(The FASEB Journal. 1999;13:305-312.)
© 1999 FASEB
A role for preadipocytes as macrophage-like cells
B. COUSINa , 1 ,
O. MUNOZb ,
M. ANDREa ,
A. M. FONTANILLESa ,
C. DANIc ,
J. L. COUSINb ,
P. LAHARRAGUEa ,
L. CASTEILLAa and
L. P ÉNICAUDa
a ESA 5018-UPS CNRS, IFR 31, CHU Rangueil, 31403 Toulouse Cédex;
b INSERM U343, Hôpital de l'Archet, BP 79, 06202 Nice Cédex 3, France; and
c CNRS UMR 6543, Université de Nice-Sophia Antipolis, 06108 Nice Cédex 2, France
 |
ABSTRACT
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Several lines of evidence have supported a link betweeen adipose tissue
and immunocompetent cells. This link is illustrated in obesity, where
excess adiposity and impaired immune function have been described in
both humans and genetically obese rodents. In addition, numerous
factors involved in inflammatory response are secreted by both
preadipocytes and macrophages. Here we show that proliferating
preadipocytes in cell lines and primary cultures, develop phagocytic
activity toward microorganisms. This is demonstrated by phagocytosis
assays and confocal microscopy. This function disappears when
preadipocytes stop proliferating and differentiate into adipocytes.
After phagocytosis, preadipocytes show microbicide activity via an
oxygen-dependent mechanism. In addition, preadipocytes as well as
adipocytes are stained with MOMA-2, a marker of monocyte-macrophage
lineage, but are negative for specific mature macrophage markers (F4/80
and Mac-1). These results suggest that preadipocytes could function as
macrophage-like cells and raise the possibility of a potential direct
involvement of adipose tissue in inflammatory processes.—Cousin, B.,
Munoz, O., André, M., Fontanilles, A. M., Dani, C., Cousin,
J. L., Laharrague, P., Casteilla, L., Pénicaud, L. A role
for preadipocytes as macrophage-like cells.
Key Words: tumor necrosis factor • cell culture • ROS • stromal cells • antigens • SOD
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INTRODUCTION
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IN HUMANS, several types of observations suggest that
obesity may be associated with altered immunity (1). Epidemiological
and clinical survey data suggest that the incidence and severity of
infectious illness are higher in obese than nonobese individuals.
Indeed, phagocytes may be particularly affected, the number of
monocytes that matured into macrophages being 50% lower in obese
compared to lean individuals (2). This relationship between excess
adiposity and immunity has been also studied in animal models of
obesity, particularly in genetically obese rodents. For example, ob/ob
mice show a decreased immunocompetence, reductions in various aspects
of cell-mediated immunity, and decreased resistance to bacterial and
viral infection (1). In addition, macrophages from Zucker sedentary
animals are significantly less able to kill phagocytized yeasts (3). It
must be emphasized that macrophages play a critical role in protecting
the host against infections via uptake and killing of pathogenic agents
such as bacteria (4).
This link between adipose tissue and immunity is reinforced by the
demonstration that adipose tissue may also be a source of factors
related not only to fat metabolism. Indeed, in both humans and rodents,
adipose tissue transcribes high levels of proteins involved in the
alternative pathway of complement such as adipsin/factor D (5), factors
C3 and B (6), acylation stimulating protein (ASP)2 (7),
together with the proteins that control their expression (8). Other
secreted proteins, such as interleukins and tumor necrosis factor alpha
(TNF
), a major mediator of inflammation, are secreted by adipocytes
and macrophages (5, 9–11). In addition, the recently discovered leptin
has been shown to be involved in both energy metabolism and
proinflammatory immune responses, especially via macrophage activity
and T lymphocyte proliferation (12, 13). These points raise a question
about the relationships that could exist between adipocytes and immune
cells, especially macrophages, which has led us to compare both cell
types. Macrophages could be characterized by specific
immunocytochemical stainings as well as functional features, including
phagocytosis and antimicrobial activity by oxygen-dependent or
independent mechanisms. We thus looked for both immunological and
functional features in preadipocytes and adipocytes from cell lines and
primary cultures.
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MATERIALS AND METHODS
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Animals
Adult female Swiss mice and Wistar rats were housed in a
conventional animal quarter. Animals were killed by decapitation.
