Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Summary

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

Abstract

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

Introduction

Monocyte transmigration is a crucial step in the development of atherosclerotic plaque that may lead to thrombosis, stroke and myocardial infarction. Atherosclerotic plaques develop from fatty streaks, generally present at sites of low oscillatory blood flow in medium to large arteries, where deposited lipid contributes to endothelial activation and localized inflammation¹. Monocytes are recruited to endothelial cells in fatty streaks via monocyte chemotactic proteins (such as CCL2) and transmigrate into the intima². Following transmigration, monocytes may form atherogenic, lipid-laden macrophages called foam cells as a consequence of lipid uptake, lipid synthesis, down-regulation of cholesterol efflux or a combination of the above factors. Monocytes may also accumulate lipids in the circulation and have a 'foamy' phenotype, possibly predisposing cells for foam cell formation^3,4. Foam cells are the defining feature of fatty streaks and early-stage atherosclerotic plaques and their formation is influenced by both lipid and inflammatory mediators⁵. Alternatively, monocytes have the ability to reverse transmigrate from the artery into the bloodstream⁶, thereby removing lipid from the intima and acting to maintain the health of the artery.

Determining the propensity of monocytes to transmigrate across arterial endothelium and form foam cells in the intima, or to reverse transmigrate and carry lipid out of the plaque, is a key requirement for understanding the role of monocyte activation in increasing atherosclerotic risk. Mouse models of CADs such as atherosclerosis are important in elucidating real-time in vivo information on fatty streak/atherosclerotic plaque development. However, these models require a genetic alteration of the natural cholesterol processing abilities of these animals usually coupled with drastic alterations in diet (such as the ApoE-/- Western-type diet model)^7,8, thereby, inducing non-physiological accumulation of circulating lipid levels which drive plaque development. These models may have limited relevance to chronic inflammatory human conditions such as HIV infection which are not associated with increased circulating cholesterol or low-density lipoprotein (LDL) levels. Furthermore, differences in monocyte biology between humans and mice make the testing of immunological questions regarding the relevance of subpopulations of monocytes (such as intermediate monocytes (CD14⁺⁺CD16⁺))⁹ difficult. This is important when studying the mechanisms driving cardiovascular disease as intermediate monocyte counts independently predict cardiovascular events^10,11. While assays exist to sequentially measure either monocyte transmigration or foam cell formation in isolation, no in vitro assay has been validated for quantifying both aspects of early atherogenesis using the same cells from clinical cohorts. Transwell models utilize a modified Boyden two-chamber system whereby cells are loaded into the top chamber and transmigrate across a porous plastic barrier or cell monolayer into a lower chamber that typically contains media with chemoattractant^12,13. Whilst widely used for analyzing leukocyte transmigration, these models do not generally incorporate a layer representing the intima, resulting in transmigrated cells migrating into solution, and do not allow for the measurement of foam cell formation or reverse transmigration of the same cells. Conversely, models of foam cell formation do not account for any transmigratory-induced changes to monocytes or effects of endothelial activation which is known to contribute to foam cell formation¹⁴. Furthermore, these systems induce foam cell formation from macrophages adhered to cell culture plates by the addition of saturating concentrations of exogenous oxidized low-density lipoprotein (oxLDL)^15,16, a key inducer of foam cell formation. LDL used in these models is often oxidized by non-physiologically-relevant processes such as CuSO₄ treatment¹⁷, therefore, questioning the physiological importance of studies using these models.

Here we describe an assay that quantifies monocyte transmigration and foam cell formation of the same cells which does not require the addition of exogenous oxLDL, thus better modelling the role of monocytes in foam cell formation. This model was originally developed by Professor William Muller (Northwestern University, Chicago)¹⁸, and has been further refined in our laboratory to assess ex vivo the atherogenicity of monocytes isolated under non-activating conditions from individuals with underlying inflammatory conditions accompanying diseases such as HIV infection¹⁹ as well as ageing²⁰, that are associated with an increased risk of atherosclerosis. This model also provides a platform for answering basic biological questions regarding the propensity of different monocyte subsets to form foam cells²⁰, the influence of endothelial activation by cytokines such as TNF on foam cell formation¹⁴, and the migratory properties of monocytes such as the depth and speed of transmigration in gels¹⁹. Furthermore, monocyte transmigration and foam cell formation can be quantified using standard microscopy, live cell imaging, flow cytometry and imaging flow cytometry, therefore, providing a versatile method to evaluate the role of monocytes in atherogenesis.

