Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

Review of the latest discoveries on the function of Rab27 GTPases and their effectors in neutrophil function, compared to their functions in other leukocytes.

Abstract

The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes.

Introduction

Exocytosis is an essential cellular mechanism for the release of secretory products and presentation of receptors and adhesion molecules. It involves vesicular trafficking through the cytoskeleton, the tethering of secretory vesicles at exocytic active sites, and vesicular docking, followed by membrane fusion events between the vesicular and the plasma membranes. This process finally leads to secretion of granule contents into the extracellular space. In leukocytes, exocytosis constitutes an important mechanism of the immune response that contributes to the creation of an antimicrobial milieu [1] and facilitates killing of invading microorganisms and infected host cells [2]. Most of our current understanding of the mechanism of exocytosis is based on static observations. However, recent evidence supports the notion that vesicles undergo highly dynamic processes before fusing with the plasma membrane. These dynamic processes include vesicular movements that increase the likelihood of significant molecular interactions between granular and motor or plasma membrane proteins, trafficking through the actin network and reversible docking. These pre-exocytic mechanisms control the rate and accuracy of the secretory event. Recent studies have shed light on the regulation of such processes and the role played by the cytoskeleton in this mechanism. In this review, we will focus on the pre-exocytic events that take place in the regulation of neutrophil (PMN) exocytosis. We will explore the role of the small GTPases from the Rab27 family and their specific effectors in neutrophil functions comparatively to their roles in other leukocytes.

Rab27a AND EFFECTORS IN HUMAN IMMUNODEFICIENCIES

GS2 is an autosomal recessive disorder caused by defects in the Rab27a gene located on chromosome 15 (15q21) [3, 4]. GS2 is a primary immunodeficiency with partial albinism [5, 6]. Patients with GS2 develop a disorder characterized by malfunctioning of CTLs, impaired NK cell function [5, 6] and defective neutrophil functions [6, 7]. At the cellular level, defects in hematopoietic cells are caused by the inability of CTLs, NK cells, and neutrophils to secrete intragranular proteins into infected cells or the extracellular milieu as a result of deficient priming and fusion of secretory organelles. GS2 patients suffer hypopigmentation, hepatosplenomegaly, neutropenia, thrombocytopenia, and immunodeficiency with recurrent and often fatal viral and bacterial infections [6]. Children with GS2 develop an uncontrolled T-lymphocyte and macrophage activation syndrome known as hemophagocytic syndrome or HLH, which usually results in death, unless the child receives a successful bone marrow transplant. Neurological manifestations, which appear in 67% of GS2 patients during the course of the disease, are secondary to the development of HLH-induced lymphocyte infiltration of the CNS, rather than a direct consequence of possible Rab27a dysfunction in the CNS [8].

FHL3 is also a major form of primary HLH caused by mutations in the UNC13-D gene located on chromosome 17 (17q25.1), which encodes for the Rab27a effector Munc13-4 [9]. Munc13-4 regulates secretion of lytic granules in CTLs and NK cells [2, 10, 11] and azurophilic and specific granules in neutrophils [12–14]. Patients with FHL3 suffer from immunodeficiency caused by defects in CTLs and NK cells, characterized by impaired priming of lytic granules prior to the fusion process with a subsequent defect in the release of cytolytic enzymes [9]. There is no hypopigmentation in FHL3, as Munc13-4 is not involved in melanosome transport. Similar to patients with GS2, individuals with FHL3 suffer from recurrent infections, and their life expectancy is very short unless successfully transplanted [15].

Two early case reports suggested that patients with GS have defects in the function of their granulocytes [6, 7]. One of those studies showed abnormal bactericidal activity in some of the patients evaluated [6], whereas the other work reported abnormalities in the phagocytic activity of neutrophils only in a subpopulation of the patients under study [7]. Although those studies supported the idea that the function of neutrophils is impaired in Rab27a deficiency, further studies were necessary to increase our knowledge of the role played by this GTPase and its effector molecules in neutrophil function. This review focuses on the findings from these studies.

