Background IgG4-related disease (IgG4-RD) is a newly recognized fibroinflammatory condition characterized by infiltration of IgG4-positive plasma cells into the pancreas, biliary tracts, or salivary glands. In addition to helper type 2 T cell-dominant immune reactions, it is reported that innate immune responses such as the activation of monocytes and basophils are involved in the pathogenesis of IgG4-RD. In the last EULAR congress, we demonstrated that neutrophil extracellular traps (NETs) activate plasmacytoid dendritic cells (pDCs), which in turn produce significantly large amounts of interferon (IFN)-α and B cell activating factor (BAFF) and lead to B cell maturation and IgG4-positive plasma cell induction in vitro. A specific stimulant that induces such innate immune responses has yet to be determined. We assume the involvement of autoantigens and autoantibodies in these responses.
Objectives Among many IgG4-RD autoantigen candidates, we focused on lactoferrin, one of the antimicrobial granule proteins that configure NETs. In this study, we aimed to determine the contribution of anti-lactoferrin antibody to NET formation and pDC activation in IgG4-RD.
Methods We enrolled 15 patients with newly diagnosed IgG4-RD. Serum levels of anti-lactoferrin antibody were measured by performing enzyme-linked immunosorbent assays (ELISA). Serum NET values were measured by using a Quant-iT PicoGreen dsDNA assay kit. Neutrophils were isolated from the peripheral blood of healthy controls and incubated with anti-lactoferrin IgG antibody or control IgG. After incubation, extracellular dsDNA was stained with Sytox, while NET formation was visualized with a laser-scanning fluorescence confocal microscopy and then quantified in a fluorometer after digestion with micrococcal nuclease.
Results Patient-derived serum showed significantly higher levels of IgG antibodies for lactoferrin than those derived from controls (mean ± standard error, 2.53±0.20 vs. 1.00±0.13 arbitrary units, p <0.01). Anti-lactoferrin antibodies of each IgG1 and IgG4 subclass were significantly elevated in the patients. Patient serum showed the tendency of higher NET values than those of controls (116.0±14.2 vs. 88.2±3.87 ng/mL, p =0.08). Neutrophils incubated with anti-lactoferrin IgG antibody formed large amounts of NETs, while NET formation was not observed in incubation with control IgG antibody. Measurement of NETs in the suspension showed significantly higher values in neutrophils stimulated with anti-lactoferrin IgG antibody than those stimulated with control IgG (305.4±61.1 vs. 77.2±4.5 arbitrary units, p =0.02).
Conclusions Here we showed that anti-lactoferrin antibodies in IgG4-RD induced NET formation. We also demonstrated that serum NET levels were higher in IgG4-RD patients than in controls. Our findings suggest that immune complexes of autoantibodies for lactoferrin and NET-derived lactoferrins stimulate neutrophils to create additional NETs. Released NETs can stimulate pDCs, which induce IgG4-producing plasma cells through IFN-α and BAFF secretion. This vicious cycle may be important to the pathogenesis of IgG4-RD. Thus, lactoferrin is possibly a key molecule and may be a potential therapeutic target in the near future.
Yamamoto M, et al. Nat Rev Rheumatol. 2013 (Epub ahead of print).
Disclosure of Interest : None declared