From the Unité Mixte de Recherche 5018 Université Paul Sabatier CNRS, IFR31, Bat L1, Centre Hospitalier
Universitaire Rangueil, 31403 Toulouse, France and the
¶ Unité Mixte de Recherche 866 Différenciation
Cellulaire et Croissance, INRA, 34060 Montpellier Cedex 01, France
Received for publication, October 23, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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Preadipocytes are present throughout adult life
in adipose tissues and can proliferate and differentiate into mature
adipocytes according to the energy balance. An increasing number of
reports demonstrate that cells from adipose lineages
(preadipocytes and adipocytes) and macrophages share numerous
functional or antigenic properties. No large scale comparison
reflecting the phenotype complexity has been performed between these
different cell types until now. We used profiling analysis to define
the common features shared by preadipocyte, adipocyte, and macrophage
populations. Our analysis showed that the preadipocyte profile is
surprisingly closer to the macrophage than to the adipocyte profile.
From these data, we hypothesized that in a macrophage environment
preadipocytes could effectively be converted into macrophages. We
injected labeled stroma-vascular cells isolated from mouse white
adipose tissue or 3T3-L1 preadipocyte cell line into the peritoneal
cavity of nude mice and investigated changes in their phenotype.
Preadipocytes rapidly and massively acquired high phagocytic activity
and index. 60-70% of preadipocytes also expressed five
macrophage-specific antigens: F4/80, Mac-1, CD80, CD86, and CD45. These
values were similar to those observed for peritoneal macrophages.
In vitro experiments showed that cell-to-cell contact
between preadipocytes and peritoneal macrophages partially induced this
preadipocyte phenotype conversion. Overall, these results
suggest that preadipocyte and macrophage phenotypes are very similar
and that preadipocytes have the potential to be very efficiently and
rapidly converted into macrophages. This work emphasizes the great
cellular plasticity of adipose precursors and reinforces the link
between adipose tissue and innate immunity processes.
White adipose tissue is known to undergo considerable changes. It
develops mainly after birth, and its mass increases greatly in obesity
(1). Conversely, it almost completely disappears during starvation or
cachexia (2). Tissue growth is due to increase in size and/or number of
mature adipocytes. These mature cells differentiate from progenitors,
i.e. preadipocytes, which are present in stroma-vascular
fraction (SVF)1 and can be
recruited throughout adult life (3). This phenomenon seems to be
reversible, and several reports describe the dedifferentiation of
mature adipocytes into a preadipocyte state (4-6).
Analysis of the literature revealed that adipocyte and
monocyte/macrophage lineages have many features in common (3, 7-14). In particular, proteins or functions known to be specific to one lineage are characterized in the other. For instance, mature adipose cells express a membrane-bound NADPH oxidase similar to that present in
specialized phagocytes (8), and preadipocytes and adipocytes secrete
numerous inflammatory cytokines such as tumor necrosis factor Recently, we reported that preadipocytes of the 3T3-L1 cell line as
well as preadipocytes in primary culture display phagocytic and
microbicidal activities similar to those of specialized phagocytic cells, i.e. macrophages (15). This activity is lower than
that of macrophages but is stimulated in inflammatory situations (16). A putative link between adipose and macrophage lineages was
strengthened by the detection on preadipocytes and adipocytes of MOMA-2
antigen, a marker of monocyte-macrophage lineage (15). However, the
most commonly used macrophage cell surface markers, F4/80 or Mac-1 (CD11b) (17), were not detected under standard conditions on preadipocytes or adipocytes.
Despite all these common features, no systematic or large scale
comparison between these two lineages has been performed to further
analyze the similarities among preadipocyte, adipocyte, and macrophage
phenotypes. This can be done by large scale gene expression data set
analysis such as transcriptome profiling. This technique is the
commonest and most powerful tool to identify genes of interest
differentially expressed according to time scale and/or treatment (18,
19). However, it has rarely been used to characterize and compare
different phenotypes (20, 21).
