Effects of the NF-κB Inhibitor Pyrrolidine Dithiocarbamate on Experimentally Induced Autoimmune Anterior Uveitis

Abstract

purpose. To determine the effect of pyrrolidine dithiocarbamate (PDTC), a nuclear factor (NF)-κB inhibitor, on chemokine and chemokine receptor expression and thus elucidate the role of NF-κB in the pathogenesis of experimental autoimmune anterior uveitis (EAAU).

methods. Uveitis was induced in Lewis rats with the injection of melanin-associated antigen into the footpad. PDTC (200 mg/kg and 100 mg/kg) was administered intraperitoneally daily, beginning 1 day after the immunization. The clinical inflammatory activity of the anterior chamber was recorded daily and scored. Immunohistochemical staining and an electrophoretic mobility shift assay assessed the effect of PDTC on NF-κB activation in the iris/ciliary body tissues. Gene expression profiles of chemokine and chemokine receptors were semiquantitatively examined by reverse transcriptase–polymerase chain reaction (RT-PCR). Aqueous chemokine levels were measured by enzyme-linked immunosorbent assay (ELISA).

results. PDTC significantly attenuated the clinical scores and monocyte/lymphocyte infiltration in rats with EAAU. PDTC effectively inhibited NF-κB activation in the iris and ciliary body, and markedly inhibited the expression of chemokine genes, including monocyte chemoattractant protein (MCP)-1, regulated-on-activation normal T-cell expressed and secreted (RANTES), and interleukin (IL)-8 and chemokine receptors genes including CCR2, CCR5, and CXCR3.

conclusions. Activation of NF-κB appears to play an important role in the pathogenesis of EAAU, through transcriptional control of MCP-1, RANTES, and IL-8 gene expression. Blocking NF-κB reduces ocular inflammation and may be an effective strategy in the treatment of acute anterior uveitis.

Acute anterior uveitis (AAU) is the most common form of uveitis in humans. It can cause significant visual problems because of its recurrent nature and secondary complications such as cataract formation, cystoid macular edema, and glaucoma. ¹ The exact mechanism of AAU has yet to be determined; thus, nonspecific corticosteroids are still the mainstay of uveitis therapy. Usually, topical corticosteroids are used in the treatment AAU; however, periocular injections and systemic steroids are necessary in recalcitrant cases. Long-term use of corticosteroid at the dose needed to suppressed ocular inflammation is likely to produce side effects and limit their application. ² Therefore, further understanding of the mechanisms of AAU and a search for a new approach of immunosuppression are important for uveitis therapy. 

Experimental autoimmune anterior uveitis (EAAU) is a well-established model of AAU that closely resembles the human disease clinically and pathologically. ³ EAAU is induced in the Lewis rat after immunization with bovine melanin-associated antigen (MAA). ⁴ This EAAU model differs from other models, in that the inflammation remains exclusively anterior, without retinal and choroid involvement. ^{5 6 7 8}  

The development of inflammatory lesions in EAAU is the result of a complex chain of events that involves recognition of specific antigens, T-cell activation, activation of cell-adhesion molecules, release of numerous chemokines, and recruitment of specific cells to the lesion. The result is inflammation and damage of the iris/ciliary body. Among these inflammatory molecules, chemokines, and their receptors regulate the trafficking and activation of specific leukocytes to the site of inflammation and are central in the pathogenesis of uveitis. ^{9 10}  

Further understanding of the molecular mechanisms of EAAU would help us to gain insight into the pathogenesis of human AAU and to develop new treatment methods. Nuclear factor (NF)-κB is a transcriptional regulatory factor that governs a host of inflammatory and immune responses and cellular growth properties by increasing the expression of specific cellular genes. ^{11 12} These include genes encoding cytokines, chemokines, chemokine receptors, and cell-adhesion molecules. ^{13 14 15 16} Activation of NF-κB and the subsequent immunologic responses are found to play major roles in the pathogenesis of certain autoimmune diseases. ^{17 18 19}  

Because the activation of chemokines and their receptors and subsequent inflammatory cellular infiltration is one of the pathologic hallmarks of EAAU and are considered to be of paramount importance in pathogenesis, ^{20 21} we hypothesized that activation of NF-κB in the iris/ciliary body promotes the expression of certain chemokines, which mediate the polymorphonuclear leukocyte and monocyte/macrophage influx into the iris/ciliary body. The consequence would be ocular inflammation in EAAU. Analyses of molecular mechanisms for the regulation of chemokine and chemokine receptors in the iris/ciliary bodies of rats EAAU should help to reveal the mechanisms of the disease process and to develop effective therapies for patients with AAU with drugs that block the activation of inflammatory signaling pathways. ²²  

The purpose of this study was twofold: first, to investigate the role of NF-κB in the activation of ocular inflammation in the iris/ciliary bodies of rats with EAAU and second, to evaluate the effect of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) ^{23 24} on the expression of chemokines and their receptors to delineate further the role of these mediators in the pathogenesis of AAU. 

