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

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.

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

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

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

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.

scholarly journals Partial chemical and functional characterization of milk whey products obtained by different processes

2012 ◽  
Vol 32 (1) ◽  
pp. 56-64 ◽  
Author(s):  
Fabiane La Flor Ziegler ◽  
Georgia Alvares Castro ◽  
Yara Maria Franco Moreno ◽  
Vanessa Oya ◽  
Maria Marluce dos Santos Vilela ◽  
...  
Keyword(s):  
Whey Protein ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Functional Characterization ◽  
Interleukin 4 ◽  
In Vivo Studies ◽  
Whey Protein Concentrate ◽  
Peripheral Blood Mononuclear

Whey protein samples (S-1 to S-5) were tested in vivo and in vitro for nutritional properties and selected bioactivities. Weanling male Wistar rats fed modified AIN-93G (12 g protein.100 g-1) diets for 21 days were used the in vivo studies. The nutritional parameters did not differ among the protein diets tested. Erythrocyte glutathione content was considered high and was higher for S-3, but liver glutathione was the same for all dietary groups. For S-3, cytokine secretion (IL-10 and TNF-α) by human peripheral blood mononuclear cells (in RPMI-1640 medium) was higher in the absence of antigen than in the presence of BCG antigen. Interleukin-4 secretion was repressed in all treatments. The IC50, whey protein concentration required to inhibit 50% of the melanoma cell proliferation, was 2.68 mg.mL-1 of culture medium for the S-3 sample and 3.66 mg.mL-1 for the S-2 sample. Based on these results, it was concluded that S-3 (whey protein concentrate enriched with TGF-β and lactoferrin) produced better nutritional and immunological responses than the other products tested.

scholarly journals Reduction of Oxidative Stress in Peripheral Blood Mononuclear Cells Attenuates the Inflammatory Response of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

2021 ◽  
Vol 22 (22) ◽  
pp. 12411
Author(s):  
Ha-Reum Lee ◽  
Su-Jin Yoo ◽  
Jinhyun Kim ◽  
Chan Keol Park ◽  
Seong Wook Kang
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cell ◽  
Inflammatory Response ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
T Cell Subsets ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Fibroblast Like Synoviocytes

The production and oxidation mechanism of reactive oxygen species (ROS) are out of balance in rheumatoid arthritis (RA). However, the correlation between ROS and T cell subsets in RA remains unclear. Peripheral blood mononuclear cells (PBMCs) from patients with RA (n = 40) and healthy controls (n = 10) were isolated from whole blood samples. Synovial tissues (n = 3) and synovial fluid (n = 10) were obtained from patients with RA. The repartition of T cell subsets and expression of ROS and cytokines were examined according to RA severity. Fibroblast-like synoviocytes (FLSs) from patients with RA were stimulated with PBMCs and the expression of inflammation-related molecules were measured by RT-PCR and cytokine array. Regulatory T cells from patients with moderate (5.1 > DAS28 ≥ 3.2) RA showed the highest expression of mitochondrial ROS among the groups based on disease severity. Although ROS levels steadily increased with RA severity, there was a slight decline in severe RA (DAS28 ≥ 5.1) compared with moderate RA. The expression of inflammatory cytokines in RA FLSs were significantly inhibited when FLSs were co-cultured with PBMCs treated with ROS inhibitor. These findings provide a novel approach to suppress inflammatory response of FLSs through ROS regulation in PBMCs.

AB0110 ADIPONECTIN INDUCES PRO-INFLAMMATORY CHEMOKINE AND CYTOKINE PRODUCTION BY PERIPHERAL BLOOD MONONUCLEAR CELLS AND FIBROBLAST-LIKE SYNOVIOCYTES FROM NON-INFLAMED SUBJECTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1355-1356
Author(s):  
Y. Zhang ◽  
J. Aldridge ◽  
G. K. Vasileiadis ◽  
A. C. Lundell ◽  
A. Rudin ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Inflammatory Responses ◽  
Multiplex Assay ◽  
Human Pbmcs ◽  
Peripheral Blood Mononuclear ◽  
Preclinical Phase ◽  
Blood Mononuclear Cells ◽  
Fibroblast Like Synoviocytes

