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(Chest. 2005;128:3148-3155.)
© 2005 American College of Chest Physicians

Interferon-{gamma} Up-regulates Expression of Cysteinyl Leukotriene Type 2 Receptors on Eosinophils in Asthmatic Patients*

Masaru Fujii, MD; Hiroshi Tanaka, MD and Shosaku Abe, MD

* From the Third Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.

Correspondence to: Masaru Fujii, MD, The Third Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1 West-16, Chuo-ku, Sapporo, 060-8543, Japan; e-mail: mafuji{at}sapmed.ac.jp


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Cysteinyl leukotrienes (cysLTs) are strong bronchoconstrictive mediators that play a key role in asthma inflammation. They act through specific receptors including cysLT type 1 receptor (CysLT1R) and cysLT type 2 receptor (CysLT2R). Although these two receptors are co-expressed on inflammatory cells, little is known about CysLT2R in patients with asthma. The aims of this study were to investigate the changes in cysLT receptors (CysLTRs) during asthma exacerbations and to determine which cytokine modulates CysLTR expression on eosinophils.

Methods: We assessed protein expression and messenger RNA of CysLT1R and CysLT2R in peripheral blood eosinophils and measured urinary leukotriene E4 levels in 36 patients with stable asthma, 23 subjects with asthma exacerbation, and 15 healthy subjects. We also evaluated the modulation of these receptors by interleukin (IL)-1ß, IL-4, IL-5, IL-13, interferon (IFN)-{gamma} and tumor necrosis factor-{alpha} in cultured eosinophils.

Results: Expression of both CysLT1R and CysLT2R on eosinophils during asthma exacerbations was significantly higher (p < 0.05) than in stable asthma and healthy subjects. A greater expression of CysLT2R in exacerbation was found in nonatopic asthmatics. Only IFN-{gamma} up-regulated cell-surface expression of CysLT2R in a dose-dependent manner and enhanced messenger RNA levels. No cytokine affected CysLT1R expression or messenger RNA level.

Conclusions: CysLT2R expression on eosinophils was increased in patients, especially in nonatopic subjects, during asthma exacerbation, and was up-regulated by IFN-{gamma}; therefore we speculate that a pathway through CysLT2R might modulate exacerbations of asthma.

Key Words: asthma exacerbation • cysteinyl leukotriene type 2 receptor • eosinophil • interferon-{gamma}


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cysteinyl leukotrienes (cysLTs) are lipid mediators that have been implicated in the pathogenesis of not only airway smooth-muscle constriction but also many inflammatory processes in patients with asthma. The effects of the cysLTs are mediated through two or more receptors, including the cysLT type 1 receptor (CysLT1R) and the cysLT type 2 receptor (CysLT2R).123456 CysLT1R is expressed on lung smooth-muscle cells, macrophages, peripheral blood eosinophils, basophils, neutrophils, monocytes, B-lymphocytes, and CD34-positive stem cells.3 CysLT2R is co-expressed with CysLT1R on eosinophils, basophils, mast cells, monocytes, and macrophages; CysLT1R is co-expressed in neutrophils only.123456 Mellor and colleagues7 showed that interleukin (IL)-4 up-regulates CysLT2R expression on cultured human mast cells and suggested that the CysLT2R response may permit the cysLTs and nucleotides generated in infection and tissue injury to elicit IL-8 generation by mast cells, potentially leading to neutrophilic infiltration, a characteristic of aerosol challenge-induced late-phase responses and of sudden death associated with asthma. Therefore, co-expression of both CysLTRs may facilitate cooperative recognition of low concentrations of ligand and enhanced signaling for cytokine generation in circumstances of inflammation or tissue injury. Despite significant progress in characterizing the structure and sequence of the CysLT2R, little is known about its cellular and molecular mechanisms and their role in airway inflammation.

Of human peripheral blood monocytes, approximately 30% express CysLT2R, while it is expressed by > 90% of purified human eosinophils.4 Massive accumulation of eosinophils in the airway wall characterizes inflammation associated with bronchial asthma. There have been no studies of CysLT2R on eosinophils in patients during asthma exacerbation.

