The major effector cells of immediate hypersensitivity reactions and allergic disease are type 2 cytokine-secreting cells (Th2 cells, Tfh cells, and ILC2s), mast cells, basophils, and eosinophils. Although each of these cell types has unique characteristics, all four secrete mediators involved in allergic reactions. Tfh cells stimulate IgE production in secondary lymphoid organs, and Th2 cells and ILC2s contribute to tissue inflammation by secreting cytokines. Mast cells, basophils, and eosinophils release their granule contents and produce other mediators, all of which are responsible for the pathologic consequences of allergic reactions. In this section, we will discuss the roles of these cell types in allergy.
Role of Th2 Cells and Innate Lymphoid Cells in Allergic Disease
Th2 cells secrete cytokines, including IL-4, IL-5, and IL-13, which promote inflammatory responses to allergens within tissues. IL-4 secreted by Th2 cells induces expression of endothelial VCAM-1 (vascular cell adhesion molecule 1), which promotes the recruitment of eosinophils and additional Th2 cells into tissues. IL-5 enhances eosinophil production in the bone marrow and activates mature eosinophils in tissues. IL-13 stimulates epithelial cells (e.g., in the airways) to secrete increased amounts of mucus, which is also a common feature of these reactions.
Consistent with a central role of Th2 cells in immediate hypersensitivity, more allergen-specific IL-4-secreting T cells are found in the blood of atopic individuals than in nonatopic persons. In atopic patients, the allergen-specific T cells also produce more IL-4 per cell than in normal individuals. In animal models, a disease resembling human asthma can be induced by generation of Th2 cells specific for an inhaled antigen or by adoptive transfer of these cells into naive mice. Accumulations of Th2 cells are found at sites of immediate hypersensitivity reactions in the skin and bronchial mucosa.
ILC2s produce some of the same cytokines as Th2 cells, specifically IL-5 and IL-13, and therefore may have similar roles in allergic reactions. Because ILCs normally reside in tissues, their cytokines may contribute to early allergic inflammation before Th2 cells are generated and migrate to the tissues. The ILC2s may also work in concert with Th2 cells later to sustain inflammation.
Mast Cells and Basophils
Mast cells, basophils, and eosinophils are myeloid cells that share some features but differ phenotypically and function ally in significant ways (Table 1). All mast cells are derived from progenitors in the bone marrow. Normally, mature mast cells are not found in the circulation. Progenitors migrate to the peripheral tissues as immature cells and undergo differentiation in response to local biochemical cues, including stem cell factor released by tissue cells, which binds to the c-KIT receptor on the mast cell precursors. Mature mast cells are found throughout the body, predominantly near blood vessels (Fig. 2A) and nerves and beneath epithelia. They are also present in lymphoid organs. Human mast cells vary in shape and have round nuclei, and the cytoplasm contains membrane-bound granules and lipid bodies. The granules contain acidic proteoglycans that bind basic dyes.
Fig1. Sequence of events in immediate hypersensitivity reactions. Immediate hypersensitivity diseases are initiated by the introduction of an allergen, which stimulates interleukin-4 (IL-4)- and IL-13-producing helper T-cell responses and immunoglobulin E (IgE) production. IgE sensitizes mast cells by binding to Fcε receptor type I (FcεRI), and subsequent exposure to the allergen activates the mast cells to secrete the mediators that are responsible for the pathologic reactions of immediate hypersensitivity. Tfh, T follicular helper cells.
Fig2. Morphology of mast cells, basophils, and eosinophils. Photomicrographs of Wright-Giemsa stained perivascular dermal mast cells (A, arrows), peripheral blood basophil (B), and peripheral blood eosinophil (C) are presented. Note the characteristic blue-staining cytoplasmic granules of the basophil and red staining of the cytoplasmic granules in the eosinophil. (A, Courtesy Dr. George Murphy. B and C, Courtesy Dr. Jonathan Hecht, Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts.)
