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Published ahead of print on August 25, 2005, doi:10.1165/rcmb.2005-0124OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 531-540, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0124OC

Localization and Upregulation of Cysteinyl Leukotriene-1 Receptor in Asthmatic Bronchial Mucosa

Jie Zhu, Yu-Sheng Qiu, David J. Figueroa, Venkata Bandi, Helen Galczenski, Kaoru Hamada, Kalpalatha K. Guntupalli, Jilly F. Evans and Peter K. Jeffery

Imperial College London at the Royal Brompton Hospital, London, United Kingdom; Merck & Co., Inc., West Point, Pennsylvania; Baylor College of Medicine, Houston, Texas; and Nara Medical University, Nara, Japan

Correspondence and requests for reprints should be addressed to Professor Peter K. Jeffery, Lung Pathology, Imperial College London, Royal Brompton Hospital, Sydney Street, London SW3 6NP, United Kingdom. E-mail: p.jeffery{at}imperial.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We have tested the hypothesis that the CysLT1 receptor is expressed by a variety of bronchial mucosal immune cells and that the numbers of these cells increase in asthma, when stable and in exacerbations. We have applied in situ hybridization and immunohistochemistry to endobronchial biopsy tissue to identify and count inflammatory cells expressing CysLT1 receptor mRNA and protein, respectively, and used double immunohistochemistry to identify the specific cell immunophenotypes expressing the receptor. Double-labeling demonstrated that bronchial mucosal eosinophils, neutrophils, mast cells, macrophages, B-lymphocytes, and plasma cells, but not T-lymphocytes, expressed the CysLT1 receptor. The numbers of CysLT1 receptor mRNA and protein positive inflammatory cells in nonsmoking, nonatopic control subjects without asthma were 13 and 16 mm–2, respectively (median values; n = 15), and were significantly greater in stable asthma (50 and 43 mm–2, respectively; n = 17; P < 0.001). Compared with stable asthma, there were further significant increases in subjects hospitalized for a severe exacerbation of their asthma (mRNA: median = 113 and protein: 156 mm–2; n = 15; P < 0.002). For the combined data of both asthma subgroups, there were strong positive correlations between the increased numbers of CD45+ leukocytes and the greater numbers of cells expressing CysLT1 receptor (mRNA: r = 0.60, P < 0.001; protein: r = 0.73, P < 0.0001). In conclusion, a variety of immunohistologically distinct inflammatory cells express the CysLT1 receptor in the bronchial mucosa and both these and the total number of leukocytes increase in mild stable disease and increase further when there is a severe exacerbation of asthma.

Key Words: asthma • cysteinyl leukotrienes • exacerbation • receptor


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Leukotriene receptor antagonists (LTRA) represent an additional and novel class of treatment for asthma (1–6), and recent data indicate a useful role for LTRA in the management of severe exacerbations (7, 8). Cysteinyl leukotrienes (CysLTs) are associated with the recruitment of eosinophils to the bronchial mucosa in mild asthma and are involved in the response to experimental allergen challenge (9–11). The role of leukotrienes has been summarized in recent reviews (12, 13) and includes: recruitment of airway inflammatory cells, bronchoconstriction, increased vascular permeability, and mucous hypersecretion. Moreover, there is accumulating evidence that CysLTs may play a role in the remodeling process of asthma, including the proliferation of airway smooth muscle (12, 14). The role for CysLTs in epidermal growth factor–induced airway smooth muscle proliferation is supported by the effectiveness of LTRAs in inhibiting these processes (14–16).

There are two pharmacologically defined G protein–coupled LT receptors described thus far: CysLT1 and CysLT2 (17, 18). More may exist, and isoforms are now being reported (19). The CysLT1 receptor is the main receptor for the actions of CysLTs in the lung and the prime target for LTRAs in the treatment of asthma (17, 18, 20) and allergic rhinitis (21–23). Figueroa and colleagues have reported CysLT1 receptor expression in histologically normal lung tissue resected from a smoker without asthma and also its expression in peripheral blood leukocytes (17, 24). The receptor has also been localized to nasal lavage cells obtained from symptomatic patients with seasonal allergic rhinitis (21) and in human nasal mucosa from patients with perennial rhinitis (22). There has been no histologic description of CysLT1 receptor gene or protein expression in the bronchial mucosa of individuals with asthma, there are no data concerning the phenotype of bronchial inflammatory cells that express it, and it is not known whether the numbers of CysLT1 receptor–positive cells are altered in asthma.

