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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 666-677, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4820

Gene Expression and Immunolocalization of 15-Lipoxygenase Isozymes in the Airway Mucosa of Smokers with Chronic Bronchitis

Jie Zhu, Iain Kilty, Helen Granger, Elizabeth Gamble, Yu Sheng Qiu, Keith Hattotuwa, Will Elston, Wai L. Liu, Alessandro Oliva, Romain A. Pauwels, Johan C. Kips, Virginia De Rose, Neil Barnes, Michael Yeadon, Stephen Jenkinson and Peter K. Jeffery

Lung Pathology, Department of Gene Therapy, Imperial College at the Royal Brompton Hospital and Department of Respiratory Medicine, London; London Chest Hospital, London, United Kingdom; Pfizer Global Research & Development, Sandwich, Kent, United Kingdom; Clinical and Biological Sciences, University of Torino, Torino, Italy; and Pathology, University Hospital, Belgium

Address correspondence to: Professor P. K. Jeffery, Lung Pathology, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. E-mail: p.jeffery{at}ic.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
15-lipoxygenase (15-LO) has been implicated in the inflammation of chronic bronchitis (CB), but it is unclear which of its isoforms, 15-LOa or 15-LOb, is primarily involved. To detect 15-LO gene (mRNA) and protein expression, we have applied in situ hybridization (ISH) and immunohistochemistry (IHC), respectively, to bronchial biopsies obtained from 7 healthy nonsmokers (HNS), 5 healthy smokers (HS), and 8 smokers with CB, and additionally include the airways of lungs resected from 11 asymptomatic smokers (AS) and 11 smokers with CB. Compared with HNS, biopsies in CB demonstrated increased numbers of 15-LOa mRNA+ cells (median: HNS = 31.3/mm2 versus CB = 84.9/mm2, P < 0.01) and protein+ cells (HNS = 2.9/mm2 versus CB = 32.1/mm2, P < 0.01). The HS group also showed a significant increase in protein+ cells (HNS = 2.9/mm2 versus HS = 14/mm2, P < 0.05). In the resected airways, 15-LOa protein+ cells in the submucosal glands of the CB group were more numerous than in the AS group (AS = 33/mm2 versus CB = 208/mm2; P < 0.001). 15-LOa mRNA+ and protein+ cells consistently outnumbered 15-LOb by ~ 7- and 5-fold, respectively (P < 0.01). Quantitative reverse transcriptase polymerase chain reaction of complementary biopsies confirmed the increased levels of 15-LOa in CB compared with that in either HNS or HS (P < 0.05). There was no difference between the subject groups with respect to 15-LOb expression. The numbers of cells expressing mRNA for 15-LOa in CB showed a positive association with those expressing interleukin (IL)-4 mRNA (r = 0.80; P < 0.01). We conclude that the upregulation of 15-LO activity in the airways of HS and of smokers with CB primarily involves the 15-LOa isoform: the functional consequences of its association the upregulation of IL-4 in chronic bronchitis requires further study.

Abbreviations: arachidonic acid, AA • asymptomatic smokers, AS • chronic bronchitis, CB • digoxygenin, Dig • hematoxylin and eosin, H&E • hydroxyeicosatetraenoic acid, HETE • healthy nonsmokers, HNS • healthy smokers, HS • immunohistochemistry, IHC • in situ hybridization, ISH • polymorphonuclear neutrophils, PMN • reverse transcriptase–polymerase chain reaction, RT-PCR • Tris-buffered saline, TBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic bronchitis (CB) is a chronic inflammatory condition of the airways characterized by cough and sputum (1). 15-lipoxygenase (15-LO) is a highly regulated lipid-peroxidating enzyme that catalyses the oxygenation of arachidonic acid, initiating the formation of a series of mediators which include the mono-, di-, and tri-hydroxyeicosatetraenoic acids (HETEs) and linoleic acid (2). Increased expression of 15-LO and its metabolites has been shown to have pro- as well as anti-inflammatory activity (3–5); these could be important in the airway inflammation of smokers with CB and, perhaps, also asthma (6–10). Nothing is known of the localization and gene/protein expression of 15-LO in healthy smokers, and little is known in smokers with CB. By comparison with healthy non-smokers, 15-HETE, a major arachidonic acid (AA) metabolite, has been reported to be significantly increased in both induced sputum and bronchial tissues of patients with CB, and also in patients with asthma (7, 8, 11). 15-HETE can promote the migration of neutrophils and mast cells into airway lumen (3), and has been reported to modulate the capacity of macrophages to phagocytose (12). 12(S)-HETE, also a product of 15-LO activity, has been shown to upregulate adhesion molecules in endothelial cells (13). Both 12(S)-HETE and 15(S)-HETE have been shown to be potent muco-secretagogues in human airway explant cultures (14), and in rat, canine, and guinea-pig airways in vivo (3, 15–17). In contrast, lipoxin A4, another 15-LO product, inhibits tumor necrosis factor-{alpha}–induced polymorphonuclear neutrophil (PMN) recruitment (4). In addition, transfection of rat kidney with human 15-LO suppresses inflammation and preserves function in experimental glomerulonephritis (5). Also, interleukin (IL)-4 has been shown to increase expression of 15-LO, 15-HETE, and 13-HODE; however, in this experimental animal study, this was associated with an inhibition of mucin secretion and an attenuated expression of mucin genes MUC5AC and MUC5B (18). Thus, the role of 15-LO in development of airway inflammation and mucus hypersecretion needs further investigation.

Following the identification of a second 15-LO isozyme, 15-LOb, we have focused in the present study on the localization of gene transcripts and protein expression for 15-LOa and 15-LOb, and on the separate quantification of these distinct isozymes in the airway walls of smokers, with the intention of identifying which of these isoforms might be the most logical target for pharmacologic modulation of inflammation and possibly of mucus hypersecretion (19–21).

Previous studies have demonstrated that IL-4 can induce 15-LOa in cultured human peripheral blood monocytes (22). The induction of total 15-LO and 15-LOa, but not the 15-LOb enzyme activity, by IL-4 in human airway epithelial cells has been demonstrated (23, 24). As we have recently reported a significant increase of IL-4 gene expression in the airways of patients with CB, as compared with both asymptomatic smokers and smokers with COPD (25), we speculated that the upregulation of IL-4 observed in patients with CB may induce 15-LO expression with the generation of 15-HETE and the consequent induction of mucus hypersecretion by glands and surface epithelial goblet cells (17). We hypothesized that the numbers of inflammatory cells expressing IL-4 or 15-LOa would be associated and similarly upregulated in the airways of smokers with CB compared with asymptomatic smokers (AS). To test this hypothesis, we have examined their association in areas of submucosal mucus-secreting glands in airway tissues resected from smokers with or without CB.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The study conformed to the declaration of Helsinki. All subjects were volunteers and gave their informed, written consent. Approval for the study was given by each of the local ethical committees. Bronchial biopsies were taken from subjects of three groups (age range: 40–69 yr) and studied in parallel: (i) healthy nonatopic nonsmokers (HNS) (n = 7), (ii) healthy smokers (HS) (n = 5), and (iii) smokers with CB (CB) (n = 8) in its stable phase without history or signs of asthma. The subjects in both the HNS and HS groups had no history of bronchopulmonary disease and had an FEV1 > 80% of predicted and an FEV1/FVC% > 70% (see Table 1).


