[Clinical research of anterior chamber inflammation after phacoemulsification surgery in patients with complicated cataract secondary to uveitis].

OBJECTIVE: To investigate anterior chamber inflammation after phacoemulsification with intraocular lens (IOL) implantation in patients with Vogt-Koyanagi-Harada (VKH) syndrome or Behçet's disease (BD). METHODS: Cohort study. Seventeen patients (20 eyes) with complicated cataracts and VKH syndrome or BD who underwent phacoemulsification with IOL implantation at Zhongshan Ophthalmic Center, Sun Yat-Sen University (SYSU) between January 2010 and June 2011 were included as the experimental group in this study. Cataract surgery was performed on these patients only when uveitis had been under control for more than three months. Thirty patients (40 eyes) with age-related cataracts who underwent phacoemulsification with IOL implantation in the same period were included as the control group. Quantitative measurements of anterior chamber aqueous flare and inflammatory cells were conducted preoperatively and postoperatively using a Laser Flare Cell Meter (LFCM). Independent t-test was used to compare patients' ages, and the energy and time of phacoemulsification between the two groups. The Student's t-test was used to assess the differences between paired data preoperatively and postoperatively. Independent t-test was also used to assess the quantitative data between groups. RESULTS: The study recruited 20 eyes in the experimental group and 40 eyes in the control group, including 11 eyes from 9 VKH patients and 9 eyes from 8 BD patients. The preoperative and postoperative flare values in the experimental group were (19.86 ± 6.47), (44.28 ± 18.47), (35.60 ± 12.65), (23.85 ± 8.41), and (13.86 ± 4.27) pc/ms, respectively, which were statistically higher than that of the control group preoperatively, and on days 1, 7, 30, and 90 after surgery (tpre = 4.643, Ppre < 0.01; t1 = 6.035, P1 < 0.01; t7 = 3.595, P7 = 0.001; t30 = 4.658, P30 < 0.01; t90 = 3.308, P90 = 0.002). Aqueous flare in Group A and Group B declined to preoperative levels on day 30 (t = 0.320, P = 0.753) and day 7 (t = 0.454, P = 0.653). For the experimental group, the inflammatory cell count on day 1 and 7 was (83.46 ± 27.08) and (27.56 ± 8.32) cells/0.5 mm(3), respectively, which was significantly higher than the preoperative level [(6.47 ± 3.56)cells/ 0.5 mm(3), t1 = 5.261, P1 < 0.01; t7 = 2.766, P7 = 0.012]. On days 30 and 90, the inflammatory cell count was (11.43 ± 4.81) and (4.82 ± 2.29) cells/0.5 mm(3), respectively, and there was no statistically significant difference in the inflammatory cell count compared with the preoperative level (t30 = 2.348, P30 = 0.042; t90 = 1.376, P90 = 0.186). For the control group, inflammatory cell count reduced to pre-operative level on day 7 (t7 = 2.464, P7 = 0.018). CONCLUSIONS: Anterior chamber inflammation reaches peak levels one week postoperatively in VKH and BD patients who receive phacoemulsification with IOL implantation. It takes three months for the inflammation to recede, and might take longer for complete restoration of the blood-aqueous barrier.

Serum levels of Th17-related cytokines in Behcet disease patients after cataract surgery.

PURPOSE: To investigate the profile of T-helper type 17 (Th17) cell-related cytokines (interleukin-23 [IL-23], IL-27, IL-17 and interferon-γ [IFN-γ]) in postoperative inflammation in patients with Behcet disease (BD) after cataract surgery. METHODS: Serum was collected from seven BD patients with complicated cataract, and from nine controls with uncomplicated cataract, before cataract surgery, and again 1, 7, 30, and 90 days after surgery. In addition, aqueous humor was collected at commencement of surgery. The protein levels of IL-23, IL-27, IL-17, and IFN-γ in the serum and in the aqueous humor were measured by an enzyme-linked immunosorbent assay. A laser flare-cell photometer was used to quantify intraocular inflammation. RESULTS: Serum IL-23, IL-27, and IFN-γ levels were significantly increased after cataract surgery in the BD versus the control patients. In the BD patients, serum levels of IFN-γ and IL-27 correlated strongly with aqueous flare values and cell counts. Remarkably, the levels of serum IL-27 were significantly associated with serum IFN-γ levels in BD patients (r=0.796; p=0.002). CONCLUSIONS: Our data indicates that serum IFN-γ and IL-27 levels are significantly elevated in BD versus control patients and are strongly associated with post-operative intraocular inflammation.