The successful cloning of the CysLT1 receptor (17) now provides the opportunity to apply molecular in situ hybridization (ISH) and immunohistochemical methods to endobronchial biopsies for the purposes of identifying the phenotypes of airway mucosal inflammatory cell that express it and counting the numbers of CysLT1 receptor–positive inflammatory cells in distinct subject groups. We examine here the hypothesis that the CysLT1 receptor is expressed by a variety of airway mucosal inflammatory cells in asthma and that the numbers of cells expressing it are increased in asthma, when stable and in association with an exacerbation. We have obtained endobronchial mucosal biopsies after obtaining informed consent, and have performed studies to locate, identify, and count cells that express the CysLT1 receptor mRNA or protein in our three subject groups: nonatopic control subjects without asthma, subjects with mild asthma in a stable phase of their disease, and subjects with asthma who were hospitalized for an acute severe exacerbation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Subjects
The study conformed to the declaration of Helsinki. The institute review boards for human studies of each hospital approved the study protocols specifically written for this project and each subject or, in the case of the intubated subjects, their surrogate gave informed, written consent. There were three subject groups. Group 1 was made up of nonsmoking surgical control subjects (henceforth referred to as NSC; n = 15) with normal lung function (i.e., FEV1 > 80% of predicted; FEV1/FVC > 70%) who were having a surgical procedure under general anesthesia for nonbronchopulmonary disease (e.g., hysterectomy or cholecystectomy). They were nonatopic (i.e., with negative skin tests for a panel of common allergen extracts). Blood eosinophil counts were normal (i.e., within the range of 0.0–0.4 x 109/liter). Group 2 consisted of patients with stable asthma (S-asthma, n = 17) (see Table 1); these were nonsmokers with intermittent, mild persistent asthma as defined clinically using Global Initiative for Asthma Guideline criteria (prebronchodilator FEV1 of >= 70% predicted and FEV1/FVC > 70%) (25). All had a clinical history of asthma of at least 1 yr duration. Their asthma was controlled using short-acting {beta}2-agonists as required and had not received regular treatment with inhaled, oral, or injected steroids or inhaled cromolyn, nedocromil, or ketotifen for more than 3 mo, or any single dose for >= 6 wk before the study start. They had not taken oral theophylline within 2 wk or long-acting {beta}2-agonist within 1 wk or short-acting {beta}2-agonist within ~ 6 h before the study start. They had not smoked for at least a year prior to the study, and their smoking history was <= 10 pack-years. Otherwise the subjects were in good health and had had no other significant disease or medication before or during the study. Each subject underwent a pre-study screening visit, in which a full clinical assessment including baseline spirometry, chest X-ray, ECG, skin prick testing to common allergens, and histamine challenge testing were performed. All the subjects demonstrated airway hyperresponsiveness to histamine with a PC20 of <= 4 mg/ml. Group 3 comprised patients with an acute severe exacerbation of asthma (E-asthma, n = 15; see Table 1), defined as a sudden worsening of asthma symptoms (i.e., wheezing, breathlessness, chest tightness, and cough) and lung function (26). Each had experienced one of the following features: continuous symptoms, frequent exacerbations or nighttime asthma symptoms, PEF or FEV1 <= 60% of predicted, an FEV1/FVC < 70%, and variability >= 30%. All required hospitalization and did not respond to usual treatment requiring emergent intubation and mechanical ventilation for respiratory failure (26). All of these patients were on {beta}2-agonist nebulizer and received between 1 and 3 doses of corticosteroids (Solumedro 60–80 mg/6–12 h) intravenously during the admission. All the patients in this group had a past history of allergy. All were on multiple daily controller medication before the exacerbation: all had received short-acting {beta}2-agonist as required, 12 of 15 had been receiving inhaled steroids, and 7 of 15 had received oral steroids (Table 1). Only one patient was a current smoker, with a 15 pack-year history. None had symptoms of chronic bronchitis and most had the reversibility (> 30%) characteristic of asthma.


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  		TABLE 1. CLINICAL CHARACTERISTICS AND TREATMENT FOR INDIVIDUALS WITH EXACERBATED ASTHMA, THOSE WITH STABLE ASTHMA, AND NONSMOKING CONTROL SUBJECTS

 
With permission, we included tissue obtained at open lung biopsy from a previous case of severe allergic asthma to examine in greater detail the specificity of CysLT1-receptor staining in bronchiolar smooth muscle and to compare airway smooth muscle CysLT1-receptor expression with that of adjacent inflammatory cells. The surgical case was a 24-yr-old male smoker (30 cigarettes/d for 5 yr) suffering variably mild or severe attacks. His clinical history revealed variable airway obstruction that responded to bronchodilator. He had airways hyperresponsiveness and peripheral blood eosinophilia (at times 14% eosinophils) and in addition chest radiographic evidence of small airway involvement (FEV1 = 41% of predicted; FEV/FVC 51%). In light of this and to make decisions about his clinical management he was referred, following informed written consent, for an open lung biopsy. The histologic findings demonstrated all the inflammatory and structural changes of asthma but, in addition, demonstrated a bronchiolar hyperplasia of lymphoreticular aggregates. The patient had received treatment with inhaled beclamethasone diproprionate (400–800 µg/d) with clenbuterol ({beta}2-agonist) (40 µg/d) and oral prednisone (5–7.5 mg/d) or theophylline (400 mg/d).

Bronchoscopy and Biopsy
Biopsies from NSC subjects were taken immediately after intubation before infusion of prophylactic antibiotics. All E-asthma patients underwent bronchoscopy, and biopsies were taken within 24 h of their hospital admission. Biopsies were taken using an Olympus, type BF P10 or BF P20D (Olympus Co., Tokyo, Japan) with alligator forceps (microvasive radial jaw single use biopsy forceps, 1.8 mm external diameter; Boston Scientific Corporation, Natick, MA) from the third, fourth, and fifth order bronchial divisions of the right or left lung. Biopsies were fixed immediately in 10% formaldehyde at room temperature. After processing, they were embedded in paraffin wax. Serial sections of 5 µm thick were cut and stained with hematoxylin and eosin (H&E) for assessment initially of their size and morphology.

