Recent progress and diverse effects in developmental immunotoxicology: overview of a symposium at the 46^th Annual SOT Meeting, Charlotte, NC

Abstract

It has long been known that the developing immune system is more sensitive and susceptible than the adult immune system to some drugs and environmental contaminants. However, notable advances have been made in the database of studies supporting developmental immunotoxicity (DIT) over the past 5 years. There is considerable evidence that responses of the immune system can be quantitatively or qualitatively different from normal adult responses when xenobiotic exposure occurs during critical periods of immune system development. Qualitative differences of DIT relative to adult exposures include examples of more persistent effects, a latency of effects, and immune dysfunction that is fundamentally different than effects observed when adults are exposed. A symposium was presented at the Society of Toxicology annual meeting to provide an update on advances in the maturing field of developmental immunotoxicology and to facilitate discussion on the range of DIT and later life effects following developmental exposure. In particular, presentations focused on implications of neuroendocrine cross-talk for DIT, the association between developmental air pollutant exposure and asthma, and recent evidence that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin may increase the risk of autoimmune responses. Several important concepts relative to DIT assessment were illustrated, i.e., (1) Screening for immunosuppression alone is not sufficient to identify all potential immunotoxic effects; (2) DIT cannot be reliably predicted from studies that only utilize adult exposures; (3) Functional testing protocols are preferred in the assessment of DIT; (4) Gender-related differences should be routinely assessed; (5) Latency (i.e., later-life adverse outcomes resulting from developmental exposures) is an important consideration that cannot be detected in adult exposure studies; and, (6) There is increasing support for DIT testing protocols with continuous exposure throughout development until the immune assay is performed.

Keywords::

• Developmental immunotoxicology
• Developmental immunotoxicity
• Children’s health
• Risk assessment

View correction statement:

Erratum

Introduction

Developmental immunotoxicity (DIT), the adverse effects on the immune system that result from pre- and/or postnatal xenobiotic exposures during immune system ontogeny, is an important area of concern for the vulnerability of the developing immune system to toxicological insult. Over the past several decades, research in this area has identified a wide variety of agents that can cause DIT effects (reviewed in Dietert and Dietert, 2007). These include environmental pollutants such as metals (arsenic, cadmium, lead, manganese, and mercury), polycyclic aromatic hydrocarbons (PAHs), polycyclic chlorinated biphenyls (PCBs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), tributyltins, environmental tobacco smoke, atrazine, and bisphenol-A, as well as pharmaceuticals such as cyclosporin A, dexamethasone, diazepam, and diethylstilbestrol.

There has been a long-standing recognition of the vulnerability of the developing immune system to toxicological insult. In 1993, the seminal review Pesticides in the Diets of Infants and Children (NRC, 1993) acknowledged that age-related susceptibility was a concern for pesticide risk assessment and recommended the need for assessment of DIT. A number of significant regulatory events followed (Table 1), addressing perspectives for both environmental risk assessment and pharmaceutical safety evaluation. Landmark legislation was promulgated for the regulation of food-use pesticides and drinking water, requiring characterization of sensitivity in susceptible populations, and specific assessment of risk for infants and children. In 1996, the Food Quality Protection Act (FQPA, 1996) required the application of a special 10-fold uncertainty factor to risk calculations to address toxicity and exposure concerns, unless sufficient data were available to characterize safety for these populations. This increased concern for childrens’ health risk assessment was also expressed in the 1997 Executive Order (President Clinton, 1997) that required United States Federal agencies to specifically address risks to children in any legislation. In a 2002 document reviewing the reference dose and reference concentration processes, EPA specifically addressed the characterization and use of developmental immunotoxicity data in risk assessment (U.S. EPA, 2002; Kimmel et al., 2005). The importance of immunological assessment for pharmaceuticals was addressed in a 2002 guidance document released by the Food and Drug Administration (U.S. FDA, 2002). Further guidance addressing preclinical assessment of endpoints (including immunotoxicity) in animal models, particularly in support of pediatric pharmaceuticals, was finalized in 2006 (U.S. FDA, 2006). This guidance addressed the need for careful consideration of the developmental ontogeny of the test species, the timing of test substance administration and endpoint assessment, and other critical aspects of study design.

