Frontline Science: Tumor necrosis factor-α stimulation and priming of human neutrophil granule exocytosis (original) (raw)

TNF-α stimulates exocytosis of secretory vesicles and gelatinase granules, and primes specific and azurophilic granule exocytosis that depend on p38 MAPK and actin reorganization.

Keywords: mass spectrometry, phosphorylation, p38 MAPK, actin cytoskeleton

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.

Introduction

Release of neutrophil granule contents plays an important role in control of microbial invasion, contributes to inflammatory tissue injury, and participates in innate and adaptive immune responses [1–5]. Tight regulation of neutrophil exocytosis is necessary to limit inflammation and damage to normal tissue, while effectively killing invading pathogens. Neutrophil granules are divided into 4 classes based on separation by density gradient fractionation and contents [6–8]. Secretory vesicles are created by endocytosis, whereas gelatinase (tertiary), specific (secondary), and azurophilic (primary) granules are formed from the _trans_-Golgi network during neutrophil maturation [2]. Neutrophil granules undergo an ordered release based on stimulus intensity [7, 8]. Secretory vesicles undergo exocytosis more easily and completely than gelatinase granules. Specific and azurophilic granules contain the most toxic components and undergo the most limited exocytosis. Neutrophils migrating into a skin blister created in normal human subjects release nearly 100% of their secretory vesicles, 40% of gelatinase granules, 20% of specific granules, and <10% of azurophilic granules [8].

The ability of neutrophils to undergo stimulated exocytosis is not static. Catz and colleagues [9, 10] reported that azurophilic and gelatinase granule exocytosis was enhanced by pretreatment with bacterial LPS. A number of cytokines, including TNF-α, GM-CSF, and IL-1β, enhance exocytosis of specific and azurophilic granules [11–13]. Adhesion to extracellular matrix proteins amplified stimulated exocytosis of specific granules [14, 15], and the cross-linking of selectins enhanced IL-8-stimulated exocytosis of specific and azurophilic granules [16]. TNF-α is an important proinflammatory cytokine that stimulates exocytosis of neutrophil granules, and TNF-α priming of respiratory burst activity is dependent on exocytosis [17–19]. Thus, defining the molecular mechanisms that control neutrophil exocytosis has the potential to provide therapeutic targets to limit tissue injury in autoimmune and inflammatory diseases. The current study examined the ability of TNF-α to stimulate and prime exocytosis of each of the 4 granule subsets and examined the role of p38 MAPK in that activity. Additionally, potential mediators of TNF-α-induced exocytosis were defined by mass spectrometry-based identification of proteins phosphorylated during TNF-α stimulation.

MATERIALS AND METHODS

Materials

Recombinant human TNF-α was from R&D Systems (Minneapolis, MN, USA). Latrunculin A, fMLF, and protease and phosphatase inhibitors were from Sigma-Aldrich (St. Louis, MO, USA). SB203580 was from EMD Millipore (Billerica, MA, USA). An MMP9 ELISA kit was from R&D Systems. ELISAs for human albumin and lactoferrin were from Abcam (Cambridge, United Kingdom). The Raf1 inhibitor, 3-(dimethylamino)-_N_-{3-[(4-hydroxybenzoyl)amino]-4-methylphenyl}benzamide, was obtained from Santa Cruz Biotechnology (Dallas, TX, USA; ZM 336372). Anti-MARCKS (used at a 1:40 dilution) and anti-Raf1 (1:400) were obtained from Abcam. Anti-ABI1 (1:40) and anti-myosin VI (1:40) were obtained from Thermo Fischer Scientific (Waltham, MA, USA).

Neutrophil isolation

Neutrophils were isolated from the blood of healthy donors using plasma-Percoll gradients followed by hypotonic lysis to remove RBCs, resuspended in KRPB without calcium and magnesium, and used immediately, as described previously [17, 18]. Microscopic evaluation of isolated cells showed that >92% of cells were neutrophils. Trypan blue exclusion indicated that >95% of cells were viable. The Institutional Review Board of the University of Louisville approved the use of human donors, who provided informed consent.

Exocytosis

To measure exocytosis of granule subsets, neutrophils were incubated with the indicated stimulus in KRPB, containing calcium and magnesium for the indicated time in the absence of cytochalasin or latrunculin, except where indicated. Neutrophils from different donors were used for each separate experiment. Neutrophils were separated from supernatant by centrifugation at 6000 g for 30 s, and protease and phosphatase inhibitor cocktails were added to the supernatant (Catalog #P2740 and #P0044; Sigma-Aldrich). The supernatant was snap frozen and stored at –80°C until ELISAs were performed or used immediately to measure MPO. Exocytosis of secretory vesicles was determined by measuring the release of albumin by ELISA [7]. Release of gelatinase granule contents was determined by ELISA for MMP9, as described previously [20]. Exocytosis of specific granules was determined by measuring the release of lactoferrin by ELISA [8]. Exocytosis of azurophilic granules was determined by measuring MPO release by NaN3-inhibitable isoluminol chemiluminescence in the presence of an exogenous supply of H2O2, as described previously [17]. The amount of MPO released was calculated from a standard curve in the presence of known amounts of pure MPO (0–20 nM).

Confocal microscopy

Neutrophils were resuspended in KRPB, supplemented with 1.2 mM Mg2+ and 0.5 mM Ca2+ at a concentration of 4 × 106 cells/ml. All experiments were performed in 1.5 ml microtubes. Cells were washed with ice-cold KRPB, and 200 μl 0.5% Triton X-100 in KRPB was added for 10 min at room temperature. Neutrophils were fixed with 10% formalin for 15 min at room temperature, permeabilized with 0.02% saponin, and incubated overnight at 4°C with primary antibody. Primary antibodies included anti-Raf1 (1:400 dilution; Abcam), FITC-labeled anti-CD35 (1:25 dilution; BD Biosciences, San Jose, CA, USA), FITC-labeled anti-CD66b (1:25 dilution; BioLegend, San Diego, CA, USA), and FITC-labeled anti-CD63 (1:25 dilution; Ancell, Bayport, MN, USA). Cells were then washed with PBS and incubated with rhodamine-labeled goat anti-rabbit IgG (1:500 dilution; Thermo Fisher Scientific) at 4°C for 1 h. Imaging was conducted on an Olympus FluoView FV10 microscope using FV10-ASW software. Single z-stack images are shown.

Phosphoprotein analysis

Neutrophils (2 × 10 7 cells/ml) were suspended in KRPB containing calcium and magnesium and incubated with or without TNF-α (2 ng/ml) for 5 or 10 min at 37°C. Following stimulation, cells were pelleted immediately at 4000 rpm for 1 min at 4°C and lysed by resuspending the pellet in ice-cold extraction buffer [20 mM Tris-HCl, pH 7.8, 10 mM HEPES, 25 mM NaCl, 2 mM EDTA, 10 mM EGTA, and 1% (w/w) protease inhibitor cocktail], followed by sonication using 3, 5 s cycles at room temperature. Cell debris and nuclei were removed by centrifugation at 700 g for 10 min at 4°C. The supernatant was transferred to ultracentrifugation tubes and centrifuged at 100,000 g for 30 min at 4°C. After centrifugation, the supernatant was stored at −80°C until used.

Samples were reduced, alkylated, and trypsinized and phosphopeptides enriched using sequential TiO2 and immobilized metal affinity chromatography chromatographic steps to purify polyphosphorylated peptides and monophosphorylated peptides, as described previously [21]. The effects of coisolation of nonphosphorylated peptides enriched in aspartic acid and/or glutamic acid residues were minimized with the use of 1 M glycolic acid as a competitive ligand for the TiO2 step. Targeted analysis of phosphopeptide fractions was achieved using a nanoflow ultra HPLC/nanospray- Linear Ion Trap-Orbitrap Elite mass spectrometer with collision-induced dissociation and electron transfer dissociation fragmentation in a bottom-up approach. Acquired data were analyzed against human Reference Sequence (HumanRef131014.fasta) and decoy databases using Sequest HT by PD1.4, considering tryptic cleavage, maximum of 2 missed cleavages per peptide, and a mass error of 50 ppm in precursor ions of a specific mass-to-charge ratio and 1.2 Da in fragmented precursor ions data. The searches considered a maximum of 4 modifications to any peptide and the modifications of cysteine (carbamidomethyl/+57.021 Da), methionine (oxidation/+15.995 Da), and serine/threonine/tyrosine (phospho/+79.966 Da). PD1.4 data were filtered first to retrieve all peptides containing serine-, threonine-, or tyrosine-containing peptides (putative kinase-targetable peptides). Peptide grouping was enable by mass and sequence. Protein grouping was enabled to consider only PSMs with confidence at least low/medium/high-confidence peptides and to consider proteins only with PSMs having a delta correlation better than 1.0. These data were filtered to eliminate entries where a low-confidence assignment was the only assignment present in any condition. If medium- or high-confidence peptides were present in 1 condition, then the peptide areas were exported for all conditions. For this purpose, medium-confidence peptides had minimal xcorr values for charge +2 > 0.9, charge +3 > 1.2, and charges +4–7 > 1.5, and for high-confidence peptides, the corresponding xcorr values were for charge +2 > 2, charge +3 > 2.5, and charges +4–7 > 3. These peptides were exported to an Excel spreadsheet for peptide grouping by mass and sequence and manual comparison of abundance between treatment and control conditions.

Statistical analysis

All data are expressed as means ± sem. Statistical analysis was performed using Student’s _t_-test or a one-way ANOVA with the Tukey-Kramer multiple-comparison test. To determine the causal impact of TNF-α on fMLF-stimulated exocytosis, a univariate AN OVA was performed. The P value for each data set is provided in Results and/or in figures. Statistical significance was set a priori at the P < 0.05 level.

RESULTS

TNF-α selectively stimulates and primes exocytosis of neutrophil granule subsets

To determine if TNF-α stimulated exocytosis or primed the ability of fMLF to stimulate exocytosis of neutrophil granules, human neutrophils were treated with 2 ng/ml TNF-α for 10 min before incubation, with or without 300 nM fMLF for 5 min. Exocytosis of each granule subset was measured by the extracellular release of granule components. Previous work in our laboratory determined that those concentrations of TNF-α and fMLF provided optimal stimulation of exocytosis [18–20]. Although the concentrations of TNF-α found in the circulation of patients with sepsis has been reported to range from 30 to 1300 pg/ml [22], preliminary studies showed that stimulation and priming of exocytosis by TNF-α in vitro were seen at 2 ng/ml TNF-α (Supplemental Fig. 1). Figure 1A shows that incubation of neutrophils with TNF-α, fMLF, or TNF-α followed by fMLF all stimulated an equivalent release of albumin. Incubation with TNF-α, followed by washing cells to remove albumin released before addition of fMLF, resulted in a failure of fMLF to stimulate further albumin release. Thus, TNF-α and fMLF appear to release most, if not all, secretory vesicles able to be mobilized under the experimental conditions, and TNF-α does not prime fMLF-stimulated secretory vesicle release.

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 ± sem 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 1B shows that TNF-α and fMLF each significantly stimulated MMP9 release. Incubation of neutrophils with TNF-α for 10 min, followed by fMLF for 5 min, resulted in a significantly enhanced MMP9 release compared with TNF-α or fMLF alone. Washing after incubation with TNF-α but before addition of fMLF reduced MMP9 release to levels not significantly different from TNF-α or fMLF, alone. Univariate analysis failed to show a significant interaction between TNF-α and fMLF for MMP9 release, suggesting that the magnitude of MMP9 release induced by fMLF did not depend on the presence of TNF-α. These results indicate that TNF-α and fMLF have an additive effect on gelatinase granule exocytosis, and TNF-α does not prime release of gelatinase granules by fMLF.

Whereas neither TNF-α nor fMLF alone stimulated a significant increase in lactoferrin release above basal levels, incubation with TNF-α before fMLF resulted in a significant increase in lactoferrin release (Fig. 1C). Washing between incubation with TNF-α and fMLF significantly attenuated the increased lactoferrin release. Univariate analysis confirmed a significant interaction between TNF-α and fMLF for lactoferrin release (P < 0.05), suggesting that the magnitude of fMLF-stimulated lactoferrin release was dependent on the presence of TNF-α. These results indicate that TNF-α primes specific granule exocytosis stimulated by fMLF.

Figure 1D shows that neither fMLF nor TNF-α alone stimulated MPO release in the absence of cytoskeletal disruption with latrunculin A. However, treatment with TNF-α, followed by fMLF, resulted in a significant increase in MPO release above basal levels. Univariate analysis confirmed a significant interaction between TNF-α and fMLF, indicating that TNF-α primed azurophilic granules for enhanced release upon subsequent fMLF stimulation.

TNF-α-stimulated protein phosphorylation

The targets of TNF-α that mediate priming and stimulation of exocytosis are unknown. To identify candidates for those targets, neutrophil proteins phosphorylated after incubation with TNF-α were identified by mass spectrometry. Human neutrophils were incubated for 5 and 10 min, with or without 2 ng/ml TNF-α. Cell proteins were extracted and phosphopeptides enriched and then identified by mass spectrometry. A total of 1224 unique, high-confidence phosphopeptides, representing 1115 unique proteins, were identified from cells stimulated with TNF-α but were not found in unstimulated cells. Phosphopeptide liquid chromatography–mass spectrometry data were deposited to MassIVE (ProteomeXchange; ProteomeXchange Accession #PXD004487; http://www.proteomexchange.org/).

Analysis of these phosphoproteins was performed using Ingenuity Pathway Analysis (Qiagen, Germantown, MD, USA). Table 1 shows the canonical pathways identified and the number of proteins in each pathway. Four of those pathways—Rho signaling, actin signaling, paxillin signaling, and integrin signaling—are involved in cytoskeletal regulation. The molecular and cellular function category with the most significant P value was Cellular Assembly and Organization. Table 2 shows the 15 most highly significant individual functions of TNF-α-induced phosphoproteins within the Cellular Assembly and Organization category. Nine of those 15 functions are related to regulation of the cytoskeleton. Four additional categories of Cellular Assembly and Organization functions were related to secretory vesicle quantity, formation, or release.

TABLE 1.

Canonical pathways of TNF-α-stimulated phosphoproteins

TABLE 2.

Cellular assembly and organization functions of TNF-induced phosphoproteins

Thirteen of the proteins phosphorylated after incubation with TNF-α are known to regulate secretory granule function (Table 3). Review of prior proteomic analyses of neutrophil granules found that only 2 of those 13 proteins were previously identified on granules: ABI1 on secretory vesicles and synaptotagmin-like 1 on azurophilic granules [23–25]. Five of those proteins—MARCKS, Ral A, Aryl hydrocarbon receptor, Raf1, and synaptotagmin-like 1—were reported previously to regulate exocytosis in granulocytes [26–33]. Syntaxin-binding protein 5 was recently shown to be required for platelet granule secretion, while inhibiting endothelial cell exocytosis [34, 35]. Regulating synaptic membrane exocytosis 1 is a component of the presynaptic active zone complex, where it regulates secretory vesicle docking, priming, and the level of granule release [36]. Thus, our data identified a number of proteins phosphorylated in response to TNF-α that are known to regulate various aspects of exocytosis.

TABLE 3.

TNF-α phosphoproteins associated with granule formation and release

The p38 MAPK pathway participates in TNF-α stimulation and priming of exocytosis

We reported previously that activation of the p38 MAPK pathway contributed to TNF-α-stimulated neutrophil exocytosis of secretory vesicles and specific granules [17–19]. To examine the role of p38 MAPK in TNF-α stimulation and priming of exocytosis, neutrophils were pretreated with 3 μM SB203580, a p38 MAPK inhibitor, before incubation with 2 ng/ml TNF-α or sequential incubation with TNF-α and 300 nM fMLF. Incubation with TNF-α, alone and TNF-α followed by fMLF significantly stimulated release of albumin (Fig. 2A) and MMP9 (Fig. 2B) that was prevented by pretreatment with SB203580. Figure 2C confirms that only sequential incubation with TNF-α and fMLF stimulated a significant increase in lactoferrin release and that release was inhibited significantly by pretreatment with SB203580. Figure 2D confirms that MPO release was only observed after sequential incubation with TNF-α and fMLF and that release was almost completely blocked by pretreatment with SB203580. These data suggest that both TNF-α-induced stimulation and priming of exocytosis are dependent on p38 MAPK activation.

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.

To identify candidate proteins that may mediate the p38 MAPK-dependent activity of TNF-α, the ability of pretreatment with 3 μM SB203580 to reduce or inhibit TNF-α-induced protein phosphorylation was determined. Of the high-confidence proteins phosphorylated by TNF-α, 89 phosphopeptides from 43 unique proteins were identified in which TNF-α-stimulated phosphorylation was at least 1.5-fold greater in the absence compared with the presence of SB203580 (Supplemental Table 1). Table 1 shows the top 15 canonical pathways of those 43 proteins and the number of proteins in each pathway. Only 3 pathways are common among the top 15 pathways for all TNF-α-induced phosphoproteins and those that are dependent on p38 MAPK activity. Included in p38 MAPK-dependent phosphoproteins were 4 of the 13 TNF-α-induced phosphoproteins determined to regulate granule formation or release: Raf1, MARCKS, ABI1, and myosin VI (Table 3). Of those proteins, none contained the consensus sequence for p38 MAPK phosphorylation, and only 1 (MARCKS) was previously reported to be a target of the p38 MAPK pathway [37].

To screen those 4 candidate targets of p38 MAPK for participation in TNF-α-mediated exocytosis, confocal microscopy examined the basal and stimulated distribution of each protein. Raf1, MARCKS, ABI1, and myosin VI were all detected in a diffuse granular pattern distributed throughout the cytoplasm that did not change with stimulation with fMLF, TNF-α, or TNF-α, followed by fMLF (data not shown). As Raf1 is an upstream regulator of ERK1/2 MAPK, the role of Raf1 and ERK1/2 in TNF-α-regulated exocytosis was examined using pharmacologic inhibitors. Figure 3A shows that fMLF, TNF-α, and TNF-α, followed by fMLF, all stimulated a significant increase in gelatinase release. Pretreatment with the Raf1 inhibitor significantly reduced gelatinase release stimulated by fMLF and fMLF plus TNF-α. Raf1 inhibition reduced TNF-α-stimulated gelatinase release so that it no longer significantly differed from basal release; however, TNF-α-stimulated gelatinase release did not significantly differ between the presence and absence of the Raf1 inhibitor. Inhibition of ERK by 50 μM PD98059 did not significantly alter gelatinase release under any of those conditions. Figure 3B shows that release of lactoferrin from specific granules after sequential stimulation with TNF-α and fMLF was significantly inhibited by Raf1 inhibition but not ERK inhibition. As the Raf1 inhibitor interfered with the MPO assay, its effect on azurophilic granule exocytosis could not be tested. To determine if Raf1 played a role in TNF-α-mediated priming of specific granule exocytosis, the effect of addition of the Raf1 inhibitor before or after incubation with TNF-α but before addition of fMLF on lactoferrin release was examined. Figure 3C shows that incubation of neutrophils with the Raf1 inhibitor before but not after addition of TNF-α prevented fMLF from stimulating a significant release of lactoferrin. The association of Raf1 with granule subsets was evaluated by determining its colocalization with markers of secretory vesicles (CD35), specific granules (CD66b), and azurophilic granules (CD63). Figure 4 shows the confocal microscopy z-stack images of distribution for CD35 and CD63. The merged images demonstrate that Raf1 colocalized with those 2 granule markers. The distribution of CD66b staining was too diffuse to localize specific granules. These data indicate that Raf1 is associated with at least some neutrophil granule subsets, plays a role in stimulated exocytosis of gelatinase granules, and is a necessary component for TNF-α-mediated priming of specific granule release. Those effects of Raf1 are independent of ERK activation.

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.

As a number of p38 MAPK-dependent phosphoproteins were predicted to be involved in actin cytoskeletal regulation, we examined the role of the actin cytoskeleton in p38 MAPK-dependent TNF-α stimulation and priming of neutrophil exocytosis. We reasoned that if p38 MAPK contributed to exocytosis through regulation of actin reorganization, then disruption of the actin cytoskeleton by treatment with latrunculin A would prevent impaired exocytosis following inhibition of the p38 MAPK pathway. Figure 5 shows the effect of pretreatment with latrunculin A on exocytosis of all 4 granule subsets stimulated by TNF-α and TNF-α plus fMLF in the presence and absence of SB203580. Latrunculin A by itself induced release of secretory vesicles, as reported previously [20], preventing evaluation of the role of the cytoskeleton. In the presence of latrunculin A, sequential addition of TNF-α, followed by fMLF, resulted in a significantly increased release of MMP9 (Fig. 5B), lactoferrin (Fig. 5C), and MPO (Fig. 5D). Disruption of actin polymerization by latrunculin A prevented inhibition of release of all 3 granule markers by SB203580, as shown in Fig. 2. We conclude from these results that the p38 MAPK pathway mediates TNF-α-induced priming of granule exocytosis through regulation of actin polymerization.

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.

DISCUSSION

The current study shows that TNF-α differentially regulates exocytosis of the 4 neutrophil granule subsets, and the data indicate that regulation is dependent on p38 MAPK-dependent reorganization of the actin cytoskeleton. TNF-α stimulated but did not prime exocytosis of secretory vesicles and gelatinase granules. Those findings are consistent with our previous study showing that TNF-α-stimulated exocytosis of secretory vesicles and gelatinase granules is a necessary component of priming of respiratory burst activity [18]. Neither TNF-α nor fMLF alone stimulated exocytosis of specific and azurophilic granules, but TNF-α primed the release of both granule subsets upon stimulation by fMLF. The requirement for exposure to 2 agonists limits the unwarranted release of highly toxic antimicrobial components from those granule subsets. Taken together with our previous report [18], our data suggest that differential regulation of exocytosis by TNF-α primes the 2 major neutrophil antimicrobial defense mechanisms for enhanced release of reactive oxygen species and toxic granule contents, while protecting against cell injury from inappropriate release of those products.

TNF-α and fMLF stimulated similar levels of secretory vesicle exocytosis, and TNF-α did not enhance fMLF-stimulated exocytosis. Those results and the effect of washing between TNF-α and fMLF stimulation indicate that the optimal concentrations of fMLF and TNF-α used in this study induced exocytosis of available secretory vesicles. Our findings support previous reports that secretory vesicles are most easily stimulated to undergo exocytosis and that optimal stimulation produces almost complete exocytosis of secretory vesicles [7, 8]. TNF-α and fMLF also stimulated similar levels of MMP9 release; however, incubation with TNF-α before stimulation with fMLF significantly enhanced fMLF-stimulated MMP9 release. Washing between incubation with TNF-α and stimulation with fMLF only partially reduced MMP9 release, and statistical analysis indicated that TNF-α did not significantly impact fMLF-stimulated gelatinase granule exocytosis. We interpret those data to indicate that TNF-α and fMLF induce an additive release of gelatinase granules, and TNF-α does not prime fMLF-stimulated gelatinase granule exocytosis.

