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Control of functional T helper cell polarization by dendritic cells

Limao Vieira, P.

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Citation for published version (APA):Limao Vieira, P. (2002). Control of functional T helper cell polarization by dendritic cells.

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Download date: 11 Feb 2021

CHAPTERR 7

Glucocorticoid ss Inhibi t Bioactiv e IL-12p70 Productio nn by In Vitro-Generate d Human Dendriti cc Cells Withou t Affectin g Their

TT cel l Stimulator y Potentia l

Pedroo L. Vieira, Pawel Kalihski, Eddy A. Wierenga, Martienn L. Kapsenberg, Esther C. de Jong

Glucocorticoidss (GC) are known to affect the immune response at several stages. However,, little is known about how GC influence the initiation of the specific immune responsee at the level of dendritic cells (DC), the highly professional APC for T cells. Therefore,, we studied whether GC modulate the cytokine production and T cell stimulatoryy function of DC. In LPS-stimulated DC, GC strongly reduced the secretion off the Tht-skewing factor IL-12p70 and, to a lesser extent, the production of the proinflammatoryy cytokines IL-6 and TNF-a. Regarding the T cell stimulatory function off DC, GC did not influence the cell surface expression of HLA-DR or the costimulatoryy molecules CD40 and CD80 and did not influence the ability of DC to takee up antigen. Consequently, GC pretreatment of DC indeed did not affect their abilityy to stimulate CD4+ Th cell proliferation in response to superantigen. However, ass a result of their defective production of bioactive IL-12, GC-pretreated DC have a reducedd ability to promote the production of IFN-y in CD4+ Th lymphocytes, as shown byy the observation that IFN-7 production could be restored by exogenous IL-12. In contrast,, GC treatment of DC enhanced the secretion of the antiinflammatory cytokinee IL-10 and the type 2 cytokine IL-5 by the T cells. It is concluded that, in additionn to their role as potent inhibitors of inflammation via the direct suppression of cytokinee production in T cells, GC may further inhibit T cell mediated inflammation indirectlyy via the suppression of IL-12 production by DC.

JJ Immunol 161:5245-5251 (1998)

99 9

Chapterr 7

INTRODUCTION N

Glucocorticoidss (GC) are potent antiinflammatory and immunosuppressive agents

thatt are widely used in the treatment of inflammatory disorders, such as autoimmune

andd allergic diseases (1). Generally, the immunosuppressive effects of GC have

beenn ascribed to their inhibitory effect on Th cell proliferation, due to the inhibition of

IL-22 production (2) and the downregulation of signal transduction through the IL-2R

(3).. In addition, several in vitro studies reported on the down regulatory effects of GC

onn the secretion of both the Th1 cell-associated cytokine IFN-y and the type 2

cytokiness IL-4 and IL-5 (4-6). In contrast, IL-4 production was reported to be

upregulatedd 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.

Thee studies on the effects of GC on APC have focused mainly on monocytes and

macrophages,, and showed the ability of GC to downregulate the secretion of several

solublee mediators such as IL-ip (7), IL-6 (7,8), IL-8 (9), IL-12p40 (10, 11), TNF-a

(12),, granulocyte-macrophage (GM)-CSF (7,13), and prostaglandins (14). Moreover,

GCC can interfere with the activation of monocytes and macrophages not only by

reducingg their phagocytic capacity and the IFN-y-induced increase of the expression

off class II MHC molecules (10, 15) but also by upregulating the expression of the cell

surfacee mannose receptor (MR) on macrophages (16).

Althoughh monocytes and macrophages can present antigen (Ag) to Th cells,

dendriticc cells (DC) are regarded as professional APC during the onset of the

immunee response, since they have the particular ability to activate naive Th

lymphocytess (17, 18). In addition, DC take up Ag very efficiently and express

constitutivelyy high levels of both costimulatory molecules of the B7 family and

adhesionn molecules required for efficient Ag-presentation (19). The secretion of DC-

derivedd immunoregulatory and immunomodulatory cytokines plays a crucial role in

thee cascade of events during the priming of naive Th cells. In this respect, IL-12 is an

importantt Th1-skewing factor, because of its ability to induce potently the production

off IFN-Y in Th cells (20, 21). IL-12 is a heterodimeric cytokine composed of a 35-kD

subunitt (p35) and a 40-kD subunit (p40) that have to be produced within the same

celll to assemble the biologically active lL-12p70 molecule (22). The levels of

bioactivee IL-12 are limited and determined mainly by regulation of the expression of

thee p35 subunit (23).