Table 1.  Regulatory landmarks in the consideration of DIT testing for risk assessment.

Year	Event	Reference
1993	NRC publication: Pesticides in the Diet of Infants and Children	(NRC, 1993)
1996	EPA legislation: Food Quality Protection Act; Safe Drinking Water Act Amendments	(FQPA, 1996; SDWA, 1996)
1997	Executive Order 13045	(President Clinton, 1997)
2002	EPA publication: A review of the reference dose and reference concentration processes	(U.S. EPA, 2002)
2002	FDA publication: Guidance for industry – immunotoxicology evaluation of investigational new drugs	(U.S. FDA, 2002)
2006	FDA publication: Guidance for industry – non-clinical safety evaluation of pediatric drug products	(U.S. FDA, 2006)

NRC = National Research Council of the National Academies of Science; EPA = U.S. Environmental Protection Agency; FDA = U.S. Food and Drug Administration.

During this same time frame, a number of critical scientific considerations were under discussion. The broad issue of age-related susceptibility was addressed in workshops conducted in 1992, 1995, and 1996 by the EPA and by the Institute of Life Sciences, Risk Science Institute (ILSI/RSI), establishing a basis for consideration of this issue in children’s health risk assessment (ILSI Risk Science Institute, 1992, 1996; Kavlock et al., 1996). Some key collaborative scientific events (which included participation by representatives from government agencies, industry, academia, and public interest groups) were conducted over the past decade to address issues specifically focused on developmental immunotoxicity (Table 2). In an EPA workshop on Identifying Critical Windows of Exposure for Children’s Health (Selevan et al., 2000), the timing of developmental exposures in relation to differential effects on immune system function was explored, emphasizing the need to consider the ontological process when assessing potential hazard. Three very important workshops were conducted from 2001-2003 to attempt to develop a consensus view on the best methods and approaches for DIT testing for immunosuppression potential, to address specific controversial or problematic issues, and to identify remaining limitations and information deficits, some of which might be addressed by further research. Although not all questions that were raised in the course of these workshops have been fully addressed, it was nevertheless possible to develop a basic workable consensus framework for DIT testing that can be utilized to design screening studies that can inform risk assessment (Holsapple et al., 2005).

Table 2.  Key collaborative scientific events that contributed to the development of a framework for DIT assessment.

Year	Event	Reference
1992	ILSI/RSI Workshop: Similarities and Differences Between Children and Adults	ILSI Risk Science Institute, 1992
1995	EPA Workshop: On Research Needs for the Risk Assessment of Health and Environmental Effects of Endocrine Disruptors	Kavlock et al., 1996
1996	ILSI/RSI Workshop: Research Needs on Age-related Differences in Susceptibility to Chemical Toxicants	ILSI Risk Science Institute, 1996
1999	EPA Workshop: Identifying Critical Windows of Exposure for Children’s Health	Dietert et al., 2000; Holladay and Smialowicz, 2000
2001	ILSI/HESI DIT Workshop	Holsapple, 2003
2001	NIEHS/NIOSH DIT Workshop	Luster et al., 2003
2003	ILSI/HESI DIT Workshop	Holsapple et al., 2005
2003	SOT Symposium: What’s So Special about the Developing Immune System?	Holsapple et al., 2004
2004	SOT Roundtable	Ladics et al., 2005
2007	SOT Symposium: Current Issues in DIT	Makris et al. (submitted)

NRC = National Research Council of the National Academies of Science; ILSE/RSI = International Life Science Institute, Research Science Institute; ILSI/HESI = International Life Science Institute, Health and Environmental Science Institute; NIEHS = National Institute of Environmental Health Science; NIOSH = National Institute of Occupational Safety and Health; SOT = Society of Toxicology.

