Lead poisoning influences TCR-related gene expression patterns in peripheral blood T-lymphocytes of exposed workers

Abstract

Previous studies have shown that occupational lead (Pb) exposure might influence human T-lymphocyte function, including such as changes in T-cell receptor (TCR) Vβ and Vγ repertoire and in expression of the TCRζ gene. Thus, the study here further investigated expression of TCRζ-related factors and the F_cɛRIγ gene (whose product has a functional role complementary to the TCRζ chain) and the Elf-1 gene whose product is involved in regulation of TCR expression. Quantitative real-time RT-PCR was used to measure expression of TCRζ, F_cɛRIγ, and Elf-1 genes in peripheral blood mononuclear cells (PBMC) isolated from 17 Pb-exposed workers. Samples were collected before and after the workers had undergone chelation therapy regimens. Twenty-three healthy individuals served as controls. The results showed that TCRζ, F_cɛRIγ, and Elf-1 gene expression in Pb-exposed workers before chelation therapy was significantly lower than in PBMC from healthy individuals. After chelation therapy, expression of TCRζ appeared to trend toward normal levels; in comparison, lower expressions of F_cɛRIγ and Elf-1 persisted. In conclusion, the previously-documented impairment of T-lymphocyte functions and T- lymphocyte-mediated immune responses seen previously in response to occupational Pb exposure might be attributable, in part, to effects on TCR signaling pathways - including those related to TCRζ and F_cɛRIγ - and to any down-regulation of membrane TCRζ expression/activity that might be associated with Pb-induced effects on Elf-1 expression.

• Chelation therapy
• Elf-1
• F_cɛ_RIγ
• gene expression
• lead poisoning
• TCRζ

Introduction

The heavy metal lead (Pb) is a widespread environmental pollutant. Pb poisoning occurring in humans either due to occupation or environment has become a great public problem. Pb is toxic to many organ systems including the immune system. The toxicity of Pb leading to immuno-suppression/-dysregulation may consequently cause allergy or autoimmunity (Fischbein et al., 1993; Sata et al., 1998). In experimental animals, Pb has been shown to target macrophages and T-lymphocytes with low–moderate levels of exposure (Chen et al., 1977; Guo et al., 1996; Hsiao et al., 2011; McCabe & Lawrence, 1991). In occupational Pb-exposed workers, Pb has been reported to lead to changes in T-lymphocyte sub-sets and the response to T-lymphocyte mitogens (Mishra et al., 2003; Pinkerton et al., 1998; Undeger et al., 1996). Our previous studies also showed that occupational Pb exposure has an influence on human T-lymphocyte functions such as changes in the T-cell receptor (TCR) Vβ and Vγ repertoire and the expression level of the genes coding for CD3ζ, T-bet, and GATA-3 (Liu et al., 2009, 2011, 2012; Niu et al., 2012).

When a T-lymphocyte encounters antigens via the TCR, information about the quantity and quality of antigen engagement is relayed to intracellular signal transduction machinery. The TCR has a short intracellular sequence that lacks signaling capacity, and the CD3 molecule contains immuno-receptor tyrosine-based activation motifs (ITAM) that couple the TCR to the signal transduction mechanism (Call & Wucherpfenning, 2005; Gouaillard et al., 2001). TCR signal transduction is mediated by ITAMs. A total of 10 ITAMs are distributed in the TCR/CD3 complex in two distinct signaling modules termed the CD3ζζ (also referred to as TCRζ) and CD3γ_ɛ/δ_ɛ (Call et al., 2004; Clevers et al., 1988). TCRζζ plays a central role in TCR signal transmission. The F_cɛ receptor type Iγ (F_cɛRIγ) chain is a member of the TCRζ chain protein family, and it is a component of the high-affinity IgE receptor F_cɛRI. There is evidence that the F_cɛRIγ chain can replace a functionally deficient TCRζ chain and facilitate TCR/CD3 complex-mediated signaling (Enyedy et al., 2001; Koyasu et al., 1992).

