• Electron paramagnetic resonance/spin trapping detection and mechanism of human neutrophil hydroxyl radical generation

      Ramos, Carroll Leslie; Rosen, Gerald M., Ph.D., J.D. (1994)
      Neutrophils are important components of the innate immune response to invading microorganisms and may also contribute to tissue damage in pathologies such as reperfusion injury and arthritis. A principle mechanism by which neutrophils damage microbes is the generation of a series of reactive oxygen intermediates. Although the production of superoxide, hydrogen peroxide and hypochlorous acid by neutrophils is well established, the endogenous capacity of these cells to generate hydroxyl radical without the addition of a supplemental iron catalyst has not been determined. Since hydroxyl radical reacts with biomolecules at diffusion controlled rates, it is important to determine if neutrophils generate this free radical and, if so, establish the mechanism by which it is formed. Kinetic and stability studies were designed to select an appropriate electron paramagnetic resonance/spin trapping system for the detection of hydroxyl radical under biological conditions. The spin trap 4-pyridyl 1-oxide-N-tert-butylnitrone (4-POBN) in combination with ethanol, in which hydroxyl radical is detected as the {dollar}\alpha{dollar}-hydroxyethyl spin adduct of 4-POBN (4-POBN-CH(CH{dollar}\sb3{dollar})OH), was found to be best suited for this application. The rate constant for the reaction of 4-POBN with {dollar}\alpha{dollar}-hydroxyethyl radical was ten to fifteen fold greater than that with other nitrone spin traps and the resulting spin adduct was stable in the presence of neutrophil secretory products. Using this technique, it was found that human neutrophils and monocytes stimulated with a phorbol ester generated hydroxyl radical. Detection did not require a transition metal catalyst and was abolished by superoxide dismutase, catalase and azide. Hydroxyl radical was not observed upon stimulation of monocyte-derived macrophages and myeloperoxidase-deficient neutrophils. However, myeloperoxidase-deficient cells supplemented with purified myeloperoxidase generated hydroxyl radical upon activation. Addition of purified myeloperoxidase to a model superoxide generating system resulted in the chloride-dependent detection of hydroxyl radical which was inhibited by superoxide dismutase, catalase and azide. Based on these findings, it was concluded that human neutrophils and monocytes generate hydroxyl radical through a myeloperoxidase-dependent mechanism which likely involves the reaction of superoxide and hypochlorous acid.
    • Formation of the reactive iron-oxo intermediate during the biosynthesis of nitric oxide by neuronal nitric oxide synthase and its possible mechanism in the generation of secondary free radicals

      Porasuphatana, Supatra; Rosen, Gerald M., Ph.D., J.D. (2001)
      Nitric oxide synthases (NOSs) are hemoproteins that catalyze the formation of nitric oxide (NO•) and L-citrulline from L-arginine by a five-electron oxidation reaction. All NOS isoforms are in the same superfamily and share structural similarities with cytochrome P-450. The generation of free radicals by NOS is primarily regulated by the binding of L-arginine. During the oxidation of L-arginine to generate NO•, the transfer of electrons from reductase to oxygenase domain of NOS leads to changes in the oxidation state of heme iron, resulting in the formation of the perferryl iron-oxo intermediate of NOS (NOS-[Fe5+=O]3+). This dissertation aims to explore the possible role of NOS-[Fe5+=O]3+ in the generation of secondary free radicals. The NOS-[Fe5+=O] 3+, which formed only when L-arginine bound to the enzyme, was proven to abstract a hydrogen atom from substrates to generate carbon-centered free radicals. It was demonstrated that the abstraction of hydrogen atom by NOS-[Fe 5+=O]3+ occurred at a carbon alpha to a heteroatom, similar to hydroxylation reaction catalyzed by cytochrome P-450. Further investigation using potassium hydrogen persulfate (KHSO5) confirmed the formation of NOS-[Fe5+=O]3+ which may be generated via the rearrangement of NOS-[Fe3+-O-O-SO 3-] following the transfer of oxygen from KHSO 5 to NOS I. The NOS-[Fe5+=O]3+ generated by KHSO5-catalyzed NOS I was shown to generate NO• from L-arginine and alpha-hydroxyethyl radical from ethanol, resembling the incidences found with the NADPH/O2 NOS as a source of substrate oxidation. Deuterium isotope effect, using unlabeled ethanol and ethyl 1,1-d2 alcohol, revealed the importance of the breakage of C1-H bond as a partial rate-limiting step for the formation of carbon-centered free radicals by NOS. Taken together, this research illustrates the contribution of L-arginine binding to the formation of NOS reactive iron-oxo complex intermediate and the role of the iron complex in the generation of carbon-centered free radicals by the hydrogen atom abstraction.
    • Role of endothelial cells in host defense

      Zhang, Bin; Rosen, Gerald M., Ph.D., J.D. (1999)
      Phagocytes, neutrophils and macrophages, are part of the non-specific host defense system in control bacterial infection by the phagocytosis and killing of these microbes through free radical-dependent and free radical-independent mechanisms. In contrast, endothelial cells, in addition to their role in maintenance of homeostasis, have classically been perceived to play a supportive role in host immune response by releasing chemoattractants that recruit phagocytes to the site of infection. Recent studies have, however, demonstrated that the endothelium is capable of responding to stimulation by cytokines as part of their activation responses in host immunity. This dissertation explores the role of endothelia as effector cells in host response. The findings from this dissertation demonstrated that endothelial cells cultured on three dimensional GelfoamRTM are activated by penicillin G to phagocytosis and kill S. aureus. Even though O2-· and NO· are known to exhibit microbicidal activity, it appears that these free radicals do not play an integral part in the observed killing of S. aureus. To further investigate the host defense role of NO·, primary cultures of endothelial cells were transduced with retroviral vector encoding NOS II gene. Upon transduction, these cells released NO· at a constant flux over a long period of time. This provided an excellent model to study the antimicrobial activity of NO· at cellular fluxes without the complications of controlling the rate of NO· from a NO·-releasing compound or those associated with cytokine treatment. When infected with either S. aureus or E. coli, NOS II transduced endothelial cells, producing NO·, phagocytosed both bacteria. However, only E. coli was sensitive to NO· dependent bacterial killing. Taken together, these studies reveal the antimicrobial roles played by endothelial cells upon activation. The mechanisms utilized by these cells include both free radical-dependent and free radical-independent pathways.