Fig 6 - uploaded by Daniel Pablo Lew
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![Submaximal secretion at suboptimal stimulation. ( A and B ) Illustration of the time course of a calculated capacitance increase over time (C t ) following a first order reaction: C t ϭ C max *(1 – e –t/ τ ) Complete secretion corresponds to 100%. Variations in the speed, τ (A), or in the amplitude, C max (B), of the reaction are modelled. ( C and D ) Measured mean capacitance traces of 4–7 patched neutrophils per [Ca 2 ϩ ] pip condition. ( E and F ) Mean rate of exocytosis over the fraction of granules secreted ( ∆ C m /C total ; total ϭ 7.5 pF) derived from the mean traces in (C) and (D). The fraction of 0.3 (30%) of the total, as obtained with 10 μ M (E) and 60 μ M [Ca 2 ϩ ] pip (F), corresponds to the maximal release of phase 1, 2.25 pF.](profile/Daniel-Lew/publication/13748367/figure/fig6/AS:282488292298754@1444361865071/Submaximal-secretion-at-suboptimal-stimulation-A-and-B-Illustration-of-the-time.png)
Submaximal secretion at suboptimal stimulation. ( A and B ) Illustration of the time course of a calculated capacitance increase over time (C t ) following a first order reaction: C t ϭ C max *(1 – e –t/ τ ) Complete secretion corresponds to 100%. Variations in the speed, τ (A), or in the amplitude, C max (B), of the reaction are modelled. ( C and D ) Measured mean capacitance traces of 4–7 patched neutrophils per [Ca 2 ϩ ] pip condition. ( E and F ) Mean rate of exocytosis over the fraction of granules secreted ( ∆ C m /C total ; total ϭ 7.5 pF) derived from the mean traces in (C) and (D). The fraction of 0.3 (30%) of the total, as obtained with 10 μ M (E) and 60 μ M [Ca 2 ϩ ] pip (F), corresponds to the maximal release of phase 1, 2.25 pF.
Source publication
We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low...
Contexts in source publication
Context 1
... effect of Ca 2 in such a system might be either a modulation of τ, i.e. the speed of the reaction, or a modulation of C max , i.e. the size of the available granule population. Figure 6A and B illus- trates the effects of modulation of τ and C max on a first order reaction. Clearly, modulation of τ has no effect on the final amplitude of the reaction. ...
Context 2
... modulation of τ has no effect on the final amplitude of the reaction. Figure 6C Figure 6F and G). This analysis showed that the maximal rate of secretion for phase 1 and 2 was reached when less than one-third of the respective granule population was released. ...
Citations
... Neutrophils have four kinds of granules; secretory vesicles, tertiary (gelatinase) granules, secondary (specific) granules and primary (azurophilic) granules. Each granule is known to be released dependent on concentrations of Ca 2+ [41][42][43] and GTP [44,45]: lower Ca 2+ promotes the release of secretory vesicles, while higher Ca 2+ induces primary granule release [41][42][43]. Likewise, GTP facilitates the hierarchical release of these granules [44,45]. ...
... Neutrophils have four kinds of granules; secretory vesicles, tertiary (gelatinase) granules, secondary (specific) granules and primary (azurophilic) granules. Each granule is known to be released dependent on concentrations of Ca 2+ [41][42][43] and GTP [44,45]: lower Ca 2+ promotes the release of secretory vesicles, while higher Ca 2+ induces primary granule release [41][42][43]. Likewise, GTP facilitates the hierarchical release of these granules [44,45]. ...
... As mentioned above, primary granules require a high intracellular level of Ca 2+ for their degranulation [41][42][43]. However, Ca 2+ entry via the plasma membrane is diminished in PMA-stimulated Hv1/VSOP-deficient neutrophils [22]. ...