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  		TABLE 1 Endobronchial biopsies: characteristics of nonsmokers, healthy smokers, and smokers with chronic bronchitis

 
Lung resections were included from two groups of smokers (> 20 pack-years) coming to surgery for lung tumor (overall age range of 50–80 yr): Group I consisted of asymptomatic smokers (AS, n = 11) with normal lung function, and Group II–consisted of smokers with CB (CB, n = 11) but with normal lung function (i.e., FEV1 > 80% of predicted; FEV1/FVC > 70%; see Table 2).


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  		TABLE 2 Surgical resection: characteristics of asymptomatic smokers and smokers with chronic bronchitis

 
All the patients had CB and were at risk for COPD as defined by the Medical Research Council (MRC) and the criteria of the Global Initiative for Chronic Obstructive Lung Disease, respectively (1, 26). The patients had < 10% reversibility or < 200 ml increase of FEV1 to inhaled ß2-agonist. No patient had a past history of either asthma or allergic rhinitis, and all were nonatopic as defined by their absence of skin reactivity to a panel of common allergens, including grass pollen sp., Parietaria sp., Artemisia sp., Cynodon sp., cat, molds, Dermatophagoides pteronyssinus, D. farinae, and Aspergillus sp. The blood eosinophil count was less than 0.5 x 109/liter.

None of the subjects had received oral or inhaled glucocorticosteroids or antibiotics in the month preceding the study. All subjects underwent full clinical examination, and received a chest radiograph, electrocardiogram, and routine blood tests. None of the patients with CB had had exacerbations (defined as increased dyspnea associated with a change in quality and/or quantity of sputum that had led the subject to seek medical attention) within the preceding year.

Bronchoscopy, Biopsies, and Lung Resections
Fiberoptic bronchoscopy was performed as previously described (27). Biopsies were taken using a Pentax Feb-19TX bronchoscope (Pentax, Tokyo, Japan) with sterile Olympus Feb-20C-1 forceps (Olympus Co., Tokyo, Japan) from the sub-carina of the basal segmental bronchi of the right lower lobe. Up to three biopsies were obtained from each subject and fixed immediately in 4% paraformaldehyde, dehydrated, and embedded in paraffin wax. Sections 5 µm thick were cut and stained with hematoxylin and –eosin (H&E). Antibodies were applied which had been raised against 15-LOa and b for immunohistochemistry. The technique of in situ hybridization (ISH) was applied using riboprobes that detected intra-cytoplasmic mRNA for 15-LOa and b, respectively. Whenever possible, two further biopsies were obtained freshly from each subject, snap-frozen in dry ice immediately, and then stored at -80°C for the later detection and quantification of 15-LOa and 15-LOb mRNA using RT-PCR.

The lung tissues obtained at surgical resection were obtained through either the Histopathology Department of University of Turin (Italy) or the Department of Respiratory Diseases at Ghent University Hospital (Belgium). Grossly normal airway tissue, taken well away from tumor, was selected to include lobar and segmental bronchi (i.e., airways with included mucus-secreting glands and cartilage). These were fixed freshly in 10% buffered formaldehyde and processed to paraffin wax. Serial sections 5 µm thick were first cut and stained with H&E. 15-LOa, 15-Lob, and IL-4 cRNA were used for the ISH procedures and were applied to tissue sections. The tissues allowed us to explore gene expression in the submucosal glands, not sampled completely by endobronchial biopsy of the more superficial mucosa.

The alveolar carcinoma A549 cell line and prostatic epithelial cells (PrEC), stimulated (or not) by 10 ng/ml of IL-4, were used as positive controls to validate 15-LOa and b, respectively. 15-LOa and 15-LOb mRNA and protein were detected by the specific mRNA probes and antibodies. The cultured cell monolayers were scraped from the culture flasks and centrifuged into cell pellets. The cell pellets were processed to paraffin wax and the blocks cut into 5-µm-thick sections, which were treated in all respects in the same way as the intact airway tissues.

Immunohistochemistry
The identification of 15-LOa and b protein producing cells within bronchial biopsies and resected airway tissues was performed using the EnVision-alkaline phosphatase (EV-AP) technique with the EV-AP Kit according to the supplier's instructions (K4017; DAKO Ltd, Cambridge, UK). The 15-LOa and b polyclonal antibodies were provided by Dr. Jenkinson (Pfizer Global Research and Development, Sandwich, Kent, UK). The selective, affinity-purified, anti-human polyclonal antibodies for both 15-LOa and 15-LOb were raised in rabbits against isozyme-specific peptide sequences (sequences used were 15-LOa = RTVGEDPQGLFC and 15-LOb = KVSWADHHPVL). The predicted selectivity was confirmed by Western blot analysis using 5 µg of purified recombinant 15-LOa, 15-Lob, and 12-LO as shown in a previous publication (23): the antibodies were selective for the two 15-LO isozymes and did not crossreact with the closely related 12-LO. IL-4 protein expression in the resected airway tissue from subjects with CB was also detected by immunohistochemistry. Briefly, the paraffin sections were incubated for 20 min with 20% normal goat serum (X0907; DAKO) and 0.1% Tween 20 (T20; British Biocell International Ltd, Cardiff, UK) in Tris-buffered saline (TBS) pH7.6. The sections were then immunostained with 15-LOa and b polyclonal antibodies or anti-human IL-4 monoclonal antibody (MAB1029; Chemicon International, Temecula, CA) for 1.5 h at room temperature. The antibodies were first titrated using the control cells and then appropriately diluted at 1:800 for 15-LOa, 1:100 for 15-Lob, and 1:40 for IL-4 in antibody diluent. Sections were washed and incubated for a further 30 min with AP-conjugated goat anti-rabbit/mouse immunoglobulin. After a further wash, bound alkaline phosphatase was detected as a red product following 20 min incubation with Naphthol AS-MX phosphate and 1 mg/ml New Fuschin. The slides were counterstained with hematoxylin to provide morphologic detail and then mounted in Aqua perm mounting medium. The alveolar carcinoma A549 cell line and prostatic epithelial cells (PrEC) were used as positive controls to validate the 15-LOa and b antibodies. We applied an irrelevant antibody: mouse IgG1, Kappa (MOPC21; Sigma, Dorset, UK M7894) for the primary layer as a negative control.

Nonisotopic ISH
We initially validated two 15-LOa (15LOaAS1 bases 1–430, 15LOaAS2 bases 845–1262) and three 15-LOb (15LObAS1 bases 1146–1523, 15LObAS2 bases 1457–1810, 15LObAS3 bases 1516–1870) antisense (AS) probes. Each probe was generated by PCR from the respective full-length coding sequences of the 15-LO isozymes. A range of sequences were investigated through the length of the 15-LOa and 15-LOb genes, with the aim of minimizing homology to other known lipoxygenase sequences. The specificity of each of these probes was then investigated by Southern blotting against 5-LO, 12-LO, 15-Loa, and 15-LOb sequences. The probes described above and chosen for the ISH studies were shown to be specific for their respective 15-LO isoform and did not crossreact with other lipoxygenases on Southern blots. Furthermore, the DNA probes were transcribed into RNA incorporating a P32 radiolabel and used to bind to full-length 15-LOa and b DNA immobilized on a nitrocellulose membrane. The resulting autoradiographs confirmed the probe specificity for each of the 15-LOa and b isoforms. The PCR samples were then cloned into Invitrogen pCR 2.1-TOPO vector (3.9 kb). We also determined the suitability of a sense (S) probe corresponding to one of the antisense probes described above (15LObS1) as a negative control. Plasmid DNA containing the 15-LO probes (Pfizer Global Research and Development) and IL-4 (318 bp in PGEM-1; Glaxo-Wellcome Biomedical, Geneva, Switzerland) were expanded in JM109 Escherichia coli–competent cells. Each of the six 15-LO cDNA probes were linearized with restriction endonuclease BamHI. The antisense IL-4 and sense IL-4 were linearized with NheI and EcoRI, respectively.

Preparation of cRNA Probes
Digoxigenin (Dig)-labeled antisense and sense complementary ribonucleic acid (cRNA) probes were generated from complementary deoxyribonucleic acid (cDNA) according to a well-tried and published method (16, 28). To check transcription efficiency and quantify the probe, 1 µl Dig-RNA was taken for electrophoresis in 1.2% agarose gel, after which it was stored at -80°C.

Pre-Hybridization
The sections were dewaxed and incubated with proteinase K to permeabilize the cells. Hybridization buffer (2x Denhardt's solution, 50 µg/ml salmon sperm DNA, 100 µg/ml yeast tRNA, 50% formamide) containing 50–100 ng/ml Dig-labeled cRNA probe, was added and incubated at 42°C overnight. Sense probes were used as the most appropriate negative control. Slides were washed in different concentrations of sodium chloride/tri-sodium citrate (SSC) and incubated in 20 µg/ml RNase A. The remaining probe hybridized with the mRNA of interest was detected with an anti-Dig antibody conjugated to alkaline phosphatase. Detection was performed by addition of 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (NBT/BCIP) substrate following an incubation of 30–60 min to give a blue/black end-product. The nuclear counterstain used was nuclear fast red.

In preliminary probe validation for ISH we found that the staining intensity of the two 15-LOa probes in A549 cells with and without 10 ng/ml IL-4 stimulation and pilot bronchial biopsies of CB were similar. Three 15-LOb probes produced similar intensity of ISH signals in the cultures of PrEC with and without IL-4 treatment, and also in cells demonstrated to be positive in the pilot bronchial biopsies. The sense probe 15-LObS1 showed no staining in IL-4–stimulated A549 cells and nonstimulated PrEC, as well as in pilot bronchial biopsies. Thus, based on our pilot observations, we chose 15LOaAS1 and 15LObAS1 probes and 15LObS1 for ISH in bronchial biopsies and lung resection tissue.

Quantitative RT-PCR
Total RNA was prepared from snap-frozen lung biopsy samples using TRIZOL Reagent (GibcoBRL, Paisley, UK) following the manufacturer's instructions. cDNA was synthesized from 1 µg of total RNA with the GeneAmp RNA PCR Kit (Perkin Elmer) in a total volume of 20 µl using random hexamer primers. Primer/probe sets were designed for each of the 15-LO isozymes to regions of the gene sequence not conserved between the known members of the lipoxygenase gene family. PCR cycle conditions were optimized in a standard PCR machine using cDNA generated from the human lung epithelial cell line A549 and transferred to the Taqman ABI Prism 7,700 sequence detector (Perkin Elmer) for quantitative PCR of biopsy samples. Taqman PCR was performed using Taqman universal PCR master mix and VIC-labeled ribosomal 18S endogenous loading control primer probe sets. FAM-labeled gene specific probes were purchased from Perkin Elmer.

The primers used were as follows. 15-Loa: forward, 5'-TTGGTTATTTCAGCCCCCATC-3'; reverse, 5'-TGTGTTCACTGGGTGCAGAGA-3'; probe, 5'-TCCCAAGTCCCACCCTCTTCCCAT-3'. For 15-Lob: forward, 5'-GAGCTTCAGGCCCGGCAGGAGAT-3'; reverse, 5'-TTCTCTAGCTCAGCCTGCAAGC-3'; probe, 5'-CTGTCCTGATCCGCCGCTGTCACTA-3'.

All primers were used at 300 nM and probe at 200 nM. PCR cycle conditions: 50°C, 2 min x1; 95°C, 10 min x1; 95°C, 15 s/60°C, 1 min x40. Data were analyzed as described in the manufacturer's instructions. Gene expression levels in each of the biopsies were expressed as a percentage of 18S rRNA expression.

Quantification and Data Analysis
Endobronchial biopsies. Slides were coded to avoid observer bias. For bronchial biopsies, areas of subepithelium excluding muscle and gland were assessed using an Apple Macintosh computer and Image 1.5 software (Apple Mac, Cupertino, CA). Protein+ and mRNA+ cells for 15-LOa and b were counted using a Leitz Dialux 20 light microscope (Leitz, Wetzlar, Germany) working at x200 magnification and fitted with an eyepiece graticule divided into 100 squares. The mRNA+ and protein+ cells present in the subepithelium were counted in the entire subepithelial area of the biopsy, excluding those areas of mucus-secreting glands and bronchial smooth muscle. A similar eyepiece graticule was used to "point count" and assess the percentage of epithelium expressing mRNA and protein for 15-LOa and b (29).

Resected airways (smokers with CB). The areas of airway wall in the lung resections, areas of interstitium in the subepithelial zone (between the external edge of the reticular basement membrane and inner aspect of bronchial muscle), areas of mucus-secreting gland, and the remainder of the airway wall (excluding cartilage) were assessed using an Apple Macintosh computer and Image 1.5 software. The mRNA+ cells and protein+ cells for 15-LOa, 15-Lob, and IL-4 were counted in the same way as for bronchial biopsies. To test the consistency of quantification and the inherent intra-observer variation of repeated counts, one section was selected and counted five times over the period of the entire study. The coefficients of variation (CV) for repeated counts of 15-LOa protein+ and mRNA+ cells by the same observer were 5% and 7%, respectively. The sections that had been reacted or hybridized with either the negative control antibody or the sense (negative) probe, respectively, were examined first to discriminate background signals from true signals.

Statistical analysis. The data for cell counts of bronchial biopsies and resection material are expressed as number of positive cells per mm2 of tissue. The data for large airway wall are expressed as the number of positive cells per mm2 tissue for the three tissue zones examined: (i) the subepithelium, (ii) that containing mucus-secreting gland acini, and (iii) the remainder of airway wall. The coefficient of variation (CV = SD/mean x 100) was used to express the error of repeat counts. Mann-Whitney U-tests were applied to test for differences between the three groups. Spearman Rank Correlation was used to determine the correlation between the number of 15-LOa/15-LOb mRNA+ cells and IL-4 mRNA+ cells, respectively. A P value of < 0.05 was accepted as indicating a significant difference in both the Mann-Whitney U-tests and Spearman Rank correlation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical Findings
Details of the healthy nonsmoker control subjectss, healthy smokers, and smokers with stable CB from whom bronchial biopsies were obtained are summarized in Table 1. The three groups were similar with regard to age: the two smoker groups were similar in their smoking history. The lung function data were similar in the three groups and were within the normal range, i.e., FEV1 > 80% of predicted and FEV1/FVC% > 70%. The characteristics of the asymptomatic smokers and those with CB from whom lungs were obtained at resection are shown in Table 2. The two groups of smokers were similar with regard to age and smoking history. The patients in these groups also had normal lung function. Their smoking history was similar to the two smoker groups sampled by endobronchial biopsy. All subjects were nonatopic, i.e., they had negative skin tests for a panel of common allergen extracts. Blood eosinophil counts were in normal range, i.e., 0.0–0.4 x 109/liter.

Bronchial Biopsies
The endobronchial biopsies were 1–2 mm in diameter and of good quality. H&E staining demonstrated that the biopsies of HNS group usually had intact pseudostratified, ciliated columnar epithelium with relatively few goblet cells. The reticular basement membrane was not thickened and there were only occasional inflammatory cells in the subepithelium. In contrast, the biopsies from the smokers with CB typically showed goblet cell hyperplasia or focal squamous metaplasia of their epithelia. Moreover, there was subepithelial infiltration by inflammatory cells in the smokers to an extent not seen in the nonsmokers.

15-LO Gene and Protein Expression
Positive control cells. IL-4–stimulated A549 and PrEC cultured cells were used as our positive controls, for both 15-LOa and 15LOb mRNA and protein expression. For 15-LOa, both ISH and immunohistochemistry (IHC) showed the absence of positivity (-) in unstimulated A549 cells and moderate positivity (++) for both ISH and IHC in the 10 ng/ml IL-4–stimulated A549 (Figure 1A). Semiquantitative analysis did not unequivocally discriminate differences between the IL-4–stimulated and nonstimulated PrEC for 15-LOb ISH and IHC positivity. Both were scored +/++ for 15-LOb protein staining intensity (Figure 1B). There was an absence of staining in the mRNA "sense," and irrelevant antibody controls both for the IL-4–treated A549 and PrEC cells.




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  		Figure 1. Immunohistochemistry for cultured positive control cells. (A) IL-4 (10 ng/ml)-stimulated A549 cells showing moderate to intense staining for 15-LOa protein in the cytoplasm of some cells (arrows). (B) Unstimulated PrEC cells showing moderate to intense staining for 15-LOb protein (arrows) (internal scale bar = 20 µm).

 
Endobronchial biopsies. The biopsies of the HNS group demonstrated mild positivity for 15-LOa mRNA and protein in some, but not all, surface epithelial cells; few subepithelial inflammatory cells were positive for 15-LOa mRNA or protein (Figures 2A and 3A). Healthy smokers showed moderate 15-LOa mRNA and protein positivity of non-goblet surface epithelial cells, and occasional submucosal gland serous cells, subepithelial inflammatory cells (Figures 2B and 3B), and smooth muscle cells. In contrast, in CB there was intense positivity for both mRNA and protein in all surface epithelial cells, where it was detected in both ciliated (Figures 2C and 3C) and nonciliated cells, including basal cells, cells of indeterminate morphology, and those that were squamous and metaplastic. There was a lack of positivity of goblet cell secretory granules per se, but the perinuclear cytoplasm was intensely mRNA-positive (Figure 3C). The intense positivity for 15-LOa mRNA and moderate positivity for 15-LOa protein was also found in many subepithelial inflammatory cells including monocytes, lymphocytes, neutrophils, and plasma cells (Figures 2C and 3C). Fibroblasts, the endothelium of blood vessels (Figure 2C), submucousal gland serous acini, and bronchial smooth muscle (not illustrated) showed moderate positivity for 15-LOa mRNA and protein.





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  		Figure 2. Nonisotopic in situ hybridization of a bronchial biopsy for 15-LOa gene expression. (A) a healthy nonsmoker showing weak expression of 15-LOa mRNA in cells present in the surface epithelium (arrowheads) and a relatively small number of 15-LOa mRNA positive subepithelial mononuclear cells (shown dark blue and arrows). (B) A healthy smoker showing upregulation of 15-LOa mRNA and moderate to intense staining of epithelial cells. There are also inflammatory cells in the subepithelial zone (arrow). (C) A smoker with chronic bronchitis demonstrating increased epithelial expression of 15-LOa mRNA and increased intensity of 15-LOa mRNA positive inflammatory cells in the subepithelial zone. The endothelium of blood vessels also express 15-LOa mRNA (arrow) (internal scale bar = 20 µm).

 




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  		Figure 3. Immunohistochemistry for 15-LOa protein in bronchial biopsies. (A) A healthy nonsmoker showing weak expression of 15-LOa protein in surface epithelium (shown stained red with the EnVision-alkaline phosphatase technique; arrowhead). (B) A healthy smoker showing moderate to intense staining for 15-LOa protein in some epithelial cells. There is weak staining of relatively few inflammatory cells (arrow) in the subepithelial zone. (C) Smokers with chronic bronchitis demonstrating intense staining of epithelial cells, the cytoplasm of ciliated cells, the peri-nuclear cytoplasmic region of goblet cells (arrowhead), and increased staining for 15-LOa protein in subepithelial inflammatory cells (arrows) (internal scale bar = 20 µm).

 
In general, the distribution of 15-LOa and 15-LOb was similar. However, in contrast, 15-LOb mRNA showed weak positivity, even in the smokers with CB (Figure 4A). There was also weak staining for 15-LOb protein (Figure 4B), occasionally present in surface (non-goblet) epithelial cells and subepithelial inflammatory cells in all three biopsy groups. Bronchial biopsies from subjects with CB, hybridized with the sense 15-LOb probe (Figure 5A) or those immunostained with an irrelevant antibody (MOPC21), were negative (Figure 5B).




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  		Figure 4. Expression of 15-LOb mRNA and protein. (A) Nonisotopic in situ hybridization for 15-LOb gene expression in the bronchial mucosa of a biopsy obtained from a smoker with bronchitis. There was weak gene expression in surface epithelial cells and a relatively small number of 15-LOb mRNA positive subepithelial inflammatory cells (arrows). (B) Immunohistochemistry for 15-LOb protein in a bronchial biopsy in a smoker with chronic bronchitis showing very weak staining throughout the surface epithelium (internal scale bar = 20 µm).

 



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  		Figure 5. In the biopsy of a smoker with chronic bronchitis there was an absence of signal using either (A) the sense control probe for ISH or (B) the irrelevant antibody for immunostaining (internal scale bar = 20 µm).

 
Resected airways (smokers with CB). H&E-stained sections showed evidence of goblet cell hyperplasia or squamous metaplasia, and the subepithelial tissue contained submucosal glands, smooth muscle, cartilage, and adventitia. Inflammatory cells were located throughout the airway wall, with relatively high numbers associated with mucus-secreting glands and also present in the subepithelium.

