Exocytosis of neutrophil granule subsets and activation of prolyl isomerase 1 are required for respiratory burst priming

Abstract

This study tested the hypothesis that priming the neutrophil respiratory burst requires both granule exocytosis and activation of the prolyl isomerase Pin1. Fusion proteins containing the TAT cell permeability sequence and either the SNARE domain of syntaxin-4 or the N-terminal SNARE domain of SNAP-23 were used to examine the role of granule subsets in TNF-mediated respiratory burst priming using human neutrophils. Concentration-inhibition curves for exocytosis of individual granule subsets and for priming of fMLF-stimulated superoxide release and phagocytosis-stimulated H2O2 production were generated. Maximal inhibition of priming ranged from 72 to 88%. Linear regression lines for inhibition of priming versus inhibition of exocytosis did not differ from the line of identity for secretory vesicles and gelatinase granules, while the slopes or the y-intercepts were different from the line of identity for specific and azurophilic granules. Inhibition of Pin1 reduced priming by 56%, while exocytosis of secretory vesicles and specific granules was not affected. These findings indicate that exocytosis of secretory vesicles and gelatinase granules and activation of Pin1 are independent events required for TNF-mediated priming of neutrophil respiratory burst.

Fig. 1

Inhibition of priming by TAT-SNAP-23 and TAT-syntaxin (Syn)-4. a Neutrophils (4 × 10⁶/ml) were incubated with or without 42 nM TAT-SNAP-23, 53 nM TAT-syntaxin-4, or 53 nM TAT-control (Ctrl) for 10 min prior incubation with or without TNF (2 ng/ml) for 10 min, followed by stimulation of superoxide release with 300 nM fMLF for 5 min. Results of superoxide release in nanomoles of reduced ferricytochrome c/4 × 10⁶ cells/5 min are presented as mean ± SEM for 5-11 experiments. b Neutrophils (4 × 10⁶ cells/ml) were incubated with 0.5 mM 2′,7′-dichlorofluorescein diacetate for 10 min at 37°C prior to a 10-min incubation with or without 42 nM TAT-SNAP-23 or 53 nM TAT-syntaxin-4, 2nd then incubated with or without TNF (2 ng/ml) for 10 min. Fifty microliters of cell suspension were sampled before and 10 min after the addition of opsonized, propidium iodide-labeled S. aureus. Uptake of labeled bacteria and oxidation of 2′,7′-dichlorofluorescein diacetate to fluorescent 2′,7′-dichlorofluorescein were measured by flow cytometry. H₂O₂ production (b) and phagocytosis (c) are expressed as mean ± SEM in mean channel fluorescence units (mcf) for 4-13 separate experiments.

Fig. 2

Concentration inhibition of granule subset exocytosis by TAT-SNAP-23. Neutrophils (4 × 10⁶/ml) were incubated without or with TAT-SNAP-23 at concentrations from 11 to 53 nM for 10 min, then treated with or without fMLF at 300 nM for 5 min. For experiments examining CD63 expression, cells were pretreated with latrunculin A (1 μM) for 30 min. Percent inhibition was calculated as 100 × (C₀ - C_TAT)/C₀, where C₀ is the value of the measured parameter in the absence of TAT-SNAP-23 and C_TAT is the value of the measured parameter in the presence of the indicated concentration of TAT-SNAP-23. a Inhibition of exocytosis of secretory vesicles was determined using plasma membrane expression of CD35. Results are presented as means ± SEM for 12 separate experiments. b Inhibition of exocytosis of gelatinase granules was determined using extracellular release of gelatinase. Results are presented as means ± SEM for 5 separate experiments. c Inhibition of exocytosis of specific granules was determined using plasma membrane expression of CD66b. Results are presented as means ± SEM for 12 separate experiments. d Inhibition of exocytosis of azurophilic granules was determined using plasma membrane expression of CD63. Results are presented as means ± SEM for 4 separate experiments.

Fig. 3

Concentration inhibition of granule subset exocytosis by TAT-syntaxin-4. Neutrophils (4 × 10⁶/ml) were incubated without or with TAT-syntaxin-4 at concentrations from 13 to 67 nM for 10 min, then treated with or without fMLF at 300 nM for 5 min. For experiments examining CD63 expression, cells were pretreated with latrunculin A (1 μM) for 30 min. Percent inhibition was calculated as described in figure 1. a Inhibition of exocytosis of secretory vesicles was determined using plasma membrane expression of CD35. Results are presented as means ± SEM for 6 separate experiments. b Inhibition of exocytosis of gelatinase granules was measured using extracellular release of gelatinase. Results are presented as means ± SEM for 5 separate experiments. c Inhibition of exocytosis of specific granules was determined using plasma membrane expression of CD66b. Results are presented as means ± SEM for 6 separate experiments. d Inhibition of exocytosis of azurophilic granules was determined using plasma membrane expression of CD63. Results are presented as means ± SEM for 8 separate experiments.

