Low E-prostanoid 2 receptor levels and deficient induction of the IL-1ß/IL-1 type I receptor/COX-2 pathway: Vicious circle in patients with aspirin-exacerbated respiratory disease.

BACKGROUND: We hypothesized that the 2 reported alterations in aspirin-exacerbated respiratory disease (AERD), reduced expression/production of COX-2/prostaglandin (PG) E2 and diminished expression of E-prostanoid (EP) 2 receptor, are closely linked. OBJECTIVE: We sought to determine the mechanisms involved in the altered regulation of the COX pathway in patients with AERD. METHODS: Fibroblasts were obtained from nasal mucosa; samples of control subjects (NM-C, n = 8) and from nasal polyps from patients with aspirin-exacerbated respiratory disease (NP-AERD, n = 8). Expression of the autocrine loop components regulating PGE2 production and signaling, namely IL-1 type I receptor (IL-1RI), COX-2, microsomal prostaglandin E synthase 1 (mPGES-1), and EP receptors, was assessed at baseline and after stimulation with IL-1ß, PGE2, and specific EP receptor agonists. RESULTS: Compared with NM-C fibroblasts, basal expression levels of IL-1RI and EP2 receptor were lower in NP-AERD fibroblasts. IL-1ß-induced IL-1RI, COX-2, and mPGES-1 expression levels were also lower in these cells. Levels of IL-1RI positively correlated with COX-2 and mPGES-1 expression in both NM-C and NP-AERD fibroblasts. Incubation with either exogenous PGE2 or selective EP2 agonist significantly increased expression of IL-1RI in NM-C fibroblasts and had hardly any effect on NP-AERD fibroblasts. Alterations in IL-1RI, COX-2, and mPGES-1 expression that were found in NP-AERD fibroblasts were corrected when EP2 receptor expression was normalized by transfection of NP-AERD fibroblasts. CONCLUSION: Altered expression of EP2 in patients with AERD contributes to deficient induction of IL-1RI, reducing the capacity of IL-1ß to increase COX-2 and mPGES-1 expression, which results in low PGE2 production. This impairment in the generation of PGE2 subsequently reduces its ability to induce IL-1RI.

Clinical and biological markers of difficult-to-treat severe chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the upper airways of which two major phenotypes exist, CRS without nasal polyps (CRSsNP) and CRS with nasal polyps (CRSwNP). Some patients with CRS have suboptimal response to current guideline treatments. These patients remain severe and uncontrolled by treatment and have a poor quality of life. It is highly important to identify both clinical and biological markers, so-called biomarkers, in this subset of patients. The presence of nasal polyps and comorbidity with asthma and with aspirin-exacerbated respiratory disease (AERD) are the most common clinical traits that have been associated to difficult-to-treat severe CRS. In addition to clinical traits, numerous biological markers, with known etiopathogenic roles in CRS, have been associated to difficult-to-treat or recalcitrant CRS. This review summarizes the existing knowledge of the clinical and biological markers associated to difficult-to-treat or uncontrolled severe CRS.

