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Kali
skiDepartment of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Regarding the T cell stimulatory function of DC, GC did not
influence the cell surface expression of HLA-DR or the costimulatory
molecules CD40 and CD80 and did not influence the ability of DC to take
up Ag. Consequently, GC pretreatment of DC indeed did not affect their
ability to stimulate CD4+ Th cell proliferation in response
to superantigen. However, as a result of their defective production of
bioactive IL-12, GC-pretreated DC have a reduced ability to promote the
production of IFN-
in CD4+ Th lymphocytes, as shown by
the observation that IFN- production could be restored by exogenous
IL-12. In contrast, GC treatment of DC enhanced the secretion of the
antiinflammatory cytokine IL-10 and the type 2 cytokine IL-5 by the T
cells. It is concluded that, in addition to their role as potent
inhibitors of inflammation via the direct suppression of cytokine
production in T cells, GC may further inhibit T cell-mediated
inflammation indirectly via the suppression of IL-12 production by
DC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and the type
2 cytokines IL-4 and IL-5 (4, 5, 6). In contrast, IL-4 production was
reported to be up-regulated by GC (2) when T cells were stimulated in
the presence of APC. Therefore, GC may not only affect T cells
directly, but also indirectly via APC.
The studies on the effects of GC on APC have focused mainly on
monocytes and macrophages and have shown the ability of GC to
down-regulate the secretion of several soluble mediators such as
IL-1ß (7), IL-6 (7, 8), IL-8 (9), IL-12p40 (10, 11), TNF- (12),
granulocyte-macrophage (GM)-CSF (7, 13), and prostaglandins (14).
Moreover, GC can interfere with the activation of monocytes and
macrophages not only by reducing their phagocytic capacity and the
IFN--induced increase of the expression of class II MHC molecules
(10, 15) but also by up-regulating the expression of the cell surface
mannose receptor (MR) on macrophages (16).
Although monocytes and macrophages can present Ag to Th cells,
dendritic cells (DC) are regarded as professional APC during the onset
of the immune response, since they have the particular ability to
activate naive Th lymphocytes (17, 18). In addition, DC take up Ag very
efficiently and express constitutively high levels of both
costimulatory molecules of the B7 family and adhesion molecules
required for efficient Ag-presentation (19). The secretion of
DC-derived immunoregulatory and immunomodulatory cytokines plays a
crucial role in the cascade of events during the priming of naive Th
cells. In this respect, IL-12 is an important Th1-skewing factor,
because of its ability to induce potently the production of IFN- in
Th cells (20, 21). IL-12 is a heterodimeric cytokine composed of a
35-kDa subunit (p35) and a 40-kDa subunit (p40) that have to be
produced within the same cell to assemble the biologically active
IL-12p70 molecule (22). The levels of bioactive IL-12 are limited and
determined mainly by regulation of the expression of the p35 subunit
(23).
Until now, the effects of GC on DC have been poorly studied. Murine studies showed that GC down-regulate the ability of in vitro-cultured DC to stimulate T cells due to either insufficient expression of B7 family costimulatory molecules or inefficient uptake and/or processing of Ag (24, 25). GC are used as topical drugs to inhibit inflammation. For that purpose, they are applied on peripheral tissues containing immature DC that are able to activate infiltrating T cells. Since modification of DC function may affect the outcome of the specific immune response, the effect of GC on human DC was studied using in vitro-generated, monocyte-derived DC as an experimental model to study DC function, which has been generally accepted since it was first reported (26). Based on their frequent clinical application and potency, we tested the synthetic GC clobetasol-17-propionate (CP) and the naturally occurring GC hydrocortisone (HC) as representatives of strong and weak GC, respectively. The effects of these GC on the ability of DC to secrete immunoregulatory and proinflammatory cytokines, to take up Ag, and to stimulate both CD4+ Th cell proliferation and the production of type 1 and type 2 cytokines in response to superantigen was investigated.
The present data show a strong inhibitory effect of GC on the ability
of human DC to secrete the Th1-polarizing factor IL-12p70 and, to a
lesser extent, the proinflammatory cytokines IL-6 and TNF-. However,
GC did not affect the uptake of Ag, the expression of class II
MHC and costimulatory molecules, or the stimulatory capacities
toward CD4+ Th cells. Due to their reduced IL-12p70 levels,
GC-pretreated DC do not promote the type 1 cytokine IFN-; rather,
these DC stimulate the secretion of the antiinflammatory cytokine IL-10
and the type 2 cytokine IL-5 by CD4+ Th lymphocytes in the
presence of superantigen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rGM-CSF (specific activity (SA) 1.11 x
107 U/mg) was a gift of Schering-Plough (Uden, The
Netherlands). Human rIFN- (SA 8 x 107 U/mg) was a
gift of Dr. P. H. van der Meide (Biomedical Primate Research
Center, Rijswijk, The Netherlands). Human rIL-4 (SA 1 x
108 U/mg) was obtained from Pharma Biotechnologie Hannover
(Hannover, Germany). Human rIL-12 (SA 1.7 x 108 U/mg)
was a gift of Dr. M. K. Gately (Hoffmann-La Roche, Nutley, NJ).
Fixed Staphylococcus aureus Cowan strain I (SAC) (Calbiochem, San Diego, CA) was used at a final concentration of 75 µg/ml. LPS (Difco, Detroit, MI) was used at a final concentration of 100 ng/ml. Superantigen S. aureus enterotoxin B (SEB) (Sigma, St. Louis, MA) was used at a final concentration of 1 ng/ml.
The GC CP (Sigma) and HC (Sigma) were kept as stock solutions of 10-2 M in 95% ethanol, stored at -80°C, and diluted into complete medium just before use. A 1:104 final dilution of 95% ethanol was included as a vehicle control in all experiments.
In vitro generation of DC from peripheral blood monocytes
Venous blood from healthy donors was collected by venipuncture in sodium-heparin-containing tubes (VT100H; Venoject, Terumo Europe, Leuven, Belgium). PBMC were isolated by density centrifugation with Lymphoprep (Nycomed, Torshov, Norway). Subsequently, PBMC were centrifuged on a Percoll (Pharmacia, Uppsala, Sweden) gradient consisting of three density layers (1.076, 1.059, and 1.045 g/ml). The light density fraction floating on the middle layer, which contained predominantly monocytes, was seeded in 24-well culture plates (Costar, Cambridge, MA) at a density of 5 x 105 cells/well. After 30 min of incubation at 37°C, nonadherent cells were removed; adherent cells were cultured in Iscove’s modified Dulbecco’s medium (Life Technologies, Paisley, U.K.) containing gentamicin (86 µg/ml; Duchefa, Haarlem, The Netherlands) and 1% FCS (HyClone, Logan, UT) and supplemented with GM-CSF (500 U/ml) and IL-4 (250 U/ml) to obtain DC as described previously (26). At day 3, the media, including the supplements, were refreshed. At day 6, CD1a+CD14- DC were obtained.
Isolation of CD4+ Th cells
Highly purified CD4+ Th cells (>98% as assessed by flow cytometry, data not shown) were obtained by incubating the PBL, which had been harvested from the lower interface of the Percoll gradient, with anti-CD4 coated Dynabeads (Dynal AS, Oslo, Norway), followed by treatment with Detachabeads (Dynal AS) according to the manufacturer’s instructions.
Stimulation of DC in the presence or absence of GC
CD1a+ DC (4 x 104 cells/well) were
stimulated in 96-well flat-bottom culture plates (Costar) in Iscove’s
modified Dulbecco’s medium containing 10% FCS in a final volume of
200 µl. The following stimuli were used: LPS (final concentration 100
ng/ml) and SAC (final concentration 75 µg/ml). Both modes of
stimulation were performed in the absence or presence of IFN-
(103 U/ml). The GC used in this study were CP and HC. Pilot
experiments demonstrated that 10-8 M CP strongly reduced
IL-12p70 production by stimulated DC, whereas a 100- to 1000-fold
higher concentration of HC was needed to observe a similar reduction.
Therefore, the concentrations of CP used for future experiments were
between 10-11 and 10-8 M whereas HC was used
in a concentration range of 10-8–10-6 M.
Supernatants were harvested after 24 h and stored at -20°C
until the levels of IL-12p70, IL-10, IL-6, and TNF- were measured by
ELISA.
GC pretreatment of DC followed by stimulation in the absence of GC
CD1a+ DC were cultured for 2 days in GM-CSF (500
U/ml) in the presence of CP (10-10 M or 10-8
M), HC (10-8 M or 10-6 M), or vehicle. To
study the stability of the observed cytokine production profiles, DC
were cultured for another 2 days in GM-CSF (500 U/ml) alone after the
removal of the GC. To stimulate the DC, the cells were
harvested, washed thoroughly, counted, and the viability was
determined. A difference in viability between GC-treated and untreated
DC was not observed in any of the experiments. DC (4 x
104 cells/200 µl) were stimulated with LPS (final
concentration 100 ng/ml) in the absence of GC. Supernatants were
harvested after 24 h and stored at -20°C until the levels of
IL-12p70, IL-10, IL-6, and TNF- were measured by ELISA.
