Phagocyte production of superoxide and hydrogen peroxide is critical to host defense but also leads to events which damage normal tissues. In vitro these two oxidant species react in the presence of a transition metal catalyst (e.g. iron) to form hydroxyl radical (OH). Whether OH is a physiologic product of phagocytes or requires an exogenous catalyst has not been clear due to the lack of a specific OH detection system. I helped develop such a system which utilizes spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry. With this system we found no evidence that neutrophils (PMNs) or mononuclear phagocytes (monocytes or monocyte-derived macrophages [MDM]) generated OH in the absence of exogenous iron. Even with iron supplementation, PMN lactoferrin and myeloperoxidase (MPO) release inhibited OH generation. Mononuclear phagocytes, however, exhibited sustained OH formation under the same conditions, presumably due to the lack of these inhibitory compounds. This application proposes to expand on these observations by examining internal and external physico-chemical factors which could influence the potential for OH generation by mononuclear phagocytes.
Five specific aims have been identified for study.
Aim 1 will determine whether the nature and dynamics of free radical generation varies with study of: 1) adherent vs non-adherent cells; 2) tissue (pulmonary) macrophages as opposed to MDM; and 3) iron-overloaded mononuclear phagocytes.
Aim 2 will examine whether iron provided by a variety of extracellular (non-phagocytosed) targets may allow OH generation to occur as a consequence of mononuclear phagocyte )2 reduction. Erythrocytes, bacteria and bacterial siderophores, and ferritin-loaded liposomes (used as models of eukaryotic cells) will be used.
Aim 3 will examine whether the phagosomal environment is associated with conditions which may alter the likelihood of OH formation occurring when targets are phagocytosed by mononuclear phagocytes.
Aim 4 utilizes recently synthesized spin trapping agents with increased lipid solubility in an attempt to increase the possible detection of OH occurring at a restricted site within the target particle. Mononuclear phagocytes take up lactoferrin and MPO and their oxidative response can be influenced by other products of inflammatory effective cells - elastase, cathepsin G, TNF, Interleukin 1 and 2, GM- CSF etc.
Aim 5 will investigate whether exposure of mononuclear phagocytes to these actors alters their potential for generation of OH. This proposal is a comprehensive program whose objective is to define the impact of endogenous and exogenous factors on the likelihood of OH formation as a consequence of mononuclear phagocytes 02 reduction. Such information would set the stage for future work to clarify the role of OH in the microbicidal activity s tissue damage associated with phagocyte oxidant production.
| Wilson, M E; Vorhies, R W; Andersen, K A et al. (1994) Acquisition of iron from transferrin and lactoferrin by the protozoan Leishmania chagasi. Infect Immun 62:3262-9 |
