International Immunology

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International Immunology, Vol. 11, No. 9, 1519-1526, September 1999
© 1999 Japanese Society for Immunology

Glucocorticoids increase the endocytic activity of human dendritic cells

Lorenzo Piemonti^1,4, Paolo Monti¹, Paola Allavena³, Biagio Eugenio Leone^2,4, Alessandra Caputo¹ and Valerio Di Carlo¹

¹ Laboratory of Experimental Surgery, Surgical Department and
² Department of Pathology, S. Raffaele Scientific Institute, 20132 Milan, Italy
³ Department Immunology and Cell Biology `Mario Negri' Institute, 20157 Milan, Italy
⁴ Department of Surgery, University of Milan, 20132 Milan, Italy

Correspondence to: L. Piemonti, Laboratory of Experimental Surgery, S. Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy

	Abstract

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We have investigated the effect of glucocorticoids (GC) on antigen uptake molecule expression and on endocytic activity of human dendritic cells (DC). Human monocyte-derived DC were differentiated in vitro for 7 days with granulocyte macrophage colony stimulating factor and IL-4 in the presence or absence of dexamethasone 10^–8 M (Dex). Dex-treated DC showed an enhancement of mannose receptor (MR)-mediated endocytosis (measured as uptake of FITC–dextran) and of fluid-phase endocytosis [measured as uptake of Lucifer yellow (LY)] The effect was dose dependent and correlated with the length of exposure to Dex. The expression of receptors involved in antigen capture was investigated by FACS analysis. Dex up-regulates MR, CD16 and CD32 expression on DC. After maturation with tumor necrosis factor- or CD40 ligand in Dex-treated DC, despite a reduction induced by maturation the endocytic activity of FITC–dextran and LY, the expression of MR, CD16 and CD32 remained higher than in control DC. In view of the fact that antigen capture was increased in cells cultured with Dex, we evaluated the ability to present soluble antigen that needs to be taken up and processed. Cells differentiated in the presence of Dex showed much lower efficiency in presenting tetanus toxin to specific autologous T cell lines. In conclusion our data suggest a new mechanism by which GC may influence immune responses. In fact with the increase in endocytic activity, Dex favors the scavenging of antigen from the external milieu, decreasing antigen concentration and availability, and simultaneously inhibiting the capacity to stimulate T cells.

Keywords: dendritic cells, endocytosis, glucocorticoids, human, mannose receptor

	Introduction

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Dendritic cells (DC) are bone marrow-derived leukocytes specialized in antigen presentation to T lymphocytes (1–4). Among antigen-presenting cells (APC), DC are considered to be the most efficient and indispensable to stimulate naive T cells. The first step of antigen presentation is antigen capture, which is followed by processing and formation of peptide–MHC complexes that are accumulated on the cell surface for presentation to immunocompetent T cells (5). Immature DC are very efficient in antigen uptake and processing (6–8), and yet have a suboptimal capacity of T cell stimulation. Maturation of DC with inflammatory cytokines [IL-1 and tumor necrosis factor (TNF)] or engagement with CD40 ligand (CD40L) (7), or exposure to yet unidentified monocyte-derived products (9,10), decreases their ability to capture antigen and confers potent co-stimulatory activity for T cells (6,7).

Immature DC can internalize large amounts of material via both fluid-phase uptake through macropinocytosis and receptor-mediated uptake (8,11). Macropinosomes, which occur at the site of membrane ruffling, require an actin-based cytoskeleton and form surface ruffles that fold back against the cell or against each other to enclose a vesicle up to 5 µm in diameter (12). In receptor-mediated endocytosis, clathrin-coated pits invaginate to form small coated vesicles; ligands adsorbed to receptors in coated pits are subsequently delivered into the intracellular compartment. DC express receptors involved in antigen uptake [e.g. mannose receptor (MR) (8), DEC 205 (13), FcRI and FcR (8)]. Receptors for antigen capturing vary in their ligand specificity and mode of delivery to the antigen-processing compartment. FcR are internalized concomitant with their ligands and targeted for degradation in the lysosomes; thus they are available for a single round of uptake only (5). In contrast to Fc receptors, MR on DC recycles constitutively while releasing its cargo and may allow internalization of ligand in successive rounds (14). In vitro, monocytes isolated from peripheral blood can differentiate into immature DC upon culture in medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4, and be maintained at a typical maturation stage associated with high endocytic and antigen-processing activity (6).

