The correlation between IL-33 and inflammation factors in rheumatoid arthritis patients in vivo and in fibroblast-like synoviocytes in vitro

Abstract Background: Interleukin-33 (IL-33) is a member of the IL-1 family of cytokines whose role remains controversial in rheumatoid arthritis (RA), because no clear conclusion has been established regarding the relationship between IL-33 and other cytokines and chemokines. The present study was conducted to evaluate the correlation of IL-33 with other cytokines and chemokines in serum and the synovia, and to explore the nature of the relationship.Results: IL-33 was found to exhibit an inverted-U-shaped correlation with multiple cytokines and chemokines in synovial fluid, including IL-6, IL-1β, CXCL8 (IL-8), CXCL9 (MIG) and CXCL10 (IP-10), but not in serum. Moreover, in vitro experiments confirmed that IL-33 also exhibits U-type dose-dependent regulation of FLS function.Conclusions: IL-33 exhibit an inverted-U-shaped correlation with multiple cytokines and chemokines in synovial fluid of RA patients. IL-33 affects the secretion of cytokines and chemokines in the synovium in a U-type dose-dependent relationship.

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Cationic Amino Acid Transporter-1 (CAT-1) Promotes Fibroblast-Like Synoviocytes Proliferation and Cytokine Secretion by Taking Up L-Arginine in Rheumatoid Arthritis

10.21203/rs.3.rs-1147918/v1 ◽

2021 ◽

Author(s):

Ying Lu ◽

Chongbo Hao ◽

Shanshan Yu ◽

Zuan Ma ◽

Xuelian Fu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Amino Acid ◽

Synovial Fluid ◽

Culture Conditions ◽

Cytokine Secretion ◽

Cationic Amino Acid Transporter ◽

Cationic Amino Acid ◽

The Relationship ◽

Fibroblast Like Synoviocytes

Abstract Background: Abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA. Methods: The concentrations of amino acids and cytokines in the synovial fluid of RA (n=9) and osteoarthritis (OA,n=9) were detected by LC-MS/MS and CBA assay, respectively. The mRNA and protein expression of CAT-1 were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo.Results: L-arginine was upregulated in the synovial fluid of RA patients and was positively correlated with elevation of the cytokines IL-1β, IL-6 and IL-8. Further examination demonstrated that cationic amino acid transporter-1 (CAT-1) was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice. Conclusion: CAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.

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AB0100 PHENOTYPIC AND FUNCTIONAL CHARACTERIZATION OF SYNOVIAL FLUID-DERIVED FIBROBLAST-LIKE SYNOVIOCYTES IN RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.3884 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1349.1-1349

Author(s):

