类风湿关节炎滑膜组织PDGF与TGF-β1的表达与意义

应用免疫组织华沙SABC对24列类风湿关节炎（RA）及4例正常滑膜组织中血小板源性生长因子（PDGF），转化生长因子β1（TGF－β1）的表达分布情况进行观察分析，结果发现20例RA滑膜组织衬里层的巨噬细胞样细胞，浸润的炎性细胞与血管内皮细胞，成纤维细胞可见PGDF的表达分析，且巨噬细胞样细胞，浸润的炎症细胞的阳性程度强于血管内皮细胞，成纤维细胞。22例RA滑膜组织衬里层与衬里下层的成纤维细胞，巨噬细胞样细胞，血管内皮细胞均见TGF－β1的阳性表达分布。两者比较，阳性细胞的染色程度，染色细胞数与分布范围大致相等。4例正常滑膜组织中PDGF，TGF－β1的表达分布均为阴性。认为RA滑膜组织中PDEFTGF－β1的表达分布较正常多且表达程度大致相等，两者协同作用，参与了RA病理过程的滑膜衬里层增生，炎性细胞浸润，血管增殖与滑膜血管翳的形成。     

Expression of membrane-type matrix metalloproteinases in synovial tissue from patients with rheumatoid arthritis or osteoarthritis

We investigated the expression of membrane-type matrix metalloproteinase (MT-MMP) and matrix metalloproteinase (MMP) mRNAs in synovial tissue from patients with rheumatoid arthritis (RA, n = 5) or osteoarthritis (OA, n = 5) by Northern blot analysis. Northern analysis demonstrated strong expression of MT1-MMP, MT3-MMP, MMP-1, and MMP-3 and weak expression of MT2-MMP and MMP-8 in synovial tissue from patients with RA or OA. MT4-MMP was not detected. No significant difference was shown in the expression of MT-MMP mRNAs between RA and OA. Synovial tissue of RA or OA patients expressed MT-MMPs as well as MMPs. These results indicate that, in addition to MMPs, MT1-MMP, MT3-MMP, and probably MT2-MMP may play a role in the degradation of bone and cartilage matrix in RA and OA. Such information may provide a clue to the development of a novel therapeutic approach targeted on the prevention of joint destruction. Received: April 30, 2000 / Accepted: September 19, 2000     

Expression of interferon beta in synovial tissue from patients with rheumatoid arthritis: comparison with patients with osteoarthritis and reactive arthritis

BACKGROUND: IFNbeta may have immunomodulatory effects in rheumatoid arthritis (RA) and its increased production in RA synovium may be a reactive attempt to inhibit inflammation. OBJECTIVE: To determine the expression of IFNbeta in the synovial tissue of patients with RA, osteoarthritis, and reactive arthritis. METHODS: Synovial biopsy specimens were obtained by arthroscopy from patients with RA and disease controls for immunohistological analysis using a monoclonal antibody specific for IFNbeta. Bound antibody was detected by an immunoperoxidase method. Stained sections were evaluated by computer assisted image analysis. Double stainings were performed with antibodies to detect CD55 positive fibroblast-like synoviocytes (FLS), CD68 positive macrophages, and CD83 positive dendritic cells (DCs) co-expressing IFNbeta. RESULTS: IFNbeta protein was abundantly expressed in the synovium of patients with RA. Digital image analysis showed a significant increase in the mean integrated optical density for IFNbeta expression in RA synovial tissue compared with disease controls. Specific up regulation of IFNbeta expression was also seen when the results were controlled for cell numbers. Phenotypic analysis showed that FLS, especially, but also macrophages and DCs may express IFNbeta in RA synovial tissue. CONCLUSIONS: The increased expression of IFNbeta in RA synovium suggests activation of an immunomodulatory mechanism that could inhibit synovial inflammation.     

