Selective Up-Regulation of Chemokine IL-8 Expression in Cystic Fibrosis Bronchial Gland Cells in Vivo and in Vitro
1998; Elsevier BV; Volume: 153; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)65633-7
ISSN1525-2191
AutoresOlivier Tabary, Jean Marie Zahm, Jocelyne Hinnrasky, Jean Paul Couetil, Pascale Cornillet, Moncef Guenounou, Dominique Gaillard, Édith Puchelle, Jacky Jacquot,
Tópico(s)Neonatal Respiratory Health Research
ResumoAccumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the ΔF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1β and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured ΔF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl− concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl− concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients. Accumulating evidence suggests that the early pulmonary inflammation pathogenesis in cystic fibrosis (CF) may be associated with an abnormal increase in the production of pro-inflammatory cytokines in the CF lung, even in the absence of infectious stimuli. We have postulated that if baseline abnormalities in airway epithelial cell production of cytokines occur in CF, they should be manifested in the CF bronchial submucosal glands, which are known to express high levels of CFTR (cystic fibrosis transmembrane conductance regulator) protein, the gene product mutated in CF disease. Immunohistochemical analyses showed that CF bronchial submucosal glands in patients homozygous for the ΔF508 deletion expressed elevated levels of the endogenous chemokine interleukin (IL)-8 but not the pro-inflammatory cytokines IL-1β and IL-6, compared with non-CF bronchial glands. Moreover, basal protein and mRNA expression of IL-8 were constitutively up-regulated in cultured ΔF508 homozygous CF human bronchial gland cells, in an unstimulated state, compared with non-CF bronchial gland cells. Furthermore, the exposure of CF and non-CF bronchial gland cells to an elevated extracellular Cl− concentration markedly increased the release of IL-8, which can be corrected in CF gland cells by reducing the extracellular Cl− concentration. We also found that, in contrast to non-CF gland cells, dexamethasone did not inhibit the release of IL-8 by cultured CF gland cells. The selective up-regulation of bronchial submucosal gland IL-8 could represent a primary event that initiates early airway submucosal inflammation in CF patients. These findings are relevant to the pathogenesis of CF and suggest a novel pathophysiological concept for the early and sustained airway inflammation in CF patients. Chronic endobronchial inflammation and bacterial infection are thought to be the main cause of mortality and morbidity in cystic fibrosis (CF), a genetic disease characterized by defective Cl− secretion and enhanced Na+absorption across airway epithelia.1Riordan JR Rommens JM Kertem B Alon N Rozmahel R Grzelczak Z Zielenski J Lok S Plavsic N Chou JL Drumm ML Iannuzzi MC Collins FS Tsui LC Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5977) Google Scholar, 2Rich DP Anderson MP Gregory RJ Cheng SH Paul S Jefferson DM McCann JD Klinger KW Smith AE Welsh MJ Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.Cell. 1990; 62: 1227-1233Abstract Full Text PDF PubMed Scopus (491) Google Scholar, 3Boucher RC Human airway ion transport (part 1).Am J Respir Crit Care Med. 1994; 150: 271-2814Crossref PubMed Scopus (320) Google Scholar Although airway inflammation in CF patients is often viewed as a response to infectious stimuli, recent findings indicate that inflammation and infection are early events in CF lung disease and suggest that airway inflammation may arise without concomitant bacterial infection.4Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DWH Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med. 1995; 151: 1075-1082PubMed Google Scholar, 5Balough KR McCubbin M Weinberger M Smits W Ahrens R Fich R The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis.Pediatr Pulmonol. 