[The blood-aqueous barrier changes after laser peripheral iridotomy or surgery peripheral iridectomy].

OBJECTIVE: To investigate the ocular blood-aqueous barrier (BAB) alteration after laser peripheral iridotomy (LPI) or surgery peripheral iridectomy (SPI) in patients with primary chronic angle-closure glaucoma (PCACG). METHODS: This was a clinical randomized controlled trial. Sixty eyes of 60 subjects with early stage of PCACG were randomly received either LPI or SPI and followed up postoperatively at day 3, week 1, 2, 3, and 4. Aqueous flare in anterior chamber was measured by FC-2000 flare-cell photometry, intraocular pressure (IOP) measured by tonometer, central corneal endothelium cell counted by endothelioscopy, peripheral anterior synechiae (PAS) detected by gonioscopy. Data were analyzed by using two-way ANOVA for repeated measures, independent samples t-test, paired t-test, nonparametric test, and Spearman rank correlation test. RESULTS: On follow-ups of pre-operative and post-operative 3 days, 1 week (w), 2w, 3w and 4w respectively, the mean aqueous flare values for LPI group were (5.47 ± 1.09), (11.96 ± 3.07), (8.08 ± 2.18), (5.68 ± 0.83), (5.80 ± 1.00), (5.69 ± 1.12) PC/ms, and for SPI group were (5.43 ± 1.13), (8.44 ± 3.22), (6.42 ± 1.77), (5.35 ± 0.71), (5.53 ± 1.26), (5.45 ± 1.23) PC/ms. During post-operative 1w the flare values in both LPI and SPI groups were significantly higher than that on pre-operation (t = -12.753, -8.101, P < 0.05; t = -5.971, -3.870;P < 0.05) and LPI group had a significantly higher mean flare value than SPI group (t = 4.329, 3.231;P < 0.05). The IOP spike in LPI group was significantly (χ(2) = 5.079, 4.022, P < 0.05) higher than that in SPI group at week 1 of post-operation. Increased IOP was positively correlated with BAB damage (r = 0.899, 0.833; P < 0.05). The numbers of medications required to maintain IOP ≤ 21 mm Hg (1 mm Hg = 0.133 kPa) at week 4 of post-operation in LPI was significantly (Z = -1.984, P < 0.05) more than that in SPI group. There were no significant differences in central corneal endothelium cell count at week 1 (t = -0.696, 0.008) and in extension of PAS at week 4 (Z = -1.270, -1.490) of post-operation when compared to pre-operation (P > 0.05). No obvious complications occurred in both groups. CONCLUSIONS: Our results demonstrated that IOP spike in both of LPI and SPI is due, at least in part, to BAB damage, which appears to be more severe in LPI group and can recover within two weeks. PAS progression and central corneal endothelium cell loss are not aggravated in 1 month after operation.

Damage to the blood-aqueous barrier in eyes with primary angle closure glaucoma.

