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Pro-Resolution Mediator Lipoxin A4 and its Receptor in Upper Airway Inflammation Shino Shimizu, Takao Ogawa, Satoshi Seno, Hideaki Kouzaki and Takeshi Shimizu Ann Otol Rhinol Laryngol 2013 122: 683 DOI: 10.1177/000348941312201104 The online version of this article can be found at: http://aor.sagepub.com/content/122/11/683
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Annals of Otology, Rhinology & Laryngology 122(11):683-689. © 2013 Annals Publishing Company. All rights reserved.
Pro-resolution Mediator Lipoxin A4 and Its Receptor in Upper Airway Inflammation Shino Shimizu, MD; Takao Ogawa, MD; Satoshi Seno, MD; Hideaki Kouzaki, MD; Takeshi Shimizu, MD Objectives: The resolution of inflammation is an active process controlled by several anti-inflammatory and pro-resolution mediators. Lipoxin A4, an endogenous lipid mediator, is a potential pro-resolution mediator that could attenuate inflammation. This study was conducted to elucidate the role of lipoxin A4 in upper airway inflammation. Methods: Nasal secretions were collected from patients with chronic rhinosinusitis with nasal polyposis, patients with allergic rhinitis, and control subjects. The concentration of lipoxin A4 was measured by enzyme-linked immunosorbent assay. Nasal tissues were obtained from nasal polyps and inferior turbinates during endonasal surgery. The mRNA expressions of lipoxygenases (LOXs), lipoxin receptor (formyl peptide receptor–like 1; FPRL-1), and cysteinyl leukotriene type 1 receptor (CysLT1R) in nasal tissues were examined by reverse-transcription polymerase chain reaction. Tissue localization of FPRL-1 was determined by immunohistochemical staining. The in vitro effect of lipoxin A4 on airway epithelial cells was also examined.
Results: A significant concentration of lipoxin A4 was found in nasal secretions, and the concentration was increased in patients with allergic rhinitis. The mRNA expressions of 5-LOX, 15-LOX-1, FPRL-1, and CysLT1R were significantly greater in nasal polyps than in inferior turbinates. FPRL-1 was localized in nasal epithelial cells. Lipoxin A4 inhibited tumor necrosis factor α–induced interleukin 8 release from airway epithelial cells via its receptor FPRL-1. Conclusions: These results indicate that lipoxin A4 may play a role in the resolution of upper airway inflammation. A low concentration of lipoxin A4 may be involved in chronic inflammation of the upper airways.
Key Words: allergic rhinitis, chronic rhinosinusitis, formyl peptide receptor–like 1, lipoxin A4, lipoxygenase, nasal polyp.
5-LOX in leukocytes. Lipoxins are generated by 5-LOX in leukocytes from 15-hydroxyeicosatetraenoic acid (15-HETE) produced by15-LOX in mucosal epithelial cells. The second pathway involves transcellular interactions between platelets and leukocytes within vessels and possibly within exudates. The intermediate leukotriene A4 is produced from arachidonic acid by 5-LOX in leukocytes, and then lipoxins are released by the transcellular conversion of leukotriene A4 to lipoxins by 12-LOX in platelets. Aspirin also triggers the generation of the epimeric form of lipoxins, aspirin-triggered lipoxins. Aspirin acetylates the active site of cyclooxygenase 2 to inhibit production of prostaglandins, but the enzyme is still able to produce 15R-HETE, which is rapidly converted to 15-epimeric lipoxin A4 or 15-epimeric lipoxin B4 by 5-LOX.1 Lipoxins have a variety of anti-inflammatory and pro-resolution activities through the direct activation of their G-protein coupled receptor, formyl peptide receptor–like 1 (FPRL-1). The partial antagonism
INTRODUCTION
The resolution of inflammation is an integral part of the physiological responses to tissue injury, infection, and allergen challenge. The resolution process is very important for maintaining tissue homeostasis and minimizing the development of chronic inflammation. The various steps of resolution are regulated by several anti-inflammatory and pro-resolution mediators, as well as by apoptosis and clearance of inflammatory cells. A breakdown of any stage of this process may lead to chronic inflammation. Lipoxins, endogenous lipid mediators typically formed during cell-cell interaction between accumulated inflammatory cells and resident cells, are actively involved in the resolution of inflammation.1 Lipoxin A4 and its positional isomer lipoxin B4 can be generated via two main lipoxygenase (LOX)– mediated pathways in human cells and tissues. The first pathway involves the sequential lipoxygenation of arachidonic acid by 15-LOX in epithelial cell and
From the Department of Otorhinolaryngology, Shiga University of Medical Science, Otsu, Japan. Correspondence: Shino Shimizu, MD, Dept of Otorhinolaryngology, Shiga University of Medical Science, Seta-tsukinowa, Otsu, Shiga 520-2192, Japan.
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Shimizu et al, Lipoxin A4 in Upper Airway Inflammation TABLE 1. PATIENT PROFILES
Patient Age No. Sex (y)
Control Allergic rhinitis Chronic rhinosinusitis with nasal polyposis AIA — aspirin-induced asthma.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
M F M M F M M F F F F M M F M M M M M M F M F F F F M F M
of cysteinyl leukotriene type 1 receptor (CysLT1R) is another potential mechanism of the anti-inflammatory action of lipoxins. Lipoxins inhibit cell proliferation and angiogenesis induced by growth factors through cross-talk between FPRL-1 and other growth factor receptors. Another potential receptor for lipoxins is the nuclear receptor aryl hydrocarbon receptor, which inhibits the immune responses of dendritic cells by up-regulating suppression of cytokine signaling.2 It has been reported that lipoxin A4 concentration was significantly suppressed in the airway fluids of patients with cystic fibrosis and severe asthma, and that a metabolically stable lipoxin A4 analog suppressed Pseudomonas aeruginosa–induced lung inflammation and allergen-driven inflammation in mice.3-6 These results indicate that impaired biosynthesis of lipoxins may be related to chronic lung inflammation, and that lipoxins may play a key role in resolution of airway inflammation. However, little is known about the role of lipoxins in upper airway
35 38 66 25 28 31 34 46 50 51 26 34 55 56 57 58 58 59 59 61 26 41 30 48 55 56 62 67 68
Blood Eosinophils (%) Polyps Asthma
Topical Nasal Steroid
