Dexamethasone inhibits dendritic cell maturation by redirecting differentiation of a subset of cells Richard Matasic´, Allan B. Dietz, and Stanimir Vuk-Pavlovic´ Stem Cell Laboratory, Mayo Clinic Cancer Center, Mayo Clinic and Mayo Foundation, Rochester, Minnesota
Abstract: To investigate how corticosteroids affect differentiation of human dendritic cells (DC) in a defined inflammatory environment, we incubated immature DC with dexamethasone in the presence of tumor necrosis factor ␣ (TNF-␣), interleukin-1 (IL-1), and prostaglandin E2. Dexamethasone inhibited differentiation into mature DC, as indicated by the reduced expression of antigen-presenting molecules, costimulatory and adhesion molecules, a marker of mature DC, and IL-12. Dexamethasone increased expression of CD14, CD36, and CD68, molecules characteristic of monocytes/ macrophages and induced CD14⫹CD83⫺ cells, a subset distinct both from immature DC and mature DC. The effects were concentration-dependent, with ID50 values between 2 and 30 nM dexamethasone. Unlike T and B cells, in DC dexamethasone induced no apoptosis, although it suppressed activated nuclear transcription factor NF-B. Dexamethasone reduced the ability of DC to stimulate proliferation of allogeneic T cells in proportion to the level of CD14⫹CD83⫺ cells in the population. CD83⫹ cells, isolated from dexamethasone-treated populations, retained the synthesis of IL-12 and the ability to stimulate proliferation of allogeneic T cells. Our data demonstrate that the dominant effect of the drug was redirecting differentiation of a subset of cells despite the presence of inflammatory cytokines. The observed ID50 values indicate that inhibition of DC differentiation might contribute significantly to in vivo immunosuppression by chronic administration of corticosteroids. J. Leukoc. Biol. 66: 909–914; 1999. Key Words: cellular differentiation · immunomodulators · cell surface molecules
INTRODUCTION Glucocorticoids suppress inflammation and immunity [1, 2]. Hence, they are widely used to treat chronic inflammatory diseases. At the cellular level, glucocorticoids reduce the response of inflammatory and immune effector cells to cytokines like tumor necrosis factor ␣ (TNF-␣) and interleukin-1 (IL-1) [1–3]. The reduced response to these cytokines is accompanied by attenuated expression of inflammatory and
immunostimulatory cytokines (i.e., TNF-␣, IL-2, and IL-12) [4] that might partake in autocrine stimulatory feedback. Glucocorticoids are known to suppress the functions of immune effector cells [5–7]. The effects of these hormones have also been studied in antigen-presenting cells, notably dendritic cells (DC) [8–14], that capture, process, and present antigens and are critical for priming and boosting immunity [15]. However, data on the effects of glucocorticoids in DC are inconsistent. For example, in rodent and human DC, corticosteroids were found to inhibit [9] and not inhibit [11, 16] the expression of MHC class II molecules, to reduce [8] and not reduce [14] the levels of costimulatory molecules, to suppress [10] and not suppress [14] DC-stimulated T cell proliferation. The reasons for the differences among observations are unclear, but it is likely that they include different histological origin and maturation status of DC. To clarify the effects of dexamethasone on DC, we used a defined in vitro system of DC differentiation. Here, monocytes are differentiated into DC in the presence of granulocytemacrophage colony-stimulating factor (GM-CSF) and IL-4 to an intermediate stage (immature DC) characterized by high rates of antigen uptake and processing, low levels of IL-12 secretion, and low levels of T cell stimulation [15]. At this stage, DC reside in tissues where they respond to stimuli from the microenvironment. Stimulation by inflammatory cytokines (such as TNF-␣ and IL-1) results in further differentiation into mature DC. Compared with immature DC, mature cells are characterized by diminished antigen uptake, increased antigen presentation, and increased expression of costimulatory molecules (e.g., CD80 and CD86). By expressing these molecules together with cytokines and chemokines, this phenotype is uniquely effective in recruitment and activation of T cells, B cells, and NK cells [15, 17]. We report that dexamethasone suppressed the function of DC without inducing any apparent apoptosis. In contradiction to Vieira et al. [14], we found that corticosteroid-treated DC did not stimulate T cell proliferation. In these cells, the levels of activated nuclear transcription factor NF-B were strongly suppressed, together with the levels of antigen-presenting molecules, costimulatory molecules, and a DC maturation marker, indicating that dexamethasone suppressed DC differentiation. We found that dexamethasone did not affect the cells equally, but that it prevented differentiation of a subset of cells
Correspondence: Stanimir Vuk-Pavlovic´, Ph.D., Guggenheim 1311A, Mayo Clinic, Rochester, MN 55905. E-mail:
[email protected] Received April 24, 1999; revised July 1, 1999; accepted July 2, 1999.
