Exocytosis Of Secondary Granules Research Articles

A Common Genetic Variant in TLR1 Enhances Human Neutrophil Priming and Impacts Length of Intensive Care Stay in Pediatric Sepsis.

Polymorphonuclear leukocytes (PMN) achieve an intermediate or primed state of activation following stimulation with certain agonists. Primed PMN have enhanced responsiveness to subsequent stimuli, which can be beneficial in eliminating microbes but may cause host tissue damage in certain disease contexts, including sepsis. As PMN priming by TLR4 agonists is well described, we hypothesized that ligation of TLR2/1 or TLR2/6 would prime PMN. Surprisingly, PMN from only a subset of donors were primed in response to the TLR2/1 agonist, Pam3CSK4, although PMN from all donors were primed by the TLR2/6 agonist, FSL-1. Priming responses included generation of intracellular and extracellular reactive oxygen species, MAPK phosphorylation, integrin activation, secondary granule exocytosis, and cytokine secretion. Genotyping studies revealed that PMN responsiveness to Pam3CSK4 was enhanced by a common single-nucleotide polymorphism (SNP) in TLR1 (rs5743618). Notably, PMN from donors with the SNP had higher surface levels of TLR1 and were demonstrated to have enhanced association of TLR1 with the endoplasmic reticulum chaperone gp96. We analyzed TLR1 genotypes in a pediatric sepsis database and found that patients with sepsis or septic shock who had a positive blood culture and were homozygous for the SNP associated with neutrophil priming had prolonged pediatric intensive care unit length of stay. We conclude that this TLR1 SNP leads to excessive PMN priming in response to cell stimulation. Based on our finding that septic children with this SNP had longer pediatric intensive care unit stays, we speculate that this SNP results in hyperinflammation in diseases such as sepsis.

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...

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.

Neutrophil adhesion to endothelial cells induced by platelet factor 4 requires sequential activation of Ras, Syk, and JNK MAP kinases

Signal transduction mechanisms associated with neutrophil activation by platelet factor 4 (PF4; CXCL4) are as yet poorly characterized. In a recent report, we showed that PF4-induced neutrophil functions (such as adhesion and secondary granule exocytosis) involve the activation of Src-kinases. By analyzing intracellular signals leading to adherence, we here demonstrate by several lines of evidence that in addition to Src-kinases, PF4 signaling involves the monomeric GTPase Ras, the tyrosine kinase Syk, and the MAP kinase JNK. Furthermore, on stimulation, GTPases Rac2 and RhoA were activated, and each was translocated to a different membrane compartment. As shown by inhibitor studies, Rac2 and JNK are located downstream of Syk and Ras. Most intriguingly, the latter 2 elements appear to control the activity of Rac2 and JNK independently of each other at different phases of the activation process. Although a first phase of Rac2 and JNK activation of up to 5 minutes is initiated by Ras, the second phase (5-30 minutes) depends predominantly on the activity of Syk. In summary, we describe that coordinated activity of Syk, Ras, and JNK mediates neutrophil adhesion to endothelial cells and that PF4 induces sequential activation of these elements.

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.

Neutrophil primary granule release and maximal superoxide generation depend on Rac2 in a common signalling pathway

Neutrophils play an integral role in innate immunity by undergoing degranulation and respiratory burst in response to inflammatory stimuli. Rac2, a monomeric GTP-binding protein, has been shown to be involved in several neutrophil functions, including primary granule release and superoxide (O(2)(-.) generation. We hypothesized that Rac2 is a common signalling molecule required for primary granule translocation and maximal O(2)(-.) production. Using bone marrow neutrophils from Rac2 knockout (KO) mice and wild type C57Bl/6 mice, we found that primary granule elastase and myeloperoxi dase release were absent in Rac2 KO neutrophils upon chemoattractant stimulation. Rac2 KO neutrophils also failed to produce maximal levels of extracellular O(2(-.) generation in response to phorbol myristate acetate (PMA). Although PMA was ineffective at eliciting primary granule mediator release, it induced secondary granule exocytosis in both WT and Rac2 KO neutrophils. Thus, the signalling pathway leading to primary granule release utilized Rac2, which was also necessary for full activation of O(2)(-.) generation in stimulated neutrophils. These findings indicate that O(2)(-.) release and secondary granule secretion may use protein kinase C (PKC) - dependent pathways, whereas primary granule exocytosis appears to rely on PKC-independent signalling events. These findings shed light on possible signalling mechanisms involved in granule secretion from activated neutrophils responding to different stimuli.

Modulation of human neutrophil inflammatory responses by nitric oxide: studies in unprimed and LPS-primed cells.

