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Published online before print March 30, 2006
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B (NF-B), pyrrolidine dithiocarbamate
* Division of Immunobiology, Institute for Genetic Medicine, and
Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Hokkaido University, Sapporo, Japan
1Correspondence: Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. E-mail: kazunori{at}igm.hokudai.ac.jp
ABSTRACT
Experimental autoimmune uveoretinitis (EAU) is a T helper type 1 cell-mediated autoimmune disease, which serves as a model of human chronic uveitis. In this model, cells of a monocyte/macrophage lineage and retinal antigen (Ag)-specific T cells infiltrate into the retina and cause inflammatory lesion, where proinflammatory cytokines and various stimuli activate a transcriptional factor, nuclear factor-
B (NF-B), which modulates inflammation and enhances immune responses. In the present study, the therapeutic effect of administration of a NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC), was examined in a murine EAU model. It was shown that PDTC ameliorated the clinical symptoms of EAU mice and significantly reduced the histopathological score compared with those in untreated mice. mRNA expressions of tumor necrosis factor 
and interleukin-1ß were suppressed in eyes of PDTC-treated EAU mice. However, when T cells from PDTC-treated EAU mice, Ag-presenting cells (APC), and the retinal Ag peptides were cocultured, these T cells showed the same level of proliferation as those from control mice. Furthermore, addition of PDTC in the culture of T cells from EAU mice, Ag, and APC completely abrogated the T cell-proliferative response and cytokine production. Pretreatment of Ag-primed T cells or APC with PDTC in vitro also reduced these responses. These results indicate that the inhibitory effect of PDTC is attributed mainly to the suppression of effector-phase responses including inflammation but not to the inhibition of T cell priming. Regulation of NF-B pathway in the lesion could be a novel target for the successful control of uveoretinitis.
Key Words: inflammation • cytokine • immunomodulation • uveitis
INTRODUCTION
Human endogeneous uveitis, including sarcoidosis, sympathetic ophthalmia, birdshot retinochoroidopathy, Vogt-Koyanagi-Harada’s disease, and Behçet’s disease, are often sight-threatening, ocular-inflammatory diseases. Experimental autoimmune uveoretinitis (EAU) serves as an animal model of human uveitis [1 ]. This experimental model has been used to evaluate the effect of immunosuppressants and immunomodulatory cytokines [2 , 3 ]. Although some of these agents are effective and currently used in clinical situations (e.g., corticosteroids, cyclosporine), we have experienced cases with no response to these treatments. Thus, using the EAU animal model, the search for novel and effective agents with new therapeutic targets is necessary for development of effective strategies to control these diseases.
EAU is thought to be a T helper cell type 1 (Th1)-dominant, autoimmune disease, and it has been anticipated that the regulation of proinflammatory cytokines, such as tumor necrosis factor (TNF-) and interleukin-1ß (IL-1ß), would lead to effective control of EAU [4
, 5
]. Indeed, it was reported that the regulation of TNF- function resulted in the suppression of inflammation in an EAU model [6
]. In addition, clinical applications of antibodies against TNF- to ocular Behçet’s disease have demonstrated the usefulness of cytokine modulation in the treatment for ocular inflammation [7
].
It is well known that nuclear factor-B (NF-B), a transcriptional factor, activates genes encoding proinflammatory cytokines in cells of innate and adaptive immunity. NF-B is present in cytoplasm as a bound form to inhibitor of B (IB), which prevents the NF-B from entering the nuclei. When cells are stimulated, IB is phosphorylated by a specific kinase, which leads to ubiquitination and the rapid degradation of IB in proteasomes. A wide range of stimuli activates NF-B, including cytokines, activators of protein kinase C, viruses, and reactive oxidative species [8
]. The release of NF-B from IB results in the transfer of this activated form of NF-B into the nucleus, where NF-B acts on the specific sequences in the promoter regions of the target genes for proinflammatory cytokines, chemokines, and enzymes. These proteins generate mediators of inflammation such as TNF-, immune receptors, or adhesion molecules, which play key roles in the initial recruitment of leukocytes to sites of inflammation. Products of the genes, which are regulated by NF-B, also cause the activation of NF-B itself. The proinflammatory cytokines, IL-1ß and TNF-, activate and are activated by NF-B. This type of positive regulatory loop appears to amplify and perpetuate local inflammatory responses.
