Phenazine-1-carboxylic acid,a secondary metabolite of Pseudomonas aeruginosa,alters expression of immunomodulatory proteins by human airway epithelial cells

Pseudomonas aeruginosa is a gram-negative bacterium that causes both acute and chronic lung disease in susceptible patient populations. P. aeruginosa secretes numerous proteins and secondary metabolites, many of which have biological effects that likely contribute to disease pathogenesis. An unidentified small-molecular-weight factor was previously reported to increase IL-8 release both in vitro and in vivo. To identify this factor, we subjected the <3-kDa fraction from P. aeruginosa-conditioned medium to HPLC analysis. A peak fraction that stimulated IL-8 release was found by mass spectrometry to have a molecular mass (MM) of 224 Da. On the basis of this MM and other biochemical properties, we hypothesized that the factor was phenazine-1-carboxylic acid (PCA). Subsequent studies and comparison with purified PCA confirmed this hypothesis. Purified PCA exhibited a number of biological effects in human airway epithelial cells, including increasing IL-8 release and ICAM-1 expression, as well as decreasing RANTES and monocyte chemoattractant protein-1 (MCP-1) release. PCA also increased intracellular oxidant formation as measured by electron paramagnetic resonance and by an intracellular oxidant-sensitive probe. Antioxidants inhibited PCA-dependent increases in IL-8 and ICAM-1, suggesting that oxidants contributed to these effects. However, in contrast to the related phenazine compound pyocyanin, PCA did not oxidize NAD(P)H at physiologically relevant pH, providing preliminary evidence that PCA and pyocyanin may have distinct redox chemistries within the cell. Thus PCA is a biologically active factor secreted by P. aeruginosa that has several activities that could alter the host immune and inflammatory response and thereby contribute to bacterial disease pathogenesis.     

Pyocyanin production by Pseudomonas aeruginosa induces neutrophil apoptosis and impairs neutrophil-mediated host defenses in vivo

Clearance of neutrophils from inflamed sites is critical for resolution of inflammation, but pathogen-driven neutrophil apoptosis can impair host defenses. We previously showed that pyocyanin, a phenazine toxic metabolite produced by Pseudomonas aeruginosa, accelerates neutrophil apoptosis in vitro. We compared wild-type and pyocyanin-deficient strains of P. aeruginosa in a murine model of acute pneumonia. Intratracheal instillation of either strain of P. aeruginosa caused a rapid increase in bronchoalveolar lavage neutrophil counts up to 18 h after infection. In wild-type infection, neutrophil numbers then declined steadily, whereas neutrophil numbers increased up to 48 h in mice infected with pyocyanin-deficient P. aeruginosa. In keeping with these differences, pyocyanin production was associated with reduced bacterial clearance from the lungs. Neutrophil apoptosis was increased in mice infected with wild-type compared with the phenazine-deficient strain or two further strains that lack pyocyanin production, but produce other phenazines. Concentrations of potent neutrophil chemokines (MIP-2, KC) and cytokines (IL-6, IL-1beta) were significantly lower in wild-type compared with phenazine-deficient strain-infected mice at 18 h. We conclude that pyocyanin production by P. aeruginosa suppresses the acute inflammatory response by pathogen-driven acceleration of neutrophil apoptosis and by reducing local inflammation, and that this is advantageous for bacterial survival.     

绿脓菌素激活MAPKs、NF-KB信号通路诱导呼吸道上皮细胞IL-8表达

采用绿脓杆菌培养上清及绿脓菌素刺激人呼吸道上皮细胞株A549和SPC-A-1,用ELISA方法检测细胞IL-8分泌水平,并使用免疫印迹(Western blot)方法观察绿脓菌素对细胞内重要的炎症信号传导途径NF—κB及丝裂原激活蛋白激酶(MAPKs)的激活作用。实验发现,绿脓杆菌培养上清及绿脓菌素可诱导呼吸道上皮细胞株IL-8分泌增加,且具有剂量依赖效应。绿脓菌素刺激细胞可使细胞内IκB—α发生降解,同时使MAPK家族蛋白分子(ERK1／2、p38、JNK)发生磷酸化。MEK1／2(ERK1／2激酶)抑制剂U0126(10μmol／L)和p38MAPK抑制剂SB203580(10μmol／L)可降低绿脓菌素诱导A549细胞IL-8的合成。以上结果显示绿脓菌素通过MAPK信号传导通路增强呼吸道上皮细胞IL-8的表达;NF-κB通路也参与了绿脓菌素调控细胞IL-8表达的过程。     

Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1

Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.     

