Usefulness of grading of neutrophil aggregate size by laser-light scattering technique for characterizing stimulatory and inhibitory effects of agents on aggregation.
We attempted to apply the particle counting method that employs laser-light scattering technique to quantify the change in numbers of neutrophil homotypic aggregates of 3 graded-sizes (small, medium and large). Ex vivo activation of human neutrophils by a chemotactic peptide, fMLP, predominantly produced small-sized aggregates (< 15 cells), and also, transiently, medium-sized aggregates (16-130 cells). Co-treatment of neutrophils with fMLP and cytochalasin B mainly produced medium-sized aggregates, with very few large-sized aggregates (> 130 cells). Interestingly, when protein kinase C was activated with phorbol 12-myristate 13-acetate small-, medium- and even large-sized aggregates of neutrophils were formed. Presence of extracellular calcium was required to produce these neutrophil aggregations. Both prostaglandin E2 (PGE2) and wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI-3K), inhibited neutrophil aggregation, whereas dbcAMP, a cell permeable analog of cyclic AMP, did not, confirming that PGE2 causes neutrophil aggregation probably through PI-3K inhibition rather than activation of adenylate cyclase. These results suggest that the application of the light scattering technique to characterize human neutrophil aggregates by both size and numbers, has advantages over conventional optical turbulent aggregometry, in that it discriminates neutrophil aggregations produced by different mechanisms of intracellular signaling.
Niwa
M
M
Department of Pharmacology, Gifu University School of Medicine, Japan. mniwa@cc.gifu-u.ac.jp
Kanamori
Y
Y
Kohno
K
K
Matsuno
H
H
Kozawa
O
O
Kanamura
M
M
Uematsu
T
T
D000730:Androstadienes / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002449:Cell Aggregation / Q000187:drug effects; D002452:Cell Count / Q000379:methods; D003571:Cytochalasin B / Q000494:pharmacology; D015232:Dinoprostone / Q000494:pharmacology; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007834:Lasers; D008027:Light; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects / Q000502:physiology; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects*; D010316:Particle Size; D010703:Phorbol Esters / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000378:metabolism; D012542:Scattering, Radiation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D010703:Phorbol Esters; D000081082:Phosphoinositide-3 Kinase Inhibitors; C008876:phorbol-12,13-didecanoate; D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003994:Bucladesine; D011493:Protein Kinase C; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin