Analyzing the Functions of Mast Cells <em>In Vivo</em> Using '<em>Mast Cell Knock-in</em>' Mice

Nicolas Gaudenzio; Riccardo Sibilano; Philipp Starkl; Mindy Tsai; Stephen J. Galli; Laurent L. Reber

doi:10.3791/52753

JoVE Journal > Immunology and Infection

Imunologia e Infecção

Analyzing the Functions of Mast Cells In Vivo Using ‘Mast Cell Knock-in‘ Mice

Published: May 27, 2015

doi:

10.3791/52753

Nicolas Gaudenzio¹, Riccardo Sibilano¹, Philipp Starkl¹, Mindy Tsai¹, Stephen J. Galli^1,2, Laurent L. Reber¹

¹Department of Pathology,Stanford University School of Medicine, ²Department of Microbiology & Immunology,Stanford University School of Medicine

Summary

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

Abstract

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the ‘mast cell knock-in’ approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

Introduction

Mast cells (MCs) are hematopoietic cells that arise from pluripotent bone marrow progenitors^1-3. Following bone marrow egression, MCs progenitors migrate into various tissues where they develop into mature MCs under the influence of local growth factors^1-3. Tissue-resident MCs are strategically located at host-environment interfaces, such as the skin, the airways and the gastrointestinal tract, where they behave as a first line of defense against external insults^3-6. MCs are often sub-classified based on their “baseline” phenotypic characteristics and their anatomic locations. In mice, two types of MCs have been described: “connective tissue-type” MCs (CTMCs) and mucosal MCs (MMCs)^1-3,7,8. CTMCs are often located around venules and near nerve fibers, and reside in serosal cavities, while MMCs occupy intraepithelial locations in the gut and respiratory mucosa^1-3.

Numerous methodologies have been applied to study biological functions of MCs^9-13. Many groups have focused on in vitro approaches using either cell lines (such as the human MC lines HMC1¹⁴ or LAD2^15,16), in vitro derived MCs (such as human peripheral blood-derived MCs¹⁷, or mouse bone marrow-derived cultured MCs [BMCMCs]¹⁸, fetal skin-derived cultured MCs [FSCMCs]¹⁹ and peritoneal cell-derived MCs [PCMCs]²⁰) or ex vivo isolated MCs from different anatomical sites. All these models are widely used to study molecular details of MC biology, such as signaling pathways involved in MC activation. However, an important aspect of MCs biology is that their phenotypic and functional characteristics (e.g., cytoplasmic granule protease content or response to different stimuli) can be modulated by anatomical location and microenvironment^2,7. Since the exact mixture of such factors that are encountered in vivo may be difficult to reproduce in vitro, we favor using in vivo approaches to gain insights into MCs functions⁹.

Several mouse strains with genetic MC deficiency exist, such as the widely used WBB6F₁–Kit ^W/W-v or C57BL/6-Kit ^W-sh/W-sh mice. These mice lack expression and/or activity of KIT (CD117), the receptor for the main MC growth factor stem cell factor (SCF)^21,22. As a result, these mice have a profound MC deficiency but also have additional phenotypic abnormalities related to their c-kit mutations (in the WBB6F₁–Kit ^W/W-v mice) or to the effects of the large chromosomal inversion that results in reduced c-kit expression (in the C57BL/6-Kit ^W-sh/W-sh mice)^9,10,12,23. More recently, several strains of mice with c-kit-independent constitutive MC deficiency have been reported^24-26. All these mice and some additional new types of inducible MC-deficient mice have been recently reviewed in detail^9,10,13.

Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This so-called ‘mast cell knock-in’ method can be used to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

Protocol

All animal care and experimentation were conducted in compliance with the guidelines of the National Institutes of Health and with the specific approval of the Institutional Animal Care and Use Committee of Stanford University.

1. Generation and Characterization of Bone Marrow-derived Cultured Mast Cells (BMCMCs).

Note: Donor BMCMCs should be generated from bone marrow cells of the same genetic background as the recipient MC-deficient mice. Male-derived donor BMCMCs are not suitable for engraftment of female mice. Female-derived donor BMCMCs will successfully engraft into both male and female recipients.