6.2 3.6 1.2 3.6 4.0 7.1 + 2.8 0 5.3 2.2 1.6 Single 2.1 Single 4.3 Multiple 8.1 Multiple + 12.1 Single + 2.4 Single 6.6 Single 3.9 Single 7.8 Single + 4.8 Multiple 21.8 Multiple AIA + 4.3 Multiple + + 15.1 Multiple AIA + 14.3 Multiple + + 7.4 Multiple AIA + 2.6 Multiple + + 8.8 Multiple + + 8.5 Multiple AIA + 11.3 Multiple + +
Systemic Steroid
+ + +
+ + + + + +
inflammation. We hypothesized that lipoxin A4 concentrations may be suppressed in chronic inflammation of the upper airways. Our aim was to elucidate whether lipoxin A4 and FPRL-1 contribute to the pathogenesis of chronic inflammation of the upper airways. To determine whether lipoxin A4 synthesis is involved in local inflammation, we first measured its concentration in nasal secretions from patients with chronic rhinosinusitis (CRS) with nasal polyposis (NP) and patients with allergic rhinitis (AR). The expressions of synthetases and receptors of lipoxins in nasal tissues were examined. We also examined the in vitro effect of lipoxin A4 on interleukin 8 secretion from airway epithelial cells. MATERIALS AND METHODS
Patients. Twenty-nine patients with nasal or paranasal sinus diseases were enrolled (age range, 25 to 68 years; mean, 47.6 years; Table 1). Allergenspecific immunoglobulin E, blood eosinophil count,
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TABLE 2. PRIMER SEQUENCES AND PROBE NUMBERS FPRL-1 CysLT1R 5-LOX 12-LOX 15-LOX-1 15-LOX-2 GAPDH
Forward Primer (5'→3') CCT CAG GAA AAT GCA CCA G ACT CCA GTG CCA GAA AGA GG AAA CAG ACC CCT GCA CAC TC GTG CAG CTT TGG CTG GTC CTT CCT GCT CGC CTA GTG TT TGA GGT CTT CAC CCT GGC TA AGC CAC ATC GCT CAG ACA C
Reverse Primer (3'→5') GCC AGC AGA CTC ATA GGA CAC GCG GAA GTC ATC AAT AGT GTC A CGG GAT TTG GTT GAG CTG GCA ACG TCA TGA TCA AAC TCC GAA GTC AGC TTC GAA CAG TGT G TTG ATG TGC AGG GTG TAT CG GCC CAA TAC GAC CAA ATC C
Probe No. 82 29 72 78 17 43 60
FPRL — formyl peptide receptor–like 1; CysLT1R — cysteinyl leukotriene type 1 receptor; LOX — lipoxygenase; GAPDH — glyceraldehyde 3-phosphate dehydrogenase.
and computed tomographic and endoscopic findings were examined in all patients. CRS was diagnosed on the basis of the clinical, endoscopic, and radiographic criteria of EPOS 2007.7 The diagnosis of asthma was based on the criteria of the Global Initiative for Asthma.8 AR was diagnosed on the basis of the Practical Guideline for the Management of Allergic Rhinitis.9 The patients were assigned to 1 of 3 groups: 19 patients with CRS with NP, 7 with AR, and 3 control patients (2 with hypertrophic rhinitis and 1 with a paranasal cyst). We did not use healthy volunteers as control subjects because the nasal secretions of healthy volunteers are too scant to collect without a stimulus. Nasal secretions were collected by suction with a Juhn Tym-Tap Collector (Medtronic, Minneapolis, Minnesota). The nasal secretions were measured, mixed with 4 volumes of phosphate-buffered saline solution by shaking for 3 hours at 4°C, and centrifuged at 500g for 30 minutes. The supernatants were stored at –80°C until use. During endonasal surgery in patients with CRS and AR, we obtained samples of nasal tissues of inferior turbinates (n = 11) and nasal polyps (n = 16) for reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemical study. We used nasal polyps to represent inflamed nasal mucosa with remarkable tissue remodeling and inferior turbinates as control mucosa. For RNA preparation, the nasal tissues were immersed in RNAlater (Ambion, Life Technologies, Carlsbad, California) at 4°C for 24 hours and stored at –80°C until use. Informed consent was obtained from all subjects before sampling. The clinical protocol was approved by the Shiga University of Medical Science Institutional Review Board for Clinical Investigation. Lipoxin A4 Concentration in Nasal Secretions. The lipoxin A4 concentration in the nasal secretions was measured in duplicate with an enzyme-linked immunosorbent assay kit for lipoxin A4 (Oxford Biochemical Research, Rochester Hills, Michigan) according to the manufacturer’s protocol. RT-PCR. Total RNA was extracted from nasal
tissues and reverse-transcribed, and then real-time PCR was performed with LightCycler 480 system II (Roche Diagnostics, Basel, Switzerland). The Taqman Master kit in combination with the Universal Probe Library (Human) was used to assess gene expression. PCR primers for Taqman/Probe Library assays were designed with the Probe Library Assay Design Center (www.roche-applied-science.com/ sis/rtpcr/upl/adc.jsp). Glyceraldehyde 3-phosphate dehydrogenase was used as a reference gene. The primer sequences and probe numbers are listed in Table 2. Analysis of the RT-PCR data was done by the DDCt method. Immunohistochemical Analysis. Nasal tissues were immersion-fixed in 4% formaldehyde in 0.1 mol/L of phosphate-buffered saline solution overnight and embedded in paraffin. Immunostaining was performed on 3-mm-thick sections with FPRL-1 rabbit anti-human polyclonal antibody (MBL International, Woburn, Massachusetts). Briefly, nonspecific binding was blocked by incubation for 30 minutes with 10% normal goat serum. The slides were washed 3 times with phosphate-buffered saline solution. The first antibody (1:100 dilution) was placed on each tissue section for 1 hour. After washing, the slides were incubated with secondary antibody (1:500 dilution; Vector Laboratories, Burlingame, California) for 1 hour. The avidin-biotin complex technique (Vectastain Elite ABC kit, Vector Laboratories) was used for visualization. The sections were developed in peroxide substrate solution containing 3,3'-diaminobenzidine and hydrogen peroxide and counterstained with hematoxylin. Negative control specimens were prepared from sections incubated without the primary antibody. Cell Culture. Normal human bronchial epithelial (NHBE) cells were purchased from Lonza (Basel, Switzerland) and cultured in supplemented bronchial epithelial growth medium (Lonza) on 6-well culture plates. After incubating the confluent cells for 24 hours in the basal medium without supplements, we exposed the NHBE cells to lipoxin A4
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Fig 1. Lipoxin A4 concentrations in nasal secretions from 7 patients with allergic rhinitis (AR) and 19 patients with chronic rhinosinusitis (CRS) with nasal polyposis (NP). Three control subjects were not healthy volunteers: 2 had hypertrophic rhinitis, and 1 had paranasal cyst. Significant concentration of lipoxin A4 was found in nasal secretions; concentration was significantly greater in nasal secretions from patients with AR than in those from patients with CRS with NP (p < 0.05).
