2.1

Mice

Female BALB/c mice (6–8 weeks) were purchased from the Chinese Academy of Sciences Shanghai Laboratory Animal Center. These mice were maintained in horizontal laminar flow cabinets and provided sterile food and water in a specific pathogen-free facility. All animals were handled in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The mice were randomly divided into 4 experimental groups (n = 12 for each group).

2.2

Nasal allergic model and 2-APB administration

According to published procedures , mice were administered 0.5 mg/mL ovalbumin (grade 5; Sigma-Aldrich, St Louis, MO) and 20 mg/mL aluminum hydroxide (Sinopharm Chemical Reagent Co, Ltd, Shanghai, China) in saline at a dosage of 0.2 mL per mouse by intraperitoneal injection. The sensitization was repeated 3 times at weekly intervals (days 1, 8, and 15) followed by daily injections of ovalbumin solution (40 mg/mL in saline) into the nostrils (0.02 mL per mouse) on days 22 to 29 (challenge) ( Fig. 1 ). Some groups of mice were administered 30 or 50 μ mol/L (0.02 mL per mouse) of 2-APB (Sigma-Aldrich) in saline into the nostril daily on challenging days (2-APB–treated mice), whereas another group of mice was not treated with it (control mice). As a negative control, a group of mice received neither sensitization nor challenge treatment except for the last challenge on day 29 (normal mice). Nasal symptoms were evaluated by counting number of sneezes and nasal rubbing frequency during 10 minutes immediately after the last ovalbumin intranasal provocation on day 29.

Protocols. Allergic rhinitis mice models were prepared by intraperitoneal immunization on days 1, 8, and 15 (priming) followed by daily intranasal challenge on days 22 to 29. Some groups of mice were treated with daily intranasal 2-APB on days 22 to 29. Normal mice were given only the last intranasal challenge on day 29.

2.3

Sample preparation

After mice were killed, the fore teeth were cut off and the lower jaw and cheek muscles were removed. Nasal-associated lymphoid tissue localized bilaterally on the posterior side of the palate was exposed by carefully peeling away the palate and was teased out with syringe needles. Nasal mucosa of each mouse was also obtained. These tissues were cut into 2 portions: one was dissected for protein isolation and the other was for mRNA extraction. All of them were frozen at −70°C.

2.4

Nasal lavage fluid

After mice were killed, 1 blunted 18-gauge needle was pointed toward the heads of a portion of mice for NLF. One injection of 1000- μ L phosphate-buffered saline (PBS) was performed, and the fluid was collected with a tube below both nares of the nose for NLF. The NLF was centrifuged for 10 minutes at 150 g at 4°C. The supernatants were stored at −70°C for LTC4, ECP, ovalbumin-specific IgE, and IL-4 assays.

2.5

Histologic examination

Twelve hours after the final nasal challenge, the heads of a portion of mice were excised and immersed in 10% neutral buffered formalin. After fixation, these heads were decalcified in 8.8% formic acid for 6 days. The specimens were embedded in paraffin, coronally sectioned at a thickness of 4 μ m, and placed onto glass slides. Each slide was allowed to dry in a 37°C incubator overnight and then baked at 60°C for 30 minutes. The tissues were deparaffinized through xylenes, rehydrated in graded alcohols, and rinsed in distilled water before the staining protocols were performed. They were then dehydrated in graded alcohols, cleared in xylene, and coverslipped. The resultant coronal nasal sections were visualized with hematoxylin/eosin (H&E) or Luna stains.

For the H&E stain, sections were immersed in Harris hematoxylin (Thermo Scientific, Waltham, MA) for 5 minutes and then rinsed in tap water. Subsequently, the sections were placed in 0.5% acid alcohol for 5 seconds, rinsed in tap water, blued in 1% ammonia, rinsed again in tap water, and rinsed a final time in deionized water. Finally, the sections were immersed in an alkaline eosin solution (pH 4.4) for 20 seconds.

