We show here that IgG antibodies, activating FcγRs, neutrophils, and PAF are the main players in ASA, whereas IgE antibodies (12
), FcεRI (5
), mast cells (5
), and histamine do not play a major role.
Several cell types have been involved in anaphylaxis; among them are mast cells and basophils. We demonstrate that neutrophils are not only sufficient to induce ASA, but play a dominant role. Indeed, neutrophil depletion, but not basophil depletion, eosinophil depletion, monocyte/macrophage inhibition or mast cell deficiency, abrogated ASA-associated death, and reduced temperature drop in WT mice. Importantly, transfer of mouse neutrophils expressing FcγRIV, but not of neutrophils expressing no activating FcγRs, restored ASA in FcR
mice. Neutrophil activation could be visualized in vivo within minutes in mice undergoing ASA. Although monocytes/macrophages were reported to be involved in a model of anaphylaxis induced by an i.v. injection of goat IgG in mice immunized with goat IgG anti-mouse IgD (33
), we could not detect any role for monocytes/macrophages in ASA in mice immunized with antigen in CFA/IFA. The depletion of eosinophils did not impair ASA either. Basophil depletion, which did not affect ASA by itself, further reduced ASA when combined with neutrophil depletion. Basophils therefore contribute to ASA together with neutrophils, but neutrophils play a dominant role. One possible explanation is that neutrophils are much more numerous than basophils in blood.
Noticeably, we observed neutrophilia following immunization of mice with antigen in CFA/IFA. We excluded that neutrophilia accounted for the dominant contribution of neutrophils to ASA by delaying antigen challenge until neutrophil numbers in immunized mice were comparable to those in naive mice. Under these conditions, neutrophils were also mandatory for ASA in 5KO mice. We found that neutrophils also predominantly contributed to IgG-induced PSA (whether induced by monoclonal IgG2b-IC or polyclonal-IC) in nonimmunized WT mice that have normal numbers of neutrophils. We also observed neutrophilia following immunizations with alum or with alum plus Pertussis toxin. In preliminary experiments, neither neutrophil depletion nor basophil depletion significantly reduced temperature drop, but a depletion of both cell types abolished ASA in mice immunized with antigen in alum (our unpublished observations). Taken together, our results demonstrate that neutrophils contribute to anaphylactic shock in 2 models of ASA, i.e., following immunization in CFA/IFA or in alum, and in 2 models of PSA, i.e., induced by polyclonal IgG-IC or monoclonal IgG2b-IC.
Two mediators, PAF and histamine, were found to play a critical role in experimental anaphylaxis. Mast cells, basophils and, apparently, neutrophils (34
) can release histamine. Histamine accounts for IgE-induced PSA but not for IgG1-induced PSA, whereas PAF accounts for IgG1-induced PSA but not IgE-induced PSA (10
). We found that PAF has a dominant role in ASA. Indeed, PAF-R antagonists, but not histamine receptor H1 antagonists, markedly reduced temperature drop and prevented death in ASA in mice immunized with antigen in CFA/IFA. In agreement with these results, ASA-associated heart rate and arterial pressure reduction were strongly impaired in PAF-R–deficient mice (15
), suggesting that ASA-associated temperature drop and mortality may also be inhibited in these mice. Indirectly supporting this assumption, ASA was virtually abrogated in cPLA2-deficient mice (our results and ref. 29
), which cannot generate several lipid-derived mediators, including PAF. Activated neutrophils (20
), monocytes/macrophages (35
), and eosinophils (36
) all produce PAF, but neutrophils were reported to be major producers (21
). We found, indeed, that neutrophils secrete PAF when stimulated by IgG2b-IC, but we also found elevated PAF levels in plasma of mice undergoing neutrophil-dependent PSA. Together our results suggest that PAF released upon neutrophil activation during ASA and PSA is responsible for anaphylactic shock.
ASA depends on activating receptors (5
) that associate with and whose expression and signaling depend on FcRγ. These are FcγRI, FcγRIIIA, FcγRIV, and FcεRI (37
). ASA was reported in IgE-deficient mice (12
), and we observed that it was comparable in WT and FcεRI-deficient mice. Similarly, the deletion of FcεRI did not affect ASA in mice immunized with antigen in alum (5
). While blocking either FcγRIIIA or FcγRIV reduced ASA, blocking both receptors abrogated ASA in WT mice. IgG antibodies seem therefore more important than IgE antibodies in ASA following immunization in CFA/IFA. FcγRIV contributed to ASA in WT mice, and it was necessary and sufficient to induce ASA in 5KO mice. Although FcγRIV are expressed by neutrophils and by monocytes/macrophages (18
), only neutrophil FcγRIV accounts for ASA in 5KO mice. A single activating FcγR on a single cell population is therefore sufficient to induce ASA. Our results demonstrate that FcγRIIIA or FcγRIV is responsible for ASA following CFA/IFA immunizations, and that each could substitute for the other to induce ASA, provided that IgG2 antibodies are produced.
That IgG1 can induce anaphylaxis was demonstrated by PSA. The only activating FcR having an affinity for IgG1 is FcγRIIIA (17
). Depletion of basophils using specific mAbs abrogated IgG1-induced PSA (10
), although mast cells and neutrophils also express FcγRIIIA. A model of basophil-deficient mice, however, challenges this result (14
). Mast cells are necessary for IgE-induced PSA, although basophils also express FcεRI. Noticeably, IgE-induced PSA was abolished in 5KO mice (data not shown). FcγRIV that can bind IgE with a low affinity (17
) is therefore insufficient to trigger this reaction. IgG1 is the predominant antibody subclass following immunization in alum, but also in CFA/IFA. IgG1 antibodies are likely to act as the main players in ASA by engaging FcγRIIIA. BSA immunizations in alum induced specific IgG1, but not specific IgG2 (Supplemental Figure 4D), indicating that FcγRIIIA, but not FcγRIV, were engaged during ASA following this immunization protocol. Supporting this conclusion, FcγRIV was not sufficient to induce ASA in 5KO mice following immunization in alum (Supplemental Figure 4E).
We found, however, that not only IgG1, but also IgG2, antibodies could induce PSA. IgG2-induced PSA could develop in 5KO mice, and neutrophils contributed to the shock. IgG2 can therefore contribute to anaphylaxis. IgG2-IC that may form in vivo upon antigen challenge following immunization in CFA/IFA may be responsible for the predominant role of neutrophils as IgG2 IC can engage FcγRIV (but also FcγRIIIA) on these cells. In the absence of IgG2 antibodies, as in ASA following immunization with antigen in alum, IgG1-IC may trigger FcγRIIIA-expressing basophils and neutrophils. It follows that basophils contribute to ASA and to PSA when IgG1-IC are present (this report and ref. 10
), but also other cells (14
), among which are neutrophils. Our results indicating that neutrophils are mandatory for polyclonal IgG-induced PSA differ from previous reports implicating mast cells (5
) and basophils (10
) in IgG1-induced PSA. They are, however, not contradictory. Indeed, monoclonal IgG1-IC can selectively engage FcγRIIIA, while IgG-IC made of IgG1 and IgG2 isotypes will engage both FcγRIIIA and FcγRIV, to induce PSA. FcR-expressing cells involved in each type of PSA may therefore be different. Taken together, these data suggest that IgE, IgG1, and IgG2 can all induce anaphylaxis when engaging FcεRI, FcγRIIIA, and FcγRIV on mast cells, basophils, and neutrophils, respectively. Although likely, it was not formally demonstrated that FcεRI or FcγRIIIA alone could induce ASA. Using the 5KO model, we provide evidence that FcγRIV alone can. Because FcγRIV is a receptor for IgG2, but not for IgG1, and is expressed by neutrophils, but not by basophils or mast cells, we are able to demonstrate here a role for the most unexpected antibody, receptor, and cell type in ASA in mice.
Human anaphylaxis is essentially active. IgG1 is the most abundant IgG subclass in human plasma, and the majority of antibodies raised by vaccinations (generally in alum) belong to this subclass. Human IgG1 binds to all activating human FcγRs (39
). Both specific IgE and IgG antibodies are found in the serum of allergic patients, but the relative concentration of the various classes is poorly known and rarely investigated. FcγRIV has no human ortholog, and FcγRIIIA are not expressed by human neutrophils, basophils, and mast cells (40
). These 3 cell types, nevertheless, express activating Fc receptors. All of them express another activating IgG receptor, FcγRIIA. Mast cells and basophils also express the high-affinity IgE receptor FcεRI in normal individuals as well as neutrophils in atopic patients (41
). These 3 cell types can produce PAF upon activation, especially neutrophils (21
). PAF could play an important role in human anaphylaxis. Indeed, plasma PAF concentration has been correlated with the severity of shocks in patients (42
). Although the cellular source of PAF in human anaphylaxis was not identified, the above-mentioned results endow neutrophils with a critical role. Supporting this assumption, IgG-IC could activate human neutrophils in vitro, and a transfer of human neutrophils restored ASA in FcR
In conclusion, we demonstrate here a previously unexpected role of neutrophils in anaphylaxis. An IgG2-induced, FcγRIV-dependent, PAF-mediated active anaphylaxis, contingent on neutrophils, could be unraveled using immunized multiple FcR-deficient mice. IgG-induced, FcγR-dependent, PAF-mediated ASA was also observed in WT mice, to which neutrophils contributed. Anaphylaxis induction is therefore a property of neutrophils, which is not trivial considering that neutrophils are the most numerous cells among blood leukocytes in humans. This may have important therapeutic consequences if indeed neutrophils can induce IgG-dependent anaphylactic reactions in humans.