Michael S. Piepenbrink

Lead and Immune Function

The heavy metal lead is a widely deposited environmental toxicant known to impact numerous physiological systems, including the reproductive, neurological, hepatic, renal, and immune systems. Studies illustrating the capacity of lead to impair immune function and/or host resistance to disease date back to at least the 1960s. However, it has only been in recent years that lead has been recognized among a new category of immunotoxicants—those that dramatically shift immune functional capacity while producing only modest changes to immune cell populations and lymphoid organs. These relatively noncytotoxic immunomodulating chemicals and drugs represent the immunotoxic hazards most difficult to identify and problematic for risk assessment using historic approaches. As a result, such environmental factors are also among the most likely to contribute to chronic immune-related disease at relevant exposure levels. This review considers the animal and human evidence that lead exposure can produce a stark shift in immune functional capacity with a skewing predicted to elevate the risk of atopic and certain autoimmune diseases. At the same time, host defenses against infectious agents and cancer may be reduced. Age-based exposure studies also suggest that levels of blood lead previously thought to be safe, that is, below 10 μg/dl, may be associated with later life immune alterations.

Perinatal Immunotoxicity: Why Adult Exposure Assessment Fails to Predict Risk

Recent research has pointed to the developing immune system as a remarkably sensitive toxicologic target for environmental chemicals and drugs. In fact, the perinatal period before and just after birth is replete with dynamic immune changes, many of which do not occur in adults. These include not only the basic maturation and distribution of immune cell types and selection against autoreactive lymphocytes but also changes designed specifically to protect the pregnancy against immune-mediated miscarriage. The newborn is then faced with critical immune maturational adjustments to achieve an immune balance necessary to combat myriad childhood and later-life diseases. All these processes set the fetus and neonate completely apart from the adult regarding immunotoxicologic risk. Yet for decades, safety evaluation has relied almost exclusively upon exposure of the adult immune system to predict perinatal immune risk. Recent workshops and forums have suggested a benefit in employing alternative exposures that include exposure throughout early life stages. However, issues remain concerning when and where such applications might be required. In this review we discuss the reasons why immunotoxic assessment is important for current childhood diseases and why adult exposure assessment cannot predict the effect of xenobiotics on the developing immune system. It also provides examples of developmental immunotoxicants where age-based risk appears to differ. Finally, it stresses the need to replace adult exposure assessment for immune evaluation with protocols that can protect the developing immune system.

The managed immune system: protecting the womb to delay the tomb

The developing immune system serves as a novel target for disruption by environmental chemicals and drugs, and one that can significantly influence later-life health risks. Specific immune maturational events occur during critical windows of pre- and early postnatal development that are not effectively modeled using adult exposure-assessment or general developmental toxicity screens. The range of postnatal health risks linked to developmental immunotoxicity (DIT) is influenced, in part, by the natural progression of prenatal-neonatal development. In this progression, the pregnancy itself imposes a Th2-bias in utero, and this produces a delay in the acquisition of Th1 functional capacity in the newborn. The status of Th1 regulatory and Th17 populations may also be important in immune function/dysfunction considerations. The necessary shift from a Th2 preferred capacity in late gestation to a more balance functional capacity in the neonate can be disrupted by xenobiotics leaving the child with increased vulnerability to a range of potential diseases. Knowledge of environmental factors that facilitate effective immune functional maturation as well as those xenobiotics capable of disrupting the process is important in strategies to reduce the incidence of diseases such as childhood asthma. Because hormesis has been shown to be an important factor in modulation of the adult immune system, it becomes even more important to understand potentially opposing dose-response effects for the immune system of the fetus, neonate, and juvenile. The direct linkage between immune dysfunction and chronic disease has become abundantly apparent in recent years. Therefore, a more comprehensive and effective approach for the protection of the developing immune system can help to reduce the incidence of later-life chronic diseases.

Developmental Immunotoxicity of Lead in the Rat: Influence of Maternal Diet

The effect of maternal dietary protein intake on lead-induced developmental immunotoxicity was studied in female Fischer 344 rats receiving lead acetate (250 ppm) or sodium acetate (control) in the drinking water during breeding and pregnancy until parturition. Dams were fed isocaloric diets (either 20% casein or 10% casein) from 2 wk prior to mating until the end of lactation. After weaning, dams and female offspring were given the 20% casein diet and regular water. Immune function was assessed in dams at 8 wk postpartum and in offspring at 13 wk of age. Dams showed no marked difference in any of the immune endpoints examined, regardless of diet or lead treatment. In contrast, lead exposure during early development produced a subsequent significant reduction of both the delayed-type hypersensitivity response and interferon γ production in adult offspring independent of maternal diet. Lead-exposed offspring from the high-dietary-protein group had significantly elevated production of both interleukin-4 and tumor necrosis factor α(TNF-α) with increased relative spleen weight and a decreased body weight compared to offspring in the lead control group. In contrast, lead-exposed offspring from dams receiving the low-protein diet had no marked change in TNF-α levels, relative spleen weight, or body weight, while interleukin-4 levels were significantly reduced compared with the lead control group. In conclusion, maternal dietary protein intake can modulate the immunotoxic effects of lead exposure during early development. This occurred at levels of protein intake and doses of lead exposure that produced no detectable effect on the maternal immune system.

Developmental Immunotoxicology of Di-(2-Ethylhexyl)phthalate (DEHP): Age-Based Assessment in the Female Rat.

Abstract Di-(2-ethylhexyl)phthalate (DEHP) helps provide flexibility to plastic products including food containers and medical IV devices. Age-based sensitivity to DEHP-induced immunotoxicity was compared in female CD strain rats following administration for 16 consecutive days in utero vs. adult exposure. Pregnant and non-pregnant rats received 0, 37.5, 75, 150, or 300 mg/kg/day of DEHP in soybean oil daily by oral gavage. In pregnant animals, this corresponded to days 6–21 of gestation. In exposed offspring, anogenital distance (AGD) was measured at one and three weeks of age. For a subset of offspring, immune assessment was conducted at 5 weeks of age following KLH immunization at 3 and 4 weeks. KLH immunizations occurred in remaining offspring at 11 and 12 weeks with immune assessment at 13 weeks. Nonpregnant adults were immunized concurrently with adult offspring and underwent immune assessment 13 weeks post-DEHP exposure. Litter size, sex ratio, and 1-week body weights were unaffected by treatment. However, AGD was altered at both ages assessed. Three-week (but not 5-week) body weights were elevated in exposed offspring. Immune organ weights, thymus histology, antibody levels (IgG and IgE), DTH reaction to KLH, total and differential leukocyte counts, ex vivo cytokine production (IL-2, -4, -10, -12, interferon-gamma), as well as TNF-alpha and nitric oxide production by macrophages were not altered by treatment at any age. Flow cytometry analysis of splenocytes showed no differences during the juvenile assessment. In conclusion, in utero exposure of female rats to DEHP alters some developmental parameters (i.e., AGD) but has no persistent effect on the immune system based on the assessment parameters. Adult female rats also had no detectable immune alteration following similar exposures.

Impact of In ovo-Administered Lead and Testosterone on Developing Female Thymocytes

The developing immune system is particularly sensitive to lead-induced immunotoxicity, but in some models, genders can differ in lead-induced immunotoxicity. Using an avian in ovo model of lead-induced T-helper disruption, the ability of in ovo administered lead and testosterone to alter thymocyte maturation among female embryos was investigated. On embryonic day (E) 8, Cornell K-strain embryos were given either testosterone (12.5 μg/egg in ethanol) or 15% ethanol in 100 μl volume. The groups then received either lead acetate (200 μg/egg) or sodium acetate (control) on E 12 of incubation. On E 20, thymocytes from 4–5 female embryos per group were analyzed by flow cytometry for cell surface markers CD3, CD4, CD8, TCR1, and TCR2. Lead alone did not induce any appreciable changes among the cell populations measured in this study. However, when testosterone treatment was followed by lead (testosterone + lead), there was a significant increase in CD4+CD8+ double-positive cells compared with either control or lead treatment groups. Testosterone, either by itself or in combination with lead, significantly reduced the percentage of cells with the CD4+CD8− phenotype when compared to the lead alone group. No change was detected with respect to the CD4−CD8+, CD4−CD8−, TCR1+, and TCR2+ phenotypes following any treatment. Therefore, sex hormonal balance in early life appears to influence the manner in which the developing thymus responds to the heavy metal lead.

Importance of Differential Expression of Marek's Disease Virus Gene pp38 for the Pathogenesis of Marek's Disease