Thus, effective regulation of NF-B activation appears to be useful for control of local or systemic inflammation, and it seems important to search for agents that prevent the activation of NF-B [9
]. Recently, it has been shown that pyrrolidine dithiocarbamate (PDTC), a class of antioxidant, is remarkably effective in inhibiting NF-B activity [10
]. The potential for modulating cell activation suggests that PDTC offers therapeutic advantage in acute and chronic inflammatory conditions, in which activation of NF-B plays a major role. We reported that NF-B played an important role in the development of inflammation at the cornea and conjunctiva [11
, 12
]. However, precise, functional roles of NF-B in development of EAU have not been elucidated yet. If NF-B activation would play a key role in initiating inflammations of EAU, down-modulation of the NF-B activity could lead to treatment of uveitis.
In the present study, we examined the distribution of NF-B p65 in the retina of EAU mice and the effect of PDTC on the distribution of NF-B p65 and development of EAU. We found that PDTC inhibited the translocation of NF-B to the nucleus, and PDTC administration to the EAU-induced mice resulted in the amelioration of EAU without suppressing the induction of autoreactive T cells.
MATERIALS AND METHODS
Mice
Female B10.BR (H-2k) mice of 6–8 weeks old and female BALB/c mice were obtained from Japan SLC (Hamamatsu, Japan) and were maintained in a specific, pathogen-free condition in the animal facility of Laboratory of Animal Experiment for Disease Model, Institute for Genetic Medicine at Hokkaido University (Sapporo, Japan). DO11.10 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All studies were conducted in compliance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research (ARVO, Rockville, MD).
Induction of EAU
B10.BR mice (designed as EAU mice) were injected subcutaneously in the upper back and flanks with 100 nmol peptides, K2, emulsified in complete Freund’s adjuvant (CFA; Difco Laboratories, Detroit, MI) containing 2.5 mg/ml heat-killed Mycobacterium tuberculosis H37Ra (Difco Laboratories). Concurrently, the mice were injected intraperitoneally (i.p.) with 0.1 µg purified Bordetella pertussis toxin (PTX; Sigma Chemical Co., St. Louis, MO) in 100 µl phosphate-buffered saline (PBS) as an additional adjuvant [13
]. The K2 peptide (ADKDVVVLTSSRTGGV, molecular weight=1603.78) corresponds to the amino acid sequence 201–216 of bovine interphotoreceptor retinoid-binding protein (IRBP), which is the immunodominant retinal autoantigen of EAU in H-2k mice [14
], and was synthesized and purified by high-pressure liquid chromatography (Hokkaido System Science, Sapporo, Japan).
PDTC treatment
From the first day of immunization, B10.BR mice received i.p. injections of PDTC (100 mg/kg; Sigma Chemical Co.) daily in 100 µl PBS. Control animals received 100 µl PBS alone.
Evaluation of EAU
Clinical assessment by funduscopic examinations of the retinal inflammation was carried out every 2 or 3 days from Day 7 after immunization. The severity of the retinal inflammation was graded on a five-point scale as described previously [15
]. Briefly, the clinical scoring was based on vessel dilatation, number of white focal lesions in vessels, and the extent of retinal vessel exudate, hemorrhage, and retinal detachment. On Days 14 and 21 after immunization, the eyes were enucleated after euthanasia with an overdose of anesthetics and fixed in 4% phosphate-buffered glutaraldehyde for 1 h and transferred into 10% phosphate-buffered formaldehyde. Fixed tissues were stained with hematoxylin and eosin, and the histological severity was graded double-blind on a scale of 0–4 as reported [1
].
Immunocytochemistry
The eyes of naïve mice, PBS-treated EAU mice, and PDTC-treated EAU mice were enucleated at Day 10 after immunization and were fixed in ice-cold 4% paraformaldehyde in 0.1 M borate buffer (pH 9.5) for 2 h and embedded in paraffin and cut into 10 µm coronal sections. Slides were dried for 1 h, rinsed twice in PBS, and then incubated with antibodies to p65 (Santa Cruz Biotechnology, CA). Binding of the primary antibody was localized by the Cy3-conjugated goat anti-rabbit immunoglobulin G (IgG; Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. Then, the Müller cell’s process was localized by monoclonal antibody (mAb) against glutamate synthetase (GS; Chemicon, Temecula, CA) [16
] and the fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), or nuclei were stained by PBS containing YO-PRO-1 (2 µM) as described previously [17
].