1995; 20: 63-70Crossref PubMed Scopus (290) Google Scholar The description of the interleukin (IL)-8 protein as an important tissue-derived chemotactic cytokine for neutrophils6Taub DT Anver M Oppenheim JJ Longo L Murphy WJ T lymphocyte recruitment by interleukin-8. Il-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo.J Clin Invest. 1996; 97: 1931-1941Crossref PubMed Scopus (245) Google Scholar, 7Richman-Eisenstat JB Jorens PG Hebert CA Ieki I Nadel JA Interleukin-8: an important chemoattractant in sputum of patients with chronic inflammatory airway diseases.Am J Physiol. 1993; 26: L413-L418Google Scholar suggests that polymorphonuclear neutrophil accumulation that takes place during lung inflammation in CF patients may also be mediated, in part, by local tissue-derived factors. The production of tumor necrosis factor (TNF)-α, IL-1β, IL-6, and IL-8 and other pro-inflammatory cytokines by airway epithelial cells and lung macrophages along with the accumulation and activation of neutrophils in the CF airways may underlie the early pathogenesis of CF lung disease.4Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DWH Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med. 1995; 151: 1075-1082PubMed Google Scholar, 5Balough KR McCubbin M Weinberger M Smits W Ahrens R Fich R The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis.Pediatr Pulmonol. 1995; 20: 63-70Crossref PubMed Scopus (290) Google Scholar, 8Dean TP Dai Y Shute JK Church MK Warner JO Interleukin-8 concentrations are elevated in bronchoalveolar lavage, sputum, and sera of children with cystic fibrosis.Pediatr Res. 1993; 34: 159-161Crossref PubMed Scopus (180) Google Scholar Before lung infection, an excessive release of pro-inflammatory cytokines and an increased number of neutrophils have been reported in the bronchoalveolar lavage fluids of CF patients.4Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DWH Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med. 1995; 151: 1075-1082PubMed Google Scholar, 5Balough KR McCubbin M Weinberger M Smits W Ahrens R Fich R The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis.Pediatr Pulmonol. 1995; 20: 63-70Crossref PubMed Scopus (290) Google Scholar Moreover, Noah et al9Noah TL Blach HR Cheng PW Wood RE Leigh MW Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis.J Infect Dis. 1997; 175: 638-647Crossref PubMed Scopus (218) Google Scholar recently demonstrated that the IL-8 levels in bronchoalveolar lavage fluids, in comparison with other pro-inflammatory cytokine levels, are markedly increased in children with CF compared with non-CF children with a bacterial infection of the lower airways. Thus, it is possible that excessive inflammation in the CF lung may be related to constitutive abnormalities in the regulation of pro-inflammatory cytokine expression by CF airway epithelial cells, independent of infectious stimuli. In this context, we have recently shown in CF mutant mice10Zahm JM Gaillard D Dupuit F Hinnrasky J Porteous D Dorin JR Puchelle E Early alterations in airway mucociliary clearance and inflammation of the lamina propria in CF mice.Am J Physiol. 1997; 272: C853-C859PubMed Google Scholar that airway inflammation may be a direct consequence of mutant CFTR (cystic fibrosis transmembrane conductance regulator) protein expression, the gene product in CF disease, as evidence for an increased number of inflammatory cells was observed in the lamina propria of CF mice in the absence of any sign of infection. All of these data favor the hypothesis that an endogenous pathway for airway inflammation may exist in CF airway cells before the manifestation of a bacteria-related infection. Submucosal gland epithelial cells are of special interest in this regard, given their ability to express the highest level of CFTR protein in comparison with other human airway epithelial cell types11Engelhart JF Yankaskas JR Ernst SA Yang Y Marino CR Boucher RC Cohn JA Wilson JM Submucosal glands are the predominant site of CFTR expression in the human bronchus.Nature Genet. 