Samples from ethmoidal and maxillary sinuses were collected during functional endoscopic sinus surgery (FESS) procedures in the Department of Otorhinolaryngology at the Ghent University Hospital. Nasal tissues were obtained from 13 patients with chronic rhinosinusitis and nasal polyposis (CRS-NP) (10 males, 3 females, age range: 30–54 years) and 11 subjects with chronic rhinosinusitis without nasal polyposis CRS (8 males, 3 females, age range: 21–53 years) who were scheduled for sinus surgery in the department of Othorinolaryngology of the Ghent University Hospital. As control group (Controls), we included 6 subjects (4 males, 2 females, age range: 21–53 years), who underwent septal surgery and removal of parts of the inferior turbinate due to anatomical variations. These patients had any acute or chronic clinical, endoscopic or imaging signs of sinusitis or polyposis; and they did not show any history of atopic, or lower airway disease.

Diagnosis of CRS was based on the presence of typical symptoms (headache, nasal obstruction and discoloured nasal drainage) longer than 12 weeks and a positive CT-Scan showing swelling of the ethmoidal and maxillary mucosa and bilateral obstruction of the osteomeatal complex but without polyp formation, visible during nasal endoscopy or during surgery. Nasal polyposis was diagnosed based on symptoms history (nasal congestion, lost of smell, changes in sense of taste and persistent postnasal drainage), clinical examination, nasal endoscopy and paranasal sinuses CT-Scan, defined as presence of visible bilateral polyps growing from the middle meatus into the nasal cavities, and affecting the ethmoidal sinuses. In the CRS-NP group, three patients had asthma and in the CRS group, there was one patient with allergic rhinitis and one with asthma. Diagnosis of asthma was based on clinical history, typical symptoms and lung (pulmonary) function tests (Spirometry and Peak Expiratory Flow), following the Global initiative for asthma (GINA) guidelines. All patients have taken aspirin or other NSAIDs without manifesting any hypersensitive reaction.

The study was approved by the ethical committee of the Ghent University Hospital, and all patients gave informed consent before their participation. The use of any oral medication with possible impact on measurements of enzymes or mediators, including systemic glucocorticoids and anti-leukotrienes, was stopped in all subjects 4 weeks before surgery. The use of topical glucocorticoids was interrupted 2 weeks before surgery.

Quantitative real time PCR was used to determine the mRNA levels of eicosanoid receptors. Nasal tissue (30 mg) was homogenized in Tri-reagent buffer (Sigma-Aldrich, MO, USA), 1 ml per 50–100 mg of tissue, in a chilled pestle mortar. Total RNA from homogenates was isolated using Tri-reagent Kit following the manufacturer's instructions (Sigma-Aldrich, MO, USA). cDNA was synthesized from 2 μg of total RNA using Oligo(dT)_12–18, random hexamers and the Superscript RNase H^-Reverse Transcriptase (Life Technologies, CA, USA), as described previously 16. Amplification reactions were performed on an iCycler iQ Real-Time PCR Detection System (Bio-Rad laboratories, CA, USA) using specific primers (Table 1), designed with the Primer3 software 17. PCR reactions contained 20 ng cDNA (total RNA equivalent) of unknown samples, 1X SYBR Green I Master mix (Bio-Rad laboratories, CA, USA) and 300 nM of primer pairs in a final volume of 25 μl. PCR protocol consisted of 1 cycle at 95°C for 10 minutes followed by 45 cycles at 95°C for 30 seconds and at 60°C for 1 minute. The expression of two housekeeping genes Beta actin (ACTB) and Hydroxymethyl-bilane synthase (HMBS) was used to normalize for transcription and amplification variations among samples. The relative number of molecules of each gene, expressed in relative expression units quantified per 20 ng of cDNA sample, was determined by the ΔCT value method as described previously 18.