Because nitric oxide (NO) can act both as a regulatory and as a toxic molecule, we have studied N-formyl-methionyl-leucyl-phenylalanine (fMLF)-stimulated responses of human neutrophils (PMNs) during various conditions of NO modulation in unprimed and bacterial lipopolysaccharide (LPS) -primed cells. Effects of various NO modulators were assessed on stimulated superoxide (O2-) generation, granule exocytosis, homotypic aggregation, and rises in intracellular free Ca2+ ([Ca2+]i). Significant differences in the effects of various NO modulators on inflammatory responses of PMNs kept in stirred suspension versus those kept under static and/or adherent conditions, were observed. L-arginine, the physiological substrate for NO synthase (NOS), and NG-nitro-L-arginine methyl ester, an inhibitor of NOS, both caused a 40-50% inhibition of LPS-induced priming of O2- generation in PMNs in stirred suspension, but not in LPS-primed PMNs under static or adherent conditions. The NO donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, completely abrogated the LPS-induced priming of O2- generation in PMNs in suspension, while causing only a 40-50% inhibition in PMNs under static or adherent conditions. The Ca2+ ionophore, A23187, prevented the LPS-induced priming of O2- generation without affecting O2- generation in unprimed PMNs. LPS priming of PMNs induced about a twofold increase in fMLF-stimulated homotypic aggregation, exocytosis of secondary granules, and rises in [Ca2+]i. In related studies, we also provide definitive evidence for enzymatic formation of NO in human PMNs and demonstrate a significant decrease in NO levels in LPS-primed PMNs. Taken together, these findings indicate that NO modulates PMN inflammatory responses and plays a protective role in priming and activation processes of inflammatory PMNs.

Human neutrophil secondary granule exocytosis is independent of protein kinase activation and is modified by calmodulin activity

Exocytosis of the secondary (2°) lysosomal granule is an important process in the activation of human neutrophils. Stored enzymes such as collagenase and gelatinase are released, and adhesion molecules from the granule membrane are inserted in the plasma membrane. This exocytosis is independent of azurophil granule release and respiratory burst activation. We investigated, using kinase and phosphatase inhibitors and activators of adenylate cyclase, common intracellular signalling mechanisms involved in exocytosis (vitamin B12 binding protein release) stimulated by different agonists. Exocytosis in response to tumour necrosis factor α (TNFα), phorbol myristate acetate (PMA) and the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was inhibited by the calmodulin antagonist N-(6-amino hexyl)-5-chloro-1-naphthalene sulphonamide (W7). Neither staurosporine, H7 nor genistein was inhibitory. In contrast, the same doses of W7 synergistically enhanced the exocytosis stimulated by the tyrosine phosphatase inhibitor sodium orthovanadate, while kinase inhibition by staurosporine or genistein dose-dependently inhibited the vanadate response. Furthermore, adenylate cyclase activation with prostaglandin E 2 or dibutyryl cyclic AMP, inhibited exocytosis in response to TNFα and FMLP, while having no effect on the release induced by vanadate or PMA. Thus, 2° granule exocytosis stimulated by receptor-bound ligands is calmodulin-dependent, and is independent of protein kinase activity. In contrast, exocytosis in response to tyrosine phosphatase inhibition is antagonised by calmodulin, since the response to vanadate was enhanced synergistically by W7. Thus, depending on the initial stimulus, calmodulin may promote or inhibit 2° granule exocytosis by human PMN.

TNF‐specific deactivation of granulocytes in vivo: A possible mechanism of self‐protection

Granulocytes (PMN) have recently been shown to be specifically deactivated towards tumor necrosis factor (TNF) by preexposure to TNF itself and to other stimuli such as endotoxin and complement C5a in vitro. When TNF (200 micrograms/m2) was infused to a male volunteer, we found ex vivo an almost complete TNF-specific deactivation of PMN adhesion (57 +/- 3* vs 6 +/- 2%**), of exocytosis of secondary granules (37 +/- 1* vs 7 +/- 1%**) and of the respiratory burst as measured by hexose monophosphate shunt activation (74 +/- 1* vs 12 +/- 3** nM/10(7) PMN/45 min). Furthermore, up- and down-regulation, respectively, of the PMN surface adhesion molecules Mac-1 and LAM-1 became TNF-resistant. Similar to the results obtained in vitro, deactivation correlated with a decrease in the number of cellular TNF receptors. In PMN of 4 patients exposed to activated complement during hemodialysis, a state of TNF-specific cross-deactivation could also be demonstrated.

Neutrophil CR3 expression and specific granule exocytosis are controlled by different signal transduction pathways.

