Phosphorylation of p65 Is Required for Zinc Oxide Nanoparticle–Induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

Background Exposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as interleukin-8 (IL-8). Objective We investigated the effect of ZnO nanoparticles on IL-8 expression and the underlying mechanisms in human bronchial epithelial cells. Methods We determined IL-8 mRNA and protein expression in primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line using reverse-transcriptase polymerase chain reaction and the enzyme-linked immunosorbent assay, respectively. Transcriptional activity of IL-8 promoter and nuclear factor kappa B (NFκB) in ZnO-treated BEAS-2B cells was measured using transient gene transfection of the luciferase reporter construct with or without p65 constructs. Phosphorylation and degradation of IκBα, an inhibitor of NF-κB, and phosphorylation of p65 were detected using immunoblotting. Binding of p65 to the IL-8 promoter was examined using the chromatin immunoprecipitation assay. Results ZnO exposure (2–8 μg/mL) increased IL-8 mRNA and protein expression. Inhibition of transcription with actinomycin D blocked ZnO-induced IL-8 expression, which was consistent with the observation that ZnO exposure increased IL-8 promoter reporter activity. Further study demonstrated that the κB-binding site in the IL-8 promoter was required for ZnO-induced IL-8 transcriptional activation. ZnO stimulation modestly elevated IκBα phosphorylation and degradation. Moreover, ZnO exposure also increased the binding of p65 to the IL-8 promoter and p65 phosphorylation at serines 276 and 536. Overexpression of p65 constructs mutated at serines 276 or 536 significantly reduced ZnO-induced increase in IL-8 promoter reporter activity. Conclusion p65 phosphorylation and IκBα phosphorylation and degradation are the primary mechanisms involved in ZnO nanoparticle-induced IL-8 expression in human bronchial epithelial cells.

volume 118 | number 7 | July 2010 • Environmental Health Perspectives Research Inhalation of zinc oxide (ZnO) particles can provoke a number of clinical responses of which the best known is metal fume fever (Gordon et al. 1992;Kuschner et al. 1995). This is accompanied by changes in composi tion of broncho alveolar lavage fluid, includ ing early increase in tumor necrosis factor α (TNFα) followed by inter leukin (IL)8 and IL6, and in numbers of poly morpho nuclear leukocytes (Blanc et al. 1993;Kuschner et al. 1995Kuschner et al. , 1997. ZnO particles in ambient air arise from incinerator emission and from wear and tear of vehicle tires (Adachi and Tainosho 2004;Bennett and Knapp 1982;Councell et al. 2004;Horner 1996;Lough et al. 2005). Previous studies have demonstrated that expo sure to Znladen (Zn in its salt and oxidized forms) ambient particles contribute to the increase in bronchitis and asthma morbidity and in lung toxicity (Adamson et al. 2000(Adamson et al. , 2003Hirshon et al. 2008;Pope 1989). It is noteworthy that engineered ZnO nano particles (< 100 nm in diameter) are cur rently being produced in high tonnage (Xia et al. 2008). Exposure to these nano particles may occur in occupational, consumer, and environmental settings (Stone et al. 2007). Inhaled nano particles can deposit along the entire respiratory tract, including airways and alveolar regions (Yang et al. 2008). Recent in vitro studies have revealed that ZnO nano particles had a stronger effect on induction of cell damage to human alveolar epithelial cells and on IL8 production from human bron chial epithelial cells and aortic endothelial cells compared with other metal oxide nano particles (Gojova et al. 2007;Park et al. 2007;Xia et al. 2008).
IL8, a member of the CXC chemokine family, is an important activator and chemo attractant for polymorphonuclear leukocytes and has been implicated in a variety of inflam matory diseases (Strieter 2002). IL8 protein is secreted at low levels from non stimulated cells, but its production is rapidly induced by a wide range of stimuli encompassing proinflamma tory cytokines (Kasahara et al. 1991), bacterial or viral products (Hobbie et al. 1997;Johnston et al. 1998), and cellu lar stressors (Fritz et al. 2005;Hirota et al. 2008;Kafoury and Kelley 2005;Sonoda et al. 1997). Expression of the IL-8 gene is regulated primarily at the level of transcription, although contributions by post transcriptional mechanisms such as mRNA stabilization have also been demon strated (Holtmann et al. 1999(Holtmann et al. , 2001Roebuck 1999;Winzen et al. 1999). The IL-8 gene is located on human chromosome 4, q12-21, and consists of four exons and three introns. Its 5´flanking region contains the usual CCAAT and TATA boxlike structures and a number of potential binding sites for several inducible transcription factors including nuclear factor kappa B (NFκB), activator protein1 (AP1), and CAAT/enhancerbinding protein (C/EBP) (Luster 1998;Roebuck 1999;Wu et al. 1997). Regulation of IL-8 gene transcriptional activa tion is stimulus and celltype specific (Brasier et al. 1998;Kasahara et al. 1991;Medin and Rothman 2006;Roebuck et al. 1999;Strieter 2002), which requires a functional NFκB element in addition to either an AP1 or a C/EBP (NFIL6) element under some con ditions of transcriptional induction (Strieter 2002). Unlike the NFκB site, the AP1 and C/EBP sites are not essential for induction but are required for maximal gene expression of the IL-8 gene (Hoffmann et al. 2002). Although ZnO induces IL8 expression in bronchial epithelial cells and IL8 plays a critical role in the pathogenesis of pulmonary disorders (Blanc et al. 1993;Kuschner et al. 1997Kuschner et al. , 1998Standiford et al. 1993), the mechanisms under lying ZnOinduced IL-8 expression have not been well characterized. In this study, we inves tigated the regulatory mechanisms underlying Background: Exposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as inter leukin8 (IL8). oBjective: We investigated the effect of ZnO nano particles on IL-8 expression and the underlying mechanisms in human bronchial epithelial cells. Methods: We determined IL-8 mRNA and protein expression in primary human bronchial epi thelial cells and the BEAS2B human bronchial epithelial cell line using reversetranscriptase poly merase chain reaction and the enzymelinked immuno sorbent assay, respectively. Transcriptional activity of IL-8 promoter and nuclear factor kappa B (NFκB) in ZnOtreated BEAS2B cells was measured using transient gene transfection of the luciferase reporter construct with or without p65 constructs. Phosphorylation and degradation of IκBα, an inhibitor of NFκB, and phosphorylation of p65 were detected using immuno blotting. Binding of p65 to the IL-8 promoter was examined using the chromatin immunoprecipitation assay. results: ZnO exposure (2−8 µg/mL) increased IL-8 mRNA and protein expression. Inhibition of transcription with actinomycin D blocked ZnOinduced IL-8 expression, which was consistent with the observation that ZnO exposure increased IL-8 promoter reporter activity. Further study demon strated that the κBbinding site in the IL-8 promoter was required for ZnOinduced IL-8 tran scriptional activation. ZnO stimulation modestly elevated IκBα phosphorylation and degradation. Moreover, ZnO exposure also increased the binding of p65 to the IL-8 promoter and p65 phosphoryla tion at serines 276 and 536. Overexpression of p65 constructs mutated at serines 276 or 536 significantly reduced ZnOinduced increase in IL-8 promoter reporter activity. ZnOinduced IL-8 expression in human bron chial epithelial cells.
Cell culture and exposure. Primary human bronchial epithelial cells. According to a pro tocol approved by the University of North Carolina Institutional Review Board, cells were obtained by cytologic brushing during bronchoscopy from healthy non smoking adult volun teers who had given informed consent; cells were frozen in liquid nitrogen until use. After thawing, the human bronchial epithe lial cells were expanded to passage 2 in bron chial epithelial growth medium (Cambrex Bioscience Walkersville, Inc., Walkersville, MD), then plated on collagencoated filter supports with a 0.4µm pore size (TransCLR; Costar, Cambridge, MA) to undergo air liq uid interface (ALI) culture in a 1:1 mixture of bronchial epithelial cell basic medium and Dulbecco's modified Eagle's mediumH with SingleQuot supplements (Cambrex), bovine pituitary extract (13 mg/mL), bovine serum albumin (1.5 µg/mL), and nys tatin (20 units). Upon confluency, all-trans retinoic acid was added to the medium, and ALI culture conditions (removal of the apical medium) were created to promote differen tiation. ZnO nano particles were suspended in moleculargrade water. Because ZnO nano particles were poorly dissolved in water, the ZnO suspension was sonicated before being added to the apical surface of the ALI culture for stimulation. BEAS2B cell line. The BEAS2B cell line was derived by transforming human bron chial cells with an adenovirus 12simian virus 40 construct (Reddel et al. 1988). We obtained BEAS2B cells from the American Type Culture Collection (ATCC, Manassas, VA). BEAS2B cells (passages 70-80) were grown on tissue culture-treated Costar plates in keratinocyte basal medium supplemented with 30 µg/mL bovine pituitary extract, 5 ng/mL human epidermal growth factor, 500 ng/mL hydrocortisone, 0.1 mM ethanol amine, 0.1 mM phospho ethanolamine, and 5 ng/mL insulin. A suspension of ZnO was added to the surface of confluent BEAS2B cells for stimulation. The doses of ZnO nano particles used in this study ranged from 2 to 8 µg/mL.

Real-time reverse transcriptase/polymerase chain reaction (RT-PCR).
Bronchial epithe lial cells grown to confluence were exposed to ZnO. Cells were washed with icecold phos phatebuffered saline (PBS) and then lysed with TRIZOL reagent (Invitrogen Corporation, Carlsbad, CA). Total RNA (100 ng), 0.5 mM nucleoside tri phosphate (Pharmacia, Piscataway, NJ), 5 µM random hexa oligo nucleotide primers (Pharmacia), 10 U/µL RNase inhibitor (Promega, San Luis Obispo, CA), and 10 U/µL Moloney murine leukemia virus RT (GIBCOBRL Life Technologies, Gaithersburg, MD) were incubated in a 40°C water bath for 1 hr in 50 µL 1× PCR buf fer to synthesize firststrand cDNAs. The reverse transcription was inactivated by heat ing at 92°C for 5 min. Quantitative PCR of IL-8 and glyceraldehyde3phosphate dehy drogenase (GAPDH) specimen cDNA and standard cDNA was performed in a 50µL final volume mixture containing TaqMan master mix (PerkinElmer, Foster City, CA), 1.25 µM probe, 3 µM forward primer, and 3 µM reverse primer. The probe annealed to the template between the two primers. This probe contained both a fluorescence reporter dye at the 5´ end (6carboxy fluorescein: emis sion λ max = 518 nm) and a quencher dye at the 3´ end (6carboxy tetramethyl rhodamine: emission λ max = 582 nm). During polymeriza tion, the probe was degraded by the 5´-3´ exo nuclease activity of the Taq DNA polymerase, and the fluorescence was detected by a laser in the sequence detector (TaqMan ABI Prism 7700 Sequence Detector System; PerkinElmer). Thermal cycler parameters included 2 min at 50°C, 10 min at 95°C, and 40 cycles of dena turation at 95°C for 15 sec and annealing/ extension at 60°C for 1 min. Relative amounts of IL-8 and GAPDH mRNA were based on standard curves prepared by serial dilution of cDNA from human BEAS2B cells. The oligo nucleotide primers and probes were purchased from Applied Biosystems (Foster City, CA).
ELISA. We assayed IL8 protein in the cell culture supernatant using with a human IL8 ELISA kit according to the manufac turer's instructions.
Chromatin immunoprecipitation (ChIP) assay. We conducted the ChIP assay using a ChIP kit (Upstate, Lake Placid, NY). Briefly, BEAS2B cells growing in 100mm dishes were treated with ZnO for 2 hr before being subjected to crosslinking with 1% formalde hyde at 37°C for 10 min. After washing with PBS, the cells were resuspended in 300 µL lysis buffer [50 mM TrisHCl (pH 8.1), 10 mM EDTA, 1% SDS, protease inhibitor cocktail]. DNA was sheared to 200-1,000 base pair small fragments by sonication. The superna tant was recovered, diluted, and precleared using salmon sperm DNA/protein A agarose. The recovered supernatant was incubated with antip65 antibody or an isotype control IgG for 2 hr in the presence of salmon sperm DNA and protein GSepharose beads. The beads were washed with lowsalt, highsalt, and lith ium chloride buffers. The immuno precipitated DNA was retrieved from the beads with 1% SDS and 0.1 mM NaHCO 3 solution at 65°C for 4 hr, then purified with a QIAquick spin column (Qiagen, Valencia, CA). The PCR was conducted on the extracted DNA using IL-8 promoterspecific primers at 95°C for 2 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. The PCR products were separated on a 1.4% agarose gel and stained with ethidium bromide.

Statistical analysis.