D. Køster ◽

J. H. Egedal ◽

M. Hvid ◽

M. R. Jakobsen ◽

U. Müller-Ladner ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Synovial Fluid ◽

Synovial Tissue ◽

Mononuclear Cells ◽

Functional Characterization ◽

Human Cartilage ◽

Fibroblast Like Synoviocytes

Background:Fibroblast-like synoviocytes (FLS) are central cellular components in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). Pathological subsets of FLS have been identified from synovial tissue. However, the synovial tissue obtained from arthroplasty procedures is acquired at late disease stages and the cellular yield obtained from synovial tissue biopsies is fairly low. Collectively, challenging the robustness of human RAin vivoandin vitromodels. FLS obtained from the synovial fluid (SF-FLS) are proposed as an alternative source of FLS, but a detailed phenotypical and functional characterization of FLS subsets from the synovial fluid has not been performed.Objectives:The aim of this study was to determine the phenotypical and functional characteristics of synovial fluid-derived fibroblast-like synoviocytes in rheumatoid arthritis.Methods:In the present study, paired peripheral blood mononuclear cells (PBMC) and SF-FLS from patients with RA were obtained (n=7). FLS were isolated from the synovial fluid by a strict trypsinization protocol1and their cellular characteristics and functionality were evaluated at passage 4. Monocultures (SF-FLS) and autologous co-cultures (SF-FLS and PBMC) were established from five patients with RA and subsequently evaluated by flow cytometry, Western blotting and multiplex immunoassays. Human cartilage-sponges (n=3) with SF-FLS and without SF-FLS (n=3) were co-implanted subcutaneously in SCID mice (n=15), mice with only cell-free human cartilage-sponges were used as controls (n=12). After 45 days, the implants were evaluated using stained sections to determine the SF-FLS invasion score based on perichondrocytic cartilage degradation. Data are expressed as median (25-75 percentile). P-values <0.05 were considered statistically significant.Results:The homogeneous subpopulations of FLS, isolated from the synovial fluid, were negative for CD34 and CD45 [98.9%, (97.5-99.7]) and positive for Thy-1 and PDPN [94.6%, (79.9-97.4]). Without stimulation, RA SF-FLS showed high and comparable levels of NFκB related pathway proteins and secreted multiple pro-inflammatory cytokines and chemokines dominated by IL-6 [2648 pg/mL, (1327-6116)] and MCP-1 [2458 pg/mL, (692-8719)]. SF-FLS increased their ICAM-1 and HLA-DR expression after encountering autologous PBMCs (p<0.01), (p<0.05). Further, SF-FLS and PBMC interacted synergistically in a co-culture model of RA and significantly increasing the secretion of several cytokines (IL-1β, IL-2, IL-6, (p<0.01)) and a chemokine (MCP-1, (p<0.01)). The invasion score of the human SF-FLSin vivowas at primary site, [1.6, (1.3-1.7)] and contralateral implantation site [1.5, (1.1-2.2)]. The invasion score of the human SF-FLS-containing implants both at primary and contralateral site were significantly higher compared with cartilage-sponges evaluated from SF-FLS-free control mice (p<0001).Conclusion:This phenotypical and functional characterization of SF-FLS, acquired and activated at the site of pathology, lays a foundation for establishingin vivoandin vitroFLS models. These FLS models will be beneficial in our understanding of the role of this cellular subset in arthritis and for characterization of drugs specifically targeting this pathological RA FLS subset.References:[1]Nielsen M. A. et al. Responses to Cytokine Inhibitors Associated with Cellular Composition in Models of Immune-Mediated Inflammatory Arthritis. ACR Open Rheumatology, 2(1):3-10.http://doi.org/10.1002/acr2.11094Disclosure of Interests:Ditte Køster: None declared, Johanne Hovgaard Egedal: None declared, Malene Hvid: None declared, Martin Roelsgaard Jakobsen: None declared, Ulf Müller-Ladner Speakers bureau: Biogen, Bent Deleuran: None declared, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Elena Neumann: None declared, Morten Aagaard Nielsen: None declared

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CATECHOL OESTROGENS AND GONADOTROPHIN SECRETION IN THE EWE: AFFINITY FOR PITUITARY OESTROGEN RECEPTORS IN VITRO AND ACTION ON GONADOTROPHIN SECRETION IN VIVO

Journal of Endocrinology ◽

10.1677/joe.0.0850503 ◽

1980 ◽

Vol 85 (3) ◽

pp. 503-509 ◽

Cited By ~ 5

Author(s):

I. J. CLARKE ◽

J. K. FINDLAY

Keyword(s):

Negative Feedback ◽

Oestrogen Receptor ◽

Dose Response Relationship ◽

Concurrent Administration ◽

Feedback Effects ◽

Dependent Relationship ◽

Gonadotrophin Secretion ◽

Dose Dependent

The binding of three catechol oestrogens, 2-OH-oestradiol-17β, 4-OH-oestrone and 2-OH-oestrone, to the ovine pituitary oestrogen receptor was measured in vitro to establish doses for the assessment of the effects of catechol oestrogens in vivo. Relative to oestradiol (100%) the compounds had receptor affinities of 30, 20 and 5% respectively. A dose of oestradiol sufficient to cause negative-feedback effects on the secretion of LH and FSH in ovariectomized ewes was established by intracarotid (i.c.) injections of 0·625–5·0 μg/dose (n = 3), and by measuring plasma levels of gonadotrophins in jugular venous samples taken at intervals of 20 min from 3 h before until 4 h after injection. A dose-dependent relationship (r = 0·88, P<0·001) was found for oestradiol and plasma LH levels. Plasma FSH was slightly (12–25%) but significantly (P<0·05) reduced by doses of 1·25–5·0 μg oestradiol, but no dose–response relationship was observed. Ovariectomized ewes (n = 4/group) were given 2·5 μg oestradiol (i.c.) simultaneously with 83 μg 2-OH-oestradiol, 125 μg 4-OH-oestrone or 500 μg 2-OH-oestrone. These doses of catechol oestrogens were chosen as being ten times that of oestradiol, with the relative affinities for oestrogen receptor taken into account. Concurrent administration of such doses of catechol oestrogens had no effect on the negative-feedback action of oestradiol in vivo. We have concluded that catechol oestrogens in the circulation probably do not modulate the action of oestradiol on release of LH or FSH; this does not preclude a possible role for them as locally produced regulators of oestrogen action.

