In situ analysis of protein S-glutathionylation in lung tissue using glutaredoxin-1-catalyzed cysteine derivatization

Abstract

Protein S-glutathionylation (PSSG) is a posttranslational modification that involves the conjugation of the small antioxidant molecule glutathione to cysteine residues and is emerging as a critical mechanism of redox-based signaling. PSSG levels increase under conditions of oxidative stress and are controlled by glutaredoxins (Grx) that, under physiological conditions, preferentially deglutathionylate cysteines and restore sulfhydryls. Both the occurrence and distribution of PSSG in tissues is unknown because of the labile nature of this oxidative event and the lack of specific reagents. The goal of this study was to establish and validate a protocol that enables detection of PSSG in situ, using the property of Grx to deglutathionylate cysteines. Using Grx1-catalyzed cysteine derivatization, we evaluated PSSG content in mice subjected to various models of lung injury and fibrosis. In control mice, PSSG was detectable primarily in the airway epithelium and alveolar macrophages. Exposure of mice to NO(2) resulted in enhanced PSSG levels in parenchymal regions, while exposure to O(2) resulted in minor detectable changes. Finally, bleomycin exposure resulted in marked increases in PSSG reactivity both in the bronchial epithelium as well as in parenchymal regions. Taken together, these findings demonstrate that Grx1-based cysteine derivatization is a powerful technique to specifically detect patterns of PSSG expression in lungs, and will enable investigations into regional changes in PSSG content in a variety of diseases.

Figure 1

In situ analysis of PSSG in mouse lung tissue using Grx1-based cysteine derivatization. A: Pattern of PSSG reactivity in control mouse lung. Red, PSSG reactivity; green, DNA content. Magnification, ×200. B: Omission of derivatization buffer as a negative control. C: Omission of MPB labeling agent as a negative control. D: Selective omission of GSH from the reaction mix as a negative control. E: Evaluation of PSSG reactivity using Grx1-based cysteine derivatization following pretreatment of tissue with diamide and GSH as a positive control. Following dewaxing and rehydration, lung tissue was incubated with 400 μmol/L diamide plus 1 mmol/L GSH for 10 minutes to cause PSSG formation. Lungs were then processed and evaluated as described in A. Insets: Top, PSSG reactivity in bronchial epithelium; bottom, PSSG reactivity in parenchymal regions (zoom = 4×). F: Assessment of Grx1 activity in vitro using a full enzymatic mix or omitting GSH as a negative control. Semiquantitative RFI values for PSSG were derived from the mean red fluorescence intensities (PSSG) in each region of interest divided by the mean green fluorescence intensity (DNA content). RFI values, mean (SEM) for bronchial epithelium and parenchymal regions based on scanning of four optical images per panel were as follows. A: Full reaction mix: bronchial epithelium, 39 (10); parenchyma, 24 (4). B: Buffer: bronchial epithelium, 9 (5); parenchyma, 10 (8). C: −MPB: bronchial epithelium, 0 (0); parenchyma, 0 (0). D: −GSH; bronchial epithelium, 6 (2); parenchyma, 3 (1). E: Diamide + GSH: bronchial epithelium, 18 (1); parenchyma, 46 (1); *P < 0.05 (analysis of variance) compared with A.

Figure 2

Comparative evaluation of PSSG reactivity in lung tissue using Grx1-based cysteine derivatization, and an anti-GSH antibody. A: Evaluation of PSSG in control mouse lung using anti-GSH antibody (red). B: Evaluation of PSSG in control mouse lung using Grx1-catalyzed cysteine derivatization (red). Green reflects DNA content. C and D: Assessment of PSSG reactivity in control mouse lung following reduction of PSSG with the thiol reducing agent, BME, before analysis of the tissue with anti-GSH antibody (C) or Grx1-based cysteine derivatization (D). Insets: Top, PSSG reactivity in bronchial epithelium; bottom. PSSG reactivity in parenchymal regions. Mean RFI values for PSSG or anti-GSH antibody reactivity based on scanning of four optical images per panel were as follows. A: Anti-GSH antibody; bronchial epithelium, 31 (2); parenchyma, 21 (4). B: Grx reaction mix; bronchial epithelium, 23 (2); parenchyma, 16 (2). C: Anti-GSH antibody plus BME; bronchial epithelium, 19 (4); parenchyma, 11(2). D: Grx reaction mix plus BME; bronchial epithelium, 2 (1); parenchyma, 2 (2).

