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

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 microM) or ionomycin (1 microM), but not OVA (1 microg/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 degranulation. 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 activation and promote resolution of IgE-mediated inflammation.