Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry

Abstract

The novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type-and stimulus-specific differences in signal transduction pathways: 1) n-formyl-methionyl-leucylphenylalanine (FMLP)/cytochalasin B-induced oxidative burst is several-fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; 2) stimulus-induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; 3) the FMLPkytochalasin Binduced calcium flux in monocytes is less sensitive to the G-protein inhibitor pertussis toxin than the flux in granulocytes; 4) in contrast to FMLPkytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; 5) the PMAinduced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLPkytochalasin B can not; 6) the G-protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and 7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLPkytochalasin B-induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10-20 nM is necessary to block PMA-induced oxidative burst. o 1992 Wiley-Liss, Inc.

Impact of Prematurity, Stress and Sepsis on the Neutrophil Respiratory Burst Activity of Neonates

“…On the contrary, we found a decreased NRBA in neonates with proven sepsis. The difference between the findings of Gessler et al [ 10] and ours could be attributed to the use of different stimulators because of the different pathways of neutrophil respira tory burst activation [28], A stimulus-specific abnormality in the respiratory burst activity of stressed neonates was also suggested by Shi geoka et al [2. 12].…”

“…As a sti mulator we used PMA. a protein kinase C activator, in order to divert various transduc tion pathways of neutrophil respiratory burst activation [28]. Also, PMA has been proved to be the most potent stimulus of oxidative burst [27],…”

Interleukin-1β induction of c-fos and collagenase expression in articular chondrocytes: Involvement of reactive oxygen species

“…Both DHR and DPI were dissolved in dimethyl sulfoxide. Cells were then fixed for 20 min in 1.5% paraformaldehyde (Sigma) and the cellular R123 fluorescence intensity of 12,000 chondrocytes was measured by flow cytometry for each sample using an argon laser with the excitation source at 488 nm (Facscalibur 4 color analyzer with CellQuest Software, [Lund-Johansen and Olweus, 1992].…”

Impaired Glutathione Peroxidase Activity Accounts for the Age-Related Accumulation of Hydrogen Peroxide in Activated Human Neutrophils

“…The preparation of neutrophils and the measurement of intracellular H2O2 accumulation were carried out using dihydrorhodamine 123 (Molecular Probes Inc., Eugene, OR) as previously described (5). Neutrophils were preincubated for 10 minutes at 37°C and then treated with 10-5 -10-7 M FMLP.…”