Insights into the regulation of TNF-a 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-a) production in lipopolysaccharide (LPS)-stimulated human mononuclear cells. INTRODUCTION: The production of TNF-a f...
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Faculdade de Medicina / USP
2008-01-01
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doaj-d8428c01cb424f3e9e1acb950d75d1772020-11-25T01:15:31ZengFaculdade de Medicina / USPClinics1807-59321980-53222008-01-0163332132810.1590/S1807-59322008000300006Insights into the regulation of TNF-a production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibitionJessica DereeJoilson O. MartinsHeidi MelbostadWilliam H. LoomisRaul CoimbraOBJECTIVE: The objective of this study was to determine the effect of nonspecific phosphodiesterase inhibition on transcription factor activation and tumor necrosis factor-alpha (TNF-a) production in lipopolysaccharide (LPS)-stimulated human mononuclear cells. INTRODUCTION: The production of TNF-a following LPS stimulation is one of the key steps in bacterial sepsis and inflammation. The mechanism by which phosphodiesterase inhibition alters TNF-a production in the presence of LPS remains unclear. METHODS: Human mononuclear cells were stimulated with LPS (1 µg/mL), in the presence and absence of Pentoxifylline (PTX; 20 mM), a nonspecific phosphodiesterase inhibitor. Western blotting of phosphorylated cytoplasmic I-kBa, 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-kBa 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. DISCUSSION: 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. CONCLUSION: 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.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1807-59322008000300006SepsisPentoxifyllineCyclic adenosine-35-monophosphate (cAMP)cAMP-response element binding protein (CREB)Nuclear factor-kB (NF-kB) |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jessica Deree Joilson O. Martins Heidi Melbostad William H. Loomis Raul Coimbra |
spellingShingle |
Jessica Deree Joilson O. Martins Heidi Melbostad William H. Loomis Raul Coimbra Insights into the regulation of TNF-a production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition Clinics Sepsis Pentoxifylline Cyclic adenosine-3 5-monophosphate (cAMP) cAMP-response element binding protein (CREB) Nuclear factor-kB (NF-kB) |
author_facet |
Jessica Deree Joilson O. Martins Heidi Melbostad William H. Loomis Raul Coimbra |
author_sort |
Jessica Deree |
title |
Insights into the regulation of TNF-a production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition |
title_short |
Insights into the regulation of TNF-a production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition |
title_full |
Insights into the regulation of TNF-a production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition |
title_fullStr |
Insights into the regulation of TNF-a production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition |
title_full_unstemmed |
Insights into the regulation of TNF-a production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition |
title_sort |
insights into the regulation of tnf-a production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition |
publisher |
Faculdade de Medicina / USP |
series |
Clinics |
issn |
1807-5932 1980-5322 |
publishDate |
2008-01-01 |
description |
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-a) production in lipopolysaccharide (LPS)-stimulated human mononuclear cells. INTRODUCTION: The production of TNF-a following LPS stimulation is one of the key steps in bacterial sepsis and inflammation. The mechanism by which phosphodiesterase inhibition alters TNF-a production in the presence of LPS remains unclear. METHODS: Human mononuclear cells were stimulated with LPS (1 µg/mL), in the presence and absence of Pentoxifylline (PTX; 20 mM), a nonspecific phosphodiesterase inhibitor. Western blotting of phosphorylated cytoplasmic I-kBa, 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-kBa 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. DISCUSSION: 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. CONCLUSION: 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. |
topic |
Sepsis Pentoxifylline Cyclic adenosine-3 5-monophosphate (cAMP) cAMP-response element binding protein (CREB) Nuclear factor-kB (NF-kB) |
url |
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1807-59322008000300006 |
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