Ag uptake assay
CD1a+ DC were incubated at 37°C for 30 min in the
presence of culture medium containing either 0.2 µg/ml of
albumin-FITC conjugate (BSA-FITC, 1:12 molar ratio; Sigma) or 0.2
µg/ml of -D-mannosylated-albumin-FITC conjugate
(manBSA-FITC, 1:2.5 molar ratio; Sigma). Negative controls were
incubated with the respective Ag at 4°C for 30 min. Ag uptake was
stopped by extensive cold washing, and cell surface fluorescence
was quenched with trypan blue (Sigma). Ag uptake was evaluated by flow
cytometry.
Induction of proliferative response in CD4+ Th cells
CD4+ Th cells (5 x 104 cells/200
µl) were cocultured in 96-well flat-bottom culture plates (Costar)
with different concentrations of vehicle- or GC-treated DC (as
indicated in Fig. 5
) coated with SEB (final concentration 1
ng/ml). Cell proliferation was assessed by the incorporation of
[3H]TdR (Radiochemical Centre, Amersham, Little Chalfont,
U.K.) after a pulse with 13 kBq/well during the last
16 h of 5-day culture after stimulation, as measured by liquid
scintillation spectroscopy.
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CD4+ Th cells (5 x 104 cells/200
µl) were cocultured with 3 x 104 vehicle- or
GC-treated DC coated with SEB (final concentration 1 ng/ml) in 96-well
flat-bottom culture plates (Costar), in either the presence or absence
of an excess of exogenous IL-12 (100 U/ml). Supernatants were harvested
after 4 days, and the levels of IFN-, IL-4, IL-5, and IL-10 were
measured by ELISA.
Cytokine measurements
Measurements of IFN-, IL-4, IL-5, IL-10, and IL-12p70 levels
in culture supernatants were performed by a specific solid-phase
sandwich ELISA as described previously (27). Measurements of IL-6 and
TNF- were performed by ELISA using pairs of specific mAbs and
recombinant cytokine standards obtained from BioSource International
(Camarillo, CA). The limits of detection of these ELISA are as follows:
IFN-, 100 pg/ml; IL-4, 60 pg/ml; IL-5, 50 pg/ml; IL-6, 20 pg/ml;
IL-10, 25 pg/ml; IL-12p70, 3 pg/ml; and TNF-, 20 pg/ml.
Analysis of expression of cell surface molecules by flow cytometry
The mouse anti-human mAbs against the following molecules were used: CD1a (OKT6; Ortho Diagnostic System, Beerse, Belgium), CD40 (EA-5; a gift of Dr. T. LeBien, University of Minnesota, Minneapolis, MN), CD80 (B7-24 (28); kindly provided by Innogenetics N.V., Ghent, Belgium), HLA-DR (L243; Becton Dickinson, San José, CA), and MR (15-2 (16); a gift of Dr. F. Noorman, Gaubius Laboratory, Netherlands Central Organization for Applied Scientific Research for Prevention and Health, Leiden, The Netherlands), followed by FITC-conjugated goat anti-mouse mAb (Jackson Immunoresearch Laboratories, West Grove, PA).
Statistical analysis
Data were analyzed for statistical significance (InStat, version
2.02, GraphPad, San Diego, CA) using ANOVA followed by
Dunnett’s multiple comparisons test. The stability of the effect of GC
on cytokine production by GC-pretreated DC at 2 days after GC
withdrawal (see Fig. 2) was evaluated using the Student’s t
test. A p value of <0.05 was considered as the level of
significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since DC are the professional effective APC and their
secretion of immunoregulatory and proinflammatory cytokines plays a
crucial role during T cell priming (17, 18), we investigated whether
cytokine production by in vitro-generated DC is affected by GC. To
address this issue, the effect of increasing concentrations of CP
and HC on IL-12, IL-6, and TNF- production was compared. As
shown in Fig. 1, the stimulation of DC
with LPS (100 ng/ml) in the presence of increasing concentrations of GC
resulted in a dose-dependent inhibition of IL-12p70, IL-6, and TNF-
production.
