Cystic fibrosis transmembrane conductance regulator recruitment to phagosomes in neutrophils

Abstract

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.

Fig. 1

CFTR expression on phagosomes isolated from peripheral blood human neutrophils. Opsonized 2-μm latex microspheres were fed to isolated human neutrophils for 15 min. After nitrogen cavitation, the cell cavitates were immunostained for CFTR expression using two mouse monoclonal antibodies (Ab): anti-CFTR-24.1 or anti-CFTR-13.1. Where indicated, Cy3 rabbit anti-human LAMP-1 was used to identify late phagosomes. The 2-μm monomeric bead/phagosome population was gated by its precise light scattering properties (a, e, arrows). Immunostaining with anti-CFTR antibodies 24.1 or 13.1 alone was performed (c, g) and compared to their corresponding isotype (Iso) antibody-negative controls (b, f). Two-color staining with anti-CFTR-24.1 and Cy3-LAMP-1 (d) or anti-CFTR-13.1 and Cy3-LAMP-1 (h) shows that most of the CFTR-positive staining was associated with LAMP-1-positive phagosomes. The percentages of particles in each quadrant of the dot plots are shown as indicated. LB-PS = Latex bead-laden phagosomes.

Fig. 2

PLB-985 cells and their derived neutrophils expressing EGFP-wt-CFTR and EGFP-ΔF508-CFTR fusion proteins. a, b Structures of lentiviral vectors bearing the EGFP-wt-CFTR or EGFP-ΔF508-CFTR fusion gene. Schematic drawing shows the structures of the two transgene constructs expressing either EGFP-wt-CFTR or EGFP-ΔF508-CFTR. LTR = Long terminal repeat; CMV = cytomegalovirus promoter. c-l Undifferentiated PLB-985-EGFP-wt-CFTR cells (c, d) and PLB-985-EGFP-ΔF508-CFTR cells (e, f) stably expressed the corresponding CFTR fusion proteins (d, f). Five days after DMSO induction, the cells differentiated into neutrophil-like cells as evidenced in part by their ability to adhere to, and spread on, the substratum (h, j) compared to the undifferentiated cells (c, e). The relative expression levels of the EGFP-CFTR fusion proteins were profiled by flow cytometry for the undifferentiated (g) and differentiated cells (l). DIC = Differential interference contrast. The red shaded area is the flow cytometric histogram for PLB-985 parental cells. The black solid line represents the fluorescent profile of the EGFP-wt-CFTR cells and the green solid line that of the EGFP-ΔF508-CFTR cells. m-r Flow cytometric analysis of differentiation of the parental PLB-985 cells and the EGFP-CFTR-derived neutrophils. PLB-985, PLB-985-EGFP-wt-CFTR and PLB-985-EGFP- ΔF508-CFTR cells were treated with (n, p, r) or without (m, o, q) 1.25% DMSO for 5 days. Then the cells were subjected to immunofluorescence staining with the antibody against human CD11b, a mature neutrophil surface marker. Flow cytometry demonstrates that >50% of the cells became mature neutrophils using the differentiation protocol. PE = Phycoerythrin. The colors refer to the online version of the figure.

Fig. 3

EGFP-wt-CFTR localizes to phagosomes. PLB-985-EGFP-wt-CFTR cells were differentiated into neutrophil-like cells with DMSO for 5 days. After phagocytosis of 3-μm opsonized latex beads, the cells were examined by confocal microscopy. The differential interference contrast image (a) of a cell with an internalized bead and the two X-Y section images from the top to the bottom of the same cell (b, c) are shown. Arrows point to the bead. To confirm the CFTR-phagosome association, a bead-phagocytosed cell (d) was immunostained for LAMP-1. The result demonstrates a typical phagosomal localization of LAMP-1, a late phagosome marker (e), which colocalized with the EGFP-wt-CFTR (f). Differentiated PLB-985-EGFP and PLB-985-EGFP-wt-CFTR cells were fed serum-opsonized 3-μm latex beads. Phagosomes were isolated and examined by fluorescence microscopy and flow cytometry. EGFP protein alone was not enriched in or targeted to the phagosomes isolated from the PLB-985-EGFP cells. Differential interference contrast (DIC) image (g), fluorescence image (h) and flow cytometry dot plot figures (i, j) are shown. Arrowheads point to the phagosomes which are negative for EGFP expression. In contrast, EGFP-wt-CFTR was associated with the phagosomes from the PLB-985-EGFP-wt-CFTR cells. Displayed are the differential interference contrast image (k), fluorescence image (l) and the flow cytometry dot plot figures (m, n). Arrows point to the phagosomes positive for EGFP-wt-CFTR (k, l). Arrowheads point to the phagosomes negative for EGFP-wt-CFTR.

Fig. 4

Significantly less ΔF508-CFTR fusion protein is associated with phagosomes. The fluorescent (a) and phase-contrast (b) micrographs show EGFP-wt-CFTR association with phagosomes by microscopy. Distinct fluorescent rings are noticeable (a, arrows). Identically prepared cells expressing EGFP-ΔF508-CFTR had little EGFP fluorescence associated with phagosomes (c, arrows). Quantification of the pixel intensity across the phagosomes was profiled. The peak fluorescence intensity of either side of the rim of each phagosome was obtained. After subtraction of the background, the mean net peak fluorescence is displayed and compared between the differentiated EGFP-wt-CFTR and EGFP-ΔF508-CFTR PLB-985 cells (e). Statistical analyses show significant differences, as marked with asterisks, by Student's t test (n = 21; p < 0.01).

Fig. 5

Enhancement of ΔF508-CFTR targeting to phagosomes by the CFTR corrector drug VRT-325. Differentiated PLB-985-EGFP-wt-CFTR and PLB-985-EGFP-ΔF508-CFTR cells were treated with 10 or 25 µM VRT-325 for 20 h and were fed the opsonized 3-μm latex beads. After homogenization, the samples were analyzed by flow cytometry to examine the EGFP fluorescence on phagosomes. The representative gating for 3-μm phagosomes is displayed (a). VRT-325 treatment did not significantly alter the percent of phagosomes positive for EGFP-wt-CFTR (b-d). However, similar drug treatment significantly increased the presentation of EGFP-ΔF508-CFTR to the phagosomes (e-g). Statistical data are displayed (h). Asterisks indicate significant differences by Student's t test (n = 5; p < 0.01).