Performance of ultrasound to monitor Achilles enthesitis in patients with ankylosing spondylitis during TNF-a antagonist therapy.

Enthesitis is considered as the primary anatomical lesion in ankylosing spondylitis (AS). We aimed to investigate the potential of ultrasound to detect early changes after TNF-a antagonist therapy of Achilles enthesitis of AS patients. One hundred AS patients with active disease, requiring TNF-a antagonist therapy, were included (etanercept n = 25, infliximab n = 25, adalimumab n = 25, non-biologic disease-modifying antirheumatic drugs (DMARDs) n = 25). Physical examination was performed to evaluate disease activity and detect Achilles enthesitis and/or retrocalcaneal bursitis. Ultrasound of the Achilles enthesitis was performed bilaterally. Follow-up examinations were performed 3 months after the initiation of therapy. Gray scale (GS) scores, Power Doppler (PD) scores, and total additive scores (TS) decreased significantly during TNF-a antagonist therapy but not in traditional non-biologic traditional DMARDs group. The bath ankylosing spondylitis disease activity index (BASDAI), bath ankylosing spondylitis metrology index (BASMI), bath ankylosing spondylitis functional index (BASFI), and Maastricht ankylosing spondylitis enthesitis score (MASES) all showed significant improvements. When three different TNF-a antagonists were analyzed separately, no significant difference was observed in GS, PD, and total scores. Subclinical Achilles enthesitis, detected only with GS ultrasound, is present in a subset of AS patients and a significant improvement can be demonstrated after 3 months of TNF-a antagonist therapy. Doppler ultrasound provides a reliable estimation to monitor the therapeutic response to TNF antagonists in AS patients with Achilles enthesitis. TNF-a antagonists have been shown to be effective in decreasing ultrasound signs of enthesitis after 3 months of therapy in AS patients.

Platelets induce a proinflammatory phenotype in monocytes via the CD147 pathway in rheumatoid arthritis.

INTRODUCTION: Activated platelets exert a proinflammatory action that can be largely ascribed to their ability to interact with monocytes. However, the mechanisms 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: The surface phenotype of platelets and the frequencies of monocyte subpopulations in the peripheral blood of RA patients were determined using flow cytometry. Platelets were sorted and co-cultured with monocytes. In addition, monocyte activation was assessed by measuring the nuclear factor κB (NF-κB) pathway. The disease activity was evaluated using the 28-joint disease activity score. RESULTS: Platelet activation, circulating intermediate monocytes (Mon2) and MPA formation were significantly elevated in RA, especially in those with active disease status. Furthermore, Mon2 monocytes showed higher CD147 expression and responded to direct cell contact with activated platelets with higher cytokine production and matrix metallopeptidase 9 (MMP-9) secretion, which increased the expression of CD147. After the addition of specific antibodies for CD147, those effects were abolished. Furthermore, the NF-κB-driven inflammatory pathway may be involved in this process. CONCLUSION: These findings indicate an important role of platelet activation and the consequent formation of MPA in the generation of the proinflammatory cytokine milieu and for the promotion and maintenance of the pathogenically relevant Mon2 monocyte compartment in RA, which is likely to play an important role in the pathogenesis of autoimmunity.

CD147 up-regulates calcium-induced chemotaxis, adhesion ability and invasiveness of human neutrophils via a TRPM-7-mediated mechanism.

OBJECTIVES: We aimed to investigate whether CD147 can up-regulate the chemotactic, adhesive and invasive properties of human neutrophils and to determine the mechanism underlying this process. METHODS: Human promyelocytic leukaemia cells (HL-60) cells and peripheral blood or synovial fluid neutrophils were isolated from RA patients. Under cyclophilin A (CypA) stimulation, chemotaxis, adhesion potential and invasion ability were assessed using chemotaxis, adhesion and invasiveness assays. Lipid raft isolation and western blot were used to determine the mechanism underlying the effects of CypA stimulation. RESULTS: CD147 up-regulates the calcium-induced chemotaxis, adhesion ability and invasiveness of human neutrophils in RA patients. Transient receptor potential melastatin 7 may be responsible for this phenomenon. CONCLUSION: These findings suggest that in RA patients, abundant CypA up-regulates the calcium-induced chemotactic, adhesive and invasive properties of neutrophils via direct binding to CD147. Cyclophilin-CD147 interactions might contribute to the destruction of cartilage and bone in RA.