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PTEN Methylation Promotes Inflammation and Activation of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

Frontiers in Pharmacology ◽

10.3389/fphar.2021.700373 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiao-Feng Li ◽

Sha Wu ◽

Qi Yan ◽

Yuan-Yuan Wu ◽

He Chen ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Articular Cartilage ◽

Dna Methyltransferase ◽

Underlying Mechanism ◽

Cartilage Destruction ◽

Specific Pcr ◽

Methylation Inhibitor ◽

Fibroblast Like Synoviocytes

Rheumatoid arthritis (RA) is characterized by a tumor-like expansion of the synovium and subsequent destruction of adjacent articular cartilage and bone. In our previous work we showed that phosphatase and tension homolog deleted on chromosome 10 (PTEN) contributes to the activation of fibroblast-like synoviocytes (FLS) in adjuvant-induced arthritis (AIA), but the underlying mechanism is not unknown. In this study, we show that PTEN is downregulated while DNA methyltransferase (DNMT)1 is upregulated in FLS from RA patients and a rat model of AIA. DNA methylation of PTEN was increased by administration of tumor necrosis factor (TNF)-α in FLS of RA patients, as determined by chromatin immunoprecipitation and methylation-specific PCR. Treatment with the methylation inhibitor 5-azacytidine suppressed cytokine and chemokine release and FLS activation in vitro and alleviated paw swelling in vivo. PTEN overexpression reduced inflammation and activation of FLS via protein kinase B (AKT) signaling in RA, and intra-articular injection of PTEN-expressing adenovirus into the knee of AIA rats markedly reduced inflammation and paw swelling. Thus, PTEN methylation promotes the inflammation and activation of FLS in the pathogenesis of RA. These findings provide insight into the molecular basis of articular cartilage destruction in RA, and indicate that therapeutic strategies that prevent PTEN methylation may an effective treatment.

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Phenotypic and functional characterization of synovial fluid-derived fibroblast-like synoviocytes in rheumatoid arthritis

Scientific Reports ◽

10.1038/s41598-021-01692-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ditte Køster ◽

Johanne Hovgaard Egedal ◽

Søren Lomholt ◽

Malene Hvid ◽

Martin R. Jakobsen ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Synovial Fluid ◽

Mononuclear Cells ◽

Functional Characterization ◽

Surface Protein ◽

Tissue Destruction ◽

Peripheral Blood Mononuclear ◽

Surface Protein Expression ◽

Fibroblast Like Synoviocytes

AbstractFibroblast-like synoviocytes (FLS) play an important pathological role in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). These cells have primarily been characterized in the RA synovial membrane. Here we aim to phenotypically and functionally characterize cultured synovial fluid-derived FLS (sfRA-FLS). Paired peripheral blood mononuclear cells (PBMC) and sfRA-FLS from patients with RA were obtained and monocultures of sfRA-FLS and autologous co-cultures of sfRA-FLS and PBMC were established. The in situ activated sfRA-FLS were CD34-, CD45-, Podoplanin+, Thymocyte differentiation antigen-1+. SfRA-FLS expressed uniform levels of NFкB-related pathway proteins and secreted several pro-inflammatory cytokines dominated by IL-6 and MCP-1. In a co-culture model with autologous PBMC, the ICAM-1 and HLA-DR expression on sfRA-FLS and secretion of IL-1β, IL-6, and MCP-1 increased. In vivo, human sfRA-FLS were cartilage invasive both at ipsilateral and contralateral implantation site. We conclude that, sfRA-FLS closely resemble the pathological sublining layer FLS subset in terms of surface protein expression, cytokine production and leukocyte cross-talk potential. Further, sfRA-FLS are comparable to tissue-derived FLS in their capabilities to invade cartilage at implantation sites but also spread tissue destruction to a distant site. Collectively, sfRA-FLS can serve as a an easy-to-obtain source of pathological sublining FLS in RA.

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Interleukin-2 secretion in vivo and in vitro by synovial fluid lymphocytes in rheumatoid arthritis

Molecular Immunology ◽

10.1016/0161-5890(88)90157-5 ◽

1988 ◽

Vol 25 (11) ◽

pp. 1209-1211

Author(s):

H. Warnatz ◽

Sabine Ruschen ◽

G. Lemm

Keyword(s):

Rheumatoid Arthritis ◽

Synovial Fluid ◽

Interleukin 2 ◽

Synovial Fluid Lymphocytes

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Evaluation of the degree of clinical rheumatoid arthritis activity based on the concentrations of cytokines TNF-alpha, IL-12, IL-15, and IL-18 in serum and synovial fluid

