Recombinant human granulocyte colony-stimulating factor as an activator of human granulocytes: potentiation of responses triggered by receptor- mediated agonists and stimulation of C3bi receptor expression and adherence

Abstract

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced superoxide release and membrane depolarization in parallel in human granulocytes stimulated by the receptor-mediated agonists, N- formyl-methionyl-leucyl-phenylalanine and wheat germ agglutinin, but not by the Ca2+ ionophore ionomycin and phorbol myristate acetate, which bypass the receptors to stimulate the cells. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL (1.3 to 2.6 nmol/L) rhG-CSF for 10 minutes at 37 degrees C. rhG-CSF produced by bacteria and mammalian cells had identical biological effects on a molar basis. rhG-CSF neither affected stimulus-induced increase in cytoplasmic free Ca2+ nor changed the number and affinity of N-formyl- methionyl-leucyl-phenylalanine receptors. The priming effect of rhG-CSF was temperature dependent and did not require new protein synthesis. rhG-CSF increased the expression of C3bi receptors on human granulocytes and enhanced granulocyte adherence to nylon fiber. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL rhG-CSF for 30 minutes at 37 degrees C. rhG-CSF had no effect on human monocytes. These findings demonstrate that rhG-CSF can selectively stimulate mature granulocyte functions.

Ex vivo expansion of mature human neutrophils with normal functions from purified peripheral blood CD34+ haematopoietic progenitor cells

“…Preparation of neutrophils. Peripheral blood neutrophils from healthy adult donors were prepared as described previously (Yuo et al, 1989b), using dextran sedimentation, centrifugation with Conray±Ficoll and hypotonic lysis of contaminated erythrocytes. Neutrophil fractions were suspended in Hanks' balanced salt solution (HBSS) and contained more than 98% neutrophils.…”

“…Determination of neutrophil functions. The superoxide (O 2 2 )-releasing capacity was determined by SOD-inhibitable reduction of ferricytochrome c as described previously (Yuo et al, 1989b). For the end-point assay, the cell suspension in HBSS was added to each well of the 48-well plate (Falcon no.…”

“…After incubation for 1 h at 378C, the reduction of ferricytochrome c was measured at 550 nm with a reference wavelength at 540 nm. In some experiments, the continuous assay was performed in a Hitachi 557 spectrophotometer (a double wavelength spectrophotometer; Hitachi, Tokyo, Japan) equipped with a thermostatted cuvette folder (378C) as described previously (Yuo et al, 1989b). The final concentration of cytochrome c was 100 mm and the final cell concentration was 1 Â 10 6 cells/ml.…”

Ex vivo neutrophil function in response to three different doses of glycosylated rHuG‐CSF (Lenograstim)

“…Previous in vitro and ex vivo studies have shown that G-CSF primes human PMN and enhances respiratory burst superoxide (O ÿ 2 ) release stimulated by the chemotactic peptide N-formylmethionylleucylphenylalanine (FMLP) Yuo et al, 1989;Sullivan et al, 1993).…”

“…Surface expression of the 2 integrin (CD11b/CD18) has been reported to be either unchanged (Spiekermann et al, 1994) or up-regulated both in vitro and ex vivo Yuo et al, 1989;Yong & Linch, 1992).…”