1992; 2: 240-248Crossref PubMed Scopus (577) Google Scholar, 12Jacquot J Puchelle E Hinnrasky J Fuchey C Bettinger C Spilmont C Bonnet N Dieterle D Dreyer D Pavirani A Dalemans W Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands.Eur Respir J. 1993; 6: 169-176PubMed Google Scholar and also their ability to provide the bulk of the fluid component of airway secretions. Recently, it was shown that CFTR Cl− channel dysfunction in CF tracheal submucosal gland cells leads to abnormal transepithelial salt and fluid secretion.13Jiang C Finkbeiner WE Widdicombe JH Miller SS Fluid transport across cultures of human tracheal glands is altered in cystic fibrosis.J Physiol. 1997; 501: 637-647Crossref PubMed Scopus (68) Google Scholar Based on these observations it is reasonable to postulate that intrinsic abnormalities related to the mutant CFTR protein in the bronchial submucosal gland cell type could lead to early inflammation in CF airways. At present, it is not known whether CF human bronchial glands in vivo could represent a major local source of pro-inflammatory cytokines and, in particular, for the IL-8 chemokine. Nor is it known whether the abnormally elevated Cl− concentration described for the airway secretions of CF patients14Joris L Dab I Quinton P Elemental composition of human airway surface fluid in healthy and diseased airways.Am Rev Respir Dis. 1993; 148: 1633-1637Crossref PubMed Google Scholar, 15Smith JJ Travis SM Greenberg EP Welsh MJ Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid.Cell. 1996; 85: 229-236Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar can modulate the inflammatory responses in airways. The concept of bronchial gland epithelial cells as an important component of airway inflammation in ΔF508 homozygote CF patients has been applied to understanding the pathogenesis of early pulmonary inflammation in CF patients.4Khan TZ Wagener JS Bost T Martinez J Accurso FJ Riches DWH Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med. 1995; 151: 1075-1082PubMed Google Scholar, 5Balough KR McCubbin M Weinberger M Smits W Ahrens R Fich R The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis.Pediatr Pulmonol. 1995; 20: 63-70Crossref PubMed Scopus (290) Google Scholar We therefore decided to address the following questions. 1) Is there evidence of elevated levels of pro-inflammatory cytokines IL-1β, IL-6, and IL-8 expressed by ΔF508 homozygous CF human bronchial submucosal glands compared with non-CF bronchial submucosal glands in vivo? And 2) If so, what are the levels and differential expression of these cytokines, in particular for the IL-8 chemokine, according to the extracellular Cl− concentration by subcultures of ΔF508 homozygous CF human bronchial gland cells compared with levels measured in non-CF bronchial gland cells in vitro? Human CF bronchial tissue was obtained from eight recipients undergoing lung transplant operations (the CF patients were all ΔF508 homozygous; four females and four males; mean age, 17.3 years; age range, 9 to 27 years). Tissue for control experiments was obtained from four non-CF patients (two males with primary pulmonary hypertension, aged 28 and 29 years, respectively, and two males with pulmonary idiopathic fibrosis, aged 40 and 61 years, respectively). For all experiments, bronchial segments were prepared within 5 hours after lung resection and were incubated in a serum-free medium composed of a 1:1 mixture of Dulbecco's modified Eagles medium (DMEM) and Ham's F-12 medium supplemented with the antibiotics colistin (200 U/ml), penicillin G (100 U/ml), and streptomycin (100 μg/ml). The upper lobar bronchi of six CF patients and three non-CF disease controls (one with primary pulmonary hypertension and two with pulmonary idiopathic fibrosis) were dissected form the pathological lung resected before transplantation, fixed with 10% formalin, and embedded in paraffin for light microscopy. Transverse bronchial sections were stained with a Giemsa solution. Areas of the submucosal connective