Fas and Fas Ligand Expression in Cystic Fibrosis Airway Epithelium

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 infectious 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 samples.

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 minutes. 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 antibody (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. Six experiments were performed for sta- HTEC lines were cultured in DMEM- tistical analysis for each antigen (Fas and HAMF12 medium (Gibco) supplemented FasL), each condition (basal condition and with 10% fetal calf serum (Gibco) and 1% cytokine pretreatment), and each cell line (NT penicillin/streptomycin/fungizon (Sigma) at and CFT-2). The Wilcoxon signed rank test ° 37 Cina5%CO2 atmosphere. was used to compare the mean fluorescence intensity at basal conditions between NT and Immunofluorescence staining and flow cytometric CFT-2 for Fas and FasL and the mean fluores- analysis cence intensity for Fas and FasL expression Surface antigen expression was studied in between basal and stimulated cells (IFN-ã or unstimulated HTEC lines and in HTEC lines TNF-á pretreatment) for each cell line. treated with TNF-á (100 U/ml, Sigma) or interferon gamma (IFN-ã) (1000 U/ml, Gen- MEASUREMENT OF SERUM SOLUBLE FAS LIGAND zyme) for 48 hours before immunofluores- Serum soluble FasL (sFasL) was measured in cence staining. The cells were removed from 21 successive patients with CF and 14 normal the dish with a solution containing versen and subjects (blood donor serum samples). Meas- then washed in PBS. Mouse anti-human Fas ures were obtained with the FasLigand ELISA monoclonal antibody (5 µg/ml, UB2, Immu- kit (MBL Co Ltd, Nagoy, Japan). FasL was notech SA) and rabbit anti-human FasL poly- measured by sandwich ELISA. Samples were clonal antibody (SC956, 10 µg/ml, Santa Cruz incubated in wells coated with the anti-FasL http://thorax.bmj.com/ Biotechnologies) were incubated with cells on monoclonal antibody 4H9 for one hour. After ice for 45 minutes with concomitant permeabi- washing, a peroxidase conjugated anti-FasL lisation with saparin for FasL staining. After monoclonal antibody (4A5) was added and washing with PBS/1% bovine serum albumin incubated for a further hour. An acid solution the cells were labelled with R-phycoerythrin was then added to terminate the enzyme reac- (RPE) conjugated anti-mouse secondary anti- tion and to stabilise the developed colour. The body at a 1:100 dilution for Fas staining and optical density (OD) of each well was fluorescein isothiocyanate (FITC) conjugated measured at 450 nm using a microplate reader. on September 26, 2021 by guest. Protected copyright. anti-rabbit antibody at a dilution of 1:50 for The concentration of sFasL was calibrated FasL staining. Cells were incubated in the dark from a dose response curve based on reference for 45 minutes on ice, washed with PBS and standards with a threshold detection concen- resuspended before analysis by fluorescence tration of 0.03 ng/ml. Because of possible activated cell sorting (FACS EPICS XL-MCL, undetectable values a Yates’ ÷2 test was used to Coulter). RPE and FITC conjugated alone and compare the partition of serum soluble FasL irrelevant isotype matched anti-human immu- concentrations between CF patients and noglobulin served as negative controls. The healthy controls.

Figure 3 TUNEL immunostaining showing (A) intranuclear DNA fragmentation (arrows) in submucosal glandular area of patient with CF; (B) control. Magniﬁcation × 40 for A, × 20 for B. 1096 Durieu, Amsellem, Paulin, et al

Results 0.4 IMMUNOHISTOCHEMISTRY

FasL was expressed with high intensity in all 0.3 Thorax: first published as 10.1136/thx.54.12.1093 on 1 December 1999. Downloaded from patients with CF, both in the ciliated epithelium and in the submucosal glandular epithe- 0.2 lium, with diVuse cytoplasmic staining (ﬁg 1A and B). Both unistratiﬁed and epidermoid metaplastic epithelium gave an intense staining 0.1

compared with controls (fig 1C). Fas antibody Serum Fas ligand (ng/ml) gave a membranous bronchial epithelial stain- 0 ing in normal and metaplastic epithelium and CF Controls in submucosal glands, both in patients with CF Figure 4 Serum concentration of soluble Fas ligand in 21 and in controls, with no significant diVerence patients with cystic fibrosis and 14 healthy controls. Threshold detectable concentration 0.03 ng/ml. between them (fig 2A and B). DNA fragmentation with intense intranuclear staining using TUNEL method was observed in some Discussion bronchial epithelial cells and submucosal Our results show that in vivo FasL expression is glands of all CF samples but not in controls (fig markedly intense in human CF bronchial 3A and B). However, the CF epithelium was epithelium compared with controls; Fas is not always well preserved and the interpret- expressed in both CF and control bronchial ation of such a diVerence is diYcult. epithelium with a similar intensity. More intense DNA fragmentation is also observed in patients with CF relative to controls but some- ANTIGEN EXPRESSION ON CULTURE CELL LINES times in impaired epithelium. In the cultures The mean fluorescence intensities for Fas and both deficient and normal CFTR genotype FasL staining measured by flow cytometric human tracheal cell lines expressed Fas and analysis are summarised in table 1. Both NT FasL but the mean fluorescence intensity for and CF-T2 cells expressed Fas with no signifi- FasL expression was higher in CF genotype cant diVerence in the mean intensity of cells. Cytokine stimulation did not modify Fas fluorescence (p = 0.11). In both cell lines Fas and FasL expression in the two cell lines exam- expression was not modified after stimulation ined. Serum soluble Fas ligand is unmeasur- by IFN-ã and TNF-á. The mean fluorescence able or is present in very low concentrations in intensity of FasL staining was significantly most patients with CF. higher for CFT-2 (p = 0.04) and pretreatment Our results are consistent with recent publi- with TNF-á or IFN-ã did not have any signifi- cations showing expression of both Fas and cant eVect. The apparent diVerence in FasL FasL in human airway epithelium.22 23 How- expression observed between NT and CF-T2 ever, in our study the immunohistochemical after IFN-ã was not statistically significant (p = staining for FasL is intense in patients with CF http://thorax.bmj.com/ 0.07). but very low in controls. The results of HTEC expression are in agreement with immunohistochemical findings, showing a significantly SERUM SOLUBLE FAS LIGAND increased expression of FasL in the CF The serum concentration of FasL was very low genotype cell line. On the other hand, Fas in CF patients with undetectable concentra- expression is the same in both human tissue tions (<0.03 ng/ml) in 13 of 21 patients. All the and HTEC lines.

