Therapeutic Targeting of Neutrophil Exocytosis

Abstract

Dysregulation of neutrophil activation causes disease in humans. Neither global inhibition of neutrophil functions nor neutrophil depletion provide safe and/or effective therapeutic approaches. The role of neutrophil granule exocytosis in multiple steps leading to recruitment and cell injury led each of our laboratories to develop molecular inhibitors that interfere with specific molecular regulators of secretion. This review summarizes neutrophil granule formation and contents, the role granule cargo plays in neutrophil functional responses and neutrophil-mediated diseases, and the mechanisms of granule release that provide the rationale for development of our exocytosis inhibitors. We present evidence for the inhibition of granule exocytosis in vitro and in vivo by those inhibitors and summarize animal data indicating that inhibition of neutrophil exocytosis is a viable therapeutic strategy.

Summary sentence:

Review the rationale for development of pharmacologic inhibitors of neutrophil granule exocytosis and the therapeutic potential of those inhibitors.

INTRODUCTION

Evidence for neutrophil participation in human disease includes the presence of neutrophils at the site of tissue injury and a reduction of organ damage in animal models after antibody-mediated or genetic depletion of neutrophils (1-4). Increasing evidence indicates that neutrophils contribute to inflammatory arthritis, acute lung injury, ischemia reperfusion injury, bullous skin lesions, inflammatory bowel disease, systemic lupus erythematosus (SLE) and other autoimmune diseases, immune complex glomerulonephritis (GN), atherosclerosis, and cancer (5-16). A role for neutrophils in initiation and progression of pathological processes leading to organ damage in those diseases makes neutrophils attractive therapeutic targets. The critical requirement for an adequate number of functional neutrophils for control of the microbial environment, as demonstrated by the risk of life-threatening infection in patients with leukopenia, limits the usefulness of therapies that globally disrupt neutrophil number or functional responses. On the other hand, therapies that target single molecular mediators of inflammation demonstrate limited efficacy likely due to redundancy within the innate and adaptive immune systems. Our strategy for interrupting neutrophil-mediated tissue injury, without impairing their ability to control microbial invasion, is to create molecular inhibitors of granule trafficking and fusion with the plasma membrane. This strategy is based on the central role granule exocytosis plays in many neutrophil responses, including recruitment to sites of inflammation, regulation of other immune cells, and cellular injury caused by generation of reactive oxygen species (ROS) and release of toxic granule cargo. We postulate that inhibition of granule exocytosis will prevent extracellular release of cytotoxic molecules while preserving neutrophil antimicrobial activity within phagosomes. This review summarizes neutrophil granule formation and contents, the role granule cargoes play in neutrophil functional responses and neutrophil-mediated diseases, and mechanisms of granule release that provide the rationale for development of exocytosis inhibitors. We review evidence of the effectiveness of granule exocytosis inhibition in vitro and in vivo and summarize studies indicating that in vivo inhibition of neutrophil exocytosis prevents organ damage in animal models of human disease.

Granulopoiesis and Granule Contents

During their maturation in the bone marrow, neutrophils acquire a heterogeneous complement of secretory granules and vesicles that provide a reservoir of antimicrobial proteins and peptides, proteases, membrane components of the NADPH oxidase and other pro-oxidant proteins, receptors, adhesion molecules, and soluble mediators of the inflammatory response (17,18). Granules are continuously formed from the Golgi beginning at the transition from myeloblast to promyelocyte stages and continuing until the segmented stage (19). Based on physical properties and expression of marker proteins, three subsets of granules are commonly accepted: azurophilic (primary), specific (secondary), and gelatinase (tertiary) granules. Synthesis of granule proteins at the time of granule subset formation is postulated to dictate their content, a hypothesis termed “targeting by timing.” Comparison of mRNA expression during neutrophil development coincides with granule subset protein distribution in the majority of proteins. Granule proteins with appropriate timing of mRNA expression include markers used to identify azurophilic (MPO, elastase, and CD63), specific (lactoferrin and neutrophil gelatinase-associated lipocalin (NGAL), and gelatinase (matrix metalloproteinase 9, MMP9) granules (20). The discrepancy in timing of mRNA expression for a substantial minority of granule proteins, however, suggests that factors in addition to timing of protein synthesis affect granule content.

Granules containing large amounts of myeloperoxidase (MPO) are earliest to form in promyelocytes and are termed peroxidase-positive granules. They are also called primary granules because they are formed first and azurophilic granules because of characteristic staining by the dye azure A. At least 850 proteins are associated with azurophilic granules by proteomic analysis (20,21), of which 135 are maximally or exclusively expressed in azurophilic granules compared to the other granule subsets (20). In addition to MPO, the dense matrix of azurophilic granules contains a large number of antimicrobial proteins, including serine proteases (proteinase-3, cathepsin G, elastase, and azurocidin), alpha-defensins, and bactericidal/permeability-increasing protein (BPI). Azurophilic granule proteases are activated by proteolytic processing prior to incorporation into granules, resulting in a highly toxic cargo capable of directly injuring cells upon release (22). Membranes of azurophilic granules contain relatively few proteins (17,20,21,23). Differences in protein synthesis during granule formation result in further heterogeneity within this subset, exemplified by the presence of defensin-positive and defensin-negative azurophilic granules. That heterogeneity may determine different membrane targets, as azurophilic granules targeting the plasma membrane are Rab27 and Slp1/JFC1-positive, while granules targeting phagosomes are Rab27 and Slp1/JFC1-negative (24).

Peroxidase-negative granules are separated into specific and gelatinase granule subsets, and further heterogeneity within those subsets is described. Peroxidase-negative granules form throughout myelocyte, metamyelocyte, and band stages. Specific (secondary) granules are the first peroxidase-negative granules formed and are defined by the presence of lactoferrin and NGAL and by the absence of gelatinase (25). Of 1024 proteins associated with specific granules, 111 are maximally expressed in that granule subset (20). In contrast, gelatinase (tertiary) granules contain gelatinase (MMP9), but lack lactoferrin and NGAL. Consistent with the targeting by timing hypothesis, a group of hybrid peroxidase-negative granules contains all three proteins. This hybrid group constitute around 60 to 70% of the total peroxidase negative granules and thus gelatinase and specific granules exist as a continuum of organelles (25). Granules containing gelatinase and ficolin-1, termed ficolin-1-rich granules, are proposed as a separate granule subset (20,26). Only 30 of 1123 proteins are maximally expressed in gelatinase granules, however, while 246 of 1193 proteins are maximally expressed in ficolin-rich granules (20). The small number of proteins maximally expressed in gelatinase granules suggests a limited value in separating gelatinase granules and ficolin-rich granules.