Fig. 4

Concentration inhibition of neutrophil priming by TAT fusion proteins. Neutrophils (4 × 10⁶/ml) were incubated with or without the indicated concentrations of TAT-SNAP-23 or TAT-syntaxin-4 for 10 min prior to priming with TNF (2 ng/ml for 10 min), followed by stimulation of superoxide release with 300 nM fMLF for 5 min or stimulation of H₂O₂ release by phagocytosis of S. aureus for 10 min. Percent inhibition was calculated as described in figure 1. a Inhibition of priming by TAT-SNAP-23. b Inhibition of priming by TAT-syntaxin-4. Results are presented as means ± SEM for 4-6 separate experiments.

Fig. 5

Disruption of the actin cytoskeleton with latrunculin A enhances exocytosis and induces priming. a Effect of pretreating neutrophils with increasing concentrations of latrunculin A on exocytosis of specific granules (CD66b) and on fMLF-stimulated superoxide release. Neutrophils (4 × 10⁶/ml) were incubated with the indicated concentrations of latrunculin A for 30 min prior to stimulation with 300 nM fMLF for 5 min. Superoxide release and CD66b expression are presented as means ± SEM for 6 separate experiments. mcf = Mean channel fluorescence units. b Concentration inhibition of latrunculin A-induced exocytosis and priming by TAT-syntaxin-4. Neutrophils were incubated with 10 M latrunculin A for 30 min and then incubated with the indicated concentrations of TAT-syntaxin-4 prior to stimulation of superoxide release and CD66b expression with 300 nM fMLF for 5 min. Results are presented as means ± SEM for 5 separate experiments.

Fig. 6

Effect of inhibition of exocytosis on translocation of p47^phox. a Representative immunoblot for p47^phox of membrane fractions obtained from unstimulated cells or cells primed with 2 ng/ml TNF for 10 min followed by stimulation with 300 nM fMLF for 5 min in the presence or absence of 47 nM TAT-SNAP-23 or 60 nM TAT-control prior to isolation of membrane fractions. b Mean ± SEM densitometric analysis of 4 separate experiments showing that TAT-SNAP-23 and TAT-control had no effect on p47^phox translocation induced by priming and stimulation.

Fig. 7

Effect of inhibition of Pin1 on priming and exocytosis. a Neutrophils (4 × 10⁶/ml) were incubated with 250 μM juglone for 30 min at 37°C prior to incubation with or without 2 ng/ml TNF for 10 min, then with or without 300 nM fMLF for 5 min. Superoxide release is presented as mean ± SEM nmol/4 × 10⁶ cells of reduced ferricytochrome c for 5 separate experiments. * p < 0.05 vs. primed cells. b Neutrophils (4 × 10⁶/ml) were incubated with 250 μM juglone for 30 min at 37°C prior to incubation with or without 2 ng/ml TNF for 10 min. Expression of CD35 (secretory vesicles) and CD66b (specific granules) is presented as mean ± SEM for 6 separate experiments. mcf = Mean channel fluorescence units.

Fig. 8

TNF priming and exocytosis are dependent on p38 MAPK, but not MAPKAPK2 (MK2). a Neutrophils (4 × 10⁶/ml) were incubated with the p38 MAPK inhibitor, SB203580 (3 μM) or MAPKAPK2 inhibitor III (10 or 30 nM) for 30 min at 37°C prior to incubation with 2 ng/ml TNF for 10 min. Exocytosis of secretory vesicles and specific granules, measured as plasma membrane expression of CD35 and CD66b, respectively, is presented as mean ± SEM of the increase above basal expression for 6 separate experiments. * p < 0.05 vs. cells stimulated by TNF alone. mcf = Mean channel fluorescence units. b Neutrophils (4 × 10⁶/ml) were incubated with SB203580 (3 μM) or MAPKAPK2 inhibitor III (10 or 30 nM) for 30 min at 37°C prior to incubation with or without 2 ng/ml TNF for 10 min then with or without fMLF (300 nM) for 5 min. Superoxide release is presented as mean ± SEM of the TNF-induced increase in superoxide release for 7 separate experiments. * p < 0.05 vs. inhibitor.