Validation of flow cytometric competitive binding protocols and characterization of fluorescently labeled ligands.

BACKGROUND: Fluorescently labeled ligands and flow cytometric methods allow quantification of receptor-ligand binding. Such methods require calibration of the fluorescence of bound ligands. Moreover, binding of unlabeled ligands can be calculated based on their abilities to compete with a labeled ligand. In this study, calibration parameters were determined for six fluorescently labeled N-formyl peptides that bind to receptors on neutrophils. Two of these ligands were then used to develop and validate competitive binding protocols for determining binding constants of unlabeled ligands. METHODS: Spectrofluorometric and flow cytometric methods for converting relative flow cytometric intensities to number of bound ligand/cell were extended to include peptides labeled with fluorescein, Bodipy, and tetramethylrhodamine. The validity of flow cytometric competitive binding protocols was tested using two ligands with different fluorescent properties that allowed determination of rate constants both directly and competitively for one ligand, CHO-NLFNYK-tetramethylrhodamine. RESULTS: Calibration parameters were determined for six fluorescently-labeled N-formyl peptides. Equilibrium dissociation constants for these ligands varied over two orders of magnitude and depended upon the peptide sequence and the molecular structure of the fluorescent tag. Kinetic rate constants for CHO-NLFNYK-tetramethylrhodamine determined directly or in competition with CHO-NLFNYK-fluorescein were statistically identical. CONCLUSIONS: Combination of spectrofluorometric and flow cytometric methods allows convenient calculation of calibration parameters for a series of fluorescent ligands that bind to the same receptor site. Competitive binding protocols have been independently validated.

Inhibition of mono-ADP-ribosyltransferase activity during the execution phase of apoptosis prevents apoptotic body formation.

The objective of this study was to understand factors responsible for apoptotic body formation and release during apoptosis. We have found that inhibition of mono-ADP ribosylation after ultraviolet (UV) light induction of apoptosis in HL-60 cells does not block caspase-3 activation, gelsolin cleavage, or endonucleolytic DNA fragmentation. However, the cytoskeletal features of apoptosis leading to apoptotic body formation and release were inhibited by meta-iodobenzylguanidine (MIBG) and novobiocin, potent inhibitors of arginine-specific mono-ADP-ribosyltransferases (mono-ADPRTs). Suppression of mono-ADP ribosylation as late as 120 min following UV irradiation blocked the depolymerization of actin and release of apoptotic bodies. This suggested that the cytoskeletal changes of apoptosis may be decoupled from the caspase cascade and that there may be a biochemical event either distal to or independent of caspase-3 that regulates apoptotic body formation. To test the hypothesis that ADP ribosylation of actin may occur with the induction of apoptosis, an in vivo assay of mono-ADPRT activity using an antibody against ADP-ribosylarginine was used. An approximately 64% increase in the ADP ribosylation of actin was observed at 2 h following exposure to UV light. When MIBG or novobiocin was present, the ADP ribosylation of actin was only 14-18% above the levels observed in control nonirradiated cells. The current study is the first to demonstrate a relationship between ADP-ribosylation of actin and the formation of apoptotic bodies.

Actin cytoskeletal function is spared, but apoptosis is increased, in WAS patient hematopoietic cells.

Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS. It was also found that the actin cytoskeleton in WAS platelets is capable of producing the hallmark cytoarchitectural features associated with activation. Further analysis revealed accelerated cell death in WAS lymphocytes as evidenced by increased caspase-3 activity. This increased activity resulted in accelerated apoptosis of these cells. CD95 expression was also increased in these cells, suggesting an up-regulation in the FAS pathway in WAS lymphocytes. Additionally, inhibition of actin polymerization in lymphocytes using cytochalasin B did not accelerate apoptosis in these cells. This suggests that the accelerated apoptosis observed in WAS lymphocytes was not secondary to an underlying defect in actin polymerization caused by mutation of the WAS gene. These data indicate that WASP does not play a universal role in signaling actin polymerization, but does play a role in delaying cell death. Therefore, the principal consequence of mutations in the WAS gene is to accelerate lymphocyte apoptosis, potentially through up-regulation of the FAS-mediated cell death pathway. This accelerated apoptosis may ultimately give rise to the clinical manifestations observed in WAS. (Blood. 2000;95:1283-1292)

Modeling activation and desensitization of G-protein coupled receptors provides insight into ligand efficacy.

