Up-regulation of Peroxisome Proliferator-Activated Receptor γ in Perennial Allergic Rhinitis

Objectives  To investigate the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) messenger RNA and protein and to localize the PPAR-γ protein in the nasal mucosa of patients with allergic rhinitis and control subjects.

Design  Prospective study.

Setting  Tertiary academic institution.

Patients  Twenty patients with perennial allergic rhinitis and 20 matched nonallergic patients.

Interventions  Inferior turbinate mucosa samples were obtained from 20 patients with perennial allergic rhinitis and 20 matched nonallegic patients. Peroxisome proliferator-activated receptor γ messenger RNA was extracted from the inferior turbinate mucosae, and then reverse transcription–polymerase chain reaction was performed. Western blot testing was used to analyze differences in PPAR-γ protein expression levels between patients with allergic rhinitis and normal controls, and the PPAR-γ protein was localized immunohistochemically.

Results  The expression levels of PPAR-γ messenger RNA and protein in the nasal mucosa was significantly increased in patients with perennial allergic rhinitis compared with controls. Peroxisome proliferator-activated receptor γ protein was expressed in the epithelium, infiltrating inflammatory cells, and submucosal glands.

Conclusions  Peroxisome proliferator-activated receptor γ is expressed in the human nasal mucosa and is up-regulated in perennial allergic rhinitis. These results suggest a possible contribution for PPAR-γ in chronic inflammation of the nasal mucosa in perennial allergic rhinitis.

Allergic rhinitis, which is one of the most common chronic diseases in the industrialized world, is often debilitating and can contribute to sinus infection, to the development of asthma, and to exacerbations in individuals who already have the disease. It is an inflammation that is induced by an immune response that involves the release of inflammatory mediators and is characterized histologically by a marked infiltration of inflammatory cells in the nasal mucosa.¹

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily of ligand-activated transcriptional factors, which include receptors for steroids, thyroid hormone, vitamin D, and retinoic acid.² There have been numerous studies implicating PPAR-γ as an important modulator of airway hyperresponsiveness in asthma.^3-5 Recently, investigative studies have supported the concept of unified airways, which suggests that allergic rhinitis and asthma are a continuum of airway allergic disease.^6,7 This theory raises interesting questions regarding a similar role for PPAR-γ in the upper airway epithelium in allergic rhinitis. A recent investigation that compared the expression of PPAR-γ in the nasal mucosa of patients with seasonal allergic rhinitis and normal controls found no significant difference in PPAR-γ expression levels between the 2 groups.⁸ However, we found no published reports concerning the expression and localization of PPAR-γ in the human nasal mucosa in perennial allergic rhinitis. Therefore, our aim was to investigate the expression of PPAR-γ messenger RNA (mRNA) and protein and to localize the PPAR-γ protein in the nasal mucosa of patients with perennial allergic rhinitis and control subjects.

Nasal mucosal biopsy specimens of the inferior turbinate were obtained from 20 patients (10 men and 10 women; age range, 23-42 years; median age, 31 years) with perennial allergic rhinitis at Korea University Hospital, Seoul, between March 2005 and September 2005. All biopsy specimens were obtained from the lower edge of the inferior turbinate approximately 2 cm posterior to the anterior margin by the same investigator (H.-M.L.), who used Blakesley forceps. The patients who were included in our study presented with a clinical diagnosis of perennial allergic rhinitis (sneezing, rhinorrhea, and/or nasal congestion on most days) for at least 2 years, had a nasal smear demonstrating eosinophilia, and had a documented positive reaction (≥3+) to 1 or more perennial allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, cockroach, molds, cat fur, or dog hair) on a commercially available skin prick test (Allergy Therapeutics, West Sussex, England) within the study period.

The patients discontinued treatment with sodium cromoglycate, antihistamines, and corticosteroids at least 4 weeks before entering the study. Also, no subjects had an infection of the respiratory tract 4 weeks before the study began. Smokers and subjects who had bronchial asthma that required the regular use of inhaled corticosteroids or systemic corticosteroids were excluded from the study.

Normal inferior turbinate biopsy specimens from 20 matched patients (10 men and 10 women; age range, 21-44 years; median age, 33 years) were used as controls (control mucosa). All the control subjects had a negative skin prick test result, had no eosinophilia on nasal smear, and had no evidence of inflammation in the nasal cavity. Informed consent was obtained from all patients and control subjects, and the study protocols were approved by the institutional review board of Korea University College of Medicine.

Tissues were cut into 2 portions. One portion was immediately frozen in liquid nitrogen and stored at −70°C for subsequent RNA processing. The other portion was fixed with 4% paraformaldehyde in 0.1M phosphate-buffered saline (pH, 7.4) overnight at 4°C and then embedded in paraffin for immunohistochemical staining.

Total RNA was extracted from the perennial allergic rhinitis mucosa specimens using a commercially available reagent (TRIzol; Gibco BRL, Grand Island, NY) according to the manufacturer's suggestions. Total RNA from each sample was reverse transcribed in 20 μL of reaction mixture containing 2.5 U of Maloney murine leukemia virus reverse transcriptase (Gibco BRL) and 50pM random hexanucleotides at 42°C for 60 minutes. Oligonucleotide primers for polymerase chain reaction (PCR) were commercially synthesized (Bioneer Co, Taejon, South Korea) based on the published sequences. Oligonucleotide primers were designed according to the following published sequences for PPAR-γ: sense primer 5′-ATG ACA GCG ACT TGG CAA TA-3′ and antisense primer 5′-GCA ACT GGA AGA AGG GAA AT-3′. Amplification of the complementary DNA was carried out using 30 cycles at 94°C for 45 seconds, 55°C for 30 seconds, and 72°C for 1 minute, followed by a final extension cycle of 72°C for 7 minutes. Specificity of the 341–base pair (bp) PCR product was verified by predicted size, restriction digestion, and DNA sequencing. As negative controls, the PPAR-γ primer or the reverse transcriptase was omitted from some reverse transcription (RT)-PCR assays. The primers 5′-GTG GAT ATT GTT GCC ATC AAT GAC C-3′ and 5′-GCC CCA GCC TTC ATG GTG GT-3′ for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were run simultaneously as an internal control (the expected size of this PCR product was 270 bp). The PCR product was analyzed by gel electrophoresis. To analyze the RT-PCR results semiquantitatively, the gel images were scanned and the intensity of the PCR products was measured using a commercially available image software package (National Institutes of Health, Bethesda, Md). The relative intensities of individual bands on a gel image were determined as the ratio of the intensity of PPAR-γ mRNA to the intensity of GAPDH (PPAR-γ/GAPDH) mRNA.

Slides were deparaffinized and then serially rehydrated in graded alcohol. For PPAR-γ immunostaining, the rehydrated sections were soaked in citrate buffer and heated in a microwave oven for 15 minutes at 500 W. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 5 minutes. Nonspecific binding was blocked with 10% normal horse serum for 20 minutes. After a brief rinse, each section was immunoreacted for 1 hour with primary antibodies at room temperature. The primary antibody was monoclonal anti–PPAR-γ (Santa Cruz Biotech, Santa Cruz, Calif). The sections were washed in phosphate-buffered saline, incubated for 1 hour with biotinylated anti–mouse IgG (Elite Kit; Vector Laboratories Inc, Burlingame, Calif), and then treated with avidin-biotin complex for 1 hour. Then, 3-3′-tetradiaminobenzidine (Vector Laboratories Inc) was applied as a chromogen. Sections were visualized after counterstaining with Mayer hematoxylin.

Tissues were homogenized, and cells were lysed in protein-extract buffer (30-mmol/L Tris-hydrochloride [pH, 8.0], 10-mmol/L edetic acid, 1% Triton X-100, 100mM sodium chloride, and 1mM phenylmethylsulfonyl fluoride) and stored at –70°C. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were then blocked for 30 minutes in TBST buffer, consisting of 20mM Tris-hydrochloride (pH, 7.6), 135mM sodium chloride, and 1% Tween 20, with 5% nonfat dry milk and incubated at 4°C overnight with monoclonal anti–PPAR-γ antibody at a dilution of 1:200. After incubation, the membrane was washed 3 times for 5 minutes in TBST buffer and then treated with peroxidase-conjugated anti–mouse IgG antibody (Jackson Immunoresearch Laboratories Inc, West Grove, Pa) for 1 hour at room temperature. The membrane was washed, and substrate was added from an enhanced chemiluminescence reagent kit (ECL; Amersham Corp, Arlington Heights, Ill) and exposed to x-ray film for 10 seconds.

A t test was used to compare the PPAR-γ mRNA expression levels between the allergic rhinitis group and the control group. Statistical analysis was performed using a commercially available statistical software package (Version 8.0; SPSS Inc, Chicago, Ill). The results were expressed as mean ± SE. Significance of a difference in the PPAR-γ/GAPDH mRNA ratio between the 2 groups was accepted at 5% level of confidence.

An RT-PCR assay was performed to detect the presence of PPAR-γ mRNA in the human nasal mucosa. Polymerase chain reaction products of the expected size corresponding to 341 bp for PPAR-γ were detected in the control mucosa and the perennial allergic rhinitis mucosa and showed 100% sequence homology with the published PPAR-γ mRNA sequence. These results indicated that the human nasal mucosa expresses PPAR-γ mRNA. On RT-PCR screening, PPAR-γ mRNAs were weakly amplified in control nasal mucosa. However, PPAR-γ mRNAs were prominently amplified in perennial allergic rhinitis mucosa (Figure 1); PPAR-γ mRNA was not expressed in the negative control. As a control for RNA integrity and the RT-PCR procedure, the same RNA samples were also analyzed for GAPDH gene expression. The GAPDH primer produced a PCR product (270 bp) from each of the tissue samples. All samples were normalized with respect to GAPDH. Semiquantitative analysis of the PCR samples showed a statistically significant difference in the PPAR-γ/GAPDH mRNA ratios between the allergic rhinitis mucosa and the control mucosa (P<.05, Figure 2).

The tissue localization of PPAR-γ protein in the nasal mucosa was analyzed by immunohistochemical staining. Positively immunostained cells appeared brown. Peroxisome proliferator-activated receptor γ protein was expressed in the epithelium, the infiltrating inflammatory cells, and the submucosal glands in both perennial allergic rhinitis mucosa and control mucosa. The expression of the PPAR-γ protein in allergic rhinitis mucosa was consistently stronger than in control mucosa (Figure 3). There was no specific localization with the negative control, confirming the specificity of the PPAR-γ antibody.

Western blot analysis showed a prominent immunoreactive band with an apparent molecular mass of approximately 67 kDa reacting with anti–PPAR-γ antibody in allergic rhinitis mucosa (Figure 4). A less prominent band of the same molecular mass was detected in the control mucosa.

In the present study, we demonstrated the localization and expression of PPAR-γ mRNA and protein in human perennial allergic rhinitis nasal mucosa. This expression was augmented in patients with allergic rhinitis compared with control subjects, particularly in the epithelium, infiltrating inflammatory cells, and submucosal acinic cells.

Peroxisome proliferator-activated receptors are ligand-activated transcription factors that belong to the nuclear receptor superfamily.² Three PPAR isoforms have been identified: (1) PPAR-α, which functions as a global regulator of fatty acid catabolism by up-regulating the transcription of genes that transport intracellular fatty acids into peroxisomes and mitochondria for their β-oxidation⁹; (2) PPAR-γ, which regulates adipogenesis and is involved in insulin sensitization and cellular differentiation as well⁹; and (3) PPAR-β/δ, which increases the level of high-density lipoproteins and can beneficially alter serum and lipid profiles.¹⁰

Numerous studies have demonstrated that PPAR-γ plays an important role in regulating inflammatory responses, acting on T cells,¹¹ eosinophils,³ monocytes/macrophages,¹² dendritic cells,¹³ and mast cells.¹⁴ In a murine model of asthma, treatment with a PPAR-γ agonist inhibited the development of allergic inflammation, including pulmonary eosinophilia and airway hyperresponsiveness.³ Benayoun et al⁴ examined the expression of PPAR-γ protein in human subjects with asthma and found elevated expression in the bronchial submucosa, airway epithelium, and smooth muscle cells compared with control subjects. They suggested that PPAR-γ might be involved in the airway inflammation that was observed in their patients. It has been established that PPAR-γ has important roles in regulating inflammatory and immune processes,¹⁵ and the enhanced PPAR-γ expression that was demonstrated in our study may reflect an inflammatory response of different cell types and structures to natural PPAR-γ ligands generated within the allergic rhinitis mucosa.

A recent study investigated the expression of PPAR-γ in the inferior turbinate mucosa in patients with seasonal allergic rhinitis and normal controls and found no significant difference in PPAR-γ expression levels between the 2 groups.⁸ However, a closer examination of the data shows that the study was performed exclusively on patients with seasonal allergic rhinitis and that all the mucosal samples were obtained 5 to 10 days after the first appearance of symptoms. Because PPAR-γ expression appears to be augmented in response to chronic inflammation, the discrepancy in the PPAR-γ expression level between that study and ours, which investigated patients with perennial allergic rhinitis with chronic symptoms, could be attributed to the duration of the inflammatory process in the patients studied. It may be possible that more than a few days of airborne allergen exposure are needed to fully activate this immune-mediated response.

In conclusion, PPAR-γ mRNA and protein are expressed in the human nasal mucosa and are up-regulated in allergic rhinitis. The PPAR-γ protein was expressed in the epithelium, infiltrating inflammatory cells, and submucosal glands in the nasal mucosa of patients with perennial allergic rhinitis and normal controls. These results suggest a possible contribution of PPAR-γ in the chronic inflammation of allergic rhinitis.

Future studies using an animal model of allergic rhinitis are required to improve our understanding of the functional properties of PPAR-γ in perennial allergic rhinitis and to explore the possibility of using PPAR-γ ligands as a novel therapeutic modality for the management of allergic rhinitis.

Correspondence: Heung-Man Lee, MD, PhD, Department of Otorhinolaryngology–Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, 80 Guro-dong, Guro-gu, Seoul, Republic of Korea 152-703 (lhman@korea.ac.kr).

Submitted for Publication: March 13, 2006; final revision received April 24, 2006; accepted May 6, 2006.

Author Contributions: Dr Lee had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Kang, Chae, and H.-M. Lee. Acquisition of data: Kang, Cinn, and Woo. Analysis and interpretation of data: Kang, S. H. Lee, and H.-M. Lee. Drafting of the manuscript: Kang and Cinn. Critical revision of the manuscript for important intellectual content: Kang, Chae, Woo, and H.-M. Lee. Statistical analysis: Hwang and Woo. Obtained funding: Hwang, S. H. Lee, and H.-M. Lee. Administrative, technical, and material support: Hwang, Chae, and S. H. Lee. Study supervision: H.-M. Lee.

Financial Disclosure: None reported.

Funding/Support: This study was supported by the Brain Korea 21 Project of the Ministry of Education and Human Resources Development, Republic of Korea, 2005.