Background/Purpose:

Polymorphonuclear granulocytes (PMNs) are the most abundant immune cell population in the synovial fluid in the joints of patients with rheumatoid arthritis (RA) and appear to play a central role in the inflammatory process leading to  destruction of the cartilage.

Upon certain types of activation, PMNs extrude their nuclear content in the form of nucleoprotein threads, known as extracellular traps (NETs), which have extensive pro-inflammatory and immune-stimulatory activities².

The focus of the present study was to investigate the responsiveness of RA neutrophils to PMA and of healthy control neutrophils to RA serum and joint fluid.

Methods:

Peripheral blood neutrophils from RA patients and control subjects were isolated and cultured with and without the addition of PMA, an established chemical trigger of NET formation. Cl-amidine, a chemical PAD4 inhibitor, was applied for inhibition studies. Spontaneous NET formation was evaluated with simultaneous immunostaining for myeloperoxidase (MPO), neutrophil elastase (NE) and DAPI, immunofluoresence microscopy and morphometry. PMNs from healthy donors were incubated with sera and synovial fluid from RA patients. NET formation by RA and control neutrophils was assessed by combined immunostainings with anti-PAD4, anti-cit-H3 and anti-H1-core histone antibodies, and DAPI. Cell-free nucleosomes were measured by ELISA, and PAD4 protein translocation from the cytoplasm to the nucleus was confirmed by Western blotting.

Results:

Freshly isolated neutrophils from patients with RA showed enhanced spontaneous NET release after 3 hours compared to the healthy controls. Intriguingly, the population of neutrophils with delobulated/diffused nuclear phenotype was also remarkably higher in RA from the outset. Moreover, neutrophils derived from RA patients exhibited even higher rates of NETosis and PAD4 nuclear translocation after chemical stimulation with PMA. The effect of PMA was practically abolished by pre-treatment with Cl-amidine, a chemical PAD4 inhibitor, which plays a key role in modifying histones and triggering NETosis. Treatment of control neutrophils with either RA serum or RA synovial fluid augmented the release of NETs significantly compared to cells treated with normal serum or synovial fluid from OA patients, respectively.

Conclusion:

RA neutrophils show enhanced NET formation that is mediated by PAD4 and is driven by factors present in the serum and synovial fluid from RA patients. Further investigations will more clearly define the role of NET formation in the innate and adaptive immune responses in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/enhanced-responsiveness-to-stimulation-and-increased-extrusion-of-extracellular-traps-by-rheumatoid-arthritis-neutrophil-granulocytes-and-of-healthy-control-neutrophils-to-serum-and-synovial/