Protein Kinase C Regulates IL-8- and fMLP-Induced Cytoplasmic Ca2+ Increase in Human Granulocytes by Receptor Modulation Measurements by Flow Cytometry

Abstract

Changes in cytosolic free Ca2+ influence important granulocyte functions like chemotactic behavior, adherence to endothelia, and phagocytosis. In the following study we used a simple reproducible procedure involving flow cytometry in combination with the fluorescent dye Fluo-3 to measure Ca2+ changes in human granulocytes. The aim of our study was to investigate the involvement of protein kinase C in regulating cytosolic free Ca2+ concentrations after stimulation of cells with IL-8 and fMLP. Both reagents induced a 5-6 fold increase in cytosolic Ca2+. Experiments conducted in Ca2+-free media showed a minor 18-29% decrease in cytosolic Ca2+ response, suggesting that intracellular Ca2+-stores are the main source for Ca2+ release after fMLP or IL-8 stimulation. Activators of protein kinase C, phorbol myristate acetate (PMA) and 1-oleyl-2-acetyl-sn-glycerol (OAG), inhibited cytosolic Ca2+-increase completely when induced by IL-8 and by 68-82% in the case of fMLP. Staurosporine, an inhibitor of protein kinase C, was able to attenuate or even abolish the PMA/OAG-effect. Our results show that changes in cytosolic Ca2+ due to IL-8 and fMLP signalling can be regulated by protein kinase C in human granulocytes. This regulatory role of protein kinase C involves some form of receptor modulation (i.e. phosphorylation, internalization, shedding).