet activation is evident in patients with RA compared with healthy control subjects. The underlying mechanism is that expression and production of various molecules by activated platelets, which participate in the interaction of plateles with endothelial cells or white cells. Monocytes playing a critical role in the pathogenesis of various disease, especially in RA. Recently, an of?cial nomenclature has been proposed for these cells that has been approved by the Nomenclature Committee of the International Union of Immunological Societies(NC-IUIS).This nomenclature de?nes three types of monocytes: classicalmonocytes(CD14++CD16-), intermediate monocytes(CD14++ CD16+), and nonclassical monocytes(CD14+CD16++). The new de?nition of monocyte subsets andmechanisms that promote dynamic changes in monocyte subsets in rheumatoid arthritis(RA) have not been clearly identified.The aim of this study was to determine whether platelet activation and the consequent formation of monocyte-platelet aggregates(MPA) might induce a proinflammatory phenotype in circulating monocytes in RA.Methods: 1. The expression of CD147, PAC-1, CD62 P and CD40 L on platelets from RA patients. We enrolled 40 patients with active RA who satisfied the ACR/EULAR RA classification criteria. Preconjugated ?uorescent antibodies were from BD Biosciences: P-selectin(CD62P)–PE, CD40L–PE, PAC-1–FITC, CD147–FITC, CD61–Per CP. Platelets were analyzed by a FACS Calibur flow cytometry and the positive cell count of PAC-1, CD62 P, CD40 L and CD147 on the surface of the cells were both determined. Furthermore, disease activity which was evaluated using DAS-28, calculate the correlation with the expression of PAC-1, CD62 P, CD40 L and CD147. 2. The percentage of monocyte subsets and the expression of CD147 in peripheral blood from RA patients. monocytes were subclassi?ed according to CD14–PE, CD16–PE-Cy5 and CCR2–Alexa Fluor expression. Results are presented as percentage of positive cells. The percentage of CD14+CD16-CCR2+(Mon1), CD14+CD16+CCR2+(Mon2) å'ŒCD14+CD16+CCR2-(Mon3) and the expression of CD147 in peripheral blood from RA patients were determined. 3. The circulating monocyte-platelet aggregate(MPA) levels in RA. Circulating MPA was defined by double positivity for CD14 and CD42 a. Flow cytometry(FCM) analysis of circulating monocyte subsets and MPAs using a four-color platform(CD14–PE, CD16–PE-Cy5, CCR2–Alexa Fluor, CD42a–FITC). 4. Platelet–Monocyte Interactions. monocytes from normal donors were incubated with or indirectly co-cultured with RA platelets.Intracellular staining of cytokines was performed using a BD Cytofix/CytopermFixation and Permeabilization kit.The proliferation of the monocyte subsets was measured using Brd U Flow Kits, Gelatin zymography method was used to detect the secretion and activation of MMP-2 and MMP-9 in the culture medium. Next, anti-CD147-antibody and Ikk inhibitor were added to coculture system to investigate their blockage effects.Results: 1. The expression of CD147, PAC-1, CD62 P and CD40 L on platelets from RA patients. Flow cytometry showed the percentage of cells that stained positive for CD147, PAC-1 and P-selectin(CD62P); the percentages of stained active and inactive RA platelets were significantly higher than those of healthy platelets, especially in active RA patients. Furthermore, the percentage of platelets that were positive for CD147 expression was considerably higher than those that were positive for PAC-1, CD62 P and CD40 L expression in the overall population that was examined. Disease activity which was evaluated using DAS-28, was also significantly and positively correlated with the expression of PAC-1, CD62 P and CD147 within the group of patients with active RA. 2. The percentage of monocyte subsets and the expression of CD147 in peripheral blood from RA patients. The frequencies of Mon2 and Mon3 monocytes in peripheral blood samples from active RA patients or inactive RA patients were higher than the percentages in the healthy control group, especially for Mon2. The expression of CD147 was increased on all three subsets of monocytes from patients with RA compared with those of the healthy control subjects. The proportion of CD147 expression on Mon2 monocytes in active or inactive RA compared with that of the healthy controls was significantly higher than the proportion of Mon1 and Mon3 cells. 3. The circulating monocyte-platelet aggregate(MPA) levels in RA. Patients with RA(active or inactive), compared with the healthy controls, had signi?cantly higher numbers of circulating MPA.Moreover, the proportion of Mon1, Mon2and Mon3 monocytes that aggregated with platelets in patients with active RA or inactive RA compared with the healthy controls were all significantly increased.The proportion of Mon2 monocytes that aggregated with platelets in active or inactive RA compared with healthy controls was significantly higher than the proportion of aggregated Mon1 and Mon3 monocytes.A linear correlation was found between the MPA levels and the percentage of Mon2 monocytes, the expression of CD147 on platelets and the expression of CD147 on Mon2 monocytes.However, the levels of MPA correlated more closely with the percentage of expressed CD147 on Mon2 monocytes than the percentage of Mon2 monocytes that were present in individual patients, whereas no such correlation was observed in healthy control subjects. The levels of MPA did not correlate with the percentages of Mon1 or Mon3 monocytes. 4. Platelet–Monocyte Interactions. We found that the percentage of Mon2 was higher when co-cultured with platelets from the RA patient group compared with the no cellular contact, CD147-blocking m Ab or control groups. Blockade of CD147 abrogated MPA formation from monocytes in the presence of platelets, and also markedly reduced the degree of the percentage of Mon2. Following coulturing with platelets, the intracellular Brd U levels were significantly increased in the Mon2 monocyte subsets.In this study, the intracellular TNF-α and IL-6 levels in Mon2 monocytes were higher compared with the levels in Mon1 and Mon3 monocytes.Following co-culturing with platelets, the intracellular Ikkβ levels were significantly increased in the Mon2 and Mon3 monocytes.the CD147-blocking m Ab-induced inhibition of CD147 expression on monocytes reduced this effect, suggesting that platelet-monocyte interactions contribute to MMP-9 secretion. To test the hypothesis that platelet-monocyte interactions evoke protease stimulation by activating NF-κB, we pretreated human isolated monocytes with an Ikk inhibitor and then incubated them with platelets. The activation of MMP-9, which was hindered by inhibition of the Ikk complex.Conclusions: In summary, the findings of this study extend the role of CD147 expression on platelets in RA. Moreover, these findings indicate an important role of Mon2 monocytes in the promotion and generation of the proinflammatory cytokine milieu, which is strongly related to and may be driven by the extent of platelet activation. Our findings shed new light on the relevance of platelet-monocyte interactions in the pathophysiology of RA, and CD147 may play an important role in this process via the NF-κB pathway. The inhibition of CD147 may be a promising target for novel therapeutic strategies in RA patients.