(0, 10, 100, and 1,000 nmol/L) for 15 minutes (Calbiochem, San Diego, California) before stimulating them with tumor necrosis factor α (TNF-α; 10 ng/ mL) for 24 hours (R&D Systems, Minneapolis, Minnesota). The NHBE cells were also preexposed to a FPRL-1 receptor antagonist, boc-2 (10 mmol/L), for 15 minutes (Boc-Phe-Leu-Phe-Leu-Phe; Scrum, Tokyo, Japan), to lipoxin A4 (100 nmol/L) for 15 minutes, and then to TNF-α (10 ng/mL) for 24 hours. The supernatants were collected and stored at –20°C until use. The concentrations of interleukin 8 in supernatants were determined with an enzyme-linked immunosorbent assay kit (R&D Systems). Statistical Analysis. All data are expressed as mean ± SD. The difference between variables was calculated by analysis of variance with a post hoc analy-
A
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B
A
B
Fig 3. mRNA expressions of A) formyl peptide receptor– like (FPRL-1) and B) cysteinyl leukotriene type 1 receptor (CysLT1R) in nasal tissues by RT-PCR were significantly greater (p < 0.05) in nasal polyps than in inferior turbinates.
sis using Fisher’s predicted least significant difference test. The Mann-Whitney U test was also used for comparison between groups. Statistical analyses were carried out with StatView 5.0 (Abacus Concepts, Piscataway, New Jersey). Probability (p) values of less than 0.05 were considered significant. RESULTS
Lipoxin A4 Concentration in Nasal Secretions. To ascertain whether lipoxin A4 synthesis is involved in local inflammation, we first measured the lipoxin A4 concentration in the nasal secretions from patients with CRS with NP and those with AR. Significant concentrations of lipoxin A4 were found in the nasal secretions, and the concentration was significantly greater in patients with AR than in patients with CRS with NP (Fig 1). There was no difference in the lipoxin A4 concentrations of patients with CRS with NP with the use of topical or systemic steroids or
C
D
Fig 2. mRNA expressions of lipoxygenases (LOXs) in nasal polyps compared to those in inferior turbinate tissue specimens by reverse-transcription polymerase chain reaction (RT-PCR). These LOXs are involved in release of lipoxin A4. mRNA expressions of A) 5-LOX and B) 15-LOX-1 were significantly greater (p < 0.05) in nasal polyps (11 specimens) than in inferior turbinates (16 specimens). mRNA expressions of C) platelet-type 12-LOX (p = 0.13) and D) 15-LOX-2 (p = 0.23) were decreased in nasal polyps.
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A
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B
Fig 4. Immunohistochemical staining of A) negative control and B) FPRL-1 from epithelial cells of nasal polyp. Scale bar — 100 μm.
with the comorbidity of bronchial asthma. LOX mRNA Expression in Nasal Tissues. 5-LOX, 15-LOX-1, 15-LOX-2, and platelet-type 12-LOX may be involved in the production of lipoxins. The mRNA expressions of 5-LOX (Fig 2A) and 15LOX-1 (Fig 2B) were significantly greater in nasal polyps than in inferior turbinates. The mRNA expressions of platelet-type 12-LOX (Fig 2C) and 15LOX-2 (Fig 2D) were lower in nasal polyps than in inferior turbinates. All nasal polyps were obtained from patients with CRS with NP, and 6 of the 16 inferior turbinate specimens were obtained from patients with AR. No differences in mRNA expression of LOXs were observed between the inferior turbinate specimens from the two patient groups. Lipoxin A4 Receptor FPRL-1 and CysLT1R mRNA Expression in Nasal Tissues. Lipoxin A4 directly activates FPRL-1 and inactivates CysLT1R. The mRNA expressions of FPRL-1 (Fig 3A) and CysLT1R (Fig 3B) were significantly greater in na-
sal polyps than in inferior turbinates. Immunohistochemical Localization of FPRL-1. Immunohistochemical study revealed that FPRL-1 was localized in the epithelial cells of nasal polyps (Fig 4B). FPRL-1 was also expressed in the epithelial cells and submucosal glands of the inferior turbinates. Effects of Lipoxin A4 Release of Interleukin 8 From Cultured NHBE Cells. The cultured NHBE cells were incubated with lipoxin A4 (0, 10, 100, and 1,000 nmol/L) for 15 minutes before 24-hour incubation with TNF-α (10 ng/mL). Lipoxin A4 partially inhibited TNF-α–induced release of interleukin 8 from cultured NHBE cells (Fig 5A). To evaluate whether FPRL-1 is involved in the lipoxin A4–induced inhibitory effect on the release of interleukin 8, we used an antagonist of FPRL-1, the boc-2 peptide. Preincubation of NHBE cells with boc-2 (10 nmol/L) for 15 minutes attenuated the lipoxin A4– induced inhibitory effect on the release of interleu-
Fig 5. Effects of lipoxin A4 (LXA4) and its receptor antagonist boc-2 on tumor necrosis factor α–induced release of interleukin 8 from normal human bronchial epithelial cells. A) Lipoxin A4 partially inhibited tumor necrosis factor α–induced release of interleukin 8 in dose-dependent manner. B) boc-2 attenuated lipoxin A4–induced inhibition of tumor necrosis factor α–induced release of interleukin 8. Asterisk — p < 0.05.
A
B
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kin 8 (Fig 5B). These results indicate that lipoxin A4 inhibited TNF-α–induced release of interleukin 8 via its receptor FPRL-1. DISCUSSION
The present study revealed that nasal secretions contain a significant concentration of lipoxin A4, and that the mRNA expressions of its synthetases 5-LOX and 15-LOX-1, and its receptor FPRL-1, were significantly greater in nasal polyps than in inferior turbinates. Our immunohistochemical study revealed that FPRL-1 is localized in nasal epithelial cells. This is the first study to show the presence of lipoxin A4 in nasal secretions and the localization of its receptor FPRL-1 in nasal mucosa.
Lipoxins are potential anti-inflammatory mediators that attenuate inflammation and inhibit the functions of neutrophils and eosinophils such as chemotaxis, adherence, transmigration, degranulation, elastase secretion, and generation of superoxide anions. Lipoxins also promote the resolution of inflammation by stimulating monocyte chemotaxis, adherence, and ingestion of apoptotic cells.2 Lipoxin A4 concentrations were found to be significantly suppressed in the airway fluids of patients with cystic fibrosis compared to patients with other inflammatory lung conditions.3 The severity of bronchial asthma was found to be related to decreased biosynthesis of lipoxin A4.4 A previous study revealed a higher concentration of lipoxin A4 in the nasal tissues of patients with CRS with NP compared to our results in nasal secretions, and lipoxin A4 was downregulated in patients with aspirin sensitivity.10 In the present study, we demonstrated a lower concentration of lipoxin A4 in the nasal secretions of patients with CRS with NP than in patients with AR. This result indicates that decreased biosynthesis of lipoxins may be involved in the pathogenesis of chronic airway inflammation.