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A) inhibited the secretion of the IL-12p70 heterodimer by
50% (p < 0.01); in contrast, no
significant reduction in either IL-6 or TNF- production was observed
at this dose. High concentrations of CP (10-8 M)
almost completely abrogated the production of IL-12p70
(p < 0.001) but affected IL-6 and TNF- to a
lesser extent. Also, HC was found to affect DC-derived cytokines (Fig. 1B), but 100- to 1000-fold higher concentrations were needed
for an effect that was comparable with that of CP. An identical down-regulation of cytokine production was observed when DC were stimulated with SAC (75 µg/ml) (data not shown). At all conditions, IL-10 production was not detectable.
Effect of GC pretreatment on cytokine secretion by DC
We questioned whether the inhibition of IL-12p70
production by GC was a direct effect during DC stimulation or could
also be found when DC were pretreated with GC. Therefore, DC were
preincubated with GC or vehicle for 2 days, washed, and stimulated with
LPS (100 ng/ml) in the absence of GC. As shown in Fig. 2 (filled bars), GC pretreatment also
inhibited the production of DC-derived cytokines upon subsequent
stimulation. The production of IL-12p70 (Fig. 2A, filled
bars) was strongly inhibited (p < 0.001) by
preincubation with either 10-10 M or 10-8 M
CP. Again, HC appeared to be much less effective and induced a similar
IL-12p70 inhibition only at 10-6 M
(p < 0.001); 10-8 M HC did
not produce any statistically significant effect. The production of
IL-6 (Fig. 2B, filled bars) and TNF- (Fig. 2C,
filled bars) was inhibited to a lesser extent compared with IL-12p70.
IL-10 production was not detectable in these culture supernatants,
which is consistent with the poor ability of DC to produce this
cytokine (27).
To assess the stability of the down-regulation of cytokine production,
GC-pretreated DC were washed extensively and kept in culture in the
absence of GC for an additional 2 days before stimulation. As shown in
Fig. 2 (open bars), IL-12p70 and IL-6 were inhibited to the same extent
compared with experiments in which they were determined directly after
GC pretreatment (filled bars). However, the additional 2 days of
culture in the absence of GC partially restored the ability of DC
pretreated with HC and low-dose CP to produce TNF-
(p < 0.001 and p < 0.01,
respectively).
GC pretreatment does not affect the expression of surface molecules on DC
We studied whether GC pretreatment of DC affects their cell
surface expression of the DC-associated marker CD1a, the class II MHC
Ag-presenting molecule HLA-DR, the costimulatory molecules CD40 and the
B7 molecule CD80, and MR. Although it has been reported previously for
monocytes and macrophages that GC interfere with the cell surface
expression of molecules involved in Ag presentation and costimulation
(10, 15), GC pretreatment of DC for 2 days did not affect the
expression of surface molecules when compared with vehicle-treated
control cells (Fig. 3).
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Using BSA-FITC and manBSA-FITC in a functional assay, we
subsequently assessed the effects of GC on the ability of DC to take up
Ag via fluid phase endocytosis or via their specific MR. Regardless of
the pretreatment conditions, neither CP nor HC affected the uptake of
either BSA-FITC (Fig. 4, left
panels) or manBSA-FITC (Fig. 4, right panels).
These results clearly show that the endocytic capacity of DC was not
affected by preculture with GC. Although MR expression has been
reported to be up-regulated by GC (16), no differences on MR-mediated
uptake were induced; therefore, this observation is in line with the
unchanged cell surface expression of MR by GC-treated DC in comparison
with control DC (Fig. 3).
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Next, we studied the effects of GC on the proliferation-inducing
capacity of DC toward Th cells. To exclude the direct effects of GC on
T cells, DC were pretreated with GC before coculture with highly
purified CD4+ Th cells in the presence of superantigen
(SEB; 1 ng/ml). In the absence of either DC or SEB, no activation of T
cells could be observed. GC-pretreated DC were as effective as
vehicle-pretreated DC in inducing T cell proliferation (Fig. 5). These results indicate that the
exposure of DC to GC does not affect their ability to stimulate T cell
proliferation in the presence of superantigen. These results are in
line with the unaffected expression of HLA-DR and costimulatory
molecules described above (Fig. 3).
