Oral immunotherapy induces IgG antibodies that act through FcγRIIb to suppress IgE-mediated hypersensitivity.

BACKGROUND: Food-induced anaphylaxis is triggered by specific IgE antibodies. Paradoxically, some subjects with significant IgE levels can ingest allergenic foods without incident. Similarly, subjects completing oral immunotherapy (OIT) tolerate food challenges despite persistent high-titer food-specific IgE. OBJECTIVE: We sought to test whether IgG antibodies induced by food immunotherapy prevent food-induced anaphylaxis and whether this occurs through the inhibitory receptor FcγRIIb. METHODS: Food allergy-susceptible Il4raF709 mice were enterally sensitized to ovalbumin (OVA). Similarly sensitized IgE-deficient (IgE(-/-)) Il4raF709 mice, which can ingest OVA without anaphylaxis, were subjected to a high-dose enteral OVA desensitization protocol (OIT). Sera from both groups were tested for the ability to activate or inhibit bone marrow mast cells (BMMCs) exposed to allergen or to passively transfer allergy to naive hosts. In parallel experiments sera obtained from patients with peanut allergy before and after undergoing OIT were interrogated for their ability to enhance or suppress peanut-induced activation in an indirect assay by using basophils from nonallergic donors. RESULTS: Il4raF709 mice exhibited strong OVA-specific IgE responses. Their sera efficiently sensitized BMMCs for activation by antigen challenge. Sera from Il4raF709/IgE(-/-) mice subjected to OVA OIT suppressed BMMC responses. This inhibition was IgG mediated and FcγRIIb dependent. Similarly, pre-OIT but not post-OIT sera from patients efficiently sensitized basophils for peanut-induced activation. IgG antibodies in post-OIT sera suppressed basophil activation by pre-OIT sera. This inhibition was blocked by antibodies against FcγRII. CONCLUSION: Food-specific IgG antibodies, such as those induced during OIT, inhibit IgE-mediated reactions. Strategies that favor IgG responses might prove useful in the management of food allergy.

Immunoglobulin E signal inhibition during allergen ingestion leads to reversal of established food allergy and induction of regulatory T cells.

Immunoglobulin E (IgE) antibodies are known for triggering immediate hypersensitivity reactions such as food anaphylaxis. In this study, we tested whether they might additionally function to amplify nascent antibody and T helper 2 (Th2) cell-mediated responses to ingested proteins and whether blocking IgE would modify sensitization. By using mice harboring a disinhibited form of the IL-4 receptor, we developed an adjuvant-free model of peanut allergy. Mast cells and IgE were required for induction of antibody and Th2-cell-mediated responses to peanut ingestion and they impaired regulatory T (Treg) cell induction. Mast-cell-targeted genetic deletion of the FcεRI signaling kinase Syk or Syk blockade also prevented peanut sensitization. In mice with established allergy, Syk blockade facilitated desensitization and induction of Treg cells, which suppressed allergy when transferred to naive recipients. Our study suggests a key role for IgE in driving Th2 cell and IgE responses while suppressing Treg cells in food allergy.

An IgE receptor mimetic peptide (PepE) protects mice from IgE mediated anaphylaxis.

Crosslinking of receptor-bound Immunoglobulin E (IgE) triggers immediate hypersensitivity reactions including anaphylaxis. Blocking the interaction of IgE with its high-affinity receptor, FcεRI, on mast cells and basophils is an attractive strategy for the treatment of allergies. This approach has seen clinical success using the anti-IgE monoclonal antibody, omalizumab. We recently designed and characterized a novel FcεRI-mimetic peptide (PepE) which contains the two key FcεRI α-chain receptor loops known to interact with the ε-heavy chain of IgE, C'-E and B-C, with an optimized linker for joining them. PepE has high specificity and affinity for IgE, blocks IgE binding to FcεRI and prevents IgE-induced mediator release from RBL2H3 cells. We have now investigated the biological effects of this peptide in vivo using a line of mice (BALB/c Il4raF709) very sensitive to IgE-mediated systemic anaphylaxis. IgE-deficient (IgE-/-) Il4raF709 mice were passively sensitized with the anti-DNP IgE monoclonal antibody (SPE-7) and subsequently challenged i.v. with DNP-BSA. Mice receiving a single dose of PepE prior to sensitization with SPE-7 IgE were fully protected from anaphylaxis while vehicle control-treated mice displayed strong reactions with significant core body temperature drops and elevated levels of mouse mast cell protease-1 (mMCP-1) in the serum. However, PepE had no effect on IgE-mediated anaphylaxis if given after IgE administration in IgE-/- mice, suggesting that PepE can block binding of free IgE to FcεRI but cannot compete with the receptor for already bound IgE in vivo. A single dose of PepE treatment did not protect IgE sufficient mice from IgE mediated anaphylaxis. However, a 3 week long course of PepE treatment protected IgE sufficient Il4raF709 mice from body temperature drops and elevation of serum mMCP-1. Our findings establish the potential of this type of structure for blocking IgE binding to mast cells in vivo and suggest that related peptides might have the potential to attenuate clinical allergic reactions.

Mast cell deficiency in Kit(W-sh) mice does not impair antibody-mediated arthritis.