Effect of lipopolysaccharide on glucocorticoid receptor function in control nasal mucosa fibroblasts and in fibroblasts from patients with chronic rhinosinusitis with nasal polyps and asthma.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the upper airways frequently associated with asthma. Bacterial infection is a feature of CRSwNP that can aggravate the disease and the response to glucocorticoid treatment. OBJECTIVE: We examined whether the bacterial product lipopolysaccharide (LPS) reduces glucocorticoid receptor (GR) function in control nasal mucosa (NM) fibroblasts and in nasal polyp (NP) fibroblasts from patients with CRSwNP and asthma. METHODS: NP (n = 12) and NM fibroblasts (n = 10) were in vitro pre-incubated with LPS (24 hours) prior to the addition of dexamethasone. Cytokine/chemokine secretion was measured by ELISA and Cytometric Bead Array. GRα, GRß, mitogen-activated protein-kinase phosphatase-1 (MKP-1) and glucocorticoid-induced leucine zipper (GILZ) expression was measured by RT-PCR and immunoblotting, GRα nuclear translocation by immunocytochemistry, and GRß localization by immunoblotting. The role of MKP-1 and GILZ on dexamethasone-mediated cytokine inhibition was analyzed by small interfering RNA silencing. RESULTS: Pre-incubation of nasal fibroblasts with LPS enhanced the secretion of IL-6, CXCL8, RANTES, and GM-CSF induced by FBS. FBS-induced CXCL8 secretion was higher in NP than in NM fibroblasts. LPS effects on IL-6 and CXCL8 were mediated via activation of p38α/ß MAPK and IKK/NF-κB pathways. Additionally, LPS pre-incubation: 1) reduced dexamethasone's capacity to inhibit FBS-induced IL-6, CXCL8 and RANTES, 2) reduced dexamethasone-induced GRα nuclear translocation (only in NM fibroblasts), 3) did not alter GRα/GRß expression, 4) decreased GILZ expression, and 5) did not affect dexamethasone's capacity to induce MKP-1 and GILZ expression. MKP-1 knockdown reduced dexamethasone's capacity to suppress FBS-induced CXCL8 release. CONCLUSION: The bacterial product LPS negatively affects GR function in control NM and NP fibroblasts by interfering with the capacity of the activated receptor to inhibit the production of pro-inflammatory mediators. This study contributes to the understanding of how bacterial infection of the upper airways may limit the efficacy of glucocorticoid treatment.

Corticosteroid treatment regulates mucosal remodeling in chronic rhinosinusitis with nasal polyps.

OBJECTIVES/HYPOTHESIS: To investigate the effect of oral plus intranasal corticosteroid (CS) treatment on nasal polyp (NP) mucosa remodeling from patients with severe chronic rhinosinusitis with nasal polyps (CRSwNP). STUDY DESIGN: Case series, retrospective study. METHODS: Patients (n = 18) with severe CRSwNP were treated with oral prednisone for 2 weeks and intranasal budesonide for 12 weeks. NP biopsies were obtained from patients biopsies before (w0) and after 2 weeks (w2) and 12 weeks (w12) of CS treatment. Matrix metalloprotease 1 (MMP-1), MMP-2, MMP-7, MMP-9, and tissue inhibitor of metalloprotease type 1 (TIMP-1) expression was evaluated by immunohistochemistry in cell and tissue structures. Epithelial damage, eosinophil infiltration, and collagen content were also examined in NP tissues before and after CS treatment. RESULTS: Compared to w0: 1) oral plus intranasal CS significantly (P < .01) increased presence of submucosal glands at w2, decreased epithelial cell hyperplasia at w12, and decreased tissue eosinophilia at w2 and w12; 2) CS treatment significantly (P < .05) increased immunoreactivity for MMP-1 and MMP-2 in the epithelium at w2, but decreased immunoreactivity for MMP-9 in the epithelium at w2 and w12; 3) at w12, CS significantly (P < .05) reduced MMP-9 immunoreactive positivity and intensity in the extracellular matrix, while increasing total collagen amount in the extracellular matrix; and 4) CS treatment significantly (P < .01) reduced the number of eosinophils and their MMP and TIMP-1 immunoreactive expression. CONCLUSIONS: CS treatment modulates NP mucosa remodeling, particularly by promoting epithelial repair, regulating tissue remodeling markers, increasing total collagen content, and reducing tissue eosinophil infiltration. LEVEL OF EVIDENCE: 4

Fluticasone furoate inhibits cytokine secretion from nasal epithelial cells and reduces eosinophil survival in an in vitro model of eosinophilic inflammation.

