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Murine Fetal Skin-Derived Cultured Mast Cells: A Useful Tool for Discovering Functions of Skin Mast Cells

      Mast cells are widely distributed throughout the body, being preferentially localized at host–environment interfaces. They have long been known as major effector cells in IgE-mediated allergic responses. However, accumulating evidence has provided many new insights into their functions. They are now known to be involved in diverse pathological processes, for example, innate and adaptive immunity. Utility of mast cell-deficient mice and mast cell-knock-in mice has provided powerful models to demonstrate compelling evidence for their in vivo relevance. Conversely, primary cultures of tissue-derived mast cells provide excellent models for in vitro studies of functions at both cellular and molecular levels. Because mast cells exhibit phenotypical and functional heterogeneity in different anatomical sites, it is important to obtain tissue-specific mast cells to clarify their function in tissue. In this regard, researchers have established several methods to prepare mast cells from different tissues, which are technically difficult to obtain at high purity and yield. To overcome these difficulties, we have developed a primary culture system to obtain large numbers of mast cells at high purity from murine fetal skin. In this review, we describe characteristics of such mast cells and their utility in mast cell biology.

      Abbreviations

      BMMC
      bone marrow-derived mast cells
      CTMC
      connective tissue-type mast cells
      ET-1
      endothelin-1
      FSMC
      fetal skin-derived mast cells
      OPN
      osteopontin
      TLR
      Toll-like receptor

      Introduction

      Over the past decade, most immunologists might have paid less attention to mast cells as important players in innate and acquired immunity and considered mast cells only as effector cells that induce acute hypersensitivity responses via IgE-mediated mechanisms. We believe that this notion has been extensively revised for such immunologists, and even for researchers handling mast cells, by the surprising findings published in the last decade or so. We now know that mast cells are involved in a huge array of pathological processes, for example, innate and adaptive immunity, tolerance induction, immune suppression, autoimmunity, chronic inflammation, tissue remodeling/wound repair, tumor progression, and detoxification. For a detailed overview on these subjects, the reader is referred to several recent excellent reviews (
      • Marshall J.S.
      Mast-cell responses to pathogens.
      ;
      • Galli S.J.
      • Kalesnikoff J.
      • Grimbaldeston M.A.
      • Piliponsky A.M.
      • Williams C.M.
      • Tsai M.
      Mast cells as “tunable” effector and immunoregulatory cells: recent advances.
      ;
      • Christy A.L.
      • Brown M.A.
      The multitasking mast cell: positive and negative roles in the progression of autoimmunity.
      ;
      • Metz M.
      • Maurer M.
      Mast cells—key effector cells in immune responses.
      ;
      • Metz M.
      • Grimbaldeston M.A.
      • Nakae S.
      • Piliponsky A.M.
      • Tsai M.
      • Galli S.J.
      Mast cells in the promotion and limitation of chronic inflammation.
      ;
      • Galli S.J.
      • Tsai M.
      Mast cells: versatile regulators of inflammation, tissue remodeling, host defense and homeostasis.
      ). It seems that mast cells have implications for a wide variety of host defense mechanisms in health and disease. Considering the expansion of the number of mast cell studies, easier access to mast cells may facilitate discovery of new functions in unexpected fields. In this review, we discuss the development of a mast cell culture system, which we recently developed, and testing of its utility for exploring undiscovered functions of mast cells.

