13 Effect of Particles on the Immune System M. Ian Gilmour NationalHealthand Environmental Effects ResearchLaboratory, U.S. Environmental Protection Agency Tina Stevens - Curriculum in Toxicology,University of North Carolina at ChapelHill RajivK.Saxena School of Life Sciences, Jawaharlal NehruUniversity CONTENTS 13.1 Overview 245 13.2 Modulation of Pulmonary Responses to Pathogens by PM 246 13.3 Toll Like Receptors and Their Regulation in the Respiratory Tract 248 13.4 Effect of Particles on AllergicImmuneResponses 249 13.5 Mechanism of Action of PM on the ImmuneResponse 250 13.6 Conclusions and FutureDirections 252 Acknowledgment 252 References 253 13.1 OVERVIEW Respiratory allergiesand infectionsare the most common form of illness in the UnitedStates and Europe,and togetherthey account for more missedschool and work days than any other types of disease (Akazawa, Sindelar, and Paltiel2003; CDC 2004). From the well-documented air pollution episodes in London, England, and Donora, Pennsylvania (Holland et al. 1979)tothe mostrecent time series analyses of multiplecities in the U.S. (Dominicietal. 2006), it is clear that elevated levels of airborneparticlesare associated with increased morbidity and mortality to respiratory infections, and increased hospital admissionsfor asthma (Koren 1995; Vigotti 1999). Asubstantial body of experimental work has alsoshownthat air pollutants such as tobacco smoke, ozone, diesel exhaust(DE), and othergases andparticlescan alter many aspects of the immune system to decrease resistance to infectionand/orexacerbate respiratoryallergies and asthma (Cohen, Zelikoff, and Schlesinger2000). Inhaled pollutants affect anumber of key host defenses,including mucociliary clearance activityinthe airways,microbial killinginthe lung - UNC funded by US EPA training agreementEPA CT 829472 245 © 2007 by Taylor & Francis Group, LLC lining fluid, pulmonary macrophage function, and the development of specific immune responses such as antibodyproduction and cell mediated immunity. In contrast, immune stimulation in the form of increased Tcell activity and reaginic (IgE) antibody formation has also been shown to occur under somecircumstances,resulting in increased incidence or severity of allergic lung disease. These results continue to be confirmed in clinical, epidemiological, and experimental studies while basicresearch activities seek mechanistic explanationsfor the effects. This chapterwill review recent research on thedifferent waysthatparticles affect the immune systemtoeither decrease resistancetoinfectious agents or increase allergicand inflammatorydisordersinthe respiratorytract. We conclude by summarizing research needsand future directions that will help in hazard identification and contribute to improved risk assessment of inhaled particles. 13.2 MODULATION OF PULMONARY RESPONSES TO PATHOGENS BY PM In aseminalreview article, Green and colleagues wrote (Green et al. 1977) that “despite the daily microbial assault that the respiratory tract experiences, the gas exchange area of the lung is main- tained in aremarkably sterile condition by the combinedantimicrobial activity of the mucociliary, phagocytic, and immune systems.” Early studies usingisolated macrophages from lung washes showed that exposure to various agentsincluding ozone, nitrogen oxides, metal compounds, and tobaccosmoke reducedthe cellsability to ingest and/or kill bacteria throughinterfering with phagocyticuptake andintracellularanti-microbialactivity (Gardner1984).Later experiments revealed additional defects, including reduced anti-microbial activity of the lung lining fluid and impaired development of specific immune responses as measured by assessment of cellular effector function, antibody productionafter immunization, Tcellphenotype changes, andcytokine production (Jakab et al. 1995). Microorganismsare killedinphagocytes by an array of digestive enzymes,toxic oxygen species, and otheranti-microbial agents. Rodents exposedtoparticulates such as carbon black (CB)(Jakab 1993), smoke (Moores, Janigan, and Hajela1993), leadoxide (Zelikoff, Parsons, and Schlesinger 1993), titanium dioxide (Gilmour et al. 1989), and road dust (Ziegler et al. 1994)exhibit reduced alveolarmacrophage (AM) phagocytosis and/or impaired pulmonary clearance of inhaled bacteria. A number of in vitro studies using both animal and humancells have also reportedthat particle matter (PM) or its toxic constituents reduces AM function. Exposuretoacrolein or benzofuranadsorbed onto CB lowered rat AM phagocytosis (Jakab et al. 1990), while humanperipheral blood monocytes had decreasedphagocytic activityafter incubation with aparticulate air sample collected from an industrial area in Germany (Hadnagy and Seemayer 1994). Possible mechanisms for reduced macro- phage phagocytosis include intracellular overloading of particulate, direct toxicity of internalized particles, and the co-production of suppressive mediators such as prostaglandinsand corticosteroids (Canningetal. 1991). Uptake of ultra fine particles, in particular, may cause cytoskeletal dysfunction (Moller et al. 2005), and impair the phagocytic activityofAMs (Lundborg et al. 2001; Renwick, Donaldson, and Clouter 2001). Less researchhas been conducted on the effect of particle exposure on the adaptiveimmune system (both local and systemic). It is known, however, that asignificant proportion of particulate matter found in urban air is derived from combustion of fossil fuels and industrial discharges and that as aresult, it contains varying amounts of metals, solvents, aromatic hydrocarbons, and other chemicalswhich modulatespecific immune function. Of themetalsinvestigated,cadmium, vanadium,chromium,lead, and nickel decrease antibody formation, antigen processing, and lym- phocyteproliferation in experimental animals (Kowolenko et al. 1988; Newcombe 1992;Cohen, Zelikoff, and Schlesinger 2000). Organic compounds, which show immunotoxic properties such as benzene, trichloroethylene, dioxins, phenols, organotonins and diesterphorbolcompounds,are also found in the atmosphere at varying concentrations (including being part of the complex adsorbate on combustion particles) and have been reported under numerous experimental conditions to reduce immune function (Saboori and Newcombe 1992; Cohen, Zelikoff, and Schlesinger 2000). Particle Toxicology246 © 2007 by Taylor & Francis Group, LLC Theeffect of particles on susceptibility to microbialpathogens hasbeenmostextensively studied with the streptococcus infectivity model. Coffin and Blommer(1967) first showed that mice exposedtoirradiated automobileexhaustwere more susceptibletoasubsequent pulmonary infection with Streptococcus zooepidemicus.Later, it was also established that the same ranking of toxicity for anumber of metal salts occurred whether the animals were exposedbyinhalation or intratracheal instillation prior to infection (Hatch et al. 1981). With this validation in place between inhalation andinstillationtechniques, subsequent work then demonstrated that instillation of 100 m gofbentonite,oil fly ash,metal salts(CdO, ZnO, NaAsO 2 ,SnCl 2 ,and CoNO 3 ), and ambientair particles collectedinGermany enhanced mortality of mice to infection by more than 50% (Hatch et al. 1985). Other particulates characterizedashaving intermediate potency ( ! 50% excess mortality) includedanambientair particle sample from Washington DC, three coal fly ash samples, powdered latex, BeO, andFe 2 O 3 ,while low-potency particlesthatdid notenhance mortality significantly at the 100 m gdose levelincludedsamplesofcoal fly ash, an ambientair sample from St. Louis and Mount St. Helens volcanic ash. More recent experimentswithinhaled concentrated air particles (CAPs)from New York City air have indicated that exposure of normal healthy micedoesnot increasesusceptibilitytobacterial infection,however the exposures worsenedexisting pulmonary infections in aged rats (Zelikoffetal. 2003). In addition to CAPs, anumber of investigations have recently focused on the immunotoxic effects of emission particles derived from the combustion of diesel and wood.The streptococcus modelhas provedtobesensitive followingexposuretorelatively lowlevels(1.9mg/m 3 )of woodsmoke (Gilmour et al. 2001), and later studies demonstrated that exposure at even lower levels (0.75 mg/m 3 )impairs pulmonary clearance of S. aureus (Zelikoffetal. 