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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.

No MeSH data available.


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Induction of rat MC lysis by rabbit serum. (A) Induction of MC lysis by rabbit serum. MCs were exposed to 10 μg/ml Thy-1 antibody in the presence of 5% native and heated-treated rabbit serum for 6 h. Cell morphology was photographed. (B) MCs were exposed to 10 μg/ml Thy-1 antibody in the presence of the indicated concentrations of rabbit serum for 6 h. Cellular supernatants were collected and assayed for LDH activities. Data shown are mean ±SE (n =4). ** P<0.01 vs. zero point control. (C) Calcein-AM/PI staining. Cells were either left untreated or treated with 5% rabbit serum in the absence or presence of 10 μg/ml anti-Thy-1 antibodies for 6 h. The viability of cells was determined using PI (red, dead cells) and calcein-AM (green, living cells) staining. (D) The percentage of PI-positive red cells per field in C. The number of PI-positive cells was counted. The data shown are mean ±SE (n =4). ** P<0.01 vs. zero control.
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f0005: Induction of rat MC lysis by rabbit serum. (A) Induction of MC lysis by rabbit serum. MCs were exposed to 10 μg/ml Thy-1 antibody in the presence of 5% native and heated-treated rabbit serum for 6 h. Cell morphology was photographed. (B) MCs were exposed to 10 μg/ml Thy-1 antibody in the presence of the indicated concentrations of rabbit serum for 6 h. Cellular supernatants were collected and assayed for LDH activities. Data shown are mean ±SE (n =4). ** P<0.01 vs. zero point control. (C) Calcein-AM/PI staining. Cells were either left untreated or treated with 5% rabbit serum in the absence or presence of 10 μg/ml anti-Thy-1 antibodies for 6 h. The viability of cells was determined using PI (red, dead cells) and calcein-AM (green, living cells) staining. (D) The percentage of PI-positive red cells per field in C. The number of PI-positive cells was counted. The data shown are mean ±SE (n =4). ** P<0.01 vs. zero control.

Mentions: We have recently reported that incubation of MCs with a rabbit serum from the Japanese White Strain Rabbit caused a complement-dependent MC lysis due to the presence of several antibodies against MCs [18]. In this study, we have used the same cell model. First, we confirmed our previous findings that incubation of MCs with the rabbit serum indeed caused cell damage, as evidenced by the appearance of cells with disrupted cell bodies (Fig. 1A), increased LDH release (Fig. 1B) and positive staining of PI (Fig. 1, C and D). The effect of the rabbit serum was comparable to that induced an anti-Thy-1 antibody plus complement, a well-reported model for induction of MC lysis [21], [22]. Also, the effect of the serum was completely abolished by treatment of the serum at 56 °C for 30 min, indicative of a mediating role of complement. Given that the serum provided both anti-MC antibodies and complement, and that it potently induced MC lysis in a way similar to Thy-1 antibody [18], we have used this model to evaluate the effects of GSH on antibody-initiated and complement-mediated cell injury.


Glutathione inhibits antibody and complement-mediated immunologic cell injury via multiple mechanisms
Induction of rat MC lysis by rabbit serum. (A) Induction of MC lysis by rabbit serum. MCs were exposed to 10 μg/ml Thy-1 antibody in the presence of 5% native and heated-treated rabbit serum for 6 h. Cell morphology was photographed. (B) MCs were exposed to 10 μg/ml Thy-1 antibody in the presence of the indicated concentrations of rabbit serum for 6 h. Cellular supernatants were collected and assayed for LDH activities. Data shown are mean ±SE (n =4). ** P<0.01 vs. zero point control. (C) Calcein-AM/PI staining. Cells were either left untreated or treated with 5% rabbit serum in the absence or presence of 10 μg/ml anti-Thy-1 antibodies for 6 h. The viability of cells was determined using PI (red, dead cells) and calcein-AM (green, living cells) staining. (D) The percentage of PI-positive red cells per field in C. The number of PI-positive cells was counted. The data shown are mean ±SE (n =4). ** P<0.01 vs. zero control.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5384323&req=5

f0005: Induction of rat MC lysis by rabbit serum. (A) Induction of MC lysis by rabbit serum. MCs were exposed to 10 μg/ml Thy-1 antibody in the presence of 5% native and heated-treated rabbit serum for 6 h. Cell morphology was photographed. (B) MCs were exposed to 10 μg/ml Thy-1 antibody in the presence of the indicated concentrations of rabbit serum for 6 h. Cellular supernatants were collected and assayed for LDH activities. Data shown are mean ±SE (n =4). ** P<0.01 vs. zero point control. (C) Calcein-AM/PI staining. Cells were either left untreated or treated with 5% rabbit serum in the absence or presence of 10 μg/ml anti-Thy-1 antibodies for 6 h. The viability of cells was determined using PI (red, dead cells) and calcein-AM (green, living cells) staining. (D) The percentage of PI-positive red cells per field in C. The number of PI-positive cells was counted. The data shown are mean ±SE (n =4). ** P<0.01 vs. zero control.
Mentions: We have recently reported that incubation of MCs with a rabbit serum from the Japanese White Strain Rabbit caused a complement-dependent MC lysis due to the presence of several antibodies against MCs [18]. In this study, we have used the same cell model. First, we confirmed our previous findings that incubation of MCs with the rabbit serum indeed caused cell damage, as evidenced by the appearance of cells with disrupted cell bodies (Fig. 1A), increased LDH release (Fig. 1B) and positive staining of PI (Fig. 1, C and D). The effect of the rabbit serum was comparable to that induced an anti-Thy-1 antibody plus complement, a well-reported model for induction of MC lysis [21], [22]. Also, the effect of the serum was completely abolished by treatment of the serum at 56 °C for 30 min, indicative of a mediating role of complement. Given that the serum provided both anti-MC antibodies and complement, and that it potently induced MC lysis in a way similar to Thy-1 antibody [18], we have used this model to evaluate the effects of GSH on antibody-initiated and complement-mediated cell injury.

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