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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.

Background: The combination of hypertonic saline (HS) and pentoxifylline (PTX) has been shown to synergistically downregulate neutrophil oxidative burst in vitro. We investigated the effects of HS/PTX on human neutrophil mitogen-activated protein kinase (MAPK) signaling and the role of Protein kinase A (PKA) in this process., Methods: Isolated neutrophils were treated with PTX (2 mmol/L), HS10 (10 mmol/L above isotonicity), and HS40 (40 mmol/L above isotonicity) alone or in combination for determination of intracellular cyclic adenosine monophosphate (cAMP) concentrations. Human neutrophils were stimulated with f-methionyl-leucyl-phenylalanine (fMLP) (1 micromol/L) before the treatments above in both the presence and the absence of PKA inhibition for Western blot analysis of MAPK p38 and extracellular signal-related kinase 1/2 (ERK 1/2) phosphorylation., Results: Concomitant exposure to HS/PTX results in an additive increase in intracellular cAMP. fMLP-induced ERK 1/2 phosphorylation was synergistically attenuated by HS/PTX. Both PTX and HS reduced p38MAPK phosphorylation. No additive effect was observed with combined treatment. Although PKA inhibition abrogated the effects of PTX, HS retained some capacity to attenuate MAPK phosphorylation., Conclusion: HS/PTX is more effective in attenuating neutrophil ERK signaling than either component alone, whereas both components alone or in combination produced comparable results with p38MAPK. Although PTX functions primarily through PKA activation, HS may suppress neutrophils through a partially PKA-independent mechanism.

Background: Blood transfusion is a risk factor for many inflammatory processes. Its supernatant fraction has been proven to activate neutrophils. We hypothesized that pentoxifylline (PTX) would attenuate stored blood-induced neutrophil activation and pro-inflammatory mediator production., Methods: Whole blood was incubated with HBSS, LPS (100 microg/mL), leukoreduced PRBC supernatant + LPS, or supernatant + LPS + PTX (2 mmol/L). TNF-alpha levels were measured by ELISA. MMP-9 was evaluated with zymography. Neutrophil CD66b expression was determined by flow cytometry in blood treated with HBSS, fMLP (1 micromol/L), supernatant + fMLP, or supernatant + fMLP + PTX., Results: TNF-alpha levels were elevated in both the LPS and supernatant + LPS groups (100%; P < 0.01 and 120%; P < 0.01, respectively). PTX administration resulted in a 106% decrease in TNF-alpha (P < 0.0001). MMP-9 levels were increased in all groups. Administration of PTX to the supernatant + LPS group generated a 33% decrease in MMP-9 levels, which was not statistically significant (P < 0.4). Upregulation of CD66b expression was seen in LPS and supernatant + LPS groups. Significant attenuation was seen with PTX (47%; P < 0.01)., Conclusions: PTX downregulates CD66b and TNF-alpha expression in supernatant-induced whole blood. Because blood transfusion can contribute to inflammatory injury, the adjunctive use of PTX may have therapeutic potential.

Background: Neutrophils play a major role as the first line in host defense after exposure to bacterial products. However, an exaggerated inflammatory response characterized by overwhelming neutrophil activation can be injurious to the host. Pentoxifylline (PTX), a nonspecific phosphodiesterase inhibitor, has been shown to attenuate neutrophil oxidative burst and decrease proinflammatory mediator synthesis. We hypothesized that PTX down-regulates neutrophil activation by decreasing the surface expression of both CD35 and CD66b, two markers of neutrophil degranulation., Materials and Methods: Venous blood was obtained from three healthy volunteers. Whole blood was incubated with HBSS (control), f-methionyl-leucyl-phenylalanine (fMLP, 1 microM/L), PTX (2 mM/L), or fMLP + PTX. CD35 and CD66b expression were measured by flow cytometry., Results: fMLP treatment caused a significant increase in CD35 and CD66b expression of when compared to controls (P < 0.01). PTX treatment revealed expression of both markers comparable to the control group. A 38% decrease in CD35 (64 +/- 12 versus 100; P < 0.01) and a 52% decrease in CD66b (48 +/- 7 versus 100; P < 0.01) expression were demonstrated in the fMLP + PTX group when compared to fMLP alone., Conclusion: In addition to the known effects of PTX on neutrophil oxidative burst, PTX also affects neutrophil degranulation, an essential step in enzyme release and subsequent tissue injury. These findings may have clinical relevance in the treatment of disease processes due to inflammation in which primed neutrophils play a role.

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 microg/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 alpha, interleukin (IL) 1beta, 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 alpha (56%), IL-1beta (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.