Asthma is believed to result from an imbalance consisting of overproduction of T-helper type 2 (Th2) cytokines (IL-4, IL-5, and IL-13) with reduced production of the T-helper type 1 (Th1) cytokine interferon (IFN)-{gamma}. However, in asthma exacerbation, IFN-{gamma}–producing T-cells in sputum are in higher proportion than in stable asthma.8 One of the common triggers of asthma exacerbations is viral infection,910 which induces airway hyperresponsiveness. We aimed to investigate the development of CysLT2R on human peripheral blood eosinophils during asthma exacerbations and to determine whether the CysLT2R expression is regulated by cytokines, including IL-1ß, IL-4, IL-5, IL-13, IFN-{gamma}, and tumor necrosis factor (TNF)-{alpha}.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Fifty-nine subjects were recruited from the outpatient section of Sapporo Medical University Hospital: 36 patients with stable asthma, and 23 patients with an asthma exacerbation. The basic characteristics of this study population are shown in Table 1 . Asthma was diagnosed according to American Thoracic Society guidelines11 as a ≥ 15% increase in FEV1 in response to a bronchodilator. Asthma severity was classified according to published guidelines.12 Moderate asthma is defined as daily symptoms with FEV1 > 60% and < 80% predicted before treatment. Mild asthma is defined as FEV1 > 80% predicted before treatment. No subjects were treated with oral steroid or leukotriene receptor antagonist. Only people with a ≥ 2 + reaction to antigen-specific serum IgE of 23 common antigens as tested by a commercial kit (CAP RAST system; Pharmacia Diagnostics; Uppsala, Sweden) were considered atopic. Exacerbations of asthma are defined as follows12: episodes of rapidly progressive increase in shortness of breath, cough, wheezing, or chest tightness, or some combination of these symptoms. In all patients with exacerbation, wheezing was audible in both lung fields. We excluded subjects with purulent sputum. Peripheral blood was collected within 12 h after the onset of asthma exacerbation, and no subjects were treated with systemic steroid before blood sampling. In all patients with asthma exacerbation, symptoms were improved by steroid therapy within 2 days and disappeared in another 3 days. In some patients, we re-examined peripheral blood eosinophils as remission period data ≥ 1 month after the symptoms disappeared.


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  		Table 1.. Baseline Characteristics of the Subjects*

 
Fifteen healthy volunteers without respiratory symptoms or objective findings of diseases, including asthma and allergic rhinitis, were also enrolled in this study as healthy control subjects (mean age, 53 ± 5 years [± SEM]; 5 male and 10 female subjects). All subjects gave written informed consent, and the study protocol was approved by the ethics committee of Sapporo Medical University School of Medicine.

Cell Separation
Eosinophils were separated from 15 mL of venous blood. Eosinophils were isolated by silica-based, colloidal media (Percoll, 1.088 g/mL; Amersham Biosciences; Piscataway, NJ) density centrifugation, followed by negative selection using anti-CD16-bound micromagnetic beads (Miltenyi BioTech; Bergisch-Gladbach; Germany). After purification, the mean eosinophil purity was consistently > 98%, and the viability was consistently > 97%, as determined by trypan blue exclusion.

Flow Cytometric Measurement of cysLT Receptor Expression
Cytofluorographic analysis was performed on saponin-permeabilized eosinophils for CysLT1R and nonpermeabilized eosinophils for CysLT2R. Samples of 5 x 105 cells were stained for 15 min at room temperature with polyclonal antibodies raised against a C-terminal peptide of the human CysLT1R, or against a N-terminal peptide of the human CysLT2R, or with an equivalent amount of a nonspecific rabbit IgG as the control antibody (all from Cayman Chemical; Ann Arbor, MI). Cells were then washed with cold phosphate-buffered saline solution and incubated for 15 min at room temperature with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma Aldrich; St. Louis, MO). Finally, cells were washed again and resuspended in phosphate-buffered saline solution before single-color immunofluorescence analysis of 10,000 cells was performed using a flow cytometer (FACScan; Becton Dickinson, Franklin Lakes, NJ). These data were analyzed using software (CellQuest; Becton Dickinson). cysLT receptor (CysLTR) expression levels were calculated with nonspecific IgG adjusted to 100% (Figs 123 ). Basal expression levels of CysLTR protein were adjusted to 100%, and CysLTR expression levels are illustrated as percentage of untreated control cells (Fig 4 ).