Protocol

NOTE: All experiments using human biological samples were performed with ethics approval from the Alfred Hospital Human Ethics Committee, Melbourne. All experiments were performed in Class II Biosafety cabinets unless specified. "Prewarmed" refers to reagents warmed to 37 °C in a waterbath.

1. Preparation of Type I Fibrous Collagen Gels: Day 1

1. Prepare polymerized collagen gels by sequentially adding and mixing 35.7 mM NaOH, 0.71 x M199, 4.58 mM acetic acid and 1.71 mg/mL type I fibrous collagen into a 5 mL polystyrene tube as per Table 1.
   NOTE: Ensure that the collagen is well-mixed by gently pipetting up and down 5 times in order to stop the formation of collagen 'pockets' in the gel mixture. Prepare 4-6 gels per test condition for microscopy or 15 gels per test condition for flow cytometry.
2. Once well mixed, aliquot 50 µL of collagen gel mixture into each well of a sterile flat-bottomed 96-well tissue culture plate. Do not use the outside rows and columns, but fill these with 200 µL of 1x Dubecco's phosphate buffered saline (PBS) to protect the gels from desiccation. Incubate plates at 37 °C/5% CO₂ for 2 h to allow the collagen to polymerize.
   Note: Place the plates directly on clean metal racks in the incubator to ensure even heat distribution.
3. Following incubation, overlay the gels with 150 µL of 1x supplemented M199 (see Table of Materials) and incubate for 5 days until use.

2. Expansion of Stored HUVEC: Day 1

1. Label and coat a 10 cm diameter Petri dish with 1 mL of 50 µg/mL fibronectin diluted in 1x PBS and incubate at room temperature for 10 min.
2. Thaw aliquots of cryopreserved primary human umbilical vein endothelial cells (HUVEC, 1.0 x 10⁶ cells) in 10 mL M20.
   NOTE: HUVEC should be prepared as previously described²¹ and used at a low passage number (< passage 4). HUVEC may be replaced with human coronary artery endothelial cells here.
3. Resuspend HUVEC in 10 mL M20 and add to fibronectin coated dish, aspirating excess fibronectin prior to addition of cells, and culture to confluence (approximately 5 days), replacing the media on Day 3.

3. Culturing HUVEC Monolayer on Collagen Gels: Day 5

1. On Day 5, detach HUVEC by aspirating culture supernatant from the Petri dish and washing away serum-containing media with 10 mL of serum-free M199.
2. Add 5 mL 0.05% trypsin/0.53 EDTA in M199 to HUVEC and incubate for 1-2 min at room temperature, shaking gently until the cells detach.
3. Once detached, quickly rinse Petri dish with 5 mL M20 and transfer the media containing cells to a 10 mL tube.
4. Centrifuge samples at 300 x g for 5 min at room temperature, aspirate supernatant, resuspend cells in 200 µL of M20, and count cells using a hemocytometer.
5. Resuspend the cells to 2.0 x 10⁵ cells/mL in M20, aspirate the M199 on the gels from the 96-well plate (step 1.3) and add 100 µL of resuspended HUVEC (2.0 x 10⁴ cells) to each collagen gel prepared above (step 1.2). Culture plates for a further 3 days at 37 °C/5% CO₂.
   NOTE: The integrity of the HUVEC monolayer may be confirmed after 3 days by silver nitrate staining²² or immunohistochemistry for tight junction proteins²³ at this stage. Additional gels must be prepared for this purpose.

4. Activation of HUVEC Monolayer and Isolation/Activation of Monocytes for Transmigration: Day 8

1. On Day 8, activate each HUVEC monolayer prior to the addition of monocytes by aspirating the M20 media overlaying the gels and adding 100 µL of 10 ng/mL human TNFα in M20 per gel. Incubate at 37 °C/5% CO₂ for 4 h.
   NOTE: Non-activated HUVEC conditions may also be included if required as controls.
2. During the 4 h HUVEC activation step, isolate monocytes from PBMCs using magnetic bead techniques to negatively select for cells as per the manufacturer's instructions. A minimum of 6.5 x 10⁶ PBMCs should be used at this step in order to reliably recover 3.0 x 10⁵ monocytes required for one condition, as monocytes typically account for approximately 10-15% of PBMCs.
   NOTE: To evaluate the effect of monocyte activation prior to monocyte transmigration and foam cell formation, monocytes can be activated at this stage. Monocytes can be isolated from thawed PBMCs if required (i.e., stored patient samples). Monocyte subsets isolated by FACS sorting can be prepared for addition to gels (Figure 1).

