and granulocyte macrophage colony stimulating factor

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International Immunology, Vol. 10, No. 5, pp. 593–599

© 1998 Oxford University Press

Dexamethasone enhances macrophage colony stimulating factor- and granulocyte macrophage colony stimulating factorstimulated proliferation of bone marrowderived macrophages Jorge LLoberas, Concepcio´ Soler and Antonio Celada Departament de Fisiologia (Immunologia), Facultat de Biologia, and Fundacio´ Pi i Sunyer, Campus Bellvitge, Universitat de Barcelona, Avenida Diagonal 645, 08028 Barcelona, Spain

Keywords: dexamethasone, cell proliferation, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor

Abstract Glucocorticoids are effective repressors of the immune system. We have examined the effect of glucocorticoids on the proliferation of murine macrophages. Dexamethasone by itself did not affect proliferation of differentiated or undifferentiated bone marrow-derived macrophages (BMM) and elicited peritoneal macrophages. However, dexamethasone enhanced the proliferation induced by macrophage colony stimulating factor (M-CSF) of these cells. The effect of dexamethasone was not restricted to M-CSF-dependent proliferation. Similarly, dexamethasone enhanced granulocyte macrophage colony stimulating factor (GM-CSF)-dependent proliferation of BMM. In agreement, macrophages transfected with the glucocorticoid receptor showed an enhancement of M-CSFdependent proliferation. The enhancement of proliferation by dexamethasone or the glucocorticoid receptor was abolished by RU 486, an antagonist of the glucocorticoid receptor. Moreover, the addition of antibodies against M-CSF inhibits the effect of dexamethasone, suggesting that dexamethasone increases the autocrine production of M-CSF. This only occurs when M-CSF or GM-CSF, which induce M-CSF, are present in the media. In tissues, dexamethasone may enhance macrophage proliferation and contribute to the resolution of the inflammatory states. Introduction In both bone marrow and tissues, macrophages proliferate in response to specific growth factors called colony-stimulating factors (CSF), such as the multi-CSF or IL-3, the granulocyte macrophage colony stimulating factor (GM-CSF) and the macrophage colony stimulating factor (M-CSF; also called CSF-1) (1). M-CSF is the major specific macrophage growth factor that controls survival, proliferation and differentiation of the monocyte–macrophage lineage from progenitors to mature cells (1). Macrophages require M-CSF for their proliferation through the G1 phase of cell cycle, but once they have entered the S phase, macrophages do not require MCSF to complete mitosis (2). M-CSF signaling is mediated by the M-CSF receptor, which is encoded by the c-fms protooncogene, a receptor with protein tyrosine kinase activity (3).

M-CSF regulates several cyclins during the G1 phase (4). It has been suggested that differentiation by M-CSF could take place in the G2/M phase (5). Furthermore, M-CSF activates several important functions of mature tissular macrophages, such as membrane transport systems, chemotaxis, phagocytosis, cytotoxicity, and the production and release by the cells of a number of biologically active molecules (1). One of the mechanisms responsible for the unregulated growth of hematopoietic cells is the autocrine production of growth factors (6). Macrophages produce several growth factors that have a positive effect on proliferation, like M-CSF (7–9), GM-CSF (10), IL-3 (11) and transforming growth factor (TGF)-β (12), but they also produce other factors, such as IFN-α, that inhibit proliferation (13–15). Some of these factors

Correspondence to: A. Celada Transmitting editor: C. Martı´nez-A

Received 21 July 1997, accepted 19 January 1998

594 Glucocorticoids in macrophage proliferation also stimulate the production of other factors. For instance, M-CSF stimulates the production of TNF (16) and then TNF can stimulate the expression of M-CSF (17,18). Glucocorticoids are important suppressors of the immune system. They influence the traffic of circulating leukocytes and inhibit many functions of leukocytes and immune accessory cells (19,20). Glucocorticoids have been shown to depress myelopoiesis (21), block monocyte to macrophage differentiation (22), inhibit IL-1, IL-6 and tumor necrosis factor (TNF)α production (23–25), and decrease microbicidal and tumoricidal activities (26,27). Glucocorticoids affect a wide variety of cellular functions by regulating the expression of multiple genes. For instance, glucocorticoids down-regulate IFN-γinduced class II MHC antigen expression (23). We found that repression of MHC class II antigen by glucocorticoids is due to an inhibition by the glucocorticoid receptor of the X box DNA binding protein (28). Recently, it has been demonstrated that dexamethasone induces a large increase in the mRNA and protein levels of the M-CSF receptor (29). Chambers et al. (30) using breast carcinomas cells showed that dexamethasone does not increase the rate of c-fms gene transcription, but stabilizes the transcripts, suggesting a post-transcriptional regulation of the M-CSF receptor. More recently, the same group showed that levels of c-fms transcripts and protein increased significantly within the first few hours of glucocorticoid treatment, when it is not attributed to prolongation of transcript half-life (31). The sequence upstream of the c-fms promotor was found to contain a potential ‘glucocorticoid response element’, the elimination of which abolished glucocorticoid stimulation. In this manuscript, we provide evidence that glucocorticoids enhance M-CSF- or GM-CSF-stimulated macrophage proliferation through the induction of autocrine production of MCSF. Methods

