9 Proinflammatory Effects of Particles on Macrophages and Epithelial Cells VickiStone School of Life Sciences, Napier University Peter G. Barlow Queen’sMedical ResearchInstitute, University of Edinburgh Gary R. Hutchison Medical ResearchCouncil,Queens MedicalResearchInstitute David M. Brown School of Life Sciences, Napier University CONTENTS 9.1 Particle-Induced Inflammation and Disease 183 9.2 The Role of EpithelialCells in Driving Particle-Induced Inflammation 185 9.3 The Effects of Particles on Macrophage Signaling Mechanisms 187 9.4 Interactions Between Macrophages and Epithelial Cells 189 9.5 Using Macrophagesand Epithelial Cells as aModel for Studying Particle-Induced Inflammation—Conclusion 191 References 191 This chapter aims to compare the ability of anumber of potentially pathogenic particlesinterms of their ability to induce inflammation and disease. The article will focus on the effects of a -quartz, asbestos,and environmental particulate air pollution particles and how thesedifferent particles activate epithelial cells and macrophages leading to inflammation. 9.1 PARTICLE-INDUCED INFLAMMATION AND DISEASE As described in the previous chapter, inflammation is considered to play akey role in driving disease induced by anumber of pathogenicrespirable particles. For example, carcinogenic and pro-fibrotic particlessuch as a -quartz and asbestos have both been showntoinduce achronic inflammatory response involving anumber of cell typesinthe lung. 1,2 The chronic inflammatory response coupled with the surface, chemical, or physical reactivity of the particles is thought to activate neutrophils and macrophages allowing inflammation to persist. 3,4 This prolonged inflam- mation can result in anumberofprocessesthatcontributetothe induction of fibrosis and carcinogenesis. Forexample,the inflammatoryprocess resultsinthe production of mitogenic 183 © 2007 by Taylor & Francis Group, LLC stimuli that induceepithelial cell proliferation (Figure9.1). 5 As cell proliferation rates increase, the chance of successfulrepair to damageddeoxyribonucleic acid (DNA)priortocell division is diminished and hence the risk of passing on amutation to daughter cells is enhanced. In addition, such particles have been demonstrated to generate reactive oxygen species (ROS) due to activity at the particle surface, 6,7 as well as from phagocytic cells (Figure9.1). Both of these processescan lead to oxidative stress and damage to macromolecules such as DNA,againincreasing the potential for mutations. Amoredetailed discussion can be found in the Chapter on Genotoxicity of Particles (Schins &Hei). Particulate air pollution particles, PM 10 (particulatematter collected through asizeselective inlet with 50% efficiency for particles of 10 m maerodynamic diameter), are associated with short-term effects such as increased hospital admissions or deathsdue to respiratory and cardiovascular causes. 8 Again, inflammation is postulated to play akey role in the mechanism by which PM 10 exacerbates pre-existing inflammatory diseases in susceptibleindividuals. 9 Thecomposition of PM 10 is complex andvariable with location andtime, however anumberofstudies have suggested that various componentssuchasthe ultrafineornanoparticle fraction (definitions providedbelow), 10,11 metals, 12–14 and endotoxin 15,16 play akey role in driving the proinflammatory effects of PM 10 . Much of theinformation that is availablerelatingtothe toxicology of ultrafineparticles (diameter less than 100 nm)has been obtained fromthe study of lowtoxicity,low-solubility nanoparticles (one diameter lessthan100 nm)suchascarbonblack, 17,18 polystyrene 19 and TiO 2 . 20 These studies andmanymoreall suggest that as particle size decreases,potential to induceoxidative stress and inflammation increases. Such results are likely to be relevanttothe toxicology of engineered nanoparticles, although this may vary if the nanoparticles are soluble, preventing biopersistance,ifthey are coated (e.g., polyethyleneglycol; PEG), 21 allowing avoidance of macrophage phagocytosisand cellular uptake, or if theparticles areextremelytoxic (e.g., a -quartz). With the recent expansion of nanotechnology, engineered nanoparticles are used in a wide variety of applications includingsunscreens,cosmetics,food, andmedicine,aswell as . . . Type