Primary Granule Exocytosis Research Articles

Rac2 regulates immune complex-mediated granule polarization and exocytosis in neutrophils.

A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase (GTPase). Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin-1A (Coro1A), a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes (IC) and relate this to intracellular Coro1A localization. We used bone marrow neutrophils (BMN) from WT and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase, MPO), secondary (lactoferrin, LTF) and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of Coro1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in MPO, MMP-2, and MMP-9, but not LTF, release, along with diminished filopodia formation, CD63 polarization, and colocalization of Coro1A with CD63 in IC-stimulated Rac2-/- BMNs. Rac2 and Coro1A were found associated with granules in CB/fMLF-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on Coro1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.

Pseudomonas aeruginosa modulates neutrophil granule exocytosis in an in vitro model of airway infection.

A population of neutrophils recruited into cystic fibrosis (CF) airways is associated with proteolytic lung damage, exhibiting high expression of primary granule exocytosis marker CD63 and reduced phagocytic receptor CD16. Causative factors for this population are unknown, limiting intervention. Here we present a laboratory model to characterize responses of differentiated airway epithelium and neutrophils following respiratory infection. Pediatric primary airway epithelial cells were cultured at the air–liquid interface, challenged individually or in combination with rhinovirus (RV) and Pseudomonas aeruginosa, then apically washed with medical saline to sample epithelial infection milieus. Cytokine multiplex analysis revealed epithelial antiviral signals, including IP‐10 and RANTES, increased with exclusive RV infection but were diminished if P. aeruginosa was also present. Proinflammatory signals interleukin‐1α and β were dominant in P. aeruginosa infection milieus. Infection washes were also applied to a published model of neutrophil transmigration into the airways. Neutrophils migrating into bacterial and viral–bacterial co‐infection milieus exhibited the in vivo CF phenotype of increased CD63 expression and reduced CD16 expression, while neutrophils migrating into milieus of RV‐infected or uninfected cultures did not. Individually, bacterial products lipopolysaccharide and N‐formylmethionyl‐leucyl‐phenylalanine and isolated cytokine signals only partially activated this phenotype, suggesting that additional soluble factors in the infection microenvironment trigger primary granule release. Findings identify P. aeruginosa as a trigger of acute airway inflammation and neutrophil primary granule exocytosis, underscoring potential roles of airway microbes in prompting this neutrophil subset. Further studies are required to characterize microbes implicated in primary granule release, and identify potential therapeutic targets.

Neutrophil-derived extracellular vesicles promote feed-forward inflammasome signaling in cystic fibrosis airways.

Cystic fibrosis (CF) airways feature high extracellular levels of the IL-1 family of proinflammatory mediators. These mediators are cleavage products of caspase-1, the final protease in the inflammasome cascade. Due to the proven chronic presence of reprogrammed neutrophils in the CF airway lumen, understanding inflammasome signaling in these cells is of great importance to understand how disease is perpetuated in this milieu. Here, we hypothesized that CF airway neutrophils contribute to chronic inflammation, in part, via the packaging of inflammasome-inducing signals in extracellular vesicles (EVs). We confirmed that CF airway fluid is enriched in IL-1α, IL-1β, and IL-18, and that CF airway neutrophils up-regulate the activating receptor IL-1R1. Meanwhile, down-modulatory signals such as IL-1R2 and IL-1RA are unchanged. Active caspase-1 itself is present in CF airway fluid EVs, with neutrophil-derived EVs being most enriched. Using a transmigration model of CF airway inflammation, we show that CF airway fluid EVs are necessary and sufficient to induce primary granule exocytosis by naïve neutrophils (hallmark of reprogramming) and concomitantly activate caspase-1 and IL-1β production by these cells and that the addition of triple-combination highly effective CFTR modulator therapy does not abrogate these effects. Finally, EVs from activated neutrophils can deliver active caspase-1 to primary tracheal epithelial cells and induce their release of IL-1α. These findings support the existence of a feed-forward inflammatory process by which reprogrammed CF airway neutrophils bypass 2-step control of inflammasome activation in neighboring cells (naïve neutrophils and epithelial cells) via the transfer of bioactive EVs.

