Neutrophil azurophilic granule exocytosis is primed by TNF-α and partially regulated by NADPH oxidase

Abstract

Neutrophil (polymorphonuclear leukocyte) activation with release of granule contents plays an important role in the pathogenesis of acute lung injury, prompting clinical trials of inhibitors of neutrophil elastase. Despite mounting evidence for neutrophil-mediated host tissue damage in a variety of disease processes, mechanisms regulating azurophilic granule exocytosis at the plasma membrane, and thus release of elastase and other proteases, are poorly characterized. We hypothesized that azurophilic granule exocytosis would be enhanced under priming conditions similar to those seen during acute inflammatory events and during chronic inflammatory disease, and selected the cytokine TNF-α to model this in vitro. Neutrophils stimulated with TNF-α alone elicited intracellular reactive oxygen species (ROS) generation and mobilization of secretory vesicles, specific, and gelatinase granules. p38 and ERK1/2 MAPK were involved in these components of priming. TNF-α priming alone did not mobilize azurophilic granules to the cell surface, but did markedly increase elastase release into the extracellular space in response to secondary stimulation with N-formyl-Met-Leu-Phe (fMLF). Priming of fMLF-stimulated elastase release was further augmented in the absence of NADPH oxidase-derived ROS. Our findings provide a mechanism for host tissue damage during neutrophil-mediated inflammation and suggest a novel anti-inflammatory role for the NADPH oxidase.

Keywords: Elastase; NADPH oxidase 2; inflammation; priming; reactive oxygen species; signaling.

Figure 1.

TNF-α elicits activation of cell surface proteins and mobilization of specific and gelatinase granules in a concentration-dependent fashion. Activation of the b₂-integrin CD11b and specific and gelatinase (tertiary) granule exocytosis occurs in response to TNF-α as measured by concentration-dependent increases in cell surface levels of active CD11b (a) and CD66b (b). Neither azurophilic granules nor secondary granules are mobilized by TNF-α alone, as evidenced by no change in CD63 (c) surface expression or lactoferrin release (d). Tertiary granule exocytosis, as measured by gelatinase (e) release, is elicited by TNF-α alone. TNF-α alone did not elicit secretion of elastase, as measured by a fluorometric activity assay (f), n = 5–9, *P < 0.05, ***P < 0.001 as compared to no agonist.

Figure 2.

p38 and ERK 1/2 MAPK involved in cell activation and exocytosis elicited by TNF-α priming. p38 MAPK inhibition (added concurrently with TNF-α) significantly diminished CD11b activation (a) and CD66b upregulation (b), whereas there was no impact on CD63 cell surface levels (c) in cells treated with TNF-α for 30 min. ERK 1/2 inhibition similarly diminished CD11b activation (d), with no effect on CD66b or CD63 cell surface levels ((e) and (f)). n = 6, *P < 0.05.

Figure 3.

TNF-α primes specific granule exocytosis and elastase release elicited by fMLF. Incubation with TNF-α markedly primes PMN for enhanced lactoferrin release in response to subsequent stimulation with fMLF (a). TNF-α alone elicits significant gelatinase release above baseline, and there is no synergistic effect of TNF-α plus fMLF (b). fMLF alone displayed a trend toward enhanced mobilization of elastase, but 30 min of priming with TNF-α markedly enhances elastase release (c), n = 9–12, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

TNF-α priming elicits direct intracellular endosomal ROS production. Intracellular ROS were measured using the OxyBurst Green-BSA probe. TNF-α (1 ng/ml) elicited time-dependent increases in intracellular fluorescence (OxyBurst GMI) (a). Incubation with 1 ng/ml TNF-α elicited a significant increase in the percentage of OxyBurst-positive cells (b), n = 6, *P < 0.05, **P < 0.01, ***P < 0.001 compared to no agonist.

Figure 5.

p38 MAPK is required for direct ROS production in response to TNF-α. ((a)–(c)) Direct ROS generation elicited by TNF-α alone is blocked in a time-dependent fashion by addition of the p38 MAPK inhibitor, SB203580. When added concurrently with TNF-α, all direct ROS is blocked, n = 7 (a) with partial inhibition when added at 15 min, n = 4 (b). (c) Inhibitor added at 30 min after peak ROS production is reached has no effect, n = 6. ((d)–(f)) Inhibition of the primed respiratory burst in response to subsequent stimulation with fMLF is correlated with the degree of direct ROS inhibition seen in (a)–(c). Significant reduction in the peak ROS generation occurs with time 0, n = 5 (d) or time 15 min, n = 4 (e), whereas inhibitor added just prior to fMLF had no effect (f), n = 5. ((a)–(f), lucigenin-enhanced chemiluminescence). ((g)–(i)) Using Amplex UltraRed as an additional complimentary assay, addition of the inhibitor at 0 or 15 min similarly elicited a significant reduction in H₂O₂ generated in response to fMLF, n = 7, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Neither p38 nor ERK 1/2 MAPKs are required for TNF-α priming of elastase release. Inhibition of p38 MAPK (a) or ERK 1/2 (b) had no effect on fMLF-mediated elastase release following priming with TNF-α. n = 6.

Figure 7.

NADPH oxidase-dependent signaling limits TNF-α priming of elastase release. CGD-PMN demonstrate enhanced elastase release in response to TNF-α alone (a). Priming of fMLF-mediated elastase release is enhanced in the absence of NADPH oxidase activity (either using DPI for inhibition, or CGD-PMN) (b). There were no differences in total measureable elastase release elicited in response to DHCB and fMLF in the setting of NADPH oxidase inhibition or genetic deficiency (c), n = 8–13 (n = 4 CGD), *P < 0.05, **P < 0.01 compared to control PMN.