penicillin, 50 µg/ml streptomycin, 20% FCS and 30% L929 cell-conditioned medium (LCM). LCM is the source of M-CSF. The cell suspensions were incubated at 37°C in a humidified 5% CO2 atmosphere. BMM were obtained after 7 days in culture in the presence of M-CSF. For experiments, cells were washed and incubated in serum-free media in the presence or absence of the indicated factors. Serum-free medium was composed of CMRL-1066 (Gibco, Gaithersburg, MD), insulin (Gibco), glutamine (Gibco), transferrin (Gibco) and sodium bicarbonate. Peritoneal elicited macrophages (PEM) were prepared as described previously (22) by peritoneal lavage of mice that had been injected i.p. 3 days previously with 1.5 ml of 10% protease peptone (Difco, Detroit, MI). Macrophage monolayers were prepared by seeding the PEM suspension (53105 cells) into flat-bottom, 24-well tissue culture plates. The cells were allowed to adhere for 2 h at 37°C before the plates were washed rigorously to remove non-adherent cells.

Proliferation assay Macrophage proliferation was measured as previously described (12) with minor modifications. Cells (53105) were incubated for 24 h in 24-well plates (3424 Mark II; Costar, Cambridge, MA) in 1 ml of medium with the indicated growth factor. Medium was aspirated and replaced by 0.2 ml of medium containing [3H]thymidine (1.0 mCi/ml). After 2 h of incubation at 37°C, the medium was removed and cells were fixed in methanol. After 3 washes with 10% trichloroacetic acid, cells were solubilized in 1% SDS and 0.3 M NaOH. Radioactivity was counted by liquid scintillation. All samples were prepared in triplicate and the results are expressed as the mean value. In some experiments cells were trypsinized and numbered with a Coulter counter (ZM model; Hialeah, FL). Each experiment was performed 3–5 times. Statistical analyses were performed using the Student’s paired t-test, comparing the results of four to five independent experiments.

Transfections Plasmids An expression construct containing the green fluorescent protein gene (Clontech, Palo Alto, CA) was used to measure transfection efficiency. The Bluescript KS1 vector was from Stratagene (La Jolla, CA), and the pBLCAT vector was from Luckow and Schutz (32). The plasmid containing the gene for the glucocorticoid receptor (DB10-7) was obtained from Dr M. Pfahl (Sidney Kimmell Cancer Research Foundation) (33,34). The pECE vector contains the SV40 promoter and enhancer (35).

Macrophages Macrophages derived from bone marrow (BMM) cultures were obtained as described (36). Six-week-old DBA/2 mice (Jackson Laboratory, Bar Harbor, ME) were killed by cervical dislocation and both femurs were dissected free of adherent tissue. The ends of the bones were cut off and the marrow tissue was eluted by irrigation with PBS. Cells were suspended by vigorous pipetting and washed by centrifugation. We cultured 107 cells in a plastic, non-tissue culture 150 mm Petri dish (Lab-Tek 4030; Miles, Naperville, IL) in 50 ml DMEM containing 2 mM L-glutamine, 1 mM Na pyruvate, 50 U/ml

Cells were removed from plates and washed twice in serumfree media. Then, cells were incubated (1.23107 in 1 ml of serum-free media) at 4°C in the cuvettes used for electroporation, with 2 µg of DNA for 15 min. Electroporation was carried out at 200 mV for 10 ms using a BTX electroporator (Transfector 100; Biotechnologies and Experimental Research, San Diego, CA). Cells were incubated for a further 15 min on ice before they were distributed in 24-well plates. In a series of preliminary experiments, we tested the appropriate conditions for transfection (10 ms at 200 mV) using a BTX electroporator (Transfector 100) (37). After transfection of any type of vector, we always found a cell mortality of ~10–20%. We also found that increasing the amount of DNA (any type) in BMM resulted in a concentration-dependent decrease in proliferation. Therefore, we chose conditions that optimized the number of cells transfected, but also permitted proliferation. To determine the transfection efficiency in some experiments, we transfected an expression construct containing the green fluorescent protein gene (38). After 2 days of culture, cells were fixed, stained and counted. Under the conditions described, 7–12% of cells were transfected as assessed by staining for green fluorescent protein.

