ALA-induced porphyrin formation and fluorescence in synovitis tissue: In-vitro and in vivo studies

The synovial inflammatory process in rheumatoid arthritis (RA) is accompanied by massive tumor-like proliferation and activation of the connective stroma. These abnormal cells actively invade and destroy the peri-articular bone and cartilage at the margins of joints where synovium and bone are attached. There is still a lack of minimally invasive synovectomy methods, which might be suitable for the smaller joints. Unfortunately, these joints are usually involved in the disease. Photodynamic therapy has been evaluated as a possible treatment modality for RA synovitis. The present study describes the differences of 5-aminolevulinic acid (5-ALA) and 5-ALA ester-induced protoporphyrin IX (PpIX) production in cell cultures obtained from patients with RA, osteoarthritis (OA) and human sarcoma cell line (HS 192.T) and in a collagen-induced arthritis model in rats. The incubation of cells with hexyl aminolevulinate (HAL) induced the same amount of fluorescence as 5-ALA and methyl aminolevulinate (MAL) at about a 100-fold lower concentration. Incubation with HAL-induced accumulation of at least twice as much porphyrins in RA- and HS 192.T-cells than 5-ALA and MAL in OA-cells. Similar levels of porphyrins were accumulated in RA and the malignant cells. In vivo, intra-articular application of 5-ALA induced a significant porphyrin accumulation in synovitis tissue as measured by in situ fluorescence spectroscopy. In contrast to our in vitro results and other reports, we could not detect enhanced fluorescence after application of up to 0.1 mg HAL.     

Temporal effects of cytokine treatment on lubricant synthesis and matrix metalloproteinase activity of fibroblast‐like synoviocytes

Fibroblast‐like synoviocytes (FLS) are major contributors to the composition and function of synovial fluid (SF). In disease, changes to important SF molecules such as hyaluronic acid (HA), lubricin, and numerous inflammatory markers contribute to a loss of SF functional properties. Previous studies characterized the ability of FLS to produce SF molecules in short‐term cultures using continuous cytokine supplementation. This study assessed the HA, lubricin, and matrix metalloproteinase‐2 (MMP‐2) secretion profile of FLS over 12 days of culture. FLS were subjected to continuous, intermittent, and sequential cytokine treatments of interleukin‐1 beta (IL‐1β), tumour necrosis factor‐alpha (TNF‐α), and transforming growth factor‐beta 1 (TGF‐β1). HA was assessed by an enzyme‐linked immunosorbent assay (ELISA) for content and agarose gel electrophoresis for molecular weight distribution. Relative lubricin content was determined by western blot. Pro MMP‐2 and active MMP‐2 were quantified by gelatin zymography. All intermittent and sequential treatments significantly increased secretion of high‐molecular‐weight (>3 MDa) HA for the duration of the culture. Sequentially treated groups elevated lubricin synthesis, whereas only groups receiving IL‐1β and TNF‐α for 2 days followed by TGF‐β1 for 1 day reduced active MMP‐2 to unstimulated control levels. These data provide important information on the long‐term functional potential of cytokine‐stimulated FLS and suggest that temporal regulation of cytokine exposure can be a powerful tool to guide healthy synovial secretions.     

The roles of IFN-γ versus IL-17 in pathogenic effects of human Th17 cells on synovial fibroblasts

Objectives

Th17 cells, while indispensable in host defense, may play pathogenic roles in many autoimmune diseases, including rheumatoid arthritis (RA). However, the mechanisms by which human Th17 cells drive autoimmunity have not been fully defined. We assessed the potential of the human Th17 CD4 T cell subset to induce expression of cell–cell interaction molecules and inflammatory mediators by fibroblast-like synoviocytes (FLS), and the roles of IFN-γ and IL-17 in these interactions.

Methods

Th1 or Th17 cells were induced from healthy adult donor CD4 T cells and were co-cultured with FLS for 48 h with/without neutralization of IFN-γ, IL-17A, or both. Alternatively, FLS were treated only with IFN-γ or IL-17 for 48 h. FLS expression of CD40, CD54, and MHC-II, as well as IL-6 and IL-8 secretion, were assessed by surface staining followed by flow cytometry and ELISA, respectively.

