An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation

Summary

We have established an ex vivo mast cell degranulation assay carried out by incubating crude peritoneal exudate cells isolated from the mice, treated with a pharmacological agent of interest and administered anti-dinitrophenol (DNP) IgE beforehand, with DNP on a carrier protein.

Abstract

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.

Introduction

Mast cells play a central role in allergy^1,2. When IgE located on the surface of mast cells via interaction with the high-affinity receptor for IgE (FcεRI) encounters a cognate allergen, a signaling cascade is elicited to prompt the release of the granules. As a result, a variety of allergy effector molecules, including monoamines (e.g., histamine, serotonin), cytokines (e.g., TNF-α), and proteolytic enzymes (e.g., tryptase, chymase), are released to cause a series of immunological, neurological and vasomuscular reactions^3,4.

A class of pharmaceuticals is called mast cell stabilizer that alleviates the allergy symptoms by attenuating the mast cell function⁵. Passive systemic anaphylaxis (PSA) is an animal model often used for probing a mast cell stabilizing activity of pharmacological agents. As the anaphylactic symptoms result primarily from the activation of mast cells following interaction of passively transferred hapten-specific IgE with the hapten on a carrier protein injected into the animal later, it is well received that a pharmacological agent of interest bears a mast cell stabilizing activity when its treatment results in amelioration of the symptoms⁶. Still, it is often imperative to directly demonstrate impairment of the mast cell function by the agent in a separate experiment to rule out the possibility that improvement of the symptoms is derived from a mechanism other than suppression of mast cell function.

Mast cell degranulation assay, which is carried out by stimulating mast cells with a chemical reagent or a specific antigen of IgE forming a complex with FcεRI on the surface of mast cells to induce exocytosis of secretory granules (i.e., degranulation), is generally used for determining a mast cell stabilizing activity of a pharmacological reagent in vitro⁷. Several types of cells are used in that assay, including the rat basophilic leukemia (RBL) cell line⁸, bone marrow-derived mast cells (BMMC)⁹, and peritoneal cell-derived mast cells (PCMC)¹⁰. While useful as a large number of cells can be easily obtained, RBL is an immortalized cancer cell line whose cellular properties are no longer akin to those of mast cells in the body. Acquiring a sufficient number of BMMC or PCMC, even though their cellular properties may more closely resemble those of mast cells in the body, is often costly and time-consuming.

A degranulation assay using purified primary mast cells is a desirable alternative¹¹. Nonetheless, the use of such an assay is not widespread as a facile method for purifying mast cells from animal tissue, particularly from mouse tissue, with a high yield, and purity is not yet available. Moreover, since the concentration and duration of treatment with a pharmacological agent to inhibit the mast cell function in vitro may not always coincide with those in vivo, results obtained with an in vitro degranulation assay may misrepresent those from an in vivo assay such as PSA, and vice versa. Hence, a novel degranulation assay, not only closely mimicking the way of mast cell activation transpiring in vivo but also accurately reflecting effects of a pharmacological reagent exerted on mast cells in vivo, is in high demand. In order to meet those needs, we devised an ex vivo mast cell degranulation assay where mast cells in peritoneal exudate cells (PECs) isolated from the mice, treated with a pharmacological agent of interest and administered IgE specific for dinitrophenol (DNP) beforehand, are stimulated with DNP-conjugated bovine serum albumin (BSA).

Protocol

All animal experiments were performed in accordance with the guideline provided by the IACUC (Institutional Animal Care and Use Committee) of Chungnam National University (Animal Protocol Number: CNU-00996).

