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(Chest. 2005;128:3724-3734.)
© 2005 American College of Chest Physicians

Expression of CXC Chemokine Receptors 1 and 2 in Human Bronchial Epithelial Cells*

Laszlo Farkas, MD; Marie-Christine Hahn; Marc Schmoczer, MD; Nico Jentsch; Kirsten Krätzel; Michael Pfeifer, MD and Christian Schulz, MD

* From the Klinik und Poliklinik für Innere Medizin II (Drs. Farkas, Schmoczer, and Schulz, Ms. Hahn, Mr. Jentsch, and Ms. Krätzel), Klinikum der Universitaet Regensburg, Regensburg; and Klinik Donaustauf (Dr. Pfeifer), Donaustauf, Germany.

Correspondence to: Laszlo Farkas, MD, Klinik und Poliklinik für Innere Medizin II, Klinikum der Universitaet Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany; e-mail: laszlo.farkas{at}klinik.uni-regensburg.de


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Introduction: CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2) have been shown to play an important role in transepithelial migration of neutrophil granulocytes during inflammation in various tissues. This study investigated the regulation of gene expression and surface expression of CXCR1 and CXCR2 in a human bronchial epithelial cell line (BEAS-2B), as well as in primary bronchial epithelial cells (PBECs) from 10 COPD patients and 10 control subjects.

Methods and results: The transcription expression of CXCR1 and CXCR2 was quantitatively assessed by means of real-time polymerase chain reaction (PCR) under various inflammatory conditions. Flow cytometry was used to measure CXCR1 and CXCR2 surface expression. There was a low baseline expression of CXCR1 and CXCR2 in real-time PCR in PBECs from COPD patients and control subjects as well as in BEAS-2B cells, and no significant regulation occurred under various inflammatory conditions in PBECs and BEAS-2B cells. Furthermore, unstimulated surface expression of CXCR1 and CXCR2 on BEAS-2B cells was very low, and no significant regulation was detectable under time-dependent inflammatory stimulation up to 24 h.

Conclusion: Various inflammatory responses that are of potential relevance in COPD pathophysiology do not affect transcription regulation and surface expression of the interleukin-8 receptors CXCR1 and CXCR2 on human bronchial epithelial cells.

Key Words: BEAS-2B • bronchial epithelium • chemokine receptor • CXC chemokine receptor 1 • CXC chemokine receptor 2 • flow cytometry • real-time amplification


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COPD is a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with a bronchopulmonary inflammation. This inflammatory response is dominated by macrophages, T-lymphocytes (predominantly CD8+), and neutrophils that are found in various parts of the lung.1 The dissociation between a predominance of neutrophils in the airway lumen and a lack of increase in these cells in the subepithelium assessed by bronchial biopsies can be found in COPD patients.2 This observation may be due to an accelerated transepithelial migration of neutrophils from the circulation into the airway lumen, which, however, has not been investigated in detail. Studies3456 have shown that alveolar macrophages and bronchial epithelial cells can express high levels of different neutrophil chemoattractant cytokines, such as interleukin (IL)-8 and growth-related oncogene (GRO)-{alpha}, which could favor this process. This, however, should also result in increased numbers of neutrophils in the subepithelium in these patients, which is not the case. This points toward further local mechanisms that might regulate the migration of neutrophils to the site of inflammation.

