Simultaneous measurement of stimulus-induced changes in cytoplasmic Ca2+ and in membrane potential of human neutrophils

The activation of human neutrophils by chemotactic peptides evokes a rapid change in membrane potential and an increase in cytoplasmic Ca2+ levels. These events are followed up to a minute later by detectable levels of microbicidal agents formed by the oxidative burst. Except for the latter, the sequence of events has remained unclear. We report here that a new fluorescent Ca2+ indicator developed by R. Tsien, Indo-1, has allowed us to resolve the temporal relationship between the rapid and transient cytoplasmic Ca2+ rise and the membrane potential change and to do so on very small samples by using a fluorescence-activated cell sorter. We have adapted a FACS 440 for simultaneous single cell membrane depolarization and cytoplasmic [Ca2+] detection in human neutrophils upon stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP). A membrane potential probe, dipentyloxacarbocyanine, allows us to determine that the membrane potential change is fMLP dose-dependent and apparently biphasic. The depolarization is maximal 40 s after stimulation. In contrast, cytosolic [Ca2+], while fMLP-dose dependent, is maximal at 10 s and already decreasing rapidly when the cell has reached its lowest potential. It can be measured with Indo-1 which has a fluorescence emission (lambda ex = 357 nm) maximum at 485 nm when Ca2+-free and 405 nm when Ca2+-liganded. The ratio of these fluorescences may then be calibrated in terms of cytoplasmic Ca2+ levels. Thus, Ca2+ release into the cytoplasm becomes the earliest evidence of neutrophil stimulation by fMLP and occurs in close association with an apparent membrane hyperpolarization.     

Activation of neutrophils: measurement of actin conformational changes by flow cytometry

Actin, which comprises approximately 10% of the weight of cytoplasmic protein of neutrophils, is the principal component of the cytoplasmic microfilament lattice. It can exist in either of two physical states, G-actin, which is monomeric, or F-actin, which is polymeric or filamentous. Actin microfilaments support many forms of cell movement. Continuous remodeling of the microfilament lattice, which seems integral to sustained movement, is possible in part because of the ability of actin to change rapidly between its monomeric G-state and its filamentous F-state. Changes in the G- and F-actin equilibrium may be studied by flow analysis using a fluorescent probe which is specific for F-actin, 7-nitrobenz-2-oxa-1,3-diazole-(NBD)-phallacidin. Alterations in neutrophil F-actin have been measured in response to chemotactic agents (e.g., formyl peptides and leukotriene B4), inhibitors of cell movement (e.g., N-ethylmaleimide and cytochalasin B), agents that promote the oxidative burst (e.g., formyl peptides and phorbol esters), and priming agents [e.g., tumor necrosis factor (TNF)]. Measurements may be taken at intervals of a few seconds, allowing comparison of rapid changes in the F-actin content to other rapidly occurring changes, such as altered membrane ion permeability and activation of cellular enzymes. The use of metabolic inhibitors has allowed dissection of some of the biochemical pathways involved in actin assembly in living cells. Although clinical studies are few thus far, the technique has also been used to study basal and stimulated F-actin levels in circulating neutrophils in neonates and in family members of patients with neutrophil-actin dysfunction.     

The viscosity of neutrophils and their transit times through small pores

Passive neutrophils from five different individuals are rapidly aspirated at constant suction pressure and at room temperature into a pipet with a diameter of 4 microns. The excess suction pressures (i.e., the pressures in excess of the small threshold pressure required to produce continuous flow into the pipet) are 5000, 10,000 and 20,000 dyn/cm2 (0.5, 1 and 2 kPa) and are comparable to those encountered in the microcirculation. The rate of entry into the pipet is modeled with a linearized version of a theory by Yeung and Evans for the newtonian flow of a neutrophil into a pipet or pore. From this theory and measurements of the cell size and its rate of entry into the pipet, we can calculate a value for the cytoplasmic viscosity. A linear (newtonian) fit of the theory to the experimental data gives a value for the viscosity of 1050 poise. A non-linear fit predicts a decrease in the "apparent viscosity" from about 1500 poise at zero excess pressure to 1000 poise at an excess aspiration pressure of 20,000 dyn/cm2. Our experiments and analysis also allow us to calculate a value for the transit time through short pores over a wide range of excess aspiration pressures and pore diameters. For example, for a pore diameter of 3 microns and an aspiration pressure of 1250 dyn/cm2, we predict a transit time of about 70 s. At 6 microns and 20,000 dyn/cm2, the predicted transit time is only about 0.04 s.     

