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Immunology
Mast cells act as key effector cells of inflammatory responses through degranulation. Mast cell degranulation is induced by the activation of cell surface receptors, such as FcεRI, MRGPRX2/B2, and P2RX7. Each receptor, except FcεRI, varies in its expression pattern depending on the tissue, which contributes to their differing involvement in inflammatory responses depending on the site of occurrence.
- mast cells
- degranulation
- IgE-targeted drugs
1. Introduction
Although mast cells are well known for their role as essential effectors involved in allergic reactions, they are difficult to find in the blood because mast cell progenitors (MCPs) migrate to tissues before maturing into mast cells [1,2]. In addition, MCPs differentiate and mature into mast cells with heterogeneous phenotypes based on the tissue environment. The traditional way of dividing mast cells into subtypes differs between mice and humans. Murine mast cells can be divided into mucosal mast cells (MMCs) or connective tissue mast cells (CTMCs) [2,3,4]. MMCs, present in the mucosal epithelium of the lung and gastrointestinal tract, express mouse mast cell protease (mMCPT)-1 and -2. Upon activation, MMCs release mostly leukotrienes with a small amount of histamine [4]. In contrast, CTMCs, which are present in the intestinal submucosa, peritoneum, and skin, express mMCPT-4, -5, -6, and carboxypeptidase A (CPA) and release high levels of histamine and prostaglandin D2 [4]. Human mast cells are classified based on the pattern of protease expression. MCT predominantly expresses tryptase and shows similar characteristics to murine MMC. MCTC expresses tryptase and chymase and is more similar to murine CTMC [3,4]. Under homeostatic conditions, the number of mast cells in vivo is meager. Upon encountering stimuli, mast cell activation occurs leading to proliferation and degranulation, inducing the onset of allergy. Mast cell degranulation is generally known to occur when IgE binds to the high-affinity IgE receptor FcεRI, but recently, other receptors involved in IgE-independent degranulation of mast cells have been described. Unlike FcεRI, the receptors involved in IgE-independent degranulation are not expressed in all mast cells. Their expression patterns vary depending on the tissue [5], allowing for the subtyping of tissue-specific mast cells based on the expression pattern of the receptors.
2. Factors Involved in the Differentiation, Proliferation, and Activation of Mast Cells
2.1. SCF (Stem Cell Factor) and IL-3
Mast cells are commonly characterized by the expression of SCF receptor KIT and a high-affinity IgE receptor FcεRI [2]. Most MCPs also express these two receptors, but unlike mature mast cells, they do not have granules in the cytoplasm [4]. When cultured in the presence of SCF and IL-3, mouse MCPs mature into mast cells with increased granule formation and FcεRI expression [3,4]. SCF plays a critical role in mast cell differentiation, proliferation, and survival, as shown by the mast cell deficiency in mice with a loss-of-function mutation in the genes of SCF or KIT [3]. In addition to mast cells, SCF participates in the development and function of multiple distinct cell lineages including hematopoietic progenitors, melanocytes, and germ cells [6]. SCF exists in both membrane-bound and soluble forms, and the SCF receptor KIT is activated only when SCF is bound as a homodimer rather than a monomer [7]. Membrane and soluble SCF each seem to have different biological functions in hematopoiesis, and soluble SCF is more important in mast cell development and survival [8,9]. In support of this finding, membrane SCF embedded in proteoliposomes or lipid nanodiscs improved revascularization in ischemic hind limbs without activating mast cells [10].