We used this technique to compare adipocyte and macrophage lineages and
found close similarities between preadipocyte and macrophage
phenotypes, suggesting that preadipocytes can convert to macrophages.
To test this hypothesis in vivo, we chose the peritoneal
cavity as an adequate environment to support macrophage phenotype after
preadipocyte transplantation. We were able to demonstrate that
preadipocytes can efficiently and rapidly convert into macrophages.
Reagents and Cell Lines--
All components for cell culture
were purchased from Invitrogen. The fluorescent nuclear marker
4',6-diamidino-2-phenylindole (DAPI) was purchased from Sigma Aldrich
Chimie. Adenovirus CMV-GFP was a gift from Prof. P. Moullier,
Laboratoire de Thérapie Génique, AFM, Nantes, France.
Antibodies: anti-F4/80 was obtained from Serotec (Cergy
Saint-Christophe, France), and all others were obtained from
Clinisciences (Mont-Rouge, France). Immunochemical reagents were
purchased from Dako (Trappes, France). Collagenase (Roche Diagnostics)
was purchased from Roche Molecular Biochemicals (Meylan, France). Ob17
and Hgfu cell lines were kindly provided by Dr. Dani, Nice, France.
J774.2 and RAW 264.7 cell lines were purchased from ECACC.
Animals and Cell Culture--
5-6-week-old Swiss nu/nu and
C57B6J mice (Harlan, France) were housed in microisolator cages in
conventional animal quarters. All animals had free access to food and
water and were sacrificed with CO2.
Peritoneal cells were obtained by washing the peritoneal cavity of mice
with 5 ml of sterile PBS and grown in DMEM/F12 containing 10%
heat-inactivated newborn calf serum (NCS). SVF and mature adipocytes
were obtained from inguinal fat depot as previously described (22).
Briefly, adipose tissue was digested with 2 mg/ml collagenase in 2%
albumin-containing DMEM/F12 medium for 30 min at 37 °C. After
filtration through a 25-µm nylon membrane to eliminate undigested
fragments, cells were centrifuged at 600 × g for 10 min to separate mature adipocytes from pellets of SVF cells. SVF cells
were counted (Coulter Z2) and plated (30,000 cells/cm2) in
DMEM/F12 10% NCS medium. Six hours after plating, cells were washed to
remove all non-adherent cells.
All cell lines were maintained before confluence in DMEM containing
10% heat-inactivated fetal calf serum and glutamine (2 mM). 3T3-F442A adipose differentiation was performed by
adding rosiglitazone (100 nM) at the first confluence day,
and then cells were fed every 2 days with the same medium.
3T3-F442A-differentiated cells were stopped 7 days after the beginning
of differentiation.
Preadipocyte Labeling and Implantation in the Peritoneal Cavity
of Nude Mice--
Culture medium containing 1 µg/ml DAPI or 70 plaque-forming units/cell adenovirus containing green fluorescent
protein (GFP) cDNA under the control of cytomegalovirus (CMV)
promoter was added for 12 h on SVF cells (6 h after plating) or on
non-confluent 3T3-L1 cells. Adequate adenovirus titer was determined
after dose response infection study. This concentration was chosen to
achieve significant labeling with limited toxic effects. The cells were then rinsed five times with DMEM + 10% fetal calf serum or DMEM/F12 + 10% NCS and twice with PBS. After trypsinization, preadipocytes were
collected and washed twice with PBS. Before injection in the peritoneal
cavity, cells were resuspended in PBS (2 × 106 in 500 µl). For control experiments, the supernatant of the last wash was
collected and filtered at 0.2 µm before injection (500 µl).
Preadipocyte and Macrophage Co-culture
Experiments--
Co-cultures were performed in DMEM/F12 + 10% NCS for
24 h. 3T3-L1 preadipocytes were labeled and washed as for in
vivo experiments. Labeled preadipocytes and non-labeled peritoneal
macrophages were plated (12,500 3T3-L1/cm2 and 25,000 macrophages/cm2) together onto glass slides (Lab-tek
slides, Nalge Nunc International, Naperville, IL) or in two chambers
separated by a track-etched membrane (pore size, 0.4 µm).
