The Complement System Is Critical in Maintaining Retinal Integrity during Aging

The complement system is a key component of innate immunity comprised of soluble components that form a proteolytic cascade leading to the generation of effector molecules involved in cellular clearance. This system is highly activated not only under general inflammatory conditions such as infections, collagen diseases, nephritis, and liver diseases, but also in focal ocular diseases. However, little is known about the role of the complement system in retinal homeostasis during aging. Using young (6-week-old) and adult (6-month-old) mice in wild type (C57BL/6) and complement knockout strains (C1q−/−, Mbl a/c−/−, Fb−/−, C3−/−, and C5−/−), we compared amplitudes of electroretinograms (ERG) and thicknesses of retinal layers in spectral domain optical coherence tomography between young and adult mice. The ERG amplitudes in adult mice were significantly decreased (p < 0.001, p < 0.0001) compared to that of young mice in all complement knockout strains, and there were significant decreases in the inner nuclear layer (INL) thickness in adult mice compared to young mice in all complement knockout strains (p < 0.0001). There were no significant differences in ERG amplitude or thickness of the INL between young and adult control mice. These data suggest that the complement system plays an important role in maintaining normal retinal integrity over time

Expression and Function of Inducible Costimulator on Peripheral Blood CD4 ؉ T Cells in Behçet&apos;s Patients with Uveitis: A New Activity Marker?

