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:233–239

© 2003 Blackwell Publishing Ltd

233

Blackwell Science, Ltd

Oxford, UK

CEIClinical and Experimental Immunology

0009-9104Blackwell Publishing Ltd, 2003

133233239Original Article

Th1/Th2 lymphocytes and dendritic cells in uveitisM. A. Frassanito et al.

Correspondence: Dr Maria Antonia Frassanito, DIMO-Section ofInternal Medicine and Clinical Oncology, P.za G. Cesare, 11 70124-Bari,Italy.

E-mail: mfrassanito@hotmail.com

Combined cyclosporin-A/prednisone therapy of patients with active uveitis suppresses IFN-

gggg

production and the function of dendritic cells

M. A. FRASSANITO†, R. DAMMACCO*, T. FUSARO†, A. CUSMAI†, S. GUERRIERO* & C. SBORGIA* *

Department of Ophthalmology and Otorhinolaryngology, Section of Ophthalmology and

†

Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy

(Accepted for publication 30 May 2003)

SUMMARY

In this study, we assessed the Th1/Th2 polarization of the immune response and the involvement of den-dritic cells (DC) and Th1 lymphocytes in the pathogenesis of uveitis. Thirty-seven patients with chronicidiopathic uveitis were enrolled: 21 of them had active uveitis and the remaining 16 were in completeremission. Patients with active uveitis were characterized as follows: 5 had intermediate uveitis, 5 panu-veitis and the remaining 11 posterior uveitis. Thirteen healthy subjects were also studied as controls.Patients with active uveitis were treated with cyclosporin-A (CsA) associated to low doses of pred-nisone (PDS) and studied at baseline and after 6 months of therapy. Analysis of cytokine-producingCD3

+

lymphocytes revealed a strong Th1 polarization of the immune response in patients with activeuveitis. Th1 lymphocytes paralleled serum IL-12 levels and the response to therapy, which greatlyreduced both IFN-

g

+

/CD3

+

lymphocytes and serum IL-12 levels, associated with a general clinicalimprovement.

In vitro

studies demonstrated that DC from untreated patients with active uveitis weremature and functionally active. In fact, they showed a higher ability to stimulate cell proliferation ofallogeneic T cells in primary mixed lymphocyte reaction (MLR) and produced larger amounts of IL-12than DC from CsA/PDS-treated patients and those in remission. These results demonstrate that CsA/PDS therapy impairs the capacity of mature DC to secrete IL-12 and inhibits their MLR activity.

Keywords

cyclosporin-A/prednisone therapy dendritic cells IL-12Th1/ Th2 lymphocytes uveitis

INTRODUCTION

Activation by cognate ligand differentiates naïve T cells into twosubsets with different cytokine production and effector functions[1,2]: Th1 lymphocytes secrete interleukin (IL)-2, interferon(IFN)-

g

, and tumour necrosis factor (TNF)-

b

, activate macroph-ages and promote cell-mediated immunity; Th2 cells produce IL-4, IL-5, IL-6, IL-13 and are involved in antibody-mediatedimmunity. The Th1/Th2 balance depends on several environmen-tal and genetic factors, and plays an important regulatory role inthe immune system. Selective defects or imbalances of the Th1/Th2 ratio are responsible for immunological disorders. Th1polarization of the immune response is involved in severalorgan-specific autoimmune diseases, whereas Th2-type responses

are mostly detected in systemic autoimmune diseases with astrong humoral component [3]. Moreover, Th1/Th2 cytokines actas autocrine and paracrine growth factors, in that they cross-regulate the development and function of each subset. Inparticular, IL-12, an immunoregulatory cytokine produced byantigen-presenting cells, promotes Th1 cell differentiation andproliferation and inhibits IL-10 and IL-4 cell release. By con-trast, IL-4 and IL-6 induce a Th2 immune response and preventTh1 cell differentiation [1,2,4].

