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The recent development of a consensus definition and proposed diagnostic criteria for anaphylaxis offers promise for research efforts and a better understanding of the epidemiology and pathogenesis of this enigmatic and life-threatening disease. This review examines basic principles and recent research advances in the mechanisms of mast cell signaling believed to underlie anaphylaxis. The unfolding complexity of mast cell signaling suggests that the system is sensitive to regulation by any of several individual signaling pathways and intermediates and that complementary pathways regulate mast cell activation by amplified signals. The signaling events underlying anaphylactic reactions have largely been identified through experiments in genetically modified mice and supported by biochemical studies of mast cells derived from these mice. These studies have revealed that signaling pathways exist to both upregulate and downregulate mast cell responses. In this review we will thus describe the key molecular players in these pathways in the context of anaphylaxis.
“Anaphylaxis is a serious allergic reaction that is rapid in onset and may cause death.” 1 This simple definition for anaphylaxis was developed through consensus by representatives of several organizations from the United States and other countries at 2 symposia sponsored by the National Institute of Allergy and Infectious Diseases and the Food Allergy and Anaphylaxis Network in 2004 and 2005 (summaries and a discussion of the symposia are available in 2 reports in the Journal of Allergy and Clinical Immunology).1,2 The goal of the symposia was to address the century-old problems of the lack of a universally accepted definition of anaphylaxis and consensus on its diagnostic criteria. This definition was developed to encourage progress in epidemiologic, clinical, and laboratory research on anaphylaxis, as well as therapeutic development and practice. In addition to defining anaphylaxis, symposia participants developed a set of 3 clinical criteria for diagnosis, presentation of any of which would indicate an anaphylactic reaction.1 The symposia participants also reviewed established methods of treatment for anaphylaxis and identified important goals for research, such as gaining a better understanding of pathophysiologic responses and the mechanisms of mast cell activation involved in anaphylaxis.1 At a symposium in 2006, researchers from the American Academy of Allergy, Asthma & Immunology and the European Academy of Allergology and Clinical Immunology met to review issues on risk assessment for anaphylaxis and identified a research agenda that included an investigation of mast cell signaling pathways.3 The present review examines recent progress in research into the molecular mechanisms of mast cell signaling in anaphylaxis.
The absence of consistent criteria for diagnosis has made it difficult to gather and analyze the data required to estimate the incidence and prevalence of anaphylaxis.4 In addition, differences in study methodologies, sample size, population representation, and environmental/allergen exposure, and incomplete and inconsistent data collection have made it a challenge to compare results from different studies.5 The American College of Allergy, Asthma and Immunology Epidemiology of Anaphylaxis Working Group met in 2004 to review the literature and to estimate the occurrence of anaphylaxis. Based on a qualitative review, the group estimated the frequency of anaphylaxis to be 50 to 2,000 episodes per 100,000 persons or a lifetime prevalence of approximately 0.05% to 2.0%; they acknowledged that because of underdiagnosis and underreporting, this estimate did not represent the true prevalence.6 The consensus definition and diagnostic criteria for anaphylaxis proposed by the National Institute of Allergy and Infectious Diseases/Food Allergy and Anaphylaxis Network symposia representatives might be used to standardize the classification of anaphylaxis and improve future epidemiologic studies.
