Constitutive production of interleukin 6/B cell stimulatory factor-2 from inflammatory synovium

Interleukin 6 (IL-6) production from synovial tissues of various diseases was examined. Augmented IL-6 production was found in inflammatory synovium not only in RA but also in other kinds of synovitis, including psoriatic arthritis and Beh?et's disease. Increased amounts of IL-6-mRNA were detected in rheumatoid synovium using a dot blot hydridization technique. Furthermore, there was a positive correlation between IL-6 production and accumulation of plasma cells in the synovium. These findings suggest that IL-6 plays an important role not only in immune response but also in active inflammation in various kinds of synovitis.     

Interleukin-15 induces interleukin-17 production by synovial T cell lines from patients with rheumatoid arthritis

IL-17-producing T cells (Th17 cells) are believed to contribute to local inflammation and joint damage in rheumatoid arthritis (RA). Limited data exist on Th17 cells located within the inflamed synovial tissue (ST) of patients with RA. Here, we aimed to generate polyclonal T cell lines (TCLs) from the RA ST and assess their cytokine production, including the effects of exogenous IL-15 on IL-17 production in vitro. For five patients with RA, polyclonal TCLs were established from ST obtained by joint surgery. Synovial TCLs were expanded and stimulated by anti-CD3/CD28 microbeads and exogenous cytokines. Cytokine production was assessed by culture supernatant analyses and intracellular flow cytometry, and TCLs were sorted based on their surface expression of CCR6. In addition to IL-17, we detected IL-6, IL-10, IFN-γ and TNF-α in the synovial TCL culture supernatants. Exogenous IL-15 increased the production of IL-17 as well as the other cytokines except IFN-γ. For IL-17, this effect was more pronounced after prolonged culture times. Intracellular flow cytometry confirmed the presence of IL-17+ and IL-17+ IFN-γ+ CD4+ T cells in the TCLs. IL-17+ and IL-17+ IFN-γ+ T cells were enriched in the CD4+ CCR6+ population. In conclusion, Th17 cells can be detected after polyclonal expansion and stimulation of RA synovial TCLs generated by joint surgery. The Th17 cells from the RA ST were enriched in the CD4+ CCR6+ population, and they were sensitive to exogenous IL-15. Th17 cells present within the synovial compartment may contribute to the RA pathogenesis and local joint damage.     

Expression of interleukin-1 and interleukin-1 receptor antagonist by human rheumatoid synovial tissue macrophages.

Interleukin-1 (IL-1) has protean effects in the pathogenesis of rheumatoid arthritis (RA). These effects include production of prostaglandins and collagenase from rheumatoid fibroblasts as well as upregulation of adhesion molecule expression on these cells. IL-1 can activate monocytes and neutrophils, as well as promote the growth of fibroblasts and endothelial cells. Recently, a novel interleukin-1 receptor antagonist protein (IRAP) has been isolated, purified, cloned, and expressed, which may modulate the effects of IL-1. In this study, we present data demonstrating that macrophages isolated from human RA synovial tissues express both IL-1 and IRAP genes. In addition, RA synovial tissue macrophages and lining cells display IL-1 and IRAP antigenic expression by immunohistochemistry. In contrast, osteoarthritis synovial tissues, as compared to RA, have fewer IL-1 and IRAP-positive macrophages. Thus, the production of IL-1 balanced by IRAP may affect the joint destruction found in these diseases.     

L-Selectin Expression on the Surface of Peripheral Blood Leucocytes from Rheumatoid Arthritis Patients is Linked to Disease Activity

Recruitment of mononuclear cells from the circulation to sites of inflammation relies on migration across vessel endothelium. T and B cells, macrophages and neutrophils infiltrate synovial tissue of rheumatoid arthritis (RA) patients. The authors have analysed the numbers of circulating CD3⁺, CD19⁺ lymphocytes, monocytes, and granulocytes expressing adhesion molecules (L-selectin, CD44 and CD11a), together with levels of expression in RA patients compared to healthy individuals. Numbers of leucocytes expressing the adhesion molecules detected were similar in RA and control groups. Lower levels of expression of L-selectin on all cells were found in RA patients compared to controls. Expression of L-selectin on T and B cells was found to correlate with disease activity in RA. The authors have observed a characteristic pattern of adhesion molecule expression in RA patients, particularly when analysing the relationships between cells. The close regulation of these molecules between RA patients and healthy individuals is discussed.     

High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints.

The CD30 is a surface molecule expressed by Th2-type lymphokine-producing T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA.     

