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Published ahead of print on March 25, 2004, doi:10.1165/rcmb.2004-0078OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 241-245, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0078OC

Expression of Functional Toll-Like Receptor-2 and -4 on Alveolar Epithelial Cells

Lynne Armstrong, Andrew R. L. Medford, Kay M. Uppington, John Robertson, Ian R. Witherden, Teresa D. Tetley and Ann B. Millar

Lung Research Group, and Academic Renal Unit, Department of Clinical Science North Bristol, University of Bristol, Southmead Hospital, Westbury on Trym, Bristol; Department of Respiratory Medicine, Bristol Royal Infirmary, Bristol; and Respiratory Medicine, National Heart and Lung Institute, Imperial College School of Medicine, Charing Cross Hospital, London, United Kingdom

Address correspondence to: Dr. Lynne Armstrong, Lung Research Group, University of Bristol, Department of Clinical Science North Bristol, Southmead Hospital, Westbury on Trym, Bristol BS10 5NB, UK. E-mail: lynne.armstrong{at}bris.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The recognition of potentially harmful microorganisms involves the specific recognition of pathogen-associated molecular patterns (PAMPs) and the family of Toll-like receptors (TLRs) is known to play a central role in this process. TLR-4 is the major recognition receptor for lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, whereas TLR-2 responds to bacterial products from gram-positive organisms. Although resident alveolar macrophages are the first line of defense against microbial attack, it is now understood that the alveolar epithelium also plays a pivotal role in the innate immunity of the lung. The purpose of the current study was to determine whether human primary type II alveolar epithelial cells (ATII) express functional TLR-2 and TLR-4 and how they may be regulated by inflammatory mediators. We have used reverse transcriptase–polymerase chain reaction and flow cytometry to determine basal and inducible expression on ATII. We have used highly purified preparations of the gram-positive bacterial product lipoteichoic acid (LTA) and LPS to look at the functional consequences of TLR-2 and TLR-4 ligation, respectively, in terms of interleukin-8 release. We have shown that human primary ATII cells express mRNA and protein for both TLR-2 and TLR-4, which can be modulated by incubation with LPS and tumor necrosis factor. Furthermore, we have demonstrated that these receptors are functional. This suggests that ATII have the potential to contribute significantly to the host defense of the human alveolus against bacteria.

Abbreviations: aquaporin, AQP • alveolar type II epithelial cells, ATII • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • Hanks' balanced salt solution, HBSS • interferon, IFN • immunoglobulin, IgG • interleukin, IL • lipoteichoic acid, LTA • lipopolysaccharide, LPS • pathogen-associated molecular patterns, PAMPs • phosphate-buffered saline, PBS • phycoerythrin, Pe • repurified lipopolysaccharide, rLPS • reverse transcriptase polymerase chain reaction, RT-PCR • surfactant protein C, SP-C • Toll-like receptor, TLR • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The appropriate recognition of potentially harmful microorganisms is particularly pertinent to the lower respiratory tract, because the lung is a primary site for the introduction and deposition of pathogenic microorganisms and a large quantity of airborne particles. Because much of the particulate matter poses no significant threat to the host, mechanisms are in place to permit activation of the immune response only when there is a genuine risk of infection. This involves the specific recognition of pathogen-associated molecular patterns (PAMPs) and the family of Toll-like receptors (TLRs) is known to play a central role in this process. These type I transmembrane proteins share significant similarities with the interleukin (IL)-1 receptor (IL-1R) and all contain the highly homologous toll/IL-1R cytoplasmic domain. The homology present in the toll/IL-1R is contrasted by significant variation in the leucine-rich repeats of the extra-cellular domains, which permits discrimination of a variety of PAMPs, including lipopolysaccharide (LPS), bacterial lipoprotein, peptidoglycans, and lipoteichoic acid (LTA) (1). Of the ten TLRs so far identified, TLR-2 and TLR-4 have been the most extensively studied. The identification of a functional mutation in the tlr4 gene, responsible for LPS hyporesponsiveness in C3H/HeJ mice (2), first suggested that TLR-4 was the major recognition receptor for the gram-negative bacterial component LPS (3), and subsequent studies have now confirmed this. By contrast, TLR-2 responds to bacterial lipoproteins from gram-positive organisms (4), as well as yeasts (5) and mycobacteria (6).