Adipose tissues were rapidly removed and processed for
immunocytochemical analysis or primary cultures.
RNA extraction and Northern blot experiments
Total RNA were extracted using the Chomczynski's method (14).
Northern blot analysis was performed as previously described and
hybridized with aP2 cDNA (15).
Glycerol phosphate dehydrogenase (GPDH) activity
GPDH activity was assayed spectrophotometrically in cell
homogenates, as previously described (16), and expressed in percent of
maximal effect. Protein content of cell homogenates were determined
according to the Bradford technique (17).
Cell culture
3T3-L1 cells were maintained until confluence (day 0) in
Dulbecco's modified Eagle's medium (DMEM) containing 10% of
heat-inactivated fetal calf serum (FCS) and 2 mM glutamine. All
components for culture were purchased from Life Technologies
(Cergy-Pontoise, France). Fully differentiated adipocytes were obtained
8–15 days after induction of differentiation, as described previously
(18). A growth curve was obtained by counting cells daily in a cell
counter (coulter Z1). Murine macrophage RAW 264.7 cell line was grown
in the same medium as were 3T3-L1 cells (19).
Embryonic myoblasts C2C12 were maintained in DMEM supplemented with 2
mM glutamine and 10% FCS. At confluence, cells were shifted into DMEM
supplemented with 2% horse serum to induce differentiation.
Primary cultures were performed from rat or mice inguinal adipose
according to Björntorp et al. (20), with minor modifications.
Isolated stromal cells were suspended in growth medium consisting of
DMEM:F12 (1:1) supplemented with 10% FCS and antibiotics until
confluence (day 0). Differentiation was then induced by replacing
growth medium with DMEM:F12 supplemented with insulin (850 nM),
triiodothyronin (2 nM), and transferrin (10 µg/ml) .
Peritoneal macrophages were obtained from Swiss mice by lavage of the
peritoneal cavity with 10 ml of 0.9% sodium chloride solution and
maintained in vitro in DMEM + 10%FCS.
Immunocytochemistry
Cells were attached to a silanized slide by a cytospin and fixed
in acetone at -20°C for 20 min. Endogenous peroxidase was removed by
a 10 min incubation in peroxidase blocking reagent. Standard
methodology was used to detect immunological staining. Blocking and
washing were performed using phosphate-buffered saline (PBS) containing
1% milk. Antibodies were obtained from Biosource (Rungis, France). For
MOMA-2 and ED2 antibodies, the dilutions used were 1:25 and 1:100,
respectively. The other antibodies (Mac-1 and F4/80) were not diluted.
The second antibody was an anti-rat immunoglobulin coupled with
horseradish peroxidase (HRP) (dilution 1:100). HRP activity was
revealed by using AEC substrate-chromogen (Dako, Trappes, France).
Negative controls were performed by omitting the first antibody.
Phagocytosis studies
Phagocytosis assays were performed by incubating cells with 10
7 zymosan particles/ml (Sigma Chemicals, St. Louis, Mo.) in
a PBS buffer for 45 min at 37°C (21). Cells were then cytocentrifuged
and stained with Meygrunwald-Giemsa before being counted under a light
microscope.
Confocal microscopy
Nonconfluent 3T3-L1 cells were incubated for 45 min with
zymosan-FITC (100 µg) (Molecular Probes Europe, Leiden, The
Netherlands), fixed in 3.7% paraformaldehyde, permeabilized, and
stained with 0.1% Triton X-100 containing 2 µg/ml propidium iodide.
Confocal microscopy was performed using an Ultima cooled confocal
scanning cytometer (Meridian Instrument, Inc., Okemos, Mich.) equipped
with an argon ion laser (Coherent Innova Interprise) coupled with an
Olympus IMT-2 inversed microscope. FITC fluorescence emission and
propidium iodide were separated using a dichroic mirror (575 nm short
pass) and measured with adequate isolation bandpass filters (530 nm for
FITC and a 605 long pass for propidium iodide). Automated captured 1
µm sections along the Z-axis were performed from the upper part to
the bottom of the cell at a magnitude of 100x.
Antimicrobial activity
Nonconfluent cells cultured in Lab-Tex tissue culture slides
were challenged with 50 µl of Candida parapsilosis(108/ml), with or without superoxide dismutase (SOD;
500U/ml), for 45 min at 37°C. Slides were stained successively with
0.01% acridine orange and 0.05% crystal violet (22), then examined
using fluorescent microscopy (Leica, DMRB). Candidacidal activity
denotes the percentage of the total number intracellular
Candida that had been killed.
