Chronic rhinosinusitis with nasal polyps and without nasal polyps is associated with increased expression of lysophosphatidic acid–related molecules Se Jin Park, M.D.,1 Young Joon Jun, M.D.,2 Ki Jeong Lee, M.D.,3 Soo Min Hwang, M.D.,3 Tae Hoon Kim, M.D.,3 Seung Hoon Lee, M.D.,3 and Sang Hag Lee, M.D3
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ABSTRACT
Background: Chronic sinusitis with nasal polyps (CRSwNPs) or CRS without NPs (CRSsNPs) is associated with expression of various cytokines. Lysophosphatidic acid (LPA) generated by autotaxin (ATX), LPA-producing enzyme, initiates signaling cascade involved in the inflammatory responses and participates in diverse biological processes through LPA receptors, including cytokine production. We analyzed the expression and distribution patterns of LPA-related molecules in nasal secretion and sinus mucosa of normal controls and patients with CRSwNPs and CRSsNPs, to evaluate the possible effects of the ATX–LPA receptor axis on the pathogenesis of CRS. Methods: LPA levels in nasal secretion and the expression and distribution patterns of ATX and LPA receptors 1–3 (LPA1–3) in sinus mucosa were investigated using ELISA, real-time polymerase chain reaction, Western blot, and immunohistochemistry. We elucidated the effect of CRS-relevant cytokines on the expression of ATX and LPA receptors, using cultured sinus epithelial cells, and investigated the effect of LPA on the expression of CRS-relevant cytokines, using sinus mucosa explant culture. Results: LPA, ATX, and LPA1–3 levels are increased in CRSwNPs and CRSsNPs. ATX and LPA1–3 were localized to superficial epithelium, submucosal glands in normal and inflammatory mucosa, but in inflammatory mucosa, they were found in inflammatory cells. LPA1–3 were noted in endothelium. Sinus mucosa explant stimulated with LPA increasingly produced IL-4, IL-5, interferon gamma, and TNF-alpha, and in cultured epithelial cells stimulated with CRS-relevant cytokines, ATX, and LPA1–3 were differentially induced. Conclusion: LPA in human sinus mucosa may play important roles in the pathogenesis of CRS, contributing to produce CRS-related cytokines. LPA-related molecules were increased in CRS, which may attribute to CRS-related cytokines. (Am J Rhinol Allergy 28, 199 –207, 2014; doi: 10.2500/ajra.2014.28.4032)
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hronic rhinosinusitis (CRS) is defined as symptomatic mucosal inflammation of the paranasal sinuses and its etiology and pathophysiology remain debatable.1,2 However, bacteria, viruses, and fungi have been theorized to play roles in inciting intense host inflammatory responses.3,4 Abnormal immune defense to these triggers, including cytokine and chemokine signaling of sinus mucosa, rather than the trigger itself, have been suggested to be ultimately responsible for the persistent inflammatory process related to chronic rhinosinusitis (CRS).3,4 Based on the clinical and pathological features, CRS is classified into CRS with nasal polyps (CRSwNPs) and CRS without NPs (CRSsNP), and both are increasingly recognized as distinct disease entities.1 CRSwNPs is characterized by Th2-biased eosinophilic inflammation with high levels of IL-5, whereas CRSsNPs is associated with high levels of interferon (IFN) ␥ and transforming growth factor (TGF) 1.3,4 However, the mechanism of inflammation in CRS has still not been fully evaluated. Lysophosphatidic acid (LPA) is a naturally occurring bioactive lysophospholipid that initiates a signaling cascade involved in inflammatory responses, including cytokine and chemokine production, and chemoattraction.5–9 Studies using human epithelial cells
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From the 1Department of Otorhinolaryngology–Head and Neck Surgery, College of Medicine, Hallym University, Chuncheon, Korea, 2Department of Otorhinolaryngology– Head and Neck Surgery, College of Medicine, Soon Chun Hyang University, Asan, South Korea, and 3Department of Otorhinolaryngology–Head and Neck Surgery, College of Medicine, Korea University, Seoul, South Korea Funded by a Grant-in-Aid for Scientific Research from the Communication Disorders Center, Korea University, Korea, and The Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2010-0004094), and the research was funded by a Korea University Grant, Korea The authors have no conflicts of interest to declare pertaining to this article Address correspondence to Sang Hag Lee, M.D., Ph.D., Department of Otorhinolaryngology–Head and Neck Surgery, College of Medicine, Korea University, 126-1 Anamdong 5-ga, Seongbuk-gu, Seoul,136-705, South Korea E-mail address: [email protected] Copyright © 2014, OceanSide Publications, Inc., U.S.A.
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showed that LPA augmented the secretion of the neutrophil chemoattractant IL-8 and the Th2 cytokines, IL-4 and IL-6.8,9 LPA can be generated from lysophosphatidylcholine by autotaxin (ATX), which has emerged as the key LPA-producing enzyme in plasma and tissues.5,10 The biological functions of LPA are mediated through activation of G protein–coupled receptors. So far, six LPA receptors have been identified, LPA1–6. The best-characterized LPA receptors are those of the endothelial differentiation gene family including LPA1, LPA2, and LPA3.5,11 The effects of ATX are mainly mediated by the enzymic formation of LPA and are dependent on the levels of LPA generated and on the types of LPA receptors involved in signal transmission, forming the ATX–LPA receptor axis.5,11 The ATX–LPA receptor axis has been shown to be up-regulated in a variety of inflammatory conditions including allergy and asthma.11,12 In human rheumatoid arthritis, the ATX gene and LPA1 are up-regulated in fibroblasts and the ATX protein is found in the synovial fluid.13,14 The conditional genetic deletion of ATX attenuated the development of the modeled disease such as pulmonary inflammation, fibrosis, and arthritis, suggesting a major role for the ATX–LPA receptor axis in chronic inflammations.11,12,15,16 In a murine asthma model, LPA1 heterozygous mice had reduced mucus production but not eosinophil infiltration, and LPA2 heterozygous mice revealed a decrease in both eosinophil infiltration and mucus production.11 Considering the evidence that eosinophils and neutrophils, in addition to the characteristic expression of cytokines, are differentially infiltrated in the sinonasal mucosa of patients with CRSsNPs and CRSwNPs, LPA-related molecules such as LPA, the LPA-producing enzyme, ATX, and LPA receptors may play a role in the pathogenesis of CRSwNPs and CRSsNPs. Therefore, the present study is 1. Measured LPA levels in nasal secretion of healthy controls and patients with CRSwNPs and CRSsNPs. 2. Determined the expression levels and distribution pattern of ATX and LPA receptors in normal ethmoid sinus mucosa of healthy control and inflammatory ethmoid sinus mucosa of patients with CRSwNPs and CRSsNPs to evaluate the possible effects of the ATX–LPA receptor axis in the pathogenesis of CRS,
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Table 1 Patient characteristics
No. of patients Age (yr) mean range No. of female/male Asthma history Skin test Smoking No. of sinus surgery SNOT-20* Computed tomography grade* Endoscopy scores* No. of sinus surgeries Total inflammatory cells# Eosinophils§ Mononuclear cells# Plasma cells§
Controls
CRSwNPs
CRsNPs
25 29.5 (18–35) 5/20 0 Negative 5/25 0 2.5 ⫾ 1.6 0 0 0 11.7 ⫾ 5.8 1.0 ⫾ 0.2 15,5 ⫾ 6.9 1.6 ⫾ 1.3
25 37.6 (17–40) 7/18 0 Negative 6/25 0 27.9 ⫾ 2.5 18 ⫾ 1.1 9.6 ⫾ 0.9 0 77.5 ⫾ 11.8 4.9 ⫾ 1,5 45.3 ⫾ 12.1 5.2 ⫾ 1.3
25 35.7 (20–45) 8/17 0 Negative 7/25 0 22.5 ⫾ 3.7 7.6 ⫾ 1.7 2.9 ⫾ 0.8 0 72.8 ⫾ 9.6 1.6 ⫾ 1.3 34.7 ⫾ 15.2 2.8 ⫾ 1.3
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*The data of CRSwNPs and CRSsNPs are significantly higher than those of controls, with differences between CRSwNPs and CRSsNPs. #The data of CRSwNPs and CRSsNPs are significantly higher than those of controls, without differences between CRSwNPs and CRSsNPs. §The data of CRSwNPs are significantly higher than those of control and CRSsNPs, without differences between CRSsNPs and controls. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; CRSsNPs ⫽ chronic rhinosinusitis without nasal polyps; SNOT-20 ⫽ 20-item Sino-Nasal Outcome Test.
