Membrane Potential Of Neutrophils Research Articles

ORAI1 and ORAI2 modulate murine neutrophil calcium signaling, cellular activation, and host defense.

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.

Complement C5a Alters the Membrane Potential of Neutrophils during Hemorrhagic Shock.

Background Polymorphonuclear granulocytes (PMN) play a crucial role in host defense. Physiologically, exposure of PMN to the complement activation product C5a results in a protective response against pathogens, whereas in the case of systemic inflammation, excessive C5a substantially impairs neutrophil functions. To further elucidate the inability of PMN to properly respond to C5a, this study investigates the role of the cellular membrane potential of PMN in response to C5a. Methods Electrophysiological changes in cellular and mitochondrial membrane potential and intracellular pH of PMN from human healthy volunteers were determined by flow cytometry after exposure to C5a. Furthermore, PMN from male Bretoncelles-Meishan-Willebrand cross-bred pigs before and three hours after severe hemorrhagic shock were analyzed for their electrophysiological response. Results PMN showed a significant dose- and time-dependent depolarization in response to C5a with a strong response after one minute. The chemotactic peptide fMLP also evoked a significant shift in the membrane potential of PMN. Acidification of the cellular microenvironment significantly enhanced depolarization of PMN. In a clinically relevant model of porcine hemorrhagic shock, the C5a-induced changes in membrane potential of PMN were markedly diminished compared to healthy littermates. Overall, these membrane potential changes may contribute to PMN dysfunction in an inflammatory environment.

Effects of DHA-rich fish oil supplementation on the lipid profile, markers of muscle damage, and neutrophil function in wheelchair basketball athletes before and after acute exercise

We investigated the effects of docosahexaenoic acid (DHA)-rich fish oil (FO) supplementation on the lipid profile, levels of plasma inflammatory mediators, markers of muscle damage, and neutrophil function in wheelchair basketball players before and after acute exercise. We evaluated 8 male basketball wheelchair athletes before and after acute exercise both prior to (S0) and following (S1) FO supplementation. The subjects were supplemented with 3 g of FO daily for 30 days. The following components were measured: the plasma lipid profile (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides), plasma inflammatory mediators (C-reactive protein, interleukin (IL)-1β, IL-1ra, IL-4, IL-6, IL-8, and tumor necrosis factor-α), markers of muscle damage (creatine kinase and lactate dehydrogenase (LDH)), and neutrophil function (cytokine production, phagocytic capacity, loss of membrane integrity, mitochondrial membrane potential, neutral lipid accumulation, phosphatidylserine externalization, DNA fragmentation, and production of reactive oxygen species (ROS)). Acute exercise increased the plasma levels of total cholesterol, LDH, IL1ra, and IL-6, led to the loss of membrane integrity, ROS production, and a high mitochondrial membrane potential in neutrophils, and reduced the phagocytic capacity and IL-6 production by the neutrophils (S0). However, supplementation prevented the increases in the plasma levels of LDH and IL-6, the loss of membrane integrity, and the alterations in ROS production and mitochondrial membrane potential in the neutrophils that were induced by exercise (S1). In conclusion, DHA-rich FO supplementation reduces the markers of muscle damage, inflammatory disturbances, and neutrophil death induced by acute exercise in wheelchair athletes.

G6PC3 Deficiency Associated with Congenital Neutropenia and Enterocolitis

Abstract 2170Glucose-6-phosphatase catalytic subunit 3 (G6PC3) deficiency causes congenital neutropenia in conjunction with various cardiac or urogenital developmental aberrations.We here describe a consanguineous pedigree of maroccan descent. Four patients presented with early-onset inflammatory bowel diseases associated with severe congenital neutropenia. Two children died in early childhood, two patients (19-years old female, 17-years old male) are alive with signs of recurrent infections (mouth ulcera, otitis, upper and lower respiratory tract infections) and refractory Crohn's like enterocolitis. Mutations in the Glucose-6-phosphate translocase (SLC37A4) gene were excluded.In an attempt to discover the underlying molecular pathophysiology, we performed SNP-based homozygosity mapping followed by in depth next-generation exome sequencing. We identified two genes with potentially deleterious homozygous sequence variations: P2RY11 (exon 13; c.C2160G, p.Y720X), encoding a protein with antiapoptotic function, and G6PC3 (exon 2; c.C323T, p.P108L). The G6PC3 mutation segregates with the disease phenotype, whereas the P2RY11 variant was also found in a healthy sibling.Functional experiments in patient's neutrophil granulocytes showed an accelerated oxidative burst capacity (OxyBurst Assay, Phagoburst Assay). Furthermore, G6PC3-deficient individuals showed a rapid dissipation of the mitochondrial membrane potential upon treatment with valinomycin, whereas the inner mitochondrial membrane potential in neutrophils from healthy individuals was maintained. We observed increased apoptosis in G6PC3-deficient neutrophils upon exposure to staurosporine. Interestingly, increased apoptosis was also seen in granulocytes from the healthy sibling with the P2RY11 sequence variant, suggesting that P2RY11 deficiency may aggravate the phenotype of G6PC3 deficiency.In summary, our study indicates that the clinical spectrum of G6PC3 deficiency may be more diverse than previously appreciated and that P2RY11 may have a modifying effect on G6PC3-deficient neutrophil granulocytes.J.D. and D. K. contributed equally to this work and should be considered aequo loco. Disclosures:Schäffer:National Institues of Health: Employment; Intramural Research Program of the National Institues of Health, NLM: Research Funding.

