Neutrophil function in whole blood and after purification: changes in receptor expression, oxidase activity and responsiveness to cytokines.
Neutrophil function and plasma membrane receptor expression was measured in cell suspensions isolated by two separate procedures and in unfractionated whole blood. When cells were prepared by a combined dextran/ficoll procedure, their ability to generate reactive oxidants in response to fMet-Leu-Phe was greater than in corresponding cells isolated by a one-step procedure on Mono-Poly Resolving Medium (M-PRM). Cells prepared by both methods could be primed in vitro by rGM-CSF, but the priming ratio was greater in cells prepared by the latter method. The ability of neutrophils in whole blood to generate reactive oxidants in response to fMet-Leu-Phe was extremely low, but this was increased by more than 10 fold if the blood was pre-incubated with rGM-CSF. Similarly, expression of CD 11b and CD 16 was very low (or undetectable) in neutrophils in whole blood, but this was rapidly increased upon priming. Activation by PMA resulted in a down regulation of CD 16 expression as the receptor was shed from the cell surface. Neutrophils isolated by either the dextran/ficoll or the M-PRM method showed increased expression of receptors compared with those in whole blood, although this expression was lower in cells isolated by the latter method. These data indicate that the isolation procedures used to obtain purified neutrophils prime both receptor expression and oxidase function, although these effects are minimalised in isolation procedures using M-PRM. Furthermore, as CD 16 expression on neutrophils in whole blood is rapidly up-regulated during priming, it seems likely that, as for complement receptors, rapidly-mobilisable intracellular stores of this receptor exist.
Watson
F
F
Dept. of Biochemistry, University of Liverpool.
Robinson
J J
JJ
Edwards
S W
SW
D000911:Antibodies, Monoclonal; D002469:Cell Separation / Q000379:methods; D015536:Down-Regulation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D016177:Macrophage-1 Antigen / Q000032:analysis; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D017463:Receptors, Complement 3b / Q000032:analysis; D017452:Receptors, IgG / Q000032:analysis*
D000911:Antibodies, Monoclonal; D016177:Macrophage-1 Antigen; D017463:Receptors, Complement 3b; D017452:Receptors, IgG; D009240:N-Formylmethionine Leucyl-Phenylalanine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D009247:NADH, NADPH Oxidoreductases