PURPOSE. Inducible costimulator (ICOS) is an important costimulatory molecule involved in T-cell activation. In this study, the role of ICOS in the pathogenesis of uveitis in Behçet&apos;s disease (BD) was investigated. METHODS. Peripheral blood mononuclear cells (PBMCs) were obtained from BD patients with uveitis in the active or remission phase and in healthy subjects. Total RNA was isolated from PMBCs, and mRNA expression was analyzed on an oligonucleotide microarray. ICOS expression on CD4 ϩ T cells was determined by flow cytometry, and the functional costimulatory effect of ICOS/B7RP-1 interaction was assessed on stimulation with concanavalin A (conA) or IRBP in the presence or absence of anti-ICOS mAb. RESULTS. As the result of microarray analysis, ICOS in PBMCs showed the greatest difference in expression in BD patients with uveitis compared with healthy control subjects. ICOS expression on CD4 ϩ T cells in BD patients with uveitis was significantly higher than that in healthy individuals, both before and after conA stimulation. Among the BD patients, ICOS expression on CD4 ϩ T cells was significantly higher in those with active uveitis than in those with remitted uveitis. Blockade of ICOS/B7-related protein-1 (B7RP-1) interaction by anti-ICOS mAb significantly decreased IFN-␥, IL-17, and TNF-␣ production by PBMCs when stimulated with conA or IRBP in BD with active uveitis. CONCLUSIONS. High ICOS expression in BD patients with uveitis contributed to the upregulation of IFN-␥, IL-17, and TNF-␣ production, suggesting that abnormal ICOS costimulation may play an immunopathologic role in the pathogenesis of uveitis in BD. (Invest Ophthalmol Vis Sci. 2010;51:5099 -5104) DOI: 10.1167/iovs.10-5286 E ndogenous uveitis such as Behçet&apos;s disease (BD), VogtKoyanagi-Harada syndrome, sympathetic ophthalmia, birdshot retinochoroidopathy, and sarcoidosis is a potentially blinding disease in humans and is responsible for 10% to 15% of the acquired blindness in Japan. 1 Although endogenous uveitis covers a spectrum of clinical entities, all forms are believed to share immunohistologic similarities characterized by the infiltration of mainly T cells. Behçet&apos;s disease (BD) is a systemic inflammatory disease characterized by oral and genital ulcers as well as ocular, cutaneous, arthritic, vascular, and neurologic lesions. 3-5 An increasing number of reports indicate that aberrant cellular immunity, such as pathogenic CD4 ϩ T-cell[b]-mediated autoimmunity via the Th1/Th17 pathway, plays a key role in the pathophysiological process in BD with uveitis, 6 -10 although the mechanisms of ocular inflammation in BD remain largely unknown. During recent years, the understanding of immunologic mechanisms involved in uveitis has advanced greatly through investigations of experimental autoimmune uveoretinitis (EAU), an animal model of human uveitis that can be induced by immunization of susceptible animals with interphotoreceptor retinoid-binding protein (IRBP) or with an eye-specific retinal antigen or by adoptive transfer of CD4 ϩ T cells specific for retinal antigens. 11 EAU resembles certain human uveitic conditions in various aspects 14,15 Although CD28 regulation has substantial effects on immunity, its function appears to reside predominantly in the control of primary, but not secondary, immune responses in various autoimmune diseases. 16 -18 The CD28 homolog inducible costimulator (ICOS) has recently been identified as a novel member of the CD28 costimulator family and is expressed by activated T cells in both humans and mice. To identify new genes that may cause or contribute to the disease process of ocular BD, we used cDNA microarrays that provide the expression profile of more than 54,000 genes, some of which are immune-related, whereas others are involved in the cell cycle, cell growth, intracellular signaling, cellular adhesion, and transport, and we compared the expression profiles of healthy individuals and patients. One of the genes found to be upregulated in patients with ocular BD is ICOS, an activation marker expressed on activated T cells that binds B7RP-1-expressing monocytes. The engagement of ICOS with B7RP-1 along with an appropriate antigen provide a positive signal that promotes T-cell differentiation, cytokine secretion, and effector function in the absence of CD28. ϩ T cells and B7RP-1 expressed on infiltrating APCs are upregulated directly after disease onset. 30 Therefore, we speculated that in active BD, pathogenic CD4 ϩ T cells that infiltrate the eye express ICOS in the inflamed eye and would be an appropriate target for the treatment of human ocular BD. Moreover, we have demonstrated that blockade of the ICOS/ B7RP-1 costimulatory pathway inhibits ocular inflammation in the effector phase of murine EAU. 30 Therefore, to investigate the role of ICOS in the pathogenesis of ocular BD, we assessed its expression on peripheral blood CD4 ϩ T cells and functional roles in patients with ocular BD. The results suggest that upregulation of ICOS is associated with the pathogenesis of uveitis in BD. MATERIALS AND METHODS PBMC Samples Thirty-five patients with BD (27 men and 8 women, mean age; 37.8 Ϯ 11.1 years) were enrolled in the study Heparinized blood samples were obtained from patients and healthy individuals, and PBMCs were isolated within 2 hours by density gradient centrifugation. The PBMCs were washed twice and resuspended at 1 ϫ 10 6 cells/mL in complete medium (RPMI 1640 supplemented with 10% fetal calf serum, 1 mM L-glutamine, 100 U/mL penicillin, and 100 g /mL streptomycin). Cell suspension was dispensed in 24-well plates (Falcon; BD Biosciences, Mountain View, CA) at 1 mL/well and incubated with or without concanavalin A (ConA; 10 g/mL) for 12 hours. Analysis of Gene Expression with cDNA Array Gene expression profile of human PBMCs was analyzed by microarray, as described previously. 