Cathepsin B regulates the appearance and severity of mercury-induced inflammation and autoimmunity.

Susceptibility and resistance to systemic autoimmunity are genetically regulated. This is particularly true for murine mercury-induced autoimmunity (mHgIA) where DBA/2J mice are considered resistant to disease including polyclonal B cell activation, autoantibody responses, and immune complex deposits. To identify possible mechanisms for the resistance to mHgIA, we exposed mHgIA sensitive B10.S and resistant DBA/2J mice to HgCl2 and assessed inflammation and pro-inflammatory responses at the site of exposure and subsequent development of markers of systemic autoimmunity. DBA/2J mice showed little evidence of induration at the site of exposure, expression of proinflammatory cytokines, T cell activation, or autoantibody production, although they did exhibit increased levels of total serum IgG and IgG1. In contrast B10.S mice developed significant inflammation together with increased expression of inflammasome component NLRP3, proinflammatory cytokines IL-1ß, TNF-α, and IFN-γ, hypergammaglobulinemia, splenomegaly, CD4(+) T-cell activation, and production of autoantibodies. Inflammation in B10.S mice was associated with a selective increase in activity of cysteine cathepsin B but not cathepsins L or S. Increased cathepsin B activity was not dependent on cytokines required for mHgIA but treatment with CA-074, a cathepsin B inhibitor, led to transient reduction of local induration, expression of inflammatory cytokines, and subsequent attenuation of the systemic adaptive immune response. These findings demonstrate that sensitivity to mHgIA is linked to an early cathepsin B regulated inflammatory response which can be pharmacologically exploited to abrogate the subsequent adaptive autoimmune response which leads to disease.

Definition of IFN-γ-related pathways critical for chemically-induced systemic autoimmunity.

IFN-γ is essential for idiopathic and murine mercury-induced systemic autoimmunity (mHgIA), and heterozygous IFN-γ(+/-) mice also exhibit reduced disease. This suggests that blocking specific IFN-γ-related pathways that may only partially inhibit IFN-γ production or function will also suppress autoimmunity. To test this hypothesis, mice deficient in genes regulating IFN-γ expression (Casp1, Nlrp3, Il12a, Il12b, Stat4) or function (Ifngr1, Irf1) were examined for mHgIA susceptibility. Absence of either Ifngr1 or Irf1 resulted in a striking reduction of disease, while deficiency of genes promoting IFN-γ expression had modest to no effect. Furthermore, both Irf1- and Ifng-deficiency only modestly reduced the expansion of CD44(hi) and CD44(hi)CD55(lo) CD4(+) T cells, indicating that they are not absolutely required for T cell activation. Thus, there is substantial redundancy in genes that regulate IFN-γ expression in contrast to those that mediate later signaling events. These findings have implications for the therapeutic targeting of IFN-γ pathways in systemic autoimmunity.

Formation of GW bodies is a consequence of microRNA genesis.

GW bodies (GWBs), or mammalian P bodies, proposed to be involved in messenger RNA storage and/or degradation, have recently been linked to RNA interference and microRNA (miRNA) processing. We report that endogenous let-7 miRNA co-precipitates with the GW182 protein complex. In addition, knockdown of two proteins, Drosha and its protein partner DGCR8, which are vital to the generation of mature miRNA, results in the loss of GWBs. Subsequent introduction of short interference RNA specific to lamin A/C is accompanied by reassembly of GWBs and concurrent knockdown of lamin A/C protein. Taken together, these studies show that miRNAs are crucial components in GWB formation.

GW bodies, microRNAs and the cell cycle.

GW bodies (GWBs) are cytoplasmic foci initially identified through the use of an autoimmune serum targeting the marker protein, GW182. GWBs were first considered as both storage centers for a specific subset of mRNAs and degradation sites for mRNAs. Interestingly, they are known to vary in size and number throughout the cell cycle and are largest in size and most abundant in number during the late S and G2 phases. Recent studies have linked RNA interference to GWBs, in that disruption or disassembly of GWBs was demonstrated to impair siRNA and miRNA silencing activity. As miRNAs are implicated in the regulation of cell cycle progression and cell proliferation, it is very likely that GWBs, the critical intracellular structures for miRNA function, may very well be also linked to this cellular process.