Abnormalities in the transcription of TCRζ may be related to its transcription factors such as E74-like factor 1 (Elf-1). Elf-1 is a transcription factor that belongs to the Ets family. In hematopoietic cells, Elf-1 mediates the induction of various genes, including those for CD4, GM-CSF, IgH enhancer, TCRα, and TCRζ (Juang et al., 2002; Tsokos et al., 2003). However, little is known about the expression pattern of the F_cɛRIγ gene and the molecular mechanism of the aberrant expression of TCRζ in workers occupationally exposed to Pb. Based on our previous finding that TCRζ gene expression level was changed after occupational Pb exposure, we investigated in the studies reported here the expression level of F_cɛRIγ and Elf-1. Because research has indicated that F_cɛRIγ mRNA expression correlates well with expression of the protein (Butkiewicz et al., 2005; Enyedy et al., 2001), we believe monitoring of the gene itself will reflect upon the status of the receptor expression on cells of the exposed worker. We believe that analysis of these factors in Pb-exposed workers (before and after chelation therapy) might be useful in helping to identify other potential Pb-mediated immunotoxicities that might occur in situ that are ultimately related to changes in the products of these genes.

Materials and methods

Samples

All samples in this study were collected at Guangzhou 12th Municipal People’s Hospital (Guangzhou Occupational Disease Hospital). All procedures were performed according to guidelines of the Medical Ethics Committee of the Health Bureau of Guangdong Province. All subjects in this study certified they were not taking any medications known to affect the immune system nor suffering from any acute/chronic disease, as these may impact on the immune system. For these studies, 23 healthy subjects (15 male, eight female; median age = 30 years, range = 21–57 years) who received an annual check-up at Guangzhou Hospital served as the control group. The characteristics of the populations examined are provided in Table 1. There were no significant differences in age and sex between the control and the PE and PT groups. Subjects in the control group had no occupational exposure to Pb and (background) Pb levels were not detectable.

Table 1. Characteristics of the study control, PE (and PT) groups.

	Control (n = 23)	PE (same as PT) (n = 17)
Age	37.2 ± 15.2	39.7 ± 6.5
Male	15	11
Female	8	6

Blood and urine samples from all Pb-exposed workers (PE group) were collected to obtain clinical verification of abnormal blood (BPb) and urinary (UPb) Pb levels. In each case, blood (3–5 ml) was collected into heparin-coated tubes at 8 am (fasting blood sample). Urine was collected as 24-h samples. BPb and UPb levels were then determined using graphite furnace atomic absorption. A diagnosis of Pb exposure was based on the Diagnostic Criteria of Occupational Lead Poisoning (GB: Z37-2002). Specifically, a Pb-exposed worker had to have had prolonged occupational exposure to Pb, as well as (a) high BPb (i.e., ≥2.9 μmol Pb/L)/UPb (≥0.58 μmol Pb/L) levels, (b) blood zinc protoporphyrin (ZPP) levels ≥2.91 μmol/L, (c) urinary δ-aminolevulinic acid (ALA) levels ≥61 μmol/L, or (d) overt clinical manifestations such as abdominal pain or distension, and/or constipation. Measures of ZPP were performed using a ZPP-3800 blood zinc protoporphyrin hematofluorometer (Kangda Company, Guangdong, China) and of ALA using a 7230G spectrophotometer (Jinghua Instruments, Shanghai, China). Apart from using these criteria, only workers with a UPb ≥0.34 μmol Pb/L in a 24-h period on each of 3 consecutive days were selected for chelation therapy. Clinical data and information about the final PE population of this study are provided in Table 2.

Table 2. Clinical data for Pb-exposed workers before and after chelation.

