To address the effect of oxidative stress on DC activation, we treated C57BL/6 BMDC with 5, 50 or 500 μm H2O2 for 60 min as described previously (17,18). Flow cytometric analysis of apoptosis and cell death as assessed by 7-AAD and Annexin V staining showed no significant decrease in cell viability (<4%) following H2O2 treatment (Figure 1A). DC were also analyzed for their expression of CCR7, CD40, CD80, CD86, class II, TLR2 and TLR4 to determine their maturation status and immunogenic profile. While no substantial increase was found in the expression of these molecules, CCR7 was found to be slightly increased upon treatment with 500 μM H2O2 (5.5 ± 0.22% and 6.51 ± 0.22% for control and 500 μM H2O2-treated, respectively; p = 0.03, data not shown). In order to determine whether upregulation of surface expression occurs at a later time point, cells were incubated for 24 h in medium following treatment and were then subjected to flow cytometric analysis. At 24 h, H2O2 treatment was not associated with any significant increase in cell death (Figure 1B). As shown in Figures 1B and C, CCR7, CD80, CD86, class II, and TLR4 were found to be significantly increased in response to 500 μM H2O2 treatment, demonstrating that free radical injury results in increased DC immunogenicity (n = 3, p < 0.05). Treated DC were also evaluated for their ability to stimulate allogeneic T cell proliferation, and H2O2-treated BMDC were found to stimulate BALB/c T cells more potently than untreated DC as measured by tritium uptake, an effect which was inhibited with prior MCI-186 treatment (Figure 1D, n = 3, p < 0.0001 for all doses of H2O2 alone). While isolation of DC from naïve hearts is problematic due to the low number of constitutive heart tissue DC, we and others have recently shown that implantation of Flt3L hybridoma cells (which produce Flt3L) increases the DC population in the heart as well as in lymphoid tissues, and DC obtained do not exhibit any phenotypical or functional alterations (13,19). To demonstrate the effect of ischemic insult in the specific context of heart tissue DC, isolated naïve C57BL/6 heart DC of mice pretreated with the Flt3L hybridoma were treated with H2O2 as above and were cocultured with allogeneic T cells. Indeed, H2O2-treated heart DC exhibited greater immunogenicity in comparison to control heart DC, as evidenced by augmented proliferation of allogeneic T cells in response to coculture with H2O2-treated DC (Figure 1E, n = 4, p < 0.0001).
Figure 1. Oxidative stress and upregulation of DC immunogenicity. (A) 60 min H202 treatment has no effect on DC death or apoptosis, as assessed by Annexin V and 7-AAD staining (n = 3–5, p = ns). (B) Cell viability and surface expression of DC markers indicative of enhanced immunogenicity were examined following 500 μM H2O2 treatment and incubation in medium for 24 h. No difference in cell viability between treated and control groups was observed, while DC expression of CCR7, CD80, CD86, class II and TLR4 was found to be significantly enhanced. (C) CCR7, CD80, CD86, class II and TLR4 expression increased significantly in response to H2O2 treatment (*p < 0.05, n = 3). (D) In an MLR, H202-treated BMDC stimulated allogeneic T cells to a greater degree than untreated DC in a dose-dependent manner, an effect inhibited by pretreatment with 10 μm MCI-186 (n = 3, *p < 0.0001). (E) 5 μm H202 treatment was also shown to augment the immunogenicity of isolated heart DC (n = 3, p < 0.0001), in which 1 × 104 C57BL/6 heart DC were incubated with 1 × 105 BALB/c CD4+ cells.
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