An involvement of neurokinin-1 receptor in FcεRΙ-mediated RBL-2H3 mast cell activation

RBL-2H3 cell line [24] was maintained as monolayer cultures in Earle’s modified Eagle’s medium (MEM), supplemented with 10 % heat-inactivated fetal bovine serum (FBS; HyClone), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified incubator under 5 % CO₂. All cell culture reagents were obtained from Gibco except where otherwise indicated.

Two different small hairpin RNAs (shRNAs) against NK1R were designed. NK1R-shRNA (NK1R-shRNA1 and NK1R-shRNA2) constructs were produced as described previously [25]. The sequences of the oligonucleotides homologous to a 21-nucleotide segment of NK1R were: (1) 5′-GCCAGUAUCUACUCCAUGAUU-3′ for NK1R-shRNA1 and 5′-CCUACAUCAACCCAGAUCUUU-3′ for NK1R-shRNA2. A scramble shRNA (negative control, Con-shRNA) was also designed. For transfection, plasmids (2.5 μg) were transfected into RBL-2H3 cells using SuperFect reagent (Qiagen) according to the manufacturer’s protocol. The transfected cells were harvested for further study 36 h later.

For determining the expression of NK1R, RBL-2H3 cells (10⁶) were lysed with 100 μl of lysis buffer (2 % SDS, 50 mM Tris–HCl pH 6.8, 4 μg/ml aprotinin, 20 μg/ml leupeptin, 2 μg/ml pepstatin, 2 μg/ml antipain, and 200 μg/ml phenylmethylsulfonyl fluoride). The lysates were boiled for 10 min and protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology) according to the manufacturer’s instructions. The cell lysates were then mixed with 5× sample buffer and fractionated onto 10 or 12 % SDS-polyacrylamide gel electrophoresis (PAGE) for NK1R analysis. After SDS-PAGE gels were run, proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (Roche Applied Science) for 90 min at 350 mA. The membrane was then blocked in TBS-Tween (0.1 %) buffer containing 5 % skim milk or bovine serum albumin (BSA, Amresco) for 2 h at room temperature (RT) followed by probing with anti-NK1R antibody (1:500–1:1,000, overnight, 4 °C; Pierce Biotechnology). The membrane was washed and incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2,000, 1 h, RT; Santa Cruz Biotechnology). Immunoreactive bands were visualized using enhanced chemiluminescence (ECL). The membrane was reprobed with anti-GAPDH antibody after incubation with stripping buffer (100 mM 2-mercaptoethanol, 2 % SDS, 62.5 mM Tris–HCl pH 6.8) for 30 min at 50 °C to control for loading. Densitometric analysis was performed by ImageJ software. Densitometry was measured in arbitrary densitometry units (ADU).

RBL-2H3 cells (2 × 10⁵) were sensitized with 0.1 μg/ml anti-dinitrophenol (DNP)-IgE (SPE-7 clone; Sigma-Aldrich) overnight and washed twice with Tyrode’s buffer (135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 5.6 mM glucose, 20 mM HEPES, and 1 mg/ml BSA at pH 7.4). The cells were then loaded with 4 μM Fluo3-AM (Dojindo, Japan) in Tyrode’s buffer for 45 min at 37 °C in a 5 % CO₂ incubator. To monitor the calcium flux, Fluo3-loaded cells were stimulated with the antigen DNP-BSA (10 ng/ml) for the indicated lengths of time, and single-wavelength measurements of Ca²⁺-bound Fluo3 at 520 nm (488 nm excitation) were performed on a laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany). Images were recorded and analyzed using the “LAS AF Lite” software package (Leica), and the elevation in fluorescence intensity of Ca²⁺-bound Fluo3, proportional to the [Ca²⁺], was calculated after subtracting the background fluorescence.

RBL-2H3 cells (3 × 10⁶) incubated overnight in complete MEM containing anti-DNP IgE (0.1 μg/ml) were washed and stimulated with DNP-BSA (10 ng/ml) for 4 h. Cell-free supernatants were collected and secreted MCP-1 was determined using a rat-specific MCP-1 ELISA detection kit (Invitrogen).

DNP-BSA-induced phosphorylation of mitogen-activated protein kinases (MAPKs) was determined by western blotting. RBL-2H3 cells were sensitized and starved overnight in serum-free MEM containing anti-DNP-IgE (0.1 μg/ml) and BSA (2 %). Stimulation was initiated by the addition of DNP-BSA (10 ng/ml) in Tyrode’s buffer at 37 °C. The reaction was stopped by placing the cells on ice. To determine the activities of MAPK molecules, the whole cell lysates were prepared and subjected to western blotting as described above. Additional protease inhibitors including 60 mM β-glycerophosphate, 2 mM sodium orthovanadate, and 10 mM sodium fluoride were added in the lysates. Anti-phospho-Erk1/2, JNK, and p38 antibodies (phospho-MAPKs) were used. To control for loading, membranes were stripped and reprobed with antibodies to total Erk1/2, JNK, and p38 (MAPKs). The intensities of the phospho-MAPKs were normalized to total MAPKs to correct for any differences in loading. All antibodies were purchased from Cell Signaling Technology unless otherwise noted and used at recommended concentrations. Aprotinin, leupeptin, pepstatin, antipain, phenylmethylsulfonyl fluoride, sodium orthovanadate, and sodium fluoride were from Sigma-Aldrich.

All data were analyzed with Prism 5 software (GraphPad Software). Data were considered statistically significant when a P value was less than 0.05, obtained with a two-tailed t test.

To determine whether NK1R plays a role in FcεRΙ-mediated RBL-2H3 cell activation, we constructed two shRNAs against NK1R (NK1R-shRNA1 and NK1R-shRNA2) and transfected them into wide-type RBL-2H3 cells to inhibit the expression of NK1R. Negative control plasmids containing scrambled shRNA (Con-shRNA) were also transfected. NK1R protein expression in RBL-2H3 cells after 36 h of transfection was assessed by western blotting (Fig. 1). Expression of NK1R-shRNA1 and NK1R-shRNA2 led to reductions in NK1R expression of 68.43 ± 2.41 and 81.1 ± 2.49 %, respectively, in RBL-2H3 cells compared with Con-shRNA. This indicates an effective knockdown of NK1R expression in RBL-2H3 cells by either NK1R-shRNA1 or NK1R-shRNA2. Hence, we named RBL-2H3 cells expressing NK1R-shRNA1 or NK1R-shRNA2 for NK1R knockdown RBL-2H3 cells and expressing Con-shRNA for control RBL-2H3 cells.

Cytokine gene expression and calcium mobilization are two remarkable events in mast cells after FcεRΙ aggregation. To evaluate the effect of NK1R on cytokine gene expression in RBL-2H3 cells following FcεRΙ stimulation, amounts of released MCP-1 were determined by ELISA. As shown in Fig. 2a, a significant reduction of MCP-1 expression was observed in NK1R knockdown RBL-2H3 cells (124.1 ± 17.7 for NK1R-shRNA1, and 99.5 ± 18.6 for NK1R-shRNA2) relative to control RBL-2H3 cells (278.2 ± 23.5 for Con-shRNA). We also monitored calcium flux in both control and NK1R knockdown RBL-2H3 cells following FcεRΙ aggregation (Fig. 2b). RBL-2H3 cells expressing either NK1R-shRNA1 or NK1R-shRNA2 showed decreased calcium mobilization compared with RBL-2H3 cells expressing Con-shRNA. These results strongly indicate an essential role of NK1R in FcεRΙ-evoked RBL-2H3 cell activation.

Efforts were made to explore the effect of NK1R on MAPK signaling downstream of the FcεRΙ in RBL-2H3 cells. After FcεRΙ stimulation, phosphorylation levels of MAPKs were determined by western blotting. For phospho-Erk1/2, phosphorylation signals were hardly detected under basal conditions in both control and NK1R knockdown RBL-2H3 cells. After the antigen DNP-BSA treatment for 5 min, control RBL-2H3 cells showed a marked increase in levels of phospho-Erk1/2. However, a much lower elevation of phosphorylation levels of Erk1/2 were observed in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells (Fig. 3a). Similar tendencies were observed on both phospho-JNK (Fig. 3b) and phospho-p38 (Fig. 3c). This significantly inhibited phosphorylation of MAPKs, which is attributable to the down-regulation of NK1R expression, indicates an involvement of NK1R in the regulation of MAPK signaling after FcεRΙ aggregation in RBL-2H3 cells.