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(Investigative Ophthalmology and Visual Science. 2008;49:3058-3064.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.07-1390
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Upregulated IL-23 and IL-17 in Behçet Patients with Active Uveitis

Wei Chi,1,2,3,4 Xuefei Zhu,1,2,3,4,5 Peizeng Yang,1,2,3 Xiaoli Liu,1,2,3 Xiaomin Lin,1,2,3 Hongyan Zhou,1,2,3 Xiangkun Huang,1,2,3 and Aize Kijlstra3,6

1From the Zhongshan Ophthalmic Center and the 2State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, People’s Republic of China; 3Uveitis Study Center, Sun Yat-sen University, and International Uveitis Study Laboratory of Guangdong Province, Guangzhou, People’s Republic of China; 5Department of Ophthalmology, The First Affiliated Hospital of Suzhou University, Suzhou, People’s Republic of China; and the 6Eye Research Institute Maastricht, Department of Ophthalmology, University Hospital Maastricht, Maastricht, The Netherlands.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
PURPOSE. Behçet disease (BD) is a systemic inflammatory disease presumably caused by an autoimmune response. The interleukin (IL)-23/IL-17 pathway has been demonstrated to be involved in the development and maintenance of certain inflammatory diseases. This study was designed to investigate the role of IL-23 and IL-17 in BD.

METHODS. IL-23p19 mRNA in peripheral blood mononuclear cells (PBMCs) was examined using RT-PCR. The levels of IL-23, IL-17, and IFN-{gamma} in sera or PBMCs were detected by ELISA. Flow cytometry was used to evaluate the frequencies of IL-17–producing and IFN-{gamma}–producing T cells and the expression of CD45RO.

RESULTS. Results showed that the expression of IL-23p19 mRNA, IL-23, IL-17, and IFN-{gamma} was markedly elevated in BD patients with active uveitis. The frequencies of IL-17–producing and IFN-{gamma}–producing T cells from PBMCs were significantly upregulated in BD patients with active uveitis. The increased IL-17 (3.10% ± 0.53%) in BD patients with active uveitis was primarily produced by CD45RO+ memory T cells. Recombinant (r) IL-23 could upregulate IL-17 production by polyclonally stimulated PBMCs, whereas interferon (IFN)-{gamma} downregulated IL-17 production.

CONCLUSIONS. These findings reveal that the levels of IL-23, IL-17, and IFN-{gamma} are elevated in BD patients with active uveitis, and they suggest that the IL-23/IL-17 pathway together with IFN-{gamma} is associated with the active intraocular inflammation in BD patients.

Behçet disease (BD) is a chronic systemic inflammatory disease characterized by recurrent uveitis, oral aphthae, genital ulcers, and skin lesions. Although the pathogenesis of BD is still unclear, several reports have suggested that autoimmunity may play a crucial role.1 2 Previous studies suggested that BD is predominated by a Th1-type immune response. Increased Th1-associated cytokines such as interferon (IFN)-{gamma}, interleukin (IL)-12, and tumor necrosis factor (TNF)-{alpha} have been documented in BD patients.3 4 However, treatment with IFN-{alpha} and anti–TNF-{alpha} could only partially prevent the progression of BD.5 Therefore, other factors seem to be involved in the development of BD.

Recent studies have shown that IL-17 is an important proinflammatory cytokine and is upregulated in certain inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease (IBD), multiple sclerosis, uveitis including BD, and Vogt-Koyanagi-Harada disease.4 6 7 8 9 10 IL-17–producing CD4+ Th cells, defined as Th17 cells, are characterized by their production of a large amount of IL-17, IL-6, and TNF- {alpha} but a low level of IFN-{gamma}.11 IL-23, a novel member of the IL-12 family, has been shown to be necessary for the development and maintenance of certain inflammatory autoimmune disease models such as IBD, autoimmune encephalomyelitis, collagen-induced arthritis, and experimental autoimmune uveitis.11 12 13 14 15 It is also demonstrated that IL-23 is able to amplify and stabilize Th17 cells in disease models and humans.11 12 13 14 15 16 These results suggest that the IL-23/IL-17 pathway may play a role in these diseases. It is not yet clear whether the IL-23/IL-17 pathway is involved in the development of BD, though an increased level of IL-17 has been found in the sera of patients with this disease.4 Therefore, the study was designed to investigate the association of IL-23 and IL-17 with the intraocular inflammation seen in BD patients.

