The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Woltman, A. M.
Right arrow Articles by van Kooten, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Woltman, A. M.
Right arrow Articles by van Kooten, C.
The Journal of Immunology, 2002, 168: 6181-6188.
Copyright © 2002 by The American Association of Immunologists

Corticosteroids Prevent Generation of CD34+-Derived Dermal Dendritic Cells But Do Not Inhibit Langerhans Cell Development1

Andrea M. Woltman2,*, Catherine Massacrier{dagger}, Johan W. de Fijter*, Christophe Caux{dagger} and Cees van Kooten*

* Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands; and {dagger} Laboratory for Immunological Research, Schering-Plough, Dardilly, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Corticosteroids (CS) have been shown to exert strong inhibitory effects on dendritic cell (DC) differentiation and function. Those studies were mostly performed with monocyte-derived DC, which represents only one subpopulation from the wide variety of DC types. In the present study the effects of the CS dexamethasone and prednisolone were investigated on the differentiation of CD34+ hemopoietic progenitor cells into 1) Langerhans cells (LC), which differentiate directly into CD1a+ DC; and 2) dermal/interstitial DC, which differentiate via a CD14+CD1a- phenotype into CD14-CD1a+ DC. CS present during the entire 11-day culture period, resulting in fully differentiated CD1a+ DC, increased the percentage of langerin+ DC within the CD1a+ population. In line with these data, CS treatment during the first 6 days of differentiation reduced the development of CD14+ dermal DC precursors and thereby seemed to support the generation of CD1a+ LC precursors. Addition of CS from day 6 onward specifically blocked the development of CD1a+ dermal DC by both inhibition of spontaneous and IL-4-induced differentiation of CD14+ DC precursors into CD1a+ DC as well as induction of apoptosis in CD14+ DC precursors. Apoptosis was not found in CD14+ macrophage precursors derived from the same CD34+ progenitors. The development and function of LC were not affected by CS, as demonstrated by a normal T cell stimulatory capacity and IL-12 production. These data demonstrate that CS interfere with the normal development of DC from CD34+ progenitors by specific induction of apoptosis in precursors of dermal/interstitial DC. In view of the different functional capacities of dermal/interstitial DC and Langerhans cells, this might affect the overall cellular immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC)3 are professional APC, which have the unique capacity to stimulate naive T lymphocytes (1). Based on their ontogeny, phenotype, tissue distribution, and function, human DC comprise at least three distinct subsets, including Langerhans cells, dermal or interstitial DC, and lymphoid DC (2, 3). Immature DC, which reside in the epithelial and connective tissues, are very efficient in the uptake of Ag both via fluid phase endocytosis and by specific receptors. On activation, immature DC undergo maturation and migrate to the lymph nodes, where they encounter naive T lymphocytes. However, it has become clear that immature DC are not just the cells specialized in Ag uptake and poorly capable of activating T cells; they actively modulate the outcome of T cell activation (4, 5, 6, 7). The balance between the induction of immune reactivity and immune modulation, including tolerance induction, seems to be determined by the nature/subtype of the DC, the state of maturation, and the secretion of soluble mediators including cytokines.

Corticosteroids (CS) are potent anti-inflammatory and immunosuppressive drugs that are widely used in the treatment of inflammatory disorders such as allograft rejection and autoimmune and allergic diseases (8). Therapeutic effects of CS have been generally ascribed to the inhibitory effect on T cells, but recently it was also shown that CS have strong inhibitory effects on DC differentiation and function. CS prevent the differentiation of CD1a-CD14+ monocytes into CD1a+CD14- immature DC in the presence of IL-4 and GM-CSF, as demonstrated by the remaining CD1a-CD14+ phenotype (9, 10). In addition, CS block subsequent LPS- or CD40 ligand (CD40L)-induced maturation without affecting cell viability, resulting in hampered T cell stimulatory capacities and decreased production levels of proinflammatory cytokines (10, 11, 12, 13).

In vitro studies of the effect of CS on human DC were mostly performed with monocyte-derived DC. However, no information is available about the effect of CS on human CD34+-derived DC. In response to GM-CSF and TNF-{alpha}, CD34+ hemopoietic progenitor cells isolated from bone marrow or cord blood (14) differentiate along two unrelated DC pathways (15): 1) the Langerhans cells, which differentiate directly from CD34+ cells into CD1a+ DC expressing langerin and E-cadherin; and 2) CD14+-cell derived DC, which are comparable with the dermal and interstitial DC and differentiate from CD34+ precursors via a CD14+CD1a- phenotype into CD14-CD1a+ DC.

In the present study we investigated the effect of the CS dexamethasone (Dex) and prednisolone (Pred) on the differentiation and survival of CD34+-derived DC by determining the effect on the expression of cell surface molecules and apoptosis. We show that CS block the development of CD14+-derived dermal/interstitial DC by the induction of apoptosis in CD14+ DC precursors and the inhibition of spontaneous and IL-4-induced differentiation into CD1a+ DC. No apoptosis was observed in CD14+ macrophage precursors or CD1a+ Langerhans cell precursors generated from the same CD34+ progenitor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cytokines and reagents

The following cytokines were used: recombinant human (rh) GM-CSF (200 ng/ml) from Schering-Plough Research Institute (Dardilly, France), rhTNF-{alpha} (2.5 ng/ml), recombinant human stem cell factor (rhSCF; 25 ng/ml), and rhM-CSF (20 ng/ml) from ITK Diagnostics (Uithoorn, The Netherlands) and IL-4 (10 ng/ml) from Sanvertech (Heerhugowaard, The Netherlands). The CS Dex (Pharmacy LUMC (Leiden, The Netherlands) and Sigma-Aldrich (St. Louis, MO)) and Pred (Pharmacy LUMC) were added at the indicated concentrations.