Positive and Negative Control Cells
Cos-7 monkey kidney cells (COS cells) transfected with CysLT1 receptor or CysLT2 receptor cDNA (17) and processed in the same manner as our paraffin-embedded biopsy tissue, were used to confirm the specificity of the CysLT1 receptor probe and antisera. They were also used as positive and negative controls for the following ISH and immunohistochemistry (IHC) procedures, respectively (17, 18, 24).

Nonisotopic ISH
CysLT1 receptor cDNA (1,033 bp) in pcDNA3.1 zeo+ vector (Merck Research Laboratories, West Point, PA) was subcloned into PGEM-3Z vector. The nucleic acid sequences of the CysLT1 receptor probe have been described previously (17). PGEM-3Z containing CysLT1 receptor cDNA was expanded in JM109 Escherichia coli–competent cells. The antisense CysLT1 and sense CysLT1 receptor probes were linearized with HindIII and XbaI, respectively.

Preparation of cRNA probes. Digoxigenin (Dig)-labeled antisense and sense complementary ribonucleic acid (cRNA) probes were generated from cDNA using T7 and SP6 RNA transcription polymerises, respectively. 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.

Prehybridization. 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 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/trisodium citrate (SSC) and incubated in 20 µg/ml RNase A. The remaining probe hybridized with the mRNA of interest was localized 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.

IHC
CysLT1 receptor protein expression in the bronchial biopsies was detected by IHC, applying the EnVision–alkaline phosphatase (EV-AP) technique according to EnVision AP Kit (DAKO Ltd, Cambridge, UK). The selective, affinity purified, anti-human polyclonal antisera for CysLT1 receptor was raised in a goat against purified full-length recombinant CysLT1 receptor (17). We replaced the primary antibody with normal goat serum as a further negative control.

The paraffin sections were blocked with 20% normal rabbit serum (DAKO) in Tris-buffered saline (TBS) pH7.6. The sections were then immunostained with CysLT1 receptor polyclonal antibodies at 1:3,000 at room temperature, washed, and incubated with rabbit anti-goat immunoglobulins in TBS with 10% normal goat serum. After washing, the sections were incubated with AP-conjugated goat anti-rabbit immunoglobulin. After a further wash, bound AP was detected as a red product following 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 alkaline phosphatase anti–alkaline phosphatase (APAAP) was used to label CD45+ inflammatory cells, eosinophils, and neutrophils using mouse anti-human CD45, leucocyte common antigen mAb (DAKO M0701), mouse anti-EG2 (Pharmacia and Upjohn Ltd, Milton Keynes, UK), and mouse anti-neutrophil elastase (DAKO Ltd), respectively.

Double immunohistofluorecence staining was performed to determine which phenotype(s) of inflammatory cell expressed the CysLT1 receptor protein and to determine the colocalization of CysLT1 receptor protein and smooth muscle actin in the E-asthma group. Immunohistochemistry for the CysLT1-receptor was similarly performed using a polyclonal goat anti–full length recombinant human CysLT1-receptor antisera previously described (17, 24). The primary anti-human antibodies for the phenotypic identification of inflammatory cells coexpressing CysLT1-receptor were mouse anti-EG2 for eosinophils, anti–neutrophil elastase for neutrophils, anti–mast cell tryptase for mast cells (DAKO), anti-CD3 for T-lymphocytes (Novocastra, Newcastle-upon-Tyne, UK), anti-CD45RO (clone OPD4) for activated CD4+ T-lymphocytes (DAKO), anti-CD8 for cytotoxic/suppressor T cells (DAKO), anti-CD64 marking cells of the monocyte lineage and anti-CD19, a marker for B cells and B-cell precursors (Pharmingen, San Diego, CA), and anti-plasma cell–specific antigen (Novocastra) for plasma cells. A marker for smooth muscle actin (Zymed Laboratories, San Francisco, CA) was used in combination with CysLT1-receptor antisera for simultaneous detection of bronchial smooth muscle and expression of this receptor. Bound antibody was detected with a combination of the appropriate fluorescein isothiocyanate or Texas red–labeled donkey secondary antisera (Jackson Immunoresearch, West Grove, PA). Sections were counterstained with DAPI. Images were acquired using a Leica TCS-SP confocal microscope with lasers giving excitation wavelengths of 351–364 nm, 488 nm, 568 nm (Leica Microsystems UK Ltd, Milton Keynes, UK).

Quantification
Slides were coded to avoid observer bias. Areas of subepithelium, excluding those areas with mucus-secreting glands and bronchial smooth muscle, were assessed using an Apple Macintosh computer and Image 1.5 software (Apple Computer, Cupertino, CA). CysLT1 receptor mRNA+ and protein+ cells, CD45+ inflammatory cells (as an overall marker of the leukocyte population), eosinophils, and neutrophils in the entire subepithelial area of the biopsy 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. Two to three bronchial biopsies for each subject were measured and counted to take account of the within subject variability: the average of the counts for each subject was used for statistical analyses.