In the present study, the mRNA expressions of 5-LOX and 15-LOX-1 were significantly greater in nasal polyps than in inferior turbinates. These results support those of previous studies that showed greater expression of mRNA in the sinonasal mucosa of patients with CRS with NP than in normal nasal mucosa.10,11 For leukotriene formation, 5-LOX in leukocytes is the key enzyme in the production of pro-inflammatory mediators such as leukotriene B4 and cysteinyl leukotrienes by converting arachidonic acid to leukotriene A4. Leukotrienes are also generated by 5-LOX in leukocytes from 15-HETE produced by 15-LOX-1. The mRNA expression of 15-LOX-1 is found in airway epithelial cells, eosinophils, and monocytes.12 Transgenic rabbits
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with increased 15-LOX-1 expression in monocytes showed an increased concentration of lipoxin at the sites of inflammation.13 The Th2 cytokines interleukin 4 and interleukin 13 induced 15-LOX-1 expression in monocytes and airway epithelial cells, and prostaglandins D2 and E2 induced 15-LOX-1 expression in epithelial cells.12 These results indicate that the expression of 15-LOX-1 in monocytes and epithelial cells is important for the transformation of lipoxins in combination with 5-LOX in leukocytes. Lipoxins are also released in combination with 5-LOX in leukocytes and with 12-LOX in platelets. Little is known about the biological role of platelettype 12-LOX in airway inflammation. In the present study, the mRNA expression of platelet-type 12LOX was suppressed in nasal polyps compared to interior turbinates. It is possible that the suppressed expression of platelet-type 12-LOX in nasal polyps may contribute to the decreased concentration of lipoxin A4 in nasal secretions from patients with CRS with NP. Both 15-LOX-1 and 15-LOX-2 are capable of metabolizing arachidonic acid to 15-HETE; however, 15-LOX-2 has limited tissue distribution, and the role of 15-LOX-2 in airway inflammation is unclear.14 In the present study, the mRNA expression of 15-LOX-2 was suppressed in nasal polyps. A lower mRNA expression of 15-LOX-2 and lesser production of lipoxin A4 have been reported in colonic mucosa of patients with ulcerative colitis and may be related to the pathogenesis of chronic inflammation.15 The lipoxin A4 receptor FPRL-1 was expressed in several types of leukocytes, such as neutrophils, monocytes, and activated T cells, as well as in resident cells, including intestinal epithelial cells, bronchial epithelial cells, synovial fibroblasts, renal mes angial cells, and astrocytes. The mRNA expression of FPRL-1 is up-regulated by various cytokines such as interferon γ, interleukin 1β, and interleukin 13 at the site of inflammation.2,16 In the present study, for the first time, we demonstrated that FPRL-1 was localized in the nasal epithelial cells. We confirmed that lipoxin A4 partially inhibited TNF-α–induced interleukin 8 release from cultured airway epithelial cells via its receptor FPRL-1. The effective dose of lipoxin A4 was equivalent to the previously reported tissue concentration of lipoxin A4 in nasal mucosa.10 These results indicate that lipoxin A4 inhibits inflammation through the direct inhibition of cytokine release from nasal epithelial cells via the FPRL-1 receptor. Lipoxins also act as partial antagonists for CysLT1R, and the mRNA expression of CysLT1R was up-regulated in nasal polyps. Lipoxins may suppress sinonasal inflammation through the inhibition of epithelial cytokine release via the FPRL-
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1 receptor and through their antagonistic effects on CysLT1R. In addition to lipoxin A4, a variety of peptide ago nists have been identified for FPRL-1, and FPRL1 signaling is cell- and agonist-specific.17 Previous studies showed that lipoxin A4/FPRL-1 signaling attenuated the nuclear accumulation of nuclear factorκB (NF-κB) and activator protein 1 (AP-1). NF-κB and AP-1 are key regulators for the production of proinflammatory cytokines such as interleukins 1β, 6, and 8 and TNF-α. In human leukocytes, lipoxin A4 partially inhibited lipopolysaccharide-induced interleukin 8 secretion through the suppression of NFκB and AP-1 activation.18 In human macrophages and intestinal epithelial cells, lipoxin A4 inhibited lipopolysaccharide-induced production of interleukin 8 and TNF-α and activation of NF-κB.19 Lipoxin A4 also attenuated lipopolysaccharide-induced produc-
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tion of interleukins 1β, 6, and 8 by the inhibition of phosphoinositol 3'-kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, NFκB, and AP-1 pathway-dependent signal transduction in pulmonary microvascular endothelial cells.20 These results indicate that similar signaling pathways may be involved in the lipoxin A4/FPRL-1– dependent inhibition of TNF-α–induced interleukin 8 secretion from human airway epithelial cells seen in the present study. In conclusion, the present study demonstrated that lipoxin A4 is an important pro-resolution mediator in upper airway inflammation and that inappropriate metabolism of arachidonic acid may be involved in the pathogenesis of chronic inflammation and nasal polyp formation in CRS. These results suggest that a lipoxin A4 analog may be a potential regulator of upper airway inflammation.
Acknowledgments: We thank the staff of the Central Research Laboratory of Shiga University of Medical Science for their important contribution toward the completion of this work.
REFERENCES
1. Haworth O, Levy BD. Endogenous lipid mediators in the resolution of airway inflammation. Eur Respir J 2007;30:98092. 2. Maderna P, Godson C. Lipoxins: resolutionary road. Br J Pharmacol 2009;158:947-59. 3. Karp CL, Flick LM, Park KW, et al. Defective lipoxinmediated anti-inflammatory activity in the cystic fibrosis airway. Nat Immunol 2004;5:388-92. 4. Levy BD, Bonnans C, Silverman ES, Palmer LJ, Marigowda G, Israel E; Severe Asthma Research Program, National Heart, Lung, and Blood Institute. Diminished lipoxin biosynthesis in severe asthma. Am J Respir Crit Care Med 2005; 172:824-30. 5. Planagumà A, Kazani S, Marigowda G, et al. Airway lipoxin A4 generation and lipoxin A4 receptor expression are decreased in severe asthma. Am J Respir Crit Care Med 2008;178: 574-82. 6. Levy BD, Lukacs NW, Berlin AA, et al. Lipoxin A4 stable analogs reduce allergic airway responses via mechanisms distinct from CysLT1 receptor antagonism. FASEB J 2007;21: 3877-84. 7. Thomas M, Yawn BP, Price D, Lund V, Mullol J, Fokkens W; European Position Paper on Rhinosinusitis and Nasal Polyps Group. EPOS Primary Care Guidelines: European Position Paper on the Primary Care Diagnosis and Management of Rhinosinusitis and Nasal Polyps 2007 — a summary. Prim Care Respir J 2008;17:79-89. 8. Global Initiative for Asthma (GINA), National Heart, Lung, and Blood Institute, eds. Global strategy for asthma management and prevention. Bethesda, Md: NHLBI, 2006. 9. Committee of the Practical Guideline for the Management of Allergic Rhinitis, eds. Practical guideline for the management of allergic rhinitis in Japan. Tokyo, Japan: Life Science Publishing, 2009. 10. Pérez-Novo CA, Watelet JB, Claeys C, Van Cauwenberge P, Bachert C. Prostaglandin, leukotriene, and lipoxin balance in chronic rhinosinusitis with and without nasal polyposis. J Allergy Clin Immunol 2005;115:1189-96.
