Abstract
The intracellular localization of cystic fibrosis transmembrane conductance regulator (CFTR) in native tissues is a major issue in the study of mutation, processing, and trafficking effects in CFTR and in the evaluation of therapeutic strategies in cystic fibrosis (CF). This work evaluated the applicability of ten different antibodies (Abs) under various fixation techniques for CFTR localization in fresh-brushed nasal epithelial cells collected from CF patients homozygous for F508del and control individuals. In parallel, the same Ab panel was also tested on BHK cell lines overexpressing wild-type or F508del CFTR. The Abs MATG1061, 169, Lis1, MP-CT1, CC24-R, MAB25031, and MAB1660 gave the best detection of CFTR in the apical region (AR) of nasal tall columnar epithelial (TCE) cells. The labeling pattern of these Abs was consistent with the postulated processing defect of F508del CFTR because only a minority of CF TCE cells present CFTR in the AR. In contrast, M3A7, MM13–4, and L12B4 weakly react with the AR and stain almost exclusively a cis-Golgi-like structure in the majority of CF and non-CF airway cells. In BHK cells, all the Abs enabled distinction between wild-type CFTR localization in cell membrane from F508del CFTR, which in these cells is exclusively located in the endoplasmic reticulum.
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Nevertheless, some studies have demonstrated mis-localization of F508del CFTR in sweat gland (Kartner et al. 1992) and in airway epithelium (Engelhardt and Wilson 1992; Puchelle et al. 1992). Kälin et al. (1999) indicate that protein levels and localization are similar between F508del and normal CFTR protein when expressed in airway (epithelial cells observed in nasal polyps) and gut but not when expressed in sweat gland (Kälin et al. 1999). Using nasal epithelial cells, freshly obtained by brushing, we have confirmed that F508del CFTR, albeit in a reduced number (22%) compared to F508del carrier (42%) and healthy cells (56%), does process through the ER and reaches the cell surface (Penque et al. 2000). The correct localization of F508del CFTR at the apical membrane in a fraction of CF airway cells is nevertheless consistent with the concept that the F508del mutation impairs the intracellular trafficking of F508del CFTR protein. However, the trafficking defect of F508del CFTR could also occur at a subcellular level other than ER retention and degradation.
In summary, recent findings clearly indicate that additional studies on native human tissues would lead to a better understanding of the processing, trafficking, and perhaps the function of normal and mutant CFTR. To establish the conditions under which normal and mutated CFTR can be studied in native tissue, we have compared by different immunofluorescence procedures, a panel of 10 commonly used CFTR Abs for CFTR detection in freshly brushed nasal epithelial cells collected from healthy individuals and from CF patients homozygous for F508del. In parallel, the same antibodies were tested in BHK cell lines stably expressing wild-type (wt) or F508del CFTR.
Materials and Methods
Individuals and Nasal Brushing Cells
After informed consent, nasal brushing cells were obtained as previously described (Penque et al. 2000) from CF patients homozygous for F508del (n = 7) and normal individuals (n = 12) with no clinical signs of CF or F508del mutation, as determined by genomic DNA analysis. Genotypes of all individuals studied were determined as described before (Duarte et al. 1996).
Cell Lines
Baby hamster kidney (BHK) cell lines expressing wt or F508del CFTR were cultivated as described (Chang et al. 1993; Haardt et al. 1999). Cells were grown in an 8-well chamber slide system (Nalge Nunc; Roskilde, Denmark) at a cell density of 2 × 104 cells/per well before being analyzed by ICC.