Activated mast cells secrete a variety of mediators that are responsible for the manifestations of allergic reactions (Table 1). These include substances that are stored in granules and rapidly released on activation and others that are synthesized on activation and secreted. The production and actions of these mediators are described later.
Table1. Mediators Produced by Mast Cells, Basophils, and Eosinophils
Subsets of mast cells have been described in mice and humans, which differ in their main location (mucosa versus connective tissue in mice), or granule protease content (tryptase only or tryptase and chymase in humans).
Basophils are blood granulocytes with structural and functional similarities to mast cells. Like other granulocytes, basophils are derived from bone marrow progenitors (which are different from the precursors of mast cells), mature in the bone marrow, and circulate in the blood (see Fig. 2B). Basophils constitute 0.5% or less of blood leukocytes. Although they are normally not present in tissues, basophils may be recruited to some inflammatory sites. Basophils contain granules that bind basic dyes, and they are capable of synthesizing many of the same mediators as mast cells (see Table 1). Like mast cells, basophils express Fcε receptor type I (FcεRI), bind IgE, and can be triggered by antigen binding to the IgE. Therefore, basophils that are recruited into tissue sites where antigen is present may contribute to immediate hypersensitivity reactions.
Neutrophils play an important role in late-phase allergic reactions. Neutrophils are recruited to the sites of allergen exposure in late-phase reactions, which occur a few hours to two days after allergen exposure (discussed later). Th2 cells and eosinophils are also recruited into these reactions. The activated neutrophils release numerous inflammatory effector molecules, including cytokines, leukotrienes, and chemokines.
Binding of IgE to Mast Cells and Basophils: The Fcε Receptor
Mast cells and basophils express a high-affinity Fc receptor specific for ε heavy chains, called FcεRI, which binds IgE. IgE, like all other antibodies, is made exclusively by B cells, yet IgE functions as an antigen receptor on the surface of mast cells and basophils. This function is accomplished by IgE bind ing to FcεRI on these cells. The affinity of FcεRI for IgE is very high (dissociation constant [Kd ] of approximately 1 × 10−10 M), higher than that of any other Fc receptor for its antibody ligand. Therefore, although the normal plasma concentration of IgE is low compared to other Ig isotypes (<5 × 10−10 M), there is full occupancy of FcεRI receptors by IgE, and the majority of mast cells are always coated with IgE, even in nonatopic individuals.
Each FcεRI molecule on mast cells is composed of an α chain that binds the Fc region of IgE and a β chain and two γ chains that are responsible for signaling (Fig.3). The amino-terminal extracellular portion of the α chain includes two Ig-like domains that form the binding site for IgE. The β chain of FcεRI contains a single immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic carboxy-terminal domain. The two identical γ chain polypeptides are linked by a disulfide bond and are homologous to the ζ chain of the T-cell antigen receptor complex. The cytoplasmic portion of each γ chain contains one ITAM. The same γ chain serves as the signaling subunit for FcγRI, FcγRIIIA, and FcαR and is called the FcR γ chain. Tyrosine phosphorylation of the ITAMs of the β and γ chains initiates the signaling cascade from the receptor that is required for mast cell activation, described shortly.
Fig3. Polypeptide chain structure of the high-affinity immunoglobulin E (IgE)–specific Fc receptor (FcεRI). IgE binds to the Ig-like domains of the α chain. The β chain and the γ chains mediate signal transduction. The immunoreceptor tyrosine activation motifs (ITAMs) in the cytoplasmic region of the β and γ chains are similar to those found in the T-cell receptor complex. LYN and FYN are tyrosine kinases that bind to the N-terminal cytoplasmic end of the β chain, and SYK is a tyrosine kinase that binds to phosphorylated ITAM motifs in the C-terminal cytoplasmic tails of the β and γ chains. These kinases participate in signaling events that activate mast cells.