The present study showed that IL-23 in the sera and IL-23p19 mRNA and IL-23 protein levels in PBMCs were upregulated in BD patients with active uveitis. The production of IL-17 and IFN-{gamma} by polyclonally stimulated PBMCs and activated T cells was significantly elevated in BD patients with active uveitis. These data suggest that upregulated IL-23 and IL-17 production is associated with active intraocular inflammation in BD patients.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 References
 
Patients
Twenty-three BD patients (18 men, 5 women) referred to us from May 2005 to September 2007, with an average age of 36.3 years, and 19 healthy persons (14 men, 5 women), with an average age of 37 years, were included in this study. The diagnosis of BD disease was based on the diagnostic criteria designed by the International Study Group for BD disease.17 18 Fourteen patients showed active recurrent intraocular inflammation. These patients had received only prednisone at a low dose (<20 mg/d), but no other immunosuppressive agents, for the treatment of uveitis for more than 2 months before blood sample collection. Blood samples were collected from all these patients with active uveitis and from healthy controls. These patients showed active recurrent intraocular inflammation evidenced by dust keratic precipitates (100%), flare and cells in the anterior chamber (100%) and vitreous cells (46.7%), and retinal vasculitis observed clinically or disclosed by fluorescein angiography (100%). The extraocular manifestations were recurrent oral aphthous lesions (100%), multiform skin lesions (66.7%), recurrent genital ulcers (44.4%), and arthritis (33.3%). Nine patients showed inactive intraocular inflammation after treatment with prednisone combined with cyclosporin A for more than 3 months. Blood samples were obtained from these patients without active uveitis at least 2 months after the termination of all medications. The experimental design is described in detail. All procedures followed the tenets of the Declaration of Helsinki, and informed consent was obtained from all patients and healthy controls.

Analysis of IL-23p19 mRNA Using RT-PCR
RT-PCR analysis was performed using the reagents and procedures described previously.10

Measurement of Cytokines by ELISA
PBMCs were stimulated with or without Staphylococcus aureus Cowan I (SAC; 0.02%; Sigma-Aldrich, St. Louis, MO) for 72 hours at a density of 2 x 106 cells/mL. IL-23 concentrations were measured by human IL-23 ELISA kit (R&D Systems, Minneapolis, MN) with a detection limit of 15 pg/mL.

For determination of IL-17 and IFN-{gamma} production, PBMCs were stimulated with or without anti-CD3 (5 µg/mL; eBioscience, San Diego, CA) and anti-CD28 antibodies (1 µg/mL; eBioscience) in the presence or absence of rIL-23 (50 ng/mL), rIL-12 (1 ng/mL), or anti-IFN-{gamma} (10 µg/mL; R&D Systems) for 72 hours. The reagents and procedures were performed as described previously.10 For determination of the influence of IL-12 or IFN-{gamma} on IL-17 production, PBMCs isolated from five healthy controls were stimulated with anti-CD3 and anti-CD28 antibodies in the presence or absence of anti–IFN-{gamma} (10 µg/mL) plus rIL-12 (1 ng/mL) or rIL-23 (50 ng/mL) or in the presence of rIFN-{gamma} (100 ng/mL; eBioscience) plus anti-IL-12p70 (10 µg/mL; R&D Systems) for 72 hours. The supernatants were used for measurement of IL-17 using ELISA.

Intracellular Cytokine Staining
In total, 2 x 106 cells/mL PBMCs were stimulated with 20 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin (Sigma) for 24 hours. During the final 4 hours, 10 µg/mL Brefeldin A (Sigma) was added to the cultured PBMCs. The stimulated PBMCs were washed in PBS and incubated with phycoerythrin (PE)-cy7–labeled anti-CD8, allophycocyanin-labeled anti-CD45RO, FITC-labeled anti-CD69, or matched isotype (ebioscience; San Diego) for 30 minutes in the dark at 4°C. These PBMCs were fixed in 4% formaldehyde, permeabilized with 0.1% saponin (Sigma), and stained with PerCP-labeled anti-CD3 (BD PharMingen, San Diego, CA), PE-labeled anti–IL-17, PE-labeled anti–IFN-{gamma} (ebioscience), or matched isotype control mAb. Cells (1 x 104) were analyzed using a FACSCalibur and CellQuest software (BD PharMingen).

