RnDSy-lu-2945

AbstractMacrophages are ubiquitously distributed throughout the body and perform many functions including inflammatory responses against pathogens by classically activated M1 macrophages and the regulation of wound healing and tissue remodeling by anti-inflammatory alternatively activated M2 macrophages. The responsibility for these pleiotropic functions lies in the expression of surface receptors unique to each subset.

As new techniques such as RNA-Seq have become available, it is becoming clear that a more robust analysis of macrophage surface marker expression is needed to understand the spectrum of macrophage activation. Concomitantly, it is also necessary to understand how the in vitro culture conditions used to generate macrophages affect the expression of various surface markers. More recently, expanded sets of M1 and M2a markers have been described for macrophages cultured in media containing fetal bovine serum (FBS) and polarized with IFN-g and LPS (M1) or IL-4 and IL-13 (M2a).1 Still, it remains unclear how these phenotypes compare to macrophages grown in media containing human serum or serum-free media and whether polarizing stimuli are necessary for the expression of various surface markers.2,3

Here we begin to delineate how the culture conditions used to generate human M1 and M2a macrophage subsets affect the expression of multiple surface markers. We directly compared the expression of fifty different surface markers on M1 and M2a macrophages cultured under various conditions. Of those fifty, we show how the presence of FBS, human AB serum or serum-free media and polarizing cytokines affected the expression of twenty of these markers such as CD200 R1 and CD32. Moreover, the inclusion of polarizing stimuli was critical to the expression of several of these markers such as CD38 and SLAMF7. These results significantly expand our knowledge of the phenotypic differences between human M1 and M2a macrophages as well as demonstrate the importance of culture conditions in generating these phenotypes.

Christopher Hammerbeck, Christine Goetz, Kristal Newman, Jody Bonnevier, Birte Aggeler | R&D Systems, 614 McKinley Place NE, Minneapolis, MN 55413

Enriched human CD14+ monocytes (solid orange histograms ) were cultured in the presence of Recombinant Human GM-CSF (50 ng/mL) or Recombinant Human M-CSF (50 ng/mL) for 6 days in an RPMI-based media containing 10% FBS (10% FBS), 10% Human AB serum (Hu AB Serum), or in StemXVivo® Serum-Free Dendritic Cell Base Media (Serum-Free). Following the 6-day maturation period, macrophages were polarized for an additional 24 hours to either the classical M1 (50 ng/mL LPS + 50 ng/mL IFN-g; solid blue histograms ) or alternatively activated M2a phenotype (20 ng/mL IL-4 + 20 ng/mL IL-13; solid purple histograms ). Unpolarized macrophages were cultured for 24 hours in media containing either GM-CSF (light blue shaded histograms ) or M-CSF (light purple shaded histograms ). All macrophages were cultured for a total of 7 days and were then compared by flow cytometry analysis. Adherent macrophages were washed with warm 1x HBSS and then removed using either warmed TrypLE™ Express Enzyme solution or by gently scraping the wells with a cell scraper. Prior to staining, cells were blocked with a combination of normal human IgG and normal mouse IgG and then incubated with primary antibodies (Table 1) for 30 minutes at 4 °C or a combination of 15 minutes at 37 °C followed by 15 minutes at room temp. Isotype control staining is represented by the solid gray histograms (). Zombie Violet™ cell viability dye labeling and doublet-exclusion were used to remove dead cells from surface marker expression analysis.

Antigen Catalog # Function

CD14 FAB3832 Clearance of gram-negative pathogens; upregulation of adhesion molecules and cytokine expression

Fcg RII/CD32 FAB1330 Phagocytosis of immune complexes; modulation of antibody production by B cells

CD38 FAB2404 Regulation of lymphocyte activation; cell adhesion; metabolism of cADPR and NAADP

B7-1/CD80 FAB140 Co-stimulation/Regulation of T cell activation

CD83 FAB1774 Co-stimulation

B7-2/CD86 FAB141 Co-stimulation/Regulation of T cell activation

C1q R1/CD93 FAB23791 Phagocytosis of apoptotic cells; leukocyte-endothelial cell adhesion

TNF RII/CD120b FAB226 Regulation of responses to TNF-a and TNF-b; anti-apoptotic signaling

SLAM/CD150 FAB1642 B cell co-stimulation, proliferation, immunoglobulin production, and signal transduction

CD163 FAB1607 Scavenger receptor; clearance of hemoglobin complexes; regulation of cytokine production by macrophages

CD200 R1 FAB3414 Suppression and regulation of macrophage-induced inflammatory damage in tissues

SR-AI/CD204 FAB2708 Clearance of apoptotic cells; co-receptor for some Toll-like receptors; facilitation of the expression of pro-inflammatory cytokines

DC-SIGN/CD209 FAB161 Cell trafficking; immunological synapse formation; T cell proliferation; capture and internalization of viral pathogens

HLA-A, B, C FAB7098 Antigen presentation

HLA-DR FAB4869 Antigen presentation

Mer FAB8912 Inhibition of Toll-like receptor-mediated innate immune responses; cellular survival; cell migration

SLAMF7/CD319 FAB1906 Regulation of natural killer cell and T cell functions

TLR2/CD282 FAB2616 Recognition of and activation of innate immune responses to lipoproteins of gram-negative and gram-positive bacteria

TLR4/CD284 FAB6248 Recognition of and activation of immune responses to bacterial LPS

B7-H1/PD-L1 FAB1561 Activation and inhibition of T cell activation

Antigen Expression profile in the presence of FBS

Expression profile differs in the presence of Hu AB serum or StemXVivo® Media

Expression profile is dependent on polarizing cytokines

Expression profile is enhanced by polarizing cytokines

CD14 Highly expressed on M2a; little to no expression on M1 Yes No No

Fcg RII/CD32 Highly expressed on M2a; little to no expression on M1 Yes No Slight

CD38 Highly expressed ONLY on polarized M1 No Yes Yes

B7-1/CD80Intermediate expression on M2a and unpolarized M1; highly expressed on polarized M1

Yes Yes Yes

CD83 Moderate expression ONLY on polarized M1 No Yes Yes

B7-2/CD86 Highest expression on polarized M1/M2a No No Yes

C1q R1/CD93 Highly expressed only on unpolarized and polarized M2a Yes No No

TNF RII/CD120b Highly expressed ONLY on polarized M1 Slight Yes Yes

SLAM/CD150 Moderate expression ONLY on polarized M1; low expression on polarized M1 Yes Yes Yes

CD163 Highly expressed only on unpolarized and polarized M2a Yes No No

CD200 R1 Highly expressed ONLY on polarized M2a Yes Yes Yes

SR-AI/CD204 Moderate expression only on unpolarized and polarized M2a Slight No No

DC-SIGN/CD209 Highly expressed ONLY on polarized M2a No Yes Yes

HLA-A, B, C Uniform expression on all macrophages No No No

HLA-DR Highest expression on polarized M1 Yes No Yes

Mer Highly expressed only on unpolarized and polarized M2a Yes No No

SLAMF7/CD319 Highly expressed ONLY on polarized M1 Yes Yes Yes

TLR2/CD282 Uniform expression on all macrophages Slight No No

TLR4/CD284 On M1 and M2a; Highest expression on polarized M1 Slight No Yes

B7-H1/PD-L1 Low on M1 and M2a; Highest expression on polarized M1 Yes Yes Slight

Figure 3.

Figure 1.

Figure 2.

Table 1. Antibodies Used to Analyze Macrophage Surface Marker Expression Table 2. Summary of Macrophage Surface Marker Expression

Isolate human PBMCs by Ficoll gradient

Enrich CD14+ monocytes using the MagCellect™ Human CD14+ Cell Isolation Kit (R&D Systems; Catalog # MAGH105)

Culture CD14+ monocytes at 1x106 cells/mL to differentiate into mature macrophages (see Fig 2)

Harvest macrophages, stain with selected antibodies (Table 1) and analyze cell surface marker expression by flow cytometry(Figure 3 and Table 2)

Platelets + Plasma

PBMCsFicoll

RBCs

Monocyte

6 days

Unpolarized M0

Polarized M2aUnpolarized M0Unpolarized M0Polarized M1

24 hr

Cell culture media •FBS-containing media (RPMI, 10% FBS, penicillin/streptomycin, Glutamax)

•Human serum-containing media (RPMI, 10% pooled normal human AB serum)

•StemXVivo® Serum-Free Dendritic Cell Base Media (R&D Systems, Catalog # CCM003)

Alternatively used differentiation kits•CellXVivo™ Human M1 Macrophage Differentiation Kit (R&D Systems, Catalog # CDK012)

•CellXVivo™ Human M2 Macrophage Differentiation Kit (R&D Systems, Catalog # CDK013)

50 ng/mL rhGM-CSF(R&D Systems,

Catalog # 215-GM)

50 ng/mL rhIFN-γ + 50 ng/mL LPS(R&D Systems,

Catalog # 285-IF)

50 ng/mL rhGM-CSF

50 ng/mL rhM-CSF(R&D Systems,

Catalog # 216-MC)

50 ng/mL rhM-CSF

20 ng/mL rhIL-4 + 20 ng/mL rhIL-13

(R&D Systems, Catalog # 204-IL;

# 213-ILB)

Monocyte

CD14 Fcγ RII/CD32 CD38 B7-1/CD80 B7-2/CD86CD83 C1q R1/CD93 TNF RII/CD120b SLAM/CD150 CD163

CD200 R1 SR-A1/CD204DC-SIGN/

CD209 TLR4TLR2HLA-A HLA-DR Mer B7-H1/PD-L1SLAMF7

10% FBS

Serum-Free

10% Hu AB Serum

M2a

M1

M2a

M1

M2a

M1

Monocyte

10% FBS

Serum-Free

10% Hu AB Serum

M2a

M1

M2a

M1

M2a

M1

References1. Beyer, M. et al. (2012) PLoS

One 7:e45466. 2. Rey-Giraud, F. et al. (2012)

PLoS One 7:e42656.3. Vogel, D.Y. et al. (2014)

Immunobiology 219:695.

Phenotypic Characterization of Human M1 and M2a Macrophages Cultured with Different Serums and in the Presence or Absence of Polarizing Cytokines

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