Mechanisms of allergy and clinical immunology
A mouse Fcγ-Fcε protein that inhibits mast cells through activation of FcγRIIB, SH2 domain–containing inositol phosphatase 1, and SH2 domain–containing protein tyrosine phosphatases

https://doi.org/10.1016/j.jaci.2007.08.051Get rights and content

Background

A human Fcγ-Fcε fusion protein (GE2) designed to inhibit FcεRI signaling by coaggregating FcεRI with the inhibitory receptor FcγRIIB has been shown to inhibit mast cell activation and block cutaneous anaphylaxis. A critical issue remained as to whether the mechanism of GE2 inhibition is competition for IgE binding or inhibitory signaling through FcγRIIB.

Objective

Our aim was to define the in vitro and in vivo mechanism of action of a mouse homolog of GE2 (mGE) and to assess the potential of human GE2 (hGE2) for therapeutic administration.

Methods

The in vitro activity of mGE on mediator release and signaling pathways was characterized in IgE-sensitized bone marrow–derived mast cells (BMMCs). The in vivo activity of mGE was examined in mouse passive cutaneous and passive systemic anaphylaxis models, and the therapeutic activity of hGE2 was evaluated in Ascaris suum–sensitized cynomolgus monkeys.

Results

mGE inhibited release of histamine and cytokines by BMMCs from wild-type mice but not by BMMCs from FcγRIIB-deficient mice. In mice mGE blocked IgE-dependent anaphylaxis mediated by mast cells with sustained efficacy. In BMMCs mGE decreased spleen tyrosine kinase and extracellular signal-regulated kinases 1/2 phosphorylation and induced FcγRIIB phosphorylation and the subsequent recruitment of SH2 domain–containing inositol polyphosphate 5′ phosphatase (SHIP) 1 and SH2 domain–containing protein tyrosine phosphatase (SHP) 1/2 phosphatases. When administered therapeutically, hGE2 protected sensitized monkeys from local anaphylaxis for 3 weeks.

Conclusion

mGE-mediated inhibition of mast cell activation is associated with inhibitory signaling through FcγRIIB that results from activation of SHIP-1 and SHP-1/2 phosphatases.

Section snippets

Cells and antibodies

Bone marrow cells from BALB/c or C57BL/6 mice were cultured for 5 to 12 weeks in complete medium (RPMI 1640; ATCC, Manassas, Va) containing 10% FBS (HyClone, Logan, Utah), 10 μg/mL gentamicin (Sigma-Aldrich, St Louis, Mo), 55 μmol/L β-mercaptoethanol (Invitrogen, Carlsbad, Calif), and recombinant IL-3 (10 ng/mL; R&D Systems, Minneapolis, Minn). FcγRIIB−/− mice on a BALB/c background were purchased from Taconic Farms (Hudson, NY). Animal experiments were approved by the Biogen Idec (San Diego,

mGE inhibits IgE-dependent BMMC activation

We generated a mouse homolog of the human GE2 fusion protein, designated mGE, consisting of mouse IgG2a-Fc and IgE-Fc regions linked by a glycine/serine peptide. By means of flow cytometry, mGE bound to To test mGE activity in vitro, BMMCs exclusively through FcεRI, which is consistent with the low affinity of FcγRIIB for monomeric IgG (data not shown). To test mGE activity in vitro, BMMCs were sensitized with IgE anti-TNP (10 μg/mL) in the presence of various concentrations of mGE. After

Discussion

We generated a mouse homolog of the previously investigated human GE2 protein designed to inhibit mast cell activation by cross-linking the inhibitory receptor FcγRIIB with FcεRI. mGE inhibitory activity was characterized on IgE-sensitized antigen-activated BMMCs and was confirmed to be similar to inhibition of human mast cells by human GE2.11 mGE was shown to inhibit BMMC activation at the level of mast cell release of both early- and late-phase mediators and required FcεRI binding. This is

References (26)

  • C.L. Kepley et al.