THE PROCESS OF EXOCYTOSIS IN NEUTROPHILS

Neutrophils play a central role in innate immunity by combating bacterial and fungal infections. They kill microorganisms by releasing microbicidal products into the phagosome or into the extracellular space [1, 16–19]. In resting neutrophils, these microbicidal molecules are segregated and stored in secretory organelles, thus protecting the host from uncontrolled activation. Mature neutrophils contain four types of exocytosable storage organelles: azurophilic granules, specific granules, gelatinase granules, and secretory vesicles [20]. Exocytosable storage organelles could be subdivided according to their granule protein content (a detailed list of neutrophil granular proteins can be found in previous reviews; for example, see ref. [20]). These secretory organelles contain a battery of molecules that contributes to the precise implementation of many neutrophil functions [21]. Azurophilic (primary) granules contain MPO and the antimicrobial peptides defensins [22], cathepsin G, and lysozyme [21]. Specific (secondary) granules contain modulators of immune and inflammatory responses, such as lactoferrin [23] and MMP-9 [24], along with the membrane component of the NADPH oxidase, cytochrome b₅₅₈. The gelatinase (tertiary) granules are similar to secondary granules in terms of protein content but are enriched in MMP-9 [25] and have lower amounts of lactoferrin [26]. Secretory vesicles contain a set of membrane proteins, including the β2-integrin family member macrophage antigen 1 (CD11b/CD18) [27], which are translocated to the plasma membrane during activation. Mobilization of these organelles replenishes the plasma membrane with diverse receptor molecules that are essential for neutrophil function. It is generally accepted that granule exocytosis during neutrophil activation is hierarchical [20]. The secretory vesicles are mobilized first, while tertiary granules, specific granules, and azurophilic granules are mobilized sequentially in response to increasingly stronger stimuli [20]. The hierarchy that characterizes the exocytosis of these granules correlates with the different roles of their secretory proteins in the processes of adhesion, migration, chemotaxis, phagocytosis, and production of ROS.

EXPRESSION OF Rab27a GTPases AND EFFECTORS IN LEUKOCYTES

Rab GTPases are low molecular weight GTP-binding proteins, which together with their specific effectors, are implicated in the regulation of vesicular trafficking. In particular, GTPases from the Rab27 family regulate the efficiency and specificity of exocytosis in nonhematopoietic and hematopoietic cells, including PMNs [28, 29], CTLs [30], NK cells [31], and mast cells [32]. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In this way, whereas Rab27 GTPases are widely expressed [28], the expression of the Rab27 effectors is much more restricted. In addition, diverse secretory organelles in a particular cell type are often regulated independently, thus increasing the complexity of the analysis of the mechanisms regulated by Rab GTPases and effectors. Finally, Rab27 GTPases participate in multiple steps of the regulation of the secretory process (i.e., priming, tethering, docking, and fusion), and in some cells, this requires consecutive engagement of multiple Rab27 effector molecules. Therefore, although the mechanisms regulated by Rab27 GTPases are governed by the general process of GTPase-effector binding, the mechanisms differ significantly from cell to cell and even between organelles in the same cells. For this reason, experimentation and/or confirmation assays using primary cells in combination with analyses of endogenous protein functions are particularly important.

Eleven molecules have been identified in mammalian cells that interact with Rab27a (or Rab27b) and are considered, in most cases, Rab27-specific effectors [33]. The expression and function of these effectors in hematopoietic cells are summarized in Table 1. The topic of the molecular basis of these interactions has been discussed extensively in previous reviews [33, 42]and will be visited here only for some of the most relevant effectors in hematopoietic cells. The Slp family of Rab27 effectors was the first identified [43]. It is composed of five members: Slp1/JFC1/exophilin7 [35], Slp2a/exophilin4, Slp3/exophilin6, Slp4-a/granuphilin-a/exophilin2 [44], and Slp5/exophilin9 [45], encoded by genes SYTL1–5, located in loci 1p36.11, 11q14.1, 6q25.3, Xq21.33, and Xp21.1, respectively. Slp1, also known as JFC1, was the first Rab27 effector identified in hematopoietic cells, and expression of Slp1/JFC1 in human neutrophils was demonstrated [35]. Proteomics analysis of neutrophil granules has confirmed the expression of Slp1/JFC1 in human neutrophils and its localization at azurophilic granules [46]. Expression of Slp1, Slp2-a, and Slp3 has also been demonstrated in CTLs [37, 38], where they were shown to localize at the plasma membrane (Slp1 and Slp2 [37]) or at perforin-containing granules (Slp3 [38]). The Slp4 and Slp5 family members have not been identified in leukocytes so far [37].

Table 1. Hematopoietic Cell-Specific Expression/Function of Rab27 Effectors.

Cell Type	Slp1/JFC1	Slp2	Slp3	Munc13-4	Mlph/Slac2-a
Neutrophil	Azurophilic granule exocytosis [13, 29]; actin remodeling [34]; NADPH oxidase [35]	NA	NA	Azurophilic and tertiary granule exocytosis [12–14]; NADPH oxidase activity [36]; phagosomal maturation [36]	NA
CTLs	Plasma membrane localization [37]	Lytic granule exocytosis [37]	Lytic granule exocytosis [38]	Lytic granule priming and fusion [9, 10]	NA
NK cells	NA	NA	NA	Lytic granule exocytosis [11, 31]	NA
DCs	NA	NA	NA	NA	NA
Mast cells	NA	NA	NA	β-Hexosaminidase secretion [10 39]	β-Hexosaminidase secretion down-regulation [39]
Platelets	Dense granule secretion [40]	NA	NA	Dense core granule secretion [41]	NA

NA, Not available.

