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Originally published online as doi:10.1189/jlb.1107768 on July 8, 2008

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(Journal of Leukocyte Biology. 2008;84:644-650.)
© 2008 by Society for Leukocyte Biology

Glucocorticoids induce an activated, anti-inflammatory monocyte subset in mice that resembles myeloid-derived suppressor cells

Georg Varga*,{dagger},1, Jan Ehrchen*,{dagger},1, Athanasios Tsianakas*,{dagger}, Klaus Tenbrock*,{ddagger}, Anke Rattenholl{dagger}, Stephan Seeliger{ddagger}, Matthias Mack§, Johannes Roth*,{ddagger} and Cord Sunderkoetter*,{dagger},2

* Institute of Immunology and Departments of
{dagger} Dermatology and
{ddagger} Pediatrics, University of Muenster, Muenster, Germany; and
§ Department of Internal Medicine II, University Hospital of Regensburg, Regensburg, Germany

2 Correspondence: Department of Dermatology, University of Muenster, Von Esmarch Str. 58, Muenster D-48149, Germany. E-mail: cord.sunderkoetter{at}ukmuenster.de

ABSTRACT

Glucocorticoids (GC) are still the most widely used immunosuppressive agents in clinical medicine. Surprisingly, little is known about the mechanisms of GC action on monocytes, although these cells exert pro- and anti-inflammatory effects. We have shown recently that GC induce a specific monocyte phenotype with anti-inflammatory properties in humans. We now investigated whether this also applies for the murine system and how this subset would relate to recently defined murine subtypes. After treatment with dexamethasone for 48 h, monocytes up-regulated scavenger receptor CD163 and Gr-1, down-regulated CX3CR1, and shared with human GC-treated monocytes functional features such as low adhesiveness but high migratory capacity. They specifically up-regulated anti-inflammatory IL-10, but not TGF-β, and in contrast to their human counterparts, they down-regulated IL-6. Although GC-induced monocytes down-regulated CX3CR1, a distinctive marker for classical/proinflammatory human and murine monocytes (CX3CR1loCCR2+Ly6Chi), they differed from this physiologically occurring subset, as they remained Ly6Cmed and unactivated (CD62 ligand++). In addition to their immunosuppressive effects, they were CD11b+Gr-1+ and expressed the IL-4R{alpha} chain (CD124), a recently described, signature molecule of tumor-induced myeloid-derived suppressor cells (MDSC). We therefore generated murine MDSC in B16 melanoma-bearing mice and indeed found parallel up-regulation of CD11b+Gr-1+ and CD124 on GC-induced monocytes and MDSC. These data allow us to speculate that the GC-induced subtype shares with inflammatory monocytes the ability to migrate quickly into inflamed tissue, where they, however, exert anti-inflammatory effects and that similarities between GC-induced monocytes and MDSC may be involved in progression of some tumors observed in patients chronically treated with GC.

Key Words: IL-10 • CD124

INTRODUCTION

Glucocorticoids (GC) affect nearly every cell of the immune system, and there is growing evidence for cell type-specific mechanisms [1 ]. In human monocytes, we have shown that GC generate a distinct phenotype with anti-inflammatory properties that can possibly influence and regulate other immune cells [2 ].

Monocytes represent a central part of innate immunity. After differentiation from stem cells in the bone marrow, monocytes enter the circulation and are present in the blood until they migrate into tissues, where they can differentiate into macrophages and dendritic cells (DC). These cells are crucial for nearly every step of an immune reaction, including the initiation of an adaptive immune response, clearance of infectious agents, as well as resolution of inflammation [3 4 5 ]. Depending on their stage of differentiation and on the mechanisms of activation, distinct subtypes of DC or macrophages with proinflammatory as well as anti-inflammatory functions have been described [4 , 6 ].

Different subpopulations have been demonstrated already at the stage of circulating monocytes. In humans, different subtypes of monocytes have been distinguished according to their expression of CD64 or CD14 and CD16 [7 ], and some subsets have described distinct functions in various disease conditions [8 ].

