Nonspecific binding of common anti-CFTR antibodies in ciliated cells of human airway epithelium

Abstract

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR.

Figure 1

Immunodetection of CFTR. (a) Cartoon showing locations of epitopes for the seven anti-CFTR antibodies used in this study. (b) Immunoblots of lysates after SDS-PAGE probed for CFTR using each antibody. BHK-WT cells were stably transfected with CFTR. Well differentiated primary bronchial epithelial (HBE) cells expressed endogenous CFTR. Protein (BHK-10 µg, HBE-40 µg) was detected after short and long exposures (Exp.1 and 2, respectively). Blots were cropped. (c) Confocal images of HBEs at the apical membrane after transduction with adenoviral mCherry-WTCFTR. Cells were cultured at the ALI for 10–15 days and immunostained on intact membrane support.

Figure 2

The bases of cilia are immunostained using CFTR antibodies 596, 528, 769 and 217, but not 450, 570 and MM13-4. (a–g) Confocal images of well differentiated HBEs co-immunostained with CFTR antibodies 596, 528, 769, 217, 450, 570 or MM13-4 and the ciliated cell marker centrin-2. Arrows indicate ciliated cells. (h) Confocal images of cells coimmunostained with 596 and antibodies against either tubulin (left, ciliated cell marker), cytokeratin 14 (middle, basal cell marker) or MUC5AC (right, goblet cell marker). (i) Representative spatial cross-correlation functions calculated via ICCS analysis between centrin-2 and CFTR immunofluorescence using 596 (left) or 450 (right) measured from image sub-regions of interest (64 × 64 pixels). (j) Average apical immunofluorescence signals with different CFTR antibodies. Intensities were normalized to the excitation power (mean ± SD, n = 12–57, ***p < 0.0001, one-way ANOVA). (k) Colocalized fraction f_ICCS extracted from ICCS analysis (mean ± SD, n = 2–28, 450 vs 596, ***p < 0.0001; 217 vs 596, **p = 0.005; 769 vs 596, **p = 0.0059; 528 vs 596, *p = 0.0313), which indicates the arithmetic mean of the fractions of CFTR and centrin-2 antibody signals that interact.

Figure 3

Super-resolution microscopy of 596 immunofluorescence reveals ring-like pattern at the bases of cilia. (a) Representative image of the apical side of a non-CF HBE cell co-immunostained with 596 and centrin-2 antibody; left side: confocal image, right side: super-resolution STED image. (b–e) Representative STED images of apical membrane fluorescence in HBE cells obtained using 596, 528, 796 and 450 antibodies.

Figure 4

Apical 596 immunofluorescence in pHBE cells isolated from a subset of F508del/F508del patients. (a) Confocal images of apical immunofluorescence using well differentiated HBE cells from two F508del/F508del patients, one 596- (F508-4, top row) and the other 596+ (F508-1, bottom row). Colocalization with centrin-2 immunofluorescence confirmed expression in ciliated cells (merge). (b) Confocal images of the apical surface of F508-4 (top row) and F508-1 (bottom row) immunostained with 596 (green) and centrin-2 (red) antibodies used for image cross-correlation spectroscopy analysis. (c) Examples of spatial cross-correlation functions calculated using ICCS analysis from sub-regions (64 × 64 pixels) of the ciliated region images used to determine colocalization values; i.e. fractions of interacting particles per beam area in the green channel (M1) and the red channel (M2), for F508-4 (top) and F508-1 (bottom). (d) Summary of apical 596 immunofluorescence intensities in F508-4 and F508-1 cells. Intensity was normalized to the excitation power, (mean ± SD, n = 28—34, ***p < 0.0001, one-way ANOVA). (e) Colocalized fraction f_ICCS extracted from ICCS analysis (mean ± SD, n = 14–26, ***p = 6 × 10⁻¹⁶, t-test). (f) Immunoblot of lysates from well differentiated F508del/F508del HBE cells probed with antibody 23C5 against the R domain of CFTR. (g, h, these images are from one blot). Short-circuit current recordings and summaries of maximal cAMP responses after 24 h pretreatment with vehicle (DMSO, black) or the corrector VX-809 (1 µM, red). Cells were exposed sequentially to amiloride (Ami; 100 µM), forskolin (FSK; 10 µM) and genistein (Gen; 50 µM). Currents were inhibited using CFTR_inh-172 (INH; 10 µM). Note that under control conditions (DMSO), F508-1 cells with apical 596 immunofluorescence had smaller stimulated currents compared to F508-4 cells that were 596-.