The importance of FcεRI in IgE-mediated immediate hyper sensitivity reactions has been demonstrated in FcεRI α chain knockout mice. When these mice are given intravenous injections of IgE specific for a known antigen followed by that anti gen, anaphylaxis does not develop or is mild, whereas a severe reaction occurs in wild-type mice treated in the same way. FcεRI expression on the surface of mast cells and basophils is increased by IgE, thereby providing a mechanism for the amplification of IgE-mediated reactions.
Whereas FcεRI on mast cells and basophils is expressed as an αβγ2 tetramer, the receptors on eosinophils are mainly αγ2 trimers and are expressed at low levels, and there is no convincing evidence that eosinophils can be activated by antigens binding to IgE that is attached to FcεRI. Another IgE receptor called FcεRII, also known as CD23, is a protein related to C-type mammalian lectins whose affinity for IgE is much lower than that of FcεRI. The biologic role of FcεRII is not known.
Activation of Mast Cells
Mast cells are activated by cross-linking of FcεRI molecules, which occurs by binding of multivalent antigens to the IgE molecules that are attached to the Fc receptors (Fig. 4). In an individual who is allergic to a particular antigen, a large proportion of the IgE bound to FcεRI on the surface of mast cells is specific for that antigen. Exposure to the antigen will cross-link sufficient IgE molecules to trigger mast cell activation. In contrast, in nonatopic individuals, the IgE molecules bound to mast cells are specific for many different antigens, all of which may have induced low lev els of IgE production. Therefore, no single antigen will cross-link enough of the IgE molecules to cause mast cell activation.
Fig4. Mast cell activation. Antigen binding to immunoglobulin E (IgE) cross-links Fcε receptor type I (FcεRI) molecules on mast cells, which induces the release of mediators that cause the hypersensitivity reaction (A and B). A photomicrograph of a resting mast cell with abundant red-staining cytoplasmic granules is shown in (C). These granules are also seen in the electron micrograph of a resting mast cell shown in (E). In contrast, the depleted granules of an activated mast cell are shown in the photomicrograph (D) and electron micrograph (F). (Courtesy Dr. Daniel Friend, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts.)
The activation of mast cells results in three types of biologic responses: secretion of preformed granule contents by exocytosis (degranulation), synthesis and secretion of lipid mediators, and synthesis and secretion of cytokines. The signaling cascades initiated by allergen-mediated FcεRI cross-linking are similar to the proximal signaling events initiated by antigen binding to lymphocytes. (Fig. 5) When FcεR1 is cross-linked by an allergen binding to the attached IgE, the LYN tyrosine kinase, which is constitutively associated with the cytoplasmic tail of the FcεRI β chain, phosphorylates the nearby ITAMs in the cytoplasmic tails of FcεRI β and γ chains. The tyrosine kinase SYK is then recruited to the ITAMs of the γ chain, becomes activated, and phosphorylates and activates other proteins in the signaling cascade, including several adaptor molecules and enzymes that participate in the formation of multicomponent signaling complexes, as described in T cells. The complex includes phospholipase Cγ (PLCγ), which catalyzes phosphatidylinositol bisphosphate breakdown to yield inositol trisphosphate (IP3) and diacylglycerol (DAG), which in turn generate Ca++ and protein kinase C (PKC) signals, respectively. FYN, a tyrosine kinase that is also constitutively associated with the cytoplasmic tail of the FcεRI β chain, phosphorylates the docking/ adaptor protein GAB2, leading to PI3-kinase activation, which also contributes to the generation of Ca++ and PKC signals. These signaling events lead to the three major responses:
• Degranulation. Activated PKC phosphorylates the myosin light-chain component of actin-myosin complexes located beneath the plasma membrane, leading to disassembly of the complex. This allows cytoplasmic granules to come in contact with the plasma membrane. The mast cell granule membrane then fuses with the plasma membrane, a process that is mediated by members of the SNARE protein family, which are involved in many other membrane fusion events. Different SNARE proteins present on the gran ule membranes and plasma membranes interact to form a multimeric complex that catalyzes fusion. The formation of SNARE complexes is regulated by several accessory molecules, including RAB3 guanosine triphosphatases and RAB-associated kinases and phosphatases. In resting mast cells, these enzymes inhibit mast cell granule membrane fusion with the plasma membrane. On FcεRI cross-linking, the resulting increase in cytoplasmic calcium concentrations and the activation of PKC block the activity of the inhibitory molecules. In addition, calcium sensor proteins respond to the elevated calcium concentrations by promoting SNARE complex formation and membrane fusion. After membrane fusion, the contents of the mast cell granules are released into the extracellular environment. This process can occur within seconds of FcεRI cross-linking and can be visualized morphologically by loss of the dense granules of mast cells (see Fig. 4). The biologic actions of the granule contents released on mast cell degranulation are described later.