Purification of cord blood CD34+ hemopoietic progenitor cells

CD34+ cells were isolated from umbilical cord blood samples and used in cultures to generate immature DC as described previously (15). In brief, CD34+ cells were isolated from mononuclear fractions through positive selection using anti-CD34-coated microbeads and Midi-Macs separation columns (both from Miltenyi Biotec, Bergish Gladbach, Germany). After cryopreservation, cells were cultured in RPMI 1640 containing 10% heat-inactivated FCS, 10 mM HEPES, 2 mM L-glutamine, 50 µM 2-ME, and penicilin/streptomycin-supplemented with GM-CSF, SCF, TNF-{alpha}, and 5% AB+ pooled human serum.

Isolation of CD1a+ and CD14+ precursors

After 6 days of culture in the presence of GM-CSF, SCF, TNF-{alpha}, and human serum, cells were collected and labeled with FITC-conjugated anti-CD1a (BD PharMingen, San Diego, CA) and PE-conjugated anti-CD14 (Leu M3; BD Biosciences, Mountain View, CA). Cells were separated according to CD1a and CD14 expression into CD1a-CD14+ and CD1a+CD14- fractions using a FACStarPlus (BD Biosciences). Sorted cells were seeded in the presence of GM-CSF.

Analysis of cell surface phenotype

Cells were harvested and washed in buffer containing 1% BSA, 1% heat-inactivated normal human serum, and 0.02% NaN3. FACS analysis was performed using nonconjugated and FITC- or PE-conjugated Abs against the following surface markers: CD1a (FITC-conjugated HI149 from BD PharMingen), CD14 (PE-conjugated Leu M3 from BD Biosciences), CD80 (mAb104; Schering-Plough, Dardilly, France), CD86 (IT2.2; BD PharMingen), and langerin (FITC-conjugated DCGM4; Schering-Plough). Staining with nonconjugated Abs was visualized using PE-conjugated goat anti-mouse Ig (DAKO, Glostrup, Denmark). The cells were assessed for fluorescence using a FACScan (BD Biosciences). Data analysis was performed using CellQuest and WinMDI software (BD Biosciences). Data from minimally three independent experiments were grouped and analyzed using paired Student’s t test to investigate the effect of CS on the development of different DC subsets. Results were considered significant at p < 0.05.

Detection of apoptosis

Phosphatidyl serine exposure was determined using annexin V-FITC (Apoptest FITC kit; Nexins Research, Kattendijke, The Netherlands) in combination with propidium iodide (PI; Molecular Probes, Eugene, OR). Cells were harvested, washed, labeled with annexin V-FITC for 30 min on ice, and subsequently taken up in 1 µg/ml PI. Annexin V/PI staining was conducted on a FACScan and analyzed using WinMDI software.

Purification of peripheral blood CD4+CD45RA+ T cells

Mononuclear cells were isolated from adult peripheral blood using Ficoll-Hypaque centrifugation. CD45RA+ T lymphocytes were then purified by immunomagnetic depletion using a mixture of mAbs MOP9 (CD14), OKT8 (CD8), 4G7 (CD19), mAb89 (CD40), UCHL-1 (CD45RO), L.243 (HLA-DR; all from Schering-Plough), ION16 (CD16), J3D3 (CD35; both from Immunotech, Westbrook, ME), NKH1 (CD56; Coulter, Miami, FL), and JC159 (glycophorine A; DAKO). After two rounds of bead depletion, the purity of CD45RA+ T cells was routinely >95%.

T cell proliferation assay

Day 10 CD1a+-derived DC were collected and, after irradiation (30 Gy), used as stimulator cells for allogeneic CD45RA+ naive T cells (2 x 104/well). Stimulator cells were added in graded doses to the T cells in 96-well round-bottom tissue culture plates (Nunc, Naperville, IL). Cultures were performed in RPMI 1640 medium supplemented with 2.5% heat-inactivated human AB+ serum, glutamine, and gentalline. Cells were pulsed with 1 µCi [3H]TdR (sp. act., 25 Ci/mmol)/well for the last 16 h of a 5-day incubation period, harvested, and counted. Results are presented as the mean ± SD obtained from triplicate cultures.

Determination of IL-12 production

Day 10 CD1a+-derived DC (1 x 105 DC/well) were cultured with or without 1 x 104 irradiated (80 Gy) CD40L-transfected L cells (16) in 24-well culture plates (Costar, Cambridge, MA). After 72 h of incubation, supernatants were harvested and tested for the amount of IL-12p40 and IL-12p70 using a two-site sandwich ELISA (DP400 and DP1200; R&D Systems, Minneapolis, MN). The detection limits of these ELISAs are 500 pg/ml for IL-12p40, and 100 pg/ml IL-12p70. Results are presented as the mean ± SD obtained from triplicate cultures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dex qualitatively alters DC development from CD34+ progenitors

To investigate the effect of Dex on the generation of DC, CD34+ progenitor cells were cultured with or without Dex for a period of 11 days. Continuous Dex treatment resulted in a decreased cell yield (44 ± 4% reduction) on day 11 compared with the control value. Dex almost completely prevented the generation of CD14+ and particularly the generation of double-positive CD14+CD1a+ cells (Fig. 1GoA). A similar decrease was found when analyzing the CD11b+ and CD11b+CD1a+ population. This latter double-positive population is thought to be the intermediate between CD14+ precursors and the fully differentiated CD1a+ dermal/interstitial-type DC (15). All fully differentiated DC express CD1a, but only Langerhans cells express langerin (17). Cultures in the presence of Dex contained more langerin+ cells (38.9 ± 2.1%; p < 0.001) within the CD1a population than the control cultures (15.5 ± 1.6%), suggesting that Dex favored the development of Langerhans cells (Fig. 1BGo).



