The reagents used in this experiment included histamine, diphenhydramine (first-generation H1R antagonist), pyrilamine (first-generation H1R antagonist), fexofenadine (second-generation H1R antagonist), and U-73122 (phospholipase C inhibitor). These five reagents used were purchased from Sigma-Aldrich (St. Louis, MO, USA). BAY11-7082 (NF-κB inhibitor) was purchased from Calbiochem (Billerica, MA, USA). Histamine, diphenhydramine, and pyrilamine were dissolved in distilled water. Fexofenadine, BAY11-7082, and pyrilamine were used in its dimethyl sulfoxide dissolved form. The final maximum concentration of dimethyl sulfoxide was below 0.1%.

Eight normal control subjects were recruited for this experiment. All patients were recruited from the Department of Otolaryngology-Head and Neck Surgery, Korea University Medical Center. Only patients without allergies, asthma or aspirin sensitivity met inclusion criteria. Likewise, none of the participants experienced treatment with anti-allergic drugs for more than 2 months. Before obtaining nasal fibroblast cultures, informed consent was thoroughly explained and received from all participants. Normal control tissues from inferior turbinate specimens were gained by patients without any sinonasal diseases by rhinoplasty surgeries. The purity of cells was confirmed by its morphological characteristics such as spindle-shaped cell morphology and flow cytometry. Nasal fibroblasts were positive for vimentin, fibroblast markers, whereas negative for E-cadherin, epithelial cell marker. Once the cell confluency reached more than 80%, cells were enzymatically digested. The nasal fibroblasts were cultured for four passages [16]. The experiment was approved by the Korea University Medical Center Institutional Review Board.

Nasal fibroblasts were seeded in 96-well culture plates at a density of 10⁴ cells/well, in culture medium and incubated for 24 hours. Fibroblasts were then treated with different concentrations of histamine or H1R antagonist for another 48 hours. Then nasal fibroblasts were incubated with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma) at a final concentration of 0.5 mg/mL. After 4 hours, MTT solution was discarded and 150 μL dimethyl sulfoxide (Sigma-Aldrich) was added to dissolve the formazan precipitate by shaking the plates for 10 minutes at mild speed on an orbital shaker. Microplate readers (F2000, Hitachi Ltd., Tokyo, Japan) were used to read the absorbance of each well at the wavelength of 540 nm.

After nasal fibroblasts were stimulated by histamine (100 μM), total RNA was extracted by a Trizol reagent (Invitrogen, Carlsbad, CA, USA). The total amount of RNA was determined by an optical system using NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA). Real-time polymerase chain reaction was performed in Quantstudio3 (Applied Biosystems, Foster City, CA, USA) using Power SYBR Green PCR Master Mix (Applied Biosystems). Relative gene expression was calculated by evaluating real-time polymerase chain reaction data using the 2 (2DDCt) method. Experiments were repeated at least 3 times and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. A primer was added to perform the polymerase chain reaction; H1R (sense sequence 5’-GTC TAA CAC AGG CCT GGA TT-3’, antisense sequence 5’-GGA TGA AGG CTG CCA TGA TA-3’) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; sense sequence, 5’-GTGGATATTGTCCCAT-3’ and antisense sequence, 5’-GCCCCAGCCTTCTTCATGGGG-3’).

The secretion levels of CXCL8 protein in nasal fibroblasts after treatment with histamine were evaluated by ELISA. Ten percentage of fetal bovine serum was used to incubate nasal fibroblasts and the supernatant media were collected. ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to quantify CXCL8 expression according to manufacturer’s instructions. Ninety-six-well microplates were coated with diluted capture antibody and incubated overnight at room temperature. They were blocked with 1% bovine serum albumin for 1 hour and then added collected-supernatant media and standards for 2 hours. Detection antibody was added for 2 hours after washing. Streptavidin-horseradish peroxidase and its substrate were used for the manifestation of color. Optical density was set to 450 nm. The microtiter plate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA) was used to determine the CXCL8 expression and each well was examined within 30 minutes.