Materials and Methods

Animals

Lewis rats, 6 to 8 weeks old and weighing 200 to 250 g, were used for the experiments. All animals were treated according to a protocol approved by the Institutional Animal Care and Use Committee of National Taiwan University and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Induction of EAAU

MAA was prepared by using a modification of the protocol of Broekhuyse et al. ⁴ The iris and ciliary body were carefully obtained from freshly extracted, pigmented bovine eyes. The tissue was gently homogenized and filtered through a wire mesh to remove cellular debris and connective tissue. The homogenate was centrifuged at 1.2 × 10⁵ g at 4°C for 15 minutes and washed once with phosphate-buffered saline (PBS) at pH 7.4. The resultant pellet was resuspended in 2% sodium dodecyl sulfate (SDS; Bio-Rad, Hercules, CA) and incubated at 70°C for 10 minutes. After centrifugation, the pellet was washed three times with water. The insoluble antigen was dried and stored at −20°C. 

Lewis rats were injected with a single dose of 100 μg of bovine MAA. The antigen was suspended in PBS, emulsified (1:1) in complete Freund’s adjuvant (Sigma-Aldrich Co., St. Louis, MO) and 1 μg purified Bordetella pertussis toxin (List, Campbell, CA). A final volume of 0.05 mL was injected intraperitoneally into the left hind footpad. 

PDTC Treatment

To examine the effect of PDTC, rats were randomly divided into three groups. Group A (n = 15) received 200-mg/kg and group B (n = 15) 100-mg/kg intraperitoneal injections of PDTC daily, beginning 1 day after immunization. Group C (n = 15) served as the control and received an intraperitoneal injection of PBS daily beginning 1 day after immunization. 

Clinical Examination

The rats were clinically observed daily with slit lamp biomicroscopy for clinical signs of ocular inflammation. Disease severity was clinically assessed with a scale ranging from 0 to 4, as described previously by Broekhuyse et al. ⁴ Zero represented a normal state. One represented slight iris-vessel dilatation and some anterior chamber cells. Two denoted iris hyperemia, with some limitation in pupil dilation, anterior chamber cells, and a slight flare. Three represented a miotic, irregular, hyperemic, and (sometimes) slightly damaged iris, with considerable flare and cells (especially with accumulation near the iris). Four denoted a seriously damaged and hyperemic iris, a miotic pupil often filled with protein, and cloudy gel-like aqueous humor (AqH). 

Tissue Preparation

Rats were killed on day 14 after immunization. The eyes harvested and quickly dissected, and the iris and ciliary body were isolated from the remaining ocular tissue under an operating microscope. 

Nuclear Protein Extract and Electrophoretic Mobility Shift Assay

Nuclear extract of rat iris/ciliary was prepared using a modified method of Dignam et al. ²⁵ The iris/ciliary body was minced on ice in 0.5 mL ice-cold buffer A composed of 10 mM HEPES (pH 7.9), 1.5 mM KCl, 10 mM MgCl₂, 1.0 mM dithiothreitol (DTT), and 1.0 mM phenylmethylsulfonyl fluoride (PMSF). The tissue was homogenized (Dounce; Bellco, Glass Co., Vineland, NJ), followed by centrifugation at 5000g at 4°C for 10 minutes. The crude nuclear pellet was suspended in 200 μL of buffer B (20 mM HEPES [pH 7.9], 25% glycerol, 1.5 mM MgCl₂, 420 mM NaCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, and 4 μM leupeptin) and incubated on ice for 30 minutes The suspension was centrifuged at 12,000g at 4°C for 30 minutes. The supernatant containing nuclear proteins was collected and kept at −70°C until use. The protein concentration was determined with a bicinchoninic acid assay kit with BSA as the standard (Pierce Biotechnology, Rockford, IL). 

