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Keywords:

  • multicolor flow cytometry;
  • chemokine receptor;
  • antibody staining;
  • monocyte

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED
  8. Supporting Information

Chemokines and their receptors play an essential role within the immune system by dictating cellular migration. In vivo, receptor–ligand interactions rarely occur in isolation as cellular recruitment and migration are complex and highly coordinated processes often involving networks of multiple chemokines and multiple receptors. Simultaneous detection of multiple chemokine receptors on the single cell level is necessary to allow immunophenotyping studies that will help understand the intricacies of these networks. Chemokine receptors undergo a basal level of ongoing internalization, intracellular trafficking, and recycling back to the cell surface, even in the absence of the ligand. In the presence of ligand, receptor–ligand interactions enhance receptor internalization, reducing the cell surface receptor concentration, making precise determination of intrinsic levels challenging. Using multicolor flow cytometry, we sought to evaluate and optimize the simultaneous detection of cell surface expression levels of CCR2, CX3CR1, and CCR5 in primary human monocytes using a single antibody panel. We observed that staining for CCR2 alone or for CX3CR1 alone showed greater expression levels than when the cells were stained with the full panel of antibodies. Fluorescent-minus-one (FMO) controls revealed that ligation of the CCR5 monoclonal antibody to the receptor interfered with detection of CX3CR1 and CCR2. Sequential addition of antibodies during the staining procedure was sufficient to restore the detection levels, suggesting close proximity and possible functional interactions between CCR2/CCR5 and CX3CR1/CCR5 in monocytes. This study highlights the importance of optimizing staining procedures and using proper controls when simultaneously evaluating expression levels of multiple chemokine receptors by flow cytometry. Concurrent assessment of multiple receptors will provide insight and greater understanding of the complex interactions involved in cellular migration. © 2013 International Society for Advancement of Cytometry


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED
  8. Supporting Information

Cell migration is an essential physiological process. From embryonic development to immune surveillance, the ability of the correct cell to be at the right place at the right time is critical. Cells must take cues from the extracellular environment and integrate these various signals in order to respond accordingly. This is achieved through chemokine receptor–ligand interactions. Cell-surface chemokine receptors (CKRs) are G protein-coupled receptors (GPCRs) that recognize and bind to secreted cytokines (chemokines). Many chemokine receptors are able to bind more than one ligand, and reciprocally, a chemokine can bind to more than one receptor, forming an intricate network of interactions. Complexity is increased when we consider the combinatorial effect of various signals, which have been shown to produce different types of results such as addition, amplification, synergy (1–3) or competition, and inhibition (4, 5).

Many inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, cardiovascular disease, and certain viral infections, including human immunodeficiency virus, have been strongly linked to monocyte function and migration (6–9). Monocytes display a broad range of adhesion molecules and chemokine receptors, including the differential expression of the chemokine receptors CCR2, CX3CR1, and CCR5, which they selectively employ to migrate (10–12). Simultaneously evaluating the expression levels of these chemokine receptors allows combinatorial subset studies that cannot be performed when chemokine receptors are assessed individually.

Multicolor flow cytometry is an indispensible tool routinely used in immunology. This technology allows researchers to look at multiple markers simultaneously on a variety of cell populations on a per-cell basis within a single experiment. Hundreds of phenotypically and functionally distinct cell types in the peripheral blood of humans have been described based on the expression of specific marker combinations (13). Chemokine receptor expression is often used to identify cell subsets and offers insight into the cell's function (14). Although CKR expression and subset identification has been performed extensively on T cells (15), much less is known about monocyte subsets defined by combinatorial chemokine receptor expression. For example, CCR7 expression on T cells is used in combination with CD45RA to identify functionally distinct CD8+ T cell subsets (16, 17). CLA and CCR4 identifies skin homing T cells (18), whereas the expression of α4β7 and CCR9 is characteristic of gut-homing T cells (19). Such detailed analysis on monocyte subsets has not been widely performed. The ability to discriminate between monocytes subsets based on the expression of multiple phenotypic markers, including chemokine receptors, is crucial to advancing our understanding of cellular immunity and the role of specific monocyte subsets in disease pathogenesis (20).