All members of the Slp family are characterized by the presence of a RBD27 (SHD) in their N-terminus and two tandem C2 domains (usually referred to as C2A and C2B domains) in their C-terminal end [33]. The SHD consists of two conserved α-helical regions, named SHD1 and SHD2 [47] or RBD27-1 and RAB27-2 [48]. Structural similarities between Slps include the presence of a conserved (S/T) (G/L)xW(F/Y)₂motif in the RBD27-2 [48]. This structure interacts with the α3-β5 loop contact interface of Rab27a [48], and mutations in the conserved tryptophan of the (S/T) (G/L)xW(F/Y)₂ motif decreases affinity for the GTPase [49, 50]. Despite these similarities, Slp3, Slp4, and Slp5 are characterized by the presence of zinc-finger motifs separating the two RBD27 motifs [33]. Slp1/JFC1 and Slp2 lack zinc-finger motifs and instead, contain a continuous RBD27 domain [33]. Comparison of the structures of the RBD27 of Slp2-a and Rabphilin3A suggested that the relative orientation of RBD27s in effectors lacking zinc-finger motifs is different from that in effectors with zinc-finger motives [48]. Further confirmation came from assays showing that removing the zinc-finger motif from Slp4-a leads to stronger interactions with Rab27a [48]. In principle, these studies suggest that the binding of Slp1/JFC1 and Slp2a to Rab27a uses a number of molecular interactions that are different from those used by Slps containing zinc-finger motives (Slp3–5).

A second group of Rab27 effectors, Slac, presents a Rab-binding SHD domain in their amino terminal but does not have C2 domains [33]. The Slac family comprises Slac2-a/Mlph and Slac2-b and Slac2-c/MyRIP, with genes located at loci 2q37.3, 11q22.3, and 3p22.1, respectively. Mlph regulates melanosome transport in melanocytes through bridging Rab27a-expressing melanosomes to the motor protein MyoVA [51, 52], and MyRIP plays a similar role in retinal pigment epithelial cells via MyoVIIa [53, 54]. In addition, a new role for Mlph in the regulation of mast cell exocytosis has been described recently [39]. A third group of Rab27 effectors includes Noc2 and Rabphilin3A (loci 17p13.3 and 12q24.13, respectively), which were described originally as Rab3-specific effectors [55]. Noc2 and Rabphilin3a have relatively conserved RBD27-containing zinc-finger motifs and bind to Rab27a with higher affinity than to Rab3 under physiological conditions [55]. Noc2 is expressed in endocrine and exocrine cells [56, 57], and immunolabeling showed localization of Noc2 on the limiting membrane of insulin-containing secretory granules in pancreatic β cells [58].

Finally, Munc13-4, the only Rab27 effector lacking SHD/RBD27 domains, contains two C2 domains, distributed in the amino and carboxy terminals, separated by two MHDs [9, 10, 59]. The RBD27a of Munc13-4 is unique. Initial studies using truncation mutants have mapped the Rab27a-interacting domain of Munc13-4 to aa 240–543 [10]. Subsequent studies used immunoprecipitation assays of overexpressed proteins in 293T cells to confirm the binding of Munc13-4 residues 1–402 to Rab27a [60], although a truncation containing the residues 240–402 of Munc13-4 was unable to pull down Rab27a in the same study, probably as a result of truncation misfolding [60]. Recent studies have now proposed that residues 240–290 of Munc13-4 are sufficient to maintain Rab27a binding and have mapped the important residues to aa 280-FQLIHK-285 [61]. This region is not completely conserved between Munc13 isoforms, and other proteins of the Munc13 family do not bind to Rab27a [61].

