Mechanisms of Allergy: Important Discoveries Bergmann K-C, Ring J (eds): History of Allergy. Chem Immunol Allergy. Basel, Karger, 2014, vol 100, pp 165–171 DOI: 10.1159/000358733
Mast Cell Research Hirohisa Saito National Research Institute for Child Health and Development, Tokyo, Japan
3
The role of mast cells in allergy remained unrevealed until the observation that they contained histamine in 1953, and then the discovery of immunoglobulin E (IgE) in 1966, nearly a century after Ehrlich’s first publication. After the discovery of IgE, myeloma-derived IgE from Peter Shackford, who made a great contribution to mankind by providing 40 liters of plasma in the year prior to his death, was distributed to many researchers. This accelerated the exploration of the mechanisms involved in allergic reactions, especially regarding the role of mast cells in IgE-mediated reactions. The identification of mast cells as a progeny of a bone marrow hematopoietic stem cell in 1977 led us to the successful in vitro culture of mast cells. Along with the development of molecular biological techniques, the structure of the high-affinity IgE receptor (FcεRI) was determined in 1989. Thus, we now understand the whole molecules the expression of which is changed when mast cells are activated via FcεRI cross-linking. However, the physiological and pathological roles of mast cells, especially where IgE is not involved, are not yet fully understood.
It will be necessary to determine the mechanisms involved in the ‘non-IgE-mediated’ steps of mast cell activation in allergic or other diseases. © 2014 S. Karger AG, Basel
As was first described in 1878 by Paul Ehrlich, mast cells are present throughout connective tissues and mucosal surfaces, particularly at the interface with the external environment such as the skin, respiratory tract and gastrointestinal tract. On the basis of their unique staining characteristics, such as metachromasia for basic/aniline dyes, and large granules, it was mistakenly believed that mast cells existed to nourish the surrounding tissue. It took almost a century after the discovery of mast cells until we understood the important role of mast cells in allergy or immunity, i.e. the presence of histamine [1] in their granules and the functional high-affinity immunoglobulin E (IgE) Fc receptors (FcεRI) on their surfaces (fig. 1) [2–4]. Since then, our knowledge about the role of mast cells in allergic diseases has rapidly progressed. Now, IgE-sensitized mast Downloaded by: UCONN Storrs 198.143.38.1 - 7/6/2015 7:43:00 PM
Abstract
Fig. 1. First identification of IgE molecules bound to mast cells. a Fluorescence micrograph of a paraffin section from a monkey skin biopsy incubated with ragweed-allergic serum and stained for bound antigen E. b Light microscope micrograph of the same area counterstained with toluidine blue [adapted from 2, fig. 8]. Binding of 125Ianti-IgE with mast cells – the microscope was focused on radioactive grains (c) or the cells (d). Mast cells in sensitized monkey skin bound 125I-anti-IgE [adapted from 4, fig. 1A, B]. Panels a and b, adapted from Hubscher et al. [2], demonstrate that some mast cells can bind allergen-specific antibody, and panels c and d, adapted from Tomioka and Ishizaka [4], confirm that mast cells can bind IgE antibody.
c
cells are known to immediately release a variety of biological mediators such as histamine or leukotrienes in response to allergen stimulation. They also synthesize and release cytokines several hours after this allergen stimulation. In this chapter, the history of mast cell research is chronologically described, mainly with reference to articles that can be obtained electronically.
Discovery of Histamine in Mast Cells/Basophils Histamine has been suspected to cause anaphylaxis since the studies by Dale and Laidlaw were published in 1910. However, anaphylactic shock was also recognized to be associated with a decrease in the histamine content of blood. This controversy was, of course, interpreted by the later discovery that histamine is stored in basophils, rapidly degenerated
166
b
d
when it is released in vivo into blood, and that basophils also release histamine during serum separation in vitro, as well as allergic reactions in vivo. Soon after the discovery of histamine in mast cells in 1953, Fawcett [5] reported that rat mast cells can release histamine when stimulated with compound 48/80, which was known as a histamine liberator. Related to histamine release in allergic reactions, in 1964 Lichtenstein and Osler [6] first demonstrated in vitro the release of histamine from human leukocytes obtained from hay fever patients by the specific antigen. Following the discovery of IgE in 1966 [7], it was confirmed that human leukocytes [8], and later basophils [9] among leukocytes, can release histamine in an IgE-dependent manner. Knowledge regarding the molecular characterization of human IgE made it possible for us to identify the IgE of other species. Thus, IgE-sensitized mast cells were demonstrated for the first time to release histamine after challenge with allergen using rat peritoneal mast cells [3].
Saito
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a
bgJ/bgJ (beige)
Bone marrows
X-ray
Identification of granulocyte type (beige or wild)
12 days Identification of mast cell type (beige or wild)
+/+
As has been nicely reviewed elsewhere [10], in 1942, H1-antihistamines, which stabilize the inactive conformation of the H1-receptor as inverse agonists, were introduced for clinical use. Now, more than 45 H1-antihistamines are available worldwide, comprising the largest class of medications used in the treatment of allergic diseases such as allergic rhinoconjunctivitis and urticaria.