CD147 induces angiogenesis through a vascular endothelial growth factor and hypoxia-inducible transcription factor 1α-mediated pathway in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an inflammatory and angiogenic disease. However, the molecular mechanisms that promote angiogenesis in RA have not been clearly identified. Our objective was to study the role of CD147 in angiogenesis and determine whether the strategy in which CD147 is suppressed might be useful in reducing angiogenesis in RA. METHODS: Correlations among expression levels of CD147, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor 1α (HIF-1α) were determined by immunohistochemistry staining. RA fibroblast-like synoviocytes (FLS) cells were cultured under various conditions, and the production of VEGF and HIF-1α was examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. The SCID mouse coimplantation model of RA (SCID-HuRAg) was established, mice were treated with CD147 monoclonal antibody, infliximab, or both CD147 and infliximab, and the volume of the grafts and the average vascular density were measured and analyzed. Western blot analyses were performed to examine the potential signaling pathways. RESULTS: The expression levels of CD147 showed significantly positive correlations with VEGF and HIF-1α levels, as well as with vascular density, in RA synovium. After small interfering RNA transfection or after addition of specific antibodies for CD147, the production of VEGF and HIF-1α were significantly reduced. The expression of VEGF and HIF-1α decreased more after CD147 inhibition than after infliximab treatment in the engrafted tissues in SCID-HuRAg mice. The phosphatidylinositol 3-kinase/Akt pathway may be involved in this process. CONCLUSION: CD147 induces up-regulation of VEGF and HIF-1α in RA FLS, further promotes angiogenesis, and leads to the persistence of synovitis. Inhibition of CD147 may be a promising target for novel therapeutic strategies.

[Effect of cyclophilin A on monocyte-derived foam cells].

AIM: To observe whether cyclophilin A (CypA)has an effect on macrophage-derived foam cells, and to investigate the involvement of CypA in the development of atherosclerosis. METHODS: The foam cell model was established through incubating the human monocyte line (THP-1 cells) with oxidized low density lipoproteins (ox-LDL). The cells were stained with fresh oil red O to study the morphology of the macrophage-derived foam cells. The cell adhesion, invasion and the production of matrix metalloproteinase (MMPs) of the macrophage-derived foam cells were detected by adhesion assay, invasion assay and gelatin zymography respectively both in the absence or presence of different concentrations of purified CypA (50, 100, 200 µg/L). Then the foam cells were respectively pre-treated with CsA, c7b8f10, HAb18 mAb, and dual treatment of c7b8f10 and HAb18 mAb respectively, to investigate the inhibitory effect on macrophage-derived foam cells. RESULTS: The adhesion, invasion and the production of MMP-9 and MMP-2 were enhanced during the differentiation of monocytes into macrophages (P<0.05). CypA, especially in the concentration of 100 ng/mL, significantly promoted the function of macrophage-derived foam cells (P<0.05). CsA, c7b8f10, HAb18 mAb, and c7b8f10- HAb18 mAb combination dramatically inhibited the function of macrophage-derived foam cells both in the absence or presence of CypA (P<0.05). The c7b8f10- HAb18 mAb combination pretreatment had the most obviously suppressive effect on macrophage-derived foam cells (P<0.05). CONCLUSION: These findings suggest that CypA up regulates the adhesion, the invasion and the expressions of MMP-2 and MMP-9 in macrophage-derived foam cells. The CypA effect is blocked by the pretreatment of the different antagonists. This research might suggest the correlation between atherosclerosis pathogenesis and the vulnerability of atherosclerotic plaques, and thus give us some good ideas for atherosclerosis therapy in future.

Expression of CD147 (EMMPRIN) on neutrophils in rheumatoid arthritis enhances chemotaxis, matrix metalloproteinase production and invasiveness of synoviocytes.