We previously reported that joint swelling, synovial thickening, and cartilage matrix depletion induced by the injection of anti-collagen monoclonal antibodies and lipopolysaccharide (LPS) in BALB/c mice are increased in the absence of inhibitory leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4; formerly gp49B1) in a neutrophil-dependent manner. Because both mast cells and neutrophils express LILRB4, we sought a mast cell requirement with mast cell-deficient mouse strains, but unexpectedly obtained full arthritis in Kit(W-sh) mice and full resistance in Kit(W/KitW-v) mice. Kit(W-sh) mice were indeed mast cell deficient as assessed by histology and the absence of IgE/mast cell-dependent passive cutaneous anaphylaxis in the ear and joint as well as passive systemic anaphylaxis. Deletion of LILRB4 in Kit(W-sh) mice exacerbated anti-collagen/LPS-induced joint swelling that was abolished by neutrophil depletion, establishing a counterregulatory role for LILRB4 in the absence of mast cells. Whereas blood neutrophil levels and LPS-elicited tissue neutrophilia were equal in Kit(W-sh) and Kit+ mice, both were impaired in Kit(W/KitW-v) mice. Although both strains are mast cell deficient and protected from IgE-mediated anaphylactic reactions, their dramatically different responses to autoantibody-mediated, neutrophil-dependent immune complex arthritis suggest that other host differences determine the extent of mast cell involvement. Thus, a conclusion for an absolute mast cell role in a pathobiologic process requires evidence from both strains.

gp49B1 deficiency is associated with increases in cytokine and chemokine production and severity of proliferative synovitis induced by anti-type II collagen mAb.

Mice with a disrupted gp49B gene, which encodes gp49B1 that is expressed on certain hematopoietic cells and has two immunoreceptor tyrosine-based inhibitory motifs (ITIM), exhibit augmented FcepsilonRI-initiated mast cell degranulation and resultant tissue edema. gp49B1-deficient (gp49B(-/-)) mice also exhibit exaggerated lipopolysaccharide (LPS)-induced intravascular neutrophil aggregation leading to cutaneous microangiopathy. To determine whether gp49B(-/-) mice exhibit elevated cytokine and chemokine levels leading to pathologic inflammation, we quantified clinical and morphologic parameters of arthritis and tissue levels of contributory mediators in gp49B(-/-) and gp49B1-sufficient (gp49B(+/+)) mice injected with anti-type II collagen monoclonal antibody (mAb) and LPS. Clinical scores for joint swelling and histological assessments of synovial thickness and cartilage matrix depletion at day 7 were significantly 2.3- to 2.5-fold greater and were more prolonged in gp49B(-/-) mice. At day 5, the amounts of IL-1beta, macrophage inflammatory protein (MIP)-1alpha, and MIP-2 were 2.1-, 2.5-, and 12-fold greater in joint extracts from gp49B(-/-) mice. A significant 2.7-fold more neutrophils infiltrated the synovium of gp49B(-/-) mice at day 7, and neutrophilia persisted with the delayed resolution of the synovitis. mAb-mediated depletion of neutrophils prevented the synovitis in both strains. Thus, gp49B1 counter-regulates the cytokine and chemokine induction and attendant neutrophilia that are all essential for synovitis and cartilage matrix depletion.

Prevention of lipopolysaccharide-induced microangiopathy by gp49B1: evidence for an important role for gp49B1 expression on neutrophils.

gp49B1 is expressed on mast cells and inhibits immunoglobulin E-dependent activation and inflammation in vivo. We now show that gp49B1 is expressed on neutrophils and prevents neutrophil-dependent vascular injury in response to lipopolysaccharide (LPS). The intradermal (i.d.) injection of LPS into gp49B1-null (gp49B-/-) but not gp49B1-sufficient (gp49B+/+) mice elicited macroscopic hemorrhages by 24 h, which were preceded on microscopic analyses by significantly more intravascular thrombi (consisting of neutrophils, platelets, and fibrin) that occluded venules and by more tissue neutrophils than in gp49B+/+ mice. However, there were no differences in the number of intact (nondegranulating) mast cells or the tissue levels of mediators that promote neutrophil recruitment. Hemorrhage was prevented by depleting neutrophils, blocking beta2 integrin-intercellular adhesion molecule 1 interactions, or inhibiting coagulation. These characteristics indicate that gp49B-/- mice are exquisitely sensitive to a local Shwartzman reaction (LSR) after a single i.d. injection of LPS, whereas in the classic LSR, a second exposure is required for increased beta2 integrin function, intravascular neutrophil aggregation, formation of occlusive thrombi, and hemorrhage. Moreover, LPS increased gp49B1 expression on neutrophils in vivo. The results suggest that gp49B1 suppresses the LPS-induced increase in intravascular neutrophil adhesion, thereby providing critical innate protection against a pathologic response to a bacterial component.

gp49B1 suppresses stem cell factor-induced mast cell activation-secretion and attendant inflammation in vivo.

We report that gp49B1, a mast cell membrane receptor with two immunoreceptor tyrosine-based inhibitory motifs (ITIM), constitutively inhibits mast cell activation-secretion induced by stem cell factor (SCF), a tissue-derived cytokine that also regulates mast cell development. The intradermal injection of SCF into the ears of gp49B1 null (gp49B(-/-)) mice elicited approximately 4- and 2.5-fold more degranulating mast cells and tissue swelling caused by edema, respectively, than in gp49B(+/+) mice. SCF did not induce tissue swelling in mast cell-deficient mice, and the responsiveness of gp49B(-/-) mice to mast cell-associated amine and lipid mediators was unaltered. When gp49B(+/+) and gp49B(-/-) mice were pretreated with antagonists of the amines, SCF-induced tissue swelling was reduced by >90% and 60%, respectively, and it was reduced by >90% in both genotypes when a cysteinyl leukotriene receptor antagonist was also provided. Hence, the dominant contribution of secretory granule amines to SCF-induced tissue swelling is the result of gp49B1-mediated inhibition of the production of cysteinyl leukotrienes by mast cells. Our findings also provide the first example of an ITIM-bearing receptor that constitutively suppresses inflammation generated in vivo independently of the adaptive immune response by a receptor that signals through intrinsic tyrosine kinase activity rather than immunoreceptor tyrosine-based activation motifs.