Signaling through G-protein coupled receptors is one of the most prevalent and important methods of transmitting information to the inside of cells. Many mathematical models have been proposed to describe this type of signal transduction, and the ternary complex (ligand/receptor/G-protein) model and its derivatives are among the most widely accepted. Current versions of these equilibrium models include both active (i.e. signaling) and inactive conformations of the receptor, but do not include the dynamics of G-protein activation or receptor desensitization. Yet understanding how these dynamic events effect response behavior is crucial to determining ligand efficacy. We developed a mathematical model for G-protein coupled receptor signaling that includes G-protein activation and receptor desensitization, and used it to predict how activation and desensitization would change if either the conformational selectivity (the effect of ligand binding on the distribution of active and inactive receptor states) or the desensitization rate constant was ligand-specific. In addition, the model was used to explore the implications of measuring responses far downstream from G-protein activation. By comparing the experimental data from the beta(2)-adrenergic, micro-opioid, D(1)dopamine, and neutrophil N -formyl peptide receptors with the predictions of our model, we found that the conformational selectivity is the predominant factor in determining the amounts of activation and desensitization caused by a particular ligand.

Regulation of oscillations in filamentous actin content in polymorphonuclear leukocytes stimulated with leukotriene B(4) and platelet-activating factor.

Stimulation of neutrophils with LTB(4) or PAF results in the production of a rapidly oscillating actin polymerization/depolymerization response. Treatment of neutrophils with inhibitors of PKC prior to stimulation with ligand resulted in a masking of the F-actin oscillations. Because myosin has been shown to be a substrate for neutrophil PKC, this protein was investigated as a potential downstream mediator of F-actin oscillations. Stimulation of neutrophils with LTB(4) resulted in myosin light chain being serine phosphorylated in a PKC-dependent manner. This phosphorylation was shown to occur in a manner that is kinetically distinct from the myosin phosphorylation induced by FMLP, a potent activator of actin polymerization that alone does not induce F-actin oscillations. Additionally, disruption of intracellular actin-myosin interactions resulted in inhibition of LTB(4)- as well as PAF-induced F-actin oscillations. These data suggest that PKC and downstream phosphorylation of myosin as well as actin-myosin interaction may play roles in mediating the production of neutrophil F-actin oscillations.

A mathematical model for ligand/receptor/G-protein dynamics and actin polymerization in human neutrophils.

A mathematical model is proposed for describing the dynamics of the chemotactic peptide-stimulated actin polymerization response in human neutrophils. The response pathway utilizes the guanine nucleotide binding protein (G-protein) signal transduction cascade common to many receptor systems and allows adaptation in the continued presence of ligand. The development of such a model is an important first step toward understanding, predicting, and ultimately manipulating neutrophil responses. The model is divided into two parts, ligand/receptor/G-protein dynamics and the actin polymerization mechanism. Fast (receptor precoupled to G-protein) and slow (free receptor) signaling pathways involving ligand/receptor/G-protein interactions produce an activated signaling molecule. The actin polymerization mechanisms utilizes an actin binding protein which complexes with actin monomer and inhibits polymerization in an unstimulated cell. During stimulation, the activated signaling molecule enhances the dissociation of monomer/binding protein complexes, allowing the actin polymerization response to occur. The fast and slow signaling pathways are predicted to have different roles in controlling the time course of this actin polymerization. Additionally, precoupled receptors are predicted to have a larger ligand association rate constant than non-precoupled (free) receptors. Model simulations agree with many of the experimentally observed characteristics of both the stimulated F-actin response and ligand/receptor binding kinetics for both the fluorescent peptide ligand CHO-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine-fluorescein (CHO-NLFNTK-fl) and the non-fluorescent peptide ligand CHO-methionyl-leucyl-phenylalanine (CHO-MLF).