FUNCTION OF Rab27 GTPases AND EFFECTORS IN NEUTROPHILS

The roles of Rab27 GTPases in neutrophil exocytosis

Functional analyses showed that Rab27a, Slp1/JFC1, and Munc13-4 are essential regulators of granule exocytosis in neutrophils [12–14, 29]. Rab27a was originally demonstrated to regulate the secretion of azurophilic granules in neutrophils [29]. This initial study showed that Rab27a localized at a subpopulation of low-density MPO-containing granules, that interference with Rab27a function impaired azurophilic granule exocytosis in granulocytes, and that neutrophils from a novel Rab27a-deficient mouse model (concrete) had impaired mobilization and defective secretion of azurophilic granule cargoes [29]. The essential function of Rab27a in the exocytosis of azurophilic granules was demonstrated further using Rab27a-inhibitory antibodies in permeabilized human neutrophils stimulated with the chemotactic factor fMLP [13]. LPS-dependent priming of fMLP-induced exocytosis was also impaired in cells with deficient Rab27a function [13]. In agreement with these results, down-regulation of Rab27a expression in the HL-60 granulocytic cell line led to decreased azurophilic granule exocytosis [29]. An independent study confirmed the localization of Rab27a at a subpopulation of azurophilic granules but failed to show Rab27a involvement in azurophilic granule secretion in response to nonphysiological stimulation induced by calcium ionophores [62]. This suggested that Rab27a is necessary to regulate trafficking events that precede the calcium-dependent fusion of the granule and plasma membranes and underscored the need of stimuli that engage the prefusion exocytic events for the analysis of Rab27a function in leukocytes [63]. Subsequent studies confirmed a central role for Rab27a in azurophilic granule exocytosis using a different mouse model of Rab27a deficiency (ashen) under physiological stimulation [63]. The involvement of Rab27a in azurophilic granule secretion regulation was demonstrated further in vivo [29, 64]. In these studies, decreased in vivo plasma levels of MPO were observed in two independent studies using two different mouse models of Rab27a deficiency (concrete and ashen) during LPS-induced systemic inflammation. Given that even under strong stimulatory conditions, i) only ∼20% of azurophilic granules are able to undergo exocytosis [65], ii) only ∼20% of total azurophilic granules express Rab27a [29], and iii) Rab27a-deficient neutrophils fail to undergo azurophilic granule exocytosis [13, 29, 63, 64], it was proposed that azurophilic granules are functionally heterogeneous and that the Rab27a-expressing subpopulation of low-density azurophilic granules constitutes the exocytosable azurophilic granule population [29]. Altogether, these studies have characterized Rab27a as a central player in the regulation of azurophilic granule exocytosis in neutrophils.

Rab27b shares 72% identity with Rab27a [66, 67], binds to Rab27a effectors, and plays an important role in the regulation of many secretory mechanisms [67–70]. However, crystal structure analyses showed that Rab27b, but not Rab27a, forms a dimmer, which mediates Rab27b-effector recognition, highlighting differences in the molecular arrangements between the two Rab27 GTPases and their effectors [71]. Neutrophils express Rab27b, and its expression is up-regulated in Rab27a-deficient cells [63]. These results suggested that the expression of Rab27b is transcriptionally or translationally linked to Rab27a expression levels. However, normal Rab27a expression levels were observed in Rab27b KO neutrophils [63]. Studies of the subcellular distribution of Rab27b show peripheral distribution and relatively low colocalization with azurophilic granules [63]. Different from Rab27a KO neutrophils, Rab27b KO neutrophils have only moderate impairment of azurophilic granule secretion [63]. These results, together with the observations that the expression of Rab27b is up-regulated in Rab27a^ash/ash neutrophils but does not compensate for the lack of Rab27a function and that the expression of Rab27a in Rab27b KO cells is similar to that observed in WT neutrophils, support the idea that these GTPases play important yet different roles during azurophilic granule exocytosis in neutrophils.

In addition to its role in azurophilic granule secretion, other studies have suggested that Rab27a regulates gelatinase/specific granule exocytosis in neutrophils [13, 62, 63]. A role for Rab27a in the secretion of MMP-9 in response to physiological stimulation was shown using permeabilized human neutrophils in the presence of inhibitory antibodies, which decreased fMLP-induced secretion in LPS-primed and unprimed neutrophils [13]. Secretory vesicles are exocytosable endocytic compartments that contain important proteins, including receptors, which are up-regulated at the plasma membrane during secretory vesicle exocytosis. This includes the β2-integrin subunit CD11b, which is also present at the membrane of other secretory organelles in neutrophils. In response to weak stimulation, CD11b is up-regulated at the plasma membrane mainly from the readily releasable secretory vesicle pool. Analyses of CD11b mobilization performed with Rab27a and Rab27b KO neutrophils suggested that these GTPases are not involved in the exocytosis of secretory vesicles [63, 64].

Mechanisms regulated by Slp1/JFC1 in vesicular trafficking and exocytosis

Slp1/JFC1 was identified in leukocytes from a human B lymphoblast cDNA library as the binding partner of the NADPH oxidase cytosolic factor p67^phox [35] and simultaneously cloned as a member of the Slp family [43]. Slp1/JFC1 is expressed in neutrophils [29, 35]. Similar to Rab27a, Slp1/JFC1 expression is up-regulated during granulocytic differentiation of promyelocytic cells [29], further supporting their role in granulocyte function. The amino terminus of Slp1/JFC1 is characterized by the presence of a RBD homologous to that present in Rabphilin3a. However, Slp1/JFC1 does not bind to Rab3a (unpublished results) [43]. Slp1/JFC1 was then shown to bind Rab27a by Seabra's and Fukuda's groups [72, 73], and this was confirmed later by our group [74]. In neutrophils, Slp1/JFC1 colocalizes with the subpopulation of secretory azurophilic granules [29]. The presence of Slp1/JFC1 in azurophilic granules was also confirmed by mass spectrometry analysis [46]. Dual-color live cell imaging was used to simultaneously visualize the localization of Slp1/JFC1 and Rab27a in a spatiotemporal manner in granulocytes [13]. This study demonstrated true colocalization of Slp1/JFC1 and Rab27a in real-time [13]. Immunoprecipitation of endogenous proteins showed that Rab27a and Slp1/JFC1 are partners in neutrophils and granulocytic cell lines [29]. Functional assays demonstrated that Slp1/JFC1 is an essential Rab27a effector in human neutrophils [13]. Interference with Slp1/JFC1 function by means of inhibitory antibodies impaired azurophilic granule exocytosis in response to the chemotactic factor fMLP [13]. An essential role for Slp1/JFC1 in azurophilic granule exocytosis was confirmed recently using neutrophils from JFC1 KO mice (Sytl1^−/−) [34]. This differs from the role of Slp1/JFC1 in CTLs, in which lack of Slp1/JFC1 expression failed to impair CTL-mediated killing of target cells [37]. However, simultaneous interference with Slp1 and Slp2a inhibited secretion in CTLs [37]. Thus, neutrophils constitute the only leukocyte identified so far for which Slp1/JFC1 is essential for regulated secretion.

