FEBS 30017 FEBS Letters 579 (2005) 5631–5634

Expression of macrophage-selective markers in human and

rodent adipocytes
Wael Khazena, Jean-Pierre MÕBikab, Céline Tomkiewiczc, Chantal Benellia, Charles Chanyb,
Ammar Achourb, Claude Foresta,*
a
Institut National de la Santé et de la Recherche Médicale, Unit 530; Université Paris Descartes, Centre Universitaire des Saints-Pères,
45 rue des Saints-Péres, 75006 Paris, France
b
Institut National de la Santé et de la Recherche Médicale, Unit 490; Université Paris Descartes, Centre Universitaire des Saints-Pères,
45 rue des Saints-Péres, 75006 Paris, France
c
Laboratoire des interférons et de la sarcolectine; 45 rue des Saints-Pères, 75006 Paris, France

Received 28 July 2005; revised 31 August 2005; accepted 1 September 2005

Available online 29 September 2005

Edited by Veli-Pekka Lehto

homologous to the secretin family of proteins [2]. Since mac-

Abstract CD14, CD68 and/or mouse F4/80 or human epidermal
growth factor module-containing mucin-like receptor 1 (EMR1)
are widely used as macrophage-specific markers. Since macro-
phages infiltrate several tissues during inflammatory processes,
CD14, CD68 and EMR1-F4/80 have been employed to discrimi-
nate between tissue-containing macrophages, like adipose tissue
(AT), and other cells. Using real-time PCR experiments, we show
that isolated adipocytes from humans and mice AT express high
levels of CD14 and CD68 mRNA, whereas EMR1-F4/80 is
mainly present in the macrophage-containing stroma-vascular
fraction. Furthermore, fibroblasts-like cells (adipoblasts), preadi-
pocytes and adipocytes from the murine cell lines, 3T3-F442A
and BFC-1, express CD14 and CD68 mRNA and protein as
determined by fluorescence-activated cell sorter, but not F4/80
which, as expected, is strongly expressed in the macrophage cell
line RAW264.7. These results reinforce the view that EMR1-F4/
80 is the best macrophage marker to date and show that CD14
and CD68 are not macrophage-specific proteins.

2005 Published by Elsevier B.V. on behalf of the Federation of
European Biochemical Societies.
Keywords: CD14; CD68; F4/80; Macrophage; Adipose tissue;
Preadipocyte cell line
rophages infiltrate several tissues during inflammatory pro-
cesses, CD14, CD68 and EMR1-F4/80 have been employed
to discriminate between tissue-containing macrophages and
other cells [6,7]. Among these tissues, adipose tissue (AT)
was recently the focus of extensive investigation. The pres-
ence of macrophages in AT was documented [6,8]. In addi-
tion, obesity and the development of obesity-related insulin
resistance were correlated with increased macrophage accu-
mulation and inflammatory activities [7,9,10]. In all these
studies, CD14, CD68 and/or F4/80 were used as macrophage
discriminative markers. However, a role for preadipocytes as
macrophage-like cells was reported, showing the plasticity of
such cells [11]. Besides, AT from various depots displayed
differential macrophage functions such as phagocytic and
microbicidal activities [12]. Furthermore, in vitro differenti-
ated adipocytes were shown to express innate immunity fac-
tors, like TLR2 and TLR4 [13]. These observations
prompted us to identify which of the previously character-
ized macrophage-selective markers could be expressed in
other cell types like fibroblasts, preadipocytes or adipocytes
and which one would be specific.