normal subjects had detectable concentrations Apoptosis dysfunction in cystic fibrosis has on September 26, 2021 by guest. Protected copyright. of sFasL ranging from 0.08 to 0.38 ng/ml recently been suggested with varying results. 2 (p<0.001,Yates’ ÷ test). The median concen- Gottlieb et al reported a “resistance” in vitro in tration was 0.125 ng/ml for normal subjects the initiation of apoptosis in a mutant CFTR and was below the threshold detection value for epithelial mouse mammary cell line caused by patients with CF (fig 4). The undetectable an inability to obtain cytoplasmic acidification concentrations were not associated with thera- compared with the wild CFTR genotype cell peutic drugs, particularly corticosteroids. line.9 On the other hand, Maiuri et al observed 10 Table 1 Mean fluorescence intensities for Fas ligand and Fas staining in human increased DNA fragmentation and increased SV40-transformed tracheal epithelial cell lines with CF (CFT-2) and normal (NT) Fas and FasL expression in CF enterocytes genotype (fluorescence activating cell sorting). Measures are expressed for unstimulated cells suggesting a possible role for Fas ligation in the (basal) and after stimulation with tumour necrosis factor-á (TNF-á ) and interferon-ã 24 (IFN-ã) (n= 6) induction of apoptosis in CF enterocytes. The high level of FasL staining observed in Basal TNF IFN p value vivo in CF bronchial epithelium suggests various hypotheses. Firstly, the implication of local Fas ligand CFT-2 Median 74 66 50 NS and chronic inflammation in the induction of Range 25–101 51–77 24–71 FasL expression: stimulation by cytokines in NT Median 42 37 38 NS this study and others did not increase Fas and Range 21–70 23–67 21–79 25 p value 0.04 FasL expression but other cytokines such as Fas IL-8 present in CF airways could be implicated CFT-2 Median 38 20 33 NS 58 Range 13–45 14–43 14–52 in vivo. Secondly, normal bronchial epithelial NT Median 21 18 22 NS cells are dependent on cell-matrix interactions Range 16–36 14–24 16–48 to prevent apoptosis26; moreover, extracellular p value NS matrix proteins can induce Fas expression in NS = non-significant diVerence (p>0.05). epithelial cells.27 The major modifications in Fas and Fas ligand expression in cystic fibrosis airway epithelium 1097

the organisation of the extracellular matrix in and FasL+ airway epithelial cells is important CF, with subepithelial fibrosis and degradation since PMNs are the predominant phagocytes 20 of collagenic and elastic fibres, mayhavean in bronchial secretions even in patients with Thorax: first published as 10.1136/thx.54.12.1093 on 1 December 1999. Downloaded from important role in controlling cell apoptosis and clinically mild lung disease.5 On the other membranous expression of proteins involved in hand, PMNs express both Fas and FasL and the regulation of cell turnover and tissue activation of the Fas pathway by Fas-FasL remodelling in CF. Thirdly, modification of interaction is one of the mechanisms of PMN FasL expression may be related to dediVeren- apoptosis.38 PMNs are the major source of the tiation of the airway surface epithelium and increased quantity of DNA found in CF epidermoid metaplasia frequently observed in sputum which essentially contributes to the CF. However, FasL expression was not intense altered viscosity of CF airway secretions.39 40 in some metaplastic areas of control samples Another hypothesis could be that FasL+ CF obtained from lobectomies for bronchial carci- airway epithelial cells activate Fas mediated noma. apoptosis of PMNs thereby inducing enhanced The high level of FasL expression in CF DNA release and alterations in mucus visco- genotype tracheal cells compared with wild elasticity. genotype cells suggests a role for deficient The mechanisms of induction of FasL CFTR in inducing FasL expression. The expression in CF airway epithelial cells remain modification of chloride secretion and cellular to be determined. Whether it contributes to polarity, the accumulation of mutant CFTR in airway tissue damage or to the clearance of endoplasmic reticulum, and the upregulation inflammatory cells is not yet known. of IL-8 expression in CF bronchial cells could induce the activation of transcriptional factors The authors thank N Billard, M Gutowski and C Dumontet for such as NK-êB. NF-êB plays an important role their technical contribution. in the regulation of inducible genes such as IL-8, TNF-á and FasL.28–30 The study was funded by a grant from Hospices Civils de Lyon. The significance of low levels of sFasL observed in patients with CF is unclear. The 1 Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of soluble trimeric form of FasL results from a complementary DNA. Science 1989;245:1066–73. metalloproteinase mediated proteolytic process 2 Welsh MJ, Anderson MP, Rich DP, et al. Cystic fibrosis, CFTR and abnormal electrolyte transport. 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