Autoantibodies to the C-terminal subunit of RLIP76 induce oxidative stress and endothelial cell apoptosis in immune-mediated vascular diseases and atherosclerosis

Abstract

Although detection of autoantibodies in the peripheral blood from patients with immune-mediated endothelial dysfunctions has so far failed to provide tools of diagnostic or pathogenetic value, putative bioindicators include anti-endothelial cell antibodies, a heterogeneous family of antibodies that react with autoantigens expressed by endothelial cells. In this study, to identify endothelial autoantigens involved in the autoimmune processes causing endothelial damage, we screened a human microvascular endothelial cell cDNA library with sera from patients with Behçet's disease. We identified antibodies to the C-terminus of Ral binding protein1 (RLIP76), a protein that catalyzes the ATP-dependent transport of glutathione (GSH) conjugates including GSH-4-hydroxy-t-2,3-nonenal, in the serum of a significant percentage of patients with various diseases characterized by immune-mediated endothelial dysfunction, including Behçet disease, systemic sclerosis, systemic lupus erythematosus and carotid atherosclerosis. These autoantibodies increased intracellular levels of 4-hydroxy-t-2,3-nonenal, decreased levels of GSH and activated C-Jun NH2 Kinase signaling (JNK), thus inducing oxidative stress-mediated endothelial cell apoptosis. The dietary antioxidant alpha-tocopherol counteracted endothelial cell demise. These findings suggest that autoantibodies to RLIP76 play a pathogenetic role in immune-mediated vascular diseases and represent a valuable peripheral blood bioindicator of atherosclerosis and immune-mediated vascular diseases.

Figure 1

The amino acid sequence and the immunochemical characterization of the N- and C-terminal regions of RLIP76. (A) The nucleotide sequence of the cloned cDNA (GenBank accession number NM 006788) was divided in 2 subunits by PCR with specific primers. The cDNA subunits were cloned in an expression vector and the N- and C-terminal regions of the protein were expressed and purified. The overlapped amino acids were squared. (B) The molecular size and the purity of the expressed proteins were confirmed by 10% SDS-PAGE stained by Coomassie blue (lane 1, N-terminal region; lane 2, C-terminal region) and serum immunoreactivity was analyzed by immunoblotting (lanes 3-6, C-terminal region; lanes 7-10, N-terminal region). Lanes 3,7: monoclonal antibody specific to 6-histidine tail; lanes 4,8: serum pool from the 2 patients with Behçet disease (BD) used in screening the library; lane 5,9: representative serum from a healthy subject; lanes 6,10: control without serum.

Figure 2

Anti-RLIP76 C-ter antibodies in patients and healthy controls. (A) Box-whisker plot of anti-RLIP76 C-ter IgG in patients with BD, SLE, SS, infectious mononucleosis, and from sex- and age-matched healthy donors (NHS). (B) Box-whisker plot of anti-RLIP76 C-ter IgG in patients with carotid atherosclerosis and from sex- and age-matched healthy donors (NHS). Median, quartiles, range, and possibly extreme values are indicated. The broken line represents the cutoff (mean + 2 SD for the healthy controls). Outliers are represented as +.

Figure 3

RLIP76 expression and localization.(A) EAhy926 were immunoprecipitated with mouse polyclonal anti-RLIP76 C-ter antibodies. The immunoprecipitates were analyzed by Western blotting, using anti–human RLIP76 C-ter antibodies. Bound antibodies were visualized with HRP-conjugated anti–human IgG and immunoreactivity was assessed by ECL. Virtually no reactivity was found with immunoprecipitates obtained using non RLIP76-specific IgG (irrelevant). (B) RLIP76 expression in vascular tissue was detected by immunohistochemistry on tissue arrays with histologic sections from normal vascular human tissue incubated with mouse anti-RLIP76 C-ter antibodies. Intense immunoreactivity was observed in vascular endothelium. (C) Immunofluorescence analysis of RLIP76 distribution in EAhy926 untreated cells (left panel) or treated with 30 μM H₂O₂ 30 minutes (right panel). Original magnification 500×, objective 100×, numeric aperture 1.4. (D,E) Flow cytometric analysis after surface staining of EAhy926 cells with antibodies to the RLIP76 C-terminus. (D) Results obtained in a representative experiment. Full light-gray histogram: untreated control cells; red histogram: H₂O₂-treated cells incubated for 30 minutes with fresh medium; black histogram: H₂O₂-treated cells incubated for 6 hours with fresh medium; blue histogram: H₂O₂-treated cells incubated for 24 hours with fresh medium. (E) Time-course evaluation of RLIP76 expression in untreated cells and in cells treated with H₂O₂ and then incubated for 30 minutes, 6 hours, and 24 hours in fresh medium (mean ± SD of the results obtained from 3 different experiments). *P < .01 by Student t test.