IPLA2 Activation Mediates Granular Exocytosis and Corrects Microbicidal Defects in ROS-Deficient and CGD Human Neutrophils.

The ubiquitous calcium-independent phospholipase A2 enzyme (iPLA2) is inhibited by calmodulin binding and known to be responsible for phospholipid remodeling housekeeping functions including granule exocytosis-associated membrane fusion in normal human neutrophils. We evaluate in human neutrophils the iPLA2 secretagogue effects using normal neutrophils, where reactive oxygen species (ROS) generation has been blocked by diphenyleneiodonium, as well as in neutrophils from chronic granulomatous disease (CGD) patients. Neutrophils were pretreated with W7, a calmodulin inhibitor known to activate iPLA2 and exocytosis of granules, and vesicles as well as intra- and extra-microbicidal activity against Staphylococcus aureus and Aspergillus fumigatus were evaluated. W7 increases exocytosis of primary, secondary, and tertiary granules and vesicles and improves neutrophil microbicidal activity against S. aureus and A. fumigatus. In neutrophils, calmodulin-mediated iPLA2 inhibition controls granule and vesicle exocytosis in the phagosome and in the extracellular microenvironment. Relieving iPLA2 inhibition results in increased exocytosis of primary, secondary, and tertiary granules and secretory vesicles with correction of defective intracellular and extracellular microbicidal activity. In CGD patients presenting ROS defective production, this increase in the non-oxidative killing pathway partially corrects their microbicidal defects.

Elastase Exocytosis by Airway Neutrophils Is Associated with Early Lung Damage in Children with Cystic Fibrosis.

Neutrophils are recruited to the airways of individuals with cystic fibrosis (CF). In adolescents and adults with CF, airway neutrophils actively exocytose the primary granule protease elastase (NE), whose extracellular activity correlates with lung damage. During childhood, free extracellular NE activity is measurable only in a subset of patients, and the exocytic function of airway neutrophils is unknown. To measure NE exocytosis by airway neutrophils in relation to free extracellular NE activity and lung damage in children with CF. We measured lung damage using chest computed tomography coupled with the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis scoring system. Concomitantly, we phenotyped blood and BAL fluid leukocytes by flow and image cytometry, and measured free extracellular NE activity using spectrophotometric and Förster resonance energy transfer assays. Children with airway inflammation linked to aerodigestive disorder were enrolled as control subjects. Children with CF but not disease control children harbored BAL fluid neutrophils with high exocytosis of primary granules, before the detection of bronchiectasis. This measure of NE exocytosis correlated with lung damage (R = 0.55; P = 0.0008), whereas the molecular measure of free extracellular NE activity did not. This discrepancy may be caused by the inhibition of extracellular NE by BAL fluid antiproteases and its binding to leukocytes. NE exocytosis by airway neutrophils occurs in all children with CF, and its cellular measure correlates with early lung damage. These findings implicate live airway neutrophils in early CF pathogenesis, which should instruct biomarker development and antiinflammatory therapy in children with CF.

Mitochondrial reactive oxygen species are involved in chemoattractant-induced oxidative burst and degranulation of human neutrophils in vitro

Activation of neutrophils is accompanied by the oxidative burst, exocytosis of various granule types (degranulation) and a delay in spontaneous apoptosis. The major source of reactive oxygen species (ROS) in human neutrophils is NADPH oxidase (NOX2), however, other sources of ROS also exist. Although the function of ROS is mainly defensive, they can also play a regulatory role in cell signaling. However, the contribution of various sources of ROS in these processes is not clear. We investigated a possible role of mitochondria-derived ROS (mtROS) in the regulation of neutrophil activation induced by chemoattractant fMLP in vitro. Using the mitochondria-targeted antioxidant SkQ1, we demonstrated that mtROS are implicated in the oxidative burst caused by NOX2 activation as well as in the exocytosis of primary (azurophil) and secondary (specific) granules. Scavenging of mtROS with SkQ1 slightly accelerated spontaneous apoptosis and significantly stimulated apoptosis of fMLP-activated neutrophils. These data indicate that mtROS play a critical role in signal transduction that mediates the major neutrophil functional responses in the process of activation.