The Fahraeus effect in sheet flows with a gap thickness of 4 to 36 microns

We measured the Fahraeus effect of blood flowing in a sheet flow model formed with two glass slides. The number of red blood cells in the sheet flow was counted to determine a sheet hematocrit Hs and the discharge hematocrit Hd was measured from blood collection. For a Hd in the range of 3 to 30 percent, we find that Hs/Hd is about .83 for a gap of 4.1 microns. When the discharge hematocrit is 30 percent, the ratio decreases to .66 as the gap approaches 7 microns and then increases as the gap becomes thicker. The results indicate that the hematocrit ratio for a gap thicker than 4.1 microns is an increasing function of the discharge hematocrit. The value of Hs/Hd found for the sheet flow models and its dependence on Hd are comparable to those of circular tubes when their diameter equals the gap thickness.     

Rheology of passive and adhesion-activated neutrophils probed by atomic force microscopy

The rheology of neutrophils in their passive and activated states plays a key role in determining their function in response to inflammatory stimuli. Atomic force microscopy was used to study neutrophil rheology by measuring the complex shear modulus G*(omega) of passive nonadhered rat neutrophils on poly(HEMA) and neutrophils activated through adhesion to glass. G*(omega) was measured over three frequency decades (0.1-102.4 Hz) by indenting the cells 500 nm with a spherical tip and then applying a 50-nm amplitude multi-frequency signal. G*(omega) of both passive and adhered neutrophils increased as a power law with frequency, with a coupling between elastic (G') and loss (G') moduli. For passive neutrophils at 1.6 Hz, G' = 380 +/- 121 Pa, whereas G' was fourfold smaller and the power law coefficient was of x = 1.184. Adhered neutrophils were over twofold stiffer with a lower slope (x = 1.148). This behavior was adequately described by the power law structural damping model but not by liquid droplet and Kelvin models. The increase in stiffness with frequency may modulate neutrophil transit, arrest, and transmigration in vascular microcirculation.     

Differential staining of neutrophils and monocytes: Surface and cytoplasmic iron-binding proteins

Summary Lactoferrin, transferrin, and ferritin were systematically visualized and semiquantified in neutrophils and monocytes/macrophages using indirect immunofluorescence and functional cytochemical techniques. They localized on cell surfaces and within the cytoplasm at the light and electron microscopical levels. In normal subjects, subpopulations of blood neutrophils and monocytes had surface lactoferrin, but little surface transferrin or ferritin was observed on these cells. Most neutrophils had brilliant granular cytoplasmic positivity for lactoferrin; variable fractions of monocytes had weak to moderate diffuse cytoplasmic lactoferrin staining localized most prominently to the cytoplasmic matrix. Most neutrophils had cytoplasmic ferritin, but few had cytoplasmic transferrin, whereas larger subpopulations of monocytes had cytoplasmic staining reactions for both proteins. To analyse maturing cells, the iron nitrilotriacetate-acid ferrocyanide method was adapted for the light microscopical analaysis of neutrophils and monocytes/macrophages in soft agar culture. Further, a combined stain that visualizes iron nitrilotriacetate-acid ferrocyanide reactivity and -naphthyl butyrate esterase activity in cells in blood and marrow smears was developed. The relative quantities and subcellular distribution of iron-binding proteins in neutrophils and monocytes/macrophages defined by the present methods can be correlated with biochemical, maturational, and functional properties of these cells.     

Cortical shell-liquid core model for passive flow of liquid-like spherical cells into micropipets.