GC-pretreated DC do not promote secretion of type 1 cytokine
IFN- by CD4+ Th cells but do stimulate secretion of
IL-10 and IL-5
Since IL-12 is a potent inducer of IFN- secretion in activated
CD4+ Th cells (20, 21), we investigated whether GC
pretreatment of DC influenced the cytokine profile of SEB-triggered
CD4+ Th cells. As shown in Fig. 6A, pretreatment with GC
reduced (p < 0.001) the ability of DC to
stimulate the production of the type 1 cytokine IFN-. In contrast,
GC-pretreated DC up-regulated T cell production of the antiinflammatory
mediator IL-10 (Fig. 6B) and the type 2 cytokine IL-5 (Fig. 6C). Interestingly, the addition of rIL-12 to these
cocultures abolished the reduction of IFN- and the up-regulation of
IL-5 production by the T cells but not the up-regulation of IL-10. In
both the absence and the presence of exogenous IL-12, IL-4 production
was not detectable in the culture supernatants. These results indicate
that the use of GC-pretreated DC as APC results in a T cell cytokine
shift toward a type 2 profile, which is largely due to the reduced
production of IL-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In contrast,
GC did not affect the level of expression of costimulatory molecules by
DC or the ability of DC to take up Ag and stimulate the proliferation
of CD4+ Th cells in vitro. Largely due to an inhibition of
IL-12p70 production, GC down-regulate the capacity of DC to induce the
secretion of the type 1 cytokine IFN- in CD4+ Th cells
while up-regulating their capacity to promote the secretion of the
antiinflammatory cytokine IL-10 and the type 2 cytokine IL-5.
GC inhibit cytokine gene transcription through the activation of their
specific cytosolic receptors (29). The actual down-regulation of
transcription of sensitive genes by GC was found to be due to the
functional inhibition of some key transcription factors. GC were shown
to inhibit the assembly of c-Fos and c-Jun to form functional activator
protein-1 (30). Furthermore, the inhibitory effect of GC on
NF-
B was shown to be mediated either via the enhancement of IB
transcription and synthesis (31, 32), which is known to bind to NF-B
and prevent its migration to the nucleus, or via a direct association
with Rel A, the 65-kDa NF-B-binding protein (33).
The direct inhibitory effect of GC on T cell-derived cytokines is well
documented, but T cells are not the only cell type in which cytokine
production is modulated by GC. Several studies reported the
down-regulatory effect of GC on the secretion of the proinflammatory
cytokines IL-1ß (7), IL-6 (7, 8), and TNF- (12) by monocytes and
macrophages. Recently, Blotta et al. (10) demonstrated the inhibitory
effect of GC on human monocyte-derived IL-12p40 with a concomitant
up-regulation of IL-10. And very recently, the GC-induced inhibition of
IL-12p70 production by human monocytes was also shown (34). However,
the effect of GC on human DC-derived bioactive IL-12p70 was not known.
Here, we report on the strong inhibition of IL-12p70 secretion by human
DC in response to GC. The positive regulation of the IL-12p40 promoter
by NF-B was reported previously (35), notwithstanding that the
regulation of the IL-12p35 promoter remains elusive. Hence, it can be
speculated that, in addition to the probable effect of GC on the
transcription of the IL-12p40 monomer, which is mediated indirectly via
the down-regulation of NF-B, GC may also affect the transcription of
IL-12p35 or interfere with the assembly of the two IL-12 subunits to
form the bioactive IL-12p70 heterodimer. Since the production of
bioactive IL-12 is determined mainly by the expression of the p35
gene (23), knowledge with regard to the regulation of IL-12p35
transcription will help to clarify the direct effects of GC on the
secretion of IL-12p70. As GC stimulate IL-10 production in human
monocytes (10), which is known to reduce IL-12 synthesis (23), it was
suggested that these GC indirectly suppress IL-12 production via the
up-regulation of IL-10. Although this inhibitory pathway may be
relevant for monocytes, in vitro-generated human DC do not produce
detectable levels of IL-10, implying that the inhibitory effect of GC
on cytokine production by DC cannot be explained by the indirect effect
of up-regulation of IL-10.
Both IL-6 and TNF- are important mediators of a wide range of
biologic activities that play a critical role in the induction of
proinflammatory and immune responses. GC inhibited IL-6 as well as
TNF- secretion by DC. The NF-B-binding site within the IL-6
promoter was suggested to be essential for the induction of IL-6
transcription (36). Likewise, NF-B is involved in the positive
regulation of TNF- gene transcription (37). Ray and Prefontaine (38)
showed that the activation of the IL-6 promoter by a combination of
NF-IL-6 and Rel A was inhibited by dexamethasone-activated GC
receptors. Since NF-B plays a critical role in inflammatory
reactions, the possible inhibition of cytokine gene transcription as a
result of the antagonism between activated GC receptors and NF-B
constitutes a likely mechanism underlying the GC-mediated
antiinflammatory and immunosuppressive mode of action.
