Lipid peroxidation-derived aldehyde-protein adducts contribute to trichloroethene-mediated autoimmunity via activation of CD4+ T cells

Abstract

Lipid peroxidation is implicated in the pathogenesis of various autoimmune diseases. Lipid peroxidation-derived aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are highly reactive and bind to proteins, but their role in eliciting an autoimmune response and their contribution to disease pathogenesis remain unclear. To investigate the role of lipid peroxidation in the induction and/or exacerbation of autoimmune response, 6-week-old autoimmune-prone female MRL+/+ mice were treated for 4 weeks with trichloroethene (TCE; 10 mmol/kg, ip, once a week), an environmental contaminant known to induce lipid peroxidation. Sera from TCE-treated mice showed significant levels of antibodies against MDA-and HNE-adducted proteins along with antinuclear antibodies. This suggested that TCE exposure not only caused increased lipid peroxidation, but also accelerated autoimmune responses. Furthermore, stimulation of cultured splenic lymphocytes from both control and TCE-treated mice with MDA-adducted mouse serum albumin (MDA-MSA) or HNE-MSA for 72 h showed significant proliferation of CD4+ T cells in TCE-treated mice as analyzed by flow cytometry. Also, splenic lymphocytes from TCE-treated mice released more IL-2 and IFN-gamma into cultures when stimulated with MDA-MSA or HNE-MSA, suggesting a Th1 cell activation. Thus, our data suggest a role for lipid peroxidation-derived aldehydes in TCE-mediated autoimmune responses and involvement of Th1 cell activation.

Fig. 1

Anti-MDA- and anti-HNE-protein adduct antibodies in the sera of MRL+/+ mice treated with TCE for 4 weeks. The values are means ± SD of eight animals in each group. Net OD values exceeding the negative control values by more than 0.2 were considered positive. The numbers of mice positive for anti-MDA-protein adduct antibodies were 0/8 and 4/8 for control and TCE-treated mice, respectively, whereas numbers of mice positive for anti-HNE-protein adduct antibodies were 0/8 and 3/8 for control and TCE-treated mice, respectively. ^* p < 0.05 vs. controls.

Fig. 2

Serum ANA levels in MRL+/+ mice treated with TCE for 4 weeks. The values are means ± SD of eight animals in each group. Net OD values exceeding the negative control values by more than 0.2 were considered positive. The numbers of mice positive for ANA were 0/8 and 5/8 for control and TCE-treated mice, respectively. ^* p < 0.05 vs. controls.

Fig. 3

Serum levels of IgM, IgG and IgG subclasses in MRL+/+ mice treated with TCE for 4 weeks. The values are means ± SD of eight animals in each group. ^* p < 0.05 vs. controls.

Fig. 4

Proliferation of splenic CD4⁺ T cells from control and TCE-treated MRL+/+ mice. Splenocytes isolated from control and TCE-treated mice were labeled with CFSE, cultured with MDA-MSA or HNE-MSA for 72h, then stained with anti-mouse CD4-PE and analyzed by flow cytometry.

Fig. 5

Quantitation of splenic CD4⁺ T cell proliferation from control and TCE-treated mice. Spenocytes were labeled with CFSE, cultured with MDA-MSA or HNE-MSA, then stained with anti-mouse CD4-PE and analyzed by flow cytometry. Proliferation is expressed as percent of proliferating CD4⁺ cells in the total CD4⁺ T cell population (PE+ cells). The values are means ± SD of eight animals in each group. ^* p < 0.05 vs. controls; ^# p < 0.05 vs. un-stimulated cells; ^** p < 0.05 vs. un-stimulated control cells.

Fig. 6

Release of IL-2 (A) and IFN-γ (B) into splenocyte cultures of control and TCE-treated MRL+/+ mice. Splenocytes were incubated with MSA alone, increasing concentrations of MDA-MSA (M1–M3), HNE-MSA (H1–H3), or anti-mouse CD3 antibody for 72h and the release of IL-2 and IFN-γ into cultures were measured by ELISA. The values are means ± SD of eight animals in each group. US: unstimulated cells. ^* p < 0.05 vs. respective controls; ^# p < 0.05 vs. MSA; ^** p < 0.05 vs. MSA controls.