• Lipid mediator production. Synthesis of lipid mediators is controlled by the cytosolic enzyme phospholipase A2 (PLA2 ) (see Fig. 5). This enzyme is activated by two signals: elevated cytoplasmic Ca++ and phosphorylation catalyzed by a MAP (mitogen-activated protein) kinase, such as ERK (extracellular receptor-activated kinase). ERK is activated as a consequence of a kinase cascade initiated through the FcεRI ITAMs, probably using the same intermediates as in T cells. Once activated, PLA2 hydrolyzes membrane phospholipids to release arachidonic acid, which is converted by cyclooxygenase or lipoxygenase into different mediators (discussed later).
• Cytokine production. Cytokine secretion by activated mast cells is a consequence of newly induced cytokine gene transcription. The biochemical events that regulate cytokine gene transcription in mast cells appear to be similar to the events that occur in T cells. Recruitment and activation of various adaptor molecules and kinases in response to FcεRI cross linking lead to nuclear translocation of NFAT (nuclear factor of activated T cells) and NF-κB (nuclear factor κB), as well as activation of AP-1 (activator protein 1) by protein kinases such as c-JUN N-terminal kinase. These transcription fac tors stimulate expression of several cytokines (IL-4, IL-5, IL-6, IL-13, tumor necrosis factor [TNF], and others) but, in contrast to T cells, not IL-2.
Fig5. Biochemical events of mast cell activation. Cross-linking of bound IgE by antigen promotes LYN phosphorylation of other signaling molecules, which leads to activation of protein tyrosine kinase SYK, which in turn causes activation of a mitogen-activated protein (MAP) kinase cascade and phospholipase Cγ (PLCγ). PLCγ catalyzes the release of inositol trisphosphate (IP3) and diacylglycerol (DAG) from membrane phosphatidylinositol 4,5-bisphosphate (PIP2). IP3 causes the release of intracellular calcium from the endoplasmic reticulum. Calcium and DAG activate protein kinase C (PKC). FYN phosphorylation of GAB2 leads to PI3K activation, which contributes to PKC activation. Calcium, MAP kinases, and PKC promote cytokine gene transcription, leading to secretion of cytokines. PKC and calcium also enhance granule exocytosis, releasing histamine and other preformed mediators. Calcium and MAP kinases combine to activate the enzyme cytosolic phospholipase A2 (PLA2), which initiates the synthesis of lipid mediators, including prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). TNF, tumor necrosis factor.
Mast cell activation through the FcεRI pathway is regulated by various inhibitory receptors, which contain immunoreceptor tyrosine-based inhibition motifs (ITIMs) within their cytoplasmic tails. One such inhibitory receptor is FcγRIIB, which coaggregates with FcεRI during mast cell activation. The ITIM of FcγRIIB is phosphorylated by LYN, and this leads to recruitment of the phosphatase called SHIP (SH2 domain–containing inositol 5-phosphatase) and inhibition of FcεRI signaling. Experiments in mice indicate that FcγRIIB inhibits mast cell degranulation in vivo. Several other inhibitory receptors are also expressed on mast cells, but their importance in vivo is not known.