Chemokine receptors undergo a basal level of ongoing internalization, intracellular trafficking, and recycling back to the cell surface, even in the absence of ligand. In the presence of ligand, receptor–ligand interactions enhance receptor internalization, reducing the cell surface receptor concentration, making precise determination of intrinsic levels challenging. Many chemokine receptors have been shown to form oligomers with members of their same receptor family, such as the CCR2/CCR2 (21) and CCR5/CCR5 (22) homodimers or with closely related receptors, such as CCR2/CCR5 (23, 24) and CCR5/CXCR4 (25) heterodimers. Flow cytometry based studies of chemokine receptors feature the use of monoclonal antibodies to these receptors. Given known interactions among CKRs, the simultaneous application of monoclonal antibodies to multiple CKRs in a single high dimensional flow cytometry experiment may lead to changes in one or more CKR expression levels, perhaps impeding the ability of the investigator to accurately gauge ex vivo levels of CKR expression from study subjects. Hence, special precautions must be taken when staining chemokine receptors due to their particular nature (26).

Our goal was to evaluate the cell–surface expression of CCR2, CX3CR1, and CCR5 on human monocytes from healthy adults within a single flow cytometry panel.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED
  8. Supporting Information

Experiments were performed between December 2011 and April 2012.

Subjects and Samples

EDTA-anticoagulated whole blood samples were collected between November 2011 and February 2012 from five normal, healthy human donors (2 males, 3 females; ages 25–35) and processed within 1 hour for PBMC isolation by ficoll-density gradient. Cells were cryopreserved in FBS+10% DMSO. We employed previously banked, de-identified specimens from a local cohort for this study, with exempt status under local IRB rules. Sampling of blood was approved by the ethical committee.

Cell Preparation and Immunophenotype Staining

Cells were thawed in warm media [RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 10 mM HEPES, 2 mM L-Glutamine (all Hyclone) and 10 μg/ml DNAse I (Sigma)], washed once and placed in a 96-well polypropylene plate for immediate staining. Cells were first stained for viability with the Yellow Amine Reactive Dye (YARD) for 15 minutes at room temperature. Afterward, the cells received the chemokine receptor antibodies (CCR2, CX3CR1, and CCR5) either simultaneously for 15 minutes at 37°C (Figs. 1 and 2), or one at a time with 5 minutes intervals at 37°C (Figs. 3 and 4), followed by addition of the remaining antibodies (CD3, CD14, CD36, CD56, CD19, CD20, CD16, HLA-DR) for 15 minutes at room temperature. Staining the chemokine receptors at physiological temperature was done in order to allow chemokine receptor cycling at the surface. In detail, sequential addition of CKR antibodies results in 30 minutes of total incubation time with 15 of those minutes at 37°C for the first antibody, while the second antibody has a 25-minute total incubation with 10 minutes at 37°C, and the third antibody has a 20-minute total incubation with 5 minutes at 37°C. The single-stain controls for the chemokine receptors had 30 minutes of total incubation time with 15 of those minutes at 37°C. Staining was performed in PBS+2%FBS. The cells were then washed twice with PBS+2%FBS and resuspended in PBS+1% paraformaldehyde. Single stain, fluorescence-minus-one (FMO) stain and full panel stains were performed. All antibodies were individually titrated for 30 minutes at room temperature and the concentrations determined to give the best separation were used for all experiments (Table 1).2