100 0

Glucocorticoidss inhibit dendritic cell-derived IL-12p70

Untill now, the effects of GC on DC have been poorly studied. Murine studies

showedd that GC downregulate the ability of in vitro cultured DC to stimulate T cells

duee to either insufficient expression of B7 family costimufatory molecules or

inefficientt uptake and/or processing of Ag (24, 25). GC are used as topical drugs to

inhibitt inflammation. For that purpose, they are applied on peripheral tissues

containingg immature DC that are able to activate infiltrating T cells. Since

modificationn of DC function may affect the outcome of the specific immune response,

thee effect of GC on human DC was studied using in vitro-generated monocyte-

derivedd DC as an experimental model to study DC function, which has been

generallyy accepted since it was first reported (26). Based on their frequent clinical

applicationn and potency, we tested the synthetic GC clobetasol-17-propionate (CP)

andd the naturally occurring GC hydrocortisone (HC) as representatives of strong and

weakk GC, respectively. The effects of these GC on the ability of DC to secrete

immunoregulatoryy and proinflammatory cytokines, to take up Ag, and to stimulate

bothh CD4+ Th cell proliferation and the production of type 1 and type 2 cytokines in

responsee to superantigen was investigated.

Thee present data show a strong inhibitory effect of GC on the ability of human DC

too secrete the Th1-polarizing factor IL-12p70 and, to a lesser extent, the

proinflammatoryy cytokines IL-6 and TNF-a. However, GC did not affect the uptake of

Ag,, the expression of class II MHC and costimulatory molecules, or the stimulatory

capacitiess toward CD4+ Th cells. Due to their reduced IL-12p70 levels, GC-pretreated

DCC do not promote the type 1 cytokine IFN-7; rather these DC stimulate the secretion

off the antiinflammatory cytokine IL-10 and the type 2 cytokine IL-5 by CD4+ Th

lymphocytess in the presence of superantigen.

MATERIAL SS AND METHODS

Cytokines ,, Antigens , and reagent s

Humann rGM-CSF (sp. act. 1.11x107 U/mg) was a gift of Schering-Plough (Uden, The

Netherlands).. Human rlFN-y (sp. act. 8x107 U/mg) was a gift of Dr. P. H. van der

Meidee (Biomedical Primate Research Center, Rijswijk, The Netherlands). Human

rlL-44 (sp. act. 1x108 U/mg) was obtained from Pharma Biotechnologie Hannover

(Hannover,, Germany). Human rlL-12 (sp. act. 1.7x108 U/mg) was a gift of Dr. M. K.

Gatelyy (Hoffmann-La Roche, Nutley, NJ).

101 1

Chapterr 7

Fixedd Staphylococcus aureus Cowan strain I (SAC) (Calbiochem, San Diego, CA)

wass used at a final concentration of 75 jig/ml. LPS (Difco, Detroit, Ml) was used at a

finalfinal concentration of 100 ng/ml. Superantigen Staphylococcus aureus enterotoxin B

(SEB)) (Sigma, St. Louis, MA) was used at a final concentration of 1 ng/ml.

Thee GC CP (Sigma) and HC (Sigma) were kept as stock solutions of 10"2 M in

95%% ethanol, stored at , and diluted into complete medium just before use. A

1:1044 final dilution of 95% ethanol was included as a vehicle control in all

experiments. .

Inn vitr o generatio n of dendriti c cell s fro m periphera l bloo d monocyte s

Venouss blood from healthy donors was collected by venipuncture in sodium-heparin

containingg tubes (VT100H; Venoject, Terumo Europe, Leuven, Belgium). PBMC were

isolatedd by density centrifugation with Lymphoprep (Nycomed, Torshov, Norway).

Subsequently,, PBMC were centrifuged on a Percoll (Pharmacia, Uppsala, Sweden)

gradientt consisting of three density layers (1.076, 1.059 and 1.045 g/ml). The light

densityy fraction floating on the middle layer, which contained predominantly

monocytes,, was seeded in 24-well culture plates (Costar, Cambridge, MA) at a

densityy of 5x105 cells/well. After 30 min of incubation at , nonadherent cells were

removed;; adherent cells were cultured in Iscove's modified Dulbecco's medium (Life

Technologies,, Paisley, U.K.) containing gentamycin (86 ng/ml; Duchefa, Haarlem,

Thee Netherlands) and 1% FCS (Hyclone, Logan, UT) and supplemented with

GM-CSFF (500 U/ml) and lL-4 (250 U/ml) to obtain DC as described previously (26).

