Particle Toxicology - Chapter 13 ppsx

A substantial body of experimental work has also shown that air pollutants such as tobacco smoke, ozone, diesel exhaust DE, and other gases and particles can alter many aspects of the im

13 Effect of Particles on the

Immune System

M Ian Gilmour

National Health and Environmental Effects Research Laboratory,

U.S Environmental Protection Agency

-Curriculum in Toxicology, University of North Carolina at Chapel Hill

Rajiv K Saxena

School of Life Sciences, Jawaharlal Nehru University

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 Allergic Immune Responses 249

13.5 Mechanism of Action of PM on the Immune Response 250

13.6 Conclusions and Future Directions 252

Acknowledgment 252

References 253

13.1 OVERVIEW

Respiratory allergies and infections are the most common form of illness in the United States and Europe, and together they account for more missed school and work days than any other types of disease (Akazawa, Sindelar, and Paltiel 2003; CDC 2004) From the well-documented air pollution episodes in London, England, and Donora, Pennsylvania (Holland et al 1979) to the most recent time series analyses of multiple cities in the U.S (Dominici et al 2006), it is clear that elevated levels of airborne particles are associated with increased morbidity and mortality to respiratory infections, and increased hospital admissions for asthma (Koren 1995; Vigotti 1999)

A substantial body of experimental work has also shown that air pollutants such as tobacco smoke, ozone, diesel exhaust (DE), and other gases and particles can alter many aspects of the immune system to decrease resistance to infection and/or exacerbate respiratory allergies and asthma (Cohen, Zelikoff, and Schlesinger 2000) Inhaled pollutants affect a number of key host defenses, including mucociliary clearance activity in the airways, microbial killing in the lung

- UNC funded by US EPA training agreement EPA CT 829472

245

lining fluid, pulmonary macrophage function, and the development of specific immune responses such as antibody production and cell mediated immunity In contrast, immune stimulation in the form

of increased T cell activity and reaginic (IgE) antibody formation has also been shown to occur under some circumstances, resulting in increased incidence or severity of allergic lung disease

These results continue to be confirmed in clinical, epidemiological, and experimental studies while basic research activities seek mechanistic explanations for the effects This chapter will review recent research on the different ways that particles affect the immune system to either decrease resistance to infectious agents or increase allergic and inflammatory disorders in the respiratory tract We conclude by summarizing research needs and 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 a seminal review 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 a remarkably sterile condition by the combined antimicrobial activity of the mucociliary, phagocytic, and immune systems.” Early studies using isolated macrophages from lung washes showed that exposure to various agents including ozone, nitrogen oxides, metal compounds, and tobacco smoke reduced the cells ability to ingest and/or kill bacteria through interfering with phagocytic uptake and intracellular anti-microbial activity (Gardner 1984) 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 production after immunization, T cell phenotype changes, and cytokine production (Jakab et al 1995)

Microorganisms are killed in phagocytes by an array of digestive enzymes, toxic oxygen species, and other anti-microbial agents Rodents exposed to particulates such as carbon black (CB) (Jakab 1993), smoke (Moores, Janigan, and Hajela 1993), lead oxide (Zelikoff, Parsons, and Schlesinger 1993), titanium dioxide (Gilmour et al 1989), and road dust (Ziegler et al 1994) exhibit reduced alveolar macrophage (AM) phagocytosis and/or impaired pulmonary clearance of inhaled bacteria A number of in vitro studies using both animal and human cells have also reported that particle matter (PM) or its toxic constituents reduces AM function Exposure to acrolein or benzofuran adsorbed onto CB lowered rat AM phagocytosis (Jakab et al 1990), while human peripheral blood monocytes had decreased phagocytic activity after incubation with a particulate 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 prostaglandins and corticosteroids (Canning et al 1991) Uptake of ultra fine particles, in particular, may cause cytoskeletal dysfunction (Moller et al 2005), and impair the phagocytic activity of AMs (Lundborg et al 2001; Renwick, Donaldson, and Clouter 2001)

Less research has been conducted on the effect of particle exposure on the adaptive immune system (both local and systemic) It is known, however, that a significant proportion of particulate matter found in urban air is derived from combustion of fossil fuels and industrial discharges and that as a result, it contains varying amounts of metals, solvents, aromatic hydrocarbons, and other chemicals which modulate specific immune function Of the metals investigated, cadmium, vanadium, chromium, lead, and nickel decrease antibody formation, antigen processing, and lym-phocyte proliferation 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 diester phorbol compounds, 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)

The effect of particles on susceptibility to microbial pathogens has been most extensively studied with the streptococcus infectivity model Coffin and Blommer (1967) first showed that mice exposed to irradiated automobile exhaust were more susceptible to a subsequent pulmonary infection with Streptococcus zooepidemicus Later, it was also established that the same ranking of toxicity for a number of metal salts occurred whether the animals were exposed by inhalation or intratracheal instillation prior to infection (Hatch et al 1981) With this validation in place between inhalation and instillation techniques, subsequent work then demonstrated that instillation of

ambient air particles collected in Germany enhanced mortality of mice to infection by more than 50% (Hatch et al 1985) Other particulates characterized as having intermediate potency (!50% excess mortality) included an ambient air particle sample from Washington DC, three coal fly ash

mortality significantly at the 100 mg dose level included samples of coal fly ash, an ambient air sample from St Louis and Mount St Helens volcanic ash More recent experiments with inhaled concentrated air particles (CAPs) from New York City air have indicated that exposure of normal healthy mice does not increase susceptibility to bacterial infection, however the exposures worsened existing pulmonary infections in aged rats (Zelikoff et al 2003)

In addition to CAPs, a number of investigations have recently focused on the immunotoxic effects of emission particles derived from the combustion of diesel and wood The streptococcus

woodsmoke (Gilmour et al 2001), and later studies demonstrated that exposure at even lower

streptococcus model has not been studied with diesel, a number of other infectivity systems have demonstrated effects with these particles Harrod and colleagues reported that inhalation exposure

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 increases lung burdens of Mycobacterium tuberculosis (Hiramatsu et al 2005), Bacillus Calmet Guerin (Saxena et al 2003b), Listeria monocytogenes (Yang et al 2001)

It is thought that diesel exhaust particles (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 involved in cellular activation (Saito et al 2002; Mundandhara, Becker, and Madden 2005) Yang et al (2001) demonstrated that the release of TH1 cytokines and reactive oxygen species (ROS) in response to L monocytogenes was deficient in bronchoalveolar lavage (BAL) cells derived from DEP exposed rats Exposure to CB did not produce a similar effect, indicating that the suppressive effect of DEP may be due to the absorbed organic 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 AMs and this effect may be secondary to the induction of oxidative 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 inhibitory effect was due to adsorbed organic compounds on DEP (Castranova et al 2001) These immunomodulatory effects have also been 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 nitric oxide in a mouse macrophage cell line is inhibited by DEP, and this effect was found to reside in the more polar aromatic hydrocarbon and resin fractions of DEP with the most potent components occurring in the n-hexane soluble fraction (Saxena et al 2003a; Shima et al 2006)

Lung epithelial cells can secrete a variety of cytokines and this response is modulated by DEP

or its associated extracts (Takizawa 2004) A gene array study showed that exposure to DEP extracts up-regulated about 50 genes in rat alveolar epithelial cells, with hemoxygenase-1 being the most prominent up-regulated (Koike et al 2004), while bronchial epithelial cells obtained from

human volunteers exposed to DEP 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 augments the expression of receptors for many bacterial and viral pathogens on lung epithelial cells (Ito et al 2006) and it has recently been demonstrated that pre-exposure to DEP increases influenza infection in human lung alveolar cells (Jaspers et al 2005) Cigarette smoke (CS) is another complex aerosol 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 increased prevalence of respiratory infections (reviewed in Johnson et al 1990) CS exposure has more recently been reported to inhibit the TH1 immune response to RSV infection in neonatal mice (Phaybouth et al 2006) and promote TH2 priming in human dendritic cells (Vassallo et al 2005)

Overall, it appears that exposure to DEP and CS, both of which consist of a complex mix of soot and organic condensate, can promote a TH2 cytokine pattern in lungs resulting in increased allergic and asthmatic-type responses (discussed below) As a consequence of the TH2 polarization and/or through other mechanisms, DEP and CS may also facilitate the growth of pathogens in lungs Interestingly, the higher load of pathogens caused by PM exposure 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 exposed to DEP and influenza display an increased viral infection accompanied with an augmented IFNg response (Jaspers et al 2005)

Unlike DEP exposure, silica particles enhance the clearance of L monocytogenes from rat lungs (Antonini et al 2000), indicating that particles may in some cases boost important parameters of the immune response Whereas DEP exposure encourages a TH2 type of cytokine profile, silica exposure promotes a TH1 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 also different between these two types of particles DEP exposed mice have a depressed systemic antibody response to sheep erythrocytes (Yang et al 2003) while elevated serum levels of IgG and IgM have been reported in silicotic rats (Huang et al 2001)

Taken together, most of these studies indicate that exposure to many airborne particulates and especially those containing toxic chemicals can affect immune function The effect in general appears to be mediated by alteration in function of macrophages and epithelial cells and is accom-panied with changes in the 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 a family of structurally homologous receptors that recognize features common to many types of pathogens (pathogen-associated molecular patterns, or PAMPs, reviewed in Takeda and Akira 2005) The role of TLRs is to activate phagocytes and tissue dendritic cells in response to pathogens This activation also leads 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 adaptive immune responses Since the respiratory tract constitutes a principal 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 a spectrum of toll-like receptors that recognize virtually all classes of

pathogens and can be stimulated to secrete a variety of immunomodulatory and chemotactic cytokines (Krutzik and Modlin 2004; Sha et al 2004; Greene and McElvaney 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 neutrophil influx in lungs and the release of MIP-1 was significantly lower in C3H/HeJ mice that have a point mutated and dysfunctional TLR4 molecule, as compared to C3H/HeN mice with a functional 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 requires the partici-pation of TLR2 This would indicate that modulation of expression and/or signaling through TLRs constitutes an important aspect of the biological effect of PM on the immune system in lungs The link between innate immune responses modulated by Toll receptors and adaptive immune responses has received much attention recently through the observation that children exposed to the TLR4 ligand, bacterial endotoxin, show significant protection against developing allergies and 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 last 30 years, the incidence of allergic disease has dramatically increased in industrialized countries Currently, the prevalence ranges from 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 people have lived

in the world, but only recently has a significant percentage of the population (particularly in developed countries) developed an allergic response to these proteins This would strongly indicate some environmental influence as opposed to a significant change in the gene pool While changes in lifestyle—including alterations in diet, activity patterns, medication use, and housing conditions— have undoubtedly had an impact on the sensitization rate, epidemiology studies have also shown that increases in ambient PM correlate with increased hospitalizations due to respiratory illness, including asthma (Ostro 1993; Dockery and Pope 1994; Atkinson et al 2001) Part of this associ-ation may be a result of allergenic pollens being bound to ambient particulates, as has been recently observed in four different European cities (Namork, Johansen, and Lovik 2006)