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into DC and allowed their development into a distinct phenotype.
Cells treated with hydrogen peroxide (1.0 mM) for 24 h were used as positive control [19].
Allogeneic T cell proliferation MATERIALS AND METHODS Isolation and cultivation of DC White blood cells, derived from healthy volunteers, were supplied in compliance with institutional guidelines and were processed individually. The cells, suspended in 50 mL of citrated plasma, were mixed with an equal volume of phosphate-buffered saline (PBS) and used for isolation of peripheral blood mononuclear cells (PBMC) by density gradient separation using Lymphocyte Separation Medium (Organon Teknika, Durham, NC). Isolated cells were washed twice with 50 mL PBS and once with cold PBS containing 0.5% bovine serum albumin (BSA) and 2.0 mM EDTA, counted with a hemocytometer, and assayed for viability by trypan blue exclusion. CD14-positive leukocytes were isolated from PBMC by immunomagnetic adsorption according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). To obtain immature DC, CD14⫹ cells were cultured at 1 ⫻ 106 cells/mL in 3.0 mL of complete medium [X-VIVO-15 medium (BioWhittaker, Walkersville, MD) supplemented with human AB serum (1.0%; Sigma, St. Louis, MO), penicillin (100 U/mL; GIBCO-BRL, Gaithersburg, MD), streptomycin (100 µg/mL; GIBCO-BRL), GM-CSF (800 IU/mL; R & D Systems, Minneapolis, MN), and IL-4 (1000 IU/mL; R & D Systems)] for 7 days. On days 3 and 5, 1.0 mL of complete medium (with GM-CSF increased to 1600 IU/mL) was added to cells. To obtain mature DC, we collected nonadherent cells on day 7, counted and replated them at 5 ⫻ 105 cells/mL in the differentiation medium [complete medium supplemented with TNF-␣ (1100 IU/mL; R & D Systems), IL-1 (1870 IU/mL; R & D Systems), and PGE2 (1.0 µg/mL; Sigma)] for 3 days [18].
Drugs and incubation conditions Stock solutions of water-soluble dexamethasone (Sigma, catalog no. D-2915) were prepared weekly at 1.0 mM in PBS, pH 7.4, and stored at 4°C. Stock solutions were freshly diluted in cell culture medium to final concentrations. Control cells were treated with an equal volume of PBS. The drug was admixed to the complete medium at the initiation of the culture of immature DC (day 0) or to the differentiation medium used to initiate the culture of mature DC (day 7).