5. Transmigration of Primary Human Monocytes: Day 8

1. To measure monocyte transmigration, remove TNFα-containing media and wash gels twice with 100 µL M199 by adding and removing media. Following washing, add 2.0 x 10⁵ freshly isolated or thawed and washed cryopreserved PBMCs or 5.0 x 10⁴ freshly isolated monocytes or purified monocyte subsets to HUVEC monolayers in 100 µL M20. Incubate for 1 h at 37 °C and 5% CO₂ to allow the forward transmigration of monocytes into the gel. It is best to allow 6 wells for every experimental condition examined.
   NOTE: To evaluate the effect of autologous serum on transmigration and foam cell formation, incubate cells with M20 containing the desired concentration of heat-inactivated donor serum and compare to conditions with a pooled human serum control. Leukocytes other than monocytes may be added at this stage. If investigating the impact of specific lipids/lipid species (e.g., HDL, oxHDL), perform all following steps with serum-free media containing 20-50 µg/mL lipids.
2. After 1 h of forward transmigration, collect the non-transmigrated cells by washing gels twice with 100 µL of prewarmed 1 mM EGTA in 1x PBS, and once with 100 µL M199 (same centrifuge conditions), pooling the supernatants from each well of the same experimental condition (usually 6 wells) into a 1.5 mL microcentrifuge tube on ice. Centrifuge the non-transmigrated cells at 4 °C, 300 x g for 5 min and resuspend in 30 µL 1x PBS.
3. Count the cells collected in the supernatant to determine the number of forward transmigrated cells.
4. The percentage of forward transmigrated cells is determined as follows:
   
5. Cover the washed gels containing transmigrated cells with 100 µL M20 and incubate for a further 48 h.
6. After 48 h, collect the supernatant and wash non-transmigrated cells twice with 100 µL prewarmed 1 mM EGTA/PBS, collecting and pooling the supernatant from each condition as in step 5.2.
   NOTE: The phenotype of reverse transmigrated cells may be tested on these cells following standard flow cytometry staining protocols.
7. Centrifuge cells at 4 °C, 300 x g for 5 min and resuspend cells in 30 µL 1x PBS. Count cells and determine the viability via trypan blue staining.
8. Determine the percentage of reverse transmigrated cells by using the following equation:
   
   NOTE: Accounting for the total number of forward transmigrated cells gives a more accurate indication of reverse transmigration as it is unlikely forward transmigration will be 100%.
9. For analysis by microscopy, fix the gels by adding 100 µL 2% formaldehyde (final concentration) to each well, cover the plates in aluminum foil and store at 4 °C until analysis. For flow cytometry, do not fix the cells at this stage. See the protocols below for microscopy (section 6) or flow cytometry (section 7) analysis.
   CAUTION: Formaldehyde is toxic and corrosive; use personal protective equipment (PPE). Care should be taken when adding formaldehyde, however, this step does not need to be performed in a fume hood due to the low concentration used.

6. Quantitation of Foam Cells and Macrophages by Microscopy (Oil-Red O Stain)

1. Remove the formaldehyde and wash the gels with 100 µL of 50% (v/v) methanol for 5 min and then 100 µL 78% (v/v) methanol for 15 min.
   NOTE: The staining steps may be performed on the laboratory bench and not in a Class II Biohazard cabinet.
2. Stain for lipid droplets by adding 100 µL of freshly prepared 0.2% (w/v) Oil-Red O (see Table of Materials for details) for 1 h at room temperature.
3. Remove excess stain by washing the gels 4 times with 100 µL of 78% (v/v) methanol, aspirating and discarding the supernatant after each wash.
4. Counterstain for 15 min at room temperature with 100 µL of Giemsa, diluted 1:10 in distilled water.
5. Aspirate the Giemsa stain and wash gels once with water.
6. To detach the gels from the plate, rim the wells using a 21 G needle, with the bevel facing outwards, around the edge of the gel.
7. To mount the gels on a microscope slide, punch two holes (6.35 mm diameter) in a 2.54 cm x 1.5 cm strip of double-sided tape. Fix the tape to a standard microscope side and remove the protective coating from the top.
   1. Add a drop of water to the holes in the tape using a transfer pipette. Using tweezers, gently transfer gels to the holes in the tape and cover with a size 1.5 glass coverslip. Press gently on the coverslip to adhere it to the tape.
8. Examine with differential interference contrast bright-field microscopy using 40X objective on an inverted microscope.
   1. Bring the HUVEC monolayer into focus at 40X magnification and scroll 'into' the gel, counting macrophages/foam cells throughout the entire depth of the gel. Repeat for three distinct fields of view located at similar distances from the edge of the gel in order to minimize possible edge effects. This will ensure gel depth is consistent between counting regions.
      NOTE: Score cells in the gel as either foam cells (defined as cells containing >1/3 of their cytoplasm as Oil-Red O stained lipid droplets) or macrophages within 2 h of mounting on slides (Figure 2). Foam cell formation is expressed as the percentage of foam cells relative to the total number of migrated cells and is expressed as the median counts in 3 fields of view examined for 6 gels per condition, therefore a median of 18 measurements per condition. An example of raw cell counts from a typical experiment is shown in Table 2.
      NOTE: Classifying a cell as a foam cell vs macrophage by microscopy is subjective and consequently can be investigator-dependent. In clinical studies, where experiments are performed over an extended period of time, it is important for all counting to be performed blinded by a single investigator. Examples of foam cells and macrophages in gels are provided in Figure 2.

7. Analysis of Transmigrated Cells by Flow Cytometry

1. To phenotypically characterize foam cells and macrophages following transendothelial migration, digest the gels by adding 100 µL of 37 °C prewarmed 1 mg/mL collagenase D diluted in M199 to each well and incubate for 20 min at 37 °C/5% CO₂.
   NOTE: Place the 96-well plates directly on clean metal racks in the incubator to ensure even heat distribution.
2. Following incubation, macerate the gels using a 200 µL pipette tip and incubate for a further 20 min at 37 °C/5% CO₂.
3. Once fully digested, filter and pool the digested replicate gels through 35 µm nylon mesh capped FACS tubes placed on ice, and wash once with FACS wash (see Table of Materials) at 4 °C, 300 x g. Resuspend the cells in 100 µL of FACS wash.
4. Incubate the resulting cells with fluorophore-conjugated antibodies specific for CD45, a live/dead cell marker and surface/intracellular phenotypic or functional markers of interest using standard flow cytometry protocols.
   NOTE: Prepare control tubes for compensation using the appropriate fluorophores and Ig compensation beads. Extracted cells may also be collected onto glass slides for immunofluorescence microscopy by cytospinning. Alternatively, we have successfully collected cells using this protocol for assessment via imaging flow cytometry.
5. Acquire cells using a flow cytometer, and perform compensation as required.
   NOTE: As foam cells/macrophages may not form individual populations using light scatter properties and may differ considerably in size and shape, set liberal forward scatter (FSC) and side scatter (SSC) gates in order to capture all migrated cells as shown in Figure 3A.
6. Following acquisition, perform the data analysis using flow cytometry software. To identify migrated cells, first select cells as negative for the live/dead marker as shown in Figure 3B. Next, perform a single-cell discrimination by creating a tight gate around the cells shown on a SSC-area vs SSC-height plot.
7. Select CD45⁺ cells and gate the major population of migrated cells present in a FSC/SSC plot as these are the macrophages/foam cells.

Results

Quantifying monocyte transmigration

Monocytes are added to the model as described in Figure 1, and six gels are prepared for each condition. Monocytes for 6 gels per donor (i.e., 5.0 x 10⁴ monocytes per gel 6 gels = 3.0 x 10⁵ monocytes per donor) are resuspended to a final volume of 600 µL of M199 media containing the required serum/isolated lipid. Cel...