    Negative regulation of FcεRI signaling by FcγRII costimulation in human blood basophils

    J Allergy Clin Immunol

    (2000)
  • T. Kawakami et al.

    Regulation of mast-cell and basophil function and survival by IgE

    Nat Rev Immunol

    (2002)
  • J.V. Ravetch et al.

    IgG Fc receptors

    Ann Rev Immunol

    (2001)
  • Cited by (36)

    • IgE and mast cells: The endogenous adjuvant

      2020, Advances in Immunology
      Citation Excerpt :

      Consistent with this alternative theory, Shp-1 has been suggested to be the mediator of FcγRIIb's inhibitory effects in human basophils (Kepley et al., 2000; Saxon, Zhu, Zhang, Allen, & Kepley, 2004; Zhu, Kepley, Zhang, Zhang, & Saxon, 2002). Interestingly, this Fcɛ-Fcγ construct diminished phosphorylation of Syk as well as Erk, which could readily explain reductions in cytokine synthesis (Mertsching et al., 2008). Shp-1 is thought to dephosphorylate several early molecules in the FcɛRI signal transduction pathway, including Syk, LAT, SLP76 and the β/γ chains of FcɛRI itself (Nakata et al., 2008; Xie, Zhang, & Siraganian, 2000).

    • Emerging trends in bispecific antibody and scaffold protein therapeutics

      2018, Current Opinion in Chemical Engineering
      Citation Excerpt :

      Administration of immunosuppressives that may limit cytokine release is another consideration for this class of bispecifics. New mechanistic areas for bispecifics are also being considered that include targeting various cell types with enhanced specificity through affinity tuning [125,126], inducing cell death in specific tissues [29], inducing enhanced or novel activity through multi-epitope binding on the same target [58–60,100•], and in some cases by bringing receptors together, inducing novel activities [91•,127–129]. These novel bispecific mechanisms add a layer of complexity to each therapeutic compared to combination IgG therapy.

    • The high-affinity immunoglobulin e receptor as pharmacological target

      2016, European Journal of Pharmacology
      Citation Excerpt :

      Co-aggregation of FcεRI with FcγRIIB was shown to inhibit FcεRI-mediated mast cell activation (Daeron et al., 1995; Tam et al., 2004) and human basophil activation (Cady et al., 2010; Cassard et al., 2012; Tam et al., 2004). More importantly, this mechanism was operative in various models of anaphylaxis in vivo in mice and monkeys (Mertsching et al., 2008; Strait et al., 2006; Zhu et al., 2005). Many different ways to co-aggregate FcγRIIB and FcεRI have been tested.

    • IgE and Mast Cells. The Endogenous Adjuvant.

      2015, Advances in Immunology
      Citation Excerpt :

      Consistent with this alternative theory, Shp-1 has been suggested to be the mediator of FcγRIIb's inhibitory effects in human basophils (Kepley et al., 2000; Saxon, Zhu, Zhang, Allen, & Kepley, 2004; Zhu, Kepley, Zhang, Zhang, & Saxon, 2002). Interestingly, this Fcɛ–Fcγ construct diminished phosphorylation of Syk as well as Erk, which could readily explain reductions in cytokine synthesis (Mertsching et al., 2008). Shp-1 is thought to dephosphorylate several early molecules in FcɛRI signal transduction pathway, including Syk, LAT, SLP76, and the β/γ chains of FcɛRI itself (Nakata et al., 2008; Xie, Zhang, & Siraganian, 2000).

    View all citing articles on Scopus

    Disclosure of potential conflict of interest: E. Mertsching, H. Hess, K. Giza, E. Negrou, K. Hathaway, J. Chung, D. Perret, and M. R. Kehry are employed by Biogen Idec. L. Bafetti, S. Perper, and M. Shields were employed by Biogen Idec when this work was performed. A. Saxon has patent rights through his employment with the University of California.

    View full text