Antibodies
A panel of the following anti-CFTR monoclonal (MAb) and polyclonal (PAb) antibodies, previously shown to recognize CFTR, was used: MATG 1061 MAb, raised against aa 503–515 in the N-terminal (Transgène; Strasbourg, France) (Puchelle et al. 1992); 169 PAb, raised against a peptide comprising amino acids (aa) 724–746 in exon 13 of the R domain of CFTR (Crawford et al. 1991); Lis1 PAb, raised against aa 1468–1480 in the C-terminus [produced in our laboratory (Farinha et al. 2002; and unpublished data)]; MP-CT1 PAb raised against a CFTR epitope in the C-terminus (Lloyd Mills et al. 1992); CC24-R PAb, raised against aa 693–716 of the R-domain (Picciotto et al. 1992); MAB25031 MAb, raised against exon 23 and 24, aa 1377–1480 of the C-terminus and MAB1660 MAb, raised against a CFTR epitope in the R-domain (R&D Systems; Minneapolis, MN); M3A7 MAb, raised against aa 1197–1480 of the C-terminal end of NBD2, L12B4 MAb, against aa 386–412 of NBD1, and MM13–4 MAb, against aa 24–35 of the N-terminus (Chemicon International; Temecula, CA). IgG concentrations and the corresponding dilutions used are given in Table 1. The microtubule organizing center (MTOC) was stained by MAb anti-γ-tubulin, (1:60). The MAb anti-β-COP (1:80) and MAb anti-Golgi 58K protein (1:60) were used to stain the Golgi apparatus (Sigma Chemical; St Louis, MO). Secondary Abs were FITC-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech; Poole UK), diluted 1:50 and FITC-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories; West Chester, PA) diluted 1:100.
Immunocytochemistry
Nasal cells were recovered from the brushes and washed in PBS as previously described (Penque et al. 2000). For formaldehyde fixation, cells were resuspended and fixed while in suspension in 4% (v/v) formaldehyde (Merck, Darmstadt, Germany; ref 104002), 3.7% (w/v) sucrose in PBS for 30 min at 4C and resuspended in cold PBS. Cells were then adhered onto silane-coated glass slides (Sigma Chemical) by centrifugation for 5 min at 2000 rpm in a Cytospin 3 centrifuge (Shandon; Life Sciences International, Cheshire, UK). The cells adhered on slides were or were not additionally fixed in methanol −20C for 5 min (formaldehyde/methanol fixation). For acetone or acetic acid fixation, both at −20C for 5 min, the cells were fixed only after being adhered onto glass slides. For the following ICC analysis, two protocols were used. The first, previously described by us (Penque et al. 2000), was applied for all Abs using different fixing solutions as described above. The second, as described by Dormer et al. (2001b), was tested for some Abs in cells that were fixed in formaldehyde.
Briefly, in our protocol, the cells on slides were permeabilized with 0.2% (v/v) Triton X-100 in PBS for 20 min, washed three times in PBS for 5 min each, and antigen blocked with 1% (w/v) BSA/PBS for 45 min before incubation overnight at 4C with one of the anti-CFTR Abs (see Table 1). The cells were then washed three times with PBS for 10 min each and incubated with the secondary Ab for 45 min at RT. Or, as in Dormer's protocol, the antigen was blocked by goat serum 1:20 for 20 min at RT before incubation with the primary Ab without cell permeabilization. The washes between the primary and secondary Ab incubations were carried out using 1% Tween-20/PBS, repeated three times for 5 min each.
In both protocols, negative controls were performed omitting the primary Ab. The slides were mounted with Vectashield (Vector Laboratories; Burlingame, CA) containing DAPI (4,6-diamino-2-phenylindole; Sigma Chemical) for nuclear staining and covered with a glass coverslip. Immunofluorescence was observed using an Olympus BX-51 with an Olympus X100 UplanApo (NA = 1.4) objective fluorescence microscope and images collected with a V-scan cooled charged-coupled device (CCD) camera (Photonic Science; Millham, UK). Fluorescence analysis and image processing were performed with Image Pro Plus 4.0 software (Media Cybernetics; Leiden, The Netherlands).
The ICC of BHK cell lines expressing wt or F508del was performed on chamber slides using our protocol (Penque et al. 2000).
Description of anti-CFTR antibodies tested in different fixation solutions
aA, acetone at −20C; F, formaldehyde at 4C; FM, formaldehyde at 4C plus methanol at −20C; AE, acetic acid/ethanol at −20C; D, Dormer et al. (2001a) protocol on formaldehyde-fixed cells.
Evaluation of Cell Types Recovered by Nasal Brushing
The freshly isolated human cells recovered from nasal brushings and spread on silane glass slides were stained by the May-Grünwald-Giemsa (MGG) method and the epithelial cells classified exactly as described by Penque et al. (2000).
Statistical Analysis
For some Abs (see Table 2 and Figure 4), about 100 TCE cells per slide were assessed for the presence or absence of CFTR labeling at the apical region, exactly as described previously (Penque et al. 2000). Data are summarized by mean ± standard deviation (SD) and by mean ± quartile deviation (QD). Statistical significance comparisons between CF and non-CF cells were made using both the parametric Student's t-test and the nonparametric Mann-Whitney U-test for two independent samples (Sokal and Rohlf 1981). Coefficients with a p value less than 0.05 were considered statistically significant.