The C2A domain of Slp1/JFC1 was initially demonstrated to bind phosphoinositides [75]. In particular, Slp1/JFC1-C2A binds to phosphatidylinositol (3,4,5)-triphosphate and localizes at the plasma membrane [75]. Plasma membrane localization of Slp1/JFC1-C2A is increased in phosphatase and tensin homolog-deficient cells and is inhibited by a net increase in intracellular calcium [75]. Together with the observation that Slp1/JFC1 binds to Rab27a through its amino terminal RBD27, these studies suggested that Slp1/JFC1 may participate in vesicular docking by bridging Rab27a-containg vesicles to phosphoinositide-enriched microdomains at the plasma membrane and that calcium increase, during the last step of the exocytic pathway, may induce the recycling of this Rab effector.

Mass spectrometry analysis of the molecular interactions of Slp1/JFC1 with endogenous neutrophil proteins revealed that Slp1/JFC1 binds to GMIP, a RhoA-GAP involved in actin remodeling [34]. The Slp1/JFC1-GMIP interaction was mapped to the C2B domain of Slp1/JFC1 [34]. Analysis of vesicular dynamics showed that Slp1/JFC1-expressing vesicles move in areas deprived of polymerized actin and that granules containing Slp1/JFC1 exclude polymerized actin from surrounding areas and do not visit areas with high-actin turnover [34]. Based on these data, it was suggested that Slp1/JFC1 is a secretory component that regulates vesicular trafficking through the cytoskeleton to facilitate vesicular docking and fusion with the plasma membrane [34]. A role for the Slp1/JFC1-binding molecule GMIP in the regulation of actin remodeling to facilitate exocytosis was supported further by the observation that down-regulation of GMIP increases actin polymerization [34]. Consequently, GMIP-deficient granulocytes have decreased vesicular trafficking, increased vesicular trapping in cortical actin, and impaired exocytosis [34]. A role for Slp1/JFC1 and the RhoA-GAP GMIP in the regulation of vesicular trafficking and exocytosis was clarified by the observations that RhoA localizes at azurophilic granules and that inactivation of the RhoA signaling pathway induces actin depolymerization and enhances exocytosis in neutrophils (ref. [34] and Fig. 1). Furthermore, using a RhoA biosensor in combination with fluorescence resonance energy transfer microscopy, it was demonstrated that RhoA colocalizes with Slp1/JFC1-positive granules and that RhoA activity surrounding areas of Slp1/JFC1-expressing granules showed a polarized distribution [34]. This suggested a possible role for RhoA in the regulation of directional movement of secretory granules [34]. Importantly, definite roles for Slp1/JFC1 and GMIP in facilitating vesicular trafficking through cortical actin during exocytosis were demonstrated using Slp1/JFC1 KO and GMIP-deficient granulocytes [34]. In the absence of either of these secretory proteins, azurophilic granules are trapped in and unable to traverse cortical actin, and therefore, neutrophil exocytosis is impaired.

Figure 1. Schematic model of GMIP and Slp1/JFC1 regulation of vesicular dynamics and exocytosis in neutrophils.

The small GTPase Rab27a recruits the effector JFC1 to secretory organelles. JFC1 interacts with the RhoA-GAP GMIP through its C2B domain [34], but whether this interaction is sufficient to recruit GMIP to secretory organelles is still obscure. RhoA localizes at azurophilic granules, and GMIP down-regulation induces RhoA activation and increases actin polymerization [34]. Down-regulation of GMIP or JFC1 increases granule entrapment in cortical actin and decreases exocytosis, while inhibition of the RhoA-downstream signaling pathway has the opposite effect [34]. In the absence of Rab27, JFC1, or GMIP, secretion is impaired.

