Frontline Science: Tumor necrosis factor-α stimulation and priming of human neutrophil granule exocytosis - PubMed (original) (raw)
Frontline Science: Tumor necrosis factor-α stimulation and priming of human neutrophil granule exocytosis
Kenneth R McLeish et al. J Leukoc Biol. 2017 Jul.
Abstract
Neutrophil granule exocytosis plays an important role in innate and adaptive immune responses. The present study examined TNF-α stimulation or priming of exocytosis of the 4 neutrophil granule subsets. TNF-α stimulated exocytosis of secretory vesicles and gelatinase granules and primed specific and azurophilic granule exocytosis to fMLF stimulation. Both stimulation and priming of exocytosis by TNF-α were dependent on p38 MAPK activity. Bioinformatic analysis of 1115 neutrophil proteins identified by mass spectrometry as being phosphorylated by TNF-α exposure found that actin cytoskeleton regulation was a major biologic function. A role for p38 MAPK regulation of the actin cytoskeleton was confirmed experimentally. Thirteen phosphoproteins regulated secretory vesicle quantity, formation, or release, 4 of which-Raf1, myristoylated alanine-rich protein kinase C (PKC) substrate (MARCKS), Abelson murine leukemia interactor 1 (ABI1), and myosin VI-were targets of the p38 MAPK pathway. Pharmacologic inhibition of Raf1 reduced stimulated exocytosis of gelatinase granules and priming of specific granule exocytosis. We conclude that differential regulation of exocytosis by TNF-α involves the actin cytoskeleton and is a necessary component for priming of the 2 major neutrophil antimicrobial defense mechanisms: oxygen radical generation and release of toxic granule contents.
Keywords: actin cytoskeleton; mass spectrometry; p38 MAPK; phosphorylation.
© Society for Leukocyte Biology.
Figures
Figure 1.. TNF-α stimulation and priming of granule subset exocytosis.
Neutrophils (4–10 × 106/ml) were incubated with or without TNF-α (2 ng/ml, 10 min), fMLF (300 nM, 5 min), TNF-α and then fMLF (TNF + fMLF), or TNF-α followed by washing before incubation with fMLF (TNF/fMLF). Supernatants were obtained by centrifugation and the release of contents from each granule subset measured. (A) Secretory vesicle exocytosis was determined by albumin release into supernatant, measured by ELISA. Results are expressed as means ±
sem
of albumin released in nanograms/4 × 106 cells from 8 separate experiments, each using neutrophils from a different individual. The P values for comparing stimulated with basal albumin release determined by ANOVA are above each bar. (B) Gelatinase granule exocytosis was determined by MMP9 release, measured by ELISA. Results are expressed as means ±
sem
of MMP9 released in nanograms/4 × 106 cells from 11 separate experiments, each using neutrophils from a different individual. The P values for comparing stimulated with basal MMP9 release determined by ANOVA are above each bar. The P value comparing TNF + fMLF versus TNF/fMLF is shown in parentheses. (C) Specific granule exocytosis was determined by lactoferrin release, measured by ELISA. Results are expressed as means ±
sem
of lactoferrin released in nanograms/4 × 106 cells for 7 separate experiments, each using neutrophils from a different individual. The P values for comparing stimulated with basal lactoferrin release determined by ANOVA are above each bar. The P value comparing TNF + fMLF versus TNF/fMLF is shown in parentheses. (D) Azurophilic granule exocytosis was determined by MPO release, measured by chemiluminescence. Results are expressed as means ±
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of MPO released in femtomole/4 × 106 cells for 4 separate experiments, each using neutrophils from a different individual. The P values for comparing stimulated with basal MPO release determined by ANOVA are above each bar.
Figure 2.. Role of p38 MAPK in TNF-α regulation of exocytosis.
Neutrophils (4–10 × 106/ml) were incubated with or without the p38 MAPK inhibitor, SB203580 (SB; 3 μM), before stimulation with TNF-α or TNF-α and then fMLF (TNF + fMLF). Supernatants were obtained by centrifugation and the release of contents from each granule subset measured. (A) Albumin release from secretory vesicles is expressed as means ±
sem
in nanograms/4 × 106 cells from 8 separate experiments, each using neutrophils from a different individual. The P values for comparing stimulated with basal albumin release determined by ANOVA are above each bar. (B) MMP9 release from gelatinase granules is expressed as means ±
sem
in nanograms/4 × 106 cells from 6 separate experiments, each using neutrophils from a different individual. The P values for comparing stimulated with basal MMP9 release determined by ANOVA are above each bar. The P value comparing MMP9 release in the presence and absence of SB203580 is in parentheses. (C) Lactoferrin release from specific granules is expressed as means ±
sem
in nanograms/4 × 106 cells for 6 separate experiments, each using neutrophils from a different individual. The P values for comparing stimulated with basal lactoferrin release determined by ANOVA are above each bar. (D) MPO release from azurophilic granules without pretreatment with latrunculin A is expressed as means ±
sem
in femtomole/4 × 106 cells for 4 separate experiments, each from a different individual. The P values for comparing stimulated with basal MPO release determined by ANOVA are above each bar.