Accumulating evidence suggests that dendritic cells (DC), themost potent antigen-presenting cells, regulate the immuneresponse, in that they stimulate quiescent, naïve and memory Band T cells, enhance tolerance by deletion of self-reactive thy-mocytes and induce anergy of mature T cells [5,6].

Uveitis is a clinically heterogeneous group of diseases inwhich intraocular inflammation often leads to decreased vision,retinal destruction and blindness [7,8]. Clinical and experimentalevidence suggests that autoimmune processes induce such inflam-mation [8]. Th1 lymphocytes appear to play a predominant role inboth human and animal models [9–14]. Genetic susceptibility to

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experimental autoimmune uveitis (EAU) in mice immunized witha uveitogenic retinal antigen, namely interphotoreceptorretinoid-binding protein (IRBP), is associated with a strong Th1immune response. T-cell deficient animals do not develop uveitis,whereas it is induced when Th1 cells from immunized mice aretransferred into T cell-deficient animals. Involvement of Th1 lym-phocytes in human uveitis has been postulated [9,10,13,14]. Areverse transcriptase-polymerase chain reaction (RT-PCR) studyof the aqueous humor has shown that Th1-type cytokines occurwithin the lesion in all forms of chronic uveitis [13].

Cyclosporin-A (CsA) is a potent inhibitor of calcium-dependent T-cell activation [15], that binds to immunophilins andsuppresses activation of the Nuclear Factor of Activated T Cells(NFAT). This factor plays a key role in the transcriptional activa-tion of cytokine genes [16]. CsA is widely used in the treatment oftransplant-recipient patients and in several T-cell mediatedautoimmune diseases [17,18], as well as in uveitis [19–21].

In this study we demonstrate that a strong Th1 polarization ofthe immune response is associated with clinically active uveitis.The percentage values of CD3

+

/IFN-

g

+

lymphocytes parallelserum IL-12 levels and the response to combined CsA/prednisone(PDS) therapy. The effect of this therapy on DC functions wasalso evaluated.

PATIENTS AND METHODS

Patients

We studied 37 patients with chronic idiopathic uveitis, defined asintraocular inflammation of unknown aetiology, lasting greaterthan 3 months. Thirteen patients were male and 24 female, andtheir mean age was 42·2 years. Twenty-one patients had activeuveitis, whereas the remaining 16 were in complete remission.Based on the recommendations of the International Uveitis StudyGroup classification system [22], patients with active uveitis weredivided as follows: 5 (23·8%) had intermediate uveitis, 5 (23·8%)panuveitis and 11 (52·4%) noninfective posterior uveitis. Thesearch for the HLA-B27 antigen did not reveal a positive associ-ation in any of them. Before giving their informed consent, allpatients and controls were fully informed of the nature of thestudy, which was approved by the Ethics Committee of the Uni-versity of Bari.

The ophthalmological diagnosis was based on a detailed clin-ical history and ocular exploration. In particular, each patient wassubmitted to a thorough ocular examination, including best-corrected visual acuity, slit-lamp with high power biomicroscopyof posterior segment, tonometry and ophthalmoscopy. Fluores-cein angiography (FA) and optical coherence tomography (OCT)were also performed in 15 cases.

Patients with active uveitis and normal renal and liver func-tions were treated with CsA. Indications for starting CsA therapyincluded one or more of the following: (a) steroid dependency;(b) toxicity from previous steroid/immunosuppressive therapy;(c) failure or inadequate response to steroids associated withother immunosuppressive drugs. All patients were also receivingdaily dosages of PDS ranging from 2·5 to 10 mg. The starting dos-age of CsA was 3–4 mg/kg/day per os (usually 200–250 mg daily),and was adjusted until a therapeutic response was achieved. Fur-thermore, topical steroid therapy was irregularly given to mostpatients. Patients with active disease were studied at baseline andagain after six months of combined CsA/PDS therapy. Patients incomplete remission had no clinical signs and were receiving no

therapy for at least 4 months. Thirteen healthy volunteers werestudied as age- and sex-matched normal controls.

