Jordi Roca-Ferrer

Comparative study of the effects of different glucocorticosteroids on eosinophil survival primed by cultured epithelial cell supernatants obtained from nasal mucosa and nasal polyps.

BACKGROUND--Supernatants from epithelial cell cultures enhance eosinophil survival in vitro, this effect being abrogated by previous incubation of eosinophils with glucocorticosteroids. This property has resulted in the development of an in vitro test to compare the potency of these drugs. A comparative study was performed with dexamethasone, methylprednisolone, deflazacort, and budesonide. METHODS--Human epithelial cell conditioned media (HECM) was generated from cultured epithelial cells obtained from healthy nasal mucosa and polyps. Eosinophils isolated from the peripheral blood were incubated with different corticosteroids for one hour before the addition of HECM. The inhibitory potency of the four steroids on the eosinophil survival index was compared using the concentration of steroid causing 50% inhibition (IC50). RESULTS--Eosinophil survival was increased by HECM from both healthy nasal mucosa and polyps. All four steroids blocked HECM-induced eosinophil survival in a dose-dependent manner. On healthy nasal mucosa methylprednisolone was the least potent (IC50 = 536 nM), deflazacort (IC50 = 264 nM) was twice as potent as methylprednisolone, while budesonide and dexamethasone were approximately nine times as potent (both IC50 = 58 nM). When potency was evaluated on the promoting effects of the HECM obtained from nasal polyps, the inhibitory potencies were lower and consequently the IC50 values were higher when compared with HECM generated from healthy nasal mucosa: methylprednisolone (IC50 = 546 nM), deflazacort (IC50 = 390 nM), dexamethasone (IC50 = 76 nM), and budesonide (IC50 = 78 nM). CONCLUSIONS--The potencies of glucocorticosteroids can be compared by evaluating their effects on the survival of eosinophils previously primed by supernatants obtained from epithelial cell culture. The different effects of steroids on eosinophils primed by HECM obtained from healthy nasal mucosa compared with HECM obtained from nasal polyps suggest that polyps might represent more active tissue which is relatively resistant to treatment with corticosteroids.

Nuclear factor‐κB activity is down‐regulated in nasal polyps from aspirin‐sensitive asthmatics

Background: We examined whether a decreased activity of nuclear factor(NF)‐κB), a transcriptional regulator of cyclooxygenase‐2 (COX‐2), could account for down‐regulation of COX‐2 in nasal polyps of aspirin‐sensitive asthmatics.

Regulation of glucocorticoid receptor in nasal polyps by systemic and intranasal glucocorticoids

Background:  Poor response of nasal polyps to glucocorticoids (GCs) may be because of abnormal expression of GC receptors (GR) α and β or to downregulation of GRα. We aimed to evaluate the in vivo regulation of GR isoforms in GC‐treated nasal polyps and to assess the relationship between clinical response to GCs and GR levels.

Upregulation of COX‐1 and COX‐2 in nasal polyps in cystic fibrosis

Background: Since abnormalities in prostanoid metabolism occur in the lower airway of patients with cystic fibrosis (CF), it is likely that they could also be detected in the nose. Methods: The degree of mRNA and protein expression of cyclo-oxygenase (COX) enzymes 1 (COX-1) and 2 (COX-2) was examined using quantitative reverse competitive polymerase chain reaction (RT-PCR) and Western blot analysis in the nasal polyps from 10 patients with CF, nasal polyps from 10 non-CF patients and 11 nasal mucosa specimens. The results are presented as 106 cDNA molecules/μg total RNA and the densitometric ratio between protein and β-actin. Results: COX-1 mRNA levels were significantly higher in CF nasal polyps (median 2.34, 25–75th percentiles 1.6–3.2) than in the nasal mucosa (0.78, 0.11–1.21), while there was no difference with non-CF nasal polyps (1.11, 0.80–3.15). COX-1 protein levels were significantly higher in CF nasal polyps (3.63, 2.71–4.27) than in nasal mucosa (1.55, 0.66–2.33) and non-CF nasal polyps (2.19, 1.72–3.68). COX-2 mRNA was significantly higher in CF nasal polyps (3.34, 2.42–7.05) than in nasal mucosa (1.69, 0.19–3.50). No differences were found in COX-2 mRNA expression between CF and non-CF polyps (1.38, 0.12–6.07). COX-2 protein levels were also significantly higher in CF nasal polyps (0.23, 0.04–0.34) than in non-CF nasal polyps (0.011, 0.009–0.016) or nasal mucosa (0.014, 0.014–0.016). Conclusions: Upregulation in the expression of COX-1 and COX-2 could explain the high production of prostanoids reported in CF. These findings raise questions regarding the potential use of selective or non-selective COX-2 non-steroidal anti-inflammatory treatment in CF.