15-LOa, 15Lob, and IL-4 mRNA and protein positivity were detected by ISH and IHC. As with the endobronchial biopsies, there was more intense expression and significantly greater numbers of 15-LOa than 15-LOb mRNA+ and protein+ cells. There was also intense gene and protein expression for IL-4. Both IL-4 and 15-LOa mRNA and protein were localized to the surface epithelium (Figures 7A and 8A), in each case particularly associated with basal cells and areas of squamous cell metaplasia. There was an abundance of strongly IL-4 and 15-LOa mRNA and protein-positive mononuclear inflammatory cells located in the subepithelial zone (Figures 7A and 8A), and also in the interstitial tissue between mucus-secreting gland acini (Figures 7B and 8B). Serous demilunes of the glands were also mildly positive. 15-LOb mRNA and protein were also detectable, but the signals were much less intense and there appeared to be fewer positive cells than that for 15-LOa. There was no or little positivity for 15-LOb in the epithelium. The sense probes for both 15-LOb and IL-4 were negative.




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  		Figure 7. Immunohistochemistry for 15-LOa protein in resected bronchial tissue of a smoker with CB. (A) epithelium and subepithelium: strong intense staining throughout surface epithelial cells and intense staining in mononuclear inflammatory cells located in the subepithelial zone (arrows). (B) Submucosa gland: strong expression of 15-LOa protein positivity in mononuclear inflammatory cells infiltrating the interstitial tissue between gland acini (arrows), in the perinuclear cytoplasm of submucosal gland mucous cells (arrowhead) and the cytoplasm of serous cells (internal scale bar = 20 µm).

 



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  		Figure 8. Graphs of counts for (A) 15-LOa and 15-LOb mRNA and (B) protein positive cells in bronchial biopsies of healthy nonsmokers (HNS), healthy smokers (HS), and smokers with chronic bronchitis (CB). The data are expressed as the number of positive cells per mm2 of a subepithelial zone (Mann-Whitney U test: horizontal bar shows median values).

 
Quantitative Histology
Bronchial biopsies. The numbers of subepithelial cells expressing mRNA or protein for 15-LOa and 15-LOb were counted in the biopsies obtained from each of the three subject groups (Figures 6A and 6B). The number of cells expressing 15-LOa and b mRNA per mm2 subepithelial area was significantly higher than the number expressing the respective 15-LOa and b protein (P < 0.05). There were always significantly greater numbers of 15-LOa than 15-LOb mRNA+ and protein+ cells (P < 0.01). Compared with the healthy nonsmoker control subjects, smokers with CB were significantly greater in their numbers of both 15-LOa mRNA+ (3-fold) (P < 0.01) and protein+ cells (10-fold) (P < 0.01). The number of 15-LOa protein+ cells in the smokers with CB was significantly higher than that of the healthy smoker group (P < 0.01); however, although higher, the number of 15-LOa mRNA+ cells in the smokers with CB was not statistically different. Compared with the healthy nonsmokers, there was a significant increase in the healthy smokers of the number of 15-LOa protein+ cells (P < 0.05) and a trend to an increase in number of 15-LOa mRNA+ cells (P = 0.07). There were, however, no significant differences in the numbers of 15-LOb mRNA+ and 15-LOb protein+ cells between the three groups. The numbers of cells expressing 15-LOa mRNA showed a strong correlation with the numbers of 15-LOa protein+ cells counted in subepithelial areas in all of biopsies obtained from healthy nonsmokers, healthy smokers, and smokers with CB (r = 0.80; P < 0.002, Spearman's rank correlation).




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  		Figure 6. Nonisotopic in situ hybridization of resected bronchial tissue from smoker with CB. The antisense probe shows strong cytoplasmic expression of 15-LOa mRNA (dark blue). (A) in mononuclear inflammatory cells located in the sub-epithelial zone. There is moderate intensity of 15-LOa gene expression in some of the epithelial cells also (arrowhead). (B) In mononuclear inflammatory cells infiltrating the interstitial tissue between submucosal gland acini (internal scale bar = 20 µm).

 
Epithelial expression of 15-LOa mRNA and protein proved difficult to quantify because its expression was so intense. However, an attempt was made to quantify the area of the epithelium with 15-LOa protein positivity by use of an eyepiece graticule and application of a validated point-counting method (29). An average of 57.1% (SEM = ±4.2%) of the epithelium of the nonsmoking control subjects expressed 15-Loa, but it was very weakly expressed (i.e., a score of 1 on a scale of 1–3). By comparison, the healthy smoker group showed that 68.5% (SEM = ±5.5%) of the epithelium was weakly or moderately positive (P < 0.05) (i.e., scoring for an intensity of 1 or 2, respectively). In contrast, strong positivity with 71.2% (SEM = ±7.6%) of the epithelium, scoring for an intensity of 1–3, was found in the smokers with CB (P < 0.05 compared with the nonsmoker group).

Resected tissue (smokers with CB). Counts were made of mRNA+ and protein+ inflammatory cell in the three zones of the airway wall. These data are expressed as the number of positive cells per mm2 in (i) a subepithelial zone, (ii) mucus-secreting glands, and (iii) the remainder of the airway wall. There were significantly higher numbers of 15-LOa protein+ cells in patients with CB than in the asymptomatic patients, in both the subepithelial (P < 0.002) and glandular compartments (P < 0.001) (see Figure 9). However, although there were similar trends between the numbers of cells expressing 15-LOa protein and 15-LOa mRNA in the mucus-secreting glands, there were no significant between-group differences with respect to 15-LOa mRNA in either the subepithelial or glandular zones.



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  		Figure 9. Counts for 15-LOa and b protein positive cells in distinct zones of the resected bronchial tissues from asymptomatic (AS) smokers and smokers with chronic bronchitis (CB). The results are expressed as the number of positive cells per mm2 subepithelial zone and glandular compartment: individual patient values and medians for the groups are shown. The Mann-Whitney U test was used to compare the differences between distinct tissue zones and subject groups. S = subepithelial zone; G = gland.

 
The counts for 15-LOa, 15-LOb, and IL-4 mRNA+ cells in CB are shown in Table 3 as median and range. 15-LOa, 15-LOb, and IL-4 mRNA+ and protein+ cells were located mainly in the mucus-secreting glandular and subepithelial zones rather than the remainder of the airway wall. In each of the three compartments there were significantly more 15-LOa than 15-LOb mRNA+ and protein+ cells (P < 0.01, Mann Whitney U test) (see Table 3 and Figure 9). In the CB group, the number of IL-4 mRNA+ and protein+ cells in the glands was greater than that in the subepithelial zone (P < 0.05). There was a significant positive correlation between the numbers of 15-LOa and IL-4 mRNA+ and also protein+ cells in both the subepithelial zone (mRNA: r = 0.82, P < 0.01; protein: r = 0.66, P < 0.04) and gland areas (mRNA: r = 0.86, P < 0.01; protein: r = 0.74, P < 0.02, Spearman rank correlation) (Figure 10). There was no association between the numbers of 15-LOb and IL-4 mRNA+ cells in any compartment.


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  		TABLE 3 Counts of 15-LOa and b mRNA+ and IL-4 mRNA+ inflammatory cells in distinct zones of the airway wall in lung resections from smokers with chronic bronchitis

 


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  		Figure 10. Association between the numbers (per mm2) of 15-LOa and IL-4 mRNA and protein positive cells in the submucosal gland interstitium of resected bronchial tissue from a subject with chronic bronchitis (Spearman's rank correlation; n = 11).