Glucocorticoids in macrophage proliferation 595

Fig. 1. Dexamethasone enhances M-CSF dependent BMM proliferation. BMM were obtained after 7 days in culture in the presence of M-CSF. Macrophages (53105 cells) were washed and incubated in serum-free medium in the absence or presence of rM-CSF and the indicated amounts of dexamethasone. (A) Thymidine incorporation was performed as indicated in Methods. Each determination was made in triplicate and the bars represent the mean 6 SD. A significant difference was observed in the presence of M-CSF when the control was compared with the samples treated with 5 and 10310–7 M of dexamethasone (P , 0.01). (B) After an overnight incubation, the number of cells was counted using a Coulter counter. Each determination was made in triplicate and the bars represent the mean 6 SD. A significant difference was observed using M-CSF when the control was compared with the samples treated with 10310–7 M of dexamethasone (P , 0.01).

Growth factors and IL Recombinant growth factors were provided as gifts from DNAX (Palo Alto, CA). In some experiments we used LCM as a source of M-CSF. The amount of M-CSF present in LCM was determined using a M-CSF standard. The growth activity of LCM was blocked by a specific mAb against M-CSF. The M-CSF blocking antibody was a gift of Dr H. S. Lin (Washington University School of Medicine, St Louis, MO) (39). Results To determine whether glucocorticoids have an effect on macrophage proliferation, we added various concentrations of dexamethasone (10–7 to 10–6 M) to murine BMM cultured in the presence of M-CSF. This growth factor allows macrophages to proliferate (.99% are Mac 11), whereas the rest of the bone marrow cells died. In the absence of M-CSF, dexamethasone did not significantly enhance macrophage proliferation. When dexamethasone was added to BMM (7 days in culture) in the presence of rM-CSF (recombinant MCSF), thymidine incorporation increased in a dose-dependent manner (Fig 1A). No further effect was seen when the amounts of dexamethasone were .10–6 M. When cells were counted in a parallel experiment, an increase in cell number in cultures treated with dexamethasone plus M-CSF was seen compared with cultures treated with M-CSF alone (P , 0.01) (Fig 1B). These data suggest that dexamethasone stimulated the MCSF-dependent proliferation of BMM. The effect of glucocorticoids on the proliferation of macrophages at different stages of maturation was also tested. The proliferation of non-adherent bone marrow cells (3 days in

culture), which represent an early stage of macrophage differentiation (40,41), was compared with elicited peritoneal macrophages, which represent a committed population of cells. The non-adherent bone marrow cells incorporated ~5 times more thymidine than the elicited peritoneal macrophages. In the presence of M-CSF, dexamethasone stimulated the proliferation of both populations of macrophages. For elicited peritoneal macrophages, in the presence of 1200 U/ ml of M-CSF we found that control cultures incorporate 25.3 6 3.13103 c.p.m. [3H]thymidine, whereas cells treated with 10–7, 5310–7 and 10–6 M dexamethasone incorporate 28.8 6 7.33103, 31.2 6 11.23103 and 33.8 6 6.53103 c.p.m. respectively. For non-adherent bone marrow cells, the controls incorporate 135.8 6 18.63103 c.p.m. [3H]thymidine, whereas in the presence of 10–7, 5310–7 and 10–6 M dexamethasone, the cells incorporate 148.1 6 22.83103, 160.3 6 18.63103 and 165.6 6 22.83103 c.p.m. respectively. To determine whether the enhancing effect of dexamethasone was specific to M-CSF-induced proliferation, we tested the growth factors GM-CSF and IL-3, as well as several other IL. The effect of adding dexamethasone to macrophages treated with either of these factors was tested. Both GM-CSF and IL-3 induced macrophage proliferation, but GM-CSF was more active than IL-3 (Figs 2 and 3). Dexamethasone enhanced the activity of GM-CSF in a dose-dependent manner but had no effect on IL-3-dependent proliferation. Other factors such as IL-1, IL-4 and IL-6, but not IL-2, had a small positive effect on BMM proliferation (data not shown). When dexamethasone was added to these factors, there was no significant change in proliferation. To determine whether the effect of dexamethasone on

596 Glucocorticoids in macrophage proliferation

Fig. 2. Effect of dexamethasone on GM-CSF-dependent macrophage proliferation. BMM were obtained after 7 days in culture in the presence of M-CSF. Cells were washed and incubated in serum-free media in the absence or presence of the indicated amounts of rGMCSF and dexamethasone. Each determination was made in triplicate and the bars represent the mean 6 SD. A significant difference was observed between the controls and the samples treated with 5 and 10310–7 M of dexamethasone in the presence of GM-CSF (P , 0.01).

Fig. 3. Effect of dexamethasone on IL-3-dependent macrophage proliferation. BMM were obtained after 7 days in culture in the presence of M-CSF. Cells were washed and incubated in serum-free media in the absence or presence of the indicated amounts of rIL-3 and dexamethasone. Each determination was made in triplicate and the bars represent the mean 6 SD. No significant differences were observed between the controls and the samples treated with dexamethasone in the presence of IL-3 (P . 0.05).

macrophage proliferation was mediated by the glucocorticoid receptor, we transfected a receptor expression plasmid (OB10-7) into macrophages. As control, macrophages were transfected with the pECE vector alone. The thymidine incorporation in cells transfected with the pECE vector was similar to that in cells transfected with the KS1 or the pBLCAT vectors. In the presence of M-CSF, thymidine incorporation in cells

transfected with the glucocorticoid receptor expression plasmid was increased in relation to the controls (P , 0.01) (Fig. 4). In the absence of added dexamethasone, macrophages transfected with the glucocorticoid receptor also present an increase of thymidine incorporation. This effect may be due to the endogenous glucocorticoids present in serum. In fact, the addition of RU486 decreases the levels of thymidine incorporation in macrophages transfected with the glucocorticoid receptor to those of the cells transfected with the control vector (Fig 4). Additional support for the involvement of the glucocorticoid receptor in the enhancement of M-CSF-induced macrophage proliferation was assessed by using the glucocorticoid receptor antagonist RU486. The binding of RU486 by the receptor results in an inactive receptor in most cases. When RU486 was added to cells together with dexamethasone, no enhancement of macrophage proliferation was observed (Fig. 4). Cells treated with both and dexamethasone had a level of thymidine incorporation that was comparable to the controls. RU486 by itself had no effect on thymidine incorporation when compared to the control. The blocking effect of RU486 was also observed when RU486 was added to cells co-transfected with a glucocorticoid receptor expression construct (Fig. 4). In this experiment, the presence of the glucocorticoid receptor increased the thymidine incorporation from 155,000 6 11,000 to 210,000 6 14,000 c.p.m. (P , 0.01). When RU486 was added the increase in thymidine incorporation by the glucocorticoid receptor was reduced to 161,000 6 10,100 c.p.m. The addition of dexamethasone to cells transfected with the glucocorticoid receptor expression construct resulted in a thymidine incorporation of 288,000 6 14,000 c.p.m. This increase in thymidine incorporation was completely reversed by the addition of RU486 (Fig. 4). These results emphasize that it is the glucocorticoid receptor that causes the enhancement of thymidine incorporation activity in our assays and that this activity can be blocked by the drug RU486. Only in the presence of M-CSF and GM-CSF did macrophages treated with dexamethasone show a significant increase in proliferation compared to the controls. These data suggest that dexamethasone is involved particularly in the mechanisms of macrophage proliferation that are M-CSF or GM- CSF dependent. When mAb to M-CSF were added to macrophages in the presence of GM-CSF, proliferation of macrophages in the presence or absence of dexamethasone was very similar (Fig. 5). Macrophages transfected with the glucocorticoid receptor in the presence of GM-CSF showed an increase in thymidine incorporation in relation to the controls (GM-CSF alone). However, antibodies against MCSF blocked the enhancement of thymidine incorporation produced by the glucocorticoid receptor or dexamethasone (Fig. 5). These data suggest that the enhancement of proliferation in macrophages treated with dexamethasone could be related to the autocrine production of M-CSF (7–9,40,41). Discussion This study was initiated to gain a better understanding of the effect of glucocorticoids on macrophages. We found that for BMM or exudative macrophages glucocorticoid enhances

Glucocorticoids in macrophage proliferation 597

Fig. 4. Transfection of the glucocorticoid receptor enhances M-CSF-dependent BMM proliferation. BMM (1.23107 cells) were transfected with 5 µg of the pECE vector or an expression vector containing the gene for the glucocorticoid receptor. Cells were incubated in the presence or absence of dexamethasone (10–6 M) or/and RU486 (antagonist of the glucocorticoid receptor). Cells were incubated in the presence of rMCSF (1200 U/ml). Each determination was made in triplicate and the bars represent the mean 6 SD. In the presence of dexamethasone a significant difference was seen between the control and transfected cells (P , 0.01).

