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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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GSH suppresses the rabbit serum-induced MC cell lysis. (A) Calcein-AM/PI staining of rabbit serum-treated cells. Cells were treated with the indicated concentrations of rabbit serum in the presence or absence of 3 mM GSH for 6 h. The viability of cells was determined using calcein (green) and PI (red) staining. (B, C) The percentage of calcein- and PI-positive cells in A. The number of calcein-positive green and PI positive red cells was counted and expressed as percentage of the total cells. Data shown are mean ±SE (n =4). ## P<0.01 vs. zero control; ** P<0.01 vs. respective control. (D) Determination of cell viability using WST assay. Cells were treated the same as above. Cell viability was evaluated by WST assay. Data shown are mean ±SE (n =4). ## P<0.01 vs. zero control; ** P<0.01 vs. respective control.
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f0010: GSH suppresses the rabbit serum-induced MC cell lysis. (A) Calcein-AM/PI staining of rabbit serum-treated cells. Cells were treated with the indicated concentrations of rabbit serum in the presence or absence of 3 mM GSH for 6 h. The viability of cells was determined using calcein (green) and PI (red) staining. (B, C) The percentage of calcein- and PI-positive cells in A. The number of calcein-positive green and PI positive red cells was counted and expressed as percentage of the total cells. Data shown are mean ±SE (n =4). ## P<0.01 vs. zero control; ** P<0.01 vs. respective control. (D) Determination of cell viability using WST assay. Cells were treated the same as above. Cell viability was evaluated by WST assay. Data shown are mean ±SE (n =4). ## P<0.01 vs. zero control; ** P<0.01 vs. respective control.

Mentions: To determine the influence of GSH on antibody/complement-initiated cell injury, we determined the extent of cell death in the presence or absence of GSH. Fig. 2 shows that MC injury induced by the rabbit serum was completely blocked by GSH. GSH effectively prevented the appearance of PI-positive dead cells. Consistently, it blocked the loss of calcein-positive living cells (Fig. 2, A-C). The protective effect of GSH was also confirmed by WST assay. GSH significantly prevented the loss of cell viability caused by the rabbit serum (Fig. 2D). These results indicate that GSH potently prevents antibody/complement-elicited cell injury.


Glutathione inhibits antibody and complement-mediated immunologic cell injury via multiple mechanisms
GSH suppresses the rabbit serum-induced MC cell lysis. (A) Calcein-AM/PI staining of rabbit serum-treated cells. Cells were treated with the indicated concentrations of rabbit serum in the presence or absence of 3 mM GSH for 6 h. The viability of cells was determined using calcein (green) and PI (red) staining. (B, C) The percentage of calcein- and PI-positive cells in A. The number of calcein-positive green and PI positive red cells was counted and expressed as percentage of the total cells. Data shown are mean ±SE (n =4). ## P<0.01 vs. zero control; ** P<0.01 vs. respective control. (D) Determination of cell viability using WST assay. Cells were treated the same as above. Cell viability was evaluated by WST assay. Data shown are mean ±SE (n =4). ## P<0.01 vs. zero control; ** P<0.01 vs. respective control.
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f0010: GSH suppresses the rabbit serum-induced MC cell lysis. (A) Calcein-AM/PI staining of rabbit serum-treated cells. Cells were treated with the indicated concentrations of rabbit serum in the presence or absence of 3 mM GSH for 6 h. The viability of cells was determined using calcein (green) and PI (red) staining. (B, C) The percentage of calcein- and PI-positive cells in A. The number of calcein-positive green and PI positive red cells was counted and expressed as percentage of the total cells. Data shown are mean ±SE (n =4). ## P<0.01 vs. zero control; ** P<0.01 vs. respective control. (D) Determination of cell viability using WST assay. Cells were treated the same as above. Cell viability was evaluated by WST assay. Data shown are mean ±SE (n =4). ## P<0.01 vs. zero control; ** P<0.01 vs. respective control.
Mentions: To determine the influence of GSH on antibody/complement-initiated cell injury, we determined the extent of cell death in the presence or absence of GSH. Fig. 2 shows that MC injury induced by the rabbit serum was completely blocked by GSH. GSH effectively prevented the appearance of PI-positive dead cells. Consistently, it blocked the loss of calcein-positive living cells (Fig. 2, A-C). The protective effect of GSH was also confirmed by WST assay. GSH significantly prevented the loss of cell viability caused by the rabbit serum (Fig. 2D). These results indicate that GSH potently prevents antibody/complement-elicited 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