T cell proliferation assay
We performed three types of T cell proliferation assay. In the first type of experiment, three EAU mice (per each group) were treated daily with PDTC or PBS alone for 10 days. T cell-enriched fractions were prepared by passing the dispersed cells from the draining lymph node (DLN; axillar, cervical, and inguinal lymph nodes) of these EAU mice over nylon-wool columns. Nylon-wool nonadherent cells (4x105/well) were cultured with 30 Gy-irradiated syngeneic splenocytes as antigen (Ag)-presenting cells (APC) and various concentrations of peptide in a 96-well flat-bottom microtiter plate for 48 h at 37°C. The cells were then pulse-labeled with 3H-thymidine (Perkin Elmer Japan, Tokyo) and incubated for 16 h. 3H-thymidine incorporation was quantitated with a direct ß-counter (Packard, Meriden, CT), and the data are presented as the mean counts per minute (CPM) minus the background (medium alone; 
CPM) as described elsewhere [15
, 18
]. Cytokines produced in the culture supernatant were quantitated as described below. In the second type of experiment, T cells were purified from PBS-treated EAU mice and then were cultured with APC and peptide K2, with or without PDTC (0.1 mg/ml). In the third type of experiment, K2-primed T cells or APC from naïve mice were cultured for 60 min in the absence or presence of PDTC (0.1 mg/ml) and washed three times. Then, these T cells from EAU mice, nontreated or pretreated with PDTC, were cultured with PDTC-pretreated APC or nontreated APC, respectively, in the presence of Ag. T cell proliferation was determined as the first experiment.
Quantitation of cytokines
Cytokines [TNF-, interferon-
(IFN-), IL-5, IL-4, and IL-2] produced in the culture supernatants were quantitated with a cytometric bead array kit (BD Biosciences, San Diego, CA) according to the manufacturer’s protocol [19
, 20
]. In some experiments, the concentrations of TNF-, IFN-, and IL-4 were measured by enzyme-linked immunosorbent assay (ELISA) kits (OptiEIA, BD Biosciences). In vivo expression of cytokines was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). The total RNA was isolated from pooled eyes of PDTC-treated or PBS-treated EAU mice (n=3 in each group) at Days 10 and 18 after immunization using the TRI Reagent (Sigma Chemical Co.). First-strand cDNA was synthesized from 5 µg of the total RNA using 200 units SuperScript III RT (Invitrogen, Carlsbad, CA) and 40 units RNAsin RNase inhibitor (Promega Corp., Madison, WI) in a total reaction mixture volume of 20 µl. The cDNA was diluted with the 80 µl dH2O, and 2 µl of the mixture was amplified by PCR. A 20-µl reaction mixture consisted of 2 µl template DNA, 2 µl each primer, 200 µM deoxy-unspecified nucleoside 5'-triphosphates, 0.2 µl Taq DNA polymerase, and 2 µl 10x ThermoPol reaction buffer (New England Biolabs, Beverly, MA). PCR product was visualized by SYBR Green nucleic acid gel stain (Molecular Probes, Eugene, OR). Reaction conditions were designed as follows: initial activation step at 95°C for 5 min, followed by 25–30 cycles with 30 s at 95°C for denaturing, 30 s at 53–60°C for annealing, and 30 s at 72°C for extension. Primer sequences: TNF- sense 5'-CCAGACCCTCACACTCAGAT-3', antisense 5'-AAC ACC CAT TCC CTT CAC AG-3'; IL-1ß sense 5'-TGA CGG ACC CCA AAA GAT GAA G-3', antisense 5'-CTG CTT GTG AGG TGC TGA TGT A-3'; ß-actin sense 5'-CAC CAT GTA CCC AGG CAT CGC G-3', antisense 5'-AGG GGC CGG ACT CAT CGT ACT-3'. Image analysis was performed using ImageGauge software (Fuji, Tokyo, Japan).
