title: Disentangling the effects of tocilizumab on neutrophil survival and function.
abstract: The synovial tissue in rheumatoid arthritis (RA) represents a hypoxic environment with up-regulated pro-inflammatory cytokines and cellular infiltrates including neutrophils. Although inhibition of the interleukin (IL)6 receptor pathway by tocilizumab is a potent treatment option for RA, it may also cause adverse effects such as an occasionally high-grade neutropenia. We analysed the impact of tocilizumab on survival, mediator secretion, oxidative burst, phagocytosis and energy availability of high-dose toll-like receptor (TLR)2/4-stimulated neutrophils (to mimic an arthritis flare) under normoxic versus hypoxic conditions. Human neutrophils were purified, pre-treated with varying doses of tocilizumab, dexamethasone or human IgG1 and high-dose-stimulated with lipopolysaccharide (LPS) alone-triggering TLR2/4-, LPS plus IL6, or left unstimulated. Cells were then incubated under normoxic (18 % O2) or hypoxic (1 % O2) conditions and subsequently analysed. Neutrophil survival and energy availability were significantly decreased by tocilizumab in a dose-dependent manner in high-dose TLR2/4-stimulated cells, but to a greater extent under normoxia as compared to hypoxia. We also found high-dose LPS-stimulated oxidative burst and phagocytosis of neutrophils to be higher under hypoxic versus normoxic conditions, but this difference was reduced by tocilizumab. Finally, we observed that tocilizumab affected neutrophil mediator secretion as a function of oxygen availability. Tocilizumab is known for both beneficial effects and a higher incidence of neutropenia when treating RA patients. Our results suggest that both effects can at least in part be explained by a reduction in neutrophil survival, a dose-dependent inhibition of hypoxia-induced NADPH oxidase-mediated oxidative burst and phagocytosis of infiltrating hypoxic neutrophils and an alteration of mediator secretion.
authors.affiliation: Department of Rheumatology and Clinical Immunology, Charité University Hospital, Charité University Medicine, Charitéplatz 1, 10117, Berlin, Germany. timo.gaber@charite.de.|German Rheumatism Research Center (DRFZ), Charitéplatz 1, 10117, Berlin, Germany. timo.gaber@charite.de.|Berlin-Brandenburg Center of Regenerative Therapies (BCRT), Augustenburger Platz 1, 13353, Berlin, Germany. timo.gaber@charite.de.
authors.forename: Timo
authors.identifier: 0000-0002-6675-3895
authors.initials: T
authors.lastname: Gaber
authors.affiliation: Department of Rheumatology and Clinical Immunology, Charité University Hospital, Charité University Medicine, Charitéplatz 1, 10117, Berlin, Germany.|German Rheumatism Research Center (DRFZ), Charitéplatz 1, 10117, Berlin, Germany.|Berlin-Brandenburg School of Regenerative Therapies (BSRT), Augustenburger Platz 1, 13353, Berlin, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Hahne
authors.affiliation: Department of Rheumatology and Clinical Immunology, Charité University Hospital, Charité University Medicine, Charitéplatz 1, 10117, Berlin, Germany.|German Rheumatism Research Center (DRFZ), Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Cindy
authors.initials: C
authors.lastname: Strehl
authors.affiliation: Department of Rheumatology and Clinical Immunology, Charité University Hospital, Charité University Medicine, Charitéplatz 1, 10117, Berlin, Germany.|German Rheumatism Research Center (DRFZ), Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Paula
authors.initials: P
authors.lastname: Hoff
authors.affiliation: Department of Gastroenterology and Hepatology, Charité University Medicine, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Dörffel
authors.affiliation: Department of Rheumatology and Clinical Immunology, Charité University Hospital, Charité University Medicine, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Eugen
authors.initials: E
authors.lastname: Feist
authors.affiliation: Department of Rheumatology and Clinical Immunology, Charité University Hospital, Charité University Medicine, Charitéplatz 1, 10117, Berlin, Germany.
authors.forename: Gerd-Rüdiger
authors.initials: GR
authors.lastname: Burmester
authors.affiliation: Department of Rheumatology and Clinical Immunology, Charité University Hospital, Charité University Medicine, Charitéplatz 1, 10117, Berlin, Germany.|German Rheumatism Research Center (DRFZ), Charitéplatz 1, 10117, Berlin, Germany.|Berlin-Brandenburg Center of Regenerative Therapies (BCRT), Augustenburger Platz 1, 13353, Berlin, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Buttgereit
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000627:therapeutic use*; D001172:Arthritis, Rheumatoid / Q000188:drug therapy*; D019069:Cell Respiration; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004734:Energy Metabolism; D006801:Humans; D000860:Hypoxia / Q000188:drug therapy*; D007167:Immunotherapy / Q000379:methods*; D053666:Interleukin-5 Receptor alpha Subunit / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000276:immunology; D010587:Phagocytosis; D016897:Respiratory Burst
chemical_list: D061067:Antibodies, Monoclonal, Humanized; C485918:IL5RA protein, human; D053666:Interleukin-5 Receptor alpha Subunit; D008070:Lipopolysaccharides; D019255:NADPH Oxidases; C502936:tocilizumab
keywords: Biological therapy; Hypoxia; Inflammation; Neutrophils