Intracellular reactions in single human granulocytes upon phorbol myristate acetate activation using confocal Raman microspectroscopy

We have obtained new evidence for the occurrence of intracellular NADPH-oxi dase activity in neutrophilic and eosinophilic granulocytes upon stimulatio n with phorbol myristate acetate (PMA), PMA activation leads to a partial t ranslocation of cytochrome b(558) from the membranes of the specific granul es to the plasma membrane. It was suggested that NADPH-oxidase activity onl y takes place in the plasma membrane, leading to an extracellular release o f oxygen metabolites because cellular self-destruction can be avoided in th is way. The effects of PMA activation were indirectly studied in recent exp eriments employing scavengers of extracellular superoxide anion and hydroge n peroxide, and support for intracellular NADPH-oxidase activity was obtain ed. In this paper we use Raman microspectroscopy as a direct method to stud y intracellular molecular reactions that result from cellular triggering by PMA. The molecular specificity of this microscopic method enables us to sh ow that intracellular reduction of both myeloperoxidase (MPO) and cytochrom e b(558) occurs in neutrophilic granulocytes, Control measurements with cyt ochrome b(558)-deficient neutrophilic granulocytes did not show a reduction of intracellular MPO. This is direct support for the occurrence of intrace llular NADPH-oxidase activity in organelles that must be in close contact w ith the azurophilic granules that contain MPO. Furthermore, a comparison wa s made with chemical reactions occurring in eosinophilic granulocytes after activation with PMA. Moreover, in these cells an intracellular reduction o f eosinophil peroxidase was observed.