CFTR Expression in Normal Airways
Four regions of the respiratory tract of a large number of subjects were examined. The nasal mucosa (upper airways contain superficial epithelium and submucosal glands. The nasal mucosa is affected in CF and is the region of the human airways most frequently used for in vivo and ex vivo functional studies. The 3rd–6th generation bronchi represent the large (and proximal) airway region. Bronchi are characterized by superficial epithelium and submucosal glands and are also severely affected by CF lung disease. Small or distal airways (bronchioles) contain only superficial epithelium and are thought to be the initial site of disease in the CF lung (Davis, 1993
). Finally, the distal lung region (respiratory bronchioles and alveoli) is lined by superficial epithelia, which in the alveolar region is of a different type than the airways and is thought not to be primarily affected in CF (Davis, 1993
CFTR mRNA Expression
ISH analyses were performed to assess the expression of CFTR mRNA in normal airway epithelia. Nasal and bronchial airways required shorter exposure time (~30 d) to the radiosensitive emulsion compared with bronchioles and distal lung (~60 d), suggesting that the level of CFTR mRNA expression decreased distally in the human lung (). In both, nasal (NT) and bronchial (BR) specimens the superficial epithelium, but not the submucosal gland acini, exhibited the most consistent and/or intense ISH signal (). In nasal epithelium, all the subjects (n = 4) exhibited a continuous ISH signal in the surface epithelium (3.25 ± 0.25 as scored by a semiquantitative index), whereas two of four subjects exhibited patchy hybridization signals in submucosal glands (1.25 ± 0.75). In all bronchial specimens (n = 5), the superficial epithelium (2.20 ± 0.48) and ciliated ducts of the glands (2.75 ± 0.49) exhibited a more continuous and robust hybridization signal than submucosal gland acini (0.70 ± 0.12). More distally, the intensity of the ISH signal was higher in the superficial epithelium of the proximal bronchioles than in the terminal bronchioles. In the alveoli, the signal was low and too diffuse and uneven to identify pneumocytes type I or II ().
Figure 1. CFTR mRNA expression in normal human airways. Representative results from CFTR ISH analyses of (A) nasal turbinate (NT, n = 4) and bronchial (BR, n = 5) epithelia and (B) distal lung (n = 3). Bright field (H&E, left) and dark field (center) of (more ...)
CFTR expression was also investigated by immunolocalization and laser confocal microscopy (LCM) techniques with recently described mAbs against human CFTR (Gentzsch etal., 2003
; Mall et al., 2004
). Briefly, these mAbs recognize human WT and ΔF508 CFTR heterologously expressed in epithelial MTE18 and BHK cells with high specificity and with 10–100 times higher sensitivity than previously published antibodies (Cohn et al., 1991
; Kalin et al., 1999
) in biochemical and immunocytochemical assays (see Figure 2 in Mall et al., 2004
). The mAbs used in this study react with different and nonoverlapping epitopes within the R-domain (570 and 217, biochemical studies) and NBD2 domain (596, 528, and 729, biochemical and immunocytochemical studies) of the CFTR molecule.
CFTR Immunolocalization in Superficial Epithelium of Proximal Airways
In nasal and bronchial airways, the superficial epithelium is tall and pseudostratified and is comprised mainly of ciliated (fluid-secreting) cells. It also contains variable numbers of intercalated goblet (mucin-secreting) cells (Burkitt et al., 1996
), which typically secrete MUC5AC mucin (Perez-Vilar et al., 2003
). CFTR immunostaining/LCM studies revealed a consistent and clear CFTR signal in the apical PM of surface columnar cells in the superficial epithelium of nasal and bronchial airways (see Figures and ). Fluorescent phalloidin was used to identify the cortical actin adjacent to the apical PM and as a test of epithelial structural integrity. CFTR immunostained cells were identified as ciliated cells by morphology (see differential interference contrast [DIC] plane) and by costaining with tubulin antibodies in LCM analyses (, inset). CFTR staining of the apical PM was observed in every ciliated cell and was present in all the subjects analyzed (n = 8 and n = 26 for nasal and bronchial specimens, respectively). Intracellular CFTR staining was low in most ciliated cells (e.g., associated with the endoplasmic reticulum, which was identified using antibodies against InsP3-receptors and/or concanavalin A, unpublished data). Of note, many of the bronchial specimens had a few lumenal cells that were intensely stained by CFTR mAbs (, inset). These cells appeared to be ciliated and were scattered throughout the surface epithelium and ciliated ducts.
