Background/Purpose:

Dysregulation of neutrophil activation and function is important in the pathogenesis of various inflammatory and autoimmune rheumatic diseases (ARDs). Neutrophil dysfunction is well recognised in rheumatoid arthritis (RA), but has also been described in systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). Integrin mediated adhesion modulates several aspects of neutrophil activation and function including: cytokine production; generation of reactive oxygen species (ROS); and the release of neutrophil extracellular traps (NETs), which are associated with disease manifestations in certain ARDs. Autoantibodies from patients with ARDs have been shown to bind to and promote the formation of NETs.  Therefore, we investigated the effects of purified IgG from patients with ARDs on ROS generation, integrin mediated neutrophil adhesion and NETosis.

Methods:

Neutrophils and IgG were isolated from patients with APS (n=9), SLE (n=5), RA (n=9) or healthy controls (HC) (n=9). ROS production of phorbol 12-myristate 13-acetate (PMA)-stimulated HC and ARD neutrophils, as well as in HC neutrophils preconditioned with ARD or HC IgG, was measured using an enzymatic assay to assess hydrogen peroxide (H₂O₂) generation. Effects of purified IgG upon neutrophil adhesion to immobilised integrin ligands, including fibrinogen (macrophage-1 antigen; Mac-1/α_Mβ₂-dependent) and fibronectin (very late antigen-4; VLA-4/α₄β₁-dependent) were then determined using a fluorescent adhesion assay. NETosis of IgG-treated HC neutrophils was measured using a novel capture ELISA.

Results:

Stimulation of HC and ARD neutrophils with PMA produced similar rates of H₂O₂ generation (n=4, p=0.2286). However, exposure of HC neutrophils to SLE (n=5; p<0.001) or RA (n=9; p<0.01) IgG increased H₂O₂ production compared to HC (n=9) IgG. Adhesion of PMA-stimulated HC neutrophils to fibrinogen was increased when incubated with RA (n=9; p<0.05) or SLE (n=5; p<0.001) IgG compared to APS (n=9) and HC (n=9) IgG, which was shown to be mediated by α_Mβ₂ through the use of a specific blocking antibody. In contrast, APS IgG increased adhesion of PMA-stimulated HC neutrophils (n=9, p<0.05) to fibronectin compared with RA (n=9), SLE (n=5) and HC (n=9) IgG, that was shown to be dependent on β₁ integrins. We also demonstrated differential effects of IgG on NETosis; with APS (n=3) and SLE (n=4) IgG eliciting greater effects than HC (n=7) and RA (n=14) IgG on spontaneous NETosis of HC neutrophils (p<0.05).

Conclusion:

Neutrophils isolated from patients with ARDs did not display different rates of ROS generation compared to controls. We found however, differential activation of neutrophils using patient IgG on HC neutrophils. In particular, RA and SLE IgG increased ROS generation and neutrophil adhesion to fibrinogen in a α_Mβ₂-dependent manner, whilst APS IgG increased neutrophil adhesion to fibronectin in a α₄β₁-dependent manner. Work is currently underway to further evaluate the intrinsic differences between ARD and HC neutrophils, and to dissect the mechanisms underlying these distinctive patterns of IgG mediated neutrophil activation and their relevance to NETosis and disease pathogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/different-autoimmune-rheumatic-diease-igg-have-differential-effects-upon-neutrophil-binding-activation-and-neutrophil-extracellular-trap-formation/