Background:Adiponectin is a cytokine mainly secreted by the adipose tissue1, whose circulating levels are paradoxically low in subjects with obesity and associate with a beneficial metabolic profile2., Recent studies have shown that adiponectin levels are elevated in both serum and synovial fluid collected from patients with rheumatoid arthritis (RA)3,4. Moreover, adiponectin is able to induce the production of interleukin (IL)-6, tumor necrosis factor (TNF), CXCL1 and CXCL8 by lymphocytes from healthy subjects5, and of IL-6 and CXCL8 by fibroblast-like synoviocytes (FLS) from patients with RA6. However, it is not clear if adiponectin is able to initiate the inflammatory processes associated with the preclinical phase of RA.Objectives:We aim to determine if adiponectin is able to induce inflammatory responses in peripheral blood mononuclear cells (PBMCs) and FLS from non-inflamed subjects.Methods:Human PBMCs were collected from healthy donors, whereas non-inflamed FLS from non-arthritic patients who underwent diagnostic arthroscopy due to previous trauma. PBMCs (1× 105cells/well in 96-well plate) and FLS (5000 cells/well in 96-well plate) were stimulated using 5 μg/ml recombinant human total adiponectin protein, and the supernatants were collected 48 hours after stimulation. Phytohemagglutinin (PHA) and TNF were used as positive controls to activate PBMCs and FLS, respectively. Using multiplex assay and ELISA, we screened the production of 13 chemokines and 12 cytokines from healthy human PBMCs and non-inflamed FLS.Results:Adiponectin was able to stimulate a distinct profile of chemokines and cytokines in PBMCs and FLS. In both healthy PBMCs and non-inflamed FLS adiponectin induced the production of CXCL1, CXCL5, CXCL8, CCL2 and IL-6. Moreover, CCL3, CCL20, CCL4, CCL17, TNF, IL-10 and GM-CSF were induced by adiponectin only in healthy PBMCs, whereas CXCL10, CCL5 and CCL11 only in non-inflamed FLS (Fig. 1 and 2).Figure 1.Adiponectin induces the production of various chemokines from PBMCs (A) and FLS (B). CXCL8 (#) was measured using ELISA, and other chemokines were measured using multiplex assay. The fold change of CXCL1 in FLS (†) was not calculated because its level before stimulation was undetectable.Conclusion:We here report that adiponectin has pro-inflammatory properties as it induced chemokine and cytokine production from human healthy PBMCs and non-inflamed FLS. As adiponectin is able to induce pro-inflammatory responses from non-inflamed cells, we suggest that this adipokine might be implicated in the preclinical phase of RA pathogenesis.References:[1]Makki K, Froguel P, Wolowczuk I. Adipose tissue in obesity-related inflammation and insulin resistance: cells, cytokines, and chemokines. ISRN Inflamm 2013;2013:139239.[2]Esser N, Legrand-Poels S, Piette J, Scheen AJ, Paquot N. Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes. Diabetes Res Clin Pract 2014;105:141-50.[3]Otero M, Lago R, Gomez R, et al. Changes in plasma levels of fat-derived hormones adiponectin, leptin, resistin and visfatin in patients with rheumatoid arthritis. Ann Rheum Dis 2006;65:1198-201.[4]Schaffler A, Ehling A, Neumann E, et al. Adipocytokines in synovial fluid. JAMA 2003;290:1709-10.[5]Frommer KW, Zimmermann B, Meier FM, et al. Adiponectin-mediated changes in effector cells involved in the pathophysiology of rheumatoid arthritis. Arthritis Rheum 2010;62:2886-99.[6]Kitahara K, Kusunoki N, Kakiuchi T, Suguro T, Kawai S. Adiponectin stimulates IL-8 production by rheumatoid synovial fibroblasts. Biochem Biophys Res Commun 2009;378:218-23.Figure 2.Adiponectin induces the production of IL-6 PBMCs (A) FLS (B) measured using ELISA.Disclosure of Interests:Yuan Zhang: None declared, Jonathan Aldridge: None declared, Georgios K. Vasileiadis: None declared, Anna-Carin Lundell: None declared, Anna Rudin Consultant of: Astra/Zeneca, Cristina Maglio: None declared

Increased Levels of Interleukin 33 in Sera and Synovial Fluid from Patients with Active Rheumatoid Arthritis

2009 ◽  
Vol 37 (1) ◽  
pp. 18-25 ◽  
Author(s):  
YASUSHI MATSUYAMA ◽  
HITOAKI OKAZAKI ◽  
HIROYUKI TAMEMOTO ◽  
HIROTAKA KIMURA ◽  
YASUYUKI KAMATA ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Disease Activity ◽  
Mononuclear Cells ◽  
Sandwich Elisa ◽  
Protein A ◽  
Peripheral Blood Mononuclear ◽  
Interleukin 33 ◽  
Activity Group ◽  
Culture Supernatants