Nuclear extracts were used for the electrophoretic mobility shift assay with an NF-κB DNA-binding protein-detection system (Pierce Biotechnology), according to the manufacturer’s protocol. Briefly, an NF-κB consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was end labeled with biotin. Nuclear protein (10 μg) was incubated with NF-κB consensus oligonucleotide for 30 minutes in a total volume of 20 μL in a binding buffer that consisted of 10 mM Tris-Cl, (pH 7.5), 1 mM MgCl₂, 50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, 4% glycerol, and 2 μg polydeoxyinosinic deoxycytidylic acid (Amersham Pharmacia Biotech, Piscataway, NJ). The specificity of the DNA/protein binding was determined by competition reactions in which a 100-fold molar excess of unlabeled NF-κB oligonucleotide was added to the binding reaction 10 minutes before the addition of the biotin probe. The reaction was stopped by adding 1 μL gel loading buffer and subjecting the sample to nondenaturing 4% polyacrylamide gel electrophoresis in 0.5× TBE (Tris, boric acid, and EDTA) buffer. 

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue sections were placed on slides, deparaffinized in a series of xylene solutions, and rehydrated through a graded series of ethanol in PBS. Endogenous peroxidase activity was blocked by the addition of 0.3% hydrogen peroxidase in methanol, and the sections were treated with 5% normal rat serum and incubated overnight with a monoclonal antibody against the p65 subunit of NF-κB (Chemicon, Temecula, CA) at 4°C. Thereafter, a biotinylated secondary antibody against mouse IgG and an avidin-biotinylated peroxidase complex (Santa Cruz Biotechnology, Santa Cruz, CA) were used with 3,3′-diaminobenzidine as a peroxidase substrate. Sections were counterstained with hematoxylin, dehydrated, and mounted. Sections stained without the primary antibody were used as a negative control. 

Preparation of RNA and cDNA

Total RNA was extracted from the iris/ciliary body with reagent (TRIzol; Invitrogen-Life Technologies Inc., Gaithersburg, MD). One microgram of total RNA from each sample was annealed for 5 minutes at 65°C with 300 ng oligo(dT) (Promega, Madison, WI) and reverse transcribed to cDNA by using 80 U Moloney murine leukemia virus reverse transcriptase (MMLV-RT; Invitrogen-Gibco, Grand Island, NY) per 50 μg reaction sample for 1 hour at 37°C. The reaction was stopped by heating for 5 minutes at 90°C. 

Polymerase Chain Reaction

PCR was performed on the resultant cDNA from each sample with specific primers for rat chemokines, chemokine receptors, and β-actin (Table 1) . The amplification was performed with a thermocycler (MJ Research, Waltham, MA). The 50-μL reaction mixture consisted of 5 μL cDNA, 1 μL sense and antisense primers, 200 μM of each deoxynucleotide, 5 μL 10× Taq polymerase buffer, and 1.25 U Taq polymerase (Promega). Conditions for amplifying each chemokine consisted of a 1-minute 94°C denaturation, and a 3-minute 72°C extension. For the annealing temperature for chemokines and receptors, 62°C to 42°C was used for MCP-1, RANTES, and MIP-1. The temperature then declined in 1°C increments, followed by 21 cycles at 55°C. The annealing temperature for CCR1, −2, −3, and −5 was from 67°C to 50°C, declining in 1°C increments and followed by 21 cycles at 60°C. At the end of amplification, the reaction mixture was heated for 10 minutes at 72°C and then cooled to 4°C. A 10-μL sample of each PCR product was separated by performing gel electrophoresis on 2% agarose containing ethidium bromide (Sigma-Aldrich) and then was analyzed under ultraviolet light against the DNA molecular length markers. The intensity of the products was determined by image analysis on computer (Digital 1D Science; Eastman Kodak, Rochester, NY), and the amount of PCR-amplifiable material was standardized against the amount of a housekeeping gene (rat β-actin). All experiments were repeated three times, and the results were identical. 

Quantification of Leukocytes in AqH

AqH was collected from the eyes with a 30-gauge needle immediately after the animals were killed on day 14 after immunization. The AqH was pooled in silicon-treated microcentrifuge tubes (Fisher Scientific, Pittsburgh, PA). A volume of 2 μL AqH from one rat was stained with 0.4% trypan-blue solution, and the number of leukocytes was counted under phase-contrast microscopy. 

