Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Summary

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Abstract

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Introduction

Our body generates 1-10 billion apoptotic cells on a daily basis. Such a large number of apoptotic cells must be cleared in a way that the immune responses remain quiescent. To ensure the clearance of apoptotic cells in a timely manner, numerous types of tissue resident cells and circulating cells develop mechanisms to engulf apoptotic cells¹. Dysfunctional regulation of apoptosis has been implicated in the onset and progression of various inflammatory disease and autoimmunity². Apoptosis also plays a critical role in the pathogenesis of cancer development and its subsequent resistance to conventional treatments^3,4. Removal of apoptotic cells generally promotes an anti-inflammatory response, which may be linked to immunological tolerance⁵. Disturbance of apoptotic cell clearance drives self-immunization and contributes to the development of systemic autoimmune diseases in both humans and mice⁶.

When cells undergo apoptosis, they expose the phosphatidylserine (PtdSer) from the inner leaflet to the outer leaflet of the membrane. PtdSer will then be recognized by phagocytes through surface receptors. Over a dozen receptors have been identified to recognize and/or facilitate the engulfment of apoptotic cells. In general, there are at least three types of surface receptors involved in the apoptotic cell clearance: tethering receptors, recognize apoptotic cells; tickling receptors, initiate engulfment; chaperoning receptors, facilitate the whole process⁷. TAM receptor tyrosine kinases (TAM RTKs) consist of Tyro-3, Axl, and Mer and are primarily expressed by myeloid cells of the immune system⁸. The primary function of TAM RTKs is to serve as tethering receptors, facilitating the phagocytic removal of apoptotic cells and debris. Our group has studied TAM mediated apoptotic cell clearance in the setting of autoimmunity for many years. The vitamin K-dependent protein growth arrest specific protein 6 (Gas6) and protein S (ProS) binds to and activates TAM receptors^9,10. Gas6 is produced in the heart, kidneys, and lungs. ProS is mainly produced in the liver¹¹. TAM recognizes of apoptotic cells in such a way that the N-terminal of Gas6/ProS binds to the PtdSer on an apoptotic cell and the C-terminal of Gas6/ProS binds to TAM receptors that anchored on the surface of phagocytes. Together with the other receptors, engulfment of apoptotic cells occurs¹². Though Mer can bind to both the ligands ProS and Gas6, we found that Gas6 appears to be the sole ligand for Mer-mediated macrophage phagocytosis of apoptotic cells, which can be blocked by anti-Mer antibody¹³. Macrophages are professional phagocytes. Rapid clearance of apoptotic cells by macrophages is important for the inhibition of inflammation and autoimmune responses against intracellular antigens. Mer receptor tyrosine kinase is critical for the macrophage engulfment and efficient clearance of apoptotic cells¹⁴. In mouse spleen, Mer mainly expresses on the marginal zone and tangible body macrophages¹³.

The protocol presented here describes a basic method to induce cell apoptosis and demonstrate ways to measure the process and the efficiency of efferocytosis. These protocols can be readily adapted to study efferocytosis by other cell types in engulfment of apoptotic cells of different origins.

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Protocol

Experimental mice were bred and maintained in our mice colony. All animal work was conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati.

1. Preparation of CFSE labeled apoptotic thymocytes

1. Euthanize two naïve C57/B6 mice by CO₂ inhalation for 10 min and dissect to open the chest cavity, remove (pull out) the thymus with curved fine-tip forceps into tissue culture petri dish containing 10 mL of RPMI1640 medium.
2. Obtain single cell suspension by grinding the whole thymus against two frosted ends of the microscope slides and then filter the suspension through 100 μm cell strainer.
3. Collect the 10 mL thymus suspension into a 50 mL tube and centrifuge at 300 x g for 5 min.
4. Remove the supernatant, resuspend in 40 mL of 1x PBS, and count cell numbers with a hemocytometer.
5. Centrifuge at 300 x g for 5 min and remove the supernatant.
6. Resuspend in 20 mL of 1x PBS in a 50 mL tube as a single cell suspension with up to 2 x 10⁸ cells (if more than 2 x 10⁸ cells, resuspend the rest of the cells in another 20 mL of 1 x PBS in a different 50 mL tube).
7. Make 5 μM of CFSE in equal volume (20 mL) of 1x PBS in a separate 50 mL tube by pipetting 40 μL of CFSE stock solution (2.5 mM) into 20 mL of 1x PBS and mix well by inverting the tube 2 - 3 times.
8. Add the 20 mL of CFSE from step 1.7 into the 20 mL of cell suspension from step 1.6 (the final concentration of CFSE is now 2.5 μM).
   NOTE: For every 20 mL cell suspension, a 20 mL of CFSE tube is needed.
9. Invert the cell and CFSE mixture tube 2 - 3 times and incubate the mixture in the dark at room temperature for a maximum of 2 min, then stop the reaction by adding 10 mL of heat-inactivated horse serum.
10. Centrifuge the 50 mL tube mixture at 300 x g for 5 min at room temperature.
    NOTE: If the CFSE labeling is successful, cell pellet will become light yellow in color.
11. Remove the supernatant and resuspend the cell pellet in 40 mL of 1x PBS and count cell numbers with a hemocytometer.
12. Centrifuge the cell suspension at 300 x g for 5 min and discard the supernatant.
13. Wash the cell pellet again with 40 mL of RPMI1640 medium.
14. Remove the supernatant and resuspend cells with RPMI1640 tissue culture medium (RPMI1640, 20 mM HEPES, 10% FBS (heat inactivated), 20 mM glutamine, and 1x Pen/Strep) at a concentration of 7 x 10⁶ cells/mL in a 100 mm tissue culture dish.
    NOTE: If more cells are obtained, a separate 100 mm tissue culture dish will be needed.
15. Add staurosporine into the cell suspension culture at a final concentration of 1 μM and culture for 4 h at 37 °C in a tissue culture incubator supplied with 5% CO₂.

