The antioxidant role of thiocyanate in the pathogenesis of cystic fibrosis and other inflammation-related diseases

Abstract

Cystic fibrosis (CF) is a pleiotropic disease, originating from mutations in the CF transmembrane conductance regulator (CFTR). Lung injuries inflicted by recurring infection and excessive inflammation cause approximately 90% of the morbidity and mortality of CF patients. It remains unclear how CFTR mutations lead to lung illness. Although commonly known as a Cl(-) channel, CFTR also conducts thiocyanate (SCN(-)) ions, important because, in several ways, they can limit potentially harmful accumulations of hydrogen peroxide (H(2)O(2)) and hypochlorite (OCl(-)). First, lactoperoxidase (LPO) in the airways catalyzes oxidation of SCN(-) to tissue-innocuous hypothiocyanite (OSCN(-)), while consuming H(2)O(2). Second, SCN(-) even at low concentrations competes effectively with Cl(-) for myeloperoxidase (MPO) (which is released by white blood cells), thus limiting OCl(-) production by the enzyme. Third, SCN(-) can rapidly reduce OCl(-) without catalysis. Here, we show that SCN(-) and LPO protect a lung cell line from injuries caused by H(2)O(2); and that SCN(-) protects from OCl(-) made by MPO. Of relevance to inflammation in other diseases, we find that in three other tested cell types (arterial endothelial cells, a neuronal cell line, and a pancreatic beta cell line) SCN(-) at concentrations of > or =100 microM greatly attenuates the cytotoxicity of MPO. Humans naturally derive SCN(-) from edible plants, and plasma SCN(-) levels of the general population vary from 10 to 140 microM. Our findings raise the possibility that insufficient levels of antioxidant SCN(-) provide inadequate protection from OCl(-), thus worsening inflammatory diseases, and predisposing humans to diseases linked to MPO activity, including atherosclerosis, neurodegeneration, and certain cancers.

Fig. 1.

Lactoperoxidase (LPO) and myeloperoxidase (MPO) catalyzed oxidation reactions. (A) LPO catalyzes oxidation of SCN⁻ to OSCN⁻ by H₂O₂. (B) MPO catalyzes two competitive oxidative reactions: Cl⁻ to OCl⁻ (upper) and SCN⁻ to OSCN⁻ (lower). (C) OCl⁻ rapidly oxidizes SCN⁻ to OSCN⁻ without enzymatic catalysis.

Fig. 2.

Cytotoxicity of MPO. Calu-3 cells were incubated in EBSS containing: no added reagents (control); GO; MPO; GO plus MPO; or GO plus MPO with the inhibitor ABAH (GO, 10 mU/mL; MPO, 1 U/mL; ABAH, 100 μM). Percentages of nonviable cells are presented as mean ± SEM (the number of independent trials is indicated in parentheses). The differences between data with and without asterisk are statistically significant (one-way ANOVA; P < 0.001).

Fig. 3.

SCN⁻ protects Calu-3 cells against MPO cytotoxicity. Cells were incubated in EBSS solution containing: no added reagents (control); GO plus MPO; or GO plus MPO and SCN⁻ (GO, 10 mU/mL; MPO, 1 U/mL; SCN⁻ at 10, 50, 100, or 400 μM). Percentages of nonviable cells are presented as mean ± SEM (the number of independent trials is indicated in parentheses). The differences between data with and without asterisk are statistically significant (one-way ANOVA; P < 0.001).

Fig. 4.

SCN⁻ protects Neuro-2A, Min6, and HAE cells against MPO cytotoxicity. Cells were incubated in EBSS containing: no added reagents (control), GO; GO plus MPO; GO plus MPO and SCN⁻ (SCN⁻, 100 or 400 μM; MPO, 1 U/mL; GO, 10 mU/mL for Neuro-2A and Min6 cells and 5 mU/mL for HEA cells). Percentages of nonviable cells are presented as mean ± SEM (the number of independent trials is indicated in parentheses). The differences between data with and without asterisk are statistically significant (one-way ANOVA; P < 0.001).

Fig. 5.

Production of OCl⁻ and OSCN⁻ catalyzed by MPO. Concentrations of OCl⁻ (●) and OSCN⁻ (▴) produced in 5 min by MPO (1 U/mL) plotted against the SCN⁻ concentration in the presence of constant 100 mM Cl⁻ (mean ± SEM, n = 4).

Fig. 6.

LPO and SCN⁻ together prevent H₂O₂ cytotoxicity. Calu-3 cells were incubated in EBSS containing: no added reagents (control), GO; GO plus SCN⁻; GO plus LPO; GO plus LPO and SCN⁻ (GO, 20 mU/mL; LPO, 1 U/mL; SCN⁻, 100 or 400 μM). Percentages of nonviable cells are presented as mean ± SEM (the number of independent trials is indicated in parentheses). The differences between data with and without asterisk are statistically significant (one-way ANOVA; P < 0.001).