Other molecular interactions of Slp1/JFC1 have been described, although whether they are relevant for leukocyte functions is still unknown. For example, overexpression and pull-down experiments showed that Slp1/JFC1 binds to the Rab-GAP EPI64, which has GAP activity for Rab27a and Rab8 [76]. Slp1/JFC1 binding to EPI64 was proposed to inhibit Rab8 function in HeLa cells [76]. However, Slp1/JFC1–EPI64 interaction is weak at endogenous protein levels [76], and neither EPI64 nor Rab8 expression was observed in leukocytes so far. In addition, Slp1/JFC1 was found to interact with Rap1GAP2, a GAP for Rap1 in platelets, where it was proposed to regulate dense granule secretion [40]. Finally, Slp1/JFC1 was proposed to regulate anterograde transport of the neurotrophin receptor TrkB in axons through interaction with collapsin response mediator protein-2 [77]. Whether these interactions are relevant for leukocyte function awaits further research.

Mechanisms regulated by Munc13-4 in vesicular trafficking and exocytosis

Munc13-4 was identified based on its homology to other members of the Munc13 family of proteins and was first cloned in rats, where it was found to be expressed predominantly in lungs at goblet cells of the bronchial epithelium and alveolar type II cells [59]. Subsequent studies characterized high levels of Munc13-4 expression at hematopoietic cells and determined that Munc13-4 is essential for cytolytic granule fusion in CTLs [9]. Munc13-4 mutations were then found responsible to be causative of FHL3 [9, 78]. In later studies, Munc13-4 was characterized as a Rab27a effector in platelets [41] and CTLs [10]. With the use of N-ethyl-N-nitrosourea-induced mutagenesis, a Munc13-4-deficient mouse (Jinx) was generated [79]. Jinx presented with deficient degranulation in NK cells and CTLs and is characterized by mouse cytomegalovirus susceptibility with high viral proliferation causing death [79], a phenotype that resembled that observed in humans with FHL3. Although Munc13-4 function was rapidly associated with a regulatory mechanism of secretory lysosome exocytosis in CTLs, NK cells, and mast cells, a role for Munc13-4 in granulocyte exocytosis was yet to be demonstrated.

In granulocytes, a pool of Munc13-4 associated with mobilizable tertiary granules is relocalized to the plasma membrane after stimulation with chemotactic peptides [12]. The effect was proposed to be mediated by increases in intracellular calcium, as the same effect was observed in neutrophils treated with ionomycin/Ca²⁺ and abolished by calcium chelators [12]. In the same work, the authors showed that the C2 domains of Munc13-4 bind to phospholipids in a calcium-independent manner, raising the question of whether calcium-induced translocation of Munc13-4-expressing vesicles to the plasma membrane occurs independently of Munc13-4 function or whether Munc13-4 is instead translocated from the cytosolic pool [12]. A direct role for Munc13-4 in secretion was demonstrated using PBL-985 granulocytes, in which Munc13-4 expression was attenuated using small interfering RNAs [12]. In those studies, a moderate but significant reduction of secretion was observed in response to fMLP [12]. In agreement with these results, a study by our group [13] showed that Munc13-4 regulates exocytosis of gelatinase and azurophilic granules in human neutrophils. In this study, endogenous Munc13-4 was found to colocalize with MMP-9, as well as with a subpopulation of exocytosable azurophilic granules [13]. In addition, inhibition of Munc13-4 using antibodies in permeabilized human neutrophils showed decreased fMLP-dependent MPO secretion in LPS-primed cells [13]. A function for Munc13-4 in neutrophil exocytosis was demonstrated further in the same work using neutrophils from the Munc13-4 null mouse model Jinx. Neutrophils from Munc13-4^jinx/jinx showed deficient MPO exocytosis and impaired mobilization of IL-10Rs, which are stored in specific granules [13]. Similar to Rab27a-deficient neutrophils, Munc13-4^jinx/jinx neutrophils showed normal mobilization of the β2 integrin subunit CD11b, indicating that Munc13-4 function in neutrophil exocytosis is selective for a group of secretory granules, excluding the readily mobilizable pool of secretory vesicles that contain CD11b [13].

