Jessica Deree

Burn-induced gut barrier injury is attenuated by phosphodiesterase inhibition: Effects on tight junction structural proteins

Loss of intestinal barrier function after burn injury allows movement of intraluminal contents across the mucosa, which can lead to the development of distant organ injury and multiple organ failure. Tight junction function is highly regulated by membrane-associated proteins including occludin and zonula occludens protein 1 (ZO-1), which can be modulated by systemic inflammation. We hypothesized that (1) burn injury leads to gut barrier injury, and (2) phosphodiesterase inhibition will attenuate these burn-induced changes. Male balb/c mice undergoing a 30% steam burn were randomized to resuscitation with normal saline or normal saline + pentoxifylline (PTX; 12.5 mg/kg). Intestinal injury was assessed by histological diagnosis and TNF-&agr; levels using enzyme-linked immunosorbent assay. Intestinal permeability was assessed by measuring the plasma concentration of fluorescein isothiocyanate-dextran after intraluminal injection in the distal ileum. Occludin and ZO-1 levels were analyzed by immunoblotting and immunohistochemistry. Thirty percent total body surface area (TBSA) burn results in a significant increase in intestinal permeability. Treatment with PTX after burn attenuates intestinal permeability to sham levels. Burn injury resulted in a marked decrease in the levels of tight junction proteins occludin and ZO-1 at 6 and 24 h. The use of PTX after burn significantly decreases the breakdown of occludin and ZO-1. Pentoxifylline also attenuates the burn-induced increase in plasma and intestinal TNF-&agr;. Confocal microscopy demonstrates that PTX attenuates the burn-induced reorganization of occludin and ZO-1 away from the tight junction. Pentoxifylline attenuates burn-induced intestinal permeability and decreases the breakdown and reorganization of intestinal occludin and ZO-1. Therefore, phosphodiesterase inhibition may be a useful adjunct strategy in the attenuation of burn-induced gut barrier injury.

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.

Pentoxifylline attenuates pulmonary inflammation and neutrophil activation in experimental acute pancreatitis.

Objectives: Acute pancreatitis (AP) is associated with a systemic inflammatory response. Pentoxifylline (PTX) has been shown to attenuate neutrophil activation and end-organ injury in shock states such as hemorrhage and sepsis. We hypothesized that PTX would down-regulate AP-induced lung injury. Methods: Sprague-Dawley rats underwent catheterization of the pancreatic duct. Acute pancreatitis (n = 7) and AP/PTX animals (n = 7) received a retrograde infusion of 3.5% sodium taurocholate and intravenous treatment with normal saline or normal saline and PTX (25 mg/kg), respectively. Pulmonary neutrophil degranulation and sequestration were determined by zymography and detection of myeloperoxidase. Nuclear factor kappa B and mitogen-activated protein kinase phosphorylation was determined by Western blot. Cytokine-induced neutrophil chemoattractant was quantified by enzyme linked immunosorbent assay. Results: Pulmonary histologic injury scores were attenuated in the AP/PTX group (P < 0.05). Plasma amylase levels remained unchanged. Pentoxifylline produced a significant decline in myeloperoxidase content and matrix metalloproteinase activity (P < 0.05). The increase in the phosphorylation of pulmonary nuclear factor kappa B, p38 mitogen-activated protein kinase, and extracellular-related signal kinase 1/2 observed after AP was not demonstrated with PTX (P < 0.05). Pentoxifylline supplementation reduced pulmonary cytokine-induced neutrophil chemoattractant levels by 50% (P < 0.05). Conclusions: Pentoxifylline significantly attenuated histologic lung injury, pulmonary neutrophil activity, and proinflammatory signaling in a severe model of AP. Therefore, PTX may serve as an adjunct for the treatment of the inflammatory complications of severe AP.

Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers.

AIMS Under normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immunostimulated intestinal cell monolayers. MAIN METHODS Caco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-alpha, IFN-gamma, IL-1), or Cytomix+PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin. KEY FINDINGS Cytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix+PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization. SIGNIFICANCE Treatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown.