Bone Marrow Extraction.
1. Pour sterile phosphate-buffered saline (PBS) into 6 well culture plate (1 well per mouse) and place on ice.
2. Soak the dissection instruments in 70% ethanol for sterilization.
3. Euthanize donor mice by carbon dioxide (CO₂) inhalation followed by cervical dislocation, then spray the mice with 70% ethanol at the sites of manipulation.
4. Use sterile dissection instruments to extract the femur, tibia and fibula bones without cutting any bone epiphyses.
5. While securing the bones with a forceps, scrape off all tissue from bones (and discard fibula) using sterile scissors (or scalpel blades). Place bones in PBS on ice until all bones are collected.
6. Carry out all remaining steps in sterile tissue culture hood. Using sterile instruments, secure bones with forceps and cut off both epiphyses to expose the medullary cavity.
7. Using 3 ml syringes and 30 G needles, and cold flushing medium (Dulbecco Modified Eagle Medium [DMEM] supplemented with 10% fetal calf serum [FCS, heat-inactivated], 2 mM L-glutamine, 1% antibiotic-antimycotic solution, 50 µM Β-mercaptoethanol), flush the red bone marrow into a Petri dish.
8. Use the same syringe to dissociate flushed bone marrow cell clusters by repeated gentle aspiration and ejection.
9. Pool femoral and tibial bone marrow from each mouse into one 15 ml centrifuge tube. Fill the tube with cold flushing medium to wash bone marrow cells and centrifuge at 400 x g for 5 min at 4 °C.
BMCMCs Culture.
1. Remove supernatant and recover pelleted bone marrow cells in 10 ml of culture medium (DMEM supplemented with 10% fetal calf serum [FCS, heat-inactivated], 2 mM L-glutamine, 1% antibiotic-antimycotic solution, 50 µM Β-mercaptoethanol and 20% WEHI-3 cell-conditioned medium [as a source of IL-3; alternatively use recombinant mouse IL-3 at 10 ng/ml]).
2. Place cells in tissue culture flask of appropriate size (e.g., T25 for 1 mouse, T75 for 2 mice, etc.).
3. 1-2 days after plating cells, transfer medium and suspension cells (leaving debris and adherent cells sticking to bottom of flask) to a new flask and add fresh culture medium (10 ml/mouse).
4. During the following weeks, feed cells every 3-4 days (add 10 ml of culture medium per mouse). Maintain cell density between 2.5 x 10⁵-1 x 10⁶ cells/ml. Transfer non-adherent cells to a new flask once a week until no adherent cells are present in the culture flask. Test maturity of cells (see below) before use for in vitro assays or engraftment into MC-deficient mice.
  NOTE: Complete differentiation of BMCMCs will take 4-6 weeks.
Assessment of BMCMC maturity.
1. Using flow cytometry.
  1. Wash cells (5 x 10⁴-5 x 10⁵ cells/condition) with ice-cold FACS buffer (PBS, 0.5% FCS) in 5 ml polystyrene round bottom tube. Centrifuge at 400 x g for 5 min at 4 °C. Aspirate supernatant.
  2. Dilute anti-mouse CD16/32 (clone 93 or 2.4G2) monoclonal antibodies 1:200 (2.5 μg/ml) in FACS buffer. Add 10 µl to each pellet and resuspend by brief vortexing. Incubate 5 min on ice to block Fc binding.
    NOTE: Protect samples from direct light for all remaining steps.
  3. Dilute phycoerythrin (PE) conjugated anti-mouse Fc εRI α (clone MAR-1) 1:200 (1 μg/ml) and fluorescein isothiocyanate (FITC) conjugated anti-mouse KIT (CD117; clone 2B8) 1:200 (2.5 μg/ml) (“staining solution”), or the respective labeled isotype control antibodies in FACS buffer (“isotype control solution”).
  4. Split half of the blocked cells from step 1.3.1.2) into an additional polystyrene round bottom tube and add 20 µl of “staining solution” in one tube and 20 µl of “isotype controls solution” in the other tube. Incubate 30 min on ice.
  5. Add 3 ml ice-cold FACS buffer and centrifuge at 400 x g for 5 min at 4 °C. Discard supernatant and resuspend the pellet in 300 µl FACS buffer containing 1 µg/ml propidium iodide (PI, for identification of dead cells). Proceed to flow cytometry analysis (see Figure 2A).
2. Using toluidine blue staining.
  1. Wash 1 x 10⁵ cells once with PBS by centrifugation at 400 x g for 5 min at room temperature, then aspirate and resuspend cells in 200 µl of PBS.
  2. Transfer the cell suspension into a prepared cytofunnel attached to a microscope slide and proceed with cyto-centrifugation using a cytocentrifuge (40 x g for 5 min).
  3. Air dry slides for 10 min and draw a wax circle around cells using a PAP pen. Add 0.1% Toluidine blue solution to cover the cells. Incubate for 1 min. Wash slides in running tap water for 1 min, then air-dry slides for 10 min.
  4. Coverslip cells using mounting medium. Proceed to light microscope analysis (see Figure 2B)