1. Introduction
The lipopolysaccharide-binding protein CD14, the macros-
ialin CD68 and the G protein-linked TM7-homologous
receptor from humans (epidermal growth factor module-con-
taining mucin-like receptor 1; EMR1) or mouse (F4/80) are
widely used macrophage markers [1,2]. CD14 binds LPS,
interacts with Toll-like receptor 4 (TLR4), thereby inducing
cytokine production and mediating phagocytic clearance of
apoptotic cells [3,4]. CD68 is a member of the lysosomal-
associated membrane protein family [5]. EMR1-F4/80 is
*
Corresponding author. Fax: +33 0 142863868.
E-mail address: claude.forest@univ-paris5.fr (C. Forest).
Abbreviations: EMR1, epidermal growth factor module-containing
mucin-like receptor 1; AT, adipose tissue; TLR, toll-like receptor;
MCP-1, monocyte chemoattractant protein-1; SVF, stroma-vascular
fraction of cells; MIF, macrophage migration inhibitory factor; FACS,
fluorescence-activated cell sorter; GAPDH, glyceraldehyde-3P dehy-
drogenase
2. Materials and methods
2.1. Animals
Animal studies were conducted according to the French Guidelines
for the Care and Use of Experimental Animals. Sixteen weeks old
Balb/c male mice (Elevage Janvier, Bagneux) were killed by decapita-
tion at 10:00 a.m. then omental (OM) and subcutaneous (SC) WAT
were removed for analyses.
2.2. Isolation of adipocytes and stroma-vascular cells from adipose tissue
Explants of SC AT were obtained from three healthy women under-
going plastic surgery. They were aged 49, 46 or 37 and had body mass
index of 26.5 ± 0.7 kg/m2. None of the subjects suffered from known
metabolic or malignant diseases nor were they taking medications
known to alter AT metabolism. All the patients gave informed written
consent. The study was carried out according to the tenets of the Hel-
sinky protocol.
SC and OM AT were isolated from mice as described in Weisberg
et al. [6]. Once digestion was complete, samples were passed through
a sterile 250-lm nylon mesh (Sefar AG, Switzerland), then centrifuged
at 1000 · g for 8 min. The adipocyte-enriched floating fraction was
further digested for 1 h with liberase 3 (Roche Applied Science),

0014-5793/$30.00
2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
doi:10.1016/j.febslet.2005.09.032
5632 W. Khazen et al. / FEBS Letters 579 (2005) 5631–5634

washed twice with Dulbecco modified EagleÕs medium (GIBCO, Invit- total and subfractionated AT isolated from a SC localization
rogen Corp.), and centrifuged at 1000 · g for 8 mins. The supernatant in mice and humans and from a profound depot in mice, the
(adipocytes) was placed in Qiazol lysis reagent (Qiagen) and kept at