PRO-PREP TM protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) was used to lyse nasal fibroblasts. The lysed materials were then placed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membranes (Millipore Inc., Billerica, MA, USA). Membranes were blocked for an hour in a 5% non-fat dry milk and were covered overnight in phospho (p)-p50, and total-p50 with 3% bovine serum albumin at 4ºC. Afterwards that, membranes were washed thoroughly for five minutes three times, and then treated with horseradish peroxide-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for one hour. Then, substrates from an ECL system (Pierce, Rockford, IL, USA) were added for visualization.

Nasal fibroblasts were seeded on coverslips and treated with histamine (100 μM). Nasal fibroblasts were fixed with 4% paraformaldehyde, and then permeabilized with 0.01% Triton X-100 in 1% bovine serum albumin for 10 minutes. After permeabilization, nasal fibroblast was blocked with 5% bovine serum albumin for 1 hour at room temperature. Nasal fibroblasts incubated with p-p50 (Sigma-Aldrich) overnight at 4ºC and then incubated with anti-rabbit Alexa 555 secondary antibodies (Invitrogen) for 1 hour. 4’-6-diamidino-2-phenylindole was applied for counterstaining of the nucleus. An LSM700 confocal laser scanning microscope (Zeiss, Oberkochen, Germany) was used for capturing and identifying stained cells.

All statistical analyses were performed via SPSS Statistics 20.0.0 version (IBM Corp., Armonk, NY, USA). Statistically significant differences between groups were evaluated by one-way analysis of variation (ANOVA) for factorial comparisons and subsequently with Turkey’s post-hoc trial. Outcomes are given as mean±standard error of the men (SEM) which were extracted from at least three independent trials. Statistical significance was defined as p<0.05.

An MTT assay was used to examine the cytotoxicity of histamine and H1R antagonists. A cell titration curve was produced from serial dilutions of nasal fibroblasts and MTT reagent. The standard curve indicated a linear response between cell number and absorption at 570 nm. Nasal fibroblasts were examined at histamine concentrations ranging from 0 to 1,000 μM with various mixtures of histamine and H1R antagonist (fexofenadine, pyrilamine, and diphenhydramine). Cell survival was not affected by histamine until the concentration reached 1,000 μM (Fig. 1A), and it was also produced by different mixtures of histamine and each H1R antagonist for 72 hours (Fig. 1B).

To investigate the CXCL8 expression in nasal fibroblasts, we measured CXCL8 expression using ELISA kit. H1R mRNA expression in nasal fibroblasts was identified by real-time polymerase chain reaction. H1R mRNA expression was increased significantly by histamine stimulation in a dose-dependent manner (Fig. 2A). ELISA was then used to examine whether histamine stimulated CXCL8 protein production in nasal fibroblasts. Nasal fibroblasts were treated with up to 200 μM histamine for 24 hours. CXCL8 protein expression increased significantly in response to histamine stimulation in a dose-dependent manner (Fig. 2B).

We investigated which histamine receptor was involved in the CXCL8 production. Nasal fibroblasts were pretreated with histamine receptor antagonists for H1R (fexofenadine, pyrilamine, diphenhydramine) and then stimulated by histamine for 24 hours. Fexofenadine, pyrilamine, and diphenhydramine significantly decreased CXCL8 production (Fig. 3A).

To find the signaling pathway for histamine-induced CXCL8 production in nasal fibroblasts, stimulation of phospholipase C as a downstream marker of CXCL8 signaling was evaluated via ELISA. Histamine-induced CXCL8 protein production decreased in a dose-dependent manner after treatment with a phospholipase C inhibitor (U73122) (Fig. 3B). CXCL8 production was significantly decreased compared to the control after 10 μM treatments with a phospholipase C inhibitor.

Next, the effect of NF-κB activation on CXCL8 production was confirmed in histamine-stimulated nasal fibroblasts. The expression levels of p-p50 were determined by western blot in nasal fibroblasts, in the absence or presence of H1R antagonist or BAY11-7082. Histamine increased expression of p-p50 and BAY11-7082 (NF-κB inhibitor) suppressed p-p50 expression (Fig. 4A). In immunocytochemical staining, histamine induced phosphorylation of p50 and translocation of p-p50 into the nucleus (Fig. 4B). The expression level of CXCL8 protein was determined by ELISA. Histamine induced expression of CXCL8 protein levels. However, BAY11-7082 inhibited expression of histamine-induced CXCL8 protein levels (Fig. 4C). These results indicated that histamine-induced CXCL8 production through NF-κB signaling pathway.