Antagonizing FcαR1 (CD89) as treatment in IgA-mediated chronic inflammation and autoimmunity

IntroductionImmunoglobulin A (IgA) is mostly considered as a non-inflammatory regulator at mucosal areas. However, previous work of our group showed that IgA can also be involved in disease pathology, because it provides a potent stimulus to activate neutrophils after crosslinking of surface CD89 (FcaRI), resulting in chronic inflammation and tissue damage. IgA (auto)antibodies and neutrophils are key players in various diseases, including blistering skin diseases and rheumatoid arthritis. Therefore, we generated an array of anti-CD89 monoclonal antibodies (mAbs) for therapeutic targeting of CD89. The biological activity of newly developed anti-human CD89 mAbs and their potential therapeutic capacity were investigated.MethodsHuman neutrophils were isolated from heparinized healthy donor blood. The ability of anti-CD89 mAbs to bind human neutrophils was investigated by flow cytometry. Furthermore, the capacity of these anti-CD89 mAbs to inhibit IgA-mediated phagocytosis, neutrophil extracellular trap (NET) release and migration was studied. To this end, neutrophils were pre-incubated with/without anti-CD89 mAbs after which they were stimulated with IgA-coated beads. The amount of phagocytosed beads, NET release and migrated neutrophils were subsequently analysed. In parallel, chemoattractant leukotriene B4 and lactoferrin (as a measure for degranulation) release were determined. Finally, the therapeutic potential of our prototypic anti-CD89 mAb clone 10E7 was in vivo tested in anti-mouse collagen XVII human IgA-treated transgenic CD89 mice, a preclinical model for autoimmune linear IgA bullous disease (LABD).ResultsOur results show that all generated anti-CD89 mAbs bound surface CD89 on neutrophils. Although these anti-CD89 mAbs bind to different epitopes on EC1 of CD89, they all have the capacity to inhibit IgA-mediated phagocytosis, neutrophil extracellular trap (NET) release and neutrophil migration. Moreover, IgA mediated leukotriene B4 and lactoferrin release are decreased in supernatant from anti-CD89 mAbs-treated neutrophils. Finally, anti-CD89 mAb clone 10E7, that was selected based on its selective binding profile on tissue micro arrays, reduced anti-mouse collagen XVII hIgA-induced neutrophil influx in an in vivo linear IgA bullous disease (LABD) mice model.ConclusionThis study clearly indicates that our newly developed anti-CD89 mAbs inhibited IgA-induced neutrophil activation and reduced anti-autoantigen IgA-induced neutrophil influx in vivo, supporting further clinical development for the treatment of LABD

IgA Complexes in Plasma and Synovial Fluid of Patients with Rheumatoid Arthritis Induce Neutrophil Extracellular Traps via FcαRI

Autoantibodies, including rheumatoid factor (RF), are an important characteristic of rheumatoid arthritis (RA). Interestingly, several studies reported a correlation between the presence of IgA autoantibodies and worse disease course. We demonstrated previously that triggering the IgA Fc receptor (FcαRI) on neutrophils results in neutrophil recruitment and the release of neutrophil extracellular traps (NETs). Because this can lead to tissue damage, we investigated whether IgA immune complexes in plasma and synovial fluid of RA patients activate neutrophils. RF isotypes were measured with ELISA, and immune complexes were precipitated using polyethylene glycol 6000. Isolated neutrophils were incubated with immune complexes, and activation and release of NETs were determined in the presence or absence of FcαRI-blocking Abs. Plasma and SF of RA patients contained IgM, IgG, and IgA RFs. Patient plasma IgA RF and IgM RF showed a strong correlation. No uptake of IgM and minimal endocytosis of IgG immune complexes by neutrophils was observed, in contrast to avid uptake of IgA complexes. Incubation of neutrophils with immune complexes resulted in the production of reactive oxygen species, as well as the release of NETs, lactoferrin, and chemotactic stimuli. Importantly, activation of neutrophils was reduced when FcαRI was blocked. Neutrophils were activated by IgA immune complexes, which suggests that neutrophils play a role in inducing joint damage in RA patients who have IgA autoantibody complexes, thereby increasing the severity of disease. Blocking FcαRI inhibited neutrophil activation and, as such, may represent an additional attractive novel therapeutic strategy for the treatment of RA

IgA enhances NETosis and release of neutrophil extracellular traps by polymorphonuclear cells via Fcα receptor I