 
Quantitative RT-PCR. Total RNA was extracted from two biopsies from each of 16 subjects: 3 healthy nonsmokers, 5 healthy smokers, and 8 patients with CB. cDNA was generated for each of the samples from 1 µg of total RNA and quantitative PCR performed using a Taqman ABI Prism 7,700 sequence detector. Relative levels of gene expression for each of the 15-LO isozymes are expressed herein as a percentage of 18S ribosomal RNA in each of the samples (Figures 11A and 11B). Analysis of the data using Student's unpaired t test shows that there was significantly greater induction of 15-LOa in the biopsy samples from patients with CB than in either the healthy nonsmokers (P < 0.05) or the healthy smokers (P < 0.05) (Figure 11B). There was no evidence for differential expression of 15-LOb mRNA levels between any of the subject groups. The mean coefficient of variation between duplicate biopsies for 15-LOa was 24.2%, and for 15-LOb was 23.9%.



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  		Figure 11. Quantitative RT-PCR analysis of 15-LO isozyme expression in different patient populations. (A) RT-PCR was carried out using isozyme-specific primers and RNA isolated from a biopsy sample as described in MATERIALS AND METHODS. The resulting DNA was fractionated on a 1% agarose gel (lane 1, 1 kb ladder; lane 2, 15-LOa–specific primers; lane 3, 15-LOa water control; lane 4, 15-LOb–specific primers; lane 5, 15-LOb water control). (B) Quantitative RT-PCR was carried out on total RNA generated from two individual biopsies from each donor for each of the 15-LO isozymes. RNA expression levels are quoted as a percentage of 18S ribosomal RNA in each sample, and median values are shown. Using the unpaired Student's t test, there is a significant increase in 15-LOa mRNA expression in patients with chronic bronchitis compared with healthy smokers and nonsmokers (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show in the present study that 15-LO mRNA and protein-expressing cells are found in the airway mucosa of smokers and nonsmokers, in both the mucosa and submucosa, and that there is significant upregulation in CB. Our study has demonstrated for the first time that although both isozymes are expressed, there are always significantly greater numbers of cells expressing15-LOa than 15-LOb and, by comparison with the airway mucosa of healthy nonsmokers, it is the expression of the 15-LOa, not 15-LOb. that is increased in healthy smokers. with a further increase in smokers who have developed CB. The increase of 15-LOa in CB is associated with an increase of IL-4, albeit the functional relevance of this still needs to be established.

Localization
15-LO expression has previously been shown in airway epithelia and skin, macrophages, eosinophils, and reticulocytes (7, 30–32). 15-LOb has been detected by Northern blot and PCR in lung epithelium, skin prostate, and cornea (20, 21), and by RT-PCR in isolated peripheral blood B lymphocytes, but not peripheral blood T lymphocytes, eosinophils, or neutrophils (21). We confirm using both bronchial biopsies and resected airway tissue that the epithelium of patients with CB shows strong positivity for both 15-LOa mRNA and protein; this is compatible with earlier studies of 15-LO in subjects with bronchitis or asthma (6). In our present study, ciliated cells were found to have an abundance of both 15-LOa mRNA and protein. We found that epithelial basal cells, nonciliated cells of indeterminate ("intermediate") morphology, and areas of squamous metaplasia also express both 15-LOa mRNA and protein. In epithelial goblet cells and submucosal gland mucous cells, although mucous granules per se did not stain, there was intense perinuclear cytoplasmic expression confirming the capacity of mucus-secreting to produce 15-LOa. There was only moderate expression of 15-LOa mRNA and protein throughout the cytoplasm of submucosal gland serous cells. In addition, in the patients with CB there was intense positivity for 15-LOa mRNA and moderate positivity for 15-LOa protein in subepithelial inflammatory cells and in those infiltrating the interstitium between the secretory acini of the mucus-secreting glands. By morphology these inflammatory cells appeared to include neutrophils, plasma cells, and lymphocyte/monocytes. Fibroblasts, the endothelium of blood vessels, and bronchial smooth muscle also showed moderate expression for 15-LOa mRNA and protein. In contrast there was weak positivity for 15-LOb.

Distinctions between 15-LOa and 15-LOb Isozymes
15-LOa and 15-LOb have been studied previously with respect to their distinct molecular structures, catalysis, kinetics, and biologic function (19–21). The homology between 15-LOa and 15-LOb is not high (31.6% at the amino acid level), with 15-LOa demonstrating a closer homology to 12-LO than to15-LOb (20). 15-LOa uses linoleic acid in preference to arachidonic acid as substrate (19). In contrast, 15-LOb has a preference for arachidonic acid, with linoleic acid being a relatively poor substrate (20). The two enzymes differ significantly in their specificity for the oxygenation of arachidonic acid. 15-LOa produces mainly 15-HETE, but also forms 12-HETE such that it accounts for 10–20% of the product of arachidonic acid peroxidation (33). In contrast, 15-LOb specifically converts arachidonic acid at C15 to 15-HETE, and no other lipid peroxidation products are detected in in vitro assays (20, 21). In addition, 15-LOa is also rapidly auto-inactivated during the conversion of arachidonic acid to 15-HETE, whereas 15-LOb continues to catalyze the reaction until substrate availability becomes rate-limiting (21). Thus, induction of 15-LOa activity may result in rapid bursts of 15-HETE production, whereas 15-LOb activity may be responsible for a lower level, chronic production of 15-HETE, as long as the arachidonic acid substrate remains available. Moreover, using normal human bronchial epithelial primary cell cultures, it has been shown that whereas 15-LOa expression and activity is induced by cytokines, such as IL-4 and IL-13, no cytokines have been identified that specifically or preferentially induce the expression or the activity of 15-LOb (23). These results suggest that 15-LOa is the regulated isozyme in the airways, whereas 15-LOb serves to maintain basal 15(S)-HETE production. Our studies herein have shown clear differences between 15-LOa and 15-LOb gene and protein expression related to smokers with CB, and established that the major 15-LO activity in the airways of smokers is that due to activity of the 15-LOa isozyme.

Increases of 15-LOa mRNA versus Protein Expression
By comparison with the healthy nonsmokers, there were significant increases in the number of 15-LOa protein+ cells in the smokers and also in those with CB. Although there were similar trends for increases in 15-LOa mRNA, the differences were not as striking nor as significant statistically. This might be due to the relatively high baseline numbers of 15-LOa mRNA+ cells and low numbers of 15-LOa protein+ cells in the healthy nonsmokers, with the result that there is greater scope for upregulation of the protein. Alternatively, there may be a constitutive level of 15-LOa mRNA expression in the airway tissues of healthy nonsmokers that is not translated into protein due to post-transcriptional inhibition (34). Supportive data for the latter suggestion comes from studies of immature rabbit reticulocytes, in which the expression of functional 15-LO is prevented at the translational level by the binding of a repressor protein to the 3' UTR of the mRNA (35). In these cells no 15-LO protein is present, in spite of a high concentration of 15-LO mRNA being detected (36). Similar regulation may occur normally in the airways and may be uncoupled in response to smoking and inflammation (34, 37). Interestingly, a previous publication has demonstrated that 15-LO mRNA and protein may not be detected in nonstimulated A549 (lung carcinoma) cells, yet the inflammatory cytokines IL-4/IL-13 can rapidly induce both 15-LO gene and protein expression these cells. Furthermore, in freshly isolated human polymorphonuclear neutrophils (PMN) from healthy volunteers, 15-LO mRNA is expressed at very low levels, but prostaglandin E2 can induce a dramatic increase in PMN 15-LO mRNA and enzyme activity (4). In the present study of airway tissues, the positive correlation between 15-LOa mRNA and protein expression, and the increases associated with smoking and disease, show that both gene and protein are upregulated but that protein translation is particularly favored and upregulated by the inflammatory response in CB.

