Molecular mechanisms of N-formyl-methionyl-leucyl-phenylalanine-induced superoxide generation and degranulation in mouse neutrophils: phospholipase D is dispensable

Abstract

Phospholipase D (PLD), which produces the lipid messenger phosphatidic acid (PA), has been implicated in superoxide generation and degranulation in neutrophils. The basis for this conclusion is the observation that primary alcohols, which interfere with PLD-catalyzed PA production, inhibit these neutrophil functions. However, off-target effects of primary alcohols cannot be totally excluded. Here, we generated PLD(-/-) mice in order to reevaluate the involvement of PLD in and investigate the molecular mechanisms of these neutrophil functions. Surprisingly, N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced these functions in PLD(-/-) neutrophils, and these functions were suppressed by ethanol. These results indicate that PLD is dispensable for these neutrophil functions and that ethanol nonspecifically inhibits them, warning against the use of primary alcohols as specific inhibitors of PLD-mediated PA formation. The calcium ionophore ionomycin and the membrane-permeative compound 1-oleoyl-2-acetyl-sn-glycerol (OADG) synergistically induced superoxide generation. On the other hand, ionomycin alone induced degranulation, which was further augmented by OADG. These results demonstrate that conventional protein kinase C (cPKC) is crucial for superoxide generation, and a Ca(2+)-dependent signaling pathway(s) and cPKC are involved in degranulation in mouse neutrophils.

Fig 1

Generation of PLD2-null mice. (A) Schematic representation of PLD2 targeting showing the partial genomic structure of the PLD2 locus (wild-type allele), a linearized PLD2 targeting vector (targeting construct), and a targeted PLD2 gene allele (targeted). A neomycin resistance cassette (neo), loxP sites (triangles), and exons (filled rectangles with the exon number) are shown. Primers used for genomic PCR (P1 to P3) and a probe fragment for Southern blotting are represented by arrows and black boxes, respectively. The relevant restriction fragments for Southern blotting are shown by horizontal lines with fragment sizes. “S” and “P” represent SmaI and PstI sites, respectively. Deletion of the PLD2 gene was verified by Southern blotting (B), genomic PCR (C), and Western blotting (D).

Fig 2

Expression of PLD isozymes in mouse neutrophils and their activation in response to fMLP. Expression of PLD1 and PLD2 in the mouse brains and neutrophils was analyzed by Western blotting (A) and RT-PCR (B). Actin (A) and GAPDH (B) were also analyzed as internal controls. (C) Wild-type, PLD1^−/−, PLD2^−/−, and PLD1/2^−/− neutrophils labeled with [³H]lyso-PAF (5 μCi/ml) were primed with CB and incubated with the indicated concentrations of fMLP in the presence of 1% EtOH at 37°C for 10 min, and then PLD activity was determined as described in Materials and Methods. Data are means ± standard errors of the means (SEM) from three independent experiments.

Fig 3

Deletion of PLD genes does not affect fMLP-induced superoxide generation and degranulation. Wild-type, PLD1^−/−, PLD2^−/−, and PLD1/2^−/− neutrophils primed with CB were incubated with the indicated concentrations of fMLP at 37°C for 10 min. Superoxide generation (A) and degranulation (B) were then determined as described in Materials and Methods. Data are means ± SEM from three independent experiments.

Fig 4

Pharmacological inhibition of PLDs does not affect fMLP-induced superoxide generation and degranulation. [³H]lyso-PAF-labeled wild-type neutrophils pretreated with the indicated concentrations of FIPI in the presence of CB and EtOH were stimulated with 10 μM fMLP at 37°C for 10 min, and then PLD activity was determined (A). Wild-type neutrophils were pretreated with 750 nM FIPI in the presence of CB and stimulated with 10 μM fMLP, and then superoxide generation (B) and degranulation (C) were determined. Data are means ± SEM from at least three independent experiments.

Fig 5

Deletion of PLD genes does not affect superoxide generation induced by fMLP in BM neutrophils and by IgG-SRBC in peritoneal neutrophils. Wild-type and PLD1/2^−/− BM neutrophils primed with CB were incubated with the indicated concentrations of fMLP at 37°C for 10 min (A). Wild-type and PLD1/2^−/− peritoneal neutrophils primed with CB were incubated with the IgG-SRBC at 37°C for the indicated times (B). Superoxide generation was then determined as described in Materials and Methods. Data are as means ± SEM from three independent experiments. *, P < 0.05 (Student's t test).

