The Effects of Asbestos Fibers on Human T Cells

Abstract

Asbestos exposure causes malignant tumors such as lung cancer and malignant mesothelioma. The effects of asbestos fibers on immunocompetent cells, however, have not been well studied. Asbestos physically comprises a fibrous substance, which differs from silica particles which are a particulate substance, although chemically it is a mineral silicate. Since silicosis patients previously exposed to silica particles often suffer from lung and autoimmune diseases, it is clear that silica exposure impairs immune tolerance. Similarly, asbestos may alter the immune system in asbestos-exposed individuals. Given that malignant tumors can result following exposure to asbestos, the attenuation of anti-tumor immunity in cases of asbestos exposure is an important area of investigation. We observed the effect of asbestos fibers on T lymphocytes, such as CD8+ cytotoxic T lymphocytes (CTLs), CD4+ helper T (Th), and regulatory T (Treg) cells, and showed that anti-tumor immunity was attenuated, as demonstrated in a system that stimulates fresh cells isolated from peripheral blood in vitro and a system that is continuously exposed to a cell line. In this manuscript, we introduce the experiments and results of studies on CTLs, as well as Th and Treg cells, and discuss how future changes in immunocompetent cells induced by asbestos fibers can be clinically linked.

Keywords: CD4+ T cell; T cell; asbestos; cytotoxic T lymphocyte; malignant mesothelioma; pleural plaque; regulatory T cell.

Figure 1

Observation of the effect of asbestos fibers on CD8+ cytotoxic T lymphocytes (CTLs). In the peripheral blood mononuclear cells (PBMCs) collected from (A) healthy volunteers (HVs), clonal expansion of CTLs was observed in the mixed lymphocyte reaction (MLR). When co-cultured with allogenic PBMCs, CD8+ cells differentiated into CTLs and proliferated, but when chrysotile and asbestos fibers were added, both differentiation and proliferation were suppressed. Furthermore, expression of granzyme B (GzB) and perforin (PFR), which execute the cell killing mechanism, and the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, which are closely related cytokines, were also reduced. (B) With HV, CD8+ cells were isolated from the peripheral blood of patients with pleural plaque (PP) and malignant mesothelioma (MM), stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin overnight, and expression levels of PFR and GzB in the chamber were observed. In the PP group, both were higher compared with HV and decreased in the MM group. (C) After the human CD8+ cell line ETV-8 was continuously exposed to chrysotile and asbestos fibers for 2 months, intracellular PFR decreased and IFN-γ production decreased.

Figure 2

Effect of asbestos fibers on Th cells. (A) CD4+ T lymphocytes were isolated from HV peripheral blood and treated with interleukin (IL)-2 together with anti-CD3 and anti-CD28 antibodies for 4 weeks to induce clonal expansion. Expression levels of CXC chemokine receptor (CXCR) 3 on the cell membrane surface changed markedly. Intracellular IFN-γ expression levels were also reduced. (B) CD4+ cells were collected from the peripheral blood of individuals with PP or MM who were actually exposed in vivo, and these cells were stimulated overnight with PMA and ionomycin in an effort to observe changes in CXCR3 on the membrane surface and intracellular IFN-γ. CXCR3 levels diminished in cells derived from HV, and the PP and MM groups. However, although IFN-γ levels were lower in the MM group, they did not differ between HV and the PP group. When several representative cytokines related to HV, PP, and MM were measured, IL-6 levels in cells derived from the PP and MM groups were higher than those of HV. There was no statistically significant difference between the PP and MM groups, although the MM group tended to be slightly higher.

Figure 3

Summary of the effect of asbestos on Treg cells using the Treg-like cell line MT-2. (A) The MT-2 line resists apoptosis after continuous exposure for as long as one year at a concentration of asbestos fibers (chrysotile or crocidolite) that induces apoptosis in less than half the cell population following transient exposure. Sublines that acquired resistance to apoptosis were established. (B) In terms of protein expression, excessive phosphorylation of β-actin occurred in sublines. (C) Cytokines cause overproduction of IL-10 and transforming growth factor (TGF)-β, which are soluble factors required for expression of typical Treg cell function, together with cell–cell contact, and functional enhancement was shown. (D) Among the transcription factors, forkhead box protein O1 (FoxO1) was significantly attenuated, and negative regulation of cyclins with positive regulation of cyclin-dependent kinase-inhibitors (CDK-Is) accelerated the cell cycle. Asbestos-exposed Treg cells were over-functional and abundant. Expression of FoxP3, an important Treg cell transcription factor, was slightly attenuated in spite of hyperactivity, although no other significant changes such as methylation were observed. (E) Furthermore, nicotinamide nucleotide transhydrogenase (NNT) was highly expressed due to the relationship between oxidative stress and oxidative phosphorylation. This may act to protect asbestos-exposed Treg cells from oxidative stress and may also play a role in their escape from cell death.