Statistical Analysis
Data are expressed as mean ± SD. Statistical analysis was performed using Student’s t-test and one-way ANOVA. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Patients and Methods
 Results
Discussion
 References
 
Increased Expression of IL-23p19 mRNA in PBMCs from BD Patients with Active Uveitis
Sequenced PCR products of IL-23p19 displayed 99.6% homology with the known IL-23p19 sequence. The intensity of the IL-23p19 mRNA transcript products was normalized by respective β-actin mRNA transcript products. Significantly increased expression of IL-23p19 mRNA was observed in BD patients with active uveitis (0.27 ± 0.04) without stimulation compared with that in BD patients without active uveitis (0.08 ± 0.03; P < 0.001) and healthy controls (0.09 ± 0.04; P < 0.001; Fig. 1A ). We further investigated the expression of IL-23p19 mRNA in PBMCs after stimulation with SAC. The results showed that IL-23p19 mRNA expression was significantly increased on SAC stimulation in patients and in controls. Furthermore, SAC-stimulated IL-23p19 mRNA was also significantly higher in BD patients with active uveitis (1.04 ± 0.37) than in BD patients without active uveitis (0.21 ± 0.08; P < 0.001) and healthy controls (0.16 ± 0.04; P < 0.001; Fig. 1B ).


Figure 1
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  		FIGURE 1. IL-23p19 mRNA in PBMCs from BD patients with (n = 8) or without (n = 6) active uveitis and healthy controls (n = 6). (A) IL-23p19 mRNA in unstimulated PBMCs. (B) IL-23p19 mRNA in SAC-stimulated PBMCs. IL-23p19 mRNA expression was significantly higher in BD patients with active uveitis (lanes 1–3) than in BD patients without active uveitis (lanes 4–6) and healthy control (lanes 7–9).

 
Elevated Levels of IL-23 in the Sera and the Supernatants of Cultured PBMCs from BD Patients with Active Uveitis
IL-23 levels of in the sera were significantly upregulated in BD patients with active uveitis compared with that in BD patients without active uveitis (P < 0.001) and healthy controls (P < 0.001) (Fig. 2A) . The expression of IL-23 in the supernatants of cultured PBMCs was significantly higher in BD patients with active uveitis than in BD patients without active uveitis (P < 0.001) and healthy controls (P = 0.004). On SAC stimulation, IL-23 production was obviously elevated in all the tested samples. Moreover, its elevation was significantly higher in BD patients with active uveitis than in BD patients without active uveitis (P < 0.001) and healthy controls (P = 0.002; Figure 2B ).


Figure 2
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  		FIGURE 2. IL-23 in sera and PBMCs of BD patients with (n = 8) or without (n = 6) active uveitis and healthy controls (n = 6). (A) IL-23 in serum. (B) IL-23 production by PBMCs after stimulation with or without SAC. IL-23 in sera and PBMCs were significantly higher in BD patients with active uveitis than in BD patients without active uveitis and healthy controls. Data are expressed as mean ± SD.

 
Increased Expression of IL-17 and IFN-{gamma} in the Supernatants of Stimulated PBMCs and Activated T Cells from BD Patients with Active Uveitis
The level of IL-17 was undetectable in the sera of all patients and controls. IL-17 levels in the supernatants of unstimulated PBMCs were undetectable or at a low level in BD patients and healthy controls. The production of IL-17 was strikingly increased on stimulation with anti-CD3 and anti-CD28 antibodies in patients and in healthy controls. Anti-CD3– and anti-CD28–stimulated PBMCs produced larger amounts of IL-17 in BD patients with active uveitis than in BD patients without active uveitis (P < 0.001) and healthy controls (P < 0.001; Fig. 3A ). IFN-{gamma} was also undetectable in the sera or the supernatants of unstimulated PBMCs from BD patients and healthy controls. On stimulation with anti-CD3 and anti-CD28 antibodies, the production of IFN-{gamma} by PBMCs was markedly increased in patients and in healthy controls. Such increases were significantly higher in BD patients with active uveitis than in BD patients without active uveitis (P = 0.008) and healthy controls (P = 0.003; Fig. 3B ).


