1. Cytosolic pH and the inflammatory microenvironment modulate cell death in human neutrophils after phagocytosis
by Raymond J. Coakley, Clifford Taggart, Noel G. McElvaney, and Shane J. O'Neill
Blood
Volume 100(9):
November 1, 2002
©2002 by American Society of Hematology

2. pHi in phagocytosing neutrophils is determined by bacterial load
pHi in phagocytosing neutrophils is determined by bacterial load.Isolated human peripheral blood neutrophils were exposed to increasing densities of heat-killed opsonized E coli while intracellular pH (pHi) was monitored by flow cytometry using cytosolic pH...
pHi in phagocytosing neutrophils is determined by bacterial load.Isolated human peripheral blood neutrophils were exposed to increasing densities of heat-killed opsonized E coli while intracellular pH (pHi) was monitored by flow cytometry using cytosolic pH-sensitive dyes and, in separate experiments, phagocytosis of fluorescently labeled bacteria was measured by flow cytometry. (A) The correlation between increasing pathogen-to-phagocyte ratio and mean number of bacteria ingested (mean channel fluorescence) by neutrophils (4 replicates each from 5 healthy donors, coefficient of determination r 2 = 0.96). (B) The effect of increasing pathogen-to-phagocyte ratios and thus mean number of bacteria ingested (0:1-50:1) on pHi over time in normal neutrophils (4 replicates each from 10 healthy donors). pHiresponses at each pathogen-to-phagocyte ratio differed significantly from the others (by MANOVA, P < .01). (C,D) The correlation between bacterial load and the change in pHi after 5 minutes and the lowest pH recorded. All data mean ± SEM.
Raymond J. Coakley et al. Blood 2002;100:
©2002 by American Society of Hematology

3. Mechanisms of pHiregulation in phagocytosing neutrophils
Mechanisms of pHiregulation in phagocytosing neutrophils.The individual and collective contributions of several putative mechanisms of proton extrusion were investigated in isolated human peripheral blood neutrophils.
Mechanisms of pHiregulation in phagocytosing neutrophils.The individual and collective contributions of several putative mechanisms of proton extrusion were investigated in isolated human peripheral blood neutrophils. Neutrophils (4 replicates each from 10 healthy donors) were exposed to opsonized heat-killed E coli at a pathogen-to-phagocyte ratio of 20:1 in the presence or absence of inhibitors. (A) The significant effect on pHifollowing phagocytosis of inhibition of passive proton conductance channels (ZnCl2, 50 μM, P < .01 versusE coli alone by 2-way ANOVA). (B) The significant effect on pHi following phagocytosis of inhibition of Na+/H+ exchange (amiloride, 0.2 mM,P < .001 by 2-way ANOVA). (C) The small but significant effect on pHi following phagocytosis of inhibition of V-ATPases (bafilomycin, 100 nM, P < .05 by 2-way ANOVA). (D) The significant effect on pHi following phagocytosis of inhibition of passive proton conductance channels, Na+/H+ exchange, and V-ATPases (P < .0001 by 2-way ANOVA). (E) At a lower pathogen-to-phagocyte ratio (3:1), inhibition of V-ATPases with bafilomycin (100 nM) has a more significant effect on pHi following phagocytosis (*P < .001 by 2-way ANOVA). All data mean ± SEM.
Raymond J. Coakley et al. Blood 2002;100:
©2002 by American Society of Hematology

4. The effect of an acidic extracellular milieu on pHi in phagocytosing neutrophils.Isolated human peripheral blood neutrophils (4 replicates from 8 healthy donors) were exposed to opsonized heat-killed E coli(pathogen-to-phagocyte ratio 20:1) in standard HCO ...
The effect of an acidic extracellular milieu on pHi in phagocytosing neutrophils.Isolated human peripheral blood neutrophils (4 replicates from 8 healthy donors) were exposed to opsonized heat-killed E coli(pathogen-to-phagocyte ratio 20:1) in standard HCO 3 − -buffered RPMI medium (pH 7.45) and a bicarbonate-free HEPES-buffered medium (pH 5.8). pHi was monitored during bacterial ingestion. Cytosolic acidification was significantly greater following phagocytosis in the acidic extracellular medium (P < .001 by 2-way ANOVA). All data mean ± SEM.
Raymond J. Coakley et al. Blood 2002;100:
©2002 by American Society of Hematology

