Wadsworth Center, New York State Department of Health, P.O. Box 509, Albany, New York 12201-0509
Received February 18, 2000; accepted May 22, 2000
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ABSTRACT |
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Key Words: lead; IgE; transplacental; lactational; neonate; allergy; immunomodulation.
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INTRODUCTION |
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There is a series of conflicting literature reports regarding maternal transfer of Pb to offspring. Miller et al. (1998) suggested that the lead concentration in the milk of exposed rats was insufficient to cause significant exposure to the nursing pup. Ryu et al. (1978) reported that the blood Pb levels of a woman employed in a Pb industry were much higher than her milk Pb levels. In contrast, Lorenzo et al. (1977) found considerably higher Pb concentrations in the milk than the blood of Pb-exposed, lactating rabbits, and a study by Keller and Doherty (1980a) reported that Pb-exposed mouse dams transmited a significantly greater amount of Pb to their offspring through their milk than by in utero exposure (transplacentally).
Little is known about the immunotoxic effects of Pb exposure during gestation and/or lactation. Previous studies have shown that exposure of adult BALB/c mice to Pb acetate induced an elevation of serum interleukin (IL)-4 (a type-2 cytokine) and IgE levels and decreased interferon-gamma (IFN-, a type-1 cytokine) levels (Heo et al., 1996
, 1997
). Also, BALB/c mice exposed to Pb acetate in their drinking water showed a significant inhibition of their cell-mediated immune (CMI) response, which included a higher mortality rate upon infection with sublethal doses of the intracellular bacteria Listeria monocytogenes, decreased IFN-
and increased IL-6 serum levels (Kishikawa et al., 1997
). Miller et al. (1998) reported that gestational Pb exposure caused an elevation of serum IgE and decreased the IFN-
levels of sera from rat offspring older than 7 weeks of age. The studies of Faith et al. (1979) and Luster et al. (1978) reported that Pb exposure of rats throughout gestation and lactation suppressed thymic weights and lymphocyte responsiveness to mitogen stimulation and delayed hypersensitivity reactions.
Due to recent reports of an increased incidence of the atopic condition including asthma in children as a posited consequence of environmental pollutants, we chose Pb as a prototypic environmental toxicant to assess its ability to affect IgE production early in life. IgE, the immunoglobulin isotype associated with mast cell degranulation, triggers the secretion of histamine and other mediators of allergic responses. Atopy is defined as IgE-mediated immune hyperresponsiveness. Total serum IgE levels are believed to be indicative of the extent of a patient's allergic disease, and elevated cord serum IgE levels have been suggested, although not without controversy, to be predictive of later atopic conditions (Edenharter et al., 1998). Additionally, a direct correlation has been made between serum IgE levels and bronchial hyper-responsiveness (BHR) (Sears et al., 1991
).
To compare the differential hazards of gestational and lactational Pb exposure, pregnant BALB/c mice were exposed to Pb in their drinking water beginning at approximately day 15 of gestation, and cross-fostering of litters at parturition was performed. Blood Pb levels were analyzed at various time points after birth to determine the efficiency of Pb transfer from dams to offspring during gestation versus lactation. Also, a variety of immune parameters were assessed during the first weeks of life to determine if Pb-induced immunomodulation was, in fact, observed due to gestational exposure, or if suckling offspring were at a higher risk of the adverse effects of Pb.
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MATERIALS AND METHODS |
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Experimental Design
Dose-response experiment.
BALB/cByJ females (8 weeks of age) were housed with BALB/cByJ males (two females, one male per cage) for 15 days, after which males were removed, and females were housed individually and begun on 0, 0.08, 0.4, and 1 mM Pb acetate in their drinking water. Lead drinking water was continued until litters were weaned at 4 weeks. Weaned mice were continued on the dose to which their mothers were exposed for an additional 2 weeks, at which time all mice were placed on tap water until assayed. Litters were sacrificed at 18 weeks after birth; thus, mice assayed at 8 weeks of age were without Pb exposure for the last 2 weeks. Results include only litters with Pb exposure begun on gestation day 1416.
Cross-fostering experiment.
BALB/cByJ female mice (8 weeks of age) were housed with BALB/cByJ males (three to four females, one male/cage) for 15 days; at this time males were removed, and females were given drinking water with 0 or 0.1 mM Pb acetate (a level about 2000-fold higher than our tap water). At parturition, several litters were cross-fostered between Pb-exposed and control dams. All cross-fostering of litters occurred between two dams who had given birth within 24 h of each other. Mice were sacrificed at 16 weeks after birth and were weaned at 4 weeks of age. Dams' Pb exposure was continued until weaning (4 weeks), after which the weaned mice were continued on control water or 0.1 mM Pb acetate.
IgE and Interleukin-4 (IL-4) ELISA
Plasma IgE and IL-4 levels were assayed by a sandwich ELISA using the method supplied by PharMingen as described (Heo et al., 1996). Briefly, Corning Easy Wash plates were coated by overnight incubation at 4°C with capture mAb (100 µl/well; 2 µg/ml). The plates were washed three times and blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (200 µl/well) for 2 h. The plates were washed again with PBS/Tween 20 two times, and the plasma or standards were added to each well. After an overnight incubation at 4°C, the plates were washed and biotinylated detecting mAb (100 µl/well; 2 µg/ml) was added and incubated at room temperature for 45 min. The plates were washed and further incubated with avidin-peroxidase (Sigma) for 30 min before detection using the substrate 2,2`-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) (Sigma). Absorbance was measured at 405 nm with an ELISA reader (Ceres UV900C: BIO TEK Instruments, Inc). The amount of IgE and IL-4 was calculated from the linear portion of the standard curves. The lower limit of detection was 12 ng/ml for IgE and 30 pg/ml for IL-4.
Spleen Cellularity
Single-cell suspensions of spleens were made by mild homogenization using single frosted slides, and cells were suspended in RPMI 1640. The suspensions were placed in 15-ml polypropylene tubes (Sarstedt), and the tubes were placed on ice for 3 min to allow debris to settle. The supernatant was then transferred into a new tube. Cell counts were performed with a Coulter Counter ZM.
Spleen and Thymus Weights
At the time of sacrifice, thymuses and spleens were removed using sterile technique and immediately placed in tared petri dishes, and wet weights were determined (Mettler PE 1600).
Blood Pb Quantification
Blood was collected by cardiac puncture and placed into plastic Microtainer tubes containing EDTA (Becton Dickinson); tubes were certified as lead-free by the Wadsworth Center's Lead Poisoning Laboratory. Pb concentrations in blood were measured using a well-established method based on electrothermal atomization atomic absorption spectrometry with Zeeman-effect background correction (Parsons and Slavin, 1993).
Flow Cytometry
Single-cell suspensions of thymuses were quantified for developmental T cell subsets (CD4/CD8, CD4+/CD8+, CD4+/CD8, and CD4/CD8+) as follows. Cells (1 x 106) were stained for 30 min with FITCanti-CD8 in combination with PEanti-CD4 (PharMingen) at 4°C in the dark. The cells were then washed once with PBS/azide and analyzed (20,000 open gated) by flow cytometry (Becton Dickinson FACScan). Separate analysis of cell viability with propidium iodide indicated no substantial differences. For analysis of lymphoid subsets in the spleen, 1 x 106 cells were treated with the following combinations: Cy-Chromeanti-CD45, FITCanti-CD3, PEanti-CD4; Cy-Chromeanti-CD45, FITCanti-CD3, PEanti-CD8; Cy-Chromeanti-CD45, FITCanti-CD3, PEanti-CD19. After the cells were stained in BD TruCountTM tubes, they were directly treated with BD FACSTM lysing solution and analyzed. The analyzed cells were first gated by side-scatter light (SS) in combination with CD45. The low SS and CD45 bright cells (lymphocytes) were assessed for the various combinations. Use of the TrueCountTM tubes allowed direct quantification of absolute cell numbers of B cells (CD3/CD19+), CD4 T cells (CD3+/CD4+), CD8 T cells (CD3+/CD8+), and NK cells (CD3/CD19).
Expression of Results and Statistical Analysis
All data are presented as mean ± SEM and were analyzed by one- or two-way analysis of variance (ANOVA) as appropriate. Data that did not meet the normal distribution criteria was log-transformed before analysis. Only when ANOVA p-value for a main effect or interaction was < 0.05, the appropriate means were separated by the Student-Newman-Keuls (SNK) post hoc test.
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RESULTS |
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Plasma IL-4 Concentrations in Pb-Exposed and Control Animals
Plasma IL-4 concentrations of the offspring of mice exposed to 0.1 mM Pb acetate gestationally and/or lactationally (14 weeks of age) as well as age-matched controls were below the detection limit (30 pg/ml) of the sandwich ELISA performed.
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DISCUSSION |
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Our results indicate that Pb readily passes through the placenta, because 1-day-old neonates had blood Pb levels similar to those of their mothers (data not shown). In addition, 1-week-old offspring who were born to dams exposed to Pb beginning at approximately day 15 of gestation and cross-fostered (nursed) within 24 h postpartum by nonexposed dams (Pb/H20 group) had higher blood Pb levels (but not significant) than age-matched controls never exposed to Pb. A better biological indicator that the 5-day gestational exposure was significant was apparent with the significant difference in splenic cellularity (Pb/PB vs H2O/Pb; Fig. 4) and splenic B-cell numbers (Pb/H2O and Pb/Pb vs H2O/Pb; Fig. 5B
). This indicates that the blood Pb levels of the offspring drop rapidly after birth if there is no further exposure lactationally, but the biological effects remain, at least on some immune parameters.
Offspring also obtain Pb from the milk of Pb-drinking dams, the H20/Pb group (Fig. 2). Interestingly, the blood Pb level appeared to plateau by 1 week, in that from 1 week of age to weaning at 4 weeks there was no further increase in the blood Pb level (Fig. 1
). This suggests a fairly consistent and constant exposure via the milk. Furthermore, with all Pb doses (Fig. 1
) when the neonates previously getting the Pb from their mothers were weaned and given their own drinking water containing Pb, their blood Pb levels declined. Thus, we observed rapid decline in blood Pb levels after gestational and/or lactational exposure to Pb, suggesting that maternal delivery of Pb gestationally and lactationally is efficient. It is also apparent that Pb uptake is greater during the fetal/neonatal period, in that adult mice drinking 1 mM Pb acetate for 2 to 4 weeks have blood Pb levels of about 3045 µg/dL, but 2-fold higher levels were achieved with 1-week-old neonates (Fig. 1
). This result emphasizes the need to better understand environmental exposures such as Pb intoxication in order to control childhood illnesses, because maternal transfer and/or neonatal absorption appears to be much more efficient than direct exposure of adults.
Keller and Doherty (1980a) reported that whole-body retention of Pb is greater in nonlactating than lactating mice, which implies that lactation provides an opportunity for enhanced Pb clearance. Furthermore, a study by Watson et al. (1997) found milk Pb levels approximately 10 times as high as corresponding blood Pb levels in lactating rats, and Keller and Doherty (1980b) found that 25% of the absorbed maternal Pb was transferred to offspring. It is possible that lactating mice retain a lower concentration of Pb because Pb may preferentially bind to milk components as opposed to other endogenous components of the lactating animal, possibly reducing organ/tissue absorption. Beach and Henning (1988) reported that greater than 80% of 203Pb in rat milk administered either in vivo or in vitro was bound to casein micelles. Their study also showed that 15- to 16-day-old pups gavaged with 203Pb-labeled rat milk had protein-bound Pb in their stomachs and small intestine when luminal contents from those areas were collected 2 h later. These studies, in accordance with results shown herein, suggest that Pb efficiently binds to milk components, resulting in a greater clearance of maternal Pb burden, which is directly transmitted to suckling offspring. The data suggest that bodily retention of Pb is greater when Pb is ingested in milk as opposed to H20. A study by Kostial and Kello (1973) reported that adult rats exposed to Pb via a milk diet had enhanced Pb absorption. However, several studies found a milk diet had no effect (Meredith et al., 1977) or decreased (Blake and Mann, 1983
; Henning and Leeper, 1984
) neonatal Pb absorption. The possible variances of intestinal absorption during early life as opposed to adulthood are another factor that may account for the observation that animals have less Pb in their bloodstream 2 weeks after weaning as opposed to before. During the third week of life in the mouse, the loss of pinocytotic absorption and lactase activity and increases in sucrase and maltase activity have been reported (Henning and Kretchmer, 1973
). Pb has been shown to be absorbed into the gastrointestinal tract of neonatal mice primarily through pinocytotic processes (Keller and Doherty, 1980c
). Therefore, the decrease in blood Pb levels of the offspring 2 weeks after weaning is most likely due to a combination of factors, which include a decrease of overall absorption and possibly increased absorption of Pb when associated with milk constituents. Previous reports also have suggested efficient lactational transfer of toxicants to pups (Gottesfeld and LaGrue, 1990; Oskarsson et al., 1995
).
After the evaluation of transplacental and lactational Pb transfer to neonates, we quantified Pb-induced effects on the lymphoid subsets in the thymus and spleen as well as plasma IgE levels. IgE has been well established as an important mediator in the onset of allergic disease, for it binds via its Fc portion to high affinity receptors (FcRI) of mast cells and basophils, triggering the release of histamine and other potent mediators. The interaction of these mediators with target cells has been directly linked to the onset of allergic asthma (Oskarsson et al., 1995
). B cells with low affinity Fc
receptors (Fc
RII, CD23) also have been implicated in asthma (Aberle et al., 1997
; Hallstrand et al., 1998
).
IgE production is directed, in part, by type 2 helper T cells (Th2 cells) (Parronchi et al., 2000). Abnormal shifts in the homeostatic Th1/Th2 balance toward a predominant Th2 response have been reported in a variety of health problems, including allergies (Umetsu and DeKruyff, 1997
), and environmental agents are known to influence these shifts (Parronchi et al., 2000
). Pb exposure has previously been shown to inhibit adult mouse Th1-related responses (IFN-
production) and enhance Th2-related responses (IL-4 and IgE production) through in vivo, ex vivo, and in vitro adult mouse studies (Heo et al., 1996
; Kishikawa et al., 1997
; McCabe and Lawrence, 1991
; Miller et al., 1998
).
Increases in the incidences of childhood allergic asthma have been found in urbanized areas (Schafer and Ring, 1997), suggestive of a contributing role from environmental toxicants found in those areas. Many of the households in these urban areas have Pb problems, allowing theoretic implication of Pb in the onset of the atopic disease. Mouse neonates (2 weeks of age) exposed to Pb pre- and/or postnatally had significantly higher plasma IgE levels than age-matched controls (Fig. 3
). Thus, it may not be too farfetched to suggest that Pb exposure either in utero or lactationally could significantly increase the risk of atopy in children; however, lactational Pb transfer may be much lower in humans, in that human milk has a substantially lower concentration of Pb than blood (Gulson et al., 1998
; Nashashibi et al., 1999
). It is of particular interest to note that only a 56 day exposure to Pb in utero and none thereafter was sufficient to significantly elevate the IgE levels of 2-week-old offspring; offspring did not have elevated blood Pb levels at this time (Fig. 2
). This implies a dramatic and sustained effect of transplacental Pb exposure on the immunocompetency of the offspring. This sustained effect was also apparent in the number of splenic B cells at 2 weeks (Fig. 5B
).