Att day 3, the media, including the supplements, were refreshed. At day 6,

CD1a+CD14"" DC were obtained.

Isolatio nn of CD4+ Th cell s

Highlyy purified CD4+ Th cells (>98% as assessed by flow cytometry, data not shown)

weree obtained by incubating the PBL, which had been harvested from the lower

interfacee of the Percoll gradient, with anti-CD4 coated Dynabeads (Dyna! AS, Oslo,

Norway),, followed by treatment with Detachabeads {Dynal AS) according to the

manufacturer'ss instructions.

102 2

Glucocorticoids inhibit dendritic cell-derived IL-12p70

Stimulatio nn of DC in the presenc e or absenc e of GC

CD1a++ DC (4x104 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

volumee of 200 pJ. The following stimuli were used: LPS {final concentration 100

ng/ml)) and SAC (final concentration 75 ug/ml). Both modes of stimulation were

performedd in the absence or presence of IFN-7 (103 U/ml). The GC used in this study

weree CP and HC. Pilot experiments demonstrated that 10"8 M CP strongly reduced

IL-12p700 production by stimulated DC, whereas a 100- to 1000-fold higher

concentrationn of HC was needed to observe a similar reduction. Therefore, the

concentrationss of CP used for future experiments were between 10'11 and 10"8 M

whereass HC was used in a concentration range of lO^-IO"6 M. Supernatants were

harvestedd after 24 h and stored at C until the levels of IL-12p70, IL-10, IL-6, and

TNF-aa were measured by ELISA.

GCC pretreatmen t of DC followe d by stimulatio n in the absenc e of GC

CD1a++ DC were cultured for 2 days in GM-CSF (500 U/ml) in the presence of CP

(10"1 M or 10*8 M), HC (10"6 M or 10"6 M), or vehicle. To study the stability of the

observedd cytokine production profiles, DC were cultured for another 2 days in

GM-CSFF (500 U/ml) alone after removal of the GC. To stimulate the DC, the cells

weree harvested, washed thoroughly, counted and the viability was determined. A

differencee in viability between GC-treated and untreated DC was not observed in any

off the experiments. DC (4x104/200pl) were stimulated with LPS (final concentration

1000 ng/ml) in the absence of GC. Supernatants were harvested after 24 h and stored

att C until the levels of IL-12p70, IL-10, IL-6, and TNF-a were measured by

ELISA. .

Agg uptak e assay

CD1a++ DC were incubated at C for 30 min in the presence of culture medium

containingg either 0.2 ng/ml of albumin-FITC conjugate (BSA-FITC, 1:12 molar ratio;

Sigma)) or 0.2 ng/ml of a-D-mannosylated-albumin-FITC conjugate (manBSA-FITC,

1:2.55 molar ratio; Sigma). Negative controls were incubated with the respective Ag at

CC for 30 min. Ag uptake was stopped by extensive cold washing, and cell surface

103 3

Chapterr 7

fluorescencee was quenched with trypan biue (Sigma). Ag uptake was evaluated by

floww cytometry.

Inductio nn of proliferativ e respons e in CD4+ Th cell s

CD4++ Th cells (5x104 cells/200uj) 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

byy 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.

Inductio nn of cytokin e productio n in CD4+ Th cell s

CD4++ Th cells (5x104 cells/200uJ) were cocultured with 3x104 vehicle- or GC-treated

DCC 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-Y, IL-4, IL-5,

andd IL-10 were measured by ELISA.

Cytokin ee measurement s

Measurementss of IFN-7, IL-4, IL-5, IL-10, and IL-12p70 levels in culture supernatants

w e r ee performed by a specific solid-phase sandwich ELISA as described previously

(27).. Measurements of IL-6 and TNF-a were performed by ELISA using pairs of

specificc mAbs and recombinant cytokine standards obtained from BioSource

Internationall (Camarillo, CA). The limits of detection of these ELISA are as follows:

IFN-Y,, 100 pg/ml; IL-4, 60 pg/ml; IL-5, 50 pg/ml; IL-6, 20 pg/ml; IL-10, 25 pg/mt;

IL-12p70,, 3 pg/ml; and TNF-a, 20 pg/ml.

