TY  - JOUR
A1  - Neuschäfer-Rube, Frank
A1  - Pathe-Neuschäfer-Rube, Andrea
A1  - Hippenstiel, Stefan
A1  - Püschel, Gerhard Paul
T1  - PGE(2) enhanced TNF alpha-mediated IL-8 induction in monocytic cell lines and PBMC
JF  - Cytokine
N2  - Background & purpose: Recent studies suggested a role of prostaglandin E-2 (PGE(2)) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNF alpha-induced IL-8 expression was studied in monocytic cell lines. Experimental approach: IL-8 mRNA and protein induction as well as IL-8 promoter activity and transcription factor activation were assessed in monocytic cell lines, primary blood mononuclear cells (PBMC) and transgenic HEK293 cells expressing the EP4 receptor. Key results: In monocytic cell lines THP-1, MonoMac and U937 PGE(2) had only a marginal impact on IL-8 induction but strongly enhanced TNFa-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNF alpha and PGE(2) than by either stimulus alone. The EP4 receptor subtype was the most abundant EP receptor in all three cell lines and in PBMC. Stimulation of THP-1 cells with an EP4 specific agonist enhanced TNF alpha-induced IL-8 mRNA and protein formation to the same extent as PGE(2). In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE(2) enhanced TNFainduced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNF alpha-mediated IL-8 mRNA induction by PGE(2) was mimicked by a PICA-activator. Furthermore in these cells PGE(2) induced expression of transcription factor C/EBPS, enhanced NF-KB activation by TNFa and inhibited TNF alpha-mediated AP-1 activation. PGE(2) and TNF alpha synergistically activated transcription factor CREB, induced C/EBPS expression and enhanced the activity of an IL-8 promoter fragment containing-223 bp upstream of the transcription start site. Conclusions and implications: These findings suggest that a combined stimulation of TNF alpha and PGE(2)/EP4 signal chains in monocytic cells leads to maximal IL-8 promoter activity, as well as IL-8 mRNA and protein induction, by activating the PICA/CREB/C/EB1313 as well as NF-kappa B signal chains.
KW  - Monocyte
KW  - Prostaglandin receptor EP4
KW  - IL-8 transcription
KW  - Signal transduction
KW  - Tumor necrosis factor alpha
Y1  - 2018
U6  - https://doi.org/10.1016/j.cyto.2018.06.020
SN  - 1043-4666
SN  - 1096-0023
VL  - 113
SP  - 105
EP  - 116
PB  - Elsevier
CY  - London
ER  - 
TY  - JOUR
A1  - Pathe-Neuschäfer-Rube, Andrea
A1  - Neuschäfer-Rube, Frank
A1  - Haas, Gerald
A1  - Langoth-Fehringer, Nina
A1  - Püschel, Gerhard Paul
T1  - Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins
JF  - Toxins
N2  - Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.
KW  - botulinum toxin
KW  - BoNT
KW  - tetanus toxin
KW  - RRR
KW  - replacement
Y1  - 2018
U6  - https://doi.org/10.3390/toxins10090360
SN  - 2072-6651
VL  - 10
IS  - 9
SP  - 1
EP  - 10
PB  - Molecular Diversity Preservation International (MDPI)
CY  - Basel
ER  -