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Cysteinyl-leukotrienes and prostaglandin D2 generated by the
5-lipoxygenase (5-LO) and cyclooxygenase (COX) pathways, respectively, cause bronchoconstriction, leukocyte recruitment, and
bronchial hyperresponsiveness in asthma. We characterized the
cellular expression of 5-LO and COX enzymes using immunohistochemistry on bronchial biopsies from 12 allergic asthmatic patients before and during seasonal exposure to birch pollen. Bronchial responsiveness (p = 0.004) and symptoms (p < 0.005) increased
and peak expiratory flow (PEF; p 
0.02) decreased in the pollen season. In-season biopsies had 2-fold more cells immunostaining for 5-LO
(p = 0.02), 5-LO-activating protein (FLAP; p = 0.04), and leukotriene
(LT)A4 hydrolase (p = 0.05), and 4-fold more for the terminal enzyme for cysteinyl-leukotriene synthesis, LTC4 synthase (p = 0.02). Immunostaining for COX-1, COX-2, and PGD2 synthase was unchanged. Increased staining for LTC4 synthase was due to increased
eosinophils (p = 0.035) and an increased proportion of eosinophils
expressing the enzyme (p = 0.047). Macrophages also increased
(p = 0.019), but mast cells and T-lymphocyte subsets were unchanged. Inverse correlations between PEF and 5-LO+ cell counts
link increased expression of 5-LO pathway enzymes in eosinophils
and macrophages within the bronchial mucosa to deterioration of
lung function during seasonal allergen exposure.
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Keywords: asthma; allergy; bronchoscopy; leukotrienes; prostaglandins
The cysteinyl-leukotrienes (cys-LTs) LTC4, LTD4, and LTE4 are important mediators of asthma produced by the 5-lipoxygenase pathway (5-LO) in mast cells and eosinophils (1). In asthmatic patients, inhaled cys-LTs cause long-lived bronchoconstriction and selective influx of eosinophils into the bronchial mucosa (2, 3). Specific LT synthesis inhibitors and cys-LT receptor antagonists reduce symptoms, improve lung function, and suppress eosinophilia in the airway and blood of patients with persistent asthma (4). Synthesis of cys-LTs may be downregulated by prostanoid products of the cyclooxygenase (COX) pathway, particularly PGE2 (5), whereas PGD2 derived from mast cells or Th2-lymphocytes may contribute to pulmonary vasodilation, bronchoconstriction, and recruitment of eosinophils, basophils, and T-lymphocytes (6).
The common substrate of the 5-LO and COX pathways is arachidonic acid. During stimulus-specific cell activation, arachidonic acid released by phospholipases, including cytosolic phospholipase A2, is translocated to the 5-lipoxygenase-activating protein (FLAP) (10) and converted in two steps to leukotriene (LT)A4 by 5-LO (11). LTA4 is converted to the dihydroxy-leukotriene, LTB4, by cells expressing LTA4 hydrolase (12), and/or to LTC4, by cells expressing LTC4 synthase, which conjugates LTA4 to reduced glutathione (13). After carrier-mediated cellular export of LTC4, the sequential cleavage of glutamate and glycine residues provides the extracellular metabolites LTD4 and LTE4, respectively. In prostanoid biosynthesis, the released arachidonic acid is directly converted by constitutive COX-1 (prostaglandin endoperoxide synthase-1) or by induced COX-2 in two steps to the intermediate prostaglandin (PG)H2 (14, 15), which is the common substrate of the terminal prostanoid synthases, including PGD2 synthase (16).
Measurements of eicosanoids and their metabolites in BAL fluid and urine after inhaled allergen challenge of atopic asthmatics, and the effects of pretreatment with antileukotriene drugs and NSAIDs, suggest that resident mast cells and infiltrating eosinophils, basophils, and monocyte-macrophages are responsible for leukotriene and prostanoid synthesis in the early and late bronchoconstrictor responses (4, 6, 17). However, the cellular expression of 5-LO and COX pathway enzymes and their responses to allergen exposure have never previously been investigated in the allergic asthmatic lung.
