Disease-causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages - PubMed (original) (raw)

Disease-causing mutations in the cystic fibrosis transmembrane conductance regulator determine the functional responses of alveolar macrophages

Ludmila V Deriy et al. J Biol Chem. 2009.

Abstract

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.

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Figures

FIGURE 1.

FIGURE 1.

CFTRinh-172-induced inhibition of phagosomal acidification in AMs isolated from WT mice. A, fluorescein-conjugated zymosan was exposed to WT AMs in the absence (control) of inhibitor or to cells that had been pretreated with 10 μ

m

CFTRinh-172 for 30 min. Images were collected on a Leica SP2 AOBS laser confocal microscope (see

supplemental material

). Particles not ingested by the cells are marked with white arrows. DIC, differential interference contrast. B, calibration curve was performed on the same cells using a three-component buffer with ionophores. Data were analyzed using ImageJ software. Single zymosan particles found in focal plane were taken as ROIs and represent data points on the curve. Data are expressed as means ± S.E. Calibration curve was fitted using Origin and the dose-response model. C, ratio of green fluorescence between particles inside versus outside cells (white arrows in A) in control cells (upper panel) and in the presence of 10 μ

m

CFTRinh-172 (lower panel). For analysis, fluorescence intensity of each inside particle was divided by the mean intensity of all outside particles. Asterisk indicates significance. Bars represent the mean of these ratios ± S.E.

FIGURE 2.

FIGURE 2.

Concentration dependence of the CFTRinh-172-induced inhibition of acidification in phagosomes and lysosomes. A, J774A.1 murine macrophages were fed with 1 mg/ml E. coli bioparticles conjugated to a pH-sensitive dye pHrodo® (Molecular Probes/Invitrogen) in the absence or presence of CFTR inhibitor at the indicated concentrations. The assay was performed in a 96-well plate format. The fluorescence was read on a Flex Station plate reader (Molecular Devices). Mean values are relative fluorescence units (RFU), calculated from four wells per sample. Error bars show standard deviation. B, AMs from WT or _cftr_−/− (KO) were incubated with dextran (10 kDa) doubly conjugated to a pH-sensitive dye pHrodo® and Rhodamine Green. The ratio of pHrodo® (pH-sensitive) to Rhodamine Green (pH-insensitive) emission was calibrated in situ using a triple buffer system, and the experimental values obtained from samples in C were determined by interpolation. C, data summary is presented as the mean ± S.E. where the number of the cells examined is indicated above each bar. Note a significant shift in the concentration of the inhibitor required to produce the maximum inhibition of acidification between freshly solubilized (“fresh”) and reconstituted from stored frozen DMSO stock (“stored”).

FIGURE 3.

FIGURE 3.

Kinetics of phagosomal acidification. A, kinetics of phagosomal acidification was determined in experiments in which zymosan particles doubly conjugated to Rhodamine Green and pHrodo® were pipetted onto cells, and particle uptake was followed with live cell video microscopy. Movies of particle uptake were analyzed and particles followed with particle tracking software. B, fluorescence ratio imaging for the particle/phagosome outlined by boxes in A was calculated and plotted normalized to initial values obtained upon particle entry into the cell as a function of time. Fluorescence ratios (proportional to acidification) were determined every 30 s following particle uptake. The acidification time course of the particle/phagosome isolated by the boxes in A is depicted in the curve with the open boxes. The data were fitted with a smooth line through the time points. The two curves represent the acidification time course of two phagosomes that showed distinctly different time courses for acidification. The time course data for the phagosome depicted with open boxes is representative of a class that acidified rapidly upon entry. The data represented by the closed boxes is representative of a phagosome that acidified with a significant lag following particle uptake. C, initiation of acidification was determined as the time at which the pH/fluorescence ratio changed to ≤90% of its initial value. The rate of acidification (τ) was determined from exponential fits to the data with the initial time point taken at a value that was ≤70% of the total change in pH. These kinetic data are summarized in the box plots for a total of 15 phagosomes from cells isolated from two WT mice. The average lag time for the onset of acidification was 72.0 ± 13 s, and the average time constant (τ) was 50.9 ± 10 s.

FIGURE 4.

FIGURE 4.

Lysosomal acidification in AM, peritoneal macrophages, and blood monocytes as a function of CFTR expression. Lysosomal pH was compared between cftr+/+ and _cftr_−/− alveolar and peritoneal macrophages, and blood monocytes were loaded with dextran doubly conjugated with FITC and TMR. Representative differential interference contrast (DIC) and fluorescent images are given in A–C. Lower panel, data comparison between cell types using ratiometric pH determination. ROIs for quantification were drawn around the whole cell. Summary data are expressed as pH means ± S.E. of the mean where the number of cells examined under each condition is shown above each data point (the number of animals from which cells were taken for each experimental point is given in parentheses). Asterisk indicates significance. Inset, ratiometric data for each cell was calibrated in vivo using a multiple buffer system, and the pH values obtained from the samples were determined by interpolation.

FIGURE 5.

FIGURE 5.

Relationship between CFTR genotype and phenotype in the regulation of lysosomal acidification. A, murine AMs were obtained from homozygous ΔF508 and G551D animals and compared with data obtained from AM isolated from WT and _cftr_−/− animals. Representative cell images from each of the cell types are shown. B, determination of intra-organelle pH in lysosome-like compartments. Fluorescence was averaged over the whole cell at a single focal plane and then background-subtracted. Summary data are expressed as pH means ± S.E. of the mean where the number of cells examined under each condition is shown above each data point (the number of animals from which cells were taken for each experimental point is given in parentheses). Asterisk means significance in all. C, ratio of TMR to fluorescein emission was calibrated in situ using a multiple buffer system, and the values obtained from the samples shown in A were determined by interpolation.

FIGURE 6.

FIGURE 6.

Capacity of macrophages to eliminate internalized bacteria is a function of AM genotype. A comparison of intracellular bacterial growth in single murine AMs was carried out using live cell microscopy. Cells were allowed to ingest EGFP-expressing P. aeruginosa for 30 min (multiplicity of infection <10). Adherent and noningested bacteria were then removed by washing and incubation with antibiotics, and live AMs were observed microscopically for ∼6 h. Representative AMs from ΔF508 CFTR mutant mouse with ingested bacteria at the initiation of the incubation period and after 6 h are seen in A. B, summary data from at least three separate experiments. C, comparison of phagocytic index across CFTR genotypes. AMs isolated from mice with different CFTR genotypes were incubated with fluorescein-conjugated zymosan A (Molecular Probes/Invitrogen) at the concentration of 0.5 mg/ml for 30 min at 37 °C with 5% CO2. Noningested particles were removed by excessive washings. The cells were visualized by confocal microscopy that allowed scanning each cell through its depth to count all ingested particles and discriminate them from those attached but not ingested by the cell. The data are represented as the means ± S.E. The number of cells analyzed is given above each bar. The phagocytic index of the WT cells was significantly different from the ΔF508 cells (p < 0.001) using the Student's t test. Asterisk indicates significance.

FIGURE 7.

FIGURE 7.

Mutations in CFTR alter the GTPγS-induced secretory response in AM. Secretion was stimulated by the intracellular introduction of 400 μ

m

GTPγS through the patch clamp pipette. A, whole cell capacitance recordings were obtained with an EPC-9 computer-controlled patch clamp amplifier (HEKA Electronik, Lambrecht, Germany) running PULSE software (HEKA). The EPC-9 includes a built-in data acquisition interface (ITC-16, Instrutech, NY). The software package controlled the stimulus and data acquisition for the software lock-in amplifier in the sine + dc mode. The temporal resolution of the capacitance data were 40 ms/point with a 1-kHz, 20-mV sine wave. The holding potential in the capacitance experiments was −10 mV. All electrophysiological experiments were performed at room temperature. A comparison of averaged secretory responses in voltage-clamped AMs over time was performed. The gray shading over the smooth line represents the S.E. of the average membrane capacitance increase as a function of mutant CFTR genotype over time. Cell number (number of mice used in parenthesis) in the WT experiments was 6(4); ΔF508 was 12(6); G551D was 6(3); _cftr_−/− KO was 3(2), and in the bafilomycin (BAF) experiments was 4(1). B, summary of the average change in membrane capacitance as well as percent change in membrane capacitance increase as a function of cell genotype. C, data summary of the time to initial secretory response thresholded at 100 fF.

FIGURE 8.

FIGURE 8.

Analysis of step size during the GTPγS-induced secretory response from wild type and ΔF508 AMs. Time-dependent changes in membrane capacitance were obtained from cultured AMs isolated from WT and ΔF508 mice following intracellular introduction of 400 μ

m

GTPγS as in Fig. 7. A, representative capacitance traces showing step changes due to small vesicle fusion (less than 0.2 pF) and large vesicle fusion (greater than 0.2 pF). B, histograms of the total number of step changes in capacitance recorded during experiments obtained from 6 (4 mice) WT and 12 (6 mice) ΔF508 cells. The number and amplitude of step changes in capacitance were obtained with an automated step analysis detection routine in IGOR (Wavemetrics, Lake Oswego, OR) written in the laboratory. Steps observed in the capacitance traces were summed for each genotype. Data were fit in the case of WT with a single Gaussian and in the events detected in the ΔF508 cells with a double Gaussian. The solid lines through the bars indicate the best Gaussian fit with the peak average above each peak. The x axis indicates both step size in picofarads and vesicle diameter in μm (in red). C, cumulative amplitude histogram of step changes in capacitance pooled from WT (solid black circles) and ΔF508 cells (solid green circles). The amplitude distribution for the ΔF508 cells is shifted to the right indicating larger vesicle release in the mutant cells (p < 0.01, Kolmogorov-Smirnov).

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