In contrast to the clear inhibitory effect of GC on DC cytokine
production, our study indicated that Ag uptake, either via endocytosis
or MR-mediated, was not affected by exposure to GC. Moreover, our data
show that the expression of the class II MHC molecule HLA-DR and the
costimulatory molecules CD40 and CD80 remained unaffected after the
preincubation of DC with GC. Indeed, pretreatment of in vitro-generated
DC with GC does not down-regulate their stimulatory potential, as
suggested by the observation that GC pretreatment did not affect the
capacity of DC to stimulate CD4+ Th cell
proliferation. Therefore, our results are in contrast with recent
reports on the reduced ability of GC-treated human monocytes (10) or
purified murine splenic DC (24) to stimulate the proliferation of T
lymphocytes. In these two studies, it was described that GC inhibit the
IFN-- (10) or 24-h-culture- (24) induced up-regulation of the
cell surface expression of molecules involved in either Ag presentation
or costimulation. However, the up-regulation of class II MHC and
costimulatory molecules as well as an increased T cell stimulatory
potential are features of the maturation of DC. The possibility cannot
be excluded that partial maturation was induced by the culturing
conditions in both studies mentioned.
In line with the fact that IL-12 induces Th1 responses via a strong
induction of IFN- secretion (20, 21), we found that GC-treated DC
with a reduced capacity to produce bioactive IL-12p70 induced less
IFN- in superantigen-stimulated CD4+ Th cells.
Previously, a similar effect was reported for GC-pretreated human
monocytes (10) and murine macrophages (11) when used to stimulate
cytokine production by CD4+ T lymphocytes. In those
studies, it was assumed that the inhibitory effect of GC on the
production of IL-12p40 by the APC was representative for the effect on
the bioactive heterodimer; however, the regulation of bioactive IL-12
production is determined mainly by the transcription of the p35 subunit
(23). Here, we report that the down-regulation of bioactive IL-12p70
production by DC in response to GC is indeed responsible for the
reduction of IFN- secretion by CD4+ T cells, and that
this reduction can be restored by the addition of exogenous bioactive
IL-12 to the cultures during stimulation. Additionally, secretion of
the antiinflammatory cytokine IL-10 and the type 2 helper cytokine IL-5
was promoted when T cells were stimulated with GC-pretreated DC,
confirming the studies of Blotta et al. (10) and DeKruyff et al. (11),
respectively. IL-12 has been shown to be involved in the
down-regulation of IL-5 secretion (27, 39) by T cells. The observed
up-regulation of IL-5 secretion by the T cells in response to
stimulation with GC-treated DC was abolished by rIL-12, indicating that
the lack of secretion of bioactive IL-12 was responsible for the
up-regulation of IL-5. Although IL-12 has been also reported to induce
T cell-derived IL-10 (40), the production of this antiinflammatory
mediator by CD4+ Th cells was not sensitive to rIL-12 in
our cultures. However, Jeannin et al. (40) made their observation with
established T cell lines in an APC-free system. Consequently, this
suggests a rather distinct regulatory pathway of IL-10 production by
IL-12 compared with the activation of resting T cells in an
APC-dependent manner as we show here.
By virtue of defective IL-12 production and the subsequent effects on cytokine production by Th cells, GC-pretreated DC should be regarded as antiinflammatory players in the Ag-specific immune system. Although GC are potent direct inhibitors of specific Th cell responses, additional modulation of the immune response may be expected through the effects of GC on DC, rendering them less supportive for inflammatory responses. In this way, the recurrent activation of T cells by GC-treated APC may lead indirectly to a suppression of the secretion of inflammatory type 1 cytokines, favoring the activation of T cells with a type 2 cytokine profile. GC pretreatment may induce increased IgE production (41, 42) and eosinophil numbers (43). Indeed, GC were shown to enhance IgE synthesis in both allergic and nonallergic individuals (44, 45). Despite this effect, therapy with GC appears to be useful in the blocking of allergen-specific reactions. This effect could probably be explained by the dominant antiinflammatory effect mediated not only via the direct inhibition of inflammatory T cell cytokines but also via the inhibition of IL-12 production in professional APC.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Esther C. de Jong, Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands. E-mail address:
3 Abbreviations used in this paper: GC, glucocorticoid(s); GM-CSF, granulocyte-macrophage CSF; MR, mannose receptor; DC, dendritic cell(s); CP, clobetasol-17-propionate; HC, hydrocortisone; SA, specific activity; SAC, Staphylococcus aureus Cowan strain I; SEB, Staphylococcus aureus enterotoxin B; manBSA, mannosylated BSA.
Received for publication April 29, 1998. Accepted for publication July 10, 1998.