Analysi ss of expressio n of cel l surfac e molecule s by flo w cytometr y

Thee 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.

LeBten,, University of Minnesota, Minneapolis, MN), CD80 (B7-24 (28); kindly

providedd by Innogenetics N.V., Ghent, Belgium), HLA-DR (L243; Becton Dickinson,

Sann José, CA), and MR (15-2 (16); a gift of Dr. F. Noorman, Gaubius Laboratory,

104 4

Glucocorticoidss inhibit dendritic cell-derived IL-12p70

Netherlandss Central Organization for Applied Scientific Research for Prevention and

Health,, Leiden, The Netherlands), followed by FITC-conjugated goat anti-mouse

mAbb (Jackson Immunoresearch Laboratories, West Grove, PA).

Statisticall analysis

Dataa were analyzed for statistical significance (InStat, Version 2.02, GraphPad, San

Diego,, CA) using ANOVA followed by Dunnett's multiple comparisons test. The

stabilityy of the effect of GC on cytokine production by GC-pretreated DC at 2 days

afterr GC withdrawal (see Fig. 2) was evaluated using the Student's Mest. A P value

off <0.05 was considered as the level of significance.

RESULTS S

Direc tt effec t of GC on cytokin e secretio n by DC

Sincee DC are the professional effective APC and their secretion of immunoregulatory

andd proinflammatory cytokines plays a crucial role during T cell priming (17, 18), we

investigatedd whether cytokine production by in vitro generated DC is affected by GC.

Too address this issue, the effect of increasing concentrations of CP and HC on IL-12,

IL-6,, and TNF-a production was compared. As is shown in Fig. 1, the stimulation of

DCC with LPS (100 ng/ml) in the presence of increasing concentrations of GC resulted

inn a dose-dependent inhibition of IL-12p70, IL-6, and TNF-a production.

a a

.125 5 I I

hoo o

500 - - B — 50 ....

255 — *** 25

1255 Figur e 1. Inhibitor y effec t of BB GC on LPS-induce d cytokin e

1000 productio n by DC. DC (4x104

. j » cells/200ul) were stimulated JJ 75 " 75 . with 100 ng/ml LPS combined

withh the indicated concentra-tionss of CP (A) or HC (B). Re-sultss are expressed as the meann percentage cytokine

oo - o (IL-12p70, ; IL-6, O; or vehiclee 10-11 10-' itr" 10*» vehicle ia08 10" 10* TNF-a, ) concentration of clobetasol-17-propionate(M)) hydrocortisone (M) triplicate cultures in the 24 h

supernatantss from one rep-resentativee experiment of three. Cultures stimulated with LPS in the presence of GC are compared withh respective cultures stimulated with LPS in the presence of vehicle (100%) SD. At 100%, IL-12p700 production (in the presence of IFN-y) was 1.8 0.4 ng/ml, IL-6 was 1.3 0.03 ng/ml, and TNF-aa was 3.5 0.03 ng/ml. Data were analyzed for statistical significance using ANOVA followed by Dun-nett'ss multiple comparisons test. 'PO.05, **P<0.01, *"P<0.001.

105 5

Chapterr 7

Evenn at the low concentration of 10"11 M, CP {Fig. 1 A) inhibited the secretion of the

IL-12p700 heterodimer by - 50% (PO.01); in contrast, no significant reduction in

eitherr !L-6 or TNF-a production was observed at this dose. High concentrations of

CPP (10"8 M) almost completely abrogated the production of IL-12p70 (P<0.001) but

affectedd IL-6 and TNF-a to a lesser extent. Also HC was found to affect DC-derived

cytokiness {Fig. 1B), but 100- to 1000-fold higher concentrations were needed for an

effectt that was comparable with that of CP.

Ann identical downregulation of cytokine production was observed when DC were

stimulatedd with SAC {75 ng/ml) (data not shown). At all conditions, IL-10 production

wass not detectable.

Effec tt of GC pretreatmen t on cytokin e secretio n by DC

Wee questioned whether the inhibition of IL-12p70 production by GC was a direct

effectt 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

stimulatedd with LPS (100 ng/ml) in the absence of GC. As is shown in Fig. 2 {filled

bars),, GC pretreatment also inhibited the production of DC-derived cytokines upon

subsequentt stimulation. The production of IL-12p70 (Fig. 2A, filled bars) was strongly

inhibitedd (PO.001) by preincubation with either 10"10 M or 10'8 M CP. Again, HC

appearedd to be much less effective and induced a similar IL-12p70 inhibition only at

10"66 M (P<0.001); 10s M HC did not produce any statistically significant effect. The

productionn of IL-6 (Fig. 2B, filled bars) and TNF-a (Fig. 2C, filled bars) was inhibited

too a lesser extent compared with lL-12p70. IL-10 production was not detectable in

thesee culture supernatants, which is consistent with the poor ability of DC to produce

thiss cytokine (27).

