developmental immunotoxicology: emerging issues

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http://het.sagepub.com/ Human & Experimental Toxicology http://het.sagepub.com/content/21/9-10/479 The online version of this article can be found at: DOI: 10.1191/0960327102ht285oa 2002 21: 479 Hum Exp Toxicol R R Dietert, J-E Lee and T L Bunn Developmental immunotoxicology: emerging issues Published by: http://www.sagepublications.com can be found at: Human & Experimental Toxicology Additional services and information for http://het.sagepub.com/cgi/alerts Email Alerts: http://het.sagepub.com/subscriptions Subscriptions: http://www.sagepub.com/journalsReprints.nav Reprints: http://www.sagepub.com/journalsPermissions.nav Permissions: http://het.sagepub.com/content/21/9-10/479.refs.html Citations: What is This? - Sep 1, 2002 Version of Record >> at UZH Hauptbibliothek / Zentralbibliothek Zürich on June 10, 2014 het.sagepub.com Downloaded from at UZH Hauptbibliothek / Zentralbibliothek Zürich on June 10, 2014 het.sagepub.com Downloaded from

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Page 1: Developmental immunotoxicology: emerging issues

http://het.sagepub.com/Human & Experimental Toxicology

http://het.sagepub.com/content/21/9-10/479The online version of this article can be found at:

 DOI: 10.1191/0960327102ht285oa

2002 21: 479Hum Exp ToxicolR R Dietert, J-E Lee and T L Bunn

Developmental immunotoxicology: emerging issues  

Published by:

http://www.sagepublications.com

can be found at:Human & Experimental ToxicologyAdditional services and information for    

  http://het.sagepub.com/cgi/alertsEmail Alerts:

 

http://het.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

http://het.sagepub.com/content/21/9-10/479.refs.htmlCitations:  

What is This? 

- Sep 1, 2002Version of Record >>

at UZH Hauptbibliothek / Zentralbibliothek Zürich on June 10, 2014het.sagepub.comDownloaded from at UZH Hauptbibliothek / Zentralbibliothek Zürich on June 10, 2014het.sagepub.comDownloaded from

Page 2: Developmental immunotoxicology: emerging issues

Developmental immunotoxicology:emerging issues

RR Dietert*, J-E Lee and TL Bunn

Department of Microbiology and Immunology and Institute for Comparative and Environmental Toxicology,Cornell University, Ithaca, New York 14853, USA

While the history of immunotoxicology research involvingexperimental animals of immature ages dates back overseveral decades, there exist remarkably little data, to date,directly comparing the impact of developmental status onimmunotoxicological risk. Given the size of the nonadulthuman population and the potential for differentialvulnerability among the various ages, this represents aserious gap of knowledge in efforts to minimize environ-mentally linked health risks. This article frames the issuessurrounding developmental immunotoxicological evalua-tions. In particular, the issues introduced include those of

potential animal models, strain/genotype selection, gender,age of exposure, and age of assessment. Recent researchresults involving early exposure to lead (Pb) and otherchemicals are discussed to highlight the nature of thedecisions that are available and the potential cost±benefitassociated with various approaches to evaluation. Human& Experimental Toxicology (2002) 21, 479 ± 485.

Key words: biomarkers; developmental immunotoxicology; im-mune maturation; relative risk; windows of vulnerability

Introduction

Immunotoxicology, as with neurotoxicology, re-presents one of the newer subdisciplines withintoxicology. Among the earliest papers associated withimmunotoxicology are several studies examining theeffects of early (nonadult) exposure to chemicals.1 ± 3

However, despite these early developmental immuno-toxicology studies and the recognition that earlyexposure studies could be informative, the area ofimmunotoxicology progressed with an extensiveresearch literature based primarily on adult exposuredata. This trend is not unique to immunotoxicologybut rather reflects the broader pattern existing withinthe toxicology literature regardless of target organ orsystem.

The result of having a predominantly adult expo-sure database has been the necessity of determiningnonadult exposure risk for the immune and otherphysiological systems based primarily on factorialsafety limits rather than on actual early exposuredata. While this approach offers the best alternativein the absence of actual nonadult data, it clearly has

major limitations. Such age-driven extrapolationscould result in either unnecessary limitation on theuse and exposure to some chemicals or, conversely,inadequate protection against problematic exposureswithin certain age groups.

This review introduces the various issues sur-rounding developmental immunotoxicity assessment.In particular, the basis for differential age-linkedsensitivities to immunotoxicants is discussed. Aframework for direct age-related comparisons is pre-sented based on recent workshop results.4 Likewise,recent experimental findings are used to emphasizethe likely existence of critical windows of exposuresas well as the importance of gender, strain/genotype,and age of assessment in direct developmental immu-notoxicological comparisons.

