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Abstract Number: 4

Pro-Inflammatory Effect of Extracellular RNA On Synovial Fibroblasts From Patients with Rheumatoid Arthritis

Birgit Zimmermann1, Silvia Fischer2, Markus Rickert3, Stefan Rehart4, Angela Lehr5, Ulf Müller-Ladner6, Klaus T. Preissner2 and Elena Neumann7, 1Internal Medicine and Rheumatology, Justus-Liebig-University of Gießen, Kerckhoff-Klinik, Bad Nauheim, Germany, 2Dept of Biochemistry, Justus-Liebig-University of Giessen, Medical School, Giessen, Germany, 3Dept of Orthopedics and Orthopedic Surgery, University Hospital Giessen and Marburg, Giessen, Germany, 4Orthopedics and Trauma Surgery, Markus-Hospital, Frankfurt, Germany, 5Dept Orthopedics and Trauma Surgery, Markus-Hospital, Frankfurt, Germany, 6Dept of Internal Medicine and Rheumatology, Justus-Liebig-University of Giessen, Kerckhoff-Klinik, Bad Nauheim, Germany, 7Department of Internal Medicine and Rheumatology, Justus-Liebig-University of Gießen, Kerckhoff-Klinik, Bad Nauheim, Germany

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: rheumatoid arthritis (RA) and synovial cells, RNA, synovial fluid

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Session Information

Title: Biology and Pathology of Bone and Joint

Session Type: Abstract Submissions (ACR)

Background/Purpose: Extracellular RNA (exRNA) is present in the serum of patients suffering of different kinds of cancer. exRNA influences physiological processes like blood coagulation and endothelial cell permeability. In this study, we analyzed the presence of exRNA as well as RNase in joints and the effects of nucleic acids and nucleases on synovial fibroblasts (SF) of patients with rheumatoid arthritis (RA). RA is a chronic inflammatory disease, characterized by the destruction of cartilage as well as bone. RASF are activated cells acting as key players in cartilage destruction. 

Methods: exRNA in synovium of RA (n=26), osteoarthritis (OA: n=26) and psoriatic arthritis (PsA: n=3) patients was stained in tissue sections with DAPI to locate DNA and SYTO® RNA Select™ Green Fluorescent Stain to locate RNA. RNase 1 was analysed in synovium (RA n=17; OA n=7) immunohistocally. RNase activity was measured in synovial fluid (RA n=14; OA n=5; PsA n=10) and supernatants of SF (RASF n=3; OASF n=3; PsA-SF n=4; healthy donors (NSF) n=3) using the Quant-iT™ RNA Assay Kit. Release of exRNA by cultured SF (RASF n=3; OASF n=3; PsA-SF n=4; NSF n=3) was measured photometrically. The secretion of IL-6 was analyzed by ELISA after treating RASF (n=3) with 10 µg or 25 µg isolated autologous RNA or DNA for 15 h. Alternatively RASF (n=3) were pre-incubated for 24 h, then 5 U/ml RNase or DNase was added. In the SCID mouse model, the effect of RNase or DNase i.v. was analyzed towards migration and cartilage invasion (RNase n=10; DNase n=9; saline control n=9).

Results: Synovium of all patient showed RNA and DNA signals co-localized within the nuclei. In RA, exRNA was detectable in the lining layer. exRNA in OA lining was present only in single defined regions. PSA showed no exRNA in lining. RNase 1 was increased in lining. RNase activity (units/mg protein) in synovial fluid was significantly increased in RA (31.6.1±4.5 p=0.023) and OA (27.0±4.5 p=0.033) compared to PsA (18.2±2.3). RNase release (units/mg protein) by cultured cells was comparable (RASF 1.13±0.24; OASF 1.16±0.07; PsA-SF 1.05±0.18; NSF 1.01±0.15). exRNA release (ng/µg protein) was stronger in RASF (RASF 0.22±0.06; OASF 0.17±0.09; PsA-SF 0.17±0.06; NSF 0.11±0.07). RASF secretion of IL-6 was increased by RNA (10 µg 135±40%; 25 µg 118±15%) or DNA (10 µg 156±35%; 25 µg 220±59%). Only RNase reduced IL-6 release (77±14% of control, p=0.136). In vivo, RASF invasion was reduced at the coimplantation site by both nucleases (control 1.92±0.24; RNase 1.22±0.21 p=0.037; DNase 0.80±0.19 p=0.002). Fibroblast migration was only reduced with DNase (control 1.51±0.28; RNase 1.28±0.39 p=0.625; DNase 0.84±0.18 p=0.042).

Conclusion: Increased exRNA in the lining and the RNase activity in the synovial tissue and fluid of OA and especially in RA patients could represent a new unbalanced regulatory system in chronic inflammation including RA. The increased exRNA but unchanged RNase release by RASF in vitro indicates that RASF contribute to secretion of pro-inflammatory exRNA. exRNA mediates an increased cytokine release by RASF and cartilage invasion which could be inhibited by RNase application. The results illustrate the pro-inflammatory and destructive effect of exRNA in RA.


Disclosure:

B. Zimmermann,
None;

S. Fischer,
None;

M. Rickert,
None;

S. Rehart,
None;

A. Lehr,
None;

U. Müller-Ladner,
None;

K. T. Preissner,
None;

E. Neumann,
None.

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