A3R Phage and Staphylococcus aureus Lysate Do Not Induce Neutrophil Degranulation.

The objective of this study was to evaluate the effects of A3R phage and Staphylococcus aureus lysate obtained after phage infection on neutrophil degranulation. The exocytosis of primary and secondary granules from neutrophils was investigated in vitro in whole blood specimens by flow cytometry based on the expression of specific markers of exocytosis (CD63 for primary granules and CD66b for secondary granules). We found that both A3R and S. aureus lysate had no significant effect on the exocytosis of primary and secondary granules. These data suggest that neither A3R virions nor any products of phage-induced lysis of S. aureus are likely to induce neutrophil degranulation in patients who are treated with phage preparations. Since neutrophil granules contain some potentially toxic proteins, our results provide an important argument for the safety of phage therapy. Moreover, these data indicate that the induction of neutrophil degranulation is not likely to contribute to antibacterial effects of phages.

TREM-like transcript 2 is stored in human neutrophil primary granules and is up-regulated in response to inflammatory mediators.

The triggering receptor expressed on myeloid cell locus encodes a family of receptors that is emerging as an important class of molecules involved in modulating the innate immune response and inflammation. Of the 4 conserved members, including triggering receptor expressed on myeloid cells 1 and 2 and triggering receptor expressed on myeloid cell-like transcripts 1 and 2, relatively little is known about triggering receptor expressed on myeloid cell-like transcript 2 expression and function, particularly in humans. In this study, experiments were performed to determine if triggering receptor expressed on myeloid cell-like transcript 2 expression is conserved between mouse and human, demonstrating that human triggering receptor expressed on myeloid cell-like transcript 2 is expressed on cells of the lymphoid, as well as myeloid/granuloid lineages, similar to murine triggering receptor expressed on myeloid cell-like transcript 2. Consistent with studies in the mouse, triggering receptor expressed on myeloid cell-like transcript 2 expression is up-regulated in response to inflammatory mediators on human neutrophils. Importantly, it was shown that triggering receptor expressed on myeloid cell-like transcript 2, in resting human neutrophils, is predominantly localized to intracellular vesicles, including secretory vesicles and primary granules; with the majority of triggering receptor expressed on myeloid cell-like transcript 2 stored in primary granules. In contrast to other primary granule proteins, triggering receptor expressed on myeloid cell-like transcript 2 is not expelled on neutrophil extracellular traps but is retained in the plasma membrane following primary granule exocytosis. In summary, these findings establish that triggering receptor expressed on myeloid cell-like transcript 2 expression is conserved between species and is likely to be important in regulating neutrophil antimicrobial function following primary granule exocytosis.

Mature cystic fibrosis airway neutrophils suppress T cell function: evidence for a role of arginase 1 but not programmed death-ligand 1.

Bacteria colonize cystic fibrosis (CF) airways, and although T cells with appropriate Ag specificity are present in draining lymph nodes, they are conspicuously absent from the lumen. To account for this absence, we hypothesized that polymorphonuclear neutrophils (PMNs), recruited massively into the CF airway lumen and actively exocytosing primary granules, also suppress T cell function therein. Programmed death-ligand 1 (PD-L1), which exerts T cell suppression at a late step, was expressed bimodally on CF airway PMNs, delineating PD-L1(hi) and PD-L1(lo) subsets, whereas healthy control (HC) airway PMNs were uniformly PD-L1(hi). Blood PMNs incubated in CF airway fluid lost PD-L1 over time; in coculture, Ab blockade of PD-L1 failed to inhibit the suppression of T cell proliferation by CF airway PMNs. In contrast with PD-L1, arginase 1 (Arg1), which exerts T cell suppression at an early step, was uniformly high on CF and HC airway PMNs. However, arginase activity was high in CF airway fluid and minimal in HC airway fluid, consistent with the fact that Arg1 activation requires primary granule exocytosis, which occurs in CF, but not HC, airway PMNs. In addition, Arg1 expression on CF airway PMNs correlated negatively with lung function and positively with arginase activity in CF airway fluid. Finally, combined treatment with arginase inhibitor and arginine rescued the suppression of T cell proliferation by CF airway fluid. Thus, Arg1 and PD-L1 are dynamically modulated upon PMN migration into human airways, and, Arg1, but not PD-L1, contributes to early PMN-driven T cell suppression in CF, likely hampering resolution of infection and inflammation.