Table 2 Sequences of PCR primers Primer (RefSeq) Autotaxin (L35594.1) LPA1 (BC030615.2) LPA2 (BC025695.1) LPA3 (NM_012152.2) IL-4 (M13982.1) IL-5 (J03478.1) IL-1 (BC008678.1) IL-13 (NM_002188.2) IFN-␥ (NM_000619.2) TNF-␣ (HQ201306.2) TGF-1 (NM_000660.4) GAPDH (NM_002046)
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Size (bp) 114 88
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Sequence
S: 5⬘-ACAACGAGGAGAGCTGCAAT-3 AS: 5⬘-AGAAGTCCAGGCTGGTGAGA-3⬘ S: 5⬘-GAAAGCATCTTGCCACAGAA-3⬘ AS: 5⬘-TTGGCCAACATGATGAAGAT -3⬘ S: 5⬘-GGTACTGCTCCTGGATGGTT-3⬘ AS: 5⬘-GCAGCATTGACCAGTGAGTT-3⬘ S: 5⬘-CACCAACTTGCTGGTTATCG-3⬘ AS: 5⬘-TTTGGTCAGGTTGCTATGGA-3⬘ S: 5⬘-TTGCTGCCTCCAAGAACACAACTG-3⬘ AS: 5⬘-TTCCTGTCGAGCCGTTTCAGGAAT-3⬘ S: 5⬘-TAGCTCTTGGAGCTGCCTACGTGTAT-3⬘ AS: 5⬘-AAGCAGTGCCAAGGTCTCTTTCAC-3⬘ S: 5⬘-TGGACAAGCTGAGGAAGA-3⬘ AS: 5⬘- CCCATGTGTCGAAGAAGATAG-3⬘ S: 5⬘-TGGTCAACATCACCCAGAACCAGA-3⬘ AS: 5⬘-AGCCTGACACGTTGATCAGGGATT-3⬘ S: 5⬘-TGCAGGTCATTCAGATGTAGCGGA-3⬘ AS: 5⬘-TGTCTTCCTTGATGGTCTCCACACTC-3⬘ S: 5⬘-AAGCCCTGGTATGAGCCCATCTAT-3⬘ AS: 5⬘-ATGATCCCAAAGTAGACCTGCCCA-3⬘ S: 5⬘-CGACTACTACGCCAAGGA-3⬘ AS: 5⬘-GAGAGCAACACGGGTTCA-3⬘ S: 5⬘-ATCATCCCTGCCTCTACTGG-3⬘ AS: 5⬘–GTCAGGTCCACCACTGACAC-3⬘
ATX ⫽ autotaxin; PCR ⫽ polymerase chain reaction; LPA ⫽ Lysophosphatidic acid receptor; LPA1 ⫽ LPA receptor 1; LPA2 ⫽ LPA receptor 2; LPA3 ⫽ LPA receptor 3; IFN ⫽ interferon.
using real-time polymerase chain reaction (PCR), Western blot, and immunohistochemistry. 3. Evaluated the effect of LPA on the cytokine expression levels, which are relevant for pathogenesis of CRS in sinonasal mucosa, using sinus mucosa explant culture. 4. Elucidated the impact of CRS-relevant inflammatory cytokines on ATX and LPA receptors expression in sinonasal mucosa, using epithelial cell culture.
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METHODS Subjects Twenty-five patients with CRSwNPs, 25 patients with CRSsNPs, and 25 controls were enrolled. Control subjects were patients undergoing endoscopic reduction because of blowout fracture and did not have sinus disease. The diagnosis of CRS was made according to the current
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Figure 1. (A) Concentration of LPA in nasal secretion of control subjects (N; n ⫽ 25) and patients with CRSwNPs (n ⫽ 25) and CRSsNPs (n ⫽ 25). (B) Real-time PCR and (C) Western blot analysis of ATX, LPA1, LPA2, and LPA3 expression in normal sinus mucosa of control subjects (N; n ⫽ 25) and inflammatory sinus mucosa of CRSwNPs (n ⫽ 25) and CRSsNPs (n ⫽ 25). The panels of Western blot show representative data (black bar, normal control; gray bar, CRSwNPs; white bar, CRSsNPs). Asterisk indicates significant difference in the expression levels of LPA, ATX, and LPA1–3 between normal control and CRSwNPs or CRSsNPs (p ⬍ 0.5). Results are expressed as mean ⫾ SD. LPA, lysophosphatidic acid; LPA1, LPA receptor 1; LPA2, LPA receptor 2; LPA3, LPA receptor 3; CRSwNPs, chronic rhinosinusitis with nasal polyps; CRSsNPs, chronic rhinosinusitis without nasal polyps; PCR, polymerase chain reaction; ATX, autotaxin.
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European Academy of Allergology and Clinical Immunology Position Paper on rhinosinusitis and NPs.18 The presence or absence of NPs was recorded based on nasal endoscopy. Endoscopic physical findings, findings on sinus coronal computed tomography scans, and patient symptoms were rated as described previously.19,20 None of the control subjects or patients with CRSwNPs and CRSsNPs had a history of asthma or a positive skin-prick test to a standard panel of aeroallergens. The patients with CRSwNPs and CRSsNPs were excluded to minimize the effects of allergy on LPA and ATX levels. Subjects were excluded if they had taken any oral or topical medication including steroid, antihistamine, or antibiotics at least 3 months before the surgery. Clinical data of patients are summarized in Table 1. The Institutional Review Boards for human beings at the Korea University Hospital approved the protocols, and the informed consent form was obtained from all patients before collecting any samples. For experimental purposes, we obtained nasal secretion using nasal lavage and ethmoid sinus mucosa during operation. Normal-appearing ethmoid sinus mucosa was obtained as normal controls during endoscopic reduction in patients with blowout fracture. Inflammatory ethmoid sinus mucosa was obtained from patients with CRSwNPs and CRSsNPs during endoscopic sinus surgery. Three pieces of ethmoid sinus mucosa were, respectively, obtained from each subject. The first and second portions were stored at ⫺80°C for subsequent RNA isolation and protein isolation. For immunohistochemistry, the third samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) and embedded in paraffin wax. A part of normal and inflammatory sinus mucosa sinus mucosa was used for isolation and culture of epithelial cells. In sections
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stained with hematoxylin and eosin, the number of total inflammatory cells, eosinophils, mononuclear cells, and the plasma cells in the subepithelial layer was counted at high-power magnification (⫻400), and five high-power fields were randomly selected.
Nasal Lavage and Analysis of LPA Nasal lavage was performed by slowly instilling 10 mL of sterile isotonic saline into each nostril using a syringe. The solution was retained for ⬃10 seconds in the nasal cavities and then was expulsed into a sterile plastic beaker. All nasal lavage samples were stored at ⫺80°C until analysis. A fraction of the nasal lavage sample (0.5 mL) was acidified with 12 N HCl to have final concentration of 0.2 N HCl and then lipids were extracted with n-butanol (2 mL) by extensive vortexing. The assay of LPA in nasal secretion was performed using ELISA as manufacturer’s instruction (Lysophosphatidic Acid Assay Kit; Echelon Biosciences, Inc., Salt Lake City, UT). An amount of 100 l of concentrated lavage fluid and anti-LPA antibody solution (4:1) was added into the wells. The secondary antibody conjugated to horseradish peroxidase was added to each well of ELISA plate. The reaction was completed by the addition of 2 N sulfuric acid and optical density was read at 450 nm in a microplate reader.
Isolation and Culture of Epithelial Cells from Normal Sinus Mucosa Human normal sinus mucosa was treated with 0.5% Dispase (GenDEPOT, Barker, TX) in a 1:1 mixture of DMEM/F12 (Lonza Inc.,
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Allendale, NJ) supplemented with penicillin G sodium and streptomycin sulfate overnight at 4°C. Epithelial cells were peeled off the underlying connective tissue and were cultured under air–liquid interface in serum-free bronchial epithelial growth medium (Lonza Walkersville, Inc., Walkersville, MD). They were stimulated with IL-4, IL-5, IL-1, IL-13, TGF-1, TNF-␣, or IFN-␥ at the concentration of 10 and 30 ng/mL, respectively, for 12 and 24 hours. These cytokines were purchased from PeproTech (Rocky Hill, NJ). The cultured cells stimulated with cytokines were harvested and analyzed for ATX and LPA receptors mRNA and proteins by using real-time PCR and Western blot.
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Figure 2. Immunohistochemical localization of (A,B, and C) ATX, (D. E. and F) LPA1, (G, H, and I) LPA2, and (J, K, and L) LPA3 in (A, D, G, and J) normal mucosa of control subjects and inflammatory sinus mucosa of (B, E, H, and K) CRSwNPs and (C, F, I, and L) CRSsNPs (arrow head, surface epithelium; thick arrow, submucosal glands; thin arrow, inflammatory cells; original magnification, ⫻100). V, vascular endothelium; LPA, lysophosphatidic acid; LPA1, LPA receptor 1; LPA2, LPA receptor 2; LPA3, LPA receptor 3; CRSwNPs, chronic rhinosinusitis with nasal polyps; CRSsNPs, chronic rhinosinusitis without nasal polyps; ATX, autotaxin.
Sinus Mucosa Explant Culture Inflammatory sinus mucosa was collected during endoscopic sinus surgery from patients with CRSwNPs or CRSwNPs and was used for tissue culture as described previously.21,22 Tissue was divided into multiple samples of 5-mm diameter, using dermal biopsy punch. All tissue samples were transferred to air–liquid interface culture plates containing 2 mL of DMEM/HamsF12 supplemented with 2 mM of l-glutamine, 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Invitrogen, Grand Island, NY). The tissues were oriented with the epithelium being exposed to the air and were cultured at 37°C with
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Figure 3. (A) Real-time PCR and (B) ELISA results of IL-4, IL-5, IL-1, IL-13, IFN-␥, TNF-␣, and TGF-1 expression levels in explant culture derived from sinus mucosa after stimulation with LPA (100 nM and 1 M) for 12 or 24 hours. Results are expressed as mean ⫾ SD. Asterisk indicates statistically significant difference in the expression levels of each cytokines between (C) nonstimulated and LPA-stimulated epithelial cells (LPA; p ⬍ 0.5). Gray bar indicates stimulation for 12 hours (n ⫽ 6), and white bar indicates stimulation for 24 hours (n ⫽ 6). PCR, polymerase chain reaction; IFN, interferon; TGF, transforming growth factor; LPA, lysophosphatidic acid.
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5% CO2 in humidified air. To evaluate the effect of LPA on the cytokine expression levels, which are relevant for pathogenesis of CRS, sinus mucosa explants culture was stimulated with LPA (100 nM and 1 M) for 12 and 24 hours and then with culture medium was harvested for real-time PCR and ELISA.
RNA Isolation and Real-Time Reverse Transcription PCR Analysis Total RNA was extracted from frozen sinus tissues and cultured epithelial cells stimulated with cytokine using Trizol reagent (Life Technologies, Rockville, MD). Total RNA (1 g) from each sample was reverse transcribed in 20 L of a reaction mixture containing 2.5 U of Moloney murine leukemia virus (RT; GIBCO BRL, Grand Island, NY). The primer sequences of each gene used in the study are listed in Table 2. Real-time PCR was performed using an iCycler (BioRad Laboratories, Hercules, CA). Each PCR reaction included 50 ng of cDNA, 12.5 L of iTAQ SYBR supermix, and 200 nM of primers. PCR conditions were 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, and 72°C extension for 20 seconds. Target gene expression was expressed as the fold increase and decrease relative to the expression of GAPDH. The mean value of the replicates for each sample was calculated and expressed as the cycle threshold (Ct). The amount of gene expression was then calculated as the difference (⌬Ct) be-
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tween the Ct value obtained for the target gene and the Ct value obtained for GAPDH. Fold change in target gene mRNA expression was determined as 2⫺⌬⌬Ct.
Immunohistochemical and Western Blot Analysis Immunohistochemical staining was performed using a peroxidaselabeled streptavidin-biotin technique. The sections were incubated overnight at room temperature with a 1:100 dilution of anti-ATX and LPA1, -2, and -3 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The negative immunohistochemical control procedure included omission of the primary antibodies and replacement of the primary antibodies by normal rabbit IgG in appropriate concentration. The color was developed using 3,3-diaminobenzidine. For Western blot analysis, the extracted protein (50 g) was separated on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to immobilon (Millipore, Bedford, MA). The blots were then incubated with anti-ATX and LPA1, -2, and -3 antibody in Trisbuffered saline overnight at room temperature. Antibody reactions were detected by using the ECL detection kit (GE Healthcare and Amersham Bioscience, Piscataway, NJ). The relative intensities of each protein signal were obtained by dividing the intensities of each protein signal by the -actin signals.
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Figure 4. (A, C, E, and G) Real-time PCR and (B, D, F, and H) Western blot of (A and B) ATX, (C and D) LPA1, (E and F) LPA2, and (G and H) LPA3 expression levels in cultured epithelial cells derived from normal sinus mucosa after stimulation with IL-4, IL-5, IL-1, IL-13, IFN-␥, TNF-␣, and TGF-1 (10 and 30 ng/mL), respectively, for 12 or 24 hours. Results are expressed as mean ⫾ SD. Asterisk indicates statistically significant difference in the expression levels of each molecule between cytokine-treated and -untreated epithelial cells (p ⬍ 0.5). Black bar indicates stimulation for 12 hours (n ⫽ 6), and white bar indicates stimulation for 24 hours (n ⫽ 6). C indicates untreated epithelial cells. PCR, polymerase chain reaction; IFN, interferon; TGF, transforming growth factor; LPA, lysophosphatidic acid; LPA1, LPA receptor 1; LPA2, LPA receptor 2; LPA3, LPA receptor 3; ATX, autotaxin.
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NPs, ATX was also found in the inflammatory cells (Fig 2, B and C). LPA1–3 were mainly distributed in the superficial epithelium, vascular endothelium, and submucosal glands in normal and inflammatory sinus mucosa. However, in inflammatory sinus mucosa of CRSwNPs and CRSsNPs, they were also found in the inflammatory cells (Fig 2, D–L).
Enzyme-Linked Immunosorbent Assay The concentrations of IL-4, IL-5, IL-1, IL-13, IFN-␥, TNF-␣, and TGF-1 were measured in supernatants from unstimulated or LPAstimulated explant cultures using ELISA kits according to the manufacturer’s instructions (R & D systems, Minneapolis, MN).
Statistical Analysis Statistical analyses were performed using SPSS for Windows (Version 16.0.0; SPSS, Inc., Chicago, IL). The age differences, 20-item Sino-Nasal Outcome Test, computed tomography score, endoscopic score, and number of inflammatory cells in the three groups were calculated by one-way ANOVA, and the Bonferroni post hoc correction was applied. One-way ANOVA, and the Bonferroni post hoc correction (␣ ⫽ 0.0167) and Tukey test were performed to establish statistically significant differences in values obtained from healthy controls and patients with CRSwNPs and CRSsNPs. The Kolmogorov-Smirnov test showed that the distribution of each data is normal. The level of significance was set at p ⬍ 0.5. Mann-Whitney U tests were performed to compare differences in expression levels of cytokine between LPA-stimulated and -nonstimulated control groups, and in expression levels of ATX and LPA1–3 between cytokinestimulated and -nonstimulated groups.
RESULTS
To identify factors that could modulate the expression of cytokines in CRS, we analyzed the expression levels of LPA in nasal lavage fluid and those of ATX and LPA1–3 in the sinus mucosa of control subjects and patients with CRSwNPs and CRSsNPs. LPA levels in nasal lavage samples from patients with CRSwNPs and CRSsNPs were significantly higher than that of control patients. However, there was no statistical difference in LPA levels between CRSwNPs and CRSsNPs (Fig 1 A). The expression levels of ATX and LPA1–3 mRNAs and proteins were determined in sinus mucosa of normal controls and patients with CRSwNPs and CRSsNPs. As shown in Fig 1, B and C, the expression levels of ATX and LPA1–3 were significantly increased in inflammatory sinus mucosa of patients with CRSwNPs and CRSsNPs, irrespective of the presence of NPs, compared with healthy nasal mucosa. However, the expression levels of ATX and LPA1–3 in inflammatory sinus mucosa did not differ significantly between CRSwNPs and CRSsNPs (Fig 1, B and C). These results indicated that the expression levels of ATX and LPA1–3 were increased in the inflammatory sinus mucosa of CRS patients, irrespective of polyp presence, compared with normal sinus mucosa. The distribution patterns of ATX and LPA1–3 in sinus mucosa of control subjects and patients with CRSwNPs and CRSsNPs were analyzed by immunohistochemistry. Immunohistochemical staining showed a similar pattern in all samples of normal sinus mucosa of control subjects and inflammatory sinus mucosa obtained from patients with CRSwNPs and CRSsNPs, respectively (Fig 2). In normal and inflammatory sinus mucosa, ATX was largely localized to the superficial epithelium and submucosal glands (Fig 2, A–C). However, in inflammatory sinus mucosa of patients with CRSwNPs and CRSs-
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To determine the possible effects of LPA contributing to the expression of cytokine in the sinonasal mucosa, the expression levels of Th1 cytokine (IL-1, IFN-␥, and TNF-␣), Th2 cytokine (IL-4, IL-5, and IL-13), and TGF-1 after stimulating sinus mucosa explant with LPA were examined with real-time PCR and ELISA. The current data showed the increased production of IL-4, IL-5, IFN-␥, and TNF-␣ in sinus mucosa at 12 and 24 hours after stimulation with LPA (Fig. 3). In contrast, the expression of IL-1, IL-13, and TGF-1 was not enhanced after stimulation with LPA (Fig. 3).
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LPA Levels in Nasal Lavage Fluid and Expression Levels and Distribution Pattern of ATX and LPA1–3 in Sinus Mucosa
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Effects of LPA on the Production of Cytokines in Sinus Mucosa
Effect of Cytokines on the Expression Levels of ATX and LPA1–3 in Sinus Mucosa
To examine which of these cytokines is most potent in inducing the ATX and LPA1–3 expression in sinus mucosa, cultured epithelial cells were incubated in the presence of cytokines for 12 or 24 hours, and then the expression levels of ATX and LPA1–3 were determined. IL-4, IL-5, IFN-␥, and TNF-␣ resulted in the increased production of ATX (Fig 4, A and B). LPA1 and LPA3 expression were enhanced by stimulation of cultured normal epithelial cells with IL-4, IL-5, IL-1, IL-13, IFN-␥, and TNF-␣, and LPA2 was induced by IL-4, IL-5, IFN-␥, and TNF-␣ (Fig 4, C–H).
DISCUSSION Several novel findings were observed in the present study. First, we showed that LPA in nasal lavage fluid and LPA-producing enzyme ATX and LPA1- 3 in sinus mucosa are constitutively expressed in normal controls and are up-regulated in patients with CRSwNPs and CRSsNPs. In normal and inflammatory sinus mucosa, ATX was largely localized to the superficial epithelium and submucosal glands. However, in inflammatory sinus mucosa, ATX was also found in the inflammatory cells. LPA1–3 in normal and inflammatory sinus mucosa were distributed in the superficial epithelium, submucosal glands, and vascular endothelium, but were additionally found in inflammatory cells infiltrated into the inflammatory sinus mucosa of CRSwNPs and CRSsNPs. Furthermore, the stimulation of cultured epithelial cells with LPA induced increased expression levels of IL-4, IL-5, IFN-␥, and TNF-␣. The expression levels of ATX and LPA1–3 were differentially increased after stimulation with cytokines. Collectively, these data suggest that LPA may play the role of an inflammatory mediator in CRS, by acting through up-regulated LPA receptors. In the present study, LPA was found in nasal lavage fluids at baseline and was significantly increased in nasal secretions in CRS, irrespective of NP presence, suggesting that LPA is a natural constituent of nasal secretion. Our results are consistent with previous studies showing the presence of LPA in various fluids, including serum, seminal plasma, cerebrospinal fluid, saliva, and in human
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bronchoalveolar lavage fluids at baseline.5 Up-regulated concentrations of LPA have been detected in the bronchoalveolar lavage fluid in lung inflammatory diseases such as asthma, pulmonary fibrosis, and in the synovial fluid of patients with rheumatoid arthritis.5,11,23 However, the mechanism of LPA generation in nasal secretion is not clear. Bioactive LPA is produced from lysophosphatidylcholine by a secreted lysophospholipase D, known as ATX, and recent studies suggest a critical role of ATX in the regulation of LPA levels in plasma and cancer tissues.24 In an acute lung injury model, increased plasma LPA levels were associated with increased ATX levels.25 In lymphoma cells, specific down-regulation of ATX decreased LPA levels.26 In the present study, ATX was constitutively expressed in normal sinus mucosa and was up-regulated in inflammatory sinus mucosa of CRSwNPs and CRSsNPs. Therefore, our data suggest the presence of the LPA-producing enzyme system in human sinus mucosa and provide the possibility that the induction of ATX may lead to the subsequent generation of LPA in CRS. Nevertheless, the mechanism that mediates the up-regulation of ATX in chronic sinusitis remains unknown. One possible mechanism is that the elevated levels of various cytokines in CRS may contribute to cytokine-induced elevation of ATX, depending on the facts that ATX is regulated by cytokines.27 Cultured epithelial cells with IL-4, IL-5, IFN-␥, and TNF-␣ resulted in up-regulated expression of ATX, which suggests that these cytokines may contribute to the induced production of ATX in the sinus mucosa of patients with CRSwNPs and CRSsNPs, thereby contributing to the increased production of LPA in nasal secretion. This suggestion is supported by the observation that ATX expression from synovial fibroblasts in the synovium was increased by TNF-␣, the major proinflammatory cytokine during pathogenic process of rheumatoid arthritis.14 In this study, we focused on the effects of LPA on expression of cytokines relevant to pathogenesis of CRS, using cultured sinus mucosa explant. Although the effects of LPA on the production of cytokine have been previously studied in a variety of cell types, the topic remains somewhat controversial. In human bronchial epithelial cells and colon cancer cells, LPA induces IL-8 expression.27,28 In ovarian and breast cancer cells, LPA was shown to be a potent inducer of IL-6 and IL-8 production.29,30 In the ureteral obstruction mouse model, blocking LPA1 attenuated the expression of TGF-1.31 In epithelial cells derived from NPs, LPA was able to induce IL-4, IL-6, and IL-8 protein levels.9 These dichotomous results concerning the effects of LPA on cytokine expression may reflect differences in tissue conditions among the various studies in the literature. Additional studies to elucidate the molecular mechanisms linking LPA action will be needed for further our understanding of these regulatory molecules. Nevertheless, these results support the hypothesis that ATX–LPA receptor axis might participate in the pathological process of CRSwNPs or CRSsNPs, contributing to the differential regulation of cytokine production in sinus mucosa. However, the question of whether the role of the ATX–LPA receptor axis is beneficial or detrimental in the pathogenesis of CRS remains to be established. Several lines of evidence indicated that LPA acts as a proinflammatory mediator in lower respiratory diseases, suggesting that LPA might accelerate inflammation in airway inflammatory diseases such as acute lung injury, pulmonary fibrosis, and asthma.5,11,32 In contrast, LPA also regulates the expression of anti-inflammatory genes in airway epithelium and may have a protective role in inflammation and remodeling.5 LPA also induces cyclooxygenase 2 and prostaglandin E2 production, which suggests that it may function as an anti-inflammatory signal in the airway.32 Other studies have also suggested that LPA might play a role in homeostasis and promote the resolution of inflammation.9,33 Considering that in the present study the stimulation of cultured mucosa with LPA resulted in increased expression of cytokines, we suggest that LPA may play a role as an inflammatory mediator in CRS. Nevertheless, additional studies are required to evaluate the functional role of the ATX–LPA receptor axis in normal and inflammatory sinus mucosa of CRS.
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The distribution patterns of LPA1–3 did not differ between normal and inflammatory sinus mucosa, suggesting that these receptor subtypes are ubiquitously expressed in normal sinus mucosa. However, we found that many inflammatory cells infiltrating into the inflammatory sinus mucosa show the immunoreactivities against LPA1–3. Therefore, it is possible that increased expression levels of LPA1–3 in inflammatory sinus mucosa are caused by the influx of inflammatory cells. LPA1 and LPA2 are expressed in neutrophils, and up-regulation of LPA1 and LPA2 expressions in neutrophils from pneumonia patients has been reported.34 Indeed, in vitro studies have shown that LPA acts as a chemoattractant for human inflammatory cells.7,35 LPA may contribute to the infiltration and activation of inflammatory cells in bronchial asthma.6 Furthermore, LPA participates in inflammatory processes by enhancing chemoattraction in neutrophils and monocytes/macrophages.6,7 Emerging evidence suggests a role for ATX– LPA in facilitating lymphocyte migration into lymphoid and chronically inflamed nonlymphoid tissues.36,37 Thus, considering that inflammatory cell infiltration or exudation into the sinus mucosa appears to play an important role in the development of CRSwNPs or CRSsNPs,38–40 we suggest that LPA, via LPA1–3, may play a significant role in inducing and/or sustaining the massive infiltration of inflammatory cells in human sinus mucosa. These suggestions are supported by the role of LPA receptors in airway inflammatory disease studied in vitro and in vivo.11,41 On the other hand, recent studies have suggested that LPA receptor expression is regulated in an inflammatory environment, such as the inflamed synovium and lung.14,41 In fibroblast-like synoviocytes from rheumatoid arthritis patients, LPA1 and 3 expression levels were higher than those in osteoarthritis.14 LPA1–3 are differentially expressed in the central nervous system and their expression is up-regulated in response to injury.42 The current study also indicated that LPA1 and LPA3 expressions were enhanced by stimulation with IL-4, IL-5, IL-6, IL-13, IFN-␥, and TNF-␣, and LPA2 was induced by IL-4, IL-5, IFN-␥, and TNF-␣. Taken together, these results offer the possibility that LPA1–3 may be up-regulated by inflammatory mediators in CRS. However, in a recent study using lung epithelial cell lines, treatment with IL-13 and IFN-␥ significantly reduced LPA1 and LPA2 mRNA levels, compared with unstimulated cells.9 Additional studies will be needed to elucidate the molecular mechanisms linking LPA-related molecules in human sinus mucosa.
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CONCLUSION In conclusion, this is the first study exploring the association between LPA-related molecules and CRS. Our results suggest that LPArelated molecules may play important roles in the pathogenesis of CRSwNPs and CRSsNPs. Additional studies to show the exact mechanism by which the ATX–LPA receptor axis induces inflammation in inflammatory sinus mucosa will provide a foundation for the development of clinical therapies for CRSwNPs and CRSsNPs.
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ABSTRACT
Background: Chronic sinusitis with nasal polyps (CRSwNPs) or CRS without NPs (CRSsNPs) is associated with expression of various cytokines. Lysophosphatidic acid (LPA) generated by autotaxin (ATX), LPA-producing enzyme, initiates signaling cascade involved in the inflammatory responses and participates in diverse biological processes through LPA receptors, including cytokine production. We analyzed the expression and distribution patterns of LPA-related molecules in nasal secretion and sinus mucosa of normal controls and patients with CRSwNPs and CRSsNPs, to evaluate the possible effects of the ATX–LPA receptor axis on the pathogenesis of CRS. Methods: LPA levels in nasal secretion and the expression and distribution patterns of ATX and LPA receptors 1–3 (LPA1–3) in sinus mucosa were investigated using ELISA, real-time polymerase chain reaction, Western blot, and immunohistochemistry. We elucidated the effect of CRS-relevant cytokines on the expression of ATX and LPA receptors, using cultured sinus epithelial cells, and investigated the effect of LPA on the expression of CRS-relevant cytokines, using sinus mucosa explant culture. Results: LPA, ATX, and LPA1–3 levels are increased in CRSwNPs and CRSsNPs. ATX and LPA1–3 were localized to superficial epithelium, submucosal glands in normal and inflammatory mucosa, but in inflammatory mucosa, they were found in inflammatory cells. LPA1–3 were noted in endothelium. Sinus mucosa explant stimulated with LPA increasingly produced IL-4, IL-5, interferon gamma, and TNF-alpha, and in cultured epithelial cells stimulated with CRS-relevant cytokines, ATX, and LPA1–3 were differentially induced. Conclusion: LPA in human sinus mucosa may play important roles in the pathogenesis of CRS, contributing to produce CRS-related cytokines. LPA-related molecules were increased in CRS, which may attribute to CRS-related cytokines. (Am J Rhinol Allergy 28, 199 –207, 2014; doi: 10.2500/ajra.2014.28.4032)
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hronic rhinosinusitis (CRS) is defined as symptomatic mucosal inflammation of the paranasal sinuses and its etiology and pathophysiology remain debatable.1,2 However, bacteria, viruses, and fungi have been theorized to play roles in inciting intense host inflammatory responses.3,4 Abnormal immune defense to these triggers, including cytokine and chemokine signaling of sinus mucosa, rather than the trigger itself, have been suggested to be ultimately responsible for the persistent inflammatory process related to chronic rhinosinusitis (CRS).3,4 Based on the clinical and pathological features, CRS is classified into CRS with nasal polyps (CRSwNPs) and CRS without NPs (CRSsNP), and both are increasingly recognized as distinct disease entities.1 CRSwNPs is characterized by Th2-biased eosinophilic inflammation with high levels of IL-5, whereas CRSsNPs is associated with high levels of interferon (IFN) ␥ and transforming growth factor (TGF) 1.3,4 However, the mechanism of inflammation in CRS has still not been fully evaluated. Lysophosphatidic acid (LPA) is a naturally occurring bioactive lysophospholipid that initiates a signaling cascade involved in inflammatory responses, including cytokine and chemokine production, and chemoattraction.5–9 Studies using human epithelial cells
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From the 1Department of Otorhinolaryngology–Head and Neck Surgery, College of Medicine, Hallym University, Chuncheon, Korea, 2Department of Otorhinolaryngology– Head and Neck Surgery, College of Medicine, Soon Chun Hyang University, Asan, South Korea, and 3Department of Otorhinolaryngology–Head and Neck Surgery, College of Medicine, Korea University, Seoul, South Korea Funded by a Grant-in-Aid for Scientific Research from the Communication Disorders Center, Korea University, Korea, and The Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2010-0004094), and the research was funded by a Korea University Grant, Korea The authors have no conflicts of interest to declare pertaining to this article Address correspondence to Sang Hag Lee, M.D., Ph.D., Department of Otorhinolaryngology–Head and Neck Surgery, College of Medicine, Korea University, 126-1 Anamdong 5-ga, Seongbuk-gu, Seoul,136-705, South Korea E-mail address: [email protected] Copyright © 2014, OceanSide Publications, Inc., U.S.A.
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showed that LPA augmented the secretion of the neutrophil chemoattractant IL-8 and the Th2 cytokines, IL-4 and IL-6.8,9 LPA can be generated from lysophosphatidylcholine by autotaxin (ATX), which has emerged as the key LPA-producing enzyme in plasma and tissues.5,10 The biological functions of LPA are mediated through activation of G protein–coupled receptors. So far, six LPA receptors have been identified, LPA1–6. The best-characterized LPA receptors are those of the endothelial differentiation gene family including LPA1, LPA2, and LPA3.5,11 The effects of ATX are mainly mediated by the enzymic formation of LPA and are dependent on the levels of LPA generated and on the types of LPA receptors involved in signal transmission, forming the ATX–LPA receptor axis.5,11 The ATX–LPA receptor axis has been shown to be up-regulated in a variety of inflammatory conditions including allergy and asthma.11,12 In human rheumatoid arthritis, the ATX gene and LPA1 are up-regulated in fibroblasts and the ATX protein is found in the synovial fluid.13,14 The conditional genetic deletion of ATX attenuated the development of the modeled disease such as pulmonary inflammation, fibrosis, and arthritis, suggesting a major role for the ATX–LPA receptor axis in chronic inflammations.11,12,15,16 In a murine asthma model, LPA1 heterozygous mice had reduced mucus production but not eosinophil infiltration, and LPA2 heterozygous mice revealed a decrease in both eosinophil infiltration and mucus production.11 Considering the evidence that eosinophils and neutrophils, in addition to the characteristic expression of cytokines, are differentially infiltrated in the sinonasal mucosa of patients with CRSsNPs and CRSwNPs, LPA-related molecules such as LPA, the LPA-producing enzyme, ATX, and LPA receptors may play a role in the pathogenesis of CRSwNPs and CRSsNPs. Therefore, the present study is 1. Measured LPA levels in nasal secretion of healthy controls and patients with CRSwNPs and CRSsNPs. 2. Determined the expression levels and distribution pattern of ATX and LPA receptors in normal ethmoid sinus mucosa of healthy control and inflammatory ethmoid sinus mucosa of patients with CRSwNPs and CRSsNPs to evaluate the possible effects of the ATX–LPA receptor axis in the pathogenesis of CRS,
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Table 1 Patient characteristics
No. of patients Age (yr) mean range No. of female/male Asthma history Skin test Smoking No. of sinus surgery SNOT-20* Computed tomography grade* Endoscopy scores* No. of sinus surgeries Total inflammatory cells# Eosinophils§ Mononuclear cells# Plasma cells§
Controls
CRSwNPs
CRsNPs
25 29.5 (18–35) 5/20 0 Negative 5/25 0 2.5 ⫾ 1.6 0 0 0 11.7 ⫾ 5.8 1.0 ⫾ 0.2 15,5 ⫾ 6.9 1.6 ⫾ 1.3
25 37.6 (17–40) 7/18 0 Negative 6/25 0 27.9 ⫾ 2.5 18 ⫾ 1.1 9.6 ⫾ 0.9 0 77.5 ⫾ 11.8 4.9 ⫾ 1,5 45.3 ⫾ 12.1 5.2 ⫾ 1.3
25 35.7 (20–45) 8/17 0 Negative 7/25 0 22.5 ⫾ 3.7 7.6 ⫾ 1.7 2.9 ⫾ 0.8 0 72.8 ⫾ 9.6 1.6 ⫾ 1.3 34.7 ⫾ 15.2 2.8 ⫾ 1.3
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*The data of CRSwNPs and CRSsNPs are significantly higher than those of controls, with differences between CRSwNPs and CRSsNPs. #The data of CRSwNPs and CRSsNPs are significantly higher than those of controls, without differences between CRSwNPs and CRSsNPs. §The data of CRSwNPs are significantly higher than those of control and CRSsNPs, without differences between CRSsNPs and controls. CRSwNPs ⫽ chronic rhinosinusitis with nasal polyps; CRSsNPs ⫽ chronic rhinosinusitis without nasal polyps; SNOT-20 ⫽ 20-item Sino-Nasal Outcome Test.
Table 2 Sequences of PCR primers Primer (RefSeq) Autotaxin (L35594.1) LPA1 (BC030615.2) LPA2 (BC025695.1) LPA3 (NM_012152.2) IL-4 (M13982.1) IL-5 (J03478.1) IL-1 (BC008678.1) IL-13 (NM_002188.2) IFN-␥ (NM_000619.2) TNF-␣ (HQ201306.2) TGF-1 (NM_000660.4) GAPDH (NM_002046)
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Sequence
S: 5⬘-ACAACGAGGAGAGCTGCAAT-3 AS: 5⬘-AGAAGTCCAGGCTGGTGAGA-3⬘ S: 5⬘-GAAAGCATCTTGCCACAGAA-3⬘ AS: 5⬘-TTGGCCAACATGATGAAGAT -3⬘ S: 5⬘-GGTACTGCTCCTGGATGGTT-3⬘ AS: 5⬘-GCAGCATTGACCAGTGAGTT-3⬘ S: 5⬘-CACCAACTTGCTGGTTATCG-3⬘ AS: 5⬘-TTTGGTCAGGTTGCTATGGA-3⬘ S: 5⬘-TTGCTGCCTCCAAGAACACAACTG-3⬘ AS: 5⬘-TTCCTGTCGAGCCGTTTCAGGAAT-3⬘ S: 5⬘-TAGCTCTTGGAGCTGCCTACGTGTAT-3⬘ AS: 5⬘-AAGCAGTGCCAAGGTCTCTTTCAC-3⬘ S: 5⬘-TGGACAAGCTGAGGAAGA-3⬘ AS: 5⬘- CCCATGTGTCGAAGAAGATAG-3⬘ S: 5⬘-TGGTCAACATCACCCAGAACCAGA-3⬘ AS: 5⬘-AGCCTGACACGTTGATCAGGGATT-3⬘ S: 5⬘-TGCAGGTCATTCAGATGTAGCGGA-3⬘ AS: 5⬘-TGTCTTCCTTGATGGTCTCCACACTC-3⬘ S: 5⬘-AAGCCCTGGTATGAGCCCATCTAT-3⬘ AS: 5⬘-ATGATCCCAAAGTAGACCTGCCCA-3⬘ S: 5⬘-CGACTACTACGCCAAGGA-3⬘ AS: 5⬘-GAGAGCAACACGGGTTCA-3⬘ S: 5⬘-ATCATCCCTGCCTCTACTGG-3⬘ AS: 5⬘–GTCAGGTCCACCACTGACAC-3⬘
ATX ⫽ autotaxin; PCR ⫽ polymerase chain reaction; LPA ⫽ Lysophosphatidic acid receptor; LPA1 ⫽ LPA receptor 1; LPA2 ⫽ LPA receptor 2; LPA3 ⫽ LPA receptor 3; IFN ⫽ interferon.
using real-time polymerase chain reaction (PCR), Western blot, and immunohistochemistry. 3. Evaluated the effect of LPA on the cytokine expression levels, which are relevant for pathogenesis of CRS in sinonasal mucosa, using sinus mucosa explant culture. 4. Elucidated the impact of CRS-relevant inflammatory cytokines on ATX and LPA receptors expression in sinonasal mucosa, using epithelial cell culture.
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METHODS Subjects Twenty-five patients with CRSwNPs, 25 patients with CRSsNPs, and 25 controls were enrolled. Control subjects were patients undergoing endoscopic reduction because of blowout fracture and did not have sinus disease. The diagnosis of CRS was made according to the current
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Figure 1. (A) Concentration of LPA in nasal secretion of control subjects (N; n ⫽ 25) and patients with CRSwNPs (n ⫽ 25) and CRSsNPs (n ⫽ 25). (B) Real-time PCR and (C) Western blot analysis of ATX, LPA1, LPA2, and LPA3 expression in normal sinus mucosa of control subjects (N; n ⫽ 25) and inflammatory sinus mucosa of CRSwNPs (n ⫽ 25) and CRSsNPs (n ⫽ 25). The panels of Western blot show representative data (black bar, normal control; gray bar, CRSwNPs; white bar, CRSsNPs). Asterisk indicates significant difference in the expression levels of LPA, ATX, and LPA1–3 between normal control and CRSwNPs or CRSsNPs (p ⬍ 0.5). Results are expressed as mean ⫾ SD. LPA, lysophosphatidic acid; LPA1, LPA receptor 1; LPA2, LPA receptor 2; LPA3, LPA receptor 3; CRSwNPs, chronic rhinosinusitis with nasal polyps; CRSsNPs, chronic rhinosinusitis without nasal polyps; PCR, polymerase chain reaction; ATX, autotaxin.
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European Academy of Allergology and Clinical Immunology Position Paper on rhinosinusitis and NPs.18 The presence or absence of NPs was recorded based on nasal endoscopy. Endoscopic physical findings, findings on sinus coronal computed tomography scans, and patient symptoms were rated as described previously.19,20 None of the control subjects or patients with CRSwNPs and CRSsNPs had a history of asthma or a positive skin-prick test to a standard panel of aeroallergens. The patients with CRSwNPs and CRSsNPs were excluded to minimize the effects of allergy on LPA and ATX levels. Subjects were excluded if they had taken any oral or topical medication including steroid, antihistamine, or antibiotics at least 3 months before the surgery. Clinical data of patients are summarized in Table 1. The Institutional Review Boards for human beings at the Korea University Hospital approved the protocols, and the informed consent form was obtained from all patients before collecting any samples. For experimental purposes, we obtained nasal secretion using nasal lavage and ethmoid sinus mucosa during operation. Normal-appearing ethmoid sinus mucosa was obtained as normal controls during endoscopic reduction in patients with blowout fracture. Inflammatory ethmoid sinus mucosa was obtained from patients with CRSwNPs and CRSsNPs during endoscopic sinus surgery. Three pieces of ethmoid sinus mucosa were, respectively, obtained from each subject. The first and second portions were stored at ⫺80°C for subsequent RNA isolation and protein isolation. For immunohistochemistry, the third samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) and embedded in paraffin wax. A part of normal and inflammatory sinus mucosa sinus mucosa was used for isolation and culture of epithelial cells. In sections
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stained with hematoxylin and eosin, the number of total inflammatory cells, eosinophils, mononuclear cells, and the plasma cells in the subepithelial layer was counted at high-power magnification (⫻400), and five high-power fields were randomly selected.
Nasal Lavage and Analysis of LPA Nasal lavage was performed by slowly instilling 10 mL of sterile isotonic saline into each nostril using a syringe. The solution was retained for ⬃10 seconds in the nasal cavities and then was expulsed into a sterile plastic beaker. All nasal lavage samples were stored at ⫺80°C until analysis. A fraction of the nasal lavage sample (0.5 mL) was acidified with 12 N HCl to have final concentration of 0.2 N HCl and then lipids were extracted with n-butanol (2 mL) by extensive vortexing. The assay of LPA in nasal secretion was performed using ELISA as manufacturer’s instruction (Lysophosphatidic Acid Assay Kit; Echelon Biosciences, Inc., Salt Lake City, UT). An amount of 100 l of concentrated lavage fluid and anti-LPA antibody solution (4:1) was added into the wells. The secondary antibody conjugated to horseradish peroxidase was added to each well of ELISA plate. The reaction was completed by the addition of 2 N sulfuric acid and optical density was read at 450 nm in a microplate reader.
Isolation and Culture of Epithelial Cells from Normal Sinus Mucosa Human normal sinus mucosa was treated with 0.5% Dispase (GenDEPOT, Barker, TX) in a 1:1 mixture of DMEM/F12 (Lonza Inc.,
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Allendale, NJ) supplemented with penicillin G sodium and streptomycin sulfate overnight at 4°C. Epithelial cells were peeled off the underlying connective tissue and were cultured under air–liquid interface in serum-free bronchial epithelial growth medium (Lonza Walkersville, Inc., Walkersville, MD). They were stimulated with IL-4, IL-5, IL-1, IL-13, TGF-1, TNF-␣, or IFN-␥ at the concentration of 10 and 30 ng/mL, respectively, for 12 and 24 hours. These cytokines were purchased from PeproTech (Rocky Hill, NJ). The cultured cells stimulated with cytokines were harvested and analyzed for ATX and LPA receptors mRNA and proteins by using real-time PCR and Western blot.
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Figure 2. Immunohistochemical localization of (A,B, and C) ATX, (D. E. and F) LPA1, (G, H, and I) LPA2, and (J, K, and L) LPA3 in (A, D, G, and J) normal mucosa of control subjects and inflammatory sinus mucosa of (B, E, H, and K) CRSwNPs and (C, F, I, and L) CRSsNPs (arrow head, surface epithelium; thick arrow, submucosal glands; thin arrow, inflammatory cells; original magnification, ⫻100). V, vascular endothelium; LPA, lysophosphatidic acid; LPA1, LPA receptor 1; LPA2, LPA receptor 2; LPA3, LPA receptor 3; CRSwNPs, chronic rhinosinusitis with nasal polyps; CRSsNPs, chronic rhinosinusitis without nasal polyps; ATX, autotaxin.