Although resident alveolar macrophages are the first line of defense against microbial attack, and are known to express TLR-2 and TLR-4 (7, 8), it has become increasingly apparent that the lung epithelium also plays a pivotal role in innate immunity. Indeed A549 carcinoma cells, which are a type II–like line, have been shown to express functional TLR-4, predominantly in the intracellular compartment (9). In terms of primary cells, human alveolar type II epithelial cells (ATII) have recently been described to express mRNA and protein for TLR-2 (8), although its functional responses were not explored.

The purpose of the current study was to determine whether human primary ATII express functional TLR-2 and TLR-4 and whether their expression can be modulated in vitro in response to inflammatory mediators. We have used reverse transcriptase (RT)-polymerase chain reaction (PCR) and flow cytometry to determine basal expression on the ATII cells. We have also looked at induction of TLR-2 and TLR-4 expression by tumor necrosis factor (TNF)-{alpha}, which is implicated in the pulmonary inflammatory response and has been shown previously to upregulate TLR-2 and TLR-4 expression on renal epithelial cells (10). We have also used highly purified preparations of LTA and LPS to look at the functional consequences (TLR-2 and TLR-4 expression, and IL-8 release) of TLR-2 and TLR-4 ligation, respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Macroscopically normal lung tissue sections (~ 15 x 5 x 5 cm) were donated by eight patients undergoing lobar resection for malignancy (three female, five male). The median age was 67 yr. Seven donors were ex-smokers and one donor had no smoking history. Ethical approval was obtained from North Bristol NHS and United Bristol Healthcare Trusts.

Isolation and Purification of ATII
ATII cells were purified according to the method of Witherden and colleagues (11). Sections were perfused with 0.9% saline to remove alveolar macrophages and digested with 0.25% trypsin (Sigma, Poole, UK). The digested tissue was chopped into pieces ~ 1–2 mm 2 in size, in the presence of newborn calf serum (Invitrogen, Paisley, UK). DNase I was then added to the suspension at 250 µg/ml in 7 ml Hanks' balanced salt solution (HBSS). The suspension was shaken vigorously for 5 min before filtering through a large mesh ({cong} 500 µm), followed by a 40-µm mesh (Fahrenheit, Milton Keynes, UK). The filtered suspension was centrifuged at 300 x g for 10 min at 4°C and the pellet resuspended in 15 ml HBSS/15 ml DCCM (React Scientific, Troon, UK) containing 100 µg/ml DNase I. The suspension was incubated in a T-75 flask for 2 h at 37°C to allow any residual contaminating macrophages to adhere. The supernatant containing nonadherent cells was then centrifuged at 300 x g for 10 min at 4°C and the pellet resuspended in complete medium (DCCM-1, 10% newborn calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin) and incubated for 2 h at 37°C to allow contaminating fibroblasts to adhere. The nonadherent cells were centrifuged at 300 x g for 10 min at 4°C and resuspended at 1 x 106 ATII cells/ml in complete media and put into 60-mm dishes (Greiner Cell Star, Stonehouse, UK) precoated with Vitrogen 100 (Cohesion Technologies, Palo Alto, CA). The ATII cells were subsequently adhered at 37°C for 24 h. The medium and any remaining contaminating cells were then removed and fresh complete medium added. The cells were then incubated at 37°C, and after 16 h the medium was removed and the cells washed with HBSS. Fresh complete medium was then added and the cells incubated for a further 24 h to establish confluent monolayers with type II morphology (ATII). ATII cell phenotype was confirmed by positive staining for alkaline phosphatase and mRNA transcripts for surfactant protein-C (SP-C) and aquaporin (AQP)3. Morphologic characteristics were confirmed by electron microscopy. ATI cell phenotype was excluded by negative staining for aquaporin 5.

RNA Extraction
Total cellular RNA was extracted from 5 x 105 ATII after culture with stimuli stated below. Cells were washed in sterile PBS and cellular RNA extracted using RNAbee (AMS Biotechnology, Abingdon, UK) according to manufacturer's instructions. Cellular RNA concentration was measured using a GeneQuant II (Amersham Biosciences, Little Chalfont, UK).