II cell proliferation PMN Macrophage Particle exposed Type I cell damage/death DNA damage Type I epithelial cell Type II epithelial cell +ROS Mitogens Alveolus Respiratory bronchiole FIGURE 9.1 Aschematic diagram of the respiratory regions of the lung depicting the effects of particles on cell proliferation and mutation. In the presence of pathogenic particles, reactive oxygen species (ROS) can damage the DNA of dividing cells leading to mutation. The ROS can also activate intracellular factors that drive cell proliferation (AP-1). These factors combined with mitogenic factors released by the inflammatory cells (neutro- phils and macrophages) stimulate typeII cell proliferation to replace the damaged type Icells. An increase in cell proliferation rate increased the possibility that mutations are not corrected and hence become permanent. Particle Toxicology184 © 2007 by Taylor & Francis Group, LLC technical applications such as electronics. This means that exposure to awider range of nanopar- ticles is likely to occur for workers, consumers, and patients via anumberofexposure routes including inhalation, ingestion, injection, anddermal adsorption. Hence, the varietyoftarget tissuesaffected by nanoparticle exposure is likely to increase. 22,23 In astudy by Brownetal. 20 usingpolystyrene beads,and by Duffin et al. 24 using carbon black, polystyrene beads and TiO 2 of varyingsizes, it was demonstrated that aclear linear relationship exists betweenthe surface area dose of the particle instilled into the rat lung and the ability to induce inflammation, as indicated by neutrophil influx 18 hafter instillation. Stoeger et al. recently identified alink between surface area and the ability of six different carbon particles to induce inflammation in the mouse lung. 25 In this study, they identified athreshold dose for the instilled particles of 20 cm 2 surface area, below which an acute proinflammatory responses couldnot be detected. Asimilar study was reported by Tran et al.usinginhalation ofTiO 2 and barium sulphateparticles into the rat lung, but in this studythe thresholddose for inducing inflammation was between200 and 300 cm 2 . 26 This tenfolddifference in threshold couldbedue to animal size and species differences. Thesubsequent translation of the thresholddose, identified by Tran et al. intoparticle surface area per surface area of lung took into consideration that deposition of the particleswould be mostprevalent in the centroa- cinar region of the lung, and found that the particle dose that initiated inflammation equated to 1cm 2 of particle surface area/1 cm 2 of lung surface area. 27 This represents auseful way of considering dose assessment in relation to toxicological impact. Asimple explanationfor the link between surface area and inflammation is that the surface/volume ratio changesdramatically at low radius and that the surface area is directly proportional to the number of atoms at the particle surface. Many papers have describedthe rolesofmacrophages andepithelial cellsindriving the inflammatory response to ultrafineparticles; 9,28 a -quartz, 3 and asbestos. 4 This review,however, will concentrate on the signaling mechanismselicited in these two celltypesonexposure to a variety of particles. 9.2 THE ROLE OF EPITHELIAL CELLS IN DRIVING PARTICLE-INDUCED INFLAMMATION When inhaled particles deposit in the upper airways, they interact with epithelial cells that are ciliatedand covered in thick, sticky mucus. This allowsthe particlestobecleared by the wafting actions of the mucociliary escalator, causingthe particles to be transported out of the lung airways to be swallowed into the stomach, or to be blown from the nose. In contrast, particles depositing in the respiratory parts of the lung, including the alveoli and terminal respiratory bronchioles, must be clearedbyphagocytic cells such as macrophages. In this region of the lung, the epithelium is not ciliated—instead the type Iepithelial cells of the alveolus are large, thin, flat structures that are designed for gaseous exchange. The type Iepithelial cells are incapable of division due to their specialized nature. If damaged by toxins or particles, the type Icells mustbereplacedbydivision of type II epithelial cells (type II cellhyperplasia) that subsequently differentiate into type Icells. Type II cells are functionally very different to type Icells, with functions that include synthesis of surfactant for the reduction of alveolar surface tension. 29 These cells are also very different in structure,possessing acuboidal structure with microvilli that extend intothe alveolar lumen. 30 The type II cells are also capable of synthesizing arange of inflammatory mediators, so they play akey role in particle induced inflammation in the lung. Particle deposition in the alveolusresults in interaction with these epithelial cells, which generate chemotactic factors(e.g., IL8) to stimulate the recruitment of phagocytic cells. Forexample, PM 10 has been showntoincrease IL8 expression by the human type II epithelial celllineA549, 31,32 as has a -quartz, 33,34 and nanoparticle carbon black. 35 Hence, this wouldsuggestthat activation of epithelial cells by particles is an essential part of theclearance process. Amoredetaileddescriptionofthe chemotacticactions of epithelial products generated by particle treatmentisprovided below. Proinflammatory Effects of Particles on Macrophages and Epithelial Cells 185 © 2007 by Taylor & Francis Group, LLC Fewstudies have demonstratedanability of ultrafine or nanoparticles to inducecytokine production by epithelial cells in vitro,unless studied at very high mass doses.This is because the surface of the particlesisvery adsorbent,and hence binds the proteins released by the cells, resulting in an underestimation of the cytokine production. Seagrave et al. have also demonstrated that IL8 can adsorbonto diesel exhaustparticulate matter. 36 This IL8 appeared to remainbiologi- cally active, as it was able to induceneutrophil shape change.Wehave recently found that this effect is not limited to cytokines such as IL8 and TNFa ,but also includesthe cytotoxicity marker lactate dehydrogenase(LDH),resulting in an underestimation of the particle-induced toxicity. 37 Hohr et al.alsodemonstrated that ultrafine TiO 2 particles adsorb myeloperoxidase protein, preventing itsaccuratedetermination in BALfluidofinstilled animals. 38 Theconsequenceof proteinadsorption onto the particle surface in terms of the protein function and the particle toxicity requiresfurther investigation; perceivable effects range from protein dysfunction to hyper-reac- tivity of cells on receiving abolus dose concentrated on the particle surface. 39 Despite the lack of evidence that nanoparticles stimulate epithelial cells to generate cytokine proteins, thereis, however,sufficient evidence to suggest that themessengerribonucleic acid (mRNA)for cytokines such as IL8 is upregulated by these particles (e.g., Schinsetal. 28 ). Thegene expression of manyproinflammatory cytokines such as IL8 and TNFa is under the control of the transcription factor nuclear factor kappa B(NF-k B). NF-k B, whennot activated, is retained in the cytoplasm of the cell by the binding of inhibitor kappa B(I k B). Phosphorylation of I k BbyIk Bkinase (IKK)results in the releaseofNF-k Band its subsequent nuclear localization. 40 Schinsetal. 28 demonstrated that treatment of the humanalveolar type II epithelial cell line (A549) with a -quartz led to apersistent depletion of I k Ballowing proinflammatorysignaling and IL8 expression to continue.Treatment of A549 cells with asbestos hasalso been showntoinduceDNA binding of NF-k Band NF-k Bdependent gene expression. 41 In adifferent study, PM 10 particles were also found to induce NF-k Bnuclear localization, DNA binding, and transcriptional activation in A549 cells. 42 However, this effect occurred in the absence of I k Bdegradation, aphenomenon that has been observed for hydrogen peroxide. 43 Ramage et al. 44 demonstrated that air pollutionparticles (PM 10 )and 14 nm carbon black particles activate the expression of heat shock protein70(HSP70) by the A549 lung epithelial cell line. HSP70 can prevent NF-k Bactivation by the stabilization of I k Bkinase. This means that HSP70 is acytoprotectiveproteinthat exists in cells as amolecular chaperone, and during oxidative stress and inflammation it can up-regulate processestoprotect the cell from damage. 45–47 HSP70 secretion has also been showntobeincreased in response to pathogenic particlessuch as asbestos. 