ARTICLE |
Correspondence to:
Andreas Claass, Klinik für Allgemeine Pädiatrie, Schwanenweg 20, D-24105 Kiel, Germany. E-mail:
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Summary |
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The hereditary disease cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Understanding of the consequences of CFTR gene mutations is derived chiefly from in vitro studies on heterologous cell cultures and on cells hyperexpressing CFTR. Data from ex vivo studies on human tissue are scarce and contradictory, a fact which is in part explained by secondary tissue destruction in most affected organs. The purpose of this study was to establish conditions under which wild-type and mutated CFTR can be studied in affected human tissue. Sweat glands carry the basic defect underlying CF and are not affected by tissue destruction and inflammation. Therefore, we used this tissue to test a panel of eight different CFTR antibodies under various fixation techniques. The antibodies were tested on skin biopsy sections from healthy controls, from CF patients homozygous for the most common mutation, F508, and from patients carrying two nonsense mutations. Of the eight CFTR antibodies, only threeM3A7, MATG 1104, and cc24met the criteria necessary for immunolocalization of CFTR in sweat glands. The labeling pattern in the CF sweat glands was consistent with the postulated processing defect of
F508 CFTR. The antibodies exhibited different sensitivities for detecting
F508 CFTR. (J Histochem Cytochem 48:831837, 2000)
Key Words: CFTR, sweat gland, immunohistochemistry, antibodies
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Introduction |
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CYSTIC FIBROSIS (CF) is the most common hereditary metabolic disorder in the white population, with an average incidence of 1:2,000 (F508 (
F508 in the CFTR gene leads to a misfolded protein, which is completely retained in the endoplasmic reticulum and degraded. This is believed to result in subcellular mislocalization of CFTR protein to be visualized by immunohistochemical staining (
In contrast to the bulk of evidence from in vitro studies, data from in vivo experiments are scarce. Direct studies on affected human tissue are hampered by several obstacles. First, the cell defect of CF causes tissue destruction in affected organs such as the airways, pancreas, and biliary system. Second, CFTR protein appears to be expressed at low densities (
Despite these difficulties, mislocalization of F508 CFTR was demonstrated in the sweat gland (
F508 CFTR in airways has been challenged by the finding that epithelial remodeling had a stronger influence on the subcellular CFTR localization than the CFTR genotype (
F508 CFTR was tissue-dependent (
The purpose of this study was to compare the different fixation techniques and antibodies as applied in the studies mentioned above and to determine the value and specificity of the CFTR antibodies commonly in use for detection of CFTR in sweat glands.
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Materials and Methods |
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Tissue Samples
Full-thickness skin biopsies were taken from the right shoulder of six healthy volunteers, four F508 homozygous CF patients, and four patients bearing two nonsense mutations within the CFTR gene (G542X/G542X; n=2; R553X/R553X; and G542X/W1282X). Informed consent was obtained from all volunteers; approval of the local ethical committee was obtained for the procedure.
CFTR Antibodies
MATG monoclonal antibodies (MAbs) were from Transgène (Strasbourg, France) with CFTR epitopes at amino acids 107117 (MATG 1016, IgG2a and MATG 1031, IgG1, unpurified), amino acids 503515 (MATG 1061, IgG2a), and amino acids 722734 (MATG 1104, IgG1). The MATG 1061 and 1104 antibodies were purified by affinity chromatography on protein G. MAb G1660, purified by DEAE fast flow column chromatography, recognizes a CFTR epitope within the R domain at amino acids 729736. MAb G2503, purified by protein ASepharose column chromatography, recognizes a CFTR epitope at the C-terminus, amino acids 14661480. Both MAbs were from Genzyme (Cambridge, MA).
Unpurified MAb M3A7 was made available by Dr. J. R. Riordan (Mayo Clinic; Scottsdale, AZ). It was raised against the region from the second nucleotide binding fold to the C-terminus of CFTR (amino acids 11951480).
The cc24 affinity-purified polyclonal rabbit antibody was donated by Dr. A. Nairn (Rockefeller University; New York, NY), and recognizes a CFTR epitope in the R-domain at amino acids 693716.
IgG concentrations and the corresponding dilutions used are given in Table 1.
