New models for analyzing mast cell functions in vivo

Abstract

In addition to their well-accepted role as critical effector cells in anaphylaxis and other acute IgE-mediated allergic reactions, mast cells have been implicated in a wide variety of process that contribute to disease or help to maintain health. While some of these roles were first suggested by analyses of mast cell products or functions in vitro, it is critical to determine whether, and under which circumstances, such potential roles actually can be performed by mast cells in vivo. This review discusses recent advances in the development and analysis of mouse models to investigate the roles of mast cells and mast cell-associated products during biological responses in vivo, and comments on some of the similarities and differences in the results obtained with these newer versus older models of mast cell deficiency.

The spectrum of ‘potential’ mast cell functions: an embarrassment of riches

Trying to figure out what mast cells (MCs) do in vivo has been challenging. This is not for want of hypotheses. Indeed, given what has been reported about MCs based on in vitro or in vivo evidence, the possibilities appear to be almost endless (Box 1). Taking such information into account, one can come up with a nearly limitless list of “potential” or “possible” MC functions – spanning many if not all aspects of health, host defense, and disease. But what, in fact, are the important functions of MCs in vivo, and how can these be identified? To answer these questions, one must first agree on terminology, including the definition of “important functions”. In Box 2, we propose working definitions of a range of possible MC contributions to biological responses or to the individual features of such processes.

Box 1: Some general observations about mast cell (MC) biology.

In vivo and in vitro studies have shown that:

• MCs are distributed throughout nearly all tissues, and often in close proximity to potential targets of their mediators such as epithelia and glands, smooth muscle and cardiac muscle cells, fibroblasts, and blood and lymphatic vessels and nerves [5].

• MCs can store and release upon degranulation and/or secrete de novo a wide spectrum of biologically active mediators (many of which also can be produced by other cell types) that individually have been shown to have potential positive or negative effects on the function of various target leukocytes or structural cells, and that thereby have the potential to influence inflammation, hemostasis, tissue remodeling, cancer, metabolism, reproduction, behavior, sleep, and many other biological responses [98–101].

• MCs can be activated to secrete biologically active products not only by IgE and specific antigen (the main mechanism which accounts for their function in allergic disorders [102]) but by a long list of other stimuli including physical agents, products of diverse pathogens [103], many innate danger signals [104], certain endogenous peptides and structurally similar peptides found in invertebrate and vertebrate venoms [62, 74, 77], and products of innate and adaptive immune responses including immune complexes of IgG, certain chemokines and cytokines (including IL-33 [105, 106]), and products of complement activation [107].

• The ability of MCs to secrete biologically active mediators can be enhanced or suppressed by many factors, including interactions with other granulocytes [108], regulatory T cells [109] or other lymphocytes [110], and certain cytokines, including the main MC development and survival growth factor, the Kit ligand stem cell factor [5, 111–113], IL-33 [114], and interferon-γ [115].

• MCs in different anatomical locations and in different species can vary in multiple aspects of phenotype, including their responsiveness to signals regulating their proliferation and function, their content of stored mediators, and their potential to produce various newly synthesized mediators [116, 117].

• The numbers, anatomical distribution, phenotype, and function of MCs can be modulated, or “tuned”, by a wide variety of genetic or environmental factors, so that the properties of MCs may be different depending on the genetic background of the host and/or the local or systemic levels of factors with effects on MC biology (including those generated during ongoing innate or adaptive immune responses or diseases) [5].

Box 2. A hierarchy of mast cell (MC) roles in biological responses.

We propose that the following working definitions of MC functions might be useful in conceptualizing how MCs can influence particular biological responses*:

1. “unique” or “non-redundant”: i.e., only MCs can perform that function;

2. “important”: that aspect of the response would be substantially different (e.g., by 50% or more) in the absence of MCs;

3. “redundant” or “overlapping”: the MC can contribute to the assessed feature of the response along with other effector or regulatory elements, but the potential contribution(s) of the MC may only be revealed if one or more of the other partially redundant or overlapping elements is impaired. One can think here of a round table supported by 10 legs that are equally spaced under its periphery. By selecting the correct ones to remove, as many as 7 of the legs could be eliminated without causing the table to fall; but remove one more leg and the table will collapse. Note that, in this simple example, by planning properly any 3 of the 10 legs could be selected to be the last ones keeping the table up – i.e., all 10 of them are equally redundant. However, in actual biological responses, some "legs" may be more important than others.

4. “non-contributory”: the MC has no role in that feature of the response.

* In particular biological responses, such as models of host defense or disease, MCs may influence some features of the response more importantly than others. Indeed, in some biological responses, MCs might have any combination of the 4 roles listed, depending on which features of the response one measures. The number of biological responses in which essentially all features of the response are fully and uniquely dependent on MCs may be small.

What kinds of experimental approaches can permit one to identify the actual contributions of MCs when investigating their potential roles in particular biological settings? The simplest would be to be able to ablate MCs selectively in vivo, e.g., with a drug or an antibody, or genetically. Moreover, one ideally would be able to ablate selectively either all MCs (producing a fully MC-deficient host, in which potential local and/or systemic effects of MCs could be tested) or only the MC populations of interest (e.g., those in the skin or lungs). Once it is established that MCs have a detectable role in a biological response, it is useful then to define how that MC role is expressed in that setting. To address this question, one ideally would be able to delete selectively elements of MC activation pathways, or MC products, or to block specifically those MC-derived products by which MCs might express that function.

To date, no agent that can solely and specifically suppress MC activation has been discovered. Notably, recent findings indicate that even the so-called “MC stabilizer” cromolyn is neither an effective nor selective inhibitor of mouse MC activation in vitro and in vivo [1]. For this reason, genetic approaches probably represent a more definitive way to identify and characterize MC functions in mice in vivo. While progress has been made in devising genetic approaches that address that goal, particularly over the last few years, each of the new approaches (as well as older models that have been widely used for many years) have known or potential limitations that must be kept in mind when interpreting the results of such work.