Statistical analysis
Results are expressed as means±SEM. The statistical
significance of differences between means was evaluated using the
paired (SOD inhibition) or unpaired (kinetics) t test.
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RESULTS
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Immunocytochemistry
Expression of specific markers of monocyte-macrophage lineage was
examined on both growing and mature 3T3-L1 cells. Indeed, macrophages
can be characterized by the expression of different surface antigens
(23–25). For example, MOMA-2 is expressed early during the development
of mononuclear phagocytes and is especially useful for identification
of macrophages in lymphoid tissues, (23, 26). F4/80 and Mac-1 (
chain of the type 3 complement receptor) are, on the contrary,
expressed on most mature macrophages (26, 27). As shown in Fig. 1A, B
),3T3-L1 cells were positive for MOMA-2, whatever the stage of
differentiation. By contrast, no staining was obtained with both F4/80
and Mac-1 monoclonal antibodies under the same conditions (Fig. 1D, F
). RAW macrophage cell line (Fig. 1C, E
) and
peritoneal macrophages (data not shown), used as positive controls,
were positive for all the antibodies tested, whereas both
undifferentiated and differentiated myoblastic C2C12 cells showed no
staining (data not shown).
Phagocytosis
To determine whether adipose cells could function as macrophages,
phagocytosis assays were performed using zymosan particles with
peritoneal macrophages and both growing and mature 3T3-L1 cells.
Confocal and optical microscopy were used, to respectively determine
whether zymosan was inside or attached to the cell and to measure the
kinetic of phagocytosis in culture. Confocal microscopy using
zymosan-FITC was performed on preadipocytes and confirmed the
intracytoplasmic localization of ingested particles (Fig. 2
).
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Figure 2. Intracellular localization of zymosan particles. Confocal sections in
the Z-axis of 3T3-L1 preadipocyte showing green zymosan-FITC particles
within the cell cytoplasm labeled with propidium iodide.
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The proliferation and differentiation process of 3T3-L1 cells were
assessed by counting cells and expression of aP2, a specific marker of
mature adipocyte (15). Northern blot confirmed the appearance of aP2
expression from 5 to 8 days after confluence and its increase until the
end of the culture, as previously described (15) (Fig. 3A
).A large increase in cell number was observed until confluence (day 0)
and reached a steady state thereafter (Fig. 3B
).
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Figure 3. Phagocytosis of zymosan particles by 3T3-L1 cell line in culture. aP2
expression (A) and growth curve (B) of 3T3-L1 in
culture. Each panel was representative of three different experiments.
C Kinetic of phagocytosis during cell culture. At day 0
(confluence), cells were switched in differentiating medium (continuous
lines) or maintained only in the presence of FCS (broken lines).
Results are the mean ±SEM of 10 independent experiments
and expressed as the percent of cells showing phagocytosis granules.
**P < 0.01; ***P < 0.001 vs. values
obtained at 24 h.
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The great majority of growing preadipocytes showed the ability to
phagocytose, this ability not being significantly different from that
of peritoneal macrophages (Fig. 3C
). By contrast, after
confluence, when the cells were induced to differentiate, the
percentage of cells able to phagocytosis decreased significantly.
Similar results were obtained with both growing and differentiated
cells attached to the plates (data not shown). This decrease in
phagocytic cell number occurred when preadipocytes stopped
proliferating, even if they did not differentiate (Fig. 3C
).
This could be reversed by replating cells at a lower density once they
reached confluence (69%±8.5 after replating vs. 30%±3.8 before).
Opsonization of zymosan did not change the percentage of phagocytic
cells (data not shown). This high capability of phagocytosis appears to
be specific for the adipose cell type, since it was also observed in
the 3T3-F442A cell line (data not shown) but not with undifferentiated
embryonic myoblastic cells (C2C12) (Fig. 3C
).
