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Comparative Study
. 2006 Oct 25;7(1):133.
doi: 10.1186/1465-9921-7-133.

Modulation of glutaredoxin in the lung and sputum of cigarette smokers and chronic obstructive pulmonary disease

Mirva J Peltoniemi  1 , Paula H Rytilä, Terttu H Harju, Ylermi M Soini, Kaisa M Salmenkivi, Lloyd W Ruddock, Vuokko L Kinnula
Affiliations
Comparative Study

Modulation of glutaredoxin in the lung and sputum of cigarette smokers and chronic obstructive pulmonary disease

Mirva J Peltoniemi et al. Respir Res. .

Abstract

Background: One typical feature in chronic obstructive pulmonary disease (COPD) is the disturbance of the oxidant/antioxidant balance. Glutaredoxins (Grx) are thiol disulfide oxido-reductases with antioxidant capacity and catalytic functions closely associated with glutathione, the major small molecular weight antioxidant of human lung. However, the role of Grxs in smoking related diseases is unclear.

Methods: Immunohistochemical and Western blot analyses were conducted with lung specimens (n = 45 and n = 32, respectively) and induced sputum (n = 50) of healthy non-smokers and smokers without COPD and at different stages of COPD.

Results: Grx1 was expressed mainly in alveolar macrophages. The percentage of Grx1 positive macrophages was significantly lower in GOLD stage IV COPD than in healthy smokers (p = 0.021) and the level of Grx1 in total lung homogenate decreased both in stage I-II (p = 0.045) and stage IV COPD (p = 0.022). The percentage of Grx1 positive macrophages correlated with the lung function parameters (FEV1, r = 0.45, p = 0.008; FEV1%, r = 0.46, p = 0.007, FEV/FVC%, r = 0.55, p = 0.001). Grx1 could also be detected in sputum supernatants, the levels being increased in the supernatants from acute exacerbations of COPD compared to non-smokers (p = 0.013) and smokers (p = 0.051).

Conclusion: The present cross-sectional study showed that Grx1 was expressed mainly in alveolar macrophages, the levels being decreased in COPD patients. In addition, the results also demonstrated the presence of Grx1 in extracellular fluids including sputum supernatants. Overall, the present study suggests that Grx1 is a potential redox modulatory protein regulating the intracellular as well as extracellular homeostasis of glutathionylated proteins and GSH in human lung.

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Figures

Figure 1
Figure 1
Number of alveolar macrophages. The number of alveolar macrophages was assessed in healthy non-smokers (NS), smokers without COPD (S), stage I–II COPD and stage IV COPD (for patient characteristics see Table 1). The means of the numbers of alveolar macrophages per square millimeter are marked with horizontal lines. * Significantly increased when compared to non-smokers (p = 0.020).
Figure 2
Figure 2
Immunohistochemical expression of Grx1 in lung specimens. Representative immunohistochemical expression of Grx1 in lung specimens from a healthy non-smoker (A), smoker without COPD (B), stage II COPD (C) and stage IV COPD (D). Grx1 was mainly expressed in alveolar macrophages in all cases. Occasional positivity was observed in the bronchial epithelium as indicated by arrows (E). No immunoreactivity was seen in the negative isotype control (F). Dilutions used were 1:3000 for goat anti-human Grx1 primary antibody and 1:300 for biotinylated rabbit anti-goat secondary antibody.
Figure 3
Figure 3
Analysis of Grx1 positive macrophages. The percentage (%) of Grx1 positive macrophages from the total number of alveolar macrophages in healthy non-smokers (NS), smokers without COPD (S), stage I–II COPD and stage IV COPD (for patient characteristics see Table 1). The means of the percentage of positive macrophages are marked with horizontal lines. * Significantly decreased when compared smokers (p = 0.021).
Figure 4
Figure 4
Correlation of tissue Grx1 with lung function parameters. Correlations between the percentage of Grx1 positive macrophages (of total macrophage population examined) and lung function parameters. The percentage of Grx1 positive macrophages was observed to significantly correlate with FEV1 (r = 0.45, p = 0.008), FEV1% (r = 0.46, p = 0.007) and FEV/FVC% (r = 0.55, p = 0.001).
Figure 5
Figure 5
Western blot analysis of Grx1 in lung homogenates. Representative Western blot analysis of Grx1 expression in the specimens (n = 32) of healthy non-smokers and smokers without COPD and in different stages of COPD (for patient characteristics see Table 2). The total amount of protein was 40 μg and the primary antibody dilution for human Grx1 was 1:2500 in all cases. The means of the intensities measured are shown as columns with error bars representing SEM. * Significantly decreased when compared to smokers (p = 0.045). ** Significantly decreased when compared to smokers (p = 0.022).
Figure 6
Figure 6
Grx1 expression in induced sputum supernatants and plasma samples. Representative Western blot analysis of Grx1 expression in the plasma (A) and induced sputum pellets (B) and supernatants (C) of healthy non-smokers (= NS), smokers (= S) and COPD patients (= C). The means of the intensities in the sputum supernatants were measured from total of 15 non-smokers, 11 smokers, 17 stable COPD (stage 0–I) and 7 acute exacerbations (for patient characteristics see Table 3) and are shown as columns (D). The primary antibody dilution for human Grx1 was 1:2500 in all cases. Error bars represent SEM. * Significantly increased when compared to non-smokers (p = 0.013).
Figure 7
Figure 7
Suggested role of Grxs in cigarette smoke induced oxidative stress. The major function of Grx is the reduction of glutathionylated proteins back to their functional states both intracellularly and in the extracellular space with concomitant release of glutathione. ROS, reactive oxygen species; RNS, reactive nitrogen species; GSH, reduced glutathione; GSSG, oxidized glutathione; GR, glutathione reductase; ELF, epithelial lining fluid.
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