Statistical Analyses
The data for cell counts of bronchial biopsies were expressed as the number of cut cell profiles with a nucleus visible (i.e., positive cells) per mm2 of the subepithelium. The coefficient of variation (CV = SD/mean x 100) was used to express the error of repeat counts. The unpaired Student's t test was used for the statistical analyses of age and lung function data. The Mann-Whitney U-test was applied to test for differences between the groups in respect of cell counts as these data were non-normally distributed. Spearman's rank correlation was used as a test for correlation between numbers of EG2-positive eosinophils, neutrophils, or CD45+ leukocytes and cells positive for CysLT1 receptor mRNA or protein. A P value of < 0.05 was accepted as indicating a significant difference in the t test or Mann-Whitney U-tests. For Spearman's rank correlation, a value of P < 0.01 was used as the threshold for statistical significance following application of Bonferoni's correction for the multiple analyses we performed.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Demography
The demographic characteristics of the NSC and subjects with S-asthma or E-asthma from whom bronchial biopsies were obtained are summarized in Table 1. NSC and E-asthma groups were similar with regard to age. However, those in the S-asthma group were significantly younger than in either the NSC and E-asthma groups (P < 0.01). Both the FEV1% of predicted and FEV1/FVC values of the E-asthma group were significantly lower than those of the NSC and S-asthma groups (P < 0.001). The subjects in the NSC and S-asthma groups, and 14 of 15 subjects in E-asthma group, were nonsmokers.

Biopsy Quality
The endobronchial biopsies were 1–2 mm in diameter and of good quality defined by examination of H&E staining on the basis of size (~ 1 mm2) and, depending on the subject group, the presence in the NSC group of intact pseudostratified, ciliated columnar epithelium with relatively few goblet cells identification. Compared with the NSC group, the biopsies from S-asthma and E-asthma subjects showed the homogenous thickening of the epithelial reticular basement membrane characteristic of asthma. The epithelium of the asthma groups typically had goblet cell hyperplasia or focal squamous metaplasia and subepithelial infiltration by inflammatory cells, especially eosinophils.

CysLT1 Receptor mRNA and Protein Expression
Both ISH (data not shown) and IHC showed moderate to strong positive staining for the CysLT1 receptor mRNA and protein in the CysLT1 receptor-cDNA transfected COS cells (Figure 1A). CysLT2 receptor-transfected cells showed only weak crossreactivity with CysLT1 receptor RNA probe (data not shown), and were negative after the CysLT1 receptor antisera (Figure 1B). In contrast, CysLT2 receptor antisera showed a strong fluorescent signal on the CysLT2 receptor–transfected COS cells. There was an absence of signal with the mRNA "sense" controls (data not shown) and no immunostaining of COS CysLT1 receptor–transfected cells when the primary antibody was replaced by normal goat serum (Figure 1C).





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  		Figure 1. Immunohistochemical staining to test the specificity of CysLT1 receptor antisera. (A) Specific recognition of CysLT1 receptor protein in CysLT1 receptor–transfected COS cells. Strong specific fluorescent signal is seen in red and DAPI stained nuclei are green. (B) CysLT2 receptor–transfected COS cells are devoid of signal when stained with CysLT1 receptor antisera. (C) Immunohistochemical negative controls. Normal goat serum replaces CysLT1 receptor antisera and shows little nonspecific staining of CysLT1 receptor–transfected COS cells (internal scale bar = 20 µm).

 
Localization of the CysLT1 Receptor
Inflammatory cells in the subepithelium stained strongly for both CysLT1 receptor mRNA and protein. There were relatively few in the NSC group(Figures 2A and 3A) and obviously more in both the S-asthma and E-asthma groups (Figures 2B and 3B). Occasionally, CysLT1 receptor–immunopositive inflammatory cells were seen to have infiltrated the surface epithelium (Figure 3B). By comparison with the negative controls, vascular endothelium across all groups was positive for both CysLT1 receptor mRNA and protein (see Figures 2A, 2B, and 3A), whereas epithelial positivity was focal and of weak intensity in the control subjects (Figures 2A and 3A) and moderate to strong in the subjects with asthma (Figures 2B and 3B). Bronchial biopsies from the E-asthma group, either hybridized with the sense CysLT1 receptor mRNA probe or those undergoing the immunostaining procedure but without the primary CysLT1 receptor antiserum, were included and did not stain (Figures 2C and 3C).





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  		Figure 2. Nonisotopic ISH of a bronchial biopsy for CysLT1-receptor mRNA expression. Positive signal is visualized as blue/black with the BCIP/NBT technique. Cells are counterstained with nuclear fast red. (A) A nonsmoking control subject showing relatively few CysLT1 receptor–positive subepithelial inflammatory cells as is the endothelium of bronchial vessels. (B) Acute severe exacerbation of asthma. Whereas there is moderate positivity in the cytoplasm of some but not all epithelial cells (arrowheads), there is strong staining of many inflammatory cells infiltrating the subepithelial zone. Blood vessel endothelium also expresses CysLT1-receptor mRNA (arrows). (C) The sense control probe shows an absence of signal in a biopsy of a case of a severe exacerbation of asthma (original magnification: x200).

 




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  		Figure 3. IHC to localize CysLT1-receptor protein in bronchial biopsies. The protein is seen as red fuchsin positivity with cell nuclei counterstained blue by hematoxylin. (A) A nonsmoking control subject showing expression of protein in only a few of the subepithelial inflammatory cells. (B) Acute severe exacerbation of asthma demonstrating an increased number of CysLT1-receptor protein–positive inflammatory cells in both subepithelium and epithelium (arrows). Some epithelial cells appear to show expression of the protein also. (C) Using normal goat serum instead of anti–CysLT1-receptor antibody, there is no positive stain for protein in the biopsy of a severe exacerbation of asthma (original magnification: x200).