11. Rostkowska-Nadolska B, Kapral M, Fraczek M, Kowalczyk M, Gawron W, Mazurek U. A microarray study of gene expression profiles in nasal polyps. Auris Nasus Larynx 2011;38: 58-64. 12. Claesson HE. On the biosynthesis and biological role of eoxins and 15-lipoxygenase-1 in airway inflammation and Hodgkin lymphoma. Prostaglandins Other Lipid Mediat 2009; 89:120-5. 13. Serhan CN, Jain A, Marleau S, et al. Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators. J Immunol 2003;171:6856-65. 14. Hsi LC, Wilson LC, Eling TE. Opposing effects of 15-lipoxygenase-1 and -2 metabolites on mapk signaling in prostate. Alteration in peroxisome proliferator–activated receptor gamma. J Biol Chem 2002;277:40549-56. 15. Mangino MJ, Brounts L, Harms B, Heise C. Lipoxin biosynthesis in inflammatory bowel disease. Prostaglandins Other Lipid Mediat 2006;79:84-92. 16. Bonnans C, Gras D, Chavis C, et al. Synthesis and anti-inflammatory effect of lipoxins in human airway epithelial cells. Biomed Pharmacother 2007;61:261-7. 17. Romano M, Recchia I, Recchiuti A. Lipoxin receptors. ScientificWorldJournal 2007;7:1393-412.
18. József L, Zouki C, Petasis NA, Serhan CN, Filep JG. Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-κB and AP-1 activation, and IL-8 gene expression in human leukocytes. Proc Natl Acad Sci U S A 2002;99:13266-71. 19. Kure I, Nishiumi S, Nishitani Y, et al. Lipoxin A4 reduces lipopolysaccharide-induced inflammation in macrophages and intestinal epithelial cells through inhibition of nuclear factor– κB activation. J Pharmacol Exp Ther 2010;332:541-8.
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Pro-Resolution Mediator Lipoxin A4 and its Receptor in Upper Airway Inflammation Shino Shimizu, Takao Ogawa, Satoshi Seno, Hideaki Kouzaki and Takeshi Shimizu Ann Otol Rhinol Laryngol 2013 122: 683 DOI: 10.1177/000348941312201104 The online version of this article can be found at: http://aor.sagepub.com/content/122/11/683
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Annals of Otology, Rhinology & Laryngology 122(11):683-689. © 2013 Annals Publishing Company. All rights reserved.
Pro-resolution Mediator Lipoxin A4 and Its Receptor in Upper Airway Inflammation Shino Shimizu, MD; Takao Ogawa, MD; Satoshi Seno, MD; Hideaki Kouzaki, MD; Takeshi Shimizu, MD Objectives: The resolution of inflammation is an active process controlled by several anti-inflammatory and pro-resolution mediators. Lipoxin A4, an endogenous lipid mediator, is a potential pro-resolution mediator that could attenuate inflammation. This study was conducted to elucidate the role of lipoxin A4 in upper airway inflammation. Methods: Nasal secretions were collected from patients with chronic rhinosinusitis with nasal polyposis, patients with allergic rhinitis, and control subjects. The concentration of lipoxin A4 was measured by enzyme-linked immunosorbent assay. Nasal tissues were obtained from nasal polyps and inferior turbinates during endonasal surgery. The mRNA expressions of lipoxygenases (LOXs), lipoxin receptor (formyl peptide receptor–like 1; FPRL-1), and cysteinyl leukotriene type 1 receptor (CysLT1R) in nasal tissues were examined by reverse-transcription polymerase chain reaction. Tissue localization of FPRL-1 was determined by immunohistochemical staining. The in vitro effect of lipoxin A4 on airway epithelial cells was also examined.
Results: A significant concentration of lipoxin A4 was found in nasal secretions, and the concentration was increased in patients with allergic rhinitis. The mRNA expressions of 5-LOX, 15-LOX-1, FPRL-1, and CysLT1R were significantly greater in nasal polyps than in inferior turbinates. FPRL-1 was localized in nasal epithelial cells. Lipoxin A4 inhibited tumor necrosis factor α–induced interleukin 8 release from airway epithelial cells via its receptor FPRL-1. Conclusions: These results indicate that lipoxin A4 may play a role in the resolution of upper airway inflammation. A low concentration of lipoxin A4 may be involved in chronic inflammation of the upper airways.
Key Words: allergic rhinitis, chronic rhinosinusitis, formyl peptide receptor–like 1, lipoxin A4, lipoxygenase, nasal polyp.
5-LOX in leukocytes. Lipoxins are generated by 5-LOX in leukocytes from 15-hydroxyeicosatetraenoic acid (15-HETE) produced by15-LOX in mucosal epithelial cells. The second pathway involves transcellular interactions between platelets and leukocytes within vessels and possibly within exudates. The intermediate leukotriene A4 is produced from arachidonic acid by 5-LOX in leukocytes, and then lipoxins are released by the transcellular conversion of leukotriene A4 to lipoxins by 12-LOX in platelets. Aspirin also triggers the generation of the epimeric form of lipoxins, aspirin-triggered lipoxins. Aspirin acetylates the active site of cyclooxygenase 2 to inhibit production of prostaglandins, but the enzyme is still able to produce 15R-HETE, which is rapidly converted to 15-epimeric lipoxin A4 or 15-epimeric lipoxin B4 by 5-LOX.1 Lipoxins have a variety of anti-inflammatory and pro-resolution activities through the direct activation of their G-protein coupled receptor, formyl peptide receptor–like 1 (FPRL-1). The partial antagonism
INTRODUCTION
The resolution of inflammation is an integral part of the physiological responses to tissue injury, infection, and allergen challenge. The resolution process is very important for maintaining tissue homeostasis and minimizing the development of chronic inflammation. The various steps of resolution are regulated by several anti-inflammatory and pro-resolution mediators, as well as by apoptosis and clearance of inflammatory cells. A breakdown of any stage of this process may lead to chronic inflammation. Lipoxins, endogenous lipid mediators typically formed during cell-cell interaction between accumulated inflammatory cells and resident cells, are actively involved in the resolution of inflammation.1 Lipoxin A4 and its positional isomer lipoxin B4 can be generated via two main lipoxygenase (LOX)– mediated pathways in human cells and tissues. The first pathway involves the sequential lipoxygenation of arachidonic acid by 15-LOX in epithelial cell and
From the Department of Otorhinolaryngology, Shiga University of Medical Science, Otsu, Japan. Correspondence: Shino Shimizu, MD, Dept of Otorhinolaryngology, Shiga University of Medical Science, Seta-tsukinowa, Otsu, Shiga 520-2192, Japan.
