Effects of synovial fluid on the respiratory burst of granulocytes in rheumatoid arthritis

Neutrophil infiltration in the synovia is an important feature of the local inflammatory process associated with rheumatoid arthritis. The present study is focused on the effects exerted in vitro by the synovial fluid versus serum on the respiratory burst of granulocytes isolated either from blood or synovial fluid of rheumatoid arthritis patients. The respiratory burst was evaluated as superoxide anion release, by lucigenin-amplified chemiluminescence. Our data show that the respiratory burst of granulocytes isolated from rheumatoid arthritis patients might trigger a significant oxidative stress both in periphery and the inflamed joint. These cells show no pathological pattern when activated in vitro by the chemotactic peptide fMLP, heterologous synovial fluid or serum. Acellular synovial fluid amplifies the superoxide anion release induced by fMLP more than the corresponding serum, indicating that a bacterian infection in the joint might enhance the oxidative damage in the inflamed synovium.     

6-Sulfanilamidoindazole arthritis: Influence of alloxan diabetes,phenylbutazone and D-penicillamine on inflammatory size,sulfhydryl groups,diphenylamine reaction and enzymes of the serum

The influence of alloxan diabetes, phenylbutazone (20 mg/kg), and D-penicillamine (30 mg/kg) on 6-sulfanilamidoindazole arthritis was investigated. Arthritis was not altered by the alloxan-diabetic stage neither by injecting alloxan during developing arthritis nor by injecting it before the beginning of 6-sulfanilamidoindazole administration. However, the arthritis was completely suppressed by phenylbutazone after both methods of administration. D-penicillamine was without significant effect. Decrease of serum sulfhydryl groups and increase of serum diphenylamine value occurring during arthritis were prevented by phenylbutazone whereas alloxan and D-penicillamine did not.     

Disease severity in K/BxN serum transfer-induced arthritis is not affected by IL-33 deficiency

Introduction

Interleukin (IL)-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous work suggested implication of the IL-33/ST2 axis in the pathogenesis of human and mouse arthritis. Here, we directly investigated the role of endogenous IL-33 in K/BxN serum transfer-induced arthritis by using IL-33 knockout (KO) mice.

Methods

Arthritis was induced by injection of complete K/BxN serum or purified IgG. Disease severity was monitored by clinical and histological scoring.

Results

K/BxN serum transfer induced pronounced arthritis with similar incidence and severity in IL-33 KO and wild-type (WT) mice. In contrast, disease development was significantly reduced in ST2 KO mice. IL-33 expression in synovial tissue was comparable in arthritic WT and ST2 KO mice, and absent in IL-33 KO mice. Transfer of purified arthritogenic IgG instead of complete K/BxN serum also resulted in similar arthritis severity in IL-33 KO and WT mice, excluding a contribution of IL-33 contained in the serum of donor mice to explain this result. We investigated additional potential confounding factors, including purity of genetic background, but the mechanisms underlying reduced arthritis in ST2 KO mice remained unclear.

Conclusions

The data obtained with IL-33 KO mice indicate that endogenous IL-33 is not required for the development of joint inflammation in K/BxN serum transfer-induced arthritis. On the contrary, arthritis severity was reduced in ST2 KO mice. This observation might relate to IL-33 independent effects of ST2, and/or reveal the existence of confounding variables affecting the severity of joint inflammation in these KO strains.     

Reduced amyloid-A-degrading activity in serum in amyloidosis associated with rheumatoid arthritis.

The ability to degrade amyloid A fibrils was studied in the serum of 31 patients with amyloidosis associated with rheumatoid arthritis, 33 patients with rheumatoid arthritis without amyloidosis, and 47 healthy controls. Fibrillar amyloid A protein and the radial diffusion method were used. The mean degrading activity in serum was significantly lower in patients with rheumatoid arthritis complicated by amyloidosis (58 +/- 19% SD of the activity in a pooled sample of sera from 100 healthy blood donors used as standard) than in patients with rheumatoid arthritis alone (78 +/- 14%; p less than 0.001) or controls (99 +/- 19%; p less than 0.001). Alpha 1-antitrypsin, concentrations of which were raised in both groups of patients, inhibited the degrading activity in serum even in low concentrations. A negative correlation between degrading activity and alpha 1-antitrypsin concentrations was observed. These findings suggest that reduced amyloid-A-degrading activity is due to inhibition rather than to deficiency of enzyme.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia Yop proteins by Western-blot

The results obtained with the use of the western-blotting showed that antibodies for released proteins YopD (33-36 kDa) were the most frequently detected antibodies in serum samples from patients suspected for yersiniosis. Reactions between serum samples studied and the YopD protein were very intense, suggesting that protein is the strongest immunogen among the utilised, released proteins Yop of Yersinia. Antibodies IgM were more often diagnosed in patients with abdominal pain in the contrary to antibodies IgA which were characteristic to patients with reactive arthritis. Detailed analysis of the results of western-blotting on serum samples obtained several times from individuals with yersinosis during the course of infection in this investigation have showed also that antibodies of the IgA class hold longer in serum of individuals with arthritis compared with individuals with yersinosis not complicated by arthritis. In joint-fluid samples obtained from patients with arthritis antibodies for particular released proteins Yop were detected in the same class of immunoglobulins like in serum samples obtained from those individuals.     

Biochemical criteria for the evaluation of drug efficiency on adjuvant arthritis and nephrotoxic serum nephritis in the rat: studies with phenylbutazone, L-Asparaginase, colchicine, lysine acetylsalicylate, and pyridinol carbamate.

The levels of serum orosomucoid, haptoglobin, and seromucoid were evaluated as possible quantitative criteria for the estimation of drug efficiency in adjuvant arthritis and nephrotoxic serum nephritis. In adjuvant arthritis, haptoglobin, seromucoid, and chiefly orosomucoid serum levels were generally very sensitive to anti-inflammatory agents such as phenylbutazone and pyridinol carbamate, and to immunosuppressive agents such as L-asparaginase. There was a significant correlation between the serum levels of these glycoproteins and the arthritis scores. In nephrotoxic serum nephritis, seromucoid levels were correlated with the proteinuria of the autologous phase and were found to be a good complementary criterion for the analysis of the efficiency of pyridinol carbamate, colchicine, iysine acetylsalicylate, and L-asparaginase.     

The involvement of Toll‐like receptor 9 in the pathogenesis of erosive autoimmune arthritis

Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll‐like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell‐dependent phase of inflammatory arthritis. In rats with pristane‐induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL‐6, α‐1‐acid‐glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9^?/? mice, streptococcal cell wall (SCW)‐induced arthritis was reduced in the T cell‐dependent phase, whereas T cell‐independent serum‐transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell‐dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis.     

Successful treatment of animal models of rheumatoid arthritis with small-molecule cyclin-dependent kinase inhibitors

Intraarticular gene transfer of cyclin-dependent kinase (CDK) inhibitors to suppress synovial cell cycling has shown efficacy in treating animal models of rheumatoid arthritis. Endogenous CDK inhibitors also modulate immune function via a CDK-independent pathway. Accordingly, systemic administration of small molecules that inhibit CDK may or may not ameliorate arthritis. To address this issue, alvocidib (flavopiridol), known to be tolerated clinically for treating cancers, and a newly synthesized CDK4/6-selective inhibitor were tested for antiarthritic effects. In vitro, they inhibited proliferation of human and mouse synovial fibroblasts without inducing apoptosis. In vivo, treatment of collagen-induced arthritis mice with alvocidib suppressed synovial hyperplasia and joint destruction, whereas serum concentrations of anti-collagen type II (CII) Abs and proliferative responses to CII were maintained. Treatment was effective even when therapeutically administered. Treated mice developed arthritis after termination of treatment. Thus, immune responses to CII were unimpaired. The same treatment ameliorated arthritis induced by K/BxN serum transfer to lymphocyte-deficient mice. Similarly, the CDK4/6-selective inhibitor suppressed collagen-induced arthritis. Both small-molecule CDK inhibitors were effective in treating animal models of rheumatoid arthritis not by suppressing lymphocyte function. Thus, the two small-molecule CDK inhibitors ameliorated arthritis models in a distinctive way, compared with other immunosuppressive drugs.     

痛风性关节炎动物模型的改良

目的通过改良痛风性关节炎动物模型,制备出更符合人类痛风性关节炎机制的动物模型。方法通过Coderre法与次黄嘌呤相结合（模型B）、Coderre法与氧嗪酸相结合（模型A）的方法建立痛风性关节炎动物模型,采用全自动生化分析仪测定血尿酸水平,观察关节滑膜组织形态学改变及大鼠不同时相步态变化,并将两种方法加以比较。结果模型B组大鼠血尿酸水平显著降低,关节滑膜组织形态学及不同时相大鼠步态改变明显,与模型A组比较有统计学意义。结论Coderre法与次黄嘌呤相结合方法建立大鼠痛风性关节炎动物模型更符合人类痛风性关节炎机制的动物模型。     

The severity of experimental arthritis is independent of IL-36 receptor signaling

Introduction

Interleukin (IL)-36 refers to three related IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, that bind to the IL-36 receptor (IL-36R). IL-36 exerts proinflammatory effects in skin and lung and stimulates T cell responses. In the present study, we examined the expression and function of IL-36R and its ligands in experimental arthritis.

Methods

Collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum transfer-induced arthritis were induced according to standard protocols. Messenger RNA levels for IL-36R and its ligands in the joints of mice with CIA were determined by RT-qPCR. Mice with CIA were injected with a blocking monoclonal anti-IL-36R, a blocking anti-IL-1RI, or their isotype-matched control antibodies at the time of arthritis onset. Anti-IL-36R or control antibodies were also injected at the time of AIA induction. Finally, IL-36R-deficient mice were examined in AIA and serum transfer-induced arthritis. The development and severity of arthritis were assessed by clinical and histological scoring.

Results

IL-36R, IL-36Ra and IL-36γ mRNA were detected in the joints of mice with CIA, but their levels did not correlate with arthritis severity. As opposed to anti-IL-1RI antibody treatment, the injection of an anti-IL-36R antibody was devoid of effect on the development and severity of CIA. The severity of joint inflammation and structural damage in AIA was also unaltered by anti-IL-36R antibody treatment. Finally, the severity of AIA and K/BxN serum transfer-induced arthritis was similar in IL-36R-deficient and wild-type mice.

Conclusions

The development and severity of experimental arthritis are independent of IL-36R signaling.     

来氟米特联合依那西普对佐剂性关节炎大鼠免疫功能的影响

目的：研究来氟米特和依那西普联合使用对佐剂性关节炎（AA）大鼠的治疗作用及其可能的作用机制。方法：建立AA大鼠关节炎模型,分为正常对照组、模型组、来氟米特组、依那西普组、来氟米特联合依那西普配伍组;采用关节炎指数评分法评价大鼠关节炎症程度,半定量RT-PCR和放射免疫法检测滑膜组织及血清中IL-1β、TNF-α表达水平,免疫组化方法检测滑膜组织中MMP－3含量。结果：①相较于AA模型组,来氟米特组、依那西普组和配伍组中大鼠的AI评分均显著下降（P〈0．01）,其中以配伍组关节炎指数为最低（P〈0．05）。②模型组大鼠血清及滑膜组织的IL-1β和TNF-α水平明显高于正常对照组（P〈0．01）,用药后各组的IL-1β和TNF-α水平均有所下降,并以配伍组降低最为明显（P〈0．01或P〈0．05）;③模型组大鼠滑膜组织MMP-3表达阳性密度显著高于正常对照组（P〈0．01）,用药后备组的MMP-3阳性密度降低（P〈0．01）,其中配伍组下降程度明显高于来氟米特组和依那西普组（P〈0．01）。结论：来氟米特和依那西普联合使用可明显减轻AA大鼠的关节炎症,降低血清和滑膜组织中IL-1β和TNF-α平,减少滑膜中MMP-3的表达,疗效优于单独使用来氟米特或依那西普。     

Leptin in septic arthritis: decreased levels during infection and amelioration of disease activity upon its administration

Weight loss is typically found during severe infections, e.g. septic arthritis. The aim of our study was to evaluate the role of leptin, regulator of food intake and energy expenditure, for the development of Staphylococcus aureus-triggered arthritis. Leptin production was found to be decreased during murine S. aureus-induced arthritis. Treatment with recombinant leptin neither restored the basal leptin levels nor affected the weight loss during the disease, but it significantly decreased the severity of septic arthritis. Exogenous leptin did not affect the staphylococcal load as measured in blood, joints and kidneys. Preceding the effects on joint manifestations, serum levels of interleukin-6 decreased in leptin-treated mice. In conclusion, the treatment with recombinant leptin reduced both the severity of joint manifestations in S.aureus-induced arthritis and the inflammatory response, as measured by serum IL-6 levels, without affecting the survival of bacteria in vivo.     

CD14 is an acute-phase protein

The origin of soluble CD14 (sCD14) in the circulation is uncertain. To examine whether CD14 could be an acute-phase protein (APP), the levels of sCD14, IL-6, and C-reactive protein were determined by ELISA in serum and synovial fluid (SF) of patients with various arthropathies, and the regulation of CD14 synthesis was examined in liver cells. In patients with crystal-mediated or immunologically mediated arthritis (rheumatoid arthritis), serum levels of sCD14 were higher than or similar to those found in infection-mediated arthritis (reactive arthritis), precluding a relation with bacteria exposure. Levels of sCD14 were similar in SF and serum, and did not correlate with the number of SF leukocytes, excluding an important source from leukocyte membrane-bound CD14, by protease-mediated shedding. In contrast, serum levels of sCD14 in patients correlated with those of C-reactive protein, a classical APP, and IL-6, a cytokine known to regulate the synthesis of APP in the liver. Serum levels of sCD14 also correlated with disease activity in rheumatoid arthritis and reactive arthritis patients. IL-6 stimulated the production of CD14 by HepG2 hepatoma cells. By real-time PCR, the inducibility of CD14 by IL-6 was also observed at the mRNA level both in HepG2 cells and human primary hepatocytes. These in vitro results were confirmed by in vivo studies in IL-6(-/-) mice injected with turpentine, an experimental model of acute-phase response. Liver levels of CD14 mRNA increased in IL-6(+/+), but not in IL-6(-/-) mice. These results indicate that sCD14 can be considered as a type 2 APP.     

Chronic inflammation inhibits GH secretion and alters the serum insulin-like growth factor system in rats

Adjuvant-induced arthritis in rats is associated with growth failure, hypermetabolism and accelerated protein breakdown. The aim of this work was to study the effects of adjuvant-induced arthritis on GH and insulin-like growth factor-I (IGF-I). Arthritis was induced by an intradermal injection of complete Freund's adjuvant and rats were killed 18 and 22 days later. IGF-I and GH levels were measured by radioimmunoassay. Pituitary GH mRNA was analyzed by northern blot and IGF binding proteins (IGFBPs) by western blot. Arthritic rats showed a decrease in both serum and hepatic concentrations of IGF-I. On the contrary, arthritis increased the circulating IGFBPs. The serum concentration of IGF-I in the arthritic rats was negatively correlated with the body weight loss observed in these animals. Arthritis decreased the serum concentration of GH and this decrease seems to be due to an inhibition of GH synthesis, since pituitary GH mRNA content was decreased in arthritic rats (p<0.01). These data suggest that the decrease in body weight gain in arthritic rats may be, at least in part, secondary to the decrease in GH and IGF-I secretion. Furthermore, the increased serum IGFBPs may also be involved in the disease process.     

Treatment with cyclophosphamide reverses acquired resistance to collagen arthritis subsequent to recovery from passive arthritis

Passive arthritis induced by anticollagen antibody was a mild, transient disease from which the animals normally recovered and the rats that had recovered from passive arthritis were resistant to develop a second phase of arthritis following a second administration of anticollagen antibody or the subsequent challenge with type II collagen. However, treatment of the recovered rats with cyclophosphamide (CY) shortly before a second administration of a serum concentrate induced a second episode of acute polyarthritis in 82% of the rats. In addition, CY treatment without further administration of a serum concentrate induced a recurrence of arthritis in 25% of the recovered rats. Similarly, treatment of the recovered rats with CY shortly before the challenge with type II collagen induced a second episode of arthritis in 83% of the rats. However, treatment with CY shortly before the challenge was unable to restore the suppressed humoral response to type II collagen. These results provide evidence that the resistant state is mediated, at least in part, by CY-sensitive events.     

Vascular endothelial dysfunction associated with elevated serum homocysteine levels in rat adjuvant arthritis: effect of vitamin E administration

We aimed to study the alterations in serum homocysteine levels and endothelium-dependent and -independent vascular relaxant responses in adjuvant-induced arthritis of the rat and to determine the effects of vitamin E administration on these changes. Arthritis was induced by a single intradermal injection of Freund's complete adjuvant into the paw. 26 days after the induction of arthritis, serum homocysteine levels and relaxant responses to acetylcholine and sodiumnitroprusside in thoracic aortas were evaluated. The relaxant responses to acetylcholine were decreased in aortas from arthritic rats, whereas the responses to sodiumnitroprusside were not significantly different when compared to the aortas from control rats. A significant increase was observed in serum homocysteine levels of the arthritic rats in comparison to those of controls. Vitamin E administration (100 mg/kg/day, i.m. for 26 days) to arthritic rats resulted in a significant increase in endothelium-dependent aortic responses to acetylcholine and a significant decrease in serum homocysteine levels with respect to the non-treated arthritic rats. However, in healthy rats, vitamin E treatment significantly decreased the acetylcholine-induced relaxant responses. We conclude that adjuvant-induced arthritis in the rat is associated with increased serum homocysteine levels and this is accompanied by a reduction in endothelium-dependent vascular responses in the thoracic aortas. Vitamin E treatment leads to normalization of the increased serum homocysteine levels and improves the endothelium-dependent relaxant responses in this experimental model.     

Copper chelation with tetrathiomolybdate suppresses adjuvant-induced arthritis and inflammation-associated cachexia in rats

Tetrathiomolybdate (TM), a drug developed for Wilson's disease, produces an anti-angiogenic and anti-inflammatory effect by reducing systemic copper levels. TM therapy has proved effective in inhibiting the growth of tumors in animal tumor models and in cancer patients. We have hypothesized that TM may be used for the therapy of rheumatoid arthritis and have examined the efficacy of TM on adjuvant-induced arthritis in the rat, which is a model of acute inflammatory arthritis and inflammatory cachexia. TM delayed the onset of and suppressed the severity of clinical arthritis on both paw volume and the arthritis score. Histological examination demonstrated that TM significantly reduces the synovial hyperplasia and inflammatory cell invasion in joint tissues. Interestingly, TM can inhibit the expression of vascular endothelial growth factor in serum synovial tissues, especially in endothelial cells and macrophages. Moreover, the extent of pannus formation, which leads to bone destruction, is correlated with the content of vascular endothelial growth factor in the serum. There was no mortality in TM-treated rat abnormalities. TM also suppressed inflammatory cachexia. We suggest that copper deficiency induced by TM is a potent approach both to inhibit the progression of rheumatoid arthritis with minimal adverse effects and to improve the well-being of rheumatoid arthritis patients.     