Polymorphonuclear cells (neutrophils) are the first cells that arrive at sites of infections. According to the current dogma, they are involved in eliminating bacteria, after which they die through apoptosis. We now demonstrate that enhanced IgA-induced phagocytosis of bacteria or beads by neutrophils led to increased cell death. Nuclear changes and positivity for the general cell death marker 7-aminoactinomycin D were observed, but the absence of annexin V membrane staining supported that neutrophils did not die via apoptosis, in contrast to neutrophils that had not phagocytosed bacteria. Moreover, increased release of neutrophil extracellular traps (NETs) was observed, which was most likely due to augmented production of reactive oxygen species after uptake of IgA-opsonized particles. Blocking the IgA Fc receptor FcαRI abrogated phagocytosis and NET formation. Thus, FcαRI triggering on neutrophils resulted in a rapid form of cell death that is referred to as NETosis, as it is accompanied by the release of NETs. As such, IgA may play a prominent role in mucosal inflammatory responses, where it is the most prominent Ab, because it enhanced both phagocytosis of bacteria and formation of NETs, which are effective mechanisms that neutrophils employ to eliminate pathogen

Antagonizing FcαR1 (CD89) as treatment in IgA-mediated chronic inflammation and autoimmunity

Introduction: Immunoglobulin A (IgA) is mostly considered as a non-inflammatory regulator at mucosal areas. However, previous work of our group showed that IgA can also be involved in disease pathology, because it provides a potent stimulus to activate neutrophils after crosslinking of surface CD89 (FcaRI), resulting in chronic inflammation and tissue damage. IgA (auto)antibodies and neutrophils are key players in various diseases, including blistering skin diseases and rheumatoid arthritis. Therefore, we generated an array of anti-CD89 monoclonal antibodies (mAbs) for therapeutic targeting of CD89. The biological activity of newly developed anti-human CD89 mAbs and their potential therapeutic capacity were investigated. Methods: Human neutrophils were isolated from heparinized healthy donor blood. The ability of anti-CD89 mAbs to bind human neutrophils was investigated by flow cytometry. Furthermore, the capacity of these anti-CD89 mAbs to inhibit IgA-mediated phagocytosis, neutrophil extracellular trap (NET) release and migration was studied. To this end, neutrophils were pre-incubated with/without anti-CD89 mAbs after which they were stimulated with IgA-coated beads. The amount of phagocytosed beads, NET release and migrated neutrophils were subsequently analysed. In parallel, chemoattractant leukotriene B4 and lactoferrin (as a measure for degranulation) release were determined. Finally, the therapeutic potential of our prototypic anti-CD89 mAb clone 10E7 was in vivo tested in anti-mouse collagen XVII human IgA-treated transgenic CD89 mice, a preclinical model for autoimmune linear IgA bullous disease (LABD). Results: Our results show that all generated anti-CD89 mAbs bound surface CD89 on neutrophils. Although these anti-CD89 mAbs bind to different epitopes on EC1 of CD89, they all have the capacity to inhibit IgA-mediated phagocytosis, neutrophil extracellular trap (NET) release and neutrophil migration. Moreover, IgA mediated leukotriene B4 and lactoferrin release are decreased in supernatant from anti-CD89 mAbs-treated neutrophils. Finally, anti-CD89 mAb clone 10E7, that was selected based on its selective binding profile on tissue micro arrays, reduced anti-mouse collagen XVII hIgA-induced neutrophil influx in an in vivo linear IgA bullous disease (LABD) mice model. Conclusion: This study clearly indicates that our newly developed anti-CD89 mAbs inhibited IgA-induced neutrophil activation and reduced anti-autoantigen IgA-induced neutrophil influx in vivo, supporting further clinical development for the treatment of LABD

Anti-FcαRI Monoclonal Antibodies Resolve IgA Autoantibody-Mediated Disease

Immunoglobulin A (IgA) is generally considered as a non-inflammatory regulator of mucosal immunity, and its importance in diversifying the gut microbiota is increasingly appreciated. IgA autoantibodies have been found in several autoimmune or chronic inflammatory diseases, but their role in pathophysiology is ill-understood. IgA can interact with the Fc receptor FcαRI on immune cells. We now established a novel IgA autoimmune blistering model, which closely resembles the human disease linear IgA bullous disease (LABD) by using genetically modified mice that produce human IgA and express human FcαRI. Intravital microscopy demonstrated that presence of IgA anti-collagen XVII, - the auto-antigen in LABD-, resulted in neutrophil activation and extravasation from blood vessels into skin tissue. Continued exposure to anti-collagen XVII IgA led to massive neutrophil accumulation, severe tissue damage and blister formation. Importantly, treatment with anti-FcαRI monoclonal antibodies not only prevented disease, but was also able to resolve existing inflammation and tissue damage. Collectively, our data reveal a novel role of neutrophil FcαRI in IgA autoantibody-mediated disease and identify FcαRI as promising new therapeutic target to resolve chronic inflammation and tissue damage