80 C for later RNA extraction. The macrophage-enriched pelleted
OM AT, the metabolism of which is known to be different
fractions of the stroma-vascular cells (SVFs) was resuspended in from that of SC [17]. For subfractionation, two consecutive
acid-phenol reagent (TRIzol; Invitrogen Corp., Cergy-Pontoise) and collagenase digestions were carried out to separate the pelleted
kept at
80 C for later RNA extraction. The same procedure was ap- SVF of cells from floating adipocytes. In mice, CD14, CD68
plied to human adipose tissue. and F4/80 mRNA were detected at higher levels in OM than
in SC (Fig. 1A). As expected, SVF from both AT localization
2.3. RNA extraction and analysis
Total RNA was extracted from 3T3-F442A [14] and BFC-1 [15] expressed the three markers at similar high levels, most prob-
cells, grown and differentiated as described [16], using TRIzol. For tis- ably detecting macrophages in this fraction (Fig. 1A). The level
sue samples (100 mg), extraction was carried out by RNeasy Mini Kit of CD14 and CD68 mRNAs in the adipocyte fraction was
(Qiagene, Courtaboeuf). RNA was quantified by spectrophotometric close to that observed in AT. This could be due to the presence
Nanodrop (ND-1000) (NYXOR BIOTECH), and the quality was as-
of remaining macrophages in the adipocyte fraction or to bona
sessed by gel electrophoresis.
The first-strand cDNA was synthesized using High Capacity cDNA fide expression of CD14 and CD68 in adipocytes. The situation
Archive (Applied Biosystems, Courtaboeuf) using 500 ng of RNA. is quite different with F4/80 transcript which was drastically re-
Samples of cDNA were diluted 1:2 in nuclease-free water. PCR ampli- duced in SC and OM adipocytes compared to the respective
fication mixtures (10 ll) contained 5 ll of Absolute SYBR Green Rox AT (Fig. 1A). This result strongly suggested that the propor-
Mix (AB gene), 0.3 ll of a mixture of 10 lM reverse and forward prim-
ers, 3.4 ll nuclease-free water and 1 ll diluted cDNA template. Quan- tion of remaining resident macrophages in the adipocyte frac-
titative RT-PCR measurements were performed on an ABI Prism tion was low and that adipocytes per se expressed CD14 and
7900 Sequence Detector system with SDS 2.1 Software (Applied Bio- CD68 but not F4/80. A similar situation occurred in human
systems). PCR cycles proceeded as follows: Taq activation (10 min, SC AT, in which CD14 and CD68 but not EMR1-F4/80
95 C), then 40 cycles of denaturation (15 s, 95 C), annealing (30 s,
mRNAs were strongly expressed in isolated adipocytes
60 C) and extension (30 s, 72 C). The melting-curve analysis showed
the specificity of the amplifications. Threshold cycle, which inversely (Fig. 1B). Others have shown a higher expression of CD68 in
correlates with the target mRNA level, was measured as the cycle
number at which the reporter fluorescent emission appears above the
background threshold. The relative mRNA levels were estimated by
the standard method using glyceraldehydes-3-phosphate dehydroge-
nase (GAPDH) as the reference gene.
Primer sequences were as follows (forward-reverse):
Human:
hGAPDH (5 0 -AACAGCCTCAAGATCAGCAA-3 0 )–(5 0 -CAGTCT-
GGGTGGCAGTGAT-3 0 ),
hCD68 (5 0 -GAACCCCAACAAAACCAAG-3 0 )–(5 0 -GATGAGAG-
GCAGCAAGATG-3 0 ),
hCD14 (5 0 -TCTCTGTCCCCACAAGTTCC-3 0 )–(5 0 -CCCGTCCA-
GTGTCAGGTTATC-3 0 ),
EMR1 (5 0 -CCAAGGGGGATAAGATGAAG-3 0 )–(5 0 -CACCAAGG-
AGATGATTAATGCC-3 0 ),
Mouse:
F4/80 (5 0 -TTTCCTCGCCTGCTTCTTC-3 0 )–(5 0 -CCCCGTCTCTG-
TATTCAACC-3 0 ),
CD68 (5 0 -AGGGTGGAAGAAAGGTAAAGC-3 0 )–(5 0 -AGAGCA-
GGTCAAGGTGAACAG-3 0 ),
CD14 (5 0 -GGCTTGTTGCTGTTGCTTC-3 0 )–(5 0 -CAGGGCTCCG-
AATAGAATCC-3 0 ),
GAPDH (5 0 -AACGACCCCTTCATTGAC-3 0 )–(5 0 -TCCACGACA-
TACTCAGCAC-3 0 ).

2.4. Flow cytometry and immunocytochemistry

Assessment of mouse adipocyte (3T3-F442A, BFC-1) and macro-
phage (RAW264.7) cell lines was performed with Alexa 488-conju-
gated CD14 antibody (Caltag laboratories, Burlingame), or Alexa
647-conjugated CD68 antibody (Serotec, Cergy Saint-Christophe)
and rat anti-F4/80 plus FITC-conjugated secondary antibodies
(Jackson immunoResearch, Cambridgeshire). Non-specific binding
was controlled with specific isotype antibodies, mouse Alexa 488-
conjugated IgG1 and rat Alexa 647-conjugated IgG2a (Caltag labo-
ratories, Burlingame). Cell suspensions were analyzed on an Epics
Elite-ESP flow cytometer (Beckman-Coulter, France). The same la-
belled cells were mounted and used for microscopic observation
using a confocal Zeiss LSM510 microscope. Each experiment was
repeated at least twice.
3. Results and discussion
We used quantitative RT-PCR experiments to study the le-
vel of expression of CD14, CD68 and EMR1-F4/80 genes in
Fig. 1. Expression of CD14, CD68 and EMR1-F4/80 mRNAs in
mouse and human AT, adipocytes and stroma-vascular cells. (A)
Mouse RNA was isolated from SC and OM AT, as well as from ADIP
and SVF cells from mice obtained following two successive treatments
with liberase 3 as described in Section 2, the final centrifugation
leading to an adipocyte-enriched fraction (ADIP) and a pelleted
macrophage-enriched fraction (SVF). CD14, CD68 and F4/80 mRNA
concentrations were evaluated by real time RT-PCR. Results are
expressed in % mRNA relative to SC AT taken as 100% for each
marker. Relative expression of each marker to the GAPDH control
gene expression in SC AT are: 1.1% for CD14, 0.2% for CD68 and
0.84% for F4/80. (B) Human RNA isolation and analysis are identical
to those for mice except that in this case, only SC AT has been studied.
Relative expression of each marker to the GAPDH control gene
expression are : 3.4% for CD14, 3.9% for CD68 and 0.5% for EMR1-
F4/80. Mean ± S.E.M. from three separate experiments is shown.
W. Khazen et al. / FEBS Letters 579 (2005) 5631–5634 5633