Figure 3
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  		FIGURE 3. IL-17 and IFN-{gamma} production by PBMCs from BD patients with (n = 8) or without (n = 6) active uveitis and healthy controls (n = 6). (A) The production of IL-17 by polyclonally stimulated PBMCs in the presence or absence of rIL-23 (50 ng/mL) or rIL-12 (1 ng/mL). (B) The production of IFN-{gamma} by stimulated PBMCs in the presence or absence of rIL-23 or rIL-12. The production of IL-17 and IFN-{gamma} was significantly higher in BD patients with active uveitis than in BD patients without active uveitis and healthy controls. Data are expressed as mean ± SD.

 
To further investigate the phenotype and frequency of IL-17–producing T cells in BD, isolated PBMCs were stimulated with or without PMA (20 ng/mL) and ionomycin (1 µg/mL) for 24 hours and then analyzed using fluorescence-activated cell sorter (FACS). The frequency of CD69, an activation marker for T cells, in CD4+ (0.50% ± 0.12%) and CD8+ T cells (0.86% ± 0.36%) was very low without stimulation. On stimulation, CD69-expressing CD4+ and CD8+ T cells were significantly increased in frequency in BD patients (CD4+CD69+/CD4 T cells, 88.44% ± 2.11%; CD8+CD69+/CD8 T cells, 84.70% ± 1.94%) and healthy controls (CD4+CD69+/CD4 T cells, 83.22% ± 6.39%; CD8+CD69+/CD8 T cells, 88.85% ± 2.34%). There was no difference concerning the frequencies of CD4+CD69+ T cells and CD8+CD69+ T cells between BD patients and healthy controls. CD4+ and CD8+ T cells scarcely produced IL-17 and IFN-{gamma} without stimulation and abundantly produced these cytokines after stimulation with PMA and ionomycin. Furthermore, the production of IL-17 and IFN-{gamma} by CD4+ and CD8+ T cells from BD patients with active uveitis was significantly higher than from BD patients without active uveitis (P < 0.001) and healthy controls (P = 0.015). Our results also showed that the percentage of IL-17–producing CD4+ T cells was significantly higher in BD patients and healthy controls than was the percentage of IL-17–producing CD8+ T cells in patients with (P < 0.001) or without (P < 0.001) active uveitis and healthy controls (P < 0.001). On the contrary, the percentage of IFN-{gamma}–producing CD8+ T cells was higher in BD patients and healthy controls than was the percentage of IFN-{gamma}–producing CD4+ T cells (P = 0.021; Figs. 4A 4B ). In view of the fact that CD4+ and CD8+ T cells are functionally and phenotypically divided into naive and memory T cells according to differential expression of the CD45 isotype on their surfaces,19 we further investigated the expression of CD45RO on these IL-17–producing T cells. The results showed that IL-17 was principally expressed by CD4+CD45RO+ and CD8+CD45RO+ memory T cells in BD patients and healthy controls after stimulation with PMA and ionomycin. The frequencies of IL-17–producing CD4+CD45RO+ memory T cells were significantly higher in BD patients with active uveitis than in BD patients without active uveitis (P < 0.001) and healthy controls (P = 0.015; Fig. 5 ).


Figure 4
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  		FIGURE 4. The percentages of IL-17–producing and IFN-{gamma}–producing T cells from BD patients with (n = 11) or without (n = 6) active uveitis and healthy controls (n = 8). Isolated PBMCs were stimulated with PMA/ionomycin for 24 hours. Cells (1 x 104) were used for FACS analysis. (A) Representative dot plots from five independent experiments illustrated the higher frequencies of IL-17–producing and IFN-{gamma}–producing CD4+ and CD8+ T cells in BD patients with active uveitis compared with those in BD patients without active uveitis and healthy controls. We first gated CD3+ T cells and then gated CD3+CD8+ T cells and CD3+CD8– T cells. Quadrant statistics represent the frequencies of IL-17– and IFN-{gamma}–producing cells within gated CD3+CD8+ T cells and CD3+CD8– T cells. (B) Percentages of IL-17–producing and IFN-{gamma}–producing CD4+ and CD8+ T cells were significantly higher in BD patients with active uveitis than in BD patients without active uveitis and healthy controls. Data are expressed as mean ± SD.

