Enriched blood IgG sialylation attenuates IgG-mediated and IgG-controlled-IgE-mediated allergic reactions

Abstract

IgE antibodies play a crucial role in allergic reactions, including systemic anaphylaxis, by binding to the high-affinity IgE Fc receptor FcεRI on mast cells and basophils and thereby inducing the release of inflammatory mediators.1Berings M. Karaaslan C. Altunbulakli C. Gevaert P. Akdis M. Bachert C. et al.Advances and highlights in allergen immunotherapy: on the way to sustained clinical and immunologic tolerance.J Allergy Clin Immunol. 2017; 140: 1250-1267Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar,2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,E1-E3 In contrast, allergen-specific IgG antibodies, induced also in response to allergen-specific immunotherapies, can suppress IgE-mediated anaphylaxis via allergen masking and particularly crosslinking FcεRI with the IgG inhibitory receptor FcγRIIB.1Berings M. Karaaslan C. Altunbulakli C. Gevaert P. Akdis M. Bachert C. et al.Advances and highlights in allergen immunotherapy: on the way to sustained clinical and immunologic tolerance.J Allergy Clin Immunol. 2017; 140: 1250-1267Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 3Finkelman F.D. Khodoun M.V. Strait R. Human IgE-independent systemic anaphylaxis.J Allergy Clin Immunol. 2016; 137: 1674-1680Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar,E1,E4-E6 However, when allergen levels are high, for example, medical drugs, IgG antibodies also have the potential to mediate anaphylaxis by crosslinking classical activating FcγRs, which is also controlled by FcγRIIB, on different innate immune cell types.2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 3Finkelman F.D. Khodoun M.V. Strait R. Human IgE-independent systemic anaphylaxis.J Allergy Clin Immunol. 2016; 137: 1674-1680Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 4Beutier H. Gillis C.M. Iannascoli B. Godon O. England P. Sibilano R. et al.IgG subclasses determine pathways of anaphylaxis in mice.J Allergy Clin Immunol. 2017; 139: 269-280.e7Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar,E5,E7-E14 Hence, analyzing or even enhancing the expression level of the inhibitory FcγRIIB might be a promising approach to predict or prevent IgG-mediated allergic reactions and also IgG-FcγRIIB–controlled-IgE-mediated allergic reactions. The intravenous immunoglobulin (IVIg), pooled human (hu) serum IgG from healthy donors, has been successfully used in high concentrations (1-2 g/kg) to treat patients with acute flares of inflammatory autoimmune diseases. Importantly, findings in animal autoimmune models have indicated that the therapeutic effect of IVIg/huIgG might be predominantly mediated via its Fc N-sialylated IgG subfraction (Fig 1, A).5Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1312) Google Scholar,6Maddur M.S. Kaveri S.V. Bayry J. Circulating normal IgG as stimulator of regulatory T cells: lessons from intravenous immunoglobulin.Trends Immunol. 2017; 38: 789-792Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,E15-E17 Elevating the fraction of sialylated bulk serum IgG antibodies to a certain critical level might be therefore sufficient to attenuate inflammatory autoimmune conditions.6Maddur M.S. Kaveri S.V. Bayry J. Circulating normal IgG as stimulator of regulatory T cells: lessons from intravenous immunoglobulin.Trends Immunol. 2017; 38: 789-792Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,E15,E17 Functionally, it has been indicated in mice that sialylated huIgG antibodies with irrelevant specificities (or sialylated huIgG1 Fc portions) can interact with the sugar-binding C-type lectin receptor SIGN-R1 (specific intercellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin-related 1) on marginal zone macrophages resulting in the expression of IL-33, which activates basophils to produce IL-4, which in turn upregulates FcγRIIB on effector macrophages in mice.5Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1312) Google Scholar,7Anthony R.M. Kobayashi T. Wermeling F. Ravetch J.V. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.Nature. 2011; 475: 110-113Crossref PubMed Scopus (461) Google Scholar,E15,E18 Here, we tested the capacity of high amounts of huIgG (1 g/kg) or lower amounts of highly sialylated murine (m) or huIgG subclass antibodies (10-50 mg/kg) with irrelevant specificities to enhance FcγRIIB expression on blood immune cells and to attenuate IgG-mediated anaphylaxis and IgG-FcγRIIB–controlled-IgE-mediated anaphylaxis in mice. IgG-mediated anaphylaxis was induced by intravenous injection of 200 μg of anti–2,4,6-trinitrophenyl (TNP) mIgG1 mAbs (clone H5),2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,8Oefner C.M. Winkler A. Hess C. Lorenz A.K. Holecska V. Huxdorf M. et al.Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs.J Allergy Clin Immunol. 2012; 129: 1647-1655Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar,9Hess C. Winkler A. Lorenz A.K. Holecska V. Blanchard V. Eiglmeier S. et al.T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.J Clin Invest. 2013; 123: 3788-3796Crossref PubMed Scopus (88) Google Scholar,E19-E21 followed by intravenous challenge with 20 or 25 μg of TNP-coupled ovalbumin 30 minutes later to allow formation of immune complexes (Fig 1, B). We first verified key cellular and molecular players of anti-TNP mIgG1-mediated anaphylaxis. We confirmed that Gr-1–expressing cells (containing monocytes, neutrophils, and eosinophils) are critical for induction of anaphylaxis (see Fig E1, A and B, in this article’s Online Repository at www.jacionline.org).4Beutier H. Gillis C.M. Iannascoli B. Godon O. England P. Sibilano R. et al.IgG subclasses determine pathways of anaphylaxis in mice.J Allergy Clin Immunol. 2017; 139: 269-280.e7Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar To assess the role of activating and inhibitory Fcγ receptors, we tested mice deficient in the signaling receptor subunit, the FcR γ−chain, of activating FcγRs (Fcerg1−/−), or in FcγRIIB (Fcgr2b−/−), respectively. Notably, murine IgG1 interacts only with the activating FcγRIII/FcR γ−chain complex but not with FcγRI- or FcγRIV-containing complexes.E12 Indeed, FcR γ−chain-deficient mice were protected from IgG1-mediated anaphylaxis (Fig E1, C), whereas animals lacking the inhibitory receptor FcγRIIB showed exacerbated symptoms compared with controls (Fig E1, D).2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,4Beutier H. Gillis C.M. Iannascoli B. Godon O. England P. Sibilano R. et al.IgG subclasses determine pathways of anaphylaxis in mice.J Allergy Clin Immunol. 2017; 139: 269-280.e7Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar To test the effect of bulk IgG Fc sialylation on IgG-mediated anaphylaxis, we injected intraperitoneally high amounts of huIgG (IVIg; 1 g/kg) or intavenously lower amounts of highly galactosylated plus sialylated (sialylated; sial) versus desialylated plus degalactosylated (degal) mIgG1 mAbs (clone MOPC-21 [50 mg/kg]E22 or clone MRC OX-7 [10 mg/kg]9Hess C. Winkler A. Lorenz A.K. Holecska V. Blanchard V. Eiglmeier S. et al.T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.J Clin Invest. 2013; 123: 3788-3796Crossref PubMed Scopus (88) Google Scholar,E23) with irrelevant specificities 23.5 hours before induction of the 30-minute anti-TNP mIgG1-mediated anaphylaxis model described above (Fig 1, B and C; see Fig E2, A and B, in this article’s Online Repository at www.jacionline.org). The unspecific huIgG as well as sialylated mIgG1 antibodies attenuated the anti-TNP mIgG1-mediated anaphylaxis, whereas the degal mIgG1 antibodies had no effect (Fig 1, D and F, and Fig E2, B and C). Notably, the IgG sialylation-mediated inhibition required SIGN-R1 (Fig 1, E and F) and FcγRIIB (Fig 1, G), whereas SIGN-R1 had no influence on the anti-TNP mIgG1-mediated anaphylaxis itself (Fig E2, D). We could not revoke the SIGN-R1–dependent effect with anti–IL-4 or anti–IL-33R blocking antibodies (data not shown), suggesting further as yet unclear SIGN-R1–dependent inhibitory pathways. Interestingly, sialylated mIgG2a mAbs (clone C1.18.4; 50 mg/kg) with irrelevant specificitiesE24 failed to suppress the anti-TNP mIgG-mediated anaphylaxis (Fig 1, H, and Fig E2, E), further indicating that, in contrast to mIgG1 or mIgG2b,2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,8Oefner C.M. Winkler A. Hess C. Lorenz A.K. Holecska V. Huxdorf M. et al.Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs.J Allergy Clin Immunol. 2012; 129: 1647-1655Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar,9Hess C. Winkler A. Lorenz A.K. Holecska V. Blanchard V. Eiglmeier S. et al.T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.J Clin Invest. 2013; 123: 3788-3796Crossref PubMed Scopus (88) Google Scholar,E20,E21,E25,E26 effector functions of mIgG2a antibodies might be less dependent on Fc glycosylation.2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,E25,E27 Instead, in vitro galactosylated plus sialylated purified serum huIgG4 attenuated the anti-TNP mIgG1-induced anaphylaxis (Fig E2, F and G). We further observed that sole intraperitoneal injection of huIgG (IVIg; 1 g/kg) or intravenous injection of sialylated mIgG1 (clone MOPC-21; 50 mg/kg), but not IgG2a (clone C1.18.4, 50 mg/kg), mAbs upregulated FcγRIIB expression on blood classical monocytes and eosinophils, which was detected by flow cytometry 24 hours later (Fig 1, I and J). FcγRIIB expression on murine neutrophils was too low to identify significant differences (data not shown). Importantly, timing, rather than antigen specificity, is critical for inhibitory effects of sialylated mIgG1 antibodies. Indeed, when applied 1 day in advance, both mIgG1 antibodies with irrelevant specificity (Fig 1, F) as well as antigen-specific (ie, anti-TNP) mIgG1 antibodies (see Fig E3, A-C, in this article’s Online Repository at www.jacionline.org),2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar when sialylated, suppressed anti-TNP mIgG1-mediated anaphylaxis. In contrast, Fc sialylation had no significant effect on the ability of antigen-specific mIgG1 antibodies to induce anaphylactic reactions in the 30-minute model (Fig E3, D and E). We therefore conclude that this delay might be critical for modulating FcγRIIB expression in an IgG Fc sialylation–dependent manner. Similarly, sialylated mIgG1 (clone MOPC-21; 50 mg/kg) with irrelevant specificity enhanced the FcγRIIB-dependent2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar inhibitory effect of antigen-specific mIgG1 antibodies in an IgE-mediated anaphylaxis model (Fig 2, A-C). These observations suggested that sialylated IgG with irrelevant specificity might also upregulate FcγRIIB expression on FcεRI-expressing blood cells. Indeed, we observed an upregulation of FcγRIIB on FcεRI-expressing blood basophils 24 hours after application of huIgG or sialylated muIgG1, but not sialylated mIgG2a antibodies, with irrelevant specificities, in a SignR1-dependent manner (Fig 2, D). Besides IVIg treatment, physiological levels of IgG Fc sialylation already vary between individuals in the blood,6Maddur M.S. Kaveri S.V. Bayry J. Circulating normal IgG as stimulator of regulatory T cells: lessons from intravenous immunoglobulin.Trends Immunol. 2017; 38: 789-792Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar which might in turn modulate the expression of Fcγ receptors on immune cells and, in turn, influence an individual’s susceptibility to IgG-mediated or IgG-FcγRIIB–controlled-IgE-mediated allergic reactions or other IgG antibody–mediated diseases. We therefore assessed in a pilot study serum IgG Fc glycosylation and the expression levels of the inhibitory receptor FcγRIIB and the activating FcγRIIA, FcγRIII(A+B), and FcεRI on peripheral human blood neutrophils, monocytes, basophils, and/or B cells of 36 volunteers (24 females and 12 males; for details, see this article’s Methods section in the Online Repository at www.jacionline.org). Because IgG sialylation levels are distinct between the 2 sexes, we analyzed male and female data separately. Intriguingly, we identified significant positive associations between the levels of serum IgG Fc sialylation and the expression levels of FcγRIIB on neutrophils in female donors and on basophils in male donors (Fig 2, E-G; see Tables E1 and E2 and Fig E4 in this article’s Online Repository at www.jacionline.org). Although a larger cohort of donors is required to clearly establish the connection between IgG sialylation and FcγRIIB expression in human leukocytes, these results are well in line with our findings in mice. The data suggest that natural or modified levels of blood IgG Fc N-sialylation regulate the expression level of the inhibitory receptor FcγRIIB on immune cells and might protect individuals via this way not only from inflammatory autoimmune conditions but also from IgG-mediated as well as IgG-FcγRIIB–controlled-IgE-mediated allergic reactions. Thus, IgG-inducing allergen-specific immunotherapy in patients suffering from IgE-mediated allergic diseases might be more promissing and successful in patients showing higher bulk serum IgG sialylation levels, which has to be investigated. Although this effect of bulk serum IgG Fc sialylation was dependent on SIGN-R1 in mice, we could not revoke this effect with anti–IL-4 or anti–IL-33R blocking antibodies, suggesting further as yet unclear SIGN-R1–dependent inhibitory pathways to upregulate FcγRIIB. Interestingly, this inhibitory effect of bulk serum IgG sialylation was mediated by mIgG1, but not mIgG2a antibodies, suggesting IgG subclass–specific roles in mice. These findings might explain earlier observations of protective effects of IVIg in the context of allergyE27-E29 and might help to develop diagnostic, prognostic, and/or therapeutic tools for controlling IgG-mediated as well as IgG-FcγRIIB–controlled-IgE-mediated allergic reactions and IgG-dependent inflammatory disorders in general. We thank Mattias Collin for providing EndoS, Birgitta Heyman for the anti-TNP IgG1 (clone H5) hybridoma cells, Rudolf Manz for the anti–GR-1 mAbs (clone RB6-8C5), and Robina Thurmann and Kathleen Kuhrwahn for technical assistance. Human blood from healthy donors was collected with the approval of and in accordance with regulatory guidelines and ethical standards set by the University of Lübeck. 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