Original Contribution
Beryllium-stimulated reactive oxygen species and macrophage apoptosis

https://doi.org/10.1016/j.freeradbiomed.2004.12.014Get rights and content

Abstract

Beryllium (Be), the etiologic agent of chronic beryllium disease, is a toxic metal that induces apoptosis in human alveolar macrophages. We tested the hypothesis that Be stimulates the formation of reactive oxygen species (ROS) which plays a role in Be-induced macrophage apoptosis. Mouse macrophages were exposed to 100 μM BeSO4 in the absence and presence of the catalytic antioxidant MnTBAP (100 μM). Apoptosis was measured as the percentage of TUNEL+ and caspase-8+ cells. ROS production was measured by flow cytometry using the fluorescence probes, dihydroethidine (DHE) and dichlorofluorescein diacetate (DCFH-DA). Be-exposed macrophages had increased TUNEL+ cells (15 ± 1% versus controls 1 ± 0.2%, P < 0.05) and increased caspase-8+ cells (18.7 ± 2% versus controls 1.8 ± 0.4%, P < 0.05). Be-induced caspase-8 activation, and a 4-fold increase in ROS formation, was ameliorated by exposure to MnTBAP. Hydrogen peroxide (30 μM) exposure potentiated Be-induced caspase-8 activation, and was also attenuated by MnTBAP. Our data are the first to demonstrate that Be stimulates macrophage ROS formation which plays an important role in Be-induced macrophage apoptosis.

Introduction

Chronic beryllium disease (CBD) is a human lung disorder caused principally by occupational exposure to the element beryllium (Be) and characterized by the formation of noncaseating interstitial lung granulomas [1], [2]. The pathogenesis of CBD is widely believed to depend upon the generation of large numbers of beryllium-antigen-specific, effector-memory CD4+ T cells that produce the proinflammatory cytokines TNF-α and IFN-γ [3], [4], [5]. More recent studies have begun to focus attention on how Be exposure affects other cells involved in modulating the immune response of the lung. Kittle et al. [6] identified TUNEL+ apoptotic cells inside CBD granulomas and found that bronchoalveolar lavage (BAL) macrophages from CBD patients undergo Be-induced apoptosis. H36.12j cells, a mouse macrophage cell line, mimic this Be-induced apoptotic response [6], [7]. Recent study shows that Be exposure induces the apoptosis of CBD BAL macrophages, but not BAL lymphocytes and CBD BAL macrophage and H36.12j cell apoptosis is accompanied by an up-regulation in the expression of activated intracellular caspases [8]. How Be exposure might trigger macrophage apoptosis remains unclear.

Oxidative stress and reactive oxygen species (ROS) contribute to the pathogenesis of varied disease processes such as atherosclerosis, chronic lung disease, macular degeneration, Alzheimer's disease, and autoimmune diabetes [9], [10], [11], [12], [13], [14]. ROS have also been shown to mediate the apoptosis of lymphocytes activated in vivo by superantigen [15], neutrophils [16], [17], endothelial cells [10], [18], drug-treated human leukemia cells [19], and in mixed neuronal and glial cells [20]. A variety of activation signals trigger ROS-induced apoptosis, including rotenone, the superantigens Staphylococcus enterotoxin A and B, TNF-α, ceramide, staurosporine, UV radiation, and the chlorinated hydrocarbon insecticide toxaphene [15], [21], [22], [23], [24], [25], [26]. Comhair et al. [27] found increased levels of the antioxidants, glutathione and glutathione peroxidase, in the epithelial lining fluid (ELF) of CBD patients. They suggested that increased antioxidant levels in the ELF represent a host response that affords the CBD lung a degree of protection against ROS generated during chronic inflammation. However, Be-induced up-regulation of ROS has not been examined.

One approach to test the hypothesis that Be exposure induces ROS-dependent apoptosis would be to inhibit this process using antioxidants that rescue a susceptible cell population from Be-induced apoptosis [19], [20], [28], [29], [30]. The porphyrin, 5,10,15,20-tetrakis(benzoic acid) porphyrin manganese(III) (MnTBAP), is a unique antioxidant scavenger that is active, stable, and nontoxic and that readily penetrates cellular membranes [31], [32]. MnTBAP possesses SOD activity in cell-free systems and protects biological systems against superoxide-mediated injury. MnTBAP also possesses catalase activity and can protect cells from hydrogen peroxide toxicity [29], [33]. Thus, if Be exposure activates the generation of ROS that in turn trigger apoptosis, then treatment with MnTBAP should inhibit or block Be-induced apoptosis.