Significance tests for comparison in the proportion of TCE cells with CFTR in the AR for different anti-CFTR Abs a
aTests were performed on formaldehyde fixed cells as described as Penque et al. (2000).
bDifferences significant at p<0.05.
Results
Previously, we showed by immunofluorescence that three currently used anti-CFTR Abs raised against different epitopes (169, MATG1061, and M13–1), were able to detect CFTR at the apical region (AR) of tall columnar epithelial (TCE) cells freshly obtained from nasal epithelia of CF and non-CF individuals (Penque et al. 2000). Here we tested different cell fixation procedures and compared, using the same protocol, whether eight additional anti-CFTR Abs (Table 1) are capable of detecting and evaluating the presence of CFTR in AR of those non-CF and CF TCE cells. Two of the Abs used previously, MATG1061 and 169, were also included in this study. Four different fixation procedures were tested: acetone −20C, formaldehyde at 4C, formaldehyde at 4C followed by an additional fixation in methanol at −20C (formaldehyde/methanol fixation), and acetic acid/ethanol fixation. Some Abs were also tested using the immunocytochemistry protocol described by (Dormer et al. 2001a) in nasal brushing cells fixation by formaldehyde fixation solution. All 10 Abs (Table 1) were also used to evaluate the CFTR localization in BHK cell lines stably expressing wt or F508del CFTR (see below).
CFTR Labeling in Nasal Cells Under Different Procedures
CFTR labeling was first tested using our protocol (Penque et al. 2000) on acetone-, formaldehyde-, formaldehyde/methanol-, or acetic acid-fixed nasal cells obtained by brushing from control individuals. Fixation of cells in suspension, i.e., before adherence onto glass slides, by either methanol, acetone, or acetic acid showed an increase in cell aggregation and shrinking, perhaps caused by cell membrane delipidation. Therefore, these fixation solutions were tested on cells already spun onto glass slides. The type of fixation tested and the dilution for each one of the CFTR antibodies is described in Table 1. The results indicated that the fixation solutions did not significantly alter the signal patterns obtained for the Abs tested (data not shown). The subsequent antibody tests were therefore performed exclusively on formaldehyde-fixed cells. This fixation solution provides better cell morphology preservation and can be used immediately after obtaining the cells while conveniently allowing storage at 4C for up to 1 week. Formaldehyde-fixed cells were also analyzed by the protocol of Dormer et al. (2001b) using some CFTR Abs from the panel (see Table 1). The results showed essentially the same distribution of CFTR localization patterns as those obtained by our protocol (see below).
CFTR Labeling in Control Nasal Brushing Cells
Considerable familiarity has been gained with the performance of each antibody. All Abs tested detected CFTR in the AR of the nasal TCE cells of normal healthy controls. However, the results shown in Figure 1 reveal some differences in the Ab sensivity for CFTR detection in this region. The MATG1061 MAb very clearly detects CFTR localized at the AR in the majority of nasal TCE cells (Figure 1B, arrowheads). With this MAb, almost no staining reaction is observed in the cytoplasm or in the nucleus of TCE cells (Figure 1B). The 169 PAb detects CFTR at the AR of TCE cells (Figure 1C, arrowheads). However, cytoplasmic labeling distribution and nuclear spot crossstaining were also observed in TCE cells with this antibody. The PAb Lis1 clearly stains the AR of TCE cells (Figure 1D, arrowhead) but also exhibits some crossreaction with cilia and nuclei of some cells (Figure 1D). The PAbs MP-CT1 and CC24-R also provide suitable labeling for CFTR detection at the AR (Figures 1E and 1F, arrowheads) despite some crossreaction with cilia (Figures 1E and 1F). Both Abs also stain a cytoplasmic region near the nucleus in a small proportion of TCE cells (Figures 1E and 1F, arrows). These labeling patterns did not significantly change when we applied the protocol of Dormer et al. (2001b) (data not shown). CFTR at the AR is also detected by either MAb MAB25031 or MAB1660 (Figures 1G and 1H). However, the intensity of the background labeling in the cytoplasm of TCE cells hinders a positive clear signal at the AR (Figures 1E and 1F). In our hands, when both Abs are combined in a single detection procedure (see Materials and Methods), the CFTR at the AR is more clearly detected because the background problem is reduced (Figure 1I, arrowhead).