Lymphocyte preparation

Peripheral blood mononuclear cells (PBMC) were isolated fromheparinized blood by Ficoll-Hypaque gradient centrifugation.After incubation for 2 h at 37

∞

C, nonadherent cells (PBL) werecollected and resuspended at 1

¥

10

6

/ml in RPMI 1640 (Biochrom,Berlin, Germany) containing 10% fetal calf serum, 2 m

M

glutamine, 100 U/ml penicillin, and 100

m

g/ml streptomycin (allfrom Biochrom).

Flow cytometry determination of intracellular cytokines

Cytokine production was analysed by intracellular immunofluo-rescence staining, as described [9]. IFN-

g

and IL-4 production wasinvestigated as type-1 and type-2 cytokines, respectively. Briefly,PBL were stimulated with 10 ng/ml phorbol myristate acetate(PMA; Sigma, St. Louis, MO, USA) and 1

m

M

ionomycin (Sigma)in round-bottomed tissue culture tubes. After 2 h at 37

∞

C, 3

m

M

monensin (Sigma) was added to prevent cytokine release andcells were incubated for an additional 3 h, washed, fixed with 4%formaldehyde for 15 min and permeabilized with phosphate buff-ered saline containing 0·5% bovine serum albumin (Sigma) and0·5% saponin (Sigma). Cultures without the addition of mon-ensine and exogenous stimuli were included as controls. Finally,phycoerythrin (PE)-conjugated anti-IL-4 or anti-IFN-

g

mono-clonal antibody (mAb) (Biosource, Fleurus, Belgium) was added.The surface phenotype of cytokine-producing cells was identifiedby incubating cell samples with fluorescein isothiocyanate(FITC)-conjugated mAb to CD3. The negative controls wereisotype-matched irrelevant antibodies. Samples were analysed byFACScan (Becton Dickinson, Mountain View, CA, USA).

Generation of mature and immature DC from peripheral adherent cells

Monocyte-derived DC were generated from the adherent fractionof PBMC [23]. Briefly, adherent cells (5

¥

10

6

/ml) were cultured infresh medium containing 400 U/ml IL-4 (Peprotech, London,UK) and 800 U/ml GM-CSF (Peprotech). On day 6, the resultingimmature DC were harvested and analysed for surface markerexpression by flow cytometry. To induce DC maturation, the cells(5

¥

10

5

/ml) were stimulated with 1 ng/ml lipopolysaccharide(LPS) (Sigma) for the final 24 h. Supernatant fluids of unstimu-lated and LPS-stimulated DC cultures were collected and testedfor cytokine release.

Phenotypic analysis of DC

Expression of specific antigens by single or double fluorescencestaining was determined by flow cytometry. FITC- or PE-conjugated mAb to CD1a (Immunotech, Marseille, France),CD14 (Sigma), CD80 (Immunotech), CD86 (Immunotech) andHLA-DR (Sigma) were used. Negative controls were set up withisotype-matched irrelevant antibodies. Samples were analysed byFACScan (Becton Dickinson). The level of antigen expression persample was also evaluated as a ratio of the mean fluorescenceintensity per sample and their mean fluorescence at the cut-offpoint (MFI).

Allogeneic mixed lymphocyte reaction (MLR)

To test their allogeneic stimulator activity, harvested DC weretreated with 0·08 mg/ml mitomycin C (Sigma) to inhibit cell

Th1/Th2 lymphocytes and dendritic cells in uveitis

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proliferation and used as stimulators in primary MLR. Then, 10

5

allogeneic PBL from a healthy subject, used as responder cells,were mixed with increasing numbers (10

2

-

10

4

cells/well) of DC inflat-bottomed 96-well culture plates for 5 days at 37

∞

C. Cells werepulsed with

3

H-thymidine (1

m

Ci/well) for 18 h before harvesting.