In this review, we will compare and contrast the advantages and potential disadvantages of both older and newer approaches to investigate the roles of MCs in vivo. We will discuss some examples of MC functions that now have been supported by evidence derived both from studies in older models (consisting of mice that have mutations affecting c-kit structure or expression and that consequently exhibit a profound MC deficiency together with a variety of other phenotypic abnormalities) and from work employing newer models in which the MC deficiency is not dependent on mutations affecting c-kit structure or expression. We also will discuss some MC functions that were proposed based on evidence obtained in “kit mutant MC-deficient mice” which have not been confirmed in initial studies employing the new “KIT-independent” models of MC deficiency. Finally, we will comment on some early results of work attempting to probe the roles of MCs in particular biological responses using more than one model system.

“Kit mutant” MC-deficient mice and “MC knock-in mice”

To date, mice whose sole abnormality is a specific lack of all populations of MCs have not been reported. However, we and others have used mice deficient for KIT, the receptor for the main MC growth and survival factor, stem cell factor (SCF) [2, 3] (which sometimes are collectively called “kit mutant mice”) to analyze the functions of MCs in vivo [4–8]. The two types of MC-deficient mice used most commonly for such studies are WBB6F₁-Kit^W/W-v and C57BL/6-Kit^W-sh/W-sh mice [5–12]. Kit^W is a point mutation that produces a truncated KIT that is not expressed on the cell surface [13]; Kit^W-v is a mutation in the c-kit tyrosine kinase domain that substantially reduces the kinase activity of the receptor [14] and Kit^W-sh is an inversion mutation that affects the transcriptional regulatory elements upstream of the c-kit transcription start site on mouse chromosome 5 [15, 16]. Both WBB6F₁-Kit^W/W-v and C57BL/6-Kit^W-sh/W-sh mice are profoundly deficient in MCs and melanocytes and have several other phenotypic abnormalities that reflect the biological distribution and functions of KIT in cells within and outside of the immune system of these mice, including some abnormalities affecting hematopoietic cells other than MCs that contribute to innate or adaptive immune responses (Box 3) [6, 8, 12, 16–18]. However, some of these “non-MC” phenotypic abnormalities differ between the two most commonly used types of kit mutant MC-deficient mice. For example, WBB6F1-Kit^W/W-v mice are anaemic, have reduced numbers of neutrophils [8, 12, 16, 18] and basophils [8, 19, 20], and are sterile [5, 6]. By contrast, C57BL/6-Kit^W-sh/W-sh mice are neither anaemic nor sterile, but have increased numbers of neutrophils [6, 8, 12, 16] and basophils [8].

Box 3: KIT expression within and outside the immune system.

Within the immune system

• KIT is highly expressed in hematopoietic stem cells (HSCs) in the bone marrow and activation of KIT by its ligand stem cell factor (SCF) contributes to regulation of the self-renewal, survival and differentiation of HSCs [48, 118–120].

• While most immune cells lose detectable KIT expression upon cell differentiation, MCs remain KIT⁺ throughout their life and SCF can promote MC maturation, survival and proliferation, as well as influence MC migration and functional responses [113, 121–124].

• KIT expression has also been described in purified eosinophils from mice infected with Schistosoma mansoni [125] and in mouse dendritic cells after stimulation with cholera toxin or house dust mite extract [126], and has been reported at low levels in some human basophils [127].

Outside the immune system

• KIT is expressed in melanocytes and germ cells [128, 129]. Both Kit^W/W-v and Kit^W-sh/W-sh mice are deficient in melanocytes but only Kit^W/W-v mice are sterile [6, 17].

• KIT is also expressed in interstitial cells of Cajal (ICC) in the gastrointestinal tract and kit mutant mice have marked reductions in ICC and have abnormal electrical pacemaker activity in the small intestine [130].

• KIT expression has been described in many other cell types in mice, such as subpopulations of sensory neurons [131], certain nerves in the CNS [132], keratinocytes [133], and tubular epithelial cells in the kidney [134]. Kit^W/Wv mice backcrossed on the A/JxB6 F₁ background display reduced naïve airway hyperresponsiveness to metacholine as compared to Kit^+/+ littermate controls in a MC-independent manner, suggesting the existence of a yet unidentified nonhematopoietic KIT⁺ cell type [135].

Differences in the biological responses in kit mutant mice compared with wild-type (WT) mice of course may reflect any one (or more) of the abnormalities that result from the alterations of KIT structure or expression in these animals, in any of the directly or indirectly affected cell lineages, and may not be due solely or even partly to their deficiency in MCs. However, at many anatomical sites, the deficiency in MCs in kit mutant mice can be selectively “repaired” by the adoptive transfer of genetically-compatible, in vitro-derived WT or mutant MCs [4–6, 10, 21]. Such in vitro-derived MCs, for example bone-marrow-derived cultured MCs (BMCMCs), can be administrated intravenously (i.v.), intraperitoneally (i.p.) or intradermally (i.d.) to create so-called ‘MC knock-in mice’. Since their description in 1985 [21], such MC knock-in mice have been widely employed to assess the importance of MCs in regulating the expression of biological responses in vivo.

However, it has long been known that, depending on the route of injection of MCs and/or the numbers of MCs injected, the numbers and/or anatomical distribution of adoptively transferred MCs after transfer to kit mutant mice can differ from those of the corresponding native MC populations in the corresponding WT mice [6, 17, 22, 23]. With direct injection of BMCMCs into the skin or peritoneal cavity of WBB6F₁-Kit^W/W-v or C57BL/6-Kit^W-sh/W-sh mice, the numbers and anatomic distribution of adoptively transferred MCs in the dermis or in the peritoneal cavity and mesentery, when assessed 4–8 weeks after MC transfer, can be similar to those of native MCs in WT mice [6, 17]. By contrast, at 4–28 weeks after injection of BMCMCs i.v. into WBB6F₁-Kit^W/W-v or C57BL/6-Kit^W-sh/W-sh mice, few or no MCs are detectable in the trachea of the mice (and numbers are much less than those in the corresponding WT mice) whereas the numbers of MCs in the periphery of the lung are substantially greater than, and the numbers of MCs around the bronchi can be similar to, those in the corresponding WT mice [6, 17, 22, 24]. Such differences in MC numbers and anatomical distribution of adoptively transferred versus corresponding WT MC populations should be taken into account when considering the results obtained in MC knock-in versus corresponding WT mice. One must also consider the possibility that the native and adoptively transferred MC populations differ in certain aspects of phenotype. Although direct comparisons of such populations have in general shown that, over time, the phenotype of the adoptively-transferred MCs comes to closely resemble that of the native population [21, 25], there have been relatively few studies of that type. Moreover, it is not currently possible to define every aspect of the phenotype of either native or adoptively transferred MC populations in situ. Therefore, one can’t formally rule out the possibility that the two MC populations might express phenotypic differences that in turn might influence the results obtained in a particular biological response.