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Shimizu et al, Lipoxin A4 in Upper Airway Inflammation TABLE 1. PATIENT PROFILES
Patient Age No. Sex (y)
Control Allergic rhinitis Chronic rhinosinusitis with nasal polyposis AIA — aspirin-induced asthma.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
M F M M F M M F F F F M M F M M M M M M F M F F F F M F M
of cysteinyl leukotriene type 1 receptor (CysLT1R) is another potential mechanism of the anti-inflammatory action of lipoxins. Lipoxins inhibit cell proliferation and angiogenesis induced by growth factors through cross-talk between FPRL-1 and other growth factor receptors. Another potential receptor for lipoxins is the nuclear receptor aryl hydrocarbon receptor, which inhibits the immune responses of dendritic cells by up-regulating suppression of cytokine signaling.2 It has been reported that lipoxin A4 concentration was significantly suppressed in the airway fluids of patients with cystic fibrosis and severe asthma, and that a metabolically stable lipoxin A4 analog suppressed Pseudomonas aeruginosa–induced lung inflammation and allergen-driven inflammation in mice.3-6 These results indicate that impaired biosynthesis of lipoxins may be related to chronic lung inflammation, and that lipoxins may play a key role in resolution of airway inflammation. However, little is known about the role of lipoxins in upper airway
35 38 66 25 28 31 34 46 50 51 26 34 55 56 57 58 58 59 59 61 26 41 30 48 55 56 62 67 68
Blood Eosinophils (%) Polyps Asthma
Topical Nasal Steroid
6.2 3.6 1.2 3.6 4.0 7.1 + 2.8 0 5.3 2.2 1.6 Single 2.1 Single 4.3 Multiple 8.1 Multiple + 12.1 Single + 2.4 Single 6.6 Single 3.9 Single 7.8 Single + 4.8 Multiple 21.8 Multiple AIA + 4.3 Multiple + + 15.1 Multiple AIA + 14.3 Multiple + + 7.4 Multiple AIA + 2.6 Multiple + + 8.8 Multiple + + 8.5 Multiple AIA + 11.3 Multiple + +
Systemic Steroid
+ + +
+ + + + + +
inflammation. We hypothesized that lipoxin A4 concentrations may be suppressed in chronic inflammation of the upper airways. Our aim was to elucidate whether lipoxin A4 and FPRL-1 contribute to the pathogenesis of chronic inflammation of the upper airways. To determine whether lipoxin A4 synthesis is involved in local inflammation, we first measured its concentration in nasal secretions from patients with chronic rhinosinusitis (CRS) with nasal polyposis (NP) and patients with allergic rhinitis (AR). The expressions of synthetases and receptors of lipoxins in nasal tissues were examined. We also examined the in vitro effect of lipoxin A4 on interleukin 8 secretion from airway epithelial cells. MATERIALS AND METHODS
Patients. Twenty-nine patients with nasal or paranasal sinus diseases were enrolled (age range, 25 to 68 years; mean, 47.6 years; Table 1). Allergenspecific immunoglobulin E, blood eosinophil count,
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TABLE 2. PRIMER SEQUENCES AND PROBE NUMBERS FPRL-1 CysLT1R 5-LOX 12-LOX 15-LOX-1 15-LOX-2 GAPDH
Forward Primer (5'→3') CCT CAG GAA AAT GCA CCA G ACT CCA GTG CCA GAA AGA GG AAA CAG ACC CCT GCA CAC TC GTG CAG CTT TGG CTG GTC CTT CCT GCT CGC CTA GTG TT TGA GGT CTT CAC CCT GGC TA AGC CAC ATC GCT CAG ACA C
Reverse Primer (3'→5') GCC AGC AGA CTC ATA GGA CAC GCG GAA GTC ATC AAT AGT GTC A CGG GAT TTG GTT GAG CTG GCA ACG TCA TGA TCA AAC TCC GAA GTC AGC TTC GAA CAG TGT G TTG ATG TGC AGG GTG TAT CG GCC CAA TAC GAC CAA ATC C
Probe No. 82 29 72 78 17 43 60
FPRL — formyl peptide receptor–like 1; CysLT1R — cysteinyl leukotriene type 1 receptor; LOX — lipoxygenase; GAPDH — glyceraldehyde 3-phosphate dehydrogenase.
and computed tomographic and endoscopic findings were examined in all patients. CRS was diagnosed on the basis of the clinical, endoscopic, and radiographic criteria of EPOS 2007.7 The diagnosis of asthma was based on the criteria of the Global Initiative for Asthma.8 AR was diagnosed on the basis of the Practical Guideline for the Management of Allergic Rhinitis.9 The patients were assigned to 1 of 3 groups: 19 patients with CRS with NP, 7 with AR, and 3 control patients (2 with hypertrophic rhinitis and 1 with a paranasal cyst). We did not use healthy volunteers as control subjects because the nasal secretions of healthy volunteers are too scant to collect without a stimulus. Nasal secretions were collected by suction with a Juhn Tym-Tap Collector (Medtronic, Minneapolis, Minnesota). The nasal secretions were measured, mixed with 4 volumes of phosphate-buffered saline solution by shaking for 3 hours at 4°C, and centrifuged at 500g for 30 minutes. The supernatants were stored at –80°C until use. During endonasal surgery in patients with CRS and AR, we obtained samples of nasal tissues of inferior turbinates (n = 11) and nasal polyps (n = 16) for reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemical study. We used nasal polyps to represent inflamed nasal mucosa with remarkable tissue remodeling and inferior turbinates as control mucosa. For RNA preparation, the nasal tissues were immersed in RNAlater (Ambion, Life Technologies, Carlsbad, California) at 4°C for 24 hours and stored at –80°C until use. Informed consent was obtained from all subjects before sampling. The clinical protocol was approved by the Shiga University of Medical Science Institutional Review Board for Clinical Investigation. Lipoxin A4 Concentration in Nasal Secretions. The lipoxin A4 concentration in the nasal secretions was measured in duplicate with an enzyme-linked immunosorbent assay kit for lipoxin A4 (Oxford Biochemical Research, Rochester Hills, Michigan) according to the manufacturer’s protocol. RT-PCR. Total RNA was extracted from nasal
tissues and reverse-transcribed, and then real-time PCR was performed with LightCycler 480 system II (Roche Diagnostics, Basel, Switzerland). The Taqman Master kit in combination with the Universal Probe Library (Human) was used to assess gene expression. PCR primers for Taqman/Probe Library assays were designed with the Probe Library Assay Design Center (www.roche-applied-science.com/ sis/rtpcr/upl/adc.jsp). Glyceraldehyde 3-phosphate dehydrogenase was used as a reference gene. The primer sequences and probe numbers are listed in Table 2. Analysis of the RT-PCR data was done by the DDCt method. Immunohistochemical Analysis. Nasal tissues were immersion-fixed in 4% formaldehyde in 0.1 mol/L of phosphate-buffered saline solution overnight and embedded in paraffin. Immunostaining was performed on 3-mm-thick sections with FPRL-1 rabbit anti-human polyclonal antibody (MBL International, Woburn, Massachusetts). Briefly, nonspecific binding was blocked by incubation for 30 minutes with 10% normal goat serum. The slides were washed 3 times with phosphate-buffered saline solution. The first antibody (1:100 dilution) was placed on each tissue section for 1 hour. After washing, the slides were incubated with secondary antibody (1:500 dilution; Vector Laboratories, Burlingame, California) for 1 hour. The avidin-biotin complex technique (Vectastain Elite ABC kit, Vector Laboratories) was used for visualization. The sections were developed in peroxide substrate solution containing 3,3'-diaminobenzidine and hydrogen peroxide and counterstained with hematoxylin. Negative control specimens were prepared from sections incubated without the primary antibody. Cell Culture. Normal human bronchial epithelial (NHBE) cells were purchased from Lonza (Basel, Switzerland) and cultured in supplemented bronchial epithelial growth medium (Lonza) on 6-well culture plates. After incubating the confluent cells for 24 hours in the basal medium without supplements, we exposed the NHBE cells to lipoxin A4
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Fig 1. Lipoxin A4 concentrations in nasal secretions from 7 patients with allergic rhinitis (AR) and 19 patients with chronic rhinosinusitis (CRS) with nasal polyposis (NP). Three control subjects were not healthy volunteers: 2 had hypertrophic rhinitis, and 1 had paranasal cyst. Significant concentration of lipoxin A4 was found in nasal secretions; concentration was significantly greater in nasal secretions from patients with AR than in those from patients with CRS with NP (p < 0.05).