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 1. Dex reduces CD14+ and CD11b+ populations and favors the emergence of CD1a+ DC precursors. CD34+ cells were cultured from days 0–6 in GM-CSF, SCF, TNF-{alpha}, and human serum and from days 6–11 only in GM-CSF. For Dex treatment of the cells, Dex (10-7 M) was added on days 0, 5, and 9. On day 11, cells were harvested, extensively washed, and used for FACS analysis after staining with specific FITC-conjugated anti-CD1a and PE-conjugated anti-CD11b and -CD14 Abs (A) or specific PE-conjugated anti-CD1a and FITC-conjugated anti-langerin Abs (B). Histograms in B represent specific staining for langerin within the CD1a+ population. Data shown are representative for four independent experiments with different donors.

 
Dex favors the emergence of CD1a+ DC precursors

The read-out on day 11 is the result of two independent differentiation pathways leading to CD1a+ DC with different functional capacities. Therefore, the effect of Dex was investigated in more detail on the individual pathways. Addition of Dex during the first 6 days markedly decreased cell yields in a dose-dependent manner compared with control cultures (Fig. 2GoA). Concerning their phenotypes, the Dex-treated cultures contained relatively more CD1a+ precursors (control, 25.4 ± 2.9%; Dex, 34.8 ± 3.4% CD1a+; p < 0.026) and fewer CD14+ precursors (control, 30.7 ± 4.5%; Dex, 22.3 ± 7.1% CD14+; p < 0.066) compared with controls. Nevertheless, Dex treatment did not prevent the differentiation of CD34+ cells along either of the two differentiation pathways (Fig. 2GoB).



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 2. Dex relatively favors the development of CD1a+ precursors. CD34+ cells were cultured in GM-CSF, SCF, TNF-{alpha}, and human serum with or without Dex. On day 6 cells were harvested, counted with trypan blue exclusion (A), and subsequently used for flow cytometric analysis (B). The number of cells obtained from Dex cultures was compared with the number of cells in control cultures. Data presented are the mean (±SD) percentages of cell yields of five independent experiments with different donors (A). The phenotype of the cells, derived from cultures with or without Dex (10-7 M), was determined by staining with specific FITC-conjugated anti-CD1a and PE-conjugated anti-CD14 Abs. The surface expressions shown are representative of five independent experiments with different donors (B).

 
Dex decreases the percentage of CD14+ precursors

Addition of Dex to day 6 CD34+-differentiated cells prevented the generation of a CD14+CD1a+ population, as observed using flow cytometric analysis after 38 h (Fig. 3GoA). Moreover, Dex induced a strong reduction in the percentage of CD14+ single-positive cells, and the intensity of the staining for CD14 was decreased. Simultaneous analysis of the expression of CD11b revealed similar Dex-induced decreases (Fig. 3GoA), whereas the percentage of CD1a+ cells was hardly affected. Similar effects were observed when the corticosteroid Pred was used (Fig. 3GoA). These effects of Dex and Pred were dose dependent, with an IC50 at 5 x 10-9 M and maximal effects at 10-7 M (Fig. 3GoB).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 3. CS reduce CD14+ and CD11b+ populations in a dose-dependent manner. A, CD34+ cells were cultured in GM-CSF, SCF, TNF-{alpha}, and human serum. On day 6 cells were harvested, extensively washed, and cultured further in the presence of GM-CSF with or without Dex or Pred (10-6 M). After 38 h cells were harvested and stained with specific FITC-conjugated anti-CD1a and either PE-conjugated anti-CD14 or -CD11b Abs. Cells were analyzed by flow cytometry; dead cells were excluded from analysis. The data shown are representative for two independent experiments with different donors. B, Cells were cultured as described above. On day 6 cells were incubated with different concentrations of Dex. After 38 h cells were analyzed for CD1a, CD14, and CD11b expression as described above. The data shown are representative for two independent experiments with different donors.

 
Dex completely prevents differentiation of CD14+ precursors into CD1a+ DC

In line with the data published about the inhibitory effect of CS on the differentiation of monocytes into DC (10), the effect of Dex on the differentiation of CD34+-derived CD14+ precursors into CD1a+ DC was investigated. FACS-sorted day 6 CD34+-derived CD14+ DC precursors were isolated (Fig. 4GoA) and cultured further in the presence of GM-CSF. Flow cytometric analysis of the cells after 48 h of incubation revealed that CD14+ precursors spontaneously differentiated toward CD1a+ DC. Culturing the CD14+ sorted cells in the presence of Dex induced a loss of CD14 and completely prevented the differentiation of CD14+ precursors into CD1a+ DC (Fig. 4GoA).



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 4. Dex blocks the differentiation of CD14+ precursors into CD1a+ DC. A, CD34+ cells were cultured in GM-CSF, SCF, TNF-{alpha}, and human serum. On day 6 CD1a-CD14+ were isolated by FACS sorting as described in Materials and Methods. Then cells were cultured further in GM-CSF with or without addition of Dex (10-7 M). After 48 h of incubation, phenotypic analysis of the cells was performed using specific FITC-conjugated anti-CD1a and PE-conjugated anti-CD14 Abs. The data presented are derived from a representative experiment of two independent experiments performed. B, CD34+ cells were cultured during the first 6 days with GM-CSF, SCF, TNF-{alpha}, and human serum, and then SCF and TNF-{alpha} were removed. On day 10 cells were harvested, extensively washed, and cultured in GM-CSF in the presence or the absence of IL-4 (10 ng/ml) and either with or without Dex (10-6 M). After 48 h cells were harvested and analyzed by flow cytometry for the expression of CD14 and CD1a. Data presented are the mean (±SD) percentage of CD14+CD1a- or CD14-CD1a+ cells in four independent experiments performed with different donors.