Measurement of Phagocytic Activity and Phagocytic
Index--
Yeast (Candida parapsilosis) was washed in PBS
and suspended at 4 × 107 yeast/ml in DMEM/F12 + 4%
NCS without antibiotics (23). After culture for 24 h on Lab-tek
slides, peritoneal cells were incubated 45 min in 1 ml of yeast
suspension at 37 °C in a CO2 chamber. Slides were then
washed three times with PBS and mounted with fluorescent mounting
medium. Mounted Lab-tek slides were observed with phased and UV lights
(UV filter: Immunocytochemistry Experiments--
For immunocytochemistry
experiments, 3T3-L1 or peritoneal cells were cytospun onto glass slides
and fixed in PBS with 3.7% paraformaldehyde for 30 min. Standard
methodology was used to detect immunological staining (15). PBS
containing 0.01% polyoxyethylenesorbitant-monolaurate (Tween 20) or
1% fatty acid free bovine serum albumin was used in DAPI-labeled or
GFP-expressing cell experiments respectively. Primary antibodies
(IgG2, rat anti-mouse) concentrations were 100 µg/ml for
anti-Mac-1, 50 µg/ml for anti-F4/80, and anti-CD86, 500 µg/ml for
anti-CD80, and 25 g/ml for anti-MHCII. A non-relevant IgG2
isotype antibody was used as negative control. The secondary antibody
was a rabbit anti-rat immunoglobulin (Fab' fragment) coupled with
rhodamin-phycoerythrin (RPE) (concentration 5 µg/ml). RPE was
visualized with fluorescent light (TX2 filter: mRNA Extraction--
RNA was isolated from cell culture,
isolated cells, or cold powdered tissue samples using Tripur reagent
(Roche Molecular Biochemicals) according to the manufacturer's
instructions. Total RNA was treated for 30 min at 37 °C with DNase
to eliminate potential DNA residues. RNA was then purified using the
phenol/chloroform extraction procedure (24). RNA quality was checked by
agarose gel electrophoresis and spectrophotometric analysis
(OD260/280).
Probe Preparation and cDNA Microarray Hybridization--
Ten
micrograms of total RNA (denatured at 70 °C for 10 min) were
reverse-transcribed for 2 h at 42 °C with 1 µl (200 units) of
Superscript II reverse transcriptase (Invitrogen), 4 µg of 25 mer/oligo(dT), 80 µCi of [ Microarray Data Analysis--
For each hybridization, the mean
of local background incremented with two S.D. was subtracted from each
spot's intensity. After this correction, all positive values were
considered as significant and transformed into log10
values. Hybridizations were then normalized by dividing the intensity
of each spot by the mean of the control spot intensity of total genomic
DNA (Tg) spotted by the manufacturer onto membrane. Profile analyses
were performed using J-Express 2.0 software (Molmine, Ref. 25) and ExpressionSieveTM 1.0 lite (BioSieve). "Random" profile
was created using the RAND Excel function (Microsoft).
Statistical Analysis--
Results are expressed as means ± S.E. The statistical significance of differences between means was
evaluated using the unpaired Student's t test. In the time
course experiments, statistical significance was evaluated using
one-way ANOVA analysis.
Microarray Analysis--
All RNA samples were purified from murine
cells to avoid mismatch recognition between the labeled probe derived
from these samples and the mouse cDNA spotted onto matrix. To
obtain the best physiological relevance, we used several cell lines for
one phenotype as well as cells freshly isolated from mouse tissues. However, for one phenotype, data from immortalized or non-immortalized cells were individualized. 3T3-L1, 3T3-F442A, Ob17, Hgfu preadipocyte, and RAW 264.7, J774.2 murine cell lines were the most often used to
investigate adipocyte and macrophage properties, respectively. Preadipocytes were enriched from SVF cells by plating and culturing for
48 h. To exclude the possibility of contaminating macrophages, we
tested for F4/80 or Mac-1-positive cells. Less than 1% of SVF cells
were expressing these antigens (data not shown). The independent hybridizations corresponding to each phenotype class were gathered together to build height representative profiles, named metaprofiles (Table I). To build them, genes were
first filtered to retain only those whom expression was detected in
more than 50% of hybridizations. The mean of their positive
hybridization values were calculated and retained. The resulting
dataset was again filtered to normalize the number of positive genes
and to exclude genes that were always negative (2040 of 5185 initial
genes) or positive (133) in all metaprofiles.