2.2. IL-4 and IL-33
On top of IgE production and Th2 cell differentiation, IL-4 also plays a vital role in inducing the proliferation of mast cells [21]. For example, intestinal mast cells were increased when mice were administered with IL-4 [22]. In addition, mice expressing Il-4rαF709 in all cells, a variant of the IL-4 receptor α chain (IL-4Rα) with enhanced IL-4R signaling, have an increased number of intestinal mast cells and show more severe anaphylactic reactions to the food allergy model compared to wild-type mice [22,23]. Furthermore, it was confirmed that IL-4 is directly involved in the expansion of mast cells in vivo [22]. The study used a mouse model whose endogenous MCPs had been removed by sublethal irradiation and reconstituted with a 50:50 mixture of bone marrow from CD45.1+ wild-type and CD45.2+ IL-4Rα−/− mice. The mesenteric lymph node (MLN) cells from ovalbumin (OVA)-sensitized Il-4rαF709 mice were adoptively transferred and then fed with OVA on days 1, 4, and 7 from the reconstitution.
MCPs and mast cells constitutively express suppression of tumorigenicity 2 (ST2; also known as IL-1RL1, T1, and IL-33R) [25,26]. ST2 forms a heterodimer with the IL-1 receptor accessory protein (IL-1RAcP; also known as IL1RAP) and exerts biological effects by binding to IL-33. IL-33, similar to IL-25 and thymic stromal lymphopoietin (TSLP), is an alarmin secreted from epithelial barrier cells when damaged by external stimuli from exposure to trauma, infection, or allergen [27]. The binding of IL-33 to ST2 on human mast cells promotes the secretion of cytokines, such as IL-5 and IL-13, and chemokines, such as CXCL8 and CCL1, and increases mast cell survival by suppressing apoptosis [28,29]. IL-33 is a particularly potent activator of type 2 innate lymphoid cells (ILC2s), which produce Th2 cytokines such as IL-4, IL-5, and IL-13 [27,30].
2.3. IL-9
IL-9-deficient and IL-9-overexpressing mice helped discover the central role of IL-9 in mast cell proliferation and goblet cell hyperplasia [41,42]. IL-9 acts directly on mast cells to promote their proliferation in vitro [43]. IL-9 is produced not only in ILC2, Th2, and IL-9-producing CD4+ T (Th9) cells but also in mast cells and is involved in both the activation and proliferation of mast cells [44]. While IL-9-deficient mice fail to develop oral antigen-induced anaphylaxis, the overexpression of intestinal IL-9 induces an anaphylactic phenotype [45]. In the same study, IL-9 was described to promote oral antigen sensitization by increasing mast cells in the small intestine and intestinal permeability. However, IL-9 signaling plays an important role only in oral antigen-induced anaphylaxis, not in parenteral antigen-induced systemic anaphylaxis [46]. IL-9-producing mucosal mast cells (MMC9s) characterized by Lin-IL-17RB- KIT+ST2+β7integrinlo, as well as MCPs by Lin-IL-17RB- KIT+ST2+β7integrinhi phenotype, can be identified in the mouse intestine [47]. MMC9 induction occurs upon a repeated oral antigen challenge, and this process requires IL-4 secretion from Th2 cells, while IL-9 mediates the proliferation of MMC9s in an autocrine manner [45]. The blocking of α4β7 integrin, which drives the intestinal migration of bone marrow MCPs, significantly reduced the number of MCPs and MMC9s in the intestine, suggesting that bone marrow MCPs give rise to MMC9s in the intestine [47]. When these two populations are cultured in the presence of SCF and IL-3, MMP9s seem to become less mature than MCPs [48].
3. Receptors Involved in Mast Cell Activation and Degranulation
3.1. High-Affinity IgE Receptor (FcεRI)
IgE plays an important role in allergic reactions and is involved in the pathogenesis of allergic diseases. Mast cells, which express the high-affinity IgE receptor FcεRI, are major effector cells for allergy responses and are degranulated by IgE. FcεRI exists as heterotetramer αβγ2 and heterotrimer αγ2 in humans [49]. In particular, the α chain with the extracellular domain is involved in IgE binding, while the β and γ chains are involved in signal transduction. FcεRI binding of the IgE–FcεRI complex cross-linked by an allergen induces degranulation signals to the mast cells [49]. In the absence of an allergen, FcεRI binds monomeric IgE, which provides anti-apoptosis signals to the mast cells, increasing mast cell survival [50,51,52].