Experimental approaches for observing homologous desensitisation and their pitfalls.

Whereas molecular mechanisms of cell desensitisation have been discussed at length in the literature, little organised information on the methods for studying desensitisation of cellular responses have been published. In this article, three commonly utilised protocols for studying homologous desensitisation of cellular responses are evaluated. These are (1) observation of attenuation of a response after an initial stimulus-induced activation, (2) repeated stimulation of cells after washing away the previous stimulus, and (3) repeated stimulation without a wash step. Advantages and limitations of each protocol are discussed and data is presented demonstrating some of the properties of the protocols.

Threshold and graded response behavior in human neutrophils: effect of varying G-protein or ligand concentrations.

Observing the qualitative characteristics of response behavior as key variables in the signal transduction cascade are changed can provide insight into the fundamental roles of these interactions in producing cellular responses. Using flow cytometric assays and pertussis toxin (PT) treatment of human neutrophils, we have shown that actin polymerization stimulated with the chemoattractants N-formyl-Met-Leu-Phe, leukotriene B4, and interleukin-8 exhibits threshold behavior in terms of G-protein number. Partial PT treatment resulted in both responding and nonresponding populations of cells upon stimulation. As PT treatment was increased, the responding population of cells continued to respond maximally, while the number of cells responding decreased. We also showed that N-formyl peptide-stimulated oxidant production exhibits threshold behavior in terms of G-protein number, and the threshold for oxidant production is significantly greater than that for actin polymerization. The threshold behavior observed with PT treatment contrasted with the graded response behavior seen when cells were stimulated with different doses of ligand. For actin polymerization, only one population of cells was observed at submaximal ligand concentrations, and as ligand concentration was decreased the whole population responded submaximally. For oxidant production, as ligand concentration was decreased there were two populations of cells, but the responding cells responded submaximally. A mathematical model incorporating receptor/ligand binding and G-protein activation was developed to account for these differences in response behavior. Our results predict that an early signal transduction event in addition to, and not initiated by G-protein activation, is necessary to account for actin polymerization and oxidant production in neutrophils.

Experimental approaches for observing homologous desensitisation and their pitfalls.

Whereas molecular mechanisms of cell desensitisation have been discussed at length in the literature, little organised information on the methods for studying desensitisation of cellular responses has been published. In this article, three commonly utilised protocols for studying homologous desensitisation of cellular responses are evaluated. These are (1) observation of attenuation of a response after an initial stimulus-induced activation, (2) repeated stimulation of cells after washing away the previous stimulus, and (3) repeated stimulation without a wash step. Advantages and limitations of each protocol are discussed and data is presented demonstrating some of the properties of the protocols.

Lipopolysaccharide protects polymorphonuclear leukocytes from apoptosis via tyrosine phosphorylation-dependent signal transduction pathways.

BACKGROUND: The present study was undertaken to determine if tyrosine phosphorylation signal transduction pathways, which are known to be activated in polymorphonuclear leukocytes (PMN) by lipopolysaccharide (LPS), play a role in priming of PMN oxidative burst and protection of PMN from apoptosis by LPS, and to determine if an interface between these two signaling pathways exists. METHODS: PMN were combined with or without 10-fold serial dilutions (0.1 ng-1 microgram/ml) of LPS and incubated at 37 degrees C/5% CO2. After 24 h PMN apoptosis was assessed using fluorescence microscopy and DNA agarose gel electrophoresis. Additional PMN were pretreated with the tyrosine kinase inhibitors genistein and herbamycin A before addition of LPS. Tyrosine phosphorylation was detected by immunoblotting. Oxidant production was quantitated by following the oxidation of a chromophore to its fluorescent product. RESULTS: LPS delayed the onset of apoptosis and prolonged the survival of PMN in a dose-dependent fashion. Both tyrosine kinase inhibitors blocked the protective effect of LPS on PMN apoptosis; however, only genistein blocked the priming effect of LPS on PMN oxidative burst. CONCLUSIONS: Tyrosine phosphorylation signal transduction pathways are central to protection of PMN from apoptosis by LPS. Although tyrosine phosphorylation pathways also play a role in priming of the oxidative burst in PMN, our data suggest that there is not an interface between these important signaling pathways.