The occurrence of neutrophils at the pannus-cartilage border is an important phenomenon for understanding the pathogenesis of rheumatoid arthritis (RA). Matrix metalloproteinases (MMPs) are predominant enzymes responsible for the cartilage degradation. The present article studied the expression of CD147 on neutrophils and its potential role in neutrophil chemotaxis, MMPs production and the invasiveness of fibroblast-like synoviocytes (FLS). The results of flow cytometry revealed that the mean fluorescence intensity of CD147 expression on neutrophils of peripheral blood from RA patients was higher than that in healthy individual. The potential role of CD147 in cyclophilin A (CyPA)-mediated cell migration was studied using chemotaxis assay and it was found that the addition of anti-CD147 antibody significantly decreased the chemotactic index of the neutrophils. Significantly elevated release and activation of MMPs were seen in the co-culture of neutrophil and FLS compared with cultures of the cells alone. An increased number of cells invading through the filters in the invasion assays were also observed in the co-cultured cells. The addition of anti-CD147 antibody had some inhibitory effect, not only on MMP production but also on cell invasion in the co-culture model. 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. Better knowledge of these findings will hopefully provide a new insight into the pathogenesis of RA.

Contribution of cyclophilin A to the regulation of inflammatory processes in rheumatoid arthritis.

INTRODUCTION: Previous studies show that cyclophilin A (CypA) acts as a strong chemotactic cytokine to neutrophils, eosinophils, and monocytes in rheumatoid arthritis (RA). METHODS: In this study, monocytes were stimulated by purified CypA and the production of matrix metalloproteinase (MMPs), the cell invasion and the release of inflammatory cytokines were detected respectively by gelatin zymography, invasion assay, and cytometric bead array FCM. RESULTS: The elevated level of inflammatory cytokine IL-8 was also detected. Results showed that CypA significantly promoted the invasion of THP-1 cells and increased the production of MMP-2 and MMP-9, which displayed a biphasic concentration dependency. In vivo experiments found that the cartilage erosion scores in CypA injection group were significantly higher than those in control group (P < 0.05). CONCLUSION: Our findings suggest that CypA significantly enhances the secretion of MMP-2 and MMP-9, the cell invasion, and the inflammatory cytokines production of monocytes. Our findings may shed some new light on the inflammatory process and the degradation of cartilage and bone in RA.

The interaction of HAb18G/CD147 with integrin alpha6beta1 and its implications for the invasion potential of human hepatoma cells.

BACKGROUND: HAb18G/CD147 plays pivotal roles in invasion by hepatoma cells, but the underlying mechanism remains unclear. Our previous study demonstrated that overexpression of HAb18G/CD147 promotes invasion by interacting with integrin alpha3beta1. However, it has never been investigated whether alpha3beta1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells. METHODS: Human SMMC-7721 and FHCC98 cells were cultured and transfected with siRNA fragments against HAb18G/CD147. The expression levels of HAb18G/CD147 and integrin alpha6beta1 were determined by immunofluorescent double-staining and confocal imaging analysis. Co-immunoprecipitation and Western blot analyses were performed to examine the native conformations of HAb18G/CD147 and integrin alpha6beta1. Invasion potential was evaluated with an invasion assay and gelatin zymography. RESULTS: We found that integrin alpha6beta1 co-localizes and interacts with HAb18G/CD147 in human hepatoma cells. The enhancing effects of HAb18G/CD147 on invasion capacity and secretion of matrix metalloproteinases (MMPs) were partially blocked by integrin alpha6beta1 antibodies (P < 0.01). Wortmannin, a specific phosphatidylinositol kinase (PI3K) inhibitor that reverses the effect of HAb18G/CD147 on the regulation of intracellular Ca2+ mobilization, significantly reduced cell invasion potential and secretion of MMPs in human hepatoma cells (P < 0.05). Importantly, no additive effect between Wortmannin and alpha6beta1 antibodies was observed, indicating that alpha6beta1 and PI3K transmit the signal in an upstream-downstream relationship. CONCLUSION: These results suggest that alpha6beta1 interacts with HAb18G/CD147 to mediate tumor invasion and metastatic processes through the PI3K pathway.

[The role of CyPA in chemotaxis of neutrophil in rheumatoid arthritis and secretion of interleukin-8].

AIM: To ascertain the effect of CyPA and IL-8 in chemotaxis of neutrophil and the level of IL-8 from the CyPA effecting of peripheral blood from RA patients. METHODS: 12 RA patients matched 4 healthy people were studied. Chemotaxis of IL-8 was measured by Boyden chamber on neutrophil of RA patients and that of 4 normal healthy people controls were studied; the level of IL-8 on neutrophil of RA patients peripheral blood after the effecting of CyPA was assessed by ELISA. Correlations between CyPA and IL-8 were observed in RA. RESULTS: The chemotaxis of neutrophil which IL-8 mixed with CyPA antibody was lower than IL-8(P<0.05); The secretion of RA peripheral blood in IL-8 was higher than normal people, as well as the secretion of RA peripheral blood in IL-8 after effecting of CyPA was higher than before (P<0.05), but the level of IL-8 after blocking CyPA was not changed. CONCLUSION: CyPA could affect the chemotaxis of IL-8 in the neutrophil of RA patients' peripheral blood. The secretion of IL-8 is accelerated by CyPA on neutrophil of RA patients'peripheral blood.