Examples of known developmentalimmunotoxicants and the generalcharacteristics of developmentalimmunotoxicants

Several review articles have described the currentliterature relative to developmental immunotoxi-cants.4 ± 7 Broad categories of developmentalimmunotoxicants are listed in Table 1 and includeseveral chemicals for which there is an extensive

© Arnold 2002 10.1191/0960327102ht285oa

*Correspondence: Dr Rodney R. Dietert, Department of Micro-biology and Immunology, Cornell University, Ithaca, New York14853, USA.E-mail: [email protected]

Received 15 July 2002; accepted 13 August 2002

Human & Experimental Toxicology (2002) 21, 479 ± 485

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adult exposure immunotoxicity database. There are atleast three criteria that would qualify a chemical orenvironmental factor to be a significant developmen-tal immunotoxicant. First, a chemical that producesan immunotoxic effect with low-level nonadultexposures similar to that seen with much higher-level adult exposures would be of concern. In thiscase, differences in the pharmacokinetics followingexposure at different ages could lead to differential ex-posure of the immune target organ. Alternatively, thedynamic process of immune maturation could makethe immune cells or immune organs more sensitiveto low concentrations of the chemical. For indirectimmunotoxic effects, the developmental status anddifferential sensitivities of other cells types (e.g.,thymic epithelium, thyroid, neurological cells) mayalso be important in the ultimate impact of earlyexposure on the immune system.

Secondly, some developmental immunotoxicantsmay be capable of producing persistent immuno-toxicity following embryonic, fetal, neonatal, and/orjuvenile exposures, while the effects are onlytransient following adult exposure. In this case,exposure to a chemical capable of disrupting acritical developmental event such as the repertoire-setting `education of T lymphocytes’ within thethymus might be expected to produce a much morepervasive and permanent immune impairment thanwould occur with adult-only exposure to the samechemical.

Finally, using the same example, it is possible thatthe spectrum of immune alterations resulting fromnonadult exposure to an immunotoxin could differfrom those of adult exposure. A recent report ondexamethasone (DEX) exposure in rats suggested thatimmune changes linked to autoimmune diseasemight differ depending upon whether the exposureoccurs in the neonate or in adults.8 Likewise, ex-posure to cyclosporin A appears to result in somedifferences depending upon the age of exposure.Fetal exposure in rodents delays the developmentof lymphoid organs and particularly blocks thymo-

cyte maturation.9 ± 11 Similar human exposuresappear to alter the course of T cell, B cell, and NKcell development.12 In contrast, adult exposures havea predominant effect on thymus size, corticomedul-lary ratios, and delayed-type hypersensitivity (DTH)response.13,14

Issues regarding age of exposure

Since no real standard has existed to facilitate de-velopmental exposures and immunotoxicologicalassessment once nonadults are utilized, it is helpfulto identify a framework that would enable mean-ingful and standardized comparisons to be drawnacross ages. A recent workshop4 utilized majorbenchmarks in immune development (initiation ofhaematopoiesis; migration of stem cells and expan-sion of progenitor cells; colonization of the bonemarrow and the thymus; maturation to immuno-competence; establishment of immune memory) toidentify a limited number of potential `windows ofexposure’ that could be examined and comparedacross both animal models and humans. These `win-dows’ are relevant to the issues at hand becausethe differences in immune development during thesedifferent periods of embryonic, neonatal, and juveniledevelopment are likely to influence immunotoxicoutcomes and, hence, the relative risk arising fromexposure.

While it would be difficult and costly to obtaincomplete datasets covering each developmentalwindow for large numbers of chemicals and environ-mental factors, the framework is very useful indesigning selective comparisons. For example, onemight hypothesize that exposure to a chemical target-ing the thymus might produce different immunotoxiceffects depending on whether exposures were tooccur only before thymus formation, during thymo-cyte selection within the thymus, or after the Tlymphocyte population was fully established anddispersed to the periphery. In fact, some limited dataon cyclosporin A and lead (Pb) support this hypoth-esis. Therefore, it is helpful if exposure regimes aredesigned with the full knowledge of the status ofimmune system development during the period inwhich the key chemical and/or metabolite is likely tobe bioavailable.