For the Luna stain, sections were immersed in working Hematoxylin-Biebrich (Sigma-Aldrich and Acros, Geel, B, Belgium, respectively) scarlet solution for 5 minutes, then dipped in 1% acid alcohol approximately 8 times, and rinsed in tap water. Then, the sections were dipped in lithium carbonate solution approximately 5 times until sections turned blue and then washed in running tap water for 2 minutes. Luna stain is specific for eosinophils and renders their cytoplasm red-brown on a blue background . The number of infiltrating eosinophils was determined microscopically in a blinded manner at a high-power field of ×400 magnification. The mouse nose has been well characterized , with squamous epithelium in the anterior part, followed by ciliated respiratory epithelium lining the midportion, and then a thick layer of neuroepithelial olfactory epithelium over the dorsoposterior regions of the nasal cavity . Thus, we distinguished olfactory epithelium easily from respiratory epithelium according to the localized region and thickness histologically and selected the latter as the observed object.

2.6

Western blotting

A portion of frozen tissues were sliced into small pieces and homogenized, then were lysed in Mouse Tissue Extract Protein Array Kit (Sigma-Aldrich), and stored at −70°C. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Sigma-Aldrich). The membrane was then blocked for 30 minutes in Tris-buffered saline with 0.2% (vol/vol) Tween 20 and 5% (wt/vol) nonfat dry milk, and incubated at 4°C overnight with primary antibody: Orai1-L1 rabbit polyclonal antibody (NewEast Biosciences, Malvern, PA) at a dilution of 1:1000. After incubation, the membrane was washed 3 times for 5 minutes in TBS–Tween 20 buffer and then treated with peroxidase-conjugated secondary antimouse IgG antibody for 1 hour at room temperature. The membrane was washed, and the antigen-antibody complexes were detected using Chemiluminescent Substrate (Sigma-Aldrich) according to the manufacturer’s instructions and exposed to x-ray film for 10 seconds. β -Actin expression was analyzed using the β -actin antibody (Sigma-Aldrich).

2.7

Enzyme-linked immunosorbent assay

The concentration of ovalbumin-specific IgE from NLF was detected using enzyme-linked immunosorbent assay (ELISA). For measurement, a sheep polyclonal antibody to mouse IgE (Serotec Ltd, Oxford, United Kingdom) was diluted with PBS (137-mmol/L sodium chloride, 8.1-mmol/L disodium hydrogen phosphate, 2.7-mmol/L potassium chloride, and 1.5-mmol/L potassium dihydrogen phosphate, pH 7.4) to 10 μ g/mL. Then, 100 μ L of the previously mentioned diluted antibody was soaked into each well of a 96-well microplate (Corning Inc, Corning, NY), and the microplate was stored overnight at 4°C. The microplate was washed 3 times with PBS and was coated with 0.5% casein-PBS for 3 hours at room temperature. After the microplate was washed with PBS, 100- μ L diluted mouse NLF and standard positive sample were added to each well, and the microplate was incubated overnight at 4°C. After the plate was washed 4 times with PBS, ovalbumin that had been biotinylated using a Biotinylation Kit (American Qualex International Inc, San Clemente, CA) was diluted with 0.5% casein-PBS and 100 μ l of it was added to each well. Then, the plate was incubated for 2 hours at room temperature. After the washing of the plate 5 times with PBS, 100 μ L of streptavidin-peroxidase (Sigma Chemical Co, St Louis, MO) was diluted to 0.5 μ g/mL by 0.5% casein-PBS and was added to each well and incubated for 1 hour at room temperature. After the plate was washed 5 times with 0.1% Tween 20 in PBS, 100 μ L of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (600 μ g/mL ABTS, 0.006% H ₂ O ₂ in 0.2-m citric acid buffer, pH 4.7; Sigma-Aldrich) was added to the wells. After incubation for 3 hours at 37°C in the dark, absorbance at 450 nm was measured using a microplate reader (Microplate Reader MTP-32; Corona Electric, Ibaraki, Japan). The value of OD ₄₅₀ was used for the specific absorbance from ABTS, was calculated by comparisons of optical density of the test sample to standard sample, and was shown in arbitrary units.