2002). While the streptococcus modelhas not been studied with diesel, anumber of other infectivitysystemshave demonstrated effects with these particles. Harrod and colleagues reported that inhalation exposure to freshlygenerated DE (1 mg/m 3 )reduced clearance of Pseudomonasaeruginosa (Harrod et al. 2005)and Respiratory syncytial virus (RSV) (Harrod et al. 2003). Other groups have similarly shown that inhalation or instillation exposure of re-entrained diesel particles at substantially higher concentrations increaseslungburdens of Myco bacterium tuberculosis (Hiramatsu et al. 2005), Bacillus Calmet Guerin (Saxena et al. 2003b), Listeria monocytogenes (Yang et al. 2001). It is thought that diesel exhaustparticles(DEP) do not directly influence the bactericidal activity of AMs(Bonay et al. 2006), but rather suppress secretion of pro-inflammatory cytokines and oxi- dative processes involvedincellular activation (Saitoetal. 2002; Mundandhara, Becker, and Madden 2005). Yangetal. (2001)demonstratedthatthe release of TH1cytokines and reactive oxygen species (ROS) in response to L. monocytogenes was deficient in bronchoalveolar lavage (BAL)cellsderived fromDEP exposed rats. ExposuretoCBdid notproduceasimilareffect, indicatingthatthe suppressiveeffectofDEP maybedue to theabsorbedorganic molecules present in DEP preparations. Yin et al. (2004) further reported that the organic components isolated from DEP inhibited production of TNF-a and IL-12 by AMsand this effect may be secondary to the inductionofoxidative stress.AMs from DEP treated rats were deficient in lipopolysaccharide (LPS) induced secretion of TNF-a and IL-1 as well as in the production of ROS in response to zymosan, and this inhibitoryeffect was due to adsorbed organic compounds on DEP (Castranovaetal. 2001). These immunomodulatoryeffects have alsobeen recreated in in vitro systems, suggesting that cell based assays may have broad screening utility. For example, Bacillus Calmette-Guerin (BCG) and LPS-induced production of nitricoxide in amouse macrophage cell line is inhibited by DEP, and thiseffect was found to reside in the more polararomatic hydrocarbon and resin fractions of DEP with the mostpotent components occurring in the n -hexane soluble fraction (Saxena et al. 2003a; Shima et al. 2006). Lung epithelial cells can secrete avarietyofcytokines and this response is modulated by DEP or itsassociated extracts (Takizawa2004). Agenearray studyshowedthatexposuretoDEP extracts up-regulated about 50 genes in rat alveolarepithelial cells, with hemoxygenase-1 being the most prominent up-regulated (Koike et al. 2004), while bronchial epithelial cells obtainedfrom Effect of Particles on the Immune System 247 © 2007 by Taylor & Francis Group, LLC humanvolunteers exposedtoDEP have increased expression of the TH2 cytokine IL-13 (Pourazar et al. 2004). Induction or modulation of cytokine response as well as modulation of uptake and survival of pathogens in macrophages and epithelial cells by PM can have important consequences on the course of disease.Influenza virus and the RSV chiefly infect epithelial cells in airways (Harrod et al. 2003). DEP exposure augmentsthe expression of receptors for manybacterial and viral pathogens on lung epithelial cells (Ito et al. 2006)and it has recentlybeen demonstrated that pre-exposure to DEP increases influenza infection in human lung alveolar cells (Jaspers et al. 2005). Cigarette smoke (CS) is another complexaerosol derived from the combustion of tobacco and its associated additives. It has been shown in numerous experimental and clinical studies that CS reduces mucociliary clearance, impairs macrophage function, reduces lymphocyte and antibody responses,and is associated with increasedprevalenceofrespiratory infections(reviewed in Johnsonetal. 1990). CS exposure has more recently been reported to inhibit the TH1 immune response to RSV infection in neonatal mice (Phaybouthetal. 