Data are presented as mean ± SE. Data were evaluated using non parametric paired ttests with the overall α level set at 0.05. Oneway analysis of variance was used to analyze the time and dosedependent trends of IL-8 mRNA and protein expression.

ZnO exposure increases IL8 expression in human bronchial epithelial cells.
To exam ine the effect of ZnO nano particles on IL-8 expression in human bronchial epithelial cells, we used ALIcultured primary human bronchial epithelial cells and BEAS2B cells. As shown in Figure 1A, exposure of ALI cultured primary human bronchial epithe lial cells to 8 µg/mL ZnO for 4 hr induced a significant increase in IL-8 mRNA levels. In BEAS2B cells, ZnO stimulation (8 µg/ mL) induced a timedependent increase in IL-8 mRNA expression (F = 47.24; p < 0.01). Exposure of BEAS2B cells to ZnO for 4 hr caused a dosedependent increase in IL-8 mRNA expression ( Figure 1C; F = 41.83, p < 0.01). In addition, ZnO exposure resulted in a dosedependent increase in IL8 protein release from BEAS2B cells after a 6hr expo sure ( Figure 1D; F = 96.14, p < 0.01). These results indicate that ZnO exposure upregu lates IL-8 expression at both mRNA and pro tein levels in human bronchial epithelial cells.
Transcriptional activation is involved in ZnO-induced IL8 expression. To examine the involvement of transcriptional regulation in ZnOinduced elevation of IL-8 mRNA, we pre treated BEAS2B cells with 10 µg/mL Act D, a potent inhibitor of RNA polymerase, before treatment with ZnO. Pretreatment with Act D for 30 min ablated ZnOinduced IL-8

Time (min)
IκBα Actin mRNA (Figure 2A), suggesting that transcrip tional regulation was required for IL-8 expres sion in ZnOexposed cells. To confirm this observation, we measured the IL-8 promoter activity through transient gene transfection of a luciferaseconjugated IL-8 promoter construct. As predicted by the Act D results, ZnO expo sure (8 µg/mL) significantly increased IL-8 pro moter reporter activity ( Figure 2B). These data indicate that ZnOinduced IL-8 gene expres sion in human bronchial epithelial cells occurs through a transcriptional mechanism. ZnO exposure induces NFκB activation. Activation of the transcription factor NFκB is required for IL-8 gene transcription activa tion in many cell types (Villarete and Remick 1996). To examine whether ZnO stimula tion increased NFκB activity, we deter mined phosphorylation and degradation of the NFκB inhibitory protein κBα (IκBα), an event indicative of the canonical NFκB activating pathway (Ghosh and Karin 2002;Karin and BenNeriah 2000). In BEAS2B cells exposed to 8 µg/mL ZnO for 15, 30, 60, or 120 min, we measured phosphorylation of IκBα on Ser32. As shown in Figure 3A, expo sure to ZnO (after pretreatment with the pro teasome inhibitor MG132) induced a modest phosphorylation of IκBα, which peaked at 15min exposure and declined thereafter. As expected, TNFα (100 ng/mL), the positive IκBα phosphorylation inducer, increased IκBα phosphorylation at 30 min exposure. In a manner consistent with this observation, ZnO stimulation in the absence of MG132 caused IκBα degradation at 30 min exposure to ZnO ( Figure 3B). These data indicated that ZnO exposure can induce modest canonical NFκB activation in BEAS2B cells. To fur ther confirm ZnOinduced NFκB activation, BEAS2B cells were transiently transfected with pNFκBluc and pSVβ-galactosidase constructs prior to ZnO treatment. As shown in Figure 3C, ZnO exposure (8 µg/mL) increased NFκB reporter activity at 6 hr of exposure, demonstrating that ZnO treatment increases NFκBdependent transcriptional activity.
NFκB is required for ZnO-induced IL8 expression. To further determine whether NFκB was involved in ZnOinduced IL-8 gene transcription, we transfected BEAS2B cells with luciferaseconjugated IL-8 promoter (p1.5IL-8luc) and κB binding site-mutated IL-8 promoter (p1.5IL-8κBluc) constructs, respectively, prior to treatment with 8 µg/mL ZnO for 6 hr. As shown in Figure 4, the luciferase reporter activity induced by ZnO stimulation was significantly reduced in the cells expressing κB binding site-mutated IL-8 promoter compared with that in the cells expressing the intact (wildtype) IL-8 pro moter, implying that NFκB is required for ZnOinduced IL-8 gene transcription.

Phosphorylation of p65 NFκB mediates
ZnO-induced IL8 mRNA expression. NFκB exerts its regulatory function through binding specific DNA sequences as homo or hetero dimers composed of members of the Rel/ NFκB family (Hayden and Ghosh 2004). The most ubiquitous NFκB complex is the hetero dimer p50/p65(RelA). We investigated whether p65 NFκB could bind to the IL-8 gene promoter in ZnOtreated BEAS2B cells incubated with 8 µg/mL ZnO for 2 hr. Cell lysates were then subjected to the ChIP assay using antip65 and isotype control anti bodies. As shown in Figure 5A, ZnO stimulation resulted in a marked increase in the binding of p65 NFκB to the IL-8 gene promoter.
Phosphorylation of specific serine residues of the p65 NFκB subunit has been shown to be important for its transcriptional activ ity (Okazaki et al. 2003). Therefore, we used phosphospecific anti bodies to meas ure the phosphorylation of p65 NFκB at ser276 and ser536 in BEAS2B cells exposed to ZnO. We observed that ZnO treatment induced a rapid increase in phosphorylation of p65 at both serine residues ( Figure 5B). Phosphorylation of p65 (Ser536) in ZnOtreated cells occurred as early as 15 min exposure and was decreased at 60 min. In contrast, phosphorylation levels of p65 (Ser276) went up more slowly but were still above the control level at 60 min. These data indicated that ZnO exposure increased p65 phosphorylation in human bronchial epithelial cells.
To determine the functional importance of p65 phosphorylation in ZnOinduced IL-8 gene transcription, we cotransfected BEAS2B cells with p1.5IL-8luc and either a wildtype p65 construct or a mutated version in which either Ser276 or Ser536 in p65 was mutated. As expected, ZnO exposure induced increased IL-8 promoter reporter activity in cells expressing wildtype p65 ( Figure 5C).
In comparison with the cells expressing wild type p65, the cells that expressed mutated p65 showed a significant reduction in IL-8 pro moter reporter activity after ZnO treatment. These data strongly suggest that phosphoryla tion of p65 is required for ZnOinduced IL-8 gene transcription.

Discussion and Conclusion
Cellular responses to environmental stimuli require rapid and accurate transmission of sig nals from cellsurface receptors to the nucleus (Karin and Hunter 1995). These signaling pathways rely on protein phosphorylation and, ultimately, lead to the activation of specific transcription factors that induce the expres sion of appropriate target genes. In the present study using human bronchial epithelial cells, we have demonstrated that exposure to the (C) IL-8 promoter reporter activity, estimated as luciferase count/β-galactosidase count, in cells grown to 40-50% confluence and co-transfected with wild-type p65 construct or a mutated version, as described in "Materials and Methods," prior to treatment with 8 µg/mL ZnO for 6 hr. Data shown are representative of three separate experiments.
*p < 0.05 compared with the p65 wild-type group. ZnO nano particles induces IL-8 gene expres sion by activating NFκB through a bimodal mechanism that involves p65 NFκB phospho rylation as well as IκBα phosphorylation and degradation. Increased expression of IL8 protein is largely dependent on transcriptional activa tion of the IL-8 gene (Wickremasinghe et al. 1999). This is corroborated by the results of the present study, which show that pretreat ment of BEAS2B cells with the transcrip tional inhibitor Act D abrogated ZnOinduced IL-8 mRNA expression as well as the activa tion of IL-8 promoter activity in ZnOtreated cells. The NFκB family of transcription fac tors is essential for inflammation, immunity, and cell proliferation. Five members of the NFκB family have been identified: NFκB1 (p50/p105), NFκB2 (p52/p100), RelA (p65), RelB, and cRel. They share a highly conserved Rel homology domain at the Nterminal end that is responsible for specific DNA binding, dimerization, and interaction with IκB. In addition, some Rel proteins such as p65 (RelA) contain one or two Cterminal transactivating domain. In mammals, the NFκB transcription factor consists of two subunits of either homo or hetero dimers of RelA/p65, cRel, and p50. The p50/RelA(p65) heterodimer is the major Rel/NFκB complex in most cell types (Gilmore 1999). In resting cells, NFκB complexes are sequestered in an inactive form in the cyto plasm of the cells through its association with an inhibitory protein belonging to the IκB family, IκBα being the prototype. Upon cell stimulation, IκBα is phosphorylated by one of a number of IκB kinases, ubiquitinylated, and degraded, thereby allowing the NFκB complex to translocate into the nucleus and regulate the expression of its target genes, such as those coding for cytokines, adhesion molecules, and chemo kines that have a crucial role in both immune and inflammatory responses (Hayden and Ghosh 2004;Karin and BenNeriah 2000;Rossi and Zlotnik 2000). Our data show that ZnO exposure induces rapid phosphorylation and degradation of IκBα, consistent with an increase in NFκB transcriptional activity and ensuing IL-8 gene transcription. However, the modest degree of phosphorylation and degra dation of IκBα induced by ZnO stimulation implied that other events might also partici pate in ZnOinduced NFκB transcriptional activation.