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Figure 1. Reduction of CCR2 and CX3CR1 detection with the full panel of antibodies compared with single stain controls. A: monocytes were identified with a singlet gate (FSC-A vs. FSC-H) for doublet exclusion followed by a size gate on the monocyte population (FSC-A vs. SSC-A). B: staining profiles of CCR2, CX3CR1, and CCR5 on monocytes. The left column displays a sample that received no antibody (unstained control). Samples in the middle column were stained with only one antibody (CCR2 or CX3CR1 or CCR5). The right column demonstrates the staining pattern of a sample that received the full panel of antibodies. Here, we see that staining with the full panel of antibodies dramatically reduced the staining intensity of CCR2 compared to the single stain control. There was a similar effect with CX3CR1 although it was less pronounced. CCR5 staining was unaffected. All samples shown have been compensated and have received identical gates for each parameter. Gate boundaries for CCR2, CX3CR1, and CCR5 were based on the unstained control. Staining for chemokine receptors (CCR2, CX3CR1, and CCR5) was performed by adding the antibodies individually (single stain) or simultaneously (full panel) for 15 minutes at 37°C, followed by addition of the remaining antibodies for 15 minutes at room temperature.

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Figure 2. Exclusion of the CCR5 antibody from the full panel restored CCR2 and CX3CR1 detection levels. A: Gating Strategy: Doublets were excluded on FSC-A vs. FSC-H plot. Monocytes were then identified using a size gate on FSC-A vs. SSC-A and dead cells (YARD bright) were excluded. We eliminated possible T cell, B cell, and NK cell contamination by gating out cells that were positive for CD3, CD56, CD19 or CD20 and kept only the HLA-DR+ cells. These cells were defined as monocytes. The different subpopulations of monocyte, as defined by CD14 and CD16 expression, can be seen. B: Staining profiles of CCR2, CX3CR1, and CCR5 on monocytes using a full antibody panel compared with FMO (fluorescence-minus-one) samples. Exclusion of the CCR5 antibody from the panel restored the staining intensity for both CCR2 and CX3CR1. All samples shown have received identical gates for each parameter. Gate boundaries for CCR2, CX3CR1, and CCR5 were based on the FMOs. Staining for chemokine receptors (CCR2, CX3CR1, and CCR5) was performed by adding the antibodies simultaneously for 15 minutes at 37°C, followed by addition of the remaining antibodies for 15 minutes at room temperature.

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Figure 3. Effect of temporal antibody staggering on the staining profiles of CCR2, CX3CR1, and CCR5 on monocytes. Staining with the CCR2 antibody first, followed by the CX3CR1 antibody then with the CCR5 antibody (top row) gave the closest results to the single stain samples (single stain frequencies from Figure 1B: CCR2 96%, CX3CR1 98%, CCR5 97%). All samples shown have received identical gates for each parameter. Gate boundaries for CCR2, CX3CR1, and CCR5 were based on the FMOs. Staining for chemokine receptors (CCR2, CX3CR1, and CCR5) was performed by adding the antibodies one at a time with 5 minutes intervals at 37°C, followed by addition of the remaining antibodies for 15 minutes at room temperature.

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Figure 4. Frequency CCR2+, CX3CR1+, and CCR5+ monocytes using temporal staggering of the full antibody panel on five healthy donors. Staining for chemokine receptors (CCR2, CX3CR1, and CCR5) was performed by adding the antibodies one at a time with 5 minutes intervals at 37°C, followed by addition of the remaining antibodies for 15 minutes at room temperature.