We hypothesized that in allergic asthmatics naturally exposed to a seasonal environmental allergen, upregulation of the expression of a specific profile of 5-LO and/or COX pathway enzymes in mast cells, eosinophils, or other cells by the lung microenvironment may be linked to poorer lung function, bronchial hyperresponsiveness, and asthma symptoms. Previous studies of eicosanoids in allergic asthma examined surrogate markers such as levels of metabolites in BAL fluid and urine, often focused on only one of the eicosanoid pathways, and used inhalation or endobronchial instillation of a large quantity of allergen in the laboratory, which does not effectively mimic natural exposure to low levels of environmental allergen over an extended period. We therefore determined the profile of immunohistochemical expression of enzymes of both the 5-LO and the COX pathways in the bronchial mucosa, the site of inflammation in the asthmatic airway, before and during natural exposure to seasonal birch pollen in 12 atopic asthmatic subjects.
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Patient Recruitment and Study Protocol
Approval for the study was obtained from the Ethics Committee at the Sahlgrenska University Hospital, Gothenburg, Sweden, and patients gave informed written consent. Twelve nonsmoking adults with mild asthma (mean age, 30.6 yr; range, 23 to 43 yr) were recruited from the Gothenberg area. All had a clinical history of seasonal birch pollen-related asthma and fulfilled the ATS criteria for asthma diagnosis during the birch pollen season (18). All subjects had a positive skin-prick test (wheal > 3 mm) to birch pollen, but none had positive skin tests to house-dust mite or molds. Six patients tested positive for cat or dog dander but had no daily contact with pets. Seven subjects allergic to grass pollen were not excluded, since the grass pollen season follows the birch pollen season.
Patients were studied pre-season in February/March and in-season
in May 1997. On neither occasion had they experienced a respiratory infection in the previous 2 mo. Patients had not used inhaled or systemic corticosteroids for at least 1 yr before the study, but they were
using inhaled 
2-agonist bronchodilators for asthma symptoms and
antihistamines (oral acrivastine and topical levocabastine) for rhinoconjunctival symptoms as required. Seven days before pre-season
and in-season bronchoscopies, monitoring of lung function was initiated with twice-daily peak expiratory flow (PEF) measurements using
Wright mini peak flow meters, and patients were asked to complete
diary cards recording their medication use (2-agonist puffs/d and antihistamines) and scores for asthma and rhinitis symptoms. Symptom
scores were on a scale of 0 to 3 where 0 = no symptoms, 1 = mild, 2 = moderate, and 3 = severe symptoms. Two days before each bronchoscopy, total serum IgE was assayed by ELISA, and bronchial responsiveness was measured as the provocation concentration of inhaled
methacholine required to decrease FEV1 by 20% from baseline
(methacholine PC20 FEV1).
Bronchoscopy and Bronchial Mucosal Biopsy
Twelve asthmatic patients underwent flexible fiberoptic bronchoscopy according to American Thoracic Society guidelines (19) on the pre-season and in-season study days. After premedication with morphine-scopolamine and inhaled salbutamol (0.2 mg), and under local anesthesia with 2% xylocaine, an Olympus BF1 bronchoscope (Olympus Corp., Lake Success, NY) was used to obtain a bronchial mucosal biopsy from the subcarina of an upper lobe segment.
Pre-season and in-season biopsies from the 12 subjects were embedded into glycol methacrylate (GMA) resin, sectioned, and immunostained for leukocyte markers (mast cells, eosinophils, monocyte-macrophages, neutrophils, and T-cells) and for enzymes of the leukotriene pathway (5-LO, FLAP, LTA4 hydrolase, LTC4 synthase) and the prostanoid pathway (COX-1, COX-2, PGD2 synthase) as described (20, 21). Immunopositive cells were counted blind on coded slides by a single observer. In six to eight representative biopsies, selected eicosanoid enzymes were also colocalized to relevant leukocyte markers by Camera lucida (22). Further details on immunostaining and antibody specificity are provided in the online data supplement to this article.
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Effect of Seasonal Allergen on Clinical Measures of Asthma Severity
Airborne birch pollen counts in Gothenburg were monitored
daily during 1997 with a Burkard volumetric trap (Figure 1).
