The Effect Of Zn～(2+) On The Expression Levels Of IL-6, IL-8 And TNF-α In Human Ⅱ Type Pulmonary Epithelial Cells

Background and ObjectivesZinc (Zn) is an essential micronutrient that plays critical roles in maintaining the normal physiological functions of the human body. Zn is necessary for the functions of more than 300 enzymes such as alkaline phosphatase, alcohol dehydratase, deoxyribonucleic acid (DNA) polymerases and Cu, Zn-superoxide dismutase (SOD) et al. Moreover, Zn is involved in cellular signaling regulation, the composition of anti-oxidant system and immune modulation. A wide range of clinical symptoms have been associated with Zn deficiency in humans. Meanwhile, a number of researches have found that Zn is the most common component of the list of soluble transition metal in the ambient particular matters (PM) and plays important roles in respiratory disorders especially in inflammatory diseases including chronic bronchitis, bronchial asthma et al. In occupational locations, a flu-like syndrome, metal fever syndrome is induced by acute inhalation of high concentrations of zinc oxide fume. It has demonstrated in vitro and vivo experiments that Zn is capable of induction of proinflammatory cytokines as tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) et al. It can be speculated that proinflammatory cytokines may be critical contributing factors to Zn-related respiratory adversities.Airway pulmonary alveolar epithelial cells are the first defending line in respiratory system protecting themselves from diverse adverse stimuli. Toxic factors including pathogens, ambient PM and allergens make destructive effects on human body through interaction with respiratory epithelial cells, making damage to the normal functions of the respiratory epithelium and disturbing the homeostasis of respiratory system. A large growing body of evidence suggests that the epithelia is not only a physical barrier, but has the potential to synthesize a variety of cytokines, e.g. IL-8, IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF). These cytokines may be implicated in the pathogenic process of a large variety of stimuli.This experiment adopts transformed pulmonary type II epithelial A549 cell line exposed to various concentrations of zinc ions or zinc oxide slurry to explore whether they can induce the A549 cells to express TNF-α, IL-6 and IL-8 and in which fashion if they can. The aim of this study is to preliminarily probe into the role of Zn in Zn-containing PM-associated pulmonary adverse effect.Materials and methods1. MaterialsThe pulmonary type II epithelial cell line A549, zinc sulphate and zinc oxide were used in our study.2. Methods2.1 Experimental groupingThere were three groups in our study, namely zinc oxide slurry group defined as A549 cells exposed to 50uM, 100μM, 250μM and 500μM zinc oxide slurry in FBS-free F-12 culture medium; zinc ion group defined as A549 cells exposed to 50μM, 100μM, and 250μM zinc (II) ion in FBS-free F-12 culture medium; blank control group defined as A549 cells not exposed to exogenetic Zn²⁺. 2.2 Assaying methods and parametersMTT assay was used to determine the effect of Zn on the A549 cell viability, semi-quantitive RT-PCR and RIA were used to assay the effect of Zn on the expression levels of IL-6, IL-8 and TNF-αmRNA and proteins in A549 cells, respectively.2.3 Statistical analysisSAS8.2 standard statistical package (SAS Institute Inc., Cary, NC) was used for data analysis. One-way ANOVA (analysis of variance) was used to compare the effect of different concentrations of Zn on A549 cells viability and on IL-6, IL-8, and TNF-αmRNA and protein expression levels in A549 cells. When one-way ANOVA was statistically significant, LSD (least significant difference) was used for multiple comparisons. And P<0.05 was considered as statistically significant.Results and Analyses1. MTT A549 cells viability assay indicated that exposure of A549 cells to 50μM, 100μM, 250μM and 500μM zinc oxide slurry in FBS-free F-12 culture medium and 50uM, 100μM, and 250μM Zn²⁺ in FBS-free F-12 culture medium for 24h had no remarkable effect on the viability of A549 cells (P>0.05). It proved that the concentrations of the agents used in our study had no significant cytotoxicity on A549 cells.2. The exposure of A549 cells to 50μM, 100μM, 250μM and 500μM zinc oxide slurry and 50μM, 100μM, and 250μM Zn²⁺ in FBS-free F-12 culture medium for 3 hours induced elevated expression levels of IL-6, IL-8 and TNF-αmRNA in a dose-dependent manner; and for 24 hours the expression levels of IL-6 and IL-8 mRNA were decreased, but the expression level of TNF-αmRNA remained increasing compared with the control group (P<0.05). The same concentration of zinc oxide slurry and Zn²⁺ had no statistically significant capacity of inducing A549 cells expression proinflammatory cytokines mRNA. These results indicated that Zn²⁺ have the capacity of inducing the expression of proinflammatory cytokines in pulmonary alveolar cells through certain pathways and have different effect on differential cytokines maybe through various pathways to affect the expression level and/or stability of mRNA.3. The exposure of A549 cells to 50μM, 100μM, 250μM and 500μM zinc oxide slurry and 50μM, 100μM, and 250μM Zn²⁺ in FBS-free F-12 culture medium induced increased levels of IL-6, IL-8 and TNF-αprotein in supernatant for 3 hours and 24 hours in a dose-time-dependent manner. It was because higher concentrations of Zn²⁺ can stimulate higher levels of proinflammatory cytokines and the same concentration of Zn²⁺ or ZnO can induce higher levels of cytokine in 24 hours than that in 3 hours. In comparison with zinc oxide slurry, the identical concentration of Zn²⁺ had a strong power to induce the proinflammatory cytokines expression. It may be because that Zn²⁺ are more soluble in water and are propitious to contact cellular membrane and to induce cells to express higher levels of cytokines.ConclusionsThis study demonstrated that the exposure of human II type pulmonary alveolar epithelial A549 cells to Zn²⁺ and zinc oxide slurry can induce the expression of IL-6, IL-8 and TNF-αproinflammatory cytokines, and human II type pulmonary alveolar epithelial cells are the important source of proinflammatory cytokines in Zn-containing ambient PM associated respiratory disorders and in metal fever syndrome induced by inhalation of zinc oxide fume.