Diesel Exhaust Particle-induced Over-expression Of Inflammatory Mediators In Human Monocytic Cells And Underlying Mechanisms

1.Background and Aims With the rapid development of Chinese economy,as well as a substantial increase in the amount of vehicles,air pollution is now becoming one of the major risk factor for residents' health.Particulate pollutants are the main pollutants in urban air.Numerous studies have indicated that ambient reparable particulate matter(PM)in air pollution is strongly associated with increased cardiovascular diseases.Previous studies have shown that long-term exposure to diesel exhaust particles(DEP),one of several major air pollutants,could induce increases in morbidity and mortality of acute coronary syndromes(ACS),which could be attributed to the ultrafine particles(UFPs)in DEP that can penetrate blood barrier into the bloodstream and vascular systems,resulting in pro-inflammation of cardiovascular system,oxidative stress,and systemic inflammation-associated formation and rupture of atherosclerosis plaques.Inflammation affects the occurrence of atherosclerosis development mainly through the promotion of white blood cell accumulation and adhesion molecule expression,foam cell plaque formation.Moreover,the inflammatory mediators can facilitate the sediment of lipids beneath arterial intima,infiltration of monocytes into the endothelium of blood vessels to form plaques,and stimulate the proliferation of artery smooth muscle in tunica media,leading to plaque rupture.In addition,the monocytes that phagocytose oxidized low density protein(ox-LDL)can strongly adhere to endothlial cells and migrate beneath endothelium to be transformed into macrophages,which further result in oxidative stress-related smooth muscle cells and endothelial cells.A large amount of reactive oxygen species(ROS)are generated following activation of monocytes and macrophages and respiratory burst.Overt production of ROS can increase the levels of intracellular calcium ions,resulting in dysfunction/apoptosis of endothlium,alter blood vessel contractivity,plaque rupture,and sudden onset of ACS.From the above,the monocyte-enriched inflammatory response plays an important role in DEP-induced toxicity,including ACS.However,the mechanisms underlying DEP-induced disorders have not been fully revealed.In this present study,the expression of inflammatory mediators in the peripheral blood mononuclear cells(PBMC)from ACS patients or a human monocyte cell line THP-1 cells exposed to DEP was determined,respectively,to clarify whether the PBMC from ACS patients were more susceptible to DEP inflammatory effects compared to those from control patients.Moreover,the mechanisms responsible for DEP-induced expression of pro-inflammatory mediators were explored.This study will provide helpful information for elucidation of DEP-induced pro-inflammatory effects and underlying mechanisms,and identification of susceptible population to DEP toxicity.2.Methods2.1 The subjects were recruited in this study in Xinxiang Central Hospital.Blood was drawn with venepuncture from a large antecubital vein.PBMC were purified using density gradient centrifugation,then treated with different concentrations of DEP(0,10,50,100?g/ m L)for 24 h.The supernatants were collected through centrifugation and subjected to ELISA measurement of interleukin-1?(IL-1?),interleukin-8(IL-8)or tumor necrosis factor-?(TNF-?),following the manufacture's instructions.2.2 The THP-1 cells were treated with different concentrations of DEP(0,10,50,100 ?g/m L)for 24 h.then subjected to ELISA measurement of interleukin-1?(IL-1?),interleukin-8(IL-8)or tumor necrosis factor-?(TNF-?),2.3 Levels of ROS were measured with flow cytometry.Specifically,THP-1 cells were incubated with DCFH-DA before stimulated with DEP for 4 h.2.4 Western blot method to detect EGFR phosphorylation expression in THP-1 after DEP exposure.2.5 THP-1 cells were incubated with 20 ?M DCFH-DA for 1 h before further treatment of cells with 10 m M N-acetyl-cysteine(NAC)for 2h.Then 100 ug/ml DEP were added for another 4 h.Intracellular ROS levels were measured as described previously.THP-1 cells were incubated with 20 ?M DCFH-DA for 1 h before further treatment of cells with 20 m M N-acetyl-cysteine(NAC)for 2 h.Then 100 ug/ml DEP were added for another 24 h.IL-8 levels were measured with ELISA.THP-1 cells were incubated with 10 ?M PD153035 for 2 h before further treatment with 100 ug/ml DEP for 24 h.Levels of IL-8 and TNF-a were measured with ELISA.3 Results3.1 Exposure to DEP increases inflammation mediator expression With the increase in the concentration of DEP,expression of IL-1? and TNF-? in the PBMC from either ACS or control patients also increases,reaching the highest at 50?g/ml(p <0.05).In contrast,IL-8 levels peak at 100?g/ml(p <0.05).At the same dose,PBMC in patients with ACS and control release the amount of IL-1??IL-8 and TNF-a is a significant difference(p <0.05).3.2 Role of ROS in DEP-induced inflammatory mediator expression As shown in Figure8,DEP exposure could increase ROS levels in THP-1 cells.Pre-treatment of THP-1 cells with antioxidant NAC could significantly suppress DEP-induced ROS production.Furthermore,NAC pre-treatment could block DEP-induced IL-8 and TNF-? expression,suggesting that oxidative stress is involved in DEP-induced IL-8 and TNF-? expression in THP-1 cells(P <0.05).3.3 Role of EGFR in DEP-induced inflammatory mediator expression As shown in Figure 10,DEP stimulation induced a dose-dependent increase in EGFR phosphorylation,implying that DEP exposure could activate EGFR.To examine whether EGFR is involved in DEP-induced IL-8 and TNF-? expression,THP-1 cells were pre-treated with the EGFR inhibitor PD153035 prior to DEP treatment.It was shown that PD153035 markedly inhibit DEP-induced IL-8 and TNF-? expression,implying that EGFR is required for DEP-induced IL-8 and TNF-? expression in THP-1 cells(P <0.05).4.Conclusions4.1 DEP stimulation can induce IL-1?,IL-8 and TNF-? expression in human PBMC.4.2 DEP could induce more inflammation mediator in the PBMCs from ACS patients than from the Control.4.3 Over-expression of IL-8 and TNF-? is observed in THP-1 cells exposed to DEP.4.4 DEP exposure could induce ROS production and EGFR phosphorylation(activation)in a dose-dependent fashion,which is required for DEP-induced IL-8 and TNF-?expression in THP-1 cells.