Expression of glucocorticoid receptors α and β in steroid sensitive and steroid insensitive interstitial lung diseases

Background: Sensitivity to glucocorticoids may be related to the concentration of glucocorticoid receptors α (GRα) and β (GRβ). A study was undertaken to assess GRα and GRβ expression in steroid insensitive interstitial lung disease (idiopathic pulmonary fibrosis (IPF)) and steroid sensitive interstitial lung diseases (sarcoidosis and cryptogenic organising pneumonia (COP)). Methods: Lung tissue was obtained from control subjects and from patients with IPF, sarcoidosis, and COP. Pulmonary function tests were carried out at the time of lung biopsy and every 3 months. GRα and GRβ expression was evaluated by both competitive RT-PCR and immunohistochemistry. Data are presented as median and 25–75th percentile. Results: GRα mRNA expression (105 cDNA copies/µg total RNA) was higher in patients with steroid sensitive interstitial lung diseases (10.0; 7.8–14.9; n = 11) than in patients with IPF (4.4; 3.2–6.6; n = 19; p<0.001). GRβ expression was at least 1000 times lower than that of GRα and did not differ between the three groups. A negative correlation was found between GRα mRNA levels and the fibrotic pathology score of the tissue (r = −0.484, p<0.01) and a positive correlation was found between GRα mRNA levels and improvement in forced vital capacity (r = 0.633; p<0.01) after treatment of patients with glucocorticoids. Immunoreactivity for GR protein was also higher in patients with sarcoidosis and COP than in those with IPF. Conclusion: The variable response of some interstitial lung diseases to steroid treatment may be the result of differences in the expression of GRα.

Corticosteroid therapy increases membrane-tethered while decreases secreted mucin expression in nasal polyps

Background:  Mucus hypersecretion is a hallmark of nasal polyposis (NP). Corticosteroids (CS) are first‐line treatment for NP, decreasing their size and inflammatory component. However, their effect on mucin production is not well‐understood. The aim of this (pilot) study was to investigate CS effect on mucin expression in NP.

Subcutaneous prostaglandin E2 restrains airway mast cell activity in vivo and reduces lung eosinophilia and Th2 cytokine overproduction in house dust mite-sensitive mice.

Background: Prostaglandin (PG) E2 is thought to exert protective effects in the lungs. Accordingly, aerosolized PGE2 prevents the experimentally induced airway response to allergen challenge in asthmatics. In vitro evidence indicating that functional PGE2 receptors (EP) are expressed on human mast cells and that PGE2 can alter cytokine production suggests that these phenomena may be involved in its beneficial effect in asthma. However, in vivo evidence is scarce. Methods: We assessed the effects of exogenous PGE2 and of the EP1/EP3 agonist sulprostone on the murine airway response to house dust mite (HDM) allergens, a model that accurately reproduces the spontaneous exposure of allergic asthma patients to aeroallergens. We also analyzed the in vivo impact of PGE2 on production in the murine airway of mast cell protease (mMCP)-1, a specific marker of lung mast cell activity, and on local production of cytokines. Results: Exogenous PGE2, but not sulprostone, reduced eosinophilic infiltration in HDM-sensitized mice by half and led to a strong reduction in airway Th2 cytokine expression. These anti- inflammatory effects were accompanied in vivo by a substantial reduction in HDM-induced upregulation of airway mMCP-1. Neither PGE2 nor sulprostone had any effect on airway hyperresponsiveness to methacholine. Conclusions: Our results indicate that the anti-inflammatory effect of PGE2 can be reproduced in vivo in HDM-sensitized mice and suggest that this protective effect is dependent in vivo on inhibition of the allergen-triggered proinflammatory activity of bronchial mast cells. Finally, the effect of PGE2 is linked to reduced upregulation of airway Th2 cytokines.

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-β1 and FMT and EMT markers were evaluated. COX-2 and α-SMA expression, PGE2 secretion and cell proliferation were measured after IL-1β and PGE2 incubation. Results Myofibroblasts from both control and IPF fibroblast cultures stimulated with IL-1β showed no COX-2 expression. IPF fibroblasts showed increased myofibroblast population and reduced COX-2 expression in response to IL-1β. TGF-β1 increased the number of myofibroblasts in a time-dependent manner. In contrast, TGF-β1 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-β1 for 72 h showed diminished COX-2 induction, PGE2 secretion and α-SMA expression after IL-1β addition. The latter decreased proliferation in fibroblasts but not in myofibroblasts. A549 cells incubated with TGF-β1 for 72 h showed down-regulated COX-2 expression and low basal PGE2 secretion in response to IL-1β. 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.

Regulation of ICAM-3 and other adhesion molecule expressions on eosinophils in vitro. Effects of dexamethasone

Background: ICAM‐3 has been recently identified as the third leukocyte‐function associated‐1 (LFA‐1) ligand. ICAM‐3 is expressed in eosinophils, but its regulation has not been studied. The objective of this study was to investigate the differential expression of ICAM‐3 and other adhesion molecules (AM) on the surface of eosinophils. We also evaluated the effects of dexamethasone on AM expression.

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.