Mechanisms of the in vitro fungicidal effects of human neutrophils against Penicillium marneffei induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Abstract

We examined the in vitro fungicidal activity of human neutrophils against conidia and yeast cells of Penicillium marneffei. Neutrophils showed a small but significant anti-fungal effect against the yeast form of P. marneffei. Treatment of neutrophils with GM-CSF significantly augmented their anti-fungal activity. In contrast, the conidia form resisted killing even by stimulated neutrophils. Neutrophil fungicidal effect was not inhibited by superoxide dismutase (SOD), while the same treatment significantly suppressed the killing of Candida albicans by GM-CSF-stimulated neutrophils. For effective killing of P. marneffei yeast cells by GM-CSF-stimulated neutrophils, direct contact between the two was essential; interference in such interaction by separation using a 0. 45-microm-pored membrane prevented such an effect. Addition of colchicine attenuated GM-CSF-stimulated neutrophil fungicidal activity in a dose-dependent manner. This effect did not appear to be mediated by interference with neutrophil mobility toward yeast cells, because similar results were obtained when the cultures were set in round-bottomed wells which facilitate their direct contact. Finally, granular extracts derived from unstimulated neutrophils significantly suppressed the growth of microorganisms. Pretreatment of neutrophils with GM-CSF markedly enhanced this effect. The fungicidal activity of granular lysates was strongly, but not completely, reduced by heat treatment. Considered together, our results indicate that GM-CSF-stimulated neutrophils killed the yeast form of P. marneffei present in close proximity, probably in a superoxide anion-independent mechanism, but through exocytosis of granular enzymes which were largely heat-labile.

Fig. 1

Contact-dependent killing of Penicillium marneffei by neutrophils. The culture was set in 24-well double chambers separated by a membrane of 0·45-μ m pores. Penicillium marneffei yeast cells (1 × 10³) were cultured in the presence of 10 ng/ml of GM-CSF in the lower chamber with or without neutrophils (1 × 10⁵) in the lower or upper chamber in a volume of 1·0 ml for 24 h, and then the number of live microorganisms was counted. Each bar represents the mean ± s.d. of triplicate cultures. PMN, Polymorphonuclear neutrophils.

Fig. 2

Effect of colchicine on the killing of Penicillium marneffei by GM-CSF-stimulated neutrophils. Penicillium marneffei yeast cells (1 × 10³/well) were cultured in 96-well flat culture plates with or without neutrophils (1 × 10⁵/well), pretreated with or without the indicated concentrations of colchicine for 30 min, in the presence or absence of 10 ng/ml of GM-CSF. After 24 h the number of live microorganisms was counted. Each bar indicates the mean ± s.d. of triplicate cultures. 1, Medium alone; 2, neutrophils; 3, neutrophils + GM-CSF; 4, neutrophils + GM-CSF + colchicine (0·1 mm); 5, neutrophils + GM-CSF + colchicine (0·25 mm); 6, neutrophils + GM-CSF + colchicine (0·5 mm). *P < 0·05.

Fig. 3

Effect of colchicine on neutrophil fungicidal activity. In 96-well flat- (a) or round- (b) bottomed culture plates, Penicillium marneffei yeasts (1 × 10³/ml) were cultured with or without neutrophils (1 × 10⁵/ml) and GM-CSF (10 ng/ml) in the presence or absence of 0·5 mm colchicine. After 24 h, the number of live microorganisms was counted. Each bar indicates the mean ± s.d. of triplicate cultures. 1, Medium alone; 2, neutrophils; 3, neutrophils + colchicine; 4, neutrophils + GM-CSF; 5, neutrophils + GM-CSF + colchicine. *P < 0·05.