Discovery of IgE Receptors on Mast Cells/Basophils Myeloma-derived IgE from Mr. Peter Shackford, who made a great contribution to mankind by providing 40 liters of plasma until his death, was purified by Kimishige Ishizaka. It was then distributed to many researchers, which accelerated the exploration of the mechanisms involved in allergic reactions, especially regarding the role of mast cells [11] and basophils [8] in IgE-mediated reactions. Metzger’s group started to identify the structural features of IgE receptors present on basophils and mast cells [12]. IgE molecules alone do not normally activate mast cells after binding to their receptors. This phenomenon seemed unique, and certainly unlike other ligand-receptor interactions. It was revealed that multivalent antigens/molecules are usually capable of activating IgE-sensitized
Mast Cell Research
Spleen colony
W/W Wv
3 mast cells whereas univalent antigens/molecules in general cannot [13]. In 1989, along with the development of molecular biological techniques, the structure of FcεRI was finally determined [14]. Today, we understand that FcεRI is a tetrameric structure comprised of an α-chain (FcεRIα) which binds IgE, a β-chain-signaling subunit (FcεRIβ), and two γ-subunits which exist as a homodimer-signaling subunit (FcεRIγ) [15]. The molecular cloning of FcεRI and the development of techniques to make mice deficient of the targeted genes have accelerated our understanding of the signal transduction pathway in IgE-dependent mast cell activation.
Discovery of Mast Cell Origin and the Culture Methods Regarding the origin of mast cells, Ehrlich suggested the source to be fibroblasts present in connective tissue. He could not imagine, of course, that these cells might derive from precursors of the hematopoietic lineage. It took 99 years until Kitamura demonstrated hematopoietic stem cells to be the origin of mast cells in 1977. Even in 1976, a textbook of hematology was published in which it was described that mast cells are derived from tissue. Kitamura et al. [16] transplanted bone marrow cells of
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Fig. 2. Identification of mast cells as a progeny of a multipotent hematopoietic stem cell. Mixed bone marrow cells obtained from a beige (bgJ/bgJ) mouse and a wild-type mouse (+/+) were injected to an irradiated wild-type mouse, following which single hematopoietic cell-derived spleen colonies were obtained after 12 days. Cells obtained from a single colony were injected to a mast cell-deficient (W/ WV) mouse. Each colony gave rise to either mast cells and granulocytes with largesized granules (beige) or those with normal granule size (wild type).
+/+ (wild type)
168
using a similar system: culture of bone marrow or spleen hematopoietic cells in the presence of supernatants derived from antigen-activated lymphocytes [21]. This factor supporting murine mast cell growth in vitro was cloned and termed as IL-3 [22]. Only basophil growth was obtained when using the same system for murine mast cell development [23], i.e. the culture of human hematopoietic cells in the supernatant of activated lymphocytes. Therefore, the discovery and cloning of human IL-3 was eagerly expected for culturing human mast cells. Human IL-3 was cloned based on the sequence of cDNA having a significant homology to that encoding murine IL-3. However, even by culturing hematopoietic cells with recombinant human IL-3, human mast cells did not grow [24]. These results disappointed all mast cell investigators at that time. In fact, human IL-3 has a significant sequence homology with murine IL-3. However, the degree of homology between human and murine IL-3 is almost similar (approximately 26–28% at amino acid sequence) to that between human IL-3 and granulocyte-macrophage colony-stimulating factor (GMCSF). Also, the receptor structure for IL-3 is distinct between human and mouse. The human has a common β-subunit that does not bind any cytokine by itself, but forms high-affinity receptors for GM-CSF, IL-3 and IL-5 with the respective α-subunit. In contrast, the mouse has two distinct β-subunits: one is specific for the IL-3 receptor and exists only on mast cells, and the other is equivalent to the human common β-subunit [25]. As was suggested in earlier studies [18, 19], LeviSchaffer et al. [26] demonstrated that fibroblasts were able to support the development of IL-3-dependent cultured murine mast cells into the mature connective-type mast cell phenotype. They also reported that mature human lung mast cells can be maintained in vitro when co-cultured with fibroblasts. Then, in 1989, Furitsu et al. [27] finally succeeded in identifying the development of human mature mast cells from hematopoietic cells when cocultured with the mouse 3T3 fibroblast cell line. In 1990, a fibroblast-derived growth factor for mast cells was cloned, termed SCF, and the results were published almost simultaneously in more than 10 original papers by different researchers. While
Saito
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beige (C57BL-bgJ/bgJ) mice with extra-large-sized granules in granulocytes or mast cells into wild-type C57BL mice which had been irradiated to eliminate bone marrow precursors. They then found mast cells with extra-large-sized granules in the wild-type mice after bone marrow transplantation. Kitamura et al. [17] also discovered the two different mice strains genetically lacking mast cells. One is Sl/Sld mice, which were later found to lack stem cell factor (SCF), and another is W/Wv mice, which lack the receptor for SCF, called c-Kit. By using these ‘natural’ mast cell-deficient mice, it was established that mast cells originate from a common progenitor shared with granulocytes, i.e. from a hematopoietic stem cell (fig. 2). Also, it was revealed that immature mast cell progenitors migrate from bone marrow into the tissue through blood circulation, unlike immature granulocytes which are kept in the bone marrow. Ginsburg and Lagunoff [18] first reported the development of mast cells in vitro in the early 1960s. However, from mouse lymph node cells, but not from bone marrow cells, mast cell growth was obtained on fibroblast layers. Since the