IL-38 抑制LPS / TLR4 诱导炎症减轻类风湿关节炎的机制研究

目的:探讨IL-38 和TLR4 在类风湿关节炎中的潜在关联及其在类风湿性关节炎中致病的机制。方法:选取2013 年1 月至2016 年2 月间本院收治的41 例类风湿关节炎患者(观察组)及45 例本院实施创伤后滑膜切除术的患者(对照组)为研究对象。收集观察组和对照组的外周血单个核细胞(PBMCs)、滑膜组织及血清。荧光定量PCR 检测PBMCs 及滑膜组织中IL-38 及TLR4 的mRNA 水平。ELISA 检测滑膜液及血清中IL-38 的表达,Western blot 检测滑膜组织中IL-38 及TLR4的表达。LPS 和/ 或IL-38 刺激RAW264.7 细胞,ELISA 检测RAW264.7 细胞上清中IL-6、IL-8 及TNF-α的含量,荧光定量PCR检测RAW264.7 细胞TLR4、IL-6、IL-8 及TNF-α的表达。NF-κB 激活-核转运试剂盒及Western blot 检测NF-κB 信号的激活水平。结果:与对照组相比,类风湿关节炎患者PBMCs、血清及滑膜组织和滑膜液中IL-38 水平显著升高,而TLR4 水平也显著升高,Pearson 相关分析显示二者呈负相关。LPS 和/ 或IL-38 刺激RAW264.7 细胞后,IL-38 能够抑制LPS 诱导的TLR4、IL-6、IL-8 及TNF-α表达,进一步的分析显示,IL-38 能抑制NF-κB 信号途径激活,因此推测IL-38 可能是通过抑制NF-κB 信号途径激活从而抑制LPS/ TLR4 信号诱导的炎症因子表达。结论:IL-38 能抑制LPS/ TLR4 诱导炎症减轻类风湿关节炎,其机制可能是通过抑制NF-κB 信号途径的激活。     

Integrins and other adhesion molecules on lymphocytes from synovial fluid and peripheral blood of rheumatoid arthritis patients.

Cell and matrix adhesion of lymphocytes participates in homing, migration and accumulation of these cells in inflamed tissues as well as in the generation of immune and inflammatory responses. In inflamed joints of rheumatoid arthritis (RA) patients, lymphocytes accumulate in the synovial membrane and the synovial fluid. In the present study we have analyzed the expression of integrins and other adhesion molecules in synovial fluid lymphocytes (RA-SFL) and paired peripheral blood lymphocytes (RA-PBL) from 21 RA patients by immunofluorescence flow cytometry. We have also investigated the expression of these adhesion molecules on peripheral blood lymphocytes obtained from 13 sex- and age-matched healthy controls (CO-PBL). RA-SFL, which consisted mostly of T cells, showed higher expression of the integrin subunits beta 1 (CD29), VLA-1 alpha, -3 alpha, -4 alpha, -5 alpha and -6 alpha when compared to RA-PBL. In turn, RA-PBL showed lower expression of these molecules than CO-PBL. The expression of the immunoglobulin-related molecules CD2, CD54 (ICAM-1) and CD58 (LFA-3) was higher on RA-SFL when compared to RA-PBL or CO-PBL, and similar results were obtained with the beta 2 integrin subunits CD11a and CD18. In contrast, L-selectin (LECAM-1) and ICAM-2 were expressed at much lower levels on RA-SFL than on RA-PBL or CO-PBL. CD44, a receptor for hyaluronic acid and collagen, was expressed by most RA-SFL, RA-PBL and CO-PBL cells but at higher density on RA-SFL. The results indicate that RA-SFL express a distinct array of adhesion molecules, similar to the one of memory T lymphocytes. This characteristic phenotype may contribute to the lymphocytic infiltration of the synovium and to the pathogenesis of RA.     

Characterization of tissue outgrowth developed in vitro in patients with rheumatoid arthritis: involvement of T cells in the development of tissue outgrowth

BACKGROUND: The aim of this study was to analyze cellular and cytokine interactions governing the development of synovial tissue outgrowth in patients with rheumatoid arthritis (RA). METHODS: A single-cell suspension of dissociated synovial tissues of RA patients was cultured for a long period to develop tissue outgrowth. The resulting tissue outgrowth was characterized by immunohistochemical staining and ELISA. RESULTS: The tissue outgrowth developed in vitro included various cell types, such as macrophage-like synovial cells, fibroblast-like synovial cells and lymphocytes. Even after prolonged cultivation, synovial cells devoid of infiltrating T lymphocytes did not form tissue outgrowth. The outgrowth contained CD3+ cells, LeuM3 (CD14)+ cells and HLA-DR+ cells. The T cells expressed lymphocyte function-associated antigen (LFA)-1 and CD2, and the synovial cells expressed intracellular adhesion molecule (ICAM)-1 and LFA-3, suggesting possible interactions via LFA-1/ICAM-1 and CD2/LFA-3. Production of T-cell derived IFN-gamma and IL-17 and synovial-cell-derived fibroblast growth factor (FGF)-1 and IL-15 was confirmed in the tissue outgrowth as well as in RA synovial tissue. These cell types stimulate each other by secreting cytokines, leading to the secretion of proinflammatory cytokines and matrix metalloproteinase (MMP)-1 by the tissue outgrowth and proliferation of both lymphocytes and synovial cells. CONCLUSION: This study emphasizes the importance of cellular interactions between T cells and synovial cells, via adhesion molecules and the secretion of cytokines with stimulatory activity towards other cell types, for the hyperactivity of RA synovial cells.     

