Insights into the regulation of TNF-α 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. INTRODUCTION: 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 mu g/mL), in the presence and absence of Pentoxifylline (PTX; 20 mM), a nonspecific phosphodiesterase inhibitor. Western blotting of phosphorylated cytoplasmic I-kappa B alpha, nuclear factor-kappa B p65 (NF-kappa B), and nuclear cAMP-response element binding protein (CREB) was performed. DNA binding of NF-kappa B and CREB was verified by electrophoretic mobility shift assay. TNF-alpha, 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-kappa B alpha phosphorylation, nuclear p65 phosphorylation, and the DNA binding activity of NF-kappa B. 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-alpha 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-kappa B 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.