Semiquantitative RT-PCR
RT-PCR was performed in a 20-µl one-step reaction using Reverse-IT RTase blend (ABgene, Epsom, UK) with 200 ng of total RNA as a template. RT was performed at 47°C for 30 min followed by 94°C for 2 min to inactivate the RT enzyme. For TLR-2 and TLR-4 amplification, PCR was performed with 30 cycles of 94°C for 20 s, 54°C for 45 s, and 72°C for 1 min. For glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene, PCR was performed with 17 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s. For SP-C and AQP3 amplification the conditions were 54°C for 30 cycles and 58°C for 32 cycles, respectively (for specific primer sequences see Table 1). Products were electrophoresed through a 1.5% agarose gel and visualized using ethidium bromide staining. mRNA quantity was determined by digital imaging densitometry (Geldoc 1000 with Quantity One software; Bio-Rad, Hemel Hempstead, UK).


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  		TABLE 1. Primer sequences for RT-PCR

 
Flow Cytometry
Flow cytometry was performed on 1 x 105 ATII that had been removed from tissue culture plates by nonenzymatic digestion (Cell dissociation solution; Sigma). Cells were incubated on ice for 2 min with 10 µg of human IgG before a 30-min incubation with 5 µl of monoclonal anti-TLR-2 antibody, anti-TLR-4 antibody (clone TL2.1 and HTA125, respectively; Cambridge Bioscience, Cambridge, UK), or mouse IgG1 control (Dako, Ely, UK). The primary antibodies were washed off in PBS/0.5% bovine serum albumin, 0.1% azide and the cells incubated for a further 30 min on ice with 2.5 µl of rabbit anti-mouse IgG1-pe (Dako). The secondary antibodies were washed off in PBS/0.5% bovine serum albumin, 0.1% azide and labeled cells were acquired on an EpicsXL flow cytometer (Beckman Coulter, High Wycombe, UK). TLR-2 and TLR-4 expression was analyzed using Expo 32 software (Beckman-Coulter).

Preparation of Purified LPS and LTA
The LTA was a kind gift of Dr. Siegfried Morath, University of Konstanz. The preparation, from Staphylococcus aureus, had been repurified by phenolic extraction to remove any residual endotoxin activity and therefore does not stimulate TLR-4 signaling (12, 13). LPS from Escherichia coli (serotype 0001:B4; Sigma) was repurified by phenolic extraction according to the method of Hirschfeld and coworkers (14), which has been shown to remove any TLR-2 stimulatory activity.

IL-8 Enzyme-Linked Immunosorbent Assay
ATII cells were initially cultured for 48 h in purified monolayers. Following this, the medium was replaced with fresh medium + 10% fetal bovine serum for a further 20 h with or without repurified LPS (rLPS) (10 µg/ml), highly purified LTA (1 µg/ml), or TNF-{alpha} (10 ng/ml). Supernatants were stored at –80°C until analysis. IL-8 levels were determined in diluted supernatants using the Pelikine IL-8 kit (sensitivity, 1–3 pg/ml; Mast Diagnostics, Bootle, UK), according to manufacturer's instructions.

Statistical Analysis
The mRNA and flow cytometry data were normally distributed as determined by the Ryan Joiner normality test. Comparisons between multiple groups were performed using one-way ANOVA, with Tukey's multiple comparison test (mct) to compare individual group differences (GraphPad Prism). A P value of < 0.05 was regarded as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Assessment of ATII Phenotype
Morphologic characteristics were confirmed by electron microscopy showing the existence of lamellar bodies and tight junctions in keeping with ATII phenotype. The cells were also positive for alkaline phosphatase expression. To confirm that the cells purified from the lung were indeed of the ATII phenotype, we also performed RT-PCR on RNA lysates. The presence of mRNA transcripts for SP-C and AQP3 confirmed type II characteristics, whereas the presence of the ATI cell phenotype in the cultures was excluded by negative expression of AQP5 (Figure 1A).



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  		Figure 1. (A) Aquaporin and SP-C mRNA expression in ATII cultures derived from three separate lung isolations. (B) Basal levels of TLR mRNA expression in ATII cultures derived from five separate lung isolations.

