Assays of Eosinophil Apoptosis and Phagocytic Uptake

Eosinophil apoptosis (programmed cell death) plays an important role in several inflammatory and allergic conditions. Apoptosis triggers various mechanisms including activation of cysteine-aspartic proteases (caspases) and is characterized by morphological and biochemical changes. These include cellular condensation, nuclear fragmentation, increased mitochondrial permeability with loss of membrane potential, and exposure BookID 483737_2_En__ChapID 10_Proof# 1 2/9/20


Introduction
Increased eosinophil accumulation at sites of inflammation is evident AU2 in a number of allergic diseases including asthma, eczema, and rhinitis [1].Recruited as part of a process including Th2 lymphocyte infiltration and immunoglobulin E (IgE)-mediated mast cell activation, eosinophils are central to disease pathogenesis [2].The subsequent release of reactive oxygen species, proteases, and inflammatory mediators including cytokines can result in increased inflammation, tissue damage, and organ dysfunction.In order to attenuate this inflammatory process eosinophil apoptosis and subsequent non-phlogistic clearance of apoptotic cells are important for ensuring efficient resolution of the inflammatory process.Dysregulation of apoptosis or efferocytosis mechanisms result in sustained inflammation and may contribute to tissue injury and chronic inflammation correlated with various inflammatory diseases [3][4][5][6].
The circulating life span of eosinophils is short with an intravascular presence of around 18-25 h prior to migration into tissues, with the thymus and gastrointestinal tract the usual destination of eosinophils in health [7].Here their life span is thought to be several days.Eosinophils isolated from peripheral blood undergo constitutive apoptosis, although at a much slower rate than the closely related neutrophil granulocyte.The eosinophil half-life in vitro is approximately 48 h following isolation [8][9][10].The process of eosinophil apoptosis is dependent upon the activation of cysteine-aspartic proteases (caspases) which are contained as inactive zymogens within the cell with eosinophils containing caspases 3, 6, 7, 8, and 9. Caspase cleavage occurs due to activation of either the extrinsic or the intrinsic pathways of apoptosis [3].The extrinsic pathway relies on the ligation of "death receptors" such as the tumor necrosis factor receptor (TNF-R), the Fas receptor (FasR), and the TNF-related apoptosis-inducing ligand receptor (TRAIL-R) [11].Cross-linking of these receptors causes clustering and, through associations with internal adaptor proteins, allows the formation of pro-caspase complexes, caspase-8 cleavage, and subsequent apoptosis.The intrinsic pathway is activated when the cell faces withdrawal of survival factors, genotoxic stress, and exposure to ultraviolet radiation or chemotherapeutic agents allowing pro-apoptotic members of the B-cell lymphoma 2 (Bcl-2) family to dissociate from their anti-apoptotic regulators and translocate to the mitochondria.Resultant increased mitochondrial membrane permeability and pore formation lead to pro-apoptotic factors such as cytochrome c to be released into the cytosol with cleavage of pro-caspase-9 to active caspase-9, thereby committing the cell to caspase-3-mediated apoptosis.The process of apoptosis results in morphological and biochemical changes including cell shrinkage, nuclear condensation, and apoptotic body formation; increased mitochondrial permeability with loss of membrane potential; DNA fragmentation; and caspase activation and externalization of phosphatidylserine on the plasma membrane.
Eosinophil life span can be modulated through alterations in the balance of pro-survival and pro-apoptotic signals and proteins.
There is evidence suggesting that Bc1-xL (an anti-apoptotic Bcl-2 family member) plays a significant role in eosinophil survival, independent of Mcl-1, a protein which is crucial in the regulation of neutrophil life span.This may, in part, explain why eosinophils undergo constitutive apoptosis at a slower rate compared to neutrophils [12].Apoptosis can be delayed by a variety of factors including cytokines (interleukin-5 (IL-5), GM-CSF, eotaxin, and Naomi N. Gachanja et al. interferon-γ), hypoxia, and bacterial exotoxins [13] and DNA which also serve to induce eosinophil migration and activation into areas of inflammation.
Conversely apoptosis can be accelerated by IL-4, FAS ligand, ligation of CD69, CD45 and CD30 cell surface receptors, and intracellular oxidant production with pharmacological agents including corticosteroids and theophyllines also driving apoptosis [14].Glucocorticoids (a class of corticosteroids) increase eosinophil apoptosis via the intrinsic pathway, with the mechanism involving changes in the phosphorylation state of Bcl-2 family members as well as inhibition of cytokine-dependent survival [5,15].Additionally, the cyclin-dependent kinase inhibitor (CDKi) R-roscovitine induces eosinophil apoptosis by mitochondrial membrane potential loss and downregulation of the key survival protein Mcl-1 [8,16].The CDKi-driven apoptosis of eosinophils in vitro is a time-, concentration-, and caspase-dependent effect [8].Similarly, flavones (polyphenolic plant-derived compounds) such as wogonin also induce eosinophil apoptosis in a time-, concentration-, and caspase-dependent manner due to augmented loss of eosinophil mitochondrial membrane potential [5].
Numerous changes in cell surface marker expression and secretion of soluble factors that occurs during apoptosis facilitate the uptake of these dying cells by surrounding phagocytes including macrophages, dendritic cells, and nonprofessional phagocytes such as epithelial cells [17,18].Important alterations to the cell membrane include phosphatidylserine exposure, changes in ICAM-1 epitopes, modification in glycosylation patterns, and charge and expression of calreticulin.Phagocytosis is a key event in macrophage phenotypic switching from a pro-inflammatory to a pro-resolving phenotype with release of anti-inflammatory cytokines and lipids (including IL-10, transforming growth factor-β (TGF-β), and resolvins) [19].Failure in clearance of apoptotic cells results in eventual disintegration of the cell membrane (termed secondary necrosis) with release of toxic intracellular contents, tissue damage, and perpetuation of the inflammatory response.
Eosinophils form approximately 1-3% of the granulocyte population in the peripheral blood of nonatopic humans; therefore enrichment following conventional granulocyte isolation methods is essential [20,21].Negative selection (anti-CD16) is most widely used although some care should be taken as eosinophils also have low levels of CD16 expression.Eosinophil life span can be influenced by several factors, therefore ensuring that eosinophil activation is prevented during isolation and culture is vital for in vitro study.Factors which may alter constitutive eosinophil life span and affect interpretation of results include the method of isolation, serum presence, temperature, pH level, oxygen tension, and cell density [3].
As described, the molecular and morphological changes that occur during the process of apoptosis allow in vitro study through a variety of different assays and approaches ranging from assessment of externalization of phosphatidylserine by flow cytometry and nuclear condensation by light microscopy to assessment of DNA fragmentation by hypodiploid peak analysis and mitochondrial membrane permeability with chromogenic dyes.This chapter therefore updates our earlier chapter on the methodologies used to examine components of the apoptotic process and subsequent phagocytic clearance of eosinophils.This protocol also requires PBS (wash buffer), 3% H 2 O 2 in methanol (blocking solution), 4% paraformaldehyde in PBS at pH 7.4 (fixation buffer; freshly prepared), and 0.1% Triton-X100 in 0.1% sodium citrate (permeabilization buffer; freshly prepared).Naomi N. Gachanja et al.