Figure 5

Apical 596 immunofluorescence in ciliated cells that are homozygous for Class I CFTR mutations. (a) Confocal images of well differentiated pHBE cells from patients homozygous for 1525-1G > A (CI-2, top row) or 621 + 1G > T (CI-1, bottom row). (b) Representative STED images of the apical surface of CI-2 (top row) or CI-1 (bottom row) cells. (c) Confocal images of the apical surface of CI-2 (top row) and CI-1 (bottom row) cells immunostained with 596 and centrin-2 antibodies used for ICCS analysis. (d) Examples of spatial cross-correlation functions calculated via ICCS analysis from sub-regions (64 × 64 pixels) of the ciliated region images used to calculate colocalization values for CI-2 (top) and CI-1 (bottom). (e) Fluorescence intensity of 596 immunostaining at the apical pole of CI-2 and CI-1 cells (mean ± SE, n = 12–23, ***p < 0.0001, one-way ANOVA). Intensity was normalized to the excitation power. (f) Colocalized fraction f_ICCS extracted from ICCS analysis (mean ± SD, n = 3–6, ***p = 5 × 10^–5, t-test). (g) Immunoblot of well differentiated HBE cells from non-CF, class I mutations, or F508del, probed for CFTR using the mAb23C5. Note that CI-1 cells are immunostained by 596 but do not have detectable CFTR protein (h, I, these images are from one blot). Representative short-circuit current recordings and after pretreatment with DMSO (black) or VX-809 (1 µM, red) for 24 h. Cells were exposed to amiloride (Ami; 100 µM), forskolin (FSK; 10 µM), genistein (Gen; 50 µM) and CFTR_inh-172 (INH; 10 µM). The histogram shows stimulations induced by FSK + Gen. (j) CFTR mRNA levels in well differentiated HBE cells from non-CF donors and patients homozygous for F508del and a Class I mutation. Data were obtained by qPCR using primers that amplify sequence near the 5’ end of the transcripts and results were normalized to GAPDH (mean ± SE, n = 3–14; non-CF vs 621 + 1G > T/621 + 1G > T, *p = 0.0360; F508del/F508del vs 621 + 1G > T/621 + 1G > T, *p = 0.02727; 1525-1G > A/1525-1G > A vs 621 + 1G > T/621 + 1G > T, *p = 0.0413; one-way ANOVA). Individual points represent independent measurements.

Figure 6

Blocking peptide WPSGGQMT abolishes 596 immunostaining at the bases of cilia. (a) Well differentiated HBEs were co-immunostained using 596 and centrin-2 antibodies without (top row) or with (bottom row) a blocking peptide corresponding to the 596 epitope. White arrows indicate ciliated cells. Zoomed-in images are shown at extreme right. (b) Rootletin isoforms containing a sequence homologous to the 596 epitope in CFTR. (c) Cartoon showing location of the pseudo-epitope in rootletin isoforms. (d) Immunoblot of well differentiated HBE cell lysates from a non-CF donor and six CF patients probed using rootletin antibody. Green and blue labels indicate donor is homozygous for F508del and a Class I mutation, respectively. Short (1) and long (2) exposures are shown. After short exposure note that rootletin was only detected in 596+ cell lysates. (e) Summary of rootletinX1 protein expression. Each point is the lysate from a different culture prepared from five non-CF donors, five 596+ donors and three 596- donors. Note that rootletinX1 band intensity is significantly lower in cells that are not immunostained using CFTR antibody 596 (mean ± SE, n = 16–17, **p = 0.0018, one-way ANOVA).

Figure 7

Cross-reactivity of 596 with rootletin and the phenotype of 596- cells. (a, b) Immunostaining of 596- (left) and 596+ (right) cells with rootletin antibody. (c) STED images of 596+ cells showing both punctate and filamentous rootletin immunofluorescence. (d) Confocal images of eGFP-tagged rootletin (lacking WSPGGQML, top row) and the isoform rootletinX1 (which has the pseudo-epitope, middle and bottom rows) expressed in CFBE41o^- cells. RootletinX1 was detected using 596 antibody (red) and this immunostaining was abolished by blocking peptide (BP). (e) Cilia length in non-CF, 596+ and 596- cells (mean ± SE, n = 119–125, ****p < 0.0001, ***p = 0.0004, one-way ANOVA). Each point represents a single cell. (f) Comparison of 596+ and 596- proliferation rates. Each point represents the mean cell count ± SE of 3 independent experiments.