Cell transfer and immunization
T cell-enriched fractions were prepared from splenocytes of DO11.10 T cell receptor [TCR; specific for ovalbumin (OVA) and restricted to H-2Ad] transgenic mice by passing the dispersed cells through the mouse T cell enrichment columns (R&D Systems, Minneapolis, MN). CD4+ and CD45RB+ cells were purified by FACS Vantage (BD Biosciences) using mAb. FITC-conjugated anti-CD4 and anti-CD45RB mAb were purchased from PeproTech (Rocky Hill, NJ). These CD4+CD45RB+ cells (5x105) were adoptively transferred to normal BALB/c mice (H-2Ad) by intravenous (i.v.) injection, and these mice were designated as (DO11.10 
BALB/c) mice. Seven days later, these (DO11.10BALB/c) mice were immunized with OVA323–339 peptide (100 nmol) in CFA and i.p.-injected with PTX (0.1 µg) in the same manner as that given to EAU mice. These mice then received daily i.p. injection of PDTC or PBS. Seven days later, responding cells from DLN were analyzed for the expressions of CD4 and KJ1-26, a clonotypic DO11.10 TCR, using FITC-conjugated anti-CD4 and phycoerythrin-conjugated KJ1-26 mAb (PeproTech). The proportion of CD4+ and KJ1-26+ cells was analyzed using a FACSCalibur flow cytometer (BD Biosciences) as described elsewhere [13
].
Western blot
BC1 cells, a murine dendritic cell (DC) line derived from BALB/c mice [21
], were seeded at a density of 1.5 x 106 cells in a 5-cm cell culture dish. After 60 min incubation, BC1 cells were pretreated with PDTC (0.1 mg/ml) for 60 min and then were stimulated with purified hamster anti-mouse CD40 mAb (HM40-3, BD Biosciences; 1 µg/ml) and recombinant murine IL-1ß (PeproTech; 40 ng/ml) for 90 min. As a control, BC1 cells were stimulated with purified hamster IgM monoclonal Ig isotype standard (BD Boisciences) and IL-1ß. A nuclear protein-enriched fraction from these cells was obtained using a nuclear extract kit (Active Motif, Carlsbad, CA). Protein concentration was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). The proteins (10 µg) were mixed with gel-loading buffer (50 mM Tris, 10% sodium dodecyl sulfate, 10% glycerol/10% 2-mercaptoethanol, 2 mg bromophenol per milliliter) at a ratio of 1:1, boiled at 95°C for 5 min. Each sample was electrophoresed in a 10% discontinuous polyacrylamide mini-gel. The protein was transferred onto a nitrocellulose membrane, which was incubated with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 60 min and then incubated with primary antibodies (1:1000) overnight at 4°C. The membranes were washed with TBS-T and then incubated with anti-Igs coupled to horseradish peroxidase (HRP; 1:2000). The immune complexes were visualized with the enhanced chemiluminescence detection reagent (Amersham, Buckinghamshire, UK). The antibodies used were anti-NF-B p65, anti-NF-B2 p100, anti-RelB (Cell Signaling Technology, Beverly, MA), and HRP-conjugate anti-rabbit Ig antibody (Dako, Carpinteria, CA). Image analysis was performed using ImageGauge software (Fuji).
Data analysis
Data are presented as mean ± SEM. Statistical analysis of EAU scoring was performed using the nonparametric Mann-Whitney U-test. Analysis of lymphocyte proliferation and cytokine production was performed using unpaired t-test. A P value was calculated for each experiment, and values of P < 0.05 were considered statistically significant.
RESULTS
PDTC treatment ameliorated EAU in mice
To examine whether the PDTC suppresses ocular inflammation, B10.BR mice were treated with PDTC after immunization with K2. One group of mice (n=10) received daily i.p. injections of 2 mg PDTC in 100 µl PBS (100 mg/kg) from the first day of immunization with K2 peptide to Day 21. A control group of mice (n=10) was given the same volume of PBS (Fig. 1a
). The retinas of EAU mice were clinically scored from Day 7 after immunization. In PBS-treated control mice, clinically diagnosable EAU began developing at approximately Day 10 and reached a plateau at Days 14–20 after immunization. In PDTC-treated mice, the clinical score of EAU followed a similar time course to that in control mice (Fig. 1a)
. However, the peak clinical severity in the PDTC-treated mice was significantly milder than that of control mice. The other group of mice (n=10) received repeated injections of PDTC until 16 days after immunization. In this group of mice, the maximal clinical scores of EAU at Day 20 were also suppressed significantly compared with those in control mice (Fig. 1b)
. It was also noted in Figure 1a
and 1b
, that the EAU onset was not influenced by the PDTC treatment in either group.