Many nonadherent cells exist as spheres in suspension and when sucked into pipets, deform continuously like liquids within the fixed surface area limitation of a plasma membrane envelope. After release, these cells eventually recover their spherical form. Consequently, pipet aspiration test provides a useful method to assay the apparent viscosity of such cells. For this purpose, we have analyzed the inertialess flow of a liquid-like model cell into a tube at constant suction pressure. The cell is modeled as a uniform liquid core encapsulated by a distinct cortical shell. The method of analysis employs a variational approach that minimizes errors in boundary conditions defined by the equations of motion for the cortical shell where the trial functions are exact solutions for the flow field inside the liquid core. For the particular case of an anisotropic liquid cortex with persistent tension, we have determined universal predictions for flow rate scaled by the ratio of excess pressure (above the threshold established by the cortical tension) and core viscosity which is the reciprocal of the dynamic resistance to entry. The results depend on pipet to cell size ratio and a parameter that characterizes the ratio of viscous flow resistance in the cortex to that inside the cytoplasmic core. The rate of entry increases markedly as the pipet size approaches the outer segment diameter of the cell. Viscous dissipation in the cortex strongly influences the entry flow resistance for small tube sizes but has little effect for large tubes. This indicates that with sufficient experimental resolution, measurement of cell entry flow with different-size pipets could establish both the cortex to cell dissipation ratio as well as the apparent viscosity of the cytoplasmic core.     

Rapid protein separation and diffusion coefficient measurement by frit inlet flow field-flow fractionation.

In this study three flow field-flow fractionation (flow FFF) channels are utilized for the separation of proteins and for the simultaneous measurement of their translational diffusion coefficients, D. One channel has a traditional sample inlet, whereas the other two incorporate a frit inlet design that permits more convenient and rapid sample introduction. The dependence of retention time on D, which leads to differential elution and the opportunity to measure D for protein peaks purified by the flow FFF process, is described theoretically and examined experimentally. Factors affecting band broadening, resolution, and optimization are also examined. The separation of proteins is achieved in the time range 4-20 min. Partial resolution is achieved in multiple runs requiring 2 min each. Values of D calculated from retention times are reported for 15 proteins. These include two protein dimers (bovine serum albumin and gamma-globulin) not ordinarily accessible to measurement. The D values from the three channels are compared with one another and with literature data. Reasonable consistency (within 3-4%) is found. High-speed repetitive runs can be used to acquire multiple values of D in time intervals as short as 1 min.     

Leukocyte rolling on P-selectin: a three-dimensional numerical study of the effect of cytoplasmic viscosity

Rolling leukocytes deform and show a large area of contact with endothelium under physiological flow conditions. We studied the effect of cytoplasmic viscosity on leukocyte rolling using our three-dimensional numerical algorithm that treats leukocyte as a compound droplet in which the core phase (nucleus) and the shell phase (cytoplasm) are viscoelastic fluids. The algorithm includes the mechanical properties of the cell cortex by cortical tension and considers leukocyte microvilli that deform viscoelastically and form viscous tethers at supercritical force. Stochastic binding kinetics describes binding of adhesion molecules. The leukocyte cytoplasmic viscosity plays a critical role in leukocyte rolling on an adhesive substrate. High-viscosity cells are characterized by high mean rolling velocities, increased temporal fluctuations in the instantaneous velocity, and a high probability for detachment from the substrate. A decrease in the rolling velocity, drag, and torque with the formation of a large, flat contact area in low-viscosity cells leads to a dramatic decrease in the bond force and stable rolling. Using values of viscosity consistent with step aspiration studies of human neutrophils (5-30 Pa·s), our computational model predicts the velocities and shape changes of rolling leukocytes as observed in vitro and in vivo.     

Differential effects of anti-inflammatory drugs on lipoxygenase and cyclo-oxygenase activities of neutrophils from a reverse passive Arthus reaction

Rat neutrophils isolated from four-hour reverse passive Arthus reaction pleural exudates actively metabolize arachidonic acid. Production of 11-hydroxy- and 15-hydroxy-icosatetraenoic acid and 12-hydroxy-heptadecatrienoic acid is inhibited by indomethacin, benoxaprofen, BW 755C, piroxicam, ibuprofen, timegadine, and naproxen, suggesting that production of these arachidonic acid metabolites occurs at similar enzymic active sites. In addition, in the presence of the calcium inophore A23187 or the non-ionic detergent, BRIJ 56, rat neutrophils also produce the lipoxygenase products 5-hydroxy-icosatetraenoic acid and leukotriene B. The production of these metabolites is calcium dependent. Moreover, the calcium ionophore A23187 and BRIJ 56 synergistically act to augment the metabolism of exogenously added arachidonic acid via lipoxygenase. The formation of these metabolites is inhibited by BW 755C, benoxaprofen and timegadine but not by other non-steroidal anti-inflammatory drugs tested. In fact, at doses which inhibit cyclo-oxygenase activity, indomethacin, naproxen, and ibuprofen stimulate arachidonic acid metabolism via lipoxygenase.     