The voltage-gated proton channel, Hv1, also termed VSOP, was discovered in 2006. It has long been suggested that proton transport through voltage-gated proton channels regulate reactive oxygen species (ROS) production in phagocytes by counteracting the charge imbalance caused by the activation of NADPH oxidase. Discovery of Hv1/VSOP not only confirmed this process in phagocytes, but also led to the elucidation of novel functions in phagocytes. The compensation of charge by Hv1/VSOP sustains ROS production and is also crucial for promoting Ca2+ influx at the plasma membrane. In addition, proton extrusion into neutrophil phagosomes by Hv1/VSOP is necessary to maintain neutral phagosomal pH for the effective killing of bacteria. Contrary to the function of Hv1/VSOP as a positive regulator for ROS generation, it has been revealed that Hv1/VSOP also acts to inhibit ROS production in neutrophils. Hv1/VSOP inhibits hypochlorous acid production by regulating degranulation, leading to reduced inflammation upon fungal infection, and suppresses the activation of extracellular signal-regulated kinase (ERK) signaling by inhibiting ROS production. Thus, Hv1/VSOP is a two-way player regulating ROS production. Here, we review the functions of Hv1/VSOP in neutrophils and discuss future perspectives.
... Ca 2+ signaling is one of the major signalings contributing to these functional responses of human neutrophils. Intracellular free Ca 2+ concentration ([Ca 2+ ] i ) mediates or essentially regulates important cellular responses in neutrophils including production and release of arachidonic acid products [14], degranulation [15,16], respiratory burst [17,18], chemotaxis [19,20]. Thus, Ca 2+ contributes essentially to the function of neutrophils during their defense against bacterial and fungal infections. ...
N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils’ functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]і) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]і in human neutrophils. We observed that NAC (1 μM ~ 1 mM) and cysteine (10 μM ~ 1 mM) increased [Ca2+]і in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucinephenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]і increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 μM) and ruthenium red (20 μM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]і increasing activity. Our results show that NAC and cysteine induce [Ca2+]і increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way.
... Ca 2+ elevations direct the remodelling of the actin cytoskeleton (9,27) as well as integrin recycling and uropod retraction during neutrophil migration (29,56). Ca 2+ elevations finely regulates the exocytosis of the different neutrophil granule populations (58,77) and the production of superoxide by NADPH oxidase located in the plasma membrane and in phagosomes (35,38,111), reviewed in (15). Ca 2+ elevations also regulate the efficiency of phagocytosis, an effect that likely reflects the Ca 2+ dependency of the maturation process (46,121) as the phagocytic ingestion phase appears to be largely Ca 2+ -independent in neutrophils, depending on the type of phagocytic receptors engaged (reviewed in (75)). ...
Phagocytic cells such as neutrophils, macrophages and dendritic cells (DCs), migrate to sites of infection or damage and are integral to innate immunity through two main mechanisms. The first is to directly neutralize foreign agents and damaged or infected cells by secreting toxic substances or ingesting them through phagocytosis. The second is to alert the adaptive immune system through the secretion of cytokines and the presentation of the ingested materials as antigens, inducing T cell maturation into helper, cytotoxic or regulatory phenotypes. While calcium signalling has been implicated in numerous phagocyte functions including differentiation, maturation, migration, secretion and phagocytosis, the molecular components that mediate these Ca(2+) signals have been elusive. The discovery of the STIM and ORAI proteins has allowed researchers to begin clarifying the mechanisms and physiological impact of Store-Operated Ca(2+) Entry (SOCE), the major pathway for generating calcium signals in innate immune cells. Here, we review evidence from cell lines and mouse models linking STIM and ORAI proteins to the control of specific innate immune functions of neutrophils, macrophages, and dendritic cells.
... For patch-clamp recordings, the neutrophils were plated on glass coverslips, in some cases coated with 1% BSA. Capacitance recordings with the time domain technique were performed as described for human neutrophils (22). Mouse neutrophils have an initial capacitance of 1.6 Ϯ 0.1 pF on albumin and 2.3 Ϯ 0.13 on glass in the presence of cytochalasin B (control cells), which probably reflects some spontaneous degranulation on glass. ...
... The capacitance values for mouse neutrophils on glass are around 25% smaller than human neutrophils (23). The cells were stimulated with either 20 M GTP␥S or 100 M Ca 2ϩ in the pipette to elicit a significant secretory response (22,23). ...