 

Figure 5
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  		FIGURE 5. The frequencies of IL-17–producing CD4+CD45RO+ and CD8+CD45RO+ T cells from BD patients with (n = 11) or without (n = 6) active uveitis and healthy controls (n = 8). IL-17 in BD patients and healthy controls was principally expressed by CD4+CD45RO+ and CD8+CD45RO+ T cells. Data shown are representative of five independent experiments.

 
Effect of IL-23 and IL-12 on the Production of IL-17 and IFN-{gamma}
To investigate the effect of IL-23 and IL-12 on IL-17 and IFN-{gamma}, we detected IL-17 and IFN-{gamma} production in the presence of rIL-23 and rIL-12. The results showed that rIL-23 could significantly promote the production of IL-17 by polyclonally stimulated PBMCs from BD patients with (P = 0.008) or without (P = 0.009) active uveitis and healthy controls (P < 0.001). Moreover, the elevation was significantly higher in BD patients with active uveitis than in BD patients without active uveitis (P < 0.001) and healthy controls (P < 0.001). On the contrary, rIL-12 significantly inhibited IL-17 production by polyclonally stimulated PBMCs from BD patients with (P = 0.041) or without (P = 0.003) active uveitis and healthy controls (P = 0.018; Fig. 3A ).

rIL-23 and rIL-12 could significantly increase the production of IFN-{gamma} by polyclonally stimulated PBMCs in BD patients with (P = 0.011 and P < 0.001, respectively) or without active uveitis (P = 0.012 and P < 0.001, respectively) and healthy controls (P = 0.008 and P < 0.001, respectively). Furthermore, the upregulation of IFN-{gamma} by rIL-12 was significantly higher than that by rIL-23 in patients with (P < 0.001) and without (P = 0.008) active uveitis and healthy controls (P < 0.001) (Fig. 3B) .

Influence of IFN-{gamma} on IL-17 Production
It has been demonstrated that IFN-{gamma} could suppress the production of IL-17 in humans and mice.10 20 21 Our further goal was to examine whether IFN-{gamma} had the regulatory effect on IL-17 production in BD patients. The results showed that, on neutralization with ant–IFN-{gamma} antibodies, IL-17 production was significantly increased in BD patients (P < 0.001) and healthy controls (P = 0.004; Figs. 6A 6B ). Furthermore, this increase in BD patients with active uveitis was significantly higher than it was in healthy controls.


Figure 6
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  		FIGURE 6. The effect of IFN-{gamma} on IL-17 production by PBMCs. (A) Polyclonally stimulated PBMCs from BD patients (n = 8) and healthy controls (n = 6) were cultured with or without anti–IFN-{gamma} (10 µg/mL) for 72 hours. IFN-{gamma} was markedly neutralized by anti–IFN-{gamma}. (B) IL-17 production by PBMCs in the presence or absence of anti-IFN-{gamma}. IL-17 production by PBMCs was significantly elevated in added anti–IFN-{gamma}. Data are expressed as mean ± SD (C) Polyclonally stimulated PBMCs from five healthy controls were cultured with rIFN-{gamma} (100 ng/mL) and anti–IL-12p70 (10 µg/mL). rIFN-{gamma} significantly inhibited the production of IL-17 when anti–IL-12p70 was added to this culture. Polyclonally stimulated PBMCs in the presence or absence of rIL-23 (50 ng/mL) or rIL-12 (1 ng/mL) were cultured with or without anti–IFN-{gamma} (10 µg/mL). rIL-23 strongly increased IL-17 production when anti–IFN-{gamma} was added. No inhibitory effect of IL-12 on IL-17 was observed when anti–IFN-{gamma} was present.