Mutant mice with constitutive MC deficiency unrelated to c-kit abnormalities

KIT has pleiotropic functions unrelated to MCs (Box 3). Therefore, even when MC engraftment results in MC numbers and anatomical distributions in the recipient kit mutant mice that are very similar to those of the corresponding WT mice, it is possible that such adoptively transferred MCs can “normalize” some of the biological responses that are abnormal in kit mutant mice because the transferred MCs compensate in the mutant mice for abnormalities in lineages other than the MC -- abnormalities that do not exist in the corresponding WT mice. To return to the example of the round table with 10 legs (Box 2), MCs may play more critical roles in some biological responses in kit mutant mice than in WT mice because that biological response has less redundancy in the kit mutant mice than is present in the WT animals.

Because of the potential complexities and caveats inherent in interpreting findings based on work employing kit mutant MC-deficient mice, several groups have sought to develop mice that are MC-deficient but which lack abnormalities related to KIT structure or expression. A common approach has been to generate mice in which Cre-recombinase (Cre) is expressed under the control of promoters thought to be “MC-specific” or at least “MC-associated” [26–29]. To date, three new strains of mutant mice with marked constitutive deficiencies in MCs have been reported (Table 1) [19, 28, 30].

Table 1.

Characteristics of “KIT-independent” mast cell (MC)-deficient mice

Defi- ciency	Mice	Construct	MC numbers	IgE-dependent MC function	Basophil numbers	Basophil bunction	Known or potential limitations for MC studies	Refs .
CONSTITUTIVE	Mcpt5-Cre; R-DTA Tg(Cma1-cre) ARoer; B6.129P2-Gt(ROSA)26 Sortm1(DTA)Lky/J	Cross between R-DTA floxed mice and transgenic mice expressing the Cre under the Mcpt5 promoter	Steady-state: marked reductions in peritoneal (98%) and skin (89–96.5%) MCs, mucosal MCs (MMCs) unlikely to be depleted	Not assessed	Not assessed (basophils thought not to express Mcpt5)	Not assessed	Probable presence of MMCs Are there cell-intrinsic defects of NK cells or other cell types?	[30]
	“Cre-Master”Cpa3Cre/+ Cpa3tm3(icre)Hrr	Gene targeting: Cre expression under the control of the Cpa3 promoter while deleting 28 nucleotides of the first exon of Cpa3 locus	Steady-state: absence of connective-tissue and mucosal MCs (in skin, peritoneum, intestine) Inflammatory conditions: remain deficient in skin MCs after PMA-induced dermatitis and in intestinal MMCs following helminth infection	Don't develop IgE-dependent models of PSA or PCA; PSA response restored by systemic engraftment of WT BMCMCs	60 % reduction in spleen basophil numbers (blood and bone-marrow basophil status not reported)	Not assessed	Cpa3 expressed in other cell types Reduced basophil numbers Are there functional abnormalities of basophils or cell-intrinsic defects in other cell types?	[19]
	“Hello Kitty”Cpa3-Cre; Mcl-1fl/fl Tg(Cpa3-cre)3Glli; B6;129-Mcl1tm3sjkJ	Cross between Mcl-1 floxed mice and transgenic mice expressing Cre under a Cpa3 promoter fragment	Steady-state: marked reductions (92–100%) in connective-tissue and mucosal MCs in the skin, trachea, lung, peritoneum, digestive tract, etc., but no reduction in small numbers of splenic MCs	Markedly reduced features of IgE-dependent models of PSA and PCA; PCA response restored by intradermal engraftment of WT BMCMCs	Reductions in basophil numbers in spleen (58%), blood (74%), and bone-marrow (75%)	Markedly reduced IgE-and basophil-dependent chronic allergic Inflammation of skin	Cpa3 expressed in other cell types Reduced basophil numbers and function Increased spleen neutrophils & mild anemia Are there cell-intrinsic defects in other cell types?	[28]
INDUCIBLE	Mcpt5-Cre; iDTR Tg(Cma1-cre) ARoer; C57BL/6-Gt(ROSA)26 Sortm1(HBEGF)Awai/J	Cross between inducible DTR floxed mice and transgenic mice expressing Cre under the Mcpt5 promoter	Steady-state: 1 week after 4 weekly i.p. and 2 s.c. DT treatments deficient in peritoneal and skin MCs (97.5%); stomach and intestinal MMCs present. Repopulation: 10% of pre-treatment skin and peritoneal MC numbers 3 weeks after the last DT treatment	Not assessed	Bone-marrow basophils not affected 1 week after 4 weekly i.p. treatments with DT	Not assessed (basophils thought not to express Mcpt5)	Presence of MMCs Are there “off target” or other side effects of repeated treatment with DT?	[30]
	“Mas-TRECK”	Transgenic mice expressing human DTR under an intronic enhancer of the Il4 gene	Steady-state: 3 days after 5 daily i.p. DT treatments: deficient in peritoneal, skin, stomach and mesenteric window MCs Repopulation: Skin MCs undetectable 12 days after the last DT treatment	Markedly reduced features of IgE-dependent models of PSA and PCA 2 days after 5 daily i.p. treatments with DT	Transient >95% reduction in blood basophil numbers 5 days after start of DT treatment and recovery 12 days after the last DT treatment	Markedly reduced features of IgE- and basophil-dependent chronic allergic inflammation of skin (induced 2 days after 5 daily i.p. treatments with DT)	Transient reduction in basophil numbers and function Are there “off target” or other side effects of repeated treatment with DT?	[50, 51]

Mcpt5-Cre; R-DTA mice

Dudeck et al. (2011) crossed MC protease (Mcpt)5-Cre transgenic mice with R-DTA^fl/fl mice [31] to generate a mouse strain in which the diphtheria toxin alpha chain (DTA) is produced only in Cre-expressing cells, thereby driving Cre-specific ablation of such cells [30]. Naive Mcpt5-Cre;R-DTA mice displayed a constitutive lack of peritoneal and ear skin MCs as well as >90% reductions in the numbers of abdominal and back skin MCs in comparison to the Cre⁻ counterparts [30]. The effect of Cre-mediated DTA expression on mucosal MCs (MMCs, which are thought not to express MCPT5) or other hematopoietic cell types in steady-state or inflammatory conditions, and the efficiency of DTA-induced deletion of connective tissue type MCs (CTMCs) during inflammatory responses associated with increased numbers of MCs, remain to be described.