 
Next, CD34+ cells were cultured for 10 days in the presence of human serum to obtain a large CD14+CD1a- population (Fig. 4GoB, left panel). Then, cells were harvested, extensively washed, and cultured for 2 more days with or without addition of IL-4 and/or Dex. Flow cytometric analysis of the cells revealed that IL-4 induced differentiation toward CD1a+ cells, which was accompanied by a complete loss of CD14+ cells (Fig. 4GoB). In analogy with the spontaneous differentiation, IL-4-driven differentiation was also blocked by addition of Dex (Fig. 4GoB). Inhibition of IL-4-induced differentiation was already observed with 10-10 M Dex and reached its maximum with 10-7 M of the drug (data not shown).

Dex prevents final differentiation of CD14+ DC precursors through specific killing

To understand the mechanisms involved in disappearance of CD14+ cells, annexin V/PI stainings were performed to follow the induction of apoptosis. Addition of Dex (Fig. 5GoA) or Pred (data not shown) to day 6 CD34+-differentiated cells induced apoptosis within 15 h, as demonstrated by an increased binding of annexin V in both PI- (early apoptotic) and PI+ (late apoptotic) populations. This Dex-induced apoptosis was dose- and time-dependently regulated (Fig. 5GoB) in a similar way as observed for its effect on cell phenotypes.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 5. Dex preferentially induces apoptosis in CD14+ DC precursors in a dose- and time-dependent manner. A and B, Cells were cultured as described in Fig. 3Go. On day 6 cells were harvested, washed, and cultured further in the presence of GM-CSF with or without Dex (10-7 M) for 15 h (A) or with different concentrations of Dex for both 15 and 38 h (B). Cells were analyzed for apoptosis using annexin V-FITC and PI. The data shown are representative for four independent experiments with different donors. C, CD34+ cells were cultured in GM-CSF, SCF, TNF-{alpha}, and human serum. On day 6 cells were harvested and separated into CD1a-CD14+ (CD14+) and CD1a+CD14- (CD1a+) populations as described in Materials and Methods. Then cells were cultured further in GM-CSF with or without Dex (10-7 M). After 48 h of incubation, cells were analyzed by flow cytometry. The percentage of apoptotic cells was determined by staining with annexin V-FITC. Data are presented as the index of Dex-induced apoptosis that represents the quotient of Dex-induced apoptosis and spontaneous apoptosis. The data shown are the mean ± SD of two independent experiments with different donors.

 
To investigate the effect of Dex on separate precursor populations, we made use of purified FACS-sorted CD1a-CD14+ and CD1a+CD14- cell populations. Dex specifically increased apoptosis in CD14+-sorted cells (Fig. 5GoC), which resulted in a markedly decreased cell survival after 96 h compared with control cultures (77.4% decreased CD14+ cell survival compared with control). Although CD1a+-sorted cells showed a higher spontaneous apoptosis, as reported previously (18), these cells were almost insensitive to Dex-induced apoptosis (Fig. 5GoC).

Dex does not induce apoptosis in CD14+ macrophage precursors

Macrophage precursors with a CD14+CD1a- phenotype can be generated by culturing CD34+ progenitors in M-CSF for 6 days. Dex treatment of those CD14+ macrophage precursors did not affect their phenotype (Fig. 6GoA). M-CSF-generated cells showed a higher spontaneous apoptosis than cells generated with GM-CSF from the same precursors. However, the M-CSF-generated precursors were completely insensitive to Dex-induced apoptosis (Fig. 6GoB). This indicates that Dex specifically induces apoptosis in CD14+ DC precursors, but not in CD14+ macrophage precursors.



View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 6. Dex does not induce apoptosis in CD14+ macrophage precursors. CD34+ cells were cultured for 6 days in the presence of M-CSF, SCF, and human serum or the standard supplements including GM-CSF, SCF, TNF-{alpha}, and human serum. On day 6 cells were harvested, extensively washed, and cultured further in either M-CSF or GM-CSF with or without Dex (10-6 M). After 48 h cells were harvested and analyzed by flow cytometry to determine their phenotype by using specific FITC-conjugated anti-CD1a and PE-conjugated anti-CD14 Abs (A) and to determine the amount of apoptotic cells using annexin V-FITC and PI (B). Phenotypic analysis was performed on PI- cells. Data shown are representative for two independent experiments with different donors.

 
Differentiated CD1a+ DC derived from CD14+ precursors are insensitive to Dex-induced apoptosis

Since CD14+ precursor DC spontaneously differentiate into CD1a+ DC, kinetic experiments were performed to investigate whether Dex could also affect the viability of fully differentiated CD14+-derived CD1a+ DC. A 24-h treatment with Dex of day 7 CD34+-derived DC induced strong apoptosis of the cells, which was decreased when the same experiments were performed with day 9 DC (Fig. 7Go). Experiments performed with day 13 DC showed a complete resistance to Dex-induced apoptosis. This decrease in Dex sensitivity was paralleled by a decrease in CD14+ cells (data not shown) and an increase in CD1a+ cells, indicating that the fully differentiated CD14+-derived CD1a+ DC are not susceptible to Dex-induced apoptosis.