Distinct subpopulations of blood monocytes are also present in the murine system, and they have been characterized by a combination of specific cell-surface signatures: Monocytes leave bone marrow being CX3CR1lowCCR2+Ly6Chigh and become CX3CR1highCCR2–Ly6Clow upon maturation in the blood [9 10 11 ]. Functionally, Geissmann et al. [10 ] and we [9 ] showed that the CX3CR1highCCR2–Ly6Clow monocytes represent mainly "resident" monocytes, which give rise to tissue macrophages and DC under steady-state conditions, and the CX3CR1lowCCR2+Ly6Chigh monocytes represent a "proinflammatory" phenotype, which selectively migrates into inflamed tissue (reviewed in refs. [4 , 12 ]).

Under pathological conditions, e.g., in certain tumors, other subtypes of monocytic cells, the so-called myeloid-derived suppressor cells (MDSC), have been identified (for review, see ref. [13 ]). In mice, they consist of CD11b+Gr-1+ cells that resemble immature monocytic/granulocytic precursor cells. By virtue of murine tumor models, these subtypes have been discovered to be involved in tumor-associated immunosuppression (for review, see ref. [13 ]).

As we have shown previously that GC induces a specific monocyte phenotype with anti-inflammatory properties in humans, we now addressed the question of whether this also applies to the murine system and how it would relate to the murine subtypes previously defined. Their identification in mice would allow us to investigate the functional capabilities of GC-induced monocytes in models of inflammation.

MATERIALS AND METHODS

Mice and murine tumor model
C57BL/6J and BALB/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed under specific pathogen-free conditions and were used between 8 and 12 weeks of age. For the tumor experiments, C57BL/6J mice were injected with 1 x 105 B16 melanoma cells s.c. in the upper hip. Fourteen days post-tumor inoculation, mice were killed, and bone marrow cells were prepared as described for the monocyte preparation. All animal studies were reviewed and approved according to federal regulations (#38/90 Bezirksregierung Münster, Germany).

Reagents and antibodies
Antibodies
Ly6C (ER-MP20), CD11b-allophycocyanin (APC), and CD124, as well as isotype controls and secondary antibodies were obtained from BD Biosciences (Heidelberg, Germany). Anti-Gr-1 (RB6-8C5) FITC antibody was purchased from EuroBioSciences (Friesoythe, Germany). The CCR2 (MC21) antibody was used as described earlier [14 ]. Anti-CD19, anti-CD11c, and anti-CD90 microbeads were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany).

Reagents
RPMI 1640 and Ficoll were purchased from Biochrom (Berlin, Germany), Transwell filter was from Costar (Bremen, Germany), and dexamethasone (DEX) was from Sigma (Taufkirchen, Germany).

Culture of B16 melanoma cells
B16M cells (B16F10 subline) were cultured in endotoxin-free DMEM (Biochrom), supplemented with 10% FCS and penicillin-streptomycin (100 U/ml penicillin and 100 mg/ml streptomycin). Cultures were maintained and propagated as described previously [15 ].

FACS staining
Cells (5x105) were incubated in 50 µl PBS/1% FCS with the indicated antibodies (1 µg/ml) for 30 min at 4°C. Cells were washed twice in 1 ml PBS/1% FCS. For intracellular staining, cells were fixed in 100 µl 1% (w/v) paraformaldehyde for 15 min at 37°C, washed twice, and permeabilized using 50 µl 1% (w/v) saponin for 10 min at 37°C. Then 1 µg of the indicated antibodies was added to the permeabilized cells and incubated for 30 min on ice. Finally, cells were washed twice, resuspended in 500 µl PBS/1% FCS, and analyzed using a FACSCalibur flow cytometer, equipped with CellQuestPro software (BD Pharmingen, Heidelberg, Germany).

