Enhancement by growth hormone of phorbol diester-stimulated respiratory burst in human polymorphonuclear leukocytes Gian Luigi Spadoni, Anna Spagnoli, Stefano Cianfarani, Domenico Del Principe, Adriana Menichelli, Stefano Di Giulio and Brunetto Boscherini Abstract. The oxidative metabolic burst of mononu- clear and polymorphonuclear phagocytes can be stimu- lated to produce free oxygen radicals. Several substances can enhance this respiratory burst activity by a priming action: recently growth hormone (rat and porcine) was demonstrated to act as a priming agent on rat peritoneal and on porcine alveolar macrophages. In our study we wanted to verify whether also human GH had a similar priming action on homologous cells, in particular on poly- morphonuclear leukocytes. To determine the oxidative activity of polymorphonuclear leukocytes, after stimula- tion with phorbol myristate acetate, a flow-cytometric assay was employed which registered the intracellular for- mation of highly fluorescent products as indicators for the intracellular formation of hydrogen peroxide. The incubation of phorbol myristate acetate-stimulated poly- morphonuclear leukocytes with GH resulted in a time\x=req-\ dependent and dose-dependent increase in fluorescence, thus demonstrating that human GH enhances in vitro the oxidative metabolic burst of these cells. The action of GH appeared to be significant after 30 min of incubation, was maximal at 60 min, and decreased after 90 min. After one hour of incubation, the first significant variation of flu- orescence appeared with GH at a concentration of 50 \g=m\g/l. The maximum effect was seen at 100 \g=m\g/l with no further increase. Specificity of GH action was demonstrated by the inhibition of its effect by the addition of monoclonal antibodies to GH. The oxidative metabolic burst of mononuclear and polymorphonuclear phagocytes can be stimulated by a variety of soluble or paniculate agents to pro¬ duce reactive oxygen intermediates such as hy- droxyl radicals, hydrogen peroxide, and super- oxide anions. These products of oxygen reduction which represent the oxygen-dependent antimicro¬ bial arsenal of these cells, play a relevant role in many cellular activities. It has been reported that the respiratory burst may be enhanced by several substances, such as interferon-y or granulocyte-macrophage colony- stimulating factor, which act as priming agents (1- 4). Recently it has been demonstrated that also growth hormone enhances the production of Su¬ peroxide anions by macrophages in response to op- sonized zymosan (5). This new property for GH was demonstrated on rat peritoneal macrophages in vivo and on porcine alveolar macrophages in vitro employing recombinant and native, rat and porcine, GH. The object of our study was to verify whether human GH had a similar priming action on homol¬ ogous cells, namely polymorphonuclear leukocytes (PMNLs), which are cells with similar oxygen-de¬ pendent intracellular killing mechanisms such as macrophages. The respiratory metabolic burst of PMNLs (6) involves the activation of NADPH oxidase to gen¬ erate 02 and NADP;H202 is produced by rapid dismutation of 02: Og+NADPH oxidase, NADP + 0¡^ H202 To determine the oxidative activity of PMNLs we stimulated these cells with a phorbol diester, namely phorbol myristate acetate (PMA), and we employed a flow-cytometric assay using 2',7'-di- chlorofluorescin-diacetate (DCFH-DA) as the Department of Pediatrics, 2nd University of Rome, Rome, Italy