Autocrine/paracrine modulation of polymorphonuclear leukocyte survival after exposure to Candida albicans.

Polymorphonuclear leukocytes (PMN) play a central role in the host response to injury and infection. These terminally differentiated phagocytes have a limited life span, after which they undergo spontaneous apoptosis. PMN life span can be significantly prolonged by several naturally occurring cytokines, and PMN are now known to be capable of cytokine production in response to various antigenic stimuli. These facts suggest the possibility that PMN possess an autocrine/paracrine mechanism for the control of their own survival. The present study was undertaken to test this hypothesis. Supernatants from PMN that had been incubated with Candida albicans for 18 h significantly decreased the number of fresh PMN demonstrating features of apoptosis and increased the percentage of viable PMN during in vitro culture. This was demonstrated by monitoring morphologic features of apoptosis with fluorescence microscopy and DNA endonuclease activity with agarose gel electrophoresis. Significant levels of tumor necrosis factor (TNF) were detectable in the supernatants of PMN that had been stimulated with C. albicans, as determined using a TNF-sensitive cell line. Neutralization of TNF biologic activity with a specific monoclonal antibody partially abrogated the supernatant-mediated prolongation of PMN survival. The present study demonstrates that PMN possess a mechanism for the modulation of their own survival, which in part may be through the production of TNF.

Calculation of diffusion-limited kinetics for the reactions in collision coupling and receptor cross-linking.

Both enzyme (e.g., G-protein) activation via a collision coupling model and the formation of cross-linked receptors by a multivalent ligand involve reactions between two molecules diffusing in the plasma membrane. The diffusion of these molecules is thought to play a critical role in these two early signal transduction events. In reduced dimensions, however, diffusion is not an effective mixing mechanism; consequently, zones in which the concentration of particular molecules (e.g., enzymes, receptors) becomes depleted or enriched may form. To examine the formation of these depletion/ accumulation zones and their effect on reaction rates and ultimately the cellular response, Monte Carlo techniques are used to simulate the reaction and diffusion of molecules in the plasma membrane. The effective reaction rate at steady state is determined in terms of the physical properties of the tissue and ligand for both enzyme activation via collision coupling and the generation of cross-linked receptors. The diffusion-limited reaction rate constant is shown to scale with the mean square displacement of a receptor-ligand complex. The rate constants determined in the simulation are compared with other theoretical predictions as well as experimental data.

Ultraviolet irradiation accelerates apoptosis in human polymorphonuclear leukocytes: protection by LPS and GM-CSF.

Polymorphonuclear leukocytes (PMN) play a central role in host response to injury and infection. Understanding factors that regulate PMN survival may therefore have a major influence on the development of novel treatment strategies for controlling life-threatening infections, as well as local and systemic inflammatory responses. Unfortunately, the presently utilized in vitro culture model of PMN apoptosis makes the examination of early biochemical events surrounding PMN apoptosis very difficult. This study demonstrates that a short course of UV irradiation (15 min) can be used to induce rapid progression of PMN through the apoptotic process with 70-90% of PMN displaying features of apoptosis by 4 h after UV exposure. Bacterial lipopolysaccharide and granulocyte-macrophage colony-stimulating factor, which are known to prolong PMN survival during in vitro culture, also protected PMN from UV-accelerated apoptosis. The UV-accelerated model of PMN apoptosis provides another valuable tool for the investigation of early signaling pathways associated with inducing or delaying PMN apoptosis.

Membrane-proximal calcium transients in stimulated neutrophils detected by total internal reflection fluorescence.