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  		Figure 1.. CysLTR expression on human blood eosinophils in each group. Top, A: Representative histograms of a single experiment. Bottom, left, B: CysLT1R. Bottom, right, C: CysLT2R. Data are expressed as box plots. Horizontal lines indicate median; squares indicate 25th and 75th percentiles, and error bars indicate 10th and 90th percentiles. Both expressions of CysLT1R and CysLT2R in patients with asthma exacerbation are significantly higher as compared with patients with stable asthma and healthy subjects.

 


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  		Figure 2.. Serial changes in CysLTR expression on human blood eosinophil in six patients with exacerbation. Left, A: CysLT1R. Right, B: CysLT2R. Both expressions of CysLT1R and CysLT2R during asthma exacerbation decrease in remission days.

 


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  		Figure 3.. CysLT2R expression on human blood eosinophils in nonatopic patients. The expression in asthma exacerbation is higher than that in stable asthma. Ab = antibody.

 


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  		Figure 4.. Modulation of CysLT2R expression on human blood eosinophil by cytokines, with control normalized to 100%. Eosinophils are cultured with 10 ng/mL of the indicated cytokines for 24 h at 37°C. Top, A: Histograms of a single experiment, treatment with (open histogram) or without (shaded histogram) the indicated cytokines. Center, B: Expression of CysLT2R induced by the indicated cytokines (n = 7). Only treatment with IFN-{gamma} up-regulates the expression of CysLT2R. Bottom, C: Expression of CysLT2R increases by the treatment with IFN-{gamma} in a dose-dependent manner (n = 5).

 
Culture
All eosinophils were obtained from patients with stable asthma. Eosinophils (5 x 105) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum at 37°C in 5% CO2 in a total volume of 1,000 µL in flat-bottomed, 24-well culture plates with and without cytokines for 24 h. We treated them using the following cytokines with a concentration of 10 ng/mL: IL-1ß, IL-4, IL-5, IL-13, IFN-{gamma}, and TNF-{alpha} (all from R&D Systems; Minneapolis, MN).

Reverse Transcriptase-Polymerase Chain Reaction Analysis of CysLTRs
Total RNA was extracted from up to 5 x 106 of eosinophils (TRIzol Reagent; Invitrogen; Carlsbad, CA). One microgram of total RNA was converted to complementary DNA by the reverse transcriptase (RT) enzyme reaction (First-Strand Complementary DNA Synthesis Kit; Amersham Pharmacia Biotech) in a total volume of 15 µL. Polymerase chain reaction (PCR) was performed in a final volume of 50 µL containing 1 µL of RT reaction product. Samples were placed in a thermal cycler (PTC-100; MJ Research; Waltham, MA). The forward primer of CysLT1R was 5'-ATGACAGCCATGAGCTTTTTC-3', and the reverse primer was 5'-CATTCTAAGGACAGAATCAA-3'. The forward primer of CysLT2R was 5'-AGCAATAACAACAGCAGGAA-3', and the reverse primer was 5'-CCA GGTCTCCAAATATCCAA-3'. The forward primer of ß-actin was 5'-GAGAAGATGACCCAGATCATGT-3', and the reverse primer was 5'-ACTCCATGCCCAGGAAGGAAGG-3'. The amplification conditions were 95°C for 1 min, 56°C for 1 min, and 72°C for 2 min at 35 cycles for CysLTRs and ß-actin. The PCR products were electrophoresed on 1% agarose gel, stained with ethidium bromide, and photographed under ultraviolet transillumination. No PCR products were obtained when RT was omitted, indicating that there was no DNA contamination.

Measurement of Urinary Leukotriene E4
Each patient provided approximately 30 mL of urine in a polypropylene container at the time of blood sampling. We have previously reported our assay method.13 In brief, using a reverse-phase, high-pressure liquid chromatography gradient system equipped with a Nova-Park C18 column (Waters Associates; Milford, MA), leukotriene E4 (LTE4) was measured with a radioimmunoassay kit (Leukotriene [3H]; DuPont New England Nuclear Research; Boston, MA). Measurements were corrected according to the creatinine content of urine.