[CD147 stimulates the angiogenesis in rheumatoid synovium via the activation of vascular endothelial growth factor].

AIM: To observe the correlation between vascular endothelial growth factor (VEGF) and CD147 expressed in the rheumatoid synovium and to investigate the effect of CD147 of cultured rheumatoid fibroblast-like synoviocytes (FLS) on the production of VEGF. METHODS: The presence of CD147 and VEGF in the rheumatoid synovium derived from 15 patients with RA and 4 patients with osteoarthritis (OA) was detected by streptavidin/peroxidase (SP) immunostaining. FLS were cultured by enzymatic digestion of synovial tissues and incubated in 24-well plates. Then different concentration of LY294002, PD98059, SP600125, SB203580 and HAb18G mAb was added to each well. VEGF in the culture supernatant was measured by sandwich ELISA. RESULTS: CD147 and VEGF in synovium from 15 patients with RA showed high expression, while CD147 and VEGF in synovium from 4 patients with OA showed low expression. Macrophages, fibroblast-like synovial cells and lymphocytes were demonstrated to express CD147 while synovial lining cells, fibroblasts surrounding microvessels and vascular smooth muscle cells were demonstrated to express VEGF. Statistic analysis indicates that VEGF production was correlated with the levels of CD147 expression. VEGFproduction was suppressed when CD147 expression was inhibited by LY294002 or HAb18G mAb. CONCLUSION: CD147 can regulate the angiogenesis in rheumatoid arthritis by VEGF. The low levels of CD147 expressed by FLS cells decrease the production of VEGF via the PI3K-Akt signaling pathway. These findings further highlight the importance of CD147 in pannus formation and angiogenesis.

[The changes of CD4(+) CD25(high) regulatory T cells in the course of rheumatoid arthritis and their significances].

AIM: To explore the percentages of CD4(+) CD25(high) regulatory T cells in peripheral blood or synovial fluid from patients with rheumatoid arthritis (RA) on different stages, and to study their correlations with the activity of the disease. METHODS: Peripheral blood lymphocytes were obtained from the patients with active RA who had received no previous disease modifying (DMARDs) therapy(n=11), from individuals with stable, well-controlled RA (n=12), from subjects whose disease were poorly improved after DMARDs therapy(n=9), and from healthy controls(n=8). The frequencies of CD4(+) CD25(high) T cells were quantified by using flow cytometry(FCM). Meanwhile, the correlations between the percentage of CD4(+) CD25(high) T cells and the level of Anti-CCP antibody, CRP, ESR and RF were also investigated. Paired blood and synovial fluid was analysed in a small group of RA and other patients. RESULTS: There was a smaller proportion of CD4(+) CD25(high) T cells in the peripheral blood of the active RA patients(mean 5.24%) and poorly-improved RA patients(mean 6.43%) than in patients with stable well-controlled RA or in healthy controls(mean 11.79% and 17.17%, respectively, P<0.01 in each case). The frequency of CD4(+) CD25(high) T cells from RA was negatively associated with Anti-CCP antibody(58.0 Ru/mL), ESR(38.8 mm/h) and CRP(2.73 mug/L), (P<0.05 for each). On contrast, The frequency of CD4(+) CD25(high) T cells from healthy controls was not significantly correlated with the level of Anti-CCP antibody(<5.0 Ru/mL), ESR(4.67 mm/h), CRP(0.15 mug/L) and RF(1.37) (all P>0.1). The percentage of CD4(+) CD25(high) regulatory T cells from synovial fluid of RA patients (24.32%) was significantly lower than that of AS patients(30.24%) (P<0.05). CONCLUSION: The results demonstrate a smaller CD4(+) CD25(high) regulatory T cell population in peripheral blood of individuals with active RA prior to disease modifying treatment and with poorly-improved RA, and this is negatively associated with the activity of the disease.