2. Engraftment of Mast Cell-deficient Mice with BMCMCs.

Ear engraftment by intradermal (i.d.) injection.
NOTE: For adoptive transfer of BMCMCs into the ear pinnae of MC-deficient mice, we recommend performing two injections of 1 x 10⁶ cells each (for a total of 2 x 10⁶ cells) per ear pinna. Intradermal engraftment of BMCMCs into the ear pinnae of MC-deficient mice has been used by many investigators in several models, including models of passive cutaneous anaphylaxis (PCA)^24,27, host defense against venoms²⁸, and chronic hypersensitivity (CHS) reactions^29,30.
1. Count and resuspend cells at 4 x 10⁷ BMCMCs/ml (1 x 10⁶ BMCMCs/ 25 µl) in cold DMEM. Transfer BMCMCs solution into a 1 ml syringe equipped with a 30 G needle. Keep on ice until injection.
2. Anesthetize 4-6 weeks old MC-deficient mice using isoflurane (2.5% v/v). Check depth of anesthesia by toe pinch, adjust isoflurane if indicated and continue to monitor breathing and toe pinch response throughout the procedure. Apply ophthalmic ointment with a Q-tip to prevent dryness of eyes.
3. With the index finger, create vertical pressure on the dorsal face of the ear pinna to expose and stretch the ventral face.
4. Perform two 25 µl i.d. injections of BMCMC solution into two different sites of the ventral face of the ear pinna, the first injection in the middle of the ear and the second injection toward the tip of the ear pinna. Wait 4-6 weeks after i.d. engraftment before performing in vivo experiments.
  Note: In our experience, by that time the number of MCs/mm² of dermis in the central part of the ear pinna generally is similar to that in the corresponding location in wild type mice, whereas the numbers of MCs/mm² in the periphery of the ear pinnae are typically substantially lower than those in the corresponding wild type mice^27,28. This should be kept in mind when designing experiments with such MC-engrafted mice (e.g., agents with possible effects on MC function should be injected into the central part of the ear pinnae, and the analysis of the effects of such treatments should also focus on that area).
Peritoneal cavity engraftment by intraperitoneal (i.p.) injection.
NOTE: Adoptive transfer of BMCMCs into the peritoneal cavity of MC-deficient mice will require one injection of 2 x 10⁶ cells per mouse. Such i.p. injections of BMCMCs into MC-deficient mice have been used by many groups to study the roles of peritoneal MCs in various models, including models of host defense against venoms³¹ or bacterial sepsis^32,33.
1. Count appropriate number of cells and resuspend at 1 x 10⁷ BMCMCs/ml (2 x 10⁶ BMCMCs/ 200 µl) in cold DMEM. Transfer BMCMCs solution into a 1 ml syringe equipped with a 25 G needle. Keep on ice until injection.
2. Perform 1 injection of 200 µl BMCMCs solution into the peritoneal cavity of 4-6 week old MC-deficient mice. Wait 4-6 weeks after i.p. engraftment before performing in vivo experiments.
Engraftment by intravenous (i.v.) injection.
NOTE:Adoptive transfer of BMCMCs by i.v. injection into MC-deficient mice will require one injection of 5 x 10⁶ cells per mouse. Intravenous (i.v.) injections of BMCMCs into MC-deficient mice have been used by many groups to study the roles of MCs in various disease models, including models of bladder infection³⁴, asthma³⁵, lung fibrosis³⁶, and antibody-mediated arthritis³⁷.
1. Count appropriate number of cells and resuspend at 2.5 x 10⁷ BMCMCs/ml (5 x 10⁶ BMCMCs/200 µl) in cold DMEM. Transfer BMCMCs solution into a 1 ml syringe equipped with a 30 G needle. Keep on ice until injection.
2. Anesthetize 4-6 weeks old MC-deficient mice using isoflurane (2.5% v/v). Check depth of anesthesia by toe pinch, adjust isoflurane if indicated and continue to monitor breathing and toe pinch response throughout the procedure. Apply ophthalmic ointment with a Q-tip to prevent dryness of eyes.
3. Perform one injection of 200 µl of BMCMC solution into the tail vein (or, alternatively, the retro-orbital vein) of a MC-deficient mouse. Wait 12 weeks after i.v. engraftment before performing in vivo experiments.