Quantification of MCP-1 and RANTES in AqH and Serum

The levels of MCP-1 and RANTES in the AqH obtained from rats were quantified on day 14 after immunization by using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The levels of MCP-1 and RANTES in the serum samples obtained from the same rats were measured as well. The ELISA was repeated twice. The sample was diluted up to 50 μL and used for the tests. Optical density was determined at A₄₅₀ (absorbance at 450 nm) with a microplate reader (Bio-Rad). The chemokine concentration was determined from standard curves by using recombinant standards supplied by the manufacturer. 

Histopathological Evaluation

Rats were killed 14 days after immunization. The eyes were enucleated and embedded in paraffin. Then, 5-μm sagittal sections were cut and stained with hematoxylin and eosin (H-E). 

Statistical Analysis

All values in the figures and text are expressed as the mean ± SD. The clinical scores between different treatment groups were evaluated with Kruskal-Wallis nonparametric methods. Differences among the chemokine and chemokine receptor mRNA expression were evaluated by Mann-Whitney test. P < 0.05 was considered significant. 

Results

Influence of PDTC on Ocular Inflammation

The effect of intraperitoneal injection of PDTC (200 and 100 mg/kg) on rats induced with MAA is shown in Figure 1 . All rats treated with MAA began to show signs of EAAU on day 9 after immunization. The daily PDTC application was associated with a significant reduction in clinical severity, compared with the eyes of PBS-treated rats (Fig 1A) . PDTC-treated eyes showed a significantly decreased number of cellular infiltrates in the anterior chamber. The results are shown in Figure 1B . 

Histologic examination revealed an infiltration of the iris/ciliary body by leukocytes (predominantly lymphocytes) on day 14 after immunization (Fig. 2A) . The number of leukocytes in the iris/ciliary body was markedly reduced after PDTC treatment (Fig. 2B) . 

Influence of PDTC on the Activation of NF-κB in the Iris/Ciliary Body

Activated NF-κB immunoreactivity in the iris/ciliary bodies of Lewis rats was studied in paraffin-embedded sections. Immunohistochemical staining with monoclonal antibody against the p65 subunit of activated NF-κB produced a diffuse staining within the iris/ciliary in the eyes with EAAU (Fig. 3A) , but only scant staining in the eyes from rats treated with PDTC (Fig. 3B) . 

Figure 4shows the DNA-binding activity of NF-κB in nuclear extracts isolated from the iris/ciliary bodies of rats with EAAU on day 14 after immunization. The gel shift assay detected a specific band in the extracts. The NF-κB/DNA binding activity was inhibited by treatment with PDTC. The specificity of the NF-κB/DNA binding complex was confirmed by the complete displacement of NF-κB/DNA complex in the presence of a 100-fold molar excess of unlabeled NF-κB probe. In contrast, a 50-fold molar excess of unlabeled AP-2 oligo probe had no effect on the DNA-binding complex. 

Effects of PDTC on Chemokine mRNA Expression in the Iris/Ciliary Body

Recognizing the critical role played by NF-κB in regulating the inducible gene expression of a number of chemokines, we studied the possible effects of PDTC on chemokine mRNA in the iris/ciliary bodies of rats with EAAU (Fig. 5) . 

CC chemokines including MIP-1, MCP-1, and RANTES were tested. Treatment with PDTC significantly reduced the expression of MCP-1 and RANTES mRNA compared with the expression levels in the eyes from untreated rats (P = 0.002 and P = 0.001, respectively). There was no significant difference in mRNA of MIP-1 between the PDTC-treated and untreated groups. 

To assess the CXC chemokines, we investigated the effects of PDTC on IL-8, SDF-1, and IP-10 mRNA expression. PDTC significantly reduced the expression of IL-8 mRNA compared with the level in eyes obtained from untreated rats (P = 0.001). There was no significant difference in expression of SDF-1 and IP-10 mRNA between the PDTC-treated and untreated groups. 

Effects of PDTC on Chemokine Receptor mRNA Expression in the Iris/Ciliary Body

To assess the CC chemokine receptors, we investigated the effects of PDTC on the expression of CCR2 and -5 mRNA. PDTC significantly lowered the expression of CCR2 and -5 (P = 0.002 and P = 0.01, respectively; Fig. 6 ). 

For CXC chemokine receptors, the effects of PDTC on CXCR1, -2, -3, and -4 mRNA expression were analyzed. PDTC significantly decreased the mRNA expression of CXCR3 (P = 0.02), but did not change the expression of CXCR1, -2, and -4 mRNA. 