A recent work by my group [14] increased insight into the mechanism regulated by Munc13-4 during azurophilic granule exocytosis. In this work, we showed that Munc13-4 regulates vesicular dynamics and LPS-induced priming of exocytosis in neutrophils. We demonstrated that Rab27a and Munc13-4 are essential components of the secretory machinery that regulates LPS-mediated priming of fMLP-induced exocytosis in neutrophils. This was accompanied by data showing that Rab27a- and Munc13-4-deficient neutrophils have a decreased number of azurophilic granules within the exocytic active zone [14, 63]. However, Rab27a was dispensable for the Munc13-4-dependent regulation of a readily releasable pool of azurophilic granules in response to LPS [14]. This suggested that not all Munc13-4 functions are Rab27a-dependent. These data are in agreement with results obtained using other hematopoietic cells. For example, despite its specific interaction with Rab27a, studies showed that Munc13-4 association with secretory lysosomes required MHDs but is independent of Rab27a binding [10]. In agreement with these studies, experiments using CTLs overexpressing recycling and late endosomal markers showed that Munc13-4 regulates lysosomal maturation into a fusion-competent lytic organelle in a Rab27a-independent manner [60]. Subsequent studies of endogenous proteins demonstrated that Rab27a or Munc13-4 recruitment to lytic granules is preferentially regulated by different receptor signals [31]. Studies using Munc13-4 point mutants that lack Rab27a binding ability demonstrated that vesicle tethering to the plasma membrane, but not secretory lysosome maturation, is a Rab27a-dependent mechanism in CTLs [61]. In neutrophils, azurophilic granule maturation was studied by our group, by analysis of the distribution of endogenously expressed secretory proteins on vesicles located in close proximity to the plasma membrane [14]. In these studies, a moderate but significant increase in the number of Munc13-4-expressing vesicles containing Rab27a was observed after stimulation with LPS, an agent that induces priming of exocytosis. This supported the idea that a mechanism involving vesicle maturation may contribute, at least in part, to LPS-dependent priming of neutrophil exocytosis [14]. However, different from that observed in CTLs [60], poor or no colocalization was observed between Rab11-positive recycle endosomes and secretory lysosomes in neutrophils [14], suggesting that maturation processes are cell-specific.

Munc13-4 was proposed originally to regulate vesicular fusion [9]. Other studies suggested that it regulates vesicular docking instead [61]. The mechanism of Munc13-4-mediated docking was studied in neutrophils using a mouse model expressing EGFP-Rab27a at endogenous levels but lacking Munc13-4 [14]. With the use of this cellular system, in combination with the analysis of vesicular dynamics by total internal reflection fluorescence microscopy, LPS stimulation, a treatment that induces priming of exocytosis but weak vesicular fusion, was found to mediate a marked reduction in the speed and displacement of Rab27a-expressing vesicles at the exocytic active site [14]. Subsequent, detailed observations demonstrated that docked granules are not motionless but rather motion-restricted [14]. These granules were able to undergo significant movement with limited displacement in small areas parallel to the plasma membrane. This suggested that putatively docked granules are able to visit subdomains of a much more restricted area of the plasma membrane than undocked granules [14]. Importantly, the mechanism of motility restriction was abolished in neutrophils lacking Munc13-4, suggesting that Munc13-4 is an essential component of the docking machinery of Rab27a-expressing organelles (ref. [14] and Fig. 2). Other studies have suggested that Munc13-4 regulates fusion instead of docking [9]. This was supported by electron micrographs showing that Munc13-4-deficient CTLs are able to align secretory lysosomes at the immunological synapse, although the granules are unable to deliver their cargo to the infected cells [9]. In vitro studies demonstrated that Munc13-4 binds to SNARE proteins through its MHD1 [80], further supporting a possible role for Munc13-4 in vesicular fusion in hematopoietic cells. It seems possible then that Munc13-4 regulates docking and fusion. Coregulation of these processes has not been demonstrated directly until recently. Using neutrophils from Munc13-4 KO mice, we showed that Munc13-4 controls the translocation and/or retention of p22^phox-containing vesicles to the plasma membrane, as well as the integration of predocked vesicles with the plasmalemma [36].

Figure 2. Munc13-4 regulates LPS-induced vesicular docking in neutrophils.

Representative images showing kinetics of granules with displacement <0.5 μm for each condition. Motility of granules with short displacement (those for which the distance between the coordinates at the beginning and the end of the analysis was <0.5 μm) was restricted significantly after LPS treatment in WT cells but not in Munc13-4-deficient neutrophils. Also, the average speed of motion-restricted granules was decreased significantly after LPS treatment in WT cells but not in Munc13-4-deficient neutrophils [14]. The diameter of the circles depicted represents 0.5 μm. This research was published originally in ref. [14]. Tg, Transgenic.