Anaphylaxis occurs rapidly and systemically, affecting 1 or more organ systems, generally where mast cells reside in relative abundance.2,7 Underlying the pathophysiology of anaphylaxis is exposure to allergen or other factors that activate mast cells or basophils, prompting degranulation and immediate (5–30 minutes) release of preformed mediators (histamine, tryptase, carboxypeptidase A, and proteoglycans), synthesis of arachidonic acid metabolites (prostaglandins, leukotrienes), and platelet-activating factor (PAF), and delayed-phase (2–6 hours) generation of cytokines (TNF-α) and chemokines resulting from increased gene expression. These mediators are thought to be responsible for the signs and symptoms that can be variably present and that relate to the respiratory tract (laryngeal edema and bronchospasm), cardiovascular system (hypotension, syncope, and arrhythmias), epidermis and dermis (urticaria and angioedema), and gastrointestinal tract (nausea, vomiting, and cramping). Histamine stimulates vasodilation, vascular permeability, heart rate, cardiac contraction, and glandular secretion. Prostaglandin D2 acts as a bronchoconstrictor, pulmonary and coronary vasoconstrictor, and peripheral vasodilator. Leukotrienes and PAF increase bronchoconstriction and vascular permeability. TNF-α is reportedly released as a preformed mediator that activates neutrophils, recruits other effector cells, and increases chemokine synthesis; it also acts as a late-phase mediator with other cytokines and chemokines, leading, in some cases, to a biphasic or protracted response.8
Anaphylaxis can occur after exposure to certain foods (eg, peanuts and tree nuts), drugs (eg, antibiotics, vaccines, and anesthetics), insect venoms (eg, hymenoptera), latex, and immunotherapy injections or without an identifiable cause (idiopathic anaphylaxis).2,9–11 Anaphylaxis is generally thought to be mediated by IgE, although there are reports of IgE-independent, IgG-dependent, or nonimmunologic mechanisms.12 The alternative pathways described arise from complement generation of anaphylatoxin, neuropeptide release, immune complex formation, cytotoxicity, or T-cell activation.3 The mechanisms underlying IgG-dependent anaphylaxis in human subjects are not well understood.13 Two mechanisms of anaphylaxis have been demonstrated in murine models: an IgE-dependent pathway that results in release of histamine and PAF and an IgG-dependent pathway that results in release of PAF, rather than histamine, as its mediator.12
Recent studies have revealed the complexity of mast cell signaling and the sensitivity of this system to regulation by individual pathways and intermediates. Murine knockout and knockdown studies have demonstrated the effect of single components resulting in either resistance or sensitivity to anaphylaxis.12 The expression of the D816V activating mutation in the tyrosine kinase KIT in human mast cells results in mast cell proliferation and mastocytosis.14 Studies of mast cell proliferation and the prevalence of episodes of hypotension in patients with mastocytosis led to the recognition of association between systemic mastocytosis and anaphylaxis; the D816V KIT mutation has been identified in mast cells from patients with mastocytosis and recurrent unexplained anaphylaxis.15–18 Omalizumab, which inhibits the binding of IgE to its high-affinity receptor, FcϵRI, was effective for treating anaphylaxis in 2 patients with systemic mastocytosis.19 The association of the activating mutation in KIT with mastocytosis and anaphylaxis indicates that there might be other mutations in mast cell signaling components that contribute to the hypersensitive phenotype and that cause a predisposition to allergic diseases, such as anaphylaxis.20 These recent research reports, as well as others, have highlighted signaling pathways and components involved in mast cell activation and degranulation that affect the regulation or threshold of activation that might serve as key events leading to mediator release and the pathophysiology of anaphylaxis.
The signaling cascades that regulate mast cell activation have been extensively investigated during the past few years and are described in depth in recent reviews.21–23 In this review the basic principles involved in the regulation of mast cell activation are described relative to the anaphylactic response and focus on the contributions of degranulation and immediately released mediators. The relevance of these studies to our understanding of the role of basophils in anaphylaxis is not clear. The models that are most widely used to study anaphylactic responses are passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA), which can be examined in genetically modified murine strains. PCA is induced in mice by means of injection of antigen-specific IgE, and then vascular extravasation is monitored at the sites of antigen injection, usually the back or ear dermis, through the release of Evans blue dye into these sites. PSA is induced by sensitization, followed by systemic injection of antigen. Responses can be monitored by measuring the levels of circulating histamine. Measurement of core temperature has also been used to monitor mast cell–mediated physiology in vivo. Although these models do not provide a complete picture of the spectrum of responses associated with human anaphylaxis, they have provided much information about the signaling molecules that regulate mast cell activation in a physiologic setting. These studies have also supported more comprehensive in vitro signaling studies conducted in bone marrow–derived mast cells (BMMCs) of knockout and transgenic mice.
