Activated neutrophil carbamylates albumin via the release of myeloperoxidase and reactive oxygen species regardless of NETosis

Abstract

Objective: An anti-carbamylated albumin antibody was detected in rheumatoid arthritis (RA) patients, and its presence was associated with serum myeloperoxidase (MPO) levels, as we reported previously. Since MPO is a key enzyme for carbamylation and is released by neutrophil extracellular traps (NETs), we aimed to demonstrate that NETosis induces carbamylation.

Methods: Human neutrophils were isolated from a healthy donor, pre-treated with or without diphenyleneiodonium (DPI, an inhibitor for the generation of reactive oxygen species (ROS)), Cl-amidine (a peptidylarginine deiminase inhibitor), 4-aminobenzoic acid hydrazide (4-ABAH, an MPO inhibitor), or GW311616A (a neutrophil elastase (NE) inhibitor), and incubated for 8 h with or without phorbol 12-myristate 13-acetate (PMA). Proteins in the medium were collected and the carbamylation of albumin was evaluated by Western blotting.

Results: The carbamylation of albumin was detected in the culture medium of activated neutrophils. NETosis was observed under the stimulation by PMA. DPI and 4-ABAH inhibited the carbamylation of albumin and NETosis. GW311616A inhibited NETosis, but not carbamylation. Neither carbamylation nor NETosis was inhibited by Cl-amidine.

Conclusion: Activated neutrophils may carbamylate ambient albumin, and this is dependent on ROS and MPO, but does not require NETosis.

Keywords:

• Anti-carbamylated protein antibodies
• anti-modified protein antibodies
• autoantibodies
• neutrophils
• rheumatoid arthritis

Acknowledgments

Experiments for Supplementary data were performed at and supported by Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA. We sincerely appreciate Dr. Mariana J Kaplan, who gave the corresponding author the resource to perform the additional experiments.

Conflict of interest

None.

Additional information

Funding

MH and MT are affiliated with a department that is financially supported by Nagahama City, Shiga, Japan, and four pharmaceutical companies (Mitsubishi Tanabe Pharma Co., Chugai Pharmaceutical Co., Ltd., UCB Japan Co., Ltd., and AYUMI Pharmaceutical Co.). MH also received research grants from Astellas and Bristol-Myers Squibb.