Induction of neutrophil apoptosis by the Pseudomonas aeruginosa exotoxin pyocyanin: a potential mechanism of persistent infection

Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 microM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.     

Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta

The mechanisms of neutrophil (PMN) recruitment to Pseudomonas aeruginosa infection remain incompletely defined. Mast cells (MC) involvement in this process has not been studied previously. In this study, we demonstrate that human cord blood-derived MC phagocytose P. aeruginosa and release mediators that activate HUVEC monolayers for supporting PMN transmigration. Pretreatment of supernatants from P. aeruginosa-MC cocultures with neutralizing anti-IL-1alpha plus anti-IL-1beta Abs, or IL-1R antagonist before addition to HUVEC for stimulation completely abrogated MC-induced PMN transmigration, while anti-TNF-alpha treatment had no effect. The expression of E-selectin and ICAM-1 on HUVEC, the latter a ligand for PMN CD11/CD18, was significantly up-regulated by P. aeruginosa-induced MC mediators. Pretreatment of human PMN with anti-CD18 mAb or pretreatment of HUVEC with a combination of three mAbs (against ICAM-1, ICAM-2, and E-selectin) inhibited by 85% the MC-dependent PMN transmigration. Moreover, P. aeruginosa-induced production of IL-1alpha and IL-1beta was down-regulated by IL-10 and dexamethasone. This study demonstrates for the first time that MC may mediate P. aeruginosa-induced PMN recruitment via production of IL-1alpha and beta. These findings have important implications for diseases involving P. aeruginosa infection and suggest novel targets for modulating P. aeruginosa-induced inflammation.     

Upregulation of HLA class II, but not intercellular adhesion molecule 1 (ICAM-1) by granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3) in synergy with dexamethasone.

In this study we have compared the effects of granulocyte macrophage colony stimulating factor (GM-CSF) on purified normal blood monocytes, with two other haemopoietic growth factors, Interleukin (IL-) 3 and Macrophage (M-)CSF on HLA class I, class II and intercellular adhesion molecule 1 (ICAM-1) expression in the presence and absence of dexamethasone (Dex). IL-3 alone, like GM-CSF, was a weak inducer of HLA class II expression but in combination with Dex markedly enhanced HLA-DR, DP and DQ expression. Similar changes were observed for HLA class I expression. The response to both IL-3 and GM-CSF was not additive in the presence of an optimal concentration of one cytokine and titrating concentrations of the other indicating that they may use common receptors and signal transduction mechanisms. Although IL-3 or GM-CSF alone also enhanced ICAM-1 expression, Dex inhibited both constitutive and the cytokine induced expression of this antigen. In contrast M-CSF, in the presence or absence of Dex, failed to enhance ICAM-1, HLA class I or II expression. These observations further highlight differences between the effects of the haemopoietic growth factors GM-CSF and IL-3 versus M-CSF in the regulation of monocyte function. Finally, the distinct effect of a combination of glucocorticoids with GM-CSF or IL-3 to induce high levels of HLA expression on human monocytes suggests they may have an important role during inflammatory conditions in vivo.     

IL-27 induces Th1 differentiation via p38 MAPK/T-bet- and intercellular adhesion molecule-1/LFA-1/ERK1/2-dependent pathways

IL-27, a novel member of the IL-6/IL-12 family, activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130 subunits, resulting in positive and negative regulations of immune responses. We recently demonstrated that IL-27 induces Th1 differentiation through ICAM-1/LFA-1 interaction in a STAT1-dependent, but T-bet-independent mechanism. In this study, we further investigated the molecular mechanisms by focusing on p38 MAPK and ERK1/2. IL-27-induced Th1 differentiation was partially inhibited by lack of T-bet expression or by blocking ICAM-1/LFA-1 interaction with anti-ICAM-1 and/or anti-LFA-1, and further inhibited by both. Similarly, the p38 MAPK inhibitor, SB203580, or the inhibitor of ERK1/2 phosphorylation, PD98059, partially suppressed IL-27-induced Th1 differentiation and the combined treatment completely suppressed it. p38 MAPK was then revealed to be located upstream of T-bet, and SB203580, but not PD98059, inhibited T-bet-dependent Th1 differentiation. In contrast, ERK1/2 was shown to be located downstream of ICAM-1/LFA-1, and PD98059, but not SB203580, inhibited ICAM-1/LFA-1-dependent Th1 differentiation. Furthermore, it was demonstrated that STAT1 is important for IL-27-induced activation of ERK1/2, but not p38 MAPK, and that IL-27 directly induces mRNA expression of growth arrest and DNA damage-inducible 45gamma, which is known to mediate activation of p38 MAPK. Finally, IL-12Rbeta2 expression was shown to be up-regulated by IL-27 in both T-bet- and ICAM-1/LFA-1-dependent mechanisms. Taken together, these results suggest that IL-27 induces Th1 differentiation via two distinct pathways, p38 MAPK/T-bet- and ICAM-1/LFA-1/ERK1/2-dependent pathways. This is in contrast to IL-12, which induces it via only p38 MAPK/T-bet-dependent pathway.     

IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-gamma.

Alteration in the surface membrane of endothelial cells (EC) is a feature of endothelial activation both at sites of inflammation in vivo and after stimulation with cytokines in vitro. The effects of stimulating EC with IL-1 or TNF include enhanced adhesiveness for polymorphonuclear leukocytes (PMN) and T cells, the induction of EC leukocyte adhesion molecule-1 (ELAM-1) expression, and the increased expression of intercellular adhesion molecule-1 (ICAM-1) and the 1.4C3 Ag. In contrast, IFN-gamma stimulation increases EC binding of T cells but not PMN and enhances ICAM-1 expression but not ELAM-1 or 1.4C3 Ag expression. Recently we have reported that the T cell-derived cytokine IL-4 also increases EC adhesiveness for T cells but not PMN. In this study we have examined the effect of IL-4 on the expression of several cytokine-inducible EC activation Ag, by using a previously described ELISA technique. IL-4 modulation of activation Ag expression was concentration dependent, optimal at around 100 U/ml, and exhibited a unique pattern compared to that seen with the other cytokines. Although, IL-4 stimulation increased 1.4C3 Ag expression (p less than 0.001), it significantly inhibited constitutive ICAM-1 expression (p less than 0.01) and did not induce ELAM-1. Furthermore, IL-4 exhibited significant synergy with IL-1 or TNF in inducing 1.4C3 Ag expression (p less than 0.001) but inhibited the increased expression of ICAM-1 produced by IL-1, TNF, or IFN-gamma (p less than 0.01) and inhibited the induction of ELAM-1 by IL-1 and TNF (p less than 0.001). In contrast, IL-4 had no effect on the expression of EC HLA-class I, -DR, -DP, or -DQ and neither enhanced nor inhibited the effect of IFN-gamma on the expression of these molecules. Finally, although IL-4 alone caused little if any shape change in EC monolayers, it strongly synergized with TNF or IFN-gamma in causing a change in shape to a more fibroblastic morphology. These observations indicate that IL-4 increases EC adhesiveness for T cells by the induction of a different adhesion molecule to ICAM-1. Furthermore, the ability of IL-4 to both enhance and inhibit the expression of activation Ag on EC already activated by IL-1, TNF, or IFN-gamma suggests that it may be important in altering the quality of inflammatory responses such as may occur during the development and maintenance of chronic or immune-mediated inflammation.     

Effect of cytokines on ICAM-1 and ZO-1 expression on human airway epithelial cells

The presence of adhesion molecules on airway epithelial cells may be important in recruiting leukocytes to the epithelium. The study aimed at investigating the effects of interleukin (IL)-4, IL-8, IL-13 and interferon-gamma (IFN-gamma) on cell viability and intracellular adhesion molecule (ICAM)-1 and zonula occludens protein (ZO)-1 expression on cultured human basal and columnar airway epithelial cells. Cycloheximide (CHX) induced cell death in both cell lines. The cytokines IL-4, IL-8, IL-13 and IFN-gamma had only minor effects on cell proliferation in the columnar 16HBE14o-cells, and inhibited the effects of CHX on cell death. IFN-gamma increased ICAM-1 expression in both cell lines. Western blot analysis showed that CHX inhibited both ICAM-1 and ZO-1 expression in the basal cell line. A combination of IL-4 and IFN-gamma appeared to break the tight junctions. IL-4 and IL-13 potentiated CHX-induced apoptosis in basal cells but not in columnar cells, possibly due to low levels of the IL-4 receptor. It is concluded that cytokines produced by airway epithelium may have a role in regulating sequestering of leukocytes to the airways during airway inflammation.     

The Pseudomonas toxin pyocyanin inhibits the dual oxidase-based antimicrobial system as it imposes oxidative stress on airway epithelial cells

The dual oxidase-thiocyanate-lactoperoxidase (Duox/SCN(-)/LPO) system generates the microbicidal oxidant hypothiocyanite in the airway surface liquid by using LPO, thiocyanate, and Duox-derived hydrogen peroxide released from the apical surface of the airway epithelium. This system is effective against several microorganisms that infect airways of cystic fibrosis and other immunocompromised patients. We show herein that exposure of airway epithelial cells to Pseudomonas aeruginosa obtained from long-term cultures inhibits Duox1-dependent hydrogen peroxide release, suggesting that some microbial factor suppresses Duox activity. These inhibitory effects are not seen with the pyocyanin-deficient P. aeruginosa strain PA14 Phz1/2. We show that purified pyocyanin, a redox-active virulence factor produced by P. aeruginosa, inhibits human airway cell Duox activity by depleting intracellular stores of NADPH, as it generates intracellular superoxide. Long-term exposure of human airway (primary normal human bronchial and NCI-H292) cells to pyocyanin also blocks induction of Duox1 by Th2 cytokines (IL-4, IL-13), which was prevented by the antioxidants glutathione and N-acetylcysteine. Furthermore, we showed that low concentrations of pyocyanin blocked killing of wild-type P. aeruginosa by the Duox/SCN(-)/LPO system on primary normal human bronchial epithelial cells. Thus, pyocyanin can subvert Pseudomonas killing by the Duox-based system as it imposes oxidative stress on the host. We also show that lactoperoxidase can oxidize pyocyanin, thereby diminishing its cytotoxicity. These data establish a novel role for pyocyanin in the survival of P. aeruginosa in human airways through competitive redox-based reactions between the pathogen and host.     

Hepatic leukocyte recruitment in response to time-limited expression of TNF-alpha and IL-1beta

The development of chronic liver diseases is mediated by sustained hepatic inflammation. Our objective was to characterize the molecular mechanisms responsible for the hepatic inflammatory response to time-limited TNF-alpha and IL-1beta expression. C57Bl/6 mice were injected with 2 x 10(7) plaque forming units intraperitoneally of an adenoviral vector containing TNF-alpha or IL-1beta (AdTNF-alpha or AdIL-1beta). A nonreplicating adenoviral vector served as control. Four days later, under ketamine and xylazine anesthesia, the liver microvasculature was examined by intravital microscopy. In the postsinusoidal venules, leukocyte rolling increased significantly in response to both AdTNF-alpha and AdIL-1beta, compared with controls. This response was significantly reduced following injection of an anti-alpha4-integrin monoclonal antibody (MAb). Postsinusoidal rolling was further reduced to baseline following injection of an anti-P-selectin or anti-L-selectin MAb. Sinusoidal adhesion was greater in mice treated with AdIL-1beta than with AdTNF-alpha. Blocking alpha4-integrin, P-selectin, or L-selectin had no significant effect on sinusoidal or postsinusoidal adhesion. In separate experiments, we administered AdTNF-alpha or AdIL-1beta to mice deficient in ICAM-1. In ICAM-1-/- mice, postsinusoidal leukocyte rolling significantly increased following expression of IL-1beta but not TNF-alpha. AdIL-1beta- but not AdTNF-alpha-mediated sinusoidal adhesion was ICAM-1 dependent. AdTNF-alpha-induced sinusoidal adhesion was significantly reduced following 4 days of anti-MIP-2 MAb and anti-KC MAb. Prolonged expression of the cytokines TNF-alpha and IL-1beta increases hepatic leukocyte-endothelial cell interactions. Interestingly, the mechanisms through which these cytokines bring about adhesion within the sinusoids differ; AdIL-1beta sinusoidal adhesion uses an ICAM-1-dependent mechanism whereas AdTNF-alpha-mediated adhesion is ICAM-1 independent but CXC chemokine dependent.     

Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.     