Background: Activated neutrophils play a central role in the pathogenesis of ARDS and multiple organ failure (MOF). Transfusion of packed red blood cells (PRBCs) is an independent risk factor in the development of ARDS and MOF. It has been postulated that factors present in the supernatant of PRBCs activate neutrophils. The magnitude of neutrophil activation is dependent on the age of the stored blood. Our laboratory and others have reported that pentoxifylline (PTX), a nonspecific phosphodiesterase inhibitor, decreases neutrophil activation. We hypothesized that adding PTX to PRBCs would attenuate blood transfusion-induced neutrophil activation., Study Design: Peripheral blood was obtained from healthy human volunteers. Oxidative burst, CD11b, and CD35 expression were measured by flow cytometry using a whole blood preparation. Whole blood was incubated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) (1 microM) alone and 42-day-old PRBC supernatant + fMLP with or without PTX (2 mmol/L)., Results: N-formyl-methionyl-leucyl-phenylalanine alone caused a significant increase in neutrophil oxidative burst (100%). The exposure of whole blood to PRBC supernatants + fMLP led to a 1.3-fold increase in neutrophil oxidative burst as compared with fMLP alone, indicating that PRBC supernatants prime neutrophils for oxidative burst by 75%. More importantly, PTX decreased neutrophil oxidative burst by 114% in supernatant + fMLP-stimulated whole blood (p < 0.001). PTX decreased CD11b expression in both fMLP (p < 0.01) and fMLP+supernatant-stimulated whole blood (p < 0.05). Supernatant from PRBCs did not have an additive effect to fMLP alone on CD11b expression. N-formyl-methionyl-leucyl-phenylalanine-induced CD35 expression was downregulated by PTX. The addition of PRBC supernatant did not increase the already upregulated fMLP-induced CD35 expression., Conclusions: Our results suggest that adding PTX to PRBC supernatant markedly decreases neutrophil activation. The lack of successful treatment strategies to effectively modulate the inflammatory response after blood transfusion indicates the need for novel therapies. Because the deleterious effects of blood transfusion on end-organ injury and MOF are associated with neutrophil activation, the adjunct use of PTX to blood transfusion may have therapeutic potential.

Background: Endotoxemia is accompanied by pro-inflammatory cytokine production, generation of reactive oxygen species, and end-organ injury. Pentoxifylline (PTX), a methylxanthine derivative and phosphodiesterase inhibitor, is known for its anti-inflammatory properties, including down-regulation of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha synthesis. Its effects on liver function and hepatic histology following acute endotoxemia have not been investigated fully. We hypothesized that PTX would preserve liver architecture and function after intravenous lipopolysaccharide (LPS) injection., Methods: Anesthetized Sprague-Dawley rats received an i.v. bolus injection of LPS (5 mg/kg), LPS + PTX (25 mg/kg), or saline (sham). Plasma concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), TNF-alpha, IL-6, and nitrite were measured at different time points after LPS injection. Liver injury was graded according to a scoring system in a blinded fashion from 0 (normal) to 4 (severe) for hepatocellular necrosis, hemorrhage, and parenchymal and sinusoidal inflammatory infiltrates. Neutrophil infiltration was measured by counting myeloperoxidase (MPO)-positive stained cells. Nuclear factor (NF)-kappaB p-65 was measured by counting positive stained nuclei of hepatocytes and Kupffer cells (KC). Inducible nitric oxide synthase (iNOS) was evaluated by positively stained KC. Data are presented as mean +/- SEM. Analysis of variance with p < 0.05 was considered statistically significant., Results: Animals treated with PTX showed a significant reduction in liver injury score and neutrophil infiltration. Treatment with PTX significantly decreased TNF-alpha, IL-6, and the concentrations of AST and ALT when compared to LPS alone. In addition, a significant decrease in NF-kappaB-positive staining in hepatocytes and KC, as well as in KC iNOS immunostaining was observed in PTX-treated animals compared to the LPS group., Conclusions: Pentoxifylline downregulates the inflammatory response significantly and decreases liver injury in acute endotoxemia.

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.

Fetal alcohol exposure causes severe neuropsychiatric problems, but mechanisms of the ethanol-associated changes in central nervous system development are unclear. In vivo, ethanol's interaction with N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid type A (GABA(A)) receptors may cause increased apoptosis in the immature forebrain. We examined whether ethanol affects survival of neonatal hippocampal neurons in primary cultures. A 6-day ethanol exposure killed hippocampal neurons with an LD50 of approximately 25 mM. Elevated extracellular potassium or insulin-related growth factor 1 inhibited cell loss. Although potentiation of GABA(A) receptors or complete block of NMDA receptors also kills hippocampal neurons, pharmacological studies suggest that ethanol's interaction with GABA(A) and NMDA receptors is not sufficient to explain ethanol's effects on neuronal survival. Ca(2+) influx in response to depolarization was depressed >50% by chronic ethanol treatment. We suggest that chronic ethanol may promote neuronal loss through a mechanism affecting Ca(2+) influx in addition to effects on postsynaptic GABA and glutamate receptors.

Neuronal activity elicits increases in intracellular Ca2+ in astrocytes, which in turn can elevate neuronal Ca2+ and potentiate the efficacy of excitatory synaptic transmission. Therefore, understanding the modulation of astrocyte Ca2+ elevations by neurotransmitters should aid in understanding astrocyte-neuronal interactions. On cultured hippocampal microislands containing only astrocytes, activation of metabotropic glutamate receptors (mGluRs) with the specific agonist 1S,3R-ACPD triggers Ca2+ elevations that are potentiated by adenosine A1 receptor activation. A1 receptor modulation of mGluR-induced Ca2+ elevations is blocked by pertussis toxin and is mimicked by the wasp venom peptide mastoparan, suggesting that potentiation occurs by means of a G(i/o) mechanism. Surprisingly, on microislands containing only astrocytes, A1 receptor antagonism or adenosine degradation suppresses mGluR-triggered Ca2+ elevations, strongly suggesting that astrocytes are a source of physiologically relevant concentrations of adenosine.