Heidi Melbostad

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-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.

Modulation of lipopolysaccharide-induced acute lung inflammation: Role of insulin.

ABSTRACT The present study was undertaken to investigate the influence of insulin on lipopolysaccharide (LPS)-induced acute lung injury. Diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 30 days) and controls were instilled with saline containing LPS (750 &mgr;g/0.4 mL) or saline alone. The following analyses were performed 6 h there after: (a) total and differential cell counts in bronchoalveolar lavage (BAL) fluid, (b) quantification of tumor necrosis factor &agr;, interleukin (IL) 1&bgr;, IL-10, and cytokine-induced neutrophil chemoattractant 1 in the BAL (enzyme-linked immunosorbent assay), (c)immunohistochemistry for intercellular adhesion molecule 1 and E-selectin on lung vessels, and (d) quantification of metalloproteinases (MMP) 2 and 9 in the BAL (zymography). Relative to controls, diabetic rats exhibited a reduction in the number of neutrophils (80%) and reduced concentrations of tumor necrosis factor &agr; (56%), IL-1&bgr; (66%), and IL-10 (35%) after LPS instillation. Cytokine-induced neutrophil chemoattractant 1 levels did not differ between groups. Increased levels of MMP-2 (90%) and MMP-9 (500%) were observed in diabetic rats compared with controls. Treatment of diabetic rats with neutral protamine Hagedorn insulin (4 IU, s.c.), 2 h before LPS instillation, completely restored the number of neutrophils and concentrations of cytokines in the BAL fluid. Despite no significant differences between diabetic and control groups, there was a remarkable increase in intercellular adhesion molecule 1 and E-selectin expression on lung vessels after insulin treatment. Levels of MMP-2 and MMP-9 did not change after treatment with insulin. Levels of corticosterone were equivalent among groups. Data presented suggest that insulin modulates the production/release of cytokines and the expression of adhesion molecules controlling, therefore, neutrophil migration during the course of LPS-induced acute lung inflammation.

LPS-stimulated PMN activation and proinflammatory mediator synthesis is downregulated by phosphodiesterase inhibition: role of pentoxifylline.

BACKGROUND Excessive production of reactive oxygen species by PMN is associated with tissue damage during inflammation. LPS interacts with the cell surface receptor CD14, which generates transmembrane signals through Toll-like protein 4 leading to mitogen activated protein kinase (MAPK) p38 activation, cytokine synthesis, PMN beta2-integrin expression and oxidative burst. Phosphodiesterase inhibition decreases proinflammatory cytokine production and tissue injury after LPS challenge. Its effects on PMN function after LPS stimulation, however, have not been fully investigated. We hypothesized that LPS-induced TNF-alpha synthesis and subsequent PMN beta2-integrin expression and oxidative burst are downregulated by concomitant treatment with the non-specific phosphodiesterase inhibitor pentoxifylline (PTX). METHODS Whole blood was incubated with HBSS (control), LPS (100 microg/mL), fMLP (1 micromol/L), LPS+PTX (2 mmol/L) and fMLP+PTX for different time intervals at 37C. Oxidative burst, CD14, and CD-11b expression were measured by flow cytometry. Serum TNF-alpha levels were measured by ELISA. In an attempt to localize the site of action of PTX (proximal or distal to PKC) cell surface receptors were bypassed by PMA stimulation (1 microg/mL) and oxidative burst was measured with and without PTX. RESULTS Up-regulation of CD14 expression was similar in LPS and LPS+PTX groups. LPS stimulation caused a significant increase in PMN oxidative burst, CD11b expression, and TNF-alpha serum levels. In addition, PMA and fMLP stimulation also caused significant increase in oxidative burst compared with controls. Concomitant addition of PTX to LPS led to a significant decrease in PMN oxidative burst (65%; p < 0.0001), PMN CD11b expression (20%; p = 0.012), and TNF-alpha levels (93%; p < 0.0001). Also, PMA- and fMLP-induced PMN oxidative burst were significantly decreased by PTX [77.5% (p < 0.0001) and 50% (p < 0.01), respectively]. CONCLUSIONS These results suggest that PTX-inhibition of oxidative burst occurs distal to PKC and may be either due to direct inhibition of NADPH oxidase or inhibition of MAPK phosphorylation, leading to decreased adhesion molecule expression and TNF-alpha synthesis. Its use in clinical scenarios in which PMN are primed may be of clinical relevance.