Statistical Analysis
Data are expressed as mean ± SEM for normally distributed data and as median and interquartile ranges (IQRs) for nonnormally distributed data. Differences among groups were analyzed by means of nonparametric Kruskal-Wallis test, followed, where significant, by the Mann-Whitney U test for comparisons among groups (Fig 1, bottom, left, B, and bottom, right, C, Fig 3). A Wilcoxon signed-rank test was used to compare CysLTR expression between asthma exacerbation (day 1) and remission days (Fig 2). For normally distributed data, the differences among groups were evaluated by analysis of variance followed by a Scheffe test (Fig 4, center, B, bottom, C, Fig 5 , right, B). Fluorescence intensity was log-transformed and described as geometric mean (log SEM). Pearson correlation was calculated to evaluate the relationship between CysLTR expression and urinary LTE4 level. A p value of < 0.05 was considered statistically significant.



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  		Figure 5.. Left, A: Effect of IFN-{gamma} (100 ng/mL for 24 h) on CysLTR messenger RNA levels in human blood eosinophils of stable asthma. Eosinophils are stimulated with IFN-{gamma} (100 ng/mL for 24 h). Total RNA is subjected to RT-PCR with the primers for ß-actin, CysLT1R, and CysLT2R. Reaction products were separated on 1% agarose gel and stained with ethidium bromide. Right, B: Scanning densitometry of gels with the data for ß-actin normalized. The densitometric intensity of each immunoreactive band was analyzed using the National Institutes of Health imaging program. The messenger RNA levels of CysLT2R are up-regulated by the treatment with IFN-{gamma} (n = 5). bp = base pair.

 

   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CysLTR Protein Expression in Human Eosinophils in Stable Asthmatics and During Exacerbation
The expression of CysLTR protein in human eosinophils was investigated by flow cytometry with a specific anti-CysLTR antibody. Both CysLT1R and CysLT2R protein expression on eosinophils are shown in Figure 1, top, A. The expression of CysLT1R protein as a percentage of nonspecific-antibody mean fluorescence intensity (MFI) was significantly different between asthma exacerbation (median, 149.8%; IQR, 125.7 to 171.1%) and stable asthma (median, 122.8%; IQR, 116.6 to 142.4%) [p = 0.04] or normal subjects (median, 123.0%; IQR, 120.1 to 132.0%) [p = 0.01; Fig 1, bottom left, B]. The expression of CysLT2R protein was significantly different between asthma exacerbation (median, 173.2%; IQR, 139.1 to 222.7%) and stable asthma (median, 150.5%; IQR, 127.7 to 185.0%) [p = 0.02] or normal subjects (median, 138.5%; IQR, 128.3 to 157.2%) [p = 0.01; Fig 1, bottom right, C]. The expression of CysLT2R protein in stable asthma was higher than in normal subjects, but these differences did not reach statistical significance.

We were able to compare the data both on exacerbation and remission days from 6 of the 23 patients with asthma exacerbation (mean age, 47 ± 7 years; 2 male and 4 female subjects). The expression of CysLT1R and CysLT2R protein in asthma exacerbation significantly decreased in the remission period (median, 151.1%; IQR, 124.2 to 163.1%, to median, 110.9%; IQR, 107.3 to 116.4%; and median, 180.2%; IQR, 168.2 to 219.6%, to median, 136.1%; IQR, 124.2 to 145.4%, respectively) [Fig 2]. No correlation was found between CysLT1R or CysLT2R expression and urinary LTE4 level (data not shown).

Next, we compared CysLT2R expression between atopic and nonatopic patients. In atopic patients, the expression of CysLT2R was not significantly different between asthma exacerbation and stable asthma (median, 175.9%; IQR, 146.6 to 223.0; vs median, 180.2; IQR, 151.5 to 215.8) [p = 0.57]; in nonatopic patients, the expression of CysLT2R in asthma exacerbation was higher than in stable asthma (median, 165.6%; IQR, 139.4 to 214.3%; vs median, 146.5%; IQR, 125.6 to 167.3%; p = 0.03) [Fig 3].