Sinus Mucosa Explant Culture Inflammatory sinus mucosa was collected during endoscopic sinus surgery from patients with CRSwNPs or CRSwNPs and was used for tissue culture as described previously.21,22 Tissue was divided into multiple samples of 5-mm diameter, using dermal biopsy punch. All tissue samples were transferred to air–liquid interface culture plates containing 2 mL of DMEM/HamsF12 supplemented with 2 mM of l-glutamine, 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Invitrogen, Grand Island, NY). The tissues were oriented with the epithelium being exposed to the air and were cultured at 37°C with
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Figure 3. (A) Real-time PCR and (B) ELISA results of IL-4, IL-5, IL-1, IL-13, IFN-␥, TNF-␣, and TGF-1 expression levels in explant culture derived from sinus mucosa after stimulation with LPA (100 nM and 1 M) for 12 or 24 hours. Results are expressed as mean ⫾ SD. Asterisk indicates statistically significant difference in the expression levels of each cytokines between (C) nonstimulated and LPA-stimulated epithelial cells (LPA; p ⬍ 0.5). Gray bar indicates stimulation for 12 hours (n ⫽ 6), and white bar indicates stimulation for 24 hours (n ⫽ 6). PCR, polymerase chain reaction; IFN, interferon; TGF, transforming growth factor; LPA, lysophosphatidic acid.
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5% CO2 in humidified air. To evaluate the effect of LPA on the cytokine expression levels, which are relevant for pathogenesis of CRS, sinus mucosa explants culture was stimulated with LPA (100 nM and 1 M) for 12 and 24 hours and then with culture medium was harvested for real-time PCR and ELISA.
RNA Isolation and Real-Time Reverse Transcription PCR Analysis Total RNA was extracted from frozen sinus tissues and cultured epithelial cells stimulated with cytokine using Trizol reagent (Life Technologies, Rockville, MD). Total RNA (1 g) from each sample was reverse transcribed in 20 L of a reaction mixture containing 2.5 U of Moloney murine leukemia virus (RT; GIBCO BRL, Grand Island, NY). The primer sequences of each gene used in the study are listed in Table 2. Real-time PCR was performed using an iCycler (BioRad Laboratories, Hercules, CA). Each PCR reaction included 50 ng of cDNA, 12.5 L of iTAQ SYBR supermix, and 200 nM of primers. PCR conditions were 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, and 72°C extension for 20 seconds. Target gene expression was expressed as the fold increase and decrease relative to the expression of GAPDH. The mean value of the replicates for each sample was calculated and expressed as the cycle threshold (Ct). The amount of gene expression was then calculated as the difference (⌬Ct) be-
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tween the Ct value obtained for the target gene and the Ct value obtained for GAPDH. Fold change in target gene mRNA expression was determined as 2⫺⌬⌬Ct.
Immunohistochemical and Western Blot Analysis Immunohistochemical staining was performed using a peroxidaselabeled streptavidin-biotin technique. The sections were incubated overnight at room temperature with a 1:100 dilution of anti-ATX and LPA1, -2, and -3 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The negative immunohistochemical control procedure included omission of the primary antibodies and replacement of the primary antibodies by normal rabbit IgG in appropriate concentration. The color was developed using 3,3-diaminobenzidine. For Western blot analysis, the extracted protein (50 g) was separated on 12% sodium dodecyl sulfate–polyacrylamide gels and transferred to immobilon (Millipore, Bedford, MA). The blots were then incubated with anti-ATX and LPA1, -2, and -3 antibody in Trisbuffered saline overnight at room temperature. Antibody reactions were detected by using the ECL detection kit (GE Healthcare and Amersham Bioscience, Piscataway, NJ). The relative intensities of each protein signal were obtained by dividing the intensities of each protein signal by the -actin signals.
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Figure 4. (A, C, E, and G) Real-time PCR and (B, D, F, and H) Western blot of (A and B) ATX, (C and D) LPA1, (E and F) LPA2, and (G and H) LPA3 expression levels in cultured epithelial cells derived from normal sinus mucosa after stimulation with IL-4, IL-5, IL-1, IL-13, IFN-␥, TNF-␣, and TGF-1 (10 and 30 ng/mL), respectively, for 12 or 24 hours. Results are expressed as mean ⫾ SD. Asterisk indicates statistically significant difference in the expression levels of each molecule between cytokine-treated and -untreated epithelial cells (p ⬍ 0.5). Black bar indicates stimulation for 12 hours (n ⫽ 6), and white bar indicates stimulation for 24 hours (n ⫽ 6). C indicates untreated epithelial cells. PCR, polymerase chain reaction; IFN, interferon; TGF, transforming growth factor; LPA, lysophosphatidic acid; LPA1, LPA receptor 1; LPA2, LPA receptor 2; LPA3, LPA receptor 3; ATX, autotaxin.
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NPs, ATX was also found in the inflammatory cells (Fig 2, B and C). LPA1–3 were mainly distributed in the superficial epithelium, vascular endothelium, and submucosal glands in normal and inflammatory sinus mucosa. However, in inflammatory sinus mucosa of CRSwNPs and CRSsNPs, they were also found in the inflammatory cells (Fig 2, D–L).
Enzyme-Linked Immunosorbent Assay The concentrations of IL-4, IL-5, IL-1, IL-13, IFN-␥, TNF-␣, and TGF-1 were measured in supernatants from unstimulated or LPAstimulated explant cultures using ELISA kits according to the manufacturer’s instructions (R & D systems, Minneapolis, MN).
Statistical Analysis Statistical analyses were performed using SPSS for Windows (Version 16.0.0; SPSS, Inc., Chicago, IL). The age differences, 20-item Sino-Nasal Outcome Test, computed tomography score, endoscopic score, and number of inflammatory cells in the three groups were calculated by one-way ANOVA, and the Bonferroni post hoc correction was applied. One-way ANOVA, and the Bonferroni post hoc correction (␣ ⫽ 0.0167) and Tukey test were performed to establish statistically significant differences in values obtained from healthy controls and patients with CRSwNPs and CRSsNPs. The Kolmogorov-Smirnov test showed that the distribution of each data is normal. The level of significance was set at p ⬍ 0.5. Mann-Whitney U tests were performed to compare differences in expression levels of cytokine between LPA-stimulated and -nonstimulated control groups, and in expression levels of ATX and LPA1–3 between cytokinestimulated and -nonstimulated groups.
RESULTS
To identify factors that could modulate the expression of cytokines in CRS, we analyzed the expression levels of LPA in nasal lavage fluid and those of ATX and LPA1–3 in the sinus mucosa of control subjects and patients with CRSwNPs and CRSsNPs. LPA levels in nasal lavage samples from patients with CRSwNPs and CRSsNPs were significantly higher than that of control patients. However, there was no statistical difference in LPA levels between CRSwNPs and CRSsNPs (Fig 1 A). The expression levels of ATX and LPA1–3 mRNAs and proteins were determined in sinus mucosa of normal controls and patients with CRSwNPs and CRSsNPs. As shown in Fig 1, B and C, the expression levels of ATX and LPA1–3 were significantly increased in inflammatory sinus mucosa of patients with CRSwNPs and CRSsNPs, irrespective of the presence of NPs, compared with healthy nasal mucosa. However, the expression levels of ATX and LPA1–3 in inflammatory sinus mucosa did not differ significantly between CRSwNPs and CRSsNPs (Fig 1, B and C). These results indicated that the expression levels of ATX and LPA1–3 were increased in the inflammatory sinus mucosa of CRS patients, irrespective of polyp presence, compared with normal sinus mucosa. The distribution patterns of ATX and LPA1–3 in sinus mucosa of control subjects and patients with CRSwNPs and CRSsNPs were analyzed by immunohistochemistry. Immunohistochemical staining showed a similar pattern in all samples of normal sinus mucosa of control subjects and inflammatory sinus mucosa obtained from patients with CRSwNPs and CRSsNPs, respectively (Fig 2). In normal and inflammatory sinus mucosa, ATX was largely localized to the superficial epithelium and submucosal glands (Fig 2, A–C). However, in inflammatory sinus mucosa of patients with CRSwNPs and CRSs-
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To determine the possible effects of LPA contributing to the expression of cytokine in the sinonasal mucosa, the expression levels of Th1 cytokine (IL-1, IFN-␥, and TNF-␣), Th2 cytokine (IL-4, IL-5, and IL-13), and TGF-1 after stimulating sinus mucosa explant with LPA were examined with real-time PCR and ELISA. The current data showed the increased production of IL-4, IL-5, IFN-␥, and TNF-␣ in sinus mucosa at 12 and 24 hours after stimulation with LPA (Fig. 3). In contrast, the expression of IL-1, IL-13, and TGF-1 was not enhanced after stimulation with LPA (Fig. 3).