Discussion

The protocol described here offers a versatile and physiologically relevant method for assessing the atherogenicity of monocytes from human clinical cohorts, by combining both monocyte transmigration and foam cell formation. This model offers advantages over alternative methods of foam cell formation as it takes into account the effect of monocyte transmigration on foam cell formation and allows the measurement of reverse transmigration⁶ in addition to the inherent propensity of monocytes to matur...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gel preparation reagents
NaOH	Sigma-Aldrich	221465-500G	0.1 M NaOH diluted in H20
10x M199	Sigma-Aldrich	M0650
AcCOOH	Sigma-Aldrich	695092-100ML	20 mM Acetic acid diluted in H20
Cultrex Bovine Collagen I	R&D Systems	3442-050-01	Type I Fibrous Collagen
Name	Company	Catalog Number	Comments
Cell culture
M199	Life Technologies	11150-059	M199 media containing Earle's salts, L-glutamine and 2.2 g/L Sodium Bicarbonate. Media supplemented with 100 µg/mL L-glutamine  and 100 U/mL penicillin/streptomycin
M20			Supplemented M199 containing 20% heat-inactivated pooled or donor serum. Individual lipid species such as LDL can be added to M199.
HUVEC			Primary human umbilical cord endothelial cells (HUVEC) can be isolated from umbilical cords donated with informed consent and ethics approval. Isolated HUVEC may also be purchased commercially.
Human coronary artery endothelial cells			Primary human coronary artery endothelial cells can be isolated from arteries donated with informed consent and ethics approval. Isolated cells may also be purchased commercially.
EDTA	BDH Merck	10093.5V	Ethylenediaminetetraacetic acid (EDTA) - 0.5 M, pH 8.0
EGTA	Sigma-Aldrich	E3889-500G	Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) - 1 mM, pH 8.0
0.05% trypsin EDTA	Gibco	25300-054	0.05% trypsin/0.53 EDTA (1X)
L-glutamine	Gibco	25030-081	L-glutamine (200 mM)
Penicillin/streptomycin	Gibco	15140-122	Penicillin/streptomycin (10,000 units/mL Penicillin and 10,000 µg/mL Streptomycin)
New born calf serum	Gibco	16010-142	New born calf serum: New Zealand origin
Fibronectin	Sigma-Aldrich	F1056-1MG	Fibronectin - 50 µg/mL aliquots prepared and stored
PBS (1x)	Gibco	14200-075	Dulbecco's Phosphate Buffered Saline (10x): Dilute to 1x with sterile H20
0.1% TNF	Gibco	PHC3015	Recombinant human TNF - Reconstituted in H20 and stored in 10 µg/mL aliquots
Low-density lipoprotein	Merck Millipore	LP2-2MG	Low-density lipoprotein (LDL)
Name	Company	Catalog Number	Comments
Microscopy
1 or 2% formaldehyde	Polysciences	4018	1 or 2% formaldehyde diluted with sterile H20
50% and 78% methanol	Ajax Finechem	318-2.5L GL	50% or 78% v/v methanol, diluted with H20
Oil Red O stain	Sigma-Aldrich	O0625-25G	Dilute to 2 mg/mL in 22% 1M NaOH and 78% (v/v) methanol
Microscope slides	Mikro-Glass	S41104AMK	Twin frosted 45 degree ground edge microscope slides (25 X 76 mm)
Cover slips	Menzel-Gläser	MENCS224015GP	22 x 40 mm #1.5 size glass cover slips
Double-sided tape	3M Scotch	4011	Super strength exterior mounting tape (25.4 mm x 1.51 m)
Giemsa stain	Merck Millipore	1.09204.0500	Giemsa's azur eosin methylene blue solution (dilute stock 1:10 in H20)
Hole punch			Hand-held single hole punch (6.35 mm punch)
Name	Company	Catalog Number	Comments
Flow cytometry
Collagenase D	Roche Diagnostics	11088858001	Collagenase D diluted in M199 media to 1 mg/mL
35 µm nylon mesh capped polystyrene FACS tubes	BD Biosciences	352235	35 µm nylon mesh capped polystyrene FACS tubes
Live/Dead Fixable Yellow Dead Cell stain	Life Technologies	L34959	Live/Dead Fixable Yellow Dead Cell stain
FACS wash			Prepare by mixing 1 X PBS-, 2 mM EDTA and 1% New born calf serum
Name	Company	Catalog Number	Comments
Plasticware
96 well plate	Nunclon	167008	Delta surface flat-bottomed 96 well plate
10 cm Petri Dish	TPP	93100	Sterile 10 cm Petri dish
1.5 mL Eppendorf tubes	Eppendorf	0030 125.150	Eppendorf tubes
Transfer pipette	Samco Scientific	222-20S	Sterile transfer pipette (1 mL, large bulb)