PURPOSE: This study investigates the inflammation in the anterior chamber in eyes with primary angle closure glaucoma (PACG) and evaluates the effect of intraocular pressure (IOP) elevation on the blood-aqueous barrier (BAB). METHODS: Thirty-five patients (35 eyes) with acute primary angle closure glaucoma (APACG), 42 patients (42 eyes) with chronic primary angle closure glaucoma (CPACG), and 50 age-matched healthy controls (50 eyes) were included in this study. The flare value and cell counts were quantified using laser flare cell photometry. Statistical analysis was performed to compare differences in flare value and cell counts between different groups and explore the relation between the inflammation and IOP. RESULTS: The mean flare value (photon counts per millisecond, ph/ms) in the APACG, CPACG, and healthy control group was 141.4±123.1, 7.7±4.1, and 4.5±1.1, respectively. The mean cell counts (cells/0.5 mm(3)) in the three groups were 126.0±67.8, 5.2±5.8, and 0.8±0.7, respectively. The flare value and cell counts in both the APACG group and the CPACG group were significantly higher than those in the healthy control group (p<0.001). Furthermore, the flare value and cell counts in the APACG group were significantly higher than those in the CPACG group (p<0.001). There were positive correlations between the IOP level and flare value (r=0.527, p<0.001), and cell counts(r=0.775, p<0.001), respectively, in the APACG group. CONCLUSIONS: Disrupted BAB and inflammation in the anterior chamber were found in eyes with both kinds of PACG. The damage of BAB was more severe in eyes with APACG than those with CPACG. The IOP elevation, especially a dramatic IOP elevation, might be the factor responsible for the change of BAB in eyes with PACG.

Production of interleukin-17 in Behcet's disease is inhibited by cyclosporin A.

PURPOSE: Behcet's disease (BD) is a systemic inflammatory disease presumably caused by an autoimmune response. Interleukin (IL)-17 has been demonstrated to be involved in the development and maintenance of certain inflammatory diseases, including BD. This study was designed to investigate the influence of cyclosporine A (CsA) on IL-17 production by peripheral blood mononuclear cells (PBMCs) from BD patients in vitro and in vivo. METHODS: Fifteen BD patients with active uveitis were involved in this study. Blood samples were taken from these patients for analysis of IL-17 and interferon (IFN)-gamma. Six patients were re-evaluated at 1 and 3 months after treatment with CsA. The levels of IL-17 and IFN-gamma in the supernatants of PBMCs from patients before treatment cultured without or with CsA at different concentrations were detected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to evaluate the frequencies of IL-17-producing and IFN-gamma-producing T cells and the expression of CD69 on CD4(+) or CD8(+) T cells before, 1, and 3 months after CsA treatment. RESULTS: The results showed that significantly higher levels of IL-17 and IFN-gamma were observed in active BD patients as compared with controls. Treatment with CsA could inhibit the production of both cytokines in association with an amelioration of intraocular inflammation. In vitro, CsA significantly inhibited the production of IL-17 and IFN-gamma by PBMCs activated with anti-CD3 and anti-CD28 antibodies or phorbol 12-myristate,13-acetate and ionomycin in BD patients with active uveitis. However, CSA did not influence the CD69 expression in CD4(+) and CD8(+) T cells induced by phorbol 12-myristate,13-acetate (PMA) ionomycin. CONCLUSIONS: Our findings showed that CsA can significantly inhibit the intraocular inflammation of BD patients and the expression of IL-17 and IFN-gamma in vivo and in vitro. The results suggested that the inhibitory effect of CsA on uveitis in BD patients may be partially mediated through inhibiting the production of IL-17 and IFN-gamma.

Elevated serum IL-23 correlates with intraocular inflammation after cataract surgery in patients with Vogt-Koyanagi-Harada disease.

PURPOSE: Patients with Vogt-Koyanagi-Harada (VKH) disease are known to have severe inflammation after cataract surgery. This study was designed to investigate the role of IL-17-producing T helper (Th17) cell-related pro-inflammatory cytokines on postoperative inflammation in VKH patients. METHODS: Serum from nine VKH patients and nine controls with age-related or congenital cataract was collected before and 1, 7, 30 and 90 days after surgery, and aqueous humor (AqH) at the commencement of surgery. Protein levels of IL-23, IL-27, IL-17 and IFN-gamma in serum and AqH were measured by ELISA. A laser flare-cell photometer was used to quantify intraocular inflammation. RESULTS: Serum IL-23 levels were significantly increased in VKH compared with control patients and peaked at 1 day postoperative, decreased rapidly in the first week, then attenuated gradually. In VKH patients, serum levels of IFN-gamma were elevated in the first week after surgery and IL-27 was upregulated in the first month. Importantly, serum IL-23 levels were strongly correlated with aqueous flare value (r=0.689; p=0.007) and cell counts (r=0.671; p=0.01) in VKH compared with control patients. CONCLUSIONS: The data indicate that serum IL-23 levels are significantly elevated in VKH compared with control patients and are strongly associated with postoperative intraocular inflammation.