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Effects of LPA on the Production of Cytokines in Sinus Mucosa
Effect of Cytokines on the Expression Levels of ATX and LPA1–3 in Sinus Mucosa
To examine which of these cytokines is most potent in inducing the ATX and LPA1–3 expression in sinus mucosa, cultured epithelial cells were incubated in the presence of cytokines for 12 or 24 hours, and then the expression levels of ATX and LPA1–3 were determined. IL-4, IL-5, IFN-␥, and TNF-␣ resulted in the increased production of ATX (Fig 4, A and B). LPA1 and LPA3 expression were enhanced by stimulation of cultured normal epithelial cells with IL-4, IL-5, IL-1, IL-13, IFN-␥, and TNF-␣, and LPA2 was induced by IL-4, IL-5, IFN-␥, and TNF-␣ (Fig 4, C–H).
DISCUSSION Several novel findings were observed in the present study. First, we showed that LPA in nasal lavage fluid and LPA-producing enzyme ATX and LPA1- 3 in sinus mucosa are constitutively expressed in normal controls and are up-regulated in patients with CRSwNPs and CRSsNPs. In normal and inflammatory sinus mucosa, ATX was largely localized to the superficial epithelium and submucosal glands. However, in inflammatory sinus mucosa, ATX was also found in the inflammatory cells. LPA1–3 in normal and inflammatory sinus mucosa were distributed in the superficial epithelium, submucosal glands, and vascular endothelium, but were additionally found in inflammatory cells infiltrated into the inflammatory sinus mucosa of CRSwNPs and CRSsNPs. Furthermore, the stimulation of cultured epithelial cells with LPA induced increased expression levels of IL-4, IL-5, IFN-␥, and TNF-␣. The expression levels of ATX and LPA1–3 were differentially increased after stimulation with cytokines. Collectively, these data suggest that LPA may play the role of an inflammatory mediator in CRS, by acting through up-regulated LPA receptors. In the present study, LPA was found in nasal lavage fluids at baseline and was significantly increased in nasal secretions in CRS, irrespective of NP presence, suggesting that LPA is a natural constituent of nasal secretion. Our results are consistent with previous studies showing the presence of LPA in various fluids, including serum, seminal plasma, cerebrospinal fluid, saliva, and in human
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bronchoalveolar lavage fluids at baseline.5 Up-regulated concentrations of LPA have been detected in the bronchoalveolar lavage fluid in lung inflammatory diseases such as asthma, pulmonary fibrosis, and in the synovial fluid of patients with rheumatoid arthritis.5,11,23 However, the mechanism of LPA generation in nasal secretion is not clear. Bioactive LPA is produced from lysophosphatidylcholine by a secreted lysophospholipase D, known as ATX, and recent studies suggest a critical role of ATX in the regulation of LPA levels in plasma and cancer tissues.24 In an acute lung injury model, increased plasma LPA levels were associated with increased ATX levels.25 In lymphoma cells, specific down-regulation of ATX decreased LPA levels.26 In the present study, ATX was constitutively expressed in normal sinus mucosa and was up-regulated in inflammatory sinus mucosa of CRSwNPs and CRSsNPs. Therefore, our data suggest the presence of the LPA-producing enzyme system in human sinus mucosa and provide the possibility that the induction of ATX may lead to the subsequent generation of LPA in CRS. Nevertheless, the mechanism that mediates the up-regulation of ATX in chronic sinusitis remains unknown. One possible mechanism is that the elevated levels of various cytokines in CRS may contribute to cytokine-induced elevation of ATX, depending on the facts that ATX is regulated by cytokines.27 Cultured epithelial cells with IL-4, IL-5, IFN-␥, and TNF-␣ resulted in up-regulated expression of ATX, which suggests that these cytokines may contribute to the induced production of ATX in the sinus mucosa of patients with CRSwNPs and CRSsNPs, thereby contributing to the increased production of LPA in nasal secretion. This suggestion is supported by the observation that ATX expression from synovial fibroblasts in the synovium was increased by TNF-␣, the major proinflammatory cytokine during pathogenic process of rheumatoid arthritis.14 In this study, we focused on the effects of LPA on expression of cytokines relevant to pathogenesis of CRS, using cultured sinus mucosa explant. Although the effects of LPA on the production of cytokine have been previously studied in a variety of cell types, the topic remains somewhat controversial. In human bronchial epithelial cells and colon cancer cells, LPA induces IL-8 expression.27,28 In ovarian and breast cancer cells, LPA was shown to be a potent inducer of IL-6 and IL-8 production.29,30 In the ureteral obstruction mouse model, blocking LPA1 attenuated the expression of TGF-1.31 In epithelial cells derived from NPs, LPA was able to induce IL-4, IL-6, and IL-8 protein levels.9 These dichotomous results concerning the effects of LPA on cytokine expression may reflect differences in tissue conditions among the various studies in the literature. Additional studies to elucidate the molecular mechanisms linking LPA action will be needed for further our understanding of these regulatory molecules. Nevertheless, these results support the hypothesis that ATX–LPA receptor axis might participate in the pathological process of CRSwNPs or CRSsNPs, contributing to the differential regulation of cytokine production in sinus mucosa. However, the question of whether the role of the ATX–LPA receptor axis is beneficial or detrimental in the pathogenesis of CRS remains to be established. Several lines of evidence indicated that LPA acts as a proinflammatory mediator in lower respiratory diseases, suggesting that LPA might accelerate inflammation in airway inflammatory diseases such as acute lung injury, pulmonary fibrosis, and asthma.5,11,32 In contrast, LPA also regulates the expression of anti-inflammatory genes in airway epithelium and may have a protective role in inflammation and remodeling.5 LPA also induces cyclooxygenase 2 and prostaglandin E2 production, which suggests that it may function as an anti-inflammatory signal in the airway.32 Other studies have also suggested that LPA might play a role in homeostasis and promote the resolution of inflammation.9,33 Considering that in the present study the stimulation of cultured mucosa with LPA resulted in increased expression of cytokines, we suggest that LPA may play a role as an inflammatory mediator in CRS. Nevertheless, additional studies are required to evaluate the functional role of the ATX–LPA receptor axis in normal and inflammatory sinus mucosa of CRS.
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The distribution patterns of LPA1–3 did not differ between normal and inflammatory sinus mucosa, suggesting that these receptor subtypes are ubiquitously expressed in normal sinus mucosa. However, we found that many inflammatory cells infiltrating into the inflammatory sinus mucosa show the immunoreactivities against LPA1–3. Therefore, it is possible that increased expression levels of LPA1–3 in inflammatory sinus mucosa are caused by the influx of inflammatory cells. LPA1 and LPA2 are expressed in neutrophils, and up-regulation of LPA1 and LPA2 expressions in neutrophils from pneumonia patients has been reported.34 Indeed, in vitro studies have shown that LPA acts as a chemoattractant for human inflammatory cells.7,35 LPA may contribute to the infiltration and activation of inflammatory cells in bronchial asthma.6 Furthermore, LPA participates in inflammatory processes by enhancing chemoattraction in neutrophils and monocytes/macrophages.6,7 Emerging evidence suggests a role for ATX– LPA in facilitating lymphocyte migration into lymphoid and chronically inflamed nonlymphoid tissues.36,37 Thus, considering that inflammatory cell infiltration or exudation into the sinus mucosa appears to play an important role in the development of CRSwNPs or CRSsNPs,38–40 we suggest that LPA, via LPA1–3, may play a significant role in inducing and/or sustaining the massive infiltration of inflammatory cells in human sinus mucosa. These suggestions are supported by the role of LPA receptors in airway inflammatory disease studied in vitro and in vivo.11,41 On the other hand, recent studies have suggested that LPA receptor expression is regulated in an inflammatory environment, such as the inflamed synovium and lung.14,41 In fibroblast-like synoviocytes from rheumatoid arthritis patients, LPA1 and 3 expression levels were higher than those in osteoarthritis.14 LPA1–3 are differentially expressed in the central nervous system and their expression is up-regulated in response to injury.42 The current study also indicated that LPA1 and LPA3 expressions were enhanced by stimulation with IL-4, IL-5, IL-6, IL-13, IFN-␥, and TNF-␣, and LPA2 was induced by IL-4, IL-5, IFN-␥, and TNF-␣. Taken together, these results offer the possibility that LPA1–3 may be up-regulated by inflammatory mediators in CRS. However, in a recent study using lung epithelial cell lines, treatment with IL-13 and IFN-␥ significantly reduced LPA1 and LPA2 mRNA levels, compared with unstimulated cells.9 Additional studies will be needed to elucidate the molecular mechanisms linking LPA-related molecules in human sinus mucosa.
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CONCLUSION In conclusion, this is the first study exploring the association between LPA-related molecules and CRS. Our results suggest that LPArelated molecules may play important roles in the pathogenesis of CRSwNPs and CRSsNPs. Additional studies to show the exact mechanism by which the ATX–LPA receptor axis induces inflammation in inflammatory sinus mucosa will provide a foundation for the development of clinical therapies for CRSwNPs and CRSsNPs.
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