Most asthmatics experience exacerbations in airway inflammation and non-specific bronchial hyper-responsiveness to a wide 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 provoking inflammation and/or bronchoconstriction Similar effects have been seen in allergic animals exposed to residual oil fly ash (ROFA) (Gavett et al 1999; Hamada et al 2002) A general increase in airway responsiveness and 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 also indications that particles may stimulate the neuroimmune junction through the release of substance P (Wong et al 2003), which in itself is a potent bronchoconstrictor and 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-linking of IgE 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 symptoms and histamine levels has been noted in dust mite-sensitive subjects when co-administered DEP and extract of house dust mite, compared to DEP or allergen extract alone At the cellular level, DEP plus IgE antibody can also act

directly on mast cells to secrete more histamine compared to DEP or anti IgE alone (Diaz-Sanchez, Penichet-Garcia, and Saxon 2000)

In addition to exacerbating existing allergic disease, there is epidemiological 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’Amato 1999; Hajat et al 1999; Nicolai 1999), and recent associations have been specifically linked to proximity to highways (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 a few human clinical studies have reported that air pollutants may indeed contribute to the increased incidence of allergic disease and asthma

Animal experiments have demonstrated that many types of particles, 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 Haar et al 2005; Nygaard, Aase, and Lovik 2005) In most cases the particles alone cause inflammation, but when administered during sensitization they also stimulate the development of allergic immune responses (in the form of increased IgE antibody, TH2 cytokines) Upon repeated challenge with antigen, these animals exhibit increased severity of allergic type disease (pulmonary eosinophils, airway hyperresponsiveness, increased mucus pro-duction, etc.) compared to control animals that received antigen exposure and vehicle control in the place of the pollutant

The relationship between particle exposure and increased allergic symptoms has been 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 a later study, atopic subjects given DEP prior to nasal immunization with a neoantigen, keyhole limpet hemocyanin (KLH), produced antigen-specific IgG, IgA, and IgE as well as IL-4 in nasal lavage fluid (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

While these specific studies used a diesel particle highly enriched in organic constituents, another body of literature also shows that the carbonaceous core of the diesel, or more inert particles like CB and PSP, can similarly induce adjuvant-like effects (Granum and Lovik 2002) Rats instilled with 100 mg of fine (FCB) or ultrafine carbon black (UFCB) had some measure of allergic adjuvancy compared to DEP particles (Singh, Madden, and Gilmour 2005), while the adjuvant effects of PSP are directly related to increase in surface area of smaller particles instilled on the same mass basis as larger particles Furthermore in another PSP study, smaller particles directly oxidized the oxidant-activated fluorophore dichlorofluoresein diacetate in a cell-free system 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 a vast variety of shapes and chemical compositions and interact with different types of cells like macrophages and epithelial cells in the respiratory tract It is therefore unlikely that a unified mechanism exists that can explain how PM exposure results in altered susceptibility

to infections on the one hand, and augmented allergic and asthmatic responses on the other Nonetheless, certain common features have been noted in the mechanism of action of many types of PM One popular theme in this research area—and indeed in many diseases in general—is that of oxidative stress and the propensity for this phenomenon to cause tissue injury and dysfunction

It is evident that inhaled particles and some of their components can injure and reduce the activity of pulmonary cells important to barrier function and the clearance of infectious agents As such, these events offer pathogens a greater chance to colonize and cause disease (El-Etr and Cirillo 2001) At a secondary level, many particles and their components (transition and heavy metals, surface free radicals, organic moieties, etc.) also affect the development of specific immune responses through alterations in antigen processing and subsequent effector function At the core

of many of these effects are oxidative reactions, which are known to alter 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 known to cause many forms of injury and inflammation and they are also produced 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 (Li and Nel 2006) have explained this paradigm in a three stage model In the first tier, oxidative stress is at a low level and the induction of antioxidant enzymes such as NAD(P)H:quinone oxidoreductase (NQO1), glutathione-S-transferase M1 (GSTM1), and heme oxygenase-1 (HO-1) are able to restore cellular redox homeostasis With continued oxidative stress these enzymes become overwhelmed and can no longer neutralize the effects of ROS When this happens, (tier 2) activation of the MAPK and NF-kB cascade induces proinflammatory responses, including production of IL-4, IL-5, IL-8, IL-10, IL-13, RANTES, MIP-1a, 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 apoptosis and necrosis (Li and Nel 2006)

The mechanism by which more generic particles cause 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 aqueous milieu, as well as cellular elements in the respiratory tract (Shi et al 2001; Aust et al 2002) Ghio, Churg, and Roggli (2004) have suggested that oxygen containing functional groups present on the surface of PM through their capacity to coordinate iron result in the generation of radicals and activation of a variety of cell signaling pathways Released reactive species and free radicals further activate and or interfere with the numerous cellular signaling pathways, resulting in a final expression of altered cytokine release profiles Suppressive effects of anti-oxidants that neutralize the ROS and the general oxidative stress have been shown to mitigate the adverse effects of PM in many systems (Whitekus et al 2002; Dick et al 2003; Takizawa 2004; Kaimul Ahsan et al 2005; McCunney 2005) It is interesting

to note that respiratory burst and release of ROS is a normal consequence of interaction between macrophages and pathogens during allergic responses and possibly even during immunological priming Clearly, augmentation of these processes by PM exposure may affect or exacerbate any intended outcome

Mechanisms up or downstream of this oxidative injury are also important and noteworthy A direct effect of ultrafine particles 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, a common theme has been that the particles cause some level of inflammation, which alters the cytokine balance in the lung For example, 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 different phenotype, while increased antigen trafficking to sub-mucosal tissue as occurs with ozone exposure (Koike and Kobayashi 2004), resulting in an amplification of allergic immune responses in susceptible individuals

The interaction of innate and adaptive immunity is also a developing area of research inspired

by the observation that low doses of bacterial endotoxin are associated with decreased allergies and

asthma in children who live on farms (Ege et al 2006) It is thought that stimulation through the Toll 4 receptor maintains mucosal immunity and a TH1 phenotype that suppresses the development

of allergic type immune development

13.6 CONCLUSIONS AND FUTURE DIRECTIONS

There are many reports showing that exposure to airborne particulates can adversely affect the immune system to increase the incidence and severity of respiratory disease While these studies provide biological plausibility for the current epidemiological findings and offer clues for the mechanisms of these effects, additional information is needed in order to more effectively manage these risks The chemical and physical characteristics of the particle that confer the observed toxicity, the shape of the dose response curve, and the impact of interactions with multiple air pollutants on the observed effects are far from clear The potential for recovery versus permanent effects, factors contributing 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 A number of approaches are required to tackle these problems

Firstly, there is a need to perform more immune testing in the many ongoing panel studies and epidemiology cohorts, and to analyze these measurements against personal exposure history and disease outcome This is clearly a large and inherently complex task and interpretation will be confou-nded by many important parameters including infection, vaccination, and antigen exposure history,

as well as other key immune modulators such as diet, activity patterns, 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 cell types are needed to complement other health outcomes examined in epidemiology cohorts and will provide vital information for risk assessors

To complement these studies, more intensive investigations in human clinical and animal studies will provide better information on the effects of individual and mixed inhaled particles

on the immune system and subsequent development of disease Inhalation studies, 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 model real life situations These can be achieved through CAPs technology and comparative testing of emission sources with the caveat that air pollution is a dynamic mix of aged particles and interactive gases In addition, examination of PM samples from different areas taking into account seasonality and source apportionment models will provide a physicochemical basis to contrast and compare health effects of PM across various regions To that end, instillation experiments like those of Steerenberg et al (2006) assessing the immune effects in animals exposed to ambient PM from several different cities in Europe supplies crucial information about which chemical components are associated with PM health effects and provide mechanistic linkage to the epidemiology studies In vitro studies can also be used as screening tools to provide relative toxicity data and mechanistic information in isolated and mixed cell systems With these research activities in place, the comparative 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 reviewed by the National Health and Environmental Effects Research Laboratory, U.S Environmental Protection Agency, and approved for publication Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does the mention of trade names or commercial products constitute endorsement or recommendation for use

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