Significance of VLA-4–VCAM-1 interaction and CD44 for transendothelial invasion in a bone marrow metastatic myeloma model

In previous work, we established the B9/BM1 syngeneic murine bone marrow metastasis model. Interleukin (IL)-6-dependent, IL-1-producing B9/BM1 cells, which colonize the vertebral and femoral marrow after i.v. injection, show great similarity in cell surface phenotype to human myeloma cells, especially the expression of 3 adhesion molecules, CD44, VLA-4 and ICAM-1. Here we investigated the function of these adhesion molecules by binding and transendothelial invasion assays using a newly established bone marrow-derived endothelial cell line (BMEC). A combination of monoclonal antibodies against CD44 and VLA-4 significantly inhibited the adherence of B9/BM1 cells to BMEC and anti-CD44 mAb especially blocked B9/BM1 transendothelial invasion of unstimulated BMEC cells. Results of additional experiments, in which the cells were treated with anti-CD44 and hyaluronidase, demonstrated that the interaction of CD44 molecules on B9/BM1 cells with hyaluronan on BMEC cells was a critical factor in both adhesion and transendothelial invasion in this model. However, stimulation of BMEC with TNFα resulted in increased invasion by B9/BM1 cells, which was completely suppressed by anti-VCAM-1 mAb, implicating a significant role of this adhesion molecule in this process during inflammation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chimeric CD4/CD44 molecules associate with CD44 via the transmembrane region and reduce hyaluronan binding in T cell lines

Cells of the immune system tightly regulate the binding ability of cell adhesion molecules. The binding of the extracellular matrix component hyaluronan to CD44 is no exception, yet the mechanisms that regulate its binding are poorly understood. In this study a chimeric CD4/CD44 molecule, containing the extracellular domain of CD4 and the transmembrane and cytoplasmic domains of CD44, was expressed in two CD44⁺ mouse T lymphoma cell lines, BW5147 and T28. This resulted in the reduced ability of endogenous CD44 to constitutively bind hyaluronan. Immunoprecipitation of the chimeric protein in 1 % Brij-96 indicated an association between the chimera and endogenous CD44. Using various chimeric CD4/CD44 molecules, the transmembrane region of CD44 was found to mediate this association. In addition, the association of chimeric CD4/CD44 molecules with endogenous CD44 correlated with reduced hyaluronan binding. Thus, the transmembrane region of CD44 is required for the association with CD44 molecules in the cell membrane and we propose that the self-association of CD44 molecules occurs on the T cell surface to promote hyaluronan binding. Cellular events altering the interactions of the transmembrane region of CD44 thus have the potential to regulate the hyaluronan binding ability of CD44.     

Recruitment of CD16+ monocytes into synovial tissues is mediated by fractalkine and CX3CR1 in rheumatoid arthritis patients

CD16+ monocytes, identified as a minor population of monocytes in human peripheral blood, have been implicated in several inflammatory diseases, including rheumatoid arthritis (RA). Fractalkine (FKN, CX3CL1), a member of the CX3 C subfamily, is induced by pro-inflammatory cytokines, while a receptor for FKN, CX3CR1, is capable of mediating both leukocyte migration and firm adhesion. Here, we investigated the role of FKN and CX3CR1 in activation of CD16+ monocytes and their recruitment into synovial tissues in RA patients. High levels of soluble FKN were detected in the synovial fluid and sera of RA patients. Circulating CD16+ monocytes showed a higher level of CX3CR1 expression than CD16- monocytes in both RA patients and healthy subjects. High level expression of CX3CR1 was also seen in CD16+ monocytes localized to the lining layer in RA synovial tissue. In the in vitro culture experiments, IL-10 induced CX3CR1 expression on the surface of monocytes, and TNFalpha induced membrane-bound FKN as well as soluble FKN expression in synovial fibroblasts. Moreover, soluble FKN was capable of inducing IL-1beta and IL-6 by activated monocytes. These results suggest that FKN might preferentially mediate migration and recruitment of CD16+ monocytes, and might contribute to synovial tissue inflammation.