Figure 3

In situ analysis of PSSG reactivity in lung tissue (A–C) or cells obtained via bronchoalveolar lavage (D–F) from mice 16 hours following oropharyngeal aspiration of PBS or LPS. PSSG was evaluated using Grx1-catalyzed cysteine derivatization as described in Figure 1. Magnification, ×200. Red, PSSG; green, DNA content. Insets in A–C: Top, PSSG reactivity in bronchial epithelium; bottom: PSSG reactivity in parenchymal regions. Insets in D–F reflect PSSG reactivity in BAL cells, as described in Figure 1. C and F: Negative control in which GSH was omitted from the derivatization reaction in lung tissue (C), or BAL cells (F). RFI values, mean (SEM), obtained from analyzing four optical images per panel were as follows. A: PBS; bronchial epithelium, 52 (13); parenchyma, 40 (12); B: LPS; bronchial epithelium, 36 (10); parenchyma, 31 (17). C: −GSH; bronchial epithelium, 5 (2); parenchyma, 2 (1). D–F: RFI values for macrophages obtained via bronchoalveolar lavage. D: PBS; 89 (14); E: LPS; 104 (10); F: −GSH, 0 (0). Note that in B, PSSG reactive cells within airspaces, which resembled macrophages were omitted from the analyses. Note that in E, neutrophils were omitted from the semiquantitative analyses. Arrows in A and B represent cells with the morphological appearance of alveolar macrophages.

Figure 4

Evaluation of PSSG in lungs of mice following inhalation of NO₂ (A–C) or >95% oxygen (D–F). Red, PSSG reactivity; green, DNA content. Magnification, ×200. Insets: Top, PSSG reactivity in bronchial epithelium; bottom, PSSG reactivity in parenchymal regions. A and D: Respective sham air exposures for the individual experiments. C and F: Negative control in which GSH was omitted from the derivatization reaction in lung tissue. RFI values; mean (SEM) values for bronchial epithelium and parenchymal regions in each inset, obtained from evaluating four to seven optical images per group, were as follows. A: Air; bronchial epithelium, 42 (5); parenchyma, 31 (7). B: NO₂; bronchial epithelium, 69 (6); parenchyma, 77 (8)*. C: –GSH control; bronchial epithelium, 10 (2); parenchyma, 3 (1). D: Air; bronchial epithelium, 33 (3); parenchyma, 40 (5). E: 95% oxygen; bronchial epithelium, 53 (13); parenchyma, 30 (5). F: −GSH control; bronchial epithelium, 7 (1); parenchyma, 12 (1)*. *P < 0.05; analysis of variance compared with parenchymal region in A.

Figure 5

Evaluation of PSSG in lungs of mice with bleomycin-induced pulmonary fibrosis using Grx1-catalyzed cysteine derivatization. A and B: Lungs were evaluated 21 days after administration of bleomycin (Bleo) or PBS as a vehicle control. Red, PSSG reactivity; green, DNA content. Magnification, ×200. C: Negative control in which GSH was omitted from the derivatization reaction in lung tissue. Insets: Top, PSSG reactivity in bronchial epithelium; bottom, PSSG reactivity in parenchymal regions. D: Semiquantitative assessment of increases in PSSG in lung tissues of mice exposed to bleomycin as compared with controls. RFI values were determined in bronchial epithelial and parenchymal regions of seven mice/group. Three optical fields per mouse were evaluated for each region. *P < 0.05 (analysis of variance) compared with PBS controls.

Figure 6

In situ analysis of Grx1 expression in the lungs of mice following inhalation of 25 ppm NO₂ (A–C) or administration of bleomycin (D–F) or vehicle controls. Red, Grx1 expression; green, DNA content. Magnification, ×200. Insets: Zoom = 4×. C and F: Negative controls in which the primary antibody was omitted.