Figure 2. CFTR localization in nasal epithelium. Representative localization of CFTR, ERM proteins, and MUC5AC in freshly excised nasal tissues; normal (NL; n = 8) and ΔF508 homozygous CF (CF; n = 7) by immunofluorescence and LCM. (A) Confocal images acquired (more ...)
Figure 3. CFTR localization in bronchial epithelium. Results in freshly excised bronchial specimens (normal n = 26; ΔF508 homozygous CF; n = 15) are presented as in . (A) CFTR is expressed in the apical PM of all ciliated cells of normal but not (more ...)
Importantly, CFTR-specific apical membrane-associated staining was not observed in other cell types, including goblet cells identified with MUC5AC antibodies (Figures and , inset), or basal cells. These results were confirmed by high resolution LCM analyses of well-differentiated bronchial cultures derived from some of the tissue specimens included in the study. These cultures developed into a pseudostratified epithelium and clearly revealed that CFTR was localized exclusively at the apical PM of ciliated cells but not in goblet cells or any other cell type (see ).
Figure 9. CFTR protein expression and function in normal versus ΔF508 homozygous bronchial epithelial cultures. Well-differentiated cultures (normal, n = 4; CF, n = 4) were subjected to protein expression and Cl– transport function analyses. (A) (more ...)
The normal bronchial specimens showed variable degrees of acute inflammation and goblet cell hyperplasia, which is expected since lung donors were mechanically ventilated and evaluated by bronchoscopy before lung harvest (Dr. S. Randell, UNC-CH, personal communication). Of note, in a few normal subjects, sections exhibiting intense inflammation and epithelial hyperplasia displayed intermediate and/or poorly differentiated cells with intracellular CFTR staining (unpublished data).
CFTR Immunolocalization in Submucosal Glands of Upper/Proximal Airways
Human submucosal glands consist of acini of serous (fluid-secreting) and mucous (mucin-secreting) cells and conducting ducts lined by an epithelium that transitions from simple to pseudostratified (similar to the superficial epithelium) as the ducts progress toward the airway lumen. It has been widely accepted that CFTR channel activity is involved in volume homeostasis of gland secretions and that CFTR protein is predominantly localized in gland acini (Engelhardt et al., 1992
; Jiang and Engelhardt, 1998
). Surprisingly, this study revealed that CFTR immunostaining signal in the gland acini was not as strong and consistent as in the superficial epithelium (Figures and ). Nasal specimens from only four of eight subjects showed specific CFTR immunostaining in the submucosal gland acini. One specimen showed specific CFTR staining in all the acinar serous cells in a tissue section (), whereas the other three specimens exhibited only a few positive serous acini. CFTR staining was localized exclusively in the apical PM of serous cells, as identified by morphology (DIC and actin staining confocal images) and by costaining with aquaporin5 (AQP5; Kreda et al., 2001
; ). AQP5 is highly expressed in human submucosal glands (Kreda et al., 2001
), and studies in the AQP5 knockout mouse indicate that AQP5 deficiency in the apical membrane of the serous glandular epithelium is associated with decreased transepithelial water permeability (Song and Verkman, 2001
Figure 4. CFTR localization in nasal and bronchial submucosal glands. Immunolocalization results in normal submucosal glands displayed as in . (A) Representative CFTR nasal (NT) immunolocalization results for 4 of 8 subjects. (B) Representative bronchial (more ...)
Figure 5. CFTR expression in bronchial submucosal gland acini. (A) CFTR immunolocalization results in bronchial submucosal gland acini displayed as in ; top panel, negative CFTR staining in normal acini (n = 20/26); middle panel, positive CFTR staining (more ...)