48 In fact, in the study by Ramage et al. 31 both PM 10 and nanoparticle carbon black stimulated increased HSP70 secretion by A549 cells—an effect that was significantly inhibited by the additionofantioxidants, suggesting arole for ROS in the particle induced upregulation and releaseofthis molecule. The roleofthe released HSP70 is notfully understood, but extracellular HSP70 has been showntoactivate macrophages leading to calcium influx, NF-k Bactivation and TNFa production, 49 and to be elevated during cardiovascular disease. 50 Thelate activation of HSP70 (as seen by Ramage et al. 31 at 6hafter celltreatment) may be associated with aproinflammatoryeffect, since release of HSP70 into the blood has been associated with aproinflammatory status.Wehypothesizethatthe differential activation of NF-k Band inhibitionvia HSP70may be treatment dependent, with relativelylow dose or lowtoxicity materials allowing HSP70 activation and upregulation of antioxidant defenses, while higher dose or toxicity materials bypass this protective mechanism leading to NF-k Bactivation and inflam- mation (Figure 9.2). As with many signaling pathways, it is likely that the two pathways suggested are not mutually exclusive and that there is either overlap or that they form part of acontinuum. Many of the cell signaling eventsdescribed above include arole for oxidative stress or ROS. For example, nanoparticle carbon blackhas been shown to deplete the antioxidant glutathione in the epithelial cell line A549. 51 While the effects of PM 10 on the glutathione content of epithelial cells in vitro have not been published, PM 10 instillation into the rat lung was found to deplete both lung Particle Toxicology186 © 2007 by Taylor & Francis Group, LLC tissue and bronchoalveolar lavage fluid glutathione content. 52 The pathologyofasbestos has also beenlinked to oxidative stress as indicated by itsability to upregulateHO-1 53 and to deplete glutathione from lung lining fluid. 7 The role of ROS in driving the proinflammatory effects of manyparticle types is evidenced by theability of antioxidants to inhibitavarietyofparticleinduced signalingevents.For example, as described above, nacystelyn prevented HSP70 nuclear localization in A549 cells treated with PM 10 or nanoparticle carbon black. 31 Brownetal. 54 also demonstrated that nacys- telyn,along with theother antioxidantssuchascurcumin, couldpreventactivation of the transcription factor NF-k Bonexposure of A549 cells to pathogenicfibres.Mannitol, ahydroxyl radical scavenger, has also been demonstrated to prevent nanoparticle carbon black induced cytotoxicity to A549 cells. 51 9.3 THE EFFECTS OF PARTICLES ON MACROPHAGE SIGNALING MECHANISMS Not all particlesentering the body are pathogenic; this is because macrophages play amajor rolein the clearance of foreign particles. Alveolar macrophage make up approximately5%ofthe total lung cell population 55 and occur at afrequency of approximatelyone macrophage per alveolus. 56 Interstitialand intravascularmacrophagesare also presentinthe lung,but theseare lesswell studied due to their inaccessibility by bronchoalveolar lavage. In order for macrophages to respond to achemotactic stimulus, to migrate, to phagocytosea particle,and then to clear that particle from the tissue,acomplex interaction of extracellular and intracellular signalingpathwaysisrequiredtocontrol thecellular response (Figure9.1 and Figure 9.2). However, these pathways are open to modulation by environmental factorsand by the toxic effects of the particle.Analteration in such signaling pathways can lead to decreased particle clearance and hence increased risk of inflammation and disease. Inflammation but antioxidant defences not upregulated DEP PM 10 NPCB Asbestos PM 10 α -Quartz HSP70 expression+ nuclear localisation Nrf-2 activation ARE activation Prevention of inflammation plus upregulated antioxidants HSP70 secretion NF-κ Bactivation AP1 activation ARE inhibition ROS ROS Epithelial cells Epithelial cells Low level exposure High level exposure FIGURE 9.2 Hypothetical mechanisms involving epithelial cells by which particles may differentially regulate proinflammatory and antioxidant defense mechanisms. PM 10 (respirable particulate air pollution) and nanoparticle carbon black (NPCB) have all been demonstrated to activate HSP70 nuclear localization, while diesel exhaust particles (DEP) have also