 
Human Primary ATII Express TLR-2 and TLR-4
We identified transcripts for TLR-2 and TLR-4 mRNA in total RNA lysates from ATII after 2 d in culture (Figure 1B). The mean expression of TLR-2 at baseline was 1.70 ± 0.20 as a ratio of GAPDH expression. TLR-4 expression was significantly lower than TLR-2 expression in the same samples at 0.43 ± 0.08 relative to GAPDH (P = 0.045) (Figure 2). We confirmed the existence of protein for both TLR-2 and TLR-4 by flow cytometry using specific antibodies. As a ratio of isotypic control antibody, TLR-2 expression was 0.91 ± 0.10, compared with 1.42 ± 0.15 for TLR-4 (Figure 3). This suggests that, in contrast to TLR-4, TLR-2 protein levels are not detectable on the surface of unstimulated ATII.



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  		Figure 2. (A) TLR-2 mRNA expression induced by rLPS in ATII cells from eight separate experiments. P < 0.0001, one-way ANOVA; ***P < 0.005 relative to medium alone (Tukey's mct). (B) TLR-4 mRNA expression induced by rLPS in ATII cells from eight separate experiments. P < 0.05, one-way ANOVA; *P < 0.05 relative to medium alone.

 


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  		Figure 3. (A) TLR-2 surface expression induced by TNF-{alpha}, determined by flow cytometry from 8 separate experiments. P < 0.05, one-way ANOVA; *P < 0.05 relative to medium alone (Tukey's mct). (B) TLR-4 surface expression induced by rLPS, determined by flow cytometry from eight separate experiments. P = 0.19, one-way ANOVA; **P < 0.01 relative to medium alone (Tukey's mct). (C) Representative basal TLR-4 surface staining (light gray) relative to isotype control (white). (D) Representative TLR-2 surface staining in response to rLPS (black) relative to basal levels (white).

 
Expression of TLR-2 and TLR-4 mRNA and Surface Protein can be Modified by Inflammatory Mediators
Culture for 2 h with rLPS led to a 2.2-fold increase in the expression of TLR-2 mRNA compared with medium alone (3.78 ± 0.17 versus 1.71 ± 0.20, P = 0.0001) (Figure 2A). Similarly, there was a 2.7-fold increase in the expression of TLR-4 mRNA in response to rLPS (1.18 ± 0.27 versus 0.43 ± 0.08, P = 0.02) (Figure 2B). Culture with TNF-{alpha} had no measurable effect on the expression of either TLR-2 or TLR-4. 20 h cultures of ATII showed no significant increase in TLR-2 surface expression after culture with rLPS (1.19 ± 0.18 versus 0.91 ± 0.10), and LTA also had no significant effect on TLR-2 surface levels. Interestingly, TNF-{alpha} increased TLR-2 surface expression 1.8-fold (1.63 ± 0.13, P < 0.05) (Figure 3A). By contrast, TLR-4 surface expression was significantly upregulated after culture with rLPS (1.89 ± 0.18 versus 1.42 ± 0.15, P < 0.01), whereas LTA and TNF had no effect on surface expression of TLR-4 protein (Figure 3B).

TLR-2 and TLR-4 Generate a Functional Response in Human Primary ATII Cells
We cultured the cells with rLPS and LTA to determine whether TLRs on ATII play a functional role in the inflammatory response to bacterial products. Using TNF-{alpha} stimulation as a positive control, we determined expression of IL-8 in our ATII culture supernatants of 4,135 ± 467 pg/ml (P < 0.01 relative to a medium alone value of 1,547 ± 88 pg/ml), which is in accordance with other studies (15) (Figure 4). We detected 2,854 ± 306 pg/ml IL-8 in response to 10 µg/ml rLPS (P < 0.01 relative to medium alone) and 2,097 ± 118 pg/ml IL-8 in response to 1 µg/ml LTA (P < 0.001 relative to medium alone). This suggests that human ATII are able to generate an inflammatory response via TLRs after ligation.