Concentration of cysteinyl leukotrienes C₄/D₄/E₄ (LTC₄/D₄/E₄), prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) were measured by Enzyme Linked Immunoassays (ELISAs) purchased from Oxford BioMedicals (Oxford, USA). Sample extraction procedures for protein removal and eicosanoid stabilization were performed according to the provider's instructions. Briefly, nasal or sinus tissues were first homogenized in ethanol (5 ml/g) for LTB₄ and LTC₄/D₄/E₄, and in 15% methanol/0.1 M sodium phosphate buffer, pH 7.5 for PGE₂ measurements and then centrifuged for 5 minutes at 3,000 rpm at 4°C. Supernatants were diluted in water, pH 3.5 and following manufacturer's instructions (Oxford BioMedicals, Oxford, USA). The detection ranges for all assays were between 0.02–10 ng/ml. The sensitivity was of 0.2 ng/ml for all assays and the intra-and inter-assay coefficient of variation less than 10%.

Soluble IL-5Rα isoform was quantified using a real time PCR, as described previously 16. Briefly, a standard curve was constructed from a plasmid containing the cDNA sequence for this receptor isoform. A fragment of this plasmid was amplified with specific primers, purified and used in equimolar 10-fold dilutions to generate a standard curve. Real time amplifications were performed in a 25 μl volume reaction containing 1X SYBR Green I Master mix (Bio-Rad laboratories, CA, USA), 300 nM of primer pairs and a set of primers specific for this hIL-5Rα isoform 16. PCR protocol consisted of 1 cycle at 95°C for 10 minutes followed by 40 cycles at 95°C for 30 seconds and at 64°C for 1 minute. Each sample was tested in duplicate. The quantity of each amplicon was calculated from the values of the standard curve and normalized by the quantities obtained for beta actin (ACTB) and hydroxymethyl-bilane synthase (HMBS).

Soluble IL-5α receptor protein concentrations were measured by a research ELISA as described previously 19 with a sensitivity of 8 pg/ml. and an intra-and inter-assay coefficient of variation less than 10%. Quantification of Eosinophil Cationic Protein (ECP) was carried out, on supernatants obtained after nasal tissue homogenization, by the UniCAP system (Pharmacia & Upjohn, Sweden), with a detection limit of < 0.5 μg/L and a coefficient of variation less than 10%.

The number of activated eosinophils was determined by staining the eosinophil granulocyte (EG₂) and semiquantitative scoring of positively stained cells on the different tissues. For that, frozen tissue sections were fixed in acetone for 10 minutes, washed in TBS buffer and incubated with (1:1000) mouse anti-human ECP/EPX monoclonal antibody (Pharmacia & Upjohn Uppsala, Sweden) for 1 hour. Then, the slides were incubated with (1:50) rabbit anti-mouse IgG for 10 minutes and developed with (1:100) alkaline phosphatase anti-alkaline phosphatase (Dako, Glostrup, Denmark) for 10 minutes at room temperature. Signal detection was performed using the New Fuchsin Substrate System, following the manufacture's instructions (Dako, Glostrup, Denmark). Semiquantitative scoring was performed by a pathologist, who was blinded for the clinical data, on a four-point scale adapted from an already validated system of semiquantitative evaluation. Zero represented the lowest and three the highest score. The analysis included all areas of the biopsies and a global score was given for each parameter.

Frozen tissue sections were fixed in acetone for 10 minutes at room temperature and washed in 1X TBS buffer. Endogenous peroxidase activity was blocked with 0.3 % hydrogen peroxidase (VWR International, Pennsylvania, USA) in PBS containing 0,001 % NaN₃ for 20 minutes at room temperature. Sections were than washed for 10 minutes with 1X TBS and incubated with foetal bovine serum during 30 minutes. Sections were then incubated for 1 hour at room temperature with primary antibodies: rabbit IgG polyclonal Antibodies for EP₁ receptor (1:250), EP₂ receptor (1:250), EP₃ receptor (1:200), EP₄ receptor (1:250), CysLT₁ (1:50) and CysLT₂ (1:50), purchased by Cayman Chemicals (MI, USA). Signal was detected with LSAB⁺ kit (HRP Rabbit/Mouse/Goat) purchased from Dako, using the AEC⁺ High Sensitivity Substrate Chromogen Kit (Dako, Glostrup, Denmark). To control for unspecific binding of the primary antibodies used in the study, parallel stainings were performed omitting the primary antibody and by substituting it with an irrelevant antibody or non-immune sera of the same species (isotype), at the same concentration as the specific antibody (antisera).

