Pertussis toxin effects on chemoattractant-induced response heterogeneity in human PMNs utilizing Fluo-3 and flow cytometry

Flow cytometric methods were utilized to determine N-formylpeptide-induced cytosolic calcium levels in human polymorphonuclear leukocytes (PMNs) detected with the calcium indicator Fluo-3. Fluo-3 was readily loaded into PMNs as the acetoxymethyl ester. At room temperature Fluo-3 extrusion was minimal (less than 10%) over a 2 h time period. Flow cytometric histograms yielded symmetric distributions indicating homogeneous labelling of the cells. Stimulation of the cells with N-formyl-met-leu-phe (FMLP) caused homogeneous activation of all cells as indicated by a shift of the fluorescence distribution to higher fluorescence levels while still maintaining a symmetrical distribution. Resting values or FMLP-induced cytosolic calcium levels were similar in cells loaded over a 20-fold range of Fluo-3-acetoxymethyl ester. The effect of graded pertussis toxin (PT) treatment on the calcium response was determined by incubating cells with different concentrations of pertussis toxin for a time period that yielded a range of ADP ribosolation levels inside the cells. When these cells were activated with FMLP, the fluorescence histograms showed that pertussis toxin treatment resulted in a conversion of cells from responders to nonresponders. The responding cells responded with maximum calcium elevations similar to controls. This behavior may reflect heterogeneous insertion of the A-protomer of PT or a very sharp threshold of coupled G-proteins required to transduce the responses.