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Table 1. BD LSRFortessa configuration and antibody panel description
LaserSolid-state 405 nm violet laser (100 mw)Solid-state coherent sapphire 488 nm blue laser (50 mw)Solid-state 561 nm yellow-green (150 mw)Solid-state 640 nm red (100 mw)
Filter505LP535LP595LP505LP635LP750LP685LP750LP
Mirror450/50515/20585/42605/40488/10488/10530/30695/40582/15780/60670/30730/45780/60
Voltage500400370370500270390510410480440500530
Conjugate/  ParameterV450V500Yellow StainQdot 605FSCSSCFITCPerCP- eFluor 710PEPE-Cy7Alexa Fluor 647Alexa Fluor 700APC-H7
Detector  (Amplification)PMT (Log)PMT (Log)PMT (Log)PMT (Log)Photo-diode (Linear)PMT (Linear)PMT (Log)PMT (Log)PMT (Log)PMT (Log)PMT (Log)PMT (Log)High Performance PMT (Log)
SignalAreaAreaAreaAreaArea and HeightAreaAreaAreaAreaAreaAreaAreaArea
Antibody/StainCCR5CD3Yellow LIVE/ DEAD Fixable Dead Cell Stain KitCD14CD36CX3CR1CD163CD56/CD19/ CD20CCR2CD16HLA-DR
Volume used  (in 60 μl total)5 μl4 μl0.1 μl0.1 μl  10 μl1 μl10 μl0.5/0.5/1 ul2 ul0.5 μl0.5 μl
Clone2D7UCHT1TÜK4CB382A9-1GHI/61B159/SJ25C1/2H7486073G8G46-6
VendorBD HorizonBD HorizonLife TechnologiesLife TechnologiesBD PharmingeneBio-scienceBD PharmingenBD PharmingenBD PharmingenBD PharmingenBD Pharmingen
Cat#562121561416L-34959Q1001355545446-6099556018557747/557835/ 560735560713560713561358
Table 2. Compensation matrix
 V450-AV500-AQdot 545-AQdot 605-AFITC-APerCP-eF710-APE-APE-Cy7-AAx647-AAx700-AAPC-H7-A
V450-A 24.8312.60.67011.640.29260.13680.057410.208100.1924
V500-A10.85 52.075.3242.8260.3848000.0906600
Qdot 545-A2.73184.34 20.190.088370.3494000.11560.076450
Qdot 605-A0.1820.46472.093 0.29880.745312.9900.0652800
FITC-A0.10397.1813.9790.3269 3.11600000
PerCP-eF710-A0.40211.0831.0530.18380.8311 05.351.69840.5513.08
PE-A4.7576.83717.745.23212.7119.14 1.6522.3361.0151.825
PE-Cy7-A00000.12480.60012.431 01.09216.45
Ax647-A01.5630.40260.24882.6941.11700.8693 44.4214.91
Ax700-A0.30440.33950.3333001.80902.722.409 28.21
APC-H7-A0.8491.0580.59230.1590.51010.32570.0887712.211.7614.435 

Flow Cytometry Reagents, Instrumentation, and Analysis

Description of the viability dye and monoclonal antibodies used is provided in Table 1 (CD36 and CD163 were included for other purposes and were not used for this analysis).

Flow Cytometry was performed on a BD LSRFortessa Special Order Research Product (Table 1). Analysis of flow cytometry data was performed using Flow Jo software (Tree Star) and software compensation was calculated (based on bead and cell compensation controls) and applied after sample collection. Biexponential transformation of the data was used to add an additional negative decade in order to enhance data visualization and gating.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED
  8. Supporting Information

We observed a discrepancy between our single-stained controls and our full panel samples. Size-gated monocytes stained with only one antibody showed a greater frequency of positivity for CCR2 and for CX3CR1, but not for CCR5 (Fig. 1). According to the single-stained controls, the majority of monocytes were positive for CCR2 (96%), CX3CR1 (98%), and CCR5 (97%). Using the full panel, those numbers decreased to 53% for CCR2 and 79% for CX3CR1 but remained at 96% for CCR5. The fluorescence-minus-one (FMO) controls (Fig. 2) revealed that omitting the CCR5 antibody from the full panel restored the cell surface expression levels of CCR2 and CX3CR1, pointing to a possible interaction between CCR5 ligation and CCR2/CX3CR1 staining profiles.

