(A) Schematic representation of the different 3D8 molecules. mediated by TRIM21. The results suggest the presence of a novel Fc-dependent signaling pathway that is activated upon internalization of IgG antibodies by human monocytes. Keywords: anti-DNA antibody, internalizing IgG, inflammatory cytokines, Fc-dependent signaling, human monocytes Introduction Antibodies can enter cells passively either by binding to constitutively internalized cell surface molecules or by forming immune complexes (ICs) with invading pathogens. Once a pathogen-antibody complex reaches the cytosol of non-immune cells, it is recognized by tripartite motif-containing 21 (TRIM21), a receptor for the antibody Fc region (1, 2). TRIM21 has Fc-dependent effector and signaling functions. The effector function, called antibody-dependent intracellular neutralization or intracellular antibody-mediated degradation, prospects to quick, VCP/p97-assisted proteasomal degradation of pathogen particles (2, 3). At the same time, TRIM21 catalyzes formation of free K63-linked ubiquitin chains, which trigger immune signaling pathways and lead to production of pro-inflammatory chemokines such as CCL5 and CXCL10 and cytokines such as TNF-, IL-6, and IFN- (4, 5). Anti-DNA autoantibodies are a hallmark of an autoimmune disease called systemic lupus erythematosus (SLE); indeed, the titer of these antibodies in serum correlates with disease activity (6, 7). These antibodies form ICs with DNA and play a key role in activating signaling pathways that trigger cytokine production (8C10). For example, DNA-containing ICs bind to Fc receptors expressed by AZ 23 plasmacytoid dendritic cells (pDCs) and are then phagocytosed and internalized; these internalized ICs are recognized by TLR9, which then induces production of type I IFN and other pro-inflammatory cytokines (8, 9). In human monocytes, DNA-ICs induce production of IL-1 by activating the NLRP3 inflammasome (10). Free anti-DNA AZ 23 autoantibodies (i.e., not a part of ICs) also activate cytokine signaling pathways, even though mechanisms are different (11C13). For example, production of IL-2 by Jurkat T cells is usually triggered via conversation with phosphoglycerate kinase 1 (11), production of IL-6 by human mesangial cells is usually brought on by antibody binding to annexin II followed by internalization (12), and production of IL-1 and IL-17A by monocytes/macrophages from SLE patients is usually brought on by conversation with TLR4, which then activates the NLRP3 inflammasome (13). Thus, in these cases cytokine signaling induced by free-form anti-DNA antibodies and by IC forms is determined by their DNA-binding properties and cross-reactivity, which are themselves ITPKB AZ 23 determined by the unique heavy-chain-variable (VH) and light-chain-variable (VL) regions of the antibody. As mentioned above, a subset of anti-DNA IgG autoantibodies enters living cells in a free form Yanase et al. (14), Seddiki et al. (15), Zack et al. (16), and Track et al. (17). The mechanism of entry is usually distinct from that used by other antibodies, which enter cells passively by covering invading pathogens. The relationship between the cytokine signaling function of TRIM21 and SLE-like autoimmune disease activity has been examined in mouse models, but the results are contradictory (18). Thus, it is still not clear whether the Fc region of internalized IgG is usually recognized by TRIM21 in the cytosol, resulting in activation of TRIM21-mediated signaling pathways and culminating in production of inflammatory cytokines that exacerbate the clinical manifestations of SLE. Here, we examined the ability of an internalizing free-form anti-DNA IgG antibody to trigger production of pro-inflammatory cytokines by human cells in an Fc-dependent manner. To do this, we used a cell-internalizable chimeric anti-DNA antibody (3D8 IgG) in which the VH and VL regions, which are derived from a mouse model of SLE (MRL-mouse), were grafted onto a human IgG1 backbone. The 3D8 single-chain variable fragment (scFv) comprises only the VH and VL regions of the 3D8 antibody, retains DNA-binding activity, and enters cells by binding to heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) expressed around the cell surface; from there it localizes to the cytosol (19). The 3D8 scFv and 3D8 scFv-Fc antibodies were used as negative and positive controls, respectively, to verify whether the Fc region of IgG triggers cytokine responses. A 3D8 IgG-N434D mutant, which does not interact.