identification of mast cells was based only on morphology, controversy ensued as to whether these cells are mast cells. Then, in 1976, Ishizaka et al. [19] confirmed mast cell growth in the long-term culture of rat thymus cells on fibroblast monolayers by demonstrating the presence of IgE receptors on the cultured cells. These observations were interpreted by the later discovery that a very small number of hematopoietic cells, which are present even in lymphoid tissues, develop into mast cells when stimulated with a lymphocytederived mast cell growth factor, interleukin (IL)-3. Also, fibroblast layers would have presumably provided growth factors such as SCF to support the full maturation of mast cells. Identification of mast cells as a progeny of a hematopoietic stem cell in 1977 [16] prompted us to utilize hematopoietic tissues for obtaining mast cells. In 1980, Denburg et al. [20] succeeded in obtaining guinea pig basophils (basophils are a dominant cell type compared to mast cells in guinea pig) in vitro by culturing bone marrow cells in the presence of supernatant derived from antigen-activated lymphocytes. Then, culture of mouse mast cells was reported by several groups almost simultaneously
a
b
3
Fig. 3. A human mast cell colony grown in
c
one of these articles was regarding the cloning of human SCF [28], mouse or rat SCF was found to similarly support human mast cell development. Although SCF could support the development of human mast cells, a substantial number of functionally mature mast cells were not always obtained. In fact, SCF expands more hematopoietic stem cells than mast cells. IL-6, another fibroblast-derived factor [29], is capable of supporting the self-renewal of hematopoietic stem cells as well as SCF, while most other cytokines, such as GM-CSF, suppress it. Therefore, SCF and IL-6 were employed for human mast cell culture [30]. IL-6 was later found to stimulate human mast cell maturation. Thus, for preventing the production of stem cell self-renewal-suppressive cytokines such as GM-CSF by macrophages, PGE2 [30], the methylcellulose culture system [31] (fig. 3) or purification of mast cell progenitors [32] are used together with SCF and IL-6. When highly purified
Mast Cell Research
d
mast cell progenitors are used, IL-3 or IL-5, but not GM-CSF, increases the number of cultured mast cells in addition to SCF and IL-6 [32].
Mast Cell Phenotypes Phenotypically distinct subsets of mast cells are present in rodents based on their distinct staining characteristics, T cell dependency and functions, namely connective tissue mast cells and mucosal mast cells [33]. In humans, two types of mast cells have been recognized based on the neutral proteases they express. TC-type mast cells (MCTC) contain tryptase together with chymase and other neutral proteases, whereas T-type mast cells (MCT) contain tryptase but lack the other neutral proteases present in MCTC [34]. MCTC preferentially dwell in connective tissue such as skin, while MCT are often found
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methylcellulose medium. Mast cell colonies (a, c) and macrophage colonies (b, d) were observed in the methylcellulose medium when adult peripheral blood cells were cultured for 6 weeks in the presence of SCF, IL-6 and IL-3. The mast cells look refractile and equally round, while macrophages look extra-large and irregularly shaped. Objective lenses were used. Original magnification ×10 (a, b), ×20 (c, d).
in mucosa such as airway epithelium. MCTC can respond to various non-immunological stimuli such as C5a or substance P, while MCT do not. Asthma can be divided into 2 subgroups (‘Th2high’ and ‘Th2-low’ asthma), based on epithelial cell gene signatures for the activity of Th2 cytokines such as IL-13. Patients with Th2-high asthma have more infiltration of mast cells into the airway epithelium. These intraepithelial mast cells express both tryptase and carboxypeptidase A3 (CPA3), but not chymase [35]. According to the classical definition [34], MCT were not supposed to express CPA3. However, according to public microarray data (http://www.nch. go.jp/imal/GeneChip/public.htm), all types of mast cells and basophils express the CPA3 transcript. Therefore, we can consider these epithelial mast cells at least as a sort of MCT. Mast cells exposed to conditioned media from IL-13-activated epithelial cells showed downregulation of chymase, but no change in tryptase or CPA3 expression [35]. This may be the reason why MCT are preferentially found in mucosa and are deficient in immunodeficiency patients [34]. This is in contrast to results demonstrating that human mast cell phenotypes, such as chymase expression, are retained over weeks, even when these mast cells are cultured in standard mast cell culture condition (supplemented with SCF and IL-6) [36].
Discovery of Cytokine Production by Mast Cells Activated mast cells can release various protein cytokines as well as low-molecular-weight mediators such as histamine. It was described in 1987 that activated mast cells express and release cytokines such as tumor necrosis factor, TNF [37]. Later, mast cell-derived TNF was shown to play a significant role
in airway hyperreactivity, T cell development (enhancement) and bacterial infection (protection). IL-4 production seems reproducible when using mouse mast cells. However, so far only a few groups have succeeded to immunohistochemically demonstrate the presence of IL-4 on human mast cells. In any case, at least in humans, basophils are more potent producers of IL-4, and IL-4 potently activates human mast cell function and maturation. Interestingly, human mast cells can produce a substantial amount of another Th2 cytokine, IL-13, in response to IgE-mediated stimuli when mast cells are pre-incubated with IL-4 [38]. Through the development of molecular biological techniques, such as microarray, and the establishment of the human mast cell culture system, we now understand that both human and mouse mast cells can express and release a variety of cytokines (mainly Th2 cytokines) and chemokines (mainly CC-chemokines and IL-8) in response to IgE- and non-IgE-associated stimuli (http://www.nch.go.jp/imal/GeneChip/public. htm) [38, 39].