Effects of PDTC on MCP-1 and RANTES Protein Levels in Ocular AqH and Serum

To address whether inhibition of MCP-1 and RANTES mRNA expression by PDTC results in inhibition of protein expression, we compared MCP-1 and RANTES protein levels in AqH. As expected, treatment with PDTC significantly reduced the levels of MCP-1 and RANTES (Fig. 7) . The levels of MCP-1 and RANTES in the serum were measured as well. Their protein levels are significantly lower than the levels measured in the AqH, in either the control or the PTDC treatment group (Fig. 7) . 

Discussion

In this in vivo study, we clearly demonstrate that there is a significant upregulation of activated NF-κB in the iris/ciliary body during the ocular inflammation that occurs during EAAU. Inhibition of this in vivo activation by PDTC ameliorates the clinical signs of EAAU. These results are entirely consistent with the suggestion that the activation of NF-κB is a critical participant in the pathogenesis of EAAU. 

We have also clearly shown that the NF-κB inhibitor PDTC effectively suppresses the expression of the chemokine genes encoding MCP-1, RANTES, and IL-8 and the expression of the chemokine receptors genes encoding CCR2, CCR5, and CXCR3. These proteins collectively contribute to the recruitment of leukocytes during inflammation. The findings indicate that NF-κB modulates inflammatory responses through its ability to induce transcription of these proinflammatory genes during the EAAU disease process. 

It is now widely accepted that the synthesis and secretion of inflammatory chemokines play important roles in the pathophysiology of ocular inflammation. ²⁶ The expression of inducible genes leading to the formation of these proteins relies on transcription factors. NF-κB plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in inflammation. There is increasing evidence that NF-κB involves in the regulation of chemokine gene expression. ¹³ However, the exact chemokines controlled by NF-κB in EAAU have remained unknown. Our results demonstrate that PDTC inhibits MCP-1, RANTES, and IL-8 gene expression, consistent with an NF-κB-dependent transcriptional regulation of these genes. It is possible that MCP-1, RANTES, and IL-8 proteins are the main chemokines involved in the pathogenesis of EAAU. MCP-1 and RANTES are potent chemoattractants for T lymphocytes, monocytes, and NK cells, all of which are the infiltrating cells observed in the iris/ciliary body in rats with EAAU. ^{27 28 29} Il-8 not only is a potent chemoattractant for neutrophils but also is critically involved in firm cellular adhesion on endothelium, which is a necessary prerequisite for transmigration of the vessel wall. ^{8 9 30 31} Therefore, inhibition of MCP-1, RANTES, and IL-8 production by PDTC may significantly block monocyte/lymphocyte infiltration and subsequent inflammatory reactions in EAAU. 

The finding that PDTC suppresses the induction of MCP-1, RANTES, and IL-8 confirms previous reports regarding the importance of NF-κB sequences in mediating the transcriptional activities of specific chemokine promoters including those of MCP-1 and RANTES. ^{32 33} Consensus binding sequences for NF-κB have been identified in the promoter regions of human and rat genes encoding MCP-1, RANTES, and IL-8. ^{32 34} Similarly, the lack of suppression of MIP-1 by PDTC observed in the present study is consistent with the absence of consensus NF-κB-binding sites in the 5′ untranslated region of genes and favors the suggestion that the transcriptional regulation of the MIP-1 gene is entirely independent of NF-κB. ^{35 36} In contrast, the lack of suppression of IP-10 by PDTC observed in the present study was surprising, since IP-10 appears to be an NF-κB regulated chemokine. ³⁷ This discrepancy occurred because the transcriptional control of IP-10 varies depending on both the stimulus and the cell type. It is likely that other non-NF-κB enhancer sequences are sufficient for induction of the IP-10 gene in EAAU. ³⁸  