Increased survival and exocytosis of toxic primary granules by neutrophils in an in vitro model of Cystic Fibrosis airway disease (INC6P.310)

Abstract Cystic Fibrosis (CF) lung disease is characterized by the massive recruitment of neutrophils (PMNs) into the bronchiolar lumen. Extracellular neutrophil elastase (NE) that is released from PMN primary granules, is a strong predictor of lung function and survival in CF patients. However, the process by which CF airway PMNs exocytose HNE is not well understood and not reproduced in animal models of CF airway disease. Methods: We used a surface collagen-coated 3D porous polystyrene scaffold (Alvetex) to mimic the airway lamina propria and grew human bronchiolar H441 cells at air-liquid interface on top. In the transepithelial migration assay, blood PMNs were placed on the basal side and allowed to migrate towards to CF airway fluid placed apically, after which PMNs were analyzed by flow cytometry for rate of migration, survival, and degranulation. Results: Using this model we show that (i) CF airway fluid induces rapid transepithelial migration and survival of neutrophils, (ii) live PMNs actively release of primary granules upon exposure to CF airway fluid, and (iii) migration towards CF airway fluid induces PMN reprogramming allowing for the activation of the inflammasome and pinocytosis. Conclusions: These results suggest a primary role for the CF microenviroment in inducing increased survival and phenotypic reprogramming of PMNs and establish our model as a robust platform for deconstructing CF airway PMN dysfunction and developing new therapies for CF inflammation.

A role for Rab27 in neutrophil chemotaxis and lung recruitment.

BackgroundNeutrophils are a critical part of the innate immune system. Their ability to migrate into infected or injured tissues precedes their role in microbial killing and clearance. We have previously shown that Rab27a can promote neutrophil migration by facilitating uropod release through protease secretion from primary granule exocytosis at the cell rear. Rab27b has been implicated in primary granule exocytosis but its role in neutrophil migration has not been investigated.ResultsHere we found Rab27b to be expressed in bone marrow derived neutrophils and Rab27b knockout (Rab27b KO) along with Rab27a/b double knockout (Rab27DKO) neutrophils exhibited impaired transwell migration in vitro in response to chemokines MIP-2 and LTB4. Interestingly, no additional defect in migration was observed in Rab27DKO neutrophils compared with Rab27b KO neutrophils. In vivo, Rab27DKO mice displayed severe impairment in neutrophil recruitment to the lungs in a MIP-2 dependent model but not in an LPS dependent model.ConclusionsThese data taken together implicate Rab27b in the regulation of neutrophil chemotaxis, likely through the regulation of primary granule exocytosis.

Exocytosis of toxic primary granules by human neutrophils is associated with high surface TRPM2 expression and pinocytosis (INC6P.401)

Abstract Polymorphonuclear neutrophils (PMNs) play a key role in host defense against microbial infection. PMN responses culminate in the mobilization of highly toxic intracellular primary granules toward either the phagosome (during phagocytosis), nucleus (during NETosis), or plasma membrane (exocytosis). Mechanisms regulating the fate of PMN primary granules remain unclear. Here, we studied the dynamics of PMN primary granule exocytosis in two relevant sets of conditions: 1- in vivo migration of blood PMNs into chronically infected airways of patients with cystic fibrosis; 2- in vitro treatment of blood PMNs with latrunculin B and fMLF. Using flow and image cytometry, we show a striking resemblance between these two sets of conditions, with a close association between primary granule exocytosis (based on surface CD63 expression) and increased surface expression of the inflammatory calcium channel TRPM2. In addition, high primary granule exocytosis associates with major membrane rearrangements, as evidenced by the loss of surface phagocytic receptors (e.g., CD16 and CD35) and high pinocytosis (based on Lucifer Yellow uptake), a process previously linked to TRPM2 activation. All the while, exocytosing PMNs maintain their nuclear integrity. Thus, high primary granule exocytosis, surface TRPM2 upregulation and high pinocytosis are a concerted set of events indicative of a functional program in activated PMNs that is distinct from phagocytosis and NETosis.

