Correlation between spontaneous oscillations of cytosolic free Ca2+ and tumor necrosis factor-induced degranulation...

By using a hemolytic plaque assay to detect release of lactoferrin and myeloperoxidase, tumor necrosis factor (TNF) was shown previously to induce secretion of these granule proteins from single adherent neutrophils. Secretion was inhibited by loading neutrophils with calcium chelators, indicating a crucial role of cytosolic free [Ca2+] in the signal transduction mechanism of TNF. In the present study, using a microfluorometer technique to follow changes in the cytosolic free [Ca2+] in single adherent neutrophils, we were not able to detect any TNF-induced [Ca2+] transients. However, these adherent cells exhibited spontaneous oscillations of their cytosolic free [Ca2+], as previously reported (Jaconi, M.E.E., Rivest, R.W., Schlegel, W., Wollheim, C.B., Pittet, D., and Lew, P.D. (1988) J. Biol. Chem. 263, 10557-10560). A close correlation was found between a reduced oscillatory activity of cytosolic free [Ca2+] and a reduced ability of TNF to induce degranulation, by reducing the extracellular [Ca2+] or loading the cells with a calcium chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). In addition, when the cells were incubated at 37 degrees C for 3 h there was a parallel decline in the spontaneous oscillatory activity of cytosolic free [Ca2+] and TNF-induced secretion of lactoferrin. Control experiments showed that phorbol 12-myristate 13-acetate-induced secretion was not affected under the same conditions, indicating that the secretory process per se was not disturbed. We conclude that TNF by itself does not give rise to any changes of the cytosolic free [Ca2+] but that the spontaneous oscillatory activity of cytosolic free [Ca2+] in adherent neutrophils is necessary for TNF-induced degranulation.