A novel fluid resuscitation strategy modulates pulmonary transcription factor activation in a murine model of hemorrhagic shock

INTRODUCTION: Combining the hemodynamic and immune benefits of hypertonic saline with the anti-inflammatory effects of the phosphodiesterase inhibitor pentoxifylline (HSPTX) as a hemorrhagic shock resuscitation strategy reduces lung injury when compared with the effects of Ringer’s lactate (RL). We hypothesized that HSPTX exerts its anti-inflammatory effects by interfering with nuclear factor kappa B/cAMP response element-binding protein (NF-κB-CREB) competition for the coactivator CREB-binding protein (CBP) in lung tissue, thus affecting pro-inflammatory mediator production. METHODS: Male Sprague-Dawley rats underwent 60 minutes of hemorrhagic shock to reach a mean arterial blood pressure of 35 mmHg followed by resuscitation with either RL or HSPTX (7.5% HS + 25 mg/kg PTX). After four hours, lung samples were collected. NF-κB activation was assessed by measuring the levels of phosphorylated cytoplasmic inhibitor of kappa B (I-κB) and nuclear NF-κB p65 by western blot. NF-κB and CREB DNA-binding activity were measured by electrophoretic mobility shift assay (EMSA). Competition between NF-κB and CREB for the coactivator CBP was determined by immunoprecipitation. Interleukin-8 (IL-8) levels in the lung were measured by ELISA. RESULTS: RL resuscitation produced significantly higher levels of lung IL-8 levels, I-κB phosphorylation, p65 phosphorylation, and NF-κB DNA binding compared with HSPTX. NF-κB-CBP-binding activity was similar in both groups, whereas CREB-CBP-binding activity was significantly increased with HSPTX. CREB-DNA binding-activity increased to a greater level with HSPTX compared with RL. DISCUSSION: HSPTX decreases lung inflammation following hemorrhagic shock compared with conventional resuscitation using RL through attenuation of NF-κB signaling and increased CREB-DNA binding activity. HSPTX may have therapeutic potential in the attenuation of ischemia-reperfusion injury observed after severe hemorrhagic shock.

Pentoxifylline modulates p47phox activation and downregulates neutrophil oxidative burst through PKA-dependent and -independent mechanisms.

Background and Aim: Pentoxifylline (PTX) has been proven to be an inhibitor of fMLP-induced neutrophil (PMN) oxidative burst and is thought to function by increasing cAMP and Protein kinase A (PKA). We hypothesized that PTX diminishes production of the neutrophil respiratory burst by both PKA-dependent and independent mechanisms. Material and Methods: Human neutrophils were isolated and stimulated with fMLP (1μM) alone or in combination with PTX (2mM). PMN activation was determined by the cytochrome C reduction method in the presence and absence of p38 MAPK (SB203580), ERK (PD98059), and PKA inhibitors (H89). Western blot analysis of Ras, Raf, p38 MAPK, ERK, and Akt was performed in PMNs exposed to fMLP and PTX. Cell membranes were fractionated to measure membrane-associated p47 phox. Treated cells were imaged using confocal microscopy to examine changes in localization of Akt and p47phox. Results: PTX produced a decrease in oxidative burst that was diminished but not abrogated by H89 exposure. The reduction in Ras, Raf, and Akt activation seen with PTX was not effected by the presence of H89. The ability of PTX to attenuate phosphorylation of p38 MAPK and ERK was significantly decreased in the presence of H89, suggesting a PKA-dependent mechanisms. Membrane fractions of neutrophils demonstrate that PTX decreased membrane-associated p47phox, thus diminishing the ability to generate oxidative burst. PTX also decreased membrane localization of Akt and p47phox by confocal microscopy. Conclusions: PTX attenuates activation of signaling molecules involved in activation of p47phox and suppress the subsequent assembly of the NADPH machinery through both PKA-dependent and PKA-independent mechanisms.

Hepatic transcription factor activation and proinflammatory mediator production is attenuated by hypertonic saline and pentoxifylline resuscitation after hemorrhagic shock.

