Limits...
Protective effect of 14-3-3 antibodies on stressed neuroretinal cells via the mitochondrial apoptosis pathway.

Bell K, Wilding C, Funke S, Pfeiffer N, Grus FH - BMC Ophthalmol (2015)

Bottom Line: We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway.The changed autoantibodies belong to the natural autoimmunity.We conclude that changes in the natural autoimmunity of patients with glaucoma can negatively impact regulatory functions.

View Article: PubMed Central - PubMed

Affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany. kbell@eye-research.org.

ABSTRACT

Background: Previous studies demonstrate changes of autoantibody concentrations against retinal and optic nerve head antigens in the serum of glaucoma patients in comparison to healthy persons. These antibodies belong to the natural autoimmunity. Previous studies showed up regulated, but also significantly down-regulated autoantibody levels. These antibodies have the ability to influence protein profiles of neuroretinal cells and possibly hold neuroprotective potential, as we have been able to demonstrate before. Aim of this study was to analyse the serum and antibody effect of glaucoma patients on neuroretinal cells in more detail and also determine the impact of antibodies found down-regulated in glaucoma patients on the pathogenesis of the neurodegenerative disease glaucoma.

Methods: Neuroretinal cells (RGC-5) were incubated with serum either from glaucoma patients or healthy controls for 24 h. Mass spectrometric analysis was performed after cell lysis. Furthermore the neuroretinal cells were preincubated with different and concentrations of 14-3-3 antibodies (0.005, 0.1, 0.5, 1, 5 and 10 μg/ml) and then stressed with H2O2, staurosporine or glutamate. Viability tests were performed with crystal violet and ROS tests with DCFH-DA. Antibody location in the cell after antibody incubation was performed with immunocytochemical methods. Additionally mass spectrometric analysis was performed with the cells after antibody incubation.

Results: Protein expression analysis with Maldi-Orbitrap MS showed changes in the expression level of regulatory proteins in cells incubated with glaucoma serum, e.g. an up-regulation of 14-3-3 and a down-regulation of Calmodulin. After preincubation of the cells with anti-14-3-3 antibody and stressing the cells, we detected an increase in viability of up to 22 % and a decrease in reactive oxygen species (ROS) of up to 31 %. Proteomic 1 analysis involvement of the mitochondrial apoptosis pathway in this protective effect and immunohistochemical analysis showed an antibody uptake in the cells.

Conclusion: We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway. Furthermore we detected a protective potential of antibodies down-regulated in glaucoma patients. The changed autoantibodies belong to the natural autoimmunity. We conclude that changes in the natural autoimmunity of patients with glaucoma can negatively impact regulatory functions.

Show MeSH

Related in: MedlinePlus

Protective effects of 14-3-3 antibody on RGC-5 stressed with H2O2. RGC-5 cells were preincubated with different concentration of 14-3-3 antibodies for 3 h and additionally stressed with 50 μM H2O2 for 1 h. This amount of H2O2 was used, as we did not want to produce a loss of viability through H2O2 our aim was to just increase ROS in the cells. The graph shows the ROS levels in % in the cells (blue line) as well as the viability (red line) of the cells after pre-incubation with different 14-3-3 antibody concentrations and stress with 50 μM H2O2 for 1 h. N in each experimental group is 4. Significantly decreased ROS levels (−31 %) were measured in the cells incubated with 10 μg/ml 14-3-3 antibodies. ROS-production was measured using DCFH-DA and expressed as percent of the control cells, which were only treated with H2O2. Significantly increased viability (+22 %) was measured for the cells incubated with 10 μg/ml 14-3-3 antibodies. Viability was measured using crystal violet and expressed as percent of the control cells additionally incubated with H2O2 (* = p < 0.05; **p < 0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4482181&req=5

Fig3: Protective effects of 14-3-3 antibody on RGC-5 stressed with H2O2. RGC-5 cells were preincubated with different concentration of 14-3-3 antibodies for 3 h and additionally stressed with 50 μM H2O2 for 1 h. This amount of H2O2 was used, as we did not want to produce a loss of viability through H2O2 our aim was to just increase ROS in the cells. The graph shows the ROS levels in % in the cells (blue line) as well as the viability (red line) of the cells after pre-incubation with different 14-3-3 antibody concentrations and stress with 50 μM H2O2 for 1 h. N in each experimental group is 4. Significantly decreased ROS levels (−31 %) were measured in the cells incubated with 10 μg/ml 14-3-3 antibodies. ROS-production was measured using DCFH-DA and expressed as percent of the control cells, which were only treated with H2O2. Significantly increased viability (+22 %) was measured for the cells incubated with 10 μg/ml 14-3-3 antibodies. Viability was measured using crystal violet and expressed as percent of the control cells additionally incubated with H2O2 (* = p < 0.05; **p < 0.01)

Mentions: An increased cell viability of 22 % (p < 0.01) was measured after preincubation of the cells with 10 μg/ml 14-3-3 ab and additional stress with H2O2 (Fig. 3), as well as a significantly decreased ROS-production of 31 % (p < 0.01) (Fig. 3). We could indicate a significantly increased viability of 12 % (p < 0.01) when incubating the cells with 0.5 μg/ml 14-3-3 ab and of 7 % (p < 0.05) when incubating the cells with 1 μg/ml 14-3-3 ab and stressing with staurosporine (Fig. 4a).Fig. 3


Protective effect of 14-3-3 antibodies on stressed neuroretinal cells via the mitochondrial apoptosis pathway.