				Before chelation				After final chelation
Case #	Gender	Age	Exposure duration (years)	ZPP (μmol/L)	ALA (μmol/L)	BPb (μmol/L)	UPb (μmol/L)	BPb (μmol/L)	UPb (μmol/L)	Course of chelation
1	Female	41	6.50	4.25	4.96	4.16	0.79	2.34	0.32	2
2	Male	42	8.25	1.46	2.31	4.77	0.41	3.99	0.21	2
3	Male	32	4.67	3.14	1.03	8.68	0.14	4.11	0.07	2
4	Male	38	5.83	3.00	1.58	7.43	0.61	3.44	0.25	3
5	Female	30	3.00	5.44	0.88	3.78	0.47	3.09	0.11	2
6	Female	44	5.58	7.03	0.69	3.77	0.46	2.64	0.11	2
7	Female	39	4.83	3.36	1.05	4.28	0.39	3.51	0.25	2
8	Male	45	3.33	6.81	6.73	4.70	0.53	2.45	0.26	4
9	Male	42	8.83	4.41	5.50	5.33	0.53	3.55	0.31	2
10	Male	27	3.83	6.92	0.65	4.35	0.49	2.20	0.23	2
11	Male	46	5.17	3.70	0.52	5.83	0.32	2.87	0.18	2
12	Male	43	2.75	7.75	9.18	3.54	0.76	2.57	0.32	3
13	Male	50	8.08	1.68	1.47	6.76	0.70	3.03	0.44	2
14	Male	48	12.08	2.06	0.04	7.81	0.32	4.06	0.27	3
15	Male	40	4.42	2.17	3.90	4.55	0.58	3.17	0.28	5
16	Female	35	3.92	0.99	0.26	2.95	0.24	1.45	0.16	2
17	Male	33	9.00	5.51	2.45	3.43	0.46	0.20	0.18	2

For the chelation therapy, each of these workers underwent a course of intravenous treatment with 1 g calcium-EDTA/day over a period of 3 days; if another round of therapy was needed (based on urinary Pb measures), this occurred after a 4-day gap. Each Pb-exposed worker (now deemed member of PT group) underwent a round of chelation therapy until clinical indices reflected an obvious improvement. In this study, there was no stand-alone non-therapy cohort (i.e., to assess effects of time-alone/time-away from exposure).

PBMC RNA extraction and cDNA synthesis

From the blood collected prior to/after the final chelation treatment, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Ficoll-Hypaque, ρ = 1.077 g/ml). The isolated PBMC (10⁷ cells/subject) were then processed with TRIzol agent (according to manufacturer protocols; Invitrogen, Carlsbad, CA) to yield their total RNA. Purity of the isolated material was confirmed by electrophoresis of a representative amount of material (0.5 μg/sample) over a 0.8% agarose gel subsequently stained with ethidium bromide. First-strand cDNA was then synthesized using random hexamers and reverse transcriptase in a Super-script II Kit (PowerScript Reverse, BD, San Jose, CA), according to manufacturer instructions. cDNA quality was confirmed by RT-PCR for amplification of the β₂-microglobulin (β₂M) gene.

TCRζ, F_cɛRIγ, and Elf-1 gene quantification by real-time RT-PCR

Real-time RT-PCR using a SYBR Green I method was employed to examine TCRζ, F_cɛRIγ, and Elf-1 gene expression levels in PBMC cDNA generated from subject cells. Primer sequences and PCR conditions were as described in Li et al. (2012). Briefly, a PCR reaction (total volume = 25 μl) was performed with ∼1 μl cDNA, 0.5 μM each primer pair, and 11.25 μl of 2.5X RealMasterMix (Tiangen, Beijing, China). After initial denaturation at 95 °C for 2 min, 45 cycles consisting of the following were performed using a MJ Research DNA Engine Opticon 2 PCR cycler (BioRad, Hercules, CA): 15 s at 95 °C; 1 min at 58.9 °C for β₂M, 60 °C for TCRζ and F_cɛRIγ, 62 °C for Elf-1; and 1 s at 82 °C for plate reading. The 2^−ΔCT× 100% method was used to represent expression of the gene of interest relative to that of internal control gene (%).