BACKGROUND Fluid resuscitation can contribute to postshock inflammation and the development of end organ injury. We have previously observed an attenuation in pulmonary and ileal inflammation when hypertonic saline and pentoxifylline (HSPTX) were concomitantly administered after hemorrhage. We hypothesized that the attenuation in hepatic injury observed with HSPTX is associated with the reduction of transcription factor activation and proinflammatory mediator production when compared with Ringers lactate (RL). METHODS Male Sprague-Dawley rats were resuscitated with racemic RL (32 mL/kg) or HSPTX (4 mL/kg 7.5% NaCl + PTX 25 mg/kg) and killed at 4 hours and 24 hours after resuscitation. Liver injury was determined by histology and serum aminotransferases. Nitrite, tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-6 were measured with enzyme-linked immunosorbent assay. High mobility group box 1, inducible nitric oxide synthase, nuclear factor (NF)-kappaB phosphorylation, and signal transducers and activators of transcription-3 phosphorylation were determined by Western blot. Transcription factor activation was verified with Electrophoretic Mobility Shift Assay. RESULTS RL resuscitation led to significant increases all measured parameters when compared with control. In contrast, HSPTX did not induce elevations in histologic liver injury or alanine aminotransferase levels. HSPTX attenuated inducible nitric oxide synthase by 23% (p < 0.01), nitrite by 25% (p < 0.05), tumor necrosis factor-alpha by 25% (p < 0.05), IL-1 by 63% (p < 0.01), IL-6 by 35% (p < 0.05), and high mobility group box 1 by 39% (p < 0.05) when compared with RL. HSPTX reduced IkappaB-alpha phosphorylation by 34% (p < 0.05), NF-kappaB p65 phosphorylation by 75% (p < 0.01), and signal transducers and activators of transcription-3 phosphorylation by 52% (p < 0.01). CONCLUSIONS The reduction in liver injury observed with HSPTX resuscitation after hemorrhage is associated with attenuation transcription factor activation and proinflammatory mediators. HSPTX has the potential to be a superior resuscitation fluid with significant immunomodulatory properties.

Pentoxifylline attenuates leukoreduced stored blood-induced neutrophil activation through inhibition of mitogen-activated protein kinases

CONTEXT Transfusion of packed red blood cells is an independent risk factor for postoperative bacterial infections and multiple organ failure. MATERIALS AND METHODS Whole blood was obtained from healthy volunteers to investigate the role of mitogen associated protein kinase (MAPK) pathways in PRBC-induced neutrophil respiratory burst, hypothesizing that the attenuating effects of pentoxifylline on this process are due to modulations in MAPK-associated signaling. RESULTS Pre-incubation of neutrophils with supernatant prior to fMLP stimulation increased p38 MAPK and ERK phosphorylation over fMLP alone pentoxifylline significantly reduced p38MAPK and ERK phosphorylation in similarly stimulated neutrophils. CONCLUSION The addition of pentoxifylline stored red blood cells attenuates neutrophil activation.

Pentoxifylline-supplemented resuscitation attenuates both early and late mediators of hepatic inflammation after hemorrhagic shock

Objective. After hemorrhage, resuscitation with racemic Ringers lactate (RL) results in hepatic injury and an increase in pro-inflammatory mediator synthesis. The aim of this study was to determine the effect of resuscitation including pentoxifylline (PTX) on mediators of hepatic inflammation after hypovolemic shock. Material and methods. Male Sprague–Dawley rats underwent 1 h of hypotension followed by resuscitation with RL or RL+PTX (25 mg/kg). Liver sections were scored for injury histologically. Nitric oxide and pro-inflammatory cytokines were measured using an enzyme-linked immunosorbent assay. Inducible nitric oxide synthase (iNOS) and high mobility group box 1 (HMGB1) content were evaluated by means of Western blotting. Results. In the presence of PTX, histologic hepatic injury was markedly less than with RL resuscitation (p<0.05). Furthermore, the addition of PTX to RL significantly attenuated the hepatic concentrations of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, iNOS, NO and HMGB1 (p<...

Phosphodiesterase inhibition downregulates intestinal injury and inducible nitric oxide synthase activity after hemorrhagic shock

Objective. Previous work suggests that supplementation of Ringers lactate (RL) resuscitation with pentoxifylline (PTX) abrogates distant organ injury after hemorrhage. The aim of this work was to determine whether PTX can effectively attenuate resuscitation-induced intestinal injury and pro-inflammatory mediator production. Material and methods. Male Sprague–Dawley rats underwent 60 min of hypotension followed by resuscitation with RL or RL + PTX (25 mg/kg). The ileum was examined for injury. Nitric acid and pro-inflammatory cytokines were measured using an enzyme-linked immunosorbent assay. Inducible nitric oxide synthase (iNOS) and phosphorylation of nuclear factor (NF)-κB and signal transducer and activator of transcription (STAT)-3 were evaluated using Western blotting. Results. RL resuscitation led to a marked increase in all parameters over the control. The addition of PTX significantly reduced histologic injury and levels of iNOS, NO, tumor necrosis factor-α, interleukin-6, and cytokine-induced ne...