Nucleotide-regulated calcium signaling in lung fibroblasts and epithelial cells from normal and P2Y2 receptor (-/-) mice

To test for the role of the P2Y2 receptor(P2Y2-R) in the regulation of nucleotide-promoted Ca2+ signaling in the lung, we generated P2Y2-R- deficient (P2Y2-R(-/-)) mice and measured intracellular Ca2+(i) responses (ΔCa2+(i)) to nucleotides in cultured lung fibroblasts and nasal and tracheal epithelial cells from wild type and P2Y2-R(-/-) mice. In the wild type fibroblasts, the rank order of potencies for nucleotide-induced ΔCa2+(i) was as follows: UTP ≥ ATP >> ADP > UDP. The responses induced by these agonists were completely absent in the P2Y2R(-/-) fibroblasts. Inositol phosphate responses paralleled those of ACa2+(i) in both groups. ATP and UTP also induced Ca2+(i) responses in wild type airway epithelial cells. In the P2Y2-R(-/-) airway epithelial cells, UTP was ineffective. A small fraction (25%) of the ATP response persisted. Adenosine and α,β- methylene ATP were ineffective, and ATP responses were not affected by adenosine deaminase or by removal of extracellular Ca2+, indicating that neither P1 nor P2X receptors mediated this residual ATP response. In contrast, 2-methylthio-ADP promoted a substantial Ca2+(i) response in P2Y2-R(-/-) cells, which was inhibited by the P2Y1 receptor antagonist adenosine 3'-5'-diphosphate. These studies demonstrate that P2Y2-R is the dominant purinoceptor in airway epithelial cells, which also express a P2Y1 receptor, and that the P2Y2-R is the sole purinergic receptor subtype mediating nucleotide-induced inositol lipid hydrolysis and Ca2+ mobilization in mouse lung fibroblasts

Characterization of NF-κB reporter U937 cells and their application for the detection of inflammatory immune-complexes

Our study tested the hypothesis that immunoglobulins differ in their ability to activate the nuclear factor-κB pathway mediated cellular responses. These responses are modulated by several properties of the immune complex, including the ratio of antibody isotypes binding to antigen. Immunoassays allow the measurement of antigen specific antibodies belonging to distinct immunoglobulin classes and subclasses but not the net biological effect of the combination of these antibodies. We set out to develop a biosensor that is suitable for the detection and characterization of antigen specific serum antibodies. We genetically modified the monocytoid U937 cell line carrying Fc receptors with a plasmid encoding NF-κB promoter-driven GFP. This clone, U937-NF-κB, was characterized with respect to FcR expression and response to solid-phase immunoglobulins. Human IgG3, IgG4 and IgG1 induced GFP production in a time- and dose-dependent manner, in this order of efficacy, while IgG2 triggered no activation at the concentrations tested. IgA elicited no response alone but showed significant synergism with IgG3 and IgG4. We confirmed the importance of activation via FcγRI by direct stimulation with monoclonal antibody and by competition assays. We used citrullinated peptides and serum from rheumatoid arthritis patients to generate immune complexes and to study the activation of U937-NF-κB, observing again a synergistic effect between IgG and IgA. Our results show that immunoglobulins have distinct pro-inflammatory potential, and that U937-NF-κB is suitable for the estimation of biological effects of immune-complexes, offering insight into monocyte activation and pathogenesis of antibody mediated diseases

Effect of loss of P2Y2 receptor gene expression on nucleotide regulation of murine epithelial Cl- transport

Extracellular nucleotides are believed to be important regulators of ion transport in epithelial tissues as a result of their ability to activate cell surface receptors. Although numerous receptors that bind nucleotides have been identified, the complexity of this receptor family, combined with the lack of pharmacological agents specific for these receptors, has made the assignment of particular receptors and ligands to physiological responses difficult. Because ATP and UTP appear equipotent and equieffective in regulating ion transport in many epithelia, we tested the hypothesis that the P2Y2 receptor (P2Y2-R) subtype mediates these responses in mouse epithelia, with gene targeting techniques. Mice with the P2Y2-R locus targeted and inactivated (P2Y2-R(-/-)) were generated, airways (trachea), gallbladder, and intestines (jejunum) excised, and Cl- secretory responses to luminal nucleotide additions measured in Ussing chambers. Comparison of P2Y2R(+/+) with P2Y2-R(-/-) mice revealed that P2Y2-R mediated most (>85-95%) nucleotide-stimulated Cl- secretion in trachea, about 50% of nucleotide responses in the gallbladder, and none of the responses in the jejunum. Dose- effect relationships for nucleotides in tissues from P2Y2-R(-/-) mice suggest that the P2Y6-R regulates ion transport in gallbladder and to a lesser extent trachea, whereas P2Y4 and/or unidentified receptor(s) regulate ion transport in jejunum. We conclude that the P2Y2 receptor is the dominant P2Y purinoceptor that regulates airway epithelial ion transport, whereas other P2Y receptor subtypes are relatively more important in other nonrespiratory epithelia