Too assess the stability of the downregulation of cytokine production, GC-

pretreatedd DC were washed extensively and kept in culture in the absence of GC for

ann additional 2 days before stimulation. As is shown in Fig. 2 (open bars), IL-12p70

andd IL-6 were inhibited to the same extent compared with experiments in which they

weree determined directly after GC pretreatment (filled bars). However, the additional

22 days of culture in the absence of GC partially restored the ability of DC pretreated

withh HC and tow dose CP to produce TNF-a (PO.001 and P<0.01, respectively).

106 6

Glucocorticoidss inhibit dendritic cell-derived IL-12p70

vehicle e

C P 1 0 , 0 M M

CPP 100BM

8M M

HCC 6M

IBB ***

*~ bb ***

H H

3*M M

00 25 50 75 100 125 0 25 50 75 100 125

%% of IL-12p70 productio n % of IL-6 productio n

00 25 50 75 100 125

%% of TNF-a productio n

Figur ee 2. Inhibitor y effec t of GC treatmen t on cytokin e productio n by DC is transien t for TNF-a DCC were treated for 2 days with the indicated concentrations of GC. After the treatment period, DC weree washed and either stimulated directly in the absence of GC with 100 ng/ml LPS (filled bars) or keptt in culture for an additional 2 days before similar stimulation (open bars). Results are expressed ass the mean percentage IL-12p70 (A), IL-6 (B) and TNF-a (C) concentration of triplicate cultures in the 244 h supernatants from one representative experiment of three. Cytokine production by GC-treated DCC is compared with the respective cytokine production by vehicle-treated control DC (100%) SD. At 100%,, IL-12p70 production (in the presence of IFN-y) was 2.0 0.2 ng/ml, IL-6 was 3.4 0.3 ng/ml, andd TNF-a was 18.7 1.3 ng/ml. Data were analyzed for statistical significance using ANOVA followedd by Dunnett's multiple comparisons test. The stability of the effect of GC on cytokine productionn by GC-treated DC at 2 days after withdrawal was evaluated using the Student's t-test. *P<0.05,, **P<0.01, ***P<0.001.

CD1aa CD80 CD40 HLA-DR MR Figur ee 3. Surfac e pheno -typ ee of GC-treate d DC. DC weree treated for 2 days with GCC and analyzed for the expressionn of cell surface moleculess by flow cytometry. AA comparison of expression intensityy by GC-treated DC withh vehicle-treated DC is shown.. Data are representa-tivee of seven independent experiments. .

vehicl e e

CPP 10-08 M

HC10 0 6M M

W WW 'WÏWib- cTSWTr

Fluorescenc ee Intensit y

GCC pretreatmen t doe s no t affec t th e expressio n of surfac e molecule s on DC

Wee studied whether GC pretreatment of DC affects their cell surface expression of

thee DC-associated marker CD1a, the class II MHC Ag-presenting molecule HLA-DR,

thee costimulatory molecules CD40 and the B7 molecule CD80, and MR. Although it

hass been reported previously for monocytes and macrophages that GC interfere with

107 7

Chapterr 7

thee cell surface expression of molecules involved in Ag presentation and

costimulationn (10, 15), GC pretreatment of DC for 2 days did not affect the

expressionn of surface molecules when compared with vehicle-treated control cells

(Fig.. 3).

GCC pretreatmen t doe s not affec t th e abilit y of DC to tak e up Ag

Usingg BSA-FITC and manBSA-FITC in a functional assay, we subsequently

assessedd the effects of GC on the ability of DC to take up Ag via fluid phase

endocytosiss or via their specific MR. Regardless of the pretreatment conditions,

neitherr CP nor HC affected the uptake of either BSA-FITC (Fig. 4, left panels) or

manBSA-FITCC (Fig. 4, right panels). These results clearly show that the endocytic

capacityy of DC was not affected by preculture with GC. Although MR expression has

beenn reported to be upregulated by GC (16), no differences on MR-mediated uptake

weree induced; therefore, this observation is in line with the unchanged cell surface

expressionn of MR by GC-treated DC in comparison with control DC (Fig. 3).