BACKGROUND: Fluticasone furoate (FF) is an intranasal corticosteroid indicated for the treatment of allergic rhinitis (AR). However, the anti-inflammatory effects of FF in the nasal mucosa have yet to be investigated thoroughly. The aim of this study was to investigate the effect of FF on eosinophil survival and cytokine secretion from nasal mucosa epithelial cells. METHODS: Epithelial cells obtained from nasal mucosa were stimulated with 10% fetal bovine serum (FBS) in the presence of FF (from 10(-12) to 10(-7)M) for 6-24 h. Cytokine [granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6 and IL-8] concentrations in supernatants were measured by ELISA. Peripheral blood eosinophils were incubated for 4 days with epithelial cell secretions in the presence or absence of FF (from 10(-12) to 10(-7)M) and survival was assessed by Trypan blue dye exclusion. Results are expressed as medians of the minimum effective concentration and IC values. RESULTS: FBS stimulated the secretion of GM-CSF, IL-6 and IL-8. FF significantly inhibited GM-CSF (up to 10(-10)M, IC25 = 12.6 pM), IL-6 (up to 10(-10)M, IC25 = 65.8 pM) and IL-8 (up to 10(-11)M, IC25 = 8.6 pM) secretion induced by FBS (n = 8). Epithelial cell secretions induced eosinophil survival from day 1 to day 4 (n = 6). This effect was significantly inhibited by FF (up to 10(-12)M) at day 3 (IC50 = 3.22 nM) and day 4 (IC50 = 1.29 nM). CONCLUSIONS: The results obtained in this in vitro model suggest that FF may reduce upper airway eosinophilic inflammation through decreasing cytokine secretion from epithelial cells and reducing eosinophil survival.

Oral plus nasal corticosteroids improve smell, nasal congestion, and inflammation in sino-nasal polyposis.

OBJECTIVES/HYPOTHESIS: To assess the effect of oral plus intranasal corticosteroids on subjective outcomes (smell and nasal congestion) and objective outcomes (tissue eosinophilia and nitric oxide) in severe nasal polyposis (NP). STUDY DESIGN: After a 4-week steroid washout period (w0), severe NP were randomized into a treatment group (n = 67) that receive oral prednisone for 2 weeks (w2) plus intranasal budesonide for 12 weeks (w12), and a control group (n = 22) that received no steroid treatment. METHODS: Barcelona Smell Test 24 (BAST-24), nasal congestion, tissue eosinophilia, and nasal nitric oxide (nNO) were assessed. RESULTS: Before treatment, patients showed a significant impairment of smell detection (30.7 ± 39.5%), identification (7.1 ± 16.1%), and forced choice (13.8 ± 23.3%) in BAST-24 compared to healthy population. At w2, the treatment group showed a significant improvement in detection, identification, and forced choice. Positive effect was also seen after 12 weeks of intranasal corticosteroids. A significant reduction of nasal congestion (1.17 ± 1.0 vs. 2.73 ± 0.5) and polyp tissue eosinophilia (10.9 ± 4.2 vs. 41.2 ± 12.2) with an increase of nNO (650 ± 317 vs. 420 ± 221 ppb) were observed at w2 compared to w0 and to the control group. These effects were also seen at w12. CONCLUSIONS: Combined oral and intranasal corticosteroids improve smell and nasal congestion and decrease nasal inflammation, as measured by reduced tissue eosinophilia and increased detection of nNO. Severity of smell loss correlates with degree of nasal congestion but not with inflammation, as measured by tissue eosinophilia or nasally exhaled nNO. Our findings suggest that improvement in smell may be related to improved conduction of odorants to the olfactory neuroepithelium.

Lung myofibroblasts are characterized by down-regulated cyclooxygenase-2 and its main metabolite, prostaglandin E2.

BACKGROUND: Prostaglandin E2 (PGE2), the main metabolite of cyclooxygenase (COX), is a well-known anti-fibrotic agent. Moreover, myofibroblasts expressing α-smooth muscle actin (α-SMA), fibroblast expansion and epithelial-mesenchymal transition (EMT) are critical to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our aim was to investigate the expression of COX-2 and PGE2 in human lung myofibroblasts and establish whether fibroblast-myofibroblast transition (FMT) and EMT are associated with COX-2 and PGE2 down-regulation. METHODS: Fibroblasts obtained from IPF patients (n = 6) and patients undergoing spontaneous pneumothorax (control, n = 6) and alveolar epithelial cell line A549 were incubated with TGF-ß1 and FMT and EMT markers were evaluated. COX-2 and α-SMA expression, PGE2 secretion and cell proliferation were measured after IL-1ß and PGE2 incubation. RESULTS: Myofibroblasts from both control and IPF fibroblast cultures stimulated with IL-1ß showed no COX-2 expression. IPF fibroblasts showed increased myofibroblast population and reduced COX-2 expression in response to IL-1ß. TGF-ß1 increased the number of myofibroblasts in a time-dependent manner. In contrast, TGF-ß1 induced slight COX-2 expression at 4 h (without increase in myofibroblasts) and 24 h, but not at 72 h. Both IPF and control cultures incubated with TGF-ß1 for 72 h showed diminished COX-2 induction, PGE2 secretion and α-SMA expression after IL-1ß addition. The latter decreased proliferation in fibroblasts but not in myofibroblasts. A549 cells incubated with TGF-ß1 for 72 h showed down-regulated COX-2 expression and low basal PGE2 secretion in response to IL-1ß. Immuno-histochemical analysis of IPF lung tissue showed no COX-2 immuno-reactivity in myofibroblast foci. CONCLUSIONS: Myofibroblasts are associated with COX-2 down-regulation and reduced PGE2 production, which could be crucial in IPF development and progression.

Differential expression of remodeling markers by tissue structure in nasal polyposis.

BACKGROUND: Data on the expression and role of matrix metalloproteinases (MMPs) and their tissue inhibitors (tissue inhibitor of metalloproteinases [TIMPs]) in chronic rhinosinusitis with nasal polyps (CRSwNPs) are contradictory, partly because or the use of different techniques of tissue analysis. The aim of this study was to establish a qualitative/semiquantitative method of analysis on the expression of these remodeling markers in different tissue structures and eosinophils in both NPs and nasal mucosa (NM). METHODS: NP tissues were obtained from patients undergoing endoscopic sinus surgery for severe CRSwNPs (n = 33) and NM tissues from patients undergoing nasal corrective surgery (n = 12). MMPs (MMP-1, MMP-2, MMP-7, and MMP-9) and TIMP type 1 (TIMP-1) expression were evaluated by immunohistochemistry in tissue structures (epithelium, glands, vessels, and extracellular matrix [ECM]) and eosinophils. Tissue eosinophilia was also analyzed in NP tissues. RESULTS: MMPs and TIMP-1 expression were found in the epithelium, glands, vessels, and ECM (in both NM and NP) and in eosinophils (only in NP). Significant (p < 0.01) findings were observed in NP compared with NM: increase in MMP-1 in ECM; decrease in MMP-2 in glands, vessels, and epithelium; decrease in MMP-7 in all tissue structures; increase in MMP-9 in ECM and decrease in epithelium and glands; and no differences in TIMP-1. NP tissues showed a clear eosinophilic inflammation compared with NM (p < 0.01). CONCLUSION: These findings suggest that (1) metalloproteases (MMP-1, MMP-2, MMP-7, and MMP-9) may play an important role in the remodeling of NPs and/or in NP formation and (2) a differential analysis of tissue structures and inflammatory cells should be performed when studying remodeling marker expression and regulation in the upper airways.

Low prostaglandin E2 and cyclooxygenase expression in nasal mucosa fibroblasts of aspirin-intolerant asthmatics.

BACKGROUND AND OBJECTIVE: Anomalies in the regulation of cyclooxygenase (COX)-1 and -2 have been described in nasal polyps of aspirin-induced asthma (AIA). Whether these anomalies are specific to nasal polyps or affect all the nasal mucosa (NM) of upper airways is still unclear. The objective of this study was to compare the COX pathway in NM of AIA patients with the NM of control subjects. METHODS: Fibroblasts were isolated from NM of five AIA patients (AIA-NM) and five control subjects (control-NM). Cells were treated with 10 ng/mL interleukin (IL)-1ß for up to 72 h. Prostaglandin E2 (PGE2 ) production was measured by enzyme-linked immunosorbent assay (ELISA), expression of COX-1 protein by Western blot and COX-2 protein by ELISA, Western blot and immunofluorescence techniques. RESULTS: IL-1ß increased PGE2 production and COX-1 protein expression in control-NM fibroblasts, but no changes were found in AIA-NM. IL-1ß provoked a significant time-dependent increase in COX-2 protein expression in control-NM fibroblasts but had a very mild effect on COX-2 protein expression in AIA-NM. CONCLUSIONS: Our data suggest that abnormalities in the COX pathway are not a phenomenon exclusive to nasal-polyp mucosa as they are also present in all the NM of AIA patients. These anomalies may be involved in the pathogenesis of airway inflammation and non-steroidal anti-inflammatory drug intolerance in asthma patients with chronic rhinosinusitis and nasal polyposis.