In contrast to the Abs described above, MAbs M3A7, MM13–4, and L12B4 hardly detect CFTR at the AR in a majority of the cell samples analyzed (Figures 1J-1L, arrowheads). Moreover, these MAbs strongly stain an intracellular structure localized close to the nucleus of TCE cells (Figures 1J-1L, arrows). This structure is also observed in basal cells. The staining pattern of this structure is very similar to those obtained with MAbs against proteins of the Golgi apparatus, such as the anti-β-COP (Figure 1M, arrow) and anti-p58 Golgi proteins (Figure 1N, arrow). Surprisingly, some control individuals (4/9 control individuals) were consistently and repeatedly negative (n=4 experiments) for those three CFTR MAbs (M3A7, MM13–4, L12B4). This observation correlated with a negative result for the anti-β-COP MAb for those same individuals despite being positive for the anti-p58 Golgi protein (results not shown). β-COP protein is the major component of the COP-I protein complex, which is critical for vesicular trafficking between the endoplasmic reticulum and the Golgi. β-COP is also a useful marker for the vesicotubular clusters at the cis face of the Golgi stack as well as the cis Golgi itself (Oprins et al. 1993). Golgi p58, a formiminotransferase cyclodeaminase, is used as a selective marker for the cytoplasmic face of the Golgi apparatus (Gao et al. 1998). In nasal TCE cells, the staining pattern of both Abs anti-β-COP and anti-p58 are very similar in location and morphology, suggesting that both Golgi structures are very close to each other in these cells. However, they seem to be distinct structures because in the same individuals no reaction was consistently obtained with anti-β-COP despite the positive reaction to anti-p58 Golgi protein.
The intracellular structure stained by M3A7, L12B4, and MM13–4 Abs is localized in a very distinct place from the microtubule organizing center (MTOC) that was detected by anti-7-tubulin MAb in the posterior region of TCE cells (Figure 1O, arrow).
Immunolocalization of CFTR, β-COP, p58K, and γ-tubulin in non-CF nasal brushing cells fixed with formaldehyde. Negative controls were performed by incubation with the secondary Abs, anti-mouse IgG (1:100;
Immunolocalization of CFTR in F508del/F508del nasal brushing cells fixed with formaldehyde. Labeling with the Abs MATG1061 (1:100;
Immunolocalization of CFTR, β-COP, and p58K in wild-type (WT) and F508del CFTR BHK-expressing cells fixed with formaldehyde. All 10 CFTR Abs tested (MATG1061 (1:100), 169 (1:100), Lis1 (1:50), MP-CT1 (1:100), CC24-R (1:100), MAB25031 (1:10), MAB1660 (1:10), MAB25031/MAB1660 (1:5) combined, M3A7 (1:50), L12B4 (1:50), and MM13–4 (1:50) label CFTR predominantly located at the plasma membrane of the WT CFTR BHK-expressing cells. In F508del CFTR BHK-expressing cells, using the same panel of CFTR Abs, the F508del CFTR is detected restricted to an area around the nucleus of the cells. In BHK cells, the staining pattern of β-COP protein, a cis-Golgi marker, localizes in small vesicles spread into the cytoplasm, while p58 protein, a Golgi apparatus marker, is concentrated in a juxtanu-clear region of the cells. Bar = 15 μm.
CFTR Labeling in F508del-Homozygous Nasal Brushing Cells
Nasal brushing cells of CF patients homozygous for F508del were examined using the 10 CFTR Abs that had previously been tested on control nasal brushing cells (Table 1).
As shown in Figure 2, all 10 CFTR Abs produced essentially the same labeling pattern in CF nasal cells compared to control samples. This means that no particular intracellular staining that could be associated with the F508del trafficking defect was observed in TCE from F508del-homozygous CF patients. The intracellular staining observed in some CF TCE cells was also observed in some non-CF cells using, e.g., the Abs Lis1, MP-CT1, CC24-R, M3A7, L12B4, or MM13–4 (Figures 3C-3E, and 3I-3K, arrows). The main difference between CF and non-CF cells seems to be the number of TCE cells presenting CFTR at the AR (see the results of statistical analysis). As observed with cells from some non-CF individuals, no reaction was found with Abs M3A7, L12B4, or MM13–4 in 2/5 F508del-homozygous CF patients.