Antimicrobial activity
In macrophages, phagocytosis is often followed by killing of
ingested microorganisms via oxidative and nonoxidative mechanisms. Such
a microbicidal activity was assessed in 3T3-L1 cells by using live
C. parapsilosis, which are especially sensitive to reactive
oxygen species (ROS). This technique was chosen because it allows us to
visualize and thus morphologically characterize cells that have
ingested yeasts. After staining with acridine orange (12, 22), both
orange (dead) and green (alive) yeasts were observed in 3T3-L1
preadipocytes (Fig. 4A
),as well as in peritoneal macrophages (Fig. 4B
). By contrast,
no orange particles were observed in the myoblast cell line C2C12
whatever the differentiation stage (Fig. 4C
).
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Figure 4. Microbicidal activity in both 3T3-L1 and peritoneal macrophages. 3T3-L1
(A), peritoneal macrophages (B), and
undifferentiated C2Cl2 (C) were
plated overnight on glass slides and incubated for 45 min with
Candida parapsilosis. Ingested yeasts revealed both bright
green and orange fluorescence, indicating live and dead
Candida within the cell cytoplasm, respectively.
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This antimicrobial activity of 3T3-L1 preadipocytes and macrophages is
partly and significantly inhibited by preincubation with SOD
(Fig. 5
).
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Figure 5. Measurement of candidacidal activity. Antimicrobial activity measured
without (plain bars) or with (hatched bars) SOD (500 U/ml) pretreatment
in 3T3-L1 preadipocytes and peritoneal macrophages (PM). Results are
the mean ±SEM of four independent experiments and are
expressed as the percent of activity observed without SOD.
*P < 0.05; **P < 0.01.
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Primary cultures
Knowing that cell lines could possess specific characteristics,
similar investigations were performed on rat and mice primary cultures,
and both types of culture gave similar results. It is noteworthy that
primary cultures were performed from stroma-vascular fraction (SVF) of
subcutaneous fat and contained different cell types, including
preadipocytes and macrophages, but no mature adipocytes. As shown in
Fig. 6A
,rat primary cell cultures exhibited phagocytosis activity. The percent
of phagocytic cells was lower than in the 3T3-L1 cell line, as
expected, due to the heterogeneity. Phagocytosis decreased slightly
during the culture, and this was concomitant with the enhancement of
GPDH activity used to assess adipocyte differentiation (Fig. 6B
).
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Figure 6. Phagocytosis and GPDH activity in rat primary cultures. A)
Percentage of cells showing phagocytic activity (bars) and ED2 staining
(line). B) GPDH activity during differentiation process,
expressed in percent of maximal activity. Results are expressed as the
mean ±SEM of five independent experiments.
*P < 0.05; **P < 0.01.
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We used ED2 antibody, a marker of tissue resident macrophages, to
determine the percent of this cell type isolated in the SVF. During the
culture, the number of cells positive for ED2 antibody did not vary
significantly and represented only 15% of the population (Fig. 6A
).
In addition, phagocytic cells showed microbicidal activity, as revealed
by orange yeasts in their cytoplasm (Fig. 7
).Similar to our observations in the 3T3-L1 cell line, this activity
showed a 50% inhibition by SOD pretreatment (Fig. 8
).
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Figure 7. Antimicrobial activity in primary cultures. Stroma-vascular fraction
was isolated from inguinal white adipose tissue, plated on glass slides
overnight, and then incubated for 45 min with Candida
parapsilosis. Staining with acridine orange/crystal violet was
performed to reveal live (green) and dead (orange) intracellular
microorganisms.
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Figure 8. Inhibition of candidacidal activity by SOD in rat primary cultures.
Candidacidal activity measured without (plain bars) or with (hatched
bars) SOD (500 U/ml) pretreatment, expressed as the percent of
antimicrobial activity observed without SOD. Results are expressed as
the mean ±SEM of five independent experiments.
*P < 0.05; **P < 0.01.
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DISCUSSION
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The results of this study demonstrated that preadipocytes exhibit
common functional features with monocyte-macrophage cells.
Macrophages are involved in a wide variety of processes, including
clearance of potentially harmful substances and killing of
microorganisms and tumor cells (4). These functions are triggered by a
specific phagocytosis activity that characterizes cells from the
mononuclear phagocyte. This main feature of macrophages is exhibited by
preadipocytes in cell lines and primary cultures, although to a lesser
extent. Indeed, primary cultures are performed from stroma-vascular
fractions that contains many cell types. In contrast, 3T3-L1 and
3T3-F442A cell lines are considered to be unipotent clonal cell lines,
studied extensively for their ability to differentiate into adipocytes,
and may represent a unique cell population.