1. Quantifying mast cell-specific molecules in the lysate of crude PECs

1. Isolate the cells from the mouse peritoneal cavity¹².
   1. Anesthetize a mouse (8 weeks old, male, BALB/C) with isoflurane. Euthanize via cervical dislocation.
   2. Place the mouse on a foam block. Wipe the abdomen with 70% ethanol.
   3. Cut the ventral skin longitudinally with blunt edge scissors. Peel off the skin of the mouse using forceps and scissors.
   4. Inject 6 mL of ice-cold Tyrode’s B buffer¹³ into the peritoneal cavity using a 10 mL syringe with a 26 G needle. Insert the needle gently to avoid pricking any organs.
   5. Massage the abdomen of the mouse for 60-90 s to collect peritoneal cells into Tyrode’s B buffer. Do it gently not to damage the blood vessels.
   6. Insert the needle (20 G) attached to a 10 mL syringe bevel up. Aspirate the fluid slowly from the peritoneal cavity (typically 5-6 mL).
   7. Remove the needle from the syringe. Dispense the peritoneal fluid into a 50 mL conical tube. Keep the tube on ice.
   8. Repeat steps 1.1.4 - 1.1.7.
   9. Centrifuge the tube at 300 x g for 5 min at 10 °C. Resuspend the cells in 1 mL of 1x red blood cell (RBC) lysis buffer. Keep the cells on ice for 3 min.
   10. Dilute the cell suspension with 2 mL of Tyrode’s B buffer. Centrifuge the tube at 300 x g for 5 min at 10 °C.
   11. Remove the supernatant. Resuspend the cells in 0.5 mL of Tyrode’s A buffer¹³.
   12. Count the cells with a hemocytometer. Adjust the cell number to 5 x 10⁶/mL with Tyrode’s A buffer.
2. Prepare mast cell-depleted PECs using a magnetic cell purification system¹⁴.
   1. Centrifuge 5 x 10⁵ crude PECs at 300 x g for 5 min at 10 °C. Resuspend the pellet in 200 µL of PBSBE cell purification buffer (0.5% BSA, 2 mM EDTA, 1 x PBS, pH 7.4).
   2. Add 1 µL of Fc blocker (0.5 mg/mL) to the cells to prevent non-specific binding of anti-c-kit monoclonal antibody (mAb) to be added next. Keep the cells on ice for 5 min.
   3. Add 1 µL of biotinylated anti-mouse c-kit mAb¹⁵ (0.5 mg/mL). Keep the cells on ice for 10 min.
   4. Add 2 mL of PBSBE buffer. Centrifuge the cells at 300 x g for 10 min at 10 °C.
   5. Remove the supernatant. Wash the cells again with 2 mL of PBSBE buffer.
   6. Resuspend the cells in 90 µL of PBSBE buffer. Add 10 µL of streptavidin-conjugated microbeads. Keep the cells on ice for 15 min.
   7. Add 2 mL of PBSBE buffer. Centrifuge the cells at 300 x g for 10 min at 10 °C.
   8. Load 500 µL of PBSBE buffer on a medium magnetic column during the spin. Allow the buffer to flow through the column.
   9. Remove the supernatant after centrifugation. Resuspend the cells in 500 µL of PBSBE buffer.
   10. Load the cells on the column. Collect the cells passing freely through the column.
   11. Wash the column with 500 µL of PBSBE buffer. Collect the cells passing through the column again.
   12. Repeat step 1.2.11.
   13. Combine the cells from steps 1.2.9-1.2.11 in one tube. Centrifuge the cells at 300 x g for 10 min at 10 °C.
   14. Resuspend the cells in Tyrode’s A buffer. Count the cells. Adjust the cell number to 5 x 10⁶ cells/mL.
3. Prepare the lysate of PECs.
   1. Plate PECs (e.g., 5 x 10⁵) in a round-bottom 96 well plate, respectively. Centrifuge the plate at 300 x g for 2 min at 10 °C to collect the cells. Remove the supernatant carefully with pipette.
   2. Add 100 µL of cell lysis buffer to the cell pellet. Resuspend the cells by pipetting up and down several times gently. Keep the plate on ice for 60 min.
   3. Centrifuge the plate at 300 x g for 5 min at 10 °C to remove cell debris. Transfer the supernatant (the cell lysate) to a new 96 well plate.
4. Measure the enzymatic activity of β-hexosaminidase¹⁶.
   1. Add the cell lysate (50 µL) to prewarmed β-hexosaminidase substrate solution (50 µL) in a 96 well microplate. Mix them gently with a pipette.
   2. Incubate the plate in a 37 °C incubator. Add 50 µL of stop solution (100 mM glycine, pH 10.7) after 30 min to terminate the enzyme reaction.
   3. Read O.D. of the reaction mixtures with a UV-visible absorbance microplate reader in dual wavelength setting; 405 nm for determining the level of the enzyme reaction and 620 nm for automatic background subtraction, respectively.
5. Measure the concentration of histamine¹⁷.
   1. Transfer 100 μL of the cleared cell lysates to the anti-histamine mAb-coated plate supplied with the histamine ELISA kit. Perform competitive histamine ELISA assay following the manufacturer’s manual.
   2. Read O.D. of the samples with a UV-visible absorbance microplate reader at 450 nm wavelength.
6. Determine the ratio of mast cells in PECs with flow cytometry¹⁸.
   1. Transfer 1 x 10⁵ crude or mast cell-depleted PECs in a round bottom 96 well plate. Centrifuge the plate at 300 x g for 2 min at 10 °C.
   2. Resuspend the cells in 50 µL of FACS buffer. Add 1 µL of anti-mouse c-kit (0.5 mg/mL) and anti-mouse IgE mAbs (0.5 mg/mL).
   3. Vortex the cells briefly. Keep the cells on ice for 20 min in the dark.
   4. Fill the wells with 150 µL of FACS buffer. Centrifuge the plate at 300 x g for 2 min at 10 °C. Discard the buffer by quickly flipping the plate.
   5. Resuspend the cells with 150 µL of FACS buffer containing propidium iodide (1 µg/mL). Analyze the cells with a flow cytometer.