 
In the E-asthma group, double immunofluorescence staining identified EG2+ eosinophils (Figures 4A–4C), neutrophils (Figure 4D–4F), mast cells (Figure 4G–4I), B-lymphocytes and plasma cells (Figures 4J–4L), and monocytes (Figures 4M–4O), and each co-expressed CysLT1 receptor protein. However, CD3+ (Figure 5), CD4+, and CD8+ T-lymphocytes were negative with the CysLT1 receptor antisera.

















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  		Figure 4. Double immunofluorescence staining for identification of co-localization of CysLT1-receptor with inflammatory cells of distinct phenotype in a bronchial biopsy from a patient with asthma with a severe exacerbation. (A, D, G, J, M) CysLT1-receptor protein immunopositivity is illustrated with Texas red fluorescence. (B) Eosinophils stained with anti-human EG2+. (E) Neutrophils stained with anti-human neutrophil elastase. (H) Mast cells stained by anti-human mast cell tryptase. (K) Plasma cells stained by anti-plasma cell-specific antibody. (N) Monocytes stained by anti-human CD64 antibody and shown by the green fluorescence of FITC. Co-expression of CysLT1-receptor with (C) EG2+ eosinophils, (F) neutrophils, (I) mast cells, (L) plasma cells, and (O) CD64+ cells are seen as yellow fluorescence (in each case, internal scale bars = 10 µm). Nuclei are counterstained blue with DAPI.

 


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  		Figure 5. Double immunofluorescence staining for CysLT1-receptor is illustrated with Texas red and CD3+ T-lymphocytes shown by green FITC fluorescence. No yellow fluorescent double-labeled cells can be seen. Nuclei are counterstained blue with DAPI (internal scale bar = 10 µm).

 
As expected, airway smooth muscle myocytes stained positively for the CysLT1 receptor but, unexpectedly, many showed relatively weak and, in some instances, no staining for either CysLT1 receptor mRNA or protein. This variability of smooth muscle myocyte positivity was seen across all subject groups. Using the same techniques applied previously to frozen lung material (17), we were able to identify, in our paraffin wax–embedded material, colocalization of the immunostaining for the CysLT1 receptor with a few, but not all, areas of immunostaining for actin, as shown in a biopsy from a patient with an exacerbation of asthma (Figures 6A–6C). In the patient with asthma from whom we had previously obtained an open lung biopsy (which allowed us to examine greater amounts of airway smooth muscle than previously possible), we found a similar pattern of variability to that seen in the relatively smaller bronchial biopsies. By comparison to the bronchiolar smooth muscle of this surgical case, the adjacent inflammatory cells were strongly positive for CysLT1 receptor mRNA (Figures 7A and 7B). In some cases, background nonspecific staining of adjacent elastic tissue was intense, such that it obscured the weak staining of the smooth muscle (Figure 7B). The morphology and elastic–van Gieson staining in the open lung biopsy material confirmed that it was the elastic component, surrounding and intermingled with the myocytes that stained rather than the area corresponding to bronchiolar smooth muscle myocytes per se (Figure 7C).





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  		Figure 6. Double immunohistochemistry for the CysLT1-receptor and smooth muscle actin, applied to a bronchial biopsy from an asthmatic patient with a severe exacerbation. (A) CysLT1-receptor protein immunopositivity is illustrated with Texas red. (B) Areas of smooth muscle actin (SMA)-positive airway smooth muscle stain FITC green. (C) Structures showing co-expression of CysLT1-receptor and (SMA) are seen as yellow (internal scale bar = 20 µm). In each case, nuclei are counterstained blue with DAPI. Note that not all muscle is double-stained (e.g., the central muscle bundle [arrow]).

 




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  		Figure 7. Human surgical biopsy in asthma. (A) ISH using oligonucleotide probes and tyramide-enhanced Texas red fluorescence for the CysLT1-receptor shows robust signals on the large influx of bronchiolar and parenchymal inflammatory cells. The smooth muscle bundle encompassed by the yellow box demonstrates the apparent absence of CysLT1-receptor expression. Nuclei are stained with DAPI blue (internal scale bar = 20 µm). (B) Higher magnification of the area outlined in A shows only focal dense red staining of smooth muscle myocytes as compared with the strong bright red/pink staining of inflammatory cells. Elastic fibers show high nonspecific dark red background staining (arrow) (internal scale bar = 20 µm). (C) Elastic Van Gieson staining (EVG) of the same biopsy shows the smooth muscle bundles clearly and adjacent elastic components (black) adjacent and intermingled with the muscle bundle. Collagen is counterstained red (internal scale bar = 20 µm).