Concentrations of LTC4, ECP, and IL-4 from NLF were evaluated using corresponding ELISA kits purchased from Ever Systems Biology Laboratory, Inc, Sacramento, CA; Panpacific Tech Co, Missouri City, TX; and Ever Systems Biology Laboratory, respectively. The ELISAs were performed in accordance with the manufacturers’ protocols.

2.8

Real-time reverse transcription polymerase chain reaction

Total RNA of a portion of samples was extracted with Trizol (Invitrogen, Carlsbad, CA) and treated with RNase-free DNase. For reverse transcription, 2 μ g of the previously mentioned RNA was reversely transcribed with random hexamers (Invitrogen), and complementary DNA (cDNA) was amplified according to the manufacturer’s instructions. Primers were designed using Primer Express Software (Applied Biosystems, Foster City, CA) from sequence available in GenBank and were synthesized (Geneland Biotech, Shanghai, China). Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect mRNAs of Orai1, LTC4 synthase (LTC4S), ECP (eosinophil-associated, ribonuclease A family, member 3 [EAR3]), germline C ɛ transcripts, and IL-4 mRNA. Orai1 primers were forward primer 5′-TTTGCCCTCATGATCAGCAC-3′ and reverse primer 5′-TTGGCGACGATGACTGATTC-3′. Leukotriene C4 synthase primers were forward primer 5′-CCTACAGGTGATCTCTGCACGA-3′ and reverse primer 5′-TATCCCTGGAAATAGCGGAGG-3′. Eosinophil-associated, ribonuclease A family, member 3 primers were forward primer 5′-TAATGCTTGCCTCATGCCTG-3′ and reverse primer 5′-TGCACAAGCCACTTGGATTT-3′. Germline C ɛ transcript primers were forward primer 5′-TGCATTGAAGGCTACGGCTA-3′ and reverse primer 5′-TTGGAGCCATTGGATTTCAG-3′. Interleukin-4 primers were forward primer 5′-ATCGGCATTTTGAACGAGGT-3′ and reverse primer 5′-TCGAAAAGCCCGAAAGAGTC-3′. Glyceraldehyde-3-phosphate dehydrogenase mRNA was also examined to control the sample-to-sample variation in RNA isolation and integrity by using a pair of primers: forward primer 5′-ACCACAGTCCATGCCATCAC-3′ and reverse primer 5′-TCCACCACCCTGTTGCTGTA-3′. After initial denaturation at 95°C for 10 minutes, the amplification profile was 15 seconds of denaturation at 95°C and 1 minute of annealing and extension at 60°C for 45 cycles. Negative control RT reaction mixtures contained no reverse transcriptase and no cDNA in the PCR amplification mixtures. For measurement, 2 μ L of diluted cDNA was amplified in a total reaction volume of 20 μ L by using a 7500 real-time PCR System (Applied Biosystems, Foster City, CA) with 20× SYBR Green mixture (Invitrogen). Specificity of PCR products was evaluated by melting curve analysis and by size in agarose gels. Using 3 dilutions of cDNA, linearity of PCR amplification was controlled. Evaluation of data was performed using the cycle threshold (ΔCT) method with glyceraldehyde-3-phosphate dehydrogenase as internal standard.

2.9

Statistical analysis

Statistical analysis was performed using a commercially available statistical software prism 4.0 (GraphPad Software Inc, San Diego, CA). Data were analyzed with the unpaired Student t test ± Welch’s correction and presented as mean ± standard error of the mean (SEM). Significance of a difference was accepted at the 5% level of confidence. P < .05 was considered statistically significant.