Assessing Morphological Changes of Apoptosis Using Light Microscopy
Apoptotic eosinophils are distinguishable from viable cells, under light microscopy, by their characteristic morphological appearances of nuclear condensation and cell shrinkage (Fig. 1a, c).
1. Suspend eosinophils (of at least 97% purity-determined by cytocentrifuge preparation as described below) at 4 Â 10 6 cells/mL in IMDM supplemented with 10% autologous serum and penicillin/streptomycin (1Â) (see Note 1).11. Air-dry slides before mounting with a drop of DPX and coverslip.View slides using a light microscope with a 40Â or 100Â (oil) objective and count >300 cells per slide (see Note 3).

Analysis of Eosinophil Morphology by Electron Microscopy
Although conventional light microscopy provides information regarding the general morphological changes that occur during apoptosis it is electron microscopy that allows detailed structural analysis of these processes.

Western Blotting for Caspases and Apoptotic Proteins
Caspases are essential throughout the apoptotic process in both the initiation and execution of the cell death process.The detection of the cleaved active forms of these proteins, or the disappearance of their inactive forms, alongside changes in the expression of other pro-and anti-apoptotic proteins in response to extrinsic and intrinsic modulators of eosinophil life span is possible through a variety of assays described below (Fig. 2b).12. Wash the membrane in TBS/0.1% Tween ® 20 for 5 min on a rocking platform.
13. Block the membrane for 1 h with 10 mL of 5% dried milk powder in TBS/0.1% Tween ® 20 at room temperature on a rocking platform.
15. Incubate with primary antibody overnight at 4 C-concentrations as per 2.4.1.in TBS/0.1% Tween ® 20 containing 5% dried milk powder (5 mL).16.Wash membrane in TBS/0.1% Tween ® 20 each for 5 min (repeat three times).17.Incubate with the corresponding secondary antibody diluted 1:2500 in TBS/0.1% Tween ® 20 containing 5% dried milk powder (5 mL) for 2 h.As discussed previously apoptosis is a caspase-dependent process; therefore assessment of caspase activity can be used as a marker of apoptotic cell death.Quantification of total caspase activity is possible using commercially available assays (homogeneous caspase assay) but their use is limited given the inability to discriminate between individual caspases.Cleavage of a fluorescently conjugated caspase substrate (e.g., VAD-fmk) to produce a fluorescent product (e.g., FITC, rhodamine 110) enables fluorescent intensity to be measured as a marker of total caspase activity.
1.These instructions are based on the use of Homogeneous Caspases Assay Kit.

Caspase Profiling Assay
Fluorometric assays for specific caspases are similar to the homogeneous assays described above but have a greater degree of specificity between the substrates of individual caspases or groups of caspases.
Fluorescently conjugated substrates specific for certain caspases are immobilized in a 96-well plate.When cell lysates are added to the wells, the level of fluorescence emitted is an indicator of the activity of that particular caspase allowing delineation of specific pathways of the apoptotic process.
1.These instructions assume the use of the ApoAlert™ Caspase Profiling Plate (see Note 5).To assess monocyte-derived macrophage phagocytosis of apoptotic eosinophils it is necessary to differentiate blood-derived human monocytes into macrophages in in vitro culture.A variety of methods exist in order to, as closely as possible, recapitulate the phenotype of tissue macrophages.Isolation by adherence utilizes monocyte ability to rapidly attach to tissue culture plastic in preference to neutrophils and lymphocytes; washing off non-adherent cells after 1 h leaves a relatively homogenous cell population for subsequent culture.Alternatively, use of a pan-monocyte isolation kit by negative selection with anti-CD14-coated magnetic beads yields a highly pure monocyte population (see Note 6).Immortalized macrophage cell lines or primary macrophages isolated from animal tissue can also be used.3. Culture monocytes for 5-7 days with media changed after day 3 in culture prior to use in subsequent experiments.

Flow Cytometry-Based Phagocytosis Assay
This assay uses a fluorescent chloromethyl dye that diffuses across cell membranes to label the cytoplasm of live eosinophils without altering their functional activity (Fig. 2c-e).These pH-sensitive dyes such as pHrodo™ or CypHer5E are minimally fluorescent at a neutral pH but fluoresce brightly in acidic conditions (i.e., within the phagolysosome).This allows distinguishing of internalized apoptotic cells that are acidified within the phagolysosome versus apoptotic cells that are adherent to the phagocyte cell surface.
Alone, this is a valid method for assessment of phagocytic uptake of apoptotic cells; however CellTracker™-green can also be used alongside in order to stain macrophages and identify the dual-Naomi N. Gachanja et al. positive population indicating ingested apoptotic cells.Efferocytosis probes can also be used to measure phagocytosed apoptotic cells by fluorescence microscopy and flow cytometry, for example a anxA5-pHrodo probe; these techniques may also have the added benefit of use in vivo [22].
1.This method assumes the use of adherent monocyte-derived macrophages in Costar ® 24-well TC-treated microplates.12. Remove the eosinophil suspension from the plate and wash macrophages with PBS three times.

2 .
In a 96-well flat-bottomed plate add 75 μL of eosinophil suspension.Add 60 μL IMDM with 10% autologous serum to each well and 15 μL of apoptosis-modifying agents (10Â concentration) or vehicle control.(NB: If two agents are used only 45 μL of IMDM is required for a total volume of 150 μL.) 3. Cover the plate with a lid and incubate at 37 C in a 5% CO 2 incubator for the required amount of time.4. Gently pipette the cell suspension in the well to resuspend adherent cells and load 200 μL (150 μL cells and 50 μL IMDM) into a cytocentrifuge chamber. 5. Cytocentrifuge at 300 rpm for 3 min.6. Air-dry for 5 min.7. Fix in methanol for 2 min.8. Stain in Diff Quik™ solution 1 or equivalent acid dye for 2 min.9. Stain in Diff Quik™ solution 2 or equivalent basic dye for 2 min.10.Rinse with distilled water.

1 .
Suspend eosinophils (at least 97% purity) at 4 Â 10 6 cells/mL in IMDM supplemented with 10% autologous serum and penicillin/streptomycin (1Â) (see Note 1). 2. In a 96-well flat-bottomed plate add 75 μL of cell suspension, 15 μL of apoptosis-modifying agents (10Â concentration) or vehicle control, and 60 μL IMDM with 10% autologous serum to each well.(NB: If two agents are used only 45 μL of IMDM is required for a total volume of 150 μL.) Eosinophil Aapoptosis 3. Cover and incubate at 37 C in a 5% CO 2 incubator for the duration of the experiment.4. Gently pipette the cell suspension in the well to resuspend adherent cells, combine the contents of 5 replicate wells together into a 500 μL Eppendorf tube, and centrifuge for 5 min at 300 Â g.

Fig. 1
Fig. 1 Determining eosinophil apoptosis by light microscopy and flow cytometry.Viable eosinophils isolated from peripheral venous human blood have eosinophilic cytoplasmic staining, abundant granules, and bilobed nuclei (white arrow) (a).Flow cytometric analysis of cell death immediately following isolation demonstrates that the majority of cells are viable (AnnV Àve /PI Àve ) (b).Apoptotic eosinophils in vitro display characteristic morphological changes of cell shrinkage, membrane blebbing, nuclear condensation, darkening of cytoplasmic staining, and nuclear condensation (black arrows) (c).Similarly increased apoptotic (AnnV +ve /PI Àve ) and necrotic (AnnV +ve /PI +ve ) staining is seen with flow cytometry (d).All images 1000Â magnification

318 12 .
Select appropriate areas for further study using a light 319 microscope.320 13.From those areas cut ultrathin (60 nm) sections and stain with 321 uranyl acetate and lead citrate.

322 14 .
View section with a Philips CM120 transmission electron Externalization of phosphatidylserine to the outer surface of the cell membrane is a key component in the apoptotic process, allowing the recognition of apoptotic cells by surrounding phagocytes.Annexin V (AnnV), in the presence of Ca 2+ , binds phosphatidylserine and when fluorescently conjugated (commonly AnnV-FITC) it can be used to identify apoptotic cells.Discrimination between viable, apoptotic, and necrotic cells is possible by using AnnV together with a nucleophilic dye such as propidium iodide (PI) in a simple flow cytometry assay.PI is excluded from cells with an intact cell membrane; however when membrane integrity is lost PI enters the cell and binds nuclear material with a consequent increase in fluorescence.Numerous other viability dyes exist that can be used in place of PI including (but not limited to) DAPI, 7AAD, and SYTOX™ dyes.

2 .
In a 96-well flat-bottomed plate add 75 μL of cell suspension, 15 μL of apoptosis-modifying agents (10Â concentration) or vehicle control, and 60 μL IMDM with 10% autologous serum to each well.(NB: If two agents are used only 45 μL of IMDM is required for a total volume of 150 μL.) Eosinophil Aapoptosis 3. Cover and incubate at 37 C in a 5% CO 2 incubator for the duration of the experiment.4. Gently pipette the cell suspension in the well to resuspend adherent cells and pipette 50 μL of the cell suspension into a flow tube with 250 μL AnnV buffer (see Note 4). 5. Incubate on ice for 5 min.

