scholarly journals Optimized “In Vitro” Culture Conditions for Human Rheumatoid Arthritis Synovial Fibroblasts

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Claudia Casnici ◽  
Donatella Lattuada ◽  
Noemi Tonna ◽  
Katia Crotta ◽  
Claudio Storini ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Proinflammatory Cytokines ◽  
Synovial Fibroblasts ◽  
In Vitro Studies ◽  
Tnf Alpha ◽  
Purinergic Signalling ◽  
Human Rheumatoid Arthritis ◽  
Cells Cultured

The composition of synovial fluid in rheumatoid arthritis (RA) is complex and strongly influences the microenvironment of joints and it is an inseparable element of the disease. Currently, “in vitro” studies are performed on RA cells cultured in the presence of either recombinant proinflammatory cytokines-conditioned medium or medium alone. In this study, we evaluated the use of synovial fluid, derived from RA patients, as optimal culture condition to perform “in vitro” studies on RA synovial fibroblasts. We observed that synovial fluid is more effective in inducing cell proliferation with respect to TNF-alpha or culture medium alone. Spontaneous apoptosis in fibroblasts was also decreased in response to synovial fluid. The expression of proinflammatory cytokines in the presence of synovial fluid was significantly elevated with respect to cells cultured with TNF-alpha or medium, and the overall morphology of cells was also modified. In addition, modulation of intracellular calcium dynamics elicited in response to synovial fluid or TNF-alpha exposure is different and suggests a role for the purinergic signalling in the modulation of the effects. These results emphasize the importance of using RA synovial fluid in “in vitro” studies involving RA cells, in order to reproduce faithfully the physiopathological environmental characteristic of RA joints.

scholarly journals 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

2006 ◽  
Vol 63 (1) ◽  
pp. 21-26 ◽  
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.

scholarly journals POS0388 INTERLEUKIN 40 (IL-40) IS UP-REGULATED IN RHEUMATOID ARTHRITIS (RA) AND ASSOCIATED WITH DISEASE ACTIVITY, LEVELS OF AUTOANTIBODIES AND CHEMOKINES

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 424.1-424
Author(s):  
A. Navrátilová ◽  
L. Andres Cerezo ◽  
H. Hulejova ◽  
V. Becvar ◽  
D. Tegzová ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
B Cells ◽  
Synovial Fluid ◽  
Disease Activity ◽  
B Cell ◽  
Synovial Tissue ◽  
Lupus Erythematosus ◽  
Synovial Fibroblasts ◽  
Clinical Activity

Background:Interleukin 40 (IL-40) is newly identified B cell - associated cytokine implicated in humoral immune responses and in B cell development. As B cells play a pivotal role in autoimmunity, we aimed to investigate the function of IL-40 in rheumatoid arthritis (RA).Objectives:The aim of our study was to determine the function of IL-40 in RA.Methods:IL-40 expression in the synovial tissue was determined by immunohistochemistry and immunofluorescence (n=4-5). IL-40 was analysed in the serum/synovial fluid of patients with RA (n=69), systemic lupus erythematosus (SLE; n=69), osteoarthritis (OA; n=44), and in healthy controls (HC; n=25). Given the association of IL-40 with B cells, we analysed the effect of rituximab therapy on the serum IL-40 in 19 patients with RA after 16 and 24 weeks of the therapy. The clinical activity of patients with RA was assessed according to the 28 joint count Disease Activity Score (DAS28). Levels of C-reactive protein (CRP) and autoantibodies were measured by routine laboratory techniques. In vitro experiments were performed in RA synovial fibroblasts (n=9). Levels of cytokines and inflammatory mediators were determined in serum, synovial fluid and supernatants using ELISA or multiplex immunoassay.Results:IL-40 was overexpressed in RA synovial tissue compared to OA, particularly by synovial fibroblasts and immune cells such as B and T lymphocytes, macrophages and neutrophils. The levels of IL-40 were significantly higher in the synovial fluid of RA patients compared to OA (33.2 (6.6-68.9) vs. 0.7 (0.1-2.4) ng/ml; p<0.0001). In addition, IL-40 was increased in the serum of RA patients compared to SLE, OA or HC (4.8 (1.7-24.9) vs. 1.4 (1.0-1.9), 1.6 (0.6-3.1) or 1.5 (0.7-2.7) ng/ml; p<0.0001 for all) and decreased after 16 (p<0.01) and 24 weeks (p<0.001) in a subgroup of rituximab treated patients with RA. IL-40 levels in RA patients correlated with autoantibodies rheumatoid factor (IgM) and anti-citrullinated protein antibody (ACPA) in the serum (p<0.0001 and p<0.01) as well as in the synovial fluid (p<0.0001 and p<0.001). IL-40 in RA synovial fluid was also significantly associated with DAS28 (p<0.05), synovial fluid leukocyte count (p<0.01), number of swollen joints (p<0.05) and neutrophil attractants IL-8 (p<0.01) and MIP-1α (p<0.01). RA synovial fibroblasts exposed to recombinant IL-40 increased secretion of IL-8 (p<0.01), MCP-1 (p<0.05) and MMP-13 (p<0.01) compared to unstimulated cells in in vitro conditions.Conclusion:Our results show for the first time that IL-40 is elevated in RA and decreases following B-cell depletion therapy. The association of IL-40 with autoantibodies and chemokines may imply its potential involvement in RA development. Moreover, IL-40 up-regulates the secretion of chemokines and MMP-13 by synovial fibroblasts, indicating its role in the regulation of inflammation and tissue destruction in RA.Acknowledgements:Supported by MHCR 023728 a SVV 260 523Disclosure of Interests:None declared

