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Evidence of a role for both anti-Hsp70 antibody and endothelial surface membrane Hsp70 in atherosclerosis - PubMed

Evidence of a role for both anti-Hsp70 antibody and endothelial surface membrane Hsp70 in atherosclerosis

Xue Leng et al. Cell Stress Chaperones. 2013 Jul.

Abstract

Although previous studies have shown that autoantigens such as Hsps have been implicated by induction of an autoimmune process in the development of atherosclerosis, the exact role of anti-Hsp70 antibody in atherosclerosis is unknown. In the present study, the levels of anti-Hsp70 autoantibodies and oxidized low density lipoprotein (OxLDL) were all significantly increased, and they were strongly correlated in an atherosclerosis model. After the endothelial cells were incubated with 20 μg/mL OxLDL for 12 h at 37 °C and followed by 90 min recovery, Hsp70 positive staining of OxLDL-treated endothelial cells was observed on the cell surface in immunostaining and flow cytometric analysis. This membrane Hsp70 was not from culture supernatant Hsp70 and binding of extracellular Hsp70 but was defined as endothelial surface membrane Hsp70. Furthermore, only in the OxLDL-treated group, but not in the untreated group, (51)Cr-labeled endothelial cells were lysed by anti-Hsp70 antibody (BD091, Ig(AS)) in the presence of either complement or peripheral blood mononuclear cells. Control antibodies, including Ig(Nor), mAb to Hsp70 (SPA-810), and mAbs to Factor VIII, α-actin, and CD3 showed no cytotoxic effects. In conclusion, anti-Hsp70 antibodies could be reacting with the endothelial surface membrane Hsp70 induced by OxLDL and were able to mediate endothelial cytotoxicity. There is a possibility that a humoral immune reaction to endothelial surface membrane Hsp70 may play an important role in the pathogenesis of atherosclerosis.

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Figures

Fig. 1
Fig. 1

Correlation between plasma anti-Hsp70 autoantibodies and OxLDL. The level of plasma OxLDL and anti-Hsp70 autoantibody in rat atherosclerosis on a high-cholesterol diet for 10 weeks. a The pathological changes of rat aortae (HE, ×200). b The level of OxLDL in plasma and c the concentration of anti-Hsp70 autoantibody were measured by ELISA as described in Materials and Methods. d Representative graph of the relationship between Hsp70 IgG autoantibody and OxLDL. e the levels of anti-Hsp70 autoantibody isotypes at 10 weeks. Values are means ± SD from three independent experiments, n = 8, *p < 0.05 compared with the control group

Fig. 2
Fig. 2

Hsp70 is present in membrane fractions depending on OxLDL-treated. RAECs were maintained with OxLDL treated at 37 °C, recovered (37 °C for 90 min in M199 medium), and lysed in TNE buffer containing 1 % Triton X-100. Membrane fractions were isolated as described in “Materials and Methods”. a RAECs were treated with 0-200 μg/mL for 12 h; b RAECs were treated with 20 μg/mL for 3, 6, 12, and 24 h. The presence of Hsp70, Hsc70 within membrane fraction was detected by Western blotting (a, b), whereas the supernatant 51Cr release was determined in a gamma counter (c, d). After OxLDL treatment, cells were washed with medium 199 three times, and 5 μCi 51Cr in 100 μL of medium 199 containing 10 % FCS was added to each well and incubated at 37 °C for 1.5 h. Cell culture supernatant was for radioactivity detection. e RAECs were incubated with 20 μg/mL OxLDL for 12 h, non-OxLDL-treated as control. The presence of Hsp70, Hsc70 in membrane fraction (M) and total cellular extracts (T) were detected by western blotting. Data are mean of three experiments. *Significant difference from controls (p < 0.01)

Fig. 3
Fig. 3

Hsp70 is present on the surface of OxLDL-treated cells. RAECs were treated (20 μg/mL OxLDL treated for 12 h) or not with OxLDL at 37 °C, recovered for 90 min at 37 °C in M199 medium, incubated with Abs against Hsp70 (BD091 (bottom panel, c, d) or SPA-810 (top panel, a, d)) at 1 h at room temperature. After three washes with PBS, the cells were incubated with Ig-FITC conjugate for 30 min, fixed with absolute methanol for 5 min, rinsed, and embedded in n-propylgalat/glycerol. The green fluorescence shows the detection of Hsp70 on the cell surface

Fig. 4
Fig. 4

Dose–response curves for CMCC. Confluent RAECs (0.5 × 106 cells/mL) in 96-well plates were treated (20 μg/mL for 12 h at 37 °C) or not with OxLDL, and recovered for 90 min at 37 °C. After three washes with medium 199, 5 μCi 51Cr in 100 μL of medium 199 containing 10 % FCS was added to each well and incubated at 37 °C for 1.5 h. After two further washes, antibodies (filled square IgAS; filled diamond IgNor; filled star IgGmouse; mAb anti-Hsp70 (filled upright triangle BD091; ex symbol SPA-810); filled circle mAb anti-Factor VIII; vertical line mAb anti-α-actin; and horizontal line mAb anti-CD3) at indicated concentrations in 100 μL of medium were added, and incubated at 37 °C for 7 h in the presence of guinea pig serum as a complement source. After incubation, supernatant radioactivity was determined in a gamma counter. The values are means of three experiments, each performed in triplicate. *p < 0.01 vs SPA-810; #p < 0.01 vs nonpretreatment

Fig. 5
Fig. 5

Dose–response curves for ADCC. The procedures for OxLDL incubated and Ab treatment were the same as described in the legend for Fig. 4. As effectors normal rat PBMC were added to the culture instead of complement. Note increased 51Cr release at higher effector/target cell ratios (a 20 μg/mL OxLDL pretreated rat aortic endothelial cells, b OxLDL-treated and untreated endothelial cells as effector/target was 50/1). Black filled diamond IgAS; gray filled circle IgNor; mAb anti-Hsp70 (filled upright triangle BD091; ex symbol SPA-810); brown filled circle mAb anti-Factor VIII; vertical line mAb anti-α-actin; open circle mAb anti-CD3; red filled diamond IgGmouse. *Significant difference from untreated cells, *p < 0.01 vs SPA-810; #p < 0.01 vs nontreatment

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