5. The effect of the inflammatory milieu from the lungs of CF patients on baseline pHi and pHi in phagocytosing neutrophils.Isolated human peripheral blood neutrophils were exposed to CF ELF (concentrated from BAL fluid from stable CF patients or healthy contr...
The effect of the inflammatory milieu from the lungs of CF patients on baseline pHi and pHi in phagocytosing neutrophils.Isolated human peripheral blood neutrophils were exposed to CF ELF (concentrated from BAL fluid from stable CF patients or healthy controls), and pHi was measured. (A) The acute alkalinizing effect on pHi of the addition of CF ELF (40 μL/mL) to neutrophils in culture. (B) The effect of preincubation with CF ELF (30 minutes, 30 μL/mL) on pHi after exposure to opsonized heat-killed E coli (EC; pathogen-to-phagocyte ratio 20:1) (*P < .02, CF versus normal ELF by 2-way ANOVA). All data mean ± SEM.
Raymond J. Coakley et al. Blood 2002;100:
©2002 by American Society of Hematology

6. Correlation of phagocytosis-induced neutrophil apoptosis with changes in pHi and intracellular oxidant generation.Isolated peripheral blood neutrophils were exposed to varying pathogen-to-phagocyte ratios, and early changes in pHi, intracellular oxidant pro...
Correlation of phagocytosis-induced neutrophil apoptosis with changes in pHi and intracellular oxidant generation.Isolated peripheral blood neutrophils were exposed to varying pathogen-to-phagocyte ratios, and early changes in pHi, intracellular oxidant production, and apoptosis were measured after 16 hours. (A) Neutrophil apoptosis is inhibited at lower pathogen-to-phagocyte ratios (*P < .01 by 2-way ANOVA), but is activated at higher ratios (**P < .005 by 2-way ANOVA). (B) Linear correlation between ΔpHi over 90 minutes and Δ% apoptosis at varying pathogen-to-phagocyte ratios. Net ΔpHi is based on a calculation of the phagocytosis-induced change in net pHi curve area above (net alkalinization) and below (net acidification) baseline pHi (baseline pHi curve is that of non–bacteria-exposed neutrophils) over time. (C) Linear correlation between intracellular oxidant generation over 90 minutes and Δ% apoptosis at varying pathogen-to-phagocyte ratios. All data mean ± SEM; r2 indicates coefficient of determination.
Raymond J. Coakley et al. Blood 2002;100:
©2002 by American Society of Hematology

7. Perturbations of pHi and the inflammatory milieu alter mechanisms of cell death in phagocytosing neutrophils.(A) The effects of amiloride (0.2 mM), ZnCl2 (50 μM), bafilomycin (100 nM), and CF ELF (40 μL/mL) on rates of apoptosis at 16 hours in phagocytosing...
Perturbations of pHi and the inflammatory milieu alter mechanisms of cell death in phagocytosing neutrophils.(A) The effects of amiloride (0.2 mM), ZnCl2 (50 μM), bafilomycin (100 nM), and CF ELF (40 μL/mL) on rates of apoptosis at 16 hours in phagocytosing neutrophils (4 replicates from 5 healthy donors) exposed to E coli at a pathogen-to-phagocyte ratio of 20:1. Inhibitors or ELF was added 10 minutes after bacteria. *P < .05; % apoptosis greater than E colialone; **P < .05; % apoptosis less than E coli. Note that bafilomycin did affect apoptosis in neutrophils exposed to lower pathogen-to-phagocyte ratios (see text). (B) The effects of amiloride, ZnCl2, CF ELF, a combination of amiloride plus ZnCl2 plus bafilomycin, and lowering of the extracellular pH on rates of necrosis in the same cells (*P < .005 versus E coli alone).
Raymond J. Coakley et al. Blood 2002;100:
©2002 by American Society of Hematology