Chemokines interact with respective G-protein-coupled receptors possessing a seven-transmembrane domain. Chemokine receptors are differentially expressed on Th1 and Th2 effector cells, resulting in the distribution of these cells in the specified environments. ^{39 40} CCR2 is the primary receptor of chemokine MCP-1 and is critical in the induction of experimental autoimmune encephalomyelitis. Mice with a CCR2 deletion are protected from dextran sodium sulfate-mediated colitis. ^{41 42} CCR5 and CXCR3 are the primary receptors of RANTES and IP-10, respectively, and are predominantly found on Th1 cells. T lymphocytes expressing CCR5 and CXCR3 are enriched in rheumatoid arthritis synovial tissue, active lesions in multiple sclerosis, and the conjunctiva in vernal keratoconjunctivitis. ^{43 44 45} Quantitative analysis of the chemokine receptor expression pattern in this study revealed significantly decreased expression of CCR2, CCR5, and CXCR3 mRNA in the iris/ciliary body after PDTC treatment. This finding probably reflects a reduced number of monocytes and T lymphocytes invading the iris/ciliary body during the acute immune-mediated inflammatory process. However, it is still possible that the CCR2, CCR5, and CXCR3 distributed on the surface of monocytes/macrophages decreased after PTDC treatment. This notion is supported by Saccani et al., ⁴⁷ who found that PDTC inhibited CCR2, CCR5, and CXCR4 expression in human monocytes. ⁴⁶ Chemokine receptors are expressed in a dynamic fashion and can be modulated by cytokines and redox status at the inflammatory sites. The control of chemokine receptor expression is a decisive mechanism for regulating chemokine action. ⁴⁸ Selective downregulation of specific chemokine receptors on a given subset of leukocytes may reduce ligand binding, ultimately contributing to the decreased recruitment of these cells to the inflammatory sites. However, further studies are warranted to elucidate this possibility in greater detail. 

In this study, with the increase of IL-8 mRNA, the IL-8 receptors CXCR1 and -2 were undetectable 14 days after immunization. Because CXCR1 and -2 are mainly distributed on the surface of neutrophils, there are two possible explanations for these observations: First, the lack of expression may be due to the decreased accumulation of neutrophils. It is possible that the accumulation and disappearance of neutrophils occurs early in the course of EAAU, therefore, on day 14 after immunization, the number of neutrophils had already decreased so that only a minimal amount of CXCR1 and -2 mRNA was detected. Second, downregulation of CXCR1 and -2 on neutrophils may account for this reduction. It can be assumed that the decrease in CXCR1 and -2 may make neutrophils unresponsive to IL-8 causing fewer of them to be attracted to the iris/ciliary body. Further study with flow cytometry and early detection in the course of EAAU is needed to clarify this question. 

PDTC represents a class of antioxidants reported to be a potent inhibitor of NF-κB. ^{49 50 51} In addition, PTDC blocks IκB-α phosphorylation, precluding the dissociation of NF-κB from IκB-α and subsequent NF-κB translocation from the nucleus in response to inflammatory stimulation. ²⁴ The potential for modulating cell activation suggests that PDTC and its analogues may offer therapeutic benefit in inflammatory conditions in which activation of NF-κB plays a major role. Despite the successful alleviation of the clinical signs of EAAU by PDTC, PDTC-treated rats display retarded body weight gain, suggesting the presence of a toxic action of PDTC. ⁵² The ultimate benefit of such targeted therapy depends on the delicate balance between inflammation suppression and interference with normal cellular functions. By selectively targeting specific NF-κB subunits that have a degree of tissue specificity, one might attain therapeutic efficacy and minimize systemic toxicity. 

In conclusion, the present study demonstrated that the activation of NF-κB is markedly induced in the iris/ciliary bodies of rats during EAAU, and that this induction plays an important role in the pathogenesis of EAAU. Inhibition of NF-κB by PDTC may prevent the activation of NF-κB and the subsequent expression of chemokines and clinical signs of inflammation. 

 Presented at the annual meeting of the Association for Research and Vision and Ophthalmology, Fort Lauderdale, Florida, April 2004.

 Supported by Grant NSC 89-2314-B002-574-M47 from the National Science Council, the Executive Yuan, Republic of China.

 Submitted for publication June 3, 2004; revised October 6, 2004; accepted November 23, 2004.

 Disclosure: C.-H. Yang, None; I.-M. Fang, None; C.-P. Lin, None; C.-M. Yang, None; M.-S. Chen, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Sequences of Rat Chemokine, Chemokine Receptors, and β-Actin Primers Used in RT-PCR