Quantitative real-time PCR
Expression of genes was determined by real-time RT-PCR as described previously [16 ]. The primers used for PCR analysis were as follows: CX3CR1, 5'-TCA TCA GCA TCG ACC GGT ACC-3', 5'-TGA CAC CGT GCT GCA CTG TC-3'; CCR2, 5'-TGA TAG TAT GCC GTG GAT GAA CTG-3', 5'-TGC AAG TTC AGC TGC CTG C-3'; CD163, 5'-GAT GAC CTG GCA TGC AAT GG-3', 5'-TCC ACT AGT CTC AGG CTC AGA TCT G-3'; IL-6, 5'-TGA GAT CTA CTC GGC AAA CCT AGT G-3', 5'-CTT CGT AGA GAA CAA CAT AAG TCA GAT ACC-3'; IL-10, 5'-GGG TTG CCA AGC CTT ATC G-3', 5'-TCT CAC CCA GGG AAT TCA AAT G-3'; TGF-β, 5'-GGA CCC TGC CCC TAT ATT TGG-3', 5'-TGT TGC AGG TCA TTT AAC CAA GTG-3'; GAPDH, 5'-GTC CAC CAG CCT GTT GCT GTA G-3', 5'-CCC ACT CTT CCA CCT TCG ATG-3'; ribosomal protein L13a (RPL), 5'-TGG TCC CTG CTC TCA AG-3', 5'-GGC CTT TTC CTT CCG TTT CTC-3'. The relative expression was calculated as 2–{Delta}comparative threshold (Ct)-specific gene/2–{Delta}Ct mean (housekeeping genes), using GAPDH and RPL as endogenous housekeeping control genes.

Preparation and purification of monocytes and DC
Monocytes were purified from bone marrow cells and from murine blood [9 ]. Erythrocytes were depleted by osmotic shock, and cells were washed with HBSS and collected by centrifugation. Cells were separated further by applying them onto a Ficoll gradient with subsequent centrifugation. Of the cells in the interphase, the remaining T cells (CD90+), B cells (CD19+), and DC (CD11c+) were removed using magnetic beads coupled to anti-CD90, anti-CD19, and anti-CD11c antibodies and using MACS technology. Finally, cells were resuspended in DMEM (Invitrogen, Karlsruhe, Germany) containing 2 mM glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 100 mg/ml penicillin/streptomycin (Biochrom), and 10% heat-inactivated FCS (Biochrom). Cells were supplemented with 50 ng/ml M-CSF and 10–7 M DEX or ethanol (EtOH) as control treatment (Ctr). DC were prepared as described in Varga and co-workers [17 , 18 ].

Adherence to plastic
For determination of cell adhesion, monocytes (2x105) were seeded into untreated, plastic tissue-culture dishes (96-well plates) and incubated for 4 h at 37°C. Nonadhering cells were removed by washing, and the remaining cells were fixed with 2% glutaraldehyde for 10 min. Wells were washed two times with H2O, and then, cells were stained with 0.5% crystal violet in 200 mM boric acid (pH 8.0) for an additional 15 min at room temperature. Finally, wells were washed three times, and cells were lysed, adding 10% acetic acid, and staining was subsequently quantified measuring OD by 560 nm in an ELISA reader.

Transmigration
Monocytes (5x105) were treated for 48 h as described and placed into the upper chamber of a Transwell filter (5 µm). The lower chamber contained monocyte medium. After 4 h, cells in the lower chamber were collected and counted. Results are expressed as percent cells that have migrated. Results represent three independent experiments (±SEM).

RESULTS

Phenotype of GC-induced monocytes
We first addressed the question of whether GC would induce a certain phenotype in murine monocytes and if this phenotype would resemble the phenotype of monocytes with anti-inflammatory properties that we had demonstrated in humans.

We treated murine monocytes with control solution or with DEX for 48 h. Afterwards, we prepared RNA and performed real-time PCR for CD163 and also for CX3CR1, as both genes were characteristic markers up- or down-regulated by GC treatment in human monocytes (CX3CR1, in addition, is a distinctive marker for a physiologically occurring human and murine subset of monocytes). As shown in Figure 1A , CD163 mRNA was up-regulated markedly upon GC treatment compared with control-treated monocytes. Up-regulation was even stronger (57.1±seven-fold induction) than we had observed previously in GC-treated human cells (4.5-fold) [2 ]. The fractalkine receptor CX3CR1 was down-regulated upon GC treatment over a 48-h period (Fig. 1B) .