2.1

Mice

Female BALB/c mice (6–8 weeks) were purchased from the Chinese Academy of Sciences Shanghai Laboratory Animal Center. These mice were maintained in horizontal laminar flow cabinets and provided sterile food and water in a specific pathogen-free facility. All animals were handled in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The mice were randomly divided into 4 experimental groups (n = 12 for each group).

2.2

Nasal allergic model and 2-APB administration

According to published procedures , mice were administered 0.5 mg/mL ovalbumin (grade 5; Sigma-Aldrich, St Louis, MO) and 20 mg/mL aluminum hydroxide (Sinopharm Chemical Reagent Co, Ltd, Shanghai, China) in saline at a dosage of 0.2 mL per mouse by intraperitoneal injection. The sensitization was repeated 3 times at weekly intervals (days 1, 8, and 15) followed by daily injections of ovalbumin solution (40 mg/mL in saline) into the nostrils (0.02 mL per mouse) on days 22 to 29 (challenge) ( Fig. 1 ). Some groups of mice were administered 30 or 50 μ mol/L (0.02 mL per mouse) of 2-APB (Sigma-Aldrich) in saline into the nostril daily on challenging days (2-APB–treated mice), whereas another group of mice was not treated with it (control mice). As a negative control, a group of mice received neither sensitization nor challenge treatment except for the last challenge on day 29 (normal mice). Nasal symptoms were evaluated by counting number of sneezes and nasal rubbing frequency during 10 minutes immediately after the last ovalbumin intranasal provocation on day 29.

Protocols. Allergic rhinitis mice models were prepared by intraperitoneal immunization on days 1, 8, and 15 (priming) followed by daily intranasal challenge on days 22 to 29. Some groups of mice were treated with daily intranasal 2-APB on days 22 to 29. Normal mice were given only the last intranasal challenge on day 29.

2.3

Sample preparation

After mice were killed, the fore teeth were cut off and the lower jaw and cheek muscles were removed. Nasal-associated lymphoid tissue localized bilaterally on the posterior side of the palate was exposed by carefully peeling away the palate and was teased out with syringe needles. Nasal mucosa of each mouse was also obtained. These tissues were cut into 2 portions: one was dissected for protein isolation and the other was for mRNA extraction. All of them were frozen at −70°C.

2.4

Nasal lavage fluid

After mice were killed, 1 blunted 18-gauge needle was pointed toward the heads of a portion of mice for NLF. One injection of 1000- μ L phosphate-buffered saline (PBS) was performed, and the fluid was collected with a tube below both nares of the nose for NLF. The NLF was centrifuged for 10 minutes at 150 g at 4°C. The supernatants were stored at −70°C for LTC4, ECP, ovalbumin-specific IgE, and IL-4 assays.

2.5

Histologic examination

Twelve hours after the final nasal challenge, the heads of a portion of mice were excised and immersed in 10% neutral buffered formalin. After fixation, these heads were decalcified in 8.8% formic acid for 6 days. The specimens were embedded in paraffin, coronally sectioned at a thickness of 4 μ m, and placed onto glass slides. Each slide was allowed to dry in a 37°C incubator overnight and then baked at 60°C for 30 minutes. The tissues were deparaffinized through xylenes, rehydrated in graded alcohols, and rinsed in distilled water before the staining protocols were performed. They were then dehydrated in graded alcohols, cleared in xylene, and coverslipped. The resultant coronal nasal sections were visualized with hematoxylin/eosin (H&E) or Luna stains.

For the H&E stain, sections were immersed in Harris hematoxylin (Thermo Scientific, Waltham, MA) for 5 minutes and then rinsed in tap water. Subsequently, the sections were placed in 0.5% acid alcohol for 5 seconds, rinsed in tap water, blued in 1% ammonia, rinsed again in tap water, and rinsed a final time in deionized water. Finally, the sections were immersed in an alkaline eosin solution (pH 4.4) for 20 seconds.

