ARTICLE |
Correspondence to: Margarida D. Amaral, Dept. of Chemistry and Biochemistry, University of Lisbon, Campo Grande, C8, P-1749- 016 Lisbon, Portugal. E-mail: mdamaral@igc.gulbenkian.pt
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Summary |
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Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl-) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations thus far identified. Our aim was to determine the histological localization of CFTR on respiratory and colon epithelia of human and murine origin with a panel of several antibodies produced against different CFTR epitopes, using an indirect immunofluorescence method. Our results on human tissues confirm the apical localization of CFTR in ciliated cells of the respiratory mucosa and show that in colon tissue CFTR is observed in both apical and basolateral membranes of epithelial cells from colon crypts. However, poor tissue preservation of colon biopsies after immunohistochemistry (IHC) raises doubts about the latter localization. Contrary to human, mouse colon epithelium (not biopsed) presents good tissue preservation and evidences many cylindrical surface cells with high apical expression of CFTR. For the antibodies' sensitivity, we demonstrate that MATG1061, 24-1, M3A7, and MPCT-1 give good results, allowing the histological localization of CFTR protein of both human and murine origin. (J Histochem Cytochem 51:11911199, 2003)
Key Words: CFTR, respiratory epithelium, colon epithelium, CFTR antibodies, indirect immunofluorescence, cystic fibrosis, histological characterization
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Introduction |
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CYSTIC FIBROSIS (CF) is the most common lethal hereditary disorder among white populations. The CF transmembrane conductance regulator (CFTR) protein, is a cAMP-activated chloride (Cl-) channel critical for the regulation of respiratory and intestinal Cl- and fluid secretion (
Most data on CFTR expression and function have been derived from experiments on CFTR-overexpressing cells, i.e., transformed cell lines. It has been shown that wild-type (wt) CFTR is directed to the apical membrane of polarized epithelial cells via the Golgi and the trans-Golgi network (TGN), possibly interacting with many proteins during its maturation (
Data from native cells/tissues are therefore still scarce and often contradictory, probably due to experimental difficulties. CFTR is generally described as a low-abundance protein in most tissues where it is endogenously expressed (
The purpose of this study was, first, to compare different CFTR immunostaining protocols and, second, to determine the specificity and sensitivity of a panel of seven anti-CFTR Abs used to immunodetect CFTR in respiratory and intestinal tissues of both human and murine origin. The immunostaining technique was therefore optimized and tested for these different anti-CFTR Abs to maximize the specific CFTR signal-to-noise ratio in IHC studies.
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Materials and Methods |
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Tissue Samples
Here we compared the histological localization of CFTR in colon and nasal epithelia of non-CF individuals and mice. Collection of human tissues was approved by the ethical committee of the hospital and informed consent was obtained from each individual.
Human Tissues.
Nasal and cartilaginous bronchial mucosa have similar structures, both consisting of the association of glands, which in turn are composed of serous and mucos-secreting cells. It has therefore been widely accepted that cell biology data obtained on nasal specimens can be extrapolated to the lower airways (9 hr, tissue samples were thoroughly rinsed with PBS and frozen in liquid nitrogen as above.
Rectal biopsy specimens from non-CF individuals (n=5) during colonoscopy were immediately immersed in DMEM/F12 medium supplemented with 250 UI/ml of penicillin, 125 µg/ml of streptomycin, and 2.5 µg/ml of amphotericin B. Then the same two protocols as above (freezing or Zamboni fixation) were applied.
Cryosections (6-8 µm thick) were obtained from frozen tissues in a cryomicrotome (ReichertJung; Bensheim, Germany) at 20C, placed onto silane-coated glass slides (MenzelGlazer; Braunschweig, Germany), air-dried, and used immediately or stored at 80C until use. IHC studies were carried out either on fresh or thawed cryosections after air-drying and rehydration for 5 min in PBS.
Mouse Samples. Mice were sacrificed after anesthesia by IP injection of nesdonal. Distal colon mucosa samples of 3 mm (length) and tracheal biopsies from the last 3 mm before bronchial bifurcation were taken. Tracheal and colon samples were rapidly removed, frozen, and sectioned as above.
Hematoxylin and Eosin Staining
One cryocut per specimen was stained with hematoxylin and eosin (H&E) for histological observation. Briefly, slides with cryocuts were soaked into Mayer's hemalum solution (Merck Diagnostica; Darmstadt, Germany) for 2 min. After three or four rapid washes in water, slides were soaked in 2% (w/v) eosin (BDH; Poole, Dorset, UK) solution for 30 sec and then rinsed again with water. Cryocuts were then dehydrated by soaking in three solutions of increasing ethanol concentrations [70% (v/v), 95%, and 100%] for 2 min each and finally in xylene also for 2 min. Slides were mounted with p-xylene-bis(N-pyridinium bromide), or DPX (BDH), covered with glass coverslips, and dried for at least 1 hr before analysis.
Antibodies
For CFTR immunostaining, we tested a panel of seven antibodies to evaluate their sensitivity and specificity towards human and murine CFTR (see Table 1). MATG1061 (Transgène, Strasbourg, France;
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Immunocytochemistry
A large part of the observed variability in CFTR immunostaining can be generally due to the protocol used. The latter should result in a good compromise between morphology preservation, achieved through adequate fixation, and avoiding epitope destruction, which usually occurs when too drastic fixation conditions are used. The fixation step is therefore very critical, because if it preserves the tissue from autolysis it also leads to a decrease in the signal due to concealment/disappearance of the antigenic motif (epitope). Before testing the specificity and sensitivity of the different anti-CFTR Abs, an optimization of critical steps (fixation, protease inhibition, dilution, and incubation time of primary Ab) was performed, using as a starting point protocols previously described for the immunolocalization of CFTR (
In these preliminary tests, we used anti-CFTR MATG1061, previously shown to produce CFTR-specific labeling in native tissues (
The adopted optimized protocol involved fixation in methanol for 10 min at -20C before labeling with anti-CFTR Ab. Finally, incubation time with primary Ab (MATG1061) was optimized by testing two different incubation times: overnight at 4C (long) and 2 hr at room temperature in a humid chamber (short). We also tested different dilutions of the primary MATG1061 Ab in the range of 1:200 1:1000 and determined that 1:200 produced the best results. For the other Abs used here, dilutions were according to previous studies or followed those indicated by the manufacturers (see Table 1).
Incubations with secondary Abs were for 45 min, also in a humid chamber at RT. Negative controls omitting the primary Ab were always performed in parallel. Sections were washed three times for 5 min in PBS between each step and finally mounted in Vectashield anti-fading medium (Vector Labs; Burlingame, CA) containing DAPI (Sigma Chemical; St Louis, MO) for nuclear labeling. Labeled sections were stored at 4C in the dark until examined on an epifluorescence Axioskop microscope (Zeiss; Gottingen, Germany) equipped with PowerGene 810 software (PSI; Chester, UK).
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Results |
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Optimization of Immunostaining Towards a Good Balance Between Tissue Preservation and CFTR Signal
As described in Materials and Methods, a preliminary study with anti-CFTR MATG1061 was performed to optimize the general immunodetection protocol concerning the effect of fixation conditions (
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Without fixation, labeling of intestinal tissue with either anti-CFTR (Fig 1E) or anti-cytokeratins Abs (Fig 1J) was still evident but tissue morphology was only poorly preserved. Loss of morphology was not observed in unfixed nasal samples (Fig 1N and Fig 1R). Overall, methanol fixation was chosen for the general CFTR immunostaining protocol as the best compromise between CFTR signal and tissue morphology for both intestinal and nasal tissues. No significant differences were observed between IHC sections incubated with or without a cocktail of protease inhibitors (data not shown). All the results obtained during the preliminary optimization study (Fig 1) are summarized in Table 2.
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IHC Localization of CFTR with Different Antibodies
Mouse Tissues.
Our objective when mouse specimens were used was to compare preservation of tissue morphology (particularly of intestinal tissue, not obtained through a biopsy procedure) and also to test the specificity of several Abs for the IHC localization of endogenously expressed murine CFTR on cryosections of different tissues. Only polyclonal (produced in rabbit) anti-CFTR Abs were tested (i.e., MPCT-1 and 169) because detection of monoclonal anti-CFTR Abs with anti-mouse IgG secondary Abs would crossreact unspecifically with sample tissues.
Trachea. With MPCT-1 PAb, intense CFTR labeling was observed in the cytoplasm and in the apical membrane of cells from the submucosal tracheal glands, as expected (white arrow in Fig 2C). Surface cells presented a characteristic apical signal (yellow arrows in Fig 2C and inset). With PAb 169, the signal observed was very faint and was detected only for the submucosal gland cells (arrow in Fig 2E). Collecting ducts were not present in the different cryosections analyzed. In the absence of primary Ab, only faint background signal was detected (Fig 2G).
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Colon. Contrary to human samples, where considerable loss of morphology occurred during CFTR immunostaining (see above), mouse colon cryosections evidenced good tissue morphology when the same protocol was used (Fig 2D). Comparison of mouse intestinal sections in which CFTR was detected by immunofluorescence (Fig 2D and Fig 2F) with similar ones stained with H&E and observed under brightfield (Fig 2B) clearly shows that morphology was still preserved in the former. Immunofluorescence signal was negative for most of the mouse colon cells, i.e., in the crypts and at the surface epithelium, when either MPCT-1 or 169 PAb was used to detect CFTR (Fig 2D and Fig 2F, respectively). An unexpectedly intense signal was observed on a few scattered epithelial cells located on the surface epithelium (arrowhead in Fig 2D) and also on some of the most superficial cells of the crypts (not shown). This intense signal was mostly cytoplasmic, with additional apical reactivity. These peculiar "CFTR high-expressor" (CHE) cells exhibited an enterocyte-like morphology. Goblet cells were CFTR-negative. Again, no signal was observed in the absence of primary Ab (Fig 2H). Results are summarized in Table 3.
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Human Tissues
It was also our objective to compare the specificity of several Abs for IHC localization of CFTR on fixed cryosections of human nasal and intestinal tissues. The optimized general immunolabeling protocol described above was used to test a panel of seven anti-CFTR Abs (Fig 3).
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Nasal Tissue. CFTR was clearly immunodetected on the luminal surface of pseudostratified epithelia of nasal polyps with MATG1061 and 24-1 MAbs (Fig 1L and Fig 3A, respectively). Although cryocuts may look different in morphology, only pseudostratified surface epithelium was analyzed for CFTR immunostaining, as can be clearly observed by the monolayer of cell nuclei in cryocuts (see DAPI staining of cell nuclei Fig 3A3C and Fig 3G3I).
Monoclonal M3A7 and polyclonal MPCT-1 Abs produced similar results to MATG1061, although the signal detected was weaker and somewhat more diffuse (Fig 3C and Fig 3G, respectively). MPCT-1 Ab also immunostained basal cells to some extent (see Fig 3G). Because the latter were shown to express very low levels of CFTR or none at all (
Intestinal Specimens. IHC analysis of intestinal cryosections was very limited by the poor preservation of tissue morphology after the IHC protocol. Indeed, tissue deterioration had not occurred before the Ab incubation, as observed by the good morphology of H&E-stained sections under brightfield (Fig 1A and Fig 1F). Therefore, no good results were obtained for human intestinal sections with any of the seven anti-CFTR Abs tested here. Diffuse apical and basolateral labeling was observed in cells from the crypts and from the surface epithelium with MATG1061 MAb (Fig 1C), 24-1 MAb (Fig 3D), 13-1 MAb (Fig 3E), and M3A7 MAb (Fig 3F). The strong unspecific background observed, however, did not allow discrimination among different cell types (e.g., goblet cells, reabsorption cells). With MPCT-1 PAb, however, apical labeling was observed when tissue morphology was preserved, a situation that sometimes but not always occurred after methanol fixation (Fig 3J). CFTR labeling was either very faint or nonspecific with 169 (Fig 3K) and CC24-R (Fig 3L) PAbs.
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Discussion |
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The present study assessed the impact of the anti-CFTR immunostaining protocol used on the results obtained in terms of both preservation of tissue morphology and the CFTR signal detected. The other contribution resulting from this study is to define which anti-CFTR Abs are sensitive and specific to detect CFTR in human and murine histological sections of nasal and intestinal origin.
Results obtained for the immunodetection and localization of CFTR on human and mouse respiratory epithelia presented here are in agreement with those from previous studies (
Exhaustive data on CFTR localization in the intestinal tract are scarce (
On the other hand, in mouse colon samples (obtained by cutting off small pieces of tissue, not biopsies) morphology was much better preserved than in colon biopsies. This leads to the suggestion that additional cell shredding may be associated with the biopsy (forceps) procedure.
In our observations of human rectal mucosa sections, CFTR is detected in both the apical region and intracellularly as
Previous mRNA in situ expression studies (
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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We thank the Association de Transfusion Sanguine Gaétan Salaün, the Conseil Régional de Bretagne, the Conseil Général du Finistère, and the French associations Vaincre la Mucoviscidose and ARC for financial support. The European CF Network (EU-QLK3-1999-00241) provided anti-CFTR antibodies and LD with a travel grant.
We are also indebted to R. Dormer, W. Guggino, H. DeJonge, and Transgène for providing antibodies, and to S. Alpiarça (Gulbenkian Institute of Science; Oeiras, Portugal) and D. Gillet (EMIU 01-15, I3S, Brest) for technical assistance. We gratefully thank Prof Paulo Ramalho (Hospital de Santa Maria, Lisbon) for performing rectoscopy procedures.
Received for publication October 3, 2002; accepted April 4, 2003.
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