Signaling Abnormalities In Neutrophil Granulocytes Of Rheumatoid Arthritis Patients Are Associated With Increased Extracellular Trap Formation and Provide a Basis For The Induction Of Anti-Citrullinated Peptide Antibodies

Chanchal Sur Chowdhury¹, Stavros Giaglis^2,3, Ulrich A. Walker⁴, Andreas Buser⁵, Paul Hasler² and Sinuhe Hahn⁶, ¹Dept. of Biomedicine, Laboratory of Prenatal Diagnostics, University Hospital Basel, Basel, Switzerland, ²Dept. of Rheumatology, Kantonsspital Aarau, Aarau, Switzerland, ³Dept. of Biomedicine, Laboratory of Prenatal Madicine, University Hospital Basel, Basel, Switzerland, ⁴Department of Rheumatology, University Hospital Basel, Basel, Switzerland, Basel, Switzerland, ⁵Division of Hematology; Swiss Red Cross Blood Transfusion Centre, University Hospital Basel, Basel, Switzerland, ⁶Dept. of Biomedicine, Laboratory for Prenatal Medicine, University Hospital Basel, Basel, Switzerland

Meeting: 2013 ACR/ARHP Annual Meeting

Keywords: citrullination, Neutrophil Extracellular Traps, PAD, rheumatoid arthritis (RA) and signal transduction

Session Information

Session Title: Rheumatoid Arthritis: Human Etiology and Pathogenesis II

Session Type: Abstract Submissions (ACR)

Background/Purpose:

Rheumatoid arthritis (RA) is often associated with anti-citrullinated peptide antibodies (ACPAs) in the serum. While ACPAs have been associated with increased inflammation, joint destruction and reduced response to therapy, the mechanisms leading to the citrullination of arginine residues by peptidyl arginine deiminase (PAD) isozymes 2 and 4 on ACPA target antigens have remained unclear. PAD2 and PAD4 are present in polymorphonuclear granulocytes (PMNs), which represent the primary part of the leukocyte infiltrate in the RA joint. Citrullination of histone by PAD4 is critical for neutrophil extracellular trap (NET) formation^1,2.

In the present study, we sought to investigate the signaling pathway leading to histone modification and NET extrusion in RA neutrophils.

Methods:

Peripheral blood neutrophils from active RA patients and control subjects were isolated. Reactive oxygen species (ROS) production was evaluated by FACS utilizing a DCFH-DA assay. Spontaneous NET formation from RA and control neutrophils was observed by scanning electron (SEM) and SytoxGreen fluoresence microscopy. Combined immunostaining with anti-myeloperoxidase (MPO), anti-neutrophil elastase (NE), anti-PAD2, anti-PAD4, anti-citH3, anti-H1-core histones antibodies and staining with DAPI were applied for detailed characterization of the process. Protein translocation was confirmed by Western blotting.

Results:

Peripheral blood PMNs from RA cases exhibited increased spontaneous NET formation in vitro assessed by fluorescence and SEM. This was associated with enhanced NE and MPO message and protein expression, elevated ROS production and translocation of PAD4 from the cytoplasm to the nucleus. Moreover, nuclear morphology and lobulation status indicated that RA PMN were in a significantly higher activation state from baseline. PAD4, together with PAD2, was found extruded and co-localized on NETs along with citrullinated histone (citH3) and DNA.

Conclusion:

Altered signalling leading to NETosis and the extracellular presence of PAD4 and PAD2 could provide a mechanism for aberrant citrullination of relevant intra- and extracellular proteins and peptides. These findings suggest a novel explanation for in vivo auto-antigen citrullination as a basis for ACPA induction, and implicate PMNs as key upstream factors in the pathogenesis of RA.

Disclosure:

C. Sur Chowdhury,
None;

S. Giaglis,
None;

U. A. Walker,
None;

A. Buser,
None;

P. Hasler,
None;

S. Hahn,
None.

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