Cytokine expression in synovial membranes of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVES--To compare, by immunohistochemistry, the cellular and cytokine profile in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial membranes (SMs). Synovium was obtained at knee arthroplasty from 10 patients with RA and 10 with OA. METHODS--Synovial membranes were stained with a panel of monoclonal antibodies (MAb) to assess cytokine expression (IL-1 alpha, IL-1 beta, IL-6, GM-CSF, TNF-alpha and EGF) and the intensity of the mononuclear cellular infiltrate (MNC). RESULTS--Significantly greater percentages of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, GM-CSF and EGF cells were detected in all areas of the rheumatoid SMs when compared with osteoarthritic SMs. Five RA but only one OA SM demonstrated focal lymphoid aggregates. Lining layer thickening was noted in RA SMs only. The intensity of the MNC and number of blood vessels were greater in the RA group. CONCLUSION--The results suggest that the differences in cytokine production by RA and OA SMs are quantitative but that the greater thickness of the synovial lining layer and higher vascularity may be specific to RA.     

Morphological features of articular cartilage and synovial membrane in osteoarthritis and rheumatoid arthritis

Osteoarthritis (OA) and rheumatoid arthritis (RA) do not show any specific morphological findings. However, there are many morphological findings which are common to each disease. In the current study, we describe morphological changes of articular cartilage and synovial membrane during progression of joint destruction in OA and RA, respectively.     

Phospholipase activity in synovial fluid from patients with rheumatoid arthritis, osteoarthritis and crystal-associated arthritis

Phospholipase activity was assayed in cell-free synovial fluid (SF) from patients with rheumatoid arthritis (RA, n = 28), osteoarthritis (OA, n = 10), and crystal-associated arthritis (C, n = 7) by measuring the release of either [14C]oleic acid or [3H]arachidonic acid from radiolabeled E. coli phospholipids. Activity measured by oleic acid release was not significantly different between the three groups of patients (RA = 571 +/- 43.3, OA = 460 +/- 54.7 and C = 718 +/- 162.6 pmol/min/mg). Arachidonic acid release was significantly (p less than 0.005) less in OA (31 +/- 7.3) than RA (61 +/- 4.7) which was similar to C (58 +/- 17.6 pmol/min/mg). Arachidonic acid release correlated significantly with the SF white blood cell count (r = 0.483, p less than 0.01). This study shows the importance of the type of substrate used to measure phospholipase activity and indicates that differences in the capacity to release arachidonic acid may exist between RA and OA disease states.     

Distribution of protein nitrotyrosine in synovial tissues of patients with rheumatoid arthritis and osteoarthritis

OBJECTIVE: Because nitric oxide related species have been found in the inflamed joints of patients with arthritis, we investigated whether protein nitrotyrosine (a marker of tissue exposure to peroxynitrite) is present in their synovial tissues. METHODS: Protein nitrotyrosine was detected immunohistochemically and by Western blot analysis. Synovial tissues removed surgically from 12 patients with rheumatoid arthritis (RA) (mean age 63.7 yrs) and 20 with osteoarthritis (OA) (mean age 66.6 yrs) were studied. RESULTS: Nitrated proteins were detected immunohistochemically in all of 18 tissues examined. Diffuse staining of the stroma was seen in all patients, with more extensive staining in RA than OA (p = 0.008). Intense staining was detected in some lymphocytes, but not in others, even within a single lymphoid aggregate. Neutrophils did not stain for nitrotyrosine. Vascular endothelial cells stained for nitrotyrosine but adjoining smooth muscle cells did not. Both cytoplasmic and nuclear staining was seen in macrophages, endothelial cells, and lymphocytes. Numerous bands of nitrated proteins were detected by Western blot analysis of 15 synovial tissue extracts. Inducible nitric oxide synthase (iNOS) was detected immunohistochemically in endothelial cells, macrophages, vascular smooth muscle cells, and synoviocytes. CONCLUSION: Nitrotyrosine-containing proteins were found in essentially all synovia from RA and OA patients. The most prominent site of nitration in all cases was the stroma. iNOS, the likely source of the nitrating species, was found in a variety of cell types.     