At this stage and to test the significance of clustering, we included a
"random" metaprofile in the dataset. In this metaprofile, the
values of hybridizations were distributed at random. The resulting dataset contained 468 genes. Before clustering, column variances were
normalized (J-Express, Plug-ins).
Interpretation of Metaprofiles with a Hierarchical Classification
Algorithm--
The metaprofiles in the dataset were clustered with a
hierarchical classification algorithm based on Pearson's correlation distance (d = 1 Phenotype of DAPI-positive (DAPI+) SVF Cells
after Peritoneal Injection--
Preadipocytes were labeled with DAPI
before injection into the peritoneal cavity. Two controls were
performed to demonstrate the absence of staining of endogenous
peritoneal cells by contaminating DAPI during injection of DAPI-labeled
cells. First, supernatants of labeling washings and highly concentrated
DAPI solution (500 µl) were directly injected into the peritoneal
cavity. Second, DAPI-labeled preadipocytes and non-labeled peritoneal
macrophages were co-cultured for 2 days in 2 chambers separated by a
track-etched membrane (pore size, 0.4 µm). No contaminating staining
was observed at any time in either case. Furthermore, we checked that
DAPI had no effect on the preadipocyte phenotype, its proliferation and
differentiation process in our conditions (data not shown).
Before injection, 28 ± 4% of SVF cells were phagocytizing with a
phagocytic index of 6 ± 1 and less than 1% were expressing the
mature macrophage antigens F4/80 or Mac-1. Twenty-four hours after
their injection into the peritoneal cavity, 15 ± 2% of
peritoneal cells were DAPI+, and the phagocytic
activity of these cells reached 92 ± 1% with a phagocytic
index of 18 ± 3. Similar results were obtained 6 days after
injection (Fig. 2, A and
B). The time course of these changes was investigated. As
soon as 24 h after cell transplantation, 61 ± 1% and
81 ± 3% of DAPI+ cells were strongly
stained with antibodies against F4/80 and Mac-1 respectively (Fig. 2,
C and D). All values were significantly higher
(p < 0.01) than those measured on non-injected SVF
cells and were not significantly different from those of peritoneal macrophages (98 ± 1% of macrophages presented phagocytic
activity with a phagocytic index of 19 ± 1; 63 ± 7% and
70 ± 4% were F4/80+ and
Mac-1+, respectively). Afterward, these values
remained at this level whatever the time of sampling.
Time Course of Phenotype Changes of Labeled 3T3-L1 Cells after
Injection into the Peritoneal Cavity--
To determine whether a
clonal preadipocyte population had similar behavior, immortalized
3T3-L1 preadipocytes were used. In basal conditions, the phagocytic
activity and index of 3T3-L1 were 26 ± 5% and 3 ± 1 yeast/cell, respectively (see below). None of these cells expressed
F4/80 or Mac-1 (data not shown). Six hours after injection, 23 ± 2% of peritoneal cells were DAPI+. Phagocytic activity and
index of these cells dramatically increased and reached 81 ± 6%
and 15 ± 1 respectively (Fig.
3A). 72 ± 3% of
DAPI+ cells expressed F4/80, and similar values
were observed for Mac-1 expression (Fig. 3B). No further
significant change of these parameters was observed whatever the time
of sampling (Fig. 3, A and B). All values were
significantly higher (p < 0.01) than those observed in
basal conditions and were not significantly different from those of
peritoneal macrophages. The expression of CD80, CD86, and MHCII was
then investigated. CD80, CD86, and MHCII antibodies stained 62 ± 4%, 63 ± 5%, and 20.1 ± 0.1% of peritoneal cells respectively (Fig. 3C). No staining with any antibodies was
detected in preadipocytes before injection. Twenty-four hours after
peritoneal injection of 3T3-L1 cells, 53 ± 10% and 68 ± 3% of DAPI+ cells expressed CD80 and CD86
respectively (Fig. 3C). The values obtained with CD80 and
CD86 were not significantly different from those of peritoneal cells.
Similar results were obtained with CD45 antigen, which is considered as
pan-hematopoietic (data not shown). Under the same conditions, MHCII
was never detected on 3T3L1 DAPI+ cells (Fig. 3C) as well as
on primary adipocyte precursors (data not shown).
To confirm these results with other labeling techniques, similar
transplantation experiments were performed with 3T3-L1 after adenovirus-mediated overexpression of GFP (GFP+). After
overnight infection with 70 plaque-forming units/cell of the CMV-GFP
adenovirus, 70% of the cultured 3T3-L1 cells expressed GFP. The
absence of free viral particles in the injection medium was checked by
injection of adenovirus-infected 3T3-L1 supernatant into the peritoneal
cavity. No GFP+ peritoneal cells were observed 1 day after
supernatant injection. One day after peritoneal injection, the changes
in GFP+ cells were similar to those observed in
DAPI+ cells (Fig. 4). These
observations confirmed the results obtained with the DAPI-labeled
cells.
Effect of Co-cultured Macrophages on 3T3-L1
Phenotype--
DAPI+ or GFP+ 3T3-L1 cells were
co-cultured with peritoneal macrophages either together on Lab-tek
slides or in two chambers separated by a track-etched membrane. After 1 day in co-culture without separation membrane, phagocytic activity of
the stained preadipocytes significantly increased and reached 45 ± 5% (Table II). In addition, after
co-culture with peritoneal macrophages, F4/80 and Mac-1 positive
preadipocytes were detected (8.3 ± 0.3% and 9 ± 1% of
DAPI+ cells (Table II). When preadipocytes were co-cultured
with C2C12 myoblasts, no change of the preadipocyte phenotype was
noted. When preadipocytes were co-cultured without cell-to-cell
contact, in the 2-chamber system, no change in phagocytic activity or
expression of macrophage antigens was observed (Table II).
To test the similarity between cells of adipocyte lineage
(i.e. preadipocytes and adipocytes) and macrophages without
focusing on specific genes, we decided to use the transcriptome
profiling approach. First, differences highlighted by our analysis
between immortalized and non-immortalized cells, whatever their
phenotype, confirmed the previous report and a posteriori
our approach (18).
The result of profiling is unambiguous. Indeed, macrophages are
genomically closer to adipocyte precursors than to adipocytes. The link
between a differentiated phenotype, i.e. macrophage, and
proliferating precursor cells excluded any artifactual clustering due
to proliferating potential. This closeness is true for phenotypes derived from cell lines as well as those derived from cells purified from mice. This argues for close resemblance between preadipocytes and
macrophages even though each metaprofile can be identified by specific
gene clusters under standard conditions. This conclusion is consistent
with our previous work (15). The strong proximity of preadipocyte and
macrophage phenotypes raises the question of whether preadipocytes have
the potential to transdifferentiate into macrophages. To test this
hypothesis, we chose to graft preadipocytes into a site that is
suitable for supporting the macrophage phenotype, i.e. the
peritoneal cavity.
Recent implantation studies have used the fluorescent nuclear marker
DAPI to trace implanted cells (26, 27). DAPI, which binds strongly to
adenine-thymidine-rich sites in nuclear DNA, is easy to use with all
cell types whatever their proliferation state, and labels 100% of
treated cells. These characteristics are very convenient for labeling
cells in primary culture, which are often refractory to transfection or
infection. An alternative would be to use adenovirus. Adenovirus has
been extensively used to transiently express protein in infected cells
(28), but it is not as effective as DAPI, especially for preadipocyte
cell lines and SVF, and leads in vivo to inflammatory
responses (29, 30). However, after injection, the labeling of host
cells caused by viral particles attached to the membrane of infected
cells cannot be excluded. For all these reasons, CMV-GFP adenovirus was
only used to check that our results were independent of the labeling technique.