Alterations of aqueous flare and cells detected by laser flare-cell photometry in patients with Behcet's disease.

PURPOSE: To quantitatively evaluate aqueous flare and cells in patients with Behcet's disease. METHODS: This study included 30 Behcet's patients (52 eyes) with active uveitis. The patients were treated with immunosuppressive agents. Aqueous flare and cells were quantified using the laser flare-cell photometry before treatment and 1 and 2 months after treatment. RESULT: Before treatment, mean aqueous flare (ph/ms) in Behcet's eyes was 25.7 ± 20.5. After treatment, flare values were significantly reduced after 1 and 2 months compared with those before treatment. No significant difference was found between flare values after 1 and 2 months. Before treatment, mean cell counts (cells/0.5 mm(3)) in Behcet's eyes were 23.2 ± 29.4. After treatment, cell counts were also significantly reduced after 1 and 2 months compared with those before treatment. Cell counts were further significantly reduced from 1 to 2 months. CONCLUSION: Both aqueous flare and cells were significantly increased in Behcet's patients with active uveitis and improved after a two-month treatment. Breakdown of the blood-aqueous barrier lasts longer than aqueous cells in these patients.

Inhibitory effect of Cyclosporin A and corticosteroids on the production of IFN-gamma and IL-17 by T cells in Vogt-Koyanagi-Harada syndrome.

Cyclosporin A (CsA) and corticosteroids are extensively used in the treatment of autoimmune diseases including Vogt-Koyanagi-Harada (VKH) syndrome. The exact immunosuppressive mechanisms of these drugs are not exactly known. Th1 and Th17 cells are important populations involved in autoimmune diseases. In this study, we investigated whether they are involved in VKH syndrome and how their function is affected by CsA and corticosteroids. The results showed that IL-17, IFN-gamma, RORgammat and T-bet were upregulated in patients with active uveitis. CsA and corticosteroids were able to downregulate all these elevated levels which correlated with the clinical improvement of the uveitis. In vitro experiments showed that CsA and dexamethasone could decrease the frequencies of Th1 and Th17 cells and inhibit IL-17 and IFN-gamma production. These data suggest that an upregulated Th1 and Th17 response is associated with active VKH syndrome. CsA and corticosteroids may exert their immunosuppressive role by downregulating Th1 and Th17 cells.

Splenic CD8+ T cells secrete TGF-beta1 to exert suppression in mice with anterior chamber-associated immune deviation.

Background CD8+ regulatory T cells (Treg) have been considered to be involved in a model of ocular-induced tolerance, known as anterior chamber-associated immune deviation (ACAID). The mechanisms of suppression by CD8+ T cells in ACAID remain only poorly understood. TGF-beta1 is considered as an inhibitory cytokine for immunosuppression in some models. The production of TGF-beta1 by CD8+ T cells in ACAID, and whether CD8+ T cells exert suppression through TGF-beta1, is unknown. Methods The suppressive effect of CD8+ T cells in ACAID mice was determined by a local adoptive transfer (LAT) assay. The production of TGF-beta1 by CD8+ T cells was measured by enzyme-linked immunosorbent assay (ELISA). Anti-TGF-beta1 antibodies were used in the LAT assay to test if they could block the inhibitory effect of CD8+ T cells. Results CD8+ T cells from ACAID mice were shown to block the delayed-type hypersensitivity (DTH) response in an antigen-specific manner in a LAT assay. These CD8+ T cells secreted TGF-beta1, and their suppression could partially be blocked by anti-TGF-beta1 antibodies. Conclusions Our study confirms that CD8+ T cells from ACAID mice possess inhibitory properties. This population exerts part of its suppressive function via the production of TGF-beta1.

[Expression of programmed death 1 and its ligands in iris-ciliary body from mice with anterior chamber-associated immune deviation].

