Autoreactive B cells are present in the lymphoid tissues of healthy individuals, but typically remain quiescent. When this homeostasis is perturbed, the formation of self-reactive antibodies can have serious pathological consequences. B cells expressing an antigen receptor specific for self-immunoglobulin-gamma (IgG) make a class of autoantibodies known as rheumatoid factor (RF). Here we show that effective activation of RF+ B cells is mediated by IgG2a-chromatin immune complexes and requires the synergistic engagement of the antigen receptor and a member of the MyD88-dependent Toll-like receptor (TLR) family. Inhibitor studies implicate TLR9. These data establish a critical link between the innate and adaptive immune systems in the development of systemic autoimmune disease and explain the preponderance of autoantibodies reactive with nucleic acid-protein particles. The unique features of this dual-engagement pathway should facilitate the development of therapies that specifically target autoreactive B cells.

Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9-MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.

Receptor crosslinking of T-cell hybridomas induces cell activation followed by apoptosis. This activation-induced cell death requires de novo synthesis of RNA and proteins, but the actual gene products that provide the death signal have not been identified. We show here that receptor crosslinking induces Fas ligand and upregulates Fas, and that the ensuing engagement of Fas by Fas ligand activates the cell-death programme. Cell death, but not activation, can be selectively prevented by a soluble Fas-immunoglobulin fusion protein. Thus, Fas and Fas ligand are the death-gene products, and their interaction accounts for the molecular mechanism of activation-induced T-cell death.

Toll-like receptors (TLRs) have a crucial role in the early detection of pathogen-associated molecular patterns and the subsequent activation of the adaptive immune response. Whether TLRs also have an important role in the recognition of endogenous ligands has been more controversial. Numerous in vitro studies have documented activation of both autoreactive B cells and plasmacytoid dendritic cells by mammalian TLR ligands. The issue of whether these in vitro observations translate to an in vivo role for TLRs in either the initiation or the progression of systemic autoimmune disease is a subject of intense research; data are beginning to emerge showing that this is the case.

Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adapter ASC and assembly of an ASC speck. ASC specks recruit and activate caspase-1, which induces IL-1β cytokine maturation and pyroptotic cell death. Here we show that after pyroptosis ASC specks accumulate in the extracellular space, where they promote further IL-1β maturation. In addition, phagocytosis of ASC specks induces lysosomal damage, nucleation of soluble ASC as well as caspase-1 and IL-1β activation in the recipient cell. ASC specks appear in bodily fluids from inflamed tissues and autoantibodies against ASC specks develop in patients and animals with autoimmune pathologies. Together, these findings reveal extracellular functions of ASC specks and a novel form of cell-to-cell communication.

Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603–607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837–847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-α, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.

The proliferative response of autoreactive rheumatoid factor (RF) B cells to mammalian chromatin-containing immune complexes (ICs) results from the sequential engagement of the B cell receptor (BCR) and Toll-like receptor 9 (TLR9). We have used ICs constructed from anti-hapten antibodies and defined haptenated dsDNA fragments to determine the form of mammalian DNA that mediates this process. Despite their relatively low abundance in mammalian DNA, we found that inclusion of hypomethylated CpG motifs in these ICs was necessary for effective activation. In the absence of antibody, the same fragments could efficiently stimulate low-affinity hapten-specific and DNA-reactive 3H9 B cells, but not RF B cells. These results extend the BCR/TLR9 coengagement paradigm to a second major class of autoreactive B cells, further confirm the critical role of the BCR in chromatin ligand delivery to TLR9, and implicate hypomethylated CpG motifs as ligand elements necessary for the initiation of systemic autoimmune disease.

Polyclonal activation has been proposed as the reason that autoantibodies are produced during autoimmune disease. This model denies a role for specific antigen selection of B cells and predicts instead a multiclonal population of unmutated or randomly mutated autoantibodies. We have found that the genetic features and clonal composition of spontaneously derived immunoglobulin G (IgG) antiself-IgG (rheumatoid factor (RF] autoantibodies derived from the autoimmune MRL/lpr mouse strain are inconsistent with both the predictions of this model and the actual outcome of experimental polyclonal activation. Instead we have found that MRL/lpr RFs are oligoclonal or even monoclonal in origin. They harbour numerous somatic mutations which are distributed in a way that suggests immunoglobulin-receptor-dependent selection of these mutations. In this sense, the MRL/lpr RFs resemble antibodies elicited by exogenous antigens after secondary immunization. The parallels suggest that, like secondary immune responses, antigen stimulation is important in the generation of MRL/lpr RFs.