Transition metal catalyzed formation of hydroxyl radical (OH) has been linked to neutrophil (PMN) microbicidal activity and tissue damage. Using spin trapping and other methodology, we and other found that PMN will not form OH unless provided with exogenous iron and that they possess two mechanisms (lactoferrin and myeloperoxidase [MPO] release) which limit their potential for OH production. These data suggest that the composite impact of exogenous catalysts, dynamics of the PMN oxidative and secretory response, and presence of factors capable of modulating that response should determine to a great extent the potential for in vivo generation of biologically significant quantities of OH. The investigation of all such possible factors is beyond the scope of any research program. Consequently, five hypotheses (specific aims) whose examination will provide major insight into the free radical biology of inflammation have been identified and will comprise Project 2. Surface adherent PMN and PMN """"""""primed"""""""" with different immunomodulatory factors exhibit alterations of cell functions.
Aim 1 examines oxygen-centered free radical formation of surface adherent PMNs as well as PMNs primed with lipopolysaccharide, y-interferon, interleukin-1, tumor necrosis factor (a and B), granulocyte-macrophage colony stimulating factor, and platelet activating factor. The endogenous capacity of these cells to generate OH as well as the dynamics of OH formation when they are provided with a catalyst will be examined using spin trapping and other recently described assays which appear to be specific for OH. MOst studies have examined PMN OH generation only in the context of an iron catalyst. Since copper can also act as an OH catalyst and is present in vivo, Aim 2 will examine the dynamics of OH generation by PMN stimulated in the presence of exogenous copper.
Aim 3 will explore the catalytic potential of several unique bacteria-derived iron chelates likely present in vivo at sites of infection. Four bacterial siderophores from Pseudomonas aeruginosa or Escherichia coli will be assessed for their ability to catalyze OH formation by both an enzymatic O2 generating system and PMN. In addition, the catalytic potential of Pseudomonas elastase induced cleavage products of transferrin will be assessed. MPO release limits of OH generation by iron supplemented PMN. Since MPO-deficient PMN are capable of killing some microbial pathogens, Aim 4 will test whether increased formation of OH (with microbial iron as the catalyst) is partially responsible for their retained microbicidal capacity. Eosinophils generate O2- and H2O2, lack lactoferrin, and possess a peroxidase (eosinophil peroxidase) distinct from MPO.
Aim 5 will determine the nature and dynamics of free radical generation by eosinophils. These results, will provide important data about the function of a related phagocyte and indirectly contribute to understanding of PMN function. Completion of these 5 aims will provide new insight into conditions necessary for in vivo generation of OH.
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