Glucocorticoids (GC) are widely used as anti-inflammatory and immunosuppressive agents in the therapy of many autoimmune and allergic diseases, and in transplantation to prevent rejection. GC affect the immune system and inflammatory response in many different way, primarily influencing monocytes/macrophages and T cells (15–20) We previously demonstrated that dexamethasone (Dex) affects the differentiation of monocytes into DC, by freezing the cells at an immature stage (21).

The aim of this work was to study the effects of GC on the endocytic activity of immature DC, as this function is critical for an optimal presentation and stimulation of T cells. Results demonstrate that Dex increases both the MR-mediated endocytosis and the membrane ruffling of DC. This increased endocytic activity, however, is not paralleled by increased stimulatory capacity.

	Methods

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Cytokines and reagents
Human recombinant GM-CSF (sp. act. 1.1x10⁴ U/µg) was from Novartis (Basel, Switzerland). Human recombinant IL-4 (sp. act. >2x10⁶ U/mg) and human recombinant TNF- (sp. act. >2x10⁷ U/mg) were from PeproTech (London, UK. Water-soluble Dex was from Sigma (St Louis, MO). Human recombinant IL-2 was from Kiron (Milan, Italy).

DC culture
Highly enriched monocytes (>80% CD14⁺) were obtained and purified from buffy coats (through the courtesy of Centro Trasfusionale, Ospedale San Raffaele, Milan, Italy) by Ficoll-Percoll gradients and by plastic adherence. Monocytes were cultured for 7 days at 1x10⁶/ml in six-well multi-well tissue culture plates (Falcon, Becton Dickinson, Somerville, NJ) in RPMI (Biochrom, Berlin, Germany) and 10% FCS (Hyclone, Logan, UT) supplemented with 50 ng/ml GM-CSF, 10 ng/ml IL-4 with or without water-soluble Dex (10^–8 M). In the control group (GM-CSF + IL-4) the cell yield was ~80% of input cells. No difference was seen in the yield of DC in Dex-treated monocytes at 10^–8 M.

DC maturation
TNF- (10 ng/ml) was added to induce maturation of DC for at least 48 h of culture. J558L cells transfected with the ligand for CD40 (J558LmCD40L) were used to induce CD40 triggering on DC. Untransfected J558L cells were used for control cultures. J558L after irradiation (10000 rad) were seeded together with DC at a 1:1 ratio in 24-well culture plates in culture medium (1x10⁶ cells/well). Cells were recovered after 48–72 h of culture.

FACS analysis
The flow cytometer used was a FACScan from Becton Dickinson (San Jose, CA). Software used was CellQuest. Cell staining was performed using mouse mAb followed by FITC-conjugated affinity-purified, isotype-specific goat anti-mouse antibodies (Ancell, Bayport, MN). The following mAb were used: 17aba (anti-CD11b), 32.2 (anti-CD32), IV.3 (anti-CD64), L243 (IgG2a anti-MHC class II) from ATCC (Rockville, MD); B73.1 (IgG2a, anti-CD16), PAM-1 (IgG1 anti-MR, as a kind gift of Dr. Biondi, Milan, Italy) (22) and W6/32 (IgG2a anti-MHC I) from Sigma; and BB1 (IgM, anti-CD 80), BU63 (IgG1, anti-CD86) and EA-5 (IgG1 anti-CD40) from Ancell. Results are expressed as percentage of positive cells or as fluorescence intensity (FI), calculated according to the formula: FI = mean fluorescence (sample) – mean fluorescence (control).