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  		Figure 4. IL-8 production induced by rLPS, LTA, and TNF-{alpha} in ATII cells from eight separate experiments. P < 0.0001, one-way ANOVA; **P < 0.01, ***P < 0.005 relative to medium alone (Tukey's mct).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study has demonstrated for the first time the existence of functionally active TLR-2 and TLR-4 on human primary ATII cells. These cells have recently been suggested to play an important role in the innate immunity of the human lung, and their ability to recognize PAMPs has been highlighted by a recent study that localized TLR-2 mRNA and protein to ATII by in situ hybridization and immunohistochemistry on human lung sections (8). There has been no equivalent study looking at the expression of TLR-4 on these cells, although TLR-4 expression has been demonstrated in the type II–like epithelial cell line A549 and in bronchial epithelium (9, 16). In a recent study by Guillot and coworkers, they were unable to localize TLR-4 to the surface of A549 cells or the bronchial epithelial cell line BEAS-2B; instead, they described a perinuclear location in association with the Golgi apparatus. However, they were able to show that intracellular TLR-4 was functionally responsive to LPS, possibly through internalization of the ligand, as has previously been described for intestinal epithelial cells (17). By contrast, we have been able to demonstrate the presence of TLR-4 on the cell surface by flow cytometry, although at a lower level than we have observed on alveolar macrophages or monocytes (unpublished observations). A549 cells, which are an adenocarcinoma line originally isolated in 1972 from a single parent cell (18), are known to differ from primary ATII cells in many respects, including IL-8 and nitric oxide production (15, 19). Many generations later they often poorly reflect the phenotype of the parent primary cell, which may explain the discrepancy with our data. Intestinal epithelial cells also do not appear to express surface TLR-4, which is possibly a modification to prevent inappropriate activation by gram-negative commensal gut organisms (20). By contrast, epithelial cells of the human bladder, which occupy a sterile environment, similar to that found in the alveolus, are known to express a significant level of functional TLR-4 on the cell surface (21). Although the upper airways of the lung are often exposed to large amounts of particulate matter, much of it containing traces of LPS, the alveolar compartment is predominantly sterile. Therefore it may be more appropriate for ATII to have TLR-4 expression more comparable to bladder rather than intestinal epithelium. Although we have successfully demonstrated functional responses to LPS and LTA, these responses depend on higher concentrations and are of a lesser magnitude to those reported in monocytes and macrophages. This could be explained by the observation in our laboratory that TLR-2 and TLR-4 surface expression on ATII is ~ 5-fold lower in both cases than that detected on alveolar macrophages (unpublished observations). Another potential explanation is the lack of CD14, which is a co-receptor for LPS signaling, on the surface of lung epithelial cells (16). Indeed, epithelial cells only respond to LPS when there is a source of exogenous sCD14, such as fetal bovine serum (16). It is also possible that other TLR downstream signaling components are implicated in this altered response, and this would warrant further investigation.

Although this study is the first to describe TLR-2 function on human primary alveolar epithelial cells, our data are supported by a previous study by Hertz and coworkers (22), who investigated TLR-2 expression and function on human primary bronchial epithelial cells. Using immunohistochemistry, they detected TLR-2 in situ on these cells, which was at a lower level than that observed in monocytes. They also demonstrated that lipoprotein was able to induce IL-8 release by bronchial epithelial cells and reduce bacterial growth in co-culture by a TLR-2–dependent mechanism. In contrast, TLR-2 has also been explored in intestinal epithelial cell lines, and as seen with TLR-4, expression and responses to LTA are diminished (23). Oral epithelial cells have also been shown to express low levels of TLR-2, which can be increased after priming with interferon (IFN)-{gamma} (24).

We have explored the regulation of TLR-2 and TLR-4 mRNA and protein in ATII and have been able to demonstrate that the expression of TLR-4 protein is moderately elevated on ATII when compared with the equivalent TLR-2 expression, whereas the reverse is true for mRNA. Although this could be a limitation of the flow cytometry detection, it could suggest that the expression of TLR-4 protein may be under tighter transcriptional regulation. Our mRNA studies have demonstrated that both TLR-2 and TLR-4 mRNA are LPS-inducible, whereas TNF-{alpha} had no effect. We chose to look at responses to TNF-{alpha}, because this cytokine is implicated in a number of inflammatory lung conditions and a previous study had demonstrated a TNF-dependent increase in TLR-2 and TLR-4 mRNA, albeit in murine renal epithelial cells (10). Our inability to detect a TNF-{alpha} response at the mRNA level may reflect species or cell-type specificity for this pathway. In terms of translation to protein, TLR-4 surface expression reflected the mRNA findings, but TLR-2 surface expression was lower, unaffected by LPS, and upregulated by TNF-{alpha}. These data suggest that TLR-4 protein levels may be determined at the transcriptional level in ATII, but that post-transcriptional mechanisms may be important for TLR-2 regulation. Studies of myeloid cells have demonstrated that the human form of TLR-2 is not inducible by LPS or TNF-{alpha}, although the murine tlr2 is LPS-responsive (25). One explanation for this species difference is that murine tlr2 promoter has only 10% homology with the human promoter, compared with 70% homology between the human and mouse TLR-4 promoter. Murine tlr2 contains binding sites for the transcription factors ets and SP-1, which are known to be inducible by LPS (26). Studies of TLR-4 regulation in mouse macrophages have shown that it is regulated both transcriptionally and post-transcriptionally. LPS can affect mRNA stability, leading to a decrease in TLR-4 mRNA expression (7). However, this is counterbalanced by an increase in the transcription rate of TLR-4 in response to LPS. There has been little research into the regulation of TLRs on nonmyeloid cells. However, it is known that there are tissue-restricted transcription factors in myeloid cells (27) (PU.1 and IFN consensus-sequence binding protein), which suggests that conclusions about epithelial regulation of TLR-2 and TLR-4 cannot easily be drawn from these studies. TLR-4 mRNA and protein expression has been shown to be unresponsive to LPS in the BEAS-2B bronchial epithelial cell line (9), but because bronchial epithelium would normally be exposed to larger amounts of endotoxin than alveolar epithelium it may be expected that their TLR-4 expression may be lower in comparison. In addition, TLR-2 and TLR-4 mRNA have been shown to be upregulated by IFN-{gamma} in oral epithelial cells (24) suggesting that interferon response elements, rather than nuclear factor-{kappa}B binding may be important in the initiation of transcription in epithelium.

There has been much debate about the specificity of TLR-2 and TLR-4 for the recognition of gram-positive and gram-negative PAMPs, and this is in part due to the impurities present in the commercial preparations (28). In this study, we chose to use highly purified LTA and LPS, which have been demonstrated to be highly specific ligands for TLR-2 and TLR-4, respectively (14, 29). We found that both ligands were able to induce IL-8 release from ATII cells, suggesting functional activity for both TLR-4 and TLR-2. The response to LPS was greater than that seen for LTA, and this may reflect the lower TLR-2 protein expression detected on the surface of ATII cells. There is now increasing evidence from studies of intestinal epithelial cells and A549 cells that the TLR-4 is located intracellularly and that LPS needs to be internalized for responses to occur (9, 17, 30). Although we have not excluded this as a possible mechanism for LPS responses in ATII, the existence of TLR-4 on the cell surface of these cells supports the formation of a cell surface complex for the initiation of LPS signaling.

In summary, we have demonstrated the existence of TLR-2 and TLR-4 mRNA and protein in human primary ATII, which can be regulated differentially by bacterial products and inflammatory mediators. This suggests that ATII play an important role in the innate immunity of the alveolus, with potential implications for our understanding of the pulmonary inflammatory response.


   Acknowledgments
 
The authors acknowledge the cardiothoracic team at the Bristol Royal Infirmary for their assistance in obtaining human lung tissue. They are also grateful to Dr. Siegfried Morath, University of Konstanz, for supplying the highly purified LTA. They also acknowledge the Action Medical Research (L.A.) and the British Lung Foundation (K.M.U.) for their financial support of this work.


   Footnotes
 
Conflict of Interest Statement: L.A. has no declared conflicts of interest; A.R.L.M. has no declared conflicts of interest; K.M U. has no declared conflicts of interest; J.R. has no declared conflicts of interest; I.R.W. has no declared conflicts of interest; T.D.T. has no declared conflicts of interest; and A.B.M. has no declared conflicts of interest.

Received in original form March 1, 2004

Received in final form March 24, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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