Neutrophil chemoattractant chemokines such as IL-8 and GRO-{alpha} belong to the ELR+-CXC chemokines that exert their effects by interacting with the CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). CXCR1 binds selectively IL-8 and granulocyte chemotactic protein-2, whereas CXCR2 binds all CXC chemokines.78 Both receptors are expressed and regulated on neutrophils, which is in line with these observations. Studies910 using IL-8 receptor knockout mice with urinary tract infections indicate that transmigration of neutrophil granulocytes only occurs when CXCR1 and CXCR2 expression on neutrophils and epithelial cells also exists beneath a chemotactic gradient of CXC chemokines. The aim of the present study was to investigate whether or not CXCR1 and CXCR2 are expressed and regulated on bronchial epithelial cells under different inflammatory conditions leading to cell activation.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Patients referred to our clinic to undergo flexible bronchoscopy for various reasons were screened for inclusion in the present study. All patients gave written informed consent, and the study was approved by the local ethics committee. Selection of COPD patients was based on the definition and classification provided by the Global Initiative for Chronic Obstructive Lung Disease (GOLD).11 In brief, inclusion criteria included patients with stable airflow limitation, FEV1 < 70% of the predicted value, bronchodilator reversibility < 10% of predicted in FEV1 after 200 µg inhaled salbutamol, and a smoking history > 10 pack-years. None of the patients with COPD had a history of atopy or evidence of atopy based on skin-prick testing for common aeroallergens. Control subjects were lifelong nonsmokers with normal lung function and no history of airway disease. Subjects who had had a respiratory tract infection or exacerbation of the airway disease within the previous 8 weeks and those receiving systemic or topical corticosteroids during the previous 12 weeks were excluded from the study. Patient characteristics can be found in Table 1 . Patients underwent fiberoptic bronchoscopy after light sedation and prior inhalation of lidocaine for local anesthesia. Bronchial epithelium was obtained by gentle brushing of segmental and subsegmental bronchi under direct visual guidance by means of a protected brush. Samples were taken from the contralateral side of the lung in patients with suspected or proven bronchial malignancy to avoid potential contamination of the sample. Brushes were immediately placed in ice-cold bronchial epithelial growth medium (Promocell; Freiburg, Germany) and transported directly to the laboratory for further processing.


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  		Table 1.. Patient Characteristics*

 
Culture of Primary Bronchial Epithelial Cells
Brushes were vortexed vigorously, and the harvested cell suspension was filtered through a 200-µm filter (Millipore; Billerica, MA) to remove mucus and cellular debris, and then treated with 4.8 U/mL of neutral protease, grade 2 (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, and cell viability was determined by trypan blue exclusion. Cells were resuspended in serum-free bronchial epithelial cell growth medium (Promocell) 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 re-fed 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). A monoclonal antifibroblast antibody (FibAS02; Dianova; Hamburg, Germany) was used to exclude contamination by fibroblasts.12 The staining was performed according to Kunz-Schughart and coworkers13 as 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 primary bronchial epithelial cells (PBECs) were stimulated with 50 ng/mL of recombinant human tumor necrosis factor (TNF)-{alpha} or 200 U/mL recombinant human interferon (IFN)-{gamma} for 4 h along with unstimulated time controls. 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 have shown that the TNF-{alpha} and IFN-{gamma} concentrations chosen to stimulate the cells induced maximum responses with respect to IL-8 (TNF-{alpha}) or intercellular adhesion molecule-1 IFN-{gamma} messenger RNA expression. 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-{alpha} concentration was used.

Culture of the Bronchial Epithelial Cell Line BEAS-2B
BEAS-2B cells were originally established from healthy human bronchial epithelium and transformed by an adenovirus 12/SV40 hybrid virus.14 They have been shown to maintain typical epithelial morphology and many epithelial functional characteristics.141516 BEAS-2B cells were cultured in tissue flasks in complete medium consisting of RPMI 1640, supplemented with 10% fetal calf serum (FCS) and antibiotics (100 µg/mL streptomycin and 100 U/mL penicillin) in a humidified carbon dioxide incubator at 37°C. Epithelial cells were trypsinized once a week after becoming > 95% confluent. The cells were generally used between passage 5–20 to avoid the generation of variation.

Isolation of Neutrophil Granulocytes
Human neutrophils, which were used as positive control, were obtained from heparinized blood of a healthy volunteer and prepared by dextran sedimentation followed by gradient centrifugation on Ficoll for 40 min at 400g and lysis of residual erythrocytes in double-distilled water as described previously.17 The cells were washed in phosphate-buffered saline solution (PBS), and resuspended at 5 x 106/mL in RPMI 1640 supplemented with 10% FCS. Neutrophils were judged to be > 95% pure by morphologic criteria and > 98% viable (according to Giemsa staining and trypan blue exclusion test, respectively). The remaining cells were typically lymphocytes.

