Increased expression of interleukin 36 in chronic rhinosinusitis and its contribution to chemokine secretion and increased epithelial permeability.

BACKGROUND: IL-36 family, a recently reported member of the IL-1 cytokine family, plays an essential role in nonspecific innate immune response to infection. This study aims at investigating the expression of IL-36 family members (α, ß, and γ) in normal and inflammatory sinus mucosa of patients with chronic rhinosinusitis (CRS), their effects on chemokine secretion and on the barrier function of epithelial and endothelial cells, and the effect of Toll-like receptors on the expression of IL-36 in epithelial cells. MATERIAL AND METHODS: The expression of IL-36 family in normal and inflammatory sinus mucosa, the production of chemokines or the expression levels of IL-36 family in epithelial cells treated with IL-36 family members or stimulated with TLR3, TLR4, TLR5, or TLR7/8 agonists were measured with real time PCR, ELISA, immunohistochemistry, or Western blot. The epithelial and endothelial permeability, and transendothelial leukocyte migration were investigated using cultured epithelial and endothelial cells. RESULTS: IL-36α, IL-36ß, and IL-36γ were localized in epithelial cells of sinonasal mucosa. Their levels increased in inflammatory mucosa of CRS patients and are up-regulated by TLR3, TLR4, or TLR5 agonists. IL-36α, or IL-36γ induced CXCL1, CXCL2, and CXCL3 production. Epithelial and endothelial permeability, transendothelial leukocyte migration were increased in cells treated with IL-36α, IL-36ß, or IL-36γ. CONCLUSIONS: These results suggest that IL-36α, IL-36ß, and IL-36γ localized in superficial epithelium may act as a responder to microbial and nonmicrobial elements through TLR and subsequently produce CXC chemokines, playing an interplay between innate and adaptive immune response.

Decreased expression of CCL17 in the disrupted nasal polyp epithelium and its regulation by IL-4 and IL-5.

BACKGROUND: In airway epithelium, thymus and activation-regulated chemokine (CCL17) and macrophage-derived chemokine (CCL22) are induced by defective epithelial barriers such as E-cadherin and attract the effector cells of Th2 immunity. However, the association between the epithelial barrier and CCL17 expression has not been studied in chronic rhinosinusitis with nasal polyp (CRSwNP). Thus, we aimed to evaluate the expression of CCL17 and its regulation by Th cytokines in nasal polyp (NP) epithelial cells. METHODS: The expression and distribution of CCL17, CCL22, E-cadherin and/or epidermal growth factor receptor (EGFR) were measured using real-time PCR, western blot, and immunohistochemistry and compared between normal ethmoid sinus epithelium and NP epithelium. In addition, the expression level of CCL17 was determined in cultured epithelial cells treated with IL-4, IL-5, IL-13, TNF-α, and IFN-γ. RESULTS: The expression of CCL17 was decreased in the NP epithelium compared to the epithelium of normal ethmoid sinus, whereas the expression of CCL22 was not decreased. E-cadherin was differentially distributed between the epithelium of normal ethmoid sinus and NP epithelium. EGFR was also decreased in NPs. Interestingly, the stimulation of cultured epithelial cells with Th2 cytokines, IL-4 and IL-5, resulted in an upregulation of CCL17 expression only in NP epithelial cells whereas the expression of CCL17 was increased in both normal epithelial cells and NP epithelial cells by Th1 cytokines. CONCLUSION: Our results suggest that the decreased expression of CCL17 in defective NP epithelium may be closely connected to NP pathogenesis and can be differentially regulated by cytokines in the NP epithelium of patients with CRSwNP.

Asymmetric expression level of clock genes in left vs. right nasal mucosa in humans with and without allergies and in rats: Circadian characteristics and possible contribution to nasal cycle.