tissue surrounding glands was selected and analyzed. A minimum of 24 microscopy fields (>450 mm2 of submucosal tissues) were examined at a magnification of ×400, and the number of inflammatory cells was quantified using a computer-assisted analysis system (CAS-200; Becton-Dickinson, Oxford, UK). The total number of inflammatory cells was counted in each delineated submucosal area. Results were expressed as inflammatory cells per square millimeter. Polymorphonuclear neutrophils could be easily recognized by their lobular nuclei and were quantified in each area examined. The percentage of neutrophils was also calculated. For the immunohistochemical analysis of bronchial tissues, frozen bronchial tissue samples were embedded in OCT (Miles Tissue Tek, Elkhart, IN), immersed in liquid nitrogen, and stored at −80°C. Bronchial cryosections (5 μm thick) deposited onto gelatin-coated glass slides were stored at −20°C after air drying and rehydrated in 0.1 mol/L PBS at pH 7.2. Sets of consecutively cryofixed sections were then blocked with PBS/1% bovine serum albumin for 10 minutes and stained for IL-1β, IL-6, IL-8, and lysozyme, a specific protein marker of bronchial gland serous-type cells.16Franken C Meijer CJLM Dijkman JH Tissue distribution of antileucoprotease and lysozyme in humans.J Histochem Cytochem. 1989; 37: 493-498Crossref PubMed Scopus (147) Google Scholar, 17Jacquot J Spilmont C Burlet H Fuchey C Buisson AC Tournier JM Gaillard D Puchelle E Glandular-like morphogenesis and secretory activity of human tracheal gland cells in a three-dimensionnal collagen gel matrix.J Cell Physiol. 1994; 161: 407-418Crossref PubMed Scopus (29) Google Scholar Monoclonal antibodies against the cytokines IL-1β, IL-6, and IL-8 (dilution, 1:100) were purchased from Biosource International (Camarillo, CA). Rabbit antiserum to human lysozyme (dilution, 1:500) was purchased from Dakopatts (Glostrup, Denmark). In all immunofluorescence experiments, bound antibodies were detected using the streptavidin-fluorescein isothiocyanate (FITC) system (Amersham International, Amersham, UK). Secondary antibodies of goat biotinyled anti-mouse and anti-rabbit IgG fractions (Boehringer Mannheim, Meylan, France) and streptavidin-FITC were used at a dilution of 1:50. Negative controls were performed using either nonimmune mouse or rabbit IgG fractions (Sigma Chemical Co., St Louis, MO). The co-localization of the IL-8 chemokine and lysozyme was performed by indirect double immunofluorescence in cryofixed CF and non-CF bronchial sections. Sections were incubated for 60 minutes at room temperature in PBS/1% bovine serum albumin containing 1 μg/ml anti-human IL-8 antibody. After staining with the IL-8 monoclonal antibody, sections were incubated for 60 minutes in PBS/1% bovine serum albumin containing 2 μg/ml lysozyme polyclonal antibody, washed in three changes of PBS/1% bovine serum albumin for 5 minutes each time followed by incubation in PBS/1% bovine serum albumin containing 2 μg/ml of both donkey anti-mouse FITC-conjugated antibody and donkey anti-rabbit Texas-red-conjugated antibody for 45 minutes. After rinsing in three changes of PBS/1% bovine serum albumin for 10 minutes each time, all specimens were counterstained with Harris hematoxylin solution for 10 seconds, mounted in citifluor antifading solution (Agar Scientific, Stansted, UK), and observed by using a Zeiss Axiophot microscope (Zeiss, Le Pecq, France) employing epifluorescence and Nomarski differential interference illumination. Human bronchial gland (HBG) cells were isolated from eight ΔF508 homozygous CF patients and from four non-CF patients as described above. Cell isolation and subcultivation procedures were performed as described previously.17Jacquot J Spilmont C Burlet H Fuchey C Buisson AC Tournier JM Gaillard D Puchelle E Glandular-like morphogenesis and secretory activity of human tracheal gland cells in a three-dimensionnal collagen gel matrix.J Cell Physiol. 