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L. Freeman, M. Hewison, S. V. Hughes, K. N. Evans, D. Hardie, T. K. Means, and R. Chakraverty Expression of 11{beta}-hydroxysteroid dehydrogenase type 1 permits regulation of glucocorticoid bioavailability by human dendritic cells Blood, September 15, 2005; 106(6): 2042 - 2049. [Abstract] [Full Text] [PDF]
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S.-Y. Dai, R. Nakagawa, A. Itoh, H. Murakami, Y. Kashio, H. Abe, S. Katoh, K. Kontani, M. Kihara, S.-L. Zhang, et al. Galectin-9 Induces Maturation of Human Monocyte-Derived Dendritic Cells J. Immunol., September 1, 2005; 175(5): 2974 - 2981. [Abstract] [Full Text] [PDF]
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M. Dorsch, Y. Qiu, D. Soler, N. Frank, T. Duong, A. Goodearl, S. O'Neil, J. Lora, and C. C. Fraser PK1/EG-VEGF induces monocyte differentiation and activation J. Leukoc. Biol., August 1, 2005; 78(2): 426 - 434. [Abstract] [Full Text] [PDF]
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S. Baumann, A. Dostert, N. Novac, A. Bauer, W. Schmid, S. C. Fas, A. Krueger, T. Heinzel, S. Kirchhoff, G. Schutz, et al. Glucocorticoids inhibit activation-induced cell death (AICD) via direct DNA-dependent repression of the CD95 ligand gene by a glucocorticoid receptor dimer Blood, July 15, 2005; 106(2): 617 - 625. [Abstract] [Full Text] [PDF]
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P. H. Tan, P. Sagoo, C. Chan, J. B. Yates, J. Campbell, S. C. Beutelspacher, B. M. J. Foxwell, G. Lombardi, and A. J. T. George Inhibition of NF-{kappa}B and Oxidative Pathways in Human Dendritic Cells by Antioxidative Vitamins Generates Regulatory T Cells J. Immunol., June 15, 2005; 174(12): 7633 - 7644. [Abstract] [Full Text] [PDF]
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 S. N. Georas Inhaled Glucocorticoids, Lymphocytes, and Dendritic Cells in Asthma and Obstructive Lung Diseases Proceedings of the ATS, November 1, 2004; 1(3): 215 - 221. [Abstract] [Full Text] [PDF]
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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]
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D. Chaussabel, R. T. Semnani, M. A. McDowell, D. Sacks, A. Sher, and T. B. Nutman Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites Blood, July 15, 2003; 102(2): 672 - 681. [Abstract] [Full Text] [PDF]
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 H. C. Heystek, A.-C. Thierry, P. Soulard, and C. Moulon Phosphodiesterase 4 inhibitors reduce human dendritic cell inflammatory cytokine production and Th1-polarizing capacity Int. Immunol., July 1, 2003; 15(7): 827 - 835. [Abstract] [Full Text] [PDF]
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R. Morita, N. Ukyo, M. Furuya, T. Uchiyama, and T. Hori Atrial Natriuretic Peptide Polarizes Human Dendritic Cells Toward a Th2-Promoting Phenotype Through Its Receptor Guanylyl Cyclase-Coupled Receptor A J. Immunol., June 15, 2003; 170(12): 5869 - 5875. [Abstract] [Full Text] [PDF]
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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]
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 A. G.S. van Halteren, E. van Etten, E. C. de Jong, R. Bouillon, B. O. Roep, and C. Mathieu Redirection of Human Autoreactive T-Cells Upon Interaction With Dendritic Cells Modulated by TX527, an Analog of 1,25 Dihydroxyvitamin D3 Diabetes, July 1, 2002; 51(7): 2119 - 2125. [Abstract] [Full Text] [PDF]
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 F. J. Barrat, D. J. Cua, A. Boonstra, D. F. Richards, C. Crain, H. F. Savelkoul, R. de Waal-Malefyt, R. L. Coffman, C. M. Hawrylowicz, and A. O'Garra In Vitro Generation of Interleukin 10-producing Regulatory CD4+ T Cells Is Induced by Immunosuppressive Drugs and Inhibited by T Helper Type 1 (Th1)- and Th2-inducing Cytokines J. Exp. Med., March 4, 2002; 195(5): 603 - 616. [Abstract] [Full Text] [PDF]
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H. Miyaura and M. Iwata Direct and Indirect Inhibition of Th1 Development by Progesterone and Glucocorticoids J. Immunol., February 1, 2002; 168(3): 1087 - 1094. [Abstract] [Full Text] [PDF]