View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 7. Differentiation of CD14+ DC precursors into CD1a+ DC is accompanied by a decreased sensitivity for Dex-induced apoptosis. CD34+ cells were cultured during the first 7 days with GM-CSF, SCF, TNF-{alpha}, and human serum. Cells were cultured further in the presence of GM-CSF. On days 7, 9, and 13 cells were harvested, analyzed by flow cytometry for the expression of CD14 and CD1a, and cultured for 24 h in the presence of GM-CSF with or without Dex (10-6 M). Then cells were harvested and analyzed by flow cytometry for the percentage of apoptosis. Data presented are the mean ± SD index of Dex-induced apoptosis of two independent experiments with different donors. The apoptosis index represents the relative increase in annexin V+ cells induced by Dex compared with control cultures, as described in Fig. 5GoC. Apoptosis detected in control cultures ranged between 7 and 18% annexin V+ cells.

 
Dex does not affect the function of Langerhans cells

Since it is clear that Dex leaves the generation of LC intact, we questioned whether LC generated in the presence of Dex are still functionally active. CD34+ progenitor cells were cultured with or without Dex and CD1a+ LC precursors were FACS-sorted on day 6. Purified CD1a+ cells were further cultured with or without addition of Dex. On day 10, CD1a+-derived cells showed clear expression of CD80 and CD86, which did not differ between control and Dex cultures (Fig. 8GoA). In addition, the percentages of langerin+ cells were similar.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 8. CD1a+-derived DC generated in the presence of Dex display normal functional activities. CD34+ cells were cultured in GM-CSF, SCF, TNF-{alpha}, and human serum in the presence or the absence of Dex (10-7 M). On day 6 CD1a+CD14- were isolated by FACS sorting. Then, cells were cultured further in GM-CSF and TNF-{alpha} with or without Dex (10-7 M). On day 10 cells were harvested and investigated for the expression of CD80, CD86, and langerin by flow cytometric analysis (A); their allogeneic CD45RA+ naive T cell stimulatory capacity (B); and their production of IL-12p40 upon CD40L stimulation (C).

 
The functional activity of CD1a+-derived DC populations was analyzed with regard to their ability to induce the proliferation of allogeneic CD45RA+ naive T cells and their capacity to produce IL-12 upon CD40L activation. CD1a+-derived DC generated in the presence of Dex induced T cell proliferation equally well as control CD1a+-derived DC (Fig. 8GoB). Furthermore, CD40L activation of both CD1a+-derived DC populations resulted in the secretion of similar amounts of IL-12p40 (Fig. 8GoC), whereas IL-12p70 could not be detected (data not shown). Thus in addition to its lack of effect on LC development, Dex has no effect on the function of LC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that CS influence the differentiation of CD34+-derived DC. Dex and Pred do not affect Langerhans cell development and function, but they block the generation of dermal/interstitial type DC as a consequence of reduced cell survival through specific apoptosis of CD14+ DC precursors and by blocking the differentiation pathway from CD14+ DC precursors into CD1a+ DC (Fig. 9Go). CD14+ macrophage precursors generated from the same CD34+ cells cultured in the presence of M-CSF were completely insensitive to CS-induced apoptosis. In theory, specific apoptosis of CD14+ DC precursors could completely account for the absence of fully differentiated CD1a+ dermal/interstitial DC. However, in view of the reported effects of CS on monocyte-derived DC (10, 12, 13), it is more likely that inhibition of differentiation and induction of apoptosis have complementary roles in this process.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 9. The influence of CS on the differentiation of CD34+ hemopoietic progenitors. In response to GM-CSF and TNF-{alpha}, CD34+ hemopoietic progenitor cells differentiate along two different pathways into Langerhans cells and dermal/interstitial DC. Although CS do not affect LC development and function, CS specifically inhibit the development of dermal/interstitial DC by 1) inhibition of the spontaneous and IL-4-driven differentiation of CD14+ precursors into CD1a+ DC, and 2) induction of apoptosis of CD14+ DC precursors. The development of CD14+ macrophage precursors from the same CD34+ cells cultured in the presence of M-CSF is completely insensitive for CS-induced apoptosis.

 
The inhibitory role of CS in isolated DC or DC function in vivo has been well documented (19, 20). In addition, several studies have demonstrated that CS affect the differentiation and function of in vitro cultured human monocyte-derived DC (10, 12, 13). Inhibition of the final maturation of monocyte-derived DC by CS has been a matter of debate (9, 11, 13, 21). Previously we demonstrated that treatment of monocytes with CS prevented their subsequent differentiation into CD1a+ DC (10). This is perfectly matched by the inhibitory effect of CS on spontaneous and IL-4-induced differentiation of CD34+-derived CD14+ precursors into CD1a+ DC. However, we as well as other researchers did not observe apoptosis-inducing activities of CS on monocyte-derived DC (11, 21, 22). The differences in sensitivity for Dex-induced apoptosis might reflect the differences in precursors, since we also found differences in Dex sensitivity of CD34+-derived dermal/interstitial DC, Langerhans DC, and macrophage precursors. Moreover, the fact that sensitivity for apoptosis decreased during differentiation of CD14+ DC precursors into fully differentiated CD1a+ dermal/interstitial DC demonstrates that also the state of differentiation or maturation of a cell determines its sensitivity to apoptosis. A possible effect of IL-4 as a survival factor in monocyte-derived DC cultures can be excluded, since IL-4 withdrawal did not induce Dex sensitivity, and the addition of IL-4 to CD34+-derived DC cultures did not prevent Dex-induced apoptosis (data not shown).

