Insights into the regulation of TNF-alpha production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition.
OBJECTIVE
The objective of this study was to determine the effect of nonspecific phosphodiesterase inhibition on transcription factor activation and tumor necrosis factor-alpha (TNF-alpha) production in lipopolysaccharide (LPS)-stimulated human mononuclear cells.


BACKGROUND
The production of TNF-alpha following LPS stimulation is one of the key steps in bacterial sepsis and inflammation. The mechanism by which phosphodiesterase inhibition alters TNF-alpha production in the presence of LPS remains unclear.


METHODS
Human mononuclear cells were stimulated with LPS (1 microg/mL), in the presence and absence of Pentoxifylline (PTX; 20 mM), a nonspecific phosphodiesterase inhibitor. Western blotting of phosphorylated cytoplasmic I-kBalpha, nuclear factor-kB p65 (NF-kB), and nuclear cAMP-response element binding protein (CREB) was performed. DNA binding of NF-kB and CREB was verified by electrophoretic mobility shift assay. TNF-a levels were determined in the supernatant of stimulated cells in the presence and absence Protein kinase A inhibition by an enzyme-linked immunosorbent assay (ELISA).


RESULTS
PTX was demonstrated to significantly reduce cytoplasmic I-kBalpha phosphorylation, nuclear p65 phosphorylation, and the DNA binding activity of NF-kB. In contrast, PTX markedly enhanced the phosphorylation and DNA binding activity of CREB. Cells concomitantly treated with PTX and LPS secreted similar levels of TNF-a in the presence and absence Protein kinase A inhibition.


CONCLUSIONS
The increased level of cAMP that results from phosphodiesterase inhibition affects cytoplasmic and nuclear events, resulting in the attenuation of NF-kB and the activation of CREB transcriptional DNA binding through pathways that are partially Protein kinase A-independent.


CONCLUSIONS
PTX-mediated phosphodiesterase inhibition occurs partially through a Protein kinase A-independent pathway and may serve as a useful tool in the attenuation of LPS-induced inflammation.
Deree
Jessica
J
Department of Trauma and Critical Care, University of California San Diego Medical Center, San Diego, CA, USA. jderee@ucsd.edu
Martins
Joilson O
JO
Melbostad
Heidi
H
Loomis
William H
WH
Coimbra
Raul
R
D015153:Blotting, Western; D017362:Cyclic AMP Response Element-Binding Protein / Q000187:drug effects / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000187:drug effects* / Q000378:metabolism; D010431:Pentoxifylline / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D018805:Sepsis / Q000188:drug therapy; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D017362:Cyclic AMP Response Element-Binding Protein; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010726:Phosphodiesterase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D010431:Pentoxifylline