Fig 6

EtOH inhibits fMLP-induced cell functions in both wild-type and PLD^−/− neutrophils. Wild-type, PLD1^−/−, PLD2^−/−, and PLD1/2^−/− neutrophils primed with CB were stimulated with the indicated concentrations of fMLP at 37°C for 10 min in the presence or absence of 1% EtOH. Superoxide generation (A to C) and degranulation (D to F) were then measured as for Fig. 3. Data are means ± SEM from three independent experiments.

Fig 7

fMLP-induced PA formation is independent of the PLD-mediated signaling pathway. Wild-type and PLD1/2^−/− neutrophils prelabeled with ³²P_i were primed with CB and stimulated with 10 μM fMLP in the presence or absence of 1% EtOH at 37°C for the indicated times, and then production of [³²P]PA in the absence of EtOH and [³²P]PEt in the presence of EtOH were measured (A and B) as described in Materials and Methods. [³²P]PEt formation (A) is shown in panel B on an enlarged scale. Wild-type neutrophils pretreated with 750 nM FIPI at 37°C for 10 min were primed with CB and stimulated with or without 10 μM fMLP at 37°C for 2 min, and then the production of [³²P]PA was measured (C). Wild-type neutrophils pretreated with the indicated concentrations of R59022 were primed with CB and stimulated with or without 10 μM fMLP at 37°C for 2 min, and then the production of [³²P]PA was measured (D). Wild-type and PLD1/2^−/− neutrophils primed with CB were incubated with or without 10 μM fMLP in the presence or absence of 1% EtOH at 37°C for 2 min, and then the production of [³²P]PA was measured (E). Means ± SEM from at least three independent experiments are shown in all panels. *, P < 0.05 (Student's t test).

Fig 8

The DG kinase inhibitor R59022 enhances fMLP-induced superoxide generation and degranulation. Wild-type neutrophils pretreated with R59022 in the presence of CB were stimulated with or without 10 μM fMLP at 37°C for 10 min, and superoxide generation (A) and degranulation (B) were measured. Data are means ± SEM from three independent experiments. *, P < 0.05 (Student's t test).

Fig 9

Pharmacological inhibition of PKC interferes with fMLP-induced superoxide generation and degranulation. Wild-type neutrophils were preincubated with the indicated concentrations of calphostin C at 37°C for 15 min. After calphostin C was photoactivated by fluorescent light at room temperature for 10 min, neutrophils were primed with CB and stimulated with 10 μM fMLP for 10 min, and then superoxide generation (A) and degranulation (B) were measured. Data are means ± SEM from three independent experiments. *, P < 0.05 (Student's t test).

Fig 10

Induction of superoxide generation by ionomycin and OADG. Wild-type neutrophils primed with CB were incubated with the indicated concentrations of OADG in the presence or absence of 1 μM ionomycin at 37°C for 7 min, and superoxide generation was measured (A). Wild-type (B) or PLD1/2^−/− (C) neutrophils primed with CB were incubated with or without 12.5 μM OADG and/or 1 μM ionomycin in the presence or absence of 30 μM R59022 at 37°C for 7 min. The cells were also stimulated with 10 μM fMLP at 37°C for 10 min. After treatment, superoxide generation was measured (B and C). Data are means ± SEM from three independent experiments. *, P < 0.05 (Student's t test).

Fig 11

Induction of degranulation by ionomycin and OADG. Wild-type neutrophils primed with CB were incubated with ionomycin and/or OADG as in Fig. 10A, and degranulation was measured (A). Wild-type (B) and PLD1/2^−/− (C) neutrophils primed with CB were incubated with ionomycin and/or OADG in the presence or absence of R59022 as for Fig. 10B and C. The cells were also stimulated with fMLP as for Fig. 10B and C. After treatment, degranulation was measured (B and C). Data are means ± SEM from three independent experiments. *, P < 0.05 (Student's t test).

Fig 12

Phosphorylation of p47^Phox by OADG and ionomycin and its inhibition by EtOH. Wild-type neutrophils were pretreated with or without R59022 and incubated at 37°C with or without OADG and/or ionomycin in the presence or absence of EtOH for 7 min. Expression levels of phospho-p47^Phox (Ser-370) and p47^Phox were then determined by Western blot analysis as described in Materials and Methods. Data are means ± SEM from three independent experiments. *, P < 0.05 (Student's t test).