Numerous peripheral tissues possess self-sustaining daily biologic rhythms that are regulated at the molecular level by clock genes such as PER1, PER2, CLOCK, and BMAL1. Physiological function of nasal mucosa exhibits rhythmic variability to a day-night environmental cycle. Nevertheless, little is known of the expression and distribution pattern of clock genes in nasal mucosa. The present study investigates the expression level and distribution pattern of PER1, PER2, CLOCK, and BMAL1 genes in nasal mucosa of healthy controls, allergic rhinitis patients, and normal rats. In human and rat nasal mucosa, the levels of these genes are asymmetrically expressed in nasal mucosa derived from right and left cavities in normal controls, allergic patients, and rat. In human nasal mucosa, the expression levels of these genes were higher in the decongested side than the congested mucosa. In rat nasal mucosa, these clock genes are expressed in a rhythmic circadian manner under the regular light/dark cycles. The expression levels of MUC5AC, a key mucin genes produced in superficial epithelium, are higher in decongested side than that congested side in human nasal mucosa. In rat nasal mucosa, MUC5AC levels showed a circadian rhythm which was associated with different expression levels in nasal mucosa derived from the right and left nasal cavities. Taken together with these results, the present study shows that the clock genes such as PER1, PER2, CLOCK, and BMAL1 are present in human and rat nasal mucosa, and suggest that these clock genes may control the pathophysiological function of nasal mucosa as circadian oscillators and affect the maintenance of the nasal cycle.

Increased expression of the aryl hydrocarbon receptor in allergic nasal mucosa, contributing to chemokine secretion in nasal epithelium.

BACKGROUND: Pollutants produced by industrial and traffic-related activities have been linked to allergic responses. These noxious agents induce their effects through the aryl hydrocarbon receptor (AhR). OBJECTIVE: We analyzed the expression and distribution pattern of AhR in normal and allergic nasal mucosa, and cytokine-driven regulation of its expression. The production levels of chemokine in cultured nasal epithelial cells were evaluated after stimulation with AhR ligand. METHODS: The expression levels and distribution pattern of AhR in normal, mild, and moderate-severe persistent allergic nasal mucosa were assessed by using real-time polymerase chain reaction, Western blot, and immunohistochemistry. The expression levels of AhR were determined in cultured nasal epithelial cells treated with T-helper 2 cytokines. In cultured epithelial cells stimulated with 2-(10H-indole-30-carbonyl)-thiazole-4-carboxylic acid methyl ester, the expression levels of granulocyte macrophage colony-stimulating factor, thymus and activation regulated chemokine, macrophage inflammatory protein 1 α, monocyte chemotactic protein 1, regulated on activation normal T-cell expressed and secreted, eotaxin, and interleukin 8 were measured with real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Expression of AhR was observed in normal and allergic nasal mucosa where it is distributed in the epithelial layer, submucosal glands, endothelial cells, and inflammatory cells. Its expression levels are increased in allergic nasal mucosa and upregulated after stimulation with T-helper 2 cytokines. The stimulation with 2-(10H-indole-30-carbonyl)-thiazole-4-carboxylic acid methyl ester resulted in increased production of chemokines in cultured epithelial cells. CONCLUSION: Analysis of the study results indicated that increased expression levels of AhR may play a role in the pathogenesis of allergic rhinitis, which contributes to chemokine production in nasal mucosa.

Increased expression of hCLCA1 in chronic rhinosinusitis and its contribution to produce MUC5AC.

OBJECTIVES/HYPOTHESIS: Mucus hypersecretion is a hallmarks of chronic rhinosinusitis. The expression of MUC5AC, a major respiratory mucin gene, is increased in chronic rhinosinusitis. The mechanisms inducing mucus hypersecretion have not been fully evaluated in chronic rhinosinusitis. Human Ca2+ -activated Cl- channel 1 (hCLCA1) is implicated in the regulation of mucus production, airway fluid, and electrolyte transport. The present study objectives was to investigate the expression of hCLCA1 in chronic rhinosinusitis and evaluate whether its level is altered by stimulation with type 1 T helper (Th1) and Th2 cytokines, and to determine the possible role of hCLCA1 on the regulation of mucin 5AC (MUC5AC) production. STUDY DESIGN: Controlled prospective study. METHODS: The expression of hCLCA1 and MUC5AC in normal and inflammatory ethmoid mucosa was determined by real-time polymerase chain reaction, immunohistochemistry, and Western blot. In cultured cells, the expression of hCLCA1 and MUC5AC was measured after stimulation with Th1 and Th2 cytokines. In a supernatant, the MUC5AC level was analyzed using enzyme-linked immunosorbent assay after treatment with niflumic acid. RESULTS: The levels of hCLCA1 and MUC5AC were increased in chronic rhinosinusitis, irrespective of nasal polyp presence, where they were distributed in superficial epithelial cells and submucosal glands. In cultured cells treated with interleukin (IL)-9, IL-4, IL-13, tumor necrosis factor-α, transforming growth factor-ß, interferon-γ, and IL-1ß, the expression of hCLCA1 and MUC5AC was increased. In cells treated with niflumic acid, the production of MUC5AC was inhibited. CONCLUSIONS: The current findings indicate that the expression of hCLCA1 is increased in chronic rhinosinusitis and may be regulated by Th1 and Th2 cytokines, possibly contributing to the production of MUC5AC. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E347-E355, 2016.