Sequential Array Cytometry: Multi-Parameter Imaging with a Single Fluorescent Channel

Heterogeneity within the human population and within diseased tissues necessitates a personalized medicine approach to diagnostics and the treatment of diseases. Functional assays at the single-cell level can contribute to uncovering heterogeneity and ultimately assist in improved treatment decisions based on the presence of outlier cells. We aim to develop a platform for high-throughput, single-cell-based assays using well-characterized hydrodynamic cell isolation arrays which allow for precise cell and fluid handling. Here, we demonstrate the ability to extract spatial and temporal information about several intracellular components using a single fluorescent channel, eliminating the problem of overlapping fluorescence emission spectra. Integrated with imaging technologies such as wide field-of-view lens-free fluorescent imaging, fiber-optic array scanning technology, and microlens arrays, use of a single fluorescent channel will reduce the cost of reagents and optical components. Specifically, we sequentially stain hydrodynamically trapped cells with three biochemical labels all sharing the same fluorescence excitation and emission spectrum. These markers allow us to analyze the amount of DNA, and compare nucleus-to-cytoplasm ratio, as well as glycosylation of surface proteins. By imaging cells in real-time we enable measurements of temporal localization of cellular components and intracellular reaction kinetics, the latter is used as a measurement of multi-drug resistance. Demonstrating the efficacy of this single-cell analysis platform is the first step in designing and implementing more complete assays, aimed toward improving diagnosis and personalized treatments to complex diseases

Pulmonary epithelial sodium-channel dysfunction and excess airway liquid in pseudohypoaldosteronism

Background: Active sodium absorption is the dominant mechanism of ion transport in airway epithelium, but its role in pulmonary physiology and airway host defense is unknown. To address this question, we studied the function of airway epithelial cells and determined the frequency of pulmonary symptoms in patients with systemic pseudohypoaldosteronism, a salt-losing disorder caused by loss-of-function mutations in the genes for the epithelial sodium channel. Methods: In nine patients 1.5 to 22 years of age who had systemic pseudohypoaldosteronism, we tested for mutations in the genes for the epithelial sodium channel, estimated the rate of sodium transport in the airway, determined the volume and ion composition of airway surface liquid, reviewed clinical features, collected laboratory data pertinent to pulmonary function, and, in three adults, measured mucociliary clearance. Results: The patients with systemic pseudohypoaldosteronism had loss-of-function mutations in the genes for the epithelial sodium-channel subunits, no sodium absorption from airway surfaces, and a volume of airway surface liquid that was more than twice the normal value. The mean (±SE) mucociliary transport rate was higher in the 3 adult patients than in 12 normal subjects (2.0 ± 0.7 vs. 0.5 ± 0.3 percent per minute, P = 0.009). Young patients (those five years of age or less) all had recurrent episodes of chest congestion, coughing, and wheezing, but no airway infections with Staphylococcus aureus or Pseudomonas aeruginosa. Older patients (those more than five years of age) had less frequent respiratory symptoms. Conclusions: Patients with systemic pseudohypoaldosteronism fail to absorb liquid from airway surfaces; the result is an increased volume of liquid in the airways. These results demonstrate that sodium transport has a role in regulating the volume of liquid on airway surfaces

Sensitive and Specific Fluorescent Probes for Functional Analysis of the Three Major Types of Mammalian ABC Transporters

An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC2(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments

Purinergic mechanism in the immune system: A signal of danger for dendritic cells

There is increasing appreciation that injured or stressed cells release molecules endowed with the ability to modulate dendritic cell maturation. The role of these molecules is thought to be that of alerting the body of an impending danger, and initiate and shape the subsequent immune response. Nucleotides are perfectly suited for this task as they are easily released upon damage of the cell membrane, rapidly diffuse in the extracellular environment and ligate specific plasma membrane receptors expressed by dendritic cells and other mononuclear phagocytes. A better knowledge of the modulation of dendritic cell responses by extracellular nucleotides may provide novel routes to enhance the immune response and increase the efficacy of vaccination

Clinically relevant mutations in the ABCG2 transporter uncovered by genetic analysis linked to erythrocyte membrane protein expression

The ABCG2 membrane protein is a key xeno- and endobiotic transporter, modulating the absorption and metabolism of pharmacological agents and causing multidrug resistance in cancer. ABCG2 is also involved in uric acid elimination and its impaired function is causative in gout. Analysis of ABCG2 expression in the erythrocyte membranes of healthy volunteers and gout patients showed an enrichment of lower expression levels in the patients. By genetic screening based on protein expression, we found a relatively frequent, novel ABCG2 mutation (ABCG2-M71V), which, according to cellular expression studies, causes reduced protein expression, although with preserved transporter capability. Molecular dynamics simulations indicated a stumbled dynamics of the mutant protein, while ABCG2-M71V expression in vitro could be corrected by therapeutically relevant small molecules. These results suggest that personalized medicine should consider this newly discovered ABCG2 mutation, and genetic analysis linked to protein expression provides a new tool to uncover clinically important mutations in membrane proteins. © 2018 The Author(s)