A point of note, however, is that such defined anaphylactic reactions observed in mice are strain dependent. The C57Bl/6 and 129/Sv strains show skewing of their immune response to TH1 and TH2, respectively. As a result, 129/Sv mice have higher levels of circulating IgE, increased expression of FcϵRI, and a greater degree of PSA reactions compared with those seen in C57BL/6 mice. Mast cells derived from the bone marrow of 129/Sv mice also show greater degranulation on FcϵRI aggregation.24 However, the phenotypes observed in specific knockout mice can be dramatically different based on the genetic background of the mice, requiring that experiments performed in different murine strains be compared with caution, a lesson for human studies on anaphylaxis.
Antigen-specific IgE binds with high affinity to FcϵRI, and in the presence of specific antigen, these complexes aggregate.23 However, other receptors expressed on mast cells, such as KIT and various G protein-coupled receptors (GPCRs), can modify this response when they are activated.21,25 The FcϵRI consists of an IgE-binding α subunit and signal-transducing β and homodimeric γ subunits (Fig 1).23 The γ homodimer is indispensible for the generation of the signals required for mast cell activation.26 However, the β chain appears to serve primarily as a modulator of the signals regulated by the γ chains.23 The ability of the γ chain to initiate downstream signaling events and the β chain to modulate these responses depend on immunoreceptor tyrosine-based activation motifs (ITAMs), which are in the cytosolic domains of the FcϵRI β and γ chains.23
After aggregation, FcϵRI coalesces with glycolipid-enriched membrane domains (lipid rafts), allowing the tyrosines within the ITAM sequences (YxxL) to become phosphorylated by the Src kinase Lyn,27 which is preferentially activated within these domains.28 Although this would suggest that Lyn is essential for the ability of mast cells to promote an anaphylactic response, studies conducted in Lyn−/− mice mice and mast cells derived from these animals have produced conflicting data. Although it was initially reported that PCA reactions did not occur in Lyn−/− mice,29 later studies reported enhanced PSA reactions in this model.30 Furthermore, although consistently increased FcϵRI-mediated cytokine production has been observed in BMMCs of Lyn−/− mice, normal,31 partially reduced,32 and enhanced30 FcϵRI-mediated degranulation has been reported in this model.
These apparent disparities have now been determined to be primarily linked to the different genetic backgrounds used to generate the Lyn−/− BMMCs. Lyn−/− BMMCs derived from the C57Bl/6 strain have a hyporesponsive degranulation phenotype when challenged with antigen, whereas Lyn−/− BMMCs derived from the 129/Sv strain have a hyperresponsive degranulation phenotype.24 The hyperresponsive phenotype was recapitulated in human mast cells in which Lyn levels were reduced after lentiviral transduction of Lyn-targeted shRNA.24 In addition to helping to clarify the role for Lyn in the anaphylactic response, these studies illustrate the potential pitfalls of relying on only 1 murine strain to study the role of specific signaling molecules in anaphylactic reactions.
The phosphorylation of the γ chains and subsequent recruitment and activation of the Zeta-chain–associated protein kinase 70 (ZAP70)-releated tyrosine kinase Syk are certainly crucial for mast cell activation,33 and indeed, PSA responses are ablated in FcϵRI γ chain knockout mice.34 Nevertheless, findings from studies of Lyn−/− BMMCs indicate that despite the reported role of Lyn in phosphorylating the FcϵRI β and γ chains, other unidentified tyrosine kinases might substitute for Lyn in these early events.
After these early receptor-proximal events, downstream signaling processes are coordinated through the assembly of macromolecular complexes through both inducible and constitutive protein-protein interactions.35 The activation of Syk (possibly in conjunction with other tyrosine kinases, such as Lyn, Fyn, and Bruton’s tyrosine kinase [Btk]) promotes inducible protein-protein interactions by phosphorylating tyrosine-containing motifs that recognize specific binding domains, such as the Src homology 2 (SH2) and PTP domains on other proteins.35 Thus formed, these signaling complexes are tethered, either directly or indirectly through cytosolic adaptor molecules, to the plasma membrane by means of interaction with transmembrane adaptor molecules, such as linker for activation of T cells (LAT) and LAT2 (non T cell activation linker [NTAL]/linker for activation of B cells [LAB]) after their phosphorylation (Fig 1).35 By this means, key intermediary signaling enzymes required for the release of the inflammatory mediators that contribute to the anaphylaxis response are recruited into the receptor-signaling complex. The central role of LAT in this process has been illustrated by the absence of PSA in LAT−/− mice and the reduced degranulation and cytokine production in mast cells derived from these animals.36
The role of LAT2, however, is more enigmatic. LAT2−/− knockout mice have an increased PSA reaction that is associated with enhanced mast cell mediator release.37,38 However LAT−/−/LAT2−/− (double-knockout) BMMCs have a greater defect in degranulation than that observed in the LAT−/− BMMCs,38 indicating that anaphylaxis might also be reduced in these animals. In human mast cells, gene knockdown studies have provided evidence that antigen-mediated degranulation and the ability of stem cell factor to enhance this response depends on LAT2 phosphorylation.39 The above data and those of other studies have led to the conclusion that, whereas LAT might control a principal pathway for the regulation of mast cell activation and hence anaphylactic reactions, LAT2 might serve to modify this response by either upregulating or downregulating the ongoing LAT-regulated signaling cascade.21 The exact circumstances under which either modification occurs is unclear.
The major signaling enzyme recruited to phosphorylated LAT40 and potentially LAT239 is phospholipase C (PLC)γ (Fig 1). There are 2 isoforms of PLCγ: PLCγ1 and PLCγ2, which are both expressed by mast cells.41 Although PLCγ activation is essential for initiating the calcium signal required for antigen-mediated mast cell activation, because of the embryonic lethality associated with the PLCγ1−/− mice, the relative contributions of the 2 isoforms to the anaphylactic response is unknown. In human mast cells activated with antigen, however, the activation of PLCγ1 is greater than that of PLCγ2,41 indicating that PLCγ1 might have a greater role in mediating human anaphylaxis. PLCγ binds to phosphorylated LAT directly, through interaction of its SH2 domain with phospho-Y132 (in the human LAT sequence).40 This interaction is stabilized through a second indirect interaction of PLCγ with LAT through the cytosolic adaptor molecules Gads and SLP76.42 Accordingly, PSA is substantially reduced in Gads−/−43 and SLP76−/−44 mice. The antigen-induced, PLCγ-mediated calcium signal that regulates degranulation and cytokine production is attenuated in BMMCs of these mice.
PLCγ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to yield diacylglycerol and inositol trisphosphate. Diacyl-glycerol regulates the activation of protein kinase C. Although various protein kinase C isoforms undoubtedly play major roles in the regulation of mast cell activation,45 there is little information about their role in mediating anaphylaxis in mice and human subjects. It is the inositol trisphosphate liberated by the action of PLCγ1 that initiates the calcium signal by binding to receptors on the endoplasmic reticulum, thereby inducing the liberation of calcium from the endoplasmic reticulum into the cytosol.46 This emptying of the intracellular calcium stores leads to an influx of extracellular calcium through store-operated calcium channels, resulting in a prolongation of the calcium signal.47 The molecular sensor for the store emptying on the endoplasmic reticulum membrane has recently been identified as Stromal interaction molecule 1 (STIM1),48,49 and the equivalent plasma-membrane calcium channel has been identified as ORAI1.50,51 On depletion of calcium stores within the endoplasmic reticulum, STIM1 oligomerizes, the endoplasmic reticulum localizes to the plasma membrane, and ORAI1 forms functional calcium channels through interaction with STIM1 and oligomerization. Hence mice deficient in either STIM152 or ORAI153 display significantly impaired PCA reactions; BMMCs from these animals have a reduced capacity to degranulate or generate cytokines after exposure to antigen. A group of transmembrane proteins termed transient receptor potential canonical channels (TRPCs) also might contribute to the influx of calcium. In the RBL2H3 rat mast cell line, TRPC5 acts in conjunction with STIM1 and ORAI1 to regulate antigen-mediated calcium flux and degranulation.54 The function of mast cells in mice that lack specific TRPC channels has yet to be described, and therefore it is currently unclear whether these channels influence anaphylactic reactions in vivo.