A fourth secretory compartment, termed secretory vesicles, was identified based on enhanced membrane-associated alkaline phosphatase after exposure of neutrophils to detergent. That observation led to the discovery of a compartment of smaller intracellular vesicles with reversed membrane orientation of alkaline phosphatase that are dispersed throughout the neutrophil cytoplasm (27-29). Secretory vesicle membranes contain numerous proteins also present in the plasma membrane, including chemoattractant and phagocytic receptors, adhesion molecules, and membrane components of the NADPH oxidase; while the matrix contains plasma proteins (18,20,28,30). Based on membrane and matrix composition, secretory vesicles are postulated to form by endocytosis during the band and segmented stages. However, there are also distinct differences in membrane composition between secretory vesicles and plasma membrane (30). Additionally, secretory vesicles acquire a distinct molecular machinery that mediates mobilization to and fusion with the plasma membrane. Those observations suggest that endocytosis alone does not account for secretory vesicle formation.

Given the heterogeneity of neutrophil granule subsets, what is the merit of granule subset classification? In addition to differences in cargo and matrix density among granule subsets, the value of classification of neutrophil granules is supported by differences in stimuli required for granule subset mobilization, in membranes targeted for fusion, and in molecules controlling membrane targeting and fusion. The hierarchical mobilization of granule subsets occurs both in vitro and in vivo, a process termed graded exocytosis. This hierarchy correlates with the timely and sequential functions executed by 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 (31-33). Gelatinase granules demonstrate a higher stimulation threshold for exocytosis than do secretory vesicles, and 30% to 40% of gelatinase granule cargo are released extracellularly in vitro and in vivo (32,33). As with secretory vesicles, gelatinase granule fuse predominantly with the plasma membrane (34). 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 (33,35). Neutrophils require a first stimulus to prepare them for release of specific granule contents upon exposure to a second stimulus. This sequential “two-hit” model for enhanced neutrophil responses is commonly referred to as “priming” (36). Transmigration in vivo induces extracellular release of 22% of specific granules (32). Specific granules fuse with nascent phagosomes as part of their general translocation to the plasma membrane (37,38). Around 20% of azurophilic granules are mechanistically equipped to release their cargo extracellularly because their granule membranes contain the regulators of exocytosis Rab27a and Slp1/JFC1 (24,39). Azurophilic granules that do not express those regulators mobilize specifically to phagosomes (37,38). Extracellular release of azurophilic granule cargo after in vitro stimulation also requires priming (33,35), and in vivo transmigration induces extracellular release of about 7% of granules (32). 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 (38-45). The continuous formation of granules during neutrophil maturation leads to overlapping contents and granule heterogeneity that blurs the lines between different granule subsets. Despite that overlap, differences among the majority of granules in each subset makes the current classification system a useful tool to explain the relationship between granule mobilization and neutrophil functional responses.

Role of Exocytosis in Neutrophil Function

Neutrophil recruitment and microbial killing is a multi-step process involving capture of neutrophils by, and progressively firmer adhesion to, vascular endothelium; transmigration across the vessel wall into the interstitial space; chemotaxis to the site of microbial invasion; phagocytosis of microorganisms; and mobilization of antimicrobial molecules (36,46,47). Based on our knowledge of granule subset content and graded exocytosis, granule mobilization is postulated to participate in each step of neutrophil activation. Experimental data generally support this hypothesis. Secretory vesicle exocytosis increases plasma membrane expression of adhesion molecules such as CD11b/CD18, which mediates firm adhesion, crawling, and migration through the endothelial cell barrier (48). Deficiency of CD11b/CD18 expression or activity results in Leukocyte Adhesion Deficiency (LAD), a rare inherited disorder that is associated with life-threatening bacterial and fungal infections due to impaired neutrophil recruitment (49). Engagement of neutrophil adhesion receptors with selectins on endothelial cells during rolling activates a constitutively expressed integrin, CD11a/CD18, leading to neutrophil arrest (50). G protein-coupled receptors bind chemokines expressed on the surface of endothelial cells, leading to a conformational change required for high affinity ligand binding by CD11a/CD18 (46), an integrin that is constitutively expressed at the plasma membrane. Signaling initiated by chemokine receptors and CD11a/CD18 induces secretory vesicle exocytosis, resulting in translocation of stored adhesion receptors, while Inhibition of exocytosis prevents CD11b/CD18 translocation and reduces neutrophil adhesion in vitro. Neutrophil crawling requires formation of new integrin-mediated adhesion bonds at the leading edge, and release of those bonds at the trailing uropod. Uropod release is associated with degradation of the extracellular domain of CD11b by proteases released by localized exocytosis (51-53), although additional, cell-intrinsic, protease-independent mechanisms also contribute to uropod detachment regulation (54). The failure of neutrophils from Chediak-Higashi syndrome patients to express the proteases necessary for uropod release may explain impaired neutrophil chemotaxis in that disease (55). Neutrophil migration through vessel walls requires exocytosis-dependent release of proteases, particularly elastase, and expression of the integrins VLA-3 and VLA-6 (56-58). The proteases contribute to neutrophil transmigration by degrading extracellular matrix (ECM) proteins in low expression regions of the vascular basement membrane and degrading VE cadherin, leading to reduced endothelial cell gap integrity (56-58). In summary, adhesion-induced secretory vesicle exocytosis replenishes adhesion molecules at the leading edge during neutrophil migration, while specific and azurophilic granule exocytosis releases proteases that promote crawling and transmigration by inducing release of the trailing uropod and by altering vascular wall integrity, respectively.

Secretory vesicle membranes contain numerous phagocytic and chemotactic receptors (20,30), which translocate to the plasma membrane upon exocytosis. Secretory vesicle exocytosis is associated with enhanced chemotaxis and phagocytosis (36), however, both responses can be dissociated from a change in receptor number (59,60). Additionally, preventing secretory vesicle exocytosis does not reduce chemotactic receptor activation of downstream signal transduction pathways (61). Thus, the role played by increased plasma membrane receptor expression following secretory vesicle exocytosis in neutrophil responses remains unclear.

Chemotaxis through interstitial ECM is postulated to result from integrin-dependent force generation as neutrophils crawl along a collagen fibril scaffold (62), although integrin-independent chemotaxis also occurs (63). Neutrophil chemotaxis through interstitial ECM is associated with increased plasma membrane expression of serine proteases and matrix metalloproteinases, but is independent of collagen degradation (64). Matrix metalloproteinases (MMPs) are necessary for neutrophil migration through collagen, possibly by altering plasma membrane expression of adhesion molecules (64). Taken together, the data indicate that granule exocytosis regulates multiple steps in neutrophil recruitment by controlling local plasma membrane expression of adhesion molecules.

Generation of reactive oxygen species (ROS) by the NADPH oxidase and release of antimicrobial proteins during granule mobilization provide synergistic antimicrobial activity (40,65). Both processes are enhanced, or primed, upon neutrophil exposure to chemokines, cytokines, or microbial and cell products (36). Recent studies indicate that priming of granule mobilization and oxidase activity are linked. Potera et al. (35) reported that TNFα-mediated priming of azurophilic and specific granule exocytosis requires NADPH oxidase activity. On the other hand, one component of priming ROS generation is cytochrome b₅₅₈ translocation to plasma membranes through mobilization of secretory vesicles and gelatinase granules and translocation to phagosomal membranes through fusion with specific granules (61,66-68). Those studies support synergistic antimicrobial activity between granule mobilization and NADPH oxidase activity and reciprocal regulation of release of granule cargo and ROS. Additionally, human neutrophils alternatively release ROS and cleave extracellular protein substrates in an oscillatory manner during migration through ECM (69). The authors propose that neutrophil pulse release of oxidants inactivate local protease inhibitors, thereby enhancing activity of proteases released during granule mobilization. Thus, NADPH oxidase activity and granule mobilization interact at many levels to determine neutrophil functional activity.

The regulation of the innate and adaptive immune systems by neutrophils has been reviewed extensively (70-79). Neutrophil granule contents participate in many regulatory activities, including those involving T cells, dendritic cells, B cells, and monocytes (70,72,73,80). The interaction between neutrophils and monocytes is particularly relevant to antimicrobial activity and inflammatory diseases. Neutrophil granule mobilization alters monocyte adhesion, migration, and bacterial phagocytosis and killing (81-85). Release of azurocidin, an inactive serine protease, and proteinase 3, an active serine protease, early in neutrophil recruitment regulates monocyte adhesion to endothelial cells and subsequent activation. The unique mechanism for secretory vesicle formation results in the presence of both serine proteases in secretory vesicles, allowing their release during transmigration (86,87). Monocyte chemotaxis is induced by gradients of cathelicidin, azurocidin, cathepsin G, and α-defensins released from neutrophil granules. Cathepsin G, proteinase 3, and neutrophil elastase also modify monocyte regulatory cytokines and their receptors (22,88).

Neutrophil granule cargoes contribute to other immune responses and provide redundancy to those responses. Serine proteases regulate immune responses and inflammation through processing of cytokines and their receptors, generating chemoattractant peptides from ECM, remodeling ECM, increasing vascular permeability, generating neutrophil extracellular traps (NETs), processing adhesion molecules, and providing an alternative pathway for apoptosis (22,88). An example of redundancy of granule content functions is provided by azurocidin release from secretory vesicles and azurophilic granules following neutrophil stimulation which increases vascular permeability by a direct action on endothelial cells (89,90). Thus, a number of granule proteins are implicated in the increased vascular permeability associated with inflammation. The hundreds of cargo molecules released extracellularly or translocated to the plasma membrane during granule exocytosis provide considerable opportunity for functional overlap. The relative importance of individual granule constituents to a specific response may be difficult to discern, and inhibition of the activity of a single molecule is unlikely to effectively treat neutrophil-dependent diseases.

Participation of Neutrophil Granule Mobilization in Disease

Neutrophil granule constituents directly injure tissue in several diseases, including autoimmune diseases (rheumatoid arthritis, bullous pemphigoid, immune complex glomerulonephritis, ANCA-associated vasculitis, and systemic lupus erythematosus) (8-12), acute lung injury due to pneumonia or sepsis (13,14), ischemia/reperfusion injury (17-19), and atherosclerosis (91,92). In this section, we summarize experimental support for tissue injury by neutrophil granule exocytosis in those diseases.

Autoimmune diseases.

Neutrophil participation in antibody-mediated glomerular diseases, immune complex glomerulonephritis (GN) and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), has been reviewed (93,94). Biopsies of human GN in which neutrophils are observed in glomeruli include anti-glomerular basement membrane (GBM) antibody GN, AAV, lupus nephritis, acute post-infectious GN, membranoproliferative GN (MPGN), and IgA-mediated GN (IgAN). The role of neutrophil granule components in AAV is most completely understood. Target antigens for ANCA, including MPO and PR3, are stored in neutrophil granules. Stimulated exocytosis during neutrophil priming leads to plasma membrane expression of ANCA antigens, thereby exposing them to circulating autoantibodies. Neutrophil-bound ANCA autoantibodies interact with FcγRs on adjacent neutrophils, stimulating ROS production and additional granule mobilization (95). Glomerular cytokine generation, neutrophil and monocyte infiltration into glomeruli, and glomerular injury in a mouse model of AAV depend on release of proteases from neutrophil specific and azurophilic granules (96). In vitro neutrophil-mediated endothelial cell injury induced by ANCA depends on serine protease release (97). Although neutrophils recruited to the glomerulus in mice with AAV show rapid generation of ROS (98), mice genetically deficient in NADPH oxidase components (gp91^phox, p47^phox) demonstrate accelerated ANCA-mediated crescentic GN, due to loss of ROS-mediated down-regulation of IL-1β (99). Clearly, neutrophil granule mobilization is a critical component of multiple steps in the pathogenesis of ANCA-mediated vasculitis.

Neutrophil granule cargo contribute to disease in other animal models of GN. Beige mice, a model of Chediak-Higashi syndrome in which neutrophils are genetically deficient in neutrophil elastase and cathepsin G but demonstrate normal ROS generation, fail to develop proteinuria after anti-GBM administration (100-102). Inhibition of neutrophil elastase prevents development of proteinuria, hematuria, and histologic evidence of injury in a rat model of anti-GBM nephritis (103). Cathepsin G and elastase degrade GBM in vitro, and elastase is present in glomeruli and urine of patients with crescentic GN and MPGN (104-106). Infusion of MMPs into isolated rat glomeruli increases albumin permeability, and active gelatinase (MMP9) is present in glomeruli of patients with several types of GN, including AAV, IgAN, acute post-infectious GN, and lupus nephritis (107,108). Neutrophil ROS generation also participates in glomerular damage. Mice genetically deficient in neutrophil NADPH oxidase or given scavengers of ROS fail to develop proteinuria after administration of anti-GBM antibodies, (100,109). Hypohalous acids, but not H₂O₂, increases albumin permeability of isolated rat glomeruli, and intrarenal generation of hypohalous acids by renal artery injection of MPO and H₂O₂ induces proteinuria in rats (110,111). The data support both a direct role of neutrophil release of granule cargo and a synergistic interaction with ROS in the pathogenesis of immune-mediated GN.

Large numbers of neutrophils are present within the synovial fluid and at the pannus/cartilage interface of joints in rheumatoid arthritis. Immunoglobulin aggregates containing rheumatoid factors (anti-immunoglobulin antibodies) at that location stimulate neutrophil exocytosis through engagement of Fcγ receptors (112). Release of granule proteolytic enzymes, including elastase, collagenase (MMP8), and gelatinase (MMP9), into inflamed joints degrades joint cartilage independently of ROS generation (113-117). Cathepsin C activates the inactive proteases contained in neutrophil granules by proteolytic processing (22). Genetic deletion or pharmacologic inhibition of cathepsin C prevent development of inflammatory arthritis in mice (118,119). Additional neutrophil granule cargo, including cathepsin G, cathelicidin, defensins, and lactoferrin, released into joints regulate monocyte recruitment, cell apoptosis, and articular cartilage gene expression of cytokines (120-122). Bullous pemphigoid represents another autoimmune disorder in which autoantibodies against hemidesmosomal proteins at the dermal-epidermal junction result in neutrophil-mediated subepidermal blistering (123). Release of elastase and MMP9 from neutrophils is required for dermal-epidermal separation (9,124,125).

In summary, neutrophils mediate tissue and organ damage in many autoimmune diseases, and granule mobilization directly and indirectly contributes to cell injury and organ dysfunction. These observations suggest that pharmacologic inhibition of neutrophil exocytosis will be an effective therapeutic intervention for autoimmune diseases, either alone or in combination with current therapies.

Sepsis and ischemia-reperfusion injury.

Sepsis is a leading cause of acute lung injury (ALI) and ischemia-reperfusion injury to other organs. Early in bacterial infection leading to sepsis, neutrophils are highly activated to mobilize granules and generate ROS (126-129). The localization of activated neutrophils within organs contributes to organ failure in sepsis. After 3 to 6 days patients develop an immunosuppressive state, including impaired neutrophil chemotaxis, reduced expression of plasma membrane receptors, impaired azurophilic and specific granule mobilization, and reduced oxidative burst (130,131).

Neutrophils are a critical component of the pathogenesis of all forms of ALI, although development of ALI in neutropenic patients indicates neutrophil-independent mechanisms exist (11,132). Neutrophils isolated from blood and bronchoalveolar lavage fluid of patients with ALI show a primed phenotype, including in vivo granule exocytosis and enhanced in vitro ROS generation upon stimulation (133). The presence of increased concentrations of neutrophil granule constituents, including elastase, MMPs, azurocidin, defensins, and cathelicidin, in blood and bronchoalveolar lavage fluid supports involvement of neutrophil granule exocytosis in ALI (134). Pharmacologic inhibition of neutrophil elastase attenuates ALI in several mouse models (135-137). Results of human studies, however, are inconsistent (138,139), likely because extracellular release of hundreds of enzymes during exocytosis are redundant mediators of cell injury. Mice deficient in MMP3 show reduced neutrophil recruitment and lung injury, while MMP9 depletion inhibits ALI without altering neutrophil recruitment (140). On the other hand, mice depleted of MMP8 show increased neutrophil recruitment and lung injury, indicating MMPs have both pro- and anti-inflammatory activities (141). Mice do not constitutively express defensins, however, when expression of human α-defensins is induced, ALI is enhanced in a model of aspiration injury (142). Direct evidence for neutrophil granule exocytosis mediating ALI is provided by induction of ALI following injection of neutrophil secretion products in neutrophil depleted animals (10). As increased microvascular permeability of alveolar capillaries is a cardinal feature of ALI, it is likely that neutrophil granule constituents previously shown to increase vascular permeability play a role (89,90). Peripheral blood neutrophils isolated from patients with ALI compromise vascular endothelial cell barrier function in vitro (143). ROS generation by neutrophils also participates in neutrophil-induced ALI (134,144,145). Thus, ROS and granule constituents act synergistically to induce tissue damage in ALI.

Neutrophil infiltration is prominent during reperfusion after ischemia of kidney, liver, myocardium, and brain (91,146-151). Inhibition of neutrophil recruitment reduces organ injury in many animal models (152-159). Data suggest that release of granule cargo and ROS generation mediate neutrophil-dependent organ damage (155,160,161). A role for ROS is supported by finding oxidant-modified proteins in organs damaged by ischemia-reperfusion injury (162-164). A role for granule exocytosis is supported by reduction in organ damage following pharmacologic inhibition or genetic deletion of neutrophil granule constituents, including elastase and MMPs (165-171). Once again, granule exocytosis and ROS generation appear to provide synergistic organ damage after ischemia-reperfusion. We predict a high probability that pharmacologic inhibition of neutrophil exocytosis will inhibit ALI. On the other hand, the more complex pathophysiology of ischemia-reperfusion injury makes the therapeutic potential of pharmacologic inhibition of neutrophil exocytosis more difficult to predict.

Atherosclerosis.

Mounting evidence indicates neutrophil exocytosis plays a significant pathophysiologic role in atherosclerotic cardiovascular disease. Neutrophils are present in atherosclerotic plaques, and a positive correlation exists between plasma levels of neutrophil granule proteins, including elastase, azurocidin, and MMP8, and coronary artery disease in humans (172-175). Neutrophils are a major cellular component of atherosclerotic lesions in ApoE^−/− mice (172), and temporary depletion reduces atherogenesis. MPO mediates endothelial damage (176-178) and is present in atherosclerotic lesions (175,179-181). MPO inhibition alters inflammation within atherosclerotic lesions and helps prevent atherosclerotic plaque rupture in murine models (182). The association of multiple granule cargoes with atherosclerotic lesions supports the concept that interfering with neutrophil exocytosis rather than inhibition of selective secretory proteins is more likely to produce positive outcomes.

Molecular mechanisms provide the rationale for neutrophil exocytosis inhibitors

Due to the importance of granule exocytosis in neutrophil activation and the toxic content of granules, neutrophil degranulation requires precise mechanisms of control. Characterization of the molecular machinery that controls the timely release of the multiple neutrophil secretory organelles provides invaluable information for the design of targeted strategies to decrease secretion (Fig.1). The efficiency and specificity of vesicular trafficking relies on Ras-like Rab small GTPases. Azurophilic granule exocytosis is stimulated by a non-hydrolyzable analog of GTP in permeabilized neutrophils (183). The small GTPase Rab27a is the central regulator of azurophilic granule exocytosis (24). As discussed above, azurophilic granules are heterogeneous and Rab27a co-fractionates with a subpopulation of low density azurophilic granules that constitute the exocytosable pool. Direct evidence for Rab27a regulation of azurophilic granule exocytosis is provided by studies performed in two different mouse models of Rab27a-deficiency (24), in permeabilized human neutrophils treated with anti-Rab27a inhibitory antibodies (39), and in Rab27a-downregulated neutrophil-like cells (24). Priming of neutrophil exocytosis by pro-inflammatory cytokines or pathogen-associated molecular patterns involves docking of Rab27a-positive vesicles with the plasma membrane and is detected by their restricted motility (184). Consequently, azurophilic granule exocytosis stimulated by LPS priming and fMLF stimulation is markedly impaired in neutrophils lacking Rab27a (24,184-186). Mice deficient in Rab27a have decreased inflammation and increased survival following LPS-induced endotoxemia, establishing a direct correlation between neutrophil secretory proteins and systemic inflammation (186).

Figure 1.

Left panel. Granules of human and murine neutrophils contain readily releasable proteases, pro-oxidant enzymes and bactericidal and cell-poring forming peptides (blue small circules). They also contain the membrane associated subunit of the NADPH oxidase (green bars), which translocates to the plasma membrane during exocytosis. The granule secretory machinery include Rab27a and its effectors (depicted here by JFC1) and v-SNAREs. Right Panel. Upon activation or during inflammation-induced exocytosis dysregulation, Rab27a effectors including JFC1 bind to Rab27a and facilitate trafficking and tethering at the plasma membrane. Subsequent engagement of SNARE proteins facilitates fusion and pore formation. This leads to the activation of the oxidase and the release of granule cargoes to the extracellular milieu leading to inflammation. Nexinhibs and TAT-peptides target the Rab27a and the SNARE machineries, respectively, thus decreasing neutrophil-mediated inflammation.

In addition to supporting azurophilic granule secretion, Rab27a regulates gelatinase and specific granule exocytosis in neutrophils (39). Specificity of Rab27a for the regulation of different granule subsets is provided by Rab-interacting proteins, named Rab effectors. Slp1/JFC1 and Munc13-4 are the two Rab27a effectors functionally characterized in neutrophils (24,39). An essential role for Slp1/JFC1 in azurophilic granule exocytosis was initially demonstrated in human neutrophils and then confirmed using neutrophils from JFC1-KO mice (39,55). On the other hand, lack of Slp1/JFC1 expression failed to impair exocytosis and target cell killing by cytotoxic lymphocytes (187). At this time, neutrophils constitute the only leukocyte for which Slp1/JFC1 is essential for regulated secretion.

Mechanistically, Slp1/JFC1 regulates vesicular trafficking through cytoskeleton reorganization which facilitates vesicular docking and fusion with the plasma membrane (188). This is mediated by the interaction of Slp1/JFC1 with Gem-interacting protein (GMIP), a GTPase-activating protein for the small GTPase RhoA (188). Slp1/JFC1 binding to GMIP inactivates RhoA in areas surrounding the granule, leading to actin depolymerization required for granule movement to the plasma membrane. Slp1/JFC1 also binds to the plasma membrane-localized phosphoinositide, phosphatidylinositol (3,4,5)-trisphosphate (PIP₃), to mediate vesicular tethering (189).

Munc13-4 is another Rab27a effector that regulates vesicular docking at the plasma membrane (190). Similar to Slp1/JFC1, interfering with Munc13-4 function inhibits azurophilic granule exocytosis in both human and murine neutrophils (39,184,191,192). Munc13-4 also regulates degranulation of gelatinase granules, but not secretory vesicles (39). Munc13-4 has two C2 domains with the ability to coordinate calcium binding. Munc13-4 binds to syntaxins in a calcium-dependent manner and is, therefore, considered a calcium sensor for the fusion of granules with target membranes. Two molecules that interfere with Munc13-4, STK24 (Serine/threonine protein kinase (STK) 24) and CCM3 (Programmed Cell Death 10), inhibit exocytosis and protect kidneys from ischemia-reperfusion injury (193). Deficiencies in either Rab27a or Munc13-4 are associated with the human immunodeficiency, Griscelli’s syndrome and Familial Hemophagocytic Lymphohistiocytosis type 3, respectively. Similar to Rab27a-deficient mice, the Munc13-4-knockout mouse model shows decreased neutrophil secretory proteins in circulation and increased survival in models of systemic inflammation (186). Munc13-4 differs from Slp1/JFC1 by regulating phagosomal maturation, endosomal functions, and bacteria killing in neutrophils (192,194), as well as exocytosis in cytotoxic lymphocytes (190,195). The different functions of Slp1/JFC1 and Munc13-4 provides the rationale for focusing on the Rab27a-JFC1 interaction to develop inhibitors of neutrophil exocytosis. This focus potentially limits the impact of secretion inhibitors on neutrophil functions that are required for anti-microbial activity and on immunity. Furthermore, targeting the Rab27a-JFC1 interaction has advantages over targeting other regulators of azurophilic granule exocytosis, for example Rac1/2 GTPases (196). Although specific inhibitors for Rac1/2 GTPases are available, they may interfere with multiple functions that Rac regulates in neutrophils and other cells (197,198).