      Brief Overview of Mast Cell Biology: Focus on Subpopulations

      Mast cells have long been known as major effector cells in acute allergic responses because the cross-linking of IgE-bound, high-affinity IgE receptors (FcεRI) on mast cells by multivalent antigens triggers the immediate degranulation and subsequent release of a wide variety of chemical and lipid mediators from these cells (
      • Rivera J.
      • Gilfillan A.M.
      Molecular regulation of mast cell activation.
      ). In addition to this rapid host reaction, mast cells are also activated by many other stimuli (for example, chemicals, toxic substances, infectious microbes, cytokines, growth factors, and hormones) with or without degranulation, leading to the differential release of multiple mediators (
      • Marshall J.S.
      Mast-cell responses to pathogens.
      ;
      • Galli S.J.
      • Kalesnikoff J.
      • Grimbaldeston M.A.
      • Piliponsky A.M.
      • Williams C.M.
      • Tsai M.
      Mast cells as “tunable” effector and immunoregulatory cells: recent advances.
      ;
      • Theoharides T.C.
      • Kempuraj D.
      • Tagen M.
      • Conti P.
      • Kalogeromitros D.
      Differential release of mast cell mediators and the pathogenesis of inflammation.
      ). Thus, mast cells have the potential to influence a huge array of host defense reactions.
      For host responses involving the function of mast cells, the anatomical location of mast cells seems to be essential. Mast cells are widely distributed throughout the body, preferentially in close proximity to epithelial surfaces (for example, skin, respiratory system, and gastrointestinal tracts) where they reside around blood vessels and peripheral nerves. They are a progeny of bone marrow-derived hematopoietic cells (
      • Kitamura Y.
      • Shimada M.
      • Hatanaka K.
      • Miyano Y.
      Development of mast cells from grafted bone marrow cells in irradiated mice.
      ). Small numbers of committed mast cell progenitors circulate in the blood and migrate to tissues, where their terminal maturation is accomplished under the influence of their final microenvironment (
      • Gurish M.F.
      • Boyce J.A.
      Mast cells: ontogeny, homing, and recruitment of a unique innate effector cell.
      ). Committed mast cell progenitors reside at special anatomical sites (for example, intestine and hair follicles) and locally proliferate with terminal maturation (
      • Kumamoto T.
      • Shalhevet D.
      • Matsue H.
      • Mummert M.E.
      • Ward B.R.
      • Jester J.V.
      • et al.
      Hair follicles serve as local reservoirs of skin mast cell precursors.
      ;
      • Gurish M.F.
      • Boyce J.A.
      Mast cells: ontogeny, homing, and recruitment of a unique innate effector cell.
      ). Proliferation, terminal differentiation, and survival of resident mast cells require growth factors, such as IL-3, stem cell factor, and/or other factors (
      • Gurish M.F.
      • Boyce J.A.
      Mast cells: ontogeny, homing, and recruitment of a unique innate effector cell.
      ). At least two types of mast cells have been historically identified in rodents based on their morphological, biochemical, and functional properties (
      • Kitamura Y.
      Heterogeneity of mast cells and phenotypic change between subpopulations.
      ). They were originally classified into connective tissue-type mast cells (CTMC) and mucosal mast cells. CTMC reside in the dermis of skin, submucosal connective tissues of the respiratory tract, joint synovia, and peritoneum. Mucosal mast cells reside in the mucosa of the gastrointestinal tract and in the lamina propria of the respiratory tract. Both cell types differ in profiles of (1) protease and glycosaminoglycan contents of their granules, (2) lipid-mediator production, and (3) responsiveness to polybasic compounds (for example, compound 48/80) and substance P (
      • Marshall J.S.
      Mast-cell responses to pathogens.
      ). Therefore, to study roles played by mast cells at any anatomical site, it is essential to characterize the phenotype and function of mast cells isolated from those given anatomical sites.
      With respect to mast cell sources for studies in mice, peritoneal mast cells and bone marrow-derived mast cells (BMMC) have been widely used. However, both cell types have some pitfalls. BMMCs are often regarded as in vitro equivalent of mucosal mast cells but they are actually immature mast cells. Mouse peritoneal mast cells represent CTMC, but it is relatively difficult to obtain large numbers at high purity from peritoneal lavage fluid. It is even more difficult and laborious to isolate large numbers of mast cells at high purity from adult skin, although this method has been established (
      • He D.
      • Esquenazi-Behar S.
      • Soter N.A.
      • Lim H.W.
      Mast-cell heterogeneity: functional comparison of purified mouse cutaneous and peritoneal mast cells.
      ).

      Establishment and Evaluation of Fetal Skin-Derived Mast Cells

      The technical breakthrough to obtain a large number of skin mast cells at high purity was made by
      • Yamada N.
      • Matsushima H.
      • Tagaya Y.
      • Shimada S.
      • Katz S.I.
      Generation of a large number of connective tissue type mast cells by culture of murine fetal skin cells.
      . They developed a new method to easily obtain relatively large numbers of fetal skin-derived mast cells (FSMC) at high purity from day 14 to day 16 murine fetal skin. Single cell suspensions were prepared from excised day 14 to day 16 fetal trunk skin specimens by limited trypsinization. After red blood cell lysis and extensive washing, crude cells (5 × 104 cells per ml) were cultured in complete RPMI in the presence of mast cell growth factors, murine recombinant IL-3 (10ngml-1), and stem cell factor (10ngml-1). Non-adherent cells and loosely adherent cells were collected 14 days after this initial culture (without changing the culture medium), and enriched for mast cells by density gradient centrifugation. The resulting mast cell preparations (about 7 × 106 cells per fetus), containing >96% CD45+ CD117+ (c-kit+) cells, were used as FSMC without further purification (Figure 1).
      Figure thumbnail gr1
      Figure 1Generation of fetal skin-derived mast cells (FSMC). Single cell suspensions derived from fetal skin are cultured at a low cell density in the presence of mast cell growth factors for 2 weeks without changing the medium. Non-adherent and loosely adherent cells are collected and enriched for mast cells by density gradient centrifugation. The resulting mast cell preparation (about 7 × 106 cells per fetus) contains > 96% CD45+ CD117+ cells, which are used as FSMC without further purification.
      • Yamada N.
      • Matsushima H.
      • Tagaya Y.
      • Shimada S.
      • Katz S.I.
      Generation of a large number of connective tissue type mast cells by culture of murine fetal skin cells.
      found that about 5% of fetal skin crude cells contained CD45+ CD49b+ CD117+ FcεRI- cells, which could proliferate in response to stem cell factor. They considered these freshly isolated cells to be immature mast cells because the FcεRI-positive cell population gradually increased the longer they were in culture. After 14 days of culture, virtually all the cells expressed high levels of functional FcεRI and thus had differentiated into mature mast cells. Compatible with these findings,
      • Meindl S.
      • Schmidt U.
      • Vaculik C.
      • Elbe-Burger A.
      Characterization, isolation, and differentiation of murine skin cells expressing hematopoietic stem cell markers.
      recently reported that the majority of lineage-negative CD45+ dermal cells from newborn and adult mouse skin were mast cell precursors. Therefore, fetal skin may provide a more suitable source than older murine skin to obtain abundant precursors of skin-derived mast cells.
      FSMCs maintained a similar surface phenotype to skin mast cells derived from adult mice. In addition, FSMC had many characteristic features of CTMC. Those include: (1) higher contents of histamine and heparin than BMMC, (2) their capacity to degranulate in response to compound 48/80 and substance P, (3) the high expression of CD49b (Table 1). Thus, FSMC, which represent CTMC, serve as an excellent model for in vitro studies of skin-derived mast cells or CTMC.
      Table 1Characteristic profiles of FSMC and BMMC1
      Table thumbnail fx1