P142 Src kinase inhibition attenuates neutrophil degranulation without impairing bacterial killing: a possible therapeutic strategy for acute lung injury?

BackgroundA key mechanism in the pathogenesis of acute lung injury (ALI) is excessive neutrophil degranulation in response to an overwhelming inflammatory or infective insult. To date, no pharmacological therapy for...

Metabolic Adaptation of Neutrophils in Cystic Fibrosis Airways Involves Distinct Shifts in Nutrient Transporter Expression

Inflammatory conditions can profoundly alter human neutrophils, a leukocyte subset generally viewed as terminally differentiated and catabolic. In cystic fibrosis (CF) patients, neutrophils recruited to CF airways show active exocytosis and sustained phosphorylation of prosurvival, metabolic pathways. Because the CF airway lumen is also characterized by high levels of free glucose and amino acids, we compared surface expression of Glut1 (glucose) and ASCT2 (neutral amino acids) transporters, as well as that of PiT1 and PiT2 (inorganic phosphate transporters), in blood and airway neutrophils, using specific retroviral envelope-derived ligands. Neither nutrient transporter expression nor glucose uptake was altered on blood neutrophils from CF patients compared with healthy controls. Notably, however, airway neutrophils of CF patients had higher levels of PiT1 and Glut1 and increased glucose uptake compared with their blood counterparts. Based on primary granule exocytosis and scatter profiles, CF airway neutrophils could be divided into two subsets, with one of the subsets characterized by more salient increases in Glut1, ASCT2, PiT1, and PiT2 expression. Moreover, in vitro exocytosis assays of blood neutrophils suggest that surface nutrient transporter expression is not directly associated with primary (or secondary) granule exocytosis. Although expression of nutrient transporters on CF blood or airway neutrophils was not altered by genotype, age, gender, or Pseudomonas aeruginosa infection, oral steroid treatment decreased Glut1 and PiT2 levels in blood neutrophils. Thus, neutrophils recruited from blood into the CF airway lumen display augmented cell surface nutrient transporter expression and glucose uptake, consistent with metabolic adaptation.

Reprogramming of a subpopulation of human blood neutrophils by prolonged exposure to cytokines

Essential cells of innate immunity, neutrophils are often considered to be a homogenous population of terminally differentiated cells. During inflammation, neutrophils are extravasated cells exposed to local factors that prolong their survival and activate their production of mediators implicated in disease progression. In this study, a phenotypically distinct subset of human neutrophils that appear after prolonged exposure to cytokines was characterized. Freshly isolated neutrophils from healthy donors were incubated with granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α and interleukin (IL)-4, three cytokines that are locally present in various inflammatory conditions. Eight to 17% of neutrophils survived beyond 72 h. This subset of non-apoptotic neutrophils, as evaluated by three different markers, was enriched by discontinuous Percoll gradient centrifugation before studying their phenotype. These viable neutrophils showed neoexpression of HLA-DR, CD80 and CD49d. Compared with freshly isolated neutrophils, they responded differentially to second signals similar to formyl-methionyl-leucyl-phenylalanine with three- to four-fold increases in production of superoxide anions and leukotrienes. These cells augmented their phagocytic index by 141%, increased their adhesion to human primary fibroblasts, but reduced their migration in response to chemotactic stimuli and decreased exocytosis of primary and secondary granules. In addition, they produced substantial amounts of IL-8, IL-1Ra and IL-1β. This neutrophil subset had a unique profile of phosphorylation of intracellular signaling molecules. In conclusion, the present identification of a novel neutrophil phenotype highlights the reprogammable character of the neutrophil. This aspect is crucial for our understanding of its contribution to disease pathogenesis and host defense.