Figure 4

Intracellular 4-HNE and GSH levels in EAhy926 endothelial cells. (A,B) Immunofluorescence analysis of the formation of 4-HNE adducts with histidine in EAhy926 cells stained with a 4-HNE specific antibody and counterstained with Hoechst dye to reveal nuclei. Panel A left, untreated cells; panel A right, H₂O₂-treated cells; panel B left, H₂O₂-treated cells incubated for 24 hours with fresh medium; panel B right, H₂O₂-treated cells incubated for 24 hours with medium containing anti-RLIP76 C-ter antibodies. Magnification 1500×, objective 100×, numeric aperture 1.4. (C) Quantitative analysis of 4-HNE adducts by flow cytometry in a representative experiment. Gray histogram, untreated control cells; red histogram, H₂O₂-treated cells; black histogram, H₂O₂-treated cells incubated for 24 hours with fresh medium; blue histogram, H₂O₂-treated cells incubated for 24 hours with medium containing anti-RLIP76 C-ter antibodies. Numbers represent median values of fluorescence intensity. (D) Quantitative time-course evaluation of 4-HNE intracellular content in untreated control cells; in cells incubated for 30 minutes, 6 hours, and 24 hours with anti-RLIP76 C-ter antibodies; in cells treated with H₂O₂; and in cells treated with H₂O₂ and then incubated for an additional 30 minutes, 6 hours, and 24 hours in fresh medium or in medium containing anti-RLIP76 C-ter antibodies. Statistical analysis performed by Student t test indicated P < .01 for: control untreated cells versus cells incubated with anti-RLIP76 C-ter antibodies for 30 minutes, 6 hours, and 24 hours; untreated cells versus H₂O₂-treated cells incubated for 30 minutes and 6 hours in fresh medium; H₂O₂-treated cells incubated in fresh medium versus H₂O₂-treated cells incubated for 6 hours and 24 hours with medium containing anti-RLIP76 C-ter antibodies. (E) Quantitative time-course evaluation of GSH intracellular content. Statistical analysis performed by Student t test indicated P < .01 for: untreated cells versus H₂O₂-treated cells incubated for 30 minutes in fresh medium; untreated cells versus cells incubated for 30 minutes, 6 hours, and 24 hours with anti-RLIP76 C-ter antibodies; H₂O₂-treated cells incubated in fresh medium versus H₂O₂-treated cells incubated for 6 hours and 24 hours with medium containing anti-RLIP76 C-ter antibodies. Data reported in panels D and E are the mean plus or minus SD of the results obtained from 3 different experiments.

Figure 5

Induction of apoptosis by anti-RLIP76 C-ter antibodies in EAhy926 endothelial cells.(A,C) Flow cytometric analysis after double staining with annexin V/propidium iodide of untreated control cells; cells incubated for 30 minutes, 6 hours, and 24 hours with anti-RLIP76 C-ter antibodies; and in cells treated with H₂O₂ and then incubated additional 30 minutes, 6 hours, and 24 hours in fresh medium or with medium containing anti-RLIP76 C-ter antibodies. (B,D) Cells were also pretreated with α-TCPH. (A,B) Results obtained from 3 independent experiments are reported as mean plus or minus SD. (C,D) Dot plots from a representative experiment. Numbers represent the percentage of annexin V single positive (early apoptosis, bottom right quadrant) or annexin V/PI double positive cells (late apoptosis, bottom right quadrant). Statistical analysis performed by Student t test indicated P < .01 for: control untreated cells versus H₂O₂-treated cells; control untreated cells versus cells incubated for 30 minutes, 6 hours, and 24 hours with anti-RLIP76 C-ter antibodies; and cells treated with H₂O₂ and then incubated in fresh medium versus cells treated with H₂O₂ and then incubated for 6 hours and 24 hours with medium containing anti-RLIP76 C-ter antibodies. P < .01 for any treatment (panel A) versus the same treatment performed after α-TCPH preincubation (panel B).