Intracellular viscosity: Methods of measurement and role in metabolism

This review is devoted to the study of intracellular viscosity. Methods of intracellular viscosity measurement in cell populations and single cells are characterized and critically evaluated. Examples of intracellular viscosity assessment in a number of various cell types and intracellular organelles are presented. The main results of the in vitro and in vivo studies on the role of viscosity in metabolism are discussed.     

Rapid screening of possible cytotoxic effects of particulate air pollutants by measurement of changes in cytoplasmic free calcium, cytosolic pH, and plasma membrane potential in alveolar macrophages by flow cytometry

BACKGROUND: Inhalable particulate dusts are involved in the genesis of several lung diseases. Besides the well-known toxic dusts, i.e., asbestos and quartz, heavy metal-containing pollutants are considered as possible harmful substances. In the present study, we compared the effect of silica chemically coated with certain metal oxides and dusts from industrial productions on cell physiological parameters of bovine alveolar macrophages (BAM). METHODS: The cytosolic free calcium concentration, [Ca2+](i), the intracellular pH (pH(i)), and the plasma membrane potential (MP) of BAM were measured by flow cytometry. The dust-induced secretion of reactive oxygen species (ROS) was measured enzymatically. RESULTS: Compared with control incubations with pure silica, the dust-induced secretion of ROS by BAM was not affected when the particles were coated with Cr(2)O(3), NiO, and Fe(3)O(4), whereas VO(2)-coated dust induced a marked increase in ROS release. This effect was not correlated to changes in [Ca2+](i), pH(i), or MP. On the other hand, Cr(2)O(3)-coated silica caused alterations in all of the three latter parameters. The same pattern of changes has been reported previously for quartz dusts (Tárnok et al.: Anal Cell Pathol 15:61-72, 1997). CONCLUSIONS: We conclude that cell physiological measurements by flow cytometry could extend the palette of tools to evaluate possible toxic effects of environmental dust samples.     

Structural insights into the cytoplasmic domain of a human BK channel

The large conductance, voltage- and Ca(2+) -activated K(+) (BK or Slo1) channel is widely expressed in mammalian cells/tissues (i.e. neurons, skeletal and smooth muscles, exocrine cells, the inner ear) and regulates action potential firing, muscle contraction and secretion. The large ionic conductance and unusual, dual stimulus-driven gating behavior of this channel have long intrigued membrane biophysicists, and recent structure/function analyses have provided increasingly detailed insights into the molecular "bells and whistles" that regulate BK channel activity. Now, in two complementary articles published by the groups of Rod MacKinnon and Youxing Jiang, high resolution x-ray crystal structures of the human BK channel's large cytoplasmic domain have been solved in both the absence and presence of bound Ca(2+), conditions which would presumably promote the resting and activated conformations of this large domain. Given the regulatory importance of the cytosolic domain on BK channel gating, these experimentally determined structures reveal a number of key insights, including: 1) the physical arrangement and interactions of the tandem RCK1 and RCK2 domains within a single channel subunit, 2) the assembly of the four large cytoplasmic domains into a symmetric, tetrameric complex, 3) the formation of the channel's "gating ring" structure, based on the assembly of the individual RCK1 and 2 domains, and 4) the structural elements underlying the regions critical for divalent metal ion binding (i.e. Ca (2+) and Mg (2+)) and their potential influence on conduction pore.     

Flow cytometric measurement of cytoplasmic pH: a critical evaluation of available fluorochromes

Three pH-sensitive fluorochromes-4-methyl-umbelliferone(4MU),2, 3-dicyano-hydroquinone (DCH), and 2',7'-bis(carboxyethyl)-5,6-carboxy fluorescein (BCECF)--were evaluated for their resolution, range, and stability of cellular fluorescence. Flow cytometric techniques for determining cytoplasmic pH (pHi) have been fully described for 4MU and DCH; BCECF has previously been used for fluorimetric estimation of pHi, and was adapted to flow cytometry. For each fluorochrome, the ratio of fluorescence intensity at two wavelengths gives a measure of pHi, which may be calibrated by obtaining the fluorescence ratios for cells suspended in buffers of varying pH in the presence of a proton ionophore. Reliable calibration proved difficult using 4MU, partly because of poor retention within cells. Both DCH and BCECF could be calibrated using a fluorescence ratio and had resolutions of 0.2 and 0.4 pH units, respectively. The fluorescence of DCH is so strongly pH dependent that there were practical difficulties in its use over a wide pH range; however, pHi measurements are possible between pH 6.0 and pH 7.5 using either DCH or BCECF. Substantial dye leakage was found for 4MU and, to a lesser extent, DCH, while BCECF was retained by cells for up to 2 hours. Despite its lower resolution BCECF had a usable range of more than 1.5 pH units and this coupled with its stable fluorescence and excitation at 488 nm rather than UV suggests a wide application.     

