Thorax 1999;54:1093–1098 1093 Fas and Fas ligand expression in cystic fibrosis airway epithelium Thorax: first published as 10.1136/thx.54.12.1093 on 1 December 1999. Downloaded from Isabelle Durieu, Carole Amsellem, Christian Paulin, Marie-Thérèse Chambe, Jacques Bienvenu, Gabriel Bellon, Y Pacheco Abstract Cystic fibrosis (CF) is a genetic disease Background—Cystic fibrosis (CF) is a caused by mutations in the cystic fibrosis genetic disease caused by mutations in the transmembrane regulator (CFTR) gene cystic fibrosis transmembrane conduct- resulting in absent or deficient expression ance regulator (CFTR) gene and defective and function of CFTR protein.12 The main expression of CFTR protein in epithelial cause of morbidity and mortality in CF is cells. The main cause of mortality in CF is linked to a chronic inflammatory and infec- linked to chronic inflammatory and infec- tious airway process which leads to progressive tious airway processes. Recent studies bronchial epithelial damage and lung have suggested perturbations in the apo- destruction.34 ptotic process in CF cell lines and entero- Defective electrolyte transport is believed cytes. A study was undertaken to to be the initial abnormality in CF disease investigate the expression of Fas and Fas resulting in alteration of the epithelial ligand (FasL) in CF bronchial epithelium secretions. Evidence of an early and intense and CF tracheal cell lines. airway inflammation, probably even in the Methods—Immunohistochemical staining absence of documented infection, has been for Fas (alkaline phosphatase anti-alkaline reported in several studies.56A possible role of phosphatase) and FasL (immunoperoxi- CFTR dysfunction in the induction and dase) was performed in eight CF bronchial maintenance of the inflammatory and infec- epithelial samples and four controls and tious process through a dysregulation of immunohistochemical DNA fragmenta- epithelial ion transport and abnormal extra- tion (TUNEL) was carried out in four CF cellular fluid has been suggested.78 It has patients and four controls. Immunofluo- recently been reported that CFTR may also be rescence staining and flow cytometric involved in the apoptotic process of epithelial analysis of Fas and FasL expression was cells.910 Apoptosis is a physiological process performed in two human tracheal epithe- essential for the maintenance of homeostasis http://thorax.bmj.com/ lial cell lines (HTEC) with normal and CF of epithelial organisation and function, and for genotype. The dosage of serum soluble clearance of inflammatory cells. Apoptosis is FasL was examined in 21 patients with CF regulated by several factors including oxidative and 14 healthy volunteers. stress,11 extracellular matrix proteins,12 and Results—FasL expression was markedly external signals such as Fas ligation.13 Fas increased in patients with CF in both the receptor (CD95/APO1) belongs to the tumour ciliated and submucosal glandular bron- necrosis factor (TNF) receptor superfamily. It Jeune équipe 21–86, chial epithelium compared with controls; is commonly expressed on lymphocytes as well on September 26, 2021 by guest. Protected copyright. Université Claude Fas was similarly expressed in bronchial as on the cell surface of various epithelial cells Bernard-Lyon, Lyon 1, samples from controls and CF patients in France mediating interactions with immune eVector I Durieu both the ciliated epithelium and submu- cells.14 Fas receptor activation by its ligand C Amsellem cosal glands. High levels of DNA fragmen- (Fas ligand) induces apoptosis of Fas bearing C Paulin tation were observed in CF but with some cells. Fas ligand (FasL) belongs to the TNF M-T Chambe epithelial cell alterations. Serum concen- family and is predominantly expressed on G Bellon trations of soluble FasL were frequently 15 Y Pacheco activated T cells. The expression of FasL in undetectable in patients with CF. In vitro, sites such as the eye and in some human Laboratoire HTEC expressed Fas and FasL in both carcinomas contributes to the existence of d’immunologie, genotypes. A higher mean fluorescence “immune privilege” of these tissues by Centre Hospitalier intensity for FasL expression was noted in inducing apoptosis in Fas bearing immune Lyon-Sud, 69495 CF genotype HTEC with median (range) cells.16 17 Its expression in epithelial tissue Pierre-Bénite, France for six experiments of 74 (25–101) for CF J Bienvenu suggests a role for FasL in controlling epithe- cells and 42 (21–70) for non-CF cells. lial tissue injury during various inflammatory Conclusion—Fas/FasL interaction is 18 Correspondence to: states. As described for TNF-á, there is a Dr I Durieu, Department of probably implicated in the human CF air- soluble form of FasL obtained by conversion Internal Medicine, Centre way apoptotic pathway. The mechanisms of membrane bound FasL through a Hospitalier Lyon-Sud, 69495 of induction of FasL expression and its Pierre-Bénite, France proteolytic process and devoid of apoptotic role in inducing tissue damage or remod- activity.19 Received 4 January 1999 elling or in controlling local inflammatory In this study we examined (1) the epithelial Returned to authors cell apoptosis remain to be determined. 