Cpa3^Cre/+ - “Cre-Master” mice

“Cre-Master” stands for “Cre-mediated mast cell eradication”. Feyerabend et al. (2011) used an elegant knock-in strategy to induce Cre expression under the control of the Cpa3 promoter while deleting 28 nucleotides of the first exon of Cpa3, which encodes for the MC-associated protease carboxypeptidase A3 (CPA3) [19]. Unexpectedly, heterozygous Cpa3^Cre/+ mice exhibited a virtually complete lack of MCs, multiple MC-associated proteases, and a MC gene expression signature, as assessed in the peritoneal cavity and skin. Skin MCs were still undetectable under inflammatory conditions that can be associated with the development of skin MCs in WBB6F₁-Kit^W/W-v mice [32] and MMCs remained absent in the intestine after helminth infection [19]. In addition, Cpa3^Cre/+ mice did not detectably respond in an IgE-dependent model of passive cutaneous anaphylaxis (PCA) and exhibited neither reduced body temperature nor mortality when subjected to an IgE-dependent model of passive systemic anaphylaxis (PSA) [33], unless they were engrafted with BMCMCs.

This profound depletion of MCs appears to be mediated by Cre-induced genotoxicity [34]. However, although CPA3 is highly expressed in MCs [35], it is also expressed in basophils [36] and some populations of T-cell progenitors and thymic T cells [27, 37], and in certain hematopoietic progenitor cells [38]. Consistent with this, the authors also detected some Cre activity in T cells [27], as well as a 60% reduction in numbers of spleen basophils [19]. While Cre expression in basophils was not sufficient to ablate the entire population, it must be kept in mind that the residual basophils may not be fully functional.

Cpa3-Cre; Mcl-1^fl/fl – “Hello Kitty” mice

Our group generated transgenic mice expressing Cre recombinase under the control of a Cpa3 promoter fragment [28] and crossed them with mice in which the gene coding the anti-apoptotic factor myeloid cell leukemia sequence 1 (Mcl-1) [39, 40] was floxed [28]. The resulting Cpa3-Cre; Mcl-1^fl/fl mice exhibited a marked kit-independent constitutive reduction in numbers of MCs (92–100% reduction in all anatomical sites tested except the spleen, that, like the spleen of the corresponding control mice, contained small numbers of MCs); Cpa3-Cre; Mcl-1^fl/fl mice also exhibited a substantial reduction in bone marrow, blood and spleen basophils (reductions of 78, 74, and 58%, respectively, in comparison to the Cpa3-Cre controls). Because these phenotypes are seen in the absence of mutations affecting c-kit structure or expression, these mice are informally called “Hello Kitty” MC- (and basophil)-deficient mice.

Assessing the responses of these markedly MC-deficient mice in three models of IgE-dependent inflammation revealed, as expected, that they were markedly deficient in two responses that previously had been characterized (in kit mutant MC-deficient mice) as IgE- and MC-dependent [41–43], specifically, IgE-dependent PCA (except at sites engrafted with WT MCs) [41] and IgE-dependent PSA [43]. However, these studies also revealed that the reduction in numbers of basophils in Hello Kitty mice, although modest compared to the deficiency in tissue MCs, was associated with a profound impairment in the animals’ ability to orchestrate a response that is IgE- and basophil-dependent, but MC-independent [28, 44]. The latter finding illustrates that mutant mice with less than full ablation of a certain type of effector cell (in this case, the basophil) may nevertheless exhibit a marked abnormality in a biological response that is particularly dependent on that cell type.

These three new types of MC-deficient mice represent welcome new tools for investigating the role of MCs in biological responses in vivo. Nevertheless, in designing experiments employing such mice (or the older models), and in interpreting the results of such work, it will be prudent to keep in mind the already identified and additional potential limitations of these models, issues that may turn out to be more important in some types of biological responses than in others (Table 1, Box 3). One potential problem common to each of the three new strains, and to the kit mutant MC-deficient mice, is that the effects on certain biological responses of a constitutive deficiency of MCs may be different than those observed when the MCs are ablated just before or during the response. The latter situation generally has more clinical relevance than the former, as in most cases one would not attempt to reduce MC numbers or functions in human subjects unless there was compelling clinical evidence that MCs are important in the pathology associated with a particular disorder.

Inducible depletion of MCs

Employing mouse models to test the hypothesis that MCs represent an important therapeutic target in a particular setting should ideally be performed using mice in which inducible and selective MCs ablation can be achieved. Depletion of MCs from mice by conventional techniques, such as the injection of depleting antibodies, is limited by the lack of surface markers that have been shown to be unique to the MC population. For example, repeated treatment with antibodies that neutralize SCF [45] or block KIT [46, 47] can result in the depletion of MCs in vivo, but can also have potential effects on other cell types such as hematopoietic stem cells [48].

One promising approach for achieving a more selective and efficient depletion of a particular cell population is the injection of diphtheria toxin (DT) into transgenic mice bearing the DT receptor (DTR) only in that particular cell type [49]. This approach was recently used by two different groups to deplete MCs in adult mice (Table 1) [30, 50, 51].

Mcpt5-Cre; iDTR mice

Dudeck et al. mated Mcpt5-Cre mice with iDTR^fl/fl mice expressing a floxed simian DTR transgene inserted into the Gt(ROSA)26Sor (ROSA26) locus, to achieve Cre-dependent expression of DTR in MCs [30]. The authors reported that a single i.p. injection of DT leads to nearly complete ablation of peritoneal MCs in Mcpt5-Cre; iDTR mice after 24 h, however they didn’t comment on MC numbers in other organs or whether there were any effects on other cell types. Repeated i.p. injections of DT (once a week for four weeks) led to complete ablation of MCs in the peritoneal cavity and abdominal skin of Mcpt5-Cre; iDTR mice as compared to Mcpt5-Cre⁻ mice, when assessed one week after the last DT injection. However, achieving complete deletion of ear skin MCs required combining repeated i.p. and subcutaneous treatment with DT [30]. Moreover, analysis of the small intestine and stomach of DT-treated Mcpt5-Cre; iDTR mice showed depletion of subepithelial CTMCs but not intraepithelial MMCs, most likely reflecting a lack of Mcpt5-Cre transgene expression in MCs of the mucosal type [30]. Nevertheless, these animals should represent a valuable tool for studying the effects of a local depletion of MCs in various acute biological processes. These mice may even be used to study the role of MCs in more chronic settings since only about 10% of peritoneal and skin MCs reappeared 3 weeks after cessation of treatment with DT under steady-state conditions. The authors reported that numbers of other major hematopoietic cells, including bone marrow basophils, were not affected by DT-treatment. However, this analysis was performed one week after the last DT injection, and it would be of interest to know whether DT injections resulted in any transient depletion of other cell types.