      New Putative Functions of Mast Cells: Contributions of FSMC

      Toll-like receptors on mast cells

      Toll-like receptors (TLRs) serve as sensors against pathogens that invade the host by recognizing pathogen-associated molecular patterns (
      • Takeda K.
      • Kaisho T.
      • Akira S.
      Toll-like receptors.
      ). Each TLR recognizes a distinct ligand, as shown by TLR-knockout mice. TLRs have been extensively studied in the context of their expression by antigen-presenting cells (that is, dendritic cells and macrophages), which induce innate immunity and subsequently acquired immunity (
      • Kaisho T.
      • Akira S.
      Toll-like receptor function and signaling.
      ). Although TLR2 and TLR4 expression and function of mast cells in response to bacterial products have been well documented (
      • Marshall J.S.
      Mast-cell responses to pathogens.
      ;
      • Stelekati E.
      • Orinska Z.
      • Bulfone-Paus S.
      Mast cells in allergy: innate instructors of adaptive responses.
      ), expression profiles of other TLRs and their functions on mast cells remain relatively unclear. Working with FSMC and BMMC,
      • Matsushima H.
      • Yamada N.
      • Matsue H.
      • Shimada S.
      TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells.
      examined TLR mRNA expression profiles and functions. TLR2 and TLR4 mRNAs were detected in both mast cell types at comparable levels. However, TLR3, TLR7, and TLR9 mRNAs were expressed by FSMC at higher levels than by BMMC (Table 1). Reflecting the differences of their TLR expression profiles, FSMC, but not BMMC, produced inflammatory cytokines (tumor necrosis factor-α and IL-6) and chemokines (RANTES, macrophage inflammatory protein-1α and -2, and MIP-2) without degranulation in response to poly(I:C), R-848, and CpG oligodeoxynucleotide, which are TLR3, TLR7, and TLR9 ligands, respectively. Because TLR3, TLR7, and TLR9 are known to recognize products associated with viruses, these in vitro findings may have implications for roles of these TLRs expressed by mast cells in viral infections. One may speculate that mast cells enhance local infiltration of a variety of immune cells by releasing inflammatory cytokines and chemokines upon viral as well as bacterial infections, presumably by being involved in the transition of innate immune responses to acquired immune responses. Further studies will be required to determine the involvement of mast cells in viral infections using mast cell-deficient mice and their reconstitution with TLR-deficient mast cells.