3. Analysis of Engrafted Mast Cell-deficient Mice.

Ear engraftment analysis.
1. At the end of the experiment, euthanize mice by CO₂ inhalation followed by cervical dislocation.
2. Isolate the ear pinnae and fix in 10% (vol/vol) buffered formalin overnight at 4 °C. Embed fixed ear pinnae in paraffin, cut 4 µm sections of ear pinnae and mount on glass slides.
3. Stain the slides with a 0.1% Toluidine blue solution for 1 min at room temperature. Wash slides in tap water for 1 min, then air-dry slides for 10 min at room temperature.
4. Coverslip slides using mounting medium, then count the number of engrafted MCs per pinnae sections using a light microscope. Evaluate the engraftment efficiency by comparing the percentage and distribution of MCs in the ear skin of engrafted mice versus wild type mice.
Peritoneal cavity engraftment analysis.
1. Evaluation of mast cells number in the peritoneal cavity.
  1. At the end of the experiment, euthanize mice by CO₂ inhalation followed by cervical dislocation. Remove carefully the ventral skin of the mice without breaking the peritoneal cavity.
  2. Inject 5 ml of cold or room temperature PBS in the peritoneal cavity using a 5 ml syringe equipped with a 25 G needle. Use cold PBS to reduce the risk of activating peritoneal cells (this is very important when evaluating peritoneal MC degranulation or levels of some MC-derived products in the peritoneal lavage fluid). Perform a massage of the abdomen for 20 sec to harvest peritoneal cells.
  3. Slowly aspirate the peritoneal lavage using a 5 ml syringe equipped with a 22 G needle. Record the volume of aspirated lavage (expect to recover up to 80% of injected volume).
  4. Transfer peritoneal lavage fluid into a 5 ml polystyrene round bottom tube and centrifuge at 400 x g for 5 min at 4 °C. Aspirate supernatant. Recover the pellet in 400 µl cold PBS.
  5. Count total number of peritoneal cells using a hemocytometer chamber. Use half of the cell suspension for a flow cytometry analysis (as described in step 1.3.1), to evaluate the percentage and marker expression of engrafted MCs in the peritoneal cavity. Multiply the total number of peritoneal cells by the percentage of MCs to obtain absolute number of peritoneal MCs.
  6. Perform a cytocentrifugation with the remaining cells as described in step 1.3.2.2. Air dry slides for 10 min at room temperature and draw a wax circle around cells using a PAP pen.
  7. Cover cells with undiluted May-Grünwald Staining solution for 5 min, followed by washing in PBS for 5 min, both at room temperature. Cover cells with Giemsa stain diluted 1:20 with deionized water and incubate 20 min at room temperature. Wash slides 2 times in tap water for 1 min, then air-dry slides.
  8. Coverslip cells using mounting medium, and calculate the percentage of engrafted MCs using a light microscope (by counting at least 400 total cells). Evaluate the engraftment efficiency by comparing the percentage of MCs in the peritoneal lavage of engrafted mice versus wild type mice.
2. Evaluation of mast cells in the mesenteric windows.
  1. Following the peritoneal lavage described in step 3.2.1, cut open the peritoneal membrane to expose the intestinal tract of the mice. Arrange 4 to 5 mesenteric windows per mouse onto a slide.
  2. Fix the slides for 1 hr in Carnoy solution (3:2:1 vol/vol/vol of ethanol, chloroform, and glacial acetic acid) at room temperature. Air-dry slides and remove the intestine (the fixed mesenteric windows will remain attached to the slides).
  3. Stain the preparations for 20 min at room temperature with Csaba (Alcian blue/Safranin O) staining solution.
    1. To make 500 ml of Csaba stain, prepare 500 ml of acetate buffer by mixing 100 ml of 1 M sodium acetate solution with 120 ml of 1 M HCl. Make up to 500 ml with deionized water and adjust pH to 1.42. Then, dissolve 90 mg Safranin [identifies ‘mature MCs’ in red], 1.8 g Alcian blue [identifies ‘immature MCs’ in blue] and 2.4 g ferric ammonium sulphate NH₄Fe(SO₄)₂ into 500 ml of acetate buffer.
  4. Coverslip slides using mounting medium, then count the number of engrafted MCs per mesenteric window using a light microscope. Evaluate the engraftment efficiency by comparing the percentage and distribution of MCs in the mesenteric windows of engrafted mice versus wild type mice.
I.v. engraftment analysis.
1. At the end of the experiment, euthanize mice by CO₂ inhalation followed by cervical dislocation, and harvest tissue of interest (e.g. lung, skin or spleen) and fix overnight in 10% (vol/vol) buffered formalin at 4 °C.
2. Embed fixed tissues in paraffin, cut 4 µm sections, and mount on glass slides. Stain the slides with a 0.1% Toluidine blue solution for 1 min at room temperature. Wash slides in tap water for 1 min, then air-dry slides for 10 min.
3. Coverslip slides using mounting medium and evaluate number and distribution of engrafted MCs per tissue sections using a light microscope.