After injection into the peritoneal cavity, most of the SVF cells as
well as 3T3-L1 preadipocytes were very rapidly converted into cells
which displayed the functional (high phagocytic capacity) and antigenic
properties (expression of F4/80, Mac-1, and CD45) of neighboring
peritoneal macrophages. This strongly argues for a phenotypic
conversion of preadipocytes into typical macrophages. To complete this
phenotype, the expression of proteins associated with
antigen-presenting function was compared with that of peritoneal macrophages. Once again, CD80 and CD86 antibodies stained similar percentages of DAPI+ and peritoneal exudated cells. The
peritoneal cavity of mouse can educate preadipocytes into phagocytosing
cell that express the immunologically significant constimulation
molecules B7.1 and B7.2 (CD80 and CD86).
The effect of peritoneal cavity on the preadipocyte phenotype is
consistent with numerous studies on cellular plasticity in which the
environmental and pathological context strongly direct the fate of
injected cells whatever their tissue origins (31-33). An intriguing
point is that differentiated cells present in the cavity,
i.e. peritoneal macrophages, can induce the phenotypic transformation of precursor cells into their own phenotype. Although this conversion is very low in vitro, it is specific because
no F4/80 or Mac-1 staining was detected when co-culture was performed with the C2C12 myoblastic cell line. Very recently, it was shown that
endothelial cells cultivated with cardiomyocytes could differentiate into cardiomyocytes (33). This suggests that differentiated cells are
themselves strong inducers of their own differentiation program. In our
study, direct contact between preadipocytes and macrophages seems to be
required to achieve this phenotypic conversion, as demonstrated by the
experiments using track-etched membrane separation. The involvement of
cell-to-cell contact is not very surprising and underlines its role in
many differentiation processes (34, 35). The nature of the observed
phenotype change is very difficult to define precisely. It in fact
corresponds to a conversion process between two lineages that were
previously thought to be independent. Although it has been shown that
preadipocyte cell lines could be induced to differentiate into other
cell types, such as osteoblasts (36), the rapid kinetics of change
could suggest a transdifferentiation process or/and a stronger lineage relationship between adipocyte progenitors and macrophages than expected.
Our study demonstrates that adipose progenitors can rapidly and
efficiently convert into typical macrophages. This raises the
possibility that the preadipocyte could have been changed into a cell
capable of costimulating a T cell. Such interaction may influence the
biology of both cell types and have grave implications in immunologic
as well as metabolic pathogenic states. Increasing cross-talk between
both fields are reported and emphasize the importance of inflammation
in various metabolic disorders as diabetes, atherosclerosis, and
obesity (16, 37-42). Overall, this underlines the plasticity of
adipose precursor cells and reinforces the developing relationship
between innate immunity and adipose tissue.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
are sensitive to lipopolysaccharide activation (14). Conversely,
aP2 and peroxisome proliferator-activated receptor
, long
described as specific to adipocyte lineage, have been detected in
macrophages (12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
e, 340-380 nm;
a, 425 nm) or
FITC (FITC filter:
e, 450-490 nm;
a, 515 nm). Phagocytic activity is the percentage of phagocytizing cells, and
the phagocytic index is the average number of engulfed yeast per
phagocytizing cell.
e, 560/40 nm;
a, 645/75 nm).
-33P]dCTP (10 mCi/ml, 2500 mCi/mmol, Amersham Biosciences), 1 mM each of dATP, dGTP,
dTTP, 3.3 mM dithiothreitol, 2 µl of H2O, and
6 µl of 5× first-strand buffer (Invitrogen) in a final volume of 30 µl. After 1 h, 1 µl of Superscript II reverse transcriptase was added to the reaction mixture. The resulting
[33P]cDNA probes were purified with ProbeQuant G-50
Micro columns (Amersham Biosciences) following the manufacturer's
instructions. After counting, probes with a total activity higher than
2 × 107 cpm were the only ones to be hybridized.