Clearly, no one prescription for selective testing isequally effective for all chemicals or environmentalfactors. For example, if a chemical does not cross theplacenta and maternal metabolites are of little con-cern, then embryonic exposure might be of lessconcern than postnatal exposure. However, evenunder these circumstances, it is conceivable that em-bryonically induced changes might arise indirectly

Table 1 Examples of known developmental immunotoxins*

General category Specific examples

Halogenated aromatichydrocarbons

2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD), polychlorinatedbiphenyl (PCB)

Polycyclic aromatichydrocarbons

7,12-Dimethylbenz[a]anthracene(DMBA), benzo[a]pyrene

Pesticides ChlordaneHeavy metals Pb, mercury (Hg), cadmium (Cd)Hormonal agents Diethylstilbestrol (DES)Therapeutics Cyclosporin A,

cyclophosphamide (CP)Mycotoxins Aflatoxin-B1

*Modified from Holladay and Luster38 and Holladay andSmialowicz.6

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as a result of shifts in various maternal systems.In contrast, a chemical or metabolite of concernthat readily crosses the placenta might dictate thatembryonic windows of immune development shouldbe a primary concern.

Experience with early exposure to Pb and im-munotoxicologic outcomes suggests that chemicalexposure throughout the duration of embryonic, neo-natal, and juvenile periods of development is the bestsingle approach for developmental immunotoxicantidentification. If exposure occurs over all windowsof development prior to adulthood, it is unlikely thatan environmental chemical, drug, or other environ-mental factor of concern would be miscategorized.However, if data are needed for comparative immu-notoxicologic risk determinations, then multipleexposure regimes with each covering different de-velopmental windows would be required. Thesedistinctions in goals and strategies may be usefulin a consideration of front-line versus secondarytesting options.

Selection of test species, strain, and gender

While the selection of age of exposure is key to alldevelopmental immunotoxicologic comparisons,additional issues involving the selection of test spe-cies, strain or genotype, and gender can be central tothe effectiveness of the evaluation. Other paperswithin this workshop will include information cover-ing many potential test species and, as a result, thisframework paper will not attempt to address thecost±benefit of each species. Rather, a few examplesof the challenge in selecting one ideal species will bementioned. Based purely on the immunological data-base and availability of immunological reagents, themouse would be a logical test species. But in terms ofthe opportunity to integrate toxicological testingacross systems (e.g., reproductive) and to have com-patibility with existing test regimes, the rat has sig-nificant advantages. However, other mammalianspecies such as the dog and the pig offer considerablerelatedness of function across certain systems to thehuman. Therefore, these species are of emerginginterest. Finally, the nonhuman primate offers phylo-genetic advantages but with elevated cost over therodent models. Obviously, an ideal situation ariseswhen immunotoxicological evaluations (includingdevelopmental immunotoxicity testing) can includedata derived from more than one vertebrate specieswith the two-species results in agreement. However, itis recognized that this may not be inherently cost-effective given the large number of chemicals andenvironmental factors that need at least minimalscreening.

Genotype selection of test animals presents twoprimary issues. First, will the strain utilized berepresentative of the species in general in termsof the immunotoxicologic response to exposure?Genotype is known to influence such factors asmetabolism, bioavailability, and target organ sus-ceptibility. Genetic-based differences in metabolismare well known for many chemicals; however, thepotential for genotype to influence bioavailabilityand/or immune system susceptibility given similarintake and metabolism represents a greater uncer-tainty at present. A second question is whether thestrain or genotype is positioned in terms of par-ticular responses and, more importantly, immunebalance such that chemically induced changes inthe balance of immune responses could be detectedin the assessment strain. This second issue is quitedivergent from the first in that it questions whetherthe specific immune response profile of the testanimal strain would enable the particular spectrumof immune changes induced by an immunotoxicantto be readily detected. For example, when Pb isadministered throughout gestation to both Fisher344 and CD strain rats, female offspring of bothstrains exhibit a significantly reduced DTH func-tion15,16 when compared to same-gender controlanimals. However, under the KLH immunization re-gime utilized, these strains differed significantly inthe balance of baseline Th1 versus Th2 responsesto the antigen. Fisher 344 rats had significantlyelevated control DTH responses over CD strain rats,while the reverse was true for control antibodyproduction. Because pulsed Pb exposure beginningin late gestation or later appears to cause a shiftfrom Th1 to Th2 function, the Pb-induced immu-notoxic changes were significant in both strains butwere more dramatic in the Fisher 344 strain. Whenthese results are considered, it raises the questionas to whether the Pb-induced changes would havebeen detectable in another strain of rat where thecontrol animals were even more skewed toward Th2responses than was seen with CD strain rats.