... Perfusion of 100 M Ca 2ϩ and/or 20 M GTP␥S into single cells via the patch-pipette induced secretion in wild-type mouse neutrophils. The Ca 2ϩ concentrations were chosen based on our previous studies in human neutrophils (22), where 100 M Ca 2ϩ was needed to elicit significant release of primary and secondary granules. ...
Article:Gelsolin is involved in IgG-mediated phagocytosis
... Mild increases in the [Ca 2+ ] i selectively promote exocytosis of secretory vesicles, and exocytosis of specific and azurophilic granules requires higher [Ca 2+ ] i [15][16][17]. Likewise, GTP is also known to mediate selective degranulation [18,19]. ...
... Ca 2+ is well known to regulate exocytosis in many cell types [59]. In neutrophils, a moderate increase in [Ca 2+ ] i induces exocytosis of secretory granules, but a large [Ca 2+ ] i increase promotes exocytosis of the secondary granules, followed by facilitation of exocytosis of azurophilic granules [13,17]. Chemoattractant-induced Ca 2+ influx has been observed in Hv1/ VSOP-deficient neutrophils [4], suggesting a possible effect on the degranulation processes. ...
Neutrophil granule exocytosis is crucial for host defense and inflammation. Neutrophils contain 4 types of granules, the exocytotic release of which is differentially regulated. This exocytosis is known to be driven by diverse mediators, including calcium and nucleotides, but the precise molecular mechanism remains largely unknown. We show in the present study that voltage-gated proton (Hv) channels are necessary for the proper release of azurophilic granules in neutrophils. On activation of NADPH oxidase by PMA and IgG, neutrophils derived from Hvcn1 gene knock-out mouse exhibited greater secretion of MPO and elastase than WT cells. In contrast, release of LTF enriched in specific granules was not enhanced in these cells. The excess release of azurophilic granules in Hv1/VSOP-deficient neutrophils was suppressed by inhibiting NADPH oxidase activity and, in part, by valinomycin, a potassium ionophore. In addition, Hv1/VSOP-deficient mice exhibited more severe lung inflammation after intranasal Candida albicans infection than WT mice. These findings suggest that the Hv channel acts to specifically dampen the release of azurophilic granules through, in part, the suppression of increased positive charges at the plasma membrane accompanied by the activation of NADPH oxidase in neutrophils.
© Society for Leukocyte Biology.
... Similar to its effect on vimentin Ser56 phosphorylation, roscovitine resulted in considerable but incomplete inhibition of β-hexosaminidase, lactoferrin, and MMP-9 secretion (Fig. 4B-D). The different extent and pattern of secretory response to GTP and inhibitory effect of roscovitine on the three granule populations is consistent with previous findings from our group (Rosales and Ernst, 1997) and others (Nusse et al., 1998), indicating that the secretory mechanisms in the different granule populations are distinct. Thus, roscovitine appears to have the greatest inhibitory effect on β-hexosaminidase secretion after 5 min of treatment while roscovitine inhibition of lactoferrin secretion was noticeable at 10 min following treatment. ...
Secretion by neutrophils contributes to acute inflammation following injury or infection. Vimentin has been shown to be important for secretion by neutrophils but little is known about its dynamics during secretion, which is regulated by cyclin-dependent kinase 5 (Cdk5). In this study, we sought to examine the vimentin dynamics and its potential regulation by Cdk5 during neutrophil secretion. We show that vimentin is a Cdk5 substrate that is specifically phosphorylated at Ser56. In response to neutrophil stimulation with GTP, vimentin Ser56 was phosphorylated and colocalized with Cdk5 in the cytoplasmic compartment. Vimentin pSer56 and Cdk5 colocalization was consistent with coimmunoprecipitation from stimulated cells. Vimentin Ser56 phosphorylation occurred immediately after stimulation, and a remarkable increase in phosphorylation was noted later in the secretory process. Decreased GTP-induced vimentin Ser56 phosphorylation and secretion resulted from inhibition of Cdk5 activity by roscovitine or olomoucine or by depletion of Cdk5 by siRNA, suggesting that GTP-induced Cdk5-mediated vimentin Ser56 phosphorylation may be related to GTP-induced Cdk5-mediated secretion by neutrophils. Indeed, inhibition of vimentin Ser56 phosphorylation led to a corresponding inhibition of GTP-induced secretion, indicating a link between these two events. While fMLP also induced vimentin Ser56 phosphorylation, such phosphorylation was unaffected by roscovitine, which nonetheless, inhibited secretion, suggesting that Cdk5 regulates fMLP-induced secretion via a mechanism independent of Cdk5-mediated vimentin Ser56 phosphorylation. These findings demonstrate the distinct involvement of Cdk5 in GTP- and fMLP-induced secretion by neutrophils, and support the notion that specific targeting of Cdk5 may serve to inhibit the neutrophil secretory process.