A novel fluorescence microscope/laser optical system was developed to measure fast transients of membrane-proximal versus bulk cytoplasmic intracellular calcium levels in cells labeled with a fluorescent calcium indicator. The method is based on the rapid chopping of illumination of the cells between optical configurations for epifluorescence, which excites predominantly the bulk intracellular region, and total internal reflection fluorescence, which excites only the region within approximately 100 nm of the cell-substrate contact. This method was applied to Fluo-3-loaded neutrophils that were activated by the chemoattractant N-formyl-met-leu-phe. Chemoattractant-activated cells showed 1) transient increases in both membrane-proximal and bulk cytosolic Ca2+ that peaked simultaneously; 2) a larger fractional change (20-60%) in membrane-proximal Ca2+ relative to bulk cytosolic Ca2+ that peaked at a time when the main Ca2+ transient was decreasing in both regions and that persisted well after the main transient was over. This method should be applicable to a wide variety of cell types and fluorescent ion indicators in which membrane-proximal ionic transients may be different from those deeper within the cytosol.

Interconverting receptor states at 4 degrees C for the neutrophil N-formyl peptide receptor.

With the aid of high time resolution kinetic data extracted from a flow cytometer, we determined that there are two N-formyl peptide receptor states for human neutrophils at 4 degrees C: a low affinity and a high affinity state. Competitive binding of FMLP, FNLP, and t-BOC with FNLPNTL-FL revealed different kinetic rate constants for two distinct reactions that control the lifetime of the low affinity ligand-receptor complex. For these ligands, the rate constant for dissociation of ligand from the low affinity receptor state (the first reaction) ranges in order of magnitude from 10(-2) to 1 s-1, and the conversion rate constant from the low affinity receptor state to the high affinity receptor state (the second reaction) ranges from 10(-4) to 10(-2) s-1. The antagonist t-BOC differed most significantly from the three agonists by having an association rate constant for the low affinity receptor on the order of 10(5) M-1 s-1; the value for all three agonists was on the order of 10(7) M-1 s-1. Characterization of the receptor conversion at 4 degrees C revealed that it is irreversible (or very slow) and independent of Gi protein and that neither receptor state is a form of receptor precoupled to Gi protein. The affinity conversion and the dissociation characteristics of each receptor state determine the duration of the signaling complex and may contribute to differences in ligand efficacy.

Receptor up-regulation, internalization, and interconverting receptor states. Critical components of a quantitative description of N-formyl peptide-receptor dynamics in the neutrophil.

High resolution kinetic data of the binding of fluorescent peptide to the N-formyl peptide receptor of neutrophils at 37 degrees C has allowed for the development of a ligand binding model that predicts statistically larger binding rate constants than those previously reported for intact neutrophils. The new model accounts for ligand association and dissociation, receptor up-regulation, ligand-receptor complex internalization, a change in receptor affinity, and the quenching of internalized fluorescent ligand. We determined that receptor up-regulation is both agonist- and temperature-induced and is inhibited by both phenylarsine oxide and pertussis toxin treatment. Model fits of ligand association to pertussis toxin-treated cells show that while receptor up-regulation was inhibited, rate constants for ligand binding, receptor affinity conversion, and internalization of ligand-receptor complexes were unaffected. Results suggest Gi-protein-mediated receptor up-regulation and Gi-protein-independent receptor affinity conversion. Simulation of ligand infusion using our model gives insight into the quantitative and dynamic relationship between the low affinity ligand-receptor complex and the actin polymerization response.

Metabolic inhibition potentiates oxidant-mediated injury of the endothelial cytoskeleton.