Expression of fibronectin splice variants and oncofetal glycosylated fibronectin in the synovial membranes of patients with rheumatoid arthritis and osteoarthritis

OBJECTIVE: The aim of this study was to define and compare the expression of fibronectin (Fn) isoforms in synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Using monoclonal antibodies specific for total Fn, extra domain (ED)-A Fn, ED-B Fn, and oncofetal glycosylated Fn, we studied the expression of the Fn isoforms in synovium. Furthermore, in situ hybridization for the detection of ED-B Fn mRNA including a double labeling technique for the detection of cell type was applied. RESULTS: Strong expression of total Fn, ED-A Fn, oncofetal glycosylated Fn and, to a lesser extent, ED-B Fn could be demonstrated in the synovial lining layer in both RA and OA. Stromal and vessel expression of Fn isoforms was more prominent in RA tissue. Pannus tissue showed strong labeling with ED-B Fn. CONCLUSION: The expression of alternatively spliced isoforms of Fn is associated with tissue remodeling and, as a partial process of this phenomenon, with neovascularization rather than underlying disease, X-ray status, or parameters of acute inflammation. In the lining layer, Fn expression correlates with hyperplasia associated with cell recruitment but not with proliferative status. Most remarkably, the expression of ED-B Fn in pannus tissue seems to be associated with the invasive phenotype described in RA tissue.     

Urinary and synovial pyridinium crosslink concentrations in patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVES--To assess urinary and synovial concentrations of hydroxypyridinium crosslinks of collagen in patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to evaluate whether a combined measurement in the two compartments could give additional information about the origin of these compounds in joint diseases. METHODS--Concentrations of hydroxypyridinoline (HP) and lysylpyridinoline (LP) were measured by high pressure liquid chromatography in urinary and synovial samples collected from 20 patients with RA and 20 patients with knee OA. Full laboratory and clinical assessments were performed. RESULTS--Urinary concentrations of both HP and LP were significantly greater in RA than in OA. Urinary HP in RA correlated with the number of swollen joints corrected for Lansbury index and with erythrocyte sedimentation rate and C reactive protein. In synovial fluid from both groups, only relatively small amounts of HP were measured, while bone type I collagen specific LP was below the limit of detection in all samples. In RA patients, but not in OA patients, there was a strong correlation between urinary and synovial concentrations of HP (r = 0.75). CONCLUSIONS--The results underline the relationship between urinary HP and disease extent and activity in RA. The findings in synovial fluid support the hypothesis of an extraskeletal origin of HP in chronic joint diseases in which cartilage and synovial turnover may be increased.     

Parvovirus associated antigen in the synovial membrane of patients with rheumatoid arthritis.

Homogenates of synovium from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were centrifuged on caesium chloride density gradients to obtain isolates of a density similar to that of parvoviruses. Six of 11 RA isolates and none of six OA isolates reacted with an antiserum raised against a rheumatoid associated, parvovirus-like agent (RA-1 virus). An anti-B19 parvovirus antiserum did not react with any of the isolates tested. Electron microscopy of negatively stained preparations of the isolates showed that small particles of diameter 10 nm were abundant in most of the RA isolates (11/13) but absent from all OA isolates. Such particles, whose identification is unknown, were also present in RA-1 positive lysates prepared from cultured RA synovial cells. These results suggest that the RA-1 virus can be directly identified in RA synovial tissue and that the virus appears to be unrelated to the human B19 parvovirus.     

Detection of stromelysin in synovial fluid and serum from patients with rheumatoid arthritis and osteoarthritis

Summary Stromelysin levels were measured using a one-step sandwich immunoassay in synovial fluid (SF) obtained from 31 patients with rheumatoid arthritis (RA) (31 samples) and 13 patients with osteoarthritis (OA) (13 samples) and in serum from 81 patients with RA (106 samples), 12 with OA (14 samples), 12 with gouty arthritis (gout) (14 samples), and 8 with osteoporosis (OP) (14 samples) to identify differences in the levels in these diseases as well as correlations with clinical parameters in RA. SF stromelysin levels were significantly higher in RA than in OA, and rose with increasing joint destruction in the former. No significant correlations were found between the SF stromelysin level in RA and various clinical parameters, except for the volume of SF which showed a correlation. Serum levels of stromelysin were highest in RA, gout, OA, and osteoporosis in decreasing order, and in RA were correlated with the Steinbrocker Stage. A significant correlation was also found between the serum stromelysin level and number of swollen joints, and correlations with the Lansbury index, ESR, CRP, WBC and Plt. The stromelysin level in SF was thought to be a useful parameter of local joint involvement and that in serum of the severity of systemic joint inflammation.     