2. Mast cell degranulation assay using crude PECs

1. Determine the extent of mast cell degranulation (% degranulation).
   1. Inject 3 BALB/C mice (8 weeks old, male) intravenously (i.v.) with 3 μg of anti-DNP mAb (mouse IgE). Isolate PECs one day after Ab injection (refer to step 1.1).
   2. Plate 90 μL of crude PECs (5.5 x 10⁶/mL) in a flat-bottom 96 well plate (total 4 wells). Incubate the plate in a 37 ^oC humidified CO₂ incubator for 30 minutes.
   3. Add 10 μL of DNP-BSA (5 ng/mL in 1x PBS) to the wells containing PECs. Incubate the plate for 10 min in a 37 °C CO₂ incubator.
   4. Centrifuge the plate at 300 x g for 5 min at 10 °C immediately after incubation. Take the supernatants (100 μL) carefully with pipette. Save them in a new round-bottom 96 well plate on ice.
   5. Add 100 µL of cell lysis buffer (0.1% Triton X-100 in 1 x PBS, pH 7.4) to the microplate wells containing PECs. Keep the plate on ice for 60 min.
   6. Carry out the β-hexosaminidase assay.
      1. Take two sets of the supernatants and the corresponding cell lysates, respectively, out of four that have been stored in ice after the degranulation assay (refer to steps 2.1.4 and 2.1.5). Split the supernatant and the cell lysate into two separate wells (50 μL each) for duplication of the assay.
      2. Add 50 μL of β-hexosaminidase substrate solution to each well. Incubate the plate at 37 °C for 30 min. Add 50 μL of stop solution (100 mM glycine, pH 10.7) to the reaction mixture.
      3. Read O.D. of the reaction mixtures (refer to step 1.4.3). Calculate the extent of mast cell degranulation as follows.
         % Degranulation = OD_supernatant /(OD_supernatant + OD_lysate) X 100%
   7. Carry out the histamine assay.
      1. Add 100 µL of 1x PBS to another two wells of the supernatants and the corresponding cell lysates saved after the degranulation to bring the total volume of the samples to 200 μL. Divide each sample into two separate wells in a histamine ELISA plate provided with the histamine ELISA kit.
      2. Perform the ELISA assay following the manufacturer’s manual. Read O.D. of the samples with a UV-visible absorbance microplate reader at 450 nm wavelength. Calculate the extent of degranulation as in 2.1.6.3.
         % Degranulation = [histamine]_sup /([histamine]_sup + [histamine]_lysate) X 100%
2. Evaluate the effects of antiallergy medications on mast cells exerted in vivo with the ex vivo mast cell degranulation assay.
   1. Administer orally (p.o.) 200 μL of dexamethasone¹⁹ (DEX) and ketotifen²⁰ (KET), respectively, to mice (6 weeks old, male) once a day for 3 days (6 mice/group).
   2. Inject the mice intravenously (i.v.) with 3 μg of anti-DNP IgE after the 3^rd treatment. Divide each group of mice into two separate cages (3 mice/cage); one for PSA assay and the other for ex vivo mast cell degranulation assay.
   3. Carry out PSA assay
      1. Inject the mice with DNP-BSA (80 μg) one day after injection of anti-DNP IgE. Measure the body temperature with a rectal thermometer every 15 min for 1 h starting immediately after injection of DNP-BSA.
      2. Take the blood one day after injection of DNP-BSA. Measure the levels of MCPT-1 in the serum with ELISA.
   4. Carry out ex vivo mast cell degranulation assay.
      1. Isolate PECs from the mice one day after injection of anti-DNP IgE. Count the numbers of isolated PECs.
      2. Plate 90 µL of crude PECs (5.5 x 10⁶/mL) in a 96 well plate (4 wells per mouse). Incubate the plates in a 37 °C humidified CO₂ incubator for 30 min.
      3. Add 10 µL of DNP-BSA (5 ng/mL). Incubate the plate for 10 min at 37 °C humidified CO₂ incubator.
      4. Centrifuge the plate at 300 x g for 5 min at 10 °C. Take the supernatants (100 µL) carefully, leaving the cells behind in the wells.
      5. Add 100 µL of cell lysis buffer to the wells. Incubate the plate on ice for 60 min.
      6. Carry out β-hexosaminidase assay and histamine ELISA assay as described in 2.1.6 and 2.1.7. Calculate% degranulation as in step 2.1.6.3.