(0, 10, 100, and 1,000 nmol/L) for 15 minutes (Calbiochem, San Diego, California) before stimulating them with tumor necrosis factor α (TNF-α; 10 ng/ mL) for 24 hours (R&D Systems, Minneapolis, Minnesota). The NHBE cells were also preexposed to a FPRL-1 receptor antagonist, boc-2 (10 mmol/L), for 15 minutes (Boc-Phe-Leu-Phe-Leu-Phe; Scrum, Tokyo, Japan), to lipoxin A4 (100 nmol/L) for 15 minutes, and then to TNF-α (10 ng/mL) for 24 hours. The supernatants were collected and stored at –20°C until use. The concentrations of interleukin 8 in supernatants were determined with an enzyme-linked immunosorbent assay kit (R&D Systems). Statistical Analysis. All data are expressed as mean ± SD. The difference between variables was calculated by analysis of variance with a post hoc analy-
A
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B
A
B
Fig 3. mRNA expressions of A) formyl peptide receptor– like (FPRL-1) and B) cysteinyl leukotriene type 1 receptor (CysLT1R) in nasal tissues by RT-PCR were significantly greater (p < 0.05) in nasal polyps than in inferior turbinates.
sis using Fisher’s predicted least significant difference test. The Mann-Whitney U test was also used for comparison between groups. Statistical analyses were carried out with StatView 5.0 (Abacus Concepts, Piscataway, New Jersey). Probability (p) values of less than 0.05 were considered significant. RESULTS
Lipoxin A4 Concentration in Nasal Secretions. To ascertain whether lipoxin A4 synthesis is involved in local inflammation, we first measured the lipoxin A4 concentration in the nasal secretions from patients with CRS with NP and those with AR. Significant concentrations of lipoxin A4 were found in the nasal secretions, and the concentration was significantly greater in patients with AR than in patients with CRS with NP (Fig 1). There was no difference in the lipoxin A4 concentrations of patients with CRS with NP with the use of topical or systemic steroids or
C
D
Fig 2. mRNA expressions of lipoxygenases (LOXs) in nasal polyps compared to those in inferior turbinate tissue specimens by reverse-transcription polymerase chain reaction (RT-PCR). These LOXs are involved in release of lipoxin A4. mRNA expressions of A) 5-LOX and B) 15-LOX-1 were significantly greater (p < 0.05) in nasal polyps (11 specimens) than in inferior turbinates (16 specimens). mRNA expressions of C) platelet-type 12-LOX (p = 0.13) and D) 15-LOX-2 (p = 0.23) were decreased in nasal polyps.
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A
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B
Fig 4. Immunohistochemical staining of A) negative control and B) FPRL-1 from epithelial cells of nasal polyp. Scale bar — 100 μm.
with the comorbidity of bronchial asthma. LOX mRNA Expression in Nasal Tissues. 5-LOX, 15-LOX-1, 15-LOX-2, and platelet-type 12-LOX may be involved in the production of lipoxins. The mRNA expressions of 5-LOX (Fig 2A) and 15LOX-1 (Fig 2B) were significantly greater in nasal polyps than in inferior turbinates. The mRNA expressions of platelet-type 12-LOX (Fig 2C) and 15LOX-2 (Fig 2D) were lower in nasal polyps than in inferior turbinates. All nasal polyps were obtained from patients with CRS with NP, and 6 of the 16 inferior turbinate specimens were obtained from patients with AR. No differences in mRNA expression of LOXs were observed between the inferior turbinate specimens from the two patient groups. Lipoxin A4 Receptor FPRL-1 and CysLT1R mRNA Expression in Nasal Tissues. Lipoxin A4 directly activates FPRL-1 and inactivates CysLT1R. The mRNA expressions of FPRL-1 (Fig 3A) and CysLT1R (Fig 3B) were significantly greater in na-
sal polyps than in inferior turbinates. Immunohistochemical Localization of FPRL-1. Immunohistochemical study revealed that FPRL-1 was localized in the epithelial cells of nasal polyps (Fig 4B). FPRL-1 was also expressed in the epithelial cells and submucosal glands of the inferior turbinates. Effects of Lipoxin A4 Release of Interleukin 8 From Cultured NHBE Cells. The cultured NHBE cells were incubated with lipoxin A4 (0, 10, 100, and 1,000 nmol/L) for 15 minutes before 24-hour incubation with TNF-α (10 ng/mL). Lipoxin A4 partially inhibited TNF-α–induced release of interleukin 8 from cultured NHBE cells (Fig 5A). To evaluate whether FPRL-1 is involved in the lipoxin A4–induced inhibitory effect on the release of interleukin 8, we used an antagonist of FPRL-1, the boc-2 peptide. Preincubation of NHBE cells with boc-2 (10 nmol/L) for 15 minutes attenuated the lipoxin A4– induced inhibitory effect on the release of interleu-
Fig 5. Effects of lipoxin A4 (LXA4) and its receptor antagonist boc-2 on tumor necrosis factor α–induced release of interleukin 8 from normal human bronchial epithelial cells. A) Lipoxin A4 partially inhibited tumor necrosis factor α–induced release of interleukin 8 in dose-dependent manner. B) boc-2 attenuated lipoxin A4–induced inhibition of tumor necrosis factor α–induced release of interleukin 8. Asterisk — p < 0.05.
