Skin prick tests (SPT) were performed with a standard panel of 10 common airborne allergens (ALK, Copenhagen, Denmark) including pollen (birch, timothy and artemisia), house dust mites (D. Pteronyssimus and D. Farinae), molds (Cladosporium and Alternaria) and animal allergens (cat, dog and horse). SPT were performed on the volar side of the forearm with saline buffer as negative and histamine chloride (10 mg/ml) as positive controls. The diameter of the wheal reactions were measured after 20 min with a ruler.

The study included 11 patients (6 women) with symptomatic birch and/or grass pollen induced intermittent allergic rhinitis and 11 healthy volunteers (7 women), serving as controls. The mean age of patients and controls was 43 (26–55) and 41 (24–55) years, respectively. The diagnosis of birch and/or grass pollen induced allergic rhinitis was based on a positive history of intermittent allergic rhinitis for at least 2 years and positive SPT to birch and/or grass. All patients were classified as having severe symptoms (itchy nose and eyes, sneezing, nasal secretion and nasal blockage) during pollen season and they had all been treated with antihistamines and nasal steroids during pollen seasons previous years. Patients had no continuous symptoms of asthma and they did not take any asthma medication. All patients presented a wheal reaction diameter >3 mm towards birch or timothy in SPT (roughly correponding to a 3+ or 4+ reaction when compared with histamine 7). Exclusion criteria included a history of perennial symptoms, upper airway infection for the last 2 weeks before the time of visit and treatment with local or systemic corticosteroids during the last 2 months. The controls were all symptom-free, had no history of allergic rhinitis and had negative SPT to the standard panel of allergens as described above. They had no history of upper airway infection for 2 weeks before the time of visit and they were all free of medication. The study was approved by the Ethics Committee of the Medical Faculty, Lund University.

Nasal lavage fluid was collected during either birch pollen (9 patients) or grass pollen season (2 patients). Patients were included when they had experienced substantial symptoms of rhinoconjunctivitis (itchy nose and eyes, sneezing, nasal secretion and nasal blockage) during at least 3 consecutive days. The majority of the patients were seen within 5–10 days after the first appearance of symptoms and a local pollen count.

Nasal lavage fluid was collected according to a previously described method 8. After clearing of excess mucus from the nose sterile saline solution of room temperature was sprayed into both nostrils, respectively. The fluid was allowed to return passively and collected in a graded tube until 7 ml was recovered. NLFs were centrifuged at 1750 rpm at 4°C for 10 min to remove the cell content and the supernatants were stored at -70°C until sample preparation.

Before concentration of the samples NLFs were thawed and centrifuged at 12300 rpm at 4°C for 20 min to remove debris. Using Vivaspin 6 and Vivaspin 500 concentrators (Vivascience, Hannover, Germany) supernatants were concentrated and desalted. The protein concentration was determined using BCA Protein Assay Kit (Pierce Biotechnology, Rockford, USA) and resulted in a protein concentration of 1572–5625 μg/ml for healthy individuals and 1833–7867 μg/ml for allergic individuals. NLFs were stored at -70°C until analysed.

Samples were mixed with rehydration solution containing 8 M Urea, 2% CHAPS, 2.8 mg/ml DTT (Sigma-Aldrich, Steinheim, Germany), 0.5% IPG Buffer (pH 3–10) (Amersham Biosciences, Uppsala Sweden) and a small amount of bromophenol blue. For analytical gels 150 μg of protein was added to a final volume of 450 μl for each sample. For the preparative gels, one for healthy and one for allergic samples, 600 μg from a pool of samples was used. To be able to load as much as 600 μg on the preparative gels pooled samples were further concentrated using Microcon YM-3 (Millipore, Billerica, USA) before added to rehydration solution. Samples were incubated for approximately 15 min in room temperature in order to completely solubilize and denature the proteins. Samples were centrifuged at 13000 rpm for 10 min and thereafter loaded onto 24 cm 3–10 non linear IPG strips (Amersham Biosciences, Uppsala, Sweden). In-gel rehydration and isoelectric focusing (IEF) was performed over night (~60000 Vh) using Ettan IPGphor Isoelectric Focusing System (Amersham Biosciences, Uppsala, Sweden). After IEF strips were stored at -70°C until analysed. The IPG strips were equilibrated in SDS equilibration buffer (75 mM Tris, 6 M Urea, 30% glycerol, 2% SDS and 0.002% bromophenol blue (Sigma-Aldrich, Steinheim, Germany)) for 2 × 15 min. DTT (10 mg/ml) (Sigma-Aldrich, Steinheim, Germany) was added to the first and iodoacetamide (25 mg/ml) (Sigma- Aldrich, Steinheim, Germany) to the second equilibration step. After equilibration strips were loaded onto laboratory-made 12.5% acrylamide second dimension gels. SDS-PAGE was performed at constant effect (10 W/gel) for about 4 h and 30 min using the Ettan DALT II system (Amersham Biosciences).