In the bronchial specimens, CFTR immunostaining was not detected in submucosal gland acini of most normal bronchial tissues (20 of 26 subjects; Figures and ). In contrast, the CFTR staining signal was stronger and consistent in the apical PM of ciliated cells lining the ciliated ducts localized in close proximity to (CFTR) negative acini in all the specimens (). Serial sections stained with ezrin or EBP50 antibodies revealed intense apical staining of both ciliated ducts and acini of all normal specimens irrespective of CFTR staining (Figures and ), suggesting that the minimal CFTR signal in gland acini could not be attributed to poor tissue preservation. Specific, but moderate CFTR immunostaining was observed only in a few submucosal serous acini of 6 normal subjects (6 of 26 subjects, ). Identical results were obtained using the two new CFTR mAbs, CFTR antibodies from other laboratories, and epitope retrieval techniques. Importantly, AQP5 staining decorated the apical PM of serous cells in all nasal and bronchial specimens, despite the absence of a CFTR signal (), indicating that the PM of serous cells was intact and protein localization for other channels was not abnormal.
In sum, in nasal and bronchial airways, the pattern of CFTR immunolocalization signals (Figures , , , ) was consistent with the pattern of CFTR mRNA expression (), suggesting that CFTR was predominantly expressed in the superficial epithelium rather than in the gland acini. Moreover, these results indicate unequivocally that CFTR was localized to the apical PM of ciliated (fluid-secreting) cells lining the superficial and gland ductal epithelia.
CFTR Immunolocalization in Small Airways
Proximal bronchioles are lined with ciliated and goblet cells organized in a pseudostratified epithelium. As in proximal airways, the CFTR immunostaining signal was clear and consistent in the apical PM of every ciliated cell of the bronchiolar epithelium (7 normal subjects, ). CFTR was not detected in Clara cells (note, Clara cells were absent in the human proximal bronchioles as previously reported by Boers et al. 1999
), nor in the PM of goblet cells identified with MUC5AC antibodies ().
Figure 6. CFTR localization in proximal bronchiolar epithelium. Representative immunolocalization results in normal (n = 7) and ΔF508 homozygous CF (n = 9) bronchioles displayed as in for (A) CFTR and (B) EBP50, ezrin, and MUC5AC. Bar, 40 μm. (more ...)
CFTR immunostaining of terminal bronchioles and alveoli was indistinguishable from the IgG control signal in all the specimens, despite well-preserved cellular structures including the PM as demonstrated by actin cytoskeleton staining (). High levels of autofluorescence emitted in most of the visible spectrum in distal lung tissues, may have masked a CFTR-specific signal, as previously suggested (Engelhardt et al., 1994
). In contrast, staining of EBP50 and ezrin was very intense in the apical domain of terminal bronchiolar epithelial cells (see below, ).
Figure 7. CFTR localization in terminal bronchioles and alveoli. Representative immunolocalization results in terminal bronchioles (TB) and alveoli (AL) of normal lungs for (A) CFTR and (B) EBP50 and ezrin. Insets: immunolocalization results in ΔF508 homozygous (more ...)
ΔF508 CFTR Expression, Maturation, and Function in CF Airways
We performed immunolocalization studies in CF airways excised at time of transplantation to investigate the fate of ΔF508 CFTR in native airways. Because of the potential for insensitivity of immunolocalization, we supplemented these studies with biochemical studies of ΔF508 CFTR protein maturation and bioelectric studies of function in freshly excised CF compared with normal tissues. Finally, to control for nonspecific effects of in situ inflammation on ΔF508 CFTR processing (Dupuit et al., 1995
), we repeated key studies in cell culture.
CFTR Immunolocalization in ΔF508 Homozygous CF Airway Epithelia
In proximal airways, nasal (7 subjects) and bronchial (15 subjects) CF specimens exhibited epithelial structures and preservation comparable to normal tissues (nasal tissues, 8 subjects; bronchial tissues, 26 subjects) as examined by H&E and actin cytoskeletal staining and confocal DIC planes (Figures and ). In contrast to the consistent and clear CFTR immunostaining signal in the apical PM of surface ciliated cells observed in all normal subjects, we failed to detect specific CFTR immunostaining in any cell type in the CF nasal and bronchial superficial epithelia of any CF subjects (Figures and ).