The interaction between C5a and sphingosine-1-phosphate in neutrophils for antineutrophil cytoplasmic antibody mediated activation

Introduction

C5a plays an crucial role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. The current study further investigated the interaction between C5a and sphingosine-1-phosphate (S1P) in neutrophils for ANCA-mediated activation.

Methods

The plasma levels of S1P from 29 patients with ANCA-associated vasculitis (AAV) in active stage and in remission were tested by enzyme-linked immunosorbent assay (ELISA). The generation of S1P was tested in C5a-triggered neutrophils. The effect S1P receptor antagonist was tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA.

Results

The plasma level of circulating S1P was significantly higher in patients with AAV with active disease compared with patients in remission (2034.2 ± 438.5 versus 1489.3 ± 547.4 nmol/L, P < 0.001). S1P can prime neutrophils for ANCA-induced respiratory burst and degranulation. Compared with non-triggered neutrophils, the mean fluorescence intensity (MFI) value for CD88 expression was up-regulated significantly in S1P-triggered neutrophils. S1P receptor antagonist decreased oxygen radical production in C5a primed neutrophils induced by ANCA-positive IgG from patients. Blocking S1P inhibited C5a-primed neutrophil migration.

Conclusions

S1P triggered by C5a-primed neutrophils could further activate neutrophils. Blocking S1P could attenuate C5a-induced activation of neutrophils by ANCA. The interaction between S1P and C5a plays an important role in neutrophils for ANCA-mediated activation.     

Rapid affinity measurement of protein-protein interactions in a microfluidic platform

A rapid screening method has been developed to determine binding affinities for protein-ligand interactions using the Gyrolab workstation, a commercial microfluidic platform developed to accurately and precisely quantify proteins in solution. This method was particularly suited for assessing the high-affinity interactions that have become typical of therapeutic antibody-antigen systems. Five different commercially available antibodies that bind digoxin and a digoxin-bovine serum albumin (BSA) conjugate with high affinity were rigorously evaluated by this method and by the more conventional kinetic exclusion assay (KinExA) method. Binding parameter values obtained using Gyrolab were similar to those recovered from KinExA. However, the total experimental time for 20 binding affinity titrations, with each titration covering 12 data points in duplicate, took approximately 4h by the Gyrolab method, which reduced the experimental duration by more than 10-fold when compared with the KinExA method. This rapid binding analysis method has significant applications in the screening and affinity ranking selection of antibodies from a very large pool of candidates spanning a wide range of binding affinities from the low pM to μM range.     

Intracellular elasticity and viscosity in the body, leading, and trailing regions of locomoting neutrophils

Toinvestigate the mechanisms underlying pseudopod protrusion inlocomoting neutrophils, we measured the intracellular stiffness andviscosity in the leading region, main body, and trailingregion from displacements of oscillating intracellulargranules driven with an optical trap. Experiments were done in controlconditions and after treatment with cytochalasin D or nocodazole. Wefound 1) in the body and trailingregion, the granules divided into a "fixed" population (too stiffto measure) and a "free" population (easily oscillated; fixedfraction 65%, free fraction 35%). By contrast, the fixed fraction inthe leading region was <5%. 2) Inthe body and trailing region, there was no difference in stiffness orviscosity, but both were sharply lower in the leading region (respectively, 20-fold and 5-fold).3) Neither cytochalasin D nornocodazole caused a decrease in stiffness, but both treatments markedlyreduced the fixed fraction in the body and trailing region to <20%and <40%, respectively. These observations suggest a discrete lattice structure in the body and trailing region and suggest that thedeveloping pseudopod has a core that is more fluidlike, in thesense of a much lower viscosity and an almost total loss of stiffness.This is consistent with the contraction/solation hypothesis ofpseudopodial formation.