Isolation of Equine Hoof Epidermal Cells for in Vitro Studies

Isolation of Equine Hoof Epidermal Cells for in Vitro Studies

Ion channel modulators mediated alterations in NO-induced free radical generation and neutrophil membrane potential

The present study investigated the effect of various ion (H+ and K+) channel modulators on nitric oxide (NO) donors (SNP and SNAP) induced free radical generation and on neutrophil membrane potential. Free radical generation was assessed by DCDHF-DA, using flow cytometry, while membrane potential was measured by a fluorescent dye, DiO-C5-(3). Neutrophil suspension in high potassium containing medium or following addition of NO donors (SNP, SNAP) to the neutrophil suspension led to free radical generation and membrane depolarization. DPI (a dual inhibitor of NADPH-oxidase and NOS), ABAH (MPO inhibitor) and BAPTA-AM (calcium chelator) significantly reduced 80 mM KCl or NO mediated free radical generation. Modulators of large (NS1619), intermediate (Chlorzoxazone) and small conductance (Apamin, chlorzoxazone) calcium activated K+ channels (TBA), voltage activated K+ channels (Kv) (4AP, 8Br-cGMP), ATP sensitive K+ channels (KATP) (Glybenclamide, pinacidil), Na+,K+-ATPase (Ouabain) and Na+/H+ exchanger (NHE, Amiloride) altered NO-induced neutrophil free radical generation response and membrane polarity. The results obtained thus suggest an association between rat neutrophil membrane depolarization and NO-dependent free radical generation.

Ehrlichia ewingiiInfection Delays Spontaneous Neutrophil Apoptosis through Stabilization of Mitochondria

The uncultivable obligate intracellular bacterium Ehrlichia ewingii, previously known only as a canine pathogen, is the most recently recognized agent of human ehrlichiosis. E. ewingii is the only Ehrlichia species known to infect neutrophils. In the blood or in ex vivo culture, neutrophils generally have a short life span. In the present study, we investigated the effect of E. ewingii infection on spontaneous apoptosis of neutrophils. E. ewingii infection significantly delayed dog neutrophil apoptosis during ex vivo culture. The inhibitory effect on neutrophil apoptosis by E. ewingii was reversible on clearance of the organism. By using the fluorescent mitochondrial dyes Mitotracker Red 580 and JC-1, we found that E. ewingii infection stabilized mitochondrial integrity by maintaining mitochondrial membrane potential in neutrophils. These results suggest that E. ewingii delays spontaneous apoptosis of neutrophils via stabilization of host cell mitochondria.

Reactive oxidants regulate membrane repolarization and store-operated uptake of calcium by formyl peptide-activated human neutrophils

Although the rapid and considerable membrane depolarization response which accompanies activation of the phagocyte NADPH oxidase is due to transmembrane electron fluxes, little is known about the involvement of reactive oxidant species (ROS) in the subsequent repolarization response. In the current study, we have investigated the effects of superoxide dismutase (SOD), catalase, methionine, and the myeloperoxidase (MPO) inhibitors, sodium azide and 4-aminobenzoyl hydrazide (ABAH), as well as those of H 2O 2 and HOCl (both at 100 μM) on the alterations in membrane potential which accompany activation of human neutrophils with the chemoattractant, FMLP (1 μM), and on store-operated uptake of Ca 2+. The generation of ROS by FMLP-activated neutrophils was monitored according to the magnitude of oxygen consumption and autoiodination, while spectrofluorimetric procedures were used to measure alterations in membrane potential and influx of Ca 2+. Treatment of the cells with H 2O 2, and HOCl, significantly impeded membrane repolarization, while sodium azide, ABAH, methionine, and catalase exerted the opposite effects, potentiating both the rates and the magnitudes of membrane repolarization and store-operated uptake of Ca 2+. These observations demonstrate that NADPH oxidase regulates neutrophil membrane potential and Ca 2+ influx not only via its electrogenic activity, but also as a consequence of the generation of ROS.