Electron microscopy
Dex-differentiated DC and control DC after exposure to 1 mg/ml FITC–dextran for 1 h were fixed in 2% paraformaldehyde for 1 h at 4°C and embedded in acryl resin (LRWhite; London Resin, London, UK). Thereafter, thin sections, mounted onto nickel grids, were immunolabeled by using anti-fluorescein mouse mAb (Boehringer Mannheim, Germany), diluted 1:100, followed by incubation in goat anti-mouse Ig-coated colloidal gold particles, 10 nm in diameter (Ylem, Milan, Italy), diluted 1:30. The sections were stained with uranyl acetate and lead citrate, and examined under a Zeiss CEM 902 electron microscope

Endocytosis
MR-mediated endocytosis was measured as the cellular uptake of FITC–dextran and quantified by flow cytometry. Approximately 2x10⁵ cells per sample were incubated in media containing FITC–dextran (1 mg/ml) (mol. wt 40,000; Sigma) for 0, 60 and 120 min. After incubation, cells were washed twice with PBS to remove excess dextran and fixed in cold 1% formalin. The quantitative uptake of FITC–dextran by the cells was determined using FACS. At least 8000 cells per sample were analyzed. Unlabeled mannose 3 mg/ml, EDTA 0.5 mM (SIGMA) and anti-MR (PAM-1) mAb were used to block MR-mediated endocytosis. Fluid-phase endocytosis through membrane ruffling was measured as the cellular uptake of 1 mg/ml of Lucifer yellow (LY) dipotassium salt (Sigma) and quantified by flow cytometry. 5-(N,N-dimethyl)-amyloride (AML) (Sigma), an inhibitor of Na⁺/H⁺ channels, was used at a final concentration of 3 mM to block uptake of the markers. AML was added to the cells at 37°C 15 min before the addition of the marker.

Antigen presentation assay
Tetanus toxin (TT)-responsive T cell lines were obtained in our laboratory by culturing mononuclear cells with TT (36 µg/ml) (Cannaught, Willowdale, Ontario, Canada) for 1 month, in the presence of IL-2. TT responsive T cells were tested at least 2 weeks after the last peripheral blood mononuclear cells stimulation and 5 days after the last addition of IL-2. DC were obtain from the same donor by culturing monocytes. After 7 days DC were preincubated with TT (6 µg/ml) for 12 h and with the maturation stimulus TNF- (10 ng/ml) for 48 h. Then DC were washed, irradiated (3000 rad) and co-cultured with TT autologous responsive T cell lines for 72 h in 96-well microtiter plates and [³H]thymidine uptake was measured during the last 18 h of culture (1 µCi/well, sp. act. 5 Ci/mmol; Amersham, Little Chalfont, UK).

Calculations and statistical analysis
Data were expressed as mean. Comparisons were performed by Student's t-test. P < 0.05 was considered statistically significant

	Results

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Addition of Dex during differentiation
We investigated the endocytic activity of DC when Dex was added during the differentiation of the cells. The endocytic activity was studied as uptake of two fluorescent markers: LY, a non-specific fluid-phase marker, and FITC–dextran, which is mainly taken up through the MR. DC cultured with GM-CSF and IL-4 in the presence of Dex added at the first day (day 0) showed a vigorous endocytosis of FITC–dextran, higher than control DC (2.3-fold increase at 1 h and 2.4-fold increase at 2 h; n = 13). The effect was dose and time of exposure dependent (Figs 1 and 2). The same behavior was seen when we used LY, a marker of fluid-phase pinocytosis (1.8 fold increase at 1 h and 1.6-fold increased at 2 h; n = 13) (Fig. 2). We also investigated the expression of antigen uptake molecules on DC differentiated in the presence of Dex (Fig. 3B). Dex up-regulated MR, IgGFc receptor II (CD32), IgGFc receptor III (CD16) and complement receptor CR3 (CD11b), while the cells cultured in the presence or absence of Dex were negative for CD64.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Effect of addition of Dex at different doses on FITC–dextran uptake and MR expression. DC were differentiated from monocytes cultured for 7 days in GM-CSF (50 ng/ml) and IL-4 (10 ng/ml) (Ctr). Dex was added at different concentrations (10–10/10–7 M) at the beginning of culture (Dex). Expression of MR (negative, not shown, was in the first decade of the logarithmic scale) was evaluated by PAM-1 mAb; endocytosis was evaluated as uptake of FITC–dextran (1 mg/ml) and measured using FACS. Results are expressed as fluorescence intensity. n = 3.