Chemokine	Sequences	Product Size (bp)
β-actin	5′-CTGGAGAAGAGCTATGAGCTG-3′	246
	5′-AATCTCCTTCTGCATCCTGTC-3′
MCP-1	5′-CTGGGCCTGTTGTTCACAGTTGC-3′	436
	5′-CTACAGAAGTGCTTGAGGTGGTTG-3′
RANTES	5′-CCATATGGCTCGGACACCA-3′	201
	5′-GCTCATCTCCAAATAGTT-3′
MIP-1	5′-GAAGGTCTCCACCACTGCCCTTGC-3′	277
	5′-TCAGGCATTCAGTTCCAGCTCAG-3′
IL-8	5′-GAAGATAGATTGCACCGA-3′	365
	5′-CATAGCCTCTCACACATTTC-3′
IP-10	5′-AATGAGAAGAGGTGTCTGAATCCG-3′	287
	5′-GCCTTGCTGCTGGAGTTACTTTTG-3′
SDF-1	5′-ATTCTTTGAGAGCCATGTCGC-3′	708
	5′-CCTTGAGCTGAGTGACTCTCG-3′
CCR2	5′-AGAATGTTAAGGAAATGGTCA-3′	341
	5′-TCACCATCATCATAGTCATACG-3′
CCR5	5′-TAAGCTCAGTCTATACCCGGT-3′	336
	5′-AAAAAATCCAAGCTCGATATT-3′
CXCR1	5′-AATTTGGAAATATCACCCGAA-3′	518
	5′-GACTGTTCCAGAACGGTATGG-3′
CXCR2	5′-TATGCTGTGGTTGTCATATACG-3′	835
	5′-TGAGAAGTCCATGGCGAAATT-3′
CXCR3	5′-TCAACATCAACTTCTACGCAG-3′	378
	5′-CTAGCCTCATAGCTCGAAAGC-3′
CXCR4	5′-CATGACAGACAAGTACCGGCT-3′	475
	5′-CAGGATAAGGATGACCGTAGT-3′

Figure 1.

 

(A) Effects of PTDC on ocular inflammation displayed by clinical scores. *P < 0.05 by Kruskal-Wallis nonparametric methods. (B) Effects of PTDC on cellular infiltration in the AqH. The number of cells is expressed as the mean ± SD. Significant differences: *P < 0.05 PDTC (200 mg/kg)-treated rats versus control; #P < 0.05 PDTC (100 mg/kg)-treated rats versus control, by Mann-Whitney test.

Figure 2.

 

Effects of PDTC on histologic (H-E stain) changes in the iris/ciliary bodies of eyes 14 days after immunization. (A) Rats treated with PBS; (B) Rats treated with PDTC (200 mg/kg) daily. Magnification, ×400.

Figure 3.

 

Immunohistochemicalstain of iris/ciliary tissue with a monoclonal antibody against the p65 subunit of NF-κB. (A) Increased NF-κB p65 staining was noted in the eyes of rats with EAAU. The sample shown is typical of those examined. (B) Eye of a PDTC (200 mg/kg)-treated rat showing significantly reduced NF-κB p65 immunoreactivity.

Figure 4.

 

Electrophoretic mobility shift assessment of the DNA-binding activity of NF-κB in the iris of control and EAAU rats. Lane 1: free probe (FP); lane 2: EAAU; lane 3: twofold EAAU; lane 4: EAAU treated with PDTC (200 mg/kg); lane 5: normal animal; lane 6: p50 subunit of NF-κB; lane 7: AP-2 consensus oligonucleotide; and lane 8: 100× unlabeled NF-κB probe.

Figure 5.

 

Effect of PDTC (200 mg/kg) on chemokine mRNA expression in the iris/ciliary bodies of Lewis rats immunized with melanin-associated antigen. The intensity of mRNA was analyzed, and the relative intensity was determined by semiquantitative PCR and compared with that of β-actin. Data are presented as the mean ± SD of results in five rats. Significant differences: *P < 0.05 versus normal; #P < 0.05 versus control by Mann-Whitney test.

Figure 6.

 

Effect of PDTC on chemokine receptor mRNA expression in the iris/ciliary bodies of Lewis rats immunized with melanin-associated antigen. The rats were treated daily with PDTC (200 mg/kg) or PBS. The intensity of mRNA was analyzed with semiquantitative PCR by comparison with that of β-actin. Data are presented as the mean ± SD in five rats. Significant differences: *P < 0.05 versus normal, #P < 0.05 versus control by Mann-Whitney test.

Figure 7.

 

Effects of PDTC on MCP-1 (A) and RANTES (B) expression in the AqH and serum. The concentrations of chemokines are expressed as the mean ± SD. Significant differences: *P < 0.05, PDTC (200 mg/kg)-treated rats versus control, by Mann-Whitney test.