Neither TNF-α nor fMLF alone stimulated exocytosis of specific or azurophilic granules, whereas sequential addition of TNF-α and fMLF stimulated exocytosis of both granule subsets. Washing cells after incubation with TNF-α significantly reduced subsequent fMLF-stimulated, specific granule exocytosis. Statistical analysis identified a significant interaction between TNF-α and fMLF, indicating that TNF-α primed the fMLF response. We interpret the loss of specific granule release with washing after exposure to TNF-α to indicate that the TNF-α-priming effect requires its continual presence. Our findings are consistent with a previous report by Bajaj et al. [38], showing that TNF-α failed to stimulate specific and azurophilic granule exocytosis in cytochalasin B-treated human neutrophils, while enhancing release stimulated by fMLF or C5a. Johnson et al. [10] reported that fMLF stimulated a modest increase in azurophilic granule exocytosis in primary murine neutrophils that was markedly increased by pretreatment with LPS. We conclude that little or no exocytosis of specific and azurophilic granules occurs unless neutrophils are exposed to agents, such as TNF-α and LPS, before a subsequent stimulus. The requirement for a priming stimulus and an activating stimulus to induce release of specific and azurophilic granule contents provides a layer of regulation that reduces the risk of exposure of normal cells to the toxic components of those granule subsets.

The molecular targets of TNF-α that mediate stimulation and priming of neutrophil exocytosis are unknown. To generate a list of candidate targets, we identified the proteins phosphorylated after incubation with TNF-α. The 1115 unique TNF-α-induced phosphoproteins were analyzed by Ingenuity Pathway Analysis (Qiagen). Four of the top 15 cannonical pathways and 9 of the top 15 Cellular Assembly and Organization functions in that analysis involved regulation of the cell cytoskeleton. The actin cytoskeleton has been reported to inhibit and facilitate regulated exocytosis. Subcortical actin has been proposed to provide a passive barrier to granule access to the plasma membrane [39], and previous studies support that role for actin in neutrophils [20, 40]. Reorganization of subcortical actin would then be required to allow granule fusion with the plasma membrane. Johnson et al. [26] reported that azurophilic granule exocytosis was dependent on localized actin depolymerization. Additionally, granule actin coats are proposed to facilitate exocytosis by mediating granule translocation or by providing a contractile force to expel granule contents [39, 41]. We previously reported the association of varying amounts of actin with all neutrophil granule subsets [23], and Mitchell et al. [40] showed that inhibition of actin polymerization blocked azurophilic granule exocytosis.

Bioinformatic analysis identified 13 TNF-α-induced phosphoproteins with functions related to secretory vesicle quantity, formation, or release (Table 3). Five of those proteins—MARCKS, Ral A, Aryl hydrocarbon receptor, Raf1, and synaptotagmin-like 1 (JFC1/Slp1)—were reported previously to regulate exocytosis in granulocytes [26–33]. The most completely characterized of those proteins is synaptotagmin-like 1 (exophilin-7), which is a Rab27 effector that facilitates azurophilic granule exocytosis. Synaptotagmin-like 1 was shown to be expressed on azurophilic granules and to regulate granule trafficking by inducing local actin depolymerization [26]. Catz and colleagues [42] showed that synaptotagmin-like 1 is phosphorylated by Akt and that phosphorylation regulates membrane binding. Of the remaining 8 proteins, syntaxin-binding protein 5 was recently shown to regulate granule secretion in platelets and endothelial cells [34, 35], and phosphorylation by Akt and PKA regulates syntaxin-binding protein 5 interaction with syntaxins [43, 44]. RIM proteins are central components of the presynaptic active zone complex, where they organize active zones and regulate docking, priming, and the level of granule release [36]. RIM proteins are phosphorylated by PKA; however, the function of that phosphorylation is unclear [45]. Huntingtin participates in vesicular trafficking and is phosphorylated by ERK and CDK5/CDK2[46].

