There is now renewed interest in the role of antibodies in autoimmunity. Recent compelling evidence indicates that autoantibodies and the effector mechanisms they induce, for example, Fc receptor ...activation of leukocytes and/or the complement cascade, are central players in the development of autoimmunity, by perpetuating inflammation and perhaps even regulating the process itself. Of increasing interest are Fc receptors, which have been more closely investigated in the past decade using recombinant proteins, gene deficient mice and mouse models of human disease. These analyses point towards major roles of Fc receptors in antibody hypersensitivity reactions and by extension autoimmune disease, and they reveal opportunities in the development of novel therapeutic approaches in the treatment of autoimmune diseases.
Recent compelling evidence indicates that autoantibodies are central players in the development of autoimmunity.
Essentials
FcγRIIa mediates life‐threatening heparin‐induced thrombocytopenia (HIT).
Most anti‐platelet factor (PF)4‐heparin IgGs are not pathogenic so diagnosis of HIT is challenging.
Dimeric ...rsFcγRIIa was used to quantify receptor‐binding activity of anti‐PF4‐heparin antibodies.
Dimeric rsFcγRIIa binding specifically correlated with occurrence of HIT.
Summary
Background
Heparin‐induced thrombocytopenia (HIT) is a major and potentially fatal consequence of antibodies produced against platelet factor 4 (PF4)–heparin complexes following heparin exposure. Not all anti‐PF4–heparin antibodies are pathogenic, so overdiagnosis can occur, with resulting inappropriate use of alternative anticoagulation therapies that have associated risks of bleeding. However, definitive platelet functional assays are not widely available for routine analysis.
Objectives
To assess the utility of dimeric recombinant soluble FcγRIIa (rsFcγRIIa) ectodomains for detecting HIT antibodies.
Patients/Methods
Plasma from 27 suspected HIT patients were tested for pathogenic anti‐PF4–heparin antibodies by binding of a novel dimeric FcγRIIa ectodomain probe. Plasmas were also tested by the use of PF4–heparin IgG ELISA, the HemosIL AcuStar HIT IgG‐specific assay, and a serotonin release assay (SRA).
Results
The dimeric rsFcγRIIa test produced no false positives and excluded four samples that were positive by IgG ELISA. In this small patient cohort, the novel assay correctly assigned 93% of the suspected HIT patients, with two of the HIT patients being scored as false negatives. The improved discrimination of the novel assay over the IgG ELISA, which scored four false positives, supports the mechanistic interpretation that binding of dimeric rsFcγRIIa detects pairs of closely spaced IgG antibodies in PF4–heparin immune complexes.
Conclusions
This study found the cell‐free, function‐based dimeric rsFcγRIIa assay to be convenient, simple, and potentially predictive of HIT. The assay had improved specificity over the IgG ELISA, and correlated strongly with the AcuStar HIT IgG‐specific assay, warranting further evaluation of its potential to identify HIT in larger patient cohorts.
The inactivation of the mouse high-affinity IgG Fc receptor FcγRI resulted in a wide range of defects in antibody Fc-dependent functions. These studies showed the primary importance of FcγRI in ...endocytosis of monomeric IgG, kinetics, and extent of phagocytosis of immune complexes, in macrophage-based ADCC, and in immune complex-dependent antigen presentation to primed T cells. In the absence of FcγRI, antibody responses were elevated, implying the removal of a control point by the deletion of FcγRI. In addition, FcR-γ chain-deficient mice were found to express partially functional FcγRI. Thus, FcγRI is an early participant in Fc-dependent cell activation and in the development of immune responses.
The substitution of plasmatic anti-RhD polyclonal antibodies by a monoclonal antibody (mAb) for preventing the hemolytic disease of the newborn (HDN) is an important issue due to supply and safety ...concerns. Since it has been suggested that FcγR are involved in the prevention of HDN, the in vitro functional properties of two anti-RhD mAbs differing through their glycosylation profiles were compared using FcγR-based assays to select a candidate mAb. T125(YB2/0), a low fucosylated antibody, bound strongly to both activating FcγRIII and inhibitory FcγRII, as opposed to its highly fucosylated counterpart. It also exerted a strong ADCC against RhD
+ RBCs and a potent FcγRIIB-mediated inhibition of cytokine release. Moreover, an in vivo RhD
+ red blood cells (RBCs) clearance assay showed that this antibody exhibits a RhD
+ RBCs clearance as potent as polyclonal anti-RhD antibodies in NOD-SCID mice. Thus, T125(YB2/O) has been selected to be tested for the prevention of anti-RhD allo-immunization.
Targeting antigen to dendritic cells (DC) in vivo might be an effective method of modulating immune responses. Given the functional specializations among DC subsets, we investigated how targeting ...different receptors on different DC subsets may influence antibody (Ab) production. We show here that targeting FIRE (F4/80‐like receptor) or CIRE (C‐type lectin receptor), two molecules expressed on the surface of immature CD8– DC in the mouse, increases Ab production 100–1000‐fold over a non‐targeted control. This response was equivalent to that achieved with CpG adjuvant. In contrast, targeting CD205, which is primarily expressed on CD8+ DC, did not elicit an Ab response unless an adjuvant was added. Strong Ab responses in FcRγ–/– mice, and with the use of F(ab')2 fragments, confirmed that FIRE and CIRE targeting was due to specific rather than FcR or complement binding. Our findings may reflect differences in the ability of CD8+ and CD8– DC subsets to stimulate immune responses in vivo. Although the consensus view is that Ag presentation on DC in their steady state leads to tolerance, the Ab enhancement from FIRE and CIRE targeting in the apparent absence of any “danger” or inflammatory signal would suggest that targeting certain DC molecules can supplant the need for external adjuvants for eliciting immune responses.
The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been ...proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.
This study defines the molecular basis of the FcalphaRI (CD89):IgA interaction, which is distinct from that of the other leukocyte Fc receptors and their Ig ligands. A comprehensive analysis using ...both cell-free (biosensor) and cell-based assays was used to define and characterize the IgA binding region of FcalphaRI. Biosensor analysis of mutant FcalphaRI proteins showed that residues Y35, Y81, and R82 were essential for IgA binding, and R52 also contributed. The role of the essential residues (Y35 and R82) was confirmed by analysis of mutant receptors expressed on the surface of mammalian cells. These receptors failed to bind IgA, but were detected by the mAb MY43, which blocks IgA binding to FcalphaRI, indicating that its epitope does not coincide with these IgA binding residues. A homology model of the ectodomains of FcalphaRI was generated based on the structures of killer Ig-like receptors, which share 30-34% identity with FcalphaRI. Key structural features of killer Ig-like receptors are appropriately reproduced in the model, including the structural conservation of the interdomain linker and hydrophobic core (residues V17, V97, and W183). In this FcalphaRI model the residues forming the IgA binding site identified by mutagenesis form a single face near the N-terminus of the receptor, distinct from other leukocyte Fc receptors where ligand binding is in the second domain. This taken together with major differences in kinetics and affinity for IgA:FcalphaRI interaction that were observed depending on whether FcalphaRI was immobilized or in solution suggest a mode of interaction unique among the leukocyte receptors.
Abstract Although anti-DNA antibodies have been decisively linked to the pathogenesis of lupus nephritis, the mechanisms have not been conclusively determined. Recently, we reported that anti-DNA ...antibodies may contribute to kidney damage by upregulation of proinflammatory genes in mesangial cells (MC), a process involving both Fc receptor-dependent and independent pathways. In investigating the mechanism by which pathogenic anti-DNA antibodies modulate gene expression in MC, we found that the pathogenic anti-DNA antibody 1A3F bound to high mobility group binding protein 1 (HMGB1), an endogenous ligand for TLR2/4 and RAGE (receptor for advanced glycation end products). Interestingly, HMGB1 treatment of MC induced a similar pattern of genes as stimulation with 1A3F. Furthermore, HMGB1 and 1A3F exhibited a synergistic proinflammatory effect in the kidney, where increased expression of HMGB1 was found in lupus patients but not in patients with other types of renal disease. TLR2/Fc and RAGE/Fc inhibited the proinflammatory effects of 1A3F on MC. Finally, we found enhanced susceptibility of lupus prone MRL- lpr / lpr (MRL/ lpr ) as compared to normal BALB/c derived MC to pathogenic anti-DNA antibody and LPS stimulation (in particular enhanced chemokine synthesis), in addition to significantly increased expression of TLR4. Our results suggest that gene upregulation in MC induced by nephritogenic anti-DNA antibodies is TLR2/4 and RAGE-dependent. Finally, HMGB1 may act as a proinflammatory mediator in antibody-induced kidney damage in systemic lupus erythematosus (SLE).
Ligand-dependent aggregation of FcγRIIa initiates multiple biochemical processes including the translocation to detergent resistant membrane domains (DRMs) and receptor tyrosine phosphorylation. ...Palmitoylation of cysteine residues is considered to be one process that assists in the localisation of proteins to DRMs. Within the juxtamembrane region of FcγRIIa there is cysteine residue (C208) that we show to be palmitoylated. Mutation of this cysteine residue results in the disruption of FcγRIIa translocation to DRMs as empirically defined by insolubility at high Triton X-100 concentrations. This study also demonstrates that the lack of lipid raft association diminishes FcγRIIa signaling as measured by receptor phosphorylation and calcium mobilisation functions suggesting that FcγRIIa signaling is partially dependent on lipid rafts.
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