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A role for preadipocytes as macrophage-like cells

^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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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.

	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.

	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 ).

View larger version (28K): [in this window] [in a new window]   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.

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 ).

View larger version (17K): [in this window] [in a new window]   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.

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 ).

View larger version (28K): [in this window] [in a new window]   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.

This antimicrobial activity of 3T3-L1 preadipocytes and macrophages is partly and significantly inhibited by preincubation with SOD (Fig. 5 ).

View larger version (33K): [in this window] [in a new window]   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.

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 ).

View larger version (23K): [in this window] [in a new window]   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.

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 ).

View larger version (28K): [in this window] [in a new window]   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.

View larger version (24K): [in this window] [in a new window]   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.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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.

View larger version (139K): [in this window] [in a new window]   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).

	ACKNOWLEDGMENTS

 
We wish to thank Dr. R. Martin for critical reading and helpful comments, and B. Barhanin for technical assistance with cell cultures.

	FOOTNOTES

 
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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