William H. Loomis

Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38.

Excessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion.

Lactated ringer's is superior to normal saline in a model of massive hemorrhage and resuscitation.

BACKGROUND Previous models comparing normal saline (NS) with lactated Ringers solution (LR) for resuscitation use only mild or moderate hemorrhage and do not address the clinical situation of massive hemorrhage and resuscitation (MHR). This work compares NS and LR by using a new rat model of MHR. METHODS NS and LR were compared by using both a traditional model of moderate pressure-controlled hemorrhage and a model of MHR. Moderate hemorrhage animals were bled to mean arterial pressure (MAP) = 60 mm Hg x 2 hour then resuscitated with crystalloid (NS or LR) for 1 hour. MHR animals were bled at a rate of 1 estimated blood volume (EBV) per hour for 2 hours with simultaneous resuscitation by using washed red blood cells (B) and crystalloid (LR+B or NS+B). MAP was kept at 60 mm Hg during the 2 hours of hemorrhage. Bleeding was then stopped, and animals were resuscitated for 1 additional hour with blood and crystalloid to MAP more than 90 mm Hg or until 10x EBV was given. Group means were compared with Students t test (p < 0.01 significant) and 2-week survival rates were compared by using Fishers exact test (p < 0.05 significant). RESULTS The moderate hemorrhage group was bled 36% of EBV. In this setting, resuscitation with NS and LR was equivalent. The final hematocrit, pH, and base excess were not different, and all animals survived in both groups. MHR animals were bled 218% of EBV. Animals resuscitated with NS+B were significantly more acidotic than animals resuscitated with equal volumes of LR+B (pH 7.14+/-.06 vs. 7.39+/-.04, respectively) and had significantly worse survival (50% vs. 100%, respectively). CONCLUSION With moderate hemorrhage, NS and LR are equivalent, but in the setting of massive hemorrhage and resuscitation, significantly more physiologic derangement and mortality occurs with NS than LR. LR is superior to NS for use in massive resuscitation.

Hypertonic Stress Increases T Cell Interleukin-2 Expression through a Mechanism That Involves ATP Release, P2 Receptor, and p38 MAPK Activation

Hypertonic stress (HS) can alter the function of mammalian cells. We have reported that HS enhances differentiated responses of T cells by increasing their ability to produce interleukin (IL)-2, a finding of clinical interest because hypertonic infusions may modulate immune function in patients. HS shrinks cells and mechanically deforms membranes, which results in ATP release from many cell types. Here we investigate if ATP release is an underlying mechanism through which HS augments T cell function. We found that mechanical stress and HS induced rapid ATP release from Jurkat T cells. HS and exogenous ATP mobilized intracellular Ca2+, activated p38 MAPK, and increased IL-2 expression. Ca2+ mobilization was attenuated in the presence of EGTA or by removal of extracellular ATP with apyrase. Adenosine did not increase IL-2 expression, as did ATP. Apyrase, inhibition of P2 receptors, or inhibition of p38 MAPK with SB203580 reduced the stimulatory effects of HS, indicating that HS enhances IL-2 expression through a mechanism that involves ATP release, P2 (perhaps P2X7) receptors, and p38 MAPK activation. We conclude that release of and response to ATP plays a key role in the mechanism through which hypertonic stress regulates the function of T cells.

Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells

The enteric nervous system may have an important role in modulating gastrointestinal barrier response to disease through activation of enteric glia cells. In vitro studies have shown that enteric glia activation improves intestinal epithelial barrier function by altering the expression of tight junction proteins. We hypothesized that severe injury would increase expression of glial fibrillary acidic protein (GFAP), a marker of enteric glial activation. We also sought to define the effects of vagal nerve stimulation on enteric glia activation and intestinal barrier function using a model of systemic injury and local gut mucosal involvement. Mice with 30% total body surface area steam burn were used as model of severe injury. Vagal nerve stimulation was performed to assess the role of parasympathetic signaling on enteric glia activation. In vivo intestinal permeability was measured to assess barrier function. Intestine was collected to investigate changes in histology; GFAP expression was assessed by quantitative PCR, by confocal microscopy, and in GFAP-luciferase transgenic mice. Stimulation of the vagus nerve prevented injury-induced intestinal barrier injury. Intestinal GFAP expression increased at early time points following burn and returned to baseline by 24 h after injury. Vagal nerve stimulation prior to injury increased GFAP expression to a greater degree than burn alone. Gastrointestinal bioluminescence was imaged in GFAP-luciferase transgenic animals following either severe burn or vagal stimulation and confirmed the increased expression of intestinal GFAP. Injection of S-nitrosoglutathione, a signaling molecule released by activated enteric glia cells, following burn exerts protective effects similar to vagal nerve stimulation. Intestinal expression of GFAP increases following severe burn injury. Stimulation of the vagus nerve increases enteric glia activation, which is associated with improved intestinal barrier function. The vagus nerve may mediate the signaling that occurs from the central nervous system to the enteric nervous system following gastrointestinal injury.

hypertonic Saline Infusion : can It Regulate Human Neutrophil Function?

