Limits...
Inhibition of the IgE-mediated activation of RBL-2H3 cells by TIPP, a novel thymic immunosuppressive pentapeptide.

Lian Q, Cheng Y, Zhong C, Wang F - Int J Mol Sci (2015)

Bottom Line: TIPP significantly suppressed the increase of intracellular calcium and the rearrangement of F-actin, attenuated the transcription of pro-inflammatory cytokines (IL-3, -4, -6, -13, TNF-α, and monocyte chemotactic protein-1 (MCP-1)), and decreased the expression of COX-2.Western blot analysis showed that TIPP had an inhibitory activity on the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and ERK kinase 1/2 (MEK1/2), and inhibited the activation of NF-κB.The data suggested that TIPP effectively suppressed IgE-mediated activation of RBL-2H3 cells via blocking MEK/ERK and NF-κB signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drugs, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China. xhrg_1042@126.com.

ABSTRACT
TIPP is a novel thymic immunosuppressive pentapeptide originally obtained from calf thymic immunosuppressive extract. The present study aimed to investigate the inhibitory activity of TIPP on IgE-mediated activation of RBL-2H3 cells. Release of β-hexosaminidase and histamine, intracellular calcium, membrane ruffling, mRNA levels of cytokines, cyclooxygenase-2 (COX-2) expression, and activation of mitogen-activated protein kinases (MAP kinases) and NF-κB were determined by colorimetric assay, fluorescence spectrophotometer, confocal fluorescence microscope, quantification PCR, and Western blot, respectively. The results showed that TIPP significantly inhibited the degranulation in IgE-antigen complex-stimulated RBL-2H3 cells without cytotoxicity. TIPP significantly suppressed the increase of intracellular calcium and the rearrangement of F-actin, attenuated the transcription of pro-inflammatory cytokines (IL-3, -4, -6, -13, TNF-α, and monocyte chemotactic protein-1 (MCP-1)), and decreased the expression of COX-2. Western blot analysis showed that TIPP had an inhibitory activity on the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and ERK kinase 1/2 (MEK1/2), and inhibited the activation of NF-κB. The data suggested that TIPP effectively suppressed IgE-mediated activation of RBL-2H3 cells via blocking MEK/ERK and NF-κB signaling pathways.

Show MeSH

Related in: MedlinePlus

Effect of thymic immunosuppressive pentapeptide (TIPP) on IgE-mediated degranulation in RBL-2H3 cells. (A) Cytotoxicity of TIPP. Results are expressed as mean ± SD (n = 5); (B–D) represent the effects of anti-DNP-IgE (monoclonal anti-dinitrophenyl antibody produced in mouse, IgE isotype) concentration, dinitrophenyl-human serum albumin (DNP-HAS) concentration, and stimulated time on IgE-mediated degranulation in RBL-2H3 cells. The amount of β-hexosaminidase in culture supernatant was determined as a biomarker of degranulation. Supernatant samples treated with 0.1% Triton X-100 (v/v) were used as a maximum of degranulation. Results are expressed as mean ± SEM (n = 3); (E,F) represent the effects of TIPP on β-hexosaminidase and histamine release under optimum conditions. Supernatant samples stimulated with IgE-antigen complex and not treated with TIPP were used as a control of 100%. Results are expressed as mean ± SEM (n = 3 for β-hexosaminidase determination and n = 6 for histamine determination). Compared to normal, ### p < 0.001; compared to control (sensitized with anti-DNP-IgE and stimulated with DNP-HSA), * p < 0.05, ** p < 0.01, and *** p < 0.001. Keto.: ketotifen.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4307361&req=5

ijms-16-02252-f002: Effect of thymic immunosuppressive pentapeptide (TIPP) on IgE-mediated degranulation in RBL-2H3 cells. (A) Cytotoxicity of TIPP. Results are expressed as mean ± SD (n = 5); (B–D) represent the effects of anti-DNP-IgE (monoclonal anti-dinitrophenyl antibody produced in mouse, IgE isotype) concentration, dinitrophenyl-human serum albumin (DNP-HAS) concentration, and stimulated time on IgE-mediated degranulation in RBL-2H3 cells. The amount of β-hexosaminidase in culture supernatant was determined as a biomarker of degranulation. Supernatant samples treated with 0.1% Triton X-100 (v/v) were used as a maximum of degranulation. Results are expressed as mean ± SEM (n = 3); (E,F) represent the effects of TIPP on β-hexosaminidase and histamine release under optimum conditions. Supernatant samples stimulated with IgE-antigen complex and not treated with TIPP were used as a control of 100%. Results are expressed as mean ± SEM (n = 3 for β-hexosaminidase determination and n = 6 for histamine determination). Compared to normal, ### p < 0.001; compared to control (sensitized with anti-DNP-IgE and stimulated with DNP-HSA), * p < 0.05, ** p < 0.01, and *** p < 0.001. Keto.: ketotifen.

Mentions: In order to evaluate the cytotoxicity of TIPP on RBL-2H3 cells, the cells were treated with different concentrations of TIPP for 24 h and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) method was used for the cytotoxicity assay. As shown in Figure 2A, TIPP had no significant cytotoxicity on RBL-2H3 cells.


Inhibition of the IgE-mediated activation of RBL-2H3 cells by TIPP, a novel thymic immunosuppressive pentapeptide.

Lian Q, Cheng Y, Zhong C, Wang F - Int J Mol Sci (2015)

Effect of thymic immunosuppressive pentapeptide (TIPP) on IgE-mediated degranulation in RBL-2H3 cells. (A) Cytotoxicity of TIPP. Results are expressed as mean ± SD (n = 5); (B–D) represent the effects of anti-DNP-IgE (monoclonal anti-dinitrophenyl antibody produced in mouse, IgE isotype) concentration, dinitrophenyl-human serum albumin (DNP-HAS) concentration, and stimulated time on IgE-mediated degranulation in RBL-2H3 cells. The amount of β-hexosaminidase in culture supernatant was determined as a biomarker of degranulation. Supernatant samples treated with 0.1% Triton X-100 (v/v) were used as a maximum of degranulation. Results are expressed as mean ± SEM (n = 3); (E,F) represent the effects of TIPP on β-hexosaminidase and histamine release under optimum conditions. Supernatant samples stimulated with IgE-antigen complex and not treated with TIPP were used as a control of 100%. Results are expressed as mean ± SEM (n = 3 for β-hexosaminidase determination and n = 6 for histamine determination). Compared to normal, ### p < 0.001; compared to control (sensitized with anti-DNP-IgE and stimulated with DNP-HSA), * p < 0.05, ** p < 0.01, and *** p < 0.001. Keto.: ketotifen.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307361&req=5

ijms-16-02252-f002: Effect of thymic immunosuppressive pentapeptide (TIPP) on IgE-mediated degranulation in RBL-2H3 cells. (A) Cytotoxicity of TIPP. Results are expressed as mean ± SD (n = 5); (B–D) represent the effects of anti-DNP-IgE (monoclonal anti-dinitrophenyl antibody produced in mouse, IgE isotype) concentration, dinitrophenyl-human serum albumin (DNP-HAS) concentration, and stimulated time on IgE-mediated degranulation in RBL-2H3 cells. The amount of β-hexosaminidase in culture supernatant was determined as a biomarker of degranulation. Supernatant samples treated with 0.1% Triton X-100 (v/v) were used as a maximum of degranulation. Results are expressed as mean ± SEM (n = 3); (E,F) represent the effects of TIPP on β-hexosaminidase and histamine release under optimum conditions. Supernatant samples stimulated with IgE-antigen complex and not treated with TIPP were used as a control of 100%. Results are expressed as mean ± SEM (n = 3 for β-hexosaminidase determination and n = 6 for histamine determination). Compared to normal, ### p < 0.001; compared to control (sensitized with anti-DNP-IgE and stimulated with DNP-HSA), * p < 0.05, ** p < 0.01, and *** p < 0.001. Keto.: ketotifen.
Mentions: In order to evaluate the cytotoxicity of TIPP on RBL-2H3 cells, the cells were treated with different concentrations of TIPP for 24 h and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) method was used for the cytotoxicity assay. As shown in Figure 2A, TIPP had no significant cytotoxicity on RBL-2H3 cells.

Bottom Line: TIPP significantly suppressed the increase of intracellular calcium and the rearrangement of F-actin, attenuated the transcription of pro-inflammatory cytokines (IL-3, -4, -6, -13, TNF-α, and monocyte chemotactic protein-1 (MCP-1)), and decreased the expression of COX-2.Western blot analysis showed that TIPP had an inhibitory activity on the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and ERK kinase 1/2 (MEK1/2), and inhibited the activation of NF-κB.The data suggested that TIPP effectively suppressed IgE-mediated activation of RBL-2H3 cells via blocking MEK/ERK and NF-κB signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Chemical Biology of Natural Products (Ministry of Education), Institute of Biochemical and Biotechnological Drugs, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China. xhrg_1042@126.com.

ABSTRACT
TIPP is a novel thymic immunosuppressive pentapeptide originally obtained from calf thymic immunosuppressive extract. The present study aimed to investigate the inhibitory activity of TIPP on IgE-mediated activation of RBL-2H3 cells. Release of β-hexosaminidase and histamine, intracellular calcium, membrane ruffling, mRNA levels of cytokines, cyclooxygenase-2 (COX-2) expression, and activation of mitogen-activated protein kinases (MAP kinases) and NF-κB were determined by colorimetric assay, fluorescence spectrophotometer, confocal fluorescence microscope, quantification PCR, and Western blot, respectively. The results showed that TIPP significantly inhibited the degranulation in IgE-antigen complex-stimulated RBL-2H3 cells without cytotoxicity. TIPP significantly suppressed the increase of intracellular calcium and the rearrangement of F-actin, attenuated the transcription of pro-inflammatory cytokines (IL-3, -4, -6, -13, TNF-α, and monocyte chemotactic protein-1 (MCP-1)), and decreased the expression of COX-2. Western blot analysis showed that TIPP had an inhibitory activity on the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and ERK kinase 1/2 (MEK1/2), and inhibited the activation of NF-κB. The data suggested that TIPP effectively suppressed IgE-mediated activation of RBL-2H3 cells via blocking MEK/ERK and NF-κB signaling pathways.

Show MeSH
Related in: MedlinePlus