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

Fang X, Hu H, Xie J, Zhu H, Zhang D, Mo W, Zhang R, Yu M - Inflamm. Res. (2012)

Bottom Line: Protein production of MCP-1 was reduced by more than 55 % in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells.In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression.NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai 200032, People's Republic of China.

ABSTRACT

Objective and design: To determine whether the neurokinin-1 receptor (NK1R) plays a role in the activation of RBL-2H3 mast cells after FcεRΙ aggregation.

Materials and methods: NK1R expression in RBL-2H3 cells was inhibited by small hairpin RNA (shRNA) against NK1R, and determined by western blotting. For activation, both NK1R knockdown and control RBL-2H3 cells were sensitized by dinitrophenol (DNP)-specific IgE and stimulated with the antigen DNP-bovine serum albumin (BSA). Following the activation of RBL-2H3 cells, monocyte chemoattractant protein (MCP-1) production and intracellular calcium flux were monitored by ELISA and confocal microscopy assay, respectively. For investigation of the signaling mechanism, phosphorylation of mitogen-activated protein kinases (MAPKs) after RBL-2H3 cell activation was assessed by western blotting.

Results: shRNA-NK1R mediated an effective inhibition of NK1R expression in RBL-2H3 cells. Protein production of MCP-1 was reduced by more than 55 % in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells. In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression.

Conclusion: NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.

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NK1R promotes FcεRΙ-induced expression of MCP-1 and calcium mobilization in RBL-2H3 cells. a Indicated RBL-2H3 cells were sensitized overnight with anti-DNP IgE (0.1 μg/ml) and stimulated with (Stimu) or without (Unstimu) DNP-BSA (10 ng/ml) for 4 h at 37 °C. Amounts of released MCP-1 in cell-free supernatants were determined by ELISA. Results are representative of three independent experiments. *P < 0.05 as compared with control RBL-2H3 cells, Student’s t test. b Sensitized RBL-2H3 cells were loaded with 4 μM Fluo3-AM and changes in dye fluorescence with time were monitored by confocal microscopy after the addition of DNP-BSA (10 ng/ml). Calcium flux is indicated by the antigen–response curve over time. Values are presented as the mean fluorescence intensity of Ca2+-bound Fluo3 of at least 20 cells. Results of two independent experiments are shown in each panel
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Fig2: NK1R promotes FcεRΙ-induced expression of MCP-1 and calcium mobilization in RBL-2H3 cells. a Indicated RBL-2H3 cells were sensitized overnight with anti-DNP IgE (0.1 μg/ml) and stimulated with (Stimu) or without (Unstimu) DNP-BSA (10 ng/ml) for 4 h at 37 °C. Amounts of released MCP-1 in cell-free supernatants were determined by ELISA. Results are representative of three independent experiments. *P < 0.05 as compared with control RBL-2H3 cells, Student’s t test. b Sensitized RBL-2H3 cells were loaded with 4 μM Fluo3-AM and changes in dye fluorescence with time were monitored by confocal microscopy after the addition of DNP-BSA (10 ng/ml). Calcium flux is indicated by the antigen–response curve over time. Values are presented as the mean fluorescence intensity of Ca2+-bound Fluo3 of at least 20 cells. Results of two independent experiments are shown in each panel

Mentions: 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.Fig. 2


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

Fang X, Hu H, Xie J, Zhu H, Zhang D, Mo W, Zhang R, Yu M - Inflamm. Res. (2012)

NK1R promotes FcεRΙ-induced expression of MCP-1 and calcium mobilization in RBL-2H3 cells. a Indicated RBL-2H3 cells were sensitized overnight with anti-DNP IgE (0.1 μg/ml) and stimulated with (Stimu) or without (Unstimu) DNP-BSA (10 ng/ml) for 4 h at 37 °C. Amounts of released MCP-1 in cell-free supernatants were determined by ELISA. Results are representative of three independent experiments. *P < 0.05 as compared with control RBL-2H3 cells, Student’s t test. b Sensitized RBL-2H3 cells were loaded with 4 μM Fluo3-AM and changes in dye fluorescence with time were monitored by confocal microscopy after the addition of DNP-BSA (10 ng/ml). Calcium flux is indicated by the antigen–response curve over time. Values are presented as the mean fluorescence intensity of Ca2+-bound Fluo3 of at least 20 cells. Results of two independent experiments are shown in each panel
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Related In: Results  -  Collection

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Fig2: NK1R promotes FcεRΙ-induced expression of MCP-1 and calcium mobilization in RBL-2H3 cells. a Indicated RBL-2H3 cells were sensitized overnight with anti-DNP IgE (0.1 μg/ml) and stimulated with (Stimu) or without (Unstimu) DNP-BSA (10 ng/ml) for 4 h at 37 °C. Amounts of released MCP-1 in cell-free supernatants were determined by ELISA. Results are representative of three independent experiments. *P < 0.05 as compared with control RBL-2H3 cells, Student’s t test. b Sensitized RBL-2H3 cells were loaded with 4 μM Fluo3-AM and changes in dye fluorescence with time were monitored by confocal microscopy after the addition of DNP-BSA (10 ng/ml). Calcium flux is indicated by the antigen–response curve over time. Values are presented as the mean fluorescence intensity of Ca2+-bound Fluo3 of at least 20 cells. Results of two independent experiments are shown in each panel
Mentions: 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.Fig. 2

Bottom Line: Protein production of MCP-1 was reduced by more than 55 % in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells.In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression.NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai 200032, People's Republic of China.

ABSTRACT

Objective and design: To determine whether the neurokinin-1 receptor (NK1R) plays a role in the activation of RBL-2H3 mast cells after FcεRΙ aggregation.

Materials and methods: NK1R expression in RBL-2H3 cells was inhibited by small hairpin RNA (shRNA) against NK1R, and determined by western blotting. For activation, both NK1R knockdown and control RBL-2H3 cells were sensitized by dinitrophenol (DNP)-specific IgE and stimulated with the antigen DNP-bovine serum albumin (BSA). Following the activation of RBL-2H3 cells, monocyte chemoattractant protein (MCP-1) production and intracellular calcium flux were monitored by ELISA and confocal microscopy assay, respectively. For investigation of the signaling mechanism, phosphorylation of mitogen-activated protein kinases (MAPKs) after RBL-2H3 cell activation was assessed by western blotting.

Results: shRNA-NK1R mediated an effective inhibition of NK1R expression in RBL-2H3 cells. Protein production of MCP-1 was reduced by more than 55 % in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells. In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression.

Conclusion: NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.

Show MeSH