Abstract
Purpose
A fungal etiology has been proposed to underlie severe nasal polyps (NP). Dectin-1 is an innate immune pattern recognition receptor which is involved in the recognition of some pathogenic fungi. We investigated the Dectin-1 levels in NP in order to evaluate the implication of such expression with respect to the development of NP.
Materials and methods
Normal inferior turbinate tissues were obtained from forty patients undergoing surgery for augmentation rhinoplasty. Nasal polyp tissues were obtained from 53 patients who underwent endoscopic sinus surgery for chronic polypoid rhinosinusitis. Real-time polymerase chain reaction and Western blot analysis were performed to evaluate the mRNA and protein level of Dectin-1, respectively. ELISA was carried out to evaluate the cytokine production (IL-4, IL-5, IL-10, and TNF-α) in NP.
Results
Real-time polymerase chain reaction and Western-blot analysis showed that Dectin-1 expression in NP was increased compared with that in normal nasal inferior turbinate tissues. ELISA results suggest that the local expression of type-1 and type-2 inflammatory cytokine is skewed toward type-2 inflammatory cytokine in NP.
Conclusions
These results suggest that Dectin-1 may play a role in the development of NP, and the production of Dectin-1, IL-4 and IL-5 (type-2 cytokines), may mainly participate in the inflammatory reaction in NP.
1
Introduction
Nasal polyps (NP) are a chronic inflammatory airway disease of the nasal and paranasal sinus mucosa . Although the long history and prevalence of NP, many questions are still not answered with respect to the pathogenesis and incidence of NP . NP is frequently associated with different local inflammatory diseases, such as chronic rhinosinusitis, Kartagener’s syndrome, and cystic fibrosis . The role of infection as an effect on or cause of NP is still debated to date. Fungal infection of the paranasal sinuses may occur in both immunocompetent and immunocompromised individuals, and studies showed that Aspergillus spp. are the most common fungal species which were found in both groups . So, a fungal etiology has been supposed to underlie severe NP in general .
Dectin-1 is a transmembrane protein which contains an immunoreceptor tyrosine-based activation-like motif within its intracellular tail and a single C-type lectin-like ligand-binding domain in the extracellular region. Dectin-1 is first detected in myeloid cells such as macrophages, dendritic cells and neutrophils . Henceforth, expression of Dectin-1 has also been reported on other cell types including eosinophils and B-cells in humans, although this receptor could not be found on these populations in mice . Dectin-1 is an innate immune pattern recognition receptor which is involved in the recognition of some pathogenic fungi . Recent studies provided evidence for immunopathogenic mechanisms underlying the orchestration of both the adaptive and the innate immune responses in NP .
To date, little is known concerning the expression of Dectin-1 in NP. We hypothesized that Dectin-1, an innate immune pattern recognition receptor, may play a role in the recognition of several pathogenic fungi in NP. The objectives of this study were to determine the expression of Dectin-1 in normal mucosa and in the NP and to evaluate the possible effects of Dectin-1 proteins on the development of NP.
2
Subjects and methods
2.1
Study design and setting
Tissues from normal inferior turbinate tissues were obtained from 40 patients (21 male and 19 female patients; mean age, 34.7 years) undergoing surgery for augmentation rhinoplasty. NP were obtained from 53 patients (27 male and 26 female patients; mean age, 35.3 years) undergoing endoscopic sinus surgery for chronic polypoid rhinosinusitis. None of these NP patients and patients undergoing surgery for augmentation rhinoplasty had a history of asthma, allergic rhinitis, or aspirin sensitivity. NP were diagnosed using standard nasal endoscopy (Karl Storz Endoscopy, Shanghai Ltd., P.R. China), and most NP were detected from the anterior end of the middle turbinate and/or the superior uncinate process mucosa. Both NP and normal inferior turbinate tissues were immediately flash frozen in liquid nitrogen and stored at − 70°C for subsequent mRNA, Western blot, and ELISA analysis. Permission to conduct the study was obtained from the Ethics Committee of the Sun Yat-sen Memorial hospital of Sun Yat-sen University, and all of the patients who participated in the study provided informed consent.
2.2
RNA extraction and real-time polymerase chain reaction (real-time PCR)
Total RNA from tissues (40 normal inferior turbinate tissues and 53 NP tissues) was extracted using Trizol Reagent (Fermentas Life Sciences, Ontario, Canada) according to the manufacturer’s instructions. To determine the levels of Dectin-1 mRNA, real-time PCR and data collection were carried out with an ABI PRISM 7900HT sequence detection system by using SYBR® Green Real-Time PCR Master Mix (Invitrogen, Carlsbad, CA). The GAPDH housekeeping gene was used as an internal control to normalize the expression levels of Dectin-1. The following primers were used: GAPDH – 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′; Dectin-1 – 5′-CTGGGAGGATGGATCAACAT-3′ and 5′- CTGACACGTGAATCCATAC ACA-3′. The PCR amplification programs consisted of an initial incubation at 50°C for 2 min followed by 40 cycles of the following steps: 95°C for 10 min, 90°C for 15 s, and 55°C for 1 min.
2.3
ELISA
The levels of inflammatory cytokines in the NP and normal inferior turbinate tissues were determined by kit enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc., Space Import–export S.r.l., Milan, Italy). The detection limits (in picograms per milliliter) of this assay were 0.5 for IL-4, 5 for IL-5 and IL-10, and 10 for tumor necrosis factor alpha (TNF-α).
2.4
Western blot analysis
Samples which were frozen in liquid nitrogen were crushed into pieces, and cells were harvested and washed with cold phosphate-buffered saline solution. The total protein was extracted using lysis buffer. Immuno-blot experiments were performed by using standard procedures. The protein concentration was routinely determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were separated by using electrophoresis on 12% SDS/polyacrylamide gels and transferred onto Biodyne A Membrane (Gelman Laboratory, Pall Corp., Ann Arbor, MI). The membrane was probed with the primary antibody, rabbit polyclonal anti-Dectin-1 (1:1000, R&D systems, Wiesbaden-Nordenstadt, Germany). Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody, anti-goat (1:5000, Sigma, St. Louis, MO). An anti-GAPDH mouse monoclonal antibody (1:4000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as an internal control. The antibody reaction was detected by using the enhanced chemiluminescence detection kit (Amersham Bioscience, Giles, U.K.), which was followed by detection of chemiluminescence on X-ray film. In order to analyze the Western Blot results, the gel images were scanned and the intensity of the Western Blot bands was measured by using Image-Pro Plus 6.0 software (Media Cybernetics, USA). The relative intensities of individual bands were determined as the ratio of the intensity of Dectin-1 protein to the intensity of GAPDH.
2.5
Statistical analysis
Statistical analysis was performed with the SPSS software (SPSS Standard version 13.0, SPSS). Data was expressed as the mean ± SD. Statistical comparisons of Dectin-1 and inflammatory cytokine expression between NP tissues and normal tissues were performed by using t test. The significance of difference between these two groups was accepted at the 5% level of confidence. A P value < 0.05 was considered significant.
2
Subjects and methods
2.1
Study design and setting
Tissues from normal inferior turbinate tissues were obtained from 40 patients (21 male and 19 female patients; mean age, 34.7 years) undergoing surgery for augmentation rhinoplasty. NP were obtained from 53 patients (27 male and 26 female patients; mean age, 35.3 years) undergoing endoscopic sinus surgery for chronic polypoid rhinosinusitis. None of these NP patients and patients undergoing surgery for augmentation rhinoplasty had a history of asthma, allergic rhinitis, or aspirin sensitivity. NP were diagnosed using standard nasal endoscopy (Karl Storz Endoscopy, Shanghai Ltd., P.R. China), and most NP were detected from the anterior end of the middle turbinate and/or the superior uncinate process mucosa. Both NP and normal inferior turbinate tissues were immediately flash frozen in liquid nitrogen and stored at − 70°C for subsequent mRNA, Western blot, and ELISA analysis. Permission to conduct the study was obtained from the Ethics Committee of the Sun Yat-sen Memorial hospital of Sun Yat-sen University, and all of the patients who participated in the study provided informed consent.
2.2
RNA extraction and real-time polymerase chain reaction (real-time PCR)
Total RNA from tissues (40 normal inferior turbinate tissues and 53 NP tissues) was extracted using Trizol Reagent (Fermentas Life Sciences, Ontario, Canada) according to the manufacturer’s instructions. To determine the levels of Dectin-1 mRNA, real-time PCR and data collection were carried out with an ABI PRISM 7900HT sequence detection system by using SYBR® Green Real-Time PCR Master Mix (Invitrogen, Carlsbad, CA). The GAPDH housekeeping gene was used as an internal control to normalize the expression levels of Dectin-1. The following primers were used: GAPDH – 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′; Dectin-1 – 5′-CTGGGAGGATGGATCAACAT-3′ and 5′- CTGACACGTGAATCCATAC ACA-3′. The PCR amplification programs consisted of an initial incubation at 50°C for 2 min followed by 40 cycles of the following steps: 95°C for 10 min, 90°C for 15 s, and 55°C for 1 min.
2.3
ELISA
The levels of inflammatory cytokines in the NP and normal inferior turbinate tissues were determined by kit enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc., Space Import–export S.r.l., Milan, Italy). The detection limits (in picograms per milliliter) of this assay were 0.5 for IL-4, 5 for IL-5 and IL-10, and 10 for tumor necrosis factor alpha (TNF-α).
2.4
Western blot analysis
Samples which were frozen in liquid nitrogen were crushed into pieces, and cells were harvested and washed with cold phosphate-buffered saline solution. The total protein was extracted using lysis buffer. Immuno-blot experiments were performed by using standard procedures. The protein concentration was routinely determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were separated by using electrophoresis on 12% SDS/polyacrylamide gels and transferred onto Biodyne A Membrane (Gelman Laboratory, Pall Corp., Ann Arbor, MI). The membrane was probed with the primary antibody, rabbit polyclonal anti-Dectin-1 (1:1000, R&D systems, Wiesbaden-Nordenstadt, Germany). Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody, anti-goat (1:5000, Sigma, St. Louis, MO). An anti-GAPDH mouse monoclonal antibody (1:4000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as an internal control. The antibody reaction was detected by using the enhanced chemiluminescence detection kit (Amersham Bioscience, Giles, U.K.), which was followed by detection of chemiluminescence on X-ray film. In order to analyze the Western Blot results, the gel images were scanned and the intensity of the Western Blot bands was measured by using Image-Pro Plus 6.0 software (Media Cybernetics, USA). The relative intensities of individual bands were determined as the ratio of the intensity of Dectin-1 protein to the intensity of GAPDH.
2.5
Statistical analysis
Statistical analysis was performed with the SPSS software (SPSS Standard version 13.0, SPSS). Data was expressed as the mean ± SD. Statistical comparisons of Dectin-1 and inflammatory cytokine expression between NP tissues and normal tissues were performed by using t test. The significance of difference between these two groups was accepted at the 5% level of confidence. A P value < 0.05 was considered significant.
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