Representative Results

An overview of the ‘mast cell knock-in’ approach is shown in Figure 1, and includes the generation of BMCMCs, the number of cells that should be engrafted i.p., i.d. or i.v. into MC-deficient mice (the number can be varied if indicated based on the experimental design) and the interval between engraftment and experiment depending on the injection site (this interval also can vary, if indicated; e.g., the content of stored mediators in MC cytoplasmic granules increases steadily with time³⁸). Figure 2 shows representative flow cytometry analyses and toluidine blue staining of BMCMCs after 1, 15 and 45 days of culture in DMEM medium containing in 20% WEHI-3 cell-conditioned medium as a source of IL-3. Note that BMCMCs cultured for 45 days are 95% pure, contain high numbers of cytoplasmic granules and can be used for engraftment experiments, while cells cultured for 15 days are not suitable for engraftment. Figure 3 shows representative successful engraftment in the ear pinnae 4 weeks after i.d. engraftment (Figure 3A), in mesenteric windows (Figure 3B) and peritoneal cavity (Figure 3C) 6 weeks after i.p. engraftment, and in the lung (Figure 3D) 12 weeks after i.v. engraftment.

Figure 1: ‘Mast cell knock-in’ mouse model for analyses of MC functions in vivo. Wild type or mutant bone marrow-derived cultured MCs (BMCMCs) are generated by culturing bone marrow cells for at least 4-6 weeks in 20% WEHI-3 cell-conditioned medium as a source of IL-3 (or alternatively in medium containing 10 ng/ml recombinant mouse IL-3). These BMCMCs can then be engrafted into MC-deficient mice to create so-called ‘mast cell knock-in’ mice. BMCMCs can be injected via different routes (intravenous [i.v.], intraperitoneal [i.p.] or intradermal [i.d.]) for local (i.d., i.p.) or systemic (i.v.) reconstitution of various MC populations. MC function(s) in various biological responses can then be analyzed by comparing the responses in wild type mice, MC-deficient mice and ‘mast cell knock-in’ mice. The contribution(s) of specific MC products can be analyzed by comparing responses of ‘mast cell knock-in’ mice engrafted with either wild type BMCMCs or BMCMCs derived from mice that lack, or express genetically-altered forms of, such products. (This is a modified version of Figure 1 from ref.¹²).

Figure 2: Evaluation of the purity of BMCMC preparations by flow cytometry and microscopy. (A) Representative flow cytometry analyses of FcεRIα and KIT (CD117) expression on the surface of 1 day (left panel), 15 days (middle panel) and 45 days (right panel) old BMCMCs. Propidium iodide (PI)-positive dead cells were excluded from the analysis (not shown). Numbers indicate the percentage of FcεRIα⁺KIT⁺ BMCMCs gated in the blue square. (B) Representative pictures of BMCMCs stained with toluidine blue, lower panel is a magnification of the regions of the upper panel defined in black dotted lines. Bars = 10 μm.

Figure 3: Evaluation of the efficiency of MC engraftment at various anatomical sites. (A) Representative pictures of 4 μm ear pinnae sections stained with toluidine blue. (B) Representative pictures of mesenteric windows stained with Safranin/Acian Blue (‘Csaba’ stain). (C) Representative pictures of cells present in peritoneal lavage fluids and stained with May-Grünwald Giemsa (MGG). (D) Representative pictures of 4 μm lung sections stained with toluidine blue. Bars = 50 μm (A, B and D) or 20 μm (C). Pictures are from C57BL/6 wild type mice (upper panel), MC-deficient Kit^W-sh/W-sh mice (middle panel) and MC-deficient mice engrafted with wild type BMCMCs: BMCMCs Kit^W-sh/W-sh (lower panel). MCs are indicated with arrows.