Fig. 5. Antibodies to M-CSF block the enhancement effect of dexamethasone or the glucocorticoid receptor on GM-CSF dependent proliferation. BMM (1.23107 cells) were transfected with 5 µg of the pECE vector or an expression vector containing the gene for the glucocorticoid receptor. Cells were incubated in the presence of GM-CSF (20 ng/ml). Cells were treated with or without dexamethasone or/and antibody to M-CSF. The working concentration of antibody was 1/100 (sufficient amount to neutralize 1000 U/ml of M-CSF). Thymidine incorporation was measured in BMM under the indicated conditions. Each determination was made in triplicate and the bars represent the mean 6 SD.

both M-CSF and GM-CSF-dependent proliferation. Also, for non-adherent bone marrow cells, glucocorticoids enhances proliferation. This suggests that glucocorticoids have an effect on proliferation independently of the degree of macrophage proliferation. In contrast, to M-CSF and GM-CSF, glucocorticoids do not influence IL-3-dependent proliferation. Macrophages treated with glucocorticoids in the presence of GM-CSF and blocking

mAb against M-CSF do not proliferate more than the controls. This suggests that in the presence of GM-CSF the enhancement of proliferation by the glucocorticoids is due to the autocrine production of M-CSF induced by GM-CSF (7). The enhancing effect of glucocorticoids on macrophage proliferation is mediated through the glucocorticoid receptor. This was demonstrated because in the presence of M-CSF, cells transfected with a receptor expression plasmid showed

598 Glucocorticoids in macrophage proliferation an increased proliferation in relation to the controls. Moreover, the effect of glucocorticoids was abolish using the glucocorticoid receptor antagonist RU486. At present we do not know all the genes regulated by glucocorticoids that could modulate macrophage proliferation in response to M-CSF. One of the possible targets for the effect of glucocorticoids is the M-CSF that is released from macrophages. The experiment using antibodies against MCSF suggests this possibility. Recent reports suggest that glucocorticoids stimulate the production of M-CSF. It has been shown that endometrial stromal cells can secrete bioactive MCSF in a progesterone-dependent manner (40,41). It seems that dexamethasone acts at the post-transcriptional level increasing the half-life of M-CSF RNA (29). Because the enhancement of proliferation requires the presence of the growth factor M-CSF, another possible candidate that may explain the effect of glucocorticoids is the MCSF receptor, which is specifically expressed on macrophages. Recently, it has been demonstrated that dexamethasone induces a large increase in the M-CSF receptor mRNA and protein levels (29). After interaction of macrophages with some molecules, such as IFN-γ or lipopolysaccharide (LPS), there are biochemical, morphological and functional modifications allow macrophages to become functionally active. This process has been called macrophage activation (42). If macrophages are proliferating under the effect of growth factors the addition of LPS or IFN-γ blocks completely their proliferative activity. This clearly shows that proliferation and macrophage activation are two different activities, for which signaling and the cascade events after interaction of the ligands with their respective receptors are totally different. Thus, it is not surprising that glucocorticoids that inhibit several functional activities in macrophages, i.e. expression of class II MHC antigens (23,28), could have opposite effects on proliferation. In fact, as we have previously discussed, it has been show that glucocorticoids increase both M-CSF production (40,41) and M-CSF receptor expression (29–31). This could explain the increased proliferation of macrophages in the presence of dexamethasone, as we show in the present manuscript. Finally, at the present time we do not know the relevance in vivo of our data. This may be difficult to assess since the number of monocytes in circulation or macrophages in bone marrow is very low. Acknowledgements We thank John Knight for the synthesis of oligonucleotides and Dr Magnus Pfahl for the plasmid containing the glucocorticoid receptor. We thank DNAX (Palo Alto, Ca) for the gift of growth factors and lymphokines, Roussel Uclaf (Romanville, France) for the RU 486, Dr H. S. Lin for the mAb against M-CSF and Deborah Vestal for editing the manuscript. This work was supported by grants from CICYT (PB94-1549) and FISS (96/2050) to A. C.

Abbreviations BMM CSF GM-CSF LCM LPS

bone marrow macrophages colony stimulating factor granulocyte macrophage colony stimulating factor L929 cell-conditioned medium lipopolysaccharide

M-CSF PEM TGF TNF

macrophage colony stimulating factor peritoneal elicited macrophages transforming growth factor tumour necrosis factor

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