Disruption of GW bodies impairs mammalian RNA interference.

The GW182 RNA-binding protein was initially shown to associate with a specific subset of mRNAs and to reside within discrete cytoplasmic foci named GW bodies (GWBs). GWBs are enriched in proteins that are involved in mRNA degradation. Recent reports have shown that exogenously introduced human Argonaute-2 (Ago2) is also enriched in GWBs, indicating that RNA interference function may be somehow linked to these structures. In this report, we demonstrate that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2. Disruption of these cytoplasmic foci in HeLa cells interferes with the silencing capability of a siRNA that is specific to lamin-A/C. Our data support a model in which GW182 and/or the microenvironment of the cytoplasmic GWBs contribute to the RNA-induced silencing complex and to RNA silencing.

Fragmentation of Golgi complex and Golgi autoantigens during apoptosis and necrosis.

Anti-Golgi complex autoantibodies are found primarily in patients with Sjögren's syndrome and systemic lupus erythematosus, although they are not restricted to these diseases. Several Golgi autoantigens have been identified that represent a small family of proteins. Common features of all Golgi autoantigens appear to be their distinct structural organization of multiple alpha-helical coiled-coil rods in the central domains flanked by non-coiled-coil N-termini and C-termini, and their localization to the cytoplasmic face of Golgi cisternae. Many autoantigens in systemic autoimmune diseases have distinct cleavage products in apoptosis or necrosis and this has raised the possibility that cell death may play a role in the generation of potentially immunostimulatory forms of autoantigens. In the present study, we examined changes in the Golgi complex and associated autoantigens during apoptosis and necrosis. Immunofluorescence analysis showed that the Golgi complex was altered and developed distinctive characteristics during apoptosis and necrosis. In addition, immunoblotting analysis showed the generation of antigenic fragments of each Golgi autoantigen, suggesting that they may play a role in sustaining autoantibody production. Further studies are needed to determine whether the differences observed in the Golgi complex during apoptosis or necrosis may account for the production of anti-Golgi complex autoantibodies.

Autoantibody to hLSm4 and the heptameric LSm complex in anti-Sm sera.

OBJECTIVE: To characterize the 15-kd human SmD-like autoantigen and its associated proteins previously shown to be recognized by IgM antibodies in patients with Epstein-Barr virus (EBV)-induced infectious mononucleosis. METHODS: The full-length complementary DNA for the 15-kd protein was expressed as recombinant protein and analyzed for reactivity using biochemical analysis and immunoprecipitation (IP). RESULTS: The 15-kd protein was determined to be the human like-Sm protein LSm4 (hLSm4). Rabbit antibody raised against the C-terminal polypeptide immunoprecipitated a 68-kd complex composed of LSm4 together with a group of smaller proteins ranging in size from 6.5 to 14 kd, consistent with the reported heptameric LSm complexes involved in U4/U6 duplex formation and messenger RNA (mRNA) decapping/degradation. About 80% of all anti-Sm sera from patients with systemic lupus erythematosus (SLE) recognized the hLSm4 in vitro translated product, while 6.7% (29 of 434) immunoprecipitated from cell extracts hLSm4 together with the other members of the hLSm complex. Four sera (0.92%) showed apparently exclusive reactivity to the hLSm complex in the absence of reactivity to Sm core proteins in the IP assay. CONCLUSION: These findings document that while IgM, but not IgG, autoantibodies to LSm4 were found in sera from patients with EBV infection, IgG autoantibodies to hLSm4 are detected in a large number of anti-Sm-positive sera from patients with SLE. Importantly, in a small number of anti-Sm sera the LSm complex can be recognized independently of the Sm core protein antigens. Our data introduce the concept that "Sm" autoantigens include Sm as well as LSm complexes involved in the maturation and degradation of mRNA.