Figure 1
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  		Figure 1. Phenotypic similarities between human and mouse GC-induced monoctyes, which were prepared as described in Materials and Methods. Resulting cells were treated with EtOH (1:10,000) or DEX (10–7 M) for the indicated times. Cells were harvested, and RNA was prepared. RT-PCR for (A) CD163 (48 h) and (B) CX3CR1 was performed as in A. The graph shows mean expression ± SD from three independent experiments. ***, P < 0.001 (Student’s t-test).

When we stained monocytes for up to 5 days in culture, we observed no significant change of phenotype over this time (data not shown).

Thus, GC-induced mouse monocytes present a rather stable subtype, which resembles the phenotype of human GC-induced monocytes with regard to the two characteristic markers CD163 and CX3CR1.

GC-regulated cytokine production by monocytes
IL-10 is a cytokine with anti-inflammatory functions characteristically induced by GC (as shown by us for human monocytes [2 ]). We found that IL-10 was also regulated significantly by GC in murine monocytes. As shown in Figure 2A , IL-10 gene transcription was already up-regulated after 4 h of GC treatment and persisted up to 48 h. Although highly reproducible for mRNA, the IL-10 protein level remained mostly below detection level. During the same time course, TGF-β, another cytokine with partially anti-inflammatory functions, was not regulated by GC (Fig. 2B) .


Figure 2
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  		Figure 2. Cytokine production of GC-induced monocytes. As in Figure 1B but RT-PCR for IL-10 (A), TGF-β (B), and IL-6 (C). Results are representative of at least three independent (±SD) experiments. (D) Supernatants of cells have been analyzed for IL-6 production using cytometric bead array (CBA) technology. Results are means of 11 independent experiments (±SEM). (E) As in D, but cells were additionally stimulated with LPS (100 ng/ml). **, P < 0.01 (Student’s t-test).

We then analyzed whether there was down-regulation of IL-6, a prototypic, proinflammatory cytokine. We detected that GC significantly down-regulated IL-6 mRNA and protein in murine monocytes (Fig. 2C 2D 2E) ; protein levels were low, but their reduction was still significant (Fig. 2D) . LPS did amplify but not render down-regulation of IL-6 by GC (Fig. 2E) . Regulation of this proinflammatory cytokine was not found in GC-induced human monocytes.

Adherence and motility of murine GC-induced monocytes
As GC-induced murine monocytes share with human GC-induced monocytes regulation of common phenotypic markers, we investigated if they would also share other functional changes, such as decreased adherence and increased mobility.

We used monocytes treated for 48 h and performed adhesion assays as described in Materials and Methods. The adherence to plastic surfaces was reduced significantly in monocytes treated with DEX (Fig. 3A ). To test if the lower adhesiveness of GC-treated monocytes would also translate in higher mobility, we used cells in a transmigration assay using a modified Boyden chamber assay. Cells were seeded onto a Transwell filter (5 µm in diameter) and allowed to migrate into the lower chamber for 4 h. GC-treated monocytes migrated spontaneously (without chemoattractant) in significantly higher numbers than their control-treated counterparts (Fig. 3B) .


Figure 3
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  		Figure 3. Adherence and mobility. (A) Cells/well (2x105), which were treated with EtOH or DEX for 48 h, were seeded into 96-well plates, and adherence was assessed after 4 h as described in Materials and Methods. Results are representative of four independent experiments (±SD). (B) Monocytes (5x105) were treated for 48 h as described and placed into the upper chamber of a Transwell filter (5 µm). The lower chamber contained monocyte medium. After 4 h, cells in the lower chamber were collected and counted. Results are expressed as percent cells that have migrated. Results represent three independent experiments (±SEM). ***, P < 0.001; **, P < 0.01 (Student’s t-test).

Thus, GC-induced murine monocytes also resemble their human counterparts with regard to their decreased adherence and increased motility.