CFTR Labeling in BHK Cells Stably Expressing Wt or F508del CFTR
Because the sensitivity in detecting CFTR in CF and non-CF TCE cells differed for the 10 antibodies, especially for the three MAbs M3A7, L12B4, and MM13–4, we examined these Abs for CFTR detection in wt- or F508del CFTR-overexpressing cells. All 10 antibodies were able to distinguish wt CFTR localization from F508del CFTR in BHK cells stably expressing wt or F508del CFTR, respectively (Figure 3). Whatever the Abs used, wt CFTR is predominantly present at the cell membrane of the cells (Figure 3 WT, arrows). The labeling pattern produced in wt cells exhibits long lines between cells, giving the idea that the cells are longer in comparison to F508del cells. F508del CFTR is mostly restricted to an area around the nucleus (Figure 3 F508del, arrowheads); therefore, cells seem rounder than wt cells. In contrast to the findings from nasal TCE cell experiments, no intracellular Golgi-like structures were detected with MAbs M3A7, L12B4 and MM13–4 in CFTR-expressing BHK cells. These Abs are therefore very sensitive and suitable for CFTR detection focused at the plasma membrane of cells expressing wt CFTR (see Figure 3 WT, arrows).
The staining pattern of β-COP protein (a cis-Golgi marker) in BHK cells consists of punctate spots spread throughout the cytoplasm. In contrast, p58 protein, another Golgi apparatus marker, localizes in a concentrated juxtanuclear region of BHK cells (Figure 3 WT and F508del, β-COP and p58). In nasal TCE cells, both Golgi proteins localize closely in a region of which the staining pattern is a unique intracellular structure transversely located in the cell above the nucleus (see Figures 1M and 1N, arrows).
Statistical Analysis of TCEs with CFTR in the AR
The anti-CFTR Abs that exhibited reproducible results concerning the sensitivity for CFTR detection at the AR were used to assess the number of CF and non-CF TCE cells that present CFTR at the AR (Table 2). Two to eight nasal brushing samples were analyzed per Ab. Altogether, we analyzed samples from non-CF subjects (n=12) and CF homozygous for F508del (n=7). More then 100 TCE cells were counted per individual for all Abs tested. The differences between the average of CF patients versus controls were statistically significant in the two tests applied (Student's t-test and Mann-Whitney) for all Abs tested (Table 2). The graphs in Figure 4 clearly illustrate distinct distributions for CF and control cells. The data showed that F508del homozygous TCE cells do have apical CFTR in a minority of TCE cells (∼18%) in comparison with non-CF TCE cells (∼56%). Nevertheless, the relative number of TCE population cells in nasal brushing samples recovered from CF and non-CF subjects and analyzed by MGG staining (not shown) are similar. These results are very consistent with ours previously described (Penque et al. 2000).
Statistical analyses of tall columnar (TCE) cells with CFTR in the apical region. Based on the percentages of TCE cells with CFTR in the apical region relative to total TCE cells in samples from nasal brushings (see Table 2) for non-CF subjects and CF patients, the graph was constructed corresponding to CFTR immunodetection with the following anti-CFTR Abs: MAB25031, MAB166, 169, Lis 1, MATG 1061, CC24-R-R, and MP-CT1. Graphs clearly display the differences in distributions for the proportion of TCE cells with CFTR in the apical region for the seven Abs tested.
Discussion
Difficulties arise in studying the intracellular localization of CFTR in native tissues because of several drawbacks, which include the poor quality of affected human organs, the differences in the experimental procedures, and the variability of the CFTR Abs used. Here we evaluated the applicability of a panel of 10 different CFTR Abs to immunolocalize CFTR in CF and non-CF native airway epithelial cells as well as in cell lines, BHK wt- or F508del CFTR-overexpressing cells. Cells recovered from the superficial nasal mucosa by brushing have been a useful native biological material in the assessment of chronic respiratory diseases, including CF (Danel et al. 1996). The nasal brushing technique is a noninvasive method that allows the easy sampling of many representative, well-preserved and dissociated cells from the superficial respiratory mucosa (Danel et al. 1996). As previously described (Bridges et al. 1991; Chapelin et al. 1996; Danel et al. 1996; Penque et al. 2000), 80–90% of nasal cells recovered by brushing are epithelial cells. In most cases, nasal polyp surface epithelium represents remodeling with a degree of differentiation, i.e., squamous metaplasia that causes a decrease of CFTR expression (Brezillon et al. 1995). In contrast, the epithelial cells contained in the nasal brush samples of individuals with no polyposis are good representatives of normal airway pseudostratified epithelium and will not, as in nasal polyps, give rise to a CFTR expression signal that could be associated with epithelial abnormalities. Fixing cells immediately after recovery from the brushes minimizes any possible alterations of protein expression related to the stress of cell dissociation.