CS-induced apoptosis is a well-documented phenomenon for lymphocytes (23), osteoclasts and osteocytes (24), and some neuronal cells (25) and is also suggested for monocytes (26). We now show that specifically the CD34+-derived CD14+ DC precursors were susceptible to CS-induced apoptosis, but not the CD34+-derived CD14+ macrophage precursors, the CD34+-derived CD1a+ Langerhans precursors, or the fully differentiated CD14+-derived CD1a+ DC. These data clearly show that although different myeloid lineages seem to be closely related to each other, and cells might retain the potential to differentiate into another subtype, some regulatory mechanisms are exclusive for specific subtypes.

The in vivo relevance of this in vitro observed effect remains to be determined. In this respect it is interesting to note that Dex induces apoptosis in rat tracheal DC in vivo (27). Moreover, spleens of mice that received a single injection of Dex contained less DC and an increased number of macrophages (28), which could be the result of both skewing in differentiation and induction of apoptosis in a specific population. In addition, glucocorticoid administration to rhesus macaques changed DC subsets in lymph nodes (29), and treatment of healthy volunteers with glucocorticoids reduced the circulating numbers of plasmacytoid DC in blood (20), which can again be the result of multiple processes, as described above. Some in vivo studies have demonstrated that topical or systemic treatment with CS might decrease the number of Langerhans cells in skin and mucosa (30, 31). However, considering the effects of CS on survival indirectly via environmental changes or effects on Langerhans cell migration (32), this is not necessarily in contrast with our findings. An important point to clarify is whether the effects described in this study are relevant only at pharmacological doses of CS or also have relevance at physiological concentrations. In humans the main CS in plasma is cortisol. In terms of potency, the IC50 of Dex (5 x 10-8 M) observed in our experiments is ~2- to 65-fold higher than the normal free plasma cortisol level. Therefore, it is not likely that the effects described on DC and macrophage development are relevant under normal conditions. However, in situations of acute stress or in some pathological conditions, endogenous CS may reach levels in the range of concentrations used in this study (33). It this case, CS may influence the development of the hemopoietic cell system and thereby influence cell-mediated immunity.

One might speculate that changes in the composition of the hemopoietic compartment significantly influence immune responses. In humans, DC originate from at least two distinct lineages, the lymphoid and myeloid lineages, that elicit distinct cellular and humoral immune responses (34). Although Langerhans cells and interstitial DC both belong to the myeloid lineage, they differ in phenotype, tissue distribution, and function. Langerhans cells lack the capacity to directly activate naive B cells (35). In addition, Langerhans cells do not express the mannose receptor used for Ag capture by other DC subsets (36) and lack DC-specific ICAM-grabbing nonintegrin, which mediates strong adhesion between DC and T cells (37). Our data show that at least T cell stimulatory capacity and IL-12 production by LC are not affected by Dex treatment. However, a detailed analysis of other cellular immune responses induced by DC generated in the presence of CS has to be performed.

In conclusion, CS suppress the development of dermal/interstitial DC without affecting the development of macrophages or the development and function of Langerhans cells from the same CD34+ progenitor cells. This extends the list of anti-inflammatory mediators, such as IL-10 (38, 39), TGF-{beta} (40), and vitamin D3 (41, 42) that actively interfere with DC development. Moreover, TGF-{beta} is a critical factor in the differentiation toward Langerhans cells (43). The fact that anti-inflammatory agents, such as TGF-{beta} and CS, relatively favor Langerhans cell development might suggest that Langerhans cells have a more regulatory role. Thus, next to their direct immunosuppressive effects on T lymphocytes, CS might skew the differentiation of hemopoietic progenitor cells into Langerhans cells and hence alter cellular immune responses.


   Acknowledgments
 
We thank Drs. M. R. Daha and L. C. Paul for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by a fellowship from the Royal Netherlands Academy of Arts and Sciences (to C.v.K.). Back

2 Address correspondence and reprint requests to Dr. Andrea M. Woltman, Department of Nephrology, Leiden University Medical Center, C3-P, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail address: a.m.woltman{at}lumc.nl Back

3 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; CS, corticosteroid; Dex, dexamethasone; Pred, prednisolone; PI, propidium iodide; rh, recombinant human; SCF, stem cell factor. Back