Vojnosanitetski pregled ◽

10.2298/vsp0601021p ◽

2006 ◽

Vol 63 (1) ◽

pp. 21-26 ◽

Cited By ~ 4

Author(s):

Ljiljana Petrovic-Rackov

Keyword(s):

Rheumatoid Arthritis ◽

Synovial Fluid ◽

Disease Activity ◽

Tnf Alpha ◽

Control Group ◽

High Degree ◽

Rheumatoid Arthritis Activity ◽

Active Disease

Bacground/Aim. Experimental in vitro and in vivo investigations in a mouse model have proved that TNF-alpha, IL-12, IL- 15 and IL-18 participate in the pathogenesis of erosive inflammatory arthritis. The aim of this research was to determine the clinical significance of cytokines in the evaluation of the activity of rheumatoid arthritis (RA). Methods. Inside a 4-year period we followed-up 64 patients with RA as newly occurred or in the phase of worsening. We observed the clinical manifestation of the disease upon wluch we divided the patients in to 3 groups: the patients with low active RA, patients with moderate active RA, and the patients with wild active RA. The control group (n = 25 patients) included the patients with osteoarthrosis (OA), and arthritis of the knee. In the samples of serum of all of the patients the concentrating of cytokines TNF-alpha, IL-12, IL-15, and IL-18 were determined using the immunoenzymatic methods in mice for human interleukines. By comparing the concentrations in 30 patients with the high, 14 patients with moderate, and 20 patients with the mild activity of RA it was determined that the patients with the high degree of the disease activity, had significantly high (p < 0.01; p < 0.05) concentrations of the examined cytokines in blood and synovial fluid as compared to the patients with the moderate and mild active disease. There was a relationship (p < 0.01) between the concentrations of cytokines in blood and synovial fluid with the quantity of the Disease Activity Score in 28 joints. Conclusions. Cytokines concentrations could be good indicators of the degree of the general activity of RA. This research could contribute to the interpretation of insufficiently well known views of the pathogenesis role and significance of citokines in an active disease.

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Anti-Rheumatoid Arthritic Effects of Paris Saponin VII in Human Rheumatoid Arthritis Fibroblast-Like Synoviocytes and Adjuvant-Induced Arthritis in Rats

Frontiers in Pharmacology ◽

10.3389/fphar.2021.683698 ◽

2021 ◽

Vol 12 ◽

Author(s):

Mei Meng ◽

Zhenggang Yue ◽

Lu Chang ◽

Yanru Liu ◽

Jinhang Hu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

P38 Mapk ◽

Ankle Joint ◽

Adjuvant Induced Arthritis ◽

Mapk Pathways ◽

Tnf Α ◽

Apoptosis Proteins ◽

Fibroblast Like Synoviocytes

In the pathogenesis of rheumatoid arthritis (RA), rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) have tumor-like characteristics, mainly manifested by hyperproliferation and resistance to apoptosis and then it will erode the bone and cartilage, eventually leading to joint destruction. Paris saponin VII (PS VII) is an active compound derived from a traditional herbal medicine named Trillium tschonoskii Maxim, which has anti-tumor, analgesic, and immunomodulatory effects. However, its anti-RA effect has not yet been reported. This study was to investigate the effect of PS VII on two rheumatoid arthritis fibroblast-like synoviocytes lines (RA-FLS and MH7A) and adjuvant-induced arthritis (AIA) in rats. In vitro, the effects of PS VII on the proliferation, cell cycle, and apoptosis of RA-FLS and MH7A cells were detected by MTT, flow cytometry, and western blot analysis. In vivo, the effect of PS VII on the weight of the rat, paw swelling, ankle joint diameter, arthritis index, serum inflammatory cytokines (TNF-α, IL-6, and IL-1β), histopathological assessment and apoptosis proteins in the synovial tissues were evaluated in AIA rats. The in vitro studies showed that PS VII inhibited the proliferation of RA-FLS and MH7A cells, induced S phase arrest and triggered cell apoptosis mainly through the mitochondrial apoptotic pathway and the regulation of JNK and p38 MAPK pathways. The in vivo studies revealed that PS VII could improve ameliorate body weight, paw swelling, ankle joint diameter, reduce the spleen and thymus index, suppress the production of TNF-α, IL-6 and IL-1β, improve histopathological changes and regulate the expressions of apoptosis proteins in AIA Rats. In conclusion, PS VII could inhibit the proliferation and trigger apoptosis of RA-FLS and MH7A cells by regulating the mitochondrial apoptosis pathway and the JNK and p38 MAPK pathways, and alleviate the symptoms of RA, signifying it to be one of the potential anti-RA therapeutics.

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