Results

Both Th1 and Th17 cells secreted IL-17 as well as IFN-γ, although IFN-γ production was much greater from Th1 cells. FLS expression of CD40, CD54, and MHC-II significantly increased upon co-culture with Th1 cells, while Th17 cells increased only the percentage of FLS that were CD54+. Both T cell subsets induced IL-6 and IL-8 secretion by RA FLS. Neutralization of IL-17A did not reduce FLS expression of CD40, MHC-II, or CD54, but did inhibit IL-6 and IL-8 secretion. Although IFN-γ was a weak inducer of IL-6 secretion and significantly inhibited IL-8 secretion from FLS when used as a single stimulus, neutralization of IFN-γ inhibited the secretion of both cytokines in Th17/FLS co-cultures with RA but not OA FLS.

Conclusion

FLS cell–cell interaction molecules and soluble inflammatory mediators are differentially regulated by IFN-γ and IL-17. The effects of IFN-γ may depend in part on the particular milieu of other co-existing cytokines and its potential to induce cell–cell interactions. The potential benefit of therapeutic neutralization of either IL-17 or IFN-γ could depend on the relative proportions of these cytokines in the synovial compartment of an RA patient.     

秦艽、威灵仙及其配伍对类风湿关节炎成纤维样滑膜细胞的抑制、促凋亡作用及对相关炎症因子的影响

目的研究秦艽、威灵仙及其配伍对类风湿关节炎成纤维样滑膜细胞(RA FLSs)的抑制、促凋亡作用及对相关炎症因子的影响。方法将购得的RA FLSs复苏后,通过光学显微鉴定和Vimentin免疫荧光鉴定并传代培养,将生长密度为80%～90%的细胞随机分为空白组、阳性药(甲氨蝶呤)组、秦艽组、威灵仙组、秦艽-威灵仙药对组5组。将中药水煎液制成质量浓度为8000μg/m L的含药培养基,将阳性药制成质量浓度为400μg/m L的含药培养基,各组分别给予相应含药培养基进行干预,以CCK-8法检测各组RA FLSs的增殖活性,以Annexin V-FITC法检测各组RA FLSs的凋亡情况,以ELISA法检测各组RA FLSs培养上清液中类风湿因子(RF)、C-反应蛋白(CRP)的含量。结果光学显微鉴定结果显示购得的RA FLSs形态符合文献报道,免疫荧光鉴定结果显示购得的RA FLSs为人源RA FLSs,且纯度大于99%; CCK-8法检测结果显示,各药物组对RA FLSs增殖均具有抑制作用,其中威灵仙组对RA FLSs的增殖抑制作用最强(P<0.01);Annexin V-FITC法检...     

Wnt调控类风湿性关节炎研究进展

类风湿性关节炎(RA)是一种主要以持续关节组织滑膜增生、软骨侵蚀、关节损坏为主要特征的累及多关节的系统性疾病,发病机制不明。滑膜衬里层成纤维样滑膜细胞(FLS)在RA发病机制中起关键作用。Wnt通路广泛参与细胞癌变和肿瘤细胞的侵袭、转移等,在肿瘤的发病机制中发挥重要调控作用。Wnt通路同样调控了RA的发生发展,下调RA Wnt和Fz表达能够抑制FLS活化、增殖,提示Wnt通路Wnt蛋白可能是潜在的RA治疗靶点。     

Parthenolide Inhibits Proliferation of Fibroblast-Like Synoviocytes In Vitro

Parthenolide is a bioactive constituent of an aromatic herb Feverfew (Tanacetum parthenium). It has been found that both parthenolide and extract of feverfew have anti-inflammatory and antinociceptive properties. Moreover, they demonstrate antiproliferative activities on different human tumour cells. The massive hyperplasia of synovial fibroblasts is the one of the most striking features of rheumatoid arthritis. It is not known whether this is due to the proliferation of synovial fibroblasts or to defective apoptosis. We investigated the effect of parthenolide on the proliferation of rabbit synoviocytes cell line HIG-82, rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and human skin fibroblasts (HSF) in vitro. Cell proliferation was assessed by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 5'-bromo-2'-deoxy-uridine methods. Parthenolide inhibited proliferation of HIG-82 and human RA-FLS. The proliferation of HSF was inhibited less effectively. The antiproliferative potential of parthenolide was demonstrated.