Preparation of Haemophilus influenzae Sonicate
Nontypeable H influenzae type B (American type culture cell 49247) were grown on culture plates for 48 h and then suspended in 5 mL of ice-cold PBS and gently shaken. The suspension was optimized for a density of 5 OD per milliliter, and bacteria were ruptured using ultrasound (two times 20 s for each sample) after incubation in distilled water for 30 min. The ruptured bacteria underwent sedimentation for 20 min at 10,000g, and the protein content in the supernatant sonicate was quantified using the Lowry method for further use in stimulatory experiments. Sterility of the sonicate was tested by incubation for 72 h on a blood agar culture plate at 37°C and 5% carbon dioxide.

Stimulation of the BEAS-2B cells
For stimulatory experiments, BEAS-2B cells were seeded at 2 x 105 in tissue flasks (25 mL) before stimulation. After 72 h, the medium was replaced by fresh complete medium. When the cells had reached > 90% confluency, the cells were washed twice with incomplete medium, adding thereafter incomplete medium containing the indicated stimuli. Neutrophils were seeded at 2 x 105 in tissue flasks (25 mL). For stimulation, cells were incubated at 37°C in RPMI 1640 medium containing 2% FCS and the following stimuli: TNF-{alpha}, 10 ng/mL; IFN-{gamma}, 100 IU/mL; lipopolysaccharide (LPS), 10 µg/mL; a cytokine mix containing TNF-{alpha}, 10 ng/mL; IFN-{gamma}, 100 IU/mL; and 10 µg/mL of LPS or H influenzae sonicate, 10 µg/mL. The cells were harvested at the indicated time points and used for further analyses.

RNA isolation
RNA was isolated using the High Pure RNA isolation kit (Roche). RNA quality and quantity were evaluated by ultraviolet spectrophotometry and agarose gel electrophoresis.

Real-Time Polymerase Chain Reaction and Real-Time Amplification
After deoxyribonuclease treatment, total complementary DNA was prepared by first-strand complementary DNA synthesis from 1 µg of total RNA according to standard protocols. Real-time polymerase chain reaction (PCR) detection of CXCR1, CXCR2, and ß-actin was carried out using the Taqman real-time amplification system using SDS 2.1 software (Applied Biosystems; Foster City, CA). The primers are listed in Table 2 . A fluorescent dye (SYBR Green; Qiagen; Valencia, CA) was used to detect PCR products. The result of real-time PCR was expressed as the threshold cycle. The threshold cycle represents the PCR cycle at which the reported fluorescence rises above a set baseline threshold when the DNA amplicon is replicating exponentially. Cytoplasmatic ß-actin was analyzed parallel to each PCR, and the resulting actin values were used as standards for presentation of CXCR1 and CXCR2 transcripts. A standard curve method (Applied Biosystems) was used to obtain relative quantification results.


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  		Table 2.. Accession Numbers and Oligonucleotide Sequences of the Primers Used for Real-time PCR

 
Measurement of Cell Activation
Cell activation according to the various stimuli was monitored by measuring IL-8 levels in the culture supernatant. IL-8 levels were assessed by using an IL-8 enzyme-linked immunosorbent assay kit.

Fluorescence Activated Cell Sorter Analysis
Cells were washed and 5 x 105 BEAS-2B or neutrophils cells were suspended in 100 µL of fluorescence activated cell sorter (FACS) buffer (PBS, 10% FCS) containing 2 µg of monoclonal antibody specific to CXCR1 (clone 42705.111) or CXCR2 (clone 48311.211) or an isotype antibody (IgG2a, clone 20102), for 40 min at 4°C. After washing using FACS buffer, the cells were incubated for 40 min at 4°C with 50 µL of FACS buffer containing fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG. The cells were washed twice and analyzed (FACScalibur using CellQuest software; Becton Dickinson; San José, CA).

Reagents
BEAS-2B cells and H influenzae type B were from the American type cell culture collection (ATCC; Manassas, VA). RPMI 1640 medium, antibiotics, FCS, PBS, and trypsin were from Invitrogen (Karlsruhe, Germany). Escherichia coli LPS serotype O55:B5, TNF-{alpha}, and IFN-{gamma} were from Sigma (Munich, Germany). PBEC culture medium was from Promocell, and RPMI 1640, FCS, and antibiotics were obtained from Invitrogen (Karlsruhe, Germany). Antibodies for FACS analysis (CXCR1, CXCR2, and IgG2a isotype control) were obtained from RnD (Minneapolis, MN). The IL-8 enzyme-linked immunosorbent assay kit was from Pierce-Endogen (Rockford, IL). The High Pure RNA isolation kit was from Roche (Mannheim, Germany). SYBR PCR master mix was from Qiagen. All other reagents (standard reagents) were obtained from Roche.

