Human Granulocyte-Macrophage Colony Stimulating Factor: An Effective Direct Activator of Human Polymorphonuclear Neutrophilic Granulocytes

Granulocyet-macrophage colony-stimulating factor (GM-CSF) was shown to modulate different granulocyte functions. In the present study we investigated the effect of purified and recombinant human GM-CSF, particularly on the oxidative metabolism of isolated human granulocytes. In addition, ultrastructural changes upon stimulation were evaluated. For detection of granulocyte activation the following assay systems were used: 1) lucigenin-dependent chemiluminescence (CL),2) superoxide-dismutase (SOD) inhibitable cytochrome C-reduction (superoxide),3) horseradish peroxidase-mediated oxidation of phenol red (hydrogen peroxide),4) release of myeloperoxidase, 5) ultrastructural detection of hydrogen peroxide-production, and 6) scanning and transmission electron microscopy (SEM and TEM, respectively). A significant CL response was seen upon stimulation with recombinant human GM-CSF at concentrations ranging from 1 to 103 U/ml. The CL response started within 5-10 min with a maximum at 60 – 90 min and lasted more than 3 h. Thereafter granulocytes were completely deactivated to restimulation with the same mediator and with Tumor Necrosis Factor, but respondend to other triggers of the oxidative burst, whereas the response to f-met-leu-phe was significantly increased, The CL signal was completely blocked by an antiserum to GM-CSF. Moreover, the response was significantly inhibited by SOD and D-mannitol, suggesting the involvement of distinct reactive oxygen species (ROS) in generating the CL response. Significant amounts of superoxide were detected within 180 min after stimulation with GM-CSF, whereas, release of hydrogen peroxide and peroxidase were only minimal as shown by functional and ultrastructural assays. Activation of granulocytes could be visualized by SEM and TEM. GM-CSF stimulated cells showed an increased adherence to the substratum developing polarized filopodia and an increased number of intercellular vesicles within 30 min after addition of the stimulus. The results clearly demonstrate that GM-CSF directly stimulates granulocytes and, particularly, their oxidative metabolism. Therefore, GM-CSF which is probably released by epidermal cells appears to be a candidate for neutrophil activation in the skin, and thereby may play a crucial role in inflammatory skin diseases

The Chemokine RANTES Is More than a Chemoattractant: Characterization of Its Effect on Human Eosinophil Oxidative Metabolism and Morphology in Comparison with IL-5 and GM-CSF

Eosinophils were shown to play a major role in the allergic inflammatory process leading to the clinical symptoms of atopic dermatitis. Only selected cytokines are capable of inducing a chemotactic response in eosinophils. In particular, the chemokine RANTES was recently shown to be a potent eosinophil chemotaxin. To examine the role of RANTES in eosinophil activation, we investigated the effect of RANTES and other chemokines on morphology and oxidative metabolism of highly purified eosinophils of normal nonatopic blood donors by assessment of functional as well as morphologic criteria. RANTES, and, to a lesser extent, MIP-1α significantly induced the production of reactive oxygen species by human eosinophils, whereas MCP-1, MIP-1β, and interleukin (IL)-8/NAP-1 had no significant effects. RANTES stimulated only a subpopulation of the normal eosinophils. With the exception of IL-8, none of the cytokines tested had any significant effect on polymorphonuclear neutrophilic granulocytes.. By scanning electron microscopy, RANTES induced characteristic changes that were completely abrogated in the presence of cytochalasin B. Based on functional and ultrastructural assays significant extracellular but not intracellular H2O2 production was detected and completely inhibited by cytochalasin B. Separation of eosinophils by discontinous density gradients revealed the existence of two hypodense eosinophil populations, one which showed significantly reduced responses upon stimulation with RANTES. RANTES-induced production of reactive oxygen species was almost completely inhibited by staurosporine, wortmannin, or pertussis toxin. Based on these data it is evident that RANTES represents a potent eosinophil-specific activator of oxidative metabolism. Besides its chemotactic activity on T cells and eosinophils, therefore, RANTES may be involved in the functional activation of eosinophils in the skin of patients with atopic dermatitis