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 ....

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 a....

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)