Statistical Analysis
Data are expressed as mean ± SD if not otherwise indicated. Baseline characteristics, relative amplification results, and FACS scan data were compared by Mann-Whitney rank analysis. Differences were considered to be significant at p < 0.05. Statistical analysis was performed using statistical software (Sigmastat 2.01; SPSS; Chicago, IL). Graphics were created using software (Sigmaplot 7.0; SPSS; Chicago, IL).


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subjects
Twenty patients (10 lifelong nonsmokers with normal pulmonary function and 10 subjects with COPD) were included in the study. All patients were referred for bronchoscopy for clinical reasons. Six patients underwent bronchoscopy for suspected peripheral or central carcinoma of the bronchus, and five patients were suspected of having pulmonary metastasis of non-lung malignancies. Seven patients were referred with unexplained or increased shortness of breath, one was suspected of having lymphoma, and one was suspected of having a pleural mesothelioma. The characteristics of both groups are shown in Table 1. The mean FEV1/FVC ratio was 51.8 ± 3.1% in the COPD group (p < 0.001) and was within normal ranges in the control group. Six patients were classified as COPD GOLD stage II, four patients were classified as COPD GOLD stage III, and one patient had COPD GOLD stage IV. All COPD patients had a smoking history of at least 20 pack-years. Bronchodilator medication in the COPD group consisted of long-acting, inhaled ß2-agonists and anticholinergics.

Messenger RNA Expression of CXCR1 and CXCR2 in Neutrophils
Neutrophils, which were used as a positive control, revealed a high amount of steady-state messenger RNA levels of CXCR1 and CXCR2. Quantitative Taqman measurements demonstrated a mean relative expression of CXCR1 and CXCR2 equal to 47.2 ± 28.2% and 51.9 ± 16.2% of ß-actin, respectively.

Messenger RNA Expression of CXCR1 and CXCR2 in BEAS-2B Cells
The unstimulated relative messenger RNA expression of both chemokine receptors was lower in BEAS-2B cells (0.012 ± 0.015% of ß-actin for CXCR1 and 0.11 ± 0.17% of ß-actin for CXCR2) and did not respond to various inflammatory stimuli (TNF-{alpha}, IFN-{gamma}, LPS, or H influenzae sonicate, as described above) using different time points (Tables 3, 4 ). We were able to show a strong cell activation under each of these conditions, measured by rising IL-8 release levels from the cells (Fig 1, 2 ).


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  		Table 3.. Stimulated Time-Dependent Relative Messenger RNA Expression Levels of CXCR1 in BEAS-2B Cells Compared to Unstimulated Messenger RNA Levels*

 

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  		Table 4.. Stimulated Time-Dependent Relative Messenger RNA Expression Levels of CXCR2 in BEAS-2B Cells Compared to Unstimulated Messenger RNA Levels*

 


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  		Figure 1.. Time-dependent IL-8 release by BEAS-2B cells normalized on 100,000 cells under stimulation using a mix of TNF-{alpha}, 10 ng/mL; IFN-{gamma}, 100 IU/mL; and LPS, 10 µg/mL ({square}) vs unstimulated control ({blacksquare}). The data are expressed as mean ± SD of six independent experiments, each done in triplicate.

 


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  		Figure 2.. Time-dependent IL-8 release by BEAS-2B cells normalized on 100,000 cells under stimulation using 10 µg/mL of H influenzae sonicate ({circ}) vs unstimulated control (•). The data are expressed as mean ± SD of six independent experiments, each done in triplicate.

