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Glutathione inhibits antibody and complement-mediated immunologic cell injury via multiple mechanisms

View Article: PubMed Central - PubMed

ABSTRACT

Antioxidant glutathione (GSH) plays an important role in the regulation of immunity. However, little is known about its effects on humoral immunity, especially its action on effector molecules like antibody and complement. Given that these molecules contain abundant disulfide bonds, we speculated that GSH might influence the action of these proteins via its thiol function. Using a model of a glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we addressed this hypothesis. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused a complement-dependent cell lysis, which was completely blocked by GSH. Moreover, GSH potently prevented the antibody-mediated agglutination of red blood cells and aggregation of antibody-sensitized microspheres. Further analysis revealed that GSH inhibited antibody binding to antigens and promoted the conversion of the antibodies to its reduced forms. GSH also potently inhibited the formation and deposition of C5b-9 in MCs and suppressed both the classic and alternative complement activation pathway. Lastly, GSH attenuated P38 activation, an oxidative sensitive kinase that partially mediated the antibody- and complement-dependent MC lysis. Depletion of GSH via inhibiting gamma-glutamylcysteine synthetase or xCT transporter augmented P38 activation and sensitized MCs to the cell lysis. Collectively, our results indicate that GSH protects cells from immunological cell damage via mechanisms involving inhibition of antibody binding to the antigens, suppression of complement activation and augmentation of cellular defense mechanism. Our study provides novel mechanistic insights into the actions of GSH in the regulation of immune responses and suggests that GSH might be used to treat certain immune disorders.

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Mediating role of p38 in complement-elicited cell death and its inhibition by GSH. (A, B) Effect of P38 in antibody/complement-initiated cell death. MCs preloaded with calcein-AM were exposed to 5% rabbit serum in the presence or absence of p38 inhibitor SB203580 (10 μM) for 6 h. Note the reduced number of PI-positive dead cells in SB203580-treated cells. The intensity of green fluorescence was determined with a fluorescence reader at 490–515 nm. The rate of cell survival relative to untreated control was calculated (Fig. 6B). # P<0.01 vs. control; ** P<0.01 vs. 5% rabbit serum control. (C) Induction of p38 phosphorylation by the rabbit serum and its prevention by GSH. MCs were incubated with 1% rabbit serum in the presence or absence of 2.5 mM GSH. The cellular lysates were subject to Western blot analysis of the level of phospho-p38. β-actin was used as loading control. (D-G) Depletion of intracellular GSH on antibody/complement-induced cell death and P38 activation. MCs were pretreated with the indicated concentration of BSO (D), or 500 μM SSA (E) for 6 h before exposing to rabbit serum for an additional 6 h. (F) MCs were pretreated with 10 mM BSO or 500 μM SSA before exposing to the indicated concentrations of rabbit serum for 15 min. The cellular lysates were subject to Western blot analysis of the level of phospho-p38. (G) Densitometric measurement of band intensity in F. Data shown are mean ±SE (n =4). # P<0.05 vs. zero control; * P<0.05 vs. respective control. H. Effect of GSH-modulating chemicals on cellular deposition of C9. Cells were pretreated with 5 mM BSO and 500 μM SSA for 6 h, followed by reaction with 10 μg/ml Thy-1 plus 10% native human serum for an additional 1 h. The cellular lysates were subjected to Western blot analysis of C9.
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f0030: Mediating role of p38 in complement-elicited cell death and its inhibition by GSH. (A, B) Effect of P38 in antibody/complement-initiated cell death. MCs preloaded with calcein-AM were exposed to 5% rabbit serum in the presence or absence of p38 inhibitor SB203580 (10 μM) for 6 h. Note the reduced number of PI-positive dead cells in SB203580-treated cells. The intensity of green fluorescence was determined with a fluorescence reader at 490–515 nm. The rate of cell survival relative to untreated control was calculated (Fig. 6B). # P<0.01 vs. control; ** P<0.01 vs. 5% rabbit serum control. (C) Induction of p38 phosphorylation by the rabbit serum and its prevention by GSH. MCs were incubated with 1% rabbit serum in the presence or absence of 2.5 mM GSH. The cellular lysates were subject to Western blot analysis of the level of phospho-p38. β-actin was used as loading control. (D-G) Depletion of intracellular GSH on antibody/complement-induced cell death and P38 activation. MCs were pretreated with the indicated concentration of BSO (D), or 500 μM SSA (E) for 6 h before exposing to rabbit serum for an additional 6 h. (F) MCs were pretreated with 10 mM BSO or 500 μM SSA before exposing to the indicated concentrations of rabbit serum for 15 min. The cellular lysates were subject to Western blot analysis of the level of phospho-p38. (G) Densitometric measurement of band intensity in F. Data shown are mean ±SE (n =4). # P<0.05 vs. zero control; * P<0.05 vs. respective control. H. Effect of GSH-modulating chemicals on cellular deposition of C9. Cells were pretreated with 5 mM BSO and 500 μM SSA for 6 h, followed by reaction with 10 μg/ml Thy-1 plus 10% native human serum for an additional 1 h. The cellular lysates were subjected to Western blot analysis of C9.