3.2. Low-Affinity IgG Receptors (FcγRs)
Although anaphylaxis is mostly associated with the cross-linking of FcεRI-bound IgE by antigens, the cross-linking of low-affinity FcγRs by antigen-bound IgG can also lead to anaphylaxis [59]. IgG-mediated systemic anaphylaxis in mice can be mediated largely through IgG1 and FcγRIII [60]. As FcγRIII is an activating receptor whereas FcγRIIB is an inhibitory receptor, IgG-mediated passive systemic anaphylaxis (PSA) is reduced in FcRIII−/− mice and is enhanced in FcRIIB−/− mice [61]. Indeed, mouse mast cells express FcγRIII and FcγRIIB, as well as FcεRI [62], and the absence of FcεRIα enhances FcγRIII-dependent mast cell degranulation and anaphylaxis [63]. Human mast cells express FcεRI and FcγRIIA, but unlike human basophils, human mast cells do not express FcγRIIB [62].
Platelet-activating factor (PAF) secreted during degranulation plays an important role in anaphylactic reactions [59]. In humans, PAF levels in the plasma increase significantly after an anaphylactic reaction and correlate strongly with the severity of the response [65]. Mast cells, neutrophils, and macrophages are some of the cell populations that produce PAF, and interestingly, they can also respond to PAF [59]. Human lung mast cells and peripheral blood (PB)-derived mast cells express the PAF receptor, but human skin mast cells do not [66].
3.3. MRGPRX2/B2
Mast cell degranulation in an IgE-independent hypersensitivity reaction is activated through Mas-related G-protein-coupled receptor X2 (MRGPRX2), not FcεRI [67,68]. While FcεRI is expressed in all mast cells, MRGPRX2 is preferentially expressed in MCTC in skin tissue [5,67,68]. In mice, MRGPRB2, the mouse ortholog of MRGPRX2, is predominantly expressed in CTMCs [69], which are the mouse equivalent of MCTC. MRGPRX2/B2 binds to various agonists, including insect venom (e.g., Mastoparan), chemical components (e.g., Compound 48/80, opioids, etc.), antimicrobial peptides (e.g., LL-37, β-defensins, etc.), neuropeptides (e.g., Cortistatin-14 (CST-14), substance P (SP), etc.), and FDA-approved drugs (e.g., Icatibant, Cetrorelix, Leuprolide, Octreotide, Sermorelin, Atracurium, Tubocurarine, Rocuronium, Ciprofloxacin, etc.) [67,69]. The homology in the amino acid sequence between MRGPRX2 and MRGPRB2 is only about 53%, and the two receptors differ in the concentration of agonists required for activation, as well as in their ligand selectivity for activation and inhibition [70]. MRGPRX2/B2 is involved in mast-cell-mediated host defense against bacterial infection.
3.4. P2X Purinoceptor 7 (P2RX7)
Unlike skin mast cells, intestinal mast cells preferentially express the extracellular ATP receptor P2RX7 and participate in inflammatory responses [77,78]. ATPs are released into the extracellular space when cells are damaged and act as a danger signal to mast cells [79]. P2RX7 expression in mast cells is regulated by retinoic acid, and retinoic acid level is regulated by a degrading enzyme, CYP26B1, whose expression is high in skin fibroblasts [77]. Thus, the mast cell expression of P2RX7 is suppressed in the skin but is strong in the intestine under homeostatic conditions. However, the inhibition of CYP26B1 receptor activity increases P2RX7 expression in skin mast cells [77].