Another important issue concerns potential gender-based differences in vulnerability to immunotoxicantexposure during fetal, neonatal, and/or juveniledevelopment. While age-based comparisons in im-munotoxicology have been relatively rare comparedwith adult exposure evaluations, only a subset ofexisting developmental immunotoxicology studieshas included direct gender comparisons. Given thesparsity of comparisons, to date, the default assump-tion should be that quantitative (e.g., dose response),if not qualitative (e.g., spectrum of immune altera-tions), differences in susceptibility are likely to existamong genders at least for some chemicals and en-vironmental factors. Bunn et al. and Lee et al. found

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that in examining female and male rats and chickensfollowing early exposures to Pb, both genders experi-enced Pb-induced immune alterations; but majordifferences existed in the nature of the changesinduced over the dose ranges and exposure agesexamined.16 ±19 For example, in the rat, late embry-onic exposed females were susceptible to subsequentsuppression of the DTH response capabilities whilemales exhibited no DTH change for the Pb dosesexamined. Therefore, potential gender-based differ-ences in developmental immunotoxicological riskmight represent a concern where planned evaluationsinclude only a single gender.

Age of assessment

One of the operational questions in developmentalimmunotoxicological testing concerns the age atwhich immune assessment should be performedfollowing early exposures. From a cost and timestandpoint, the earlier the age at which testing couldbe done reliably, the better. There are three potentialproblems with single age assessment. First, if thetesting is done at a very early age shortly afterexposure ends, then it may not be clear whetherimmunotoxic changes would persist into later agesincluding adulthood. Secondly, there is the possibil-ity that some changes might not be fully manifesteduntil later ages. For example, at least some of thechanges induced by early exposure in rats to Pb werenot detected in juveniles but were evident in fullymature offspring.16 The majority of Pb-inducedchanges were present at both ages of assessmentand Pb clearly would have been identified as a de-velopmental immunotoxicant based on juvenile ratassessment; but one unanswered question is whethersome immunotoxicants could be miscategorized ifonly juvenile assessment data were available. Thereverse issue is whether adult assessment followingfetal, weanling, or juvenile exposure is sufficient todetect a transitory immune alteration that, never-theless, would present a significant health risk. Todate, little information exists that would resolve thisquestion.

Immune balance

One of the more recent paradigms of basic immu-nology is that levels or the balance of key cytokinescan help determine the actual spectrum of im-mune responses mobilized in response to diseasechallenges.20 ± 23 This is central for overall healthmaintenance since different disease challengesrequire different subsets of immune responses to

provide the host with adequate protection. The iden-tification, in some species, of discrete T-helper cellpopulations capable of directing the flavor of animmune response toward certain cell-mediated orhumoral responses has been central to the conceptof immune balance.21 Additionally, it is known thatantigen-presenting cell function can be important inthe type of T-helper populations generated duringspecific responses.24 ± 26 Given this fundamentalproperty of immune balance, it is not sufficient tosimply consider whether chemicals or other environ-mental factors might obliterate immune functionalcapacity. Instead, it also should be determinedwhether the spectrum of the response has been sig-nificantly altered as a result of chemical exposure.

While the 1970s through early 1990s saw anemphasis within immunotoxicological evaluationand hazard identification placed on immunosuppres-sion, it is clear that any comprehensive examinationof environment ±immune health risk associationsalso must consider the risk of hypersensitivity andautoimmunity. Figure 1 illustrates these points. Addi-tionally, it seems more likely that the incidence ofchemicals, drugs, and other environmental factorscapable of inducing massive immunosuppression atrelevant doses is much lower than the incidencecapable of causing biologically and/or clinically rele-vant shifts in immune balance. Therefore, as hasoccurred with adult exposure assessment in immu-notoxicology, developmental immunotoxicologyassessment should address the possibility that envi-ronmentally induced changes in immune responsebalance may occur. In effect, changes in immune bal-ance mean that only selected immune responsesmight be suppressed while other categories of host

Figure 1 The figure illustrates the need to consider not simplyenvironmentally induced immunosuppression as a potentiallysignificant immunotoxic change but also alterations that mightincrease the risk of hypersensitivity reactions and/or autoimmun-ity. In effect, any significant deviation from a normal balancedimmune capability would be of concern and should be capable ofbeing detected through developmental immunotoxicologicalassessment

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defense±immune responses may be elevated, as in thecase of some autoimmune diseases. Any assessmentsystem or strategy should be capable of detectingsuch changes.