scholarly journals Splicing machinery is impaired in rheumatoid arthritis, associated with disease activity and modulated by anti-TNF therapy

2021 ◽  
pp. annrheumdis-2021-220308
Author(s):  
Alejandro Ibáñez-Costa ◽  
Carlos Perez-Sanchez ◽  
Alejandra María Patiño-Trives ◽  
Maria Luque-Tevar ◽  
Pilar Font ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Disease Activity ◽  
Synovial Fibroblasts ◽  
Data Set ◽  
Altered Expression ◽  
Healthy Donors ◽  
Inflammatory Profile ◽  
Splicing Machinery

ObjectivesTo characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response.MethodsLeucocyte subtypes from 129 patients with RA and 29 healthy donors (HD) were purified, and 45 selected SM elements (SME) were evaluated by quantitative PCR-array based on microfluidic technology (Fluidigm). Modulation by anti-tumour necrosis factor (TNF) therapy and underlying regulatory mechanisms were assessed.ResultsAn altered expression of several SME was found in RA leucocytes. Eight elements (SNRNP70, SNRNP200, U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1 and SRSF10) were equally altered in all leucocytes subtypes. Logistic regressions revealed that this signature might: discriminate RA and HD, and anti-citrullinated protein antibodies (ACPAs) positivity; classify high-disease activity (disease activity score-28 (DAS28) >5.1); recognise radiological involvement; and identify patients showing atheroma plaques. Furthermore, this signature was altered in RA synovial fluid and ankle joints of K/BxN-arthritic mice. An available RNA-seq data set enabled to validate data and identified distinctive splicing events and splicing variants among patients with RA expressing high and low SME levels. 3 and 6 months anti-TNF therapy reversed their expression in parallel to the reduction of the inflammatory profile. In vitro, ACPAs modulated SME, at least partially, by Fc Receptor (FcR)-dependent mechanisms. Key inflammatory cytokines further altered SME. Lastly, induced SNRNP70-overexpression and KHDRBS1-overexpression reversed inflammation in lymphocytes, NETosis in neutrophils and adhesion in RA monocytes and influenced activity of RA synovial fibroblasts.ConclusionsOverall, we have characterised for the first time a signature comprising eight dysregulated SME in RA leucocytes from both peripheral blood and synovial fluid, linked to disease pathophysiology, modulated by ACPAs and reversed by anti-TNF therapy.

scholarly journals AB0108 IRAK4 INHIBITION SUPPRESSES PROINFLAMMATORY CYTOKINE PRODUCTION FROM HUMAN MACROPHAGES STIMULATED WITH SYNOVIAL FLUID FROM RHEUMATOID ARTHRITIS PATIENTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1353.2-1353
Author(s):  
A. Yadon ◽  
D. Ruelas ◽  
G. Min-Oo ◽  
J. Taylor ◽  
M. R. Warr
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Signaling Pathways ◽  
Proinflammatory Cytokines ◽  
Proinflammatory Cytokine ◽  
Cytokine Production ◽  
Interleukin 1 ◽  
Proinflammatory Cytokine Production ◽  
Human Macrophages ◽  
Percent Suppression