1994; 161: 407-418Crossref PubMed Scopus (29) Google Scholar, 18Maizieres M Kaplan H Millot JM Bonnet N Manfait M Puchelle E Jacquot J Neutrophil elastase promotes rapid exocytosis in human airway gland cells by producing cytosolic Ca2+ oscillations.Am J Respir Cell Mol Biol. 1998; 18: 32-42Crossref PubMed Scopus (21) Google Scholar In brief, cells were isolated by enzymatic digestion from bronchial submucosa and grown onto type I collagen-coated 25-cm2 tissue culture flasks in a DMEM/Ham's F12 mixture (50/50%, v/v) supplemented with 1% Ultroser G (a serum substitute from Sepracor, Villeneuve-la-Garenne, France), glucose (10 g/L), and sodium pyruvate (0.33 g/L). Penicillin G (100 U/ml) and streptomycin (100 μg/ml) were also added. The culture medium was replaced every 3 days. After 2 weeks in primary culture at 37°C under 5% CO2 in air, cells were treated with 0.25% trypsin, 0.5 mmol/L EDTA in a Ca2+- and Mg2+-free PBS solution. The removal of contaminated fibroblasts from the primary culture was carried out using a selective trypsination procedure as previously described.17Jacquot J Spilmont C Burlet H Fuchey C Buisson AC Tournier JM Gaillard D Puchelle E Glandular-like morphogenesis and secretory activity of human tracheal gland cells in a three-dimensionnal collagen gel matrix.J Cell Physiol. 1994; 161: 407-418Crossref PubMed Scopus (29) Google Scholar Cells were then grown in 25-cm2 tissue culture flasks. After 4 weeks, third-passage HBG cells had proliferated and exhibited characteristics of homogeneous submucosal epithelial and secretory gland cells, including two protein markers specific to the glandular serous-type cell, these being lysozyme and secretory leukocyte proteinase inhibitor (SLPI) also known as antileukoprotease.17Jacquot J Spilmont C Burlet H Fuchey C Buisson AC Tournier JM Gaillard D Puchelle E Glandular-like morphogenesis and secretory activity of human tracheal gland cells in a three-dimensionnal collagen gel matrix.J Cell Physiol. 1994; 161: 407-418Crossref PubMed Scopus (29) Google Scholar, 18Maizieres M Kaplan H Millot JM Bonnet N Manfait M Puchelle E Jacquot J Neutrophil elastase promotes rapid exocytosis in human airway gland cells by producing cytosolic Ca2+ oscillations.Am J Respir Cell Mol Biol. 1998; 18: 32-42Crossref PubMed Scopus (21) Google Scholar The phosphorylation-regulated Cl− channel activity of CFTR was assessed using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) as previously described.19Brezillon S Zahm JM Pierrot D Gaillard D Hinnrasky J Millard H Klossek JM Tümmler B Puchelle E ATP depletion induces a loss of epithelium functional integrity and down-regulates CFTR (cystic fibrosis transmembrane conductance regulator) expression.J Biol Chem. 1997; 272: 27830-27838Crossref PubMed Scopus (32) Google Scholar The CF and non-CF HBG cells grown on type I collagen-coated glass coverslips were loaded with 3.5 mmol/L SPQ in a hypotonic chloride buffer (1:1 mixture of distilled water and a chloride buffer containing 130 mmol/L NaCl, 2.4 mmol/L K2HPO4, 10 mmol/L d-glucose, 1 mmol/L CaSO4, 1 mmol/L MgSO4, and 10 mmol/L HEPES, pH 7.4) for 10 minutes at 37°C. Cells were then rinsed twice and incubated with the chloride buffer for 15 minutes at 37°C. Cells were placed in a temperature-controlled chamber (37°C) on the stage of an inverted microscope (Zeiss IM35) and incubated in a nitrate buffer in which NaCl was replaced by 103 mmol/L NaNO3. Cyclic AMP (cAMP) stimulation was achieved by exposing the cells to 25 μmol/L forskolin (Sigma Chemical Co.). The chloride secretion of approximately 60 to 80 cells was estimated by measurement of SPQ fluorescence variations obtained using an excitation light wavelength at 365 nm and emission light wavelength at >395 nm through a 32× planachromat objective. A software-driven shutter in the excitation light path was used to automatically illuminate cells for 2 seconds and simultaneously record the fluorescent image every minutes for 15 minutes. Mean variations in SPQ fluorescence after cAMP stimulation were plotted against time over the 15-minute