human OM AT compared to SC AT [18]. Considering that F4/
80 is macrophage-specific, we found that in rodent SC, adipo-
cyte CD14 and CD68 mRNAs correspond, respectively, to
11.1% and 10.8% of those mRNAs in the macrophage-contain-
ing SVF of cells. The same calculation for OM AT gives 9.1%
and 16.2%. In human SC adipocytes, the percentages found
were much higher than in rodent SC adipocytes, i.e. 35.4%
and 62.2% for CD14 and CD68, respectively.
SVF contains macrophages plus other cell types, for instance
fibroblasts and preadipocytes. To discriminate between these
cell types, we decided to use two distinct model cell systems
of fibroblasts-like cells (adipoblast) differentiation into adipo-
cytes, the 3T3-F442A and the BFC-1 mouse cell lines. As
shown in Fig. 2A, CD14 and CD68 mRNAs (but not F4/80,
data not shown), were present in both cell lines throughout dif-
ferentiation, demonstrating that fibroblast-like cells (adipo-
blasts, 2 days before confluence; day
2), preadipocytes
(confluence; day 0) and adipocytes (day 13), all expressed
CD14 and CD68 transcripts, although at various levels, with
a peak at day 0 and day 2. Like in mice AT in vivo, CD14
mRNA was present at higher concentration than CD68
(Fig. 2A). We chose cells at day 2 to evaluate the presence of
CD14, CD68 and F4/80 by fluorescence-activated cell sorter
(FACS). Fig. 2B shows that about 25% of 3T3-F442A and
BFC-1 cells expressed both CDs. As expected from the RT-
PCR studies, F4/80 was absent from both lines. The highly po-
sitive RAW264.7 cells, a control macrophage cell line from
mice, showed antibody specificity for the three markers. F4/
80 was present in RAW264.7 only. Immunofluorescence stud-
ies with the same antibodies as those used for FACS pointed
out the presence of CD14 and CD68 at the cell surface of both
lines (Fig. 2B, inset).