Bell K, Wilding C, Funke S, Pfeiffer N, Grus FH - BMC Ophthalmol (2015)

Protective effects of 14-3-3 antibody on RGC-5 stressed with H2O2. RGC-5 cells were preincubated with different concentration of 14-3-3 antibodies for 3 h and additionally stressed with 50 μM H2O2 for 1 h. This amount of H2O2 was used, as we did not want to produce a loss of viability through H2O2 our aim was to just increase ROS in the cells. The graph shows the ROS levels in % in the cells (blue line) as well as the viability (red line) of the cells after pre-incubation with different 14-3-3 antibody concentrations and stress with 50 μM H2O2 for 1 h. N in each experimental group is 4. Significantly decreased ROS levels (−31 %) were measured in the cells incubated with 10 μg/ml 14-3-3 antibodies. ROS-production was measured using DCFH-DA and expressed as percent of the control cells, which were only treated with H2O2. Significantly increased viability (+22 %) was measured for the cells incubated with 10 μg/ml 14-3-3 antibodies. Viability was measured using crystal violet and expressed as percent of the control cells additionally incubated with H2O2 (* = p < 0.05; **p < 0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482181&req=5

Fig3: Protective effects of 14-3-3 antibody on RGC-5 stressed with H2O2. RGC-5 cells were preincubated with different concentration of 14-3-3 antibodies for 3 h and additionally stressed with 50 μM H2O2 for 1 h. This amount of H2O2 was used, as we did not want to produce a loss of viability through H2O2 our aim was to just increase ROS in the cells. The graph shows the ROS levels in % in the cells (blue line) as well as the viability (red line) of the cells after pre-incubation with different 14-3-3 antibody concentrations and stress with 50 μM H2O2 for 1 h. N in each experimental group is 4. Significantly decreased ROS levels (−31 %) were measured in the cells incubated with 10 μg/ml 14-3-3 antibodies. ROS-production was measured using DCFH-DA and expressed as percent of the control cells, which were only treated with H2O2. Significantly increased viability (+22 %) was measured for the cells incubated with 10 μg/ml 14-3-3 antibodies. Viability was measured using crystal violet and expressed as percent of the control cells additionally incubated with H2O2 (* = p < 0.05; **p < 0.01)
Mentions: An increased cell viability of 22 % (p < 0.01) was measured after preincubation of the cells with 10 μg/ml 14-3-3 ab and additional stress with H2O2 (Fig. 3), as well as a significantly decreased ROS-production of 31 % (p < 0.01) (Fig. 3). We could indicate a significantly increased viability of 12 % (p < 0.01) when incubating the cells with 0.5 μg/ml 14-3-3 ab and of 7 % (p < 0.05) when incubating the cells with 1 μg/ml 14-3-3 ab and stressing with staurosporine (Fig. 4a).Fig. 3

Bottom Line: We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway.The changed autoantibodies belong to the natural autoimmunity.We conclude that changes in the natural autoimmunity of patients with glaucoma can negatively impact regulatory functions.

View Article: PubMed Central - PubMed

Affiliation: Experimental Ophthalmology, Department of Ophthalmology, University Medical center of the Johannes Gutenberg University, Langenbeckstraße 1, 55131, Mainz, Germany. kbell@eye-research.org.

ABSTRACT

Background: Previous studies demonstrate changes of autoantibody concentrations against retinal and optic nerve head antigens in the serum of glaucoma patients in comparison to healthy persons. These antibodies belong to the natural autoimmunity. Previous studies showed up regulated, but also significantly down-regulated autoantibody levels. These antibodies have the ability to influence protein profiles of neuroretinal cells and possibly hold neuroprotective potential, as we have been able to demonstrate before. Aim of this study was to analyse the serum and antibody effect of glaucoma patients on neuroretinal cells in more detail and also determine the impact of antibodies found down-regulated in glaucoma patients on the pathogenesis of the neurodegenerative disease glaucoma.

Methods: Neuroretinal cells (RGC-5) were incubated with serum either from glaucoma patients or healthy controls for 24 h. Mass spectrometric analysis was performed after cell lysis. Furthermore the neuroretinal cells were preincubated with different and concentrations of 14-3-3 antibodies (0.005, 0.1, 0.5, 1, 5 and 10 μg/ml) and then stressed with H2O2, staurosporine or glutamate. Viability tests were performed with crystal violet and ROS tests with DCFH-DA. Antibody location in the cell after antibody incubation was performed with immunocytochemical methods. Additionally mass spectrometric analysis was performed with the cells after antibody incubation.

Results: Protein expression analysis with Maldi-Orbitrap MS showed changes in the expression level of regulatory proteins in cells incubated with glaucoma serum, e.g. an up-regulation of 14-3-3 and a down-regulation of Calmodulin. After preincubation of the cells with anti-14-3-3 antibody and stressing the cells, we detected an increase in viability of up to 22 % and a decrease in reactive oxygen species (ROS) of up to 31 %. Proteomic 1 analysis involvement of the mitochondrial apoptosis pathway in this protective effect and immunohistochemical analysis showed an antibody uptake in the cells.

Conclusion: We found significant effects of serum antibodies on proteins of neuroretinal cells especially of the mitochondrial apoptosis pathway. Furthermore we detected a protective potential of antibodies down-regulated in glaucoma patients. The changed autoantibodies belong to the natural autoimmunity. We conclude that changes in the natural autoimmunity of patients with glaucoma can negatively impact regulatory functions.

Show MeSH
Related in: MedlinePlus