Proteomic Profiling of Macrophages by 2D Electrophoresis

Summary

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Abstract

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Introduction

Macrophages are heterogeneous and plastic cells that are able to acquire distinct functional phenotypes. In vivo, these cells respond to a large variety of micro environmental signalssuch as microbial products, cytokines, etc.¹. In vitro, the pro-inflammatory phenotype (M1) of macrophage can be induced by lipopolysaccharide (LPS) and the anti-inflammatory phenotype (M2) by some cytokines such as interleukin-4 (IL-4). Moreover, macrophages can switch from an activated M1 to M2 phenotype, and conversely, upon specific signals².

Depending on the phenotype, macrophages will have different functions. M1 macrophages are cells that produce pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), to kill microorganisms or tumor cells³. In contrast, M2 macrophages prevent these inflammatory response like in wound healing and fibrosis by producing anti-inflammatory factors such as TGF-β^3,4.

Human peripheral blood mononuclear cells from healthy donors were isolated by Ficoll density gradient centrifugation as previously described⁵ using a technique adapted from Boyum⁶. Macrophages in culture can be differentiated into M1 or M2 phenotype after 6 days of primary culture⁷.

Analysis of protein expression or protein changes between the two subtypes of macrophages under various controlled stimuli, such as host pathogenicity or microbial toxins, will be helpful to decipher the functionality of the pro- and anti-inflammatory macrophages.

Proteomics are unique tools for direct monitoring of proteins that are specifically up- or down-regulated in human cultured macrophages under various stimuli. Fluorescent dyes have resolved some of the limitations of 2D gel electrophoresis, such as low sensitivity and image analysis⁸. The dyes reacting with cysteine residues have increased the detection sensitivity compared to those reacting with lysine residues⁹. In a previous study, we demonstrated the usefulness of DIGE saturation labelling for the analysis of scarce samples¹⁰ compared to the classical silver-stained 2D electrophoresis¹¹. This technology is helpful in rapidly analysing protein modifications between the two subtypes of macrophages or between untreated and treated macrophages from the same subtype.

The advantages of this proteomic technique are having access to the information of protein size and post-translational modifications by analysing the 2D gel¹². It should be taken into account that this is not a high throughput technique, limiting the number of samples that can be analysed. The development of high throughput assays based on mass spectrometry as reviewed recently¹³ can improve this.

Here we present how to perform 2D DIGE analysis from the protein extraction of cultured macrophages through the processes of electrophoresis, isoelectrofocusing, and SDS-PAGE as well as information on the usefulness of adequate 2D software.

Protocol

The protocol follows the guidelines of our institutions human research ethics committee. Buffy coat from healthy human donors were obtained from the Regional Blood Transfusion Center (Lille, France). Samples obtained from the buffy coats are declared as an Inserm collection (n°DC2010-1209).

1. Material and Culture Media Preparation

1. Dilute 10x Phosphate Buffer Saline (PBS) in sterile distilled water to obtain 1x PBS.
2. Make RPMI 1640 medium supplemented with gentamicin (40 μg/ml) and L-glutamine (2 mM), with and without 10% pooled human serum.

2. Primary Cultures of Monocyte-derived Macrophages (MDM)

1. Dilute buffycoat (25 ml) with 1x PBS. Carefully load on a Ficoll/leucosep tube and centrifuge at 1,600 x g for 20 min, at room temperature.
2. Collect monocytes at the interface into a new tube. Wash 3x with 10 ml 1x PBS containing 0.1% ethylenediaminetetraacetic acid (EDTA) by successive centrifugations at 1,000 x g, 370 x g and 160 x g for 10 min each, and then once in 1x PBS alone at 160 x g for 10 min.
3. Resuspend the cell pellet in 5 ml RPMI-1640 medium without serum and seed the cells in 35 mm dishes at a density of 1 x 10⁶ cells per dish.
4. After sedimentation for 90 min in the incubator, discard the supernatant containing the non-adherent cells. Wash the adherent cells, consisting of monocytes, 3x with 1 ml PBS; then add 1 ml of fresh medium containing 10% (v/v) human serum to the previously serum free cells.
5. After 6 days of culture, to yield alternative differentiated macrophages (M2), add recombinant human IL-4 (15 ng/ml) and maintain for 6 days. Then, treat differentiated macrophages with lipopolysaccharide (100 ng/ml) at day 12 for 4 hr to obtain M1 macrophages.

3. Extraction of M1 and M2 Macrophage Proteins for 2D Electrophoresis

1. Wash macrophages three times with 25 mM Tris, pH 7.4, and scrap in buffer containing 30 mM Tris pH 8, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 2 M thiourea and 7 M urea.
2. Lyse cells using a mixer suitable for 1.5 ml microcentrifuge tubes for 5 min in ice and store at -20 °C.
3. Determine protein concentration using a commercial Bradford reagent. Store 100 µl aliquots of the proteins at -20 °C until use.

4. Isoelectrofocusing

NOTE: Perform all labelling procedures in the dark.