Mice	Available Backgrounds	MC numbers (steady state)	Other phenotypes (reviewed in^9,10,13)	Reported engraftments with BMCMCs
*Kit^W/W-v*	(WB/Re x C57BL/6)F₁ (Jackson Laboratories – WBB6F₁/J-KitW/KitW-v/J)	Absence of connective-tissue and mucosal MCs	Anemia, reduced basophil and neutrophil numbers, deficiencies in melanocytes and interstitial cells of Cajal, sterile, etc.	i.v.^35,37; i.p.^31,62; i.d.^28,29; i.c.⁵⁰^,51; i.a.⁴⁹; fp.i.⁶³
*Kit^W-sh/W-sh*	C57BL/6 (Jackson Laboratories – B6.Cg-KitW-sh/HNihrJaeBsmJ) Single nucleotide polymorphism analysis performed at Jackson laboratories shows that these mice are only ~87% C57BL/6-genetic background.	Absence of connective-tissue and mucosal MCs	Moderate increase in basophil and neutrophil numbers, increased numbers of myeloid-derived suppressor cells⁶⁴, deficiencies in melanocytes and interstitial cells of Cajal	i.v.^23,34-36; i.p.^31,62; i.d.^28,29; i.a.⁴⁹
	C57BL/6J⁶² (Jackson Laboratories – B6.Cg-KitWsh/HNihrJaeBsmGlliJ) These mice have been backcrossed >11 times on the C57BL/6J background.
	BALB/c⁶⁵ (C.B6-Kit^W-sh)
*Mcpt5-Cre;* *R-DTA*	C57BL/6²⁵ (Tg(Cma1-cre) ARoer; B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky/J)	Marked reductions in peritoneal (98%) and skin (89-96.5%) MCs, mucosal MCs unlikely to be depleted	Probable presence of mucosal MCs; reporter mice reveals Cre-mediated deletion of ‘floxed’ YFP transgene in ~30% spleen NK cells⁶⁶	none
*Cpa3^Cre/+*	C57BL/6²⁶ (B6.129P2-Cpa3tm3(icre)Hrr)	Absence of connective-tissue and mucosal MCs	Cpa3 expressed in other cell types; reduced basophil numbers	i.v.²⁶
*Cpa3^Cre/+*	BALB/c²⁶ (B6.129P2/OlaHsd.BALB/c-Cpa3tm3(icre)Hrr)	Absence of connective-tissue and mucosal MCs		i.v.²⁶
*Cpa3-Cre;* *Mcl-1^fl/fl*	C57BL/6²⁴ (Tg(Cpa3-cre)3Glli; B6;129-Mcl1^tm3sjkJ)	Absence of connective-tissue and mucosal MCs	Cpa3 expressed in other cell types; increased spleen neutrophils & mild anemia; reduced basophil numbers	i.d.²⁴; i.a.⁴⁹ i.v. (our unpublished data)

Table 1: Strains of mice with constitutive MC deficiencies. Several strains of mice with c-kit-dependent or c-kit-independent constitutive MC deficiency are available. In principle, all of these strains can be used to generate ‘mast cell knock-in’ mice (although, to the best of our knowledge, this has not yet been reported for Mcpt5-Cre;R-DTA mice). However, each of these strains has other phenotypic abnormalities and limitations that should be kept in mind when interpreting results obtained with these mice. Some examples of successful MC engraftment can be found in the references. (This is a modified and updated version of Table 1 from ref.⁹).

Discussion

Almost 30 years after its initial description³⁸, the ‘mast cell knock-in’ approach continues to provide valuable information about what MCs can do or can’t do in vivo. The functions of MCs were long thought to be limited to their role in allergy. Data generated using the ‘mast cell knock-in’ approach have changed this view, by providing evidence that MCs can, among other functions, play critical roles in host defense against certain pathogens^4,39 or venoms^28,31, or can even suppress certain immune responses^29,34,40.

In our protocol description, we decided to focus on the generation and engraftment of bone marrow-derived cultured MCs (BMCMCs), because large numbers of these cells can be generated in vitro from the bone marrow of wild type or mutant mice. However, MCs can also be cultured directly from embryonic stem cells (embryonic stem cell-derived cultured MCs [ESCMCs])⁴¹ and, when genetically compatible, these cells can also be used for engraftment into MC-deficient mice. This alternative approach is particularly interesting for studying the role of a protein whose deficiency induces embryonic lethality in mice, and therefore for which BMCMCs deficient for this protein cannot be generated. Both BMCMCs and ESCMCs can also be transduced in vitro with lentiviruses encoding genes of interest or shRNA to silence genes of interest, before engraftment of these cells into MC-deficient mice^31,33.