Fig. 2. Expression of CD14, CD68 and F4/80 in 3T3-F442A, BFC-1 and RAW264.7. (A) RNA was isolated from 3T3-F442A and BFC-1 cells at
various days relative to confluence (day 0). CD14 and CD68 mRNA concentrations were evaluated by quantitative RT-PCR. Results are expressed in
% mRNA relative to CD68 mRNA taken as 100% at confluence. Relative expression of CD68 to the GAPDH control gene expression at confluence
are: 1.1% for 3T3-F442A and 0.4% for BFC-1. Mean ± S.E.M. from three separate experiments is shown. (B) Representative flow cytometry analysis
of 3T3-F442A, BFC-1 and RAW264.7 cells using specific CD14, CD68 or F4/80 antibody as described in Section 2. Insert: photomicrographs of
membrane-labelled CD14 (green) and CD68 (red) for 3T3-F442A and BFC-1.
5634
Taken together, our results demonstrate that among the
three expected macrophage-specific markers, only EMR1-F4/
80 shows a strict specificity. The observation that EMR1-F4/
80 is neither expressed in human and murine adipocytes nor
in 3T3-F442A and BFC-1 cells at different development stages
(fibroblasts, preadipocytes and adipocytes) makes us consider
this protein as the best macrophage marker. Both CD14 and
CD68 are expressed in fibroblasts-like cells (adipoblasts), pre-
adipocytes and adipocytes in addition to macrophages. Recent
studies have shown that CD68 antibodies overlap in specificity
with fibroblasts [19]. This observation reinforces our demon-
stration that CD14 and CD68 are not macrophage-specific
markers. AT is an important mediator of inflammation and in-
nate immunity [20,21]. Various AT depots display differential
phagocytic and microbicidal activities depending on their local-
ization [12] and preadipocytes have the potential to be con-
verted into macrophages [22]. We demonstrate here that
adipocytes also present macrophage-like characters. Other
studies show the presence of MCPI in human adipose tissue
[18,23] and expression of the macrophage migration inhibitory
factor (MIF) in murine adipose tissue [8] and 3T3-L1 cell line
[24]. Additionally, markers, originally described as adipocyte-
specific, are present in macrophages [25,26]. The role of
CD14 and CD68 in the adipocyte differentiation process re-
mains an opened issue to be addressed.
Acknowledgements: This work was supported by the Institut National
de la Recherche Médicale, the Université René Descartes and by grants
from the Association pour la Recherche contre le Cancer and the Asso-
ciation de Langue Française pour lÕEtude du Diabète et des Maladies
Métaboliques-GlaxoSmithKline (to C.F.). W.K. is recipient of a fel-
lowship from the Centre français pour lÕaccueil et les échanges interna-
tionaux (EGIDE). We thank Dr. Louis Benelli (Institut Chirurgie
Plastique Clinique Hartman, Paris) for his helpful involvement with
human studies.
References
[1] Taylor, P.R., Martinez-Pomares, L., Stacey, M., Lin, H.H.,
Brown, G.D. and Gordon, S. (2005) Macrophage receptors and
immune recognition. Annu. Rev. Immunol. 23, 901–944.
[2] Martinez-Pomares, L., Platt, N., McKnight, A.J., da Silva, R.P.
and Gordon, S. (1996) Macrophage membrane molecules: mark-
ers of tissue differentiation and heterogeneity. Immunobiology
195, 407–416.
[3] Devitt, A., Moffatt, O.D., Raykundalia, C., Capra, J.D., Simmons,
D.L. and Gregory, C.D. (1998) Human CD14 mediates recogni-
tion and phagocytosis of apoptotic cells. Nature 392, 505–509.
[4] Triantafilou, M. and Triantafilou, K. (2002) Lipopolysaccharide
recognition: CD14, TLRs and the LPS-activation cluster. Trends
Immunol. 23, 301–304.
[5] Ramprasad, M.P., Terpstra, V., Kondratenko, N., Quehenberger,
O. and Steinberg, D. (1996) Cell surface expression of mouse
macrosialin and human CD68 and their role as macrophage
receptors for oxidized low density lipoprotein. Proc. Natl. Acad.
Sci. USA 93, 14833–14838.
[6] Weisberg, S.P., McCann, D., Desai, M., Rosenbaum, M., Leibel,
R.L. and Ferrante Jr., A.W. (2003) Obesity is associated with
macrophage accumulation in adipose tissue. J. Clin. Invest. 112,
1796–1808.
[7] Curat, C.A., Miranville, A., Sengenes, C., Diehl, M., Tonus, C.,
Busse, R. and Bouloumie, A. (2004) From blood monocytes to
adipose tissue-resident macrophages: induction of diapedesis by