Increasing evidence from biochemi cal and genetic experiments strongly suggests that optimal induction of NFκB target genes also requires posttranslational modifications of NFκB p65 (Perkins 2006;Schmitz et al. 2004). For example, the acetylation of p65 has been proposed to facilitate the retention of the NFκB complex in the nucleus (Ashburner et al. 2001;Chen et al. 2001). The phosphorylation of p65 can result in a conformational change that increases its DNA binding activity and ability to recruit histone acetyltransferases such as cAMP response elementbinding (CREB) binding protein and p300 and to displace p50histone deacetylase1 complexes from DNA, leading to increased transcriptional activity (Ashburner et al. 2001;Chen et al. 2001). The NFκB p65 can be phosphorylated at multiple sites either in the Nterminal Rel homology domain or Cterminal transa ctivating domain (Viatour et al. 2005). The bestcharacterized phosphorylated residues on p65 are Ser276 and Ser536. In the present study, we observed that ZnO exposure increased the binding of p65 to the IL-8 gene promoter and also increased the phosphorylation of p65 at Ser276 and Ser536 in BEAS2B cells. Previous studies have shown that p65 can be phosphorylated by a variety of cytoplasmic and nuclear kinases in a stimu lus and cell typespecific manner (Jamaluddin et al. 2007;Schmitz et al. 2004;Viatour et al. 2005;Zhong et al. 2002). Further study will be required to elucidate the mechanisms responsi ble for ZnOinduced phosphorylation of p65.
The role of particle dissolution in ZnO induced toxic effects has been investigated; however, the results differ with particle size and cell types. A study using human aortic endothelial cells showed that ZnO nano particles (20-70 nm diameter) can be inter nalized into cells and that ZnOinduced inflammatory response is due to the presence of the particles rather than ZnOreleased Zn 2+ (Gojova et al. 2007). In contrast, a recent study using Raw264.7 cells and BEAS2B cells proposed that the toxicity of ZnO nano particles (13 nm) in both cell types is related to particle dissolution that could happen in culture medium and intracellular endosomes (Xia et al. 2008). In separate experiments (data not shown) we observed that ZnO par ticles were poorly dissolved in water and that the phago cytosis inhibitor cyto chalasin D partially blocked ZnOinduced IL-8 expres sion in BEAS2B cells, implying that ZnO particle internalization and subsequent disso lution may be involved in ZnOinduced IL-8 expression.
Human inhalation studies have shown that exposure to ZnO in welding fumes induced early increase in TNFα protein con centration and subsequent elevation of IL8 and IL6 protein levels in broncho alveolar lavage fluid (Blanc et al. 1993;Kuschner et al. 1997). Moreover, TNFα has been shown to induce IL8 production from BEAS2B cells (Fujisawa et al. 2000). These observations lead to an assumption that ZnOinduced IL-8 expression may be mediated through an autocrine mechanism that involves TNFα. However, we have observed that TNFα neutralizing antibody had minimal inhibi tory effect on ZnOinduced IL-8 mRNA and protein expression even though it blocked TNFαinduced IL-8 mRNA expression by > 90% (data not shown), implying that TNFα is not involved in ZnOinduced IL-8 expression in BEAS2B cells.
ZnO usually exists in the form of ultra fine particles in ambient and workplace air; thus, exposure to ZnO particles is associated with adverse effects in environmental and occupational settings. Characterization of the under lying mechanisms of ZnO toxicity may provide helpful information in the prevention and treatment of pulmonary and systemic dis orders related to inhalation of ultrafine ZnO particles.