In fact, findings concerning exposure to Pb sup-port many of these concepts. Muller et al., McCabeet al., and McCabe and Lawrence, as well as otherresearchers, have shown that one hallmark ofPb-induced immunotoxicity resulting from adultexposure of mice is depression of the Th1-depend-ent function, the DTH reaction.27 ± 29 Pb exposure ofmature animals can cause a shift away from Th1toward Th2 capabilities.30,31 At the cellular andorgans levels, Pb-induced immunotoxic changes aremodest at best. Yet, functionally, the impact isprofound with major changes in immune responseprofiles and host defense capabilities resulting fromPb exposure.32 ± 34 Therefore, environmental chemi-cals can cause shifts in immune balance. Additionally,meaningful shifts in immune balance do not necessa-rily have to be accompanied by major losses ofimmune populations and gross changes in immuneorgans.

One reason why the issue of immune balance isparticularly important in developmental immuno-toxicology is linked to the concept of ontogeny ofimmune capabilities. Peden has presented the casethat, in humans, Th2-associated functions are the de-velopmental default capabilities in early gestationaldevelopment.7 The concept is that Th1-associatedreactions, which could be potentially problematic inthe allogeneic maternal ± fetal interaction, do notdevelop in the offspring until the latter portions ofgestation. From the standpoint of immunomodulation,this means that following exposure to any environ-mental factors capable of interfering with appropriateTh1 development, the offspring would be left with arobust Th2 function, a deficit in Th1-associatedcapacity, and a potentially serious imbalance. The

following section provides support for this generalconcept.

Critical windows of exposure ---- the issue ofdifferential risk

With early exposure to low levels of Pb throughoutgestation,35,36 the immunotoxic outcomes are similarto those seen following higher-level adult exposures;however, with more targeted exposures, it appearsthat timing is an important issue. Our recent obser-vations in both rats37 and chickens18 are summarizedin Table 2 and suggest that very early embryonicexposure to Pb, while producing some persistentimmune changes, does not inherently depress Th1-associated function. However, exposure later inembryonic development produces the classical re-duction in Th1-associated function in the offspring.This supports the argument of Peden regarding ontog-eny of immune capabilities.7 If crucial elementsrequired for Th1 functional capacity have yet toappear at the time Pb is introduced into the earlyembryo (first half of gestation in the rat and up tonine days of incubation in the chicken), Pb-induceddamage of Th1-linked targets apparently does notoccur.

Therefore, based on the example of early Pb expo-sure, it is possible that exposure to certain chemicalsor environmental factors over brief but differentperiods of development could result in not onlydifferent dose± response profiles for persistent im-munotoxicity, but also differences in the spectrumof resulting immunotoxicity. While it had beenassumed that differential immunotoxic risk waslikely for adult versus nonadult exposures in gen-eral, it had been less clear whether more narrowwindows of differential vulnerability could existsolely within early development. The current data

Table 2 Comparison of persistent immune changes following early exposure to Pb in female rats and chickensa

Species and exposure Altered immune parameters

Reduced DTHb

activationIncreased thymic

weight d

Altered cytokinebalancee

Depressedc

macrophage

RatFull gestational exposure + NDf + +First half of gestation ¡ ¡ ¡ ¡Last third of gestation + + + ¡

ChickenEmbryonic day 5 ¡ ¡ ND +Embryonic day 7 ¡ ND ND +Embryonic day 9 ¡ ND ND +Embryonic day 12 + ¡ ND ¡aInformation taken from Bunn et al.,17,19,37Lee et al.,18and Chen et al.36bDTH response to KLH in rats and BSA in chickens. cCapacity toproduce nitric oxide. dThymic weight relative to body weight. eRefers to either IL-10/IL-12 comparisons or IL-4/IFN-g comparisons asmeasures of Th2/Th1-related cytokine balance. fNot done.

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for early Pb exposures raise the possibility that, forsome chemicals, exposures during different develop-mental windows of gestation will result in differentialrisk of immunotoxicity to the developing immunesystem.

Clearly, it will be helpful to enlarge the develop-mental immunotoxicological database by havingmore investigations conducted using a broader arrayof toxicants. Additionally, direct comparisons ofexposures during different windows of immunedevelopment can aid the identification of compara-tive risk of immunotoxicity and ultimately improve

the protection of the potentially most vulnerablepopulations.

Acknowledgements

The authors thank Shawna Lindsay and ForrestSanders for their help with the preparation of thismanuscript. The research results described in thepaper involving the authors’ laboratory were sup-ported by the NIEHS (Superfund), the USDA (regionalgrant), and the Chemical Manufacturer’s Association.

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