15-LO, IL-4, Inflammation, and Mucus Hypersecretion
Our recently published work in smokers with CB has demonstrated an increase in IL-4 mRNA expression in proximal airways resected from smokers with CB compared with asymptomic smokers. We have reported a positive correlation between the number of IL-4 mRNA+ cells and the total number of inflammatory cells in both the subepithelium and glandular compartments (25). Similarly, the number of inflammatory cells expressing 15-LOa protein in both subepithelium and gland areas of airway from CB are significantly higher than that of asymptomic smokers. In addition, we have demonstrated for the first time that there is also a significant positive correlation between the numbers of 15-LOa mRNA+/protein+ cells and IL-4 mRNA+/protein+ cells in both subepithelial zone and the submucosal mucus-secreting glands. In contrast, there was no correlation between the numbers of 15-LOb and IL-4 mRNA+ cells in the same tissue compartments. These findings are compatible with the hypothesis that IL-4 may upregulate the expression of the 15-LOa isozyme in the airways of subjects with CB.

The mechanism of IL-4 induction of 15-LOa expression in A549 lung epithelial cells has been studied recently, and appears to act through phosphorylation of STAT6 and transcriptional activation of the 15-LOa gene (24). The functional consequences of increased 15-LOa production in the airway mucosa is as yet unclear. IL-4 and 15-HETE possess a wide range of biologic functions, and they might interact quite differently in normal and diseased conditions. As already indicated, 15-LO may have paradoxical proinflammatory and anti-inflammatory effects; similarly, IL-4 and 15-LO may stimulate or reduce the secretion of mucins (3, 5, 14, 15, 18). Although our data do not allow a statement about the functional consequences of 15-LOa upregulation in smokers and in individuals with CB, the positive relationships between increased IL-4 and increased 15-LOa in both surface and glandular epithelium is of interest. The association between the number of inflammatory cells expressing IL-4 and 15-LOa, and their localization in the subepithelial zone and inter-acinar interstitium of mucus-secreting glands, provide clues for understanding the roles of 15-LOa and IL-4 in the pathogenesis of airway inflammation and mucus hypersecretion in smokers with CB. However, further work is required to establish whether the changes we have discovered are biologically relevant to smokers and to those that go on to develop airway inflammation and mucus hypersecretion.

In conclusion, our endobronchial biopsy– and resected airway tissue–based molecular and immunohistochemical findings demonstrate upregulation of 15-LO in the airway mucosa of smokers with or without CB and indicate that it is the 15-LOa isozyme that is the major regulated isoform. 15-LOa, but not 15-LOb, shows a positive and strong association with increases of IL-4 expression in the submucosal mucus-secreting glands and subepithelial compartments of the airway wall in patients with CB. The biologic significance of this interesting association needs to be understood, but may hold the potential for specific intervention aimed at controlling the inflammation and/or the mucus hypersecretion that characterizes smokers who go on to develop CB.


   Acknowledgments
 
The authors are grateful to Pfizer Global Research and Development (Sandwich, Kent, UK) and the British Lung Foundation for their financial support.

Received in original form January 30, 2002

Received in final form July 17, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pauwels, R., N. Anthonisen, P. J. Barnes, A. S. Buist, P. Calverley, Clark, Y. Fukuchi, L. Grouse, J. C. Hogg, C. Jenkins, D. S. Postma, K. F. Rabe, S. D. Ramsey, S. I. Rennard, R. Rodriguez-Roisin, N. Siafakas, S. D. Sullivan, and W. C. Tan. 2001. Global Initiative for Chronic Obstructive Lung Disease. National Institutes of Health; National Heart, Lung, and Blood Institute. p. 1–30.
  2. Schewe, T., and H. Kuhn. 1991. Do 15-lipoxygenases have a common biological role? Trends Biochem. Sci. 16:369–373.[Medline]
  3. Johnson, H. G., M. L. McNee, and F. F. Sun. 1985. 15-Hydroxyeicosatetraenoic acid is a potent inflammatory mediator and agonist of canine tracheal mucus secretion. Am. Rev. Respir. Dis. 131:917–922.[Medline]
  4. Levy, B. D., C. B. Clish, B. Schmidt, K. Gronert, and C. N. Serhan. 2001. Lipid mediator class switching during acute inflammation: signals in resolution. Nat. Immunol. 2:612–619.[Medline]
  5. Munger, K. A., A. Montero, M. Fukunaga, S. Uda, T. Yura, E. Imai, Y. Kaneda, J. M. Valdivielso, and K. F. Badr. 1999. Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis. Proc. Natl. Acad. Sci. USA 96:13375–13380.[Abstract/Free Full Text]
  6. Shannon, V. R., P. Chanez, J. Bousquet, and M. J. Holtzman. 1993. Histochemical evidence for induction of arachidonate 15-lipoxygenase in airway disease. Am. Rev. Respir. Dis. 147:1024–1028.[Medline]
  7. Profita, M., A. Sala, L. Riccobono, E. Pace, A. Paterno, S. Zarini, L. Siena, A. Mirabella, G. Bonsignore, and A. M. Vignola. 2000. 15(S)-HETE modulates LTB(4) production and neutrophil chemotaxis in chronic bronchitis. Am. J. Physiol. Cell Physiol. 279:C1249–C1258.[Abstract/Free Full Text]
  8. Profita, M., A. Sala, L. Riccobono, A. Paterno, A. Mirabella, A. Bonanno, D. Guerrera, E. Pace, G. Bonsignore, J. Bousquet, and A. M. Vignola. 2000. 15-Lipoxygenase expression and 15(S)-hydroxyeicoisatetraenoic acid release and reincorporation in induced sputum of asthmatic subjects. J. Allergy Clin. Immunol. 105:711–716.[Medline]
  9. Bradding, P., A. E. Redington, R. Djukanovic, D. J. Conrad, and S. T. Holgate. 1995. 15-lipoxygenase immunoreactivity in normal and in asthmatic airways. Am. J. Respir. Crit. Care Med. 151:1201–1204.[Abstract]
  10. Kasahara, Y., R. M. Tuder, C. D. Cool, and N. F. Voelkel. 2000. Expression of 15-lipoxygenase and evidence for apoptosis in the lungs from patients with COPD. Chest 117:260S[Free Full Text]
  11. Kumlin, M., M. Hamberg, E. Granstrom, T. Bjorck, B. Dahlen, H. Matsuda, O. Zetterstrom, and S. E. Dahlen. 1990. 15(S)-hydroxyeicosatetraenoic acid is the major arachidonic acid metabolite in human bronchi: association with airway epithelium. Arch. Biochem. Biophys. 282:254–262.[Medline]
  12. Miller, Y. I., M. K. Chang, C. D. Funk, J. R. Feramisco, and J. L. Witztum. 2001. 12/15-lipoxygenase translocation enhances site-specific actin polymerization in macrophages phagocytosing apoptotic cells. J. Biol. Chem. 276:19431–19439.[Abstract/Free Full Text]
  13. Grossi, I. M., L. A. Fitzgerald, L. A. Umbarger, K. K. Nelson, C. A. Diglio, J. D. Taylor, and K. V. Honn. 1989. Bidirectional control of membrane expression and/or activation of the tumor cell IRGpIIb/IIIa receptor and tumor cell adhesion by lipoxygenase products of arachidonic acid and linoleic acid. Cancer Res. 49:1029–1037.[Abstract/Free Full Text]
  14. Marom, Z., J. H. Shelhamer, F. Sun, and M. Kaliner. 1983. Human airway monohydroxyeicosatetraenoic acid generation and mucus release. J. Clin. Invest. 72:122–127.
  15. Yanni, J. M., M. H. Foxwell, L. L. Whitman, W. L. Smith, and J. C. Nolan. 1989. Effect of intravenously administered lipoxygenase metabolites on rat tracheal mucous gel layer thickness. Int. Arch. Allergy Appl. Immunol. 90:307–309.[Medline]
  16. Yeadon, M., R. C. Price, and A. N. Payne. 1995. Allergen-induced glycoconjugate secretion in guinea-pig trachea in vivo: modulation by indomethacin, BW B70C and ZD-2138. Pulm. Pharmacol. 8:53–63.[Medline]
  17. Shelhamer, J. H., Z. Marom, F. Sun, M. K. Bach, and M. Kaliner. 1982. The effects of arachinoids and leukotrienes on the release of mucus from human airways. Chest 81:36S–37S.[Free Full Text]
  18. Jayawickreme, S. P., T. Gray, P. Nettesheim, and T. Eling. 1999. Regulation of 15-lipoxygenase expression and mucus secretion by IL-4 in human bronchial epithelial cells. Am. J. Physiol. 276:L596–L603.
  19. Brash, A. R., M. Jisaka, W. E. Boeglin, M. S. Chang, D. S. Keeney, L. B. Nanney, S. Kasper, R. J. Matusik, S. J. Olson, and S. B. Shappell. 1999. Investigation of a second 15S-lipoxygenase in humans and its expression in epithelial tissues. Adv. Exp. Med. Biol. 469:83–89.[Medline]
  20. Brash, A. R., W. E. Boeglin, and M. S. Chang. 1997. Discovery of a second 15S-lipoxygenase in humans. Proc. Natl. Acad. Sci. USA 94:6148–6152.[Abstract/Free Full Text]
  21. Kilty, I., A. Logan, and P. J. Vickers. 1999. Differential characteristics of human 15-lipoxygenase isozymes and a novel splice variant of 15S-lipoxygenase. Eur. J. Biochem. 266:83–93.[Medline]
  22. Conrad, D. J., H. Kuhn, M. Mulkins, E. Highland, and E. Sigal. 1992. Specific inflammatory cytokines regulate the expression of human monocyte 15-lipoxygenase. Proc. Natl. Acad. Sci. USA 89:217–221.[Abstract/Free Full Text]
  23. Brown, C. D., I. Kilty, M. Yeadon, and S. Jenkinson. 2001. Regulation of 15-lipoxygenase isozymes and mucin secretion by cytokines in cultured normal human bronchial epithelial cells. Inflamm. Res. 50:321–326.[Medline]
  24. Shankaranarayanan, P., P. Chaitidis, H. Kuhn, and S. Nigam. 2001. Acetylation by histone acetyltransferase CREB-binding protein/p300 of STAT6 is required for transcriptional activation of the 15-lipoxygenase-1 gene. J. Biol. Chem. 276:42753–42760.[Abstract/Free Full Text]
  25. Zhu, J., S. Majumdar, Y. S. Qiu, T. Ansari, A. Oliva, J. C. Kips, R. A. Pauwels, V. De Rose, and P. K. Jeffery. 2001. IL-4 and IL-5 gene expression and inflammation in the mucus-secreting glands and subepithelial tissue of smokers with chronic bronchitis: lack of relationship with CD8+ cells. Am. J. Respir. Crit. Care Med. 164:2220–2228.[Abstract/Free Full Text]
  26. Medical Research Council Report. 1965. Definition and classification of chronic bronchitis (for clinical and epidemiological purposes). Lancet 1:775–779.[Medline]
  27. Laitinen, L. A., M. Heino, A. Laitinen, T. Kava, and T. Haahtela. 1985. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am. Rev. Respir. Dis. 131:599–606.[Medline]
  28. Wilkinson, D. G. 1992. In situ Hybridization: A Practical Approach. Oxford University Press, Oxford.
  29. Aherne, W. A., and M. S. Dunnill. 1982. Point counting and morphometry. In W. A. Aherne and M. S. Dunnill, editors. Morphometery. E. Arnold, London. p. 33–45.
  30. Nadel, J. A., D. J. Conrad, I. F. Ueki, A. Schuster, and E. Sigal. 1991. Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells. J. Clin. Invest. 87:1139–1145.
  31. Sigal, E., S. Dicharry, E. Highland, and W. E. Finkbeiner. 1992. Cloning of human airway 15-lipoxygenase: identity to the reticulocyte enzyme and expression in epithelium. Am. J. Physiol. 262:L392–L398.[Abstract/Free Full Text]
  32. Levy, B. D., M. Romano, H. A. Chapman, J. J. Reilly, J. Drazen, and C. N. Serhan. 1993. Human alveolar macrophages have 15-lipoxygenase and generate 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid and lipoxins. J. Clin. Invest. 92:1572–1579.
  33. Bryant, R. W., J. M. Bailey, T. Schewe, and S. M. Rapoport. 1982. Positional specificity of a reticulocyte lipoxygenase: conversion of arachidonic acid to 15-S-hydroperoxy-eicosatetraenoic acid. J. Biol. Chem. 257:6050–6055.[Free Full Text]
  34. Kuhn, H., D. Heydeck, R. Brinckman, and F. Trebus. 1999. Regulation of cellular 15-lipoxygenase activity on pretranslational, translational, and posttranslational levels. Lipids 34:S273–S279.
  35. Ostareck-Lederer, A., D. H. Ostareck, N. Standart, and B. J. Thiele. 1994. Translation of 15-lipoxygenase mRNA is inhibited by a protein that binds to a repeated sequence in the 3' untranslated region. EMBO J. 13:1476–1481.[Medline]
  36. Thiele, B. J., H. Andree, M. Hohne, and S. M. Rapoport. 1982. Lipoxygenase mRNA in rabbit reticulocytes. Its isolation, characterization and translational repression. Eur. J. Biochem. 129:133–141.[Medline]
  37. Thiele, B. J., M. Berger, A. Huth, I. Reimann, K. Schwarz, and H. Thiele. 1999. Tissue-specific translational regulation of alternative rabbit 15-lipoxygenase mRNAs differing in their 3'-untranslated regions. Nucleic Acids Res. 27:1828–1836.[Abstract/Free Full Text]



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