Effects of hydrogen peroxide on the metabolism of human rheumatoid and osteoarthritic synovial fibroblasts in vitro.

The effects of hydrogen peroxide (H2O2) on the metabolism of cultured human synovial fibroblasts derived from joints of four patients with rheumatoid arthritis and three with osteoarthritis have been investigated. The exposure of rheumatoid cell cultures to this oxygen derived species at sublethal concentrations (1-100 mumol/l) induced a dose related inhibition of both hyaluronic acid (HA) and DNA synthesis. In contrast, in osteoarthritic cell lines a biphasic response was shown. At low concentrations of H2O2 (less than 10 mumol/l) a stimulatory effect on HA synthesis was noted, whereas in the presence of higher concentrations (greater than 10 mumol/l) a significant inhibition of synthesis occurred. These deleterious effects of H2O2 were partially reduced by the addition of catalase to the culture media. The finding that both HA and DNA synthesis were inhibited at concentrations of H2O2 less than those which caused loss of cell integrity (greater than 200 mumol/l) suggests oxidation of intracellular components, such as glyceraldehyde-3-phosphate dehydrogenase, and subsequent depletion of ATP concentrations.     

Expression of CD44 in normal and rheumatoid synovium and cultured synovial fibroblasts.

OBJECTIVE--To determine if expression of CD44, the principal receptor for hyaluronan, was altered in rheumatoid (RA) synovium and cultured rheumatoid synovial fibroblasts. METHODS--Synovium was obtained from normal adult human joints (n = 4) and from joints of patients with RA (n = 5). Specific monoclonal antibodies to CD44 were used in immunofluorescence of whole synovium and cultured synovial fibroblasts and in quantitative Western blotting and ELISA of CD44 in cultured synovial fibroblasts. RESULTS--CD44 was restricted to the lining layer in normal synovium but present, in reduced concentrations, throughout rheumatoid synovium. Cultured rheumatoid cells were 19% larger in area and showed far fewer and less extensive CD44-positive cytoplasmic extensions, together with reduced staining intensity compared with normal. Quantitative Western blotting normalised for cell protein showed a 75% reduction (normal = 1754 (835), rheumatoid = 409 (84) mean (SD) arbitrary units) in the amount of CD44 in rheumatoid cells compared with normal, and enzyme linked immunosorbent assay (ELISA) of cultured cell monolayers normalised for cell number indicated a 29% reduction (normal = 0.707 (0.110), rheumatoid = 0.504 (0.103), mean (SD) optical density at 405 nm). CONCLUSIONS--Rheumatoid synovial cells showed altered morphology and reduced CD44 expression compared with normal cells. CD44, by means of modulated associations with the cytoskeleton, may be involved in cell shape change.     

Microsatellite analysis in rheumatoid arthritis synovial fibroblasts

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic disease characterised by irreversible destruction of the affected joints. As aggressive transformed-appearing synovial fibroblasts are commonly found at the site of invasion of the rheumatoid synovium into the adjacent cartilage and bone, the presence of microsatellite instability (MSI) and expression of mismatch repair enzymes as a possible mechanism in the alteration of these cells was examined. METHODS: DNA was extracted from the synovial fibroblasts and blood of 20 patients with long term RA undergoing joint replacement, and the presence of MSI was studied at 10 microsatellite loci. In addition, immunohistochemistry was performed to evaluate the expression of the two major mismatch repair enzymes (hMLH1 and hMSH2) in rheumatoid synovium. RESULTS: MSI could not be detected in any of the fibroblast cell populations derived from the 20 different rheumatoid synovial samples. In addition, strong expression of mismatch repair enzymes could be seen in numerous cells, including fibroblasts, throughout the synovium. CONCLUSIONS: Applying the currently used and established markers for MSI, the data show for the first time that MSI does not appear to have an important role in alteration of rheumatoid synovial fibroblasts into an aggressive phenotype. On the other hand, strong mismatch repair enzyme synthesis in rheumatoid synovium supports the hypothesis of continuing DNA repair, presumably due to long term, inflammation induced DNA damage.     

Hyaluronic acid production in vitro by synovial lining cells from normal and rheumatoid joints.