19 April 1999 (Thorax 1999;54:1093–1098) expression of Fas and Fas ligand in bronchial Revised version received samples obtained from CF patients and 18 August 1999 Accepted for publication Keywords: cystic fibrosis; apoptosis; Fas/APO1; Fas non-CF patient controls; (2) the expression of 24 August 1999 ligand Fas/FasL in two human tracheal epithelial cell 1094 Durieu, Amsellem, Paulin, et al lines, one of them with a CF genotype and the a 1:500 dilution was applied for one hour. other with the normal CFTR genotype; and Detection of antibody was visualised with 3,3'- (3) the serum concentrations of soluble FasL in diaminobenzidine in Tris buVer. Tissue sec- Thorax: first published as 10.1136/thx.54.12.1093 on 1 December 1999. Downloaded from patients with CF and healthy controls. tions incubated with the peroxidase conjugated secondary antibody served as negative control. Methods FasL antibody was used in the presence or IMMUNOHISTOCHEMICAL STUDY absence of the corresponding blocking peptide Patients (amino acid residues 2–19, Santa Cruz Bio- Between 1993 and 1997 eight patients with CF technologies) to confirm the specificity of the (2M:6F) of mean age 16 (range 6–30) staining. underwent bronchial biopsies (five patients) or To detect DNA fragmentation in bronchial lobectomy (three patients) because of repeated epithelium the terminal deoxynucleotidyl infectious complications with localised bron- transferase (TdT) mediated dUTP nick end chiectasis. Patient characteristics and recruit- labelling (TUNEL) method using an in situ ment have been previously described.20 Four cell death detection kit, fluorescein, and patients carried ÄF508/ÄF508 mutations and peroxidase antifluorescein antibody (Boeh- four ÄF508/other mutations. Forced expiratory ringer Mannheim) was used in four patients volume in one second (FEV1) varied from 26% with CF and controls. to 86% predicted and forced vital capacity (FVC) from 32% to 99% predicted. Two FAS AND FAS LIGAND EXPRESSION IN AIRWAY CELL patients had chronic sputum colonisation with LINES Staphylococcus aureus, the others with Pseudo- Cultures of cell lines monas aeruginosa. Two human fetal SV40 transformed tracheal Four male subjects who underwent lobec- epithelial cell (HTEC) lines were studied—one tomy for bronchial carcinoma were used as with the CF genotype CFT-2 homozygous for controls using non-neoplastic bronchial sam- ples. Bronchial tissues The fibreoptic bronchial biopsy specimens and selected samples of bronchi obtained from sur- gical lobectomies were embedded in OCT compound (Tissue Tek), snap frozen in liquid nitrogen, and stored at –80°C until cryosec- tioning. Immunohistochemistry: antigen expression and detection of DNA fragmentation http://thorax.bmj.com/ Five µm cryostat tissue sections were mounted onto poly-L-lysine coated slides and stored at –20°C. The slides were air dried for 30 minutes and fixed in 4% paraformaldehyde for 30 min- utes. Tissue sections were incubated with anti- Fas monoclonal antibody (UB2, Immunotech SA, Marseille, France) at a 1:500 dilution in PBS/Triton 2% for one hour at room tempera- on September 26, 2021 by guest. Protected copyright. ture. PBS/Triton 2% was substituted for the primary antibody for the negative controls. Samples were then washed extensively in Tris buVer 0.1 M and incubated for 30 minutes with a rabbit anti-mouse secondary antibody at a dilution of 1:25. After washing, samples were incubated with a mouse monoclonal alkaline phosphatase anti-alkaline phosphatase anti- body (Sigma Immuno Chemicals, St Louis, Missouri, USA) at a dilution of 1:50 for 30 minutes sheltered from light. Detection of antibody was visualised with the FastTM Fast Red TR naphthol substrate (Sigma Immuno Chemicals). Tissue sections incubated with mouse monoclonal alkaline phosphatase anti- alkaline phosphatase antibody alone served as negative controls. For FasL staining, tissue sections were incu- bated for 30 minutes in H2O2 0.3% washed in Tris buVer and incubated with anti-FasL rabbit polyclonal antibody (SC956; Santa Cruz Bio- Figure 1 Fas ligand immunoperoxidase staining showing technologies, Santa Cruz, California, USA) at diVuse staining in (A) the ciliated epithelium and (B) the glandular area of a patient with CF and (C) very low a dilution of 1:100 for one hour. After washing epithelial (ep) staining in control patient. Magnification × a peroxidase conjugated secondary antibody at 20 for A and B, × 40 for C. Fas and Fas ligand expression in cystic fibrosis airway epithelium 1095 Thorax: first published as 10.1136/thx.54.12.1093 on 1 December 1999. Downloaded from Figure 2 Fas alkaline phosphatase anti-alkaline phosphatase immunostaining showing (A) positive staining in both ciliated (*) and glandular epithelium (*) of patients with CF and (B) positive staining in control patient. Magnification × 20 for A, × 40 for B. ÄF508 mutations and one wild CFTR geno- mean (SD) fluorescence intensity was ana- type cell line (NT) used as a control.21 The two lysed.