Granule protein processing and regulated secretion in neutrophils

Abstract

Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines, and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First, we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking, and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection.

Keywords: Rho GTPase; cytokine; exocytosis; protein sorting; secretion.

Figure 1

Neutrophil morphology as observed by electron microscopy. Neutrophils were isolated from human peripheral blood, and processed for electron microscopy after incubation for 15 min at 37°C with vehicle (A,B) or 10 μM cytochalasin B and 5 μM fMLF (C,D) as described in Ref. (22). (A,B) The cytosol of a resting cell is filled with vesicles, with primary granules (P) staining intensely dark with DAB, while secondary (S) and tertiary (T) granules show more translucent staining. Secretory vesicles (SV) are in close proximity to the Golgi complex (G). Few mitochondria (M) are observed. (C,D) After stimulation only a few dense primary granules (P) remain and many empty vesicles (EV) appear. A double membrane autophagosome (AP) was observed to form around a primary granule; however, the relevance of this observation is unknown. Scale (A,C) = 8 μm × 8 μm, 9,100× magnification; (B,D) = 2.4 μm × 2.4 μm, 27,600× magnification.

Figure 2

Neutrophil Golgi complex as observed by electron microscopy. Neutrophils processed for electron microscopy as described in Ref. (22) show a small stacked Golgi with an increase in electron dense vesicle (arrows) after stimulation. Conditions were incubation with vehicle (A) or 10 μM cytochalasin B and 5 μM fMLF (B) for 15 min at 37°C. Scale, 2.4 μm × 2.4 μm, 27,600×magnification.

Figure 3

Schematic of neutrophil protein sorting pathways. Three prominent trafficking pathways for granule proteins are depicted: AP-1/GGA-dileucine based sorting, AP-3/AP-4-tyrosine-based sorting, and selective clustering-based sorting. Soluble hydrolases contain M6P signals and are sorted via the M6P-receptors, which contain dileucine signals. These complexes are shuttle to the sorting endosome through GGA and AP-1-dependent pathways. In the endosome, a drop in pH results in dissociation of the receptor–cargo complex with the retrieval of the M6P-receptor by an AP-1 dependent pathway. Serine proteases (neutrophil elastase, proNE) and trans-membrane proteins (CD63) contain tyrosine signals and are sorted directly to granules via AP-3/AP-4. Many granule proteins contain neither dileucine nor tyrosine signals and are thought to sort to granules via a clustering-based pathway since this occurs for many granule proteins that are highly concentrated. CD63 trafficking is unique as it can be found on several neutrophil membranes and may act to selectively retrieve granule proteins that escape normal trafficking via constitutive secretion.

Figure 4

Schematic of neutrophil signaling pathways regulating degranulation. Two pathways that regulate granule mobilization are depicted: upstream kinase cascade and downstream fusion machinery. Activation of neutrophils through surface receptors triggers the activation of a kinase cascade. Central downstream effectors of these kinases target cytoskeletal remodeling, these include Vav, which activates Rac, paxillin, which facilitates microtubule polarization, and the generation of PI(3,4,5)P3, which facilitates polarization and actin remodeling. Note that Rac is activated at multiple points in the signaling pathways (Vav*, P-Rex*) and hence may be needed for several immune cell functions in addition to degranulation. Granule tethering is regulated by Rab27 recruitment of Munc13-4. Munc13-4 is a calcium-sensitive link between Rab function and the fusion machinery of SNAREs. The Munc13-4:SNARE interaction requires calcium flux and is targeted by PKC, which are activated in the upstream kinase cascade.