Received for publication October 17, 2001. Accepted for publication April 16, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
  2. Hart, D. N.. 1997. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 90:3245.[Free Full Text]
  3. Cutler, C. W., R. Jotwani, B. Pulendran. 2001. Dendritic cells: immune saviors or Achilles’ heel?. Infect. Immun. 69:4703.[Free Full Text]
  4. Reid, S. D., G. Penna, L. Adorini. 2000. The control of T cell responses by dendritic cell subsets. Curr. Opin. Immunol. 12:114.[Medline]
  5. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, A. H. Enk. 2000. Induction of interleukin 10-producing, nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192:1213.[Abstract/Free Full Text]
  6. Thomson, A. W., L. Lu. 1999. Dendritic cells as regulators of immune reactivity: implications for transplantation. Transplantation 68:1.[Medline]
  7. Finkelman, F. D., A. Lees, R. Birnbaum, W. C. Gause, S. C. Morris. 1996. Dendritic cells can present antigen in vivo in a tolerogenic or immunogenic fashion. J. Immunol. 157:1406.[Abstract]
  8. Wilckens, T., R. De Rijk. 1997. Glucocorticoids and immune function: unknown dimensions and new frontiers. Immunol. Today 18:418.[Medline]
  9. Piemonti, L., P. Monti, P. Allavena, M. Sironi, L. Soldini, B. E. Leone, C. Socci, V. Di Carlo. 1999. Glucocorticoids affect human dendritic cell differentiation and maturation. J. Immunol. 162:6473.[Abstract/Free Full Text]
  10. Woltman, A. M., J. W. Fijter, S. W. A. Kamerling, L. C. Paul, M. R. Daha, C. Van Kooten. 2000. The effect of calcineurin inhibitors and corticosteroids on the differentiation of human dendritic cells. Eur. J. Immunol. 30:1807.[Medline]
  11. Vanderheyde, N., V. Verhasselt, M. Goldman, F. Willems. 1999. Inhibition of human dendritic cell functions by methylprednisolone. Transplantation 67:1342.[Medline]
  12. de Jong, E. C., P. L. Vieira, P. Kalinski, M. L. Kapsenberg. 1999. Corticosteroids inhibit the production of inflammatory mediators in immature monocyte-derived DC and induce the development of tolerogenic DC3. J. Leukocyte Biol. 66:201.[Abstract]
  13. Rea, D., C. Van Kooten, K. E. van Meijgaarden, T. H. Ottenhoff, C. J. Melief, R. Offringa. 2000. Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10. Blood 95:3162.[Abstract/Free Full Text]
  14. Reid, C. D., A. Stackpoole, A. Meager, J. Tikerpae. 1992. Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34+ progenitors in human bone marrow. J. Immunol. 149:2681.[Abstract]
  15. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, I. Durand, M. Cella, A. Lanzavecchia, J. Banchereau. 1997. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor {alpha}. II. Functional analysis. Blood 90:1458.[Abstract/Free Full Text]
  16. Garrone, P., E. M. Neidhardt, E. Garcia, L. Galibert, C. Van Kooten, J. Banchereau. 1995. Fas ligation induces apoptosis of CD40-activated human B lymphocytes. J. Exp. Med. 182:1265.[Abstract/Free Full Text]
  17. Valladeau, J., O. Ravel, C. Dezutter-Dambuyant, K. Moore, M. Kleijmeer, Y. Liu, V. Duvert-Frances, C. Vincent, D. Schmitt, J. Davoust, et al 2000. langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules. Immunity 12:71.[Medline]
  18. Canque, B., S. Camus, M. Yagello, J. C. Gluckman. 1998. Special susceptibility to apoptosis of CD1a+ dendritic cell precursors differentiating from cord blood CD34+ progenitors. Stem Cells 16:218.[Medline]
  19. Verhoeven, G. T., J. M. Van Haarst, H. J. De Wit, P. J. Simons, H. C. Hoogsteden, H. A. Drexhage. 2000. Glucocorticoids hamper the ex vivo maturation of lung dendritic cells from their low autofluorescent precursors in the human bronchoalveolar lavage: decreases in allostimulatory capacity and expression of CD80 and CD86. Clin. Exp. Immunol. 122:232.[Medline]
  20. Shodell, M., F. P. Siegal. 2001. Corticosteroids depress IFN-{alpha}-producing plasmacytoid dendritic cells in human blood. J. Allergy Clin. Immunol. 108:446.[Medline]
  21. Matasic, R., A. B. Dietz, S. Vuk-Pavlovic. 1999. Dexamethasone inhibits dendritic cell maturation by redirecting differentiation of a subset of cells. J. Leukocyte Biol. 66:909.[Abstract]
  22. Woltman, A. M., J. W. De Fijter, S. W. Kamerling, S. W. van der Kooij, L. C. Paul, M. R. Daha, C. Van Kooten. 2001. Rapamycin induces apoptosis in monocyte- and CD34-derived dendritic cells, but not in monocytes and macrophages. Blood 98:174.[Abstract/Free Full Text]
  23. Schwartzman, R. A., J. A. Cidlowski. 1994. Glucocorticoid-induced apoptosis of lymphoid cells. Int. Arch. Allergy Immunol. 105:347.[Medline]
  24. Weinstein, R. S., R. L. Jilka, A. M. Parfitt, S. C. Manolagas. 1998. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids: potential mechanisms of their deleterious effects on bone. J. Clin. Invest. 102:274.[Medline]
  25. Mitchell, I. J., A. J. Cooper, M. R. Griffiths, D. J. Barber. 1998. Phencyclidine and corticosteroids induce apoptosis of a subpopulation of striatal neurons: a neural substrate for psychosis?. Neuroscience 84:489.[Medline]
  26. Schmidt, M., H. G. Pauels, N. Lugering, A. Lugering, W. Domschke, T. Kucharzik. 1999. Glucocorticoids induce apoptosis in human monocytes: potential role of IL-1{beta}. J. Immunol. 163:3484.[Abstract/Free Full Text]
  27. Brokaw, J. J., G. W. White, P. Baluk, G. P. Anderson, E. Y. Umemoto, D. M. McDonald. 1998. Glucocorticoid-induced apoptosis of dendritic cells in the rat tracheal mucosa. Am. J. Respir. Cell. Mol. Biol. 19:598.[Abstract/Free Full Text]
  28. Moser, M., T. De Smedt, T. Sornasse, F. Tielemans, A. A. Chentoufi, E. Muraille, M. Van Mechelen, J. Urbain, O. Leo. 1995. Glucocorticoids down-regulate dendritic cell function in vitro and in vivo. Eur. J. Immunol. 25:2818.[Medline]
  29. Koopman, G., A. G. Dalgleish, B. S. Bhogal, A. G. Haaksma, J. L. Heeney. 2001. Changes in dendritic cell subsets in the lymph nodes of rhesus macaques after application of glucocorticoids. Hum. Immunol. 62:208.[Medline]
  30. Belsito, D. V., T. J. Flotte, H. W. Lim, R. L. Baer, G. J. Thorbecke, I. Gigli. 1982. Effect of glucocorticosteroids on epidermal Langerhans cells. J. Exp. Med. 155:291.[Abstract/Free Full Text]
  31. Ashworth, J., J. Booker, S. M. Breathnach. 1988. Effects of topical corticosteroid therapy on Langerhans cell antigen presenting function in human skin. Br. J. Dermatol. 118:457.[Medline]
  32. Cumberbatch, M., R. J. Dearman, I. Kimber. 1999. Inhibition by dexamethasone of Langerhans cell migration: influence of epidermal cytokine signals. Immunopharmacology 41:235.[Medline]
  33. Sternberg, E. M., G. P. Chrousos, R. L. Wilder, P. W. Gold. 1992. The stress response and the regulation of inflammatory disease. Ann. Intern. Med. 117:854.
  34. Pulendran, B., J. Banchereau, E. Maraskovsky, C. Maliszewski. 2001. Modulating the immune response with dendritic cells and their growth factors. Trends Immunol. 22:41.[Medline]
  35. Dubois, B., B. Vanbervliet, J. Fayette, C. Massacrier, C. Van Kooten, F. Briere, J. Banchereau, C. Caux. 1997. Dendritic cells enhance growth and differentiation of CD40-activated B lymphocytes. J. Exp. Med. 185:941.[Abstract/Free Full Text]
  36. Mommaas, A. M., A. A. Mulder, R. Jordens, C. Out, M. C. Tan, P. Cresswell, P. M. Kluin, F. Koning. 1999. Human epidermal Langerhans cells lack functional mannose receptors and a fully developed endosomal/lysosomal compartment for loading of HLA class II molecules. Eur. J. Immunol. 29:571.[Medline]
  37. Geijtenbeek, T. B., R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, G. J. Adema, Y. van Kooyk, C. G. Figdor. 2000. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3. receptor that supports primary immune responses. Cell 100:575.[Medline]
  38. De Smedt, T., M. Van Mechelen, G. De Becker, J. Urbain, O. Leo, M. Moser. 1997. Effect of interleukin-10 on dendritic cell maturation and function. Eur. J. Immunol. 27:1229.[Medline]
  39. Buelens, C., V. Verhasselt, D. De Groote, K. Thielemans, M. Goldman, F. Willems. 1997. Interleukin-10 prevents the generation of dendritic cells from human peripheral blood mononuclear cells cultured with interleukin-4 and granulocyte/macrophage-colony-stimulating factor. Eur. J. Immunol. 27:756.[Medline]
  40. Yamaguchi, Y., H. Tsumura, M. Miwa, K. Inaba. 1997. Contrasting effects of TGF-{beta}1 and TNF-{alpha} on the development of dendritic cells from progenitors in mouse bone marrow. Stem Cells 15:144.[Medline]
  41. Griffin, M. D., W. Lutz, V. A. Phan, L. A. Bachman, D. J. McKean, R. Kumar. 2001. Dendritic cell modulation by 1{alpha},25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo. Proc. Natl. Acad. Sci. USA 98:6800.[Abstract/Free Full Text]
  42. Piemonti, L., P. Monti, M. Sironi, P. Fraticelli, B. E. Leone, E. Dal Cin, P. Allavena, and C. Di, V. 2000. Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells. J. Immunol. 164:4443.
  43. Borkowski, T. A., J. J. Letterio, A. G. Farr, M. C. Udey. 1996. A role for endogenous transforming growth factor {beta}1 in Langerhans cell biology: the skin of transforming growth factor {beta}1 null mice is devoid of epidermal Langerhans cells. J. Exp. Med. 184:2417.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 The Journal of RheumatologyHome page
N. BRAUN, P. FRITZ, A. RIETH, W. SCHROTH, M. KIMMEL, D. BIEGGER, D. ZAKIM, and M. D. ALSCHER
Predictors for Treatment Success and Expression of Glucocorticoid Receptor in Giant Cell Arteritis and Polymyalgia Rheumatica
J Rheumatol, October 1, 2009; 36(10): 2269 - 2276.
[Abstract] [Full Text] [PDF]