 
Our results showed that rIL-12 induced a large amount of IFN-{gamma} production and suppressed IL-17 production. IFN-{gamma} could markedly suppress the production of IL-17. We further investigated whether IL-12 exerted its inhibitory effect on IL-17 through IFN-{gamma}. The results showed that rIFN-{gamma} could significantly inhibit IL-17 production when anti–IL-12p70 was used in this culture (P = 0.009). rIL-12 did not suppress the production of IL-17 after IFN-{gamma} was neutralized, demonstrating that the suppressive effect of IL-12 on IL-17 is mediated by IFN-{gamma}. Furthermore, our study showed that a larger amount of IL-17 was produced when rIL-23 plus anti–IFN-{gamma} was used in this culture (Fig. 6C) .


   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
The present study showed that IL-23p19 mRNA in PBMCs, IL-23 in serum and supernatants of PBMCs, and IL-17 and IFN-{gamma} production in supernatants of PBMCs were all markedly increased in BD patients with active uveitis. Significantly upregulated IL-17– and IFN-{gamma}–producing T cells were also observed in BD patients with active uveitis. IL-17 was mainly expressed by CD45RO+ memory T cells. It was also demonstrated that IL-23 could promote IL-17 production, whereas IFN-{gamma} downregulated IL-17 production. All these findings suggest that IL-23 and IL-17 are associated with active intraocular inflammation in BD patients.

IL-23 has been demonstrated to play a pivotal role in some inflammatory autoimmune disease models and to be associated with certain human diseases.11 12 13 14 15 22 23 Accordingly, the role of IL-23 in the pathogenesis of BD was investigated in this study. First, our study showed that IL-23 was upregulated in the sera of BD patients with active uveitis compared with BD patients without active uveitis and controls. Second, IL-23p19 mRNA and IL-23 protein in PBMCs were augmented in BD patients with active uveitis. Third, our study revealed that SAC-stimulated PBMCs produced heightened IL-23p19 mRNA and IL-23 in BD patients with active uveitis. All these results suggest that upregulated IL-23 is associated with the active intraocular inflammation seen in BD.

Given that IL-17 is one of primary effectors involved in the mechanism of IL-2312 24 and that an increased expression of IL-23 in BD patients with active uveitis is observed in our experiments, we tested the expression of IL-17 in BD patients. Our results showed that IL-17 production by polyclonally stimulated PBMCs and activated T cells was markedly elevated in BD patients with active uveitis compared with that in patients without active uveitis and in controls. Moreover, our study revealed that most IL-17–producing T cells were CD4+CD45RO+ and CD8+CD45RO+; the former were predominant in patients and healthy controls. These results are consistent with those reported by Shin et al.19 In their study, IL-17 mRNA is demonstrated to be primarily expressed in CD4+CD45RO+ and CD8+CD45RO+ memory T cells from humans after polyclonal stimulation. Given that human CD45RO+ T cells are defined as antigen-experienced memory T cells,19 25 it is reasonable to presume that IL-17 is mainly produced by CD4+CD45RO+ memory T cells. More important, our results showed that the frequencies of IL-17–producing CD4+ CD45RO+ and CD8+CD45RO+ T cells were higher in BD patients with active uveitis than in BD patients without active uveitis and healthy controls. In view of the high pathogenicity and the crucial effect of IL-17–producing Th cells in the development and maintenance of certain autoimmune diseases,9 10 11 it is likely that a large amount of IL-17–producing CD4+CD45RO+ memory T cells correlates with the active intraocular inflammation in BD patients.