Mentions: Apart from antibody and complement, the cellular defense against C5b-9-induced cell injury is another determinant of cell destiny. Previous studies have shown that the cell lysis induced by complement was partially mediated by induction of oxidative stress and activation of stress-sensitive kinases [25]. Under our experimental settings, we have demonstrated a partially mediating role of P38 [18]. As GSH is one of the major antioxidants against oxidative stress, we speculated that GSH might also work through modulation of the oxidative sensitive kinase. To test this hypothesis, we first confirmed our previous findings that inhibition of P38 with a specific chemical inhibitor SB2203580 significantly attenuated the rabbit serum-induced cell death (Fig. 6, A and B). Intriguingly, GSH also suppressed P38 activation under both basal and serum-stimulated conditions. To further establish the role of GSH, we used BSO (an inhibitor of gamma-glutamylcysteine synthetase) [26], and SSA (an inhibitor of the XCT transporter) [27] to deplete intracellular GSH. As shown in Figs. 6D to 6G, depletion of GSH with these agents promoted P38 activation and sensitized cells to the antibody/complement-initiated cell death. Of note, depletion of intracellular GSH with BSO and SSA did not greatly affect the cellular deposition of C9 (Fig. H), suggesting that these chemicals did not affect the formation of the membrane attack complex. Collectively, these observations indicate that GSH also exerts a protective role against antibody- and complement-initiated cell injury via modulation of oxidative sensitive P38.


Glutathione inhibits antibody and complement-mediated immunologic cell injury via multiple mechanisms
Mediating role of p38 in complement-elicited cell death and its inhibition by GSH. (A, B) Effect of P38 in antibody/complement-initiated cell death. MCs preloaded with calcein-AM were exposed to 5% rabbit serum in the presence or absence of p38 inhibitor SB203580 (10 μM) for 6 h. Note the reduced number of PI-positive dead cells in SB203580-treated cells. The intensity of green fluorescence was determined with a fluorescence reader at 490–515 nm. The rate of cell survival relative to untreated control was calculated (Fig. 6B). # P<0.01 vs. control; ** P<0.01 vs. 5% rabbit serum control. (C) Induction of p38 phosphorylation by the rabbit serum and its prevention by GSH. MCs were incubated with 1% rabbit serum in the presence or absence of 2.5 mM GSH. The cellular lysates were subject to Western blot analysis of the level of phospho-p38. β-actin was used as loading control. (D-G) Depletion of intracellular GSH on antibody/complement-induced cell death and P38 activation. MCs were pretreated with the indicated concentration of BSO (D), or 500 μM SSA (E) for 6 h before exposing to rabbit serum for an additional 6 h. (F) MCs were pretreated with 10 mM BSO or 500 μM SSA before exposing to the indicated concentrations of rabbit serum for 15 min. The cellular lysates were subject to Western blot analysis of the level of phospho-p38. (G) Densitometric measurement of band intensity in F. Data shown are mean ±SE (n =4). # P<0.05 vs. zero control; * P<0.05 vs. respective control. H. Effect of GSH-modulating chemicals on cellular deposition of C9. Cells were pretreated with 5 mM BSO and 500 μM SSA for 6 h, followed by reaction with 10 μg/ml Thy-1 plus 10% native human serum for an additional 1 h. The cellular lysates were subjected to Western blot analysis of C9.
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f0030: Mediating role of p38 in complement-elicited cell death and its inhibition by GSH. (A, B) Effect of P38 in antibody/complement-initiated cell death. MCs preloaded with calcein-AM were exposed to 5% rabbit serum in the presence or absence of p38 inhibitor SB203580 (10 μM) for 6 h. Note the reduced number of PI-positive dead cells in SB203580-treated cells. The intensity of green fluorescence was determined with a fluorescence reader at 490–515 nm. The rate of cell survival relative to untreated control was calculated (Fig. 6B). # P<0.01 vs. control; ** P<0.01 vs. 5% rabbit serum control. (C) Induction of p38 phosphorylation by the rabbit serum and its prevention by GSH. MCs were incubated with 1% rabbit serum in the presence or absence of 2.5 mM GSH. The cellular lysates were subject to Western blot analysis of the level of phospho-p38. β-actin was used as loading control. (D-G) Depletion of intracellular GSH on antibody/complement-induced cell death and P38 activation. MCs were pretreated with the indicated concentration of BSO (D), or 500 μM SSA (E) for 6 h before exposing to rabbit serum for an additional 6 h. (F) MCs were pretreated with 10 mM BSO or 500 μM SSA before exposing to the indicated concentrations of rabbit serum for 15 min. The cellular lysates were subject to Western blot analysis of the level of phospho-p38. (G) Densitometric measurement of band intensity in F. Data shown are mean ±SE (n =4). # P<0.05 vs. zero control; * P<0.05 vs. respective control. H. Effect of GSH-modulating chemicals on cellular deposition of C9. Cells were pretreated with 5 mM BSO and 500 μM SSA for 6 h, followed by reaction with 10 μg/ml Thy-1 plus 10% native human serum for an additional 1 h. The cellular lysates were subjected to Western blot analysis of C9.
Mentions: Apart from antibody and complement, the cellular defense against C5b-9-induced cell injury is another determinant of cell destiny. Previous studies have shown that the cell lysis induced by complement was partially mediated by induction of oxidative stress and activation of stress-sensitive kinases [25]. Under our experimental settings, we have demonstrated a partially mediating role of P38 [18]. As GSH is one of the major antioxidants against oxidative stress, we speculated that GSH might also work through modulation of the oxidative sensitive kinase. To test this hypothesis, we first confirmed our previous findings that inhibition of P38 with a specific chemical inhibitor SB2203580 significantly attenuated the rabbit serum-induced cell death (Fig. 6, A and B). Intriguingly, GSH also suppressed P38 activation under both basal and serum-stimulated conditions. To further establish the role of GSH, we used BSO (an inhibitor of gamma-glutamylcysteine synthetase) [26], and SSA (an inhibitor of the XCT transporter) [27] to deplete intracellular GSH. As shown in Figs. 6D to 6G, depletion of GSH with these agents promoted P38 activation and sensitized cells to the antibody/complement-initiated cell death. Of note, depletion of intracellular GSH with BSO and SSA did not greatly affect the cellular deposition of C9 (Fig. H), suggesting that these chemicals did not affect the formation of the membrane attack complex. Collectively, these observations indicate that GSH also exerts a protective role against antibody- and complement-initiated cell injury via modulation of oxidative sensitive P38.

View Article: PubMed Central - PubMed

ABSTRACT

Antioxidant glutathione (GSH) plays an important role in the regulation of immunity. However, little is known about its effects on humoral immunity, especially its action on effector molecules like antibody and complement. Given that these molecules contain abundant disulfide bonds, we speculated that GSH might influence the action of these proteins via its thiol function. Using a model of a glomerular mesangial cell (MC) lysis induced by antibodies plus complement, we addressed this hypothesis. Exposure of rat MCs to anti-Thy-1 antibody plus complement or anti-MC rabbit serum caused a complement-dependent cell lysis, which was completely blocked by GSH. Moreover, GSH potently prevented the antibody-mediated agglutination of red blood cells and aggregation of antibody-sensitized microspheres. Further analysis revealed that GSH inhibited antibody binding to antigens and promoted the conversion of the antibodies to its reduced forms. GSH also potently inhibited the formation and deposition of C5b-9 in MCs and suppressed both the classic and alternative complement activation pathway. Lastly, GSH attenuated P38 activation, an oxidative sensitive kinase that partially mediated the antibody- and complement-dependent MC lysis. Depletion of GSH via inhibiting gamma-glutamylcysteine synthetase or xCT transporter augmented P38 activation and sensitized MCs to the cell lysis. Collectively, our results indicate that GSH protects cells from immunological cell damage via mechanisms involving inhibition of antibody binding to the antigens, suppression of complement activation and augmentation of cellular defense mechanism. Our study provides novel mechanistic insights into the actions of GSH in the regulation of immune responses and suggests that GSH might be used to treat certain immune disorders.

No MeSH data available.


Related in: MedlinePlus