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* From the Klinik und Poliklinik für Innere Medizin II, Bereich Pneumologie (Drs. Schulz, Wolf, and Schroll, and Ms. Krätzel, and Ms. Köhler), Klinikum der Universität Regensburg, Regensburg; and Klinik Donaustauf (Dr. Pfeifer), Donaustauf, Germany.
Correspondence to: Christian Schulz, MD, Medizinische Klinik und Poliklinik für Innere Medizin II, Klinikum der Universität Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany; e-mail: christian.schulz{at}klinik.uni-regensburg.de
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- and interferon (IFN)-
-induced expression and release of the neutrophil chemoattractants interleukin (IL)-8 and growth-related oncogene (GRO)- in primary bronchial epithelial cells (PBECs) from smokers without airflow obstruction and patients with COPD. In addition, the expression of both TNF--receptor subtypes—p55 TNF-receptor subtype (TNF-R55) and p75 TNF-receptor subtype (TNF-R75)—was quantified in PBECs.
Design: PBECs from eight smokers without airflow limitation and eight patients with COPD were stimulated with 50 ng/mL of TNF and 200 U/mL of IFN- for 4 h along with unstimulated time controls. The transcriptional expression and protein release were quantitatively assessed by means of real-time polymerase chain reaction and enzyme-linked immunosorbent assay.
Results: Basal level messenger RNA (mRNA) expression and protein release of IL-8 and GRO- were not significantly different between both groups, although a trend toward higher IL-8 levels was seen in patients with COPD. TNF- induced significantly higher mRNA amounts of IL-8 (p = 0.005) and GRO- (p = 0.007) in patients with COPD. This was accompanied by higher protein release data for IL-8 (p = 0.005) and GRO- (p = 0.007). IFN- had no significant effect on the mRNA expression and protein release of IL-8 and GRO- in either group. TNF-R55 and TNF-R75 were detectable in PBECs. However, no significant differences were found between both groups with respect to steady-state mRNA levels of TNF--receptor subtypes.
Conclusion: PBECs from patients with COPD show significantly higher TNF--induced release of the neutrophil chemoattractant CXC-chemokines IL-8 and GRO- compared to smokers without airflow limitation. This increased activation of PBECs may contribute to the predominance of neutrophils seen in the airway lumen of patients with COPD.
Key Words: bronchial epithelium • COPD • growth-related oncogene- • interleukin-8 • smoking
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), which implies a type 1 cytokine profile.6 Although the mucosal inflammation and infiltration is without prominence of neutrophils,35 patients with COPD have a higher numbers of neutrophils in induced sputum samples than smokers without airflow obstruction.789 In agreement with these results, significant positive correlations between the levels of the neutrophil attractants growth-related oncogene (GRO)- or interleukin (IL)-8 and neutrophil numbers in induced sputum are found in patients with COPD.89 However, the mechanisms that contribute to the increased bronchial inflammatory load associated with COPD are not precisely known. Most likely, activation of the bronchial epithelium, which is the first line of defense encountered by cigarette smoke or other environmental factors, could promote and perpetuate an inflammatory reaction in the airways. Bronchial epithelial cells have the potential to synthesize and release a wide variety of inflammatory mediators, such as IL-8, tumor necrosis factor (TNF)-, or IL-1ß.1011 Differences in the activation of bronchial epithelial cells may contribute to a faster and more pronounced transit of neutrophils from the airway wall and vasculature into the lumen, which could explain differences in bronchial inflammatory load between smokers without airway obstruction and patients with COPD. With respect to the results of Saetta and colleagues,6 it can be speculated that the type 1 cytokine profile may cause an increased activation of bronchial epithelial cells.
In the present study, we therefore hypothesized that there may be differences in the basal level and induced expression and release of the neutrophil chemoattractant CXC-chemokines IL-8 and GRO- between bronchial epithelial cells from patients with COPD and smokers without airway obstruction. To test this hypothesis, we quantitatively evaluated the unstimulated, TNF-- and IFN--induced messenger RNA (mRNA) and protein levels in primary bronchial epithelial cells (PBECs) from smokers without airflow limitation and smoking patient with COPD.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients underwent fiberoptic bronchoscopy under light sedation. Bronchial epithelium was obtained by gentle brushing of segmental and subsegmental bronchi under direct visual guidance by means of a protected brush. Each patient underwent 20 to 30 brushes of the bronchial epithelium. Brushing was accomplished by a very light gliding motion along the surfaces of the airways. In cases where lung cancer was suspected, brushes were performed from the contralateral side to preserve maximum distance to the tumor. Brushes were immediately placed in ice-cold culture medium and transported directly to the laboratory for further processing.