The high phagocytosis ability of preadipocytes seems to depend on the
proliferative state of these cells. Indeed, when cell growth was
stopped by confluence, phagocytosis activity decreased, and this could
be reversible by replating cells at a lower density. This potential
link between proliferation and phagocytosis was suggested by previous
studies using 3T3-T cell lines. This subclone of NIH-3T3 has been shown
to differentiate into adipocytes in some specific conditions. In the
presence of human plasma, 3T3-T could exhibit both phagocytosis
granules and nonspecific esterase activity, a marker of mature
macrophages (28).
In macrophages, phagocytosis is often followed by killing of ingested
microorganisms via oxidative and nonoxidative mechanisms. The
antimicrobial activity observed in preadipocytes could be comparable to
the oxidative burst measured in similar conditions in macrophages (22).
Indeed, this activity is partly dependent on SOD, indicating that ROS
are involved in this phenomenon. This is in agreement with previous
reports demonstrating that, as for macrophages, preadipocytes possess
an H2O2 generating system (29, 30). The
antimicrobial activity remaining after SOD treatment could be dependent
on other killing systems, including elastase, other proteases, and
lysozomal enzymes.
Various populations of macrophages exist in particular locations,
arising from different lineages and having specific functions (23–25).
We have used three different markers of monocyte/macrophage lineage to
characterize 3T3-L1 cells. The absence of staining of preadipocytes
with Mac-1, which mediates uptake of opsonized microorganisms or
particles (23), is in agreement with the absence of effect of zymosan
opsonization. The immunological characterization of 3T3-L1 suggests
that they could be related to macrophage lineage, but preadipocytes
seem clearly different from mature resident macrophages. This
hypothesis could also be proposed for primary cultures. Since
preadipocytes do not seem to be stained with mature macrophage markers,
we used an antibody to ED2 antigen that recognizes resident macrophages
(24, 26, 31) to distinguish between both cell types present in such
cultures. Only 30% of phagocytic cells are ED2 positive; this means
that the large majority of phagocytic cells (70%) could not be
identified as mature macrophages.
Apart the fact that adipose cells secrete numerous factors related to
inflammatory processes, the relationship between adipose cells and
macrophages is supported by recent studies. Indeed, an inverse
relationship between adipocyte and macrophage differentiation has been
described. Peroxisome proliferator-activated receptor-
(PPAR), a
transcription factor that activates adipocyte differentiation, is a
negative regulator of macrophage activity (32, 33). In contrast, the
adipogenic actions of PPAR agonists are antagonized by several
cytokines, including TNF (34, 35). Thus, it appears that when
differentiation into adipocytes is enhanced, an inhibition of
macrophage activation occurs. The converse is also true.
According to the evidence presented in this study, a new physiological
role is proposed for preadipocytes. First, like macrophages,
preadipocytes could be involved in the removal of dying or defective
cells, leading to a permanent tissue remodeling. Second, they could be
one of the factors in the inflammatory response and could be
particularly involved in some pathological conditions such as obesity.
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Figure 1. Immunocytochemical characterization of 3T3-L1 cells. MOMA-2 staining on
undifferentiated (A) or differentiated (B) 3T3-L1
cells. Immunocytochemistry using F4/80 (C; D) and Mac-1 (E; F)
monoclonal antibodies on preadipocytes (D; F) and
peritoneal macrophages (C, E).
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ACKNOWLEDGMENTS
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We wish to thank Dr. R. Martin for critical reading and helpful
comments, and B. Barhanin for technical assistance with cell cultures.
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FOOTNOTES
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1 Correspondence: CNRS UPRESA 5018, CHU Rangueil, 1 Av. J. Poulhès, 31403 Toulouse Cédex, France. E-mail: cousinb{at}rangueil.inserm.fr 
2 Abbreviations: ASP, acylation stimulating protein; DMEM,
Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GPDH,
glycerol phosphate dehydrogenase; HRP, horseradish peroxidase; PBS,
phosphate-buffered saline; PPAR, peroxisome proliferator-activated
receptor-; ROS, reactive oxygen species; SOD, superoxide dismutase;
SVF, stroma-vascular fraction; TNF, tumor necrosis factor.
Received for publication July 29, 1998.
Revision received October 5, 1998.
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ABSTRACT
INTRODUCTION