 
Quantification of Leukocytes and Cells Expressing the CysLT1 Receptor
The coefficients of variation (CV) for repeat counts of cells immunopositive for CysLT1 receptor mRNA and protein by one observer were 5% and 6%, respectively. The data for the counts of the numbers of sub-epithelial cells expressing mRNA or protein are summarized in Figure 8. Compared with NSC, the values for the S-asthma group were significantly greater for both CysLT1 receptor mRNA+ (3.8-fold) (P < 0.001) and protein+ cells (2.7-fold) (P < 0.001). The numbers of CysLT1 receptor mRNA+ and protein+ cells in exacerbations of asthma were significantly higher than those of the stable asthma group, 2.3-fold (P < 0.002) and 3.6-fold (P < 0.001), respectively (Figure 8). There were significantly greater numbers of CD45+ leukocytes in the S-asthma group (median [range] = 634.7 [199.2–1,469.2]) than in the NSC group (median [range] = 144.6 [42.5–534.4]; P < 0.0001). Compared with the S-asthma group, the E-asthma group showed a further significant increase in the number of CD45+ leukocytes (median [range] = 992.7 [367.8–3,393.8] P < 0.02).



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  		Figure 8. Graphs of counts for CysLT1-receptor mRNA and protein positive cells in bronchial biopsies of non-smoker control (NSC), S-asthma, and E-asthma groups. The data are expressed as the number of positive cells per mm2 of subepithelium. Dots show individual counts and horizontal bars show median values (Mann Whitney U test).

 
Correlations
The number of cells expressing CysLT1 receptor mRNA per mm2 subepithelial area in each groups correlated positively with the number expressing the receptor protein (r = 0.53–0.67, P < 0.01 Spearman Rank Correlation). There were no significant associations between the numbers of EG2-positive eosinophils or neutrophils and the numbers of cells expressing CysLT1 receptor mRNA or protein in either the S-asthma or E-asthma groups. However, there were strong positive correlations between the numbers of CD45+ leukocytes and the numbers of cells expressing CysLT1 receptor mRNA or protein when the data for the S-asthma and E-asthma groups were combined (mRNA: r = 0.60, P < 0.0001; Protein: r = 0.73, P < 0.001) (Figure 9) and the significant positive correlations remained for the protein when each asthma subgroup was considered alone (S-asthma: r = 0.50, P < 0.005; E-asthma: r = 0.75, P < 0.002).



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  		Figure 9. Corrrelations between the numbers of CD45+ inflammatory cells and CysLT1 receptor mRNA+ or CysLT1 receptor protein+ cells, expressed as the number of cells/mm2 of subepithelium in both stable asthma and that associated with a severe asthma exacerbation (Spearman rank correlation; n = 32).

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
These data are the first in asthma to demonstrate both mRNA and protein expression for the CysLT1 receptor in bronchial mucosal inflammatory cells. Apart from bronchial (CD3+) T-lymphocytes (including CD4 and CD8 subsets) that do not express the CysLT1 receptor, we have identified a variety of other bronchial inflammatory cells, including eosinophils, neutrophils, mast cells, macrophages, B-lymphocytes, and plasma cells that do express it. Moreover, in a comparison with nonsmoking control subjects without asthma, we show that there are greater numbers of CysLT1 receptor–positive cells in stable asthma and further significant increases in individuals with asthma hospitalized for an acute severe exacerbation. Some of these data have been presented by us previously, but only in preliminary abstract form (27).

The CysLT1 receptor was first characterized by Lynch and colleagues, and receptor mRNA was shown by ISH of normal lung to be expressed in both inflammatory and smooth muscle cells (17). In addition, following the development of a CysLT1 receptor–specific antisera, IHC of snap-frozen histologically normal lung biopsy samples showed localized the receptor protein to interstitial macrophages and bronchiolar smooth muscle (24). CysLT1 receptor expression was also reported in peripheral blood: pregranulocyte CD34+ (progenitor) cells, eosinophils, monocytes, and pre–B cells but not CD4+ nor CD8+ T cells. Double labeling immunohistochemical techniques were subsequently applied to nasal turbinate tissue from patients suffering persistent nasal obstruction, refractory to treatment, demonstrating that mast cells, neutrophils, and vascular endothelial cells also expressed the CysLT1 receptor (22). Moreover, the immunopositivity for both CysLT1 and CysLT2 receptor protein was detected on surface and gland epithelium of nasal biopsies from patients with aspirin-sensitive and aspirin-tolerant rhinosinusitis, although expression of CysLT2 significantly exceeded that of CysLT1 (28).

Until now, there have been no studies of asthmatic bronchi, the most relevant target tissue for the anti-asthma actions of LTRAs, and there has been no quantitative histologic report of cells expressing this receptor in individuals with asthma. On the basis of our present description of the presence of CysLT1 receptor–positive cells in asthmatic bronchial mucosa, we concur with the previously reported colocalization studies of nonasthma tissues and of peripheral blood cells and, in addition, confirm the absence of its localization to T-lymphocytes, including the CD4 and CD8 T cell subsets present in bronchial tissue. This is the first report of CysLT1 receptor positivity in asthmatic bronchial tissue and the first to describe its colocalization to plasma cells.