 

View larger version (27K): [in this window] [in a new window]   Fig. 2. Effect of addition of Dex at different times on FITC–dextran and LY uptake. DC were differentiated from monocytes cultured for 7 days with GM-CSF and IL-4. Dex 10–8 M was added at different times of culture (right column): in the last 24 h (day 6; n = 3), after 3 days of culture (day 3; n = 3) or at the beginning of culture (day 0; n = 13). Endocytosis was evaluated as uptake of FITC–dextran (1 mg/ml) or LY and measured by FACS. Results are expressed as FI. *P < 0.05.

 

View larger version (23K): [in this window] [in a new window]   Fig 3. Effect of Dex on receptors involved in antigen uptake. Cells were labeled with the designed mAb and then with FITC-labeled goat anti-mouse Ig. Results are expressed as fluorescence intensity. (A) Control DC were cultured for 14 days in GM-CSF and IL-4 (ctr). Dex-DC were cultured for 7 days in GM-CSF (50 ng/ml) and IL-4 (10 ng/ml), then Dex 10–8 M was added for 7 days (dex). (B) DC were differentiated from monocytes cultured for 7 days with GM-CSF 50 ng/ml and IL-4 10 ng/ml (ctr). Dex (10–8 M) was added at the beginning of culture (dex). n = 13; *P < 0.05.

 
Effect of Dex on the endocytic activity of differentiated DC
DC were differentiated from blood monocytes by in vitro culture with GM-CSF and IL-4 for 7 days. Dex 10^–8 M was added for an additional 7 days. The endocytic activity of Dex-treated DC was compared to that of DC cultured for 14 days with GM-CSF and IL-4 only. Cells cultured with Dex showed a higher endocytosis of LY than control DC (Fig. 2). The same behavior was seen when FITC–dextran was used. We also investigated the expression of some receptors involved in antigen capture (Fig 3A): MR, and IgGFc receptors CD16, CD32 and CD64. MR was never detected on freshly isolated monocytes but was induced by GM-CSF and IL-4, as described (5). Dex up-regulated MR and IgGFc receptor II (CD32) expression on DC. The cells in the presence or absence of Dex were negative for CD64 and had very low levels of CD16.

Effects of maturation on endocytic activity of Dex-differentiated DC
It has been demonstrated that some inflammatory cytokines such as TNF- and IL-1 or CD40 triggering are maturation factors for cultured DC, which result in a decrease of endocytic activity. As expected, the addition of TNF- or CD40L for 48 h after 7 day culture resulted in a lower internalization of FITC–dextran and LY in control DC and in Dex-differentiated DC. The endocytic activity of FITC-dextran (Fig. 4) and LY (data not shown), despite a reduction induced by maturation, remained higher in Dex-treated DC than in control DC. Antigen uptake molecule expression after maturation with both TNF- and CD40L showed a reduction of MR and CD32 intensity, and, for Dex-differentiated cells, of CD16.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effect of Dex on endocytic activity of DC cultured with maturation stimuli. DC were differentiated from monocytes cultured for 7 days in GM-CSF (50 ng/ml) and IL-4 (10 ng/ml) in the absence (A) or presence (B) of Dex 10–8 M. DC were treated with TNF- 10 ng/ml for the last 48 h of culture. J558L cells transfected with the ligand for CD40 (J558LCD40L) were co-cultured with DC at a 1:1 ratio for 48–72 h. Endocytosis was evaluated as uptake of FITC–dextran (1 mg/ml) and measured by FACS. Results are expressed as FI. n = 6; *P < 0.05.