 
Messenger RNA Expression of CXCR1 and CXCR2 in PBECs
We found a low baseline messenger RNA expression of CXCR1 and CXCR2 in both subjects with normal lung function (0.0020 ± 0.0014% of ß-actin for CXCR1 and 0.75 ± 0.63% of ß-actin for CXCR2) and those with COPD (0.0024 ± 0.0010% of ß-actin for CXCR1 and 0.65 ± 0.53% of ß-actin for CXCR2), which did not differ significantly between the two groups. There was no significant increase after stimulation with TNF-{alpha} or IFN-{gamma} as indicated above (Fig 3, 4 ). There were no significant differences in unstimulated or stimulated expression of CXCR1 and CXCR2 within each group or between the two groups.



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  		Figure 3.. Relative changes in messenger RNA expression of CXCR1 in PBECs from subjects with normal lung function (n = 10) and COPD patients (n = 10) after 4-h stimulation with either TNF-{alpha} (50 ng/mL) or IFN-{gamma} (200 IU/mL) vs unstimulated control. Each box plot represents the data from 10 subjects, each done in triplicate. Data are presented as mean and interquartile range. The vertical bars represent the fifth and 95th percentiles. Within the boxes, the bold horizontal bars mark the mean value and the lighter horizontal bars mark the median value. • indicates outliers. n.s. = not significant (significance was established at p < 0.05).

 


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  		Figure 4.. Relative changes in messenger RNA expression of CXCR2 in PBECs from subjects with normal lung function (n = 10) and COPD patients (n = 10) after 4-h stimulation with either TNF-{alpha} (50 ng/mL) or IFN-{gamma} (200 IU/mL) vs unstimulated control. Each box plot represents the data from 10 subjects, each done in triplicate. Data are presented as mean and interquartile range. The vertical bars represent the fifth and 95th percentiles. Within the boxes, the bold horizontal bars mark the mean value, and the lighter horizontal bars mark the median value. • indicates outliers. See Figure 3 for expansion of abbreviation (significance was established at p < 0.05).

 
Surface Expression of CXCR1 and CXCR2 in Neutrophils
In unstimulated neutrophils, which were used as a positive control, surface expression detected by FACS scan was 99.4 ± 0.47% of cells gated for CXCR1 and 97.8 ± 1.67% of cells gated for CXCR2. Data are expressed as mean ± SD from three independent experiments each done in duplicate. Representative FACS scan results can be seen in Figure 5 , top left, A, and Figure 6 , top left, A.



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  		Figure 5.. Flow cytometry analysis of surface expression of CXCR1 on unstimulated neutrophil granulocytes (top left, A) that were used as positive control. Also shown are representative surface expression data of CXCR1 for unstimulated BEAS-2B (top right, B), BEAS-2B cells stimulated for 24 h with a mix of TNF-{alpha}, 10 ng/mL; IFN-{gamma}, 100 IU/mL; and LPS, 10 µg/mL (bottom left, C), and BEAS-2B cells stimulated for 24 h with 10 µg/mL of H influenzae sonicate (bottom right, D) [thin line indicates isotype antibody; bold line indicates anti-CXCR1 antibody].

 


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  		Figure 6.. Flow cytometry analysis of surface expression of CXCR2 receptors on unstimulated neutrophil granulocytes (top left, A) that were used as positive control. Also shown are representative surface expression data of CXCR2 for unstimulated BEAS-2B (top right, B), BEAS-2B cells stimulated for 24 h with a mix of TNF-{alpha}, 10 ng/mL; IFN-{gamma}, 100 IU/mL; and LPS, 10 µg/mL (bottom left, C), and BEAS-2B cells stimulated for 24 h with 10 µg/mL of H influenzae sonicate (bottom right, D) [thin line indicates isotype antibody; bold line indicates anti-CXCR1 antibody].

 
Surface Expression of CXCR1 and CXCR2 in BEAS-2B Cells
FACS analysis revealed a low, if any, surface expression of CXCR1 and CXCR2 on BEAS-2B cells, since only 3.9 ± 3.6% and 2.5 ± 2.3% of cells gated stained positive for CXCR1 and CXCR2, respectively. Stimulation of BEAS-2B cells with either a cytokine mix containing TNF-{alpha} at 10 ng/mL, IFN-{gamma} at 100 IU/mL, and LPS at 10 µg/mL, or H influenzae sonicate at 10 µg/mL had no effect on CXCR1 and CXCR2 expression (Fig 5, 6). Previous results have shown that both conditions lead to a strong activation of BEAS-2B cells as assessed by IL-8 release (Fig 1, 2).


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study aims to investigate for the first time (to our knowledge) the transcription regulation and surface expression of CXCR1 and CXCR2 in human bronchial epithelial cells. The neutrophil chemoattractant chemokine IL-8 binds with high affinity to both receptors, whereas CXCR2 binds several other neutrophil chemoattractants, eg, GRO-{alpha} and epithelial cell-derived neutrophil-activating peptide 78.78 Previous cell culture and animal studies910 have shown that both receptors can facilitate neutrophil transmigration through epithelial cell layers. Therefore, we hypothesized that under inflammatory conditions, a bronchoepithelial up-regulation of both receptors occurs, thereby facilitating the recruitment of neutrophils to the airway lumen. If so, this could be a possible mechanism for the increased numbers of neutrophils seen in the airway lumen of COPD patients. However, using various and well-defined inflammatory stimuli, no regulation and rather low expression levels of both CXCR1 and CXCR2 were found in human bronchial epithelial cells.

We used BEAS-2B cells, a well-established human bronchial epithelial cell line, and PBECs from COPD patients and healthy control subjects as a cell culture model to investigate CXCR1 and CXCR2 expression on both the transcription and protein level. IFN-{gamma}, TNF-{alpha}, LPS, a cytokine mix, and a sonicate of nontypeable H influenzae were used to activate the bronchoepithelial cells. For all stimulating conditions used in the cell culture model, an association with COPD pathophysiology has previously been shown.1819202122

Even under different inflammatory stimulations that induced a strong activation of BEAS-2B cells as demonstrated by IL-8 release, no significant variation in messenger RNA or membrane expression of CXCR1 and CXCR2 was found. When we continued to investigate the transcription expression of CXCR1 and CXCR2 in human PBECs under stimulation with TNF-{alpha} or IFN-{gamma}, we could not detect a significant up-regulation in the PBECs from healthy donors or from patients with COPD under these stimulating conditions, although we have previously been able to show that there is a relevant increase in the release of IL-8 using these stimulating conditions.23 In contrast to the lack of up-regulation of these chemokine receptors in the respiratory epithelium as seen in the present study, Godaly et al9 showed a significant increase in the epithelial surface expression of CXCR1 and CXCR2 receptors in the A498 human kidney epithelial cells following an in vitro infection with E coli. Pretreatment of the infected epithelial cells with anti-CXCR1 antibodies significantly reduced the neutrophil transmigration in a Transwell assay across E coli-infected epithelial cell layers.9 According to these results, it is likely that the membrane expression of CXCR1 on bladder epithelial cells is essential for the transepithelial migration of neutrophils, which was confirmed by the same authors in a murine IL-8 receptor knockout animal model. In addition, Bäckhed et al24 demonstrated a several-fold up-regulation of CXCR1 and CXCR2 in human gastric cell line AGS by Helicobacter pylori stimulation. Furthermore, an increased messenger RNA expression of CXCR2 in psoriatic epidermis specimens compared to control has been shown.25 In opposition to these findings, Momma et al26 found that CXCR1 and CXCR2 were expressed at very low levels in human retinal pigment epithelial cells and were not affected by human cytomegalovirus infection. These controversial results may indicate that epithelial cells from different organs might show different expression levels of the chemokine receptors, and epithelium in different tissues can react differently to various inflammatory stimuli.

The major portion of infiltrating leukocytes in exacerbations and in the severe disease state of COPD has been shown to be formed by neutrophils.2728 However, the high counts of neutrophils in lavage fluids from subjects with chronic bronchitis do not correspond to their numbers found in the bronchial subepithelium.4 One possible explanation for this discrepancy is the rapid transepithelial migration of neutrophils to the airway lumen, such that accumulation of these cells is not apparent in tissue analysis. Despite the fact that the mechanism of neutrophil accumulation in the bronchial lumen in COPD patients is not completely understood, there is evidence that cytokines may play a role: the concentration of IL-10, an anti-inflammatory cytokine, is decreased in sputum of smokers with COPD,29 whereas the amount of IL-8 and TNF-{alpha} is increased.3 Furthermore, an up-regulation of E-selectin and intercellular adhesion molecule-1 on subepithelial vessels and on bronchial epithelium of subjects with COPD has been demonstrated, suggesting a mechanism of leukocyte recruitment from the circulation and for their migration into the airway lumen through the epithelium.30 In addition, Qiu and coworkers31 showed an increased expression of the chemokine receptors CXCR1 and CXCR2 in the subepithelium and an up-regulation of CXCR2 in the epithelium of bronchial biopsy specimens of severely exacerbated COPD patients using in situ hybridization techniques.