MATG 1061 MAb and 169 PAb were previously tested by us using nasal brushing cells (Penque et al. 2000). In this study we confirmed that using a variety of fixing solutions, both Abs are suitable for evaluation of CFTR in the apical region (AR) of CF and non-CF nasal TCE. MATG1061 showing markedly less background staining than 169. Although MATG1061 has been applied successfully in several studies of airway cells (Puchelle et al. 1992; Jacquot et al. 1993; Brezillon et al. 1995; Dupuit et al. 1995; Kälin et al. 1999), Claass et al. (2000) demonstrated that MATG1061 was nonspecific for CFTR in the sweat glands. However, these authors employed an IHC analysis using an enhanced enzymatic alkaline phosphatase-anti-alkaline phosphatase (APAAP) (Cordell et al. 1984) and not an immunofluorescence one. This method could increase nonspecific binding of the MATG1061 in the skin sections.
Using PAb MP-CT1 in nasal brushing cells, Dormer et al. (2001b) recently confirmed our previous results (Penque et al. 2000) showing that a reduced number of CF TCE cells from CF-homozygous F508del patients have F508del CFTR at the AR. The majority of TCE cells from those CF patients presents a characteristically perinuclear location of F508del CFTR. Using MP-CT1 PAb in formaldehyde-fixed cells, we confirmed the results of Dormer et al. (2001b) concerning the percentage of non-CF and F508del/F508del TCE cells that present CFTR in the AR. Using MP-CT1 PAb (or any of the other nine Abs) we did not observe CF TCE cells with a particular perinuclear F508del CFTR localization, even with the use of different fixation solutions and/or Dormer's methodology (Harris et al. 2001) (see CF repository in http://central.igc.gulbenkian.pt/cftr/index.html). Some cytoplasmic staining is also observed in non-CF cells using those Abs. We could not detect apparent differences in the localization of CFTR and F508del CFTR in the nasal TCE cells. TCE cells that did not show CFTR labeling at AR, whether from CF or non-CF samples, showed weak widespread staining throughout the cell. Occasionally, we observed an intracellular structure barely stained by the Ab MP-CT1, but also by CC24-R and Lis1, and more prevalently stained by the Abs M3A7, L12B4, and MM13–4 (see arrows in Figures 1 and 2) but those structures occur indistinctly in the apical CFTR-positive or -negative cells of both CF and non-CF samples (see below). The MP-CT1 Ab also showed some crossreaction with cilia under all conditions tested. The same result was observed with the PAb CC24-R, whose epitope is located in the R-domain of CFTR (Picciotto et al. 1992) and Lis1, raised against the C-terminus (Farinha et al. unpublished data). However, PAbs showed good sensitivity in detection of both wt and F508del CFTR in the AR of nasal TCE cells. The immunostaining patterns of CC24-R and Lis1 PAbs were similar to that of MP-CT1 PAb, irrespective of the fixing solution used. In our experiments, CC24-R PAb did not exhibit more sensitivity for detecting F508del CFTR as observed by (Claass et al. 2000) in sweat glands from patients homozygous for F508del. F508del CFTR is undetectable in sweat glands using several CFTR Abs but is observed intra-cellularly using CC24-R PAb (Claass et al. 2000).
The MAbs MAB25031 and MAB1660, previously produced by Genzyme (24–1 MAb and 13–1 MAb) and now commercially available from R&D Systems, have been used for ICC analysis in CFTR-overexpressing cell lines (Gregory et al. 1990; Denning et al. 1992). The MAB25031 MAb has also been used to localize CFTR in airway cells (Dupuit et al. 1995; Penque et al. 2000; Castillon et al. 2002). In the study performed in skin sections (Claass et al. 2000), both MAbs MAB25031 and MAB1660 produced nonspecific labeling in sweat glands at the concentrations tested. Using a higher concentration, we show that, although the labeling produced by both Abs was only faintly visible (MAB25031 slightly better than MAB1660), both MAbs are able to detect CFTR at the AR of CF and non-CF TCE cells. The sensitivity could be improved by the combination of both MAbs in the same reaction. Both MAbs, individually or combined, can distinguish wt from F508del CFTR in BHK cells overexpressing wt or F508del CFTR. However, the combination of both Abs did not improve the reaction in BHK cells (data not shown).
