Shuichiro Nakabo1,2, Koichiro Ohmura1, Shuji Akizuki1, Kosaku Murakami1, Ran Nakashima1, Motomu Hashimoto3, Hajime Yoshifuji1, Masao Tanaka3, Tsuneyo Mimori1. 1. Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 2. Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA. 3. Department of Advanced Medicine for Rheumatic Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
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.
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.
Entities:
Keywords:
Anti-carbamylated protein antibodies; anti-modified protein antibodies; autoantibodies; neutrophils; rheumatoid arthritis
Authors: Katherine Cook; Wei Xue; Suha Atabani; Peter Symonds; Abdullah Al Omari; Ian Daniels; Sabaria Shah; Ruhul Hasan Choudhury; Daisy Weston; Rachael Metheringham; Victoria Brentville; Lindy Durrant Journal: Front Immunol Date: 2022-04-08 Impact factor: 8.786
Authors: Katherine W Cook; Wei Xue; Peter Symonds; Ian Daniels; Mohamed Gijon; David Boocock; Clare Coveney; Amanda K Miles; Sabaria Shah; Suha Atabani; Ruhul H Choudhury; Poonam Vaghela; Daisy Weston; Rachael L Metheringham; Victoria A Brentville; Lindy G Durrant Journal: J Immunother Cancer Date: 2021-07 Impact factor: 13.751