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B into the nucleus in EAU retinaB in normal and inflammatory ocular tissues, the retinas from EAU mice and normal mice were stained with an antibody against the NF-B subunit p65. EAU was generated in B10.BR mice as described previously [14
], and the mice were treated with PDTC or PBS. Retinas were obtained from eyes of these EAU mice on Day 10 after immunization with peptide K2. Retinas were also sampled from normal B10.BR mice.
When the retinas of normal mice were stained with anti-p65, the positive staining was detected in the same region stained with GS, suggesting that p65 was distributed in the GS-positive Müller cell process (Fig . 3a
and 3b
). Conversely, the nuclei of the GS-positive cells in the retina of EAU mice were positively stained with anti-p65 (Fig. 3c
and 3d)
. These results demonstrate that the NF-B p65 subunit is translocated into the nuclei in inflamed retina and suggest that this translocation is involved in activation of various genes encoding proinflammatory cytokines in ocular inflammation. It is notable that the staining of p65 was not detected in the retina of PDTC-treated mice compared with that of control mice (Fig. 3e)
. These results indicate that PDTC treatment suppress NF-B translocation into the nuclei of the EAU retina.
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and IL-1ß, in eyes of EAU mice using RT-PCR. It has been reported that productions of these cytokines are elevated in aqueous humor from eyes developing EAU [22
]. The RNA was extracted from the eyes (pooled from three mice) enucleated at Day 10 or 18 after immunization. No significant expressions of these cytokines were detected in eyes at Day 10 (before onset of EAU), whereas distinguished bands of TNF- and IL-1ß messages were detected at Day 18 after induction of EAU (Fig. 4a
). It should be noted that eyes of PDTC-treated mice expressed significantly less TNF- and IL-1ß mRNA than those of control eyes (Fig. 4a
and 4b)
. These results indicate that PDTC ameliorates EAU by down-regulating the expression of these proinflammatory cytokine genes in situ, probably by regulating NF-B transport to the nuclei in immunocompetent cells and glial cells of uveas and retinas.
|
, IFN-, and IL-4, -5, and -2 in the culture supernatants were quantitated by cytometric bead array methods as described in Materials and Methods. The production levels of TNF- and IFN- were rather up-regulated in the T cells from PDTC-treated EAU mice compared with those from control mice, whereas those of IL-4 and IL-2 showed no differences between experimental group and control group (Fig. 5b)
.
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BALB/c) mice were then immunized with OVA323–339 peptides, i.p. injected with PTX, and treated daily with PDTC or PBS. When the proportion of DO11.10, KJ1-26+CD4+ T cells in DLN was analyzed 7 days after immunization, similar proportions of DO11.10 T cells were observed in PBS-treated (0.81%) and PDTC-treated (0.61%) mice (Fig. 6
). These findings demonstrate that the priming process with peptide Ag is not the primary target by the PDTC-mediated suppression.
|
B translocation into the nucleus of CD40-stimulated DCB is located in the cytoplasm as a heterodimer of the structurally related proteins p50, p52, RelA, c-Rel, and RelB, and nuclear translocation of the NF-B reflects its activation [24
]. Indeed, we demonstrated that NF-kB p65 was distributed in the nuclei of retina in EAU mice but not in control mice (Fig. 3)
. We then examined the effect of PDTC on the translocation of the NF-B subunit to the nuclei using BC1 cells, a mouse myeloid DC line [21
]. BC1 cells were pretreated with PDTC for 60 min and then were incubated with IL-1ß (40 ng/ml) and anti-CD40 mAb (1 µg/ml) for 90 min. Nuclear protein extracts of these cells were subjected to Western blot to determine the levels of the NF-B subunit, p65, RelB, and p52. These three subunit expressions were increased in the nuclear fraction upon stimulation with IL-1ß and anti-CD40 mAb (Fig. 7
). However, the expression of these subunits in the nuclei was suppressed by PDTC pretreatment. These findings indicate that PDTC pretreatment suppresses NF-B translocation into the nucleus of DC and suggest that PDTC treatment suppresses the DC activation in vivo.