 
Dex-differentiated DC do not show a different mechanism in FITC–dextran uptake.
To confirm that uptake of FITC–dextran occurs through a membrane activity, we inhibited the membrane movements using AML, an inhibitor of Na⁺/H⁺ channels. In both groups AML completely abrogated the FITC–dextran uptake. To investigate the role of MR in DC endocytosis, we tested the inhibitory effect of EDTA (0.5 mM), mannan (a bacterial polysaccharide that binds with high affinity with the MR) and anti-MR antibody (PAM-1). As shown in Fig. 5, the uptake of FITC–dextran was inhibited to a comparable extent in Dex-differentiated DC and control DC by EDTA, mannan and anti-MR mAb, while the same treatments did not affect the uptake of LY. Thus ligand specificity, calcium dependency and a specific antibody identify MR as the most important receptor involved in endocytosis of FITC–dextran in both Dex-differentiated DC and in control DC. Immunoelectron microscopy was performed to investigate the intracellular localization of FITC–dextran, (Fig. 6). Dex-treated dendritic cells showed many cytoplasmic dense granules, 50 nm in mean diameter, immunolabeled by anti-fluorescein antibody localized in subplasmalemmal areas as well as in deeper paranuclear sites. Control DC showed a lower number of immunolabeled cytoplasmatic dense granules and also a more regular external surface.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Inhibition of FITC–dextran uptake by AML, EDTA, unlabeled mannan and specific MR mAb. DC were differentiated from monocytes cultured for 7 days with GM-CSF 50 ng/ml and IL-4 10 ng/ml (ctr). Dex (10–8 M) was added at the beginning of culture (dex). Endocytic activity was evaluated after 1 h as uptake of FITC–dextran and measured by FACS. MR endocytosis was blocked using AML (3 mM, added to the cells at 37°C 15 min before the addition of the marker), unlabeled mannan (3 mg/ml), EDTA or a mAb specific for MR (PAM-1). Results are expressed as percent of FITC–dextran uptake compared to untreated DC. n = 3.

 

View larger version (74K): [in this window] [in a new window]   Fig. 6. Electron microscopy. (A) A Dex-treated dendritic cell shows many cytoplasmic dense granules, 50 nm in mean diameter, immunolabeled by anti-fluorescein antibody (see inset), localized in subplasmalemmal areas as well as in deeper paranuclear sites. The cell shows a cytoplasmatic projection, in which labeled dense granules are seen (arrow). (B) A control DC shows a lower number of immunolabeled cytoplasmic dense granules. Note also a more regular external surface. Uranyl acetate and lead citrate. Original magnification x7000. Bar = 1 µm.

