Spontaneous secretion of peptidyl-arginine deiminase 2 and cell surface exposure of peptidyl-arginine deiminase 4 by neutrophils

Abstract Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA) and elevated levels of protein citrullination can be found in the joints of RA patients. Although the pathophysiological mechanisms and the cell type(s) responsible for the increase in protein citrullination remain incompletely understood, the neutrophil is emerging as a prime suspect. Here we report that fully viable resting neutrophils from healthy donors have enzymatically active PAD4 exposed on their surface and that they spontaneously secrete enzymatically active PAD2. Activation of the neutrophils by several stimulatory agents, such as immune complexes, tumor necrosis factor a, phorbol myristate acetate, and agonists of Toll-like receptors1, 5, 7, and 9, increased the immunoreactive amount of PAD4 on the cells. However, immune complex, LPS and PMA stimulation reduced PAD2 secretion. The presence of extracellular PAD2 and PAD4 was not caused by NETosis, apoptosis, or lysis of the cells, as verified by gating on live cells and adding NETosis inhibitor in flow cytometry experiments. Neutrophils from the blood of RA patients also exposed PAD4 on their surface at levels similar to those from healthy subjects. Our data add a novel pathway for the extracellular citrullination of proteins by PAD2 and PAD4 in RA and suggest that this pathway is part of normal neutrophil biology also in the absence of disease.