Mouse Genefilters (GF400 from ResGen, Invitrogen) were used for
profiling expression analysis. Genefilters contain 5185 genes (75% are
expressed sequence tags (ESTs)). The membranes were pretreated with
boiled 0.5% SDS for 10 min. Prehybridization was performed for 4 h at 42 °C in MicroHyb hybridization solution (ResGen) with 5 µg
of denatured mouse Cot-1 and 4 µg of poly(dA) as blocking reagents.
The column-purified and denatured probes were then added and hybridized
at 42 °C for 16 h. After hybridization, the membranes were
successively washed twice for 10 min at 50 °C in roller tubes
containing 2× standard saline citrate solution (SSC) and 1% SDS. The
last two washes were carried out in a plastic box with horizontal
shaking: 2× SSC and 1% SDS for 20 min at 50 °C and 0.5× SSC and
1% SDS for 15 min at 55 °C. The membranes were then exposed to low
energy phosphorimage screens for 2 days. Images were acquired using
PhosphorImager 445 SI (Molecular Dynamics, Amersham Biosciences,
Sunnyvale, CA), and analyzed with IMAGENE 4.0 (Biodiscovery, Marina del
Rey) and Excel (Microsoft) software. Each hybridization was performed
with independent RNA samples (cell preparation and RNA extraction).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Experimental design of metaprofiles
r) and average
linkage. The clustering algorithm separated metaprofiles into two
distinct clusters (Fig. 1). The first
cluster included liver and random metaprofiles. As expected, the two
negative controls random and liver metaprofiles were the most isolated
profiles with a low similarity between them (d > 0.8).
The second cluster contained all cellular metaprofiles. Thus, the
macrophage phenotypes, which corresponded to a differentiated phenotype, were closer to the preadipocyte metaprofiles
(d
0.5) than to the metaprofiles of differentiated
adipocytes (from fatty tissue or also preadipocyte cell lines)
(d > 0.5). Within the subcluster, preadipocyte or
macrophage metaprofiles were systematically paired according to the
nature of the sample (immortalized cell lines versus
non-immortalized cells). This common behavior suggested a strong
similarity between the two phenotypes, although the identification of
gene clusters preferentially expressed in one metaprofile demonstrated that they were not identical. However, because of this close
resemblance, the question of putative conversion of preadipocytes into
macrophages emerged from this analysis. To address this question, we
decided to transplant labeled preadipocytes into an environment
suitable for supporting the macrophage phenotype (i.e. the
peritoneal cavity).
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Fig. 1.
Metaprofile comparison with hierarchical
clustering algorithm. Metaprofiles are ordered along the
x-axis, and genes are ordered along the y-axis.
Trees represent the proportional distance measured between each
metaprofile (top) or each gene (left).
Metaprofile-specific gene clusters are identified by red
bars and their corresponding letters (right).
Letters are shown at the top of their corresponding
metaprofile.
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Fig. 2.
Phagocytic activity and antigenic features of
DAPI-labeled SVF cells after peritoneal injection. A,
phagocytosis of peritoneal cells 24 h after peritoneal injection
of DAPI-labeled SVF cells. Contrast phase light and UV filter were
used. Blue nuclei correspond to cells stained by DAPI.
Closed arrowhead, phagocytizing DAPI+ cells.
B, the phagocytic activity of DAPI+ SVF cells
was measured 24 h or 6 days after peritoneal injection. Phagocytic
activity represents the percentage of DAPI+ phagocytizing
cells per total DAPI+ cells. Results are means ± S.E.
of at least three independent experiments. All results differed
significantly (**, p < 0,01) from values obtained with
SVF cells before the injection. C, immunocytochemistry was
performed 24 h after peritoneal injection of labeled cells with
anti-F4/80 antibody. The secondary antibody was coupled to RPE. UV
filter (top), TX2 filter (bottom). Closed
arrowhead, DAPI+ F4/80+ cells;
D, the percentages of F4/80+ and
Mac-1+ cells per total DAPI+ cells were
measured 24 h and 6 days after peritoneal injection of
DAPI-labeled cells. Results are means ± S.E. of at least three
independent experiments. All results differed significantly (**,
p < 0.01) from values obtained with SVF cells before
injection.
