Iris Garcia

Randomized, Prospective Trial of Antioxidant Supplementation in Critically Ill Surgical Patients

ObjectiveTo determine the effectiveness of early, routine antioxidant supplementation using α-tocopherol and ascorbic acid in reducing the rate of pulmonary morbidity and organ dysfunction in critically ill surgical patients.Summary Background DataOxidative stress has been associated with the develo

Porphyromonas gingivalis Lipopolysaccharide Is Both Agonist and Antagonist for p38 Mitogen-Activated Protein Kinase Activation

ABSTRACT Lipopolysaccharide (LPS) is a key inflammatory mediator. It has been proposed to function as an important molecule that alerts the host of potential bacterial infection. Although highly conserved, LPS contains important structural differences among different bacterial species that can significantly alter host responses. For example, LPS obtained from Porphyromonas gingivalis, an etiologic agent for periodontitis, evokes a highly unusual host cell response. Human monocytes respond to this LPS by the secretion of a variety of different inflammatory mediators, while endothelial cells do not. In addition, P. gingivalis LPS inhibits endothelial cell expression of E-selectin and interleukin 8 (IL-8) induced by other bacteria. In this report the ability of P. gingivalis LPS to activate p38 mitogen-activated protein (MAP) kinase was investigated. It was found that p38 MAP kinase activation occurred in response to P. gingivalis LPS in human monocytes. In contrast, no p38 MAP kinase activation was observed in response to P. gingivalis LPS in human endothelial cells or CHO cells transfected with human Toll-like receptor 4 (TLR-4). In addition, P. gingivalis LPS was an effective inhibitor of Escherichia coli-induced p38 MAP kinase phosphorylation in both endothelial cells and CHO cells transfected with human TLR-4. These data demonstrate that P. gingivalis LPS activates the LPS-associated p38 MAP kinase in monocytes and that it can be an antagonist for E. coli LPS activation of p38 MAP kinase in endothelial and CHO cells. These data also suggest that although LPS is generally considered a bacterial component that alerts the host to infection, LPS from P. gingivalis may selectively modify the host response as a means to facilitate colonization.

Hypertonic Preconditioning Inhibits Macrophage Responsiveness to Endotoxin

Hypertonic saline has been shown to modulate cell shape and the response of components of the innate immune response. However, the effect of hypertonic saline on the macrophage remains unknown. We hypothesized that hypertonic preconditioning would impair subsequent inflammatory mediator signaling through a reduction in stress fiber polymerization and mitogen-activated protein kinase activity after LPS stimulation. Rabbit alveolar macrophages were stimulated with 100 ng/ml of LPS. Selected cells were preconditioned with 40–100 mM of NaCl, mannitol, or urea for 4 h and returned to isotonic medium before LPS stimulation. Cellular protein was harvested and subjected to Western blot analysis for the dually phosphorylated active forms of p38 and extracellular signal-related kinase (ERK) 1/2. TNF production was determined by an L929 bioassay, and stress fiber polymerization was evaluated by confocal microscopy. Preconditioning of macrophages with NaCl or mannitol resulted in dose-dependent reduction in ERK 1/2 phosphorylation with no effect on p38 phosphorylation. Urea preconditioning had no effect on either mitogen-activated protein kinase. A dose-dependent attenuation of TNF production was seen with NaCl and mannitol preconditioning (p < 0.05), but not with urea. NaCl and mannitol preconditioning resulted in failure of LPS-induced stress fiber polymerization, whereas urea did not. Extracellular hypertonic conditions (i.e., NaCl and mannitol) have an immunomodulatory effect on macrophages, demonstrated through failure of optimal stress fiber polymerization, ERK 1/2 activity, and TNF production. Intracellular hypertonic conditions (i.e., urea) had no significant effect. Hypertonic saline or mannitol resuscitation, therefore, may help protect against multiple-organ dysfunction syndrome as a result of this reduced proinflammatory responsiveness.

Modulation of macrophage responsiveness to lipopolysaccharide by IRAK-1 manipulation.