Flow cytometry and cell sorting Cells were harvested by scraping, followed by rinsing the dishes with PBS. Cells (between 1 ⫻ 105 and 1 ⫻ 106) were incubated for 10 min at room temperature in 500 µL of a 1:10 dilution of mouse serum (Jackson Immunoresearch Laboratories, West Grove, PA), washed, resuspended in PBS, aliquotted into tubes containing antibody solutions, adjusted to a final volume of 50 µL, and incubated on ice for 20 min in the dark. Cells were washed with 2.0 mL PBS, fixed in 400 µL paraformaldehyde (1.0%), processed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA), and analyzed by CellQuest analysis software (Becton Dickinson). Data were analyzed for geometric mean fluorescence intensity and for the percentage of marker-positive cells (determined as cells brighter than controls stained with isotype and/or nonspecific antibodies). Unfixed cells were incubated with antibody as above, sorted by the use of a FACSVantage cell sorter (Becton Dickinson), washed, and replated. Immunoglobulin G isotype control (MOPC21/3421) was obtained conjugated to fluorescein or phycoerythrin (all antibodies from BioSource, Camarillo, CA, unless noted otherwise). Antibodies specific for HLA-DR (clone B-F1), CD1a (B-B5), CD14 (B-A8), CD40 (EA-5), CD58 (IC3; PharMingen, San Diego, CA), CD68 (KP1; Accurate, Westbury, NY), and CD86 (BU63; Ancell, Bayport, MN) were used in the fluoresceinated form. Antibodies specific for HLA Class I molecules (G46-2.6; PharMingen), CD14 (3G8; PharMingen), CD16 (3G8, PharMingen), CD36 (CB38-NL07; PharMingen), CD54 (8.4A6), CD80 (L307.4; Becton Dickinson), and CD83 (HB15A; Immunotech, Westbrook, ME) were conjugated to phycoerythrin. Apoptosis was quantified by membrane-associated annexin V (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instructions.
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DC were washed twice in RPMI-1640 containing 10% human AB serum, penicillin, streptomycin, and 2-mercaptoethanol (50 µM; GIBCO-BRL) and irradiated by 3000 rad from a cesium-137 source. CD3-positive target leukocytes were prepared from PBMC by immunoadsorption (R & D Systems), pooled from nine individuals, and resuspended at 1 ⫻ 106 cells/mL in RPMI-1640 supplemented as above. Target cell suspension, 100 µL, was incubated with DC for 3 days at 37°C in 5% CO2. During the last 12 h, the cells were pulsed with 1.0 µCi [3H]thymidine (Amersham, Arlington Heights, IL) and then harvested and evaluated for incorporated radioactivity.
Measurement of secreted IL-12 Conditioned medium was collected from each well, centrifuged to remove debris, and stored at ⫺70°C. After equilibration to room temperature, 50 µL were withdrawn for IL-12 measurement. IL-12 concentration was determined by sandwich-enzyme-linked immunosorbent assay (ELISA; Endogen, Westbury, MA) specific for the p40 unit according to the manufacturer’s instructions. All measurements were performed in triplicate.
Electrophoretic mobility shift assay The assay was performed according to Pahl and Bauerle [20]. Twelve hours after stimulation with the cocktail of inflammatory mediators with or without dexamethasone, cells were collected by scraping, washed with PBS, and lysed for 30 min on ice using a buffered high-salt detergent [20 mM HEPES, pH 7.9, 350 mM NaCl, 20% wt/vol glycerol, 1% wt/vol NP-40 detergent, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 0.1% phenylmethylsulfonyl fluoride (PMSF), 1% aprotinin]. The lysate was centrifuged at 13,000 µg for 5 min in the cold. Protein concentrations were measured by the Micro BCA Protein Assay (Pierce, Rockford, IL). Equal amounts of the lysate, containing 20 µg of protein, were used to assess NF-B activation by binding to a 32P-labeled NF-B-specific oligonucleotide using the manufacturer’s protocol (Promega, Madison, WI). For controls, either unlabeled competitor or unlabeled noncompetitor oligonucleotide was included in the reaction mixture. Samples were incubated at room temperature for 25 min, electrophoresed on 6% polyacrylamide gels, and visualized by autoradiography.
Statistical analysis All experiments were repeated with samples from 2–12 individual donors. The probability that the mean values of two experimental groups were identical was tested by the two-tailed Student’s t test for paired samples. The level of significance was set at P ⫽ 0.05. The flow cytometry histograms shown were obtained with cells from a single donor representative of multiple experiments (each with cells from 3–12 donors) that yielded statistically significant differences. Where applicable, data are reported as means ⫾ standard deviation.