While the small GTPase Rab27a is widely expressed in tissues with secretory activity and plays a general role in secretory cells (199), the specificity of the regulatory mechanisms mediated by this GTPase is defined by its effector proteins. Twelve proteins that operate as putative Rab27a effectors have been described that are not ubiquitously expressed and regulate specific cellular functions in a discrete number of cell types (Reviewed in 200,201). For instance, in melanocytes, melanosome trafficking is regulated by the Rab27a effector Slac2-a/melanophilin (202) and by the motor protein MyoVa (203). Munc13-4, a Rab27a effector known to regulate exocytosis of neutrophil granules (204), also controls exocytosis of lytic granules in cytotoxic T-lymphocytes (CTLs) (190) and therefore, interference with Munc13-4 function would likely affect both neutrophil and CTL exocytosis. Although CTLs’ lytic granules are proposed to be regulated by both Slp1/JFC1 and Slp2, likely by redundant mechanisms, individual knockouts of either Slp2-a or JFC1/Slp1 fail to impair CTL-mediated killing of targets (205). These data support that interference with JFC1 or with the Rab27a-JFC1 interaction does not affect CTL function. Regulation of insulin secretion by Rab27a is mediated by the effectors granuphilin (206) and Noc2 (207). While JFC1 is proposed to exert some level of control on a minor subpopulation of secretory granules that have not been docked to the plasma membrane in β cells, JFC1 knockdown has no effect on the distribution or exocytosis of insulin granules, JFC1-null mice do not show glucose intolerance, and JFC1-null β cells show normal insulin secretion in response to glucose (208). Based on these data, interference with Rab27a-JFC1 binding is unlikely to affect insulin metabolism. Similarly, Weibel-Palade body exocytosis is regulated by Slp4-a and MyRIP (209) but not by JFC1. Rab effectors can also be promiscuous. In particular, JFC1 interacts with the small GTPase Rab8a in addition to Rab27a (210). Tyr-122 in Rab27a, a residue involved in the interaction of this GTPase with its effector and recognized by neutrophil exocytosis inhibitors (Nexinhibs) (211), is not conserved in Rab8a. Thus, small-molecule inhibitors of the Rab27a-Slp1/JFC1 interaction are not expected to affect Rab8a-Slp1/JFC1 interaction. Again, highlighting the view that focusing on protein-protein, Rab-effector-specific interactions increases the specificity of the inhibitor and decreases the likelihood of undesired effects. Finally, our group demonstrated that the Rab27a-JFC1 interaction is important for the secretion of PSA by prostate carcinoma cells (212); however, it is unlikely that interference with this mechanism has deleterious consequences under conditions of neutrophil-mediated inflammation in which the use of Rab27a-JFC1 inhibitors would be clinically relevant. In conclusion, although Rab27a plays important functions in several secretory systems, targeting the Rab27a-JFC1 interaction, which is dominant in neutrophils but redundant or irrelevant in other cells, reduces the likelihood of off-target effects.

SNAREs, fusion machines as targets to control secretion

The specificity of the fusion process is regulated by vesicle-associated proteins, vesicle(v)-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins and their counterpart target(t)-SNAREs at the plasma membrane (SNARE hypothesis, reviewed in (213,214). T-SNAREs consist of syntaxins and SNAPs (synaptosome-associated protein), and vSNARE consist of VAMPs (vesicle-associated membrane proteins, also called synaptobrevins). SNAREs form a coiled-coil structure with four α-helices contributed by three different molecules (215). This structure is believed to generate the force to bring two lipid bilayers into proximity, overcoming electrostatic repulsion and facilitating membrane fusion. It is proposed that the movement of the charged COOH terminus of the vSNARE within the membrane in response to the forces generated by zippering of the SNARE complex induces pore formation (213), which is preceded by conformational changes in the SNAP protein at the sites of fusion (216).

Several SNARES are associated with neutrophil granules and plasma membrane, including SNAP-23, syntaxin-4, syntaxin-6, VAMP-1, VAMP-2, and VAMP-7. SNAP-23 and syntaxin-6 regulate specific granule exocytosis (217). VAMP2 regulates the exocytosis of gelatinase and specific granules but not azurophilic granules, while syntaxin-4 interacts with SNAP-23 to regulate exocytosis of several neutrophil granule subsets (218). VAMP-1 and VAMP-7 regulate the secretion of azurophilic granules (219), while SNAP-23 appears not to be involved (220). The identification of SNAREs that regulate neutrophil granule secretion provides the rationale for development of inhibitory peptides targeting these fusion regulators.

Targeting granule trafficking and docking to inhibit neutrophil exocytosis

Considerations for selectivity and specificity of protein-protein interaction inhibitors

Trafficking molecules, including SNAREs and Rab GTPases, are broadly expressed in eukaryotic cells. Thus, limiting inhibition of granule trafficking to neutrophils is unlikely in humans or animal models, leading to the possibility of significant side effects. To minimize non-specific effects, the characterization of molecular interactions regulating vesicular trafficking in neutrophils provides the basis for design of peptide and small molecule inhibitors. Because the specific function of a Rab GTPase protein in a particular biological setting is defined by its effectors, a higher level of selectivity is achieved by focusing on protein-protein interactions between specific Rabs and effectors. Similarly, SNARE-inhibitor peptides may gain selectivity by interfering with interactions between specific SNARE proteins. The development of selective and potent, cell-active protein-protein interaction inhibitors is most effective in interactions where the binding partners share a relatively small molecular interface, because large interaction interfaces may have binding energies too high or interface structures too complex to permit disruption by the binding of small molecules (221,222). The small Rab27a-effector binding interface predicts an ability to disrupt interactions with small molecules with adequate binding energy (223). The larger area of interaction among SNARE domains (~78 amino acids) suggests that larger molecules are needed to disrupt SNARE protein interaction. Protein-protein interaction assays that maintain the natural cellular environment for these interactions, i.e. intact organelles, significantly improves specificity and increases success in finding inhibitors effective in intact cells. Importantly, targeting protein-protein interactions provides opportunities to directly target pathways that drive disease development (224). Targeting protein-protein interactions may also increase specificity over the use of enzyme and kinase inhibitors due to the high conservation of binding sites between family members (224).

Small-molecule inhibitors of Rab GTPases and effectors for the control of secretion.

Azurophilic granules contain and secret the most toxic, pro-inflammatory cargoes of human neutrophils. Rab27a regulates azurophilic granule exocytosis through interaction with the effector Slp1/JFC1 while interacting with a different effector to control secretion of other neutrophil granule subsets. The Rab-binding domain of Slp1/JFC1 is formed by two synaptotagmin-like protein-homology domains and contains a TGDWF motif. The Rab27a-Slp1/JFC1 interaction is essential for neutrophil azurophilic granule exocytosis, as a point mutation at tryptophan 83 in the TGDWF motif of the Slp1/JFC1 Rab-binding domain disrupts Rab27a binding and inhibits secretion. An important reason for targeting these modulators of vesicular trafficking is that they are not major regulators of phagocytosis in neutrophils (24,192). Thus, interference with the Rab27a-Slp1/JFC1 interaction does not affect one of the most important neutrophil-mediated innate immune response mechanisms.

To identify inhibitors of neutrophil exocytosis, we developed an assay to screen molecular libraries to identify novel small molecule inhibitors of the specific binding between the small GTPase Rab27a and JFC1 (211). The assay is based on the principle of time-resolved FRET (TR-FRET) using a highly stable fluorescence donor, terbium cryptate, and green fluorescent protein (EGFP) as the acceptor. The assay is performed using cell lysates expressing JFC1 with a Myc-tag in its amino-terminal domain and EGFP-Rab27a. We next use a terbium-conjugated anti-Myc antibody, which specifically binds to the tag moiety in Myc-JFC1. FRET signal is triggered by the close proximity between the donor (terbium) and acceptor (EGFP) in response to the specific binding of Myc-JFC1 to EGFP-Rab27a (211). To increase the likelihood of identifying specific inhibitors with physiological significance, the reactions are performed with lysates obtained by non-denaturing methods, and the integrity of the organelles is maintained throughout the analysis. Thus, the method measures the binding of JFC1 to Rab27a on intact vesicles, i.e. their natural environment, an approach that has many advantages over alternative methods that measure protein-protein interactions in solution.

Using this approach and high-throughput screening analysis, counterscreens and orthogonal validation, we identified Nexinhibs, small-molecule inhibitors of the JFC1-Rab27a interaction which constitute the first neutrophil-specific exocytosis inhibitors (211) (Fig. 1). A binding pocket for Nexinhib20 with the highest druggability score was identified within a Rab27a domain associated with JFC1 family effector binding (211). This pocket contains a Rab27a domain associated with the binding to the synaptotagmin-like homology domains of Slp/JFC1 family members. Structural modeling identified Tyr-122 in Rab27a as a NEI20-interacting residue, an important observation because this tyrosine mediates Rab-effector specificity (223). Initial structure-activity relationship analysis of Nexinhibs identified potentially important groups in three compounds, Nexinhibs 4, 20 and 20 analog (211). Nexinhibs inhibit azurophilic granule exocytosis without affecting phagocytosis, NET production or cell viability (211). Because exocytosis of azurophilic granules is not selective for their cargoes, inhibition of azurophilic granule secretion prevents the release not only of MPO but also the secretion of the serine proteases, including proteinase 3, and the upregulation of important adhesion molecules and integrins. Thus, Nexinhibs have potential applications in approaches aimed at decreasing neutrophil exocytosis and tissue infiltration.

Nexinhibs decrease the upregulation of the adhesion molecules and the galectin-3 receptor CD66b at the plasma membrane (211). Furthermore, the upregulation of the NADPH oxidase membrane-associated subunit cytochrome b₅₅₈ and the production of extracellular superoxide anion was also attenuated by Nexinhibs (211). Those data support the idea that ROS production and exocytosis are mechanistically linked and that interfering with exocytosis has additional anti-inflammatory benefits.

The effect of Nexinhibs on phagocytosis was analyzed using opsonized latex beads, flow cytometry analysis of fluorescent particle internalization, quantitative analysis of phagocytose opsonized zymosan particles and phagocytosis of live Pseudomonas aeruginosa (211). These studies showed that Nexinhibs do not interfere with the process of phagocytosis. Furthermore, NETosis, induced by live P. aeruginosa, was also not affected by Nexinhib treatments.

LPS administration induces high levels of azurophilic granule proteins in plasma (24,186). Rab27a-deficient mice have reduced levels of neutrophil secretory proteins in circulation after LPS insult (24,186), and those mice are protected from LPS-induced death (186). In addition, Rab27a^−/− (ashen) mice show decreased neutrophil infiltration into tissues (186). Jfc1^−/− mice, whose azurophilic granule exocytosis is also impaired (188), show reduced plasma levels of neutrophil secretory proteins and increased survival to LPS insult. The efficacy of Nexinhib20 was tested in this model of LPS-induced systemic inflammation in mice. Given before LPS injection, Nexinhib20 significantly prevents LPS-induced neutrophil exocytosis and decreases plasma levels of neutrophil secretory proteins (208). Furthermore, Nexinhib20-treated mice showed decreased tissue infiltration by inflammatory neutrophils in kidney and liver (211), a phenotype also observed in the Rab27a^−/− model (186).

Neutrophil exocytosis inhibitors are also potentially useful in autoinflammatory diseases induced by genetic alterations. Neutrophils from the Nlrp3^A350V inducible mouse model (MWS CreT) recapitulates human patients with the A352V mutation in NLRP3 observed in the Muckle-Wells sub-phenotype (MWS) of cryopyrin-associated periodic syndrome (CAPS) are characterized by normal gelatinase granule exocytosis but exacerbated azurophilic granule cargo secretion even under non-stimulated conditions (225). The increased azurophilic granule exocytosis in MWS neutrophils is attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20. Although in vivo validation is pending and this is a multifactorial syndrome, it is possible that decreasing neutrophil exocytosis may have beneficial effects alone or in combination with other available therapies. Thus, Nexinhibs offer a targeted approach to attenuate systemic inflammation in both acquired and genetically induced inflammatory processes not only by decreasing secretion, but also reducing neutrophil infiltration.

Peptide inhibitors of SNARE interactions.

Based on the SNARE hypothesis described above, we postulated that peptide aptamers derived from SNARE domains would compete for binding between intact SNARE proteins, thereby preventing the interaction of SNARE proteins required for fusion between granule and target membranes (Fig 1). To promote cell uptake, cell penetrating peptides were linked to SNARE domain-mimicking peptides. Fusion proteins containing the 11 amino acid cell penetrating peptide of HIV TAT and the N-terminal or C-terminal SNARE domains of SNAP-23 or the SNARE domain of syntaxin-4 were created and in vitro activity was characterized (61,67). All fusion proteins readily enter neutrophils in vitro. Optimal concentrations of fusion proteins containing the N-terminal SNAP-23 SNARE domain (TAT-SNAP-23) inhibited fMLF-stimulated exocytosis of secretory vesicles, gelatinase granules, and specific granules by 60% to 80%, but failed to inhibit azurophilic granule exocytosis. TAT-SNAP-23 bound to a combination of recombinant GST-VAMP-2 and recombinant syntaxin-4 in vitro, supporting the hypothesis that inhibition of exocytosis occurred by blocking interaction of SNARE proteins. Fusion proteins containing the syntaxin-4 SNARE domain (TAT-STX-4) inhibited exocytosis of all 4 granule subsets by 50% to 80%. The C-terminal SNAP-23 SNARE domain fusion protein failed to inhibit neutrophil granule exocytosis. TAT-SNAP-23 and TAT-STX-4 inhibited increased plasma membrane expression of cytochrome b₅₅₈ induced by TNFα and platelet activating factor and prevented priming of respiratory burst activity. Those data showing that granule mobilization is required for priming of respiratory burst activity are consistent with data with Nexinhibs and support the theme that granule exocytosis and ROS generation are synergistic responses. The effect of TAT-SNAP-23 on ROS generation and bacterial killing within phagosomes was examined by measuring bleaching of GFP-expressing S. aureus by MPO-catalyzed hypochlorous acid generation and S. aureus survival in vitro in human neutrophils (61). Neither were affected by TAT-SNAP-23 treatment, indicating that the conditions for intraphagosomal bacterial killing remain intact. In vitro studies using TAT-SNAP-23 and TAT-STX-4 also showed that exocytosis regulates neutrophil intracellular killing of Listeria monocytogenes and plays a role in T-cell suppression by neutrophils in ovarian cancer (226,227). A previous study showed that TAT-fusion proteins readily entered neutrophils, but the majority of ingested protein were degraded in an endosomal compartment (228). Although this intracellular degradation could significantly impact dosing in vivo, an optimal concentration for in vitro studies and effective in vivo dose remain remarkable constant for multiple preparations of both fusion proteins.