The calcium signal is terminated after depolarization of the membrane through the calcium-activated, nonselective cation channel transient receptor cation channel, subfamily M (TRPM4), which downregulates calcium influx.55 Consequently, mice deficient in TRPM4 have exaggerated PCA reactions, and BMMCs of these mice show enhanced degranulation and cytokine production.55 After TRPM4-dependent termination of calcium influx, excess cytosolic calcium is restored to resting levels by the shunting of excess calcium ions into the endoplasmic reticulum, mitochondria, and extracellular stores by Ca2+-AT-Pase pumps (for a review of calcium regulation, see Ma and Beaven56).
In addition to PLCγ, the other major signaling enzyme that is essential for intermediary signaling in mast cells is phosphoinositide 3-kinase (PI3K; Fig 1). PI3K regulates multiple processes within mast cells that are required not only for degranulation and cytokine production but also for mast cell proliferation, differentiation, and survival.57 PI3K is a family of enzymes that catalyze the conversion of PIP2 to phosphatidylinositol 3,4,5-trisphosphate, thereby providing docking sites for pleckstrin homology domain–containing proteins.57 Several classes of PI3K exist. However, mast cell activation is primarily regulated by class 1A and class 1B PI3Ks.57 Class 1A PI3Ks are linked to receptors, such as FcϵRI, that signal through tyrosine kinases, whereas class 1B PI3Ks are linked to G protein–coupled receptors (GPCRs). Null transgenic mice, knockout mice, or both have been generated for the class 1A PI3K P110δ and the class 1B PI3K p110γ. Studies conducted in BMMCs of transgenic mice expressing inactive p110δ revealed that this isoform has a major role in stem cell factor–mediated mast cell responses, such as proliferation, chemotaxis, and enhancement of antigen-induced mediator release,58 as well as the ability of antigen to promote degranulation and cytokine production.58 Similarly, studies conducted in BMMCs of p110γ-deficient mice demonstrated a role for this isoform in the potentiation of antigen-mediated degranulation by GPCR agonists, such as adenosine.59 Surprisingly, the antigen-mediated degranulation response was also attenuated in these cells,59,60 indicating that, at least in culture, part of the antigen-mediated degranulation response is secondary to the release of GPCR agonists, which then act on a cell-surface GPCR to enhance degranulation.
The relative in vivo roles of the p110δ and p110γ isoforms in anaphylaxis have been controversial. A study published in 2002 reported that antigen-mediated PSA was virtually absent in p110γ-deficient mice.59 A subsequent in vivo study, however, indicated that it was the p110δ isoform that was responsible for the anaphylactic response.58 A study published in 2008 compared the effects of blocking p110δ and p110γ by administering selective inhibitors of each isoform to wild-type mice. This study supported a role for PI3Kδ, but not PI3Kγ, in anaphylaxis.60 The reasons for the disparity between the various reports remain unclear, and thus we await further experimentation to clarify this issue. However, it is clear that PI3K-regulated pathways are essential for antigen-mediated mast cell activation.57
PI3K must be recruited to the membrane-associated receptor-signaling complex to function in mast cell activation. This localization is regulated by the inducible interaction of the p85 or p55 adaptor subunits of PI3K with the cytosolic adaptor molecule GAB2 after its phosphorylation by the Src family tyrosine kinase Fyn. Antigen-mediated PI3K activation in mast cells also appears to be positively regulated by Ras guanyl nucleotide–releasing protein (RasGRP1)61 and negatively regulated by regulator of G protein signaling (RGS13).62 Hence anaphylactic reactions are attenuated in GAB2−/−,63 Fyn−/−,64 and RasGRP1−/−61 mice and enhanced in RGS13−/− mice.62 Similarly, the ability of antigen to induce degranulation, cytokine production, or both is substantially reduced in BMMCs developed from GAB2−/−, Fyn−/−, and RasGRP1−/− mice and enhanced in BMMCs developed from RGS13−/− mice. The actions of PI3K are reversed by the inositol phosphatases PTEN and SH2-containing inositol phosphatase 1 (SHIP1), which dephosphorylate PIP2 at the D3 and D5 positions, respectively.57 Activation of SHIP1 thorough coligation of FcγRIIb with FcϵRI with fusion proteins effectively blocks anaphylactic reactions in mice.65 The role of PTEN in the anaphylactic response has not been assessed in vivo.
Studies on the relative roles of LAT, LAT2, GADS, GAB2, PI3K, PLCγ, and Fyn in mast cells have led to the concept of complementary pathways regulating mast cell activation.21,64 Therefore it is assumed that these complementary pathways would operate for the regulation of anaphylactic reactions in vivo. It is proposed that at early stages of mast cell activation, the LAT-GADS-SLP76-PLCγ1 pathway plays a predominant role after the activation of Syk, whereas the pathways that lead to the activation of PI3K might serve to prolong and amplify ongoing PLCγ-dependent responses.21 It is not clear how the LAT-GADS-SLP76-PLCγ1 pathway affects the activation of PI3K. However, based on the observation that the reduced PSA in Vav1−/− mice is associated with a decrease in PLCγ phosphorylation and PI3K activation,66 Vav1 might have a role in this interaction. On the other hand, PI3K appears to modulate the activation of PLCγ through the Tec kinase Btk. Btk contains a pleckstrin homology domain, and thus is recruited to the plasma membrane and activated in a PI3K-dependent manner after FcϵRI aggregation through its binding to membrane-associated PIP2.45 Defects in the Btk gene are associated with X-linked immunodeficiency in mice and X-linked agammaglobulinemia in human subjects. Mice with X-linked immunodeficiency have a defective PCA reaction, and mast cells from these mice, Btk−/− mice, or both have partially reduced degranulation and cytokine responses.67 These defects can be attributed to a similar decrease in the PLCγ1-mediated calcium response observed in these cells.
The lipid metabolite sphingosine-1-phosphate (S1P) has recently been shown to have a role in anaphylactic reactions in mice.68 Formation of S1P from sphingosine is catalyzed by sphingosine kinase (SphK; Fig 1), which exists in 2 forms: SphK1 and SphK2. Both forms have been reported to be expressed in human and murine mast cells,68,69 although 1 study failed to find expression of SphK2 in human BMMCs.70 Studies conducted in Sphk1- and SphK2-knockout mice demonstrated that each kinase contributes to anaphylactic reactions by inducing the generation of S1P.68 However, whereas SphK2 appears to function within mast cells to regulate the calcium flux necessary for activation of protein kinase C and anaphylaxis, SphK1 appears to contribute to anaphylaxis by generating circulating S1P, which acts on surface receptors.71 Mast cells express as least 2 receptors for S1P: S1P1 and S1P2. After binding of S1P, these receptors regulate mast cell chemotaxis and degranulation, respectively.71 In contrast, intracellular pools of S1P have been proposed to contribute to the calcium signal required for mast cell activation, although the mechanism responsible is largely unclear.
Advances in mast cell signaling research have improved our understanding of the pathophysiology of anaphylaxis. The identification of signaling pathways and components that amplify signals or alter the threshold of activation of mast cells, leading to degranulation and mediator release, has the promise of identifying novel approaches for prevention and treatment of anaphylaxis. The discovery of polymorphisms and mutations in components that regulate mast cell signaling might lead to ways to identify subjects who are most susceptible to life-threatening episodes of anaphylaxis caused by mast cell activation.
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To provide excellent reviews on key aspects of allergic disease to those who research, treat, or manage allergic disease.
Physicians and researchers within the field of allergic disease.
The American Academy of Allergy, Asthma & Immunology (AAAAI) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. The AAAAI designates these educational activities for a maximum of 1 AMA PRA Category 1 Credit™. Physicians should only claim credit commensurate with the extent of their participation in the activity.
Authors: Dean D. Metcalfe, MD, Richard D. Peavy, PhD, and Alasdair M. Gilfillan, PhD
This CME activity is supported by an educational grant from Merck & Co., Inc.
The authors have declared that they have no conflict of interest.
Supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
The Editors wish to acknowledge Seema Aceves, MD, PhD, for preparing this glossary.