In CF bronchial tissues, morphological differences associated with chronic infection were present in most of the CF specimens, e.g., goblet cell hyperplasia, consistent with lungs from CF subjects undergoing lung transplantation. Accordingly, in most cases, more than one bronchial specimen from each of the 15 ΔF508 homozygous CF patients was analyzed for CFTR immunolocalization and bronchial areas with clearly identifiable epithelial morphology (e.g., presence of ciliated cells) were utilized in the study. Epithelial areas presenting advanced metaplasia and/or tissue damage were not included in the comparative analyses between both groups. In most CF bronchial specimens, metaplasic tissues showed an absence of CFTR expression in the apical PM of superficial cells, but in a few CF subjects, intracellular CFTR staining was observed in some intermediate and/or nondifferentiated cells (unpublished data). Staining for MUC5AC revealed that the number and distribution of goblet cells varied according to the degree of inflammation in CF specimens. CF nasal tissues displayed moderate signs of epithelial inflammation and goblet cell hyperplasia (), whereas bronchial tissues showed extensive goblet cell hyperplasia (). Interestingly, MUC5AC staining was also observed in the “mucus plaques” lining the surface of many bronchial CF specimens ().
Submucosal glands in all CF subjects also displayed an absence of specific CFTR immunostaining signal in any gland cell type () despite intense staining for AQP5 in the apical domain of acinar serous cells (). Speculation has been raised about AQP5 role in submucosal gland secretory functions and whether AQP5 expression could be altered in CF (Verkman et al., 2003
). In this study, the intensity of AQP5 staining in serous cells was not different in CF versus normal tissues, suggesting that the expression levels of AQP5 protein are not altered in CF. Interestingly, although not detected in normal glands, MUC5AC immunostaining was observed in scattered gland mucous cells of CF submucosal glands ().
In the CF proximal () and terminal bronchioles and alveoli (, insets), no specific CFTR staining was detected in any epithelial cell type (9 subjects) despite well-preserved epithelial cell integrity as demonstrated by actin cytoskeleton staining and DIC images (Figures and ). MUC5AC staining indicated the presence of goblet cells in proximal bronchioles and was again observed in the “mucus plaques” lining the surface of CF proximal bronchioles ().
In sum, CFTR immunolocalization studies indicate that ΔF508 homozygous CF subjects exhibited undetectable levels of CFTR protein in the apical PM of epithelial cells throughout all the airway regions.
cAMP-dependent Cl– Secretion in ΔF508 Homozygous CF Nasal Tissues
CFTR Cl– channel activity was examined in freshly isolated nasal tissues that were also subjected to CFTR protein localization (see above) and maturation studies (see below). Both indices of Na+ transport, i.e., basal and amiloride-sensitive equivalent short circuit current (Isc), were significantly increased in nasal epithelia from five ΔF508 homozygous CF subjects compared with seven normal specimens. Importantly, after blockade of Na+ transport, forskolin/IBMX induced a significant Cl– secretory response in normal (31.0 ± 8.1 μA.cm2) but not ΔF508 homozygous CF nasal tissues (0.5 ± 0.8 μA.cm2; ). These data indicate that CFTR-mediated Cl– secretion is absent in ΔF508 homozygous CF nasal epithelia.
Figure 8. CFTR function and protein expression in nasal and bronchial epithelia from normal and ΔF508 homozygous subjects. CFTR Cl– channel activity in response to forskolin/IBMX was examined in freshly excised nasal tissues from normal (n = 6) (more ...)
