Original contributionEvidence of a defective thiol status of alveolar macrophages from COPD patients and smokers
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.
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