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

REVIEW

Murine Fetal Skin-Derived Cultured Mast Cells: A Useful Tool for Discovering Functions of Skin Mast Cells Hiroyuki Matsue1, Naotomo Kambe1 and Shinji Shimada2 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. Journal of Investigative Dermatology (2009) 129, 1120–1125; doi:10.1038/jid.2009.44; published online 26 February 2009

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 1

Department of Dermatology, Graduate School of Medicine, Chiba University, Chiba, Japan and 2Department of Dermatology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan Correspondence: Dr Hiroyiki Matsue, Department of Dermatology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 2608670, Japan. E-mail: [email protected] 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

Received 30 January 2008; revised 12 April 2008; accepted 22 April 2008; published online 26 February 2009

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cells that induce acute hypersensitivity responses via IgEmediated 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, 2004; Galli et al., 2005; Christy and Brown, 2007; Metz and Maurer, 2007; Metz et al., 2007; Galli and Tsai, 2008). 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 IgEbound, high-affinity IgE receptors (FceRI) 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 and Gilfillan, 2006). 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, 2004; Galli et al., 2005; Theoharides et al., 2007). 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 et al., 1977). Small numbers of committed mast cell progenitors circulate in the blood and migrate to tissues, where their terminal maturation is accomplished under the & 2009 The Society for Investigative Dermatology

H Matsue et al. A Tool for Exploring New Mast Cell Functions

influence of their final microenvironment (Gurish and Boyce, 2006). Committed mast cell progenitors reside at special anatomical sites (for example, intestine and hair follicles) and locally proliferate with terminal maturation (Kumamoto et al., 2003; Gurish and Boyce, 2006). Proliferation, terminal differentiation, and survival of resident mast cells require growth factors, such as IL-3, stem cell factor, and/or other factors (Gurish and Boyce, 2006). At least two types of mast cells have been historically identified in rodents based on their morphological, biochemical, and functional properties (Kitamura, 1989). 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, 2004). 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 et al., 1990). 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 et al. (2003). 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 IL3 (10 ng ml1), and stem cell factor (10 ng ml1). Nonadherent 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 496% CD45 þ CD117 þ (c-kit þ ) cells, were used as FSMC without further purification (Figure 1). Yamada et al. (2003) found that about 5% of fetal skin crude cells contained CD45 þ CD49b þ CD117 þ FceRI

Days 14–16

Fetal skin Trypsinization single-cell suspensions (5 × 104 cells ml–1)

Culture for 2 weeks (IL-3 + SCF) Percoll gradient centrifugation FSMCs (purity: >96%) (~7 x 106 cells per fetus) Figure 1. Generation 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 4 96% CD45 þ CD117 þ cells, which are used as FSMC without further purification.

cells, which could proliferate in response to stem cell factor. They considered these freshly isolated cells to be immature mast cells because the FceRI-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 FceRI and thus had differentiated into mature mast cells. Compatible with these findings, Meindl et al. (2006) recently reported that the majority of lineage-negative CD45 þ 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. 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 et al., 2003). 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 www.jidonline.org 1121

H Matsue et al. A Tool for Exploring New Mast Cell Functions

Table 1. Characteristic profiles of FSMC and BMMC1 Alcian blue/Safranin red double staining

FSMC

BMMC

Red

Blue

+

Heparin

Chondroitin sulfate

Histamine content

+++

+

Granule proteases

+++

+

Granules

Degranulation induced by Ionomycin

+

+

Compound 48/80

+



Substance P

+



ET-1

+



OPN





IgE cross-linking

+

+

+

+

+

ND

CD45

+

+

CD117

+

+

FceRI

+

+

CD49b

+



CD77



+

TLR1, 2, 3, 4, 6, 7, 9

TLR2, 4, 6





Enhanced by OPN Reduced by ET-1 (at low concentration) Phenotype

TLR expression Spontaneous ET-1 ET-1 induced by Ionomycin

+



TLR ligands2

+



Spontaneous OPN

+



Ionomycin

+

+

IgE cross-linking

+

+

OPN induced by

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FSMC

BMMC





OPN augmented or induced by TLR ligands3

+++

Major proteoglycans

Table 1. Continued

BMMC, bone marrow-derived mast cells; ET-1, endothelin-1; FSMC, fetal skin-derived mast cells; LPS, lipopolysaccharide; ND, not determined; ODN, oligodeoxynucleotide; OPN, osteopontin; PGN, peptidoglycan; TLR, Toll-like receptor. All photographs were reproduced with the permission of the publisher (from Yamada et al., 2003). 1 Data presented in this table are summarized from the following four papers: Matsushima et al. (2004a, b), Nagasaka et al. (2008), and Yamada et al. (2003). 2 TLR ligands include PGN for TLR2, poly(I:C) for TLR3, LPS for TLR4, and CpG ODN for TLR9. 3 TLR ligands include poly(I:C) for TLR3, LPS for TLR4, R848 for TLR7, and CpG ODN for TLR9.