Local activation of the macrophage by endotoxin is essential for the eradication of invasive gram-negative infections. Circulating endotoxin at lower concentrations results in immune cell activation at distant sites leading to tissue injury. Although the cellular mechanisms involved in these potentially dissimilar events are incomplete, it appears that the proximal kinase IRAK-1 plays a role. Thus, sense and antisense IRAK-1 oligonucleotides were used to determine the role IRAK-1 plays in macrophage activation by systemic (1–100 ng/mL) and local (1000 ng/mL) concentration of lipopolysaccharide (LPS) within THP-1 cells. Within the sense group, 1–1000 ng/mL of LPS within the sense group resulted in cellular activation of ERK-1/2, p38, and JNK/SAPK and the nuclear activation of NF-&kgr;B and AP-1. This activation was associated with proinflammatory cytokine production and cellular spreading. Systemic concentrations of LPS within the antisense group were associated with significant attenuation of intracellular signaling, cytokine production, and cellular spreading compared with the sense group. Local concentrations of LPS within the antisense group, however, were associated only with a delay in intracellular signaling, with no effect on cytokine production or cell spreading compared with the sense group. Based on these results, it appears that IRAK-1 is essential to macrophage activation at systemic, but not local, concentrations of LPS. These data suggest that redundant pathways exist that are functional at higher concentrations of LPS. Therefore, IRAK-1 appears to be the central kinase involved in the activation of the macrophage at distant sites during septic shock but is not necessary for activation in areas of local infection.

Interactions of Calcium/Calmodulin-dependent protein kinases (CaMK) and Extracellular-Regulated Kinase (ERK) in monocyte adherence and TNFα production

ABSTRACT The circulating monocyte possesses a markedly different functional phenotype relative to the macrophage (M&phis;). The adhesive interactions encountered by the monocyte, en route to the inflammatory focus, generate signals that culminate in the expression of a pro‐inflammatory M&phis; phenotype, marked by enhanced cytokine production. Previously, we demonstrated that calcium and calmodulin are essential for maximal M&phis; activation and, in particular, TNF&agr; production. These effects are likely to be mediated through signal transduction kinases that require the calcium/calmodulin complex. Here, we investigated the effect of adherence on calcium/calmodulin‐dependent protein kinase (CaMK) II and IV activation of the extracellular‐signal regulated kinase (ERK) 1/2 cascade and on lipopolysaccharide (LPS)‐induced TNFa production by human monocytes. Adherence activated ERK 1/2 and led to an 8‐fold potentiation in LPS‐induced TNF&agr; production over similarly stimulated non‐adherent cells. Inhibition of CaMK II prior to adherence prevented ERK 1/2 activation and attenuated by up to 40%, the TNF&agr; response to subsequent LPS stimulation. CaMK II inhibition after adherence, however, failed to modify cytokine release. Inhibition of CaMK IV, both after adherence and in non‐adherent monocytes, significantly inhibited LPS‐induced ERK 1/2 activation and abrogated TNF&agr; production by up to 75%. These data suggest that the function of CaMK II in TNF&agr; production by adherent monocytes occurs during adhesion, is mediated in part by activation of ERK 1/2, and appears to “prime” the monocyte for enhanced cytokine production. CaMK IV, through activation of ERK 1/2, appears to have a direct role in the LPS signal transduction for TNF&agr; production.

Antioxidants attenuate endotoxin-induced activation of alveolar macrophages*

BACKGROUND Endotoxin (lipopolysaccharide [LPS]) stimulation of tissue-fixed macrophages induces the generation of toxic oxidants. However, recent studies also implicate redox changes in both the signal transduction pathways for cytokine genes and the generation of physiologically active arachidonic acid metabolites. Because cytokines and arachidonic acid metabolites initiate and perpetuate deleterious systemic inflammatory responses, we tested whether macrophage activation could be modulated by antioxidants. METHODS Rabbit alveolar macrophages were obtained by bronchoalveolar lavage, isolated, treated with the antioxidants vitamin E or N-acetylcysteine (NAC), and stimulated with an optimal dose of LPS (10 ng/ml). Assays were performed for tumor necrosis factor (TNF), procoagulant activity, and prostaglandin E2. Total cellular RNA was extracted for Northern blot analysis of TNF messenger RNA. RESULTS Exposure of the macrophage to the antioxidants vitamin E and NAC inhibited TNF production, accumulation of TNF messenger RNA, procoagulant activity expression, and prostaglandin E2 production. CONCLUSIONS Macrophage signal transduction of LPS is dependent on the generation of reactive oxygen intermediates that can be blocked both at the level of the lipid membrane (vitamin E) and at the intracellular level (NAC). This suggests a potential therapeutic role for antioxidants is disease states such as adult respiratory distress syndrome and multiple organ failure syndrome, which are characterized by excessive macrophage activation.

Slow channel calcium inhibition blocks proinflammatory gene signaling and reduces macrophage responsiveness

BACKGROUND This study investigates the possible intracellular mechanisms responsible for calcium antagonist protection in tissue-fixed macrophages, a central modulator of the proinflammatory phenotype. METHODS Rabbit alveolar macrophages were exposed to lipopolysaccharide in the presence of different specific calcium antagonists. Cellular and nuclear protein were extracted and analyzed by Western blot for the phosphorylated forms of PYK2, ERK 1/2, and p38, and nuclear translocation of NF-kappaB and AP-1. Tumor necrosis factor-alpha (TNF-alpha) expression was measured by an L929 bioassay on cellular supernatants. Statistical analysis was performed by unpaired Students t tests. RESULTS Cells pretreated with 100 to 500 micromol/L of diltiazem or 50 to 100 micromol/L of verapamil, both slow channel calcium blockers, led to dose-dependent reductions in lipopolysaccharide-induced PYK2 and ERK 1/2 phosphorylation, and nuclear translocation of AP-1 when compared with controls (p < 0.05). Neither inhibitor had any significant effect on p38 or NF-kappaB translocation. EGTA an extracellular calcium chelator, had no significant effect on any intracellular process studied. A dose-dependent reduction in TNF-alpha production was demonstrated with diltiazem and verapamil (p < 0.05), with no effect induced by EGTA. CONCLUSION Slow channel calcium influx is essential for optimal intracellular signaling through PYK2 and ERK 1/2. This reduced intracellular signaling correlated with reduced AP-1 translocation and TNF-alpha production. Extracellular calcium chelation had no significant effect on intracellular signaling or TNF-alpha production. This study further elucidates the protective mechanism of action of calcium channel blockade by diltiazem and verapamil by reducing intracellular calcium release and down-regulating the excessive proinflammatory phenotype.

Role of the mTOR Pathway in LPS-Activated Monocytes: Influence of Hypertonic Saline

BACKGROUND As heightened protein synthesis is the hallmark of many inflammatory syndromes, we hypothesize that the mammalian target of rapamycin (mTOR) pathway, which control the cap-dependent translation initiation phase, was activated by lipopolysaccharide (LPS). In addition, we studied the effect of hypertonic saline solution (HTS) on the mTOR cascade in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS PBMCs were isolated from healthy volunteers and treated with LPS. Cells were pretreated with phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors, or with HTS. Supernatants were harvested 20 h following LPS treatment, and interleukin-10 (IL-10), interleukin-6 (IL-6) and tumor necrosis alpha (TNFα) were analyzed by ELISA. Immunoblot experiments were performed for components of the PI3K/Akt/mTOR pathway at various time points. RNA was extracted after 90 min for real-time RT-PCR quantification. RESULTS The mTOR pathway is activated in PBMCs within 1 h of LPS stimulation. Pretreatment with rapamycin, a specific inhibitor of mTOR, resulted in a significant decrease of IL-10 and IL-6 translation and expression but did not affect the LPS-induced TNFα production. Both the mTOR pathway and the LPS-induced IL-6 production were down-regulated by HTS pretreatment. CONCLUSIONS The PI3k/Akt/mTOR cascade modulates LPS-induced cytokines production differentially. IL-10 and IL-6 expression are both up-regulated by activation of the mTOR pathway in response to LPS in PBMCs, while TNFα is not controlled by the mTOR cascade. Meanwhile, pretreatment of PBMCs with a HTS solution suppresses mTOR activity as well as LPS-induced IL-6, suggesting a more central role for mTOR as a regulator of the immuno-inflammatory response.

Oxidants augment endotoxin-induced activation of alveolar macrophages

Endotoxin (lipopolysaccharide) stimulation of macrophages (Mø) induces the generation of toxic reactive oxygen intermediates (ROI); however, recent studies implicate intracellular redox changes in signal transduction pathways for cytokines. To test whether oxidant stress modulates Mø activation, rabbit alveolar Mø were exposed to the following: diamide (oxidizes intracellular glutathione); glucose oxidase (generates hydrogen peroxide); or xanthine oxidase (generates superoxide), before lipopolysaccharide. Supernatants were assayed for tumor necrosis factor (TNF) and cell lysates were assayed for procoagulant activity (PCA). TNF mRNA was analyzed by Northern blot. Mø exposure to diamide and glucose oxidase augmented TNF production, PCA expression, and TNF mRNA accumulation; however, xanthine oxidase exposure inhibited TNF production while augmenting PCA expression. Mø signal transduction can be enhanced by increasing cellular oxidant stress. The differential response of TNF versus PCA suggests the existence of distinct redox-sensitive signal transduction pathways. These data define a mechanism by which oxidants generated during inflammation may modulate Mø function.

Microfluidic Leukocyte Isolation for Gene Expression Analysis in Critically Ill Hospitalized Patients

BACKGROUND Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood. METHODS We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique. RESULTS When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals. CONCLUSIONS The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting.