Chai et al. BMC Microbiology 2014, 14:26http://www.biomedcentral.com/1471-2180/14/26

RESEARCH ARTICLE Open Access

Pseudomonas pyocyanin stimulates IL-8expression through MAPK and NF-B pathways indifferentiated U937 cellsWenshu Chai1*, Jia Zhang2, Yan Duan1, Dianzhu Pan1, Wei Liu1, Ying Li1, Xue Yan1 and Baiyi Chen3*

Abstract

Background: Pyocyanin (PCN), an extracellular product of Pseudomonas aeruginosa and a blue redox activesecondary metabolite, plays an important role in invasive pulmonary infection. However, the detailed inflammatoryresponse triggered by PCN infection in inflammatory cells (particularly macrophages), if present, remains to beclarified. To investigate the effects of PCN on macrophages, the ability of PCN to induce inflammation reaction andthe signaling pathway for IL-8 release in PCN-induced differentiated U937 cells were examined.

Results: It was found that PCN increased IL-8 release and mRNA expression in Phorbol 12-myristate 13-acetate (PMA)differentiated U937 cells in both a concentration- and time-dependent manner by reverse transcription-polymerasechain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). P38 and ERK MAPKs were activated after10 min of induction with PCN and their levels returned to baselines after 30 min by Western blotting. It was also foundthat within 10 min of PCN incubation, the level of p-I-B in the cytosol was increased, which returned to baseline levelafter 60 min. Meanwhile, the level of p-p65 was increased in the nuclear extract and cytosol, and maintained high intotal cell lysates. The results were further confirmed by the observation that p38, ERK1/2 and NF-B inhibitors inhibitedPCN-induced NF-B activation and attenuated PCN-induced IL-8 expression in U937 cells as a function of theirconcentrations. Moreover, it was shown that PCN induced oxidative stress in U937 cells and N-acetyl cysteine,an antioxidant, was able to inhibit PCN-induced IL-8 protein expression.

Conclusions: It is concluded that PCN induces IL-8 secretion and mRNA expression in PMA-differentiated U937cells in a concentration- and time- dependent manner. Furthermore, p38 and ERK MAPKs and NF- signalingpathways may be involved in the expression of IL-8 in PCN-incubated PMA-differentiated U937 cells.

Keywords: Pyocyanin, IL-8, U937 cell, p38, ERK, NF-B

BackgroundPseudomonas aeruginosa (P. aeruginosa), an opportunisticpathogen, causes infections associated with high inci-dences of morbidity and mortality in immunocomprom-ised hosts. P. aeruginosa colonizes the lower respiratorytract in patients resulting in bronchiectasis, cystic fibrosis,and chronic obstructive pulmonary disease [1-3]. Thepathogen has a broad host range, which produces a large

* Correspondence: cws1964@aliyun.com; chenbaiyi63@163.com1Department of Respiratory Diseases, the First Affiliated Hospital of LiaoningMedical University, Jinzhou, Liaoning, 121001, China3Department of Infectious Diseases, the First Affiliated Hospital of ChinaMedical University, Shenyang, Liaoning 110001, ChinaFull list of author information is available at the end of the article

2014 Chai et al.; licensee BioMed Central LtCommons Attribution License (http://creativecreproduction in any medium, provided the orwaiver (http://creativecommons.org/publicdomstated.

number of extracellular products including elastase andalkaline protease, LasA protease, hemolysin, rhamnolipid,and pyocyanin (PCN). These extracellular productsalter host cell function and may contribute to diseasepathogenesis.Among recognized virulence factors, the redox-active

phenazine PCN, a blue redox active secondary metabol-ite, plays an important role in invasive pulmonary infec-tion. Early studies have shown that PCN causes multipleeffects on human cells, such as inhibition of cell respiration,ciliary function, epidermal cell growth, and prostacyclin re-lease. Furthermore, PCN alters calcium homeostasis, caus-ing damage to human cells. Recent studies have confirmedthat PCN can alter the hosts immune response and in-crease IL-1 and TNF- secretion induced by monocytes.