Figure 6

Apoptotic pathway induced by anti-RLIP76 C-ter in EAhy926 cell line. (A) Flow cytometric data were obtained in the absence (first row) or in the presence (second row) of 30 μM pan-caspase inhibitor zVAD. The numbers in each panel refer to the percentage of cells containing caspase 3 in its active form. Results obtained in a representative experiment are reported. (B,C) Graphs showing the mean plus or minus SD of the percentages of cells with the active form of caspase 3 obtained from 3 different experiments done without (panel B) or with (panel C) zVAD. Statistical analyses indicate a significant (P < .01) decrease in caspase 3 activity in cells pretreated with zVAD before anti-RLIP76 C-ter antibody exposure. (D) Quantitative flow cytometric analysis investigating the JNK activation state 6 hours after the various treatments with a polyclonal antibody able to identify JNK (pT183/pY185). The numbers in each panel refer to the percentage of cells containing JNK in its active form. Results obtained in a representative experiment are reported. (E) Time-course analysis of the activation state of JNK. Statistical analyses of the results obtained from 3 independent experiments (reported as mean ± SD) indicate a significant difference (P < .01) between cells treated with RLIP76 C-ter antibodies, alone or in combination with H₂O₂, and both untreated and H₂O₂-treated cells at any time point considered.

Figure 7

Effects of anti-RLIP76 C-ter antibodies on human microvascular primary cells. (A) Flow cytometric analysis of HMVEC-L cells, either untreated or H₂O₂-treated cells, after surface staining with antibodies to RLIP76. Mean plus or minus SD of the results obtained from 3 different experiments. *P < .01 by Student t test. (B,C) Quantitative flow cytometric analysis of 4-HNE adducts and GSH intracellular content in: untreated control cells, cells treated with H₂O₂ for 30 minutes, cells incubated with anti-RLIP76 C-ter antibodies for 24 hours, and cells treated with H₂O₂ for 30 minutes and then incubated for an additional 24 hours in medium containing anti-RLIP76 C-ter antibodies. Data reported in panels B and C are the mean plus or minus SD of the results obtained from 3 different experiments. Student t test indicated P < .01 for: control untreated cells versus H₂O₂-treated cells; untreated cells versus cells incubated with anti-RLIP76 C-ter antibodies for 24 hours; and untreated cells versus H₂O₂-treated cells incubated for 24 hours with medium containing anti-RLIP76 C-ter antibodies. (D) Dot plots from a representative experiment performed 48 hours after different treatments. Numbers represent the percentage of annexin V single positive (early apoptosis, bottom right quadrant) or annexin V/PI double positive cells (late apoptosis, bottom right quadrant). (E) Flow cytometric analysis of apoptosis after double staining with annexin V/propidium iodide of untreated control cells; cells treated with H₂O₂ and then incubated at different time points with fresh medium or with medium containing anti-RLIP76 C-ter antibodies. Cells were also treated for the same times with anti-RLIP76 C-ter antibodies given alone. Results obtained from 3 independent experiments are reported as mean plus or minus SD. Student t test indicated P < .01 for: control untreated cells versus cells incubated for 24 and 48 hours with anti-RLIP76 C-ter antibodies, and cells treated with H₂O₂ and then incubated in fresh medium versus cells treated with H₂O₂ and then incubated for 24 and 48 hours with medium containing anti-RLIP76 C-ter antibodies. (F) Quantitative flow cytometric analysis of apoptosis in cells treated with anti-RLIP76 C-ter antibodies for 24 and 48 hours pretreated or not with zVAD or α-TCPH as indicated in “Methods.” As control, cells were also treated at the same time points with zVAD or α-TCPH given alone. Results obtained from 3 independent experiments are reported as mean plus or minus SD. (G) Quantitative flow cytometric analysis of the JNK activation state obtained with a polyclonal antibody specific for active form of JNK (pT183/pY185) in cells treated with anti-RLIP76 C-ter antibodies in the presence or absence of α-TCPH. * represents P < .01 by Student t test.