A
B
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kin 8 (Fig 5B). These results indicate that lipoxin A4 inhibited TNF-α–induced release of interleukin 8 via its receptor FPRL-1. DISCUSSION
The present study revealed that nasal secretions contain a significant concentration of lipoxin A4, and that the mRNA expressions of its synthetases 5-LOX and 15-LOX-1, and its receptor FPRL-1, were significantly greater in nasal polyps than in inferior turbinates. Our immunohistochemical study revealed that FPRL-1 is localized in nasal epithelial cells. This is the first study to show the presence of lipoxin A4 in nasal secretions and the localization of its receptor FPRL-1 in nasal mucosa.
Lipoxins are potential anti-inflammatory mediators that attenuate inflammation and inhibit the functions of neutrophils and eosinophils such as chemotaxis, adherence, transmigration, degranulation, elastase secretion, and generation of superoxide anions. Lipoxins also promote the resolution of inflammation by stimulating monocyte chemotaxis, adherence, and ingestion of apoptotic cells.2 Lipoxin A4 concentrations were found to be significantly suppressed in the airway fluids of patients with cystic fibrosis compared to patients with other inflammatory lung conditions.3 The severity of bronchial asthma was found to be related to decreased biosynthesis of lipoxin A4.4 A previous study revealed a higher concentration of lipoxin A4 in the nasal tissues of patients with CRS with NP compared to our results in nasal secretions, and lipoxin A4 was downregulated in patients with aspirin sensitivity.10 In the present study, we demonstrated a lower concentration of lipoxin A4 in the nasal secretions of patients with CRS with NP than in patients with AR. This result indicates that decreased biosynthesis of lipoxins may be involved in the pathogenesis of chronic airway inflammation.
In the present study, the mRNA expressions of 5-LOX and 15-LOX-1 were significantly greater in nasal polyps than in inferior turbinates. These results support those of previous studies that showed greater expression of mRNA in the sinonasal mucosa of patients with CRS with NP than in normal nasal mucosa.10,11 For leukotriene formation, 5-LOX in leukocytes is the key enzyme in the production of pro-inflammatory mediators such as leukotriene B4 and cysteinyl leukotrienes by converting arachidonic acid to leukotriene A4. Leukotrienes are also generated by 5-LOX in leukocytes from 15-HETE produced by 15-LOX-1. The mRNA expression of 15-LOX-1 is found in airway epithelial cells, eosinophils, and monocytes.12 Transgenic rabbits
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with increased 15-LOX-1 expression in monocytes showed an increased concentration of lipoxin at the sites of inflammation.13 The Th2 cytokines interleukin 4 and interleukin 13 induced 15-LOX-1 expression in monocytes and airway epithelial cells, and prostaglandins D2 and E2 induced 15-LOX-1 expression in epithelial cells.12 These results indicate that the expression of 15-LOX-1 in monocytes and epithelial cells is important for the transformation of lipoxins in combination with 5-LOX in leukocytes. Lipoxins are also released in combination with 5-LOX in leukocytes and with 12-LOX in platelets. Little is known about the biological role of platelettype 12-LOX in airway inflammation. In the present study, the mRNA expression of platelet-type 12LOX was suppressed in nasal polyps compared to interior turbinates. It is possible that the suppressed expression of platelet-type 12-LOX in nasal polyps may contribute to the decreased concentration of lipoxin A4 in nasal secretions from patients with CRS with NP. Both 15-LOX-1 and 15-LOX-2 are capable of metabolizing arachidonic acid to 15-HETE; however, 15-LOX-2 has limited tissue distribution, and the role of 15-LOX-2 in airway inflammation is unclear.14 In the present study, the mRNA expression of 15-LOX-2 was suppressed in nasal polyps. A lower mRNA expression of 15-LOX-2 and lesser production of lipoxin A4 have been reported in colonic mucosa of patients with ulcerative colitis and may be related to the pathogenesis of chronic inflammation.15 The lipoxin A4 receptor FPRL-1 was expressed in several types of leukocytes, such as neutrophils, monocytes, and activated T cells, as well as in resident cells, including intestinal epithelial cells, bronchial epithelial cells, synovial fibroblasts, renal mes angial cells, and astrocytes. The mRNA expression of FPRL-1 is up-regulated by various cytokines such as interferon γ, interleukin 1β, and interleukin 13 at the site of inflammation.2,16 In the present study, for the first time, we demonstrated that FPRL-1 was localized in the nasal epithelial cells. We confirmed that lipoxin A4 partially inhibited TNF-α–induced interleukin 8 release from cultured airway epithelial cells via its receptor FPRL-1. The effective dose of lipoxin A4 was equivalent to the previously reported tissue concentration of lipoxin A4 in nasal mucosa.10 These results indicate that lipoxin A4 inhibits inflammation through the direct inhibition of cytokine release from nasal epithelial cells via the FPRL-1 receptor. Lipoxins also act as partial antagonists for CysLT1R, and the mRNA expression of CysLT1R was up-regulated in nasal polyps. Lipoxins may suppress sinonasal inflammation through the inhibition of epithelial cytokine release via the FPRL-
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1 receptor and through their antagonistic effects on CysLT1R. In addition to lipoxin A4, a variety of peptide ago nists have been identified for FPRL-1, and FPRL1 signaling is cell- and agonist-specific.17 Previous studies showed that lipoxin A4/FPRL-1 signaling attenuated the nuclear accumulation of nuclear factorκB (NF-κB) and activator protein 1 (AP-1). NF-κB and AP-1 are key regulators for the production of proinflammatory cytokines such as interleukins 1β, 6, and 8 and TNF-α. In human leukocytes, lipoxin A4 partially inhibited lipopolysaccharide-induced interleukin 8 secretion through the suppression of NFκB and AP-1 activation.18 In human macrophages and intestinal epithelial cells, lipoxin A4 inhibited lipopolysaccharide-induced production of interleukin 8 and TNF-α and activation of NF-κB.19 Lipoxin A4 also attenuated lipopolysaccharide-induced produc-
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tion of interleukins 1β, 6, and 8 by the inhibition of phosphoinositol 3'-kinase (PI3K), p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, NFκB, and AP-1 pathway-dependent signal transduction in pulmonary microvascular endothelial cells.20 These results indicate that similar signaling pathways may be involved in the lipoxin A4/FPRL-1– dependent inhibition of TNF-α–induced interleukin 8 secretion from human airway epithelial cells seen in the present study. In conclusion, the present study demonstrated that lipoxin A4 is an important pro-resolution mediator in upper airway inflammation and that inappropriate metabolism of arachidonic acid may be involved in the pathogenesis of chronic inflammation and nasal polyp formation in CRS. These results suggest that a lipoxin A4 analog may be a potential regulator of upper airway inflammation.
Acknowledgments: We thank the staff of the Central Research Laboratory of Shiga University of Medical Science for their important contribution toward the completion of this work.
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