Figure 3.. Role of Raf1 in TNF-α regulation of gelatinase and specific granule exocytosis.
Neutrophils (4–10 × 106/ml) were incubated with or without the Raf1 inhibitor, 3-(dimethylamino)-N-{3-[(4-hydroxybenzoyl)amino]-4-methylphenyl}benzamide (140 nM), or the ERK inhibitor, PD98059 (50 μM), before stimulation with TNF-α (2 ng/ml, 10 min), fMLF (300 nM, 5 min), or TNF-α and then fMLF (TNF + fMLF). Supernatants were obtained by centrifugation and the release of MMP9 and lactoferrin measured by ELISA. (A) MMP9 release from gelatinase granules is expressed as means ±
sem
in nanograms/4 × 106 cells from 6 separate experiments, each using neutrophils from a different subject. The P values comparing stimulated versus basal MMP9 release for each condition are shown above each bar. The P values comparing release in the presence and absence of an inhibitor are shown in parentheses above the appropriate bars. (B) Lactoferrin release from specific granules is expressed as means ±
sem
in nanograms/4 × 106 cells for 6 separate experiments, each using neutrophils from a different individual. The P values comparing stimulated versus basal lactoferrin release for each condition are shown above each bar. (C) Neutrophils (4 × 106/ml) were incubated with or without the Raf1 inhibitor, 3-(dimethylamino)-N-{3-[(4-hydroxybenzoyl)amino]-4-methylphenyl}benzamide (140 nM), before (Raf1Inh + TNF + fMLF) or after (TNF + Raf1Inh + fMLF) incubation with TNF-α (2 ng/ml, 10 min). Neutrophils were then stimulated with fMLF (300 nM, 5 min). Supernatants were obtained by centrifugation and the release of lactoferrin measured by ELISA. Lactoferrin release is expressed as means ±
sem
in nanograms/4 × 106 cells for 6 separate experiments, each using neutrophils from a different individual. The P values comparing stimulated versus basal release for each condition are shown above each bar. The P value comparing stimulation in the presence and absence of the Raf1 inhibitor is in parentheses.
Figure 4.. Colocalization of Raf1 with neutrophil granule subsets.
Neutrophils were plated on confocal microscope slides and permeabilized with Triton X-100 before fixation and staining. Cells were stained for Raf1 using rabbit anti-Raf1 and rhodamine-conjugated goat anti-rabbit (shown in red) and for a marker of secretory vesicles (CD35; A–C) or a marker of azurophilic granules (CD63; D–F) using FITC-labeled anti-CD35 and anti-CD63. The panels show photomicrographs from z-stacks of staining for the granule marker only (A and D), for Raf1 only (B and E), and of the merged pictures (C and F). The merged images show that Raf1 colocalizes with secretory vesicles and azurophilic granules.
Figure 5.. Role of actin reorganization in mediating p38 MAPK regulation of exocytosis.
For all experiments, neutrophils (4–10 × 106/ml) were incubated with latrunculin A (1 μM) for 15 min before incubation, with or without the p38 MAPK inhibitor, SB203580 (3 μM) for 15 min. Cells were then stimulated with or without TNF-α or TNF-α and then fMLF (TNF + fMLF). Supernatants were obtained by centrifugation and the release of contents from each granule subset measured. (A) Albumin release from secretory vesicles is expressed as means ±
sem
in nanograms/4 × 106 cells from 8 separate experiments, each using neutrophils from a different individual. (B) MMP9 release from gelatinase granules is expressed as means ±
sem
in nanograms/4 × 106 cells from 6 separate experiments, each using neutrophils from a different individual. (C) Lactoferrin release from specific granules is expressed as means ±
sem
in nanograms/4 × 106 cells for 6 separate experiments, each using neutrophils from a different individual. (D) MPO release from azurophilic granules is expressed as means ±
sem
in femtomole/4 × 106 cells for 4 separate experiments, each using neutrophils from a different individual. The P values for all granule markers comparing stimulated versus basal release are shown above each bar. The P values comparing stimulation in the presence and absence of SB203580 are shown in parentheses above the appropriate bars.
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