RESULTS Dexamethasone inhibits DC differentiation To determine how dexamethasone affects differentiation of immature DC into mature DC, we introduced dexamethasone together with the differentiation medium to immature DC for 3 days and measured the levels of selected membrane molecules as a function of dexamethasone concentration. The expression of membrane molecules was dependent on dexamethasone concentration (data not shown); the levels of CD14 and CD36 were up-regulated and those of CD40 and CD80 were downregulated (Fig. 1). The midpoint values for these changes were between 2 and 20 nM dexamethasone with the full effect at 500 http://www.jleukbio.org
Fig. 1. Levels of membrane molecules in immature DC matured for 3 days with the differentiation medium (containing TNF-␣, IL-1, and PGE2) in the absence (open histograms) and in the presence of dexamethasone (500 nM, shaded histograms).
nM. Qualitatively similar effects were observed using hydrocortisone (data not shown). Histograms in Figure 1 show the effects of dexamethasone (500 nM) on the distribution of membrane molecules in the population. Unlike the unimodal distribution in control cells, distribution of HLA class I, CD14, CD16, CD36, CD54, CD80, and CD83 became markedly bimodal. This change in marker distribution indicated that the observed effects of dexamethasone were specific, i.e., that dexamethasone modified the expression of HLA class I, CD14, CD16, CD36, CD54, CD80, and CD83 in a fraction of maturing cells (Fig. 1).
with 1000 nM dexamethasone (Fig. 2; P ⫽ 0.004 relative to cells treated with 500 nM dexamethasone). Thus, dexamethasone induced no apoptosis, implying that the effects shown in Figures 1 and 2 resulted from specific action of dexamethasone on cell differentiation, rather than from nonspecific effects such as cell killing.
Dexamethasone switches induction of membrane molecules in maturing DC
We found no change in cell number and trypan blue exclusion as a function of dexamethasone (data not shown). Because cells committed to apoptosis still can exclude trypan blue, we determined the levels of phosphatidylserine accessible to annexin-V binding, an early marker of apoptosis [21]. Compared with the positive control (mature DC treated with 1.0 mM hydrogen peroxide, 100% annexin-V binding), annexin-V bound to 17.4 ⫾ 1.0% of untreated mature DC, to 14.3 ⫾ 0.8% of cells treated with 500 nM dexamethasone (P ⫽ 0.0007 relative to untreated cells) and to 10.2 ⫾ 0.4% of cells treated
The bimodal distribution of membrane molecules in Figure 1 implies that dexamethasone induced heterogeneity into the originally unimodal cell population. To ascertain the character of the dexamethasone-induced subset(s), we studied the expression of CD14, a monocyte marker, and CD83, the marker of matured DC. We determined the relationship of these cell markers in doubly labeled immature DC, mature DC, and cells matured in the presence of dexamethasone (Fig. 3). Typically, immature DC were CD14⫺CD83⫺ (Fig. 3A). Mature DC were characterized by the absence of CD14 and by high levels of CD83 (CD14⫺CD83⫹; Fig. 3B). When matured in the presence of dexamethasone, a population of CD14⫺CD83⫹ cells was maintained, but was also accompanied by a population of CD14⫹CD83⫺ cells. Thus, differentiation medium in the
Fig. 2. Annexin-V-binding DC matured in the differentiation medium in the absence (thin line) and the presence of dexamethasone, 1000 nM (thick line).
Fig. 3. Contour diagram of cells analyzed for coexpression of CD14 and CD83. (A) Immature DC; (B) mature DC; (C) DC matured in the presence of dexamethasone (700 nM).
Dexamethasone does not change DC viability
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presence of dexamethasone resulted in development of immature DC into two populations (CD14⫺CD83⫹ and CD14⫹CD83⫺; Fig. 3C), one of which (CD14⫹CD83⫺) is distinct both from immature DC (Fig. 3A) and mature DC (Fig. 3B).
