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. 2012 Sep;18(9):1401-6.
doi: 10.1038/nm.2862.

Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcγRIIB and dectin-1

Christian M Karsten  1 , Manoj K Pandey, Julia Figge, Regina Kilchenstein, Philip R Taylor, Marcela Rosas, Jacqueline U McDonald, Selinda J Orr, Markus Berger, Dominique Petzold, Veroniqué Blanchard, André Winkler, Constanze Hess, Delyth M Reid, Irina V Majoul, Richard T Strait, Nathaniel L Harris, Gabriele Köhl, Eva Wex, Ralf Ludwig, Detlef Zillikens, Falk Nimmerjahn, Fred D Finkelman, Gordon D Brown, Marc Ehlers, Jörg Köhl
Affiliations

Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of FcγRIIB and dectin-1

Christian M Karsten et al. Nat Med. 2012 Sep.

Abstract

Complement is an ancient danger-sensing system that contributes to host defense, immune surveillance and homeostasis. C5a and its G protein–coupled receptor mediate many of the proinflammatory properties of complement. Despite the key role of C5a in allergic asthma, autoimmune arthritis, sepsis and cancer, knowledge about its regulation is limited. Here we demonstrate that IgG1 immune complexes (ICs), the inhibitory IgG receptor FcγRIIB and the C-type lectin–like receptor dectin-1 suppress C5a receptor (C5aR) functions. IgG1 ICs promote the association of FcγRIIB with dectin-1, resulting in phosphorylation of Src homology 2 domain–containing inositol phosphatase (SHIP) downstream of FcγRIIB and spleen tyrosine kinase downstream of dectin-1. This pathway blocks C5aR-mediated ERK1/2 phosphorylation, C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo, including peritonitis and skin blisters in experimental epidermolysis bullosa acquisita. Notably, high galactosylation of IgG N-glycans is crucial for this inhibitory property of IgG1 ICs, as it promotes the association between FcγRIIB and dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert anti-inflammatory properties beyond their impact on activating FcγRs.

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Figures

Figure 1
Figure 1. IgG1 immune complexes (IC) inhibit C5a-mediated inflammatory responses in vivo and in vitro by an FcγRIIB-dependent mechanism
Peritoneal migration of neutrophils in response to C5a (a) or OVA/anti-OVA-IC (b) ± 107.3-IC in WT, Fcer1g−/− or Fcgr2b−/−mice. Impact of 107.3-IC on (c) CD11b expression and (d) iC3b-dependent adhesion in neutrophils from Fcer1g−/− and Fcgr2b−/− mice. C5a-mediated migration of BM-derived neutrophils (e) and increase in [Ca2+]i in BM-derived cells (f) in WT, Fcer1g−/− or Fcgr2b−/− mice ± 107.3-IC. (g) Gating of BM neutrophils according to their FSC/SSC pattern (left dot-plot) and the expression of the Gr-1 marker (1st histogram on the left). Dose-dependent impact of 107.3-IC on C5a-mediated ERK1/2 phosphorylation in Gr-1+ WT BM neutrophils. (h) Effect of a C5a receptor antagonist (C5aRA) on C5a-mediated ERK1/2 phosphorylation. White histogram: ERK phosphorylation in the absence of C5a (background); grey histogram: treatment with C5a (5 × 10−7M; 1 min); magenta histogram: C5a (5 × 10−7M; 1 min) ± 107.3 IC (g) or C5aRA (h) at the indicated concentrations. Results in (c) and (g) are representative of at least three independent experiments. Values in (a) and (b), and (d-f) are means ± s.e.m. (n=5-10/group). *P<0.05, **P<0.01, ***P<0.001.
Figure 2
Figure 2. The inhibitory effect of IgG1 IC depends on Dectin-1
(a) Impact of 107.3-IC on C5a-mediated migration of BM neutrophils when Dectin-1 is blocked by laminarin (upper panel) or curdlan (lower panel) (n=3/group). (b), C5aR(CD88) and Dectin-1 expression (lower left contour-plot; isotype control shown in the lower right plot) on Ly-6Ghi BM-derived neutrophils (upper left histogram) as determined by flow cytometry. FcγRIIB expression of Ly-6Ghi neutrophils is shown in the upper right histogram. (c) BM cells that migrated towards C5a (10−8 M for 30 min) were stained on the membrane for expression of Ly-6G, Dectin-1 and C5aR and analyzed by confocal microscopy. All migrated cells are Ly-6Ghi neutrophils that express Dectin-1 and C5aR. Impact of 107.3-IC treatment on C5a-mediated chemotaxis of (d) BM-derived neutrophils from 129 wt and from Clec7a−/− mice or (e) a Clec7a−/− neutrophil cell line transfected with the Dectin-1B isoform (D1B) or with vector alone (pMXs-IZ) (d and e, n=3/group). Values are means ± s.e.m. *P<0.05, *P<0.01, *** P<0.001.
Figure 3
Figure 3. IgG1 IC activate Src kinase and promote phosphorylation of tyrosine and Syk downstream of Dectin-1 and of SHIP downstream of FcγRIIB
C5a-mediated chemotaxis of BM neutrophils ± 107.3-IC in the presence or absence of (a) specific blockade of tyrosine phosphorylation by genistein, (b) Src kinase inhibitors I or (c) II. (d) C5a-mediated chemotaxis of BM neutrophils in cells from conditional Syk knockout mice (Sykflox/flox) ± tamoxifen treatment. (e) Impact of the Syk tyrosine kinase blockade by R406 on C5a-mediated BM neutrophil chemotaxis in the presence of 107.3-IC. Values in (a-c and e) are means ± s.e.m.(n=3-5/group). *P<0.05, **P< 0.01, ***P<0.001. (f, g) Impact of 107.3-IC treatment on phosphorylation of (f) Syk (p-Syk) or (g) SHIP (p-SHIP) in BM-neutrophils from WT, Fcgr2b−/− or Clec7a−/− mice. Depicted are immunofluorescence signals in the absence of 107.3 IC (1st rows in f and g) or obtained 3 min after incubation with 107.3 IC (2nd - 4th rows in f and g). Co-localization of P-Syk and p-SHIP with FcγRIIB and Dectin-1 in response to 107.3 IC treatment is evidenced by white fluorescent neutrophils in the 2nd row of (f) and (g). The inserts show co-localization (white spots) using z-stacks of images acquired by confocal microscopy and isosurfaced using the IMARIS image analysis software. Results are representative of two (Clec7a−/−) or three independent experiments.
Figure 4
Figure 4. High Fc glycan galactosylation is critical for the inhibitory effect of Ig1 IC in vivo and drives the association of FcγRIIB and Dectin-1
(a) Peritoneal migration of neutrophils in response to C5a ± 107.3-IC in WT, Fcgr2b−/− or Clec7a−/− mice. (b) Impact of curdlan pretreatment on peritoneal neutrophil migration in response i.p injection of C5a ± HiGalH5-IC. (c) Development of cutaneous lesions (1st row), subepidermal split formation (2nd row), linear deposition of rabbit anti-type VII collagen (3rd row) and C3 (4th row) at the basement membrane in experimental EBA (d 12) in the PBS, HiGalH5- and LoGal-H5-IC treatment groups. Black arrows point towards dermal-epidermal separation. Scale bars, 100μm. (d) Clinical disease severity in the PBS, LoGalH5- and HiGalH5-IC-treated animals during the course of experimental EBA (d 4-12) as quantified by assessment of the area under the curve (AUC); values in (a), (b) and (d) are means ± s.e.m. *P<0.05, **P<0.01. (a and b, (n=5/group), (c, n=9-12/group). Time dependent change in fluorescence intensity of Cy3-HiGal- (e) and Cy3-LoGalH5-IC (f) during acceptor photobleach in the presence (top pictures) or absence (bottom pictures) of acceptor staining on BM cells. Arrows indicate the change of fluorescence intensity as compared with the starting value (marked by the black line). (g) FRET efficiency following acceptor bleaching in BM cells treated with either Cy3-HiGalH5- or Cy3-LoGalH5-IC as donor and anti-Dectin-1 AbAlexa647 as acceptor. As a negative control, Cy3-HiGalH5-IC was used as donor and anti-TLR4 AbAlexa647 as acceptor. n=number of cells evaluated.
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