Home page
 Ann Rheum DisHome page
J A Hardin
Dendritic cells: potential triggers of autoimmunity and targets for therapy
Ann Rheum Dis, November 1, 2005; 64(suppl_4): iv86 - iv90.
[Full Text] [PDF]

Home page
 BloodHome page
X. Chen, T. Murakami, J. J. Oppenheim, and O. M. Z. Howard
Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking
Blood, October 1, 2005; 106(7): 2409 - 2416.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
C. van Kooten and A. M. Woltman
CD40 brings the shuttle down
Blood, September 15, 2005; 106(6): 1895 - 1896.
[Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
G. J. Kahaly, S. Pitz, G. Hommel, and M. Dittmar
Randomized, Single Blind Trial of Intravenous versus Oral Steroid Monotherapy in Graves' Orbitopathy
J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5234 - 5240.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. E. Wiley, M. Cwiartka, D. Alvarez, D. C. Mackenzie, J. R. Johnson, S. Goncharova, L. Lundblad, and M. Jordana
Transient Corticosteroid Treatment Permanently Amplifies the Th2 Response in a Murine Model of Asthma
J. Immunol., April 15, 2004; 172(8): 4995 - 5005.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. M. Woltman and C. van Kooten
Functional modulation of dendritic cells to suppress adaptive immune responses
J. Leukoc. Biol., April 1, 2003; 73(4): 428 - 441.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Woltman, A. M.
Right arrow Articles by van Kooten, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Woltman, A. M.
Right arrow Articles by van Kooten, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS