Effects of environmental lead exposure on T-helper cell-specific cytokines in children

Abstract

Lead (Pb) may alter T-lymphocyte reactivity in situ by preferentially enhancing the development of T-helper 2 (T_H2)- and inhibiting T_H1-lymphocyte development. These effects could result in dysregulation of the presence/availability of T_H1- and T_H2-associated cytokines. The aim of this study was two-fold, that is, to assess whole blood Pb levels in schoolchildren from Taiwanese communities that varied in degree of potential for Pb exposure and then ascertain if there were relationships between Pb exposure and changes in levels of key T_H1 and T_H2 cytokines. Grades 5 and 6 students were selected from four different community schools, i.e., one from: urban area with new homes; urban area with old homes; rural site with old homes; and area located near an oil refinery. Students at each site were further divided into healthy and respiratory allergy subgroups. Blood was collected and whole blood Pb levels and serum interferon (IFN)-γ, interleukin (IL)-12, -4, and -5 levels were determined. The results indicate no differences in whole blood Pb levels (<4 µg/dl) among students from urban and rural sites; these values were similar in the healthy and allergic subjects. Serum T_H1 and T_H2 cytokine levels also did not differ among/within the groups. In contrast, refinery children had significantly increased Pb levels (5.2–8.8 µg/dl) relative to any of the other sets’ levels. Of these, children with allergies had serum T_H2 cytokine levels significantly higher and T_H1 cytokine levels significantly lower than their healthy counterparts. Oddly, though having elevated Pb levels, healthy refinery students did not display altered T_H1 or T_H2 cytokine levels relative to control student values. From this, we conclude that substantively increased whole blood Pb levels may promote T_H cell dysregulation and alter the availability of key T_H1 and T_H2 cytokines, effects that could ultimately contribute to development of pulmonary allergic diseases.

Keywords::

• Lead
• asthmas
• allergic rhinitis
• T-helper cell
• cytokine

Introduction

Blood lead concentrations in the human population have declined since the elimination of leaded gasoline, lead solder in canning, and lead-containing paints. However, many focal environmental sources of lead remain, including certain industries such as ceramics, mining, and smelting, as well as companies involved in battery repair and/or the recycling of batteries and electronics (Patrick, 2006). Environmental lead exposure occurs predominantly by ingestion of lead-contaminated household dust or soil in older houses that contain lead-based paint; the degree of exposure often increases with deterioration or renovation of the houses. Smelters and tap water are two other important potential sources of lead exposure (Brown et al., 2006). The current screening threshold established by the Centers for Disease Control and Prevention for an elevated blood lead level in a child is 10 µg/dl (Bellinger, 2004), but this value should not be interpreted as a level below which adverse effects do not occur. Some data suggest that deleterious effects can occur even at serum levels well below 10 µg/dl (Tellez-Rojo et al., 2006).

Human neurodevelopmental problems, including deficits in cognitive and academic skills, have been shown to be related to blood lead levels of <10 µg/dl or even lower than <5 µg/dl (Lamphear et al., 2000). A hallmark of lead-induced immunotoxicity is a pronounced shift in the balance of T-helper (T_H) lymphocyte function towards T_H2 responses at the expense of T_H1 function (Dietert et al., 2004). Lower degrees of lead exposure cause suppression of the T_H1 cytokine interferon (IFN)-γ and the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and leads to enhanced production of IL-4 and/or IL-10 (Iavicoli et al., 2006). These effects of lead exposure can induce and maintain a T_H2-immune response, which may contribute to increased susceptibility to pathologic agents, allergic hypersensitivity, and/or T_H2-dominated autoimmune diseases (Hemdan et al., 2005).

Although the exact mechanism for the stimulation of immunoglobulin-E-(IgE) producing B-lymphocytes remains to be elucidated, higher lead concentrations can lead to increases in IgE levels (Lutz et al., 1999; Karmaus et al., 2005). Since the magnitude of environmental lead exposure in Taiwanese children has been poorly investigated, the percentage of school-age children that have blood lead levels ≥10 µg/dl is not known. As mentioned above, these levels have been associated with a decline in IQ and an increase in frequency of atopic diseases. Only one study has reported a mean blood lead level of 5.50 µg/dl in children in Kao-Hsiung City, Taiwan (Wang et al., 2002). Therefore, the determination of blood lead levels among children will facilitate identification of potential neurotoxic hazards and lead to the possibility of reducing allergic diseases in this at-risk population.

Materials and methods

Primary school students in Grades 5 and 6 (N = 214) from four different communities with potentially different risk for environmental lead exposure were assessed in this study. Students were organized into four groups, i.e., students from: a new urban community with houses <10 years old (Group I, N = 50); an urban community with houses >10 years old (Group II, N = 50); a rural community with houses <10 years old (Group III, N = 50); and, a community located near an oil refinery (Group IV, N = 50) in the Tao-Yua area, New Taipei City. A consort statement was given to the school administrators in order to explain the project background and provide information on the progress of all participants in the trial from the time they were randomized into the study until the end of the study. The participants or their parents provided informed consent prior to assessment and drawing of blood samples. The students selected from each school were subdivided into two groups: an allergic group and nonallergic group, based on the criteria established by the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire and doctor diagnoses.

Whole blood lead concentrations were measured in the laboratory of a teaching hospital. Commercial ELISA kits (R&D System, Inc., Minneapolis, MN) were used to measure the levels of the T_H1 cytokines IL-12 and IFNγ and the T_H2 cytokines IL-4 and IL-5. The circulating titres of IgE specific to common allergens in Taiwan, including dust mites, German cockroaches, and both cat and dog dander, were measured using an ImmunoCAP^® system (a radio allergo-sorbent test [RAST]; Phadia, Upsalla, Sweden). The Statistical Package for Social Sciences (SPSSTM Version 12 for Windows^™; SPSS, Inc., Chicago, IL) software was used for the T_H1- and T_H2-cytokine analyses. A p-value ≤ 0.05 was considered statistically significant.

Results

A total of 214 students were included in this study. The study subjects were allocated into sets of 50 students each in Groups I (25 nonallergy and 25 allergy), II (25 nonallergy and 25 allergy), III (25 nonallergy and 25 allergy), and IV (27 nonallergy and 23 allergy).

There were no significant differences in the whole blood lead concentrations among the students in Groups I, II, and III (range = 3.16–3.83 µg/dl; Table 1). Furthermore, within each Group, there were no significant differences in whole blood lead levels between the students in the allergy and nonallergy subgroups. In contrast, whole blood lead levels among the students living near the oil refinery (Group IV) were significantly higher than in the other three groups. Values in this group ranged from 5.23 µg/dl among healthy students to 8.80 µg/dl in students in the allergy subgroup (Table 1). These whole blood lead values represent relative increases of 51.6 and 155.1% over the average values from the other three groups (if pooled outcomes utilized, i.e., mean = 3.45 µg/dl).

Table 1.  Whole blood lead concentration in students of four different community schools.

Group I		Group II		Group III		Group IV
Healthy (25)	Allergic (25)	Healthy (25)	Allergic (25)	Healthy (25)	Allergic (25)	Healthy (30)	Allergic (34)
3.36 ± 0.25	3.36 ± 0.27	3.83 ± 0.21	3.83 ± 0.28	3.16 ± 0.25	3.16 ± 0.22	*5.23 ± 0.36	*8.80 ± 0.45

Group I = New urban community (N = 50); Group II = Urban, house age >10 years (N = 50); Group III = Rural area (N = 50); and Group IV = Community located near a refinery (N = 64).

All values shown are Mean (± SD) in µg/dl.

*Value is significantly different (at p < 0.05) from corresponding subgroup values in all other student groups.

The serum levels of the T_H1 cytokines IFNγ and IL-12 and the T_H2 cytokines IL-4 and IL-5 were similar among the healthy and allergic students in Groups I, II, and III (Table 2). Furthermore, the levels of these cytokines were seen not to have been altered to any significant extent among the healthy students from the refinery site (even though they did have significantly greater lead burdens [see above]).

Table 2.  Serum levels of T_H1 and T_H2 cytokines in participants from four different community schools.

	Group I		Group II		Group III		Group IV
	Healthy (25)	Allergic (25)	Healthy (25)	Allergic (25)	Healthy (25)	Allergic (25)	Healthy (30)	Allergic (34)
IFNγ	9.01 ± 2.33	9.15 ± 2.38	9.14 ± 2.28	9.21 ± 2.31	9.17 ± 2.11	9.20 ± 2.17	9.11 ± 2.28	*0.20 ± 0.14
IL-12	4.51 ± 1.37	4.92 ± 1.48	4.55 ± 1.33	4.59 ± 1.39	4.43 ± 1.46	4.48 ± 1.41	4.57 ± 1.24	*0.16 ± 0.11
IL-4	6.83 ± 3.28	6.90 ± 3.26	6.78 ± 3.48	6.91 ± 3.51	6.92 ± 3.27	6.90 ± 3.56	6.93 ± 3.38	*45.30 ± 27.50
IL-5	4.05 ± 2.26	4.10 ± 2.38	4.03 ± 2.11	4.12 ± 2.24	4.11 ± 2.17	4.15 ± 2.29	4.00 ± 2.17	*25.70 ± 11.50

Group I = new urban community (N = 50); Group II = urban, house age >10 years (N = 50); Group III = rural area (N = 50); and Group IV = community located near a refinery (N = 64).

All values shown are Mean (± SD) in µg/dl.

*Value is significantly different (at p < 0.05) from all other subgroup values.