... 28 A rise in intracellular calcium concentration plays a central role in signaling downstream of chemokine ligation and adhesion function and we and others have detected Ca 2+ flux to be an indicator of the level of PMN activation during recruitment on inflamed endothelium under shear stress. 15,25,45,59,60,77,84,85 A combination of shear stress and E-selectin engagement is a requirement for transition of LFA-1 to an extended conformation and subsequent maximal activation of b 2 -integrins in rolling PMN. 42,76 Moreover, it is the superposition of activation via ESLs and G-protein coupled receptors that elicit optimal levels of calcium flux on arrested PMN. ...
Application of mechanical force to bonds between selectins and their ligands is a requirement for these adhesion receptors to optimally perform functions that include leukocyte tethering and activation of stable adhesion. Although all three selectins are reported to signal from the outside-in subsequent to ligand binding, E-selectin is unique in its capacity to bind multiple sialyl Lewis x presenting ligands and mediate slow rolling on the order of a micron per second. A diverse set of ligands are recognized by E-selectin in the mouse, including ESL-1, CD44 (HCELL), and PSGL-1 which are critical in transition from slow rolling to arrest and for efficient transendothelial migration. The molecular recognition process is different in humans as L-selectin is a major ligand, which along with glycolipids constitute more than half of the E-selectin receptors on human polymorphonuclear neutrophils (PMN). In addition, E-selectin is most efficient at raising the affinity and avidity of CD18 integrins that supports PMN deceleration and trafficking to sites of acute inflammation. The mechanism is only partially understood but known to involve a rise in cytosolic calcium and tyrosine phosphorylation that activates p38 MAP kinase and Syk kinase, both of which transduce signals from clustered E-selectin ligands. In this review we highlight the molecular recognition and mechanical requirements of this process to reveal how E-selectin confers selectivity and efficiency of signaling for extravasation at sites of inflammation and the mechanism of action of a new glycomimetic antagonist targeted to the lectin domain that has shown efficacy in blocking neutrophil activation and adhesion on inflamed endothelium.
... Whatever the Ca 2+ role, the overall process may sense it with affinities that vary from nerve terminals (K m , about 200 µM;Heidelberger et al., 1994) to endocrine cells (K m , about 20 µM;Proks et al., 1996). Within the same cell, different granule populations may even be secreted with widely different Ca 2+ affinities (Nüsse et al., 1998), which are likely to be related to the involvement of distinct Ca 2+ sensors: of these several have been postulated, e.g., synaptotagmin, annexins, the S-100 proteins, and even calmodulin (Geppert et al., 1994;Lang et al., 1997;Peters and Mayer, 1998;Schafer and Heizmann, 1996). The relatively poor Ca 2+ affinity of the system opens the problem of whether Ca 2+ can ever reach the supra-µM levels necessary to activate it (problems of this type have already been alluded to when discussing other systems in previous sections). ...
Referee: Guiseppe Inesi, M.D., Ph.D., Professor and Chairman, School of Medicine, Dept. of Biochemistry and Molecular Biology, Univeristy of Maryland, Baltimore.
