MONOCYTE/MACROPHAGE FREE RADICAL BIOLOGY ASSESSED BY EPR

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.