been shown to activate the antioxidant response element (ARE), allowing antioxidant upregulation and prevention of inflammation. Conversely, asbestos, PM 10 and a -quartz have all been shown to activate NF-k Bleading to cytokine gene expression and inflam- mation. The two pathways are not suggested to be mutually exclusive, but may be acontinuum or overlap. Proinflammatory Effects of Particles on Macrophages and Epithelial Cells 187 © 2007 by Taylor & Francis Group, LLC For mostparticles, the site from which particlesmustbecleared is usually the respiratory system.However, nanoparticles have been demonstrated to translocate to other organs. 57,58 It is well documented that intravenous injection of avarietyofparticles, including nanoparticles, results in their accumulation within the reticulo-endothelial cells including the Kupffer macrophage cells of theliver andmacrophages of the spleen. 59,60 Hence it is possible thatnanoparticles which translocate across the pulmonary barrierwill be taken up by this system of tissue macrophages. Indeed, the studies demonstrating particle translocation from the lung do exhibit accumulation in organssuch as the liver. 61 Furthermore, due to the diverse applications devised for nanoparticles, exposure routes will alsoinclude ingestion as components of foods and injection as components of medicines. Adescriptionofcellular uptake and translocation mechanisms for nanoparticles are described and discussed in the chapter by Rothen-Rutishauser et al. Many studies have demonstrated akey role for macrophages in driving the proinflammatory response to pathogenic particles. Of these studies,alarge proportion have been conducted in vitro usingboth primary cells and cell lines, so the results generated—although focused initially on the lung—are now relevanttoany potentially exposedtissue type.Since nanoparticles are knownto activate macrophages in vitro (described below), it is likely thatregardlessofthe tissue type, macrophages can be activatedbynanoparticle exposure, but that clearance will not be fully effec- tive, leading to aproinflammatorystatus. However, this response may not be auniversal reaction to nanoparticles, since modificationwithagentssuchasPEG is known to preventuptakebythe reticulo-endothelial system. 62 Moghimi and colleagues have published anumber of studies demon- strating how drug deliveryparticles such as liposomes and polystyrene beads can be modified to avoidmacrophage uptake. 63–66 Non-ionic surfactantsand polymeric macromolecules have proven to be very successfulfor thispurpose. Surfactantsdecrease thevan derWaals forcesthatare responsible for particle aggregation, and actually increasethe repulsive forces. Polymer-coated particles are thought to avoidmacrophage uptake by prevention of opsinization of the particle surface.While this field of research is fairly well advanced for nanomedicine, this data requires examination in order to allow application to othertypesofnanoparticles. In vitro studies with macrophages have demonstrated that nanoparticle carbon black and PM 10 activate theexpressionofproinflammatory mediators such as tumornecrosis factoralpha (TNFa ). 67–69 Furthermore, thesestudies have investigatedthe signaling mechanismsactivated by the nanoparticles and PM 10 ,identifying that both particle typesactivate calcium signaling 70,71 via amechanism involving ROS. This would suggestthat oxidative stress or ROS are important in both the epithelial cell (as described above) and macrophage responses to pathogenic particles. The signals induced then activate transcriptionfactors such as NF- k B, leadingtothe subsequent increased production of TNFa proteinproduction. 15,50 To date, most of the studies relating to the induction of inflammation and nanoparticles have concentrated on the upregulation of inflammation. However, anumber of signaling pathways are activated by oxidative stress that can protect the cell. For example, activation of the antioxidant response element (ARE) by Nrf-2 leads to the upregulationofantioxidant defenses in response to ROS production. ARE is agenetic sequence found in the promoter of many genes that controls the expression of antioxidant defense pathways, including enzymes such as heme-oxygenase-1 (HO-1) and glutathione S-transferase (GST). 