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Chai et al. BMC Microbiology 2014, 14:26 Page 2 of 12http://www.biomedcentral.com/1471-2180/14/26

PCN can also inhibit the bodys specific immune responseto clear out pathogens, extend the time limit or prevent theinfection of bacterial clearance, and increase secretion ofinflammatory mediators in the body that can produce ad-verse reactions. Studies have also shown that PCN andits precursor, promethazine-1-carboxylic acid, changethe hosts immune response by adjusting the RANTES[4] and IL-8 levels, and that in a variety of respiratorycell lines and primary cell cultures, PCN stimulationcan cause the release of IL-8, IL-1 and IL-6 [5], accom-panied by increased levels of IL-8 mRNA. PCN alsoacts in synergy with IL-1, IL-1 and TNF- to induceIL-8 expression in human airway epithelial cell lines[6-8]. In contrast to its effects on IL-8 expression,PCN inhibits cytokine-dependent expression of themonocyte/macrophage/T-cell chemokine RANTES. It ispossible that the inhibition could cause inflammation ofmononuclear macrophage and T cell influx to subside.Alveolar macrophages are significant defense cells and

inflammation regulatory cells which switch on multipli-city mediators of inflammation and cytokines and thencause acute lung injury. Although lung macrophageshave the capacity to participate in the host response toP. aeruginosa, the role of alveolar macrophages in acuteP. aeruginosa infection has not been clearly defined. Themolecular mechanism by which these factors exert theireffects is poorly understood. Human medullary systemcell line U937 cells share characteristics with monoblastsand pedomonocytes. The human U937 promonocyticcell line was selected as the cell model since it is widelyused to study the differentiation of promonocytes intomonocyte-like cells [9-11]. Therefore, in this study, U937cells were induced and differentiated into macrophageswith phorbol 12-myristate 13-acetate (PMA) and used tostudy PCN effects on human macrophages.Pseudomonas infections are characterized by a marked

influx of polymorphonuclear cells (PMNs) (neutrophils)[12]. Increased release of IL-8, a potent neutrophilchemoattractant, in response to PCN may contribute tothe marked infiltration of neutrophils and subsequentneutrophil-mediated tissue damage that are observed inPseudomonas-associated lung diseases [7]. Previous stud-ies by other investigators have identified a Pseudomonassecretory factor with the properties of PCN that increasesIL-8 release by airway epithelial cells both in vitro [13]and in vivo [14]. Based on these studies, we examined theeffect of PCN on IL-8 release in vitro using the humanmonocyte model (PMA-differentiated human promonocy-tic cell line U937) in synergy with inflammatory cytokines.The reasons for specific focus on IL-8 and nuclear factor-B (NF-B) pathway for IL-8 modulation are that IL-8 isan established enhancer of neutrophil function [5,6,8],while NF-B is a transcription factor believed to play a keyrole in IL-8 expression [15].

Meanwhile, a number of studies have also shown thatthe mitogen-activated protein kinases (MAPKs, includingERK, JNK and p38) signal transduction pathways mediatea variety of stimulating factors-induced IL-8 expression[4,16-18]. NF-B is a ubiquitous pleiotropic transcriptionfactor. Studies have shown that NF- activation is a con-tributing factor for a variety of lung diseases and lung in-flammation [19-21]. Pyrrolidine dithiocarbamate, a metalchelator and antioxidant, can inhibit the activation of NF-kB specifically by suppressing the release of the inhibitorysubunit Ik-B from the latent cytoplasmic form of NF-kB.Recent studies have indicated that maximal IL-8 proteinexpression requires activation of NF-B as well as MAPKs[17]. However, the precise relationship between NF-Btransactivation and MAPK activation remains unclear. Inaddition, few cellular pathways that are affected by PCNare known. Hence, the present study was designed to test-ify whether PCN can provoke the activation of macro-phages, and whether NF-B and MAPKs are involved inthis possible process.

MethodsChemicals and reagentsRPMI-1640, fetal bovine serum (FBS), and antibioticswere purchased from GIBCO BRL (Grand Island, NY).Phospho-specific p38 MAPK and p38, and phospho-specific ERK1/2 and ERK1/2 were from New England-Biolabs (Bevely, MA). Stocks of the selective p38 MAPKinhibitor SB203580, and stocks of the selective ERK1/2inhibitor PD98059 were purchased from Calbio-chem-Behring (Za Jolla, CA). Phospho-NF-B p65 (Ser276)antibody was purchased from Cell Signaling Technology(CST, Danvers, MA) and anti-p-IB- (Ser32) from SantaCruz Biotechnology (Santa Cruz, CA) . IL-8 assay kit andTNF- were purchased from R&D Systems (Minneapolis,MN). PMA was purchased from Merck Biosciences(San Diego, CA). PMS (phenazinem ethosulfate, molecularformula: C14H14N2O4S) was from AMRESCO (Solon, OH).NF-B inhibitor PDTC, PCN, N-acetylcysteine, LDH, SOD,CAT, and MDA assay kits were purchased from SigmaChemical Co. (St. Louis, MO). All other reagents, unlessspecified, were purchased from Sigma Chemical Co.

Cell culture and differentiationU937 cells were purchased from ATCC (American TypeCulture Collection, Rockville, MD) and were cultured at37C in a humidified atmosphere with 5% CO2 in RPMI1640 medium supplemented with 10% FCS and 50 g/mLgentami