In addition to allergen-induced cross-linking of FcεRI, many other inflammatory stimuli can activate mast cells in the absence of allergens or synergize with allergens. The complement fragments C3a and C5a can cause mast cell degranulation, and this is why they were named anaphylatoxins. Other stimuli induce selective activation of mast cells to produce arachidonic acid metabolites, cytokines, and chemokines, but not degranulation. These stimuli include Toll-like receptor (TLR) ligands; substances released from injured cells, including ATP; fungal glucans; antimicrobial peptides; cytokines such as stem cell factor (SCF), IL-3, IL-4, IL-9, and IL-33; leukotrienes; and several chemokines. These additional IgE-independent modes of mast cell activation are likely important for the physiologic role of mast cells as sentinel cells of innate immunity, initiating inflammatory responses to infection or tissue injury.
Neuropeptides, including substance P, somatostatin, and vasoactive intestinal peptide, induce mast cell histamine release and may mediate neuroendocrine-linked mast cell activation. The nervous system is known to modulate allergic reactions, and neuropeptides may be involved in this effect, including by direct activation by allergens, releasing substance P from sensory neurons. The flare produced at the edge of the wheal in elicited immediate hypersensitivity reactions is in part mediated by the nervous system, as shown by the observation that it is markedly diminished in skin sites lacking innervation. Similar impacts have been shown for sensory neurons and increased airway con striction with allergen exposure in asthma models. Cold temperatures and intense exercise also trigger mast cell degranulation, but the mechanisms involved are not known.
Mast cell degranulation can also be stimulated by many different cationic substances, collectively called secretagogues. These include endogenous inflammatory peptides, drugs known to cause adverse allergy-like reactions, and the compounds 48/80 and mastoparan used experimentally as pharmacologic triggers for mast cells.
Many agents activate mast cells independent of FcεRI by binding to a receptor called MAS-related G protein–coupled receptor-X2 (MRGPRX2), which is highly expressed only by skin and other tissue mast cells and neurons of the dorsal root ganglion, but not mucosal mast cells. Ligands that bind to MRGPRX2 and thereby activate mast cells include several antibiotics and anesthetic drugs, components of insect venoms, antimicrobial peptides, molecules secreted by eosinophil and neuropeptides. MRGPRX2 is considered to be a likely mediator of some allergy like reactions to drugs and other pathologic conditions characterized by urticaria (superficial dermal edema with itching).
Mast cells also express Fc receptors for IgG heavy chains, and the cells can be activated by cross-linking bound IgG. This IgG-mediated reaction is the likely explanation for the finding that Ig ε chain knockout mice are not completely resistant to antigen-induced mast cell–mediated anaphylaxis. However, the high-affinity IgE receptor, FcεRI, has a higher affinity for IgE than Fcγ receptors have for IgG, IgE is the major antibody class that binds to mast cells, and hence, IgE is the antibody isotype involved in most immediate hypersensitivity reactions.
Mast cell activation is not an all-or-nothing phenome non, and different types or levels of stimuli may elicit partial responses, with production of some mediators but not others. Such variations in activation and mediator release may account for variable clinical presentations.
Several uncommon disorders of mast cells illustrate how their increased functions can lead to disease. Systemic mastocytosis (SM) is a clinically heterogeneous disease associated with mutations in the tyrosine kinase c-KIT, which controls mast cell development. SM is a clonal mast cell disorder that is associated with an increased number of mast cells with dysregulated function and cell morphology. These mast cells can infiltrate multiple organs (e.g., skin, liver, spleen, bone marrow, and GI tract) and release increased amounts of mediators, including tryptase, yielding atopic symptoms including idiopathic ana phylaxis and skin, hematopoietic, lung, and gastrointestinal symptoms. There are targeted kinase inhibitors that may be efficacious in treating SM, and more severe forms may require other medications, including chemotherapy. There are also many other related mast cell disorders, including mast cell activation syndrome, which is less well defined and is not associated with c-KIT mutations, and hereditary alpha-tryptasemia (HaT).
Mediators Derived from Mast Cells
The functions of mast cells are mediated by soluble molecules released from the activated cells (Fig. 6; see also Table 2). These mediators may be divided into preformed mediators, which include vasoactive amines, and newly synthesized media tors, which include lipid mediators and cytokines.
Fig6. Biologic effects of mediators of immediate hypersensitivity. Mast cell and basophil media tors include vasoactive amines and enzymes stored preformed in granules, as well as cytokines and lipid mediators, which are largely newly synthesized on cell activation. The biogenic amines and lipid mediators induce vascular leakage, bronchoconstriction, and intestinal hypermotility, all components of the immediate response. Cytokines and lipid mediators contribute to inflammation, which is part of the late-phase reaction. Enzymes probably contribute to tissue damage. Activated eosinophils release preformed cationic proteins and enzymes that are toxic to parasites and host cells. Some eosinophil granule enzymes probably contribute to tissue damage in chronic allergic diseases. LTC4 , Leukotriene C4 ; PAF, platelet-activating factor; PGD2 , prostaglandin D2 ; TNF, tumor necrosis factor.
Vasoactive Amines. Many of the biologic effects of mast cell activation are mediated by vasoactive amines that are released from cytoplasmic granules and act on blood vessels and smooth muscle. Vasoactive amines are low-molecular weight compounds that contain an amine group and act directly on blood vessels. In human mast cells, the major mediator of this class is histamine, but in some rodents, serotonin may also be important. Histamine acts by binding to target cell receptors, and different cell types express distinct classes of histamine receptors (e.g., H1, H2, H3) that can be distinguished by their sensitivity to different pharmacologic inhibitors. The actions of histamine are short lived because histamine is rapidly removed from the extracellular milieu by amine-specific transport systems. Histamine binding to cellular receptors initiates intracellular events, such as phosphatidylinositol breakdown to IP3 and DAG, and these products cause different changes in different cell types. Histamine actions on endothelium include contraction of the endothelial cells, leading to increased interendothelial spaces, increased vascular permeability, and leakage of plasma into the tissues. Histamine also stimulates endothelial cells to synthesize vascular smooth muscle cell relaxants, such as prostacyclin (PGI2 ) and nitric oxide, which cause vasodilation. These actions of histamine produce the wheal-and-flare response of immediate hypersensitivity (described later). H1 receptor antagonists (commonly called antihistamines) can inhibit the vascular responses to intradermal allergen. Histamine also causes contraction of intestinal and bronchial smooth muscle. Thus, histamine may contribute to the increased peristalsis and bronchospasm associated with ingested and inhaled allergens, respectively. However, in some allergic disorders, and especially in asthma, antihistamines are not effective at suppressing the reaction. Moreover, bronchoconstriction in asthma is more prolonged than are the effects of histamine, indicating that other mediators are important in some forms of allergy.
Granule Enzymes and Proteoglycans. Neutral serine pro teases, including tryptase and chymase, are the most abundant protein constituents of mast cell secretory granules and may contribute to tissue damage in immediate hypersensitivity reactions. Tryptase is present in all human mast cells and is not known to be present in any other cell type. Therefore, the presence of tryptase in human biologic fluids is interpreted as a marker of mast cell activation, and serum tryptase assays have been used to diagnose anaphylaxis and other disorders associated with mast cell activation. Chymase is found in some human mast cells and its presence or absence is one criterion for characterizing human mast cell subsets, as discussed earlier. The functions of these enzymes in vivo are not established; however, several activities demonstrated in vitro suggest important bio logic actions. For example, tryptase cleaves and activates collagenase, thereby causing tissue damage, whereas chymase can convert angiotensin I to angiotensin II, which causes transient vasoconstriction. Chymase also degrades epidermal basement membranes and stimulates mucus secretion. Other enzymes found within mast cell granules include carboxypeptidase A and cathepsin G. Basophil granules also contain several enzymes, some of which are the same as those in mast cell granules, such as neutral proteases.
Proteoglycans, including heparin and chondroitin sulfate, are also major constituents of mast cell and basophil granules. These molecules are composed of a polypeptide core and multiple unbranched glycosaminoglycan side chains that impart a strong net negative charge to the molecules. Within the granules, proteoglycans serve as storage matrices for positively charged amines, proteases, and other mediators and prevent their accessibility to the rest of the cell. The mediators are released from the proteoglycans at different rates after granule exocytosis, with vasoactive amines dissociating more rapidly than tryptase or chymase. In this way, the proteoglycans may control the kinetics of immediate hypersensitivity reactions.
Lipid Mediators. Mast cell activation results in the rapid de novo synthesis and release of lipid mediators that have a variety of effects on blood vessels, bronchial smooth muscle, and leukocytes. The most important of these mediators are derived from arachidonic acid, which is generated by PLA2-mediated hydrolysis of membrane phospholipids, as discussed earlier. Arachidonic acid is then metabolized by either the cyclooxygenase or lipoxygenase pathways to produce mediators of allergic reactions.
The major arachidonic acid–derived mediator produced by the cyclooxygenase pathway in mast cells is prostaglandin D2 (PGD2 ). Released PGD2 binds to receptors on smooth muscle cells and acts as a vasodilator and a bronchoconstric tor. PGD2 also promotes neutrophil chemotaxis and accumulation at inflammatory sites. PGD2 synthesis can be prevented by cyclooxygenase inhibitors, such as aspirin, and nonsteroidal antiinflammatory agents, such as ibuprofen. These drugs may paradoxically exacerbate asthmatic bronchoconstriction in certain settings because they shunt arachidonic acid toward pro duction of leukotrienes, discussed next.
The major arachidonic acid–derived mediators produced by the lipoxygenase pathway are the leukotrienes, especially LTC4 and its degradation products LTD4 and LTE4 , all of which are called cysteinyl leukotrienes. LTC4 is made mainly by mast cells in mucosa and by basophils, but not by mast cells in connective tissues. Mast cell–derived leukotrienes bind to specific receptors on smooth muscle cells, different from the receptors for PGD2 , and cause prolonged bronchoconstriction. When injected into the skin, these leukotrienes produce a long-lived wheal-and flare reaction.
A third type of lipid mediator produced by mast cells and basophils, as well as several other cell types, is platelet-activating factor (PAF), named for its discovery as an inducer of rabbit platelet aggregation. PAF is synthesized as a derivative of membrane phospholipids. It has direct bronchoconstricting actions, causes retraction of endothelial cells, and relaxes vascular smooth muscle. However, PAF is hydrophobic and is rapidly destroyed by a plasma enzyme called PAF hydrolase, which limits its biologic actions. Individuals with an inherited deficiency of PAF hydrolase are at high risk for developing early-onset asthma. Levels of PAF and its metabolites are elevated in ana phylaxis. In rodent models, pharmacologic inhibitors of PAF receptors ameliorate some aspects of immediate hypersensitivity in the lung, but PAF antagonists have not proved useful in clinical trials. PAF also may be important in late-phase reactions, in which it can activate inflammatory leukocytes.
Cytokines. Mast cells produce many cytokines that contribute to allergic inflammation (the late-phase reaction). These cytokines include TNF, IL-1, IL-4, IL-5, IL-6, IL-9, IL-13, CCL3, CCL4, and various colony-stimulating factors, such as IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). As mentioned earlier, mast cell activation induces transcription and synthesis of these cytokines. Th2 cells that are recruited into the sites of allergic reactions also produce some of these cytokines. The cytokines that are released from activated mast cells, Th2 cells, and possibly ILC2s are mainly responsible for the inflammation associated with the late-phase reaction. TNF activates endothelial expression of adhesion molecules and together with chemokines accounts for neutrophil and monocyte recruitment to the reaction site. In addition to allergic inflammation, mast cell cytokines also contribute to innate immune responses to infections. For example, as we will discuss later, mouse models indicate that mast cells are required for effective defense against some bacterial infections, and this effector function is mediated largely by TNF.
Eosinophils
Eosinophils are bone marrow–derived granulocytes that are abundant in the inflammatory infiltrates of late-phase reactions and are involved in many of the pathologic processes in allergic diseases. GM-CSF, IL-3, and IL-5 promote eosinophil differentiation from myeloid precursors in the bone marrow and, after maturation, they circulate in the blood. Eosinophils are normally present in peripheral tissues, especially in mucosal linings of parts of the respiratory, gastrointestinal, and genitourinary tracts. The granules of eosinophils contain basic proteins that bind acidic dyes such as eosin (see Table 2 and Fig. 2C). As mentioned earlier, eosinophils express very low levels of FcεRI, and the receptor lacks a signaling chain, so its function in these cells is unclear.
Cytokines produced by Th2 cells and ILC2s promote the activation of eosinophils and their recruitment to late-phase reaction sites. Both Th2 cells and ILC2s are sources of IL-5, which stimulates bone marrow production of eosinophils and is a potent activator of mature eosinophils that enhances the ability of these cells to release granule contents. In the absence of this cytokine (e.g., in IL-5 knockout mice), there is a deficiency of eosinophil numbers and functions. In patients with allergic asthma (characterized by overactive type 2 immune reactions), blockade of IL-4, IL-13, or IL-5 signaling ameliorates symptoms. Eosinophils are recruited into late-phase reaction sites, as well as sites of helminthic infection, and their recruitment is mediated by a combination of adhesion molecule interactions and chemokines. Eosinophils bind to endothelial cells expressing E-selectin and VCAM-1, the ligand for the VLA-4 integrin. Eosinophil recruitment and infiltration into tissues also depend on the chemokine eotaxin (CCL11), which is produced by epithelial cells at sites of allergic reactions and binds to the chemokine receptor CCR3 expressed on eosinophils. In addition, the complement product C5a and the lipid mediators PAF and LTB4 produced by mast cells also function as chemoattractants for eosinophils.
Upon activation, eosinophils release granule proteins that are toxic to microbes and may injure normal tissues. The gran ule contents of eosinophils include lysosomal hydrolases found in other granulocytes as well as eosinophil-specific proteins that are particularly toxic to helminthic organisms, including major basic protein and eosinophil cationic protein. These two cationic polypeptides have no known enzymatic activities, but they damage the integument of helminths and the cell walls of bacteria, as well as cells in normal tissues. In addition, eosinophilic granules contain eosinophil peroxidase, which is distinct from the myeloperoxidase found in neutrophils and catalyzes the production of hypochlorous or hypobromous acid. These products are also toxic to helminths, protozoa, and host cells.
Activated eosinophils, like mast cells and basophils, pro duce and release lipid mediators, including PAF, prostaglandins, and the cysteinyl leukotrienes. These eosinophil-derived lipid mediators may contribute to the pathologic processes of allergic diseases. Eosinophils also produce a variety of cytokines, including IL-4 and TGF-β, which likely promote inflammatory responses and fibrosis receptively. Eosinophils produce large amounts of the protein galectin-10 compared to other cell types and, when released from activated and dying eosinophils, the galectin-10 aggregates to form crystals that can be seen in microscopic tissue sections, historically called Charcot-Leyden crystals. These crystals are often found at sites of chronic type 2 inflammation, such as the bronchi of allergic asthma patients. Experimental evidence indicates that Charcot-Leyden crystals have a variety of proinflammatory activities, including inflammasome activation and priming of DCs to induce Th2 cell differentiation. These effects are reduced in experimental animals by treatment with antibodies that specially block crystallization of galectin-10.