Future Perspective Today we even understand the whole molecular pattern, the expression of which is changed when mast cells are activated via FcεRI cross-linking. However, the physiological and pathological roles of mast cells, especially where IgE is not involved, are not fully understood yet. It will be necessary to determine the mechanisms involved in the ‘non-IgEmediated’ phase of mast cell activation in allergic or other diseases. Also, we need to understand the relative role of mast cells in allergic or innate-type inflammation compared to the roles of other immune and tissue-derived cells.
References
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3 Bach MK, Bloch KJ, Austen KF: IgE and IgGa antibody-mediated release of histamine from rat peritoneal cells. 1. Optimum conditions for in vitro preparation of target cells with antibody and challenge with antigen. J Exp Med 1971;133:752–771.
4 Tomioka H, Ishizaka K: Mechanisms of passive sensitization. 2. Presence of receptors for IgE on monkey mast cells. J Immunol 1971;107:971–978. 5 Fawcett DW: Cytological and pharmacological observations on the release of histamine by mast cells. J Exp Med 1954;100:217–224.
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1 Riley JF, West GB: The presence of histamine in tissue mast cells. J Physiol 1953; 120:528–537. 2 Hubscher T, Watson JI, Goodfriend L: Target cells of human ragweed-binding antibodies in monkey skin. 1. Immunofluorescent localization of cellular binding. J Immunol 1970;104:1187–1195.
19 Ishizaka T, Okudaira H, Mauser LE, Ishizaka K: Development of rat mast cells in vitro. 1. Differentiation of mast cells from thymus cells. J Immunol 1976;116:747–754. 20 Denburg JA, Davison M, Bienenstock J: Basophil production. J Clin Invest 1980; 65: 390–399. 21 Schrader JW, Lewis SJ, Clark-Lewis I, Culvenor JG: The persisting (P) cell: histamine content, regulation by a T cell-derived factor, origin from a bone marrow precursor, and relationship to mast cells. Proc Natl Acad Sci USA 1981;78:323–327. 22 Ihle JN, Keller J, Oroszlan S, Henderson LE, Copeland TD, Fitch F, Prystowsky MB, Goldwasser E, Schrader JW, Palaszynski E, Dy M, Lebel B: Biologic properties of homogeneous interleukin 3. 1. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, p cell-stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity. J Immunol 1983;131:282–287. 23 Razin E, Rifkind AB, Cordon-Cardo C, Good RA: Selective growth of a population of human basophil cells in vitro. Proc Natl Acad Sci USA 1981;78:5793–5796. 24 Saito H, Hatake K, Dvorak AM, Leiferman KM, Donnenberg AD, Arai N, Ishizaka K, Ishizaka T: Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins. Proc Natl Acad Sci USA 1988;85:2288–2292. 25 Miyajima A: Molecular structure of the IL3, GM-CSF and IL-5 receptors. Int J Cell Cloning 1992;10:126–134. 26 Levi-Schaffer F, Austen KF, Caulfield JP, Hein A, Gravallese PM, Stevens RL: Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes. J Immunol 1987;139:494–500. 27 Furitsu T, Saito H, Dvorak AM, Schwartz LB, Irani AM, Burdick JF, Ishizaka K, Ishizaka T: Development of human mast cells in vitro. Proc Natl Acad Sci USA 1989; 86:10039–10043. 28 Martin FH, Suggs SV, Langley KE, Lu HS, Ting J, Okino KH, Morris CF, McNiece IK, Jacobsen FW, Mendiaz EA, Birkett NC, Smith KA, Johnson MJ, Parker VP, Flores JC, Patel AC, Fisher EF, Erjavec HO, Herrera CJ, Wypych J, Sachdev RK, Pope JA, Leslie I, Wen D, Lin CH, Cupples RL, Zsebo KM: Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 1990;63:203–211.
Prof. Hirohisa Saito National Research Institute for Child Health and Development 2-10-1 Okura, Setagaya-ku Tokyo, 157-8535 (Japan) E-Mail saito-hr @ ncchd.go.jp
Mast Cell Research
29 Van Damme J, Cayphas S, Van Snick J, Conings R, Put W, Lenaerts JP, Simpson RJ, Billiau A: Purification and characterization of human fibroblast-derived hybridoma growth factor identical to T-cell-derived Bcell stimulatory factor-2 (interleukin-6). Eur J Biochem 1987;168:543–550. 30 Saito H, Ebisawa M, Tachimoto H, Shichijo M, Fukagawa K, Matsumoto K, Iikura Y, Awaji T, Tsujimoto G, Yanagida M, Uzumaki H, Takahashi G, Tsuji K, Nakahata T: Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells. J Immunol 1996;157:343–350. 31 Saito H, Kato A, Matsumoto K, Okayama Y: Culture of human mast cells from peripheral blood progenitors. Nat Protoc 2006; 1: 2178–2183. 32 Kirshenbaum AS, Goff JP, Semere T, Foster B, Scott LM, Metcalfe DD: Demonstration that human mast cells arise from a progenitor cell population that is CD34+, c-kit+, and expresses aminopeptidase N (CD13). Blood 1999;94:2333–2342. 33 Enerbäck L: Mast cells in rat gastrointestinal mucosa. 1. Effects of fixation. Acta Pathol Microbiol Scand 1966;66:289–302. 34 Irani AMA, Schecter NM, Craig SS, DeBlois G, Schwartz LB: Two types of human mast cells that have distinct neutral protease compositions. Proc Natl Acad Sci USA 1986;83:4464–4469. 35 Dougherty RH, Sidhu SS, Raman K, Solon M, Solberg OD, Caughey GH, Woodruff PG, Fahy JV: Accumulation of intraepithelial mast cells with a unique protease phenotype in TH2-high asthma. J Allergy Clin Immunol 2010;125:1046–1053. 36 Kashiwakura J, Yokoi H, Saito H, Okayama Y: T cell proliferation by direct cross-talk between OX40 ligand on human mast cells and OX40 on human T cells: comparison of gene expression profiles between human tonsillar and lung-cultured mast cells. J Immunol 2004;173:5247–5257. 37 Young JD, Liu CC, Butler G, Cohn ZA, Galli SJ: Identification, purification, and characterization of a mast cell-associated cytolytic factor related to tumor necrosis factor. Proc Natl Acad Sci USA 1987;84:9175–9179. 38 Bischoff SC: Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nat Rev Immunol 2007;7:93–104. 39 Nakajima T, Inagaki N, Tanaka H, Tanaka A, Yoshikawa M, Tamari M, Hasegawa K, Matsumoto K, Tachimoto H, Ebisawa M, Tsujimoto G, Matsuda H, Nagai H, Saito H: Marked increase in CC chemokine gene expression in both human and mouse mast cell transcriptomes following Fcε receptor I cross-linking: an interspecies comparison. Blood 2002;100:3861–3868.
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6 Lichtenstein LM, Osler AG: Studies on the mechanisms of hypersensitivity phenomena. 9. Histamine release from human leukocytes by ragweed pollen antigen. J Exp Med 1964;120:507–530. 7 Ishizaka K, Ishizaka T, Hornbrook MM: Physico-chemical properties of human reaginic antibody. 4. Presence of a unique immunoglobulin as a carrier of reaginic activity. J Immunol 1966;97:75–85. 8 Ishizaka T, Ishizaka K, Johansson SG, Bennich H: Histamine release from human leukocytes by anti-γE antibodies. J Immunol 1969;102:884–892. 9 Ishizaka T, De Bernardo R, Tomioka H, Lichtenstein LM, Ishizaka K: Identification of basophil granulocytes as a site of allergic histamine release. J Immunol 1972; 108: 1000–1008. 10 Simons FER, Simons KJ: Histamine and H1antihistamines: celebrating a century of progress. J Allergy Clin Immunol 2012;128: 1139–1150. 11 Ishizaka T, Ishizaka K, Orange RP, Austen KF: The capacity of human immunoglobulin E to mediate the release of histamine and slow reacting substance of anaphylaxis (SRS-A) from monkey lung. J Immunol 1970;104:335–343. 12 Kulczycki A Jr, Isersky C, Metzger H: The interaction of IgE with rat basophilic leukemia cells. 1. Evidence for specific binding of IgE. J Exp Med 1974;139:600–616. 13 Ishizaka K, Ishizaka T: Immune mechanisms of reversed type reaginic hypersensitivity. J Immunol 1969;103:588–595. 14 Blank U, Ra C, Miller L, White K, Metzger H, Kinet JP: Complete structure and expression in transfected cells of high affinity IgE receptor. Nature 1989;337:187–189. 15 Gilfillan AM, Tkaczyk C: Integrated signalling pathways for mast-cell activation. Nat Rev Immunol 2006;6:218–230. 16 Kitamura Y, Shimada M, Hatanaka K, Miyano Y: Development of mast cells from grafted bone marrow cells in irradiated mice. Nature 1977;268:442–443. 17 Kitamura Y, Yokoyama M, Matsuda H, Ohno T, Mori KJ: Spleen colony-forming cell as common precursor for tissue mast cells and granulocytes. Nature 1981; 291: 159–160. 18 Ginsburg H, Lagunoff D: The in vitro differentiation of mast cells: cultures of cells from immunized mouse lymph nodes and thoracic duct lymph on fibroblast monolayers. J Cell Biol 1967;35:685–697.
Mast Cell Research Hirohisa Saito National Research Institute for Child Health and Development, Tokyo, Japan
3
The role of mast cells in allergy remained unrevealed until the observation that they contained histamine in 1953, and then the discovery of immunoglobulin E (IgE) in 1966, nearly a century after Ehrlich’s first publication. After the discovery of IgE, myeloma-derived IgE from Peter Shackford, who made a great contribution to mankind by providing 40 liters of plasma in the year prior to his death, was distributed to many researchers. This accelerated the exploration of the mechanisms involved in allergic reactions, especially regarding the role of mast cells in IgE-mediated reactions. The identification of mast cells as a progeny of a bone marrow hematopoietic stem cell in 1977 led us to the successful in vitro culture of mast cells. Along with the development of molecular biological techniques, the structure of the high-affinity IgE receptor (FcεRI) was determined in 1989. Thus, we now understand the whole molecules the expression of which is changed when mast cells are activated via FcεRI cross-linking. However, the physiological and pathological roles of mast cells, especially where IgE is not involved, are not yet fully understood.
It will be necessary to determine the mechanisms involved in the ‘non-IgE-mediated’ steps of mast cell activation in allergic or other diseases. © 2014 S. Karger AG, Basel
As was first described in 1878 by Paul Ehrlich, mast cells are present throughout connective tissues and mucosal surfaces, particularly at the interface with the external environment such as the skin, respiratory tract and gastrointestinal tract. On the basis of their unique staining characteristics, such as metachromasia for basic/aniline dyes, and large granules, it was mistakenly believed that mast cells existed to nourish the surrounding tissue. It took almost a century after the discovery of mast cells until we understood the important role of mast cells in allergy or immunity, i.e. the presence of histamine [1] in their granules and the functional high-affinity immunoglobulin E (IgE) Fc receptors (FcεRI) on their surfaces (fig. 1) [2–4]. Since then, our knowledge about the role of mast cells in allergic diseases has rapidly progressed. Now, IgE-sensitized mast Downloaded by: UCONN Storrs 198.143.38.1 - 7/6/2015 7:43:00 PM
Abstract
Fig. 1. First identification of IgE molecules bound to mast cells. a Fluorescence micrograph of a paraffin section from a monkey skin biopsy incubated with ragweed-allergic serum and stained for bound antigen E. b Light microscope micrograph of the same area counterstained with toluidine blue [adapted from 2, fig. 8]. Binding of 125Ianti-IgE with mast cells – the microscope was focused on radioactive grains (c) or the cells (d). Mast cells in sensitized monkey skin bound 125I-anti-IgE [adapted from 4, fig. 1A, B]. Panels a and b, adapted from Hubscher et al. [2], demonstrate that some mast cells can bind allergen-specific antibody, and panels c and d, adapted from Tomioka and Ishizaka [4], confirm that mast cells can bind IgE antibody.
c
cells are known to immediately release a variety of biological mediators such as histamine or leukotrienes in response to allergen stimulation. They also synthesize and release cytokines several hours after this allergen stimulation. In this chapter, the history of mast cell research is chronologically described, mainly with reference to articles that can be obtained electronically.
Discovery of Histamine in Mast Cells/Basophils Histamine has been suspected to cause anaphylaxis since the studies by Dale and Laidlaw were published in 1910. However, anaphylactic shock was also recognized to be associated with a decrease in the histamine content of blood. This controversy was, of course, interpreted by the later discovery that histamine is stored in basophils, rapidly degenerated
166
b
d
when it is released in vivo into blood, and that basophils also release histamine during serum separation in vitro, as well as allergic reactions in vivo. Soon after the discovery of histamine in mast cells in 1953, Fawcett [5] reported that rat mast cells can release histamine when stimulated with compound 48/80, which was known as a histamine liberator. Related to histamine release in allergic reactions, in 1964 Lichtenstein and Osler [6] first demonstrated in vitro the release of histamine from human leukocytes obtained from hay fever patients by the specific antigen. Following the discovery of IgE in 1966 [7], it was confirmed that human leukocytes [8], and later basophils [9] among leukocytes, can release histamine in an IgE-dependent manner. Knowledge regarding the molecular characterization of human IgE made it possible for us to identify the IgE of other species. Thus, IgE-sensitized mast cells were demonstrated for the first time to release histamine after challenge with allergen using rat peritoneal mast cells [3].
Saito
Downloaded by: UCONN Storrs 198.143.38.1 - 7/6/2015 7:43:00 PM
a
bgJ/bgJ (beige)
Bone marrows
X-ray
Identification of granulocyte type (beige or wild)
12 days Identification of mast cell type (beige or wild)
+/+
As has been nicely reviewed elsewhere [10], in 1942, H1-antihistamines, which stabilize the inactive conformation of the H1-receptor as inverse agonists, were introduced for clinical use. Now, more than 45 H1-antihistamines are available worldwide, comprising the largest class of medications used in the treatment of allergic diseases such as allergic rhinoconjunctivitis and urticaria.
Discovery of IgE Receptors on Mast Cells/Basophils Myeloma-derived IgE from Mr. Peter Shackford, who made a great contribution to mankind by providing 40 liters of plasma until his death, was purified by Kimishige Ishizaka. It was then distributed to many researchers, which accelerated the exploration of the mechanisms involved in allergic reactions, especially regarding the role of mast cells [11] and basophils [8] in IgE-mediated reactions. Metzger’s group started to identify the structural features of IgE receptors present on basophils and mast cells [12]. IgE molecules alone do not normally activate mast cells after binding to their receptors. This phenomenon seemed unique, and certainly unlike other ligand-receptor interactions. It was revealed that multivalent antigens/molecules are usually capable of activating IgE-sensitized
Mast Cell Research
Spleen colony
W/W Wv
3 mast cells whereas univalent antigens/molecules in general cannot [13]. In 1989, along with the development of molecular biological techniques, the structure of FcεRI was finally determined [14]. Today, we understand that FcεRI is a tetrameric structure comprised of an α-chain (FcεRIα) which binds IgE, a β-chain-signaling subunit (FcεRIβ), and two γ-subunits which exist as a homodimer-signaling subunit (FcεRIγ) [15]. The molecular cloning of FcεRI and the development of techniques to make mice deficient of the targeted genes have accelerated our understanding of the signal transduction pathway in IgE-dependent mast cell activation.
Discovery of Mast Cell Origin and the Culture Methods Regarding the origin of mast cells, Ehrlich suggested the source to be fibroblasts present in connective tissue. He could not imagine, of course, that these cells might derive from precursors of the hematopoietic lineage. It took 99 years until Kitamura demonstrated hematopoietic stem cells to be the origin of mast cells in 1977. Even in 1976, a textbook of hematology was published in which it was described that mast cells are derived from tissue. Kitamura et al. [16] transplanted bone marrow cells of
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Fig. 2. Identification of mast cells as a progeny of a multipotent hematopoietic stem cell. Mixed bone marrow cells obtained from a beige (bgJ/bgJ) mouse and a wild-type mouse (+/+) were injected to an irradiated wild-type mouse, following which single hematopoietic cell-derived spleen colonies were obtained after 12 days. Cells obtained from a single colony were injected to a mast cell-deficient (W/ WV) mouse. Each colony gave rise to either mast cells and granulocytes with largesized granules (beige) or those with normal granule size (wild type).
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using a similar system: culture of bone marrow or spleen hematopoietic cells in the presence of supernatants derived from antigen-activated lymphocytes [21]. This factor supporting murine mast cell growth in vitro was cloned and termed as IL-3 [22]. Only basophil growth was obtained when using the same system for murine mast cell development [23], i.e. the culture of human hematopoietic cells in the supernatant of activated lymphocytes. Therefore, the discovery and cloning of human IL-3 was eagerly expected for culturing human mast cells. Human IL-3 was cloned based on the sequence of cDNA having a significant homology to that encoding murine IL-3. However, even by culturing hematopoietic cells with recombinant human IL-3, human mast cells did not grow [24]. These results disappointed all mast cell investigators at that time. In fact, human IL-3 has a significant sequence homology with murine IL-3. However, the degree of homology between human and murine IL-3 is almost similar (approximately 26–28% at amino acid sequence) to that between human IL-3 and granulocyte-macrophage colony-stimulating factor (GMCSF). Also, the receptor structure for IL-3 is distinct between human and mouse. The human has a common β-subunit that does not bind any cytokine by itself, but forms high-affinity receptors for GM-CSF, IL-3 and IL-5 with the respective α-subunit. In contrast, the mouse has two distinct β-subunits: one is specific for the IL-3 receptor and exists only on mast cells, and the other is equivalent to the human common β-subunit [25]. As was suggested in earlier studies [18, 19], LeviSchaffer et al. [26] demonstrated that fibroblasts were able to support the development of IL-3-dependent cultured murine mast cells into the mature connective-type mast cell phenotype. They also reported that mature human lung mast cells can be maintained in vitro when co-cultured with fibroblasts. Then, in 1989, Furitsu et al. [27] finally succeeded in identifying the development of human mature mast cells from hematopoietic cells when cocultured with the mouse 3T3 fibroblast cell line. In 1990, a fibroblast-derived growth factor for mast cells was cloned, termed SCF, and the results were published almost simultaneously in more than 10 original papers by different researchers. While
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beige (C57BL-bgJ/bgJ) mice with extra-large-sized granules in granulocytes or mast cells into wild-type C57BL mice which had been irradiated to eliminate bone marrow precursors. They then found mast cells with extra-large-sized granules in the wild-type mice after bone marrow transplantation. Kitamura et al. [17] also discovered the two different mice strains genetically lacking mast cells. One is Sl/Sld mice, which were later found to lack stem cell factor (SCF), and another is W/Wv mice, which lack the receptor for SCF, called c-Kit. By using these ‘natural’ mast cell-deficient mice, it was established that mast cells originate from a common progenitor shared with granulocytes, i.e. from a hematopoietic stem cell (fig. 2). Also, it was revealed that immature mast cell progenitors migrate from bone marrow into the tissue through blood circulation, unlike immature granulocytes which are kept in the bone marrow. Ginsburg and Lagunoff [18] first reported the development of mast cells in vitro in the early 1960s. However, from mouse lymph node cells, but not from bone marrow cells, mast cell growth was obtained on fibroblast layers. Since the identification of mast cells was based only on morphology, controversy ensued as to whether these cells are mast cells. Then, in 1976, Ishizaka et al. [19] confirmed mast cell growth in the long-term culture of rat thymus cells on fibroblast monolayers by demonstrating the presence of IgE receptors on the cultured cells. These observations were interpreted by the later discovery that a very small number of hematopoietic cells, which are present even in lymphoid tissues, develop into mast cells when stimulated with a lymphocytederived mast cell growth factor, interleukin (IL)-3. Also, fibroblast layers would have presumably provided growth factors such as SCF to support the full maturation of mast cells. Identification of mast cells as a progeny of a hematopoietic stem cell in 1977 [16] prompted us to utilize hematopoietic tissues for obtaining mast cells. In 1980, Denburg et al. [20] succeeded in obtaining guinea pig basophils (basophils are a dominant cell type compared to mast cells in guinea pig) in vitro by culturing bone marrow cells in the presence of supernatant derived from antigen-activated lymphocytes. Then, culture of mouse mast cells was reported by several groups almost simultaneously
a
b
3
Fig. 3. A human mast cell colony grown in
c
one of these articles was regarding the cloning of human SCF [28], mouse or rat SCF was found to similarly support human mast cell development. Although SCF could support the development of human mast cells, a substantial number of functionally mature mast cells were not always obtained. In fact, SCF expands more hematopoietic stem cells than mast cells. IL-6, another fibroblast-derived factor [29], is capable of supporting the self-renewal of hematopoietic stem cells as well as SCF, while most other cytokines, such as GM-CSF, suppress it. Therefore, SCF and IL-6 were employed for human mast cell culture [30]. IL-6 was later found to stimulate human mast cell maturation. Thus, for preventing the production of stem cell self-renewal-suppressive cytokines such as GM-CSF by macrophages, PGE2 [30], the methylcellulose culture system [31] (fig. 3) or purification of mast cell progenitors [32] are used together with SCF and IL-6. When highly purified
Mast Cell Research
d
mast cell progenitors are used, IL-3 or IL-5, but not GM-CSF, increases the number of cultured mast cells in addition to SCF and IL-6 [32].
Mast Cell Phenotypes Phenotypically distinct subsets of mast cells are present in rodents based on their distinct staining characteristics, T cell dependency and functions, namely connective tissue mast cells and mucosal mast cells [33]. In humans, two types of mast cells have been recognized based on the neutral proteases they express. TC-type mast cells (MCTC) contain tryptase together with chymase and other neutral proteases, whereas T-type mast cells (MCT) contain tryptase but lack the other neutral proteases present in MCTC [34]. MCTC preferentially dwell in connective tissue such as skin, while MCT are often found
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methylcellulose medium. Mast cell colonies (a, c) and macrophage colonies (b, d) were observed in the methylcellulose medium when adult peripheral blood cells were cultured for 6 weeks in the presence of SCF, IL-6 and IL-3. The mast cells look refractile and equally round, while macrophages look extra-large and irregularly shaped. Objective lenses were used. Original magnification ×10 (a, b), ×20 (c, d).
in mucosa such as airway epithelium. MCTC can respond to various non-immunological stimuli such as C5a or substance P, while MCT do not. Asthma can be divided into 2 subgroups (‘Th2high’ and ‘Th2-low’ asthma), based on epithelial cell gene signatures for the activity of Th2 cytokines such as IL-13. Patients with Th2-high asthma have more infiltration of mast cells into the airway epithelium. These intraepithelial mast cells express both tryptase and carboxypeptidase A3 (CPA3), but not chymase [35]. According to the classical definition [34], MCT were not supposed to express CPA3. However, according to public microarray data (http://www.nch. go.jp/imal/GeneChip/public.htm), all types of mast cells and basophils express the CPA3 transcript. Therefore, we can consider these epithelial mast cells at least as a sort of MCT. Mast cells exposed to conditioned media from IL-13-activated epithelial cells showed downregulation of chymase, but no change in tryptase or CPA3 expression [35]. This may be the reason why MCT are preferentially found in mucosa and are deficient in immunodeficiency patients [34]. This is in contrast to results demonstrating that human mast cell phenotypes, such as chymase expression, are retained over weeks, even when these mast cells are cultured in standard mast cell culture condition (supplemented with SCF and IL-6) [36].
Discovery of Cytokine Production by Mast Cells Activated mast cells can release various protein cytokines as well as low-molecular-weight mediators such as histamine. It was described in 1987 that activated mast cells express and release cytokines such as tumor necrosis factor, TNF [37]. Later, mast cell-derived TNF was shown to play a significant role
in airway hyperreactivity, T cell development (enhancement) and bacterial infection (protection). IL-4 production seems reproducible when using mouse mast cells. However, so far only a few groups have succeeded to immunohistochemically demonstrate the presence of IL-4 on human mast cells. In any case, at least in humans, basophils are more potent producers of IL-4, and IL-4 potently activates human mast cell function and maturation. Interestingly, human mast cells can produce a substantial amount of another Th2 cytokine, IL-13, in response to IgE-mediated stimuli when mast cells are pre-incubated with IL-4 [38]. Through the development of molecular biological techniques, such as microarray, and the establishment of the human mast cell culture system, we now understand that both human and mouse mast cells can express and release a variety of cytokines (mainly Th2 cytokines) and chemokines (mainly CC-chemokines and IL-8) in response to IgE- and non-IgE-associated stimuli (http://www.nch.go.jp/imal/GeneChip/public. htm) [38, 39].
Future Perspective Today we even understand the whole molecular pattern, the expression of which is changed when mast cells are activated via FcεRI cross-linking. However, the physiological and pathological roles of mast cells, especially where IgE is not involved, are not fully understood yet. It will be necessary to determine the mechanisms involved in the ‘non-IgEmediated’ phase of mast cell activation in allergic or other diseases. Also, we need to understand the relative role of mast cells in allergic or innate-type inflammation compared to the roles of other immune and tissue-derived cells.
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Prof. Hirohisa Saito National Research Institute for Child Health and Development 2-10-1 Okura, Setagaya-ku Tokyo, 157-8535 (Japan) E-Mail saito-hr @ ncchd.go.jp
Mast Cell Research
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