Decreased expression of E-cadherin and ZO-1 in the nasal mucosa of patients with allergic rhinitis: Altered regulation of E-cadherin by IL-4, IL-5, and TNF-alpha.

BACKGROUND: Allergic rhinitis is a chronic nasal inflammatory disease mediated by an immunoglobulin E mediated process to environmental allergens. Although atopy is a potent predisposing risk factor for allergic rhinitis, local tissue susceptibilities are inevitable for disease expression. The nasal epithelium maintains tissue homeostasis by providing a physical barrier controlled by epithelial junctional proteins. However, the expression of epithelial junctional proteins has not been studied in patients with allergic rhinitis. We sought to elucidate the expression and the regulation of epithelial junctional proteins in the nasal epithelium of patients with allergic rhinitis. METHODS: The expression of E-cadherin and zonula occludens (ZO) 1 in epithelium of turbinate was measured by using real-time polymerase chain reaction, Western blot, and immunohistochemical assays, and was compared between control subjects and patients with allergic rhinitis. In addition, the expression levels of E-cadherin and ZO-1 were determined in cultured epithelial cell treated with interleukin (IL) 4, IL-5, tumor necrosis factor (TNF) alpha, and interferon gamma. RESULTS: The expression and the immunoreactivity of E-cadherin and ZO-1 were decreased in the nasal epithelium of patients with allergic rhinitis. Interestingly, the stimulation of cultured epithelial cells with IL-4, IL-5, and TNF-alpha resulted in downregulation of E-cadherin expression only in cultured epithelial cells of patients with allergic rhinitis, whereas E-cadherin expression in cultured epithelial cells of controls was not affected by stimulation with the same panel of cytokines. CONCLUSION: Decreased expression of epithelial junctional proteins was found in patients with allergic rhinitis. The disruption of epithelial integrity by IL-4, IL-5, and TNF-alpha in vitro indicated a possible role for these cytokines in the pathogenesis of patients with allergic rhinitis.

Macrolides increase the expression of 11ß-hydroxysteroid dehydrogenase 1 in human sinonasal epithelium, contributing to glucocorticoid activation in sinonasal mucosa.

BACKGROUND AND PURPOSE: The anti-inflammatory and immunomodulatory effects of macrolides include the ability to decrease mucus secretion and inhibit inflammatory mediators in chronic rhinosinusitis. Nevertheless, their mechanisms of action remain to be determined. Here we have investigated the effects of macrolide antibiotics (clarithromycin, azithromycin and josamycin; representating the 14-, 15- and 16-membered macrolides) on endogenous steroids in human sinonasal epithelial cells and mouse nasal mucosa. EXPERIMENTAL APPROACH: The effects of macrolides on the expression of steroid-converting enzymes [11ß-hydroxysteroid dehydrogenase (11ß-HSD1 and 11ß-HSD2)], steroid-synthesizing enzymes (3ß-HSD, CYP21, CYP11B1 and CYP11A1) and cortisol levels were assessed in cultured human epithelial cells. In control and adrenalectomized mice , these enzymes and corticosterone levels were evaluated in nasal mucosa and serum after administration of macrolides. KEY RESULTS: The expression levels of 3ß-HSD, CYP21, 11ß-HSD1 and CYP11B1 increased in human epithelial cells treated with clarithromycin and azithromycin, whereas the expression levels of 11ß-HSD2 and CYP11A1 were not affected. Josamycin had no effects on the expression of these enzymes. Cortisol levels increased in epithelial cells treated with clarithromycin or azithromycin. The expression of 3ß-HSD, CYP11A1, CYP21, CYP11B1 and 11ß-HSD1 was upregulated in nasal mucosa of mice treated with clarithromycin or azithromycin, but not in adrenalectomized mice. CONCLUSIONS AND IMPLICATIONS: This study provides evidence that 14- and 15-membered macrolide antibiotics may affect the expression of steroid-synthesizing and steroid-converting enzymes in human sinonasal epithelial cells and mouse nasal mucosa, increasing the endogenous cortisol levels in sinonasal mucosa.