A second approach to this hypothesis would be to determine if compounds that are activated by the generation of ROS are also inhibited by MnTBAP. The dyes, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and dihydroethidine (DHE) have been used to measure intracellular generation of hydrogen peroxide and superoxide, respectively [17], [18]. Intracellular DCFH-DA is metabolized to DCFH by esterases. In the presence of H2O2, DCFH is oxidized to 2′-7′-dichlorofluorescein (DCF+) and fluorescence is measured by flow cytometry. Limitations have been noted as to the use of DCF+ fluorescence as a specific marker for quantitative intracellular H2O2 formation [34]. DHE can undergo two-electron oxidation to form DNA-binding ethidium bromide (EB+) in a reaction that, while relatively specific for superoxide anion, has also been noted to have some quantitative limitations [35]. For the present study, if Be exposure increases the levels of ROS there should be a corresponding increase in DCF+ and EB+ fluorescence, and MnTBAP should inhibit the Be-induced, ROS-dependent activation of these compounds.

Determining if Be exposure induces ROS in CBD is hampered in part by the need to obtain cells from the anatomical site of disease by bronchoscopy, an invasive procedure, and in part by the lack of an adequate animal model of CBD. Based on our previous studies showing that mouse macrophage cell lines mimic the Be-induced apoptosis observed in CBD BAL macrophages [6], [7], [8], we tested the hypothesis that Be exposure triggers macrophage apoptosis mediated by increased ROS using alveolar macrophages from mice and mouse macrophage cell lines. Our study shows for the first time that Be exposure enhances the level of ROS in mouse alveolar macrophages and in macrophage cell lines, suggesting that increased production of ROS plays a key role in Be-induced macrophage-programmed cell death.

Section snippets

Chemicals and reagents

Carrier-free 7BeCl2, specific activity 2.6 mCi/mg at 1.66 mCi/ml, was purchased from Oak Ridge National Laboratory (Oak Ridge, TN) and counts per minute were determined using a Packard Cobra Auto-gamma counter (Downers Grove, IL). Beryllium sulfate (Brush Wellman, Inc., Cleveland, OH) and aluminum sulfate (Sigma, St. Louis, MO) were maintained at 4°C as stock solutions of 1 × 10−3 M BeSO4 and 1 × 10−3 M Al2(SO4)3 in water and diluted to final concentrations of 10 or 100 μM, as specified in the

MnTBAP does not inhibit 7BeCl2 uptake

As a control, to ensure that MnTBAP did not attenuate Be-induced macrophage apoptosis by simply chelating the metal from culture supernatant and thereby prevent Be uptake, we determined the counts per minute (cpm) in H36.12j culture supernatant and in cytoplasmic and nuclear extracts of cells exposed to 1.0 mCi of 7BeCl2 in the absence and presence of 500 μM MnTBAP. After 24 h, 7BeCl2 + 500 μM MnTBAP-treated culture supernatant had 6724 cpm with 361 cpm in the cytoplasmic extract and 87 cpm in

Discussion

In this study, we tested the hypothesis that Be stimulates the formation of ROS which plays a role in Be-induced macrophage apoptosis. We tested this hypothesis in mouse alveolar macrophages and in mouse macrophage cell lines that mimic the ability of human BAL macrophages to undergo Be-induced apoptosis [6], [7], [8]. Beryllium-induced macrophage apoptosis was significantly attenuated by a catalytic antioxidant, enhanced in the presence of exogenous ROS, and occurred with the ROS-dependent

Acknowledgments

The authors thank Dr. Terry A. Potter, Dr. Kristie Gribe, William J. Townend, Shirley Sobus, and Alexas Jonth for their technical assistance.

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    This study was supported by RO1 ES-06358, PO1 ES11810, RO1 ES012504, RO1 HL 62410, K08 HL03887, and MO1 RR00051 from the National Institutes of Health.

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