Friederike Jönsson

Mouse and human neutrophils induce anaphylaxis

Anaphylaxis is a life-threatening hyperacute immediate hypersensitivity reaction. Classically, it depends on IgE, FcεRI, mast cells, and histamine. However, anaphylaxis can also be induced by IgG antibodies, and an IgG1-induced passive type of systemic anaphylaxis has been reported to depend on basophils. In addition, it was found that neither mast cells nor basophils were required in mouse models of active systemic anaphylaxis. Therefore, we investigated what antibodies, receptors, and cells are involved in active systemic anaphylaxis in mice. We found that IgG antibodies, FcγRIIIA and FcγRIV, platelet-activating factor, neutrophils, and, to a lesser extent, basophils were involved. Neutrophil activation could be monitored in vivo during anaphylaxis. Neutrophil depletion inhibited active, and also passive, systemic anaphylaxis. Importantly, mouse and human neutrophils each restored anaphylaxis in anaphylaxis-resistant mice, demonstrating that neutrophils are sufficient to induce anaphylaxis in mice and suggesting that neutrophils can contribute to anaphylaxis in humans. Our results therefore reveal an unexpected role for IgG, IgG receptors, and neutrophils in anaphylaxis in mice. These molecules and cells could be potential new targets for the development of anaphylaxis therapeutics if the same mechanism is responsible for anaphylaxis in humans.

Mouse and human FcR effector functions

Mouse and human FcRs have been a major focus of attention not only of the scientific community, through the cloning and characterization of novel receptors, and of the medical community, through the identification of polymorphisms and linkage to disease but also of the pharmaceutical community, through the identification of FcRs as targets for therapy or engineering of Fc domains for the generation of enhanced therapeutic antibodies. The availability of knockout mouse lines for every single mouse FcR, of multiple or cell‐specific—‘à la carte’—FcR knockouts and the increasing generation of hFcR transgenics enable powerful in vivo approaches for the study of mouse and human FcR biology. This review will present the landscape of the current FcR family, their effector functions and the in vivo models at hand to study them. These in vivo models were recently instrumental in re‐defining the properties and effector functions of FcRs that had been overlooked or discarded from previous analyses. A particular focus will be made on the (mis)concepts on the role of high‐affinity IgG receptors in vivo and on results from antibody engineering to enhance or abrogate antibody effector functions mediated by FcRs.

Fcγ Receptors Inhibit Mouse and Human Basophil Activation

Besides high-affinity IgE receptors (FcεRI), human basophils express activating (FcγRIIA) and inhibitory (FcγRIIB) low-affinity IgG receptors. IgG receptors (FcγR) were also found on mouse basophils, but not identified. We investigated in this study FcγR and the biological consequences of their engagement in basophils of the two species. We found the following: 1) that mouse basophils also express activating (FcγRIIIA) and inhibitory (FcγRIIB) low-affinity FcγR; 2) that activating FcγR can activate both human and mouse basophils, albeit with different efficacies; 3) that negative signals triggered by inhibitory FcγR are dominant over positive signals triggered by activating FcγR, thus preventing both human and mouse basophils from being activated by IgG immune complexes; 4) that the coengagement of FcεRI with inhibitory and activating FcγR results in a FcγRIIB-dependent inhibition of IgE-induced responses of both human and mouse basophils; 5) that FcγRIIB has a similar dominant inhibitory effect in basophils from virtually all normal donors; and 6) that IL-3 upregulates the expression of both activating and inhibitory FcγR on human basophils from normal donors, but further enhances FcγRIIB-dependent inhibition. FcγR therefore function as a regulatory module, made of two subunits with antagonistic properties, that prevents IgG-induced and controls IgE-induced basophil activation in both mice and humans.

The high-affinity human IgG receptor FcγRI (CD64) promotes IgG-mediated inflammation, anaphylaxis, and antitumor immunotherapy.

Receptors for the Fc portion of IgG (FcγRs) are mandatory for the induction of various IgG-dependent models of autoimmunity, inflammation, anaphylaxis, and cancer immunotherapy. A few FcγRs have the ability to bind monomeric IgG: high-affinity mouse mFcγRI, mFcγRIV, and human hFcγRI. All others bind IgG only when aggregated in complexes or bound to cells or surfaces: low-affinity mouse mFcγRIIB and mFcγRIII and human hFcγRIIA/B/C and hFcγRIIIA/B. Although it has been proposed that high-affinity FcγRs are occupied by circulating IgG, multiple roles for mFcγRI and mFcγRIV have been reported in vivo. However, the potential roles of hFcγRI that is expressed on monocytes, macrophages, and neutrophils have not been reported. In the present study, we therefore investigated the role of hFcγRI in antibody-mediated models of disease and therapy by generating hFcγRI-transgenic mice deficient for multiple endogenous FcRs. hFcγRI was sufficient to trigger autoimmune arthritis and thrombocytopenia, immune complex-induced airway inflammation, and active and passive systemic anaphylaxis. We found monocyte/macrophages to be responsible for thrombocytopenia, neutrophils to be responsible for systemic anaphylaxis, and both cell types to be responsible for arthritis induction. Finally, hFcγRI was capable of mediating antibody-induced immunotherapy of metastatic melanoma. Our results unravel novel capabilities of human FcγRI that confirm the role of high-affinity IgG receptors in vivo.