For the Luna stain, sections were immersed in working Hematoxylin-Biebrich (Sigma-Aldrich and Acros, Geel, B, Belgium, respectively) scarlet solution for 5 minutes, then dipped in 1% acid alcohol approximately 8 times, and rinsed in tap water. Then, the sections were dipped in lithium carbonate solution approximately 5 times until sections turned blue and then washed in running tap water for 2 minutes. Luna stain is specific for eosinophils and renders their cytoplasm red-brown on a blue background . The number of infiltrating eosinophils was determined microscopically in a blinded manner at a high-power field of ×400 magnification. The mouse nose has been well characterized , with squamous epithelium in the anterior part, followed by ciliated respiratory epithelium lining the midportion, and then a thick layer of neuroepithelial olfactory epithelium over the dorsoposterior regions of the nasal cavity . Thus, we distinguished olfactory epithelium easily from respiratory epithelium according to the localized region and thickness histologically and selected the latter as the observed object.

2.6

Western blotting

A portion of frozen tissues were sliced into small pieces and homogenized, then were lysed in Mouse Tissue Extract Protein Array Kit (Sigma-Aldrich), and stored at −70°C. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Sigma-Aldrich). The membrane was then blocked for 30 minutes in Tris-buffered saline with 0.2% (vol/vol) Tween 20 and 5% (wt/vol) nonfat dry milk, and incubated at 4°C overnight with primary antibody: Orai1-L1 rabbit polyclonal antibody (NewEast Biosciences, Malvern, PA) at a dilution of 1:1000. After incubation, the membrane was washed 3 times for 5 minutes in TBS–Tween 20 buffer and then treated with peroxidase-conjugated secondary antimouse IgG antibody for 1 hour at room temperature. The membrane was washed, and the antigen-antibody complexes were detected using Chemiluminescent Substrate (Sigma-Aldrich) according to the manufacturer’s instructions and exposed to x-ray film for 10 seconds. β -Actin expression was analyzed using the β -actin antibody (Sigma-Aldrich).

2.7

Enzyme-linked immunosorbent assay

The concentration of ovalbumin-specific IgE from NLF was detected using enzyme-linked immunosorbent assay (ELISA). For measurement, a sheep polyclonal antibody to mouse IgE (Serotec Ltd, Oxford, United Kingdom) was diluted with PBS (137-mmol/L sodium chloride, 8.1-mmol/L disodium hydrogen phosphate, 2.7-mmol/L potassium chloride, and 1.5-mmol/L potassium dihydrogen phosphate, pH 7.4) to 10 μ g/mL. Then, 100 μ L of the previously mentioned diluted antibody was soaked into each well of a 96-well microplate (Corning Inc, Corning, NY), and the microplate was stored overnight at 4°C. The microplate was washed 3 times with PBS and was coated with 0.5% casein-PBS for 3 hours at room temperature. After the microplate was washed with PBS, 100- μ L diluted mouse NLF and standard positive sample were added to each well, and the microplate was incubated overnight at 4°C. After the plate was washed 4 times with PBS, ovalbumin that had been biotinylated using a Biotinylation Kit (American Qualex International Inc, San Clemente, CA) was diluted with 0.5% casein-PBS and 100 μ l of it was added to each well. Then, the plate was incubated for 2 hours at room temperature. After the washing of the plate 5 times with PBS, 100 μ L of streptavidin-peroxidase (Sigma Chemical Co, St Louis, MO) was diluted to 0.5 μ g/mL by 0.5% casein-PBS and was added to each well and incubated for 1 hour at room temperature. After the plate was washed 5 times with 0.1% Tween 20 in PBS, 100 μ L of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (600 μ g/mL ABTS, 0.006% H ₂ O ₂ in 0.2-m citric acid buffer, pH 4.7; Sigma-Aldrich) was added to the wells. After incubation for 3 hours at 37°C in the dark, absorbance at 450 nm was measured using a microplate reader (Microplate Reader MTP-32; Corona Electric, Ibaraki, Japan). The value of OD ₄₅₀ was used for the specific absorbance from ABTS, was calculated by comparisons of optical density of the test sample to standard sample, and was shown in arbitrary units.

