Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose
Citrullinated proteins are important autoantigens in the inflamed joints of patients with rheumatoid arthritis (RA). Several of these proteins are derived from proteins generally found in the extracellular space, such as fibrinogen and type II collagen. It is therefore important to understand the source of extracellular peptidyl deiminases, (PAD) the enzymes responsible for citrullination. After entering the joints of RA patients, neutrophils are activated and release DNA in an active process termed NETosis. As this process involves histone citrullination by PAD4 we hypothesized that neutrophils undergoing NETosis in the rheumatoid joint release enzymatically active peptidyl arginine deiminases.
Methods
Extracellular DNA was quantified in the synovial fluid (SF) of patients with RA (n=27) and osteoarthritis (OA) (n=15). Presence of decondensed DNA in association with neutrophil elastase was studied on smear preparations of RA SF and synovial tissue. Release of PAD2 and PAD4 was examined during in vitro induced NETosis using Western Blotting and verified by mass spectrometry and immunofluorescence. NETS isolated from in vivo and in vitro activated neutrophils were isolated and probed on western blots. PAD activity in the supernatant of in vitro stimulated neutrophils and in the SF of patients with RA (n=7) and OA (n=9) was determined by a conversion assay involving citrullination of target peptides.
Results
Extracellular DNA was detected in the SF from RA patients at significantly higher levels than in OA SF (p<0.001) and correlated with SF neutrophil cell counts (n=15, R2=0.68, p=0.002) and with PAD activity (n=14, R2=0.32, p=0.03) in the SF. Immunofluorescence revealed the co-localization of neutrophil elastase with decondensed DNA in RA SF and within neutrophil precipitates on the surface of the synovial lining layer. Furthermore, PAD activity was detected at significantly higher levels in the SF of RA patients when compared to SF from OA patients (p<0.001) and the isoenzymes PAD2 and PAD4 were both found to be present in the SF of RA patients. Significantly higher PAD activity could also be detected in the supernatant of in vitro stimulated neutrophils when compared to unstimulated cells (p<0.05). Western blotting revealed the release of free PAD2 and PAD4 into the supernatant and also the association of both isoenzymes with isolated NETs. The loss of nuclear PAD4 signal during NETosis was confirmed by immunofluorescence staining.
Conclusion
These results demonstrate the enzymatic activity of PADs in the synovial fluid of RA patients. Combined with our findings from in vitro stimulated neutrophils the data suggest that neutrophils undergoing NETosis are a source of this extracellular activity. The correlation of the measured PAD activity with DNA levels and neutrophil cell counts in the synovial fluid of RA patients are in line with this hypothesis. This study highlights the possibility that activated neutrophils recruited into the joints could continuously release enzymatically active PADs which contribute to the continuous generation of citrullinated autoantigens and thus drive an inflammatory response in the joint.
Disclosure:
J. Spengler,
None;
B. Lugonja,
None;
A. Creese,
None;
J. Ytterberg,
None;
K. Lundberg,
None;
M. Milward,
None;
M. Pearson,
None;
C. Buckley,
None;
A. Filer,
None;
K. Raza,
None;
P. Cooper,
None;
I. Chapple,
None;
D. Scheel-Toellner,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/release-of-enzymatically-active-peptidyl-arginine-deiminases-pads-by-neutrophils-allows-generation-of-citrullinated-extracellular-autoantigens-in-the-synovial-fluid-of-patients-with-rheumatoid-arthr/