Because the upregulation of IL-23 and IL-17 in BD patients with active uveitis was observed in our study, we further tested the influence of IL-23 on IL-17 production. Our results showed that rIL-23 could promote the production of IL-17 by polyclonally stimulated PBMCs from BD patients and healthy controls. Moreover, rIL-23 promoted higher IL-17 in BD patients with active uveitis than in patients without active uveitis and healthy controls. These results suggested that upregulated IL-23 in BD patients with active uveitis may exert its role by promoting IL-17 production. The upregulated IL-23 and IL-17 production in BD patients with active uveitis after nonspecific stimulation in this study is consistent with the results in VKH patients with active uveitis and patients with psoriasis.10 23 Upregulated IL-23 or IL-17 has also been observed in patients with IBD22 or scleritis and uveitis,9 respectively. It is well known that these human autoimmune diseases display different clinical manifestations and pathologic features and that different autoantigens are involved in them. It is likely that IL-23/IL-17 may be a common pathway for these autoimmune diseases. Targeted manipulation of the IL-23/IL-17 pathway may provide insight into a new strategy for these diseases. However, it must be pointed out that upregulated IL-23 and IL-17 production in BD patients with active uveitis was found with nonspecific stimulation. Studies have shown that autoimmune responses to a number of antigens, such as S-antigen and interphotoreceptor retinoid-binding protein, are involved in the development of this disease.1 2 26 A recent study by us (not yet published) did not show any increased production of IL-17 by PBMCs from BD patients or healthy controls on stimulation with S-Ag peptides (data not shown). Nevertheless, this result does not exclude the possibility that another antigen-driven IL-23/IL-17 pathway is involved in this disease. More studies are needed to address this problem.

Because IFN-{gamma} was considered an important mediator involved in the development of BD,3 4 a study was also performed to detect the expression of this cytokine, and importantly, to evaluate the influence of IL-23 on its production. Our results showed a significantly upregulated expression of IFN-{gamma} in BD patients with active uveitis, confirming previous reports. Interestingly, our results also showed that IL-23 could significantly promote IFN-{gamma} production. These results are consistent with those observed by us in Vogt-Koyanagi-Harada disease10 and with those reported by Hoeve et al.20 and suggest that IL-23 may also exert its function through induction of IFN-{gamma}. Given that IL-12 is classically considered an inducer of IFN-{gamma} production,27 we also tested its effects on this cytokine. The study revealed a similar result. It was also found that IL-12 and IFN-{gamma} could inhibit the expression of IL-17. Because IFN-{gamma} is the downstream cytokine of IL-12, we tested whether IL-12 inhibited IL-17 production directly or by the induction of IFN-{gamma}. Our study showed that IL-12 exerted its inhibitory effect on IL-17 through IFN-{gamma}. This finding is similar with that reported by Harrington et al.28 All these results suggest that a negative modulatory mechanism by IFN-{gamma} is present in BD patients and that this mechanism may contribute, to a certain extent, to the outcome of the inflammation seen in these patients.

In conclusion, our study showed elevated production of IL-23, IL-17, and IFN-{gamma} by PBMCs and increased frequencies of IL-17–producing and IFN-{gamma}–producing T cells in BD patients with active uveitis. It also showed that IL-17 was principally produced by CD45RO+ memory T cells. All these results suggest that the IL-23/IL-17 pathway is associated with active uveitis in BD patients and that this pathway may be involved in the pathogenesis of BD. Studies on the manipulation of this pathway may warrant its role and also provide a strategy for the treatment of this disease.


   Footnotes
 
4 These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors. Back

Supported by the Fund for Innovative Research Groups of Guangdong Province (05200176), the Key Project of Natural Science Foundation (30630064), and the International Cooperation Project of Guangdong Province (3030901005077).

Submitted for publication October 30, 2007; revised December 6, 2007, and January 21, 2008; accepted May 12, 2008.

Disclosure: W. Chi, None; X. Zhu, None; P. Yang, None; X. Liu, None; X. Lin, None; H. Zhou, None; X. Huang, None; A. Kijlstra, 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.

Corresponding author: Peizeng Yang, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, People’s Republic of China; peizengy{at}126.com.


   References
Top
 Abstract
 Patients and Methods
 Results
 Discussion
 References
 

  1. Yamamoto JH, Minami M, Inaba G, Masuda K, Mochizuki M. Cellular autoimmunity to retinal specific antigens in patients with Behçet’s disease. Br J Ophthalmol. 1993;77(9)584–589.[Abstract/Free Full Text]
  2. de Smet MD, Dayan M. Prospective determination of T-cell responses to S-antigen in Behçet’s disease patients and controls. Invest Ophthalmol Vis Sci. 2000;41(11)3480–3484.[Abstract/Free Full Text]
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