Dexamethasone-treated DC are ineffective in allogeneic mixed leukocyte reaction We used an allogeneic mixed leukocyte reaction to determine the effect of dexamethasone on the ability of DC to stimulate T cell proliferation. Immature DC were only marginally effective, but mature DC effectively stimulated T cell proliferation (Fig. 4). DC matured in the presence of dexamethasone-stimulated T cells less effectively than mature DC (Fig. 4); the level of inhibition was dependent on dexamethasone concentration (data not shown). We isolated CD83⫹ cells from untreated DC populations and from dexamethasone-treated populations. Dexamethasonetreated CD83⫹ cells and untreated CD83⫹ cells were similarly effective in stimulating T cell proliferation (Fig. 4). Thus, the reduction of the ability of unsorted dexamethasone-treated DC to stimulate T cell proliferation is effected by the reduction in the fraction of CD83⫹ differentiated cells.
Dexamethasone affects IL-12 expression by DC We measured the levels of IL-12 secreted by cells matured in the absence and the presence of dexamethasone. After 2 days of incubation, the cells were washed free of the drug and CD83⫹ cells were sorted and replated for 1 day in the absence of dexamethasone in the cytokine-free medium or in the differentiation medium. Untreated DC secreted high amounts of IL-12 under both conditions; dexamethasone (500 nM) reduced the amounts of IL-12 to less than one-quarter and one-half, respectively (Fig. 5). However, CD83⫹ cells isolated from the
Fig. 5. Levels of IL-12 in the media of unfractionated DC and CD83⫹ DC matured in the absence or the presence of dexamethasone, 500 nM. After 2 days of incubation with or without dexamethasone, cells were sorted (CD83, Dex-CD83), washed, and replated without dexamethasone in the cytokine-free medium (lighter shading) or in the differentiation medium (darker shading) for 24 h. All differences among groups were statistically significant. MDC, mature DC; CD83, sorted CD83-positive cells; Dex, dexamethasone-treated.
dexamethasone-treated populations secreted much more IL-12 than the unfractionated populations (Fig. 5). Thus, the dexamethasone-induced reduction in the levels of IL-12 secreted by the unfractionated cells must be borne, in large part, by the increase in the fraction of CD83⫺ cells.
Dexamethasone suppresses levels of activated NF-B We measured the levels of activated NF-B by gel mobility shift assay to determine whether inhibited differentiation was accompanied by a reduction in the levels of this transcription factor. In immature DC, the levels of activated NF-B were low. In cells incubated with the differentiation medium for 12 h, the levels of activated NF-B were high, whereas in the presence of dexamethasone, these levels were strongly reduced (data not shown).
DISCUSSION
Fig. 4. Allogeneic T cell proliferation stimulated by immature DC (open triangles), mature DC (open circles), CD83-positive cells isolated from mature DC (filled circles), DC matured in the presence of dexamethasone, 500 nM (open squares), and CD83-positive cells isolated from cells matured in the presence of dexamethasone, 500 nM (filled squares). At the DC:T cell ratios of 1:18 and 1:54, T cell proliferation stimulated by isolated CD83-positive cells differed significantly from the proliferation stimulated by the respective unsorted cells (P ⬍ 0.05).