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A. Boonstra, F. J. Barrat, C. Crain, V. L. Heath, H. F. J. Savelkoul, and A. O'Garra 1{alpha},25-Dihydroxyvitamin D3 Has a Direct Effect on Naive CD4+ T Cells to Enhance the Development of Th2 Cells J. Immunol., November 1, 2001; 167(9): 4974 - 4980. [Abstract] [Full Text] [PDF]
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 B.N. Lambrecht, J-;B. Prins, and H.C. Hoogsteden Lung dendritic cells and host immunity to infection Eur. Respir. J., October 1, 2001; 18(4): 692 - 704. [Abstract] [Full Text] [PDF]
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F. Van Laethem, E. Baus, L. A. Smyth, F. Andris, F. Bex, J. Urbain, D. Kioussis, and O. Leo Glucocorticoids Attenuate T Cell Receptor Signaling J. Exp. Med., April 2, 2001; 193(7): 803 - 814. [Abstract] [Full Text] [PDF]
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G.A.W. Rook, G. Seah, and A. Ustianowski M. tuberculosis: immunology and vaccination Eur. Respir. J., March 1, 2001; 17(3): 537 - 557. [Abstract] [Full Text] [PDF]
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K. D. Kim, Y.-K. Choe, I. S. Choe, and J.-S. Lim Inhibition of glucocorticoid-mediated, caspase-independent dendritic cell death by CD40 activation J. Leukoc. Biol., March 1, 2001; 69(3): 426 - 434. [Abstract] [Full Text]
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J. Banchereau, B. Pulendran, R. Steinman, and K. Palucka Will the Making of Plasmacytoid Dendritic Cells in Vitro Help Unravel Their Mysteries? J. Exp. Med., December 18, 2000; 192(12): f39 - f44. [Full Text] [PDF]
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J. M. Bakker, A. Kavelaars, P. J. G. H. Kamphuis, P. M. Cobelens, H. H. van Vugt, F. van Bel, and C. J. Heijnen Neonatal Dexamethasone Treatment Increases Susceptibility to Experimental Autoimmune Disease in Adult Rats J. Immunol., November 15, 2000; 165(10): 5932 - 5937. [Abstract] [Full Text] [PDF]
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 R. W. BAKER, B. R. WALKER, R. J. SHAW, J. W. HONOUR, D. S. JESSOP, S. L. LIGHTMAN, A. ZUMLA, and G. A. W. ROOK Increased Cortisol . Cortisone Ratio in Acute Pulmonary Tuberculosis Am. J. Respir. Crit. Care Med., November 1, 2000; 162(5): 1641 - 1647. [Abstract] [Full Text]
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D. Rea, C. van Kooten, K. E. van Meijgaarden, T. H. M. Ottenhoff, C. J. M. Melief, and R. Offringa Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10 Blood, May 15, 2000; 95(10): 3162 - 3167. [Abstract] [Full Text] [PDF]
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P. L. Vieira, E. C. de Jong, E. A. Wierenga, M. L. Kapsenberg, and P. Kalinski Development of Th1-Inducing Capacity in Myeloid Dendritic Cells Requires Environmental Instruction J. Immunol., May 1, 2000; 164(9): 4507 - 4512. [Abstract] [Full Text] [PDF]
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J. Komi and O. Lassila Nonsteroidal anti-estrogens inhibit the functional differentiation of human monocyte-derived dendritic cells Blood, May 1, 2000; 95(9): 2875 - 2882. [Abstract] [Full Text] [PDF]
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C. E. Demeure, H. Tanaka, V. Mateo, M. Rubio, G. Delespesse, and M. Sarfati CD47 Engagement Inhibits Cytokine Production and Maturation of Human Dendritic Cells J. Immunol., February 15, 2000; 164(4): 2193 - 2199. [Abstract] [Full Text] [PDF]
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 J. G. I. van Rietschoten, H. H. Smits, D. van de Wetering, R. Westland, C. L. Verweij, M. T. den Hartog, and E. A. Wierenga Silencer Activity of NFATc2 in the Interleukin-12 Receptor beta 2 Proximal Promoter in Human T Helper Cells J. Biol. Chem., September 7, 2001; 276(37): 34509 - 34516. [Abstract] [Full Text] [PDF]
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; or
10-6 M HC, 
). After the treatment period, GC were
washed away, and different concentrations of DC were used to stimulate
5 x 104 CD4+ Th cells in the presence of
1 ng/ml SEB. The proliferative response was determined at day 5 of
coculture by [3H]TdR incorporation. Data are presented as
mean cpm ± SD of triplicate cultures. Results are representative
of six independent experiments.