We certainly cannot rule out that not all factors that might play a role in COPD and acute exacerbation, eg, persisting virus infections, different pathogenic bacteria, bacterial colonization, genetic susceptibility, or other exogenous factors, were included in the cell culture model used. The underlying reason for COPD exacerbation was not shown in detail by Qiu and coworkers,31 so we do not know whether the majority of these patients suffered from an exacerbation due to Gram-negative bacteria or other microbial agents. Furthermore, the cell culture model used did not include other types of inflammatory cells that play an important role in the pathogenesis of COPD: macrophages, neutrophils, or lymphocytes.

Although no induction of CXCR1 or CXCR2 expression in the bronchial epithelium could be shown in the present study, some limitations should be taken into consideration. The bronchial epithelial cell line BEAS-2B is a cell line established by transformation of human bronchial epithelium by infection with adenovirus 12/SV40 hybrid virus and, despite the fact that they still possess some characteristics of bronchial epithelial cells, eg, the ability to undergo squamous differentiation,15 it cannot be ruled out that the respective genes (CXCR1 and CXCR2) might be mutated, such that the ability to up-regulate this receptor might have been lost. It should be mentioned in this context, however, that BEAS-2B cells have been used as representative cells for the bronchial epithelium many times in a variety of different investigations, including analysis of Toll-like receptors,32 analysis of eotaxin expression,33 and analysis of the expression of ß2-adrenergic receptors.34

Therefore, we added additional data comparing the CXCR1 and CXCR2 messenger RNA expression in primary bronchial cells from patients with COPD and control patients without airway obstruction. We found a similar low baseline expression in both groups as well as no induction of the messenger RNA expression of the two genes after stimulation with TNF-{alpha} and IFN-{gamma}. Nevertheless, the initial hypothesis that inflammatory conditions found in COPD facilitate bronchoepithelial expression of IL-8-receptors CXCR1 and CXCR2 is not supported.

In conclusion, there is little, if any, bronchoepithelial cell surface expression of the IL-8 receptors CXCR1 and CXR2, and it does not respond to various well-defined proinflammatory conditions that are of potential relevance in COPD pathophysiology. The mechanisms responsible for bronchoepithelial up-regulation of mainly CXCR2 in patients with severe COPD exacerbation, as has been recently shown by Qiu et al,31 remain unclear and should be the subject of further studies.


   Footnotes
 
Abbreviations: CXCR1 = CXC chemokine receptor 1; CXCR2 = CXC chemokine receptor 2; FACS = fluorescence activated cell sorter; FCS = fetal calf serum; GOLD = Global Initiative for Chronic Obstructive Lung Disease; GRO = growth-related oncogene; IFN = interferon; IL = interleukin; LPS = lipopolysaccharide; PBEC = primary bronchial epithelial cell; PBS = phosphate-buffered saline solution; PCR = polymerase chain reaction; TNF = tumor necrosis factor

The work was performed at the Klinik und Poliklinik für Innere Medizin II, Klinikum der Universität Regensburg, Regensburg, Germany.

This study was supported by a grant SCHU 1606/2-1 from the Deutsche Forschungsgemeinschaft.

Received for publication August 31, 2004. Accepted for publication June 14, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Saetta, M, Turato, G, Maestrelli, P, et al (2001) Cellular and structural bases of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 163,1304-1309[Free Full Text]
  2. Saetta, M, Di Stefano, A, Maestrelli, P, et al Activated T-lymphocytes and macrophages in bronchial mucosa of subjects with chronic bronchitis. Am Rev Respir Dis 1993;147,301-306[ISI][Medline]
  3. Keatings, VM, Collins, PD, Scott, DM, et al Differences in interleukin-8 and tumor necrosis factor-{alpha} in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996;153,530-534[Abstract]
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