SUMMARY.  The role of pp38 in the pathogenesis of Mareks disease (MD) has not been fully elucidated. Previously, we reported the presence of two splice variants (Spl A and Spl B) for pp38. We also reported that the wild-type pp38 (WT), as well as the mutated pp38 (MUT), altered the oxidative phosphorylation pathway in infected cells. To determine whether the different forms of pp38 are important for the pathogenesis of MD, we generated RB-1B–based bacterial artificial chromosome (BAC) clones expressing pp38MUT, pp38Spl A, and pp38Spl B. Infectious viruses were recovered from these BAC clones in chick kidney cells (CKC). The Spl A and Spl B viruses had significantly smaller plaque sizes and replicated to a lesser degree in CKC than the WT and MUT viruses. Two in vivo experiments were conducted by inoculating 7-day-old P2a chicks with 1000 plaque-forming units of each virus. In the first experiment, chicks were sacrificed at 4, 8, 11, and 15 days postinfection (PI). WT and MUT viruses had similar viremia levels using virus isolation and quantitative real-time PCR (qPCR) assays, whereas Spl A and Spl B viruses had significantly lower viremia levels than WT and MUT viruses. In the second experiment, we showed that tumor development and MD mortality were similar in the WT- and MUT-infected chickens, with all birds MD positive at 5 wk PI. In contrast, chickens infected with Spl B and Spl A had a significantly lower MD incidence at 11 wk PI, when the experiment was terminated. RESUMEN.  Importancia de la expresión diferencial del gene pp38 del virus de Marek en la patogénesis de la enfermedad de Marek. El papel del gene pp38 en la patogénesis de la enfermedad de Marek (con las siglas en inglés MD) no ha sido aclarado completamente. Previamente se reportó acerca de la presencia de dos variantes alternativas de corte y empalme (Spl A y Spl B) para el gene pp38. También se informó de que el gene pp38 de tipo silvestre (WT), así como la forma mutante de pp38 (MUT), alteraron la vía de la fosforilación oxidativa en las células infectadas. Para determinar si las diferentes formas del gene pp38 son importantes para la patogénesis de la enfermedad de Marek, se generaron clones del virus RB-1B mediantes clonación en un cromosoma bacteriano artificial (BAC) que expresaban a los virus infecciosos pp38MUT, pp38Spl A y pp38Spl B. Se recuperaron partículas infecciosas a partir de estos clones BAC en cultivos de células renales de pollo (CKC). Los virus Spl A y Spl B producían placas significativamente más pequeñas y se replicaban en un grado menor en los cultivos de células renales en comparación con el virus silvestre y su forma mutante. Se llevaron a cabo dos experimentos in vivo mediante la inoculación de pollos P2a de siete días de edad con 1000 unidades formadoras de placa de cada virus. En el primer experimento, los pollos fueron sacrificados a los 4, 8, 11, y 15 días después de la infección. Los virus silvestre y mutante produjeron niveles similares de viremia que se determinaron mediante el aislamiento del virus y ensayos de PCR cuantitativos en tiempo real, mientras que los virus Spl A y Spl B mostraron niveles significativamente más bajos de viremia en comparación con los virus silvestre y mutante. En el segundo experimento, se demostró que el desarrollo de tumores y la mortalidad por la enfermedad de Marek fue similar en los pollos infectados con los virus silvestre o mutante, con todas las aves positivas a la enfermedad de Marek a las 5 semanas después de la inoculación. En contraste, los pollos infectados con los virus Spl B y Spl A mostraron una incidencia de la enfermedad de Marek significativamente menor a las once semanas después de la inoculación, cuando se concluyó el experimento.

Immune Complex Vaccines for Chicken Infectious Anemia Virus

SUMMARY. Infection of maternal, antibody-negative chickens with chicken infectious anemia virus (CIAV) can cause clinical disease, while infection after maternal antibodies wane often results in subclinical infection and immunosuppression. Currently, vaccines are not available for vaccination in ovo or in newly hatched chickens. Development of CIAV vaccines for in ovo use depends on the ability to generate vaccines that do not cause lesions in newly hatched chicks and that can induce an immune response regardless of maternal immunity. Immune complex (IC) vaccines have been successfully used for control of infectious bursal disease, and we used a similar approach to determine if an IC vaccine is feasible for CIAV. Immune complexes were prepared that consisted of 0.1 ml containing 105.4 tissue culture infective dose 50% of CIA-1 and 0.1 ml containing 10 to 160 neutralizing units (IC Positive [ICP]10 to ICP160), in which one neutralizing unit is the reciprocal of the serum dilution required to protect 50% of CU147 cells from the cytopathic effects caused by CIA-1. Virus replication was delayed comparing ICP80 and ICP160 with combinations using negative serum (IC Negative [ICN]80 or ICN160). In addition, the number of birds with hematocrit values <28% were decreased with ICP80 or ICP160 compared to ICN80 or ICN160. Seroconversion was delayed in ICP80 and ICP160 groups. To determine if ICP80 or ICN160 protected against challenge, we vaccinated maternal, antibody-free birds at 1 day of age and challenged at 2 wk or 3 wk of age with the 01-4201 strain. Both ICP80 and ICP160 protected against replication of the challenge virus, which was measured using differential quantitative PCR with primers distinguishing between the two isolates. Thus, in principle, immune complex vaccines may offer a method to protect newly hatched chicks against challenge with field virus. However, additional studies using maternal, antibody-positive chicks in combination with in ovo vaccination will be needed to determine if immune complex vaccines will be useful to protect commercial chickens.