Culture of PBECs
Brushes were vortexed vigorously, and the harvested cell suspension was filtered through a 200-µm filter (Millipore; Billerica, MA) filter to remove mucus and cellular debris, and then treated with 4.8 U/mL Dispase II (Roche; Mannheim, Germany) to eliminate cell clumping. The cells were then centrifuged at 150g for 10 min, and the pellet was resuspended in cell culture medium. Cell number was determined using a hemocytometer. Cell viability was determined by Trypan blue exclusion. Cells were resuspended in serum-free bronchial epithelial cell growth medium (Promocell; Freiburg, Germany) supplemented with a variety of growth factors, including bovine pituitary extract (0.052 mg/mL), recombinant human epidermal growth factor (0.5 ng/mL), insulin (5 µg/mL), hydrocortisone (0.5 µg/mL), epinephrine (0.5 µg/mL), triiodothyronine (6.5 ng/mL), transferrin (0.01 mg/mL), and retinoic acid (0.1 ng/mL). In addition, the medium contained penicillin G (100 U/mL), streptomycin (100 µg/mL), and amphotericin B (0.25 µg/mL) [Invitrogen; Karlsruhe, Germany]. Cultures were maintained in a humidified atmosphere at 37°C in air/carbon dioxide (95/5% volume/volume); 0.5 x 105 cells were plated on six-well culture dishes and were refed three times a week until they were confluent. The identity of the epithelial cells was confirmed in all cultures by light microscopy and in randomly selected cultures by immunocytochemical staining for cytokeratin expression using a pancytokeratin antibody (clone KL1) directed against cytokeratin types 1, 2, 5, 6, 7, 8, 11, 14, 16, and 18 (Immunotec; Marl, Germany). The monoclonal antifibroblast antibody FibAS02 (Dianova; Hamburg, Germany) was used to exclude contamination by fibroblasts.13 The staining was performed according to Kunz-Schughart and coworkers14 as has been described in detail previously.
Confluent cultures were growth arrested for 24 h by leaving out epidermal growth factor and pituitary extract from the medium and washed two times with freshly prepared bronchial epithelial cell growth medium. Then PBECs were stimulated with 50 ng/mL of recombinant human TNF- (Sigma-Aldrich; Munich, Germany), or 200 U/mL recombinant human IFN- (Roche) for 4 h along with unstimulated time controls. Thereafter, supernatants were collected and stored at – 80°C until the analyses for IL-8 and GRO- were performed. In addition, cell densities were determined by counting Trypan blue-stained, trypsinized aliquots from each well and were corrected for final volume. The stimulation was limited to 4 h to avoid secondary effects. In preliminary experiments, concentration response curves had shown that the TNF- and IFN- concentrations chosen to stimulate the cells induced maximum responses with respect to IL-8 (TNF-) or intercellular adhesion molecule (ICAM)-1 (IFN-) mRNA expressions. Furthermore, apoptosis assays using flow cytometry-derived side scatter images clearly demonstrated that under the conditions used in the present study, no induction of apoptosis was detectable in human bronchial epithelial cell lines (BEAS-2B) and PBECs, which had to be taken into consideration since a rather high TNF- concentration was used.
Enzyme-linked Immunosorbent Assay Kits
Commercially available enzyme-linked immunosorbent assay (ELISA) kits for IL-8 (Pierce-Endogen; Rockford, IL) with a lower detection limit of 25.6 pg/mL, and GRO- (R&D Systems; Minneapolis, MN) with a lower detection limit of 31.2 pg/mL were used to determine cytokine levels in cell culture supernatants. Cytokine levels were measured according to the instructions of the manufacturer. Specific cytokine concentrations were expressed as picograms of cytokine per milliliter and 104 cells.
RNA Isolation
RNA was isolated using the High Pure RNA Isolation Kit (Roche Diagnostics; Mannheim, Germany). RNA quality and quantity were evaluated by ultraviolet spectrophotometry and agarose gel electrophoresis.