BSA-FITCC manBSA-FIT C

ii 'ik-

>> '1

0) )

"It. .

- ^ \ \

A A

vehicl e e

CPP 10-10 M

CPP 1008 M

HCC 10-08M

HC10 0 6 M M

Figur ee 4. Intracellula r accumulatio n off FITC-conjugate d Ag by GC-treate dd DC. DC were treated for 2 dayss with the indicated concentrations off GC and subsequently pulsed for 30 minn in medium containing either 0.2 ug/mll BSA-FITC (left panels) or 0.2 ng/mll manBSA-FITC (right panels). Floww cytometry assessment of Ag-uptakee at C (open profiles) is com-paredd with negative uptake controls at

CC (filled profiles). Data are repre-sentativee of five independent experi-ments. .

Fluorescenc ee Intensit y

GCC pretreatmen t doe s no t affec t th e abilit y of DC to stimulat e proliferatio n of

CD4++ Th cell s

Next,, we studied the effects of GC on the proliferation-inducing capacity of DC

towardd Th cells. To exclude the direct effects of GC on T cells, DC were pretreated

108 8

Glucocorticoidss inhibit dendritic cell-derived IL-12p70

withh GC before coculture with highly purified CD4* Th cells in the presence of

superantigenn (SEB; 1 ng/ml). In the absence of either DC or SEB, no activation of T

cellss could be observed. GC-pretreated DC were as effective as vehicle-pretreated

DCC in inducing T cell proliferation (Fig. 5). These results indicate that the exposure of

DCC to GC does not affect their ability to stimulate T cell proliferation in the presence

off superantigen. These results are in line with the unaffected expression of HLA-DR

andd costimulatory molecules described above (Fig. 3).

Figur ee 5. GC pretreatmen t of DC does not im-pai rr thei r abilit y to stimulat e proliferatio n of CD4** Th cells . DC were treated for 2 days with GCC (vehicle, ; 10"8 M CP, O; or 10"6 M HC, . Afterr the treatment period, GC were washed away andd different concentrations of DC were used to stimulatee 5x104 CD4* Th cells in the presence of 1 ng/mll SEB. The proliferative response was deter-minedd at day 5 of coculture by [3H]-TdR incorpo-ration.. Data are presented as mean cpm SD of triplicatee cultures. Results are representative of six independentt experiments.

GCC pretreate d DC do no t promot e th e secretio n of th e typ e 1 cytokin e IFN-y by

CD4++ Th cell s bu t do stimulat e th e secretion of IL-10 and IL-5

Sincee IL-12 is a potent inducer of IFN-y secretion in activated CD4+ Th cells (20, 21),

wee investigated whether GC pretreatment of DC influenced the cytokine profile of

SEB-triggeredd CD4+ Th cells. As is shown in Fig. 6A, pretreatment with GC reduced

(PO.001)) the ability of DC to stimulate the production of the type 1 cytokine IFN-y. In

contrast,, GC-pretreated DC upregulated T cell production of the antiinflammatory

mediatorr IL-10 (Fig. 6B) and the type 2 cytokine IL-5 (Fig 6C). Interestingly, the

additionn of rlL-12 to these cocultures abolished the reduction of IFN-y and the

upregulationn of IL-5 production by the T cells but not the upregulation of IL-10. In

bothh the absence and the presence of exogenous IL-12, IL-4 production was not

detectablee in the culture supernatants. These results indicate that the use of GC-

pretreatedd DC as APC results in a T cell cytokine shift toward a type 2 profile, which

largelyy due to the reduced production of IL-12.

0.011 0.1 1 10 100

DCC concentration (cells/well x10"3)

109 9

Chapterr 7

++ IL-12 - - - +IL-12

Figur ee 6. GC-pretreate d DC do not promot e IFN-y secretio n but stimulat e IL-10 and IL-5 secretio nn by CD4+ Th cells . DC were treated for 2 days either with vehicle (filled bars), 10'8 M CP (hatchedd bars), or 10'6 M HC (open bars). After the treatment period, GC were washed away; 3x104

DCC were used to stimulate 5x10 CD4+ Th cells in the presence of 1 ng/ml SEB, either in the absence orr presence of rlL-12 (100 U/ml). Cytokine production was determined by ELISA in day-4 supematants.. Data are presented as the mean percentage of cytokine production SD of triplicate cultures.. At 100%, IFN-y was 22.2 0.04 ng/ml, IL-10 was 1.4 0.3 ng/ml, and IL-5 was 0.4 0.05 ng/ml.. Results are representative of three independent experiments. Data were analyzed for statistical significancee using ANOVA followed by Dunnett's multiple comparisons test. *P<0.05, " P O . 0 1 , "*P<0.001. .

DISCUSSION N

Thee present study shows that GC are potent inhibitors of production of the bioactive

IL-12p700 heterodimer and, to a lesser extent, of the proinflammatory cytokines IL-6

andd TNF-a. In contrast, GC did not affect the level of expression of costimulatory

moleculess by DC or the ability to take up Ag and stimulate the proliferation of CD4+

Thh cells in vitro. Largely due to an inhibition of IL-12p70 production, GC

downregulatee the capacity of DC to induce the secretion of the type 1 cytokine IFN-y

inn CD4+ Th cells while upregulating their capacity to promote the secretion of the

antiinflammatoryy cytokine IL-10 and the type 2 cytokine IL-5.

GCC inhibit cytokine gene transcription through the activation of their specific

cytosolicc receptors (29). The actual downregulation of transcription of sensitive genes

byy 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

activatorr protein-1 (30). Furthermore, the inhibitory effect of GC on N F - K B was shown

too be mediated either via the enhancement of IKBOC transcription and synthesis (31,

32),, which is known to bind to N F - K B and prevent its migration to the nucleus, or via

aa direct association with Rel A, the 65-kD N F - K B binding protein (33).

§§ 5 0 0 -

11 400 D D ££ 300 a a JJ 200 LL L

ZZ ioo

++ IL-12

110 0

Glucocorticoidss inhibit dendritic cell-derived IL-12p70

Thee direct inhibitory effect of GC on T cell-derived cytokines is well documented,

butt T cells are not the only cell type in which cytokine production is modulated by

GC.. Several studies reported the downregulatory effect of GC on the secretion of the

proinflammatoryy cytokines ll_-1(3 (7), IL-6 (7, 8) and TNF-a (12) by monocytes and

macrophages.. Recently, Blotta et al. (10) demonstrated the inhibitory effect of GC on

humann monocyte-derived IL-12p40 with a concomitant upregulation of IL-10. And

veryy recently, the GC-induced inhibition of IL-12p70 production by human monocytes

wass also shown (34). However, the effect of GC on human DC-derived bioactive

IL-12p700 was not known.

Here,, we report on the strong inhibition of IL-12p70 secretion by human DC in

responsee to GC. The positive regulation of the IL-12p40 promotor by NF-KB was

reportedd previously (35), notwithstanding that the regulation of the IL-12p35 promoter

remainss elusive. Hence, it can be speculated that, in addition to the probable effect of

GCC on the transcription of the IL-12p40 monomer, which is mediated indirectly via the

downregulationn of NF-KB, GC may also affect the transcription of IL-12p35 or

interferee 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

expressionn of the p35 gene (23), knowledge with regard to the regulation of IL-12p35

transcriptionn will help to clarify the direct effects of GC on the secretion of IL-12p70.

Ass GC stimulate IL-10 production in human monocytes (10), which is known to

reducee IL-12 synthesis (23), it was suggested that these GC indirectly suppress IL-12

productionn via the upregulation of IL-10. Although this inhibitory pathway may be

relevantt for monocytes, in vitro-generated human DC do not produce detectable

levelss of IL-10, implying that the inhibitory effect of GC on cytokine production by DC

cannott be explained by the indirect effect of upregulation of IL-10.

Bothh IL-6 and TNF-a are important mediators of a wide range of biological

activitiess that play a critical role in the induction of proinflammatory and immune

responses.. GC inhibited IL-6 as well as TNF-a secretion by DC. The NF-KB binding

sitee within the IL-6 promoter was suggested to be essential for the induction of IL-6

transcriptionn (36). Likewise, NF-KB is involved in the positive regulation of TNF-a

genee transcription (37). Ray and Prefontaine (38) showed that the activation of the

IL-66 promoter by a combination of NF-lL-6 and Rel A was inhibited by

dexamethasone-activatedd GC receptors. Since NF-KB plays a critical role in

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Chapterr 7

inflammatoryy reactions, the possible inhibition of cytokine gene transcription as a

resultt of the antagonism between activated GC receptors and NF-KB constitutes a

likelyy mechanism underlying the GC-mediated antiinflammatory and immunosuppres-

sivee mode of action.