GC-induced monocytes are distinct from physiologically occurring subtypes
Under physiological conditions, there are distinct subpopulations of murine blood monocytes. Among them, CX3CR1lowCCR2+Ly6Chigh proinflammatory monocytes (released from the bone marrow) developed into CX3CR1highCCR2–Ly6Clow resident monocytes via a stage of CX3CR1medCCR2–/+Ly6Cmed cells [9 , 10 ]. As CX3CR1 was down-regulated on GC-induced murine monocytes (Fig. 1B) , we wondered if the GC-induced monocyte subtype would belong to one of these distinct, physiologically occurring subtypes. We therefore tested the regulation of other subset-defining surface molecules such as CCR2 (reviewed in refs. [4 , 12 , 14 ]) and Ly6C [9 ]. As shown in Figure 4A and 4B , GC treatment of monocytes led to down-regulation of CCR2 (mRNA and protein) over a period of 48 h, indicating that GC induces down-regulation of this surface receptor.


Figure 4
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  		Figure 4. GC-induced murine monocytes exhibit a distinct phenotype. Monocytes were prepared as described in Materials and Methods. Resulting cells were treated with EtOH (1:10,000) or DEX (10–7 M) for the indicated amounts of times. (A) Cells were harvested, and RNA was prepared. RT-PCR for CCR2 was performed as described. Graphs show mean expression ± SD from three independent experiments. (B) Cells were harvested after 48 h culture and stained for CCR2, (C) Ly6C, and (D) Gr-1 and were analyzed by flow cytometry. The histogram is representative of at least four independent experiments. Filled graph, Secondary antibody (sec. ab); black line, control monocytes (CtrMo); red line, GC-induced monocytes.

We have recently described Ly6C as a marker that distinguishes three monocytic subtypes—high on precursor cells and becoming down-regulated when monocytes mature in the blood [9 ]. As shown in Figure 4C , Ly6C is down-regulated moderately on monocytes upon GC treatment. On the other hand, Gr-1 was up-regulated significantly by GC (Fig. 4D , P=0.0342, and data not shown) so that GC treatment results in Ly6Cmed and Gr-1hi cells.

The expression of additional surface molecules of monocytes and macrophages, e.g., CD11b, CD115, and F4/80, was measured by flow cytometry and summarized in Table 1 . The results reveal an up-regulation of CD62L, indicating that these monocytes had not been acutely activated by GC or other stimuli during our experimental procedure. This also confirms that these Gr-1hi cells belong to the monocyte/macrophage lineage by constitutive expression of CD11b, CD115, and F4/80.


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  		Table 1. Phenotype of GC-Induced Monocytes

Taken together, murine GC-induced monocytes are pure CX3CR1lowCCR2–Ly6CmedCD62Lhigh monocytes and thus, present a nonactivated phenotype distinct from the known, physiologically occurring monocyte subsets (Table 1 , and see Go Fig. 6 ).


Figure 5
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  		Figure 5. GC-induced murine monocytes resemble MDSC. (A) Monocytes from naïve or B16 melanoma-bearing mice (14 days) were stained with anti-CD11b and anti-Gr-1 antibodies and were subsequently analyzed by FACS. Dot-plots are representative of two independent experiments. (B) As in A but stained with anti-CD124. One experiment out of two is shown. (C) Monocytes were treated for 48 h with EtOH or with DEX. Cells were then harvested and stained with the indicated antibodies. Subsequently, cells were analyzed by flow cytometry (FACS). DEX-treated monocytes (DEX-Mo) are shown. Result is representative of four independent experiments. (D) As in C but stained for CD124. iso, .


Figure 6
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  		Figure 6. GC-induced monocytes (M) have a distinct phenotype.

GC-induced monocytes resemble MDSC
As the combined expression of CX3CR1, CCR2, Ly6C, and CD62L differed from the expression on the physiologically occurring monocyte subsets, we wondered if GC-induced murine monocytes would be rather related to murine monocyte subsets, as have been described under pathophysiological conditions. In particular, the combination of anti-inflammatory activities with expression of Gr-1+ (Fig. 5C) indicated similarities to MDSC. These are CD11b+Gr-1+ cells with immunosuppessive qualities that have been found to develop in the presence of malignant tumors (for review, see refs. [13 , 19 ]). They have recently been found to express as a surface signature the IL-4R{alpha} chain (CD124), which also has been implicated to be important for their immuno-regulatory function [19 ]. We therefore investigated whether GC-induced monocytes would have further similarities to these tumor-induced MDSC.