period. The mean number of cells analyzed per non-CF and CF HBG cell monolayer culture was at least 65. Data are presented as the relative fluorescence, this being 100X (Ft/Fo), where Ft is the fluorescence intensity at time t and Fo is the fluorescence intensity at time 0. Total cellular RNA was extracted from 25-cm2 culture flasks of confluent third-passage ΔF508 homozygous CF HBG and non-CF HBG cells using an acid guanidinium/phenol/chloroform method (Trizol, GIBCO BRL, Gaithersburg, MD). For Northern analyses, aliquots of 15 μg of total RNA (determined by spectrophotometry, 260-nm wavelength) were denatured and size fractionated by electrophoresis through a 1.0% agarose/7.0% formaldehyde gel. The integrity of the RNA was confirmed by observing under ultraviolet (UV) light the 28 S and 18 S ribosomal bands after ethidium bromide staining. For Northern blots, the RNA was transferred onto a nylon membrane (Hybond N; Amersham International) by capillary transfer and UV cross-linked to the membrane. Filters were hybridized at 50°C with a 32P end-labeled oligonucleotide probe with the sequence 5′GTT-GGC-GCA-GTG-TGG-TCC-ACT-CTC-AAT-CAC-3′ using a random prime DNA labeling kit (Boehringer Mannheim). Membranes were hybridized for 15 hours at 50°C, washed twice for 10 minutes in 2X SSC, 0.01% SDS, 10 minutes in 1X SSC, 0.1% SDS at room temperature and 15 minutes in 0.1X SSC, 0.1% SDS at 50°C, and finally autoradiographed for 1 week at −70°C. Autoradiogram signal strengths of hybridized mRNA were quantified by scanning densitometry (GS 690 imaging densitometer and Molecular Analyst software; Bio-Rad, Richmond, CA). Adjustments for small differences in loading were made using densitometry of the ethidium bromide fluorescence of the 28 S band of the gel photographed before blotting. Before the exposure of cells to either low (85 mmol/L), intermediate (135 mmol/L), or high (170 mmol/L) Cl−concentration, third-passage confluent monolayers of ΔF508 homozygous CF and non-CF HBG cells were incubated for 16 hours in an Ultroser G-free RPMI 1640 medium in 95% air/5% CO2 to ensure that cells were in a quiescent state. At the end of the 16-hour period, the culture supernatants from an additional 6-hour period were collected and stored at −80°C until tested for the presence of the cytokines IL-1β, IL-6, IL-8, and IL-10, as described below. In this way, individual monolayers of CF and non-CF HBG cells were exposed for an additional 6-hour period to Cl− solutions containing either 85 mmol/L, 135 mmol/L, or 170 mmol/L Cl−, respectively. Immediately after each period of cell exposure, supernatants were collected and stored at −80°C until tested for the presence of cytokines. The three chloride-containing solutions used in this study (85 mmol/L Cl−, 135 mmol/L Cl−, and 170 mmol/L Cl−) contained 1 mmol/L CaCl2, 20 mmol/L KCl, and either 60 mmol/L NaCl, 105 mmol/L NaCl, or 148 mmol/L NaCl, pH 7.4, respectively, as previously reported.15Smith JJ Travis SM Greenberg EP Welsh MJ Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid.Cell. 1996; 85: 229-236Abstract Full Text Full Text PDF PubMed Scopus (901) Google Scholar In another set of experiments, at the end of the first 16-hour period in the Ultroser G-free RPMI 1640 medium, the production of IL-8 by third-passage confluent cultures of CF and non-CF HBG cells was analyzed in response to exposure to glucocorticoids previously solubilized in Ultroser G-free RPMI 1640 medium (1, 5, and 10 μmol/L dexamethasone; Sigma Chemical Co.). From individual monolayers of ΔF508 homozygous CF and non-CF HBG cells, IL-8 secretion was measured over the subsequent 6-hour culture period in the absence or the presence of the different concentrations of dexamethasone. Cytokine concentrations in the culture supernatants of third-passage confluent ΔF508 homozygous CF and non-CF HBG cells were determined by following the manufacturer's instructions in commercially available ELISA kits (Biosource International). The ELISAs for IL-1β, IL-6, IL-8, and IL-10 