Consistent with our previous studies [17–19], the current study indicates that p38 MAPK plays a critical role in TNF-α-stimulated exocytosis of secretory vesicles and gelatinase granules. The ability of SB203580 to block exocytosis of specific and azurophilic granules during sequential addition of TNF-α and fMLF suggests that p38 MAPK also plays a significant role in TNF-α priming of exocytosis. Subtraction analysis of phosphoproteins identified 43 proteins in which TNF-α-mediated phosphorylation was reduced by inhibition of p38 MAPK, 4 of which—ABI1, MARCKS, myosin VI, and Raf1—also regulated vesicular formation and release (Table 3). All 4 of those proteins were identified in human neutrophils by confocal microscopy; however, none showed a change in their pattern of expression with stimulation. Additionally, none of those 4 proteins contained a consensus sequence for p38 MAPK phosphorylation. Thus, it is likely that 1 or more of the kinases downstream of p38 MAPK regulate neutrophil exocytosis. We previously reported that 1 of those downstream kinases—MAPK-activated protein kinase 2—was not involved in TNF-α-stimulated exocytosis or priming of respiratory burst activity [18]. As 3 of those 4 proteins were known to interact with the actin cytoskeleton, we postulated that a mechanism of action of p38 MAPK activation was through regulation of the actin cytoskeleton. We predicted that disruption of the actin cytoskeleton would block the effect of p38 MAPK inhibition on TNF-α-mediated stimulation and/or priming of exocytosis. Our results showed that the inhibitory effect of SB203580 was lost in cells pretreated with latrunculin A, supporting a role for p38 MAPK in TNF-α stimulation and priming of exocytosis through regulation of the actin cytoskeleton. The availability of a pharmacologic inhibitor allowed us to confirm the role of Raf1 in gelatinase and specific granule exocytosis. Our data suggest that Raf1 plays a role in gelatinase granule exocytosis stimulated by TNF-α or by fMLF and in TNF-α priming of fMLF-stimulated, specific granule exocytosis. Further studies are required to define the molecular mechanisms by which phosphorylation of Raf1 and other molecular targets of TNF-α regulate actin cytoskeletal reorganization and mediate stimulation and priming of exocytosis.

AUTHORSHIP

K.R.M. and R.A.W. conceived of the project, analyzed data, and wrote the manuscript. S.M.U. assisted in the conception of the project and data interpretation. M.L.M. performed mass spectrometry and analyzed proteomic data. T.M.C. and S.T. performed experiments and statistical analysis. M.T.B. designed and performed confocal microscopy experiments and interpreted data.

Supplementary Material

Supplemental Data

ACKNOWLEDGMENTS

This work was supported by a Merit Review Award (BX001838) from the Department of Veterans Affairs (to K.R.M.) and by the U.S. National Institutes of Health (K99/R00 HL087924 to S.M.U.).

Glossary

ABI1

Abelson murine leukemia interactor 1

CDK

cyclin-dependent kinase

KRPB

Krebs-Ringer phosphate buffer

MARCKS

myristoylated alanine-rich protein kinase C substrate

MMP9

matrix metalloproteinase 9, gelatinase

MPO

myeloperoxidase

PD1.4

Proteome Discoverer 1.4

PKA/C

protein kinase A/C

PSM

peptide-to-spectrum match

RIM

Rab3-interacting molecule

Footnotes

The online version of this paper, found at www.jleukbio.org, includes supplemental information.

SEE CORRESPONDING EDITORIAL ON PAGE 4

DISCLOSURES

The authors declare no conflicts of interest.

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