Our findings help us to understand why several phenotypes of inflammatory cell, such as neutrophils and eosinophils, are recruited to the bronchial mucosa in response to experimental inhalation of leukotrienes (9). Moreover, there is a positive correlation between the recruitment of increasing numbers of leukocytes and those expressing CysLT1 receptor. This argues in favor of the tissue accumulation in asthma of greater numbers of a variety of CysLT1 receptor–positive leukocytes through recruitment rather than by novel expression or upregulation of this receptor on pre-existing mucosal cells. Moreover, the lack of association between the numbers of cells expressing CysLT1 receptor mRNA or protein and the numbers of neutrophils or eosinophils indicates that cells other than these are being recruited to contribute to the statistically significant correlation we report. Our study did not aim nor was it designed to determine the functional consequences of the increased numbers of CysLT1 receptor–positive leukocytes. However, our study does demonstrate that the targets for leukotriene receptor antagonists (LTRA) are present in the bronchial mucosa, and that they increase in asthma and in association with exacerbations. Clinically we consider it reasonable to speculate that treatment with LTRA in these circumstances might well attenuate any functional consequence of an increase in their number.

Previously we have reported that bronchial smooth muscle expresses the CysLT1 receptor (17). However, on closer inspection in the present study, we found that while a few myocytes did so, others often only weakly expressed the receptor or did not express either its mRNA or protein at all. This contrasted with the intense reaction product seen in adjacent inflammatory cells. We were initially surprised at this finding because the CysLT1 receptor is a member of the Gq-coupled receptor subfamily, which includes the H1 histamine, B2 bradykinin, and endothelin (ET)A-receptors identified in cultured airway smooth muscle cells (29). Studies in vivo or ex vivo have demonstrated that these receptors bind their agonists to induce airway smooth muscle contraction (29). We have not found reference to another G protein–coupled receptor that mediates contraction of airway smooth muscle but that is not expressed via IHC and ISH. Thus, while these results may challenge our expectations and current dogma, we wish to keep an open mind and consider these results to be of interest to others in the field. Clearly there is a need for follow-up work with a focus on these findings in muscle.

We conclude that our data support our hypothesis and highlight, for the first time, the variety of CysLT1 receptor–positive inflammatory cells that may be regulated by the actions of CysLTs or inhibited by the effects of LTRAs in the asthmatic bronchial mucosa. These targets include: eosinophils, neutrophils, mast cells, monocytes, B- (but not T-) cells, and plasma cells. Moreover, in association with an increased recruitment of leukocytes, there is a significant increase of CysLT1 receptor–positive cells in the bronchial mucosa of individuals with asthma and a further increase in those experiencing a severe exacerbation of their asthma.


   Acknowledgments
 
The authors thank Merck & Co. Inc. for an educational grant, Edward Inett and Vasile Laza-Stanca for invaluable help in capturing the double-staining images of inflammatory cells, and Sheng Yang-Qiu for help with the figure presentation.


   Footnotes
 
Supported by a research grant from Merck & Co., Inc. USA.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0124OC on August 25, 2005

Conflict of Interest Statement: J.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Y.-S.Q. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.J.F. is a Merck & Co. USA employee. V.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.G. is an employee of Merck & Co. USA. K.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.K.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.F.E. is an employee of Merck & Co. USA. P.K.J. has been reimbursed by GlaxoSmithKline (GSK), AstraZeneca (A-Z), and Merck, Sharpe & Dohme (Merck) for attending many conferences and has participated as a paid speaker in scientific meetings or courses organized and financed by various pharmaceutical companies (such as GSK, A-Z, Merck, and Boehringer Ingelheim); he has served as a consultant to GSK & Novartis; P.K.J. has received research grants from several pharmaceutical companies over many years and currently holds research grants from GSK, Merck, and A-Z, the first of which includes a grant for a multicenter clinical trial. His institution has received unrestricted grants from a wide variety of pharmaceutical companies.