We therefore applied the murine model with B16 melanoma to generate MDSC. On Day 14 after injection of 1 x 105 B16 melanoma cells s.c. into mice, there was marked growth of melanoma. Mice were killed, and monocytes were prepared (from bone marrow) to compare their expression of CD11b, Gr-1, and CD124 with that of GC-induced monocytes. As shown in Figure 5A , a significantly higher proportion of monocytes from tumor-bearing mice was CD11b+ and Gr-1+ in comparison with monocytes from controls (data not shown). A similar difference applied for GC-induced murine monocytes (Fig. 5C) in comparison with control monocytes (data not shown). In particular, CD124 was up-regulated markedly and significantly on monocytes from tumor-bearing mice compared with cells from naïve mice (Fig. 5B) , demonstrating the presence of MDSC in the murine B16 melanoma model. Remarkably, significant up-regulation of CD124 was also found in GC-treated monocytes compared with control-treated monocytes (Figs. 5D and 6 ).

Taken together, GC-induced murine monocytes are CD11b+Gr-1+, and they up-regulate the IL-4R{alpha} chain CD124, thus revealing the same phenotype as tumor-induced MDSC.

DISCUSSION

We have shown previously that GC do not generally suppress gene transcription in human monocytes but rather, up-regulate molecules that are associated with anti-inflammatory features, e.g., IL-10 and CD163 [2 ]. In this study, we wondered whether this also applies for the murine monocytes. This would be important, as it would allow us to investigate the functional capabilities of GC-induced monocytes in vivo with the help of murine models of inflammation.

We revealed that GC-induced murine monocytes present a rather stable subtype, which resembles their human counterpart with regard to two characteristic features, i.e., increased expression of CD163 and down-regulation of the fractalkine receptor CX3CR1.

Up-regulation of scavenger receptor CD163 is a phenotypic hallmark of GC action on human monocytes. Here, we show that this up-regulation is even more prominent in GC-induced murine monocytes.

GC-induced human monocytes up-regulate mediators with anti-inflammatory properties including IL-10 [2 , 20 , 21 ]. Up-regulation of anti-inflammatory IL-10 in general is a well-known phenomenon after treatment with GC [2 , 20 , 21 ]. We therefore analyzed whether murine GC-induced monocytes would also exert functions that are involved in down-regulating inflammatory processes or even in immunosuppression.

Indeed, IL-10 mRNA was up-regulated (Fig. 2A) . As in their human counterparts, this up-regulation was rather selective, as at the same time, TGF-β, another regulatory cytokine, was not induced (Fig. 2B) .

We also found down-regulation of IL-6 (Fig. 2C and 2D) , a cytokine with proinflammatory properties that has also been reported to exhibit anti-inflammatory capacities in some circumstances [22 ].

Thus, GC-induced murine monocytes share with their human counterparts a particular pattern of surface markers and up-regulation of IL-10, and there is additional down-regulation of IL-6 that indicates species-specific mechanisms (for summary, see Table 2 ).


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  		Table 2. Comparison of Human and Murine GC-Induced Monocytes

Murine GC-induced monocytes also presented a low capacity to adhere and a higher motility (Fig. 3) [2 ], indicating that they are able to transmigrate through endothelial barriers more rapidly. Therefore, GC not only induce monocytes that possibly exert anti-inflammatory activity, but they may also induce increased migration of these anti-inflammatory monocytes to sites of inflammation in vivo.

As CX3CR1 was down-regulated on GC-induced murine monocytes (Fig. 1B) and as low expression of CX3CR1 is a marker of the recently described, physiologically occurring subtypes of murine and human monocytes, we wondered if the GC-induced monocyte subtype would resemble or belong to one of these subtypes. In mice, it was shown that CX3CR1lowCCR2+Ly6Chigh monocytes are released from the bone marrow and recruited to the tissue in the presence of inflammatory stimuli ("classical/proinflammatory" monocytes). In the absence of inflammation, they eventually mature into CX3CR1highCCR2–Ly6Clow cells in the blood via a stage of CX3CR1medCCR2–/+Ly6Cmed cells and replenish resident tissue phagocytes or DC in the context of homeostasis [4 , 9 , 12 ].

We found that GC-induced monocytes present a nonactivated phenotype distinct from the known physiologically occurring monocyte subsets (Fig. 6) . They share with the more mature monocyte the reduced expression of Ly6C and a nonactivated state shown by the presence of CD62L. Yet, they do present low expression of CX3CR1 and high expression of Gr-1, as found on inflammatory monocytes recruitable to inflamed tissue (Fig. 1 and Tables 1 and 2 ). It is speculative, if they present an (originally) inflammatory monocyte, which is endowed with the prerequisites to migrate quickly into inflamed tissue (CCR2+CX3CR1low, generally high motility, and low adhesive properties), where they could then exert anti-inflammatory actions.

As the combined expression of CX3CR1, CCR2, and Ly6C differed from the expression on the physiologically occurring monocyte subsets, we wondered if GC-induced monocytes would rather be related to subsets described under pathophysiological conditions.

In particular, the combination of anti-inflammatory or even immunosuppressive activities with expression of CD11b+ and Gr-1+ (Fig. 5C) prompted us to directly test a close relation between GC-induced monocytes and tumor-induced MDSC, which were isolated from the peripheral blood of cancer-bearing humans as well as of mice, and they are involved in tumor-associated immunosuppression [13 ].

We took advantage of the fact that MDSC have recently been distinguished by expression of the IL-4R{alpha} chain (CD124; for review, see ref. [13 ]). We investigated expression of CD124 in GC-induced murine mononcytes and monocytes from mice bearing B16 melanoma. We were able to demonstrate that the IL-4R{alpha} chain CD124 was indeed up-regulated by the increased population of CD11b+ Gr-1+ monocytes in B16 melanoma-bearing mice. Remarkably, CD124 was also up-regulated on monocytes upon treatment with GC, thus making them phenotypically indistinguishable from MDSC. Further analysis revealed that GC-induced monocytes were phenotypicallay indistinguishable from MDSC with respect to expression of CD11b, Gr-1, and IL-4R{alpha} chain.

This could have important implications for the better understanding of GC-induced monocytes as well as MDSC differentiation and function, as we demonstrate that important phenotypic features of MDSC can be achieved by treatment with a single, defined mediator such as GC. MDSC are believed to be important for tumor-associated immunosuppression, and they have been shown to be involved in tumor progression. It is therefore tempting to speculate that the similarities between GC-induced monocytes and MDSC may present a mechanism that is involved in the higher incidence and faster progression of tumors in patients treated with GC for prolonged periods [23 ]. It will be exciting to see whether further analysis of GC- and tumor-induced monocytes reveals closer similarities with respect to their immunosuppressive functions.

In summary, we demonstrate that GC induce a distinct and durable anti-inflammatory phenotype in mouse monocytes that is phenotypically identical to MDSC.

ACKNOWLEDGEMENTS

This study was supported by a grant of the Deutsche Forschungsgemeinschaft (DFG; SU 195/3-1), Innovative Medizinische Forschung, Münster (IMF; SU 220606 to A. T. and C. S.), and Interdisciplinary Research Center Münster (IZKF Sun2/019/07; to A. T.). G. V. and J. E. designed research, performed research, and wrote the paper; K. T. designed research; A. T., A. R., S. S., and M. M. performed research; and J. R. and C. S. designed research and wrote the paper. We thank A. Stadtbaeumer, E. Nattkemper, and U. Nordhues for excellent technical support and S. Varga-Braun for critical reading of the manuscript.

FOOTNOTES

1 These authors contributed equally to this work. Back

Received November 18, 2007; revised May 22, 2008; accepted June 9, 2008.

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