1. Reduce 5 µg of each sample (M1 and M2 macrophages) adjusted to 9 μl with lysis buffer with 2 mM Tris(2-carboxyethyl)phosphine (TCEP) for 1 hr at 37 °C.
2. Add cyanine 3 (Cy3) to M1 extract and M2 sulfhydryl-reactive dye extract at a concentration of 0.8 nM/μg protein. Add cyanine 5 (Cy5) to M1 and M2 extracts at a concentration of 0.8 nM/μg protein and incubate for 30 min at 37 °C.
3. Stop the reaction with the addition of an equal volume of sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 130 mM dithiothreitol (DTT) and 2% pharmalytes.
4. Mix Cy3-labeled M1 samples with Cy5-labeled M2 samples in one hand and Cy3-labeled M2 samples with Cy5-labeled M1 samples in another hand.
5. Rehydrate an Immobilized pH gradient (IPG) strip (240 mm, pH 3-10 linear gradient) with 450 μl of labelled mixed samples in buffer containing 7 M urea, 2 M thiourea and 4% CHAPS on a isoelectric focusing (IEF) cell system for 24 hr without applying any current.
6. Perform focusing at 300 volts (V) for 3 hr, and then at a gradient to 1,000 V for 6 hr, at a gradient to 8,000 V for 3 hr and finally at 8,000 V for 3 hr.

5. Second Dimension

NOTE: Perform all electrophoresis procedures in the dark.

1. Incubate the IPG strips in equilibration buffer containing 0.1 mM Tris-HCl (pH 8), 6 M urea, 2% (w/v) sodium dodecyl sulfate (SDS) and 30% (v/v) glycerol for 10 min.
2. Transfer the equilibrated IPG strips for the second dimension (SDS-polyacrylamide gel electrophoresis (PAGE)) onto 12.5% PAGE gels and seal with low-melting agarose.
3. Carry out electrophoresis at 20 °C using an Ettan-Daltsix system at a constant voltage of 70 V overnight followed by 300 V until the bromophenol blue front reaches the bottom of the gel.

6. Image Acquisition and Bioinformatic Analysis

1. Scan gels cast between the two low-fluorescence glass plates with a DIGE Imager scanner at excitation/emission wavelengths of 532/580 nm for Cy3 and 633/670 nm for Cy5 to yield images with a pixel size of 100 μm.
2. Perform image analysis with commercial software as previously detailed¹².
3. Calculate and normalize spot volumes in each image. Assign a normalized spot volume as a proportion of the total value of each spot detected in the gel.
4. Analyse the differences in protein spot volumes for each type of macrophages by comparing the normalized spot volume value between the two groups (M1 and M2). Consider the difference between the spot volumes to be significant if the change is 1.5 fold (p <0.05, one-way ANOVA analysis).

Results

To perform appropriate differential proteomic analysis, the processing of samples to be analysed should be verified.

In the example presented, the cell culture quality of macrophages is required for morphological and molecular aspects as previously published⁵. The differentiation of monocytes into macrophages and the homogeneity of the culture was followed by phase-microscopy. Figure 1 showed an example of primary cultures of M1 and M2 macrophages. As shown, we veri...

Discussion

The protocol described herein details a method to analyse the impact of various stimuli of the two subtypes of macrophages, M1 (pro-inflammatory) and M2 (anti-inflammatory). Primary cultures of M1 and M2 macrophages were obtained from the differentiation of monocytes as previously published⁷.

The procedure of 2D DIGE gel electrophoresis requires specialized materials and equipment, such as IEF cell for isoelectrofocusing, low-fluorescence plates for the SDS-PAGE in order to scan twi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640	Invitrogen	31870-074
PBX 10 X	Invitrogen	14200-083
L-glutamine-200 mM-100X	Invitrogen	25030-024
gentamycin 10 mg/ml	Invitrogen	15710-049
human serum	Invitrogen	34005100
Ficoll d = 1,077	ATGC	L6115
Leucosep	Dutscher	16760
6-wells plate PRIMARIA	Becton Dickinson	353846
IL-4	Promocell	B-61410
lipopolysaccharide	Sigma-Aldrich	L-2654
Giemsa	Fluka	48900
EDTA MM372,2	Research Organics	3.00E+01
Filter 0.22 µm	Millipore	SCGPTORE
100 ml cylinder	Corning	430182
TCEP	Interchim	UP242214
Bradford reagent	Bio-Rad	5000006
Cy3+Cy5-reactive dye	GE Healthcare	25-8009-83
IPG strip 3-10 24cm	GE Healthcare	17-6002-44
Protean IEF cell	Bio-Rad	165-4000
Low-melting agarose	Invitrogen	15517-014
Ettan-Daltsix system	GE Healthcare	80-6485-08
Ettan DIGE Imager scanner	GE Healthcare
Progenesis Samespot	Non linear dynamics
50 ml tubes	any supplier	n/a
15 ml tubes	any supplier	n/a
CHAPS	Sigma-Aldrich	C5070
Urée	Merk	108484-500
Thiourée	Sigma-Aldrich	T7875
DTT	Bio-Rad	1610611
APS	Sigma-Aldrich	A3678
TEMED	Sigma-Aldrich	T9281
Tris Base	Sigma-Aldrich	T1503
Tris HCl	Sigma-Aldrich	T3253
Pharmalytes 3-10	GE Healthcare	17-0456-01
SDS	Sigma-Aldrich	L3773
Bromophenol blue	Sigma-Aldrich	114391
Glycerol	Sigma-Aldrich	G6279
Acrylamide 40%	Bio-Rad	161-0148
2D clean Up	GE Healthcare	80-6454-51
Glycine	Sigma-Aldrich	G7126
Diméthylformamide	Sigma-Aldrich	22705-6
electrode wicks	Bio-Rad	165-4071