Background:Rheumatoid arthritis (RA) is characterized by chronic, uncontrolled joint inflammation and tissue destruction. Macrophages are thought to be key mediators in both the initiation and perpetuation of this pathology.1,2The RA synovium contains a complex inflammatory milieu that can stimulate macrophage-dependent production of proinflammatory cytokines through multiple signaling pathways.1,2Existing evidence indicates that toll-like receptors (TLRs) and interleukin-1 receptors (IL-1R) along with their agonists, damage-associated molecular patterns (DAMPs) and IL-1β, are highly expressed in RA joints and are important mediators of synovial macrophage activation and proinflammatory cytokine production.1-9IRAK4 (interleukin-1 receptor-associated kinase 4) is a serine/threonine kinase that facilitates TLR and IL-1R signaling in many cell types, including macrophages.10IRAK4 inhibition represents an opportunity to reduce proinflammatory cytokine production in the joints of patients with RA.Objectives:To investigate the effect of a highly selective IRAK4 inhibitor on proinflammatory cytokine production from human macrophages stimulated with synovial fluid from patients with RA.Methods:Primary human monocytes from 2 independent donors were differentiated for 6 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate human monocyte-derived macrophages (hMDMs). hMDMs were then pretreated with an IRAK4 inhibitor for 1 hour and subsequently stimulated for 24 hours with RA synovial fluid from 5 patients. Culture supernatants were then assessed for secretion of proinflammatory cytokines by MesoScale Discovery.Results:RA synovial fluid stimulation of hMDMs resulted in the production of several proinflammatory cytokines, including IL-6, IL-8, and TNFα. Pretreatment of hMDMs with an IRAK4 inhibitor resulted in the dose-dependent inhibition of IL-6, IL-8, and TNFα production, with an average EC50± SD of 27 ± 31, 26 ± 41, and 28 ± 22 nM, respectively. Maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 76 ± 8.8, 73 ± 15, and 77 ± 13, respectively. To evaluate the specific IRAK4-dependent signaling pathways mediating this response, hMDMs were pretreated with inhibitors of TLR4 (TAK242) and IL-1R (IL-1RA) prior to stimulation with RA synovial fluid. Both TAK242 and IL-1RA inhibited proinflammatory cytokine production. For TAK242, maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 39 ± 25, 48 ± 24, and 50 ± 21, respectively. For IL-1RA maximal percent suppression ± SD of IL-6, IL-8, and TNFα were 18 ± 18, 20 ± 23, and 16 ± 18, respectively. The broad range of inhibition across each stimulation highlights the complexity and variability in the signaling pathways mediating proinflammatory cytokine production from hMDMs stimulated with RA synovial fluid, but demonstrates that RA synovial fluid can stimulate proinflammatory cytokine production in hMDMs, at least partly, through IRAK4-dependent pathways.Conclusion:This work demonstrates that IRAK4 inhibition can suppress proinflammatory cytokine production from macrophages stimulated with synovial fluid from patients with RA and supports a potential pathophysiological role for IRAK4 in perpetuating chronic inflammation in RA.References:[1]Smolen JS, et al.Nat Rev Dis Primers.2018;4:18001.[2]Udalova IA, et al.Nat Rev Rheumatol.2016;12(8):472-485.[3]Joosten LAB, et al.Nat Rev Rheumatol.2016;12(6):344-357.[4]Huang QQ, Pope RM.Curr Rheumatol Rep.2009;11(5):357-364.[5]Roh JS, Sohn DH.Immune Netw.2018;18(4):e27.[6]Sacre SM, et al.Am J Pathol.2007;170(2):518-525.[7]Ultaigh SNA, et al.Arthritis Res Ther.2011;13(1):R33.[8]Bottini N, Firestein GS.Nat Rev Rheumatol.2013;9(1):24-33.[9]Firestein GS, McInnes IB.Immunity.2017;46(2):183-196.[10]Janssens S, Beyaert R.Mol Cell.2003;11(2):293-302.Disclosure of Interests:Adam Yadon Employee of: Gilead, Debbie Ruelas Employee of: Gilead, Gundula Min-Oo Employee of: Gilead, James Taylor Employee of: Gilead, Matthew R. Warr Employee of: Gilead

scholarly journals Prostacyclin-IP signaling and prostaglandin E2-EP2/EP4 signaling both mediate joint inflammation in mouse collagen-induced arthritis