Ion channel clustering enhances weak electric field detection by neutrophils: apparent roles of SKF96365-sensitive cation channels and myeloperoxidase trafficking in cellular responses

We have tested Galvanovskis and Sandblom's prediction that ion channel clustering enhances weak electric field detection by cells as well as how the elicited signals couple to metabolic alterations. Electric field application was timed to coincide with certain known intracellular chemical oscillators (phase-matched conditions). Polarized, but not spherical, neutrophils labeled with anti-K(v)1.3, FL-DHP, and anti-TRP1, but not anti-T-type Ca(2+) channels, displayed clusters at the lamellipodium. Resonance energy transfer experiments showed that these channel pairs were in close proximity. Dose-field sensitivity studies of channel blockers suggested that K(+) and Ca(2+) channels participate in field detection, as judged by enhanced oscillatory NAD(P)H amplitudes. Further studies suggested that K(+) channel blockers act by reducing the neutrophil's membrane potential. Mibefradil and SKF93635, which block T-type Ca(2+) channels and SOCs, respectively, affected field detection at appropriate doses. Microfluorometry and high-speed imaging of indo-1-labeled neutrophils was used to examine Ca(2+) signaling. Electric fields enhanced Ca(2+) spike amplitude and triggered formation of a second traveling Ca(2+) wave. Mibefradil blocked Ca(2+) spikes and waves. Although 10 microM SKF96365 mimicked mibefradil, 7 microM SKF96365 specifically inhibited electric field-induced Ca(2+) signals, suggesting that one SKF96365-senstive site is influenced by electric fields. Although cells remained morphologically polarized, ion channel clusters at the lamellipodium and electric field sensitivity were inhibited by methyl-beta-cyclodextrin. As a result of phase-matched electric field application in the presence of ion channel clusters, myeloperoxidase (MPO) was found to traffic to the cell surface. As MPO participates in high amplitude metabolic oscillations, this suggests a link between the signaling apparatus and metabolic changes. Furthermore, electric field effects could be blocked by MPO inhibition or removal while certain electric field effects were mimicked by the addition of MPO to untreated cells. Therefore, channel clustering plays an important role in electric field detection and downstream responses of morphologically polarized neutrophils. In addition to providing new mechanistic insights concerning electric field interactions with cells, our work suggests novel methods to remotely manipulate physiological pathways.

Surfactant modulates calcium response of neutrophils to physiologic stimulation via cell membrane depolarization.

Pulmonary surfactant (PS) reduces inflammation in the lung by poorly understood mechanisms. We have observed that surfactant-associated proteins (SAP) insert monovalent cation channels in artificial membranes. Neutrophils are primary mediators of acute pulmonary inflammation, and their functions are activated by increases in cytosolic ionized calcium concentration ([Ca2+]) and by changes in membrane potential. We hypothesize that PS inserts SAP-dependent cation channels in neutrophils, causing membrane depolarization, altered [Ca2+] response, and depressed activation. Human neutrophils were isolated, exposed to PS+SAP (1% Survanta), PS-SAP (1% Exosurf), or buffer, and washed before activating with selected stimulants. PS+SAP reduced phorbol ester- and formyl peptide-stimulated adherence and aggregation by 38% (p < 0.05) and 54% (p < 0.02), respectively. PS+SAP also inhibited the formyl peptide-induced [Ca2+] response of neutrophils (p < 0.01), but only in the presence of external Ca2+. Further characterization of this inhibition demonstrated that PS+SAP blocked formyl peptide-induced influx of both Ca2+ and Mn2+, and that this inhibition was present during activation by other neutrophil stimulants (IL-8, immune complexes). Prior depolarization of neutrophils with gramicidin-D similarly inhibited the [Ca2+] response of neutrophils to formyl peptide, and analysis of neutrophil membrane potential by 3,3'-dipentyloxaearbocyanine iodide (diOC5(3)) fluorescence revealed that PS+SAP induced rapid neutrophil depolarization. In contrast, PS-SAP exhibited little effect on neutrophil function, [Ca2+], or membrane potential. We conclude that PS+SAP decreases neutrophil adherence and aggregation responses, blocks Ca2+ influx after physiologic stimulation, and decreases membrane potential. We speculate that these effects are caused by membrane depolarization via SAP-dependent cation channel insertion, and that all of these effects contribute to the antiinflammatory properties of PS+SAP.

The Membrane Potential of Resting and Activated Neutrophils: Determinants and Significance

When activated, neutrophils display a number of biological responses, including chemotaxis, exocytosis and a respiratory burst. Concomitantly, these cells undergo marked changes in their transmembrane potential. The purpose of this brief manuscript is to critically review the existing information regarding the determinants of the resting membrane potential of neutrophils and the mechanisms proposed to underlie the depolarization recorded upon activation. In addition, the possible functional significance of the membrane potential changes is also discussed succinctly.

Neutrophil hyperpolarization in response to a chemotactic peptide.