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Fig. 3.
Phenotype modifications of DAPI-labeled
3T3-L1 cells after peritoneal injection. A, time course of
phagocytic activity and index of DAPI+ 3T3-L1 cells.
Phagocytic activity represents the percentage of phagocytizing cells
per total DAPI+ cells (broken line, left y
axis). Phagocytic index represents the average number of engulfed
yeast per phagocytic cell (continuous line, right y
axis). Results are means ± S.E. of at least three
independent experiments. All results differed significantly (**,
p < 0.01) from values obtained with non-confluent
3T3-L1 cells. Peritoneal macrophages were used as positive controls.
B, expression of macrophage-specific markers by peritoneal
DAPI+ cells. Six hours after the peritoneal injection of
labeled cells, immunocytochemistry experiments were performed with
primary antibodies and RPE-coupled secondary antibody. Values represent
the percentage of F4/80+ and Mac-1+ cells per
total DAPI+ cells. Results are means ± S.E. of at
least three independent experiments. All results differed significantly
(**, p < 0.01) from values obtained with non-confluent
3T3-L1 cells (t , 0 h). C, additional
characterization of peritoneal DAPI+ cells. Twenty-four
hours after peritoneal injection of labeled cells, immunocytochemistry
experiments were performed with primary antibodies anti-CD80,
anti-CD86, and anti-MHCII. Values represent the percentage of positive
cells per total DAPI+ cells. Results are means ± S.E.
of at least three independent experiments. The means differed
significantly (**, p < 0.01) from values obtained with
non-confluent 3T3-L1 controls.
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Fig. 4.
3T3-L1 phenotype before and after peritoneal
injection. A, phagocytosis of DAPI-labeled 3T3-L1 cells
under standard conditions. B, phagocytosis of peritoneal
cells 6 days after peritoneal injection of DAPI-labeled 3T3-L1 cells.
Contrast phase light and UV filter were used. Blue
nuclei correspond to cells stained by DAPI. Closed
arrowhead, DAPI+ phagocytizing cells, open
arrowhead, DAPI+ non-phagocytizing cells.
C, phagocytosis of peritoneal cells 24 h after
peritoneal injection of GFP-expressing 3T3-L1. Contrast phase light
(top) and FITC filter (bottom). Closed
arrowhead, GFP+ phagocytizing cell. D,
immunocytochemistry was performed 24 h after peritoneal injection
of DAPI-labeled 3T3-L1 with anti-F4/80 antibody. The secondary antibody
was coupled to RPE. DAPI staining (blue light,
top), RPE fluorescence (red light,
bottom). Closed arrowhead, DAPI+
F4/80+ cells; open arrowhead, DAPI+
F4/80 cells. E, immunocytochemistry was
performed 24 h after peritoneal injection of GFP expressing 3T3-L1
with anti-F4/80 antibody. The secondary antibody was coupled to RPE.
GFP expression (green light, top), RPE
fluorescence (red light, bottom). Closed
arrowhead, GFP+ F4/80+ cells.
Macrophage contact is necessary for specific conversion of
preadipocytes into macrophages
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank C. Dani for helpful discussions. Adenovirus CMV-CFP was donated by the Association Française contre les Myopathies.
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FOOTNOTES |
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* This work was supported by Grant 4CS01F from Génopôle Toulouse and Association Française contre les Myopathies/INSERM.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from the Ministry of National Education, Research and Technology.
To whom correspondence should be addressed. Tel.:
33-5-62-17-08-91; Fax: 33-5-62-17-09-05; E-mail:
casteil@toulouse.inserm.fr.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M210811200
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ABBREVIATIONS |
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The abbreviations used are: SVF, stroma-vascular fraction; DAPI, 4',6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; NCS, newborn calf serum; GFP, green fluorescent protein; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate.
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