TI:  H2O2 Release from Human Granulocytes during Phagocytosis.
     Relationship to Superoxide Anion Formation and Cellular 
     Catabolism of H2O2: Studies with Normal and Cytochalasin 
     B-Treated Cells

DT:  July 13, 1977

AU:  R.K Root, J. Metcalf

SO:  J. Clin. Invest, Vol. 60, Dec 1977, 1266-1279

AB:  Normal and cytochalasin B-treated human granulocytes have 
     been studied to determine some of the interrelationships 
     between phagocytosis-induced respiration and superoxide and 
     hydrogen peroxide formation and release into the 
     extracellular medium by intact cells.  By using the 
     scopoletin fluorescent assay to continuously monitor 
     extracellular hydrogen peroxide concentrations during 
     contact of cells with opsonized staphylococci, it was 
     demonstrated that the superoxide scavengers ferricytochrome 
     c and nitroblue tetrazolium significantly reduced the amount 
     of H2O2 released with time from normal cells but did not 
     abolish it.  This inhibitory effect was reversed by the 
     simultaneous addition of superoxide dismutase (SOD), whereas 
     the addition of SOD alone increased the amount of detectable 
     H2O2 in the medium.  The addition of sodium azide markedly 
     inhibited myeloperoxidase-H2O2-dependent protein iodination 
     and more than doubled H2O2 release, including the residual 
     amount remaining after exposure of the cells to 
     ferricytochrome c, suggesting its origin from an 
     intracellular pool shared by several pathways for H2O2 
     catabolism.

     When cells were pretreated with cytochalasin B and opsonized 
     bacteria added, reduced oxygen consumption was observed, but 
     this was in parallel to a reduction in specific binding of 
     organisms to the cells when compared to normal.  Under the 
     influence of inhibited phagosome formation by cytochalasin 
     B, the cells released an increased amount of superoxide and 
     peroxide into the extracellular medium relative to oxygen 
     consumption, and all detectable peroxide release could be 
     inhibited by the addition of ferricytochrome c.  Decreased 
     H2O2 production in the presence of this compound could not 
     be ascribed to diminished bacterial binding, decreased 
     oxidase activity, or increased H2O2 catabolism and was 
     reversed by the simultaneous addition of SOD.  Furthermore, 
     SOD and ferricytochrome c had similar effects on another 
     H2O2-dependent reaction, protein iodination, in both normal 
     and cytochalasin B cells.  When oxygen consumption, O2-, and 
     H2O2 release were compared in the presence of azide under 
     identical incubation conditions, the molar relationships for 
     normal cells were 1.00:0.34:0.51 and for cytochalasin B-
     treated cells 1.00:0.99:0.40, respectively.  Nonopsonized, 
     or opsonized but disrupted, bacteria did not stimulate any 
     of these metabolic functions.

     The results indicate that with normal cells approximately 
     50% of H2O2 released during phagocytosis is derived directly 
     from O2- by dismutation, the remainder appearing from an 
     (intra)cellular source shared by azide-inhibitable heme 
     enzymes.  With cytochalasin B treatment the evidence is 
     consistent with the derivation of all H2O2 from an O2- 
     precursor which is released from the cell surface.  
     Furthermore, when activated by phagocytic particle binding, 
     the neutrophil O2- generating system appears to make more of 
     this compound than can be accounted for by dismutation to 
     H2O2.  This establishes conditions for the direct 
     participation of both compounds in the microbicidal and 
     cytocidal activity of these cells.