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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 is a novel thymic immunosuppressive pentapeptide originally obtained from calf thymic immunosuppressive extract.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.

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.

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(A) Confocal microscopic observation of the binding of TIPP with RBL-2H3 cells. Pictures represent location of TIPP, nucleus, membrane and merged, respectively. Magnification: 63×; (B–D) represent the concentration-, time- and temperature-dependent characterization of the TIPP binding.
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ijms-16-02252-f006: (A) Confocal microscopic observation of the binding of TIPP with RBL-2H3 cells. Pictures represent location of TIPP, nucleus, membrane and merged, respectively. Magnification: 63×; (B–D) represent the concentration-, time- and temperature-dependent characterization of the TIPP binding.

Mentions: The mode of TIPP binding with RBL-2H3 cells was tested. The confocal fluorescence microscope results showed that TIPP bound with the cell membrane and could get into cells (Figure 6A). The binding kinetics was determined by flow cytometry analysis. A higher mean fluorescent intensity was obtained with increases in TIPP concentration, time, and temperature, which indicated the effect of concentration-, time- and temperature-dependence in the binding between TIPP and RBL-2H3 cells (Figure 6B–D).


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)

(A) Confocal microscopic observation of the binding of TIPP with RBL-2H3 cells. Pictures represent location of TIPP, nucleus, membrane and merged, respectively. Magnification: 63×; (B–D) represent the concentration-, time- and temperature-dependent characterization of the TIPP binding.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-02252-f006: (A) Confocal microscopic observation of the binding of TIPP with RBL-2H3 cells. Pictures represent location of TIPP, nucleus, membrane and merged, respectively. Magnification: 63×; (B–D) represent the concentration-, time- and temperature-dependent characterization of the TIPP binding.
Mentions: The mode of TIPP binding with RBL-2H3 cells was tested. The confocal fluorescence microscope results showed that TIPP bound with the cell membrane and could get into cells (Figure 6A). The binding kinetics was determined by flow cytometry analysis. A higher mean fluorescent intensity was obtained with increases in TIPP concentration, time, and temperature, which indicated the effect of concentration-, time- and temperature-dependence in the binding between TIPP and RBL-2H3 cells (Figure 6B–D).

Bottom Line: TIPP is a novel thymic immunosuppressive pentapeptide originally obtained from calf thymic immunosuppressive extract.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.

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