**3. Results**

#### *3.1. Characteristics of Study Workers and Controls*

Table 1 provides the characteristics of the control group and worker group. Participants in the control group were men, while the worker group had nine men and five women. Half of the study workers were exposed to Pb for more than 10 years. Age, BMI, hemoglobin, hematocrit, and smoking history of both groups did not di ffer. Thus, demographic characteristics of the two groups were apparently homologous. The median blood Pb concentration in the worker group was 37.07 μg/dL. This blood Pb concentration was 8.7-fold higher than the controls (*p* < 0.001). It was also higher than the OSHA exposure limit for Pb-exposed workers of 25 μg/dL [24].

#### *3.2. Profiling of Cytokine Production and Other Immunologic Parameters*

Table 2 provides a profile of the immunologic parameters measured. These included the percentage of helper T (Th) lymphocytes (CD3+CD4+) together with cell proliferation index and cytokine production in response to stimulation by a mitogen. The median percentage of Th lymphocytes in workers was similar to that of controls (60.74% versus 56.31%, *p* = 0.395). In the cell proliferation and cytokine production assay, peripheral blood mononuclear cells (PBMCs) from study subjects were used, with PHA as a stimulant mitogen. The median PHA-stimulated cell proliferation index was 1.14 in workers and 1.50 in controls (*p* = 0.226). The median level of interferon-gamma (IFN-γ) generated in response to PHA stimulation was 12.14 pg/mL in workers and 20.07 pg/mL in controls (*p* = 0.103), while the median level of interleukin-4 (IL-4) was 175.58 pg/mL in workers and 155.65 pg/mL in controls (*p* = 0.060). Interestingly, the median ratio of IL-4/IFN-γ was approximately 2-fold higher in workers compared with controls (15.03 versus 7.01, *p* = 0.026). Hence, there was a shift towards humoral

immune response in Pb-exposed workers, evident from an increase in IL-4 production concomitant with a decrease in IFN-γ production, which has a suppressive effect on cell-mediated immunity.


**Table 1.** Demographic data of control and worker groups.

Age, BMI, hemoglobin, hematocrit, and blood Pb concentration are presented as median ± standard deviation (SD).—means no data or not determined. 1 The *p*-values for age, BMI, and blood Pb were derived from the Mann–Whitney U test. The *p*-value for smoking history was derived from chi-square test. Significant different at *p*-value < 0.05.


**Table 2.** Profiling of immunologic parameters.

1 The *p*-value was derived from the Mann–Whitney U test, significant difference at *p*-value < 0.05.

#### *3.3. E*ff*ects of High Pb Exposure on Innate Immunity*

Figure 1 provides results of an investigation on innate immunity, where white blood cells separated from the buffy coats of fresh heparinized blood samples from subjects who were tested for their phagocytic activity. As the effect of smoking on the percentage of phagocytic cells was minuscule, data from non-smokers and smokers in the group of workers or controls were pooled. The median percentage of phagocytic active cells was 8.4% lower in workers than controls (89.92% vs. 98.11%, *p* = 0.019).

**Figure 1.** Effects of high Pb exposure on innate immunity. Histograms show the percentages of phagocytic cells in one Pb-exposed worker (**A**) and one control subject (**B**). Boxplots compare percentages of phagocytic cells in controls versus workers, non-smokers, and smokers included (**C**). Outliers were the data points below or above 1.5× interquartile range. The *p*-value was derived from the Mann–Whitney U test, in which outliers were included.

#### *3.4. E*ff*ects of High Pb Exposure on the Populations of Cytoxic and Regulatory T Lymphocytes*

Figure 2 provides boxplots that compare the percentages of cytotoxic T (Tc) lymphocytes (CD3+CD8+) in controls and workers, stratified by smoking status. As an effect of smoking on percentage of Tc lymphocyte was statistically insignificant, data from non-smokers and smokers in the group of workers or controls were pooled. The median percentage of Tc lymphocytes in the worker group was 33.9 % lower than the control group (24.30% versus 36.76%, *p* = 0.004).

**Figure 2.** Effects of high Pb exposure on the percentage of cytotoxic T lymphocytes. The flow cytometry strategies (dot plots) show side scatter vs. CD3 (PerCP) (**A**), isotype control stained (**B**), the cytotoxic T (Tc) lymphocytes in one control subject (**C**), and one Pb-exposed worker (**D**). Boxplots compare percentages of Tc lymphocytes in controls versus workers, non-smokers, and smokers included (**E**). Outliers were the data points below or above 1.5× interquartile range. The *p*-value was derived from the Mann–Whitney U test, in which outliers were included.

Figure 3 provides boxplots that compare the percentages of regulatory T (Treg) lymphocytes (CD4+CD25brightCD127dim) in controls and workers, stratified by smoking status. As the effect of smoking on the percentage of Treg lymphocytes was statistically insignificant, data from non-smokers and smokers in the group of workers or controls were pooled. The median percentage of Treg lymphocytes in the worker group was 2.7-fold higher than the control group (6.10% versus 2.28%, *p* < 0.001). Pb-exposed workers appeared to have a decreased percentage of Tc cells concomitantly with an increased percentage of Treg cells, thereby suggesting suppression of cellular immunity.

**Figure 3.** Effects of high Pb exposure on the percentage of regulatory T lymphocytes. The flow cytometry strategies (dot plots) show side scatter vs. CD4 (FITC) (**A**), isotype control stained (**B**), the regulatory T (Treg) lymphocytes in one control subject (**C**) and one Pb-exposed worker (**D**). Boxplots compare percentages of regulatory Treg lymphocytes in controls versus workers, non-smokers, and smokers included (**E**). Outliers were the data points below or above 1.5× interquartile range. The *p*-value was derived from the Mann–Whitney U test, in which outliers were included.

#### *3.5. Blood Pb in Relation to Cytokines amd Immunologic Parameters*

Table 3 provides results of the Spearman's rank correlation analysis of blood Pb concentration and seven immunologic parameters measured for an entire group (controls plus workers, *n* = 30). Blood Pb concentration did not correlate with phagocytic activity, proliferative response, IFN-γ, or % T helper cells (*p* ≥ 0.05). However, blood Pb concentrations showed significant positive correlations with the percentages of Treg cells (*r* = 0.843, *p* < 0.001) and IL-4 (*r* = 0.473, *p* = 0.041), while showing an inverse correlation with the percentages of Tc cells (*r* = −0.563, *p* = 0.015). Thus, Pb-exposed workers were found to have elevated IL-4 production levels together with an elevated percentage of Treg cells, while having a decreased percentage of Tc cells.

**Table 3.** The Spearman's rank correlation analysis of blood Pb concentrations and seven immunologic parameters.