3

H-thymidine uptake was evaluated in a

g

-counter (BeckmanInstruments, Palo Alto, CA, USA). Negative controls, repre-sented by PBL without cultured DC, gave a base count of1300

±

600 cpm. The ability of DC to stimulate cell proliferationwas expressed as stimulation index: cpm responder cells

+

stimu-lator cells/cpm responder cells.

Cytokines measurement

IL-10 and IL-12p70 levels were measured in serum and cell cul-ture supernatant fluids using an enzyme amplified-sensitivityimmunoassay (Biosource), according to the procedure suggestedby the manufacturer. Briefly, samples were added to appropriatewells of a microtitre plate coated with a blend of mAb against dis-tinct epitopes of cytokines to ensure a highly sensitive assay. Afterincubation, appropriate horseradish peroxidase (HRP)-labelledmAbs, namely anti-IL-10 and anti-IL-12p70, were added. Boundenzyme-labelled antibodies were detected through a chromoge-nic reaction by the addition of tetramethylbenzidine and H

2

O

2

inacetate citrate buffer. Plates were read at 450 nm in a microplate

reader (Bio-Rad, Hercules CA, USA). A standard curve was usedto quantify cytokine levels. The detection limits were 0·5 pg/mland 1 pg/ml for IL-12p70 and IL-10, respectively.

Statistical analysis

Results are expressed as the mean

±

the standard deviation (SD).Statistical analysis was performed by means of the nonparametricpaired Wilcoxon rank-sum test, with a value of

P

<

0·05 as the sig-nificance cut-off.

RESULTS

Th1 immune response and uveitis

In patients with active uveitis, cytofluorimetric analysis of intrac-ellular cytokine production by peripheral CD3

+

lymphocytesshowed a strong polarization of the immune response toward aTh1 pathway. As shown in Fig. 1a, the percentage values of IFN-

g

-producing cells were significantly increased in patients withuntreated active uveitis (41·5

±

8·7%) compared with patients incomplete remission (11·7

±

4·2%) and controls (9·8

±

3·5%). Fur-thermore, Th1 lymphocytes paralleled the response to CsA/PDS(Fig. 1b): in fact, a consistent reduction of IFN-

g

-producing cellswas observed in patients with active uveitis after CsA/PDS ther-apy for 6 months (19·7

±

9·3%). This reduction was associated

Fig. 1.

Th1 polarization of immune response in uveitis. Cytokine production by CD3

+

lymphocytes was analysed by intracellular immun-ofluorescence staining. IFN-

g

and IL-4 production resembled a Th1- and Th2-type cytokine, respectively. (a) Untreated patients with activeuveitis showed elevated percentage values of Th1 lymphocytes compared with patients in remission and healthy subjects. No differencesin Th2 cells were observed. (b) CsA/PDS therapy for 6 months significantly reduced percentage values of CD3

+

/IFN-

g

+

lymphocytes. Valuesare expressed as mean

±

standard deviations.

CD3+/IFN-g +

CD3+/IL-4+

0

10

20

30

40

Per

cent

age

of p

ositi

ve c

ells

Healthysubjects(No. 13)

Uveitisin remission

(No. 16)

Untreatedactive uveitis

(No. 21)

P < 0.01

Untreatedactive uveitis

(No. 21)

CsA/PDS-treatedactive uveitis

(No. 21)

P < 0.01

(a) (b)

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with a striking improvement in visual acuity, binocular indirectophthalmoscopy score, vitreous haze, and intraocular inflamma-tion.

Effect of CsA/PDS therapy on IL-10 and IL-12p70 serum levels

Since IL-10 and IL-12p70 are immunoregulatory cytokines withopposite effects on the Th1 immune response [24], their serumlevels were analysed by ELISA in patients and controls (Table 1).