6 .
Add PI (1 μL of 1 mg/mL solution) to each sample immediately prior to running the sample on a flow cytometer.7. Analyze on a flow cytometer using FL-1/FL-2 channel analysis.Viable cells are dual AnnV/PI negative; apoptotic cells are AnnV positive, and PI negative; necrotic cells are dual AnnV/ PI positive (Fig. 1 b, d).
pores within the mitochondrial membrane that occurs during the intrinsic apoptotic process results in loss of mitochondrial membrane potential (ΔΨM) and facilitates the movement of proteins into the cytoplasm, in particular cytochrome c, with resultant caspase activation.Changes in the mitochondrial membrane potential of eosinophils can be measured using Mito-Capture™, a cationic dye which in viable cells accumulates and polymerizes within mitochondria and fluoresces in the red (FL-2) channel-indicated by a fluorescence emission shift from green (535 nm) to red (590 nm).During apoptosis, when ΔΨM is compromised, the dye remains monomeric within the cytoplasm and fluoresces in the green (FL-1) channel.This mitochondrial depolarization can be quantified by flow cytometry as an increase in FL-1 fluorescence (Fig. 2a), or by plate-based fluorometric assay as a decrease in the red/green fluorescence intensity ratio.1.For each sample dilute 0.5 μL MitoCapture™ reagent in 500 μL pre-warmed (37 C) MitoCapture™ incubation buffer in a 1.5 mL Eppendorf tube.(NB: This protocol relies on the use of a MitoCapture™ mitochondria permeability detection kit.) 2. Suspend eosinophils (at least 97% purity) at 4 Â 10 6 cells/mL in IMDM (10% autologous serum).

3 .
In a 96-well flat-bottomed plate add 75 μL of cell suspension, 15 μL of apoptosis-modifying agents (10Â concentration) or vehicle control, and 60 μL IMDM with 10% autologous serum to each well.(NB: If two agents are used only 45 μL of IMDM is required for a total volume of 150 μL.) 4. Cover and incubate at 37 C in a 5% CO 2 incubator for the duration of the experiment.5. Add 150 μL of cell suspension to 500 μL diluted MitoCap-ture™ reagent.6. Incubate on shaking heat block at 37 C, 300 rpm, for 15 min.

7 .
Centrifuge at 300 Â g for 5 min and discard supernatant.8. Resuspend cells in 300 μL MitoCapture™ incubation buffer.9. Analyze using a flow cytometer with increased fluorescence in FL-1 channel indicating loss of ΔΨM and increased apoptosis.

Fig. 2
Fig. 2 Assessing intracellular events during apoptosis and phagocytic clearance of apoptotic cells.Loss of mitochondrial membrane potential due to increased membrane permeability during apoptosis is measured by increased fluorescence of Mitotracker™ dye.Representative histogram (a) of control (blue)-and dexamethasone (red)-treated eosinophils after 20-h in vitro culture-apoptotic cells indicated by gate.Changes in the expression of intracellular regulators of apoptosis can be assessed by western blot (b).Caspase inhibitor zVAD delays apoptosis, maintains Mcl-1 expression, and prevents caspase-3 cleavage while pro-apoptotic cyclindependent kinase inhibitors (CDKi) cause Mcl-1 downregulation and caspase-3 cleavage.Measurement of phagocytosis of apoptotic cells by monocyte-derived macrophages (gated) is demonstrated by increased CellTracker™-green fluorescence from stained apoptotic cells in dexamethasone-treated cells (d) relative to control (c) (black arrows).Overlay histogram demonstrates population of macrophages containing apoptotic cells (e, gated)

8 .
Transfer volume equivalent to 30 μg protein into fresh Eppendorf tubes and make up to total volume of 30 μL with PBS (without cations) and 8 μL of 4Â sample buffer.9. Heat at 95 C for 5 min.10.Load samples onto a 12% polyacrylamide (or equivalent) gel including molecular weight standards.Run at 110 V until the dye front reaches the bottom of the gel.11.Transfer proteins onto the PVDF membrane at 80 V for 1 h at 4 C.