Asthmatic patients (n = 12) experienced significant deteriorations in bronchial responsiveness during the birch pollen season compared with pre-season values, with median PC20 FEV1
for methacholine falling from 14.0 to 2.7 mg/ml (p = 0.001, n = 12, Wilcoxon), accompanied by significant falls in morning and
evening PEF (Figure 2). Asthma symptom scores rose from
0.18 ± 0.09 to 0.68 ± 0.10 (p = 0.004), and there was a trend to
increased use of inhaled 2-agonists from 0.04 ± 0.04 to 0.23 ± 0.07 puffs/d (p = 0.19). Seasonal allergen exposure also significantly increased rhinitis symptom scores from 0.23 ± 0.07 to
1.07 ± 0.10 (p < 0.0001), accompanied by a significant rise in
the use of oral and topical antihistamines (p = 0.017).
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Leukocyte Markers in Bronchial Biopsies
Immunohistochemical analysis of paired bronchial biopsies obtained from 12 asthmatics showed that mean eosinophil counts more than doubled, from 15.5 ± 3.3 to 35.0 ± 10.4 cells/mm2 (p = 0.035), and CD68+ macrophage counts rose from 8.3 ± 1.8 to 14.9 ± 2.6 cells/mm2 (p = 0.019), compared with pre-season biopsies (Figure 3). Neutrophil counts showed a nonsignificant trend towards a rise in-season (p = 0.08), whereas tryptase-positive (AA1) mast cell counts were stable. There were no significant changes in CD3 T-lymphocytes or in CD4, CD8, or CD25 T-cell subsets (Table 1).
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Eicosanoid Pathway Enzyme Expression in Bronchial Biopsies
Immunohistochemistry of bronchial biopsies from the 12 asthmatic subjects revealed clear cell-associated staining for the eicosanoid pathway enzymes. During the pollen season, mean counts of 5-LO+ cells almost doubled, from 31.7 ± 7.9 to 58.2 ± 8.2 cells/mm2 (p = 0.02, n = 12), and FLAP+ cell counts more than doubled, from 8.7 ± 2.9 to 18.1 ± 3.7 cells/mm2 (p = 0.04) (Figure 4). There was a smaller, but significant, rise in the counts of LTA4 hydrolase+ cells (p = 0.05), and counts of cells expressing LTC4 synthase nearly quadrupled, from 1.06 ± 0.25 to 3.87 ± 1.03 cells/mm2 (p = 0.021) (Figure 4). In contrast, counts of cells immunostaining for COX-1, COX-2, and PGD2 synthase remained stable in-season (Figure 5).
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Cellular Localization of Eicosanoid Pathway Enzymes
Using the camera lucida technique (21, 22) on adjacent thin sections of biopsies from a subgroup of six to eight asthmatics selected as representative of the full group of 12 subjects, immunostaining for 5-LO, FLAP, LTC4 synthase, COX-1, and COX-2 was colocalized to eosinophils, mast cells, and macrophages, both before and during the birch pollen season.
In pre-season biopsies, 5-LO, FLAP, and LTC4 synthase were each relatively evenly distributed between the relatively low numbers of eosinophils, mast cells, and macrophages (Figure 6). In contrast, mast cells represented the majority both of COX-1+ cells (53.8%) and of COX-2+ cells (53.2%), with eosinophils accounting for only 13.8% and 22.5%, respectively.
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During the birch pollen season, three-quarters of the rise in 5-LO+ cell counts in paired biopsies from eight representative patients could be accounted for by increases in the counts of 5-LO+ eosinophils (from 9.1 ± 3.2 to 17.9 ± 4.7 cells/mm2; p = 0.18, n = 8) and of 5-LO+ macrophages (from 6.6 ± 1.6 to 12.7 ± 2.8 cells/mm2; p = 0.05) (Figure 6), with no significant change in the numbers of 5-LO+ mast cells. The rise in FLAP+ cells in paired biopsies from eight representative patients in-season could be entirely accounted for by increases in the numbers of FLAP+ eosinophils (from 1.4 ± 0.8 to 7.8 ± 2.9 cells/mm2; p = 0.09) and FLAP+ mast cells (from 1.5 ± 0.4 to 7.7 ± 2.6 cells/ mm2; p = 0.03) (Figure 6). In addition, the proportion of mast cells immunostaining for FLAP rose in-season from 8.4 to 17.4% (p = 0.06), and the proportion of eosinophils expressing FLAP also tended to rise in-season, from 7.9 to 14.2% (p = 0.09), but there were no changes in FLAP expression in macrophages.