37 Briefly, total RNA was isolated from pooled PBMCs obtained from seven patients with BD and active uveitis by an extraction method (Isogen Nippon Gene, Tokyo, Japan), and portions (2 g) of the preparation were subjected to amplification of mRNA with T7 RNA polymerase. Biotin-labeled cRNA (10 g) synthesized from the amplified RNA was subjected to hybridization with the Human Whole Genome Bioarray chip (Amersham Biosciences, Piscataway, NJ) that contains oligonucleotides corresponding to a total of approximately 55,585 human genes. Detection and digitization of hybridization signals were performed (G2565; Agilent Technologies, San Diego, CA) and analyzed (CodeLink Expression Analysis software version 4.0; Amersham Biosciences). The microarray data of various costimulatory molecules are summarized in Reagents FITC-conjugated anti-human CD4 (L3T4) was purchased from eBioscience (San Diego, CA), and anti-human ICOS (DX29) from BD PharMingen (San Diego, CA). ConA was obtained from Vector Laboratories (Burlingame, CA). Preparation of IRBP Fresh swine IRBP was purified according to the method described by Fukai et al. Immunofluorescence and Flow Cytometry The results of ICOS gene expression obtained from microarray were confirmed by flow cytometry. PMBCs were isolated by density gradient centrifugation of heparinized blood samples obtained from patients and healthy individuals. Each PBMC sample was divided into two aliquots: one for direct flow cytometry analysis of ICOS expression and the other for flow cytometry analysis after activation with conA at 10 g/mL for 12 hours. ICOS analysis was performed by using the following procedures: A cocktail of FITC-conjugated anti-CD4 and PE-conjugated anti-ICOS was added to the PBMCs. After the cells were washed with PBS, the stained cells (live-gated based on forward-and sidescatter profiles and propidium iodide exclusion) were passed through a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA) and the results analyzed (CellQuest program; BD Biosciences). ICOS expression on human CD4 ϩ T cells was calculated by gating for the CD4 ϩ T cell population, and 10,000 cells were analyzed in each experiment. Cytokine Assay Purified PBMCs (5 ϫ 10 5 /well) from BD patients with active uveitis were cultured in 96-well microtiter plates, with or without stimulation with conA (3 g/mL) or IRBP (10 g/mL) plus anti-ICOS mAb (10 g/mL) or control IgG at 37°C for 48 hours. The unstimulated PBMCs served as negative control samples. After stimulation, cell-free supernatants were collected at 48 hours and assayed for IFN-␥, TNF-␣, IL-2, and IL-6 by cytometric bead array (CBA) kits (BD PharMingen) and IL-17 by ELISA (Human IL-17 Quantikine ELISA kit; R&amp;D Systems, Minneapolis, MN), according to the manufacturers&apos; instructions. The minimum levels detected by CBA kits in either the culture supernatant or serum/plasma were 2.6 pg/mL for IL-2, 3.0 pg/mL for IL-6, 7.1 pg/mL for IFN-␥, and 2.8 pg/mL for TNF-␣; the minimum level in the IL-17 ELISA was 7.8 pg/mL. In a preliminary study, serial concentrations of conA (1, 3, 5, and 10 g/mL) or IRBP (1, 5, and 10 g/mL) were used in the assays. The results showed that optimal concentrations of cytokines were obtained at a concentration of 3 g/mL for conA and 10 g/mL for IRBP, as previously described. Statistical Analysis Data were analyzed (JMP 5; SAS Institute, Inc., Cary, NC) and the results expressed as the mean Ϯ SD. Statistical analysis was performed by using a paired or unpaired t-test. P Ͻ 0.05 was considered significant. RESULTS Analysis of Microarray Results in BD Patients with Active Uveitis To identify the genes involved in the pathogenesis of ocular BD, we compared phosphorescent images of the arrays hybridized with cDNA probes generated from RNA preparations from BD patients with active uveitis and healthy individuals. The array membrane that we used contained 360 positive bacterial controls and 384 negative controls. All positive controls were detected on the microarray. No signals were observed for the negative spots, indicating that the hybridization was highly specific. The DNA screening can evaluate more than 54,000 genes for a single sample. Among these genes, we focused on the immune-related ones, especially those of costimulatory molecules. Among the seven genes of costimulatory molecules, ICOS showed the greatest difference in expression in BD patients with active uveitis compared with that in healthy control subjects ICOS Expression on CD4 ؉ T Cells of BD Patients with Uveitis Flow cytometry was performed to confirm the results of gene array analysis. ICOS expression (median fluorescence intensity [MFI] of ICOS on CD4 ϩ T cells; Comparison of ICOS Expression on CD4 ؉ T Cells in BD Patients with Active or Inactive Uveitis Next, we compared ICOS expression on CD4 ϩ T cells in BD patients with active or inactive uveitis. ICOS expression (MFI of ICOS on CD4 ϩ T cells, Cytokine Production Stimulated with ConA and Uveitogenic Antigen To determine whether ICOS has functional costimulatory activity, we first measured the amounts of pathogenic Th1 and Th17 cytokines (IFN-␥, IL-17, TNF-␣, IL-6, and IL-2) produced by nonspecific stimulation (conA) of PBMCs obtained from BD patients with active uveitis. When ICOS costimulation was blocked with anti-ICOS mAb, the amounts of IFN-␥, IL-17, and TNF-␣ (Figs. 3A-C) produced by PBMCs were both significantly reduced. Since PBMCs from BD patients are known to be sensitized to the two most uveitogenic retina-specific antigens, S-antigen and IRBP, 1 we then examined the amounts of pathogenic Th1 and Th17 cytokines produced by stimulation with swine IRBP protein as the specific antigen. DISCUSSION The precise pathogenesis of uveitis associated with BD is unknown. Accumulated data obtained from animal models such as EAU suggest that Th1 and Th17 cells have major roles IOVS, October 2010, Vol. 51, No. 10 New Activity Marker for Ocular Behçet&apos;s Disease 5101 Downloaded from iovs.arvojournals.org on 06/30/2019 in its pathogenesis. We have previously shown that blockade of the ICOS ϩ costimulatory pathway has an ameliorating effect during the effector phase of EAU, by suppressing the expansion and effector function of pathogenic Th1 cells. In conclusion, our data provide additional evidence of the potential utility of ICOS expression on CD4 ϩ T cells as a marker of disease activity and as a promising therapeutic target for ocular BD via its inhibition of Th1 and Th17 cytokines. As the population studied was small and heterogeneous, further studies are needed to confirm the findings