449 19 .
Develop using enhanced chemiluminescence according to the 450 manufacturer's instructions.

451 20 .
Strip and re-probe blot with β-actin or GAPDH as a loading

3 .
In a black 96-well microplate load 100 μL of cell suspension (1 Â 10 5 cells per well), with apoptosis-modifying agents and incubate at 37 C for the duration of the experiment.4. Dilute stock caspase substrate 1:10 in incubation buffer and add 100 μL freshly prepared 1Â caspase substrate to each well.Include negative and positive controls (media alone and cell lysate).

2 . 4 .
Suspend eosinophils (at least 97% purity) at 2 Â 10 6 cells/mL in IMDM (10% autologous serum).In 2 mL Eppendorf tubes pipette 1 mL of cell suspension (2 Â 10 6 cells) and apoptosismodifying agents and incubate at 37 C for the desired length of time.Eosinophil Aapoptosis 3. Centrifuge at 220 Â g for 5 min at 4 C and aspirate the supernatant.Resuspend the cell pellet in 400 μL ice-cold 1Â lysis buffer; incubate on ice for 10 min.5.While cells are incubating add 10 μL DTT per 1 mL 2Â reaction buffer, and then pipette 50 μL to each well of the 96-well caspase profiling plate.6. Cover the plate and incubate for 5 min at 37 C. 7. Vortex the cell lysate and add 50 μL of lysate to duplicate wells of each caspase substrate.8. Cover the plate and incubate for 2 h at 37 C. 9. Use a plate reader to measure fluorescence (excitation: 380 nm, emission 460 nm).

3. 8
Assessing Nuclear Changes During Apoptosis: Gel Electrophoresis of DNA Activation of the apoptotic process results in endonucleasemediated cleavage of DNA.After early large-scale degradation of DNA (50-200 kbp) endonuclease activity generates singlenucleosome or oligonucleosomal fragments of around 180 bp (or multiples thereof).This cleavage process creates discrete sized lengths of DNA which, when run through gel electrophoresis, produce a characteristic "laddering" effect that is distinct from the "smear" generated by the random DNA cleavage that occurs during cell necrosis.1. Suspend eosinophils (at least 97% purity) at 5 Â 10 6 cells/mL in IMDM (10% autologous serum).In 2 mL Eppendorf tubes pipette 1 mL of cell suspension (2 Â 10 6 cells) and apoptosismodifying agents and incubate at 37 C for the desired length of time.2. Extract genomic DNA using Wizard ® Genomic DNA Purification Kit. 3. Run the DNA (23 μL DNA mixed with 7 μL loading dye) on a 2% agarose gel containing GelRed (5 μL in 50 mL) in 1Â TBE buffer at 110 V. 4. Run until the dye front reaches the end of the gel and visualize the gel under ultraviolet illumination.

3. 9
Hypodiploid DNA Content Endonuclease-mediated cleavage of nuclear DNA during apoptosis causes an apparent decrease in DNA content of tritonpermeabilized cells.Nuclear staining using propidium iodide allows detection of this "hypodiploid" cell population.This technique works well with eosinophils, as they are terminally differentiated and do not undergo proliferation meaning only two peaks are visible when DNA content is measured: diploid (viable) cells and hypodiploid (apoptotic) cells.11.Add 50 μL of nucleotide mixture to two negative control wells.12. Make the TUNEL reaction mixture by adding the enzyme solution (50 μL) to 450 μL nucleotide mixture.13.Treat the two positive control wells with DNase I for 10 min at room temperature to introduce DNA strand breaks.14.Wash the plate twice in PBS (200 μL per well) and then resuspend in TUNEL reaction mixture (50 μL per well).15.Cover the plate and incubate at 37 C for 60 min.16.Wash twice in PBS (200 μL per well) and then transfer to flow cytometry tubes for analysis of fluorescence levels (FL-1).

14 .
Collect the detached macrophages by pipetting vigorously and place in a flow cytometer tube on ice.Eosinophil Aapoptosis