Spontaneous Secretion of the Citrullination Enzyme PAD2 and Cell Surface Exposure of PAD4 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 patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA.

Insights into the study and origin of the citrullinome in rheumatoid arthritis

“…Two potential sources of extracellular PADs have been suggested to be responsible for this process. These include PADs secreted from activated neutrophils, and PADs released during neutrophil death . Nevertheless, while there is convincing evidence that soluble PADs are found in RA synovial fluid, the mechanisms involved in extracellular PAD activation are not fully understood.…”

“…Indeed, neither endogenous nor recombinant PADs have catalytic activity in synovial fluid unless reducing agents, such as dithiothreitol or reduced glutathione, are exogenously added . Similarly, PADs secreted from activated neutrophils, during PMA‐induced NET formation, or from necrotic neutrophils are only active in the presence of high amounts of dithiothreitol . In some cases, supraphysiologic amounts of calcium (>1.5 mM, with 1‐1.5 mM normally present in extracellular compartments) are also added exogenously to increase extracellular PAD activity .…”

“…95,96 Similarly, PADs secreted from activated neutrophils, during PMA-induced NET formation, or from necrotic neutrophils are only active in the presence of high amounts of dithiothreitol. 73,94,95 In some cases, supraphysiologic amounts of calcium (>1.5 mM, with 1-1.5 mM normally present in extracellular compartments) are also added exogenously to increase extracellular PAD activity. 73,94,96 Importantly, however, these conditions do not co-exist in vivo during physiologic conditions and whether these conditions are altered in the rheumatoid joint is unknown.…”

“…73,94,95 In some cases, supraphysiologic amounts of calcium (>1.5 mM, with 1-1.5 mM normally present in extracellular compartments) are also added exogenously to increase extracellular PAD activity. 73,94,96 Importantly, however, these conditions do not co-exist in vivo during physiologic conditions and whether these conditions are altered in the rheumatoid joint is unknown. Nevertheless, while extracellular citrullination is likely a very inefficient process, the presence of extracellular PAD substrates supports the existence of mechanisms that may facilitate extracellular citrullination in RA.…”

Peptidylarginine deiminases and deiminated proteins at the epidermal barrier

“…In some cases, other PADs were located in the nucleus although they have no nuclear localization signal . Viable neutrophils from healthy donors were recently reported to have active PAD4 exposed on their external surface …”