IFN-{gamma}–Induced CysLT2R Protein Expression in Human Eosinophils
Eosinophils were treated for 24 h with 10 ng/mL of IL-1ß, IL-4, IL-5, IL-13, IFN-{gamma}, and TNF-{alpha}. The expression of CysLT1R protein was not significantly affected by treatment of eosinophils with any cytokine (data not shown). We found that IFN-{gamma} but not IL-4, IL-5, or IL-13 markedly increased CysLT2R expression (Fig 4, top, A, and center, B). As compared with basal expression (MFI normalized to 100%), CysLT2R expression was increased to 238.7 ± 24.4% in cells treated with IFN-{gamma}. In parallel experiments, TNF-{alpha} and IL-1ß had no effect on CysLT2R protein expression. We also found that treatment of eosinophils with IFN-{gamma} significantly enhanced the expression of CysLT2R in a dose-dependent manner (Fig 4, bottom, C). As compared with basal expression without IFN-{gamma} treatment, CysLT2R expression was increased to 227.8 ± 21.1%, 256.0 ± 47.6%, and 374.4 ± 46.8% in cells treated with IFN-{gamma} at 10, 100, and 1,000 ng/mL, respectively. Although the magnitude of the increase in CysLT2R expression induced by IFN-{gamma} varied among donor cells, IFN-{gamma} increased CysLT2R expression as compared to untreated cells in all tested concentrations.

IFN-{gamma}–Induced CysLT2R Messenger RNA Expression in Human Eosinophils
To assess whether increased CysLT2R protein expression was associated with augmented CysLT2R messenger RNA levels, cells were stimulated with 100 ng/mL of IFN-{gamma}, and their steady-state levels of CysLT2R messenger RNA were analyzed (Fig 5). Human eosinophils constitutively expressed low levels of CysLT2R messenger RNA and IFN–augmented transcript levels after 24 h of stimulation. Scanning densitometry analysis showed that IFN-{gamma} augmented CysLT2R messenger RNA levels 2.5-fold over baseline levels (Fig 5, right, B). The present study shows that IFN-{gamma} significantly up-regulated steady-state messenger RNA expression for CysLT2R, not for CysLT1R.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found that CysLT2R expression on peripheral blood eosinophils from patients with asthma exacerbation was higher than in those with stable asthma. CysLT2R expression level decreased after asthma therapy. From these results, CysLT2R expression may partly reflect disease activity in mild and moderate asthma. In nonatopic patients, the expression of CysLT2R in asthma exacerbation was higher than in stable asthma but not for atopic patients. Therefore, CysLT2R expression might mainly contribute to asthma exacerbation in nonatopic patients. And we demonstrated for the first time that IFN-{gamma} alone up-regulated the expression of CysLT2R protein and messenger RNA levels, but other inflammatory and Th2 cytokines (IL-1ß, IL-4, IL-5, IL-13, TNF-{alpha}) had no effect. CysLT2R has been identified in heart Purkinje-conducting fiber cells, adrenal chromaffin cells, vascular smooth-muscle cells, peripheral blood eosinophils, monocytes, macrophages, basophils, and mast cells.245 Mellor et al7 showed that CysLT2R expression levels on human mast cells were up-regulated by IL-4. However, another study14 reported that CysLT2R expression levels were not increased in human bronchial smooth-muscle cell by IL-4, IL-13, transforming growth factor-ß, and IFN-{gamma}. It was suggested that CysLT2R expression might be controlled differently in each cell.