In contrast, among the students with known respiratory allergies in Group IV, serum IFNγ and IL-12 concentrations were significantly decreased, while those of serum IL-4 and IL-5 were significantly increased, relative to values noted with all the other groups assessed. Specifically, values for serum IFNγ and IL-12 in these children were found to be, respectively ≈ 0.20 and 0.16 pg/ml, as compared to an average level of 9.15 and 4.58 pg/ml in all the other groups of children analyzed (Table 2). These represent near-total collapses, i.e., decrements of 97.8% and a 96.5% for IFNγ and IL-12 levels, respectively, in the formation/release of each cytokine. On the other hand, serum IL-4 and IL-5 levels in these children were seen to be ≈ 45.30 and 25.70 pg/ml, respectively, as compared to average levels of 6.87 and 4.09 pg/ml in all the other groups (Table 2). These values reflect increases of ≈559% and a 528% in formation/release, respectively, of IL-4 and IL-5 in these particular children.

Discussion

Lead-contaminated house dust is the major source of lead intake in early childhood (Lamphear et al., 2002). Lead exposure also occurs predominantly in children living in older houses that contain lead-based paint, and the levels of lead exposure increase with deterioration or renovation of the houses (Campbell and Osterhoudt, 2000). Pediatric lead poisoning is more common than adult lead poisoning; its effects may occur at low blood levels and present with subclinical symptoms. However, the periods of enhanced vulnerability to lead exposure in childhood have not been identified. Thus, physicians are required to have a high index of suspicion when dealing with pediatric patients (Garza et al., 2006).

Recent studies have implicated low-level exposure and blood lead levels previously considered acceptable as causative factors for cognitive dysfunction, neurobehavioral disorders, neurologic damage, hypertension, and renal impairment (Patrick, 2006). Toxic lead can modulate the immune response of animals as well as humans. Both stimulation and suppression of the immune response have been shown; these effects are dependent on the T_H1 and T_H2 responses, with the preferential activation of T_H2 cells and inhibition of T_H1 cells considered an underlying cellular mechanism of the immune response alterations (Singh et al., 2003; Heo et al., 2004).

It has been reported that lead exposure enhances IL-4 production and inhibits IFNγ production in wild-type BALB/c mice, and enhances delayed-type hypersensitivity (DTH) responses in IFNγ-deficient mice (Gao et al., 2006). These changes are likely to occur through release of Type-2 cytokines and enhancement of expression of major histocompatibility complex (MHC) Class II antigens. IFNγ reduces T_H2-mediated allergic responses. In a mouse model, a presence of IFNγ during pregnancy confers the fetus protection against allergen provocations in adult life (Lima et al., 2005). Another animal study (Iavicoli et al., 2006) showed a significant increase in IL-4 production-and a profound decrease in IFNγ and IL-2 production-when high dietary lead levels (40 and 400 ppm) were present. At the lowest dietary lead level (0.02 ppm) tested in that study, increased IFNγ and IL-2 production-along with a significant decrease in IL-4 production-was observed. Taken together, these findings provided evidence of induction of alterations in cytokine availability/production that was clearly dependent on blood lead concentration.

As blood lead content increases, there is a notable shift towards T_H2 production (Heo et al., 1996). This is reversed at lower blood lead levels, when T_H1 development is favored (Iavicoli et al., 2006). Serum IgE titers significantly correlated with blood lead levels; serum IgE titres were increased in battery manufacturing workers with concentrations ≥30 µg/dl compared to in those at <30 µg/dl (Heo et al., 2004). A statistically significant relationship between IgE titre and blood lead concentration was also noted in children exposed to environmental lead (Lutz et al., 1999). This relationship may explain why the incidence of childhood atopic responses, including asthma, has risen considerably in recent years particularly in areas containing high levels of environmental pollutants.

The mean blood lead level in the general Taiwanese population is 8.6 µg/dl in males and 6.7 µg/dl in females (Wu et al., 1997). A subsequent report showed that the mean blood lead level in children in Kao-Hsiung City was 5.50 µg/dl (Wang et al., 2002). Thus, the potential for toxic effects from exposure to lead are likely to still exist in Taiwan. The results of the current study show that the blood lead concentration among students from Groups I, II, and III are not statistically different, even between the nonallergy and allergy subgroups. Serum IFNγ, IL-12, IL-4, and IL-5 levels were also normal (for 5–6-year-old children) and not different between the healthy and allergy groups. However, the whole blood lead levels in the schoolchildren in Group IV were significantly higher compared to any of the other children. The fact that serum levels of the T_H2 cytokines IL-4 and IL-5 were also significantly higher (and those of T_H1 cytokines IFNγ and IL-12 significantly lower) in the Group IV allergy group as compared to those in any other children—including healthy counterparts with elevated blood lead levels—suggests that substantively-increased whole blood Pb levels may promote T_H cell dysregulation and alter the availability of key cytokines. Such effects could ultimately contribute to the development of asthma, allergic rhinitis, or other respiratory allergic diseases.

Declaration of interest

The authors report no conflicts of interest. The authors are alone responsible for the content and writing of the paper.