**4. Discussion**

In this study, we investigated Pb exposure levels in occupational settings together with adverse effects of such exposure levels on the function of the body's immune system. The blood Pb concentrations in study workers showed a wide range, 25.11 to 58.47 μg/dL, while the blood Pb concentrations in age-matched farmers (a control group) were in a narrow range, 3.02 to 4.24 μg/dL. The most likely source of Pb in the control group is the diet, as Pb is present in virtually all foodstuffs [27,28]. The effects of Pb on lymphocyte proliferation, natural killer (NK) cell cytotoxicity, and IFN-γ production by PBMCs have been seen in occupationally exposed persons [29]. In parallel, some effects of Pb on the immune response have been seen in animal models, such as the innate immune system in zebra fish [30] and the humoral and cell-mediated immune responses in mice [31,32].

Compared with a former report [23], Pb exposure levels among boatyard workers in our study workers remained as high as previously observed. In addition, we observed a decrease in phagocytic activity of the neutrophils from boatyard workers. This finding agreed with a previous report [31]. The decreased phagocytic activity may be attributable to the action of IL-1, as it is known to be involved in the stimulation of neutrophils and the recruitment activated cells into the site of injury [33]. However, IL-1 was up-regulated after exposure to mixtures of Pb and arsenic [30].

IFN-γ, produced by CD8+ Th1 cells, represents a component of cell-mediated immunity known for its anti-viral and anti-parasitic propensities [34]. IFN-γ inhibits the proliferation of Th2 cells and acts in synergy with other cytokines, notably TNF-<sup>α</sup>, to impede the proliferation of normal and transformed cells [34]. In a recent review, in vivo, in vitro, and ex vivo studies have been used to confirm that IFN-γ production is inhibited by Pb [35]. It is of relevance that the levels of IFN-γ produced by PBMCs from workers showed a tendency to be lower than those from controls. In an early study [36], Pb acetate was found to be involved in immediate hypersensitivity reactions (degranulation of rat mast cells) and allergic hypersensitivity, which was mediated by Th2 [36]. Exposure to low-dose Pb led to a decrease in IFN-γ Th1 cytokine and proinflammatory cytokines TNF-α and IL-1 while inducing IL-4 and/or IL-10 to maintain a Th2 immune response [37]. It has been suggested that Pb may increase susceptibility to infection and the incidence of allergic hypersensitivity [35,37].

IL-4 is a Th2 cytokine secreted by activated Th2 and NKT cells. It is a potent inducer of naïve CD4+ T cells and directs their differentiation into Th2 effector cells. IL-4 is known as a marker of humoral immunity [38]. In the present study, PBMCs from workers showed a tendency to produce more IL-4 together with less IFN-γ than controls. Consequently, the IL-4/IFN-γ ratio produced by PBMCs from workers was 2-fold higher than the control group. This indicated toxicity of Pb on cytokine production, which is consistent with previous studies in the following ways. In BALB/c mice, levels of the *IL-4* gene were increased after exposure to heavy metals, Pb included [39]. Elevated IL-4 after Pb exposure may cause an induction of type 2 helper T (Th2) cells and M2 macrophages [40]. In effect, the increased production of IL-4 seen in boatyard workers might confer upon them increased susceptibility to infection, allergic hypersensitivity, and/or autoimmune diseases dominated by Th2.

A significant increase in Treg observed among workers in the present study is similar to previous experimental studies [41,42]. Up-regulation of IL-2RB in lymphocytes after exposure to heavy metals was observed in addition to the higher expression of Treg differentiation [43,44]. It is suggested that Pb may activate TGF-β, one of the main regulatory cytokines [41], which in turn stimulates the differentiation of regulatory T cells, promoting FoxP3 expression [45]. The link between Treg cells and cytokine suppression was confirmed by a study targeting the PI3-AKT pathway that caused the inhibition Treg proliferation [46]. In the present study, a significant reduction in Tc lymphocytes (CD3+CD8+) was observed in boatyard workers. Likewise, a significant decrease in the percentage of CD4+ Th cells was noted in a study of Pb-exposed children [47].