The chemotactic peptide formylmethionyl-leucyl-phenylalanine (fMLP), at concentrations below 10(-9) M, elicits a sustained increase in the human neutrophil's membrane potential within 10 s of its addition. This hyperpolarization, detected with the fluorescent cationic potentiometric probes, 3,3'-dipentyloxacarbocyanine (diO-C5-(3)), and 1,1'-dipropyl-3,3,3',3'-tetramethylindocarbocyanine iodide (diI-C3-(3)), and with the anionic probe bis-(1,3-diethylthiobarbituric)trimethine oxonol (bis-oxonol), is immediately followed by a large depolarization when [fMLP] greater than 10(-9) M. By extracellular substitution of sodium ions with potassium ions or choline or by pretreatment of the cells with ionophores, we report here that the hyperpolarization is primarily dependent on an intact potassium ion gradient and is accompanied by a concurrent acidification of the cytoplasm (approximately 0.05 pH unit) Although the latter occurs simultaneously with a large, transient increase in cytosolic Ca2+ at [fMLP] greater than 10(-10) M, it occurs without a detectable increase in cytosolic Ca2+ at [fMLP] less than 10(-10) M. The hyperpolarization is neither affected nor initiated by the chemotactic peptide antagonist tert-butyloxycarbonyl-methionyl-leucyl-phenylalanine, whereas the depolarization is completely inhibited. Neutrophils isolated from patients with X-linked chronic granulomatous disease exhibit normal hyperpolarizations and cytosolic Ca2+ increases in response to chemotactic peptides but exhibit no depolarization or oxidative burst. The hyperpolarization appears earlier in the ontogeny of differentiating myeloid precursor cells than either the rise in cytosolic Ca2+ or the depolarization response. Together, these findings indicate that an increase in transmembrane potential is one of the earliest events in the neutrophil response to chemotactic peptides, coinciding temporally with increases in cytoplasmic Ca2+ and H+ concentrations but preceding detectable oxidative burst activity.

Voltage-dependent and Ca2(+)-activated ion channels in human neutrophils.

To investigate the regulation of membrane voltage and transmembrane ion fluxes in human neutrophils, we studied plasma membrane currents using the whole-cell patch-clamp method. We observed three distinct ion channel currents: (a) a voltage-dependent K+ current, (b) a Ca2(+)-activated K+ current, and (c) a Ca2(+)-activated Cl- current. The voltage-dependent K+ current was found in cells at rest. Its conductive properties suggested an inwardly rectifying channel. The channel was activated at membrane potentials more positive than -60 mV, suggesting that it may determine the resting membrane potential of neutrophils. Activation of neutrophils by the Ca2+ ionophore ionomycin led to an increase in whole-cell K+ and Cl- currents. The Ca2(+)-activated K+ channel differed from the voltage-dependent K+ channel because it was insensitive to voltage, because it rectified outwardly, and because the voltage-sensitive K+ channel was Ca2(+)-independent. The Ca2(+)-activated Cl- channel showed outward rectification and no apparent voltage dependency. The Ca2(+)-activated K+ and Cl- channels may play a role in cell volume homeostasis and/or cellular activation.

26] Ionic events relevant to neutrophil activation

This chapter discusses various ionic events relevant to neutrophil activation. A variety of ionic events are described in neutrophils in response to stimulation by physiological and nonphysiological agonists. Increased rates of the transport of sodium, potassium, calcium, and hydrogen, together with transient changes in the intracellular levels of these ions, are reported. Stimulated changes in the membrane potential of neutrophils are also described in this chapter. The measurement of the rates of transport of various ions in neutrophils, as in other cells, relies on the general principles of compartmental analysis. In view of the potential complexity of the behavior of multicompartment systems, the general outline of the latter is provided. In practice, the following parameters are measured: (1) the initial rate of uptake or influx, (2) the initial rate of efflux, in which case, the exit from the cells of a labeled ion with which the cells had previously been loaded can be monitored, and (3) steady-state measurements.

Plasma membrane potential of neutrophils generated by the Na + pump

The plasma membrane potential of human neutrophils was monitored using the anionic dye oxonol-V. The cells maintain a potential of −75 ± 17 mV when suspended in physiological saline solutions. The cells are scarcely depolarized by extracellular K + and the depolarization induced by the chemotactic peptide fMet-Leu-Phe is of similar magnitude for cells suspended in 5 or 155 mM K +. Neutrophils are, however, depolarized by suspension in K +-free media or after treatment with ouabain. Neutrophils catalyse Na +-H + exchange and possess other electroneutral ion transport systems. We propose that the neutrophil membrane potential is generated by an electrogenic Na + pump, that osmotic stability is achieved by electroneutral ion transport systems and that electrical stability is maintained by anion leakage. Similar mechanisms may also operate in other biological membranes.