Dissimilar biosynthesis of interleukin-6 by different areas of synovial membrane of patients with rheumatoid arthritis and osteoarthritis.

In this study the IL-6 production was studied by synovial cells isolated from patients with either rheumatoid arthritis (RA) or osteoarthritis (OA). The kinetics of spontaneous IL-6 production differs in both groups. Furthermore, the induction of IL-6 by bacterial lipopolysaccharide (LPS) in synovial cell cultures of RA is much more rapid than in those of OA patients. On the other hand, more PGE2 was detected in culture supernatants from synovial adherent cells of OA than in those of RA patients. We also compared the IL-6 production and the amount of IL-6 mRNA in fragments derived from the areas of synovial tissue showing macroscopic signs of intensive inflammation (area A), with those from relatively intact (area B) synovial sites. In synovial fragments, but not in isolated adherent cells at area A the in vitro IL-6 production starts earlier in RA than in OA. In the area A, significantly more CD14+, CD43+ and HLA-DR+ cells were detected than in the other compartment less involved in local inflammatory events.     

Distribution of mast cells in human synovial tissue of patients with osteoarthritis and rheumatoid arthritis

Mast cells are demonstrated in synovial membranes of patients with osteo-arthritis and rheumatoid arthritis using a new staining principle based on interaction of heparin in mast cell granules with peroxidase labeled avidin. It was found that mast cell numbers in the subsynovial layer of patients with rheumatoid arthritis were significantly lower than those in patients suffering from osteo-arthritis. This decrease can be mainly attributed to patients with rheumatoid arthritis whose synovitis was characterized by a distinct histomorphological pattern consisting of lining cell ulcers and granulation tissue. However, when mast cell numbers in rheumatoid arthritis and osteo-arthritis patients were compared without respect to mast cell distribution in the subsynovial layer or the stratum fibrosum, no statistical differences between the diseases could be observed.     

Characteristics of the protease activity in synovial fluid from patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: To clarify which proteases are specifically activated in the lesions of rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: The activity levels of the serine proteases of the coagulation and fibrinolytic systems, and of elastase and collagenase as controls, in synovial fluid from 27 RA patients and 28 OA patients were measured using fluorogenic synthetic substrates which had methylcoumarylamide (MCA) at their COOH-termini. The thrombin-antithrombin III complex (TAT) content was also measured by ELISA. RESULTS: Among the proteases, thrombin-like activity was the highest in both RA and OA. The profiles of protease activity were similar in RA and OA, but their activities were in general significantly higher in RA than in OA (p < 0.01). The levels of both thrombin-like activity and TAT were about 7.5-fold higher in RA than in OA, while the levels of CRP and fibrinogen were only about 2-fold higher. Biochemical characterization of the thrombin-like activity in the synovial fluid of RA patients showed that this activity was due to thrombin. Thrombin-like activity positively correlated with the TAT concentration in RA (r = 0.750, p < 0.0001), but not in OA. CONCLUSION: Activation of the coagulation system was more marked in RA than in OA, strongly suggesting that in RA there is an imbalance between thrombin and its inhibitors, and that thrombin is more closely linked to the pathogenesis of RA than to that of OA. Our results also show that analysis of the synovial fluid may be useful to estimate the activation of the coagulation system in RA, but not that of the fibrinolytic system.     

类风湿关节炎患者血清滑液和滑膜组织白细胞介素-18的水平及意义

目的 研究类风湿关节炎（RA）患者血清、滑液中白细胞介素－18（IL－18）蛋白及滑膜组织IL－18mRNA表达水平，探讨其在RA致病中的作用。方法 应用双抗夹心酶免疫吸附（ELISA）法和细胞生物法分别测定RA患者血清、滑液中IL－28蛋白水平和生物活性，同时还检测NO、前列腺素E2的含量；有用半定量RT－PCR法检测膜组织IL－18m RNAG表达水平。以骨关节炎（OA）病人及因外伤截肢的正常人作对照。结果 RA患者血清、滑液中IL－18蛋白水平和生物活性均显著高于对照组，滑液中量及活化性比血清高；RA滑膜组织IL－18mRNA表达水平也明显高于对照组。结论 过度表达的IL－18参与了RA的致病过程,；选择性地抑制IL－18生物活性，将是RA治疗的新途径。