The expression of adiponectin receptor is altered in mild and moderate/severe persistent allergic nasal mucosa.

BACKGROUND: Adiponectin, one of the adipokines, has been implicated in the inflammatory process in patients with allergic rhinitis. The level of adiponectin is affected by immunotherapy. Considering the fact that adiponectin receptors (AdipoRs) mediate intracellular signaling events in response to the binding of adiponectin, the role of AdipoRs in healthy and allergic nasal mucosa should be determined. This study investigates the level of expression and distribution pattern of AdipoR1 and AdipoR2 in healthy, mild, and moderate/severe persistent allergic nasal mucosa to understand the role of adiponectin in allergic rhinitis. METHODS: The level of expression and distribution pattern of AdipoR1 and AdipoR2 were evaluated in healthy, mild, and moderate/severe persistent allergic nasal mucosa, using semiquantitative reverse-transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. RESULTS: AdipoR1 was expressed in healthy, mild, and moderate/severe persistent allergic nasal mucosa where it was commonly localized to the vascular endothelium. However, AdipoR2 was not expressed in any samples of nasal mucosa tested in the present study. Semiquantitative RT-PCR and Western blot analysis showed that the level of expression of AdipoR1 mRNA and protein was decreased in mild and moderate/severe persistent allergic nasal mucosa in comparison with healthy nasal mucosa, but not significantly different between mild and moderate/severe persistent allergic nasal mucosa. CONCLUSION: These results indicate that AdipoR1 may play an important role in the pathogenesis of allergic nasal mucosa, suggesting a role for AdipoR1 in vascular dysfunction in mild and moderate/severe persistent allergic nasal mucosa.

Overexpression of the superoxide anion and NADPH oxidase isoforms 1 and 4 (NOX1 and NOX4) in allergic nasal mucosa.

BACKGROUND: The purpose of this study was to investigate the expression and distribution of superoxide anion, NADPH oxidase (NOX)1, and NOX4 in healthy, allergic nasal mucosa and nasal polyps to evaluate the possible influence of oxidative stress on the development of allergic rhinitis and nasal polyps. METHODS: The expression and distribution of superoxide anion, NOX1 and NOX4 were evaluated in healthy and allergic nasal mucosa and nasal polyps, using dihydroethidium fluorescence, semiquantitative reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot. RESULTS: NOX1 and NOX4 were localized mainly in the epithelial layer, submucosal glands, vascular endothelium, and inflammatory cells in healthy and allergic nasal mucosa and nasal polyps. The cellular source that generated superoxide anion is also localized in the epithelial cells, submucosal glands, vascular endothelium, and inflammatory cells, demonstrating the similar sites of expression of NOX1 and NOX4 in healthy and allergic nasal mucosa and nasal polyps. NOX1 and NOX4 mRNA and proteins and superoxide anions had increased levels of expression in allergic nasal mucosa and nasal polyps compared with healthy nasal mucosa. CONCLUSIONS: These results indicate that NOX1 and NOX4 may play an important role in reactive oxygen species production, contributing to the oxidative stress in allergic rhinitis and nasal polyp tissues.

Up-regulation of heparanase in the ethmoid sinus mucosa of patients with chronic sinusitis.