We usually use 20% WEHI-3-conditioned medium as a source of IL-3 for the culture of BMCMCs. However, recombinant IL-3 (10 ng/ml, as described in step 1.2.1) can also be used, and addition of recombinant stem cell factor (SCF) to the culture medium can substantially enhance the numbers of BMCMCs generated^42,43. Depending on the study, 10 to 100 ng/ml of recombinant SCF have been used, in addition to IL-3, to generate BMCMCs^36,44,45. One should keep in mind that commercially available murine recombinant SCF preparations from different suppliers may differ in their potency in influencing the development of BMCMCs. It is also important to recognize that details of approach used to generate BMCMCs (such as whether one adds recombinant SCF to IL-3-containing medium, the duration of the culture period, etc.) may influence the phenotype and function of such cells. For example, it has been reported that BMCMCs chronically exposed to SCF have increased levels of histamine and certain proteases^44,46, but display a marked attenuation of FcεRI-mediated degranulation and cytokine production in vitro⁴⁵. Finally, in addition to IL-3, WEHI-3-conditioned medium contains many biologically active molecules that may affect MC functions. BMCMCs obtained with WEHI-3-conditioned medium are therefore likely to differ from BMCMCs obtained with recombinant IL-3 (or with recombinant IL-3 plus SCF). There have been few studies of whether or for how long any such differences in the phenotypes of BMCMCs generated in different types of culture medium are retained after the cells’ engraftment into different anatomical sites in vivo, and additional studies of this type may be of interest. However, regardless of the chosen culture conditions, the same culture medium recipe should be used to generate all of the BMCMCs to be used for engraftment in experiments from which results will be pooled for analysis. Moreover, MCs should be cultured for at least 4 to 6 weeks before their engraftment into MC-deficient mice, in order to reach a purity of 95-98% (Figure 2). This is to reduce the possibility that the presence, in the “BMCMC populations”, of hematopoietic cells other than those committed to the mast cell lineage (which are present in the cultures at early intervals after placing the bone marrow cells in vitro) might result in the appearance of donor-derived cells in addition to MCs in the ‘mast cell knock-in’ mice.

We present here a detailed protocol for engrafting MC-deficient mice with wild type or mutant BMCMCs intraperitoneally (i.p.), intravenously (i.v.) or intradermally (i.d.) in the ear pinna, since these routes of injection have been used by many investigators. However, BMCMCs have also been successfully engrafted into the back skin⁴⁷, in the footpad⁴⁸, intra-^{articularly49} or intra-cranially^50,51. The number of BMCMCs to engraft, as well as the interval between engraftment and experiment, can vary depending on the route of injection and the targeted organ to engraft (Figure 1). It is very important to respect such intervals after engrafting the BMCMCs before starting the experiment in order to allow sufficient time for BMCMCs (which are not fully mature MCs) to become more mature in vivo. Because the content of mediators stored in the MC’s cytoplasmic granules can continue to increase during the course of the cell’s lifetime^38,52, for certain experiments one may wish to increase the interval between MC engraftment and the initiation of the experiment to assess MC function.

Depending on the route of injection and/or the numbers of BMCMCs injected, the numbers and/or anatomical distribution of the adoptively transferred MCs can differ from those of the corresponding native MC populations in wild type mice^12,23,53,54. MC-deficient mice engrafted i.p. or i.d. with BMCMCs can have about the same numbers and distribution of MCs than the native MC population in wild type mice, in the peritoneal cavity and mesentery and in the dermis, respectively when assessed 4 to 8 weeks after MC transfer^12,23. Intravenous transfer of BMCMCs does not lead to normal MC numbers and/or distribution in most tissues. For example, no or very few MCs are found in the skin of such i.v.-engrafted ‘mast cell knock-in’ mice. At 4-28 weeks after i.v. injection of BMCMCs into MC-deficient mice, numbers of MCs in the trachea are substantially lower than those in the corresponding wild type mice. By contrast, the numbers of MCs in the periphery of the lung are typically greater than those in the corresponding wild type mice^12,23,53,55. I.v. transfer of BMCMCs also results in high levels of MCs in the spleen, whereas very few native MCs are typically found in this organ in wild type mice^23,56. Importantly, previous reports demonstrated that i.v. injection of BMCMCs into MC-deficient mice fails to result in engraftment of the MC populations in specific anatomical sites such as the spinal cord, lymph nodes or heart^54,57. Several groups have also noted that such i.v. engraftment does not result in engraftment of the intestinal mucosal MC (MMC) population^23,58-60. Such differences in MC numbers and/or distribution of adoptively-transferred MCs versus native MCs must be taken into account when interpreting data obtained using the MC ‘mast cell knock-in’ model⁹.

Several strains of MC-deficient mice exist, and choosing which one(s) to use in a particular project is important. c-kit mutant MC-deficient mice, such as Kit^W/W-v or Kit^W-sh/W-sh mice, have been traditionally used by many investigators. However, such mice suffer from many c-kit-related phenotypic abnormalities beside their profound MC deficiency (Table 1). In recent years, several strains of mice with c-kit-independent constitutive MC deficiency have been reported^24-26. Some of these mice also exhibit other phenotypic abnormalities beside their MC deficiency (Table 1), and additional abnormalities might also be discovered as the phenotype of these newly described strains is still under investigation. All these mice and some additional new types of MC-deficient mice have been recently reviewed in detail^9,10,13.