REGULATION OF NEUTROPHIL-SPECIFIC FUNCTIONS BY Rab27a GTPase AND ITS EFFECTORS

Accurate vesicular trafficking is necessary for the regulation of most neutrophil functions, including exocytosis, phagocytosis, chemotaxis, and the production of ROS. For example, the activation of the NADPH oxidase at the plasma membrane is associated with the process of exocytosis [81]. Importantly, Rab27a KO and Munc13-4 KO neutrophils have deficient extracellular ROS production in response to soluble stimuli [36, 63]. This is associated with deficient fusion of flavocytochrome b₅₅₈-containing vesicles with the plasma membrane in Munc13-4 KO neutrophils [36]. Interestingly, intraphagosomal ROS production induced by opsonized zymosan or serum-opsonized live bacteria, including Pseudomona aeruginosa and Staphylococcus aureus, is not affected in Rab27a-deficient cells [36, 63, 82]. This correlates with the observations that neither Rab27a nor Slp1/JFC1 is recruited to the phagosomal membrane during particle engulfment [29], that absence of Rab27a does not affect phagosomal maturation during serum-opsonized bacterial phagocytosis, and that delivery of p22^phox to the phagosomal membrane is not impaired in Rab27-deficient neutrophils under these experimental conditions [36]. Other studies suggested that Rab27a plays an inhibitory rather than a positive regulatory role in phagocytosis by macrophages [83]. This is in disagreement with results seen in DCs, in which Rab27a plays a role in phagosomal maturation, NADPH oxidase delivery to the phagosome, and antigen presentation [84]. In neutrophils or macrophages, participation of Rab27a in phagocytosis was only observed during engulfment of IgG-opsonized red blood cells [82, 85] but not IgG-opsonized zymosan, which not only engages FcγRs but also binds and signals through mannose receptors and CR3 [85] or serum-opsonized live bacteria [36, 82], suggesting that Rab27a is not essential for phagocytosis in neutrophils and macrophages under conditions of systemic infection that involve complex opsonization. Importantly, we demonstrated that different from Rab27a, Munc13-4 is essential for phagosomal maturation during engulfment of serum-opsonized bacteria in neutrophils [36]. Under our experimental conditions, Munc13-4 regulates the delivery of azurophilic granules and multivesicular bodies to the phagosomal membrane, highlighting Rab27a-independent functions for Munc13-4 during the innate immune response [36]. In addition, a function for Rab27a in the regulation of chemotaxis was first shown in T cells [86] and later confirmed in neutrophils [87], where it was suggested that Rab27a-mediated protease release facilitates neutrophil recruitment by allowing uropod detachment. Finally, neither Rab27a nor its effectors are essential for NET formation or delivery of granular cargo to NETs [36, 88]. In fact, Munc13-4 KO cells have a significant increase in the number of cells producing NETs in response to bacterial insult [36]. Importantly, despite this compensatory mechanism, Munc13-4 KO neutrophils have a significantly impaired bactericidal activity that mainly accounts for defects in phagosomal maturation that lead to impaired intraphagosomal killing but also for deficient NET-associated killing [36].

ROLE OF Rab27a AND EFFECTORS IN SYSTEMIC INFLAMMATION

Genetic defects in the Rab27a and the Munc13-4 genes lead to immunodeficiencies in humans, characterized by frequent viral and bacterial infections. However, the responses of Rab27a- or Munc13-4-deficient organisms to systemic inflammation are different. A recent study presented evidence supporting these distinct roles for Rab27a and Munc13-4 in the regulation of systemic inflammation induced by LPS [64]. In this work, neutrophil retention in liver sinusoids, a hallmark of LPS-induced systemic inflammation, was impaired in Rab27a-deficient but not Munc13-4 KO mice [64]. This correlated with decreased expression of CD44, a mediator of neutrophil attachment to synusoids, in Rab27a- but not Munc13-4-deficient neutrophils [64]. With the use of a model of local bronchiolar inflammation induced by MIP-2 intranasal administration, others showed that neutrophil infiltration into lungs is deficient in Rab27a KO mice [87]. The defect was attributed to abnormal chemotactic response of Rab27a-deficient neutrophils. However, lung infiltration by neutrophils was normal in Rab27a KO mice treated systemically with LPS [64]. It is possible then that some of the inflammatory pathways activated during systemic inflammation can bypass the Rab27a-dependent mechanisms used by neutrophils to migrate into lungs. Importantly, mice respond to LPS insult with elevated levels of MPO in plasma [29, 64], but mice deficient in Rab27a or Munc13-4 have reduced plasma MPO levels after LPS administration [29, 64], and similar to MPO-deficient mice [89], Rab27a KO and Munc13-4 KO mice are protected from LPS-induced death [64]. Although Rab27a KO mice showed other protective effects, including decreased TNF-α secretion [64], these effects were absent in Munc13-4 KO mice, supporting the idea that decreased MPO secretion, similar to MPO deficiency [89], is associated with increased survival in endotoxemia. However, as the effect on survival was less manifested in mice lacking effectors than in those lacking Rab27a [64], it is likely that Slp1/JFC1 and Munc13-4 have some redundant functions in vivo. Altogether, these data support a proinflammatory role of Rab27a and effectors.

CONCLUDING REMARKS

The mechanism of vesicular trafficking has gained great attention in the past decade. It is now clear that vesicular transport is a process of fundamental importance in the regulation of leukocytes and the innate immune response and that Rab27a and its effectors are major contributors to this regulatory process. Future experiments are expected to define selective and specific functions for Rab27a effectors, as well as establish potential opportunities for therapeutic intervention for the treatment of inflammatory processes and possibly, immunodeficiencies.

AUTHORSHIP

S.D.C. wrote the manuscript and created figures.