S-glutathionylation regulates inflammatory activities of S100A9

Abstract

Reactive oxygen species generated by activated neutrophils can cause oxidative stress and tissue damage. S100A8 (A8) and S100A9 (A9), abundant in neutrophil cytoplasm, are exquisitely sensitive to oxidation, which may alter their functions. Murine A8 is a neutrophil chemoattractant, but it suppresses leukocyte transmigration in the microcirculation when S-nitrosylated. Glutathione (GSH) modulates intracellular redox, and S-glutathionylation can protect susceptible proteins from oxidative damage and regulate function. We characterized S-glutathionylation of A9; GSSG and GSNO generated S-glutathionylated A8 (A8-SSG) and A9 (A9-SSG) in vitro, whereas only A9-SSG was detected in cytosol of neutrophils activated with phorbol myristate acetate (PMA) but not with fMLP or opsonized zymosan. S-Glutathionylation exposed more hydrophobic regions in Zn(2+)-bound A9 but did not alter Zn(2+) binding affinity. A9-SSG had reduced capacity to form heterocomplexes with A8, but the arachidonic acid binding capacities of A8/A9 and A8/A9-SSG were similar. A9 and A8/A9 bind endothelial cells; S-glutathionylation reduced binding. We found little effect of A9 or A9-SSG on neutrophil CD11b/CD18 expression or neutrophil adhesion to endothelial cells. However, A9, A9-SSG and A8/A9 promoted neutrophil adhesion to fibronectin but, in the presence of A8, A9-mediated adhesion was abrogated by glutathionylation. S-Glutathionylation of A9 may protect its oxidation to higher oligomers and reduce neutrophil binding to the extracellular matrix. This may regulate the magnitude of neutrophil migration in the extravasculature, and together with the functional changes we reported for S-nitrosylated A8, particular oxidative modifications of these proteins may limit tissue damage in acute inflammation.

FIGURE 1.

Characterization of A9-SSG. A, two peaks with retention times of 16.3 min and 16.9 min were observed when recombinant human A9 (black) was separated by C4 RP-HPLC. Separation of GSNO- (gray) and GSSG-treated (dashed line) A9 generated an additional peak with retention time of 15.95 min (peaks 1 and 2, respectively). Deconvoluted masses were derived from the ESI mass spectra (insets) of multiply charged ions of peaks 1 (B) and 2 (C). In GSNO-treated A9, masses corresponding to A9 (13,255 Da) and likely to A9-SNO (13,285 Da; +30 Da) were observed. GSNO and GSSG both generated a major component of 13,560 Da, equivalent to the theoretical mass of A9-SSG, and a minor component of 13,577 Da, that may correspond to oxidation of a single Met residue in A9-SSG.

FIGURE 2.

C4 RP-HPLC separation of A9 (retention time, 13.1 min; peak 1) and A9* (retention time 14 min; peak 2) from neutrophil cytosol. A, shown are the first 20 N-terminal amino acid residues of A9 and A9* protein sequences, depicting the start Met (Met¹ and Met⁵). B, a representative chromatogram of one of five donors is shown. The deconvoluted mass spectrum of peak 1 (C) indicated a major component of 13,152 Da, corresponding to A9. The major component of the deconvoluted mass spectrum of peak 2 (D) indicated a molecular mass of 12,690 Da, corresponding to A9*. Components with masses of 13,169 Da and 12,705 Da likely correspond to oxidation of a single Met residue in A9 and A9*, respectively.

FIGURE 3.

C4 RP-HPLC separation of neutrophil cytosol and identification of A9 adducts. A, an additional component (‡) with a retention time of 15.7 min was generated when cytosol of neutrophils treated with PMA (gray) or PMA plus ionomycin (dashed line) were separated and compared with untreated (black) cells. Several A9 adducts (Table 1) were observed when the ESI mass spectra (insets) of the peak (‡) from PMA (B)- and PMA plus ionomycin (C)-treated neutrophils were deconvoluted. A component of 13,457 Da was observed in both and may correspond to A9-SSG. Results are representative of separations of neutrophil cytosol preparations from three normal donors.

FIGURE 4.

Immunofluorescence staining of A9 and GSH in neutrophils. A, A9 (upper panels; red) and GSH (middle panels; green) were detected in resting neutrophils, predominantly in the cytoplasm, and around the cell membrane. An overlay of the two images (lower panels) with 4′,6′-diamidino-2-phenylindole staining for nuclei (blue) indicates co-localization of A9 with GSH. Negative control antibodies, rabbit IgG, and IgG2a isotype control, showed no background fluorescence (inset). B, A9 and GSH were detected in the cytoplasm and around the cell membrane of PMA plus ionomycin-treated neutrophils; the merged image indicates co-localization of A9 with GSH. C, A9 was observed in the cytoplasm and around the cell membrane of fMLP-treated neutrophils, whereas GSH was mainly cytoplasmic, and little co-localization was obvious. D, in neutrophils activated by opsonized zymosan, A9 and GSH were located predominantly around the cell membrane and the phagosome containing zymosan. The merged image indicates co-localization of A9 with GSH. Scale The bar = 28 μm; magnification, 1000×. Results are representative of cells from two donors that had undergone the same treatments.

FIGURE 5.

Zn²⁺-induced structural changes in A9 and A9-SSG. A larger change in fluorescence intensity was observed in A9-SSG (●) compared with A9 (○) upon the addition of increasing doses of Zn²⁺. Zn²⁺ binding affinities of the two forms were similar; A9-SSG had a Zn²⁺ binding constant (K_d) of 8.9 ± 0.1 μm, and A9 had a K_d of 11 ± 0.4 μm. Results shown are the averages of duplicate wells and are representative of three experiments.

FIGURE 6.

A9 and A9-SSG induce integrin-dependent neutrophil adhesion to fibronectin. A, treatment of dextran-sedimented white blood cells with fMLP (1 μm; black bars) significantly increased expression of CD11b, CD18, and CBRM (*, p < 0.05) compared with untreated controls (white bars), assessed by flow cytometry. A9 (horizontal bars) and A9-SSG (vertical bars) treatments increased CD11b, CD18, and CBRM expression, but effects were not significant when compared with untreated controls. Values represent the mean fluorescence intensities ±S.D. of neutrophils from six donors; quantitation is given under “Experimental Procedures”. B, fMLP (checkered bars), A9 (horizontal bars), and A9-SSG (vertical bars) significantly induced neutrophil adhesion to fibronectin in a dose-dependent manner. A8/A9 (black bar) also stimulated neutrophil adhesion, but A8/A9-SSG (dark gray bar) and A8 (light gray bar) had no effect. Data represent CI values ± S.D. of neutrophils from three donors; quantitation is given under “Experimental Procedures.” *, p < 0.01, fMLP, A9, A9-SSG, and A8/A9 compared with the untreated control; **, p < 0.01, A8/A9-SSG compared with A8/A9.

FIGURE 7.

Effect of A9, A9-SSG, A8/A9, and A8/A9-SSG on HMEC-1 binding and activation. A, significantly more A9 and A8/A9 (black bars) bound HMEC-1 cells than their glutathionylated forms (A9-SSG and A8/A9-SSG; gray bars). Data are normalized to total mean absorbance and represent the means ± S.D. of duplicate measurements from five experiments; quantification is given under “Experimental Procedures.” *, p < 0.01, A9-SSG compared with A9, and A8/A9-SSG compared with A8/A9. B, IL-8, ICAM-1, and VCAM-1 mRNA were induced in HMEC-1 cells treated with fMLP but not with A8, A9, A9-SSG, A8/A9, or A8/A9-SSG. HMEC-1 cells were harvested 6 h post-treatment, and mRNA levels were quantitated. Data are normalized to β-actin and represent the means ± S.D. of triplicate measurements of at least two experiments. *, p < 0.05, fMLP compared with untreated control. TNF, tumor necrosis factor-α.