Untreated patients with active disease showed elevated IL-12p70 levels (187·0

±

30·4 pg/ml), in keeping with the view thatTh1 cells are involved in uveitis. Serum IL-12p70 levels paralleledperipheral CD3

+

/IFN-

g

+

lymphocytes, in that they were signifi-cantly higher compared to those in patients receiving CsA/PDS(53·6

±

22·9 pg/ml), patients in remission (32·5

±

17·3 pg/ml) andthe controls (28·1

±

6·0 pg/ml). CsA/PDS therapy drasticallyreduced IL-12 whereas serum IL-10 levels were within the normal

range in each group. Thus, resolution of uveitis was apparentlymediated by CsA/PDS suppression of IL-12p70 and IFN-

g

pro-duction and not by the suppressive effect of IL-10.

Patient-derived DC are functionally active

Since IL-12 is produced following activation and maturation ofDC [25], the

in vitro

ability of patient-derived DC to produce IL-12 was analysed in the next set of experiments. DC were devel-oped from peripheral adherent cells in the presence of GM-CSFand IL-4, and stimulated with LPS. In particular, supernatant flu-ids from DC cultured in presence or absence of LPS were col-lected and tested for IL-12p70 release (Fig. 2). Unstimulated DCfrom untreated patients produced significantly more cytokinethan DC from patients in remission, healthy subjects and CsA/PDS-treated patients, whose ability to produce IL-12p70 wasdrastically reduced. Stimulation of DC with LPS strongly up-regulated IL-12p70 production in each patient group and inhealthy subjects, though LPS-stimulated DC from untreatedpatients showed the highest ability to release the cytokine. Thesedata paralleled the serum IL-12p70 levels observed in patientsand controls, and suggested that patient-derived DC were func-tionally active.

Phenotypic analysis of DC

Immunophenotypic analysis was performed on immature andmature DC generated

ex vivo

from the adherent cell fraction ofPBMC. As shown in Table 2, patients and normal immature DCwere positive for CD1a, CD80. LPS stimulation induced a signif-icant up-regulation of costimulatory molecules and HLA-DRantigen expression, evaluated as percentage of positive cells andmean fluorescence intensity. CD86 and HLA-DR antigen expres-

Table 1.

Serum cytokine levels in patients with uveitis and in healthy subjects

IL-10 (pg/ml)

IL-12p70 (pg/ml)

Untreated active uveitis (No. 21) 2·5

±

0·9 187·0

±

30·4†CsA/PDS-treated active uveitis (No. 21) 1·9

±

1·3 53·6

±

22·9Uveitis in complete remission (No. 16) 3·4

±

0·7 32·5

±

17·3Healthy subjects (No. 13) 2·7

±

0·5 28·1

±

6·0

†

P

<

0·005

Fig. 2.

IL-12p70 production by unstimulated and LPS-stimulated dendritic cells (DC). Monocyte-derived DC were generated fromadherent PBMC of patients with untreated active uveitis, on CsA/PDS therapy, in remission, and healthy subjects, cultured for 6 days inpresence of GM-CSF (800 U/ml) and IL-4 (400 U/ml). Immature DC (1

¥

10

5

/well) were stimulated in 96-well flat-bottom culture plateswith LPS (1 ng/ml) for 24 h. Supernatant fluids of DC cultured in absence and presence of LPS were collected and tested for IL-12p70content by ELISA. IL-12p70 levels were significantly increased in supernatant fluids of unstimulated and LPS-stimulated DC fromuntreated patients with active uveitis. CsA/PDS therapy inhibited IL-12p70 production. Values are expressed as mean

±

standard deviation.*

P

<

0·05.

Untreated active uveitis

Active uveitis on CsA/PDS

Uveitis inremission

Healthy subjects

*

*

0 500 1000 1500 2000 2500 3000 3500

Unstimulated DC

LPS-stimulated DC

IL-12p70 levels (pg/ml)

Th1/Th2 lymphocytes and dendritic cells in uveitis

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sion was higher in unstimulated DC derived from untreatedpatients with active uveitis compared with that observed in ustim-ulated DC from patients receiving CsA/PDS, patients in remis-sion and controls.

Effect of CsA/PDS therapy on MLR activity

Finally, the ability of the cultured patient-derived DC to stimulatecell proliferation was determined by coculturing increasing num-bers of unstimulated and LPS-stimulated DC with allogeneicPBL. Unstimulated DC from untreated patients with active uvei-

tis showed the highest MLR stimulator capacity: in fact, they elic-ited a stronger cell proliferation than those from patientsreceiving CsA/PDS, those in remission and normal donors(Fig. 3a). The higher values in untreated patients indicate that DCare mature active cells, whereas the reduced MLR ability of DCfrom treated patients suggests that CsA/PDS administrationinhibits accessory capacity of DC.

LPS-stimulation increased MLR activity of DC from eachpatient group and controls, though the highest values wereobserved using mature DC from untreated patients (Fig. 3b).

Table 2.

Phenotypic analysis of immature and mature dendritic cells

Immature DC (% positive cells*) (MFI†) Mature DC (% positive cells*) (MFI†)

CD1a CD14 CD80 CD86 HLA-DR CD1a CD14 CD80 CD86 HLA-DR

Active uveitis 83

±

5·7 4

±

3·0 90

±

3·2 43

±

6·7 95

±

4·1 87

±

6·3 4

±

2·4 93

±

6·5 90

±

5·4 98

±

2·4No. 21 (13) (3) (17) (27) (43) (16) (1) (23) (32) (58)

CsA-treated uveitis 87

±

9·5 2

±

1·2 87

±

4·7 38

±

8·3 63

±

5·5 88

±

5·7 6

±

8·1 91

±

4·9 89

±

6·8 97

± 1·8No. 21 (15) (2) (12) (20) (21) (14) (2) (20) (23) (42)

Remission uveitis 95 ± 7·2 3 ± 1·5 83 ± 6·8 23 ± 7·0 47 ± 5·3 93 ± 7·0 1 ± 0·9 88 ± 5·3 87 ± 8·2 95 ± 1·5No. 16 (18) (2) (10) (10) (13) (17) (2) (18) (25) (33)

Healthy subjects 93 ± 8·4 2 ± 1·8 93 ± 4·1 25 ± 6·9 55 ± 4·9 97 ± 8·8 3 ± 2·2 95 ± 2·8 91 ± 5·6 95 ± 2·2No. 13 (14) (3) (13) (12) (12) (18) (1) (21) (35) (40)

*Values are expressed as mean ± standard deviation; †MFI: mean fluorescence intensity

Fig. 3. Allostimulatory activity (MLR assay) of DC. Monocyte-derived DC were generated from adherent PBMC of patients withuntreated active uveitis (▲), on CsA/PDS therapy (�), in remission (�) and healthy subjects (�). Increased numbers of (a) unstimulatedand (b) LPS-stimulated DC (1 ¥ 102-1 ¥ 104/well) were tested for their capacity to stimulate allogenic PBL. Negative controls, representedby PBL without the addition of cultured DC, gave a base counts less than 200cpm. The ability of DC to stimulate cell proliferation isexpressed as stimulation index. Values are expressed as mean ± standard deviation. *P <0·05.

1 5025 75 100

0

20

40

80

100

Number of DC x 102

0

20

40

60

80

1 5025 75 100

Number of DC x 102

Stim

ulat

ion

inde

x

*

*

*

*

*

(a) (b)

*

Active uveitis on CsA/PDS

Uveitis in remission

Healthy subjects

Untreated active uveitis

*

*

*

*

Immature DC Mature DC

238 M. A. Frassanito et al.

© 2003 Blackwell Publishing Ltd, Clinical and Experimental Immunology, 133:233–239

DISCUSSION

Th1/Th2 cytokine production by peripheral blood lymphocyteswas analysed by an intracellular cytokine staining method inwhich CD3+ cells differentiate in IFN-g - or IL-4-producing cellsafter PMA/ionomycin stimulation. PMA is a powerful pharmaco-logical stimulus that activates all cytokine genes [26]. Therefore,the different pattern of cytokine expression observed in eachgroup after PMA/ionomycin stimulation is consistent with phys-iologically regulated cytokine expression [27,28].

