A Comparison of Reactive Oxygen Species Generation by Rat Peritoneal Macrophages and Mast Cells Using the Highly Sensitive Real-Time Chemiluminescent Probe Pholasin: Inhibition of Antigen-Induced Mast Cell Degranulation by Macrophage-Derived Hydrogen Peroxide 1 Emily J. Swindle,* John A. Hunt, † and John W. Coleman 2 * Mast cells and macrophages live in close proximity in vivo and reciprocally regulate one another’s function in various ways. Although activated macrophages possess a powerful reactive oxygen species (ROS) generating system, there is conflicting evidence regarding whether mast cells can produce ROS. We used the highly sensitive real-time chemiluminescent probe Pholasin to examine ROS release by peritoneal macrophages and mast cells isolated from OVA-sensitized rats. Macrophages stimulated with PMA (0.8 M) or ionomycin (1 M), but not OVA (1 g/ml), released high-level ROS, levels of which peaked after 3–7 min and declined to baseline levels within 1 h. Superoxide was identified as the major ROS species induced by PMA but not by ionomycin. In contrast, purified mast cells stimulated with PMA released low-level ROS, which was entirely due to the contaminating (2%) macrophages, and did not release any detectable ROS in response to ionomycin or OVA at concentrations that induced degran- ulation. Stimulation of mixed cell populations with PMA to induce macrophage ROS release led to 50% inhibition of serotonin release from mast cells stimulated 5 min later with OVA. The PMA-induced inhibitory factor was identified as hydrogen peroxide. In conclusion, activated rat peritoneal macrophages but not mast cells produce ROS, and macrophage-derived hydrogen peroxide inhibits mast cell degranulation. The latter could be an important mechanism whereby phagocytic cells regulate mast cell acti- vation and promote resolution of IgE-mediated inflammation. The Journal of Immunology, 2002, 169: 5866 –5873. M ast cells are key effectors of specific and innate im- munity. In specific immunity, they respond to Ag via receptor-bound IgE to release multiple mediators in- cluding histamine, eicosanoids, and cytokines, which together con- tribute to immediate allergic reactions and inflammatory cell re- cruitment (1, 2). In innate immunity, mast cells release TNF in response to direct interaction with bacterial adhesion molecules, and this cytokine induces neutrophil recruitment with subsequent bacterial clearance through phagocytosis (3–5). A key element of phagocytosis and killing of bacteria, and possibly of importance also in mediating and regulating inflammation, is the production of reactive oxygen species (ROS) 3 (6 – 8). In activated macrophages and neutrophils, ROS are generated by NADPH oxidase in a pro- cess called the respiratory burst. This key enzyme catalyzes the generation of superoxide (O 2 - ) and hydrogen peroxide (H 2 O 2 ) using electrons provided by the hexose monophosphate shunt (9 – 11). NADPH oxidase of phagocytic cells can also be activated by protein kinase C agonists such as PMA (12). Because mast cells are pivotal to neutrophil-mediated defense against certain bacteria (3–5) and both neutrophils and macrophages phagocytose and kill bacteria by ROS production (9 –11), the possible contribution of mast cells themselves to ROS production is an important issue. Although mucosal mast cells can phagocytose and kill bacteria, evidently via generation of ROS (13), the question of whether mast cell ROS generation accompanies degranulation is less clear cut. Several studies have reported that IgE/Ag (14), calcium ionophore (15), or compound 48/80 (14 –19), which cause rodent peritoneal mast cell degranulation, concurrently induce release of ROS. In terms of measuring the kinetics of extracellular ROS release, there are prob- lems associated with the use of luminol and 2-methyl-6(4-methoxy- phenyl)-3,7-dihydroimidazol[1,2-a]pyrazin-3-one (MCLA), which were used by several of the above studies (14, 17–19). For exam- ple, luminol can act as a source of superoxide in the presence of univalent oxidants to yield a chemiluminescent product that is su- peroxide dismutase (SOD) and catalase inhibitable: this would lead to artifactual production of superoxide (20). Artifactual su- peroxide is also generated from MCLA, the form of luciferin spe- cific for superoxide (as used in Refs. 14 and 17–19), in the pres- ence of cell-derived NADH (21). Hence, concerns are raised about the reliability of ROS detection using these methods. Therefore, it is important to reevaluate ROS production by mast cells using alternative techniques of high sensitivity. Pholasin is a glycoprotein derived from the marine bivalve mol- lusc Pholas dactylus (22, 23). It is similar to luminol in that both are photoproteins that react with ROS to form a chemiluminescent product. Pholasin can react with a variety of ROS to form oxy- pholasin and light as a byproduct; the protein reacts only once with *Department of Pharmacology and Therapeutics and † U.K. Centre for Tissue Engi- neering, Department of Clinical Engineering, University of Liverpool, Liverpool, United Kingdom Received for publication July 22, 2002. Accepted for publication September 11, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a grant from The Wellcome Trust. 2 Address correspondence and reprint requests to Dr. John W. Coleman, Department of Pharmacology and Therapeutics, Sherrington Buildings, University of Liverpool, Liverpool, Liverpool L69 3GE, U.K. E-mail address: coleman@liv.ac.uk 3 Abbreviations used in this paper: ROS, reactive oxygen species; MCLA, 2-methyl- 6(4-methoxyphenyl)-3,7-dihydroimidazol[1,2-a]pyrazin-3-one; SOD, superoxide dismutase; DPI, diphenyleneiodonium; [ 3 H]serotonin, 5-[1,2-[ 3 H](N)]-hydroxytryp- tamine creatinine sulfate; ALU, actual light units; AUC, area under the curve; RBL- 2H3, rat basophilic leukemia 2H3. The Journal of Immunology Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00