In paired biopsies from six representative patients, the rise in LTC4 synthase+ cells in-season was entirely accounted for by a significant increase in LTC4 synthase+ eosinophils, from 0.2 ± 0.1 to 4.2 ± 2.0 cells/mm2 (p = 0.048) (Figure 6). The proportion of eosinophils staining for LTC4 synthase rose significantly, from 1.5 ± 1.1 to 9.0 ± 2.6% (p = 0.047), whereas the proportions of mast cells and macrophages staining for LTC4 synthase did not change (Figure 6, lower panel ).
In paired biopsies from six representative subjects, counts of cells expressing COX-1 and COX-2 did not change significantly in-season compared with pre-season values, and their distribution remained predominantly (45% to 55%) in mast cells, the total counts of which also did not significantly change (Figure 3). However, there was a significant decrease in the proportion of mast cells expressing COX-1, falling from 71.6 ± 4.6% pre-season to 46.1 ± 7.1% in-season (p < 0.0001), and a trend for the proportion of mast cells expressing COX-2 to fall from 38.7 ± 7.0% to 21.2 ± 5.0% (p = 0.18). These changes in COX expression within the mast cell populations were not seen in eosinophils or macrophages.
Relationship of Leukocyte Counts and Eicosanoid Enzyme Immunostaining to Clinical Measures of Asthma Severity
Among the clinical measures of disease severity, only PEF values showed meaningful relationships to leukocyte markers in
bronchial biopsies. In the full group of 12 asthmatic subjects,
in-season PEF values correlated inversely with macrophage
counts (PEFA.M.: 
= 
0.78, p = 0.003; PEFP.M.: = 0.76, p = 0.004), but not significantly with counts of other cell types. Significant relationships were observed between PEF and the
counts of cells immunostaining for leukotriene enzymes. Pre-season PEFA.M. and PEFP.M. values correlated inversely with
counts of 5-LO+ cells ( = 0.59, p = 0.04, and = 0.61, p = 0.036, respectively; n = 12) (Figure 7), particularly with counts of
5-LO+ eosinophils ( = 0.80, p = 0.017, and = 0.78, p = 0.023). Pre-season PEFA.M. and PEFP.M. values also correlated
inversely with FLAP+ cells ( = 0.62, p = 0.032, and = 0.64, p = 0.026).
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During the pollen season, PEFA.M. and PEFP.M. values
again correlated significantly with counts of 5-LO+ cells ( = 0.65, p = 0.023, and = 0.66, p = 0.021, respectively, n = 12) (Figure 7), particularly with counts of 5-LO+ macrophages
( = 0.64, p = 0.09 and = 0.62, p = 0.1) and weakly with
5-LO+ eosinophils ( = 0.40, p = 0.2, and = 0.43, p = 0.16). There were no significant relationships in-season between PEF values and immunostaining for COX-1 and COX-2.
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The aim of this study was to investigate the cellular expression of central enzymes of the cysteinyl-leukotriene and prostanoid biosynthetic pathways at the bronchial mucosal site of airway inflammation in seasonal allergic asthma, and to relate their expression to lung function.
The 12 birch pollen-sensitive asthmatics recruited to the study were investigated before and during the well-defined birch pollen season in Sweden (Figure 1). To preclude the masking of seasonal changes by concomitant exposure to perennial allergens, as observed in previous studies of natural allergen (23), subjects were either not sensitive or avoided exposure to common perennial allergens. The seasonal impact of allergic inflammation on the airway was confirmed by the increased responsiveness to methacholine and by reductions in morning and evening peak expiratory flow in-season (Figure 2).
The profile of inflammatory leukocyte changes observed in the bronchial biopsies during birch pollen exposure extends the previous finding of elevated eosinophil counts in BAL fluid during a season of high birch pollen counts (24), but it is distinct in important aspects from that observed after allergen bronchoprovocation in the laboratory. Our data confirm the findings of an influx of eosinophils, neutrophils, and monocyte-macrophages in bronchial biopsies and BAL fluid after allergen challenge (25), but contrast with reports of increases in mast cell and T-cells (28, 29). The lack of significant changes in these cells may reflect the mild disease experienced by the asthmatics in this study, but it is noteworthy that their absence did not preclude rises in eosinophil and macrophage counts, increased airway reactivity, and decreased pulmonary airflow (Figures 2 and 3).
