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Asparaginase immune complexes induce Fc-γRIII-dependent hypersensitivity in naive mice.

ABSTRACT: Asparaginase (ASNase) is an important drug for the treatment of leukemias. However, hypersensitivity to ASNase can increase the risk of leukemia relapse. Two mechanisms of ASNase hypersensitivity have been identified in mice. The existence of a pathway involving anti-ASNase IgG and Fc-γ receptor III (Fc-γRIII) implies that IgG and ASNase immune complexes (ICs) could directly induce hypersensitivity. The aim of this study was to detect ASNase ICs in mice after hypersensitivity reactions and determine their role in hypersensitivity. Protein G beads were used to detect plasma ASNase ICs by flow cytometry. Anti-ASNase IgG was purified from the plasma of sensitized mice, and ASNase ICs were prepared ex vivo at various ratios of ASNase to anti-ASNase IgG. The levels of ASNase ICs detected after hypersensitivity reactions correlated with reaction severity (R² = 0.796; P = 0.0005). ASNase ICs prepared ex vivo required high levels of anti-ASNase IgG for formation, and binding to naive and sensitized immune cells depended on soluble anti-ASNase IgG, antigen:antibody ratio, and Fc-γRIII. Similarly, basophil activation by ASNase ICs depended on the antigen:antibody ratio and Fc-γRIII. Consistent with the ex vivo results, naive mice receiving ASNase ICs developed hypersensitivity reactions. Our data demonstrate that ASNase ICs can directly contribute to the onset and severity of ASNase hypersensitivity.-Rathod, S., Ramsey, M., DiGiorgio, D., Berrios, R., Finkelman, F. D., Fernandez, C. A. Asparaginase immune complexes induce Fc-γRIII-dependent hypersensitivity in naive mice.

SUBMITTER: Rathod S

PROVIDER: S-EPMC6766650 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy sometimes associated with thrombosis. The hallmark of HIT is antibodies to the heparin/platelet factor 4 (PF4) complex that cause thrombocytopenia and thrombosis through platelet activation. Despite the clinical importance, the molecular mechanisms and late consequences of immune platelet activation are not fully understood. Here, we studied immediate and delayed effects of the complexes formed by human PF4 and HIT-like monoclonal mouse anti-human-PF4/heparin IgG antibodies (named KKO) on isolated human platelets in vitro. Direct platelet-activating effect of the KKO/PF4 complexes was corroborated by the overexpression of phosphatidylserine (PS) and P-selectin on the platelet surface. The immune platelet activation was accompanied by a decrease of the mitochondrial transmembrane potential (??m), concurrent with a significant gradual reduction of the ATP content in platelets, indicating disruption of energy metabolism. A combination of PS expression and mitochondrial depolarization induced by the PF4-containing immune complexes observed in a substantial fraction of platelets was considered as a sign of ongoing platelet death, as opposed to a subpopulation of activated live platelets with PS on the plasma membrane but normal ??m. Both activated and dying platelets treated with KKO/PF4 formed procoagulant extracellular microvesicles bearing PS on their surface. Scanning and transmission electron microscopy revealed dramatic morphological changes of KKO/PF4-treated platelets, including their fragmentation, another indicator of cell death. Most of the effects of KKO/PF4 were prevented by an anti-Fc?RII monoclonal antibody IV.3. The adverse functional and structural changes in platelets induced by the KKO/PF4 complexes were associated with strong time-dependent activation of calpain, but only trace cleavage of caspase 3. The results indicate that the pathogenic PF4-containing HIT-like immune complexes induce direct prothrombotic platelet activation via Fc?RIIA receptors followed by non-apoptotic calpain-dependent death of platelets, which can be an important mechanism of thrombocytopenia during HIT development.

Project description:ObjectiveAutoantibodies are detected in most patients with rheumatoid arthritis (RA) and can be of the IgM, IgG or IgA subclass. Correlations between IgA autoantibodies and more severe disease activity have been previously reported, but the functional role of IgA autoantibodies in the pathogenesis of RA is ill understood. In this study, we explored the effect of IgA immune complexes on osteoclast mediated bone resorption.MethodsAnti-citrullinated peptide antibody (ACPA) and anti-carbamylated protein (anti-CarP) antibody levels of the IgA and IgG isotype and rheumatoid factor (RF) IgA were determined in synovial fluid (SF) of RA patients. Monocytes, neutrophils, and osteoclasts were stimulated with precipitated immune complexes from SF of RA patients or IgA- and IgG-coated beads. Activation was determined by neutrophil extracellular trap (NET) release, cytokine secretion, and bone resorption.ResultsNET formation by neutrophils was enhanced by SF immune complexes compared to immune complexes from healthy or RA serum. Monocytes stimulated with isolated SF immune complexes released IL-6 and IL-8, which correlated with the levels of ACPA IgA levels in SF. Osteoclasts cultured in the presence of supernatant of IgA-activated monocytes resorbed significantly more bone compared to osteoclasts that were cultured in supernatant of IgG-activated monocytes (p=0.0233). Osteoclasts expressed the Fc receptor for IgA (FcαRI; CD89) and Fc gamma receptors. IgA-activated osteoclasts however produced significantly increased levels of IL-6 (p<0.0001) and IL-8 (p=0.0007) compared to IgG-activated osteoclasts. Both IL-6 (p=0.03) and IL-8 (p=0.0054) significantly enhanced bone resorption by osteoclasts.ConclusionIgA autoantibodies induce release of IL-6 and IL-8 by immune cells as well as osteoclasts, which enhances bone resorption by osteoclasts. We anticipate that this will result in more severe disease activity in RA patients. Targeting IgA-FcαRI interactions therefore represents a promising novel therapeutic strategy for RA patients with IgA autoantibodies.

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Asparaginase immune complexes induce Fc-γRIII-dependent hypersensitivity in naive mice.

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