human mature adipocytes. Diabetes 53, 1285–1292.
[8] Skurk, T., Herder, C., Kraft, I., Muller-Scholze, S., Hauner, H.
and Kolb, H. (2004) Production and release of macrophage
W. Khazen et al. / FEBS Letters 579 (2005) 5631–5634
migration inhibitory factor from human adipocytes. Endocrinol-
ogy 146, 1006–1611.
[9] Xu, H., Barnes, G.T., Yang, Q., Tan, G., Yang, D., Chou, C.J.,
Sole, J., Nichols, A., Ross, J.S., Tartaglia, L.A. and Chen, H.
(2003) Chronic inflammation in fat plays a crucial role in the
development of obesity-related insulin resistance. J. Clin. Invest.
112, 1821–1830.
[10] Wellen, K.E. and Hotamisligil, G.S. (2003) Obesity-induced
inflammatory changes in adipose tissue. J. Clin. Invest. 112,
1785–1788.
[11] Cousin, B., Munoz, O., Andre, M., Fontanilles, A.M., Dani, C.,
Cousin, J.L., Laharrague, P., Casteilla, L. and Penicaud, L. (1999)
A role for preadipocytes as macrophage-like cells. FASEB. J. 13,
305–312.
[12] Villena, J.A., Cousin, B., Penicaud, L. and Casteilla, L. (2001)
Adipose tissues display differential phagocytic and microbicidal
activities depending on their localization. Int. J. Obes. Relat.
Metab. Disord. 25, 1275–1280.
[13] Lin, Y., Lee, H., Berg, A.H., Lisanti, M.P., Shapiro, L. and
Scherer, P.E. (2000) The lipopolysaccharide-activated toll-like
receptor (TLR)-4 induces synthesis of the closely related
receptor TLR2 in adipocytes. J. Biol. Chem. 275, 24255–
24263.
[14] Green, H. and Kehinde, O. (1976) Spontaneous heritable changes
leading to increased adipose conversion in 3T3 cells. Cell 7, 105–
113.
[15] Forest, C., Doglio, A., Ricquier, D. and Ailhaud, G. (1987) A
preadipocyte clonal line from mouse brown adipose tissue. Short-
and long-term responses to insulin and beta-adrenergics. Exp.
Cell. Res. 168, 218–232.
[16] Plee-Gautier, E., Aggerbeck, M., Beurton, F., Antoine, B.,
Grimal, H., Barouki, R. and Forest, C. (1998) Identification of
an adipocyte-specific negative glucose response region in the
cytosolic aspartate aminotransferase gene. Endocrinology 139
(12), 4936–4944.
[17] Lafontan, M. and Berlan, M. (2003) Do regional differences in
adipocyte biology provide new pathophysiological insights?.
Trends Pharmacol. Sci. 24 (6), 276–283.
[18] Bruun, J.M., Lihn, A.S., Pedersen, S.B. and Richelsen, B. (2005)
Monocyte chemoattractant protein-1 release is higher in visceral
than subcutaneous human adipose tissue (AT): implication of
macrophages resident in the AT. J. Clin. Endocrinol. Metab. 90,
2282–2289.
[19] Inoue, T., Plieth, D., Venkov, C.D., Xu, C. and Neilson, E.G.
(2005) Antibodies against macrophages that overlap in specificity
with fibroblasts. Kidney Int. 67, 2488–2493.
[20] Berg, A.H. and Scherer, P.E. (2005) Adipose tissue, inflammation,
and cardiovascular disease. Circ. Res. 96, 939–944.
[21] Pond, C.M. (2005) Adipose tissue and the immune system.
Prostagl. Leukot. Essent. Fatty Acids 73, 17–30.
[22] Charriere, G., Cousin, B., Arnaud, E., Andre, M., Bacou, F.,
Penicaud, L. and Casteilla, L. (2003) Preadipocyte conversion to
macrophage. Evidence of plasticity. J. Biol. Chem. 278, 9850–
9855.
[23] Christiansen, T., Richelsen, B. and Bruun, J.M. (2005) Monocyte
chemoattractant protein-1 is produced in isolated adipocytes,
associated with adiposity and reduced after weight loss in morbid
obese subjects. Int. J. Obes. Relat. Metab. Disord. 29, 146–150.
[24] Hirokawa, J., Sakaue, S., Tagami, S., Kawakami, Y., Sakai, M.,
Nishi, S. and Nishihira, J. (1997) Identification of macrophage
migration inhibitory factor in adipose tissue and its induction by
tumor necrosis factor-alpha. Biochem. Biophys. Res. Commun.
235, 94–98.
[25] Ricote, M., Li, A.C., Willson, T.M., Kelly, C.J. and Glass,
C.K. (1998) The peroxisome proliferator-activated receptor-
gamma is a negative regulator of macrophage activation.
Nature 391, 79–82.
[26] Makowski, L., Brittingham, K.C., Reynolds, J.M., Suttles, J. and
Hotamisligil, G.S. (2005) The fatty acid-binding protein, aP2,
coordinates macrophage cholesterol trafficking and inflammatory
activity. Macrophage expression of aP2 impacts peroxisome
proliferator-activated receptor gamma and IkappaB kinase activ-
ities. J. Biol. Chem. 280, 12888–12895.