2006)and promote TH2 priming in humandendritic cells (Vassallo et al. 2005). Overall, it appears that exposure to DEP and CS, both of which consist of acomplex mix of soot and organic condensate, can promote aTH2 cytokine patterninlungs resulting in increased allergic and asthmatic-type responses (discussed below). As aconsequence of the TH2 polarization and/or through othermechanisms, DEP and CS may also facilitatethe growth of pathogens in lungs. Interestingly, the higherload of pathogens causedbyPMexposure may eventually result in a stronger TH1 immune response, as seen in the BCG models of infection where bacterial load as well as IFNg response are boosted by DEP (Saxena et al. 2003b). This has also been observed in vitro where cultured lung epithelial cells exposedtoDEP and influenza display an increased viral infection accompanied with an augmented IFNg response (Jaspers et al. 2005). UnlikeDEP exposure, silica particles enhancethe clearance of L. monocytogenes from rat lungs (Antoninietal. 2000), indicating that particles may in some cases boost important parameters of the immune response. Whereas DEP exposure encouragesaTH2 type of cytokine profile, silica exposure promotes aTH1 profile of cytokine release (Davis, Pfeiffer, and Hemenway 2000; Garn et al. 2000). In contrast to this apparent short term benefit,chronic exposure to silica still predisposes the host to pulmonary tuberculosis (TeWaternaude et al. 2006). Antibody responses in DEP and silica exposed animals are alsodifferent betweenthese two typesofparticles. DEP exposedmice have adepressed systemic antibody response to sheep erythrocytes (Yangetal. 2003)while elevated serumlevels of IgG and IgM have been reportedinsilicotic rats(Huangetal. 2001). Taken together, most of these studies indicate that exposure to many airborne particulatesand especiallythose containing toxic chemicals canaffectimmunefunction. Theeffectingeneral appears to be mediated by alteration in function of macrophages and epithelial cells and is accom- panied with changesinthe spectrum of cytokine release.Altered cytokine milieu may in turn modulate the subsequent adaptive immune responses. 13.3 TOLL LIKE RECEPTORS AND THEIR REGULATION IN THE RESPIRATORY TRACT Toll like receptors (TLRs) constitute afamily of structurally homologous receptors that recognize features common to many types of pathogens (pathogen-associated molecular patterns, or PAMPs, reviewed in Takeda andAkira 2005). TheroleofTLRsistoactivatephagocytes andtissue dendritic cellsinresponsetopathogens. This activation alsoleads to production of important mediators of innate immunity (cytokines and chemokines), as well as the promotion of surface expression of co-stimulatory molecules essential for the induction of adaptiveimmune responses. Since the respiratory tract constitutes aprincipal portal of entry for inhaled microbes, TLR bearing cells in the lung play an important role in responding to pathogens.Both pulmonary macrophages and epithelial cells express aspectrumoftoll-like receptors that recognize virtually all classes of Particle Toxicology248 © 2007 by Taylor & Francis Group, LLC pathogensand canbestimulatedtosecreteavariety of immunomodulatory andchemotactic cytokines(Krutzik andModlin2004; Shaetal. 2004; Greene andMcElvaney 2005).Several reports indicate that PM may modulate the expression of TLRs in lung. Expression of TLR2 that recognizes mycobacterial components is depressed in the lungs of smokers,indicating that the immune response to tuberculosis-like organisms may be sub-optimal (Droemann et al. 2005). DEP induced neutrophilinflux in lungs and the releaseofMIP-1 was significantly lower in C3H/HeJ mice that have apoint mutated and dysfunctional TLR4 molecule, as comparedtoC3H/HeN mice with afunctional TLR4 molecule (Inoue et al. 2006). TLRs may also be involved in the response of lung epithelial cells to PM. Becker et al. (2005) showed that IL-8 release by lung epithelial cells in response to ambient PM requiresthe partici- pation of TLR2. This would indicatethat modulation of expression and/or signaling through TLRs constitutes an important aspectofthe biological effect of PM on the immune system in lungs. The link betweeninnate immune responses modulated by Tollreceptorsand adaptive immune responses has receivedmuch attention recently through the observation that children exposedto the TLR4 ligand, bacterial endotoxin, show significant protectionagainst developing allergiesand asthma (Schaub, Lauener, and von Mutius 2006), and is discussed in Section 13.4. 13.4 EFFECT OF PARTICLES ON ALLERGIC IMMUNE RESPONSES Over the last30years, the incidence of allergic disease hasdramatically increased in industrialized countries. Currently, the prevalence rangesfrom 25 to 40% for allergic rhinitis and 6%–12%for allergic asthma (CDC 2004). Major environmental sensitizers, such as cockroach and dust mite feces, animal dander, molds, and seasonal pollens have been ubiquitous as long as peoplehave lived in theworld,but onlyrecentlyhas asignificantpercentage of thepopulation (particularly in developed countries) developed an allergic response to these proteins. This would stronglyindicate some environmental influence as opposedtoasignificantchange in the gene pool. While changesin lifestyle—including alterations in diet, activitypatterns, medication use, and housing conditions— have undoubtedlyhad an impact on the sensitization rate, epidemiology studies have also shown that increases in ambient PM correlate with increased hospitalizationsdue to respiratory illness, includingasthma (Ostro 1993; Dockery and Pope 1994; Atkinson et al. 2001). Part of this associ- ation may be aresult of allergenicpollens beingbound to ambientparticulates, as has been recently observed in four different European cities (Namork, Johansen, and Lovik 2006). Most asthmatics experience exacerbations in airwayinflammation and non-specific bronchial hyper-responsiveness to awide range of inhaled substances, including CS, DE, hypertonic saline, and even cold air. These challenges are not antigenic in nature, but rather they behave as irritants in provokinginflammation and/or bronchoconstriction.Similar effectshavebeenseeninallergic animals exposed to residual oilflyash (ROFA) (Gavett et al. 1999; Hamada et al. 2002).A general increaseinairwayresponsivenessand lung injury by exposure to PM may be an additive effect on top of pre-existing inflammation or through the development of increased tissue sensi- tivity. There are alsoindications that particles may stimulate the neuroimmunejunction through the releaseofsubstanceP(Wong et al.2003),which in itself is apotentbronchoconstrictorand inflammatory mediator (Joos et al. 2003). Particles can act directly on cells important in the effector phase of allergic reactions. Type I hypersensitivity reactions, such as those occurring in allergic asthma,are caused by the cross- linkingofIgE molecules on the surface of mast cells. This signal induces the cells to degranulate and release preformed histamines, and synthesize prostaglandins, leukotrienes and immunomodu- latory cytokines. An increase in the severity of allergic symptomsand histamine levels has been noted in dust mite-sensitivesubjectswhenco-administered DEP and extract of house dust mite, comparedtoDEP or allergen extract alone. At the cellular level, DEP plus IgE antibody can also act Effect of Particles on the Immune System 249 © 2007 by Taylor & Francis Group, LLC directly on mastcells to secretemore histamine comparedtoDEP or anti IgE alone (Diaz-Sanchez, Penichet-Garcia, and Saxon 2000). In addition to exacerbating existing allergic disease,thereisepidemiological evidence that certain air pollutants including ozone and DE are associated with the development of new disease (Wade and Newman 1993; Rusznak,Devalia, and Davies 1994; D’Amato1999; Hajat et al. 1999; Nicolai 1999), and recentassociationshavebeenspecificallylinked to proximitytohighways (Brunekreef et al. 1997; Delfino et al. 2003; Gauderman et al. 2005). While these effects need to be confirmed with better personal exposure information, investigations in animals and in afew humanclinical studies have reported that air pollutants may indeed contribute to the increased incidence of allergic disease and asthma. Animal experimentshave demonstrated that manytypesofparticles, including ambient PM, DEP, ROFA, CB particles, and polystyrene particles (PSP), can act as immunologic adjuvants when administered with an antigen via intraperitoneal, intranasal, intratracheal, and inhalation routes of exposure (Takafuji et al. 1989; Fujimaki et al. 1997; Maejima et al. 1997; Lambert et al. 2000; Van Zijverden et al. 2000; de Haaretal. 2005; Nygaard, Aase, and Lovik 2005). In most cases the particles alone cause inflammation, but whenadministered during sensitization they also stimulate thedevelopmentofallergicimmune responses (inthe form of increasedIgE antibody, TH2 cytokines).Upon repeated challengewithantigen,these animals exhibit increasedseverityof allergic type disease (pulmonary eosinophils, airway hyperresponsiveness, increased mucus pro- duction, etc.) comparedtocontrol animals that receivedantigen exposure and vehicle control in the place of the pollutant. Therelationship betweenparticleexposureand increased allergic symptoms hasbeen examined in limited human studies with both allergic and non-allergic subjects. Individuals with allergic rhinitis and mild asthma exposed to 0.3 mg of DEP intra-nasally had significantly enhanced IgE antibody production in the nasal mucosa (Diaz-Sanchez et al. 1994). In alater study, atopic subjects givenDEP prior to nasal immunization with aneoantigen, keyhole limpet hemocyanin (KLH), produced antigen-specificIgG,IgA,and IgEaswellasIL-4innasallavagefluid (Diaz-Sanchez et al. 1999), while subjects given KLH alone only produced IgG and IgA, indicating that the DEP acted as an adjuvant to promote primary allergic sensitization. Whilethese specificstudies used adiesel particle highly enrichedinorganic constituents, another body of literature alsoshowsthat the carbonaceous core of the diesel, or more inert particles likeCBand PSP,can similarly induceadjuvant-like effects (Granum andLovik 2002). Rats instilled with 100 m goffine (FCB) or ultrafine carbon black (UFCB) had some measureofallergic adjuvancy compared to DEP particles (Singh, Madden, and Gilmour 2005), while the adjuvant effects of PSP are directly related to increaseinsurface area of smaller particles instilled on the same mass basisaslarger particles. Furthermoreinanother PSP study, smaller particlesdirectly oxidized theoxidant-activatedfluorophoredichlorofluoreseindiacetate in acell-freesystem compared to larger particles of the same chemical makeup (Brown et al. 2001), supporting the notion that the effect is related to surface area and particle number rather than mass. 13.5 MECHANISM OF ACTION OF PM ON THE IMMUNE RESPONSE Particles come in avast variety of shapesand chemical compositions and interact with different typesofcells like macrophages and epithelial cells in the respiratory tract. It is therefore unlikely that aunified mechanism exists that can explain how PM exposure results in altered susceptibility to infectionsonthe one hand, andaugmented allergicand asthmaticresponses on the other. Nonetheless, certain common features have been noted in the mechanismofactionofmany typesofPM. Onepopular themeinthisresearch area—and indeed in many diseases in general—is that of oxidativestressand thepropensity forthisphenomenon to causetissue injury and dysfunction. Particle Toxicology250 © 2007 by Taylor & Francis Group, LLC It is evident that inhaled particles and some of their components can injure and reduce the activityofpulmonary cells important to barrierfunction and the clearance of infectious agents. As such, theseevents offer pathogens agreater chance to colonizeand cause disease (El-Etr and Cirillo 2001). At asecondary level, many particlesand their components (transition and heavy metals, surface free radicals, organicmoieties, etc.)alsoaffect the developmentofspecific immune responses through alterations in antigen processing and subsequent effector function. At the core of many of these effects are oxidative reactions, which are knowntoalter homeostatic balance and have dramatic effects on molecular, cellular, and tissue function, including host defenses. There is also good evidence that ROS are involved in the adjuvant effect of diesel particles on the induction of TH2 type immune responses (Nel 2005). ROS are knowntocause many forms of injury and inflammation and they are alsoproduced by inflammatory cells induced during both sensitization and effector phases of allergic lung disease.Thiol antioxidants suppress DEP or DEP extract induced ROS in macrophage cell lines and inhibit DEP-enhanced allergic responses in mice (Whitekus et al. 2002). Nel and colleagues (Liand Nel 2006)have explained this paradigm in a three stage model. In the first tier, oxidative stress is at alow leveland the induction of antioxidant enzymessuchasNAD(P)H:quinone oxidoreductase (NQO1),glutathione-S-transferase M1 (GSTM1), and hemeoxygenase-1 (HO-1) are able to restorecellular redox homeostasis. With continuedoxidative stress theseenzymes becomeoverwhelmed and can no longer neutralize the effects of ROS. When this happens, (tier 2) activation of the MAPK and NF-kB cascade induces proinflammatoryresponses, including production of IL-4, IL-5, IL-8, IL-10,IL-13,RANTES, MIP- 1 a ,MCP-3, GM-CSF, TNF-a ,ICAM-1, and VCAM-1. At higher levels of oxidative stress (tier 3), the permeability of the mitochondria is compromised and disruption of the electron transfer chain results in cellular apoptosisand necrosis(Li and Nel 2006). The mechanism by which more generic particlescause immune effects is also thought to be due to oxidative stress through the presence of surface free radicals that are generated by the interaction of PM with the aqueousmilieu, as well as cellular elements in the respiratory tract (Shi et al. 2001; Aust et al. 2002). Ghio, Churg, and Roggli (2004) have suggestedthat oxygen containing functional groups present on the surface of PM through their capacity to coordinate iron result in the generation of radicalsand activation of avariety of cellsignaling pathways. Released reactive species and freeradicals further activate and or interferewith the numerous cellular signaling pathways, resulting in afinal expression of altered cytokine releaseprofiles. Suppressive effects of anti-oxidants that neutralize the ROS and the general oxidative stress have been shown to mitigatethe adverse effects of PM in many systems (Whitekus et al. 2002; Dick et al. 2003; Takizawa2004; Kaimul Ahsan et al. 2005; McCunney2005). It is interesting to note that respiratory burst and release of ROS is anormal consequence of interaction between macrophages and pathogens during allergic responses and possiblyeven during immunological priming. Clearly,augmentation of these processesbyPMexposure may affect or exacerbate any intendedoutcome. Mechanisms up or downstream of this oxidative injury are also important and noteworthy. A direct effect of ultrafineparticles on the cytoskeleton of macrophages (Moller et al. 2005)and overwhelming of the cellular processes by an overload of PM (Oberdo ¨ rster 1995)are illustrative examples. In the allergy/adjuvant models, acommon theme has been that the particles cause some level of inflammation, which altersthe cytokine balance in the lung. Forexample, allergic adju- vancy effects of ROFA can be replicated by direct administration of TNF-a ,and are also reversed by treatment with anti-TNF-a antibodies (Lambert et al. 2000). Pulmonary injury also results in the recruitment of antigen presenting cells which may polarize subsequent immune responses to a differentphenotype,while increasedantigen traffickingtosub-mucosaltissueasoccurswith ozone exposure (Koike and Kobayashi2004), resulting in an amplification of allergic immune responses in susceptible individuals. The interaction of innate and adaptive immunity is alsoadeveloping area of research inspired by the observation that low doses of bacterial endotoxinare associated with decreased allergiesand Effect of Particles on the Immune System 251 © 2007 by Taylor & Francis Group, LLC asthma in children who live on farms (Ege et al. 2006). It is thought that stimulation through the Toll 4receptormaintains mucosal immunity and aTH1 phenotype that suppressesthe development of allergic type immune development. 