Given the limitations of each of the MC-deficient strains currently available, we recommend attempting to test hypotheses about MC function using more than one model of MC deficiency⁹. In our laboratory, we generally perform pilot experiments in 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 engraftment experiments to ascertain the role of MCs and assess the potential roles of certain MC-derived products. Finally, it should be noted that several strains allowing inducible depletion of MCs or Cre recombinase-mediated deletion of “floxed” genes in MCs have also been recently described^25,49,61. These strains have been reviewed in detail elsewhere^9,10,13 and represent promising alternative – or complementary – approaches to study MC functions in vivo.

Declarações

The authors have nothing to disclose.

Acknowledgements

N.G. is the recipient of fellowships from the French “Fondation pour la Recherche Médicale FRM” and the Philipp Foundation; R.S. is supported by the Lucile Packard Foundation for Children’s Health and the Stanford NIH/NCRR CTSA award number UL1 RR025744; P.S. is supported by a Max Kade Fellowship of the Max Kade Foundation and the Austrian Academy of Sciences and a Schroedinger Fellowship of the Austrian Science Fund (FWF): J3399-B21; S.J.G. acknowledges support from National Institutes of Health grants U19 AI104209, NS 080062 and from Tobacco-Related Disease Research Program at University of California; L.L.R. acknowledges support from the Arthritis National Research Foundation (ANRF) and National Institutes of Health grant K99AI110645.

Materials

1% Antibiotic-Antimycotic Solution	Corning cellgro	30-004-Cl
3 ml Syringe	Falcon	309656
35 mm x 10 mm Dish	Corning cellgro	430588
5 ml Polystyrene Round Bottom Tube	Falcon	352058
Acetic Acid Glacial	Fisher Scientific	A35-500
Alcian Blue 8GX	Rowley Biochemical Danver	33864-99-2
Allegra 6R Centrifuge	Beckman
Anti-mouse CD16/32 (clone 93) Purified	eBioscience	14-0161-81
2-Mercaptoethanol	Sigma Aldrich	M7522
BD 1 ml TB Syringe	BD Syringe	309659
BD 22G x1 (0.7 mm x 25 mm) Needles	BD Precision Glide Needle	205155
BD 25G 5/8 Needles	BD Syringe	305122
BD 30G x1/2 Needles	BD Precision Glide	305106
Blue MAX Jr, 15 ml Polypropylene Conical Tube	Falcon	352097
Chloroform	Fisher Scientific	C298-500
Cytoseal 60 Mounting Medium	Richard-Allan Scientific	8310-4
Cytospin3	Shandon	NA
DakoCytomation pen	Dako	S2002
Dulbecco Modified Eagle Medium (DMEM) 1x	Corning cellgro	15-013-CM
Ethanol	Sigma Aldrich	E 7023-500ml
Fetal Bovine Serum Heat Inactivated	Sigma Aldrich	F4135-500ml
FITC Conjugated IgG2b K Rat Isotype Control	eBioscience	14-4031-82
Fluorescein Isotiocyanate (FITC) Conjugated Anti-mouse KIT (CD117; clone 2B8)	eBioscience	11-1171-82
Formaldehyde	Fisher Scientific	F79-500
Giemsa Stain Modified	Sigma Aldrich	GS-1L
Isothesia	Henry Schein Animal Health	29405
May-Grunwald Stain	Sigma Aldrich	MG-1L
Multiwell 6 well plates	Falcon	35 3046
Olympus BX60 Microscope	Olympus	NA
Paraplast Plus Tissue Embedding Medium	Fisher Brand	23-021-400
PE Conjugated IgG Armenian Hamster Isotype Control	eBioscience	12-4888-81
Phosphate-Buffered-Saline (PBS) 1x	Corning cellgro	21-040-CV
Phycoerythrin (PE) Conjugated Anti-mouse FceRIa (clone MAR-1)	eBioscience	12-5898-82
Propidium Iodide Staining Solution	eBioscience	00-6990-50
Recombinant Mouse IL-3	Peprotech	213-13
Safranin-o Certified	Sigma Aldrich	S8884
Tissue culture flasks T25 25 cm²	Beckton Dickinson	353109
Tissue culture flasks T75 75 cm²	Beckton Dickinson	353110
Toluidine Blue 1 % Aqueous	LabChem-Inc	LC26165-2
Recombinant Mouse SCF	Peprotech	250-03

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Gaudenzio, N., Sibilano, R., Starkl, P., Tsai, M., Galli, S. J., Reber, L. L. Analyzing the Functions of Mast Cells In Vivo Using ‘Mast Cell Knock-in‘ Mice. J. Vis. Exp. (99), e52753, doi:10.3791/52753 (2015).