In BAL fluid and bronchial mucosal biopsies of symptomatic asthmatics, there is increased expression of cytokines and chemokines that can enhance the recruitment and reduce the apoptosis of eosinophils and/or monocytes in vitro, including interleukin-5, eotaxin, and RANTES (22, 30, 31). Cys-LTs also elicit eosinophilia in the bronchial mucosa when inhaled by asthmatics, and leukotriene modifier drugs reduce eosinophil counts in the BAL fluid and blood of asthmatics (3, 4). Because the eosinophil is itself a source of LTC4 in vitro (32), a cycle of cys-LT synthesis and eosinophil recruitment may contribute to sustained airway obstruction.
During the pollen season, highly significant increases were observed in bronchial mucosal counts of cells immunostaining for LT pathway enzymes (Figure 4). Expression of LTC4 biosynthetic enzymes was colocalized by camera lucida to mast cells, eosinophils, and macrophages, which are known to synthesize LTC4 in vitro (32). Increases in 5-LO+ and FLAP+ cells were mostly attributable to increased eosinophils and macrophages, whereas for LTC4 synthase, the increase was entirely attributable to eosinophils (Figure 6), suggesting that increased prevalence of LTC4 synthase-positive eosinophils may be the most important factor increasing the potential of the allergic asthmatic airway to generate cys-LTs. Furthermore, an increased proportion of the eosinophil population immunostained for FLAP and LTC4 synthase in-season compared with pre-season (Figure 6). Because the expression of 5-LO, FLAP, and LTC4 synthase and the capacity for cys-LT synthesis are induced in human eosinophils in vitro by IL-3, IL-4, and IL-5 (33, 35, 36), our data may indicate that the cytokine microenvironment also induces their expression and activity within eosinophils in the seasonal allergic asthmatic lung. In contrast, the increase in counts of cells immunostaining for LTA4 hydrolase is probably related to the significant increase in macrophages and the trend towards increased neutrophil counts. Increased potential for the synthesis of LTB4 by these cells may contribute to influx of leukocytes, including eosinophils, in the seasonal asthmatic airway.
We have previously reported 19-fold increments in LTC4 synthase immunostaining in biopsies from patients with moderately severe aspirin-intolerant asthma (21), but the 2- to 4-fold increases in 5-LO, FLAP, and LTC4 synthase immunostaining in-season in the present study are consistent with the relatively mild asthma experienced by our subjects. Similar 2-fold increments in 5-LO and FLAP immunostaining were seen in bronchial biopsies of adults with mild persistent asthma compared with those in normal subjects (37). Biopsies from patients with moderately severe persistent asthma have fivefold elevated counts of LTC4 synthase+ cells (21). 5-LO and FLAP immunostaining increased 2- to 3-fold in normal subjects after infection with human rhinovirus serotype 16, and this correlated with scores for cold symptoms (37). Although relatively modest, the in-season changes in the present study are thus consistent with those seen in persistent asthma of varying severities and with virus-induced airway inflammation. The changes are specific to the 5-LO pathway not to the COX pathway, and are temporally associated with deteriorations in lung function, methacholine responsiveness, and asthma symptom scores in the pollen season.
Assays of cys-LTs in urine, induced sputum, and BAL fluid have been extensively used to show activation of the 5-LO pathway after bronchoprovocation challenges in susceptible subjects such as allergen (17) and aspirin (21). In the absence of laboratory bronchoprovocation, they are less consistent in showing elevated basal production of cys-LTs in asthma, with the notable exception of aspirin-intolerant patients. In the present study, the mild allergic disease experienced by the subjects, the long time-scale of the study, the low levels of environmental aeroallergen, and the lack of bronchoprovocation to activate the 5-LO pathway acutely meant that measurements of cys-LTs in BAL or other biologic fluids were unlikely to be sufficiently sensitive to reflect immunopathologic changes in 5-LO pathway enzyme expression in the bronchial mucosa. Instead, the functional relevance of these changes is supported by the significant inverse correlations of 5-LO immunostaining with morning and evening PEF, both before and during the pollen season (Figure 7). In-season, the relationship was particularly influenced by the numbers of 5-LO-positive eosinophils and macrophages. These cells also expressed the majority of FLAP and LTC4 synthase, and it is likely that activation of the LTC4 biosynthetic pathway within these cells in a principal factor leading to airway dysfunction during the birch pollen season.