Inn contrast to the clear inhibitory effect of GC on DC cytokine production, our study

indicatedd that Ag uptake, either via endocytosis or MR-mediated, was not affected by

exposuree to GC. Moreover, our data show that the expression of the class II MHC

moleculee HLA-DR and the costimulatory molecules CD40 and CD80 remained

unaffectedd after the preincubation of DC with GC. Indeed, pretreatment of in vitro-

generatedd DC with GC does not downregulate their stimulatory potential, as

suggestedd by the observation that GC pretreatment did not affect the capacity of DC

too stimulate CD4+ Th cell proliferation. Therefore, our results are in contrast with

recentt reports on the reduced ability of GC-treated human monocytes (10) or purified

murinee splenic DC (24) to stimulate the proliferation of T lymphocytes. In these two

studiess it was described that GC inhibit the IFN-y- (10) or 24-h-culture- (24) induced

upregulationn of the cell surface expression of molecules involved in either Ag

presentationn or costimulation. However, the upregulation of class II MHC and

costimulatoryy molecules as well as an increased T cell stimulatory potential are

featuress of maturation of DC. The possibility cannot be excluded that partial

maturationn was induced by the cuituring conditions in both studies mentioned.

Inn line with the fact that IL-12 induces Th1 responses via a strong induction of

IFN-yy secretion (20, 21), we found that GC-treated DC with a reduced capacity to

producee bioactive IL-12p70 induced less IFN-y 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

onn the production of IL-12p40 by the APC was representative for the effect on the

bioactivee heterodimer; however, the regulation of bioactive IL-12 production is

determinedd mainly by the transcription of the p35 subunit (23). Here, we report that

thee downregulation of bioactive IL-12p70 production by DC in response to GC is

indeedd responsible for the reduction of IFN-y secretion by CD4+ T cells, and that this

reductionn can be restored by the addition of exogenous bioactive IL-12 to the cultures

duringg stimulation. Additionally, secretion of the antiinflammatory cytokine IL-10 and

112 2

Glucocorticoidss inhibit dendritic cell-derived !L-12p70

thee type 2 helper cytokine IL-5 was promoted when T cells were stimulated with GC-

pretreatedd 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 downregulation of IL-5

secretionn (27, 39) by T cells. The observed upregutation of IL-5 secretion by the T

cellss in response to stimulation with GC-treated DC was abolished by rlL-12,

indicatingg that the lack of secretion of bioactive IL-12 was responsible for the

upregulationn of IL-5. Although IL-12 has been also reported to induce T cell derived

IL-100 (40), the production of this antiinflammatory mediator by CD4+ Th cells was not

sensitivee to rlL-12 in our cultures. However, Jeannin et al. (40) made their

observationn with established T ceil lines in an APC-free system. Consequently, this

suggestss a rather distinct regulatory pathway of IL-10 production by IL-12 compared

withh the activation of resting T cells in an APC-dependent way as we show here.

Byy virtue of defective IL-12 production and the subsequent effects on cytokine

productionn by Th cells, GC-pretreated DC should be regarded as antiinflammatory

playerss in the Ag-specific immune system. Although GC are potent direct inhibitors of

specificc Th cell responses, additional modulation of the immune response may be

expectedd through the effects of GC on DC, rendering them less supportive for

inflammatoryy responses. In this way, the recurrent activation of T cells by GC-treated

APCC 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

pretreatmentt may induce increased IgE production {41, 42) and eosinophil numbers

(43).. Indeed, GC were shown to enhance IgE synthesis in both allergic and

nonallelicc individuals (44, 45). Despite this effect, therapy with GC appears to be

usefull in the blocking of allergen-specific reactions. This effect could probably be

explainedd by the dominant antiinflammatory effect mediated not only via the direct

inhibitionn of inflammatory T cell cytokines but also via the inhibition of IL-12

productionn in professional APC.

Acknowledgment s s Wee thank J. Wormmeester for logistic help. This work was financially supported by Fundacaoo para a Ciência e a Tecnologia, Lisbon, Portugal (grant PRAXIS XXI/BD/9195/966 to P.L.V.) and Yamanouchi Europe BV, Leiderdorp, The Netherlandss (E.C.d.J.). E.A.W. received an independent fellowship from the Royal Netherlandss Academy of Arts and Sciences.

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