OBJECTIVE: To study the expression and possible implication of programmed death 1 (PD-1) and its ligands in the iris-ciliary body from mice with anterior chamber-associated immune deviation (ACAID). METHODS: This was an experimental study. Twenty-four BALB/c mice were divided into ACAID group, negative controls, positive controls and phosphate-buffer saline controls. ACAID was evaluated by delayed-type hypersensitivity response. Expressions of PD-1 and its ligands (PD-L1, PD-L2) in the iris-ciliary body from ACAID mice were determined by real-time polymerase chain reaction and immunohistochemical studies. RESULTS: Delayed-type hypersensitivity was not detected in ACAID group, indicating that ACAID was induced successfully. The expression of PD-1 mRNA in ACAID group was higher than that in all other groups (F = 248. 109, P < 0.05). The positive controls showed a high expression of PD-L1 mRNA (F = 179. 033, P < 0.05). PD-1 and PD-L1 positive cells with round or oval shapes were found in whole iris-ciliary body, especially in the iris base and pupil margin. Expression of PD-1 and PD-L1 proteins were similar to their expression at mRNA levels. Neither mRNA nor protein of PD-L2 was detected in the iris-ciliary body in all groups. CONCLUSION: An increased expression of PD-1 in the iris-ciliary body of ACAID mice indicates that this is involved in the induction of ACAID.

[Comparison of different power-density krypton laser combined with Nd:YAG laser peripheral iridotomy].

PURPOSE: To compare the influence of krypton laser with different power densities combined with Nd:YAG laser peripheral iridotomy (LPI) on the intraocular pressure, blood-aqueous barrier and inflammatory of anterior chamber as well as the therapeutic effect. METHODS: Using a laser flare cell meter and Goldmann tonometer, the level of aqueous protein, the number of cells in the anterior chamber and intraocular pressure of 31 patients (62 eyes) who underwent krypton laser with different power densities combined with Nd:YAG laser peripheral iridotomy were examined and recorded preoperatively and postoperatively. RESULTS: The mean preoperative and 1-hour, 3-day, 7-day, 1-month postoperative intraocular pressure (IOP) of the high power-density group were (15.68 +/- 2.41), (27.13 +/- 3.48), (20.97 +/- 5.27), (16.35 +/- 1.14) and (15.06 +/- 2.02) mmHg, while those of the low were (15.35 +/- 1.78), (22.77 +/- 3.26), (16.26 +/- 2.41), (15.68 +/- 2.06) and (15.06 +/- 1.36) mmHg. The mean preoperative and 3-day, 7-day, 1-month postoperative flare intensity of the high power-density group were (4.65 +/- 1.50), (10.41 +/- 2.47), (7.31 +/- 2.31) and (6.15 +/- 2.16) pc/ms, while those of the low were (4.45 +/- 1.19), (6.47 +/- 1.11), (4.81 +/- 0.55) and (4.98 +/- 1.48) pc/ms. The number of aqueous cells of the high was (0.47 +/- 0.42), (36.22 +/- 9.16), (18.54 +/- 3.60) and (6.29 +/- 0.98), while that of the low was (0.58 +/- 0.52), (24.73 +/- 6.09), (10.61 +/- 1.70) and (2.96 +/- 1.35). The mean 1-hour and 3-day postoperative IOP of the high was higher than that of the low. Both the mean flare intensity and the mean number of aqueous cells of the high power-density group were higher than those of the low. The differences were of statistical significance (P < 0.05). The mean flare intensity of the high power-density group in the 1-month postoperative follow-up was still higher than the baseline. The mean number of aqueous cells of both the high and the low power-density groups in the 1-month postoperative follow-up was still higher than the baseline. During 1-month follow-up, no obvious visual damage, diffuse corneal endothelial burns or corneal decompensation, lens injury and closure of the peripheral iris incision were observed. CONCLUSION: When krypton laser combined with Nd:YAG laser peripheral iridotomy is under consideration, relatively low power-density krypton laser is recommended because it can achieve the similar therapeutic effects as high power-density krypton laser but leads to less complications and a briefer recovery. More follow-ups are needed after LPI, because the number of aqueous cells in 1-month follow-up was still abnormal.

MALDI-TOF/TOF-MS reveals elevated serum haptoglobin and amyloid A in Behcet's disease.

Behcet's disease (BD) is a multisystemic autoimmune disease with unclear etiology and pathogenesis. To screen aberrant serum proteins in BD, serum samples were obtained from eight male BD patients with active uveitis and eight male healthy volunteers with informed consent. The serum samples from active BD patients and normal controls were pooled. Highly abundant serum proteins (albumin and IgG) were depleted from these two samples using an affinity capture based kit. The obtained samples were subjected to two-dimensional gel electrophoresis (2-DE). Protein spots were visualized with the "blue silver" staining. Differently expressed proteins were subsequently identified by matrix-assisted laser desorption /ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Western blot and enzyme-linked immunosorbent assay (ELISA) were performed using the serum samples from 18 patients with active BD, 6 patients with inactive BD, 22 patients with Vogt-Koyanagi-Harada (VKH) syndrome, and 20 healthy volunteers to validate the results of 2-DE and MS. Proteomic profiles of the pooled samples were compared, and approximately 800 protein spots were observed in each of the gels. Expression levels of four of the protein spots in active BD were significantly higher than those in the normal controls. Mass spectrometric protein identification revealed that the four protein spots corresponded to two proteins: haptoglobin (Hp) and serum amyloid A (SAA). Western blot and ELISA showed that Hp was only overexpressed in active BD but not in inactive BD, VKH syndrome, or healthy controls. An obvious band of SAA was detected in 72.2% of the serum samples from BD patients, whereas a vague band of this protein was found in 10.0% of the tested normal samples and 9.1% of VKH samples. Our results revealed a significantly increased expression of Hp and SAA in serum of active BD patients. These two proteins may be involved in the development of BD.

S-antigen specific T helper type 1 response is present in Behcet's disease.

PURPOSE: To investigate the frequency and phenotypic and functional characteristics of S-antigen (S-Ag) specific T cells in patients with Behcet's disease (BD). METHODS: Blood was taken from 23 active BD patients, 12 inactive BD patients, and 14 healthy controls. The clinical features of the patients were summarized. T cell response against 40 mixed S-Ag peptides was identified by interferon gamma (IFN-gamma) enzyme-linked immunospot assay (ELISPOT). CD69 and CD45RO were used to characterize the phenotype of S-Ag specific T cells. The functional property of S-Ag specific T cells was investigated by measuring the production of cytokines. RESULTS: Response to the mixed S-Ag peptides was found in 56.5% and 25% of active and inactive BD patients, respectively. The responsiveness to S-Ag peptides was unrelated to the clinical features of the patients. About 65.8% of IFN-gamma(+) CD4(+) T cells in active BD patients expressed CD69 and CD45RO concomitantly. S-Ag peptides significantly induced a production of IFN-gamma and tumor necrosis factor (TNF)-alpha but not interleukin (IL)-2, IL-4, and IL-17 by peripheral blood mononuclear cells (PBMCs) in active BD patients with a response to S-Ag. CONCLUSIONS: S-Ag specific T cells are present in certain active BD patients, and most of them are activated memory CD4(+) T cells. These T cells may be involved in the pathogenesis of BD via producing Th1-dominant cytokines.

Upregulated IL-23 and IL-17 in Behçet patients with active uveitis.

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.

Involvement of Foxp3-expressing CD4+ CD25+ regulatory T cells in the development of tolerance induced by transforming growth factor-beta2-treated antigen-presenting cells.

A growing body of evidence has shown that professional antigen-presenting cells (APC) treated with transforming growth factor-beta (TGF-beta) can induce a systemic antigen (Ag)-specific tolerance, similar to anterior chamber-associated immune deviation (ACAID). However, the exact mechanism for immune tolerance induced by TGF-beta-treated APC has not been elucidated. In this study, we showed that intravenous injection of ovalbumin (OVA)-pulsed APC treated with TGF-beta(2) induced a peripheral tolerance as evidenced by an impaired delayed-type hypersensitivity (DTH) response. CD4(+) T cells from mice receiving an intravenous injection of TGF-beta(2)-treated APC pulsed with OVA could adoptively transfer a specific tolerance to naïve mice. An increased frequency of FoxP3-expressing CD4(+) CD25(+) T cells was observed in mice with tolerance. CD4(+ )CD25(+) T cells from TGF-beta(2)-treated APC-injected mice produced a large amount of TGF-beta(1) and exhibited an in vitro antigen-specific suppressive activity. CD4(+) CD25(+) T cells from TGF-beta(2)-treated APC-injected mice were able to inhibit the antigen-specific DTH response significantly when adoptively transferred to naïve mice. These results indicate that FoxP3-expressing CD4(+) CD25(+) T cells might be actively involved in the development of tolerance induced by TGF-beta(2)-treated APC.

Association of the CTLA-4 gene with Vogt-Koyanagi-Harada syndrome.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), a critical negative regulator of the T cell response, has been shown to be associated with a variety of autoimmune diseases. In this study, we investigated the association of CTLA-4 gene polymorphisms (- 1661A/G; - 318C/T; + 49G/A, and CT60) with Vogt-Koyanagi-Harada (VKH) syndrome in Chinese Han patients and normal controls. The results showed that the frequency of the G allele at the + 49 site was significantly higher in VKH patients than that observed in healthy controls (71.6% versus 62.8%, P = 0.0046, Pc = 0.037). Three haplotypes were identified from the four SNPs. The frequency of haplotype - 1661A:- 318C:+ 49G:CT60G, the most prevalent haplotype both in patients and controls, was significantly higher in patients than that in controls (70.1% versus 60.0%, P= 0.0013, n= 16, Pc = 0.021). These results suggest that CTLA-4 genetic polymorphisms are associated with the susceptibility to VKH syndrome.

Increased expression of Foxp3 in splenic CD8+ T cells from mice with anterior chamber-associated immune deviation.

PURPOSE: To examine the expression of Foxp3 on CD8+ T cells in the spleen during anterior chamber-associated immune deviation (ACAID). METHODS: Ovalbumin (OVA) was injected into the anterior chamber (AC) of C57BL/6 mice, and the delayed-type hypersensitivity (DTH) response was measured to evaluate the development of ACAID. The suppressive effect of CD8+ T cells in ACAID mice was determined by a local adoptive transfer (LAT) assay. Flow cytometry was used to assay the frequency of CD8+ Foxp3+ T cells from normal mice, ACAID mice, and control mice receiving an AC injection of PBS (PBS-AC-injected mice). These frequencies were also tested after polyclonal or specific antigen stimulation. The mRNA level of Foxp3 in CD8+ splenocytes from different groups with or without stimulation were determined by reverse transcription-polymerase chain reaction. RESULTS: OVA injection into the AC induced ACAID, and CD8+ T cells from ACAID mice inhibited the ear-swelling response by OVA-primed responder cells in LAT assay. Flow cytometry analysis showed that the frequency of CD8+ Foxp3+ cells in splenocytes was upregulated in ACAID mice following polyclonal or specific antigen stimulation. Foxp3 mRNA was only detected in CD8+ T cells from ACAID mice after polyclonal stimulation. CONCLUSIONS: An upregulated Foxp3 expression in CD8+ T cells is associated with the development of ACAID, suggesting an involvement of CD8+ Foxp3+ T cells in this model of immune tolerance.

CD4+CD25+Tregs express an increased LAG-3 and CTLA-4 in anterior chamber-associated immune deviation.

BACKGROUND: Regulatory CD4(+)CD25(+) T cells have been proven to be essential for maintenance of peripheral tolerance and autoimmune diseases. ACAID is a model of immune privilege in the eye. Relatively little is known about the role and phenotype of these regulatory CD4(+)CD25(+) T cells in ACAID. METHODS: Injection of OVA into the anterior chamber of BALB/C mice was performed to induce ACAID. The frequencies of splenic CD4(+)CD25(+) Tregs and the expression of CTLA-4 and LAG-3 on these cells were determined by flow cytometry. Magnetic cell sorting was used to isolate CD4(+)CD25(+) and CD4(+)CD25(-)T cells. The function of CD4(+)CD25(+) T cells was detected by in vitro immunosuppression assays and in vivo adoptive transfer. RESULTS: ACAID was successfully induced following an i.c. injection of OVA. Frequencies of CD4(+)CD25(+) and Tregs were significantly increased in ACAID mice as compared to those in controls. The CD4(+)CD25(+) T cells stimulated with OVA in ACAID mice showed a stronger suppressive ability in vitro than those seen in non-ACAID mice. CD4(+)CD25(+) T cells from ACAID mice, but not from non-ACAID mice, were able to suppress DTH responses in an antigen-specific manner following adoptive transfer. The frequencies of CTLA-4 or LAG-3 on Tregs in ACAID mice were higher as compared with those in naive mice. CONCLUSION: Splenic CD4(+)CD25(+)Foxp3(+)T cells expressing CTLA4 and LAG3 play an important role in the induction of ACAID.