“Mas-TRECK” mice

Otsuka et al. and Sawaguchi et al. described a new transgenic strain, named “Mas-TRECK” (for Mast cell-specific enhancer-mediated Toxin Receptor-mediated Conditional cell Knock out), in which expression of the human DTR gene is under the control of an intronic enhancer (IE) element of the Il-4 gene [50, 51]. They previously reported that this IE element was essential for IL-4 expression in MCs but not basophils, natural killer (NK) T cells or Th2 cells [52]. Repeated i.p. treatment of Mas-TRECK mice with DT for 5 consecutive days completely depleted MCs in the skin, peritoneal cavity, stomach and mesenteric windows, as assessed 3 days after the last injection, and abrogated IgE-dependent PCA and PSA reactions [50]. They also showed that skin MCs remain depleted for at least 12 days after cessation of DT treatment [51]. However, DT treatment in these mice also leads to a transient depletion of blood basophils and virtually completely inhibited the development of a model of basophil-dependent, IgE-mediated chronic allergic inflammation of the skin [44, 50]. Other major types of leukocytes (dendritic cells, B, T, NKT cells, eosinophils and neutrophils) did not express DTR mRNA and were not affected by DT treatment, although numbers of these cells were only reported for analyses done 12 days after the end of DT treatment [50, 51].

Mutant mice with deletion of MC-associated products

If a mediator is selectively expressed by MCs (to prove this, expression needs to be analyzed in other cell types under both baseline and pathological conditions), its role can be investigated in vivo by testing animals in which that mediator has been knocked out. However, many of these highly MC-associated (if not truly MC-selective) mediators (such as MC-associated proteases) show strong interdependence in terms of proper storage in the cytoplasmic granules and this clearly must be kept in mind when interpreting results obtained with mice genetically deficient in such mediators (Table 2). Analyzing to what extent MCs represent important sources of products that can also be derived from other cell types, such as leukotrienes, prostaglandins, cytokines, chemokines, and growth factors, would require deletion of that product specifically in MCs. In this regard, the newly developed “MC-specific Cre” mice [26–29, 53] may allow for specific deletion of “floxed” genes in MCs.

Table 2.

Genetic deletion of mast cell (MC)-associated products

Mutant mice	Gene name	Phenotype and/or limitations	Refs.
HDC−/−	Histidine decarboxylase	No histamine produced (mice should be maintained under histamine free diet, since histamine can also be acquired through ingestion) Decreased MC numbers Altered storage of various proteases in MC granules Histamine can also be produced by other cell types, including some other hematopoietic cells such as basophils or neutrophils	[63, 64]
Mcpt1−/−	MC protease 1	Markedly reduces esterase activity in intestinal mucosal MCs Histochemical and ultrastructural changes in granules of mucosal MCs	[65]
Mcpt4−/−	MC protease 4 (chymase)	Does not affect the number or morphology of MCs in multiple anatomical sites tested Increased tryptase activity in peritoneal MCs	[66, 67]
Mcpt5−/−	MC protease 5	Markedly reduced storage of CPA3 and CPA activity in peritoneal MCs Increased tryptase activity in peritoneal MCs Decreased chymase activity in peritoneal MCs	[66, 68, 69]
Mcpt6−/−	MC protease 6	Does not affect the number or morphology of MCs in multiple anatomical sites tested Does not affect histamine and MCPT4 levels in peritoneal MCs	[70, 71]
Mcpt7−/− (= C57BL/6)	MC protease 7	C57BL/6 mice are unable to express MCPT7 because of a point mutation in the exon/intron 2 splice of the Mcpt7 gene Does not affect the number or morphology of MCs in multiple anatomical sites tested	[72]
Cpa3−/−	Carboxypeptidase A3	Markedly reduced storage of MCPT5 in MC granules	[73]
Cpa3Y356L,E378A	Carboxypeptidase A3	Inactive CPA3 due to two point mutations in the catalytic domain Doesn’t affect storage of MCPT5 in MC granules	[62, 74]
NDST-2−/−	N-deacetylase/N-sulfotransferase-2	MCs are unable to synthesize heparin Decreased numbers of connective tissue-type MCs Reduced storage of other proteases (MCPT4, MCPT5, CPA3) in MC granules	[75, 76]

Dudeck et al. (2011) were, to our knowledge, the first to take advantage of this system in order to reduce secretion of MC-derived interleukin (IL)-10 in vivo by crossing Mcpt5-Cre transgenic mice [26] with Il-10^fl/fl mice [30]. Other researchers used this approach with Mcpt5-Cre mice in order to drive expression of a gain-of-function mutation of KIT (Kit^D814V) [54] or to specifically delete SH2 domain-containing phosphatase-2 (SHP2) gene [55] in CTMCs. Recently, a new mouse strain expressing Cre under the control of the high affinity receptor for IgE, β chain promoter (FcεRI-β Cre) has been generated and used to delete the phosphatase and tensin homolog (Pten) gene in the MC compartment [53].

Two critical issues have to be taken into consideration when interpreting the results obtained using a Cre/lox approach. First, Cre activity must be efficient in, and ideally selectively restricted to, MCs, both in naive animals and under inflammatory conditions, since MC promoter-driven Cre expression may vary depending on the conditions and models tested. In this regard, using a reporter mouse is a valuable tool for the assessment of Cre-mediated recombination under different conditions in vivo.