Pyocyanin alters redox homeostasis and carbon flux through central metabolic pathways in Pseudomonas aeruginosa PA14

The opportunistic pathogen Pseudomonas aeruginosa produces colorful, redox-active antibiotics called phenazines. Excretion of pyocyanin, the best-studied natural phenazine, is responsible for the bluish tint of sputum and pus associated with P. aeruginosa infections in humans. Although the toxicity of pyocyanin for other bacteria, as well as its role in eukaryotic infection, has been studied extensively, the physiological relevance of pyocyanin metabolism for the producing organism is not well understood. Pyocyanin reduction by P. aeruginosa PA14 is readily observed in standing liquid cultures that have consumed all of the oxygen in the medium. We investigated the physiological consequences of pyocyanin reduction by assaying intracellular concentrations of NADH and NAD+ in the wild-type strain and a mutant defective in phenazine production. We found that the mutant accumulated more NADH in stationary phase than the wild type. This increased accumulation correlated with a decrease in oxygen availability and was relieved by the addition of nitrate. Pyocyanin addition to a phenazine-null mutant also decreased intracellular NADH levels, suggesting that pyocyanin reduction facilitates redox balancing in the absence of other electron acceptors. Analysis of extracellular organic acids revealed that pyocyanin stimulated stationary-phase pyruvate excretion in P. aeruginosa PA14, indicating that pyocyanin may also influence the intracellular redox state by decreasing carbon flux through central metabolic pathways.     

Hyperglycemia increases the levels of vascular cellular adhesion molecule-1 and monocyte-chemoattractant-protein-1 in the diabetic endothelial cell

Hyperglycemia is the major cause of diabetic angiopathy. Aim of our study was to evaluate the impact of high glucose on cell growth and function of human "diabetic" endothelial cells (EC). Incubation of non-diabetic EC with glucose moderately inhibited cell growth and increased the expression of ICAM-1 and E-selectin. In the disease-specific EC, glucose treatment resulted also in moderately inhibited cell growth by 5-10%, increased basal expression of VCAM-1 by 10-20%, and an enhanced release of monocyte-chemoattractant-protein-1 (MCP-1) by 40-70%. The expression of ICAM-1 and E-selectin and the release of IL-6 and IL-8 was not affected. The usage of our disease-specific EC model might evaluate the impact of systemic factors of diabetic patients in the progression of endothelial dysfunction, and may be suitable to develop relevant therapeutic regimens.     

The Role of Reactive Oxygen Intermediates in the Regulation of Cytokine-Induced ICAM-1 Surface Expression on Endothelial Cells

ICAM-1 upregulation by endothelial cells plays a pivotal role in many disease processes, but signalling mechanisms leading to increased expression are poorly understood. In the current study we investigated the regulatory capacity of reactive oxygen intermediates (ROIs) in ICAM-1 activation by stimulating endothelial cells with the pro-inflammatory cytokines IL-1β, TNFα, IFNγ, IL-2, and IL-4 prior to antioxidant treatment. ICAM-1 was expressed constitutively and upregulated on ECV304 by IL1-β, IL2, and IFNγ and on SKHEP-1 by IFNγ, IL1-β, and TNFα. Phenanthroline (PHE) and disulfiram (DIS) showed the greatest ability to inhibit cytokine-stimulated ICAM-1 expression and in a dose-dependent manner. The α,α-diphenyl-β-picrylhydrazyl (DPPH) conversion assay showed that PHE and DIS had zero ability to scavenge free radicals and thus no known antioxidant activity. However, both are known metal chelators and our findings therefore suggest a unique role for metal ions in the control of cytokine-induced ICAM-1 expression on endothelial cells.     

Inhibition of quorum sensing in Pseudomonas aeruginosa by N-acyl cyclopentylamides

N-octanoyl cyclopentylamide (C8-CPA) was found to moderately inhibit quorum sensing in Pseudomonas aeruginosa PAO1. To obtain more powerful inhibitors, a series of structural analogs of C8-CPA were synthesized and examined for their ability to inhibit quorum sensing in P. aeruginosa PAO1. The lasB-lacZ and rhlA-lacZ reporter assays revealed that the chain length and the ring structure were critical for C8-CPA analogs to inhibit quorum sensing. N-decanoyl cyclopentylamide (C10-CPA) was found to be the strongest inhibitor, and its concentrations required for half-maximal inhibition for lasB-lacZ and rhlA-lacZ expression were 80 and 90 microM, respectively. C10-CPA also inhibited production of virulence factors, including elastase, pyocyanin, and rhamnolipid, and biofilm formation without affecting growth of P. aeruginosa PAO1. C10-CPA inhibited induction of both lasI-lacZ by N-(3-oxododecanoyl)-L-homoserine lactone (PAI1) and rhlA-lacZ by N-butanoyl-L-homoserine lactone (PAI2) in the lasI rhlI mutant of P. aeruginosa PAO1, indicating that C10-CPA interferes with the las and rhl quorum-sensing systems via inhibiting interaction between their response regulators (LasR and RhlR) and autoinducers.