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Immunohistochemistry
Biopsy specimens were cryofixed in liquid nitrogen. Four-µm cryosections were air-dried, fixed in acetone for 10 min at room temperature (RT) and stored at -70C until use. Some sections were additionally fixed for 5 min at -20C in methanol, as indicated.
Immunohistochemical analysis was performed using the enhanced enzymatic alkaline phosphataseanti-alkaline phosphatase (APAAP) method (
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Results |
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A pilot study on paraffin-embedded skin sections yielded no sweat gland labeling with any of the eight antibodies against CFTR. Therefore, the antibodies were tested on cryofixed skin sections. Fixation techniques were modified by fixing the air-dried sections solely in acetone or subsequently in methanol at -20C, as frequently practiced with MATG antibodies. The results of all CFTR antibodies are summarized in Table 1.
Labeling in Acetone-fixed Control Skin Sections
The CFTR antibodies were first tested on acetone-fixed skin sections of healthy controls. Signals were observed with the CFTR antibodies MATG 1061, MATG 1104, M3A7, and cc24. The results are shown in Fig 1A and Fig 1D. M3A7 stained only the reabsorptive epithelium of sweat glands. Intensive labeling of the luminal surface of the duct epithelium was noted but also of the perinuclear regions of basal and luminal duct cells. The secretory cells were negative as were all other structures of the skin sections. Occasionally, weak IgG-crossreactive material was observed in the secretory cells. The results with the MATG 1104 antibody were comparable, provided it was applied at final concentrations below 10-3g/liter (corresponding to dilutions above 1:1000). At higher concentrations, crossreactive material in the connective tissue was observed, but epithelial labeling was absent. With cc24, similar signal patterns were obtained. The antibody gave some additional background staining of all epithelial cells, including the whole sweat gland, but clearly distinguished from the intense sweat duct labeling. MATG 1061 differed from these three antibodies in that it preferentially stained the secretory coils and, to a lesser extent, the reabsorptive ducts of the sweat glands. Competition experiments were performed with MATG 1104 and cc24. Sweat duct labeling was suppressed by the specific antigenic peptides (Fig 2). With the antibodies G1660, G2503, MATG 1016, and MATG 1031, no signals above background level were observed.
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Labeling in Methanol-fixed Control Skin Sections
The antibodies were subsequently tested on skin sections that were fixed in methanol at -20C (Fig 3). All antigenic sites, as detected by M3A7 were completely abolished by this procedure. The labeling by MATG 1061 was altered in that strong luminal signals were observed in both the secretory and reabsorptive sweat gland epithelium. However, labeling was not confined to the luminal membranes but extended to intraluminal material. Crossreactive material was also observed in the connective tissue, most prominent in the direct periglandular region. The same signal pattern was observed with the MATG 1104 antibody on methanol-fixed tissue, irrespective of antibody concentration.
Labeling in F508 CF Patients' Skin Sections
Skin sections of CF patients were examined using the four antibodies that had proved to produce labeling of control sweat glands (Fig 4). M3A7, MATG 1104, and cc24 were incubated on acetone-fixed sections, whereas MATG 1061 was tested on methanol-fixed sections.
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M3A7 and MATG 1104 failed to label sweat glands from patients homozygous for the F508 deletion. In contrast, intracytoplasmic staining was clearly observed above background level in the reabsorptive sweat ducts after incubation with cc24. A distinct zone at the luminal sweat duct membranes remained unlabeled (Fig 4C). MATG 1061 produced the same staining pattern as in control tissue.
Labeling in Skin Sections from CF Patients with Nonsense Mutations in the CFTR Gene
All four antibodies labeled sweat glands in skin sections. However, the results on cellular localization, on the labeling of secretory epithelial cells, and on the labeling of F508 sweat glands were contradictory. To address these issues, the antibodies were tested on skin sections from patients with nonsense mutations in the CFTR gene. In these patients, the mutations cause CFTR protein synthesis to cease prematurely. Fig 5 shows that M3A7 and MATG 1104 produced no labeling in the sweat glands from these patients, and cc24 gave only the background staining that was noted in all epithelial cells. However, the MATG 1061 antibody labeled luminal epitopes in sweat glands and connective tissue. The signals obtained with this antibody in skin sections must therefore be considered to be unspecific.
Thus, we were able to select three different antibodies for immunohistochemical detection of wild-type CFTR in sweat glands. However, the sensitivity in detecting F508 CFTR differed for the three antibodies.
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Discussion |
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We have undertaken a study on the feasibility of CFTR antibodies currently in use for immunohistochemistry on human tissue. Skin sections were chosen for three reasons. First, they are easy to obtain, with minimal discomfort for patients. Second, they contain structures that are expected and have been shown to express CFTRthe sweat glandsas well as structures free of CFTR, which can serve as internal controls (e.g., blood vessels, nerves). Finally, the structure of the sweat gland is not affected by secondary effects such as inflammation and remodeling. We have examined eight CFTR antibodies under two fixation techniques, including those that were used in the studies by
The Genzyme antibodies G1660 and G2503 are commercially available and have been used for immunocytochemical analysis in CFTR-hyperexpressing cells (
A variety of CFTR antibodies that were raised against defined oligopeptides can be obtained through Transgène. The MATG 1016 and MATG 1031 antibodies are directed against the extracellular loop of the first transmembrane domain of CFTR (
In contrast, we were not able to achieve specific labeling by MATG 1061. In an attempt to reconcile these results with the data from airway epithelium, we added a methanol fixation step according to the literature protocols (
Tissue from CF patients carrying two nonsense mutations can therefore be expected to represent perfect "biological" negative controls. We could trace five patients in Germany, of whom four were willing to cooperate in the study. This biological negative control clearly showed that the MATG 1061 labeling was nonspecific for CFTR in the sweat glands. These results stress the importance of valid controls. Methodically negative controls, including the competition studies, were not sufficient to demonstrate or rule out the specificity of the labeling. M3A7 was produced by Dr. N. Kartner and is directed against a large epitope ranging from the second nucleotide binding fold to the C-terminal end of CFTR. Its specificity in detecting CFTR in sweat glands has been documented in the studies by F508 CFTR was not detectable with either the M3A7 or the MATG 1104 monoclonal antibody. Absence of CFTR-reactive signals in sweat glands from
F508 homozygous patients was also shown by
F508 CFTR only with the cc24 antibody, whose epitope is located in the regulatory domain of CFTR. This polyclonal antibody was produced in New Zealand White rabbits against a synthetic peptide of the R domain (
F508 CFTR and produced no unspecific signals on the biological controls, except for the faint background staining that was observed in all controls. It clearly differed from the specific signals in that it was invariably observed irrespective of the patient's genotype. The increase in sensitivity of the antibody is obviously accompanied by a slight decrease in specificity, which can easily be compensated for by the appropriate controls, as demonstrated.
Of eight CFTR antibodies established in the literature, three were suitable for immunodetection of wild-type CFTR, and one of F508 CFTR, in sweat glands. As discussed in Kartner's publication, M3A7 gains sensitivity on antibody purification (
F508 CFTR lies in their sensitivity for CFTR rather than in differences between the wild-type and
F508 CFTR proteins. All three epitopes are situated outside the
F508 deletion, which is located in the first nucleotide binding fold.
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Acknowledgments |
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We thank A. Nairn, J. R. Riordan, and Transgène for providing antibodies, and H. Bolte for technical assistance with cryosections.
Received for publication January 13, 2000; accepted January 19, 2000.
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Literature Cited |
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Bedwell DM, Kaenjak A, Benos DJ, Bebok Z, Bubien JK, Hong J, Tousson A, Clancy JP, Sorscher EJ (1997) Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nature Med 3:1280-1284[Medline]
Brézillon S, Dupuit F, Hinnrasky J, Marchand V, Kälin N, Tümmler B, Puchelle E (1995) Decreased expression of the CFTR protein in remodeled human nasal epithelium from non-cystic fibrosis patients. Lab Invest 72:191-200[Medline]
Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O'Riordan CR, Smith AE (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63:827-834[Medline]
Cohn JA, Melhus O, Page LJ, Dittrich KL, Vigna SR (1991) CFTR: development of high-affinity antibodies and localization in sweat gland. Biochem Biophys Res Commun 181:36-43[Medline]
Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford KAF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32:219-229[Abstract]
Dalemans W, Hinnrasky J, Slos P, Dreyer D, Fuchey C, Pavirani A, Puchelle E (1992) Immunocytochemical analysis reveals difference between the subcellular localization of normal and Phe508 recombinant CFTR. Exp Cell Res 201:235-240[Medline]
Demolombe S, Baró I, Laurent M, Hongre A-S, Pavirani A, Escande D (1994) Abnormal subcellular localization of mutated CFTR protein in a cystic fibrosis epithelial cell line. Eur J Cell Biol 65:214-219[Medline]
Denning GM, Ostedgaard LS, Cheng SH, Smith AE, Welsh MJ (1992) Localization of cystic fibrosis transmembrane conductance regulator in chloride secreting epithelia. J Clin Invest 89:339-349[Medline]
Dupuit F, Kälin N, Brézillon S, Hinnrasky J, Tümmler B, Puchelle E (1995) CFTR and differentiation markers expression in non-CF and F 508 homozygous CF nasal epithelium. J Clin Invest 95:1601-1611
Gregory RJ, Cheng SH, Rich DP, Marshall J, Paul S, Hehir K, Ostedgaard L, Klinger KW, Welsh MJ, Smith AE (1990) Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 347:382-386[Medline]
Hamosh A, Rodenstein BJ, Cutting GR (1992) CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Hum Mol Genet 1:542-544[Medline]
Hanaoka K, Devuyst O, Schwiebert EM, Wilson PD, Guggino WB (1996) A role for CFTR in human autosomal dominant polycystic kidney disease. Am J Physiol 270:C389-399
Howard M, Frizzell R, Bedwell DM (1996) Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nature Med 4:467-469[Medline]
Kälin N, Claass A, Sommer M, Puchelle E, Tümmler B (1999) F508 CFTR protein expression in tissues from patients with cystic fibrosis. J Clin Invest 103:1379-1389
Kartner N, Augustinas O, Jensen TJ, Naismith AL, Riordan JR (1992) Mislocalization of F508 CFTR in cystic fibrosis sweat gland. Nature Genet 1:321-327[Medline]
Lukacs GL, Mohamed A, Kartner N, Chang XB, Riordan JR, Grinstein S (1994) Conformational maturation of CFTR but not its mutant counterpart (F508) occurs in the endoplasmic reticulum and requires ATP. EMBO J 13:6076-6086[Abstract]
McCaughan KK, Brown CM, Dalphin ME, Berry MJ, Tate WP (1995) Translational termination efficiency in mammals is influenced by the base following the stop codon. Proc Natl Acad Sci USA 92:5431-5435[Abstract]
Picciotto MR, Cohn JA, Bertuzzi G, Greengard P, Nairn AC (1992) Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 267:12742-12752
Pind S, Riordan JR, Williams DB (1994) Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the CFTR. J Biol Chem 269:12784-12788
Puchelle E, Gaillard D, Ploton D, Hinnrasky J, Fuchey C, Boutterin MC, Jacquot J, Dreyer D, Pavirani A, Dalemans W (1992) Differential localization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis airway epithelium. Am J Respir Cell Mol Biol 7:485-491[Medline]
Riordan JR, Rommens JM, Kerem B-S, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou J-L, Drumm ML, Iannuzzi MC, Collins FC, Tsui L-C (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066-1073[Medline]
Shoshani T, Kerem E, Szeinberg A, Augarten A, Yahav Y, Cohen D, Rivlin J, Tal A, Kerem B (1994) Similar levels of mRNA from the W1282X and the delta F508 cystic fibrosis alleles, in nasal epithelial cells. J Clin Invest 93:1502-1507[Medline]
Trezise AE, Buchwald M (1991) In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator. Nature 353:434-437[Medline]
Ward CL, Kopito RR (1994) Intracellular turnover of cystic fibrosis transmembrane conductance regulator: inefficient processing and rapid degradation of wild-type and mutant proteins. J Biol Chem 269:25710-25718
Welsh MJ, Tsui L-C, Boat TF, Beaudet AL (1995) Cystic fibrosis. In Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. New York, McGrawHill, 3799-3876
Will K, Dörk T, Stuhrmann M, von der Hardt H, Ellemunter H, Tümmler B, Schmidtke J (1995) Transcript analysis of CFTR nonsense mutations in lymphocytes and nasal epithelial cells from cystic fibrosis patients. Hum Mutat 5:210-220[Medline]