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

Deree J, Martins JO, Melbostad H, Loomis WH, Coimbra R - Clinics (Sao Paulo) (2008)

Bottom Line: PTX was demonstrated to significantly reduce cytoplasmic I-kBalpha phosphorylation, nuclear p65 phosphorylation, and the DNA binding activity of NF-kB.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Trauma and Critical Care, University of California San Diego Medical Center, San Diego, CA, USA. jderee@ucsd.edu

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.

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Related in: MedlinePlus

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.
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f4-cln63_3p0321: 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.

Mentions: Phosphorylation of nuclear CREB was used as a marker for CREB activation. LPS stimulation caused a negligible increase in CREB phosphorylation when compared to control (Figure 4A). PTX alone caused a marked increase in CREB phosphorylation (P < 0.01 vs. HBSS). When LPS and PTX exposure occurred simultaneously, CREB phosphorylation was significantly higher than with LPS stimulation alone (543 ± 92 vs. 100 ± 0; P < 0.01). In addition, the amount of CREB phosphorylation seen with concomitant LPS and PTX treatment was less than that seen with PTX alone, although this difference was not statistically significant (P = 0.08).


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

Deree J, Martins JO, Melbostad H, Loomis WH, Coimbra R - Clinics (Sao Paulo) (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664230&req=5

f4-cln63_3p0321: 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.
Mentions: Phosphorylation of nuclear CREB was used as a marker for CREB activation. LPS stimulation caused a negligible increase in CREB phosphorylation when compared to control (Figure 4A). PTX alone caused a marked increase in CREB phosphorylation (P < 0.01 vs. HBSS). When LPS and PTX exposure occurred simultaneously, CREB phosphorylation was significantly higher than with LPS stimulation alone (543 ± 92 vs. 100 ± 0; P < 0.01). In addition, the amount of CREB phosphorylation seen with concomitant LPS and PTX treatment was less than that seen with PTX alone, although this difference was not statistically significant (P = 0.08).

Bottom Line: PTX was demonstrated to significantly reduce cytoplasmic I-kBalpha phosphorylation, nuclear p65 phosphorylation, and the DNA binding activity of NF-kB.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Trauma and Critical Care, University of California San Diego Medical Center, San Diego, CA, USA. jderee@ucsd.edu

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.

Show MeSH
Related in: MedlinePlus