Original contribution
Evidence of a defective thiol status of alveolar macrophages from COPD patients and smokers

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Abstract

In increasing numbers of pulmonary diseases an association with a loss of intracellular thiols, mainly glutathione, is postulated. Therefore, the quantitative measurement of thiols within different viable cells is a possible metabolic parameter for cellular function and defense capacity of all pulmonary immune cells including alveolar macrophages (AM), that are highly compromised by oxidative stress. In this study the cellular thiol content was determined using fluorochrom conjugated chloromethyl derivatives (5-chloromethylfluorescein diacetate, CMFDA) in flow cytometry. The procedure was evaluated in vitro using biochemical techniques for glutathione quantification. Based on this approach, AM obtained from bronchoalveolar lavage (BAL) of smokers and patients with chronic obstructive pulmonary disease (COPD) showed a significant thiol deficiency compared to a nonsmoker/non-COPD group. The cellular thiol expression of AM from smokers and COPD patients reached only 50 and 53% of the control group. Lowest thiol concentrations (47% of control) were detected within the smoker+/COPD+ group. This intracellular thiol deficiency significantly correlated with reduced lung function (FEV1, PaO2). With regard to the tightly regulated thiol metabolism of immune cells, these results imply the onset of functional disturbances in thiol deficient AM. The determination of the cellular thiol content of AM, obtained from BAL by flow cytometry, presents a simple and reliable tool to monitor the effect of therapeutic measures focusing on the stabilization of the cellular thiol status.

Introduction

Oxidative stress and/or imbalances of the thiol-disulfide status and glutathione metabolism have been documented for a number of pulmonary diseases [1], [2]. In cases of nicotine abuse-related lung injuries, which lead to variable degrees of inflammation in the lower respiratory tract, reactive oxygen intermediates (ROI)-induced disturbances of the thiol metabolism play a pivotal role [3]. Moreover, depletion of glutathione is the major trigger of increased epithelial (airspace) permeability caused by the condensate of cigarette smoke [4]. Chronic obstructive pulmonary disease (COPD) and emphysema are known to be associated with impaired glutathione metabolism as well as a pathological production of endogenous oxidants by xanthine oxidase [5].

The progression of COPD partially depends on an imbalance of proteases and antiproteases. The latter, as alpha 1-antitrypsin, are highly susceptible to oxidation and are inactivated by inhalative (exogenous), inflammatory, and molecular-derived (endogenous) ROI. Therefore a defective thiol metabolism is thought to support lung tissue destruction [6]. However, little is known about the cellular thiol status of alveolar macrophages under these conditions.

The involvement of thiols in pulmonary diseases and the control of therapeutic effects on stabilization of the thiol metabolism require diagnostic methods as screening procedure for the cellular thiol content. In the present study the cellular thiol concentration of alveolar macrophages (AM) from bronchoalveolar lavage fluid (BAL) was determined by flow cytometry using fluorochrom conjugated chloromethyl derivatives. The cellular thiol status of smoking and nonsmoking COPD patients was compared to that of smokers and healthy controls.

Section snippets

Materials

Five-chloromethylfluorescein diacetate (CMFDA), 5-(and-6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine (CMTMR) and 5-chloromethyleosin diacetate (CMEDA) were obtained from Molecular Probes (Eugene, OR, USA). Buthionine sulfoximine (BSO), DMSO, 5,5′-dithio-bis-(2-nitrobencoic acid) (DTNB), N-ethyl maleimide (NEM), NADPH, glutathione reductase (yeast), reduced and oxidized glutathione, EDTA and 5-sulfosalicylic acid were purchased from Sigma (Deisenhofen, Germany). Glutathione Assay Kit

Flow cytometric determination of thiols in alveolar macrophages

Flow cytometric analysis of alveolar macrophages is complicated by a strong autofluorescence pattern, particularly in smokers. Compared to nonsmokers, non-specific fluorescence signals were found to be more then 20-fold higher (Fig. 1A). This problem was avoided by means of the thiol-reactive dye CMFDA, which exhibits highest fluorescence quantum yields resulting in a high signal-noise distance. In Figs. 1B and 1C two representative histograms display the CMFDA fluorescence signal of AM from

Discussion

As the role of oxidative stress in the pathogenesis of several pulmonary diseases is better understood, the determination and modulation of antioxidant systems, particularly the cellular thiol status of immune cells, are of increasing interest. Therefore the analysis of thiols in routinely obtained materials as BALF is reasonable. Conventional biochemical methods exhibit excellent precision and reliability but are restricted by limited cell counts and are expensive as screening protocols.

Acknowledgements

The excellent technical assistance of Anke Nehring and Ilona Skupin is gratefully acknowledged. The authors thank Josephine Lightowler for critical reading of the manuscript.

References (20)

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