In general, a shift toward to Th2-dominant lymphocytic inflammation exists in asthma, but the role of IFN-{gamma} in asthma airway inflammation has not been clearly established. Viral infection, especially rhinovirus, is one of the common triggers of asthma exacerbations.910 Airway inflammation by viral infections is accompanied by marked leukocyte trafficking and production of cysLTs and Th1 cytokines such as IFN-{gamma}.15 In young children with acute episodes of virus-induced wheezing, cysLTs and IFN-{gamma} levels are significantly increased in sputum.15 IFN-{gamma} is also known to activate and prolong the in vitro survival of eosinophils and to enhance the release of cysLTs.1617 Hessel and colleagues18 reported that IFN-{gamma} has an effect on the induction of airway hyperresponsiveness because treatment with antibodies to IFN-{gamma} completely abolished the development of airway hyperresponsiveness in ovalbumin-challenged animals. Thus, IFN-{gamma} has been implicated in both virus- and allergen-induced bronchial hyperresponsiveness.1819 Medoff and colleagues20 reported that IFN-{gamma}–inducible protein 10 was up-regulated in allergic pulmonary inflammation and that this contributes to the airway hyperreactivity and Th2 inflammation seen in a mouse model of asthma, and hypothesized that inducible protein 10 contributes to airway hyperreactivity and airway inflammation by increasing the recruitment of Th2 lymphocytes and eosinophils. Based on these observations, the complex inflammatory mechanism of asthma cannot be completely compartmentalized into Th1 or Th2 inflammation.

The limitation of this study was that we did not measure serum viral antibodies in 23 patients with asthma exacerbation. But among them, eight patients have self-reported symptoms of upper respiratory tract infection (URI) [ie, fever elevation, sore throat, nasal discharge, or increased serum C-reactive protein, but no purulent sputum] before the exacerbation. The expression of CysLT2R in self-reported symptoms of URI-positive patients was higher than in those without symptoms of URI (median, 202.3%; IQR, 148.5 to 221.0%; vs median, 157.2%; IQR, 134.1 to 173.1%) [p = 0.05]. Therefore, we hypothesized that viral infection in the upper respiratory tract increased IFN-{gamma} in the airway and induced CysLT2R expression on accumulated eosinophils, which might modulate asthma exacerbation.

In the present study, CysLT1R expression on peripheral blood eosinophils from patients with asthma exacerbation increased, and recent similar results demonstrated that the number of CysLT1R-positive cells in the bronchial biopsy specimen increased in asthma exacerbation as compared with stable.21 Our data also revealed that IL-1ß, IL-4, IL-5, IL-13, IFN-{gamma}, and TNF-{alpha} failed to up-regulate the expression of CysLT1R protein. This result agrees with a previous study22 using human peripheral blood eosinophils. In addition, CysLT1R expression may be subject to cytokine-mediated regulation; in HL-60 cells, IL-5 up-regulated CysLT1R messenger RNA expression with enhanced CysLT1R protein expression and function.23 However, in our study, IL-5 did not up-regulate CysLT1R protein expression in human peripheral blood eosinophils. The discordance between the two studies can probably be explained by the difference between transformed cells and normal cells.

In summary, our data demonstrate that CysLT2R expression on eosinophils was higher during asthma exacerbation than in stable asthma. This trend was more prominent in nonatopic subjects with asthma. Ex vivo studies revealed that the CysLT2R expression was up-regulated by IFN-{gamma}, but not by IL-1ß, IL-4, IL-5, IL-13, and TNF-{alpha}. We suggest that functions involving CysLT2R might modulate asthma exacerbations.


   Acknowledgements
 
The authors thank Manabu Fujita, PhD, Discovery Research Laboratories II, Ono Pharmaceutical Company, Ltd, for assistance with PCR analysis.


   Footnotes
 
Abbreviations: cysLT = cysteinyl leukotriene; CysLTR = cysteinyl leukotriene receptor; CysLT1R = cysteinyl leukotriene type 1 receptor; CysLT2R = cysteinyl leukotriene type 2 receptor; IFN = interferon; IL = interleukin; IQR = interquartile range; LTE4 = leukotriene E4; MFI = mean fluorescence intensity; PCR = polymerase chain reaction; RT = reverse transcriptase; Th1 = T-helper type 1; Th2 = T-helper type 2; TNF = tumor necrosis factor; URI = upper respiratory tract infection

Supported by Ono Pharmaceutical Company, Ltd, Osaka, Japan.

Received for publication March 1, 2005. Accepted for publication May 9, 2005.


   References
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 Abstract
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 Discussion
 References
 

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