Elevated serum levels of CXCL9/ monokine induced by interferon-gamma and CXCL10/interferongamma-inducible protein-10 in ocular sarcoidosis. Invest Ophthalmol Vis Sci

PURPOSE. Sarcoidosis is a chronic multisystem granulomatous disorder characterized by an accumulation of activated CD4 ϩ T cells and monocytes/macrophages in involved organs. Chemokines are required for the extravasation of leukocytes to the inflammation site. This study was undertaken to determine which chemokines are augmented in the serum of patients with active ocular sarcoidosis. METHODS. Seventeen patients with diagnosed ocular sarcoidosis, 28 with suspected ocular sarcoidosis, 16 with Behçet&apos;s disease, 17 with Vogt-Koyanagi-Harada disease, and 18 healthy subjects were studied. Serum levels of CCL2, CCL5, CXCL8, CXCL9, and CXCL10 were simultaneously measured by cytometric bead array using flow cytometer. In addition, serum CXCL9 and CXCL10 levels in the patients with diagnosed or suspected ocular sarcoidosis were compared with respect to ocular disease activity, the presence of bilateral hilar lymphadenopathy (BHL), and laboratory data. RESULTS. Serum levels of both CXCL9 and CXCL10 were markedly elevated in the patients with diagnosed or suspected ocular sarcoidosis compared with patients with other types of uveitis and healthy subjects. Although CCL2 and CXCL8 were detected in the serum of all subjects, the levels were extremely low with no significant differences between groups. Elevation of serum CXCL9 and CXCL10 in ocular sarcoidosis correlated significantly with ocular disease activity and ACE (angiotensin converting enzyme) levels and was unrelated to the presence of BHL, erythrocyte sedimentation rate, white blood cell count, serum IgG, or serum lysozyme. CONCLUSIONS. The results demonstrated that serum levels of CXCL9 and CXCL10 were elevated markedly in the patients with ocular sarcoidosis and correlated with ocular disease activity and ACE level. (Invest Ophthalmol Vis Sci

Detection of autoantigens in experimental autoimmune uveoretinitis (EAU)

Shown is a two-dimensional gel image of murine retinal proteins stained with SYPRO Ruby. Approximately 2,000 spots were observed. Among the SYPRO Ruby stained protein spots, the 2D-WB positive spots either in control or EAU were randomly numbered. The numbers and the positions of the seven candidate autoantigens in EAU on a SYPRO Ruby stained gel are shown in panel . The spot numbers of the candidate autoantigens are common between panel , , