Insights into the regulation of TNF-alpha production in human mononuclear cells: the effects of non-specific phosphodiesterase inhibition

Abstract

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 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.

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.

Figure 1

Phosphodiesterase inhibition attenuates LPS-induced TNF-α production. Isolated human mononuclear cells (5 x 10⁶) were incubated with LPS (1 μg/mL) in the presence and absence of Pentoxifylline (PTX; 20 mM). TNF-α concentrations in supernatant were measured by ELISA (pg/mL) up to 18 hours after stimulation (n = 4 per group). PTX downregulated TNF-α production by mononuclear cells in a rapid and sustained manner (*, P < 0.05 vs. LPS / PTX).

Figure 2

Phosphodiesterase inhibition attenuates LPS-induced I-κBα phosphorylation. Isolated human mononuclear cells (5 x 10⁶) were incubated with LPS (1 μg/mL) and Pentoxifylline (PTX; 20 mM). I-κBα phosphorylation was assessed by western blotting, as described in Materials and Methods. Results are expressed as a percentage of LPS stimulation ± SEM (n = 4 per group). LPS-stimulation markedly enhanced I-κBα phosphorylation over that of the control (*, P < 0.05 vs. HBSS). The addition of PTX resulted in a marked reduction in I-κBα phosphorylation (†, P = 0.02 vs. LPS).

Figure 3

Effects of phosphodiesterase inhibition on LPS-induced NF-κB phosphorylation. Isolated human mononuclear cells (5 x 10⁶) were incubated with LPS (1 μg/mL) in the presence and absence Pentoxifylline (PTX; 20 mM). A, Nuclear NF-κB p65 phosphorylation was assessed by western blotting, and results are expressed as a percentage of LPS stimulation ± SEM (n = 4 per group). LPS-stimulation markedly enhanced NF-κB phosphorylation over that of the control (*, P < 0.01 vs. HBSS). The addition of PTX resulted in diminished NF-κB phosphorylation (†, P = 0.03 vs. LPS). B, NF-κB DNA-binding was determined by EMSA, as described in Materials and Methods. NF-κB-DNA binding was upregulated following LPS treatment and attenuated following the addition of PTX in a manner that was similar to NF-κB p65 subunit phosphorylation.

Figure 4

Effects of phosphodiesterase inhibition on LPS-induced CREB phosphorylation. Isolated human mononuclear cells (5 x 10⁶) were incubated with LPS (1 μg/mL) in the presence and absence Pentoxifylline (PTX; 20 mM). A, CREB phosphorylation was assessed by western blotting, and the results are expressed as a percentage of LPS stimulation ± SEM (n = 4 per group). Incubation of cells with PTX alone resulted in a concentration-dependent upregulation of CREB phosphorylation (*, P < 0.05 vs. HBSS). The concomitant administration of LPS slightly attenuated phosphorylation, which nonetheless remained significant when compared to that observed in cells treated with LPS alone (†, P < 0.01 vs. LPS). B, CREB-DNA binding was determined by EMSA, as described in Materials and Methods. CREB-DNA binding after LPS stimulation was similar to that of the control. PTX upregulated CREB-DNA binding in the presence and absence of LPS in a manner that was similar to CREB phosphorylation.

Figure 5

Phosphodiesterase inhibition downregulates LPS-induced TNF-α production independent of Protein kinase A (PKA). Isolated mononuclear cells (5 x 10⁶) were preincubated with the PKA inhibitor, H89 (10 μM), for one hour prior to stimulation with LPS (1 μg/mL) or LPS (1 μg/mL) and Pentoxifylline (PTX; 20 mM) for an additional 30 minutes. The concentration of TNF-α present in the supernatant was measured by ELISA. PTX downregulated LPS-induced TNF-α expression (*, P < 0.01 vs. LPS). The addition of H89 did not significantly alter the effects of PTX on TNF-α production after LPS stimulation.