 
Increase in antigen uptake capacity does not result in increase in antigen presentation activity
In view of the fact that antigen capture was increased in cells cultured with Dex, we evaluated the ability to present soluble antigen that needs to be taken up and processed. Cells differentiated in the presence of Dex showed much lower efficiency in presenting TT to specific autologous T cell lines (Fig. 7A). In vitro exposure of APC to TNF- for 48 h after antigen pulsing increased T cell proliferation with both APC cultures, but the ability of cells cultured with Dex was always lower than control DC (Fig. 7B). To explain a possible mechanism responsible for the inhibition of T cell response, we investigated the expression of molecules involved in the activation of T lymphocytes such as MHC and co-stimulatory molecules (Fig. 8). Before TNF- exposition, Dex-treated DC showed higher expression of MHC I, MHC II and CD80, and lower expression of CD86 and CD40 molecules than control DC. After maturation with TNF-, MHC I remained higher in Dex-treated DC, CD86 and CD 40 remained lower, and no differences in MHC II and CD 80 expression were seen.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Effect of Dex on the presentation of soluble TT to specific T cell lines. Monocytes were cultured with GM-CSF and IL-4 in the presence (dex) or absence (ctr) of Dex (10–8 M). After 7 days, cultures were pulsed with TT (6 µg/ml) for 12 h. After antigen pulsing, APC were cultured for 48 h in medium alone (A) or with TNF 10 ng/ml (B). Cells were then washed and mixed, at different stimulator:responder ratios, with 105 autologous TT-specific T cells. Autologous monocytes as stimulators are also shown (Mo). Proliferation was assessed as [3H]thymidine uptake during the last 18 h of a 3 day experiment. n = 3.

 

View larger version (37K): [in this window] [in a new window]   Fig. 8. Effect of Dex on DC phenotype. Membrane phenotype analysis of cells cultured for 7 days in GM-CSF (50 ng/ml) ± IL-4 (10 ng/ml) with or without Dex (10–8 M). DC maturation: DC were treated with TNF- 10 ng/ml for the last 48 h of culture. Cells were labeled with the designed mAb and then with FITC-labeled goat anti-mouse Ig. Results are expressed as FI, calculated as described in text. n = 6, *P < 0.05 versus control.

 

	Discussion

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In this study we show that Dex increased the efficacy of antigen uptake by DC through at least two means: by non-specific endocytosis, as shown by LY uptake, as well as by up-regulation of MR-mediated endocytosis, as shown by FITC–dextran uptake. Increased MR-mediated endocytosis was achieved when Dex was added to GM-CSF and IL-4 from the initiation of the culture or after differentiation. MR is a surface 175 kDa C-type lectin containing eight carbohydrate recognition domains with broad specificity for sugars (23). It mediates phagocytosis of mannose-coated particles such as yeasts and endocytosis of mannosylated glycoprotein in macrophages (24). In DC MR mediates antigen uptake and strongly enhances HLA class II-restricted antigen presentation (7,25,26). In contrast to Fc receptors and mIg which are degraded together with their cargo, the MR is not targeted to lysosomes but rapidly recycles between the plasma membrane and an intracellular, early endocytic compartment (14). Increased vesicle trafficking and membrane recycling coupled with increased expression of MR (up to 2 times more fluorescence intensity) is a probable explanation for the overall greater efficiency of Dex-differentiated DC at capturing FITC–dextran. The increased MR and FcR expression combined with an enhanced membrane turnover provide Dex-treated DC with a more efficient endocytic function. The endocytosis of FITC–dextran induced by Dex was greatly reduced by competition with unlabeled mannose, by EDTA and by an antibody to the human MR. The data obtained when we used a functional anti-MR mAb (PAM-1) showed that only a small part of FITC–dextran is internalized via an alternative pathway. To confirm that Dex also stimulated other mechanisms of endocytosis we evaluated the uptake of LY which is internalized mostly by membrane ruffling and macropinocytosis. Indeed Dex increased capture of LY compared to untreated cells. Thus Dex stimulates both MR-dependent and fluid-phase endocytosis. Despite an increase in endocytic activity, DC differentiated in the presence of Dex showed a decrease in their capacity to present a soluble antigen to autologous T cells. A possible mechanism responsible for the inhibition of T cell response is the different expression of molecules involved in the activation of T cells. Firstly, in both immature and mature states, Dex-treated DC showed a lower level of two co-stimulatory molecules, CD86 and CD40, relevant for T cell activation. Secondly, immature Dex-treated DC showed a higher expression of MHC molecules. The higher MHC expression on membrane before the maturation may result in a lower disposability of MHC molecules, and a lower capacity to process and form peptide–MHC complexes in the intracellular space. In fact an increase in antigen uptake activity may result in higher antigen presentation if DC effectively process antigen, and present peptide in the context of both class I and class II molecules. Dex-treated DC showed a paradoxical behavior: a simultaneous increment in antigen uptake activity and MHC molecules expression on the cellular membrane that may impair the capacity to present soluble antigens. Thus, our data suggest a new mechanism by which GC may influence immune responses. Dex increases the expression of MR and Fc receptors on DC. MR play a role in the uptake of mannosylated proteins that are especially abundant in a variety of microorganism, in contrast to the situation in higher eukaryotes, where mannose residues are usually buried within the carbohydrate moieties of glycoproteins and therefore not available for binding to the MR. Fc receptors mediate the clearance of immunocomplexes and antigen uptake after antibody production. With the increase in endocytic activity Dex favors the scavenging of antigen from the external milieu, decreasing antigen concentration and availability, and simultaneously inhibits the capacity to stimulates T cells. In addition MR may play a role as a scavenger for secreted lysosomal enzymes, such as -hexosaminidase and ß-glucuronidase, bearing high mannose-type carbohydrates (28). Curiously Morel et al. (29) and Longoni et al. (30) described that IL-10, an anti-inflammatory and immunosuppressive cytokine, like Dex, increases the capacity to capture antigen in human DC with a concomitant decreased stimulatory activity. So we can speculate that this mechanism may play a role in the immunosuppressive effect of these two reagents. Our result are apparently in contrast with a recent study on murine airway DC, in which Holt et al. (31) speculate that Dex may inhibit antigen uptake. In murine airway DC, in marked contrast to the results obtained with mixed lymphocyte reactions, effective presentation of ovalbumin was almost completely inhibited if antigen pulsing was performed in the presence of steroid. In the study, antigen uptake capacity of DC was not directly measured, and the deficit in antigen presentation may be a problem of processing and not in uptake of antigen. In fact in our study, despite a higher endocytic activity, steroid also inhibited effective presentation of TT.