and macrophages), which induce innate immunity and subsequently acquired immunity (Kaisho and Akira, 2006). Although TLR2 and TLR4 expression and function of mast cells in response to bacterial products have been well documented (Marshall, 2004; Stelekati et al., 2007), expression profiles of other TLRs and their functions on mast cells remain relatively unclear. Working with FSMC and BMMC, Matsushima et al. (2004b) 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-a and IL-6) and chemokines (RANTES, macrophage inflammatory protein-1a 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 et al., 1988). 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

H Matsue et al. A Tool for Exploring New Mast Cell Functions

failure (Nambi et al., 2001; Schiffrin, 2001; Taddei et al., 2001). 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 et al., 1994; Rockey and Chung, 1996; Goldie and Henry, 1999; Hocher et al., 2000; Mullol and Picado, 2000). Working with FSMC (Matsushima et al., 2004a), we found that ET-1 was able to (a) induce degranulation by FSMC via ETA in a dose-dependent manner (108 to 106 M), (b) significantly augment degranulation by IgE-sensitized FSMC using the above dose ranges, (c) at its lower concentration (1010 M), significantly suppress degranulation induced by FceRI aggregation, (d) induce proinflammatory cytokine production (tumor necrosis factor-a and IL-6), (e) significantly enhance VEGF production and transforming growth factor-b1 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 multifunctional 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 et al., 2004). Because ET-1 has high homology (470%) to sarafotoxins, the most potent toxic components of certain venoms, they hypothesized that mast cells might also be protective in envenomation. Indeed, Metz et al. (2006b) 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 et al., 2006a). 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 et al., 2001), wound healing (Liaw et al., 1998), dystrophic calcification, and coronary restenosis (Rangaswami et al., 2006). Immunologists found that it is expressed and functions in many immune cell types (Rangaswami et al., 2006). 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 et al., 2000; Diao et al., 2004; Renkl et al., 2005; Shinohara et al., 2005, 2006). For instance, OPN plays an important role in efficient TH1 immune responses in many experimental settings. Ashkar et al. (2000) 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 et al., 2005). T-bet-dependent OPN expression by activated T cells skewed CD4 þ and CD8 þ T cells toward TH1 and Tc1 CD8 þ T cells, respectively (Shinohara et al., 2005). In a more recent report, intracellular OPN in plasmacytoid dendritic cells regulated their IFN-a production, thus being involved in TH1 immunity to viral infection (Shinohara et al., 2006). 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 et al., 2004). OPN is also involved in autoimmune diseases, including multiple sclerosis and its animal model (Chabas et al., 2001; Jansson et al., 2002), and rheumatoid arthritis and its animal model (Yumoto et al., 2002; Yamamoto et al., 2003; Xu et al., 2005). 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 and Mathis, 2002; Gruber, 2003; Levi-Schaffer and Piliponsky, 2003; Marshall, 2004; Theoharides and Conti, 2004; Galli et al., 2005). 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 et al., 2008). OPN was found to (1) be spontaneously produced by FSMC and be inducible in BMMC in response to stimuli (for example, FceRI aggregation and ionomycin), (2) significantly augment mast cell degranulation by FceRI aggregation, and (3) www.jidonline.org 1123

H Matsue et al. A Tool for Exploring New Mast Cell Functions

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 and Paus (2008) recently discussed why this newly discovered property of mast cellderived 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 et al. (2006) used FSMC as a mast cell type to investigate integrin aIIbb3 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 et al. (2006) found that C5-epimerase-deficient FSMC distorted the heparin Osulfation 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 et al. (2005) 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

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.

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ACKNOWLEDGMENTS

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We especially thank our ex-colleague, Dr Nobuo Yamada, who established the FSMC cell culture system during his postdoctoral fellowship in National

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