Real-time Polymerase Chain Reaction (Light Cycler)
One microgram of total RNA was reversely transcribed into complementary DNA (cDNA) [20 µL] according to standard protocols. In brief, cDNA probes were synthesized in a 20-µL reaction with 1 µg of total RNA, 0.5 µg of oligo(dT)12–18, 40 U of ribonuclease inhibitor (Rnasin; Promega; Madison, WI), 4 µL 5x reverse transcriptase buffer, 0.5 mmol/L deoxynucleoside triphosphate, and 200 U of Moloney murine leukemia virus reverse transcriptase enzyme (Invitrogen; Karlsruhe, Germany). After the reaction, water was added to a final volume of 150 µL cDNA solution. Real-time polymerase chain reaction (PCR) was performed in a Light Cycler (Roche). All PCR experiments were done using the Light Cycler DNA Master SYBR Green I kit (Roche Molecular Biochemicals; Mannheim, Germany). Each reaction (20 µL) contained 5 µL of cDNA solution, 2.5 mM MgCl2, 1 pmol of each primer, and 2 µL of Fast Starter Mix (containing buffer, deoxynucleoside triphosphates, Sybr Green dye, and Taq polymerase) [Roche Molecular Biochemicals]. The amplification program consisted of 1 cycle at 95° with 5-min hold ("hot start") followed by 45 cycles of 95°C for 15 s, 62°C annealing temperature for 5 s, and 72°C extension for 10 s. The amplicon was verified by melting curve analysis following the PCR reaction. A negative control without cDNA was run with every PCR to assess specificity of the reaction. For verification of the correct amplification product, PCR reactions were analyzed on an ethidium bromide-stained 2% agarose gel. Analysis of data were performed using Light Cycler software version 3.5. Efficiency of the PCR was determined by analyzing a dilution series of cDNA. Using the analysis mode of the Light Cycler software package (Roche Molecular Biochemicals), the slope of log concentrations of the cDNA dilutions was calculated. Efficiency was calculated as follows: efficiency = 10-1/slope. Only primer pairs showing an amplification efficiency between 1.95 and 2.02 were used for quantification. The measured efficiencies were used for calculation of the single mRNA values. Cytoplasmic ß-actin was analyzed in parallel to each PCR, and the resulting actin values were used as standards for presentation of the specific transcripts as indicated. The increase in mRNA abundance on TNF- or IFN- stimulation was calculated as fold increase of the basal level. In each patient, the upregulation of the ICAM-1 and IFN--inducible protein-10 (IP-10) were used as positive controls to verify the effectiveness of IFN-. Oligonucleotide primers were designed according to the published sequences (Table 1 ).
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test (Statistical Package for the Social Sciences, Version 11.5.1 for Windows; SPSS; Chicago, IL); p < 0.05 denotes a significant statistical difference. |
Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was seen in both groups. Although there was a trend toward higher IL-8 abundance in patients with COPD, no statistically significant differences were found in the unstimulated situation (p = 0.28). Four-hour stimulation with TNF- induced 2- to 70-fold increases in IL-8 mRNA transcripts. Using the housekeeping gene ß-actin as an internal standard, the median levels in IL-8 message rose to 4.6% (range, 3.1 to 9.9) in smokers without airflow limitation and 12.9% (range, 5.4 to 36.9) in patients with COPD (p = 0.005). Similar results were found for the induction of GRO- message. Quantitative assessment using the ß-actin values as the internal standard revealed a relative abundance of 7.3% (range, 3.8 to 11.2) and 16.2% (range, 8.1 to 81.1) of GRO- mRNA (p = 0.007) in smokers without airflow limitation and patients with COPD, respectively. Although IFN- revealed several hundred-fold increases in ICAM-1 and IP10 mRNA expression in each patient (data not shown), neither IL-8 nor GRO- responded significantly to IFN- stimulation. Table 3
summarizes the light cycler measurements.
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Protein Concentrations. Figures 1, 2
summarize the median release of IL-8 and GRO- under unstimulated, TNF--stimulated, and IFN--stimulated conditions. The protein data confirm the results from the light cycler measurements showing that TNF led to significantly higher IL-8 (16.0 pg/mL/104 cells; range, 9.3 to 39.5) and GRO- (7.0 pg/mL/104 cells; range, 5.3 to 22.8) releases in PBECs from patients with COPD compared to smokers without airflow obstruction, where the IL-8 and GRO- levels rose to 8.1 pg/mL/104 cells (range, 3.6 to 13.2) and 2.7 pg/mL/104 cells (range, 0.7 to 9.6), respectively. As shown in Table 4
, the TNF--induced protein release of IL-8 and GRO- correlated highly with each other. In addition, correlations were performed between TNF--induced protein release data and those lung function parameters indicating the degree of airflow limitation. For all correlations tested, inverse relationships between protein release and airway obstruction were found. With the exception of FEV1/FVC (percentage predicted) vs GRO- (p = 0.06, r = – 0.49), all correlations reached the level of significance (Table 4). Furthermore, no differences were seen with respect to the unstimulated protein release, and both cytokines responded nonsignificantly to IFN- stimulation, which reflects the mRNA results as well.
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-induced cytokine responses, the expression levels of the TNF-receptor subtypes—p55 TNF-receptor subtype (TNF-R55) and p75 TNF-receptor subtype (TNF-R75)—were quantified at the steady-state mRNA level by means of light cycler measurements. As shown in Figure 3
, both TNF receptors were detectable in PBECs. The expression levels of TNF-R55 compared to TNF-R75 were 5,670-fold and 4,730-fold higher in smokers without airflow limitation and patients with COPD, respectively. No significant differences in the expression levels between groups were detected for TNF-R55 (p = 0.8) nor for TNF-R75 (p = 0.6).
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced and IFN--induced mRNA expression and protein release of the CXC-chemokines IL-8 and GRO- were examined in PBECs of smokers without airflow limitation and patients with COPD. We demonstrate that primary airway epithelial cells from patients with COPD show a significantly higher degree of activation in response to TNF-, resulting in significantly increased expression of both chemokines. In contrast, IFN- had no effect on the release of IL-8 and GRO- in either group, although clear upregulations of ICAM-1 and IP10 were seen. Since no differences were found in the steady-state mRNA expression levels of both TNF-receptor subtypes, this indicates differences in the function of bronchial epithelial cells between patients with COPD and smokers without airflow limitation.
IL-8 and GRO- both possess an additional structural designation, namely the amino-acid motif E-L-R-CXC just proximal to their first two cysteine residues. These E-L-R-CXC chemokines act primarily as neutrophil chemoattractants.16 Since neutrophils seem to play an important role in the development and progression of COPD in smokers, these two chemokines are of potential relevance with respect to the specific bronchopulmonary inflammation seen in COPD.35 Keatings and colleagues8 showed that in induced sputum the neutrophil cell count was significantly higher in patients with COPD than in smokers without airflow obstruction. In addition, neutrophil percentages were significantly correlated with the degree of airflow limitation, and the highest IL-8 concentrations were found in the patients with the highest number of neutrophils.8 Studies917 confirmed that the degree of airway obstruction is significantly correlated with the level of IL-8 and GRO- in sputum samples or BAL fluid derived from patients with COPD. However, the cellular sources have yet to be determined. Because the bronchial epithelium is the first line of defense for inhaled substances, an increased activation of airway epithelial cells may explain, at least in part, why COPD develops only in some smokers. We have shown a trend toward higher unstimulated bronchoepithelial IL-8 mRNA expression in patients with COPD. These findings were accompanied by significantly higher TNF--induced expression of IL-8 and GRO- at both the transcriptional and protein level in these patients. Given the latter observation, these results are likely to be of pathophysiologic relevance. Our results are in general agreement with the observations of others18 who also described functional differences between epithelial cells from smokers and ex-smokers with and without COPD. de Boer and colleagues18 described significantly higher bronchiolar epithelial IL-8 mRNA and protein levels in patients with COPD in comparison to smokers and ex-smokers without airway obstruction. However, these levels did not correlate with the numbers of macrophages, mast cells, or neutrophils within the lamina propria or epithelium of the bronchioles. The dissociation between a predominance of neutrophils in the bronchiolar lumen assessed by BAL or induced sputum and lack of increased numbers of these cells in the mucosa is a well-described observation.3519 Taking the results of the present study into consideration, an alternative explanation for these findings could be that the higher expression of IL-8 and GRO- in patients with COPD may lead to an increased and faster transit of neutrophils from the airway wall and vasculature into the bronchial lumen. We cannot rule out the possibility that the findings of the present study represent secondary characteristics of COPD. However, the exposure to cigarette smoke and the cumulative inhalative burden were not significantly different between smokers with and without airflow obstruction. Therefore, the greater TNF--induced IL-8 and GRO- chemokine release, which was used as a surrogate parameter to assess bronchial epithelial cell activation, is probably part of the COPD pathology itself. In addition, the significant correlations found between protein expression data and degree of airway obstruction seem to support the idea that these findings are of functional relevance.
Increased numbers of CD8+ T-cells are present in the parenchyma and airways of patients with COPD.2021 It has been shown that these cells produce IFN-, which is in accordance with a predominant type 1 response.6 Therefore, the possibility exists that higher concentrations of IFN- contribute to the pronounced bronchoepithelial activation and release of IL-8 and GRO- found in patient with COPD. However, contradictory results have been published regarding IFN--induced activation of bronchoepithelial cells measured by means of IL-8 release.2223 Using second- or third-passage cultured human bronchial epithelial cells, Striz et al23 described a significant secretion of IL-8 after stimulation with 10 ng/mL of IFN-. In contrast, a study by van der Velden and colleagues22 showed that IFN- had no effect on the release of IL-8 in human bronchial epithelial cells despite increased expressions of ICAM-1 and human leukocyte antigen class II molecules. The latter is in agreement with the present study since neither patients with COPD nor smokers without airway obstruction showed a significant stimulation of the IL-8 or GRO- release in unpassaged PBECs from 16 different patients. Since the median increases of the ICAM-1 or IP10 mRNA expression levels were > 500-fold of the basal levels, it is very unlikely that the IFN- concentration used in the present study was too low to induce cytokine release. The latter findings also contradict that high concentrations of IFN- caused false-negative results, a phenomenon known as the prozone effect.Furthermore, similar effects regarding IL-8 production were found in cultured proximal tubular epithelial cells from human kidneys,24 and in vivo inhalation of IFN- in normal human volunteers did not induce an up-regulation of IL-8 gene transcripts in alveolar macrophages.25 In summary, these results indicate that IL-8 and GRO- are not induced in airway epithelial cells by a predominant type 1 immune response with increased secretion of IFN-. This, however, should not imply that IFN--producing Tc1 cells cannot effect airway epithelial cell function through other mechanisms (eg, ICAM-1 expression, modulation of cell-cell contact).
It is well established that the release of IL-8 from bronchial epithelial cells can be increased via an increase in intracellular cyclic adenosine monophosphate content.2627 Hence, it can be argued that the treatment of the patient with COPD with long-acting ß-agonists, anticholinergics, or slow-release theophylline may have affected the results of the present study. We cannot entirely rule out the possibility of treatment effects. However, bronchodilator medication was withheld 24 h prior to bronchoscopy, and PBECs were kept in culture for several days until they were confluent, which included repetitive changes of culture medium. Therefore, it seems unlikely that the differences seen in the present study between smokers with and without COPD are due to treatment differences.
In conclusion, we have demonstrated an increased TNF-- but not IFN--induced expression of the neutrophil chemoattractant CXC-chemokines IL-8 and GRO- at the transcriptional and protein levels in PBECs derived from patients with COPD compared to smokers without airway obstruction. In view of the fact that the expression of the TNF--receptor subtypes TNF-R55 and TNF-R75 was not different between groups, this points toward a COPD-specific activation of PBECs, which may contribute to chronic airway inflammation and progress in chronic airflow limitation. This might explain in part why only a small proportion of smokers acquire COPD.
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Footnotes |
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= growth-related oncogene-; ICAM = intercellular adhesion molecule; IFN = interferon; IL = interleukin; IP10 = interferon- inducible protein-10 kDa; mRNA = messenger RNA; PBEC = primary bronchial epithelial cell; PCR = polymerase chain reaction; TNF = tumor necrosis factor; TNF-R55 = p55 tumor necrosis factor receptor subtype; TNF-R75 = p75 tumor necrosis factor receptor subtype This study was supported by a grant from the Dr. Karl Wilder Stiftung, Berlin, Germany.
Received for publication February 28, 2003. Accepted for publication December 2, 2003.
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References |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996;153,530-534[Abstract]
and MCP-1 in sputum samples from patients with COPD. Thorax 2002;57,590-595 differentially regulate release of monocyte chemotactic protein-1 and interleukin-8 by human bronchial epithelial cells. Eur Cytokine Netw 1998;9,269-277[ISI][Medline]
and on cytokine production and phenotypic pattern of human bronchial epithelial cells. Int J Immunopharmacol 2000;22,573-585[CrossRef][ISI][Medline]
in normal human airways. Am Rev Respir Dis 1993;148,1677-1682[ISI][Medline]
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