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Method Article

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

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DOI:

10.3791/52753

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May 27th, 2015

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

In This Article

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 ā€˜mast cell knock-inā€™ 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 progenitors1-3. Following bone marrow egression, MCs progenitors migrate into various tissues where they develop into mature MCs under the influence of local growth factors1-3. 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 insults3-6. MCs are often sub-classified based on their ā€œbaselineā€ phenotypic characteristics and their anatomic locations. In mice, two types of MCs have been described: ā€œconnective tissue-typeā€ 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 mucosa1-3.

Numerous methodologies have been applied to study biological functions of MCs9-13. Many groups have focused on in vitro approaches using either cell lines (such as the human MC lines HMC114 or LAD215,16), 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 microenvironment2,7. 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 reported24-26. All these mice and some additional new types of inducible MC-deficient mice have been recently reviewed in detail9,10,13.

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 ā€˜mast cell knock-inā€™ 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.

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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.

  1. 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 ĀµM Ī’-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 Ā°C.
  2. 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 ĀµM Ī’-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.
  3. 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 Ā°C. Aspirate supernatant.
      2. Dilute anti-mouse CD16/32 (clone 93 or 2.4G2) monoclonal antibodies 1:200 (2.5 Ī¼g/ml) in FACS buffer. Add 10 Āµl 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 ĪµRI Ī± (clone MAR-1) 1:200 (1 Ī¼g/ml) and fluorescein isothiocyanate (FITC) conjugated anti-mouse KIT (CD117; clone 2B8) 1:200 (2.5 Ī¼g/ml) (ā€œstaining solutionā€), or the respective labeled isotype control antibodies in FACS buffer (ā€œisotype control solutionā€).
      4. Split half of the blocked cells from step 1.3.1.2) into an additional polystyrene round bottom tube and add 20 Āµl of ā€œstaining solutionā€ in one tube and 20 Āµl of ā€œisotype controls solutionā€ 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 Ā°C. Discard supernatant and resuspend the pellet in 300 Āµl FACS buffer containing 1 Āµg/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 Āµl 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.

  1. 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) reactions29,30.
    1. Count and resuspend cells at 4 x 107 BMCMCs/ml (1 x 106 BMCMCs/ 25 Āµl) 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 Āµl 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 mice27,28. 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).
  2. 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 sepsis32,33.
    1. Count appropriate number of cells and resuspend at 1 x 107 BMCMCs/ml (2 x 106 BMCMCs/ 200 Āµl) 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 Āµl 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.
  3. 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 Āµl) 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 Āµl 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.

  1. 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 Ā°C. Embed fixed ear pinnae in paraffin, cut 4 Āµm 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.
  2. 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 Ā°C. Aspirate supernatant. Recover the pellet in 400 Āµl 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-GrĆ¼nwald 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 ā€˜mature MCsā€™ in red], 1.8 g Alcian blue [identifies ā€˜immature MCsā€™ 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.
  3. 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 Ā°C.
    2. Embed fixed tissues in paraffin, cut 4 Āµm 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.

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Results

An overview of the ā€˜mast cell knock-inā€™ 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 time...

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Discussion

Almost 30 years after its initial description38, the ā€˜mast cell knock-inā€™ approach continues to provide valuable information about what MCs can do or canā€™t do in vivo. The functions of MCs were long thought to be limited to their role in allergy. Data generated using the ā€˜mast cell knock-inā€™ approach have changed this view, by providing evidence that MCs can, among other functions, play critical roles in host defense against certain pathogens4,39

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Disclosures

The authors have nothing to disclose.

Acknowledgements

N.G. is the recipient of fellowships from the French ā€œFondation pour la Recherche MĆ©dicale FRMā€ and the Philipp Foundation; R.S. is supported by the Lucile Packard Foundation for Childrenā€™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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1% Antibiotic-Antimycotic SolutionCorning cellgro30-004-Cl
3 ml SyringeFalcon309656
35 mm x 10 mm DishCorning cellgro430588
5 ml Polystyrene Round Bottom TubeFalcon352058
Acetic Acid GlacialFisher ScientificA35-500
Alcian Blue 8GXRowley Biochemical Danver33864-99-2
Allegra 6R CentrifugeBeckman
Anti-mouse CD16/32 (clone 93) PurifiedeBioscience14-0161-81
2-MercaptoethanolSigma AldrichM7522
BD 1 ml TB SyringeBD Syringe309659
BD 22 G x 1 (0.7 mm x 25 mm) NeedlesBD Precision Glide Needle205155
BD 25 G 5/8 NeedlesBD Syringe305122
BD 30 G x 1/2 NeedlesBD Precision Glide305106
Blue MAX Jr, 15 ml Polypropylene Conical TubeFalcon352097
ChloroformFisher ScientificC298-500
Cytoseal 60 Mounting MediumRichard-Allan Scientific8310-4
Cytospin3ShandonNA
DakoCytomation penDakoS2002
Dulbecco Modified Eagle Medium (DMEM) 1xCorning cellgro15-013-CM
EthanolSigma AldrichE 7023-500ml
Fetal Bovine Serum Heat InactivatedSigma AldrichF4135-500ml
FITC Conjugated IgG2b K Rat Isotype ControleBioscience14-4031-82
Fluorescein Isotiocyanate (FITC) Conjugated Anti-mouse KIT (CD117; clone 2B8)eBioscience11-1171-82
FormaldehydeFisher ScientificF79-500
Giemsa Stain ModifiedSigma AldrichGS-1L
IsothesiaHenry Schein Animal Health29405
May-Grunwald StainSigma AldrichMG-1L
Multiwell 6 well platesFalcon35 3046
Olympus BX60 MicroscopeOlympusNA
Paraplast Plus Tissue Embedding MediumFisher Brand23-021-400
PE Conjugated IgG Armenian Hamster Isotype ControleBioscience12-4888-81
Phosphate-Buffered-Saline (PBS) 1xCorning cellgro21-040-CV
Phycoerythrin (PE) Conjugated Anti-mouse FceRIa (clone MAR-1)eBioscience12-5898-82
Propidium Iodide Staining SolutioneBioscience00-6990-50
Recombinant Mouse IL-3Peprotech213-13
Safranin-o CertifiedSigma AldrichS8884
Tissue culture flasks T25 25 cm2Beckton Dickinson353109
Tissue culture flasks T75 75 cm2Beckton Dickinson353110
Toluidine Blue 1% AqueousLabChem-IncLC26165-2
Recombinant Mouse SCFPeprotech250-03

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