The down-regulation of neutrophil adhesion molecule expression after hemorrhagic shock may reduce neutrophil-mediated organ injury. Hypertonic saline (HS) blocks neutrophil activation, and HS infusion in animals reduces organ injury. In this study, we investigated whether HS infusion in healthy human volunteers can affect neutrophil function. Healthy human volunteers were administered either 4 mL/kg of a 7.5% HS (n = 6) or normal saline (NS, 0.9%; n = 5) over 15 min. Mean arterial pressure (MAP) and plasma sodium levels were measured. Blood samples were obtained before and 1 h after fluid administration. Cells were stimulated with fMLP or left untreated. Neutrophil phagocytosis and expression of CD11b and L-selectin was determined with flow cytometry. HS infusion caused a 7 +/- 2 mM rise in plasma Na+ levels that was sustained at 6 +/- 1 mM for 60 min. MAP was affected only in one subject. HS and NS infusion had little effect on neutrophil phagocytosis. After HS infusion, CD1lb expression of unstimulated neutrophils was 26 +/- 6% lower than before HS infusion, and that of fMLP-stimulated cells was 12 +/- 2% lower compared to pre-infusion values. NS infusion had no significant effects on neutrophil CD11b expression. L-selectin expression of unstimulated cells after HS infusion was 9 +/- 3% higher than in the pre-infusion samples. These data suggest that HS infusion could indeed affect human neutrophils by suppressing CD11b expression. Although modest in healthy subjects, this effect may be more pronounced in trauma patients where reduced neutrophil-endothelial cell interactions might lessen neutrophil-mediated tissue damage.

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.

Acute lung injury in endotoxemic rats is associated with sustained circulating IL-6 levels and intrapulmonary CINC activity and neutrophil recruitment--role of circulating TNF-alpha and IL-beta?

Endotoxemia initiates a cytokine response that is thought to mediate the syndromes of sepsis and multiple organ failure. This study measured cytokine levels in the blood and airways of rats at critical time points during the development of lung injury induced by chronic endotoxin (LPS) infusion in the rat. Tumor necrosis factor-alpha (TNF), interleukin-1-beta (IL-1), and interleukin-6 (IL-6) were measured in the blood and bronchoalveolar lavage fluid (BALF) of endotoxemic and control animals. BALF was also studied for the percentage of neutrophil (PMN) count and chemotactic activity. Lung histology was determined at 72 h following infusion of LPS. Chronic endotoxemia of > or = 48 h but not < or = 24 h resulted in severe acute lung injury (ALI). Circulating levels of TNF and IL-1 were only transiently elevated, whereas IL-6 remained elevated in the endotoxemic rats. TNF, IL-1, and IL-6 levels in BALF were only transiently elevated. Chemotactic activity, levels of cytokine-induced neutrophil chemoattractant (CINC), and the percentage of PMN counts in BALF all increased significantly by 36 h. Other potential chemoattractants; leukotriene B4 and transforming growth factor-beta were not elevated in BALF. In conclusion, severe ALI requires a minimum of 48 h LPS infusion in this model and is associated with high levels of circulating IL-6, increased CINC activity, and an increased percentage of PMN count in BALF. Local inflammatory events may be as important as the systemic cytokine milieu in mediating ALI. The signal for these local events does not appear to depend solely on the transient elevations of circulating TNF and IL-1 at the onset of endotoxemia, although sustained high levels of IL-6 may be important.

Hypertonic/hyperoncotic fluids reverse prostaglandin E2 (PGE2)-induced T-cell suppression.

In recent years, hypertonic, and hyperoncotic fluids have been examined for their potential to replace conventional isotonic fluids. This study describes the effects of commonly used intravenous fluids on immune function. The action of increased concentrations of hypertonic saline (HTS), hypertonic saline-dextran (HSD), dextran (Dx), albumin (ALB), and hydroxyethylstarch (HET) on in vitro proliferation of phytohemagglutinin-stimulated normal and prostaglandin E2-suppressed human peripheral blood mononuclear cells was tested. At clinically relevant levels, HTS, HSD (20-40 mM hypertonicity), and ALB (2.5 mg/mL) enhanced T-cell proliferation by 65, 75, and 70%, respectively. Dx and HET had little effect. HTS also reversed prostaglandin E2-suppressed (10 ng/mL) T-cell proliferation to normal levels, and HSD enhanced T-cell proliferation by 40%, in contrast to Dx, ALB, and HET which had minimal effects. The results suggest that hypertonic/hyperoncotic solutions might improve prostaglandin-mediated suppression of T-cell function in patients and may be a useful adjunct to reduce the risk of infection.

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.

Role of p38 MAPK in burn-induced intestinal barrier breakdown.

BACKGROUND Severe burn results in intestinal barrier breakdown, which may lead to the generation of a systemic inflammatory response and distant organ injury. Intestinal barrier integrity is regulated, in part, by the tight junction protein myosin light chain kinase (MLCK). Previous studies in cell culture have shown that activation of p38 MAPK plays an important role in modulating intestinal barrier function. We hypothesized that (1) severe burn up-regulates p38 MAPK activation and results in increased intestinal permeability via augmented expression of MLCK, and (2) inhibition of p38 MAPK will prevent the burn-induced increase in MLCK expression, resulting in improved intestinal barrier integrity. MATERIALS AND METHODS Male Balb/c mice were subjected to a 30% total body surface area (TBSA) full thickness steam burn, then randomized to receive an intraperitoneal injection of a p38 MAPK inhibitor (SB203580, 25 mg/kg) or vehicle. In vivo intestinal permeability to 4kDa FITC-Dextran was measured. Expression of phosphorylated p38 MAPK, total p38 MAPK, MLCK, and phosphorylated MLC from intestinal extracts was assessed by immunoblotting. RESULTS Severe burn increased intestinal permeability, which was associated with activation of p38 MAPK, and increased expression of MLCK. Treatment with SB203580 significantly attenuated burn-induced intestinal permeability (212 microg/mL versus 81 microg/mL, P<0.05), and decreased expression of intestinal MLCK resulting in decreased phosphorylation of MLC. CONCLUSION p38 MAPK plays an important role in regulating burn-induced intestinal permeability through activation of MLCK. Inhibition of p38 MAPK may be an important therapeutic target aimed at attenuating intestinal barrier breakdown by preventing the burn-induced alterations in tight junction proteins.