72,73 NF-k Bactivation leads to increased expression of proin- flammatory cytokines, while Nrf-2 activationleads to theinductionofantioxidant defense mechanisms, including HO-1. Li et al. 74 demonstrated that both organic and inorganicextracts of diesel exhaustparticulatesinduced the expression of HO-1 andGST in macrophages via a transcription factor Nrf-2. In other studies,the oxidant tert-butyl hydroperoxide and lipopolysac- charide (LPS) have both been demonstratedtoactivate theARE in macrophages. 75 TheARE actually containsabinding site for the transcriptionfactor AP-1. We have previously demonstrated that nanoparticle carbon blackincreases AP-1DNA binding. In the study by Ng et al. 57 AP-1 binding to the ARE inhibited its activation thus preventing the upregulation of antioxidant defenses. Particle Toxicology188 © 2007 by Taylor & Francis Group, LLC This wouldsuggestthat in macrophages exposedtoenvironmental particles or nanoparticles there is the potential for activation of pathways that both increaseinflammation (via NF-k Band AP-1) and increaseantioxidant defense mechanisms (via Nrf-2). Furthermore, these pathways have thepotential to interact with AP-1inhibiting ARE andtherefore preventing upregulation of antioxidant defense mechanismsbyNrf-2. One of the primary roles of macrophages is to phagocytose foreign material. Nanoparticle uptake by macrophages has been observed in anumber of studies 76–78 and in thesestudies the uptake of the nanopartices has been associated with asubsequent decrease in the ability of the macrophagestotakeupeitherfluorescent2m mpolystyrene beads, 76 yeast, 79 or fluorescent E.Coli. 62 Similarly, macrophages recoveredfrom the lungs of rats instilled with PM 10 particles exhibited adecrease in the uptake of fluorescent polystyrene beads ex vivo. 80 These studies would suggestthat particle clearance is not efficient in the presence of nanoparticles, allowing the particles to persist in the lung, drive inflammation, and perhaps cross the epithelial barrier and gain access to the lung interstitium 20 and blood. 57 However, macrophages obviously have alimited capacity to phagocytose particles of any type. In thefuture, it wouldbeinteresting to try to estimate therelativeability of macrophages to phagocytoseparticles of different sizes, andthentoidentify whether the maximumtolerated volume of particle uptake with particlesofdifferent size is comparable.With the data generated thus far,itisdifficult to determine whether the nanoparticles per se specifically inhibitthe further uptake of larger particles, or simply “fill” or “overload” the cells preventing further particle uptake. It is worth noting that in the study by Renwick et al. 58 the lower doses of nanoparticles actually induced asmall increase rather than adecrease in bead uptake. 81 The effects of asbestos on macrophage are alsowell studied. Theability of asbestos to induce proinflammatorycytokine production by macrophages has been related to the fibre length, with longer fibres beingmore effective. 82 Thelength of fibres also impacts the ability of macrophages to take up asbestos by phagocytosis. Fibers of longer than 15 m mare not easily ingestedand lead to a process knownasfrustrated phagocytosis. 83 Asbestos also induces an increased influx of calcium into the cytoplasm of macrophages, althoughthis could be as aconsequence of cell death rather than aspecific signaling event that controlscytokine production. 84 Treatment of macrophages with asbestos fibres has also been shown to deplete glutathione and to activate NF-k BDNA,aneffect that was inhibited by antioxidants. 85 Particles of a -Quartz are thought to be highly cytotoxic to macrophages, such that uptake of a -quartz leads to inhibition of macrophage function, macrophage celldeath, and subsequent releaseofthe a -quartz particle load back into the lung tissue. 3 Forinstance, a -quartz has been shown to inducecalcium elevation in macrophages, but as apart of the cell death process rather than as aspecific signaling event. 86,87 Several studies have demonstrated an ability of a -quartz to induceTNFa production by macrophages, 88,89 suggesting that in addition to simplykilling themacrophages, theseparticles must also be able to activate thecellsleadingtoa proinflammatoryresponse. 9.4 INTERACTIONS BETWEEN MACROPHAGES AND EPITHELIAL CELLS Of course, in the body, particlesnever encounter one cell type at atime, and instead the response is aculmination of complex interactions betweenparticles and manycell types. There are anumber of ways in which such interactions can be studied, including: (i) Animal models (ii) Lung slices or isolated organs (iii) Co-cultures (iv) Conditioned media from individual cell types and transferring these to other cells in culture Proinflammatory Effects of Particles on Macrophages and Epithelial Cells 189 © 2007 by Taylor & Francis Group, LLC With options (i)–(iv), attributingthe inflammatory or signaling responsesobservedto any particularcelltypes can be difficult,but they have the advantage of providing amore physiologically relevantresponse. Our ownstudies using conditioned media suggestthat exposure of epithelial cells to nanopar- ticlecarbon black results in the generation of chemotactic factorsthat stimulate the migration of macrophages. 90 These results indicate that the effects of nanoparticles are not limited to macro- phages and that acomplexinteraction between macrophages and epithelial cells plays akey role in driving the inflammatory response. Oncethe macrophages migrate to and locate the nanoparticles, it mightbeexpected that these particles would be taken up by phagocytosis and subsequently clearedfrom the lung tissue by migration of the macrophage onto the mucociliary escalator, or by migration to thelymph nodes. However, there maybeadiscordinthe effectiveness of this mechanismwhennanoparticlesare involved. Hypothetically, if particles caninduceepithelial cells to secrete macrophage chemoattractants, aprolonged exposure of epithelial cells to nanopar- ticles may result in hypersecretion of chemoattractantsinto the alveolar space. It is possible that this may disrupt the normal chemotactic gradient within the lung and result in particle-laden macro- phages remaining within the respiratory regioninsteadofmigrating to the mucociliary escalator for clearance. The impact of nanoparticles on the chemotactic gradient withinthe lung clearly requires further investigation. Our recent studies also suggestthat in response to PM 10 macrophages make substances that activate epithelial cells, resulting in an upregulationofthe adhesion molecule ICAM-1. 91 These adhesion molecules are important in vivo as they allow interaction with migratingleukocytes, facilitating their traffic through the tissue. Othermethodsofmacrophage recruitment to sitesofparticledepositioncouldinvolve activation of the alternative complement cascade, resulting in the local generation of the chemo- tactic proteinC5a. Barlow et al. 60 demonstrated that carbon black nanoparticles could inducethe generation of macrophage chemotactic factorsinblood serum. Although serum is not generally present in the alveolar space, in timesofprolonged inflammation, the lung vasculature may be compromised, allowing seepage of serumproteins into the alveolarspace.Ifnanoparticles were to comeinto contact with these proteins, it is feasible that an excessgeneration of chemotactic substances could be generated, resulting in heightened inflammation. This observation may also have ramifications with regard to particle translocation from the lung into the blood stream and systemic activation of complement could have serious effects on the cardiovascular system. Ourown studies have alsoshown that macrophages fromratsinstilled with PM 10 show a decreasedpotential to migrateexvivo 80 .Thiscould prove deleterious to particleclearance in the lung as particle-laden macrophages may be unable to migrate towards the mucociliary escalator to be removed from the lung. This wouldagain result in prolonged macrophage retention and increased inflammation as aresult. Hutchison et al. 92 treated either an alveolarepithelial cellline (A549) or amonocytic cell line (MM6) with PM 10 .The PM 10 was collectedinnear vicinity of asteel plant in the U.K. The PM 10 sampleswere found to activate proinflammatory (interleukin 1(IL-1B) and TNFa )and pro-fibrotic (TGF b )cytokine gene expression by macrophages, with thosesampleshighest in metal content beingmost effective. Thesame PM 10 sampleswere not effective at inducing expression of proin- flammatory cytokines such as IL8, IL6, or TNFa by epithelial cells. In the studybyHutchison et al. 92 the secretory products of the epithelial cells were recovered and used to treat the macrophage cell line in order to ascertain whetherthese products wouldelicit changesinmacrophage activity. Despite the lack of IL8, IL6, or TNFa production measured in this experiment, the supernatant from these cells was very potent at inducing the expression of anumber of proinflammatory mediators (IL8 and granulocyte-macrophage colony-stimulating factor)bythe macrophages. In fact, the supernatant from the epithelial cells treated with the PM 10 of highest metal content was so potent that it inhibited macrophage cytokine production, probably due to toxicity. These findings suggestcellular interactions are taking place, however the factors driving Particle Toxicology190 © 2007 by Taylor & Francis Group, LLC these responses by epithelial cells are currently unknown. Previous studies usingconditioned media have highlightedthe importanceofcytokine/chemokine release and the role these molecules have in driving effects both locally and systemically, particularly in the field of particle toxicology. Schmidt et al. publishedastudy in which macrophages were treated with silica, aknow agent of pulmonary fibrosis. 93 Thesilica treatment stimulated releaseofIL-1B, which was subsequently found to modulate proliferation of fibroblasts, providing evidence that the particles induced release of cytokines from one cell type to impact on the function of another celltype.Albrecht et al. also reportedsimilar effects attributed to unknown factorsinmedia createdfrom lavage of quartz treated animals. 94 The results of this study indicate that stimulation of NF-k Bwas partially TNFa inde- pendent, suggestingother mediatorswere involvedinNF-k Bactivationand theassociated inflammation. Due to the complex overlapping and redundant nature of inflammatory signaling, it is often difficult to attributespecificeffectstoone molecule; however, while studies using conditioned media are not always useful to determine exactlywhich factorsare responsible for the cellular response, they are useful for providing information on the combined and overall effects of the particle induced mediators. 9.5 USING MACROPHAGES AND EPITHELIAL CELLS AS AMODEL FOR STUDYING PARTICLE-INDUCED INFLAMMATION—CONCLUSION As outlined above, awide varietyofstudies indicate that different pathogenic particlesinduce oxidativestress and signaling mechanismsinboth epithelial cells and macrophages that drive the inflammatory response leading to disease. Many of the studies conducted to investigate the role of these two cell types in driving the inflammatory response have been conducted in vitro. In many cases, the in vitro cultured cells reflect closely the potency of the particlesobserved using in vivo models with respect to oxidative stress and the activation of proinflammatory signaling events. With the rapid expansion of nanotechnology and the potential for increased exposure to awide variety of particles of unknown toxicity, it will be essential to exploit such in vitro protocols in order to prevent excessive animal testing. It is therefore essential that asystematic assessment of the relevance and reliability of anumber of in vitro models be conducted. Such models are likelyto include single celltypes, cell lines, primary cells, and mixed cultures. With such models,itis essential to prevent artifactsdue to the use of particles(e.g., protein adsorption).Relevant controls (e.g., low-toxicity particles) mustalsobeincluded, and of course, it will be necessary to compare the results to in vivo responses,usinghistorical data where appropriateand possible. 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Napier University CONTENTS 9. 1 Particle- Induced Inflammation and Disease 183 9. 2 The Role of EpithelialCells in Driving Particle- Induced Inflammation 185 9. 3 The Effects of Particles on Macrophage. nuclear factor (NF)-kappaB: Requirement of Ras/mitogen-acti- vated protein kinases in the activation of NF-kappaB by oxidants, Am. J. Respir. Cell Mol. Biol., 20, 94 2, 199 9. 44. Ramage, L. and. enzymes such as heme-oxygenase-1 (HO-1) and glutathione S-transferase (GST). 72,73 NF-k Bactivation leads to increased expression of proin- flammatory cytokines, while Nrf-2 activationleads to