In contrast to the 5-LO pathway enzymes, immunostaining for COX-1, COX-2, and PGD2 synthase did not change in the birch pollen season (Figure 5). These enzymes were predominantly expressed in mast cells, the populations of which remained static (Figure 3). However, the proportion of mast cells immunostaining for COX-1 and COX-2 fell, whereas the proportion immunostaining for FLAP tended to rise. In murine bone-marrow-derived mast cells in vitro, the expression of 5-LO, FLAP, and LTC4 synthase is promoted by IL-3, whereas expression of COX-1, COX-2, and PGD2 synthase requires stem cell factor (c-kit ligand) (38, 39). Our biopsy data suggest that the cytokine microenvironment in the seasonal asthmatic lung may impair the ability of mast cells to generate prostanoids but promote their potential to generate cys-LTs. Mast-cell-derived PGD2 contributes modestly to early bronchoconstriction in atopic subjects challenged with inhaled allergen (6), but the overall lack of changes in COX-1, COX-2, and PGD2 synthase immunostaining and the absence of meaningful relationships with clinical measures of asthma severity suggest that PGD2 does not contribute significantly to airway dysfunction in seasonal allergic asthma.
The significantly increased immunodetection of each protein of the 5-LO/FLAP/LTC4 synthase pathway in this study contrasts with our previous investigation in bronchial biopsies from patients with aspirin-intolerant asthma (AIA) (21). In that study, AIA biopsies had significantly more cells expressing LTC4 synthase but without increased detection of the other 5-LO pathway proteins, and the selective increase in LTC4 synthase immunostaining correlated with elevated BAL fluid cys-LT levels and with bronchial responsiveness to inhaled aspirin (21). Much of the increased prevalence of LTC4 synthase+ cells was accounted for by a higher proportion of eosinophils immunostaining for the enzyme in AIA biopsies (50%) than in the aspirin-tolerant asthma biopsies (21%). In the present study of adults with mild asthma, the fraction of eosinophils immunostaining for LTC4 synthase increased in-season from 1.5% to 9%. Taken together, our data suggest that the proportion of eosinophils expressing LTC4 synthase may be closely related to the contribution of cys-LT to asthma severity.
In summary, our present data and those of our previous study (21) argue strongly that clinical and pathophysiologic subtypes of asthma can be traced back to mechanisms involved in lipid mediator generation. Immunopathologic changes in bronchial biopsies from seasonal asthmatics were restricted to the LT pathway, not the prostanoid pathway, and mainly associated with airway eosinophils and macrophages. Both before and during the birch pollen season, counts of cells expressing 5-LO showed marked inverse correlations with peak expiratory flow, suggesting that airway dysfunction in seasonal asthma is closely linked to an increased capacity of the airway to generate leukotrienes.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. Tony Sampson, Medical Specialties, Level F South Block (825), Southampton General Hospital, Southampton, S016 6YD, UK. E-Mail: aps{at}soton.ac.uk
(Received in original form August 24, 2000 and accepted in revised form September 27, 2001).
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.orgAcknowledgments: The writers acknowledge the generous gift of antibodies to 5-LO, FLAP, and LTA4 hydrolase from Dr. Jilly Evans (Merck & Co., West Point, PA). They also thank Dr. Sue Wilson, Mrs. Janet Underwood, and Mrs. Angela Tuck for advice on immunohistochemical techniques.
Supported by grants from the Wessex Medical Trust (Southampton, UK), by Programme Grant G8604034 from the UK Medical Research Council, by the Freemasons Grand Lodge 250th Anniversary Fund (Royal College of Surgeons of England, London, UK), by the Swedish Foundation for Healthcare Sciences & Allergy Research, by the Swedish Asthma & Allergy Association, by Grants A1-31599 & HL-361110 from the National Institutes of Health, by the Hyde & Watson Foundation (USA), and by a grant-in-aid from Merck & Co (New Jersey, USA).
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