Signal transduction pathways (MAPKs, NF-κB, and C/EBP) regulating COX-2 expression in nasal fibroblasts from asthma patients with aspirin intolerance.

BACKGROUND: Recent studies have revealed that cyclooxygenase-2 (COX-2) expression is down-regulated in aspirin-induced asthma (AIA). Various signal pathways (MAPKs, NF-κB and C/EBP) are involved in COX-2 regulation. OBJECTIVE: To investigate the regulation of COX-2 expression through MAP-kinase pathway activation and nuclear factor translocation in aspirin-induced asthma (AIA). METHODS: Fibroblasts were isolated from specimens of nasal mucosa (NM, N = 5) and nasal polyps (NP, N = 5). After IL-1ß (1 ng/ml) incubation, COX-2 and phosphorylated forms of ERK, JNK and p38 MAPK were measured by Western blot. MAPK's role in IL-1ß-induced COX-2 expression was assessed by treating cells with ERK (PD98059), JNK (SP600125) and p38 MAPK (SB203580) inhibitors (0.1-10 µM) prior to IL-1ß exposure. NF-κB and C/EBP nuclear translocation was measured by Western blot and TransAM® after IL-1ß (10 ng/ml) exposure. RESULTS: No differences were observed in the MAPK phosphorylation time-course between NM and NP-AIA fibroblasts. The p38 MAPK inhibitor at 10 µM significantly reduced IL-1ß-induced COX-2 expression in NM fibroblasts (85%). In NP-AIA fibroblasts the COX-2 inhibition (65%) at 1 and 10 µM was not statistically significant compared to non-treated cells. ERK and JNK inhibitors had no significant effect in either the NM or NP-AIA cultures. The effect of IL-1ß on NF-κB and C/EBP subunits' nuclear translocation was similar between NM and NP-AIA fibroblasts. CONCLUSIONS: These results suggest that p38 MAPK is the only MAPK involved in IL-1ß-induced COX-2 expression. NM and NP-AIA fibroblasts have similar MAPK phosphorylation dynamics and nuclear factor translocation (NF-κB and C/EBP). COX-2 downregulation observed in AIA patients appears not to be caused by differences in MAPK dynamics or transcription factor translocation.

Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts.

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)(2) (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-ß-induced collagen mRNA expression and interleukin (IL)-1ß-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1ß/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

Impact of cell culture methods on the outcomes of the in vitro inflammatory response in nasal polyps.

BACKGROUND: In vitro culture of nasal polyp cells is frequently used in the investigation of inflammatory mechanisms and effect of treatments in nasal polyposis. Research outcomes may, however, be influenced by the culture methodology used. METHODS: Nasal polyp and nasal mucosa in vitro fibroblast cultures were pre-treated with foetal bovine serum (FBS)-free culture medium or medium supplemented with either FBS or charcoal-stripped (cs) FBS. Cells were then stimulated with FBS or csFBS, with or without different doses of dexamethasone for 4 and 24h. IL-6, IL-8, GM-CSF and VEGF release and cell viability were measured. RESULTS: The highest cytokine levels were found in growth-arrested cells stimulated with 10% FBS. csFBS poorly stimulated cytokine release. Nasal polyp released larger IL-8 amounts than nasal mucosa fibroblasts. Dexamethasone decreased cytokine production dose- and time-dependently in both nasal mucosa and nasal polyp fibroblasts. The IC25 of IL-8 inhibition by dexamethasone was higher in nasal polyp than in nasal mucosa fibroblasts. Cell viability did not differ among treatments. CONCLUSIONS: Cytokine production by in vitro cultured nasal fibroblasts is affected by the culture conditions used and is inhibited by dexamethasone in both fibroblast types. Our results highlight the importance of culture methodology on nasal polyp research outcomes.