2006 ◽  
Vol 203 (2) ◽  
pp. 325-335 ◽  
Author(s):  
Tetsuya Honda ◽  
Eri Segi-Nishida ◽  
Yoshiki Miyachi ◽  
Shuh Narumiya
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Joint Inflammation ◽  
Synovial Fibroblasts ◽  
The Other ◽  
Receptor Subtype ◽  
Significant Suppression ◽  
Wild Type ◽  
Collagen Induced Arthritis

Prostaglandin (PG)I2 (prostacyclin [PGI]) and PGE2 are abundantly present in the synovial fluid of rheumatoid arthritis (RA) patients. Although the role of PGE2 in RA has been well studied, how much PGI2 contributes to RA is little known. To examine this issue, we backcrossed mice lacking the PGI receptor (IP) to the DBA/1J strain and subjected them to collagen-induced arthritis (CIA). IP-deficient (IP−/−) mice exhibited significant reduction in arthritic scores compared with wild-type (WT) mice, despite anti-collagen antibody production and complement activation similar to WT mice. IP−/− mice also showed significant reduction in contents of proinflammatory cytokines, such as interleukin (IL)-6 in arthritic paws. Consistently, the addition of an IP agonist to cultured synovial fibroblasts significantly enhanced IL-6 production and induced expression of other arthritis-related genes. On the other hand, loss or inhibition of each PGE receptor subtype alone did not affect elicitation of inflammation in CIA. However, a partial but significant suppression of CIA was achieved by the combined inhibition of EP2 and EP4. Our results show significant roles of both PGI2-IP and PGE2-EP2/EP4 signaling in the development of CIA, and suggest that inhibition of PGE2 synthesis alone may not be sufficient for suppression of RA symptoms.

Kynurenic Acid in Synovial Fluid and Serum of Patients with Rheumatoid Arthritis, Spondyloarthropathy, and Osteoarthritis

2013 ◽  
Vol 40 (6) ◽  
pp. 903-909 ◽  
Author(s):  
Jolanta Parada-Turska ◽  
Wojciech Zgrajka ◽  
Maria Majdan
Keyword(s):  
Rheumatoid Arthritis ◽  
Regression Analysis ◽  
Synovial Fluid ◽  
Blood Cell ◽  
Red Blood Cell ◽  
High Performance ◽  
Kynurenic Acid ◽  
Linear Regression Analysis ◽  
Cell Counts

Objective.Previously we demonstrated that kynurenic acid (KYNA), an endogenous metabolite of kynurenine, is present in the synovial fluid of patients with rheumatoid arthritis (RA). KYNA inhibits proliferation of synoviocytesin vitro. The goal of our study was to compare KYNA concentrations in synovial fluid and blood of patients with RA, inflammatory spondyloarthropathies (SpA), and osteoarthritis (OA).Methods.Serum and synovial fluid samples were obtained from 189 patients with RA, 56 patients with SpA, and 32 patients with OA. KYNA was separated using a high-performance liquid chromatography system and measured fluorometrically.Results.KYNA concentration in synovial fluid obtained from patients with RA and SpA was significantly lower than that in patients with OA (p < 0.05). The concentration of KYNA in serum of patients with RA, SpA, and OA did not differ among all groups studied. The positive correlation between KYNA content in synovial fluid and serum was found in patients with RA (p < 0.05). Univariate linear regression analysis demonstrated that fibrinogen was significantly associated with KYNA in synovial fluid (p < 0.05), and red blood cell counts, morning stiffness, and pain scores were significantly associated with KYNA level in serum (all p < 0.05). Multivariate regression analysis revealed correlation between the following independent variables: hemoglobin level, hematocrit, red blood cell count in conjunction with age and KYNA content in synovial fluid. A lack of correlation was observed between KYNA content in synovial fluid of patients with RA and other clinical and laboratory measures of disease activity.Conclusion.Our data show a local deficit of KYNA in inflammatory states.

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