Second dimension gels were fixed in 30% ethanol and 10% acetic acid over night, washed 4 × 30 min in 20% ethanol and stained with the fluorescent dye ruthenium II tris-bathophenantroline disulfonate (1 μM) for about 6 h. Thereafter gels were destained in 40% ethanol and 10% acetic acid over night and washed with double distilled water for about 4 × 30–60 min 9. All incubation and washing steps were performed with gentle agitation. Gels were kept dark in double distilled water at 4°C until scanned. The gels were automatically scanned using a robotic system together with a 9410 Typhoon scanner (488 nm laser) from Amersham Biosciences 10 and the gel images were analysed using the computer softwares Image master 2D Platinum (Amersham Biosciences) and Ludesi 2D Interpreter (Ludesi AB, Lund, Sweden). The volume in each spot was calculated as integrated optical density over the spot's area. The amount of protein in each spot was expressed as %VOL (ppm), that is the volume for the spot divided with the total volume for all spots in the gel.

Using the Ettan spot handling workstation (Amersham Biosciences) selected spots were automatically cut from the preparative gels, destained and enzymatically digested with trypsin (porcine Sequencing Grade Modified Trypsin, Promega, Madison, USA). The tryptic peptides were then spotted onto a MALDI target plate 11. The MALDI target plates were loaded in a Micromass M@ldi MALDI-TOF mass spectrometer (Waters, Milford, USA) for analysis of the peptide masses.

Peptide masses retrieved from MALDI-TOF analysis spectra were submitted to a database (IPI human 1.38) 12 by using the search engine PIUMS 13. The following matcher parameters were used: constant modification of cysteine by carbamidomethylation, variable modification of methionine by oxidation and maximum 1 missed cleavage for trypsin. A protein hit was considered significant if the PIUMS quality score was ≥ 4.7, which corresponds to an expectation value of 0.01. A search in IPI human 1.38 was also done using the search engine Mascot and the results from this search were compared with the results from PIUMS.

NLFs were mixed with SDS sample buffer, heated at 95–100°C for 5 min and centrifuged at 10 000 rpm for 10 min. Equal amounts of the samples were loaded onto NuPAGE Bis-Tris 4–12% gel (Invitrogen, Carlsbad, USA), separated by electrophoresis (Mini vertical gel system, Thermo EC, Waltham, USA), and blotted to Immobilon-P PVDF membranes (Millipore, Billerica, USA). Membranes were blocked in buffer 1 (Tris-HCl 10 mM pH 7.4, NaCl 0.9% and dry milk 5%) and then incubated overnight with primary antibody (1 μg/ml) against psoriasin, galectin-3 (Abcam, Cambridge, UK), Wnt-2B (Zymed, South San Francisco, USA) and alpha enolase (Santa Cruz, Santa Cruz, USA), respectively. Membranes were washed 2 times with buffer 1 followed by incubation for approximately 2 h with HRP conjugated secondary antibody (50 ng/ml). After 2 washes with buffer 2 (Tris-HCl 10 mM pH 7.4, NaCl 0.9% and Tween 20 0.05%) membranes were incubated for 5 min in SuperSignal West Pico solution (Pierce Biotechnology, Rockford, USA). The chemiluminescence was detected using MAN-X X-ray system (Fujifilm Science Imaging systems, USA). Developed films for quantitative analysis were scanned and analysed in ImageQuant (Molecular dynamics, Sunnyvale, USA). There were no antibodies available against hypothetical protein MGC33648 and the relevant fragment of intersectin-2. Hence, these proteins could not be assessed with western blot.

All values were expressed as mean values ± SEM. Statistical analysis of the protein expression was performed in Ludesi 2D interpreter (Ludesi AB) using one-way ANOVA.

Of the spots picked from the 2D-gels and submitted to MALDI-TOF analysis 61 spots were identified (figure 1 and table 1). 21 of these were identified as separate proteins. The majority of the proteins has previously been identified in NLF 5614 but this is the first study where psoriasin, galectin-3, alpha enolase, intersectin-2, Wnt-2B and hypothetical protein MGC33648 have been recognized. The occurrence of psoriasin, galectin-3, Wnt-2B and alpha enolase was confirmed with western blot (figure 2).

14 spots exhibited a clear difference in the protein content when the material from allergic and non-allergic individuals was compared (table 2). 8 of these spots were identified as different fragments of albumin and all these were up-regulated in allergic individuals (1.8- to 2.6-fold). Wnt-2B (splice isoform 1), transthyretin and Ig gamma-2 chain c region were also found to be up-regulated in allergic compared to non-allergic individuals (2.5-, 1.6- and 2.1-fold, respectively). In contrast, prolactin-inducible protein and two forms of psoriasin were found to be down-regulated in allergic individuals (2.0-, 2.0- and 3.4-fold, respectively). The psoriasin levels in nasal lavage fluids from three patients with allergic rhinitis and three controls were also assessed using western blot analysis (figure 3A). Quantitative analysis revealed reduced levels among the allergic patients; 19962 ± 5410 for the non-allergic individuals compared to 6834 ± 2258 for the allergic individuals (figure 3B).