were sensitive to a level of 5 pg/ml. In experiments where defined amounts of particular recombinant human cytokines (used as standards) were added to supernatants, the total recovery of each of the cytokines was always close to 100%. Analysis of TNF-α was not performed, however, due to the lack of supernatant samples after analysis of the cytokines described above. Cell viability was confirmed by trypan blue exclusion after all experimental interventions. The uniformity of the cell monolayer was determined by quantifying the cell number per well. Total cellular protein concentrations were measured using the Bradford method (Bio-Rad Laboratories). All results are expressed as pg/ml/106 cells. Results are expressed as means ± SD. Each data point was performed in triplicate at least, and each cell culture experiment was performed at least three times. Differences in cytokine levels were analyzed by the Student's t-test for paired and unpaired samples. The mean number of the inflammatory cells and, in particular, the number of polymorphonuclear neutrophils found in six ΔF 508 homozygous CF patients and in three non-CF disease controls are reported in Table 1. In CF patients, the mean number of inflammatory cells was higher (1856 ± 721 cells/mm2; n = 6) as compared with non-CF disease controls (910 ± 352 cells/mm2;n = 3), but the difference was not significant between the two groups. In the submucosal periglandular areas examined, a predominance of mononucleated cells was observed in the two groups. Although the mean number of neutrophils in the CF patient group (116 ± 65 cells/mm2) was higher than that obtained from non-CF disease control group (65 ± 16 cells/mm2), the difference was not significant. Interestingly, when expressed in percentage of total inflammatory cells, the percentage of neutrophils was low and similar in the two groups of subjects analyzed (6 ± 2% in CF and 8 ± 4% in non-CF disease controls).Table 1Individual Counts for Inflammatory Cells and Polymorphonuclear Neutrophils in Bronchial Submucosal Connective Tissues Surrounding Secretory Glands in CF Patients and Non-CF Disease ControlsPatientsAge (years)SexTotal inflammatory cellsNeutrophilsΔF508Homozygous CFCF121M1283 ± 125459 ± 56CF226F2051 ± 1441202 ± 171CF323F754 ± 99941 ± 28CF49F2104 ± 131490 ± 114CF527M2794 ± 2154138 ± 100CF616M2141 ± 1232141 ± 98Non-CF controlsHypertension28M793 ± 48652 ± 39Idiopathic fibrosis61M1301 ± 56259 ± 39Idiopathic fibrosis40F631 ± 34984 ± 65The values represent the number of cells per square millimeter of tissue examined and are expressed as mean ± SD of at least 24 microscopic fields (magnification, ×400) of transverse bronchial sections analyzed for each patient in both the two CF and non-CF groups. M, male; F, female. Open table in a new tab The values represent the number of cells per square millimeter of tissue examined and are expressed as mean ± SD of at least 24 microscopic fields (magnification, ×400) of transverse bronchial sections analyzed for each patient in both the two CF and non-CF groups. M, male; F, female. To evaluate the possibility that the endogenous expression of pro-inflammatory cytokines is up-regulated in CF bronchial submucosal gland epithelial cells, we monitored the immunoreactivity of cytokines IL-1β, IL-6, and IL-8 in consecutive serial bronchial tissue sections obtained from eight ΔF508 homozygous CF patients and from four non-CF disease patients. Figure 1 provides a representative set of the eight CF and four non-CF cryofixed bronchial sections and shows the expression and localization of IL-8 protein by immunofluorescence. The surface epithelium of CF bronchial tissues (Figure 1, a and b) and of non-CF bronchial tissues (data not shown) demonstrated a notable absence of immunoreactivity to the IL-8 antibody. An examination of bronchial submucosal cryosections at low magnification revealed an intense immunoreactivity for IL-8 inside the submucosal glandular structures lying
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