Concentrations of LTC4, ECP, and IL-4 from NLF were evaluated using corresponding ELISA kits purchased from Ever Systems Biology Laboratory, Inc, Sacramento, CA; Panpacific Tech Co, Missouri City, TX; and Ever Systems Biology Laboratory, respectively. The ELISAs were performed in accordance with the manufacturers’ protocols.

2.8

Real-time reverse transcription polymerase chain reaction

Total RNA of a portion of samples was extracted with Trizol (Invitrogen, Carlsbad, CA) and treated with RNase-free DNase. For reverse transcription, 2 μ g of the previously mentioned RNA was reversely transcribed with random hexamers (Invitrogen), and complementary DNA (cDNA) was amplified according to the manufacturer’s instructions. Primers were designed using Primer Express Software (Applied Biosystems, Foster City, CA) from sequence available in GenBank and were synthesized (Geneland Biotech, Shanghai, China). Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to detect mRNAs of Orai1, LTC4 synthase (LTC4S), ECP (eosinophil-associated, ribonuclease A family, member 3 [EAR3]), germline C ɛ transcripts, and IL-4 mRNA. Orai1 primers were forward primer 5′-TTTGCCCTCATGATCAGCAC-3′ and reverse primer 5′-TTGGCGACGATGACTGATTC-3′. Leukotriene C4 synthase primers were forward primer 5′-CCTACAGGTGATCTCTGCACGA-3′ and reverse primer 5′-TATCCCTGGAAATAGCGGAGG-3′. Eosinophil-associated, ribonuclease A family, member 3 primers were forward primer 5′-TAATGCTTGCCTCATGCCTG-3′ and reverse primer 5′-TGCACAAGCCACTTGGATTT-3′. Germline C ɛ transcript primers were forward primer 5′-TGCATTGAAGGCTACGGCTA-3′ and reverse primer 5′-TTGGAGCCATTGGATTTCAG-3′. Interleukin-4 primers were forward primer 5′-ATCGGCATTTTGAACGAGGT-3′ and reverse primer 5′-TCGAAAAGCCCGAAAGAGTC-3′. Glyceraldehyde-3-phosphate dehydrogenase mRNA was also examined to control the sample-to-sample variation in RNA isolation and integrity by using a pair of primers: forward primer 5′-ACCACAGTCCATGCCATCAC-3′ and reverse primer 5′-TCCACCACCCTGTTGCTGTA-3′. After initial denaturation at 95°C for 10 minutes, the amplification profile was 15 seconds of denaturation at 95°C and 1 minute of annealing and extension at 60°C for 45 cycles. Negative control RT reaction mixtures contained no reverse transcriptase and no cDNA in the PCR amplification mixtures. For measurement, 2 μ L of diluted cDNA was amplified in a total reaction volume of 20 μ L by using a 7500 real-time PCR System (Applied Biosystems, Foster City, CA) with 20× SYBR Green mixture (Invitrogen). Specificity of PCR products was evaluated by melting curve analysis and by size in agarose gels. Using 3 dilutions of cDNA, linearity of PCR amplification was controlled. Evaluation of data was performed using the cycle threshold (ΔCT) method with glyceraldehyde-3-phosphate dehydrogenase as internal standard.

2.9

Statistical analysis

Statistical analysis was performed using a commercially available statistical software prism 4.0 (GraphPad Software Inc, San Diego, CA). Data were analyzed with the unpaired Student t test ± Welch’s correction and presented as mean ± standard error of the mean (SEM). Significance of a difference was accepted at the 5% level of confidence. P < .05 was considered statistically significant.