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

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.

Therapeutic targeting of neutrophil exocytosis

“…This hierarchy correlates with the timely and sequential functions executed by the cargoes of different granules in the neutrophil response. Secretory vesicles are most readily released in response to stimuli, and complete mobilization of secretory vesicles to the plasma membrane occurs with in vitro stimulation and during in vivo transmigration into human skin chambers . Gelatinase granules demonstrate a higher stimulation threshold for exocytosis than secretory vesicles, and 30–40% of gelatinase granule cargoes are released extracellularly in vitro and in vivo .…”

“…Secretory vesicles are most readily released in response to stimuli, and complete mobilization of secretory vesicles to the plasma membrane occurs with in vitro stimulation and during in vivo transmigration into human skin chambers . Gelatinase granules demonstrate a higher stimulation threshold for exocytosis than secretory vesicles, and 30–40% of gelatinase granule cargoes are released extracellularly in vitro and in vivo . As with secretory vesicles, gelatinase granule fuse predominantly with the plasma membrane .…”

“…Specific granules are the third most responsive granule subset. Stimulation of human neutrophils in vitro with a single physiologic agent does not produce release of specific granule cargo . Neutrophils require a first stimulus to prepare them for the release of specific granule contents upon exposure to a second stimulus.…”

“…Azurophilic granules that do not express those regulators mobilize specifically to phagosomes . Extracellular release of azurophilic granule cargo after in vitro stimulation also requires priming, and in vivo transmigration induces extracellular release of about 7% of granules . Despite the limited number of azurophilic granules that engage in exocytosis per cell, the large number of neutrophils undergoing exocytosis during inflammation and infection provides sufficient cargo release to induce vascular and tissue damage .…”

Priming and de‐priming of neutrophil responses in vitro and in vivo

“…Secretory vesicles contain FPR1, CD11b and cytochrome b 558 . Priming alone leads to exocytosis of secretory and gelatinase vesicles, with subsequent increased release of specific and azurophilic granule contents on exposure to activating agents . Several signalling pathways regulating primed ROS production also influence degranulation.…”