In the study by Dudeck et al. (2011), Mcpt5-Cre⁺ mice were crossed to the Cre excision reporter mice ROSA26 Stop^flox EYFP (R26Y) and EYFP expression was assessed by flow cytometry in naive animals, confirming a highly efficient recombination in peritoneal and skin MCs but also revealing an unexpected recombination in a small population of blood NK cells. Lilla et al. (2011) crossed transgenic Cpa3-Cre mice [28] with a mT/mG reporter line [56], thus revealing the steady-state detection of Cre expression in a small population of basophils, eosinophils and neutrophils in addition to MCs [28]. The breeding of Cpa3-Cre mice with particular ‘floxed’ mice might therefore result in gene inactivation in certain populations of granulocytes (as well as in MCs) in double transgenic mice and this might limit the ability of this approach to reveal specific roles of MC-derived products in settings in which such other cells also may have important roles. Transgenic mice expressing Cre recombinase under the control of the conserved baboon alpha-chymase promoter (Chm:Cre) [29] displayed Cre expression specifically in lung and colon tissues by using Chm:Cre/ROSA26R reporter mice. However, in the lung of Chm:Cre/ROSA26R naive mice, 26% of Cre positive cells were KIT negative, strongly suggesting that Cre activity might not be fully MC-specific.

Second, the Cre-mediated gene inactivation should be demonstrated in MCs and only in MCs. Dudeck et al. used an elegant, sensitive method of single-cell PCR in order to assess the specificity and efficiency of Cre-mediated Il-10 gene inactivation in several cell types using nested primers [30, 57]. The authors showed thereby an efficient inactivation of the functional Il-10 locus in peritoneal and skin MCs, but not in peritoneal B cells, macrophages or skin T cells from Mcpt5-Cre⁺; Il-10^fl/fl mice, or any cell type tested in Mcpt5-Cre⁻; Il-10^fl/fl mice. However, blood NK cells, which exhibited some Cre-mediated recombination, apparently have not been tested for Il-10 gene inactivation [30].

“Kit mutant” vs. “KIT-independent” MC-deficient mice in models of host defense and disease: Concordance, controversies, and opportunities

As reviewed above, individual mouse models of MC deficiency, or models to alter the expression of MC-associated products, differ in their features and may vary in their advantages and limitations for studies of MC function. The newer models of MC deficiency are particularly attractive because they lack the KIT-related phenotypic alterations associated with kit mutant MC-deficient mice. However, because the newer models only recently have been described, it is likely that there is still more to be learned about their phenotype, including features that may influence the interpretation of experiments designed to investigate MC functions. In addition, one often can select from among a wide variety of experimental protocols and conditions to investigate particular hypotheses about MC functions in examples of host defense or disease.

Both factors, i.e., (1) the choice of MC-deficient mouse model(s) and (2) the selection of particular experimental conditions for investigating the roles of MCs in various types of biological responses, can influence the results of such work. Moreover, the choice of experimental protocol may be particularly important in biological responses in which the MC is more likely to have a redundant rather than unique (non-redundant) role (Box 2). This rather obvious point was evident even before the introduction of the new models of MC deficiency, as is illustrated by the results of efforts to employ kit mutant MC-deficient mice to investigate the roles of MCs in cutaneous contact hypersensitivity (CHS) responses or in allergic inflammation of the airways (Table 3).

Table 3.

Examples of consistent or discrepant conclusions about mast cell (MC) functions based on in vivo studies in MC-deficient mice

Model/disease	Mutant mice used	Test response	Main findings/conclusions	Refs.	Possible reason(s) for any discrepancies in results*
IgE-dependent passive anaphylaxis	MC knock-in kit mutant mice	PCA and/or PSA	WT BMCMCs (i.d.) restored ability of MC-deficient KitW/W-v mice to express robust PCA response	[41]	Does not apply (to date, all results are concordant)
	Cre-Master		PCA & PSA could not be elicited in kit mutant mice; WT BMCMCs (systemic engraftment) restored ability of MC-deficient mice to express PSA responses	[19]
	Hello Kitty		WT BMCMCs (i.d.) restored ability of MC-deficient mice to express robust PCA response	[28]
	Mas-TRECK		PCA & PSA could not be elicited in MC-deficient mice	[50]
Innate resistance to venoms and/or their toxic components	MC knock-in kit mutant mice	Toxicity of venoms of 3 snakes & honey bee and sarafotoxin 6b (S6b)	MCs enhanced resistance to each venom and to S6b in kit mutant mice, and potato carboxypeptidase inhibitor reduced resistance to snake venoms and S6b in WT mice	[77]	Does not apply (to date, all results are concordant)
	KitW/W-v, Cpa3Y356L,E378A	Toxicity of S6b	CPA3 can cleave S6b and neutralize this venom component	[74]
	MC knock-in kit mutant mice, Mcpt4−/− mice	Toxicity of Gila monster & 2 scorpion venoms and helodermin	MCs and MC chymase (MCPT4) can reduce toxicity of each venom and of helodermin and VIP	[62]
“Asthma” (allergic inflammation and hyper-responsiveness of the airways)	KitW/W-v	17 day-long model, sensitization with adjuvant (alum)	Possible contributory role for MCs (but no studies in MC knock-in mice)	[78]	Importance of MC roles in such mice may vary based on differences in doses of antigen used for sensitization and/or challenge and/or amount of adjuvant, etc. (i.e., “strength” of the protocol in inducing involvement and activation of immune cells in addition to MCs).
	KitW/W-v	30 day-long model, sensitization with adjuvant (alum)	Redundant or no roles for MCs (no studies in MC knock-in mice)	[79]
	MC knock-in KitW/W-v	20 day-long model, sensitization with adjuvant (alum)	Contributory roles for MCs with low dose (but not with high dose) antigen challenge	[80]
	KitW/W-v	31-day long model, sensitization with adjuvant (alum)	Redundant or no role for MCs with high dose antigen challenge (no studies in MC knock-in mice)	[81]	Differences in the models (duration of model; sensitization with or without use of adjuvant [alum]) that may influence the “strength” of the protocol in inducing involvement and activation of immune cells in addition to MCs
	MC knock-in KitW/W-v	44 day-long model, sensitization w/o1 adjuvant	Contributory role for MCs in AHR2 and airway inflammation
	MC knock-in kit mutant mice	9 week-long model, sensitization w/o1 adjuvant	Important roles for MCs in multiple features of the pathology	[82]
		47 day-long model, sensitization w/o1 adjuvant		[83]
	KitW-sh/W-sh, C57BL/6 background	47-day long model, sensitization w/o1 adjuvant	Possible contributory role for MCs (but no studies in MC knock-in mice)	[84]	Differences in genetic backgrounds (C57BL/6 vs. BALB/c) can influence importance of roles of MCs in this model in KitW-sh/W-sh mice.
	C.B6-KitW-sh/W-sh, BALB/c background		Full development of ‘asthmatic’ features in the MC-deficient mice
	Mas-TRECK	28-day long model, sensitization w/o1 adjuvant	Inducing MC depletion prior to challenge reduced AHR2, but had no detectable effect on eosinophil infiltration	[50]	Differences in roles of MCs as revealed by MC engraftment into kit mutant mice prior to sensitization (by other groups) vs. by MC-depletion in DT treated Mas-TRECK mice after sensitization (this study) Roles of adoptively transferred MCs in kit mutant mice may differ from those of MCs in Mas-TRECK mice.
Antibody-dependent arthritis	MC knock-in KitW/W-v	K/BxN [83] serum transfer model	Important role for MCs in enhancing	[85]	Important role for neutrophils in the model in WT mice may result in “redundant” contributions of MCs being masked in presence of neutrophilia (in KitW-sh/W-sh mice) or normal levels of neutrophils (in Cre-Master mice) but being revealed in presence of neutropenia (in KitW/W-v mice) Differences in genetic backgrounds of WBB6F1-KitW/W-v mice vs. that (C57BL/6) in other types of MC-deficient mice MCs have redundant or no roles in this model.
	KitW/W-v	Anti-collagen antibodies		[12]
	KitW-sh/W-sh	K/BxN [83] serum transfer model	Full development of features of arthritis in MC-deficient mice	[86, 87]
		Anti-collagen antibodies		[12]
	Cre-Master	K/BxN [83] serum transfer model		[19]
Experimental allergic encephalomyelitis (EAE)	MC knock-in KitW/W-v	MOG35–553 in CFA4 containing M. tuberculosis (s.c.) and PTX5 (i.v.)	Important role for MCs in exacerbating pathology	[88]	Different doses of peptide and/or adjuvant (in some experiments) or effects of other factors that might influence these responses* (as possible explanations for differences in results obtained by different groups using KitW/W-v mice or among different types of MC-deficient mice) Differences in genetic backgrounds of WBB6F1-KitW/W-v mice vs. that (C57BL/6) in other types of MC-deficient mice Important roles for other immune cells in the models in WT mice (and perhaps enhancement of certain T cell functions in KitW-sh/W-sh mice [85–87]) may result in “redundant” contributions of MCs only being revealed (in some but not all [85] studies) in the context of the immune cell defects present in KitW/W-v mice. In EAE models, MCs engrafted into kit mutant mice exhibit different roles than do native MCs in WT mice (i.e., MCs may have redundant or no roles in these models in WT mice).
	KitW/W-v and KitW-sh/W-sh		Full development of the disease in MC-deficient mice	[19, 89]
	KitW-sh/W-sh		Exacerbation of the disease in MC-deficient mice	[90, 91]
	Cre-Master		Full development of the disease in MC-deficient mice	[19]
Cutaneous contact hypersensitivity (CHS)	KitW/W-v	Sheep red blood cells or picryl chloride-induced CHS	Reduced development of CHS responses in MC-deficient mice	[92]	Differences in details of CHS protocols (e.g., types and/or concentrations of haptens, types of vehicles [in some experiments]) or effects of other factors that might influence these responses* Different times of assessment and types of features analyzed to quantify responses (in some experiments).
		Picryl chloride- or Ox6-induced CHS	Redundant roles or no roles for MCs in the CHS responses	[58]
		TNCB7-induced CHS	MCs can enhance elicitation of the CHS response (included MC knock-in experiments)	[93]
		Ox6-induced CHS, sensitization with low-dose Ox6	Reduced development of active or passive models of CHS responses in MC-deficient mice	[59]
	MC knock-in KitW/W-v	Ox6-induced CHS, sensitization with low-dose Ox6	MCs can facilitate elicitation of the CHS response	[94]
		Ox6-induced CHS, sensitization with high-dose Ox6	MCs can down regulate the CHS response
	Mas-TRECK	DNFB8-induced CHS	Reduced CHS response when MCs are absent or depleted by DT treatment	[51]
	MC knock-in kit mutant mice	DNFB8 and urushiol-induced CHS	Increased CHS responses and skin pathology when MCs or MC-derived IL-10 are absent	[11]	Differences in details of CHS protocols or effects of other factors that might influence these responses* MCs engrafted into kit mutant mice, and IL-10 derived from such engrafted MCs, exhibit different roles in CHS in kit mutant mice than do native MCs in WT mice (i.e., MCs and MC-derived IL-10 may suppress the responses in KitW-sh/W-sh mice (e.g., perhaps because certain T cell functions are abnormal in these mice [85–87) but have redundant or no roles in the responses analyzed in WT mice)
	kit mutant mice	DNFB8-induced CHS	kit-mutant MC-deficient mice have increased CHS responses	[30]
	Mcpt5-Cre; R-DTA, Mcpt5-Cre; iDTR, Mcpt5-Cre; Il10fl/fl	DNFB8-induced CHS	Reduced swelling in first 2 days of CHS response when MCs are absent or depleted by DT treatment; no detectable role for MC-derived IL-10 in the CHS response	[30]

¹w/o; without

²AHR; airway hyperreactivity

³MOG; myelin oligodendrocyte glycoprotein

⁴CFA; complete Freund’s adjuvant

⁵PTX; pertussis toxin

⁶Ox; oxazolone

⁷TNFB; trinitrochlorobenzene

⁸DNFB; 1-fluoro-2,4-dinitrobenzene

^*In light of recent work indicating that immune responses in mice can be influenced by differences in the host’s endogenous microflora [95–97], it is possible that this factor (or other differences affecting animals housed in different institutions) can influence the importance of the MC’s role in innate or adaptive immune responses, and thereby contribute to the differences in results obtained when similar or identical protocols for inducing such responses are tested in different laboratories.

