Roles of phospholipase D in phorbol myristate acetate-stimulated neutrophil respiratory burst

Abstract

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10-fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA-stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA-stimulated respiratory burst. Using 1-butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA-stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA-stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA-stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA-stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2-dioctanoyl-sn-glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA-stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA-stimulated respiratory burst.

Fig 1

The inhibition of fMLP- and PMA-stimulated respiratory burst by U73122. Neutrophils (1 × 10⁷/ml) were incubated with various concentrations of U73122 for 10 min. and then stimulated with fMLP (100 nM) or PMA (100 nM). The respiratory burst was measured as chemiluminescence burst. Data are means ± S.D. from three independent measurements.

Fig 2

The activities of PLD in PMA- or fMLP-stimulated human neutrophils treated without or with U73122. Neutrophils (1 × 10⁷/ml) were incubated with various indicated concentrations of U73122 at 37 °C for 10 min. and then stimulated with fMLP (100 nM) or PMA (100 nM). 10 min. after stimulation the cells were lysed for the assay of PLD activity. Data are means ± S.D. of three independent assays.

Fig 3

The inhibition of PMA stimulated respiratory burst by 1-butanol. Neutrophils (1 × 10⁷/ml) were incubated with various indicated concentrations of 1-butanol at 37 °C for 10 min. and then stimulated with PMA (100 nM). The respiratory burst was measured as chemiluminescence burst. Data are means ± S.D. from three independent measurements.

Fig 4

The effect of GF109203X on the respiratory burst and PLD activity in PMA-stimulated neutrophils. Neutrophils (1 × 10⁷/ml) were incubated with various indicated concentrations of GF109203X at 37°C for 10 min. and then stimulated with PMA (100 nM). The respiratory burst was measured as chemiluminescence burst (A). The PLD activity was assayed 10 min. after stimulation (B). Data are means ± S.D. of three independent experiments.

Fig 5

The effect of DOG on the respiratory burst in PMA-stimulated neutrophils treated with or without U73122. Neutrophils (1 × 10⁷/ml) were incubated with or without U73122 (1 μM) at 37°C for 10 min. DOG (100 nM) was added with or without PMA (100 nM) to the cells. The respiratory burst was measured as chemiluminescence burst. Data are means ± S.D. of three independent experiments.