Results

Determining the optimal number of PECs for ex vivo mast cell degranulation assay

Mast cells (c-kit⁺·IgE⁺ double positive cells)¹⁵ represent only about 2% of PECs (Figure 1A). Estimating the maximum levels of mast cell-specific molecules to be detected in the culture supernatants on the assumption that 100% of the granules were released by mast cells in PECs, we measured the amounts of β-hexos...

Discussion

The finding that mast cell degranulation assay can be carried out with a relatively small number of crude mouse PECs is significant. Even though PECs must be an excellent source of primary mouse mast cells, it is demanding to purify mast cells in PECs. Although a density gradient media such as Percoll²⁵ has been successfully used for purification of mast cells from rat PECs, its use for purification of mouse peritoneal mast cells has been limited presumably for the difference in the densities of r...

Materials

Name	Company	Catalog Number	Comments
1 mL syringe		1757589701
1.5 mL micro tube	Hisol	MT-15003
10 mL syringe		1757593161
15 mL conical tube	Thermo Fisher scientific	14-959-53A
20xPBS	Tech & Innovation	BPB-9121-500mL
4-nitrophenyl-N-acetyl-β-D-glucosaminide	SIGMA	N9376
5 mL polystyrene round-bottom tube	Life sciences	352003
50 mL conical tube	Thermo Fisher scientific	14-959-49A
Aluminium Fiol	BioFact	TS1-3330
Anti-mouse CD117(c-kit)	Biolegend	135129	keep at 2-8°C
Anti-mouse IgE mAbs	Thermo Fisher scientific	11-5992-81	keep at 2-8°C
Antiti-DNP-IgE	SIGMA	D8406-.2MG	keep at -20°C
Centrifuge	HANIL	396150
D-(+)-gluouse	SIGMA	G8270
Dexamethasone	SIGMA	D2915-100MG
DNP-BSA	Invitrogen	2079360	keep at -20°C
EDTA	Biofact	PB131-500
Fetal Bovine serum	Thermo Fisher scientific	11455035
Gelatin	SIGMA	G1890
Glycine	JUNSEI	27185-0350
hemocytometer	ZEISS	176045
HEPES	Thermo Fisher scientific	15630130
Histamine ELISA kit	Abcam	GK3275957-4	keep at 2-8°C
Hotplate stirrer	Lab teach	zso-9001
Isoflurance	Troikaa	I29159
ketotifen fumarate salt	SIGMA	K2628
MCPT-1 ELISA kit	Thermo Fisher scientific	88-7503-22	keep at 2-8°C
Mouse Fc block	BD Biosciences	553141	keep at 2-8°C
Propidium iodiole	SIGMA	81845	keep at 2-8°C
RBC lysis buffer	Biolegend	420301
Round-bottom 96 well	SPL-life sciences	30096
Single use syringe filter	Startoriusag	16555
Streptavidin microbeads	MilteryiBiotec	130-048-101	keep at 2-8°C
Triton X-100	JUNSEIchemical	49415-1601
TWEEN 20	SIGMA	9005-64-5
Water bath	CHANGSHINSCIENCE	190107