Biochemical Analyses of Freshly Excised CF versus Normal Epithelia
Nasal and bronchial tissues utilized in immunolocalization (and functional) studies were also subjected to Western blot analysis to investigate CFTR protein maturation. CFTR is a glycoprotein that separates into two well-defined bands in a denaturing gel. The slower band is the fully glycosylated mature protein or band “C” (180–190 kDa), which corresponds to the CFTR protein localized in the PM and membrane compartments in transit to the PM. The faster band or band “B” (~160 kDa) corresponds to the core-glycosylated immature protein that has not progressed beyond the endoplasmic reticulum. Both mature (band C) and immature (band B) CFTR protein bands were observed in tissues from normal individuals (nasal, 3 subjects; bronchial, 10 subjects). In contrast, CF tissues from ΔF508 homozygous CF individuals (nasal, 3 subjects; bronchial, 10 subjects) exhibited only band B, i.e., the immature protein form of CFTR (). These results indicate that CF tissues express undetectable levels of mature protein in epithelial cells.
Interestingly, both normal and CF tissues exhibited only a low level of core-glycosylated CFTR protein (), which corresponded to the absence of specific intracellular immunostaining of ciliated cells in normal and CF airways (). In contrast, heterologous overexpression systems often display a higher level of core-glycosylated CFTR protein and intracellular immunostaining in cells expressing either WT or ΔF508 CFTR (Mall et al., 2004
). This phenomenon probably reflects either higher protein synthetic rates in heterologous cells or that core-glycosylated CFTR protein may be processed more efficiently into mature protein and/or disappear faster in native tissues.
Bronchial Epithelial Cultures
CFTR expression/function studies were performed in well-differentiated bronchial cultures derived from some specimens included in the immunolocalization study. Immunofluorescence and high-resolution LCM studies of normal cultures revealed that CFTR was expressed at the apical PM of columnar cells in this pseudostratified epithelium (). CFTR localization was identified exclusively in the apical PM of ciliated cells but not in goblet cells or any other cell type (). Similar studies revealed that CF cultures also exhibited a pseudostratified epithelium, but CFTR was not detected in the apical PM of any cell type (). Normal cultures exhibited mature CFTR protein (180–190 kDa) by Western blot, whereas ΔF508 homozygous cultures exhibited only immature CFTR protein (~160 kDa; ).
CFTR Cl– channel activity was measured in bronchial cultures as forskolin (cAMP)-dependent Cl– secretion after amiloride pretreatment. In normal cultures, forskolin induced a large secretory current (ΔIsc = 26.1 ± 2.9 μA/cm2, range 14.2–38.1 μA/cm2, 4 subjects). In contrast, forskolin failed to induce Cl– secretion in any CF culture (Isc =–1.9 ± 0.5 μA/cm2, range –0.3 to –4.0 μA/cm2, 4 subjects, p < 0.01; ).
In sum, ΔF508 homozygous CF bronchial cultures produced identical results to those in native bronchial tissue. There were undetectable levels of mature CFTR protein in the apical PM of epithelial culture cells, and CFTR-mediated Cl– secretion was absent in ΔF508 homozygous CF airway cultures.
Expression of ERM Proteins in Native Airway Epithelia from Normal and ΔF508 Homozygous CF Individuals
Nasal, Bronchial, and Bronchiolar Superficial Epithelia
The distribution and intensity of EBP50 and ezrin immunofluorescence was indistinguishable in normal and CF specimens from nasal (), bronchial (), and proximal bronchiolar () regions. Both proteins colocalized to the apical region of columnar cells, overlapping mostly with the cortical actin staining signal and to a high contrast line observed in the DIC plane characteristic of microvilli. The highest intensity of EBP50 and ezrin staining was in the microvilli of ciliated cells.
Airway Submucosal Glands
Ezrin and EBP50 antibodies produced intense apical staining in ciliated and collecting ducts and in acini of the submucosal glands in all normal and CF nasal and bronchial specimens (Figures and ).
Terminal Bronchioles and Alveoli
EBP50 and ezrin colocalized to the apical PM of all epithelial cuboidal cells of the terminal bronchioles and alveolar epithelial cells in both normal and CF specimens (). In both, normal and CF specimens the intensity of the staining signals were similar in all airway regions for ezrin but appeared to be decreased in the alveoli for EBP50.