      Endothelin-1 on mast cells

      Endothelin-1 (ET-1) was originally discovered as a potent vasoconstrictive peptide derived from endothelial cells (
      • Yanagisawa M.
      • Kurihara H.
      • Kimura S.
      • Tomobe Y.
      • Kobayashi M.
      • Mitsui Y.
      • et al.
      A novel potent vasoconstrictor peptide produced by vascular endothelial cells.
      ). Earlier studies focused on its potential as a mediator in the cardiovascular system. For instance, ET-1 was found to be involved in a variety of vascular diseases, including essential systemic hypertension, pulmonary hypertension, coronary vasospasm, and cardiac failure (
      • Nambi P.
      • Clozel M.
      • Feuerstein G.
      Endothelin and heart failure.
      ;
      • Schiffrin E.L.
      Role of endothelin-1 in hypertension and vascular disease.
      ;
      • Taddei S.
      • Virdis A.
      • Ghiadoni L.
      • Sudano I.
      • Magagna A.
      • Salvetti A.
      Role of endothelin in the control of peripheral vascular tone in human hypertension.
      ). Subsequent studies revealed that ET-1 was also involved in the pathogenesis of non-vascular diseases, including inflammatory diseases (for example, asthma and allergic rhinitis) and fibrotic diseases (for example, systemic sclerosis, pulmonary fibrosis, and hepatic fibrosis) (
      • Vancheeswaran R.
      • Azam A.
      • Black C.
      • Dashwood M.R.
      Localization of endothelin-1 and its binding sites in scleroderma skin.
      ;
      • Rockey D.C.
      • Chung J.J.
      Endothelin antagonism in experimental hepatic fibrosis. Implications for endothelin in the pathogenesis of wound healing.
      ;
      • Goldie R.G.
      • Henry P.J.
      Endothelins and asthma.
      ;
      • Hocher B.
      • Schwarz A.
      • Fagan K.A.
      • Thone-Reineke C.
      • El-Hag K.
      • Kusserow H.
      • et al.
      Pulmonary fibrosis and chronic lung inflammation in ET-1 transgenic mice.
      ;
      • Mullol J.
      • Picado C.
      Endothelin in nasal mucosa: role in nasal function and inflammation.
      ).
      Working with FSMC (
      • Matsushima H.
      • Yamada N.
      • Matsue H.
      • Shimada S.
      The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.
      ), we found that ET-1 was able to (a) induce degranulation by FSMC via ETA in a dose-dependent manner (10-8 to 10-6M), (b) significantly augment degranulation by IgE-sensitized FSMC using the above dose ranges, (c) at its lower concentration (10-10M), significantly suppress degranulation induced by FcεRI aggregation, (d) induce proinflammatory cytokine production (tumor necrosis factor-α and IL-6), (e) significantly enhance VEGF production and transforming growth factor-β1 mRNA expression, and (f) ET-1 was able to be produced by FSMC in response to TLR ligands (that is, TLR2, TLR3, TLR4, and TLR9 ligands) (Table 1). On the contrary, BMMC did not respond to ET-1 and did not produce ET-1 in response to these TLR ligands. These findings indicate that multi-functional effects of ET-1 on mast cells may participate in the pathogenesis of a wide array of diseases, including allergic, inflammatory, angiogenic, and fibrotic diseases.
      Our findings lacked in vivo relevance of ET-1-induced activation and degranulation by mast cells. Soon thereafter, Maurer et al. made a landmark discovery that linked ET-1 and sepsis. They found that ET-1-dependent degranulation by mast cells resulted in protease release that degraded ET-1 and reduced its levels. This release protected normal mice, but not mast cell-deficient mice, from the lethal effects of peritoneal sepsis that was induced after an intraperitoneal injection of ET-1 (
      • Maurer M.
      • Wedemeyer J.
      • Metz M.
      • Piliponsky A.M.
      • Weller K.
      • Chatterjea D.
      • et al.
      Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.
      ). Because ET-1 has high homology (>70%) to sarafotoxins, the most potent toxic components of certain venoms, they hypothesized that mast cells might also be protective in envenomation. Indeed,
      • Metz M.
      • Piliponsky A.M.
      • Chen C.C.
      • Lammel V.
      • Abrink M.
      • Pejler G.
      • et al.
      Mast cells can enhance resistance to snake and honeybee venoms.
      ) elegantly demonstrated that mast cells could defend against toxins of snakes or bees by releasing proteases (mainly carboxypeptidase A) that cleave and detoxify those toxins, thus providing protection from the pathological consequences of snake bites or bee stings. This discovery surprised us because mast cells were commonly considered to participate in anaphylactic shock by elaborating a wide array of mediators leading to tissue damage. Detoxification is thus a novel aspect of innate immunity played by mast cells.
      Another in vivo relevance of the ET-1 system in mast cells was recently reported by the same group (
      • Metz M.
      • Lammel V.
      • Gibbs B.F.
      • Maurer M.
      Inflammatory murine skin responses to UV-B light are partially dependent on endothelin-1 and mast cells.
      ). They found that ET-1 potently induced degranulation by mast cells that were purified from the ear skin of adult mice in vitro and that ET-1 induced skin inflammation in vivo by activating skin-resident mast cells. Both activations were mediated via ETA receptors. The in vitro data were consistent with our data that ET-1 induced degranulation by FSMC via ETA receptors, which supports the notion that FSCM represent skin-derived mast cells. In addition, UVB-mediated increase of ET-1 levels in the skin was associated with UV-induced skin inflammation evoked, in part, by the activation of mast cells via ligation of ET-1 to ETA receptors on mast cells. Therefore, the ET-1 system in mast cells serves as a double-edged sword depending on physiological and pathological conditions.

      Osteopontin on mast cells

      Osteopontin (OPN) is an arginine–glycine–aspartate (RGD)-containing glycoprotein, which participates in bone remodeling (
      • Denhardt D.T.
      • Noda M.
      • O’Regan A.W.
      • Pavlin D.
      • Berman J.S.
      Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival.
      ), wound healing (
      • Liaw L.
      • Birk D.E.
      • Ballas C.B.
      • Whitsitt J.S.
      • Davidson J.M.
      • Hogan B.L.
      Altered wound healing in mice lacking a functional osteopontin gene (spp1).
      ), dystrophic calcification, and coronary restenosis (
      • Rangaswami H.
      • Bulbule A.
      • Kundu G.C.
      Osteopontin: role in cell signaling and cancer progression.
      ). Immunologists found that it is expressed and functions in many immune cell types (
      • Rangaswami H.
      • Bulbule A.
      • Kundu G.C.
      Osteopontin: role in cell signaling and cancer progression.
      ). Those include macrophages, activated T cells, myeloid dendritic cells, plasmacytoid dendritic cells, and natural killer T (NKT) cells; thus it can exhibit many potential effects on the immune system (
      • O’Regan A.W.
      • Nau G.J.
      • Chupp G.L.
      • Berman J.S.
      Osteopontin (Eta-1) in cell-mediated immunity: teaching an old dog new tricks.
      ;
      • Diao H.
      • Kon S.
      • Iwabuchi K.
      • Kimura C.
      • Morimoto J.
      • Ito D.
      • et al.
      Osteopontin as a mediator of NKT cell function in T cell-mediated liver diseases.
      ;
      • Renkl A.C.
      • Wussler J.
      • Ahrens T.
      • Thoma K.
      • Kon S.
      • Uede T.
      • et al.
      Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotype.
      ;
      • Shinohara M.L.
      • Jansson M.
      • Hwang E.S.
      • Werneck M.B.
      • Glimcher L.H.
      • Cantor H.
      T-bet-dependent expression of osteopontin contributes to T cell polarization.
      ,
      • Shinohara M.L.
      • Lu L.
      • Bu J.
      • Werneck M.B.
      • Kobayashi K.S.
      • Glimcher L.H.
      • et al.
      Osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells.
      ). For instance, OPN plays an important role in efficient TH1 immune responses in many experimental settings.
      • Ashkar S.
      • Weber G.F.
      • Panoutsakopoulou V.
      • Sanchirico M.E.
      • Jansson M.
      • Zawaideh S.
      • et al.
      Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity.
      demonstrated that OPN contributed to a host defense against pathogens (for example, herpes simples) and to the TH1-dependent granuloma formation by a TH1-driving cytokine, IL-12, secreted by macrophages. OPN induced maturation of myeloid dendritic cells toward a TH1-promoting phenotype (
      • Renkl A.C.
      • Wussler J.
      • Ahrens T.
      • Thoma K.
      • Kon S.
      • Uede T.
      • et al.
      Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotype.
      ). T-bet-dependent OPN expression by activated T cells skewed CD4+ and CD8+ T cells toward TH1 and Tc1 CD8+ T cells, respectively (
      • Shinohara M.L.
      • Jansson M.
      • Hwang E.S.
      • Werneck M.B.
      • Glimcher L.H.
      • Cantor H.
      T-bet-dependent expression of osteopontin contributes to T cell polarization.
      ). In a more recent report, intracellular OPN in plasmacytoid dendritic cells regulated their IFN-α production, thus being involved in TH1 immunity to viral infection (
      • Shinohara M.L.
      • Lu L.
      • Bu J.
      • Werneck M.B.
      • Kobayashi K.S.
      • Glimcher L.H.
      • et al.
      Osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells.
      ). In addition to its function as a TH1 cytokine, OPN also exerts its effects on inflammatory processes. For example, NKT cell-derived OPN is a major mediator of Concanavalin A-induced hepatitis by augmenting NKT cell activation and triggering neutrophil infiltration (
      • Diao H.
      • Kon S.
      • Iwabuchi K.
      • Kimura C.
      • Morimoto J.
      • Ito D.
      • et al.
      Osteopontin as a mediator of NKT cell function in T cell-mediated liver diseases.
      ). OPN is also involved in autoimmune diseases, including multiple sclerosis and its animal model (
      • Chabas D.
      • Baranzini S.E.
      • Mitchell D.
      • Bernard C.C.
      • Rittling S.R.
      • Denhardt D.T.
      • et al.
      The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease.
      ;
      • Jansson M.
      • Panoutsakopoulou V.
      • Baker J.
      • Klein L.
      • Cantor H.
      Cutting edge: attenuated experimental autoimmune encephalomyelitis in eta-1/osteopontin-deficient mice.
      ), and rheumatoid arthritis and its animal model (
      • Yumoto K.
      • Ishijima M.
      • Rittling S.R.
      • Tsuji K.
      • Tsuchiya Y.
      • Kon S.
      • et al.
      Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice.
      ;
      • Yamamoto N.
      • Sakai F.
      • Kon S.
      • Morimoto J.
      • Kimura C.
      • Yamazaki H.
      • et al.
      Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis.
      ;
      • Xu G.
      • Nie H.
      • Li N.
      • Zheng W.
      • Zhang D.
      • Feng G.
      • et al.
      Role of osteopontin in amplification and perpetuation of rheumatoid synovitis.
      ). Thus, OPN is now believed to participate in complex processes of a wide array of inflammatory and immune diseases. Mast cells are also equipped with potent capacities to participate in a wide array of pathological processes (for example, innate and adaptive immune responses, autoimmune responses, tissue remodeling/wound repair, and tumor progression by releasing a wide array of mediators;
      • Benoist C.
      • Mathis D.
      Mast cells in autoimmune disease.
      ;
      • Gruber B.L.
      Mast cells in the pathogenesis of fibrosis.
      ;
      • Levi-Schaffer F.
      • Piliponsky A.M.
      Tryptase, a novel link between allergic inflammation and fibrosis.
      ;
      • Marshall J.S.
      Mast-cell responses to pathogens.
      ;
      • Theoharides T.C.
      • Conti P.
      Mast cells: the Jekyll and Hyde of tumor growth.
      ;
      • Galli S.J.
      • Kalesnikoff J.
      • Grimbaldeston M.A.
      • Piliponsky A.M.
      • Williams C.M.
      • Tsai M.
      Mast cells as “tunable” effector and immunoregulatory cells: recent advances.
      ). We hypothesized that mast cells may produce OPN, which may affect their function and participate in such diverse pathological processes. To this end, we recently examined effects of OPN on FSMC, and BMMC and OPN production by these mast cell types (
      • Nagasaka A.
      • Matsue H.
      • Matsushima H.
      • Aoki R.
      • Nakamura Y.
      • Kambe N.
      • et al.
      Osteopontin is produced by mast cells and affects IgE-mediated degranulation and migration of mast cells.
      ). OPN was found to (1) be spontaneously produced by FSMC and be inducible in BMMC in response to stimuli (for example, FcεRI aggregation and ionomycin), (2) significantly augment mast cell degranulation by FcεRI aggregation, and (3) promote chemotaxis of mast cells (Table 1). Our results have also revealed that OPN secreted by FSMC was biologically active and had no effect on the development of FSMC. Conversely, ample experimental evidence of the involvement of OPN in the pathogenesis of many inflammatory and immune diseases may provide a key to elucidate a link between mast cells and those diseases. However, further studies will be required to define the role of OPN as a pleiotropic mediator that regulates many functions of mast cells, presumably by participating in multiple aspects of inflammatory and immune diseases. In addition, based on our findings,
      • Bulfone-Paus S.
      • Paus R.
      Osteopontin as a new player in mast cell biology.
      recently discussed why this newly discovered property of mast cell-derived OPN accounts for the recent concept that OPN may serve as a multipurpose environmental damage-response protein.

      Other evidence of utility of FSMC for mast cell research

      We hope that FSMC will become more popular for mast cell studies because there are no technical difficulties in obtaining large numbers at high purity. Indeed,
      • Oki T.
      • Kitaura J.
      • Eto K.
      • Lu Y.
      • Maeda-Yamamoto M.
      • Inagaki N.
      • et al.
      Integrin alphaIIbbeta3 induces the adhesion and activation of mast cells through interaction with fibrinogen.
      used FSMC as a mast cell type to investigate integrin αIIbβ3 expression. Another advantage of FSMC is that we can generate cells from gene knockout mice even if these mice result in neonatal lethality. In other words, we can generate certain gene-deficient FSMC from intercrossing heterozygous mice. Using this technique,
      • Feyerabend T.B.
      • Li J.P.
      • Lindahl U.
      • Rodewald H.R.
      Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells.
      found that C5-epimerase-deficient FSMC distorted the heparin O-sulfation pattern, providing new information of heparin biosynthesis in mast cells. Because we can reconstitute dermal mast cells in mast cell-deficient mice several weeks after FSMCs are intradermally injected (unpublished data), this reconstitution method may provide a faster means to investigate in vivo functions of particular gene-deficient dermal mast cells.

      Concluding remarks

      • Galli S.J.
      • Kalesnikoff J.
      • Grimbaldeston M.A.
      • Piliponsky A.M.
      • Williams C.M.
      • Tsai M.
      Mast cells as “tunable” effector and immunoregulatory cells: recent advances.
      considered mast cells as “tunable” effector and immunoregulatory cells. Mast cells may be central for initiating, tuning, and regulating inflammatory and immune responses observed in complex tissue reactions in health and disease. These new findings obtained from mouse systems keep providing new ideas to test their relevance in humans and conceptual frameworks to develop new agents to enhance or silence mast cell functions for human health. We hope that FSMC will provide an easily accessible tool for a broad range of immunologists to discover novel roles played by mast cells, especially CTMC, in their research field. Such approaches may ultimately provide new, unexpected ideas to control many diseases by manipulating mast cell functions.

      Conflict of Interest

      The authors declare no conflict of interest.

      ACKNOWLEDGMENTS

      We especially thank our ex-colleague, Dr Nobuo Yamada, who established the FSMC cell culture system during his postdoctoral fellowship in National Institutes of Health (Bethesda, MD) under the supervision of Dr Stephen I. Katz. We also gratefully acknowledge the work of our co-workers, especially Drs Matsushima and Nagasaka. The work on FSMC was partly supported by grants (17390310, 17659334, 18390311, and 19659281) from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government. Because we focused on FSMC in this brief review, we only cited a fraction of the relevant literature. We, therefore, apologize to any colleagues whose works are not appropriately acknowledged in this review.

      REFERENCES

        • Ashkar S.
        • Weber G.F.
        • Panoutsakopoulou V.
        • Sanchirico M.E.
        • Jansson M.
        • Zawaideh S.
        • et al.
        Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity.
        Science. 2000; 287: 860-864
        • Benoist C.
        • Mathis D.
        Mast cells in autoimmune disease.
        Nature. 2002; 420: 875-878
        • Bulfone-Paus S.
        • Paus R.
        Osteopontin as a new player in mast cell biology.
        Eur J Immunol. 2008; 38: 338-341
        • Chabas D.
        • Baranzini S.E.
        • Mitchell D.
        • Bernard C.C.
        • Rittling S.R.
        • Denhardt D.T.
        • et al.
        The influence of the proinflammatory cytokine, osteopontin, on autoimmune demyelinating disease.
        Science. 2001; 294: 1731-1735
        • Christy A.L.
        • Brown M.A.
        The multitasking mast cell: positive and negative roles in the progression of autoimmunity.
        J Immunol. 2007; 179: 2673-2679
        • Denhardt D.T.
        • Noda M.
        • O’Regan A.W.
        • Pavlin D.
        • Berman J.S.
        Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival.
        J Clin Invest. 2001; 107: 1055-1061
        • Diao H.
        • Kon S.
        • Iwabuchi K.
        • Kimura C.
        • Morimoto J.
        • Ito D.
        • et al.
        Osteopontin as a mediator of NKT cell function in T cell-mediated liver diseases.
        Immunity. 2004; 21: 539-550
        • Feyerabend T.B.
        • Li J.P.
        • Lindahl U.
        • Rodewald H.R.
        Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells.
        Nat Chem Biol. 2006; 2: 195-196
        • Galli S.J.
        • Kalesnikoff J.
        • Grimbaldeston M.A.
        • Piliponsky A.M.
        • Williams C.M.
        • Tsai M.
        Mast cells as “tunable” effector and immunoregulatory cells: recent advances.
        Annu Rev Immunol. 2005; 23: 749-786
        • Galli S.J.
        • Tsai M.
        Mast cells: versatile regulators of inflammation, tissue remodeling, host defense and homeostasis.
        J Dermatol Sci. 2008; 49: 7-19
        • Goldie R.G.
        • Henry P.J.
        Endothelins and asthma.
        Life Sci. 1999; 65: 1-15
        • Gruber B.L.
        Mast cells in the pathogenesis of fibrosis.
        Curr Rheumatol Rep. 2003; 5: 147-153
        • Gurish M.F.
        • Boyce J.A.
        Mast cells: ontogeny, homing, and recruitment of a unique innate effector cell.
        J Allergy Clin Immun. 2006; 117: 1285-1291
        • He D.
        • Esquenazi-Behar S.
        • Soter N.A.
        • Lim H.W.
        Mast-cell heterogeneity: functional comparison of purified mouse cutaneous and peritoneal mast cells.
        J Invest Dermatol. 1990; 95: 178-185
        • Hocher B.
        • Schwarz A.
        • Fagan K.A.
        • Thone-Reineke C.
        • El-Hag K.
        • Kusserow H.
        • et al.
        Pulmonary fibrosis and chronic lung inflammation in ET-1 transgenic mice.
        Am J Respir Cell Mol Biol. 2000; 23: 19-26
        • Jansson M.
        • Panoutsakopoulou V.
        • Baker J.
        • Klein L.
        • Cantor H.
        Cutting edge: attenuated experimental autoimmune encephalomyelitis in eta-1/osteopontin-deficient mice.
        J Immunol. 2002; 168: 2096-2099
        • Kaisho T.
        • Akira S.
        Toll-like receptor function and signaling.
        J Allergy Clin Immun. 2006; 117: 979-987
        • Kitamura Y.
        Heterogeneity of mast cells and phenotypic change between subpopulations.
        Annu Rev Immunol. 1989; 7: 59-76
        • 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
        • Kumamoto T.
        • Shalhevet D.
        • Matsue H.
        • Mummert M.E.
        • Ward B.R.
        • Jester J.V.
        • et al.
        Hair follicles serve as local reservoirs of skin mast cell precursors.
        Blood. 2003; 102: 1654-1660
        • Levi-Schaffer F.
        • Piliponsky A.M.
        Tryptase, a novel link between allergic inflammation and fibrosis.
        Trends Immunol. 2003; 24: 158-161
        • Liaw L.
        • Birk D.E.
        • Ballas C.B.
        • Whitsitt J.S.
        • Davidson J.M.
        • Hogan B.L.
        Altered wound healing in mice lacking a functional osteopontin gene (spp1).
        J Clin Invest. 1998; 101: 1468-1478
        • Marshall J.S.
        Mast-cell responses to pathogens.
        Nat Rev Immunol. 2004; 4: 787-799
        • Matsushima H.
        • Yamada N.
        • Matsue H.
        • Shimada S.
        The effects of endothelin-1 on degranulation, cytokine, and growth factor production by skin-derived mast cells.
        Eur J Immunol. 2004; 34: 1910-1919
        • Matsushima H.
        • Yamada N.
        • Matsue H.
        • Shimada S.
        TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells.
        J Immunol. 2004; 173: 531-541
        • Maurer M.
        • Wedemeyer J.
        • Metz M.
        • Piliponsky A.M.
        • Weller K.
        • Chatterjea D.
        • et al.
        Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.
        Nature. 2004; 432: 512-516
        • Meindl S.
        • Schmidt U.
        • Vaculik C.
        • Elbe-Burger A.
        Characterization, isolation, and differentiation of murine skin cells expressing hematopoietic stem cell markers.
        J Leukoc Biol. 2006; 80: 816-826
        • Metz M.
        • Grimbaldeston M.A.
        • Nakae S.
        • Piliponsky A.M.
        • Tsai M.
        • Galli S.J.
        Mast cells in the promotion and limitation of chronic inflammation.
        Immunol Rev. 2007; 217: 304-328
        • Metz M.
        • Lammel V.
        • Gibbs B.F.
        • Maurer M.
        Inflammatory murine skin responses to UV-B light are partially dependent on endothelin-1 and mast cells.
        Am J Pathol. 2006; 169: 815-822
        • Metz M.
        • Maurer M.
        Mast cells—key effector cells in immune responses.
        Trends Immunol. 2007; 28: 234-241
        • Metz M.
        • Piliponsky A.M.
        • Chen C.C.
        • Lammel V.
        • Abrink M.
        • Pejler G.
        • et al.
        Mast cells can enhance resistance to snake and honeybee venoms.
        Science. 2006; 313: 526-530
        • Mullol J.
        • Picado C.
        Endothelin in nasal mucosa: role in nasal function and inflammation.
        Clin Exp Allergy. 2000; 30: 172-177
        • Nagasaka A.
        • Matsue H.
        • Matsushima H.
        • Aoki R.
        • Nakamura Y.
        • Kambe N.
        • et al.
        Osteopontin is produced by mast cells and affects IgE-mediated degranulation and migration of mast cells.
        Eur J Immunol. 2008; 38: 489-499
        • Nambi P.
        • Clozel M.
        • Feuerstein G.
        Endothelin and heart failure.
        Heart Fail Rev. 2001; 6: 335-340
        • O’Regan A.W.
        • Nau G.J.
        • Chupp G.L.
        • Berman J.S.
        Osteopontin (Eta-1) in cell-mediated immunity: teaching an old dog new tricks.
        Immunol Today. 2000; 21: 475-478
        • Oki T.
        • Kitaura J.
        • Eto K.
        • Lu Y.
        • Maeda-Yamamoto M.
        • Inagaki N.
        • et al.
        Integrin alphaIIbbeta3 induces the adhesion and activation of mast cells through interaction with fibrinogen.
        J Immunol. 2006; 176: 52-60
        • Rangaswami H.
        • Bulbule A.
        • Kundu G.C.
        Osteopontin: role in cell signaling and cancer progression.
        Trends Cell Biol. 2006; 16: 79-87
        • Renkl A.C.
        • Wussler J.
        • Ahrens T.
        • Thoma K.
        • Kon S.
        • Uede T.
        • et al.
        Osteopontin functionally activates dendritic cells and induces their differentiation toward a Th1-polarizing phenotype.
        Blood. 2005; 106: 946-955
        • Rivera J.
        • Gilfillan A.M.
        Molecular regulation of mast cell activation.
        J Allergy Clin Immun. 2006; 117: 1214-1225
        • Rockey D.C.
        • Chung J.J.
        Endothelin antagonism in experimental hepatic fibrosis. Implications for endothelin in the pathogenesis of wound healing.
        J Clin Invest. 1996; 98: 1381-1388
        • Schiffrin E.L.
        Role of endothelin-1 in hypertension and vascular disease.
        Am J Hypertens. 2001; 14: 83S-89S
        • Shinohara M.L.
        • Jansson M.
        • Hwang E.S.
        • Werneck M.B.
        • Glimcher L.H.
        • Cantor H.
        T-bet-dependent expression of osteopontin contributes to T cell polarization.
        Proc Natl Acad Sci USA. 2005; 102: 17101-17106
        • Shinohara M.L.
        • Lu L.
        • Bu J.
        • Werneck M.B.
        • Kobayashi K.S.
        • Glimcher L.H.
        • et al.
        Osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells.
        Nat Immunol. 2006; 7: 498-506
        • Stelekati E.
        • Orinska Z.
        • Bulfone-Paus S.
        Mast cells in allergy: innate instructors of adaptive responses.
        Immunobiology. 2007; 212: 505-519
        • Taddei S.
        • Virdis A.
        • Ghiadoni L.
        • Sudano I.
        • Magagna A.
        • Salvetti A.
        Role of endothelin in the control of peripheral vascular tone in human hypertension.
        Heart Fail Rev. 2001; 6: 277-285
        • Takeda K.
        • Kaisho T.
        • Akira S.
        Toll-like receptors.
        Annu Rev Immunol. 2003; 21: 335-376
        • Theoharides T.C.
        • Conti P.
        Mast cells: the Jekyll and Hyde of tumor growth.
        Trends Immunol. 2004; 25: 235-241
        • Theoharides T.C.
        • Kempuraj D.
        • Tagen M.
        • Conti P.
        • Kalogeromitros D.
        Differential release of mast cell mediators and the pathogenesis of inflammation.
        Immunol Rev. 2007; 217: 65-78
        • Vancheeswaran R.
        • Azam A.
        • Black C.
        • Dashwood M.R.
        Localization of endothelin-1 and its binding sites in scleroderma skin.
        J Rheumatol. 1994; 21: 1268-1276
        • Xu G.
        • Nie H.
        • Li N.
        • Zheng W.
        • Zhang D.
        • Feng G.
        • et al.
        Role of osteopontin in amplification and perpetuation of rheumatoid synovitis.
        J Clin Invest. 2005; 115: 1060-1067
        • Yamada N.
        • Matsushima H.
        • Tagaya Y.
        • Shimada S.
        • Katz S.I.
        Generation of a large number of connective tissue type mast cells by culture of murine fetal skin cells.
        J Invest Dermatol. 2003; 121: 1425-1432
        • Yamamoto N.
        • Sakai F.
        • Kon S.
        • Morimoto J.
        • Kimura C.
        • Yamazaki H.
        • et al.
        Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis.
        J Clin Invest. 2003; 112: 181-188
        • Yanagisawa M.
        • Kurihara H.
        • Kimura S.
        • Tomobe Y.
        • Kobayashi M.
        • Mitsui Y.
        • et al.
        A novel potent vasoconstrictor peptide produced by vascular endothelial cells.
        Nature. 1988; 332: 411-415
        • Yumoto K.
        • Ishijima M.
        • Rittling S.R.
        • Tsuji K.
        • Tsuchiya Y.
        • Kon S.
        • et al.
        Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice.
        Proc Natl Acad Sci USA. 2002; 99: 4556-4561