高脂食物对动脉硬化合并类风湿关节炎 小鼠肠道菌群的影响

目的分析高脂食物对动脉硬化合并类风湿关节炎小鼠肠道微生物的影响,了解动脉硬化合并类风湿关节炎小鼠肠道微生物的变化。方法 8周龄ApoE~(-/-)小鼠饲喂高脂食物和普通食物至17周龄来诱发动脉硬化症状,再通过给17周龄ApoE~(-/-)小鼠腹腔注射抗6-磷酸葡萄糖异构酶(glucose-6-phosphate isomerase,GPI)抗体呈阳性的K/BxN血清,从而诱导其产生类风湿关节炎症状。通过Illumina HiSeq平台对各组小鼠粪便进行16S rDNA V4区测序,分析动脉硬化合并类风湿关节炎小鼠肠道微生物的变化。结果 ApoE~(-/-)小鼠饲喂高脂食物后,其血清低密度脂蛋白胆固醇(LDL-C)浓度和血清总胆固醇(TC)浓度均显著升高,主动脉内膜斑块面积比喂普通食物的ApoE~(-/-)小鼠显著增加,表明ApoE~(-/-)小鼠饲喂高脂食物后引起更显著的动脉硬化症状。再通过腹腔注射抗GPI抗体呈阳性的K/BxN血清,各组ApoE~(-/-)小鼠均出现关节肿胀,饲喂高脂食物的ApoE~(-/-)小鼠其踝关节宽度和临床评分(clinical score)低于饲喂普通食物组小鼠。OTU数、Shannon指数和Simpson指数显示高脂食物和K/BxN血清处理组ApoE~(-/-)小鼠肠道菌群多样性降低,Firmicutes/Bacteroidetes值升高,t-test分析显示在属水平上,Prevotellaceae_UCG-001显著降低,Ruminiclostridium_6显著升高。t-test分析和Firmicutes/Bacteroidetes比值显示ApoE~(-/-)小鼠肠道菌群结构紊乱。结论高脂食物使ApoE~(-/-)小鼠的肠道菌群组成和结构发生改变,导致ApoE~(-/-)小鼠的动脉硬化症状加重,类风湿关节炎症状减轻。提示肠道微生物组成和结构的改变,可能与动脉硬化合并类风湿关节炎发病机制相关。     

Serum Vaspin Levels Are Associated with the Development of Clinically Manifest Arthritis in Autoantibody-Positive Individuals

Objectives

We have previously shown that overweight may increase the risk of developing rheumatoid arthritis (RA) in autoantibody positive individuals. Adipose tissue could contribute to the development of RA by production of various bioactive peptides. Therefore, we examined levels of adipokines in serum and synovial tissue of subjects at risk of RA.

Methods

Fifty-one individuals positive for immunoglobulin M rheumatoid factor (IgM-RF) and/or anti-citrullinated protein antibodies (ACPA), without arthritis, were included in this prospective study. Levels of adiponectin, vaspin, resistin, leptin, chemerin and omentin were determined in baseline fasting serum samples (n = 27). Synovial tissue was obtained by arthroscopy at baseline and we examined the expression of adiponectin, resistin and visfatin by immunohistochemistry.

Results

The development of clinically manifest arthritis after follow-up was associated with baseline serum vaspin levels (HR1.5 (95% CI 1.1 to 2.2); p = 0.020), also after adjustment for overweight (HR1.7 (95% CI 1.1 to 2.5); p = 0.016). This association was not seen for other adipokines. Various serum adipokine levels correlated with BMI (adiponectin r = -0.538, leptin r = 0.664; chemerin r = 0.529) and systemic markers of inflammation such as CRP levels at baseline (adiponectin r = -0.449, omentin r = -0.557, leptin r = 0.635, chemerin r = 0.619, resistin r = 0.520) and ESR (leptin r = 0.512, chemerin r = 0.708), p-value<0.05. Synovial expression of adiponectin, resistin and visfatin was not associated with development of clinically manifest arthritis.

Conclusions

In this exploratory study, serum adipokines were associated with an increased inflammatory state in autoantibody-positive individuals at risk of developing RA. Furthermore, serum vaspin levels may assist in predicting the development of arthritis in these individuals.