Characterisation of degranulation and phagocytic capacity of a human neutrophilic cellular model, PLB-985 cells

Exocytosis of neutrophil granules is a major event that converts circulating neutrophils into fully activated cells capable of chemotaxis, phagocytosis and destruction of pathogens. The PLB-985 cell line is a suitable neutrophilic cellular model which is utilised to study the different functional responses of neutrophils. In this study, we characterised the differentiation of PLB-985 cells toward the granulocytic pathway, using three different inducing agents: dbcAMP, DMSO and DMF. The differentiation efficiency was monitored by observation of cell morphology with electron microscopy, and by analysis of the expression of receptors such as FPRL1 and FcγRIIA, the distribution or release of granule markers, phagocytic capacity, as well as measurement of fMLF-induced calcium fluxes. Exocytosis and phagocytosis in differentiated cells were weaker as compared to neutrophils. fMLF stimulated primary granule exocytosis in cells differentiated with dbcAMP, DMSO and DMF, whereas the release of the contents of tertiary granules, as well as that of secretory vesicles, was only observed in dbcAMP-differentiated cells. DMSO-differentiated cells exhibited the highest phagocytic capacity. Altogether our results reinforce the fact that depending on the differentiating agent used, PLB-985 cells represent a useful model to study neutrophil functions and to bypass difficulties inherent to these primary cells.

Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (< or = 10 microM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca(2+) ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca(2+) ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

Involvement of Munc18 isoforms in the regulation of granule exocytosis in neutrophils

Human neutrophil granule exocytosis mobilizes a complex set of secretory granules. This involves different combinations of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins to facilitate membrane fusion. The control mechanisms governing the late fusion steps are still poorly understood. Here, we have analyzed SNARE-interacting Sec1/Munc18 (SM) family members. We found that human neutrophils express Munc18-2 and Munc18-3 isoforms and that Munc18-2 interacts with the target-SNARE syntaxin 3. Munc18-2 was associated preferentially with primary granules but could also be found with secondary and tertiary granules, while Munc18-3 was majorily associated with secondary and tertiary granules. Ultrastructural analysis showed that both Munc18-2 and Munc18-3 were often located in close proximity to their respective SNARE-binding partners syntaxin 3 and syntaxin 4. Both isoforms were also found in plasma membrane fractions and in the cytosol, where they associate with cytoskeletal elements. Upon stimulation, Munc18-2 and Munc18-3 redistributed and became enriched on granules and in the plasma membrane. Munc18-2 primary granule exocytosis can be blocked by introduction of Munc18-2-specific antibodies indicating a crucial role in primary granule fusion. Our results suggest that Munc18-2 acts as a regulator of primary granule exocytosis, while Munc18-3 may preferentially regulate the fusion of secondary granules.

The Immunostimulatory Peptide WKYMVm‐NH2 Activates Bone Marrow Mouse Neutrophils via Multiple Signal Transduction Pathways

G-protein-coupled receptors play a major role in the activation of the innate immune system, such as polymorphonuclear neutrophils. Members of the formyl peptide receptor family recognize chemotactic peptides as well the amyloïd-beta peptide and fragments of the human immunodeficiency virus envelope and may thus be implicated in major pathologies. The peptide WKYMVm-NH2 probably activates the receptor FPRL1 and its mouse orthologues Fpr-rs1 and Fpr-rs2. We examined the stimulation of C57BL6 mouse neutrophils by WKYMVm-NH2 and the effects of several inhibitors for intracellular signalling pathways (wortmannin, LY 294002, staurosporin, H-89, U 73122, thapsigargin and SKF 96365). We show here that WKYMVm-NH2 is a powerful stimulator of primary and secondary granule exocytosis as well as superoxide production. The signalling pathway involves phosphoinositide 3-kinase, protein kinase C, phospholipase C and store-operated calcium influx. Studies with peptide antagonists suggest that WKYMVm-NH2 preferentially activates exocytosis via FPRL1 and not FPR, the major receptor for N-formylated peptides such as fMLF. However, the signalling pathways activated by WKYMVm-NH2 in mouse neutrophils are similar to those activated by fMLF in human neutrophils. Thus, the effect and the signalling pathways of the two agonists and their receptors are at least partially overlapping.

A rapid, direct assay to measure degranulation of primary granules in neutrophils from kidney of fathead minnow ( Pimephales promelas Rafinesque, 1820)

A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,3′,5,5′-tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of β-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content.