13.6 CONCLUSIONS AND FUTURE DIRECTIONS There are many reports showing that exposure to airborneparticulates can adversely affect the immune system to increasethe incidence and severity of respiratory disease.While thesestudies providebiologicalplausibilityfor thecurrent epidemiologicalfindings andoffer cluesfor the mechanismsofthese effects, additionalinformation is needed in ordertomoreeffectively manage these risks. The chemical andphysical characteristics of theparticlethatconfer the observed toxicity, the shape of the dose response curve,and the impact of interactions with multipleair pollutantsonthe observed effects are far from clear. The potential for recovery versus permanent effects, factorscontributing to susceptibility (particularly age and genetic predisposi- tion), and effects of chronic low level exposures versus acute higher level exposures are all areas that require more study.Anumber of approaches are requiredtotackle these problems. Firstly, thereisaneed to perform moreimmune testing in the many ongoingpanel studies and epidemiology cohorts, and to analyze these measurements against personal exposure historyand disease outcome.Thisisclearly alargeand inherentlycomplex task and interpretation will be confou- nded by many important parameters including infection, vaccination, and antigen exposure history, as well as other key immune modulatorssuch as diet, activitypatterns, alcohol use, etc.Nevertheless, general markers of immune competence such as total and antigen specific antibody levels,immune cell activity, and cytokine profiling in various celltypesare needed to complement other health outcomes examined in epidemiology cohorts and will provide vital information for risk assessors. To complement thesestudies,moreintensive investigationsinhuman clinical andanimal studies will provide better information on the effects of individual and mixed inhaled particles on the immune system and subsequent development of disease. Inhalationstudies, which either harness or create realistic pollution exposures, will identify the effect of both acute and chronic PM exposure on healthy and diseased animals to modelreal life situations. These can be achieved through CAPs technology and comparative testing of emission sources with the caveat that air pollution is adynamic mix of aged particles and interactive gases. In addition, examination of PM samplesfrom different areas taking into account seasonality and source apportionment models will provideaphysicochemical basistocontrastand comparehealth effectsofPMacrossvarious regions. To that end, instillation experiments like thoseofSteerenberg et al. (2006) assessing the immune effects in animalsexposed to ambient PM from several different cities in Europe supplies crucial information about which chemical components are associated with PM health effects and providemechanistic linkagetothe epidemiology studies. In vitro studiescan also be used as screening toolstoprovide relativetoxicity data andmechanistic informationinisolatedand mixedcellsystems.Withthese researchactivities in place, thecomparative toxicity and mechanisms of action of PM can be studied and more meaningfully applied to hazard identification and risk assessment processes. ACKNOWLEDGMENT The authors appreciate the editorial comments of Dr.Maryjane Selgrade, U.S. EPA. This paper has been reviewedbythe NationalHealthand Environmental Effects ResearchLaboratory, U.S. Environmental Protection Agency, and approved for publication. 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Environmental Chemicals with Immunotoxic Properties, Raven Press Ltd, New York, 1992 Saito, Y., Azuma, A., Kudo, S., Takizawa, H., and Sugawara, I., Long-term inhalation of diesel exhaust affects cytokine expression in murine lung tissues: comparison between low- and high-dose diesel exhaust exposure, Exp Lung Res., 28, 493–506, 2002 Saxena, Q B., Saxena, R K., Siegel, P D., and Lewis, D M., Identification of organic . of the MAPK and NF-kB cascade induces proinflammatoryresponses, including production of IL-4, IL-5, IL-8, IL-10,IL -1 3, RANTES, MIP- 1 a ,MCP-3, GM-CSF, TNF-a ,ICAM-1, and VCAM-1. At higher levels. NehruUniversity CONTENTS 13. 1 Overview 245 13. 2 Modulation of Pulmonary Responses to Pathogens by PM 246 13. 3 Toll Like Receptors and Their Regulation in the Respiratory Tract 248 13. 4 Effect of Particles on. TNF-a and IL-12 by AMsand this effect may be secondary to the inductionofoxidative stress.AMs from DEP treated rats were deficient in lipopolysaccharide (LPS) induced secretion of TNF-a and IL-1