The role of toll-like receptors in systemic autoimmune disease

Abstract

The AM14 B cell receptor, derived from an autoimmune Fas-deficient mouse, is a rheumatoid factor specific for IgG2a. B cells expressing the AM14 receptor have been effectively activated by immune complexes (ICs) consisting of IgG2a associated with endogenous TLR ligands that essentially serve as autoadjuvants. One source of endogenous ligand is DNAse-sensitive material, presumably chromatin, released from dead/dying mammalian cells. The chromatin response was blocked by endosome acidification inhibitors, eliminated in MyD88-deficient cells and markedly reduced, in TLR9-deficient cells; residual activity appeared to depend on a TLR(s) other than TLR9. Sources of TLR9-reactive mammalian DNA have been explored. AM14 B cells also responded to ICs consisting of IgG2a bound to RNA-associated autoantigens. These responses were eliminated in MyD88-deficient cells but unaffected by TLR9 deficiency. Thus, activation depends on BCR-directed delivery of the ICs to vesicular compartments that contain an appropriate TLR. Intriguingly, such BCR/TLR co-engagement leads to the expression of gene products not elicited by independent activation of the BCR (anti-IgM F(ab′)₂) or TLR9 (CpG s-ODN) alone. The effects of nucleic acid-associated ICs on both B cells and antigen presenting cells indicate that the TLR9 subfamily may be a key target for therapeutic intervention in patients with systemic autoimmune diseases.

Introduction

Systemic autoimmune diseases such as SLE are frequently associated with the production of high titers of autoantibodies specific for nuclear and/or cytoplasmic cellular components. Such antibodies subsequently form immune complexes (ICs), deposit in the blood vessel walls, joints, and glomeruli, and thereby contribute to the vasculitis, arthritis, and glomerulonephritis characteristic of systemic disease. Remarkably, in both animal models and human disease, the autoantibody repertoire consistently targets a relatively limited set of DNA and RNA associated autoantigens [1], [2]. A better understanding of the mechanisms by which these particular molecules activate autoreactive B cells is critical to the design of more effective therapeutics.

A number of recent studies have linked the antigenicity of particular autoantigens to the process of apoptotic cell death and subsequent conformational changes of many of the prominent autoantigens. Apoptosis can be induced by environmental insults or by cytotoxic effector cells; in either case, many potential autoantigens are cleaved by caspases or granzymes, or undergo post-translational modifications such as oxidation, citrullination, or phosphorylation [3], [4]. It has been proposed that such alterations may lead to the creation of neo-epitopes that are then recognized as foreign by the adaptive immune system. The process of epitope spreading can then effectively break tolerance to the actual self-determinants. However, it has recently been shown that autoantibodies specific for granzyme B target proteins are just as prevalent in granzyme B-deficient autoimmune mice as in granzyme B-sufficient autoimmune prone mice [5]. It follows that autoantigen cleavage may be a fingerprint of apoptosis but not necessarily a trigger for autoantibody production.

Alternatively, some aspect of the apoptotic process may directly prime the innate immune system to initiate an adaptive response, perhaps by converting autoantigens into “autoadjuvants”, or by promoting the redistribution or aberrant accumulation of pre-existing autoadjuvants. In this context, normally benign self-determinants may appear foreign. The potential adjuvanticity of mammalian nucleic acids was originally revealed by the pivotal studies of Ronnblom et al. who first demonstrated the potent effects of DNA- and RNA-containing ICs on IFNα-producing dendritic cells [6], [7]. Our laboratory has extended this analysis by taking advantage of a B cell receptor transgenic line designated AM14 [8].

AM14 B cells recognize IgG2a with relatively low affinity and can be activated in vitro only by IgG2a presented as a multivalent IC. Importantly, the type of antigen contained in the IC plays a critical role in the cell activation process. IgG2a mAbs specific for histones, nucleosomes, or DNA bind to DNAse sensitive material released from dead or dying cells that is present in the supernatant of primary B cell cultures [9], [10]. These “chromatin” ICs are potent ligands for AM14 B cells under conditions in which IgG2a bound to protein antigens, or protein ICs, have little effect. The stimulatory capacity of the chromatin ICs can be explained by their ability to effectively engage both the AM14 B cell receptor and one or more chloroquine/bafilomycin sensitive receptors. Ongoing studies from our laboratory have focused on the identification or the relevant receptor(s) and the nature of the mammalian DNA ligand.