BACKGROUND: Heparanase (HPA) is known to be involved in tissue remodeling of various organs with inflammatory diseases. The aim of this study was to investigate the level of expression and the pattern of distribution of HPA in normal human sinus mucosa, inflammatory sinus mucosa, and nasal polyps to evaluate the possible effect of HPA on the tissue remodeling of chronic inflammatory sinus mucosa and nasal polyps. METHODS: Normal sinus mucosa was obtained from the ethmoid sinus during endoscopic reduction in 25 patients with blowout fractures. Inflammatory sinus mucosa and nasal polyps were obtained from 25 patients undergoing endoscopic sinus surgery for chronic sinusitis with nasal polyps. The levels of expression and the pattern of distribution of HPA were evaluated in normal human sinus mucosa, inflammatory sinus mucosa, and nasal polyps, using reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, and Western blot analysis. RESULTS: HPA mRNA and protein were detected in inflammatory sinus mucosa and nasal polyps but not in normal sinus mucosa. HPA was mainly localized in the vascular endothelium, epithelium, submucosal glands, and inflammatory cells of inflammatory sinus mucosa. In nasal polyps, inflammatory cells and vascular endothelium showed immunopositivity in the entire portion, whereas glands and epithelial cells did not show positivity. CONCLUSION: Our results indicate that HPA is not constitutively expressed in normal sinus mucosa and is upregulated in chronic inflammatory sinus mucosa and nasal polyps, suggesting that HPA may play an important role in the tissue remodeling in chronic sinusitis.

Expression and distribution patterns of the stem cell marker, nestin, and the stem cell renewal factor, BMI-1, in normal human nasal mucosa and nasal polyps.

CONCLUSIONS: These results suggest that nestin and BMI-1 are candidates for stem cell markers and renewal factors in human nasal mucosa, may contribute to tissue homeostasis and differentiation in the epithelium and submucosal glands of normal nasal mucosa, and may play a role in proliferation of nasal polyps. OBJECTIVES: The stem cell marker, nestin, and the stem cell renewal factor, BMI-1, have been identified in a variety of inflammatory and normal tissues, implicating these markers in tissue regeneration. MATERIALS AND METHODS: We investigated the expression and distribution of nestin and BMI-1 in normal nasal mucosa and nasal polyps, using RT-PCR, immunohistochemistry, and Western blotting. RESULTS: Nestin and BMI-1 were localized to the epithelium and submucosal glands of normal nasal mucosa and nasal polyps. The expression of nestin was confined to the plasma membrane and cytoplasm, whereas BMI-1 showed a nuclear staining pattern. In normal nasal mucosa and nasal polyps, nestin and BMI-1 expression was strongest in the basal portion of the epithelial layer, and decreased toward the upper portion. In the submucosal glands, weak to strong expression was commonly detected in the glandular acini. There was no significant difference in the level of expression of nestin and BMI-1 between normal nasal mucosa and nasal polyps.

Down-regulation of carbonic anhydrase isoenzymes in nasal polyps.

OBJECTIVES/HYPOTHESIS: Carbonic anhydrase (CA) is a zinc metalloenzyme that participate in the biological processes of various fluid transporting epithelia, including ion and water transport. CA may thus play a role in the pathophysiology of normal nasal mucosa and nasal polyp. We evaluated the expression and pattern of distribution of mRNAs and proteins for CA isoenzymes in normal nasal mucosa and polyps. STUDY DESIGN: This was a controlled, prospective study. METHODS: The expression levels of 11 isoenzyme genes (I, II, III, IV, VA, VB, VI, VII, IX, XII, XIV) were evaluated in normal mucosa and polyps using semiquantitative reverse-transcriptase polymerase chain reaction. The expression and pattern of distribution of CA I, II, and IX were also investigated using immunohistochemistry and Western blot. RESULTS: mRNAs for the 11 CA isoenzymes were detected in all normal nasal mucosa and polyps tested. Their expression levels were decreased in nasal polyp in comparison with normal nasal mucosa. CA I was detected in the epithelial cells, endothelial cells, and submucosal gland of normal nasal mucosa, but only in the endothelial cells in nasal polyp. CA II in normal mucosa was intensely expressed in the submucosal gland cells and CA IX was noted in the cytoplasm and plasma membrane of epithelial cells and submucosal glands. However, these findings were not found in nasal polyp. Western blot also showed differences in their expression levels. CONCLUSIONS: These results indicate that CA may participate in the physiology of normal nasal mucosa, but also suggest that altered their expression in nasal polyps may cause impaired electrolyte and water transport across the epithelial cells.