The MAbs M3A7 and L12B4 produced by (Kartner et al. 1992) are now commercially available, as well as MM13–4, from Chemicon. In many studies, M3A7 and L12B4 showed specificity for CFTR immunoblotting, immuprecipitation, and immunocytochemistry in CFTR-overexpressing cells (Lukacs et al. 1994; Jensen et al. 1995; Kopito 1999; Farinha et al. 2002). This result is confirmed here for BHK cells overexpressing wt or F508del CFTR. Although, Kartner et al. (1992) originally showed the mislocalization of F508del CFTR in sweat duct biopsies from CF patients (in comparison with wt CFTR in control skin biopsies), they failed to show CFTR localization with M3A7 and L12B4 in airway tissue. In all conditions tested here, M3A7, L12B4, and MM13–4 hardly detected CFTR at the apical region of nasal epithelial cells, confirming the previous observation by Kartner et al. (1992). Our data show that these three MAbs react preferentially and very strongly with an intracellular structure similar in location and morphology to the Golgi apparatus. Moreover, some CF and non-CF individuals always show negative reaction for those CFTR Abs. This observation correlated with a negative result for anti β-COP Abs in those same individuals despite being positive for the anti-p58 Golgi protein. By using M3A7 and L12B4, Kartner et al. (1992) showed individual variation concerning the detection of dense staining bodies throughout the sweat coil of some CF and F508del carrier individuals. Kartner et al. (1992) attributed this variation to the ability of some cells to completely degrade trapped F508del in the cell. Because we observed intracellular structures also labeling non-CF nasal TCE cells that do not express F508del CFTR, the explanation advanced by Kartner et al. (1992) does not fit our data. Nevertheless, it has been described that most normal CFTR never reaches the cell surface, being degraded intracellularly (see Kopito 1999 for review). Therefore, some non-CF (and non-F508 carrier) individuals could also show some variability in the ability to degrade normal CFTR. Although this is an interesting possibility, more experiments should be performed to clarify these observations. Caution must be taken in interpreting results with those Abs while examining fresh nasal epithelium.
In nasal TCE cells, the staining pattern obtained with anti-β COP and anti-p58 Abs suggests that cis-Golgi vesicles and Golgi structures are too close to be distinguished by immunofluorescence methods. In contrast, in BHK cells both structures are clearly distinct in location and morphology. This finding is probably due to differences in the state of polarization of cells and should be considered when trafficking studies are performed in these cells.
In summary, all the CFTR Abs tested are able to distinguish wt from F508del CFTR location in BHK cells overexpressing wt or F508del CFTR. However, in native nasal epithelial cells obtained by brushing, 7/10 Abs tested, MATG 1061 (provided by Transgène), 169, Lis1, MP-CT1, CC24-R, MAB1660, and MAB2560 (the last two commercially available from R&D Systems) exhibited sensitivity for CFTR detection in the AR. These Abs can be used to assess CFTR localization in the AR of native respiratory cells because they provide a broad consensus, i.e., the Abs show that F508del-homozygous nasal TCE cells do not have apical CFTR in a minority of cells in comparison to non-CF nasal TCE cells, which exhibit CFTR in the majority of cells.
Footnotes
Acknowledgements
Supported in part by a PRAXIS XXI PCOTI/1999/MGI/35737 research grant. ICO is recipient of an FCT doctoral fellowship. AE was recipient of a travel grant from the European CF Network (EU-QLK3–1999–00241).
We thank Dr C. Barreto (HSM, Lisbon), CF patients and control individuals for their cooperation, Dr W. Guggino (Baltimore, MD) for 160 Ab, Dr H. DeJonge (Rotterdam, The Netherlands), for CC24-R Ab, Dr R.L. Dormer (Cardiff, UK), for the MP-CT1 Ab, Transgène (Strasbourg, France) for the MATG1061 Ab, C. Cunha (FCUL, Lisbon) for technical assistance, and H. Davidson (Edinburgh, UK) for discussion and revision of the manuscript.