Phenotypes, distribution, and morphological features of antigen-presenting cells in the murine cornea following intravitreal injection.

PURPOSE: To study the phenotypes, distribution, and morphologies of different antigen-presenting cells (APCs) in the murine cornea. METHODS: Intravitreal injection of fluorescently tagged ovalbumin (OVA) or antibodies to MHC-II (I-A(d)), F4/80, CD11c, B7-1, and B7-2 was performed to label cells in the murine cornea. Light and transmission electron microscopy were used to examine corneal histology. Intravital microscopy, epifluorescence microscopy, and confocal microscopy were used to evaluate the labeled cells. In vitro staining was performed to validate the in vivo staining and localize the labeled cells. Three-dimensional rotatable images were taken to evaluate relationships between two differently labeled cells. RESULTS: Histological examination revealed no observable change in the cornea following intravitreal injection. In vivo staining showed that OVA+ cells and cells positive for MHC-II, F4/80, CD11c, B7-1, or B7-2 were noted throughout the cornea with a decreasing density from limbus toward the central cornea. Two populations with distinct morphological features were identified among these APCs. Labeled cells were found beneath the epithelium or in the shallow stroma in the central and paracentral cornea, but in all layers in the peripheral cornea. A number of F4/80+ and CD11c+ cells were also positive for OVA, MHC-II, B7-1, or B7-2. Rotatable images showed a close contact between two differently labeled cells. CONCLUSIONS: Intravitreal injection of labeled antibodies can be adapted to visualize labeled cells in the cornea. APCs with distinct morphologies, phenotypes, and distribution may contribute to the immunologically privileged feature of the cornea.

IL-23 promotes CD4+ T cells to produce IL-17 in Vogt-Koyanagi-Harada disease.

BACKGROUND: Vogt-Koyanagi-Harada (VKH) disease is a systemic refractory autoimmune disease. IL-23 has been thought to play a critical role in autoimmune disease through inducing the development of IL-17-producing CD4(+) T cells. OBJECTIVE: To investigate the expression of IL-23 and IL-17 and the influence of IL-23 on IL-17 production in patients with VKH disease. METHODS: Blood samples were taken from 25 patients with VKH disease and 16 healthy controls. Peripheral blood mononuclear cells (PBMCs) were subjected to analysis of IL-23p19 mRNA and IL-23 protein expression using RT-PCR and ELISA, respectively. The IL-17 levels in the supernatants of PBMCs and CD4(+) T cells cultured in the absence or presence of recombinant (r)IL-23, rIL-12, or anti-IFN-gamma were determined by ELISA. RESULTS: The patients with VKH disease with active uveitis showed an elevated level of IL-23p19 mRNA in PBMCs, higher IL-23 in the serum and supernatants of PBMCs, and increased production of IL-17 by polyclonally stimulated PBMCs and CD4(+) T cells. Recombinant IL-23 significantly enhanced IL-17 production, whereas rIL-12 and IFN-gamma inhibited IL-17 production. More importantly, IL-17 production was significantly increased in patients with active uveitis in the presence of rIL-23. Both rIL-23 and rIL-12 enhanced IFN-gamma production. CONCLUSION: The results suggest that IL-23-stimulated production of IL-17 by CD4(+) T cells may be responsible for the development of uveitis seen in patients with VKH disease. CLINICAL IMPLICATIONS: This study provides a new insight into the mechanism involved in the development of VKH disease.