Organ cultures and primary cell cultures were established from synovial tissue collected from patients with rheumatoid arthritis. Hyaluronic acid measured by the incorporation of [3H]glucosamine into the polysaccharide was found to be synthesised in the cultures immediately after transfer from in-vivo to in-vitro conditions. This was in contrast to the primary cultures established from cells isolated from normal joints. The latter cells did not synthesise any detectable hyaluronate. 90-100% of the cells in primary culture were found to be esterase positive, indicating their macrophage nature. The molecular weight of the hyaluronate produced by the pathological cells was low (approximately 50 000) compared with the molecular weight of hyaluronate found in joint fluid from normal or rheumatoid joints. Cell lines of fibroblasts established from rheumatoid joints and studied after four or seven passages also produced hyaluronate of low molecular weight. It is known that similar cell lines from normal joints produce a high molecular weight polymer.     

DNA hypomethylation in rheumatoid arthritis synovial fibroblasts

Objective

Rheumatoid arthritis synovial fibroblasts (RASFs) are phenotypically activated and aggressive. We undertook this study to investigate whether the intrinsic activation of RASFs is due to global genomic hypomethylation, an epigenetic modification.

Methods

Global genomic hypomethylation was assessed by immunohistochemistry, flow cytometry, and L1 promoter bisulfite sequencing. The levels of Dnmt1 were determined in synovial tissue and cultured SFs by Western blotting before and after treatment with cytokines and growth factors. Normal SFs were treated for 3 months with a nontoxic dose of the DNA hypomethylation drug 5‐azacytidine (5‐azaC), and changes in gene expression were revealed using complementary DNA arrays. The phenotypic changes were confirmed by flow cytometry.

Results

In situ and in vitro, RASF DNA had fewer 5‐methylcytosine and methylated CG sites upstream of an L1 open‐reading frame than did DNA of osteoarthritis SFs, and proliferating RASFs were deficient in Dnmt1. Using 5‐azaC, we reproduced the activated phenotype of RASFs in normal SFs. One hundred eighty‐six genes were up‐regulated >2‐fold by hypomethylation, with enhanced protein expression. These included growth factors and receptors, extracellular matrix proteins, adhesion molecules, and matrix‐degrading enzymes. The hypomethylating milieu induced irreversible phenotypic changes in normal SFs, which resembled those of the activated phenotype of RASFs.

Conclusion

DNA hypomethylation contributes to the chronicity of RA and could be responsible for the limitation of current therapies.

     

表皮生长因子对类风湿关节炎滑膜细胞的作用

目的 观察表皮生长因子（ＥＧＦ）对类风湿关节炎（ＲＡ）滑膜细胞的作用和细胞中有丝分裂素激活的蛋白激酶（ＭＡＰＫ）的激活情况。方法 ３～５代体外培养的ＲＡ滑膜细胞,＾３Ｈ－ＴｄＲ掺入检测ＥＧＦ对细胞ＤＮＡ合成的影响;ＥＧＦ刺激后裂解细胞,收获蛋白,Ｗｅｓｔｅｒｎ ｂｌｏｔ检测ＭＡＰＫ的活化。结果 ＥＧＦ刺激组和对照组每分钟计数值差异有显著性（Ｐ〈０．００１）。ＥＧＦ能引起细胞内明显ＭＡＰＫ活化。结论     

The effect of rheumatoid synovial fluid macrophages on DNA,glycosaminoglycan and collagen synthesis by synovial fibroblasts

Summary The effects of soluble factors secreted by peripheral blood monocytes and rheumatoid synovial fluid macrophages were tested on human synovial fibroblast cultures. Both monocytes and macrophages liberated factors which reduced DNA synthesis (³H-thymidine incorporation) by synovial fibroblasts. Monocyte and macrophage factors stimulated hyaluronic acid synthesis. The activation obtained with rheumatoid synovial macrophages was considerably greater than that with monocytes. Foetal bovine serum was found to have a clear stimulatory effect on the synthesis of collagen and other proteins by fibroblasts. The effects of monocyte and macrophage factors on protein synthesis in synovial fibroblasts were small: collagen synthesis was slightly increased relative to other extracellular proteins.     

Properties of rheumatoid and normal synovial tissue in vitro and cells derived from them

Summary Using organ and cell culture techniques for tissues and cells derived from human sources, we have investigated cellular interactions involving synovial tissue. Normal synovium in culture produced less prostaglandin E (PGE) and collagenase than cultures of rheumatoid synovial fragments. When synovial tissue was dissociated by enzymatic digestion, monolayers of adherent cells were established in primary culture. The adherent cells rapidly lost the ability to synthesize large amounts of PGE and collagenase and rheumatoid synovial cells became indistinguishable from normal synovial cells. Supernatants from cultured human mononuclear blood cells contained activities (Mononuclear cell factor(s)=MCF) which stimulated PGE and collagenase production by either normal or rheumatoid synovial cells. Conditioned medium from cultures of either normal or rheumatoid synovial fragments (Synovial factor(s)=SF) also stimulated production of PGE and collagenase by these human cells. Both MCF and SF also stimulated the production of PGE by cells isolated from human trabecular bone. Since both normal and rheumatoid synovial cells respond similarly to these factors, there appears to be little specificity with regard to whether the target cells are derived from normal or pathological sources. Furthermore, since both normal and rheumatoid synovium are able to produce similar amounts of stimulatory activity, inflammatory cells are not solely responsible for these phenomena. Normal synovium must therefore contain cells which can be recruited to participate in these potential cellular interactions. Destruction of joint structures may be mediated by factors of the type studied here, which may be produced when there is failure of the mechanisms that prevent them from being synthesised or released.     

Effects of interleukin-6 on the metabolism of connective tissue components in rheumatoid synovial fibroblasts

Objective. High levels of interleukin-6 (IL-6) have been found in the synovial fluid of patients with rheumatoid arthritis (RA). We undertook the present study to investigate the role of IL-6 in this disease. Methods. We examined the effects of IL-6, in comparison with IL-1, on the biosynthesis of extracellular matrix macromolecules and of matrix-degrading proteinases in rheumatoid synovial fibroblasts. Results. In rheumatoid synovial fibroblasts, IL-6 by itself enhanced the production of plasminogen activator, its inhibitor, and tissue inhibitor of metalloproteinases, whereas it did not modulate the biosynthesis of precursor of matrix metalloproteinase 1 (proMMP-1) (tissue collagenase), proMMP-3 (stromelysin), or connective tissue components. However, IL-1–induced production of proMMP-1 and proMMP-3 was preferentially augmented by IL-6. Conclusion. These results suggest that in RA, IL-6 may participate along with IL-1 in fine tuning of the catabolism of connective tissue components, by modulating the balance between connective tissue–degrading enzymes and their inhibitors.     

In vitro conditions affecting the synthesis of sulfated proteoglycans by normal and rheumatoid synovial cells in culture

In vitro conditions affecting synthesis of sulfated proteoglycans by cell suspensions derived from monolayer cell cultures of normal and rheumatoid synovial tissue were examined. The capacity of cells to synthesize proteoglycans was estimated by the incorporation of ³⁵S–sulfate into cetylpyridinium chloride–precipitable material. Synthesis of sulfated proteoglycans was maximal during log phase, and after 2–3 hours of recovery from disaggregation. Normal synovial cells appeared to be more sensitive to changes in serum concentration than were rheumatoid synovial cells, but rheumatoid synovial cells were more sensitive to changes in cell density. The proportion of newly synthesized extracellular proteoglycans increased with the duration of incubation in ³⁵S–sulfate.     

Plasminogen activator activity of rheumatoid and nonrheumatoid synovial fibroblasts

Extracellular and cell associated plasminogen activator (PA) activities were measured in a series of synovial fibroblast cell lines, derived by outgrowth and passage from both rheumatoid and nonrheumatoid tissue. In early passages of these cell lines under standardized culture conditions, the enzyme activity was low for most lines but relatively high in a few. There was no significant difference in the PA levels of the cell lines from either source. In addition, cells from both groups increased their PA activity in response to conditioned medium from peripheral blood mononuclear cells. It is concluded that the presence of fibrin-like material in rheumatoid joints cannot be simply explained by a relative defect in PA production by cells obtained after outgrowth and passage from rheumatoid explant material.     

Plasma membrane glycoproteins of cultured rheumatoid synovial fibroblasts

Fibroblast cultures were started from synovial tissue samples of 12 rheumatoid arthritic, 9 non-rheumatoid synovitic and 6 control patients. External galactose units of plasma membrane glycoproteins of confluent cells were labelled using the galactose oxidase-tritiated borohydride method. These surface-labelled cells were analyzed for possible differences in their glycoproteins by electrophoresis in SDS-containing polyacrylamide gradient gels. Total cell lysates were separated into 50-60 polypeptide bands. Fluorography of the gels revealed about 20 labelled plasma membrane glycoproteins. Some strain-specific differences were detected between the samples in all the groups, but no correlation with rheumatoid arthritis could be demonstrated.     

The effects of synovial fluid macrophages and interleukin-1 on hyaluronic acid synthesis by normal synovial fibroblasts

Summary The effects of peripheral blood monocyte and rheumatoid synovial fluid macrophage conditioned media were studied on hyaluronic acid (HA) metabolism of normal synovial fibroblasts. Both media stimulated HA synthesis about two-fold compared to controls (1% fetal calf serum). The activated mononuclear phagocyte conditioned media did not contain HA-degrading activity in these experiments. The effects of various concentrations of interleukin-1 (IL-1) on HA synthesis and proliferation of synovial fibroblasts were studied. Even at very low concentrations (0.1 IU IL-1/ml) HA synthesis was stimulated. With increasing concentrations HA synthesis did not increase but proliferation was stimulated. Stimulated fibroblasts synthesized mainly high molecular weight HA. Thus with IL-1-activation, normal synovial fibroblasts could not produce increased amounts of abnormal HA with decreased molecular weight.     

甲氨蝶呤对类风湿关节炎滑膜细胞增生及细胞周期的影响

目的研究甲氨蝶呤(MTX)对类风湿关节炎(RA)滑膜细胞增生及细胞周期的影响,揭示MTX治疗RA的机制。方法应用四唑氮化合物(MTS)/黄嘌呤氧化酶(PMS)比色法和流式细胞术分别测定MTX处理和未处理RA患者滑膜细胞的细胞增生水平和细胞周期。结果加入不同浓度MTX(0.2~2滋mol/L),RA患者滑膜细胞接种后第4天起增生水平均显著低于未处理的RA患者滑膜细胞(P<0.05);细胞周期分析显示加入不同浓度MTX(0.2~2滋mol/L)后第5、8天,RA患者滑膜细胞停留在G1期的比例要比未处理的RA患者滑膜细胞明显增加(P<0.05),而S、G2期的细胞比例比未处理的RA患者滑膜细胞则明显减少(P<0.05);MTX能以剂量依赖性明显抑制滑膜细胞的增生水平。结论MTX在治疗RA时,主要起到对RA患者滑膜细胞的增生及增生相关病理过程的抑制。     

Effects of interleukin-6 on the metabolism of connective tissue components in rheumatoid synovial fibroblasts.

OBJECTIVE. High levels of interleukin-6 (IL-6) have been found in the synovial fluid of patients with rheumatoid arthritis (RA). We undertook the present study to investigate the role of IL-6 in this disease. METHODS. We examined the effects of IL-6, in comparison with IL-1, on the biosynthesis of extracellular matrix macromolecules and of matrix-degrading proteinases in rheumatoid synovial fibroblasts. RESULTS. In rheumatoid synovial fibroblasts, IL-6 by itself enhanced the production of plasminogen activator, its inhibitor, and tissue inhibitor of metalloproteinases, whereas it did not modulate the biosynthesis of precursor of matrix metalloproteinase 1 (proMMP-1) (tissue collagenase), proMMP-3 (stromelysin), or connective tissue components. However, IL-1-induced production of proMMP-1 and proMMP-3 was preferentially augmented by IL-6. CONCLUSION. These results suggest that in RA, IL-6 may participate along with IL-1 in fine tuning of the catabolism of connective tissue components, by modulating the balance between connective tissue-degrading enzymes and their inhibitors.