There are at least two ways to view the fact that work by different groups (or even different experiments performed by the same group [58, 59]) support “different conclusions” about the importance of MCs in various biological responses. The first is that this constitutes a “controversy” regarding the roles of MCs in that type of biological response [60, 61]. The second (which is not necessarily incompatible with the first) is that such discrepancies identify interesting opportunities for understanding the basis for the differences, and thereby to gain additional insights into the regulation of these biological responses.

For example, in the case of both CHS and allergic airway inflammation, current findings are compatible with the conclusion that the ability of the MC to enhance particular features of the models is most readily detected when one attempts to elicit “relatively weak” responses, particularly in mice of suitable genetic background (Table 3). In the case of allergic inflammation of the airways, inducing reactions with relatively low doses of antigen for sensitization and challenge may more closely mimic clinical settings than do protocols which use strong adjuvants for sensitization and large amounts of antigen for airway challenge. With respect to models of antibody-dependent arthritis, it seems plausible, although not yet formally proven, that the relative neutrophil deficiency of WBB6F₁-Kit^W/W-v mice (as well as perhaps other abnormalities affecting hematopoietic cells in addition to MCs and neutrophils) contributes to the inability of these mice fully to develop the features of the pathology (Table 3).

In more general terms, it seems reasonable both to think that evolution has engineered redundancy into the mechanisms needed to sustain many critical biological processes in order to ensure that they remain robust, as well as to propose that such mechanistic redundancy also applies to many pathological processes. Indeed, we speculate that there may be only a small number of biological responses in which MCs are uniquely critical (e.g., as an important source of proteases that may be highly expressed in MCs), to the extent that little or no response would be detectable in the absence of MCs under any conditions of testing. Accordingly, in many types of complex biological responses in which the roles of MCs may be partially overlapping with those of other effector elements, we expect that the choice of experimental model, including the intensity of the stimulus used to elicit the response, may be critical in determining whether the contributions of MCs will be sufficiently important that their absence will be reflected in a significant impairment of the response. Similarly, it seems reasonable to propose that the more critical the MC’s contributions to a particular biological response, the more likely one will find abnormalities in that response when it is tested in each of the different types of MC-deficient mice.

For these reasons, we recommend attempting to test hypotheses about MC function using more than one model of MC deficiency. In our lab, we used to perform pilot experiments for any new project in both WBB6F₁-Kit^W/W-v and WCB6F₁-Mgf^Sl/Sl-d mice. We then switched to testing WBB6F₁-Kit^W/W-v and C57BL6-Kit^W-sh/W-sh mice and we are now transitioning to doing pilot experiments in C57BL6-Kit^W-sh/W-sh and Cpa3-Cre; Mcl-1^fl/fl mice. If we obtain concordant results in both types of MC-deficient mice, we then proceed to further studies, which can include using mice genetically deficient in particular MC products of interest. An example is our recent study of the roles of MCs and the MC-associated chymase, MCPT4, in enhancing resistance to the venoms of the Gila monster and two scorpions, and to the endogenous peptide, VIP [62].

By contrast, if tests in two different types of MC-deficient mice yield discordant findings, we generally do not continue. While pursuing such a project (or such an “opportunity”) might reveal interesting information about why the MC’s role may be “revealed” in one type of MC-deficient mouse and not in the other, there is also the risk that we might not succeed in explaining the discrepancy and thereby consume in an ultimately futile effort resources which could be used for more promising lines of inquiry. While in many settings it may not be practical also to test multiple models or protocols of the type of host defense or disease of interest, one certainly should keep in mind, as discussed above, that the roles of MCs in influencing different features of particular biological responses clearly can vary based on the selection of experimental conditions to examine.

Although we are in early days with respect to some of the newer models that can be employed for MC research, there already are examples where work in older and newer models have provided either very similar or rather discordant evidence for particular MC functions. Some of the possible reasons for why discrepant results have been obtained regarding the roles of MCs in particular biological responses are noted in Table 3. We have summarized in Box 4 some conclusions about the nature and importance of the roles of MCs in different types of biological responses that we think are compatible with current evidence derived from work in multiple older and newer models of MC-deficient mice and/or in mice deficient in certain MC-associated products.

Box 4. Concordant and discordant findings regarding mast cell (MC) functions in vivo.

1. Findings in multiple model systems (including kit mutant MC-deficient mice and MC knock-in mice, and in the newer MC-deficient mice with normal KIT) support the conclusion that MCs have important and perhaps in some cases even non-redundant roles in many acute, IgE-dependent responses [12, 19, 28, 41–43, 50, 136] but are not required for development of a more chronic IgE-dependent cutaneous response that is dependent on basophils [28, 44].

2. Studies in both various kit mutant MC-deficient mice and in MC-associated protease-deficient mice are consistent in supporting an important role for mast cells [62, 77] and their proteases (namely, CPA3 [74] and MCPT4 [62]) in reducing the pathology and mortality induced by the venoms of certain reptiles and arthropods.

3. Different models of MC deficiency have yielded different results when the possible contributions of MCs have been examined in certain complex biological responses. In each of these responses, it is likely that multiple types of immune cells may have partially redundant or overlapping roles. Such responses include antibody-dependent arthritis [12, 85–87, 137], EAE [19, 60, 88–91], and cutaneous contact hypersensitivity [11, 30, 51, 58, 59, 92–94, 138, 139]. In models of airway inflammation and airway hypersensitivity, it has been reported that both the details of the model system [50, 78–83, 140], and mouse strain background [84], can influence the extent to which MCs are important for the development of various features of the responses; this is also likely to be true regarding the ability to discern the importance of the MC’s contributions to many other biological responses.

Concluding remarks

This is an exciting time in MC research. The continued availability of “old” models (including “MC knock-in kit mutant mice” and various MC protease-deficient mice), combined with the introduction of several promising “new” models of MC deficiency or for MC-targeted deletion of MC products, offers a wealth of opportunities to enhance progress in solving the long-standing “riddle of the mast cell”, at least in mice. Some of this work may even suggest new approaches for the treatment of diseases in which MCs have been implicated. However, based on the results obtained so far with both the older and newer models for MC research, we think that the most robust conclusions about what MCs can do (or don’t do) in various biological responses in vivo, and regarding the importance of such MC contributions, are likely to be derived from investigations that employ multiple informative model systems. This approach increases the cost of such work, but permits one to exploit the attractive features of the various models while keeping in mind the known and potential limitations in each of them.