In the past modulation of MR has been extensively studied in murine and human macrophages. Macrophages activation by cytokines or bacterial pathogens (e.g. IFN-, lipopolysaccharide and phorbol myristate acetate) led to a rapid decrease of MR activity (32,33); on the other hand, Dex potently enhanced MR expression and activity (32,34,35). Thus Dex appears to have a similar action on other APC-like macrophages.

GC are known to down-regulate the capacity of monocytes and/or macrophages to secrete IL-1, IL-6, TNF-, IL-10 and MIP-1, and to regulate several immunologically relevant activities of these cells including secretion of prostaglandins, bactericidal and fungicidal activities, phagocytosis and pinocytosis, and expression of surface receptors for complement and Ig (15–20). It is also know that GC regulate the cytokine secretion of T lymphocytes and inhibit their mitogenic potential (15–20). Based on these observations, the activities of GC have been attributed primarily to their influence on monocytes/macrophages and on T cells. Our data suggest a new mechanism and a new cell target by which GC may influence the immune response in humans. Because DC, rather than macrophages and monocytes, are responsible for the initiation of T cell-mediated immunity, the action of Dex on these cells may play a relevant role in the immunosuppressive and tolerogenic activity of this drug.

	Acknowledgments

 
This work was supported by grants of CNR (Finalized Project Biotechnology no. 97.01301. PF 49) and Istituto Superiore di Sanità, Italy.

	Abbreviations

 

AML	5-(N,N-dimethyl)-amyloride
APC	antigen-presenting cell
CD40L	CD40 ligand
Dex	dexamethasone
DC	dendritic cell
FI	fluorescence intensity
GC	glucocorticoid
GM-CSF	granulocyte macrophage colony stimulating factor
LY	Lucifer yellow
MR	mannose receptor
TT	tetanus toxin

	Notes

 
Transmitting editor: G. Doria

Received 15 March 1999, accepted 31 May 1999.

	Reference

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