The aim of this study was to determine if exposure of cells to oxidants and metabolic inhibition, conditions which are present during ischemia-reperfusion, act synergistically to produce cytoskeletal disruption. Adherent bovine pulmonary artery endothelial cells were subjected to metabolic inhibition by incubating the cells in glucose-free buffer containing 650 nM oligomycin for 2 hr. Cells were rescued from metabolic inhibition by washing the cells with buffer containing 5.5 mM glucose and were simultaneously exposed to 0, 25, 100, or 5000 microM H2O2. At various time points during recovery from metabolic inhibition the microfilaments and microtubules were stained for microscopic evaluation. Intracellular ATP levels were determined by the luciferin/luciferase assay. Cells that were not metabolically inhibited showed minimal microfilament disruption at lower doses of H2O2. Cells that were subjected to metabolic inhibition but not exposed to H2O2 showed microfilament disruption after 2 hr of metabolic inhibition, but normal microfilament architecture was seen in over 95% of the cells by 1 hr after recovery from metabolic inhibition. Cells that were metabolically inhibited and then exposed to doses of H2O2 as low as 25 microM showed marked microfilament disruption at 1 and 2 hr after the metabolic inhibition was relieved. The microtubules were distorted, but did not depolymerize except when exposed to concentrations of H2O2 > or = 5000 microM. Metabolic inhibition appeared to selectively potentiate the effect of subsequent oxidant exposure and the potentiation largely affected microfilament architecture with secondary effects on microtubule morphology and endothelial cell shape.

Cell polarization as a possible mechanism of response termination.

Neutrophils stimulated with a low concentration of chemotactic peptide N-formyl-met-leu-phe respond with a brief pulse of actin polymerization and a persistent change in morphology from round to polarized. The recovery of the actin polymerization response is unlikely to be due to usual receptor desensitization mechanisms. We hypothesized that cell polarization and redistribution of signaling components effect the depolymerization phase of the actin response. In this report we show that the overall actin depolymerization is equivalent to the reduction in the volume occupied by F-actin while its concentration at the front of the polarized cell remains undiminished. To test whether the confinement of F-actin to a small volume can be caused by receptor redistribution, we observed receptors and F-actin in the same cell and found that their localization was different. To explain our findings, we propose a model based on the affinity of a signaling component, other than the receptor, for the areas of the cell rich in F-actin.

Rapid oscillations of actin polymerization/depolymerization in polymorphonuclear leukocytes stimulated by leukotriene B4 and platelet-activating factor.

We previously showed that activation of polymorphonuclear leukocytes by leukotriene B4 (LTB4) and platelet-activating factor produces a rapidly oscillating actin polymerization/depolymerization response. In this study, we show that 1) oscillations are not due to the stimulated cyclic release of autocoids that could bind to cell surface receptors and activate subsequent cycles; 2) oscillations are not related to oscillations of ligand binding; and 3) the particular kinetic pattern is a property of the receptor, not of the binding constants of the ligand. The major conclusion of these studies is that the oscillations are a property of the intrinsic signaling pathways triggered by these chemoattractants. We also questioned whether increased actin nucleation activity was induced by LTB4 and found that, although LTB4 induced a transient actin nucleation response, there was not a direct correlation between oscillations of the actin polymerization/depolymerization and the actin nucleation activity. This suggests that processes other than actin nucleation, such as release of monomeric actin from monomer sequestering proteins and regulation of depolymerization, are likely to be involved.

Ligand-receptor interaction rates in the presence of convective mass transport.

The rate of binding of a ligand to receptors on the cell surface can be diffusion limited. We analyze the kinetics of binding, diffusion-limited in a stationary liquid, in the presence of convective mass transport. We derive a formula that expresses the reaction kinetics in terms of the mass transfer coefficient. A moderately transport-limited kinetics is not readily recognizable from the shape of the binding curve and may lead to erroneous estimates of the rate coefficients. We apply our results to practically important cases: a cell suspension in a stirred volume of liquid and a confluent cell colony under a laminar stream. Using typical numbers characterizing the ligand-receptor interactions, we show that stirring and perfusion can be important factors determining the reaction rates. With the confluent colony, the early reaction kinetics requires a different treatment, and we provide it for the case of low receptor occupancy. We show that, even with a fast perfusion, a cell monolayer can transiently generate a zone of depletion of the ligand, and that would affect the early stages of the reaction. Our results are expressed in a simple analytical form and can be used for the design and interpretation of experimental data.