All data was analyzed using the MedCalc software version 6.0 (Mariakerke, Belgium). Results are presented in Box-and-Whisker plots, where the central box represent the values from the lower and upper interquartile range, and the middle line the median. Data comparison within different patient groups was performed using the Kruskal-Wallis test (H-test). The Wilcoxon test (or Mann-Whitney U test) for unpaired samples was applied to evaluate the statistical differences between two patient groups. Spearman's rank correlation analysis was performed to determine statistical significance of differences between two parameters in a classification group. P values equal or less than 0.05 was regarded as significant.

Expression of leukotrienes and prostanoid receptors analyzed by quantitative real time PCR showed an up-regulation of CysLT₁ and CysLT₂ receptors in CRS patients compared to controls, but only CysLT₁ was significantly higher in CRS-NP in comparison to CRS patients as showed in Figure 1a. EP₂ and EP₄ mRNA was up-regulated in both CRS groups when compared with normal subjects (Figure 1b). In contrast, EP₁ and EP₃ expression was similar in controls and CRS patients but significantly down-regulated in the CRS-NP group (Figure 1b). Concentrations of BLT₁ and BLT₂ receptors were similar in the three groups of patients (data not shown).

Expression profile for the four EP and the two cysLTs receptors analyzed in each individual group of patients is illustrated in Figures 2 and 3. In the control group, expression of EP receptors did not show any differences. However, in the CRS and EP₁ and EP₃ receptors were down-expressed compared to EP₂ and EP₄ and this difference was more accentuated in the CRS-NP subjects. On the other hand CysLT₁ and CysLT₂ mRNA levels were similar in CRS and normal mucosa, however significantly higher concentrations of CysLT₁ compared to CysLT₂ were observed in the CRS-NP group.

Levels of eicosanoids and eosinophilic inflammatory markers are summarized in table 2. While concentrations of LTC₄/D₄/E₄, were significantly higher in CRS-NP compared to CRS and controls, no differences were observed in LTB₄ levels between the groups. PGE₂ concentrations however, were similar in CRS and control (p > 0.10) but statistically lower in the nasal polyp tissue (p = 0.001). Real time PCR for sol-IL-5α R showed a significantly increase in CRS-NP subjects compared to CRS and in CRS compared to control subjects. Accordingly, sol-IL-5Rα protein was also statistically higher in the CRS-NP compared to CRS and in this group compared to control tissue. ECP was significantly increased in CRS compared to the control group (p < 0.05) and even more in CRS-NP (p < 0.02).

Immunohistochemistry results demonstrated a strong infiltration of inflammatory cells in the nasal polyp compared to the CRS and inferior turbinate tissues (data not shown) and the median score for EG₂ positive cells was significantly higher in NP tissue compared to the control and CRS tissues as summarized in Table 2.

The Spearman's rank correlation analysis showed a strong correlation between both cysLTs receptors mRNA with sol-IL-5Rα protein concentrations (CysLT₁: rho = 0,574, p = 0.01; CysLT₂: rho = 0,523; p < 0.05), ECP (CysLT₁: rho = 0,544, p = 0.02; CysLT₂: rho = 0,413; p = 0.03) and the total number of activated eosinophils (CysLT₁: rho = 0,546, p = 0.02; CysLT₂: rho = 0,614; p = 0.03). No correlations were found between the levels of prostanoid receptors and eosinophilic inflammation markers.

Immunohistochemical staining for CysLT₁ and CysLT₂ receptors in the nasal tissue is represented in Figure 3. Immunoreactivity of both cysLTs receptors was observed in inflammatory cells in the lamina propria in both CRS groups. In addition, these receptors were expressed in the sub-epithelial layer of the nasal mucosa and to a lesser extend in the epithelium. Prostanoid-E receptors were mainly expressed in the epithelium and in mucosal glands (Figure 4). Immunoreactivity for EP₂ and EP₄ was higher in inflammatory cells compared to epithelium, contrary to EP₁ and EP₃ staining, which was localized mainly in epithelial and sub-epithelial regions and blood vessels.