2. Preparation of peritoneal macrophages

1. Inject two C57B6 mice (or any gene-manipulated mice in the lab) intraperitoneally with 1 mL of 3% aged thioglycollate at day 0.
2. Euthanize the mice at day 5 as in step 1.1, cut and peel open the abdominal skin but leave the peritoneum intact. Flush the peritoneal cavity by quickly pushing 10 mL of wash buffer (RPMI1640, 2% FBS, 0.04% EDTA) into the peritoneal cavity using a 10 mL syringe attached with a 18 G needle.
3. Retrieve the wash buffer slowly with the same needle/syringe, and collect the wash buffer into a 50 mL tube (details refer to Janssen lab article¹⁵).
4. Wash the peritoneal gavage twice with 1x PBS, resuspend the peritoneal macrophages in RPMI1640 tissue culture medium at a density of 2 x 10⁶ cells/mL and aliquot 500 μL into each well of the 24-well plate. Leave the plate in a tissue culture incubator for 2 h.
5. Remove the floating cells by aspirating and replacing with 500 μL of fresh culture medium, twice.
6. Optionally, add TAM receptor tyrosine inhibitor, RXDX-106, at concentrations indicated in the figure legends into each well of the macrophage culture and incubate for another 2 h.

3. Co-culture of peritoneal macrophages with apoptotic thymocytes

1. Collect apoptotic thymocytes from step 1 and wash three times with RPMI1640 medium. The efficiency of apoptotic induction can be measured at this stage with the Annexin V/7-AAD kit.
2. Distribute 0 - 12 x 10⁶ cells (in 500 μL medium) into each well of the macrophage cultures from protocol #2, according to the experimental arrangement (e.g., see Figure 1). This makes the whole culture volume of 1 mL in each well of the 24-well plate. Add the blocking antibody into the culture immediately before the addition of apoptotic thymocytes.
3. Culture the cell mixture at 37 °C for 4 h in a tissue culture incubator supplied with 5% CO₂.
4. Wash each well of the culture with 1x PBS (containing 500 μM EDTA) twice to remove the free-floating apoptotic cells.
5. Stain the plate-bound macrophages with CD11b-PE at this stage.
   1. Wash the plate-bound macrophages with staining buffer (1x PBS, 1% BSA) once.
   2. Add 200 μL of staining buffer containing 2 μL CD11b-PE into each well.
   3. Incubate the plate at 4 °C for 20 min.
   4. Wash each well of plate three times with the staining buffer.
   5. Add 200 μL of staining buffer and proceed the plate for image analysis under the fluorescent microscope.
6. Alternatively, detach the plate-bound macrophages by adding 1 mL of 1% lidocaine in PBS and incubating for 10 min at 37 °C.
7. Detach plate-bound macrophages with repeat pipetting.
8. Transfer macrophage suspension into an individual 5 mL round-bottom FACS tube from each well of the 24-well plate.
9. Centrifuge at 300 x g for 5 min.
10. Remove the supernatant and add 200 μL of staining buffer containing 2 μL of CD11b-PE (1:200 dilution in staining buffer).
11. Incubate the cell suspension in staining buffer at 4 °C for 20 min.
12. Wash the cell suspension twice with staining buffer.
13. Add 200 μL of staining buffer and proceed for FACS analysis with a flow cytometer and analyze for the percentage of CFSE positivity macrophages.

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Results

Analysis of peritoneal macrophage-mediated engulfment of apoptotic thymocytes. Peritoneal macrophages and apoptotic cells were prepared and co-cultured as described in the protocol. Macrophages were detached and stained with PE conjugated anti-CD11b antibody for 20 min on ice. Macrophages were then washed and processed in a flow cytometer. As seen, there is no CFSE positive macrophage in the bottom right quadrant when no apoptotic cells were added into the culture (Figure 1<...

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Discussion

Apoptosis is a highly conserved cell death process that involves many signal cascades and induces protein expression, secretion, and transportation. Apoptosis is often associated with cellular morphology changes¹⁷. Apoptotic cells actively release cytokines and chemokines that attract phagocytes to migrate to the site and initiate the process of engulfment, an extremely complex pathway under tight control¹⁸. On the other hand, necrotic cell death releases danger signals tha...

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Materials

Name	Company	Catalog Number	Comments
Ack lysing buffer	GIBCO	A10492
Annexin V/7-AAD	BD Pharmingen	559763
Anti-Mer antibody	R&D Systems	BAF591
CD11b-PE (clone M1/70)	BD Pharmingen	553311
CFSE	Invitrogen	C1157
DMSO	Sigma-Aldrich	D-2650
EDTA (0.5 mM)	GIBCO	15575-020
FACS tubes	BD Biosciences	352017
Frosted slides	Fisher Scientific	12-552-343
Horse Serum (Heat-inactivated)	Invitrogen	26050088
Lidocaine	Sigma-Aldrich	L-5647	Prepare 1% buffer in 1x PBS
PBS, 1x	Corning	21040CV
RPMI-1640	Corning	10040CV
RXDX-106	Selleck Chemicals	CEP-40783
Staurosprine (100mg)	Fisher Scientific	BP2541-100	Add 214.3 ml of DMSO into 100mg to make 1mM stocking solution
Thioglycolate Medium Brewer Modified	BD Biosciences	243010	Prepare 3% thioglycolate buffer in 1´PBS, autoclaved, and store in the dark for 3 months.