The potential for TAT-SNAP-23 to alter disease was tested in rodent models. Intravenous administration of TAT-SNAP-23 reduced ALI induced by pulmonary immune complex deposition in rats (229). Although neutrophil recruitment into lungs was not altered, interstitial lung edema and plasma leakage into alveoli were significantly inhibited by treatment. Neutrophils isolated from bronchoalveolar lavage fluid contained TAT-SNAP-23 and demonstrated evidence for impaired exocytosis. Subsequently, TAT-SNAP-23 administration was shown to reduce lung injury in mice with sepsis- or shock-induced ALI (230). In those models TAT-SNAP-23 reduced neutrophil recruitment into the lungs and blocked priming of respiratory burst activity in vitro and in vivo. As SNARE proteins are responsible for membrane trafficking in all cells and SNAP-23 is ubiquitously expressed, the potential for unwanted systemic effects was an important consideration. Neither mice nor rats showed observable side effects after TAT-SNAP-23 administration, specifically no alterations in state of consciousness, motor function, activity level, or food and water intake occurred. An in vitro and in vivo evaluation of the TAT-fusion proteins inhibitor effect on granule trafficking in other cells is required to identify potential non-specific effects.

The effect of TAT-SNAP-23 treatment of neutrophil-dependent, acute immune complex GN in mice was recently examined. TAT-SNAP-23 administration prevented proteinuria 24 hr after administration of anti-GBM antibody, despite similar glomerular antibody deposition, mesangial cell proliferation, and neutrophil infiltration into glomeruli (231,232). Cultivation of neutrophil granule contents with podocytes caused disruption of the actin cytoskeleton, which is an in vitro correlate of podocyte foot process effacement and loss of glomerular filtration barrier function that results in proteinuria. Thus, granule constituents released extracellularly are directly responsible for a component of neutrophil-mediated glomerular injury. The ability of TAT-SNAP-23 to prevent ALI in 3 different animal models and to prevent immune complex GN validates our hypothesis that pharmacologic inhibition of neutrophil exocytosis is a viable therapy for neutrophil-dependent diseases. As TAT-SNAP-23 does not alter azurophilic granule exocytosis in vitro, the results suggest that specific and gelatinase granules or secretory vesicles contain cargo responsible for disease.

Differences in the effects of exocytosis inhibition on neutrophil recruitment may be related to differences in organ vasculature. Both lung and glomeruli represent specialized vascular beds in which neutrophils adhere to capillaries. In systemic vasculature neutrophil adhesion and transmigration occurs at post-capillary venules. As the mechanisms of neutrophil recruitment differ between post-capillary venules and specialized vascular beds, the effect of inhibition of exocytosis on neutrophil recruitment may vary based on the site of inflammation.

The concept that SNARE domain mimicking peptides fused to cell permeability peptides inhibits exocytosis was recently extended to mast cells (233). The authors fused 17 amino acid sequences from the amino-terminus, carboxyl-terminus, and central portion of SNARE domains derived from SNAP-23, syntaxin-4, VAMP2, VAMP4 and VAMP8 with a cell permeability sequence. The peptides from the amino-terminus of syntaxin-4, VAMP2, and VAMP8 inhibited stimulated release of histamine and/or β-hexosaminidase from RBL-2H3 mast cells by 40% to 50%. Daily administration of VAMP2 and VAMP8 peptides for 4 weeks inhibited atopic dermatitis in a mouse model. Although these studies indicate a lack of selectivity of SNARE inhibition for neutrophils, the long-term administration of SNARE inhibitors did not produce significant side effects that would limit their therapeutic potential. Further studies are needed to define the cells and organs affected by different SNARE inhibitors in vivo.

Concluding remarks

The characterization of neutrophil trafficking pathways identified specific molecular targets and interactions that proved to be druggable either with small-molecules or inhibitory peptides with the common objective of inhibiting secretion and decreasing inflammation. These inhibitors were further demonstrated to regulate neutrophil exocytosis in vitro and to modulate neutrophil secretion in in vivo models of human disease. This research highlights the importance of targeting neutrophils in biological meaningful settings and provides evidence that the use of neutrophil exocytosis inhibitors will likely spur the development of new targeted specific inhibitors of neutrophil-mediated inflammation with clinical significance.

ABBREVIATIONS:

ALI

acute lung injury

ANCA

anti-neutrophil cytoplasmic antibody

AAV

ANCA-associated vasculitis

MMP8

collagenase

CAPS

cryopyrin-associated periodic syndrome

ECM

extracellular matrix

TAT-SNAP-23

fusion proteins containing cell penetrating peptide of HIV TAT and the N-terminal SNAP-23 SNARE domain

TAT-STX-4

fusion proteins containing cell penetrating peptide of HIV TAT and the syntaxin-4 SNARE domain

GMIP

Gem-interacting protein

GBM

glomerular basement membrane

GN

glomerulonephritis

IgAN

IgA-mediated GN

LAD

Leukocyte Adhesion Deficiency

MMP9

matrix metalloproteinase 9

MPGN

membranoproliferative glomerulonephritis

MWS

Muckle-Wells sub-phenotype

MPO

myeloperoxidase

NETs

neutrophil extracellular traps

NGAL

neutrophil gelatinase-associated lipocalin

Nexinhib

neutrophil-specific exocytosis inhibitor

ROS

reactive oxygen species

SNARE

soluble N-ethylmaleimide-sensitive factor attachment protein receptor

SNAP

synaptosome-associated protein

SLE

systemic lupus erythematosus

TR-FRET

time-resolved FRET

VAMP

vesicle-associated membrane protein