Objective.To determine levels of interleukin 33 (IL-33) in serum and synovial fluid (SF) and their clinical associations in patients with rheumatoid arthritis (RA). To evaluate the ability of activated peripheral blood mononuclear cells (PBMC) and fibroblast-like synoviocytes (FLS) from RA patients to release IL-33.Methods.Sera were obtained from 59 patients with RA, 10 patients with infectious diseases, and 42 healthy volunteers. SF samples were obtained from 15 patients with RA and 13 with osteoarthritis. IL-33 levels were measured using a sandwich ELISA after removal of rheumatoid factor with protein A-Sepharose beads. FLS were stimulated with IL-1ß and tumor necrosis factor, and treated with or without chemical damage. PBMC were stimulated with anti-CD3/CD28 antibodies. The levels of IL-33 were measured in the culture supernatants and cell lysates by ELISA or immunoblotting.Results.Serum IL-33 levels were significantly higher in RA patients, especially in the high disease activity group compared to the moderate or low activity group. IL-33 levels in SF were elevated in all 15 RA patients measured. IL-33 levels were higher in SF samples than in sera in 7 RA patients measured simultaneously. The 30-kDa IL-33 precursor was detected in the culture supernatants of damaged FLS but was not detected in those of activated PBMC and non-damaged FLS.Conclusion.IL-33 levels were elevated in sera and SF samples from patients with RA, and correlated with disease activity. IL-33 was produced mainly in inflamed joints; IL-33/ST2L signaling might play an important role in joint inflammation of human RA.

scholarly journals Functional characterization of adherent synovial fluid cells in rheumatoid arthritis: Destructive potential in vitro and in vivo

2003 ◽  
Vol 48 (7) ◽  
pp. 1873-1880 ◽  
Author(s):  
Michel Neidhart ◽  
Christian A. Seemayer ◽  
Klaus M. Hummel ◽  
Beat A. Michel ◽  
Renate E. Gay ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Functional Characterization ◽  
Synovial Fluid Cells ◽  
Destructive Potential

Increased Interleukin 21 (IL-21) and IL-23 Are Associated with Increased Disease Activity and with Radiographic Status in Patients with Early Rheumatoid Arthritis

2010 ◽  
Vol 37 (10) ◽  
pp. 2014-2020 ◽  
Author(s):  
TUE KRUSE RASMUSSEN ◽  
THOMAS ANDERSEN ◽  
MALENE HVID ◽  
MERETE LUND HETLAND ◽  
KIM HØRSLEV-PETERSEN ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Disease Activity ◽  
Plasma Levels ◽  
Mononuclear Cells ◽  
Early Stage ◽  
Peripheral Blood Mononuclear ◽  
Cellular Expression ◽  
Interleukin 17A ◽  
Sample Set

Objective.To investigate the levels of the T helper (Th)17-related cytokines interleukin 17A (IL-17A), IL-21, and IL-23 and their association with disease activity in rheumatoid arthritis (RA).Methods.In a longitudinal sample set from patients with early RA (< 6 months; n = 40), we measured the plasma cytokine levels of IL-17A, IL-21, and IL-23 and analyzed for correlation with disease activity in 28 joints (Disease Activity Score 28-joint count; DAS28), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and total Sharp score (TSS). In a transverse sample set of patients with chronic RA (> 8 years), using paired peripheral blood mononuclear cells and synovial fluid mononuclear cells, we investigated the cellular expression of IL-17A, IL-21, and IL-23R.Results.Patients with early-stage RA had significantly increased plasma levels of IL-21 and IL-23, but not IL-17A, compared to patients with chronic RA and healthy volunteer controls. Plasma levels of IL-21 and IL-23 after 12 months of treatment correlated with DAS28 and ESR, but not to TSS. Changes in IL-23 plasma levels from time of diagnosis to 12 months correlated with change in DAS28 and with TSS scores at 2 years. The numbers of CD4+ T cells producing IL-21 were significantly increased in the synovial fluid of patients with chronic RA, with only marginal coexpression of IL-21 and IL-17A.Conclusion.Our results show a significant association between plasma levels of IL-21 and IL-23 and disease activity in RA, supporting the hypothesis that IL-21 and IL-23 are important pathogenic factors of this disease.

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