Received in original form April 5, 2005

Accepted in final form August 10, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Holgate ST, Peters-Golden M. Introduction: the anti-inflammatory role of cysteinyl leukotriene receptor antagonists in asthma. J Allergy Clin Immunol 2003;111:S1–S4.[Medline]
  2. Krawiec ME, Wenzel SE. Leukotriene inhibitors and non-steroidal therapies in the treatment of asthma. Expert Opin Pharmacother 2001;2:47–65.[Medline]
  3. Drazen JM. Anti-leukotrienes as novel anti-inflammatory treatments in asthma. Adv Exp Med Biol 2002;507:217–221.[Medline]
  4. Coffey M, Peters-Golden M. Extending the understanding of leukotrienes in asthma. Curr Opin Allergy Clin Immunol 2003;3:57–63.[Medline]
  5. Parameswaran K, Liang H, Fanat A, Watson R, Snider DP, O'Byrne PM. Role for cysteinyl leukotrienes in allergen-induced change in circulating dendritic cell number in asthma. J Allergy Clin Immunol 2004;114:73–79.[CrossRef][Medline]
  6. Parameswaran K, Watson R, Gauvreau GM, Sehmi R, O'Byrne PM. The effect of pranlukast on allergen-induced bone marrow eosinophilopoiesis in subjects with asthma. Am J Respir Crit Care Med 2004;169:915–920.[Abstract/Free Full Text]
  7. Camargo CA Jr, Smithline HA, Malice MP, Green SA, Reiss TF. A randomized controlled trial of intravenous montelukast in acute asthma. Am J Respir Crit Care Med 2003;167:528–533.[Abstract/Free Full Text]
  8. Cylly A, Kara A, Ozdemir T, Ogus C, Gulkesen KH. Effects of oral montelukast on airway function in acute asthma. Respir Med 2003;97:533–536.[CrossRef][Medline]
  9. Laitinen LA, Laitinen A, Haahtela T, Vilkka V, Spur BW, Lee TH. Leukotriene E4 and granulocytic infiltration into asthmatic airways. Lancet 1993;341:989–990.[CrossRef][Medline]
  10. Nakamura Y, Hoshino M, Sim JJ, Ishii K, Hosaka K, Sakamoto T. Effect of the leukotriene receptor antagonist pranlukast on cellular infiltration in the bronchial mucosa of patients with asthma. Thorax 1998;53:835–841.[Abstract/Free Full Text]
  11. Macfarlane AJ, Dworski R, Sheller JR, Pavord ID, Barry KA, Barnes NC. Sputum cysteinyl leukotrienes increase 24 hours after allergen inhalation in atopic asthmatics. Am J Respir Crit Care Med 2000;161:1553–1558.[Abstract/Free Full Text]
  12. Currie GP, Srivastava P, Dempsey OJ, Lee DKC. Therapeutic modulation of allergic airways disease with leukotriene receptor antagonists. Q J Med 2005;98:171–182.
  13. Wenzel SE. The role of leukotrienes in asthma. Prostaglandins Leukot Essent Fatty Acids 2003;69:145–155.[CrossRef][Medline]
  14. Woodruff PG, Dolganov GM, Ferrando RE, Donnelly S, Hays SR, Solberg OD, Carter R, Wong HH, Cadbury PS, Fahy JV. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med 2004;169:1001–1006.[Abstract/Free Full Text]
  15. Panettieri RA, Tan EM, Ciocca V, Luttmann MA, Leonard TB, Hay DW. Effects of LTD4 on human airway smooth muscle cell proliferation, matrix expression, and contraction In vitro: differential sensitivity to cysteinyl leukotriene receptor antagonists. Am J Respir Cell Mol Biol 1998;19:453–461.[Abstract/Free Full Text]
  16. Jeffery PK. The roles of leukotrienes and the effects of leukotriene receptor antagonists in the inflammatory response and remodelling of allergic asthma. Clin Exp Allergy Rev 2001;1:148–153.[CrossRef]
  17. Lynch KR, O'Neill GP, Liu Q, Im DS, Sawyer N, Metters KM, Coulombe N, Abramovitz M, Figueroa DJ, Zeng Z, et al. Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 1999;399:789–793.[CrossRef][Medline]
  18. Heise CE, O'Dowd BF, Figueroa DJ, Sawyer N, Nguyen T, Im DS, Stocco R, Bellefeuille JN, Abramovitz M, Cheng R, et al. Characterization of the human cysteinyl leukotriene 2 receptor. J Biol Chem 2000;275:30531–30536.[Abstract/Free Full Text]
  19. Back M. Functional characteristics of cysteinyl-leukotriene receptor subtypes. Life Sci 2002;71:611–622.[CrossRef][Medline]
  20. Evans JF. Cysteinyl leukotriene receptors. Prostaglandins Other Lipid Mediat 2002;68–69:587–597.
  21. Figueroa DJ, Borish L, Baramki D, Philip G, Austin CP, Evans JF. Expression of cysteinyl leukotriene synthetic and signalling proteins in inflammatory cells in active seasonal allergic rhinitis. Clin Exp Allergy 2003;33:1380–1388.[CrossRef][Medline]
  22. Shirasaki H, Kanaizumi E, Watanabe K, Matsui T, Sato J, Narita S, Rautiainen M, Himi T. Expression and localization of the cysteinyl leukotriene 1 receptor in human nasal mucosa. Clin Exp Allergy 2002;32:1007–1012.[CrossRef][Medline]
  23. Wilson AM, O'Byrne PM, Parameswaran K. Leukotriene receptor antagonists for allergic rhinitis: a systematic review and meta-analysis. Am J Med 2004;116:338–344.[CrossRef][Medline]
  24. Figueroa DJ, Breyer RM, Defoe SK, Kargman S, Daugherty BL, Waldburger K, Liu Q, Clements M, Zeng Z, O'Neill GP, et al. Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes. Am J Respir Crit Care Med 2001;163:226–233.[Abstract/Free Full Text]
  25. Global Initiative for Asthma. NHLBI/WHO workshop report. National Institutes of Health, Bethosda, MD, USA; 1995. p. 1–10.
  26. Global Initiative for Asthma. NHLBI/WHO workshop report: National Institutes of Health, Bethosda, MD, USA; 2002. p. 68–76.
  27. Zhu J, Oiu Y, Bandi V, Guntupalli KK, Figueroa D, Galczenki H, Evans J, Jeffery PK. Localisation and quantification of gene and protein expression for the CysLT1 receptor increases in bronchial biopsies of stable asthma and in severe exacerbations [abstract]. Am J Respir Crit Care Med 2004;169:A196.
  28. Corrigan C, Mallett K, Ying S, Roberts D, Parikh A, Scadding G, Lee T. Expression of the cysteiny leukotriene receptors cysLT1 and cysLT2 in aspirin-sensitive and aspirin-tolerant chronic rhinosinusitis. J Allergy Clin Immunol 2005;115:316–322.[Medline]
  29. Billington CK, Penn RB. Signaling and regulation of G protein-coupled receptors in airway smooth muscle. Respir Res 2003;4:1–23.[CrossRef][Medline]



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