The present study demonstrates a strong Th1 polarization ofthe immune response in active uveitis: elevated CD3+/IFN-g +

lymphocyte percentages were observed in patients with active dis-ease and paralleled serum IL-12 levels. Our results are consistentwith clinical and experimental studies, showing the involvementof IFN-g-producing cells in the pathogenesis of uveitis [9–14,29].

Th1 polarization of immune response is regulated by severalfactors, including the genetic background, the antigen concentra-tion, and in particular the cytokine environment [1,2]. In vitro andin vivo studies indicate that IL-12 is a key molecule in the cytok-ine cascade which regulates the immune response and emphasizeits role in several immunological diseases [30–32]. Seder et al. [32]demonstrated a differential role of IL-12 in the maintenance ofimmune response in infectious vs. inflammatory autoimmune dis-eases. They suggested that the continuous presence of IL-12 isessential to sustain an abnormal Th1 pathogenic response. Thus,IL-12 is important both as the primer of a Th1 immune responseand the subsequent driver of the pathogenic process [28].

DC are potent antigen-presenting cells that, depending oftheir maturation stage and their localization, perform differentfunctions within the immune system [5,6,33,34]. Immature DC arecharacterized by a high ability for antigen capture and processing,but a low T cell stimulatory ability. Exposure to microorganismsand bacterial cell products such as LPS induces maturation of DCthat is associated with a reduced endocytic and phagocytic capac-ities, enhanced production of inflammatory cytokines andchemokines, acquisition of migratory functions and increasedability to stimulate T cells. Based on their physiological role, it hasbeen suggested that DC are involved in uncontrolled immuneresponses associated with chronic inflammatory conditions [34].Our data have demonstrated that unstimulated DC from patientswith untreated active disease produce large amounts of IL-12 anddisplay an elevated MLR stimulatory capacity. These in vitro dataare in line with in vivo results (elevated serum IL-12 levels andTh1 polarization), and suggest that patient-derived DC aremature and functionally active cells. CsA therapy, associated tolow doses of PDS, reduced both peripheral Th1 lymphocytes andserum IL-12 levels, coupled with a striking clinical improvement.In addition, unstimulated DC from CsA/PDS-treated patientsshowed a reduced ability to produce IL-12 and induce allogeneiccell proliferation.

These data are consistent with other experimental observa-tions of the in vitro ability of CsA [35] and corticosteroids [36,37]to affect DC maturation and functions. Exposure of DC to CsAduring maturation inhibits their allostimulatory capacity andinflammatory cytokine production, namely TNF-a and IL-12, inresponse to LPS without a significant effect on the phenotypicmarker expression [38].

The pathogenesis of ‘idiopathic’ uveitis is uncertain and pos-sibly multifactorial. Based on these data, we speculate thatenhanced DC function is able to induce an abnormal immune

response, characterized by the expansion of Th1 lymphocytes andelevated serum IL-12 levels. Support to the pathogenetic role ofIL-12 stems from recent observations that anti-IL-12 mAb treat-ment of mice immunized with the uveitogenic antigen IRBPinhibits the development of Th1 cells and induces suppressive Th2lymphocytes [39]. Furthermore, IL-12-deficient mice are resistantto IRBP-induced EAU [40]. These mice developed a normalimmune response, but had an impaired Th1 response. Transfer ofprimed lymphocytes of wild-type mice, cultured with IRBP,induced EAU in syngeneic wild-type recipients, but was unable toinduce EAU in IL-12-deficient mice. Endogenous IL-12 is thusseen as essential for induction and expression of EAU, whichclosely resembles human uveitis [40].

ACKNOWLEDGEMENT

This study was supported by the Ministry of Education, Universities andResearch (MIUR, ‘Molecular Engineering-CO3’ fund), Italy.

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