Statistical analysis

An independent samples t test was performed to compare mean gene expression levels between the PE/PT group and control groups. Paired sample t tests were performed to compare mean gene expression levels between the PE and PT groups. All data are expressed as the mean ± SD. A p value < 0.05 was considered statistically significant.

Results

Blood (BPb) and urine (UPb) lead levels

Prior to chelation therapy, blood and urine lead levels in the exposed workers were seen to be 4.06 ± 1.57 (BPb) and 0.38 ± 0.22 (UPb) μmol Pb/L. Following the entire course of chelation therapy/therapies, there were significant decreases in both values, i.e., BPb was now 2.25 ± 1.00) μmol Pb/L and UPb was (0.16 ± 0.10) μmol Pb/L (Figure 1). BPb and UPb values for the control subjects were each no more than, respectively, 0.97 and 0.12 μmol Pb/L.

Figure 1. Level of lead in blood and urine from Pb-exposed workers. Values shown are from each subject (in terms of μmol/L) before (PE) and after (PT) chelation therapy.

Expression of TCRζ, F_cɛRIγ, and Elf-1 genes in PBMC

TCRζ, F_cɛRIγ, and Elf-1 gene expression levels in PBMC from the Pb-exposed workers, both before after chelation therapy - as well as in cells from healthy controls - are shown in Figure 2. In the PE cells, expression of TCRζ (1.05 ± 0.53), F_cɛRIγ (1.73 ± 1.43), and Elf-1 (4.19 ± 2.97%) genes was significantly decreased in comparison with that in cells from the healthy controls (TCRζ = 1.57 ± 0.75, F_cɛRIγ = 18.65 ± 13.06, and Elf-1 = 7.83 ± 2.04%). Chelation therapy led to increased TCRζ and decreased F_cɛRIγ and Elf-1 expression levels compared with the PE values, but these changes were not significant. Even with chelation, the expression levels of F_cɛRIγ (1.37 ± 1.33) and Elf-1 (3.61 ± 2.13%) remained significantly decreased in comparison to levels in control host PBMC. In contrast, expression of TCRζ (1.32 ± 0.93%) now no longer significantly differed from control PBMC values.

Figure 2. Expression levels of TCRζ, F_cɛRIγ, and Elf-1. Levels (%) of (A) TCRζ, (B) F_cɛRIγ, and (C) Elf-1 in PMBC recovered from workers before (PE) and after (PT) chelation therapy or from healthy controls (HC).

Effect of number of chelation rounds on TCRζ, F_cɛRIγ, and Elf-1 gene expression in PBMC

All Pb-exposed workers underwent chelation therapy until their clinical indices had obvious improvement. A comparison of the above-noted outcomes in the context of the different numbers of courses of chelation therapies had little-to-no impact on expression of the measured genes. In workers who underwent two courses, expression of TCRζ in the PE and PT cells were decreased in comparison with that in cells from healthy controls, but these changes were not significant (Table 3). In Pb-exposed workers, only expression of TCRζ in the PE cells was significantly decreased compared to in cells from controls. In all cases, chelation therapy led to increased TCRζ expression levels compared with pre-therapy PE values, albeit the changes were not significant. After chelation, expression levels of TCRζ no longer significantly differed from control levels. In the case of the single individual that underwent five rounds of therapy, the highly unusual PT values was most likely a result of individual variation in response to the therapy (as the PE value was in line with those in all other Pb-exposed workers).

Table 3. Relative TCRζ expression level (%) in PE/PT PBMC as a function of number of therapy rounds.

Number of courses of chelation therapy	PE	PT
2 (n = 12)	1.2 ± 0.5	1.4 ± 1.0
3 (n = 3)	0.6 ± 0.6*	0.9 ± 0.2
4 (n = 1)	0.7	0.9
5 (n = 1)	0.8	2.4

*p = 0.05 versus control group level.