[Cyclooxigenase-2 levels are increased in the lung tissue and bronchial tumors of patients with chronic obstructive pulmonary disease]. / La ciclooxigenasa-2 está regulada al alza en el pulmón y en los tumores bronquiales de pacientes con enfermedad pulmonar obstructiva crónica.

INTRODUCTION: The expression of cyclooxygenase 2 (COX-2) is usually increased in inflammation and cancer. This study examines the expression of COX-2 in the lung of chronic obstructive pulmonary disease (COPD) patients with lung cancer. METHODS: We studied 44 male patients with bronchial cancer (27 squamous carcinoma and 17 adenocarcinoma). Samples were obtained from the pulmonary parenchyma, from the bronchial mucosa adjacent to the tumor and from the tumor itself. Lung tissue specimens from 14 patients with pneumothorax were used as control. The mRNA and the COX-1 and COX-2 proteins were assessed by RT-PCR and Western blot, respectively. RESULTS: COX-1 and COX-2 mRNA levels were significantly higher in the lung parenchyma of COPD patients than in the control subjects. COX-2 mRNA levels were also higher in the lung parenchyma than in both tumor and airway tissue samples procured from COPD patients. There were no differences in the COX-2 mRNA levels between squamous carcinoma and adenocarcinoma. In contrast, COX-2 protein levels were significantly higher in tumors than in lung parenchyma and airways. COX-2 protein levels were higher in adenocarcinoma compared with squamous carcinoma. CONCLUSION: This study shows that in COPD, the pathway of cyclooxygenase is activated and associated with an increase in the expression of COX-2 in lung tumors. These observations suggest that COX-2 is possibly involved in the association between COPD and cancer.

Reduced expression of COXs and production of prostaglandin E(2) in patients with nasal polyps with or without aspirin-intolerant asthma.

BACKGROUND: Researchers have debated whether regulation of the COX enzymes (COX-1 and COX-2), which mediate production of prostaglandins (PGs), affects the pathogenesis of nasal polyps (NPs) and aspirin-intolerant asthma (AIA). OBJECTIVE: We investigated the roles of PGE(2), COX-1 and COX-2, and PGE(2) receptors in the development of NPs and AIA by measuring their expression in fibroblasts derived from nasal mucosa (NM) and NPs. METHODS: Fibroblasts were isolated from the NM of subjects without asthma who had septal deviation, turbinate hypertrophy, or both (control subjects, n = 7); NPs of aspirin-tolerant nonasthmatic patients (n = 7); and NPs of patients with asthma who were intolerant of aspirin (n = 7). Polyp samples were collected during endoscopic surgery. Cultures were stimulated with IL-1ß (10 ng/mL) for 72 hours. We used ELISA, immunoblotting, and immunofluorescence analyses to measure secretion of PGE(2), expression of COX-1 and COX-2, and expression of the PGE(2) receptors EP1 to EP4. RESULTS: Compared with NM from control subjects, PGE(2) concentrations were significantly lower in IL-1ß-stimulated fibroblasts from patients with NPs who were tolerant to aspirin and even lower in polyps from patients with AIA. Similarly, IL-1ß exposure induced the expression of COX-1 and COX-2 in fibroblasts from NM of control subjects, had only moderate effects on fibroblasts from NPs of aspirin-tolerant nonasthmatic patients, and almost no effect on fibroblasts from NPs of patients with AIA. IL-1ß also induced expression of EP2 in fibroblasts from control NM but not in fibroblasts from NPs of aspirin-tolerant nonasthmatic patients or those with AIA. CONCLUSION: Alterations in the COX pathway (ie, reduced production of PGE(2) and lack of upregulation of COX-1, COX-2, and EP2 under conditions of inflammation) are associated with NPs in patients with or without AIA.

Mometasone and desloratadine additive effect on eosinophil survival and cytokine secretion from epithelial cells.