Further discourse on DIT assessment has also been conducted through a variety of scientific symposia and forums, a few key examples of which are listed in Table 2. The 2004 roundtable session explored the important concept of incorporating DIT assessments into other toxicological protocols, such as a single- or multi-generation reproductive toxicity study. In a symposium conducted at the 2003 Society of Toxicology annual meeting, presentations explored the unique nature of the developing immune system, and evidence was presented suggesting that the early life immune system was a critical toxicologic target. However, the symposium participants concluded that: “comparative scientific data clearly demonstrating that the developing immune system does in fact show greater vulnerability to chemical perturbations are still very limited.” The overall conclusion of the symposium was that additional DIT studies were needed to more clearly demonstrate the extent of differential age-related sensitivity and to guide potential regulatory decisions in better protecting children’s health (Holsapple et al., 2004).

In 2007, a symposium entitled “Current Issues in Developmental Immunotoxicology” was held at the 46^th Annual Meeting of the Society of Toxicology (SOT) in Charlotte, NC. This symposium was sponsored by the SOT Immunotoxicology Specialty Section and was co-sponsored by the SOT Reproductive and Developmental Toxicology, Neurotoxicology, and Risk Assessment Specialty Sections. The 2007 symposium addressed a number of issues relevant to the assessment of DIT and the use of these data in human health risk assessment. The overall topics represented a logical continuation of activities and scientific advancements in this field. It moved beyond description and support of the concept of increased susceptibility and age-based sensitivity of the immune system to draw attention to implications of developmental immunotoxicology on later-life immune dysfunction. In addition, the 2007 DIT symposium presented a broad focus spanning the full range of immunotoxicity research areas, rather than concentrating on immunosuppression alone as is often done in this discipline.

Overview of the DIT symposium

The presentations given at the 2007 symposium illustrated a significant advance in the database of studies supporting DIT compared to those available in 2003. DIT has been shown for the four major classes of immunotoxicity including unintended immunosuppression, unintended immunostimulation, hypersensitivity, and autoimmunity. To capture this broad scope, the DIT symposium utilized experts from the immunotoxicology research community as well as experts from outside fields that perform DIT-related research that is normally published in pediatric or neuroendocrine journals and can therefore be overlooked by immunotoxicologists monitoring more traditional immunotoxicology journals. During the symposium, Dr. Rodney Dietert highlighted several areas of research with increased evidence for the implications of DIT on disease and health in later life. Dr. Denise Bellinger presented the implications of neuroendocrine cross-talk for developmental immunotoxicology with an emphasis on stress in the pre- and perinatal environment. A presentation was given by Dr. Lester Kobzik that brought data together from an increasing number of studies demonstrating the strength and complexity of the association between developmental air pollutant exposure and asthma. Recent evidence that developmental exposure to TCDD may increase the risk of autoimmune responses was presented by Dr. Steven Holladay that extended the range of immune effects beyond immunosuppression previously associated with developmental exposure to TCDD. In the final presentation, Dr. Yung Yang examined the regulatory framework as it currently exists to drive developmental immunotoxicology research and risk assessment. Their insights are captured in the subsequent series of papers and briefly summarized here.

In the paper by Dr. Rodney Dietert (Cornell University), the enhanced vulnerability of the developing immune system for environmental insult is based on unique immune maturational events that occur during critical windows of vulnerability in early life. The semi-allogeneic pregnancy state, with suppression of graft rejection and associated skewing of the fetal and neonatal immune system, also influences the specific nature of DIT outcomes. In the exposed offspring, targeted immunosuppression can co-exist with an increased risk of allergic and/or autoimmune disease. Because with DIT immune dysfunction rather than profound immunosuppression is the greater concern, testing approaches should emphasize multi-functional assessment. Beyond T-lymphocytes, dendritic cells and macrophages are sensitive targets. The last-trimester fetus and the neonate are normally depressed in T_H1-dependent functions and postnatal acquisition of needed T_H1 capacity is a major concern with DIT. With this in mind, assessment should include a measure of T_H1-dependent cell- mediated immunity [cytotoxic T-lymphocyte (CTL) activity or delayed-type hypersensitivity (DTH) response] in conjunction with a multi-isotype T-dependent antibody response (TDAR) and evaluation of innate immunity (e.g., NK activity). Other parameters such as immune histology, immunophenotyping, cytokine responses and organ weights can be useful when included with immune functional evaluation.

The paper by Drs. Fedulov and Kobzik (Harvard School of Public Health) focuses on the link between the perinatal environment and risk of developing asthma in the offspring. As emphasized in Dr. Kobzik’s presentation in the DIT symposium, the paper calls attention to the fact that maternal asthma, rather than paternal, is a well established risk factor for children developing the disease. The paper summarizes several animal research models of the maternal transmission phenomenon for asthma risk. Recent data from Drs. Kobzik and colleagues demonstrated that intranasal exposure of pregnant mice to particles increased airway hypersensitivity symptoms in their offspring independent of the asthmagenic properties of the particles (i.e., exposure to control inert TiO₂ and known asthmagens such as diesel exhaust particles both promoted symptoms). The paper presents the hypothesis along with supporting data that pregnancy alters the innate immune response such that common environmental airborne particles become environmental stressors that increase the risk of developing asthma in the offspring.

The paper by Drs. Bellinger, Lubahn, and Lorton (Loma Linda University of Medicine) expands on the presentation given by Dr. Bellinger at the 2007 DIT symposium. They review the effects of maternal and perinatal stress on immune function in offspring as well as implications for DIT. The paper summarizes neuroendocrine control of the immune system through glucocorticoids, catecholamines, and other mediators. Drs. Bellinger et al. highlight animal data on the consequences of both prenatal and postnatal stress on the immune system. They also present the limitations of animal models of early life stress to predict human risk of functional immune effects. The authors spell out the potential implications for interactions and shared mechanisms between chemical, physical, and psychosocial stress on the immune system. Dr. Bellinger and colleagues conclude that psychosocial stress during prenatal and early postnatal development increases the vulnerability of offspring to the effects of immunotoxicants.

The paper by Drs. Gogal and Holladay (Virginia Tech) reviews current data supporting the hypothesis that developmental exposure to TCDD increases the risk of postnatal autoimmune responses and reflects the presentation given by Dr. Holladay at the 2007 DIT symposium. The paper presents data on immune effects of TCDD that suggest an autoimmune mechanism and therefore DIT effects in addition to immunosuppression, which is commonly associated with developmental exposure to TCDD in the immunotoxicology literature. The paper outlines evidence demonstrating that developmental TCDD alters normal fetal thymocyte selection in a number of ways including down-regulation of thymic MHC Class I and II molecule expression, altered relative CD4 and CD8 surface antigen expression, and a shift in T-cell receptor (TCR) expression during the initiation of development of neonatal central tolerance. Together with published studies, the data from Drs. Gobel and Holladay support the potential for increased risk of autoimmunity following developmental exposure to TCDD as a result of impaired normal thymic deletion of autoreactive T-lymphocytes.

The presentation by Dr. Yung Yang (U.S. EPA) provided a concise overview of the current regulatory framework as it applies to DIT research and the implications of DIT data for risk assessment. A brief summary of the presentation is provided here and a separate manuscript is not included in this issue of the Journal of Immunotoxicology. Dr. Yang highlighted major regulatory actions that contribute to an increased focus on the developmental period for immunotoxicity research and risk assessment (see Table 1) and suggested these actions contributed to the shift away from reliance on classical guideline toxicity studies utilizing adult animals for risk assessment of children and infants to testing approaches designed to assess the vulnerability of the developing immune system to toxicants.

Data obtained from laboratory studies designed to investigate DIT demonstrate that exposure during critical developmental windows are associated with increased sensitivity (causing immunotoxicity at lower doses in the young than in adults) and increased persistence of effects (causing long-lasting immunotoxicity at doses that either do not affect immune function or cause short-term effects in adults) (e.g., see review by Luebke et al., 2006). Dr. Yang also highlighted recent changes in the approach to DIT testing that emphasize continuous exposure from the developmental and prenatal periods through the juvenile stage when testing is performed after the animal acquires a functionally mature immune system and an adult-level immune response. This is in marked contrast to older testing regimes in which the developmental exposure was followed by a non- exposure period that amounted to a recovery phase prior to testing after the animal had attained an adult-level immune response.

Summary/Conclusion

It is clear from the presentations given at the symposium on “Current Issues in Developmental Immunotoxicology” at the annual SOT meeting and the succeeding series of papers that DIT includes the full range of immunotoxicity seen in adult studies from immunosuppression to autoimmunity. It is also obvious that considerable advances have been made in the strength of the database supporting DIT over the past 5 years. This is particularly evident by contrasting the state of the science evident in the 2007 SOT symposium summarized here and in the following papers, with the 2003 SOT symposium entitled “Children’s Health Risk – What’s So Special About the Developing Immune System?” While the 2003 symposium was concerned with supporting early life exposure as a critical developmental period for the immune system (Holsapple et al., 2004), the 2007 symposium moved beyond the description and support of DIT to explore the broader implications of DIT on later life immune function (Makris et al., Submitted). The following papers illustrate a significant advance in the overall database of studies supporting DIT compared to those available in 2003.

In conclusion, the 2007 symposium and subsequent papers demonstrate the strength of the database of studies supporting DIT and represent some of the broad range of consequences of DIT on later life immune function. In addition, there are several general concepts that repeatedly emerged in discussions at the symposium and are reflected in these papers.

• Assays for immunosuppression alone are not sufficient to detect DIT

  DIT effects have been demonstrated in multiple aspects of immunotoxicity and it is common for DIT effects to be targeted rather than pervasive. Therefore, a robust DIT assessment strategy requires multiple functional assays to investigate multiple aspects of the immune response.

• Adult immunotoxicity testing is not sufficient to detect potential DIT

  It is well established that there is an increased sensitivity and susceptibility of the immature immune system to toxicological insult that may result in greater dose-sensitivity or persistence of adverse outcome.

• Functional testing protocols are preferred in the assessment of DIT

  DIT outcomes and later life effects such as asthma, autoimmunity, and mild to moderate immunosuppression may not be detected by standard toxicological screening methods that do not include specific immune function assays.

• Gender-based differences in DIT are relatively common

  While gender differences in DIT might be predicted for a subset of endocrine disrupting chemicals, it is also seen for chemicals that are not classic mimics or blockers of gonadal steroids.

• Adult immunotoxicity testing would not detect potential DIT-associated latency

  DIT testing is required to examine the potential of latency or later life triggering of immune dysfunction following early life exposure.

• The preferred DIT testing protocol has moved toward continuous exposure

  Developmental exposure with later life evaluation of immune function can be characterized as recovery study, and there is increasing support for continuous exposure throughout development up until the immune assay is performed.

DIT includes a range of potential effects (unintended immunosuppression, unintended immunostimulation, hypersensitivity, and autoimmunity) and therefore a comprehensive testing regime must reflect the range of potential immunotoxicity. The disease outcomes resulting from early life exposures, including asthma, autoimmunity, and immunosuppression are of critical importance in the assessment of risks to human health. DIT appears to be linked to an increased risk of a wide spectrum of infectious diseases, cancer, autoimmunity, asthma, and atopy (Dietert and Dietert, 2007). Adverse consequences of a compromised immune system resulting from developmental exposure may be expressed at any point throughout an individual’s lifespan, not just during development. Increased research into the identification and characterization of hazards to the developing immune system should be undertaken in light of the breadth of later life immune effects associated with developmental exposure.