In the workers who underwent two or three courses of therapy, even though their clinical indices had reached normal levels, expression of F_cɛRIγ and Elf-1 genes was still significantly decreased in comparison with that in cells from the healthy controls (Tables 4 and 5). As a function of the numbers of courses of chelation therapy, F_cɛRIγ and Elf-1 expression levels were not significantly impacted and expression levels of each remained significantly decreased compared to levels in control PBMC. Again, in the case of the single individual that underwent five therapy rounds, the highly unusual PT Elf-1 value was most likely a result of individual variation in response to the chelation therapy; in the case of F_cɛRIγ, both sets of values were in line with the other Pb-exposed workers.

Table 4. Relative Elf-1 expression level (%) in PE/PT PBMC as a function of number of therapy rounds.

Number of courses of chelation therapy	PE	PT
2 (n = 12)	3.9 ± 3.1*	3.6 ± 2.2*
3 (n = 3)	3.2 ± 1.4*	2.7 ± 1.6*
4 (n = 1)	4.5	3.3
5 (n = 1)	9.6	6.9

*p ≤ 0.05 versus control group level.

Table 5. Relative F_cɛRIγ expression level (%) in PE/PT PBMC as a function of number of therapy rounds.

Number of courses of chelation therapy	PE	PT
2 (n = 12)	1.6 ± 1.4*	1.2 ± 1.3*
3 (n = 3)	0.9 ± 0.6*	2.3 ± 1.9*
4 (n = 1)	3.5	0.6
5 (n = 1)	3.9	1.8

*p ≤ 0.05 versus control group level.

Correlation among TCRζ, F_cɛRIγ, and Elf-1 expression levels in PBMC

A significant negative-correlation was observed between expression levels of TCRζ and F_cɛRIγ genes in cells isolated from the healthy control subjects (r = −0.559, p = 0.03, Figure 3A). A negative correlation was found with the cells from the Pb-exposed workers before chelation therapy, though the difference was not statistically significant (r = −0.072, p = 0.784, Figure 3B). This negative correlation was no longer apparent following chelation therapy (r = 0.110, p = 0.675, Figure 3C).

Figure 3. Correlation analyses for TCRζ and F_cɛRIγ expression levels. (A) HC, (B) PE, and (C) PT groups. A significant negative correlation was found for the HC group and a non-significant negative correlation was found for the PE group. There was no correlation for the PT group.

A significant positive correlation between TCRζ and Elf-1 gene expression levels was found for the PBMC of the healthy control hosts (r = 0.583, p < 0.022, Figure 4A). Although a positive correlation was noted for in PBMC TCRζ and Elf-1 expression for Pb-exposed workers both before and after chelation therapy, the differences were not statistically significant (r = 0.424, p = 0.090; Figure 4(B); and r = 0.435, p = 0.081; Figure 4C).

Figure 4. Correlation analyses for TCRζ and Elf-1 expression levels. (A) HC, (B) PE, and (C) PT groups. A significant positive correlation was found in the HC group; non-significant positive correlations were found for the PE and PT groups.

In the context of the clinical measurements, there was no correlation between blood Pb levels and TCRζ (r = −0.208, p = 0.238), Elf-1 (r = −0.071, p = 0.691), or F_cɛR1γ (r = −0.236, p = 0.178) values for the PE subjects. Similarly, there was no correlation between urine Pb levels and TCRζ (r = −0.053, p = 0.764), Elf-1 (r = −0.255, p = 0.146), or F_cɛR1γ (r = −0.226, p = 0.198) values.

Discussion

The TCR/CD3 complex plays a central role in T-lymphocyte activation; alterations of any sub-units in the complex may impact on T-lymphocyte activation levels (Chen et al., 2009; Fischer et al., 2005). Decreased TCRζ gene expression has been described in Pb-exposed workers (Liu et al., 2009). Absence of the TCRζ chain not only influences TCR expression on the cells and the number of single-positive circulating T-lymphocytes it also impairs proliferative responses and mature T-lymphocyte activation (Li, 2008; Mozaffari et al., 2004). It may be interesting to examine whether TCRζ expression could recover in Pb-exposed workers after chelation therapy. In this study, the TCRζ gene expression was compared among 17 Pb-exposed workers before and after chelation therapy and against those in PBMC from healthy individuals. Similar to what was seen in our previous study (Liu et al., 2009), it was seen here that TCRζ gene expression in cells from Pb-exposed workers before chelation treatments was significantly lower than in healthy control cells, but was increased as a result of chelation.

It is known that F_cɛRIγ, which is unlike the TCRζ chain, mediates signaling through ZAP-70 by associating with the phosphorylated protein kinase Syk (Enyedy et al., 2001; Shiue et al., 1995; Taylor et al., 1997) that can substitute when there are TCRζ deficiencies. In the study here, F_cɛRIγ gene expression and its correlation with TCRζ expression was analyzed in Pb-exposed workers before and after chelation therapy. Expression of F_cɛRIγ was significantly lower (relative to in control subject cells) in Pb-exposed workers, pre- and post-therapy. This finding suggested to us that, in the case of TCRζ down-regulation, F_cɛRIγ expression in the Pb-exposed workers was incapable of being up-regulated to contribute to transduction of TCR signaling in a manner similar to that used by a conserved functional motif ITAM. The present study also analyzed the correlation between TCRζ and F_cɛRIγ gene expression; F_cɛRIγ expression lost its negative correlation with TCRζ expression in the Pb-exposed workers, both before and after chelation therapy. This suggested that apparent defects in T-lymphocyte-mediated immunity were involved in the immuno-dysregulation seen in Pb-exposed workers (Liu et al., 2009, 2011, 2012; Niu et al., 2012; Valentino et al., 2007).

The mechanism of abnormal of TCRζ gene expression in T-lymphocytes of Pb-exposed workers is unclear. To gain insight into the molecular mechanism of the lower TCRζ gene expression, expression levels of Elf-1, which could bind to the TCRζ promoter and is involved in transcriptional regulation of the TCRζ gene and promotes its transcription in T-lymphocytes, were assessed (Juang et al., 2008; Kulkarni et al., 2009). The data showed a significantly lower expression of Elf-1 in Pb-exposed workers before and after chelation therapy. Moreover, the loss of Elf-1 expression was positively correlated with the change in TCRζ expression level in the PBMC from these workers (both before and after the therapy). It is, therefore, possible that decreased Elf-1 expression contributed to down-regulation of TCRζ expression in Pb-exposed workers.

Conclusions

We believe this to be the first study to describe TCRζ-related genes, i.e., F_cɛRIγ and Elf-1, in Pb-exposed workers. Our findings suggest that the T-lymphocyte immune impairment caused by occupational lead exposure might be associated, at least in part, with abnormal TCR signaling pathways. This impairment is mainly related to the down-regulation of downstream molecules in TCR signaling pathways, e.g., TCRζ and F_cɛRIγ, and down-regulation of TCRζ might be mainly associated with changes in expression of Elf-1. Although there was an improvement in the clinical indices of the Pb-exposed workers after chelation therapy, further investigation is needed to follow-up on functional outcomes/immune cell recovery, including on T-cell activation, TCR signaling, alterations in Th1/Th2 balance, etc. in these workers following chelation therapy. In addition, our future studies need to include a time-alone (time-away from Pb exposure) group to confirm that the outcomes we measured here were in fact due solely to the chelation therapy.

Declaration of interest

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

This study was sponsored by grants from the National Natural Science Foundation of China (No. 81370605), the Natural Science Foundation of Guangdong Province (No. S2012010008794), the Fundamental Research Funds for the Central Universities (No. 21612425), and the Post-doctoral Science Foundation Project of China (No. 20070410840).