We sought to determine if a delay in the addition of the CCR5 antibody would negate the observed decrease of CCR2 and CX3CR1 staining. To test this, we staggered the addition of the CKR antibodies with 5-minute incubation. We attempted four different combinations of CKR antibody additions. One combination added CCR2 first, followed by CX3CR1, then CCR5. Another started with CX3CR1, followed by CCR2, then CCR5. The third combination started with CCR5, followed by CCR2, then CX3CR1. We also repeated the simultaneous addition of CCR2, CX3CR1, and CCR5.

In Figure 3, we can clearly see that staining with CCR2 first restores the staining levels close to the single stain level. Although CCR2 positivity is lower (90% compared with 96% in the single-stained well), it is much higher than in the wells where CX3CR1 was added first (69%), where CCR5 was added first (31%), or when the antibodies were added simultaneously (51%). CX3CR1 expression levels seemed unaffected by prior addition of CCR2 (97%) compared with the single stain (98%). There was a decrease in CX3CR1 when CCR5 was added first (64%) or when the antibodies were added simultaneously (84%). CCR5 levels stayed above 95% for all conditions.

Sequential staining starting with CCR2, followed by CX3CR1, then CCR5 gave the closest results to the single stain controls. We used this optimized staining procedure on five healthy volunteers (Fig. 4). All chemokine receptors showed a frequency of positivity greater than 80%. CCR5 had the highest overall frequency (median 96.3) followed by CX3CR1 (median 93.5). CCR2 had a lower frequency of positivity (median 84.3) suggesting a possible greater sensitivity of CCR2 to the effects of multiple chemokine receptor staining.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED
  8. Supporting Information

Interactions between CCR5 and CCR2 have been previously reported and evidence for CCR2/CCR5 dimerization is well established. Indeed, others have observed cross-inhibition of ligand binding between CCR5 and CCR2b (23). In our case, it is possible that ligation of CCR5 by the CCR5 antibody causes steric hindrance leading to a decreased binding ability of the CCR2 antibody. This implies close proximity of CCR5 and CCR2, presumably, in the form of a heterodimer. Alternatively, ligation of the CCR5 antibody could cause a conformational change in the CCR2/CCR5 heterodimer, which would mask the CCR2 epitope recognized by the antibody. Interestingly, we see a similar, but less pronounced effect with CX3CR1, indicating a possible close interaction between CCR5/CX3CR1 and also CCR2/CX3CR1. Evidence for dimerization of CX3CR1 with CCR5 or CCR2 has not, to our knowledge, been previously presented. Our study was not intended to resolve which mechanism may have led to the observed effect, but rather to empirically document an ordering of chemokine receptor staining that led to preservation of levels that are detected in single-stained samples.

Given the importance of chemokine receptors and cell migration in the setting of many inflammatory diseases, (27, 28), it becomes apparent that multiple chemokine receptors will need to be studied simultaneously instead of being viewed as single entities in order to identify specific cell subsets that may be involved in pathogenesis. For example, the monocyte subset defined as CX3CR1loCCR2+ has been shown to get actively recruited into inflamed tissues, whereas the CX3CR1hiCCR2 subset is characterized by CX3CR1-dependent recruitment to noninflamed tissues (29). However, both these subsets have been shown to be present in atherosclerotic plaques and differentially utilize CCR2, CCR5, and CX3CR1 to enter the plaque, where they likely play distinct roles (10, 30). Ongoing advances in flow cytometry facilitating the concurrent detection of greater numbers of markers will serve as an indispensable platform for identification of cell subsets involved in disease, and may lead to the development of targeted therapeutic interventions with increased specificity (31, 32). However, because of the particular nature of chemokine receptors (continuous cell-surface cycling, homo- and heterodimerization), special precautions must be taken during the staining procedure. These include staining at physiological temperature to allow receptor cycling (26), and as we demonstrate here, the sequential addition of antibody staining reagents. Inclusion of proper controls is critical to ensure an accurate depiction of expression levels.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
MIFlowCyt_Checklist.doc84KSupporting Information: MIFlowCyt
Table_2-Compensation_Matrix.docx16KSupporting Information

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