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At concentrations akin to those obtained in immunosuppression protocols [22], dexamethasone inhibited differentiation into mature DC, resulting in impaired DC function. This inhibition took place despite the presence of high concentrations of TNF-␣, IL-1, and prostaglandin E2 that are documented for highly potent induction of differentiation [18]. Inhibited differentiation leaves the cells devoid of antigen-presenting molecules, costimulatory molecules, and cytokines required for initiation and maintenance of the immune response. Dexamethasone did not inhibit differentiation by reducing the overall cell metabolism, but it selectively reduced the expression of molecules pertinent to immunostimulation and it http://www.jleukbio.org
up-regulated expression of other molecules. These dexamethasone effects, even at high concentrations, took place in the absence of any apparent cell loss. Differentiation of DC is controlled by activated NF-B [23, 24]. In cells treated with TNF-␣, activated NF-B protects from apoptosis [3, 25, 26], whereas inhibition of NF-B leads to apoptosis in normal and malignant B cells [27, 28] and T cells [28]. In human DC, dexamethasone markedly reduced the levels of activated NF-B without apoptosis, demonstrating that in these cells NF-B does not protect from programmed cell death. Similar conclusions were recently reached for a murine DC line in which selective inhibition of NF-B prevented differentiation, but did not trigger apoptosis [24]. The presence of dexamethasone maintained a fraction of CD14-selected cells that did not mature, but did express markers not characteristic of DC. This finding indicates that one set of cells fully matured into DC and that the other set became refractory to differentiation medium in the presence of the drug. It is interesting that molecules expressed on dexamethasone-treated cells (CD14, CD36, CD68) are characteristic of the monocyte/macrophage lineage [29], implying the existence of a differentiation checkpoint affected by inflammatory cytokines and dexamethasone. In other words, if dexamethasone overrides the signals from the differentiation medium, it directs the cell toward the monocyte/macrophage phenotype. This notion is fully in line with the current view of DC differentiation and the role of dexamethasone in macrophage development [for review see ref. 30]. Taken together, our results show that the effects of dexamethasone depend on concentration and cell differentiation stage. Our experimental system (incubation with both dexamethasone and cytokines) allowed us to monitor differentiation-specific effects. Our findings support the model of dexamethasoneinhibited antigen presentation and costimulation [8–10] and extend it to imply that the defective presentation and costimulation result from inhibited differentiation. Our results are in contrast to those by Vieira et al. [14] on the effects of glucocorticoids on changes in expression of membrane markers and T cell proliferation. The differences in data might stem predominantly from differences in the differentiation stage of DC. Vieira et al. grew cells in GM-CSF and IL-4 for 7 days and obtained CD1a⫹, CD14⫺ immature DC. They further incubated the cells with the glucocorticoid for 2 days and found no change in membrane marker expression and the ability of cells to stimulate T cell proliferation. Thus, Vieira et al. studied the effects of the glucocorticoid on the rather static immature DC and could not detect cytokine effects on the process of differentiation. When they stimulated immature DC with lipopolysaccharide, steroid effects were similar to those observed in this study. Similar to our data, Moser et al. [10] found that dexamethasone reduced the expression of costimulatory molecules in murine splenocytes and their ability to initiate a T cell response. These effects of dexamethasone could be countered by GM-CSF [10]. Because our experiments were conducted in the presence of GM-CSF, the effects of dexamethasone in human cells in vivo might be much larger than observed in this study. Likewise, Piemonti et al. [31] found that dexamethasone
suppressed development of mature phenotype and cytokine secretion in human DC. We studied the model of immature DC driven to differentiation by inflammatory mediators. Under these conditions, we demonstrated that the commitment of the immature DC to differentiation could be controlled by dexamethasone. Dexamethasone increased the fraction of immature DC that cannot mature, but rather develop a phenotype distinct from both immature and mature DC. It is interesting that despite the high concentrations of inflammatory mediators employed in this study, the ID50 values for dexamethasone effects in DC were low. The high sensitivity of immature DC to dexamethasone indicates the possibility that inhibition of DC differentiation contributes significantly to in vivo immunosuppression borne by chronic corticosteroid administration.
ACKNOWLEDGMENTS The Stem Cell Laboratory is generously supported by a grant from Mrs. Adelyn L. Luther, Singer Island, FL, and by the Mayo Clinic Cancer Center. A. B. D. is a scholar of the Glen and Florence Voyles Foundation, Terre Haute, IN. R. M. is the recipient of a postdoctoral fellowship from the Melvin S. Cohen Foundation, Inc., Eau Claire, WI. We thank Mrs. Peggy A. Bulur and Mr. Gaylord J. Knutson for expert assistance and Dr. Franklyn G. Prendergast for continuing interest and encouragement.
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