Human FcγRIIA induces anaphylactic and allergic reactions

IgE and IgE receptors (FcεRI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (FcγRIIIA and FcγRIV) expressed by neutrophils, rather than on IgE and FcεRI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express FcγRIIIA or FcγRIV, but FcγRIIA. We therefore investigated the possible role of FcγRIIA in allergy by generating novel FcγRIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, FcγRIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking FcγRIIA in vivo abolished these reactions. We identified mast cells to be responsible for FcγRIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for FcγRIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after FcγRIIA engagement. IgG and FcγRIIA may therefore contribute to allergic and anaphylactic reactions in humans.

Cutting Edge: The Murine High-Affinity IgG Receptor FcγRIV Is Sufficient for Autoantibody-Induced Arthritis

K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient Wsh mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.

Neutrophils mediate antibody-induced antitumor effects in mice

Tumor engraftment followed by monoclonal antibody (mAb) therapy targeting tumor antigens represents a gold standard for assessing the efficiency of mAbs to eliminate tumor cells. Mouse models have demonstrated that receptors for the Fc portion of immunoglobulin G (FcγRs) are critical determinants of mAb therapeutic efficacy, but the FcγR-expressing cell populations responsible remain elusive. We show that neutrophils are responsible for mAb-induced therapy of both subcutaneous syngeneic melanoma and human breast cancer xenografts. mAb-induced tumor reduction, abolished in neutropenic mice, could be restored in FcγR-deficient hosts upon transfer of FcγR+ neutrophils or upon human FcγRIIA/CD32A transgenic expression. Finally, conditional knockout mice unable to perform FcγR-mediated activation and phagocytosis specifically in neutrophils were resistant to mAb-induced therapy. Our work suggests that neutrophils are necessary and sufficient for mAb-induced therapy of subcutaneous tumors, and represent a new and critical focal point for optimizing mAb-induced immunotherapies that will impact on human cancer treatment.

Human Basophils Express the Glycosylphosphatidylinositol-Anchored Low-Affinity IgG Receptor FcγRIIIB (CD16B)

Basophils express not only high-affinity IgE receptors, but also low-affinity IgG receptors. Which, among these receptors, are expressed by human basophils is poorly known. Low-affinity IgG receptors comprise CD32 (FcγRIIA, FcγRIIB, and FcγRIIC) and CD16 (FcγRIIIA and FcγRIIIB). FcγRIIA, FcγRIIC, and FcγRIIIA are activating receptors, FcγRIIB are inhibitory receptors, FcγRIIIB are GPI-anchored receptors whose function is poorly understood. Basophils were reported to express FcγRII, but not FcγRIII. We aimed at further identifying basophil IgG receptors. Basophils from normal donors and from patients suffering from an allergic skin disease (atopic dermatitis), allergic respiratory diseases (allergic rhinitis and asthma), or a nonallergic skin disease (chronic urticaria) were examined. We found that normal basophils contain FcγRIII transcripts and express FcγRIIIB, but not FcγRIIIA, which were detected on 24–81% basophils from normal donors and on 12–100% basophils from patients. Noticeably, the proportion of FcγRIIIB+ basophils was significantly lower in atopic dermatitis patients than in other subjects. This decreased FcγRIII expression was not correlated with an activated phenotype of basophils in atopic dermatitis patients, although FcγRIIIB expression was down-regulated upon basophil activation by anti-IgE. Our results challenge the two dogmas 1) that basophils do not express FcγRIII and 2) that FcγRIIIB is exclusively expressed by neutrophils. They suggest that a proportion of basophils may be lost during enrichment procedures in which FcγRIII+ cells are discarded by negative sorting using anti-CD16 Abs. They unravel an unexpected complexity of IgG receptors susceptible to modulate basophil activation. They identify a novel systemic alteration in atopic dermatitis.

Contribution of Human FcγRs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies.

The biological activities of human IgG antibodies predominantly rely on a family of receptors for the Fc portion of IgG, FcγRs: FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, FcγRIIIB, FcRL5, FcRn, and TRIM21. All FcγRs bind IgG at the cell surface, except FcRn and TRIM21 that bind IgG once internalized. The affinity of FcγRs for IgG is determined by polymorphisms of human FcγRs and ranges from 2 × 104 to 8 × 107 M−1. The biological functions of FcγRs extend from cellular activation or inhibition, IgG-internalization/endocytosis/phagocytosis to IgG transport and recycling. This review focuses on human FcγRs and intends to present an overview of the current understanding of how these receptors may contribute to various pathologies. It will define FcγRs and their polymorphic variants, their affinity for human IgG subclasses, and review the associations found between FcγR polymorphisms and human pathologies. It will also describe the human FcγR-transgenic mice that have been used to study the role of these receptors in autoimmune, inflammatory, and allergic disease models.

Mast Cells and Company

Classically, allergy depends on IgE antibodies and on high-affinity IgE receptors expressed by mast cells and basophils. This long accepted IgE/FcεRI/mast cell paradigm, on which the definition of immediate hypersensitivity was based in the Gell and Coomb’s classification, appears too reductionist. Recently accumulated evidence indeed requires that not only IgE but also IgG antibodies, that not only FcεRI but also FcγR of the different types, that not only mast cells and basophils but also neutrophils, monocytes, macrophages, eosinophils, and other myeloid cells be considered as important players in allergy. This view markedly changes our understanding of allergic diseases and, possibly, their treatment.