BACKGROUND: Although antihistamines and topical corticosteroids are used in combination to treat allergic rhinitis, their additive effect has not been yet demonstrated. The aim was investigate the antiinflammatory additive effect of mometasone and desloratadine on cytokine and sICAM-1 secretion by epithelial cells, and on eosinophil survival stimulated by human epithelial cells secretions from nasal mucosa and polyps. METHODS: Epithelial cells obtained from nasal mucosa or polyps were stimulated with 10% fetal bovine serum in presence of mometasone (10(-11) M-10(-5) M) with/without desloratadine (10(-5) M). Cytokine and sICAM-1 concentrations in supernatants were measured by ELISA. Peripheral blood eosinophils were incubated during 4 days with epithelial cell secretions with (10(-11) M-10(-5) M) and/or desloratadine (10(-5) M) and survival assessed by Trypan blue. Results are expressed as percentage (mean ± SEM) compared to control. RESULTS: Fetal bovine serum stimulated IL-6, IL-8, GM-CSF and sICAM-1 secretion. In mucosa and polyp epithelial cells, mometasone inhibited this induced secretion while desloratadine inhibited IL-6 and IL-8. The combination of 10(-5) M desloratadine and 10(-9) M mometasone reduced IL-6 secretion (48 ± 11%, p < 0.05) greater extent than mometasone alone (68 ± 10%) compared to control (100%). Epithelial cell secretions induced eosinophil survival from day 1 to 4, this effect being inhibited by mometasone. At day 4, the combination of mometasone (10(-11) M) and desloratadine (10(-5) M) provoked an increased inhibition of eosinophil survival induced by cell secretions (27 ± 5%, p < 0.01) than mometasone (44 ± 7%) or desloratadine (46 ± 7%) alone. CONCLUSIONS: These results suggest that the combination of desloratadine and mometasone furoate have a greater antinflammatory effect in an in vitro model of eosinophil inflammation than those drugs administered alone.

Nuclear translocation of the glucocorticoid receptor in fibroblasts of asthmatic patients with nasal polyposis insensitive to glucocorticoid treatment.

BACKGROUND: Nasal polyposis (NP) is treated with topical glucocorticoids (GC). Some patients require endoscopic nasal surgery because GC treatment is ineffective. To exert its function, the GC needs to bind with the GC receptor (GR) and the GC-GR complex moves to the cell nucleus. Our aim was to establish whether the poor response to GC is due to an alteration in the translocation of the GR to the nucleus. METHODS: We performed nasal fibroblast cell cultures from seven healthy controls and 12 patients with NP and asthma. Fibroblasts were incubated with budesonide or dexamethasone (10(-7) M) for different times (30 min to 4 h) and GR translocation was analyzed by immunocytochemistry. RESULTS: Both GC induced GR translocation in every group, doubling its concentration in the cell nucleus (30 min) compared to baseline. There were no differences in GR translocation between controls and patients, nor differences related to the severity of asthma or intolerance to non-steroidal anti-inflammatory drugs (NSAIDs). Atopic subjects showed a decrease in GR translocation with budesonide (1 h, 3 h and 4 h, P<0.05) and dexamethasone (30 min and 2 h, P<.05). CONCLUSIONS: The insensitivity to GC treatment does not appear to be due to an alteration in GR translocation to the nucleus. Neither does the severity of asthma or intolerance to NSAIDs appear to alter GR translocation. The association between atopy and the alteration in GR function deserves further investigation.

Lower sensitivity of nasal polyp fibroblasts to glucocorticoid anti-proliferative effects.

BACKGROUND: Treatment with glucocorticoids (GCs) is the cornerstone of nasal polyp (NP) therapy, but some patients respond poorly to them. Fibroblasts are involved in both inflammation and remodelling of NP. We aimed to evaluate whether NP fibroblasts are less sensitive to GCs' anti-proliferative and anti-inflammatory effects, compared to nasal mucosa (NM) fibroblasts. METHODS: Fibroblasts were obtained from NP (n = 8) from asthmatic patients undergoing endoscopic surgery and NM (n = 8) from patients undergoing nasal corrective surgery. Fibroblasts were stimulated with DMEM at 0.5% or 5% FBS, or TGF-ß (5 ng/ml), with or without dexamethasone (10(-11) to 10(-5)M) for different times. Cell proliferation, collagen mRNA expression and IL-6 and IL-8 release were measured. RESULTS: After 3-days, dexamethasone dose-dependently inhibited proliferation of NM (p < 0.001) but not that of NP fibroblasts. Dexamethasone (10(-6)M) reduced by 25% the proliferation of NM fibroblasts. Dexamethasone also inhibited proliferation of NM (p < 0.01) but not that of NP fibroblasts at 5-days. TGF-ß induced collagen-1α1, -1α2, and -3α1 mRNA levels in both NM and NP fibroblasts (p < 0.05), and dexamethasone did not alter TGF-ß-induced collagen mRNA levels in either fibroblast type at 24 h. Dexamethasone dose-dependently decreased (p < 0.05) FBS-induced IL-6 and IL-8 release in both NM and NP fibroblasts at 4 h, although at 10(-8)M, dexamethasone inhibited cytokine production in NM (p < 0.05) but not in NP fibroblasts. CONCLUSIONS: This impaired sensitivity of nasal polyp fibroblasts to in vitro glucocorticoid effects concurs in part with the poor clinical response that these nasal polyp patients show to glucocorticoid treatment.

The importance of smell in patients with bronchiectasis.

BACKGROUND: The aim of the study was to evaluate the sense of smell in patients with bronchiectasis. METHODS: Prospective controlled study was performed on 91 patients with bronchiectasis. Bronchiectasis patients were sub-classified depending on: the presence of chronic rhinosinusitis, with or without nasal polyps, and the bronchiectasis ethiology. Olfactory function was evaluated by means of the Barcelona Smell Test (BAST-24) olfactometry for detection, identification, and forced choice for the first and fifth cranial nerve dependent odours in comparison to a group of 120 healthy volunteers. RESULTS: Most patients with bronchiectasis (80.2%) satisfied EP(3)OS criteria of chronic rhinosinusitis (CRS), and 26.4% presented nasal polyps (NP). Smell detection, identification, and forced choice tests were significantly (p < 0.001) worse in bronchiectasis patients than healthy controls for both the 1st and 5th CN. Among subgroups, patients with CRS presented a significant (p < 0.05) reduction in smell detection compared to both healthy controls and patients without CRS. Patients with both CRS and NP presented a significant (p < 0.01) reduction in both smell detection and forced choice compared to patients with CRS and without NP. Patients with bronchiectasis and primary humoral immunodeficiency had a poorer smell detection (p < 0.001) and forced choice (p < 0.001) compared with post-infective and idiopathic bronchiectasis patients. CONCLUSIONS: Patients with bronchiectasis have a moderate loss of smell with a higher impairment in patients with CRS, being maximal in patients with NP. Patients with immunodeficiency bronchiectasis showed high prevalence of CRS, and therefore marked impairment on the sense of smell. The mechanism could be explained through a mixed ethiology (obstruction/inflammation).

Importance of glucocorticoid receptors in upper and lower airways.

Glucocorticoids (GCs) are the most common and effective drugs for treating inflammatory airway diseases, but some patients respond poorly to them. GC effects are mediated through the glucocorticoid receptor (GR). We present an update on the GR gene, the GR alpha and GR beta splicing variants, their translational and post-translational modifications, as well as their alterations in disease. GR alpha is ubiquitously expressed and is responsible for the induction and repression of target genes. GR beta acts as a dominant negative inhibitor of GR alpha-mediated transactivation and transrepression in certain cell types. The GR beta message is expressed at low levels in numerous tissues and its protein is only expressed in specific cell types. Increased GR beta expression has been reported in bronchial asthma, nasal polyposis and inflammatory bowel diseases (IBD), and after incubation of cells with certain proinflammatory stimuli. In addition to GR beta, other mechanisms explaining GC resistance include alterations in GR binding to ligand, nuclear translocation, and binding to GRE, and/or a defective cross-talk with transcription factors and cofactors.