Antibodies in action: the role of humoral immunity in the fight against atherosclerosis.

The sequestering of oxidation-modified low-density lipoprotein by macrophages results in the accumulation of fatty deposits within the walls of arteries. Necrosis of these cells causes a release of intercellular epitopes and the activation of the adaptive immune system, which we predict leads to robust autoantibody production. T cells produce cytokines that act in the plaque environment and further stimulate B cell antibody production. B cells in atherosclerosis meanwhile have a mixed role based on subclass. The current model is that B-1 cells produce protective IgM antibodies in response to oxidation-specific epitopes that work to control plaque formation, while follicular B-2 cells produce class-switched antibodies (IgG, IgA, and IgE) which exacerbate the disease. Over the course of this review, we discuss further the validation of these protective antibodies while evaluating the current dogma regarding class-switched antibodies in atherosclerosis. There are several contradictory findings regarding the involvement of class-switched antibodies in the disease. We hypothesize that this is due to antigen-specificity, and not simply isotype, being important, and that a closer evaluation of these antibodies' targets should be conducted. We propose that specific antibodies may have therapeutical potential in preventing and controlling plaque development within a clinical setting.

Auto-Antibody Production During Experimental Atherosclerosis in ApoE-/- Mice.

Current models stipulate that B cells and antibodies function during atherosclerosis in two distinct ways based on antibody isotype, where IgM is protective and IgG is inflammatory. To examine this model, we generated ApoE-/- Aid-/- mice, which are unable to produce IgG antibodies due to the absence of activation-induced deaminase (AID) but maintain high plasma cholesterol due to the absence of apolipoprotein E (APOE). We saw a dramatic decrease in plaque formation in ApoE-/- Aid-/- mice compared to ApoE-/- mice. Rigorous analysis of serum antibodies revealed both ApoE-/- and ApoE-/- Aid-/- mice had substantially elevated titers of IgM antibodies compared to C57BL/6J controls, suggesting a more complex dynamic than previously described. Analysis of antigen specificity demonstrated that ApoE-/- Aid-/- mice had elevated titers of antibodies specific to malondialdehyde-oxidized low density lipoprotein (MDA-oxLDL), which has been shown to block macrophage recruitment into plaques. Conversely, ApoE-/- mice showed low levels of MDA-oxLDL specificity, but had antibodies specific to numerous self-proteins. We provide evidence for a hierarchical order of antibody specificity, where elevated levels of MDA-oxLDL specific IgM antibodies inhibit plaque formation. If the level of MDA-oxLDL specific IgM is insufficient, self-reactive IgM and IgG antibodies are generated against debris within the arterial plaque, resulting in increased inflammation and further plaque expansion.

Directing Quinone Methide-Dependent Alkylation and Cross-Linking of Nucleic Acids with Quaternary Amines.

Polyamine and polyammonium ion conjugates are often used to direct reagents to nucleic acids based on their strong electrostatic attraction to the phosphoribose backbone. Such nonspecific interactions do not typically alter the specificity of the attached reagent, but polyammonium ions dramatically redirected the specificity of a series of quinone methide precursors. Replacement of a relatively nonspecific intercalator based on acridine with a series of polyammonium ions resulted in a surprising change of DNA products. Piperidine stable adducts were generated in duplex DNA that lacked the ability to support a dynamic cross-linking observed previously with acridine conjugates. Minor reaction at guanine N7, the site of reversible reaction, was retained by a monofunctional quinone methide-polyammonium ion conjugate, but a bisfunctional analogue designed for tandem quinone methide formation modified guanine N7 in only single-stranded DNA. The resulting intrastrand cross-links were sufficiently dynamic to rearrange to interstrand cross-links. However, no further transfer of adducts was observed in duplex DNA. An alternative design that spatially and temporally decoupled the two quinone methide equivalents neither restored the dynamic reaction nor cross-linked DNA efficiently. While di- and triammonium ion conjugates successfully enhanced the yields of cross-linking by a bisquinone methide relative to a monoammonium equivalent, alternative ligands will be necessary to facilitate the migration of cross-linking and its potential application to disrupt DNA repair.

Oxidative quenching of quinone methide adducts reveals transient products of reversible alkylation in duplex DNA.

ortho-Quinone methides (ortho-QM) and para-quinone methides are generated by xenobiotic metabolism of numerous compounds including environmental toxins and therapeutic agents. These intermediates are highly electrophilic and have the potential to alkylate DNA. Assessing their genotoxicity can be difficult when all or some of their resulting adducts form reversibly. Stable adducts are most easily detected but are not necessarily the most prevalent products formed initially as DNA repair commences. Selective oxidation of ortho-QM-DNA adducts by bis[(trifluoroacetoxy)iodo]benzene (BTI) rapidly quenches their reversibility to prevent QM regeneration and allows for observation of the kinetic products. The resulting derivatives persist through standard enzymatic digestion, chromatography, and mass spectral analysis. The structural standards required for this approach have been synthesized and confirmed by two-dimensional NMR spectroscopy. The adducts of dA N(6), dG N1, dG N(2), and guanine N7 are converted to the expected para-quinol derivatives within 5 min after addition of BTI under aqueous conditions (pH 7). Concurrently, the adduct of dA N1 forms a spiro derivative comparable to that characterized previously after oxidation of the corresponding dC N3 adduct. By application of this oxidative quenching strategy, the dC N3 and dA N1 adducts have been identified as the dominant products formed by both single- and double-stranded DNA under initial conditions. As expected, however, these labile adducts dissipate within 24 h if not quenched with BTI. Still, the products favored by kinetics are responsible for inducing the first response to ortho-QM exposure in cells, and hence, they are also key to establishing the relationship between biological activity and molecular structure.