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and IFN- productions (Fig. 8d)
. No IL-4 production was detected in any cultures (data not shown). These results suggest that PDTC affects not only primed T cells but also APC. DISCUSSION
NF-B has drawn considerable attention because of the unique mechanism of activation, the active role in cytoplasmic/nuclear signaling, and the rapid response to pathogenic stimulations [25
]. Thus far, however, only few reports have presented the state of NF-B in the retina during EAU. In the present study, we demonstrated that NF-B p65 was present in Müller’s cell processes in normal retina but translocated into the nucleus when EAU was induced. The translocation of NF-B p65 in the retinal Müller’s cells during EAU suggests that NF-B plays a role in the inflammatory responses. It was reported that susceptibility to EAU was correlated with the extent of TNF- production by Müller’s cells under in vitro conditions [26
]. Activation of NF-B leads to TNF- production, which plays a key role in initiating or perpetuating local immune responses. Thus, it seems rational that regulation of NF-B activation could be a strategy for the control of EAU. In fact, our immunohistochemical analysis demonstrated that NF-B p65 expression was suppressed in nuclei of the retina from PDTC-treated EAU mice.
Our present results clearly showed that EAU was ameliorated by the treatment with PDTC. It has been reported that PDTC inhibited NF-B activation in several cell systems and protected animals from septic shock in vivo [27
28
29
]. In other models for ocular inflammation, PDTC treatment suppressed endotoxin-induced uveitis (EIU) and experimental autoimmune anterior uveitis (EAAU) in rat [30
, 31
]. EIU is induced in the Lewis rat by i.v. injection of lipopolysaccaride, and the inflammation is characterized by neutrophil infiltration into the anterior segment of the eye, up-regulation of cytokine production in aqueous humor [32
33
34
35
36
37
]. EAAU is induced by immunization with bovine melanin-associated Ag, and the pathogenesis is associated closely with the Ag-specific T cell activation [38
39
40
41
]. Both animal models resemble human acute anterior uveitis, as the inflammation is more prominent in the anterior segment of the eye than that in the posterior segment.
Conversely, EAU is induced in several strains of susceptible animals by immunization with ocular Ag, which seems to be a model for posterior segment uveitis. EAU is considered to be a Th1-mediated autoimmune disease and is characterized by the initial infiltration of mononuclear cells in the retinal perivascular sites, followed by the infiltration of phagocytes in the outer retina and uveal tract [42 , 43 ]. Thus, EAU is different from EIU and EAAU in the pathogenesis or the localization of inflammatory lesions. In addition, a longer time course of EAU in comparison with EIU suggests that EAU can be a model for chronic ocular inflammation. Thus, our present results suggest that PDTC have therapeutic benefits, not only in acute inflammation but also in chronic ocular inflammation. It has been reported in other cell systems that PDTC is effective on acute and chronic inflammation [44 ].
EAU is induced by Th1 cells, which produce the Th1-specific cytokines [45
]. Indeed, T cells from the DLN of EAU mice responded vigorously to immunogen peptides and produced TNF-, IFN-, and other cytokines. We examined whether the suppression of EAU by PDTC was attributed to inhibition of T cell priming. We found that the generation and expansion of K2-primed T cells were not influenced by PDTC administration. Furthermore, rather increased productions of Th1 cytokines were observed in the culture supernatant of K2-primed T cells from PDTC-treated mice, K2 peptides, and APC compared with T cells from nontreated EAU mice. It was also shown that IL-5 production was slightly high in T cells from PDTC-treated mice compared with those from PBS-treated EAU mice. We postulate that K2-primed effector T cells were not reactivated sufficiently in the presence of APC, which had been inactivated by PDTC in vivo. When these T cells were cultured for 48 h ex vivo in the presence of intact APC, proliferation and cytokine productions by these T cells might be induced quickly in a manner of rebound reaction. In contrast, it is clear that testing the effect of PDTC added in vitro to T cells from EAU mice completely abrogated T cell proliferation and cytokine production. This suggests that regulation of the immune response by NF-B in vivo could involve more complex signaling pathways acting on different cell types than in vitro.
To confirm directly that PDTC does not influence priming and expansion of Ag-specific T cells, we performed an adoptive transfer experiment, where naïve T cells from OVA-specific TCR transgenic mice were i.v.-injected into the host mice. These (DO11.10BALB/c) mice were treated with PDTC after immunization with OVA peptide, and flow cytometric analysis was carried out using KJ1-26, a clonotypic mAb. No significant difference was shown in the proportion and the actual number of CD4+KJ1-26+ cells from DLN between control and PDTC-treated host. This finding permits us to conclude that PDTC have little influence on the T cell priming and clonal expansion. These results appear to be consistent with the observation that the onset of EAU was not delayed by PDTC administration.
PDTC, which was reported originally as an antioxidant agent, inhibits NF-B considerably [10
]. Thus, it was considered that the possible molecular mechanisms of PDTC action on NF-B might involve interference with scavenging of reactive oxygen radicals [46
], chelation of divalent metal ions [47
], changes in intracellular thiol levels [48
], or a combination of these effects. Recently, however, it was shown that PDTC blocked NF-B activation by inhibiting the IB-ubiquitin ligase activity, independent of antioxidative functions [49
]. In addition, it has been reported that PDTC inhibits the degradation of IB and subsequent translocation of NF-B subunits to the nucleus [44
]. Thus, it seems important to examine whether PDTC also inhibits the translocation of the p65 in EAU retina. We could demonstrate this by immunohistochemical analysis. In addition, we could show that mRNA expressions of TNF- and IL-1ß, both of which are regulated by NF-B, were indeed suppressed significantly in eyes from PDTC-treated EAU mice compared with those in control mice. Thus, it seems that the suppression of the proinflammatory cytokine gene expressions by PDTC is mediated through inhibition of NF-B translocation.
In the present study, we also analyzed the direct effect of PDTC on the activation of NF-B in APC using BC1 cells. The BC1, an immature DC line, was established from splenocytes of BALB/c mice. Using BC1 cells, a number of important findings have been reported and verified [21
, 50
51
52
53
]. DC is the most potent APC and plays major roles in the regulation of immune responses to various Ag [54
, 55
]. It is reported that CD40/CD40 ligand interactions on DC and T cells are critical for EAU development [56
]. We found that PDTC pretreatment of BC1 cells, which were stimulated with IL-1ß and anti-CD40 mAb in vitro, suppressed the translocation of p65, RelB, and p52 into the nuclei.
In addition, Ag-specific T cell proliferation and cytokine production in the culture of Ag-primed T cells, Ag, and APC were suppressed by addition of PDTC. When Ag-primed T cells or APC were pretreated with PDTC, the T cell proliferation and cytokine production were also suppressed. A recent report described that PDTC treatment of DC led to an arrest in the DC maturation [57 ]. Thus, these results demonstrate that PDTC inhibits not only Ag-primed T cells but also APC, including DC and macrophages, which may result in inefficient stimulation of effector T cells in the site of ocular inflammation.
In conclusion, we demonstrated that PDTC regulated NF-B activation and the resultant cytokine production and suggested that this agent could have a therapeutic advantage in ocular autoimmune disease. Further analysis of the actual target molecules and cells, which PDTC acts, is needed to reveal the precise mechanism underlying the PDTC action.
ACKNOWLEDGEMENTS
This study was supported in part by a grant for research on sensory and communicative disorders from The Ministry of Health, Labor, and Welfare Japan, by grants-in-aid for scientific research on priority areas from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) Japan, and by a grant-in-aid for scientific research from Japan Society for the Promotion of Science (JSPS).
Received August 14, 2005; revised January 30, 2006; accepted February 15, 2006.
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) or PBS alone (•). Daily injections were performed throughout the observations (a) or for 16 days after immunization (b). Results are presented as the mean clinical score for all eyes of each group of mice (10 mice per group) ± SEM. Black arrowheads indicate daily injection of PDTC or PBS. Significance was determined using Mann-Whitney U-test (**, P <0.01).
) or presence (
) of PDTC (0.1 mg/ml). (b) Cytokine production by T cells in the culture supernatant. Cytokine production was quantitated as described in the legend to