... They demonstrated that primary granules require a strong stimulation and the highest intracellular Ca 2+ concentration for exocytosis, while secondary and tertiary granules are mobilized already by weaker stimulation . Consistently, Nusse et al., using the patch-clamp technique, identified two phases of secretion with high and low Ca 2+ affinity within individual human neutrophils (Nusse et al., 1998). The high-affinity component consisted of secondary and tertiary granules, while the low-affinity component consisted of MPO positive (primary) granules. ...
Polymorphonuclear leukocytes (PMN) can release opioid peptides which bind to opioid receptors on sensory neurons and inhibit inflammatory pain. This release can be triggered by chemokine receptor 1/2 (CXCR1/2) ligands. Our aim was to identify the granule subpopulation containing opioid peptides and to assess whether MAPK mediate the CXCR1/2 ligand-induced release of these peptides. Using double immunofluorescence confocal microscopy, we showed that beta-endorphin (END) and Met-enkephalin (ENK) were colocalized with the primary (azurophil) granule markers CD63 and myeloperoxidase (MPO) within PMN. END and ENK release triggered by a CXCR1/2 ligand in vitro was dependent on the presence of cytochalasin B (CyB) and on p38 MAPK, but not on p42/44 MAPK. In addition, translocation of END and ENK containing primary granules to submembranous regions of the cell was abolished by the p38 MAPK inhibitor SB203580. In vivo CXCL2/3 reduced pain in rats with complete Freund's adjuvant (CFA)-induced hindpaw inflammation. This effect was attenuated by intraplantar (i.pl.) antibodies against END and ENK and by i.pl. p38 MAPK inhibitor treatment. Taken together, these findings indicate that END and ENK are contained in primary granules of PMN, and that CXCR1/2 ligands induce p38-dependent translocation and release of these opioid peptides to inhibit inflammatory pain.
... The activity of sPLA 2 in the extracellular medium after stimulation of the neutrophils with A23187 was not higher than that detected in the extracellular medium after stimulation with fMLP. These results suggest that although an increase in [Ca 2+ ]i is required for degranulation [40,41], the moderate increase stimulated by fMLP is sufficient for sPLA 2 -V secretion, and a further increase of [Ca 2+ ]i does not affect this secretion. Analysis of the different cellular fractions and immunofluorescence microscopy revealed that sPLA 2 -X is found on the plasma membranes after stimulation and colocalized with the plasma membrane component of the NADPH oxidase, gp91 phox (Fig. 2C-E). ...
Neutrophils and differentiated PLB-985 cells contain various types of PLA(2)s including the 85 kDa cytosolic PLA(2) (cPLA(2)), Ca(2+)-independent PLA(2) (iPLA(2)) and secreted PLA(2)s (sPLA(2)s). The present study focuses on the behavior of sPLA(2)s in neutrophils and PLB cells and their relationship to cPLA(2)alpha. The results of the present research show that the two types of sPLA(2) present in neutrophils, sPLA(2)-V and sPLA(2)-X, which are located in the azurophil granules, are differentially affected by physiological stimuli. While sPLA(2)-V is secreted to the extacellular milieu, sPLA(2)-X is detected on the plasma membranes after stimulation. Stimulation of neutrophils with formyl-Met-Leu-Phe (fMLP), opsonized zymosan (OZ) or A23187 resulted in a different kinetics of sPLA(2) secretion as detected by its activity in the neutrophil supernatants. Neutrophil priming by inflammatory cytokines or LPS enhanced sPLA(2) activity detected in the supernatant after stimulation by fMLP. This increased activity was due to increased secretion of sPLA(2)-V to the supernatant and not to release of sPLA(2)-X. sPLA(2) in granulocyte-like PLB cells exhibit identical characteristics to neutrophil sPLA(2), with similar activity and optimal pH of 7.5. Granulocyte-like cPLA(2)alpha-deficient PLB cells serve as a good model to study whether sPLA(2) activity is regulated by cPLA(2)alpha. Secretion and activity of sPLA(2) were found to be similar in granulocyte-like PLB cells expressing or lacking cPLA(2)alpha, indicating that they are not under cPLA(2)alpha regulation.