It appears most likely that the IgE in the plasma of the neonates is endogenous, for the possibility of maternal transfer is not consistent with the results, and it has previously been established that IgE does not cross the placenta (Averech et al., 1994; Carretti, 1974
). Therefore, mice exposed to Pb until the day of birth would, theoretically, not have IgE levels dissimilar from control animals. Thus, the only other potential source of maternal IgE would have to be from the milk. Cord serum IgE levels are higher in mothers with allergy than without. This trend is not found in the children of allergic fathers (Halonen et al., 1992
), but it is not clear whether IgE or IgE-promoting factors in human or mouse milk are influential. Although high cord serum IgE levels have been connected with later allergic disease (Oldak, 1997
), the stimulatory factors, including antigens leading to this increase, have not been delineated (Furuhashi et al., 1997
; Lilja et al., 1997
). In any case, IgE transfer in milk cannot explain our results, as the neonates exposed only gestationally to Pb actually had the highest levels of IgE (Fig. 3
).
It is of interest to note that the Pb-induced elevation of plasma IgE was seen despite the fact that both gestational and/or lactational Pb-exposure caused a significant reduction of splenic white blood cells at both 2 and 3 weeks of age (Fig. 4). B cells (CD19) were the most substantially depleted subset. Together these results suggest a selective direct or indirect activation of B cells by Pb, in that the activation would require isotype class switching. The reduced number of B cells in the Pb-exposed animals at 2 weeks must have an elevated proportion of IgE producing B cells or B cells with greater ability to secrete IgE on a per-cell basis. A study of 2- to 4-year-old children found those at a genetically determined high risk for atopy had considerably fewer immunocompetent T-cell precursors in their blood as compared to their low-risk counterparts (Holt et al., 1992
). It is possible that a lag in immunological development (observed as delayed splenic lymphogenesis; Fig. 4
) is a precursor to the onset of allergic disease, which in this case may have been initiated by Pb exposure.
The presence of an environmental stressor such as Pb during pregnancy and/or breast feeding may be influential, as Pb can enhance cytokines that promote IgE production, and during pregnancy the maternal immune system is already shifted preferentially toward these cytokines (Lin et al., 1993; Nilsson et al., 1997
). This imbalance also exists in the neonate and normally slowly returns to a more neutral balance (Blanco Quiros, 1998
). IL-4 is a known promoter of IgE production, and Pb is known to enhance IL-4 levels in adult mice. Unfortunately, the serum levels of IL-4 in all neonates were undetectable; however, IL-4 is a potent cytokine whose plasma levels are usually very low. Elevated IL-4 and IgE have been assessed in rats exposed to Pb gestationally (Miller et al., 1998
). A low but elevated microenvironmental concentration in a lymphoid organ may be adequate to promote the IgE production, which may remain skewed into adulthood. These posited shifts in cytokines could present a window of enhanced susceptibility to atopic diseases.
Pb is only one of numerous environmental agents, including infectious agents, that may combine to promote a predominant type 2 immune response and/or alter the ability of neonates to recover their proper immune balance postpartum. It has recently been reported that the key factor in the propagation of allergic disease could be the efficiency of mechanisms governing immune deviation (Prescott et al., 1998). Pb alone is an unlikely candidate to explain the recent increase in asthma incidence, as environmental Pb exposure levels have been declining. However, other agents with which there could be synergy may be increasing. The above data present a plausible explanation for the increases of atopy including asthma found in areas where environmental toxicants such as Pb are present. These findings warrant future research addressing the onset and severity of airway sensitivity and/or other allergic symptoms of mammalian systems exposed to Pb and/or other environmental promoters of type 2 immunity during early life.
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ACKNOWLEDGMENTS |
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NOTES |
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