Neutrophils express receptors (CR3) for the complement fragment C3bi. CR3 expression can be increased by exposure of the cells to chemotactic factors such as FMLP or to the calcium ionophore A23187. It has been suggested that CR3 moieties are stored in the membrane bounding either the secondary or the tertiary (gelatinase containing) granules. To help define the mechanisms mediating CR3 up-regulation, the effects of several inhibitors upon CR3 expression and secondary granule exocytosis were investigated. Pertussis toxin inhibited FMLP-induced (but not A23187-induced) CR3 expression and exocytosis, indicating that an early step in FMLP-induced CR3 expression is activation of a pertussis toxin-sensitive G protein. However, CR3 expression and exocytosis appeared to be controlled by separate mechanisms distal to G protein activation because 1) DBcAMP and the protein kinase inhibitor H-7 inhibited or stimulated exocytosis, respectively, without affecting CR3 expression; 2) the calmodulin (chlorpromazine and trifluoperazine) and myosin L chain kinase (ML9) inhibitors had greater effects on exocytosis than on CR3 expression; and 3) the kinetics of CR3 expression and exocytosis differed markedly. Thus, although G protein activation is a common early step in both processes, there is a bifurcation of the two processes distally. The mechanisms mediating CR3 up-regulation and tertiary granule exocytosis were also investigated. Extracellular Ca2+ was essential for tertiary granule exocytosis, but not for CR3 up-regulation. We conclude that because the mechanisms controlling CR3 up-regulation and exocytosis diverge soon after the binding of a chemotactic ligand to its receptor, that at least the bulk of increased CR3 expression is not simply a by-product of secondary and tertiary granule exocytosis but is the result of the mobilization of CR3 moieties from an intracellular pool of uncertain identity.

Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels.

It has long been assumed that a rise in cytosolic free Ca2+, [Ca2+]i, is a necessary and sufficient event for the stimulation of a variety of cellular processes. The development of a technique which allows monitoring of [Ca2+]i in small intact cells has led to a critical revision of this simple postulate. We have recently shown that in neutrophils, Ca2+-ionophore-induced elevations of [Ca2+]i, quantitatively similar to those caused by chemotatic peptides, are ineffective in stimulating cell responses, which suggests that an additional signal is required for receptor-mediated activation. Here we show that subthreshold concentrations of phorbol myristate acetate (PMA) and of a Ca2+ ionophore can quantitatively mimic the effect of a physiological agonist. However, PMA at higher concentrations can trigger NADPH-oxidase activity, exocytosis and protein phosphorylation, even when [Ca2+]i is lowered 10-20 times below the normal resting level. These results strongly suggest that activation of protein kinase C is sufficient, by itself, to induce NADPH-oxidase activation and exocytosis of secondary granules in neutrophils.

Pathogenesis of foreign body infection. Evidence for a local granulocyte defect.

Implanted foreign bodies are highly susceptible to pyogenic infections and represent a major problem in modern medicine. In an effort to understand the pathogenesis of these infections, we studied the phagocytic function in the vicinity of a foreign body by using a recently developed guinea pig model of Teflon tissue cages subcutaneously implanted (Zimmerli, W., F.A. Waldvogel, P. Vaudaux, and U.E. Nydegger, 1982, J. Infect. Dis., 146:487-497). Polymorphonuclear leukocytes (PMN) purified from tissue cage fluid had poor bactericidal activity against a catalase-positive microorganism. When compared with blood or exudate PMN, they exhibited a significant reduction in their ability to generate superoxide in response to a particulate or a soluble stimulus (72 and 57%, respectively, P less than 0.001). Not only their total contents in myeloperoxidase, beta-glucuronidase, lysozyme, and B12 binding protein were significantly reduced (by 62, 21, 47, and 63%, respectively, P less than 0.01), but also their capability for further secretion of residual B12 binding protein upon stimulation. Ingestion rates of endotoxin-coated opsonized oil particles were reduced by 25% (P less than 0.05). In an effort to reproduce these abnormalities in vitro, fresh peritoneal exudate PMN were incubated with Teflon fibers in the presence of plasma. Interaction of PMN with the fibers led to significant increases in hexose monophosphate shunt activity and exocytosis of secondary granules (P less than 0.01). PMN eluted after such interaction showed defective bactericidal activity, oxidative metabolism, and granular enzyme content similar to those observed in tissue cage PMN. The local injection of fresh blood PMN into tissue cages at the time of, or 3 h after, inoculation with 100 microorganisms (Staphylococcus aureus Wood 46) reduced the infection rate from 50 to 56 cages to 1 of 21 (P less than 0.001) and 3 of 8 cages (P less than 0.001), respectively. These results suggest that the in vivo as well as in vitro interaction of PMN with a nonphagocytosable foreign body induces a complex PMN defect, which may be partly responsible for the high susceptibility to infection of foreign bodies.