3.5. Adhesion G-Protein-Coupled Receptor E2 (ADGRE2)
ADGRE2, also known as EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2) or CD132, belongs to an adhesion G-protein-coupled receptor family [80]. It consists of a large extracellular domain (α subunit) and a seven-transmembrane domain (β subunit). Although the α and β subunits of ADGRE2 are initially translated into a single polypeptide precursor, they are non-covalently linked by autocatalytic cleavage [80]. ADGRE2 is primarily expressed in myeloid leukocytes [81] but also on the surface of lung mast cells and the HMC1 human mast cell line [82].
The endogenous ligand of ADGRE2 is dermatan sulfate, which is the predominant glycosaminoglycan in the skin [80]. Despite the binding of ADGRE2 to dermatan sulfate itself not inducing a detectable mast cell activation response, mechanical force added to dermatan sulfate binding induces mast cell degranulation [80]. When mast cells attached to dermatan sulfate are stimulated by mechanical vibration, the α subunit of ADGRE2 dissociates from the β subunit, leading to mast cell activation [83].
4. Development of Drugs Targeting the Inhibition of Mast Cell Degranulation
4.1. Inhibition of IgE-Dependent Degranulation
Most early biological drugs developed for allergy treatment were anti-IgE Abs that inhibit the binding of IgE to FcεRI on the effector cells. Efforts have been made to strategically block the binding of IgE to FcεRI without affecting the binding of IgE to the low-affinity IgE receptor CD23 to increase the treatment’s potency. CD23 is expressed in a variety of cells, including T cells, B cells, polymorphonuclear leucocytes, monocytes, follicular dendritic cells, intestinal epithelial cells, and bone marrow stromal cells [85]. In B cells, CD23 is involved in the suppression of IgE production [85]. Presently, a fusion protein that links the FcεRIα domain to the Fc domain of Abs is being developed as an IgE blocking agent.
Various types of biologic drugs have been developed, but many have been discontinued for various reasons. For example, quilizumab is a humanized monoclonal Ab (mAb) that recognizes only membrane IgE (mIgE), not serum IgE. It has an afucosylated Fc domain to enhance its binding to FcγRIIIA and functions by eliminating mIgE-positive B cells via NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC) [86,87]. However, in phase 2 clinical trials with patients with allergic asthma or chronic spontaneous urticaria (CSU), quilizumab only partially reduced serum IgE levels and did not significantly improve symptoms. Compared to omalizumab, XmAb7195 is an anti-IgE mAb with 5.3-fold higher IgE binding affinity and 400-fold higher binding affinity to the inhibitory IgG receptor FcγRIIB due to two point mutations in the IgG1 Fc domain [86,87].
4.1.1. Omalizumab
Omalizumab (Xolair) is the only anti-IgE agent approved by the FDA to date. It is a humanized IgG1 mAb that blocks IgE binding to FcεRI by recognizing the Cε3 domains of IgE [86]. Omalizumab improves allergic symptoms by reducing the free IgE levels in the blood and FcεRI expression on effector cells. However, injection site reactions and systemic anaphylaxis have been reported from the clinical trials of omalizumab [88,89,90]. More recently, a study on omalizumab reported that these adverse events occur through the interaction between the IgG1 Fc domain and IgG receptor FcγR using an FcγR-humanized mouse model expressing hFcγRI, hFcγRIIaH131, hFcγRIIbI232, hFcγRIIcstop13, hFcγRIIIav158, and hFcγRIIIbNA2 [91]. Omalizumab is currently approved for the treatment of moderate to severe persistent allergic asthma patients aged 6 years and older, CSU patients aged 12 years and older, and nasal polyp patients aged 18 years and older [92].
4.1.2. Ligelizumab
Ligelizumab (QGE031), similar to omalizumab, is a humanized IgG1 mAb that recognizes the Cε3 domains of IgE but has a higher binding affinity to IgE than omalizumab [86,87]. Ligelizumab inhibits the binding of IgE to FcεRI more effectively than omalizumab. However, ligelizumab is less effective than omalizumab in suppressing the binding of IgE to the low-affinity IgE receptor CD23 [102]. Due to the fact that IgE binding to CD23 on B cells induces the inhibition of IgE production, ligelizumab is more effective than omalizumab in suppressing IgE production in B cells [102]. Unlike the expected, clinical trials of ligelizumab have not demonstrated a greater clinical benefit of ligelizumab over omalizumab. Ligelizumab showed better efficacy than omalizumab on inhaled and skin allergen provocation responses in patients with mild allergic asthma but not superior to the placebo or omalizumab in patients with severe asthma [103,104]. So, its development in asthma was eventually discontinued.
4.1.3. UB-221
UB-221 is an IgG1 mAb (a humanized mouse 8D6 Ab) and its binding affinity (KD) to the Cε3 domain of IgE is 5.85 × 10−11 M, which is approximately a four times stronger affinity than omalizumab (KD = 2.25 × 10−10 M) but about four times weaker than ligelizumab (KD = 1.61 × 10−11 M) [108]. Unlike omalizumab and ligelizumab, UB-221 does not interfere with IgE binding to CD23 [108]. This allows UB-221 to effectively inhibit IgE synthesis in peripheral blood mononuclear cells (PBMCs) in an experimental setting where the IgE synthesis is stimulated by IL-4 and anti-CD40 Ab. UB-221 can simultaneously inhibit the IgE production of B cells and degranulation of effector cells such as mast cells.
4.1.4. IgETRAP
Presently, the fusion protein IgETRAP (GI-301), in which the IgD/IgG4 hybrid Fc domain is linked to the α chain of FcεRI, is being developed as an IgE blocking agent [37]. The α chain of FcεRI of IgETRAP has a high IgE affinity, about 69 times stronger than that of omalizumab. In addition, the Fc domain of IgETRAP has no binding sites for FcγRs and complement component 1q (C1q), unlike the IgG1 Fc domain used for all other anti-IgE agents, and therefore, IgETRAP does not induce ADCC, complement-dependent cytotoxicity (CDC), or IgG-mediated anaphylaxis [37]. A combination approach with omalizumab has been sought to mitigate the potential risk of developing anaphylaxis to OIT. Considering that the use of omalizumab also has a potential risk of anaphylaxis [91], a non-IgG1 agent such as IgETRAP may be a better combination option for OIT. In fact, IgETRAP reduced free IgE levels in the sera of CSU patients more effectively than omalizumab in vitro and better controlled high blood IgE levels in cynomolgus monkeys when administered subcutaneously [37]. In an immunogenicity assay conducted by EpiScreen using CD8+-depleted PBMCs from at least 50 individuals with various HLA types, IgETRAP showed a T cell response in <10% of donors, comparable to the response observed from omalizumab and trastuzumab (anti-HER2 receptor mAb; Herceptin) used as negative controls [37].
4.2. Inhibition of IgE-Independent Degranulation
Though no news on the development of MRGPRX2-targeting drugs is available to date, the involvement of MRGPRX2 in the IgE-independent mast cell degranulation pathway suggests MRGPRX2 as a potential target for anti-allergy drug development. One study showed the selective depletion of MRGPRX2+ mast cells in the skin using photosensitizer-conjugated anti-MRGPRX2 Ab injection and near-infrared irradiation [111]. Moreover, several studies reported the inhibition of MRGPRX2-mediated mast cell degranulation and the alleviation of inflammatory symptoms using various approaches, such as immunomodulatory single-strand oligonucleotide (ssON), DNA aptamer, chemicals, and tripeptide QWF [112,113,114,115]. These treatments inhibit mast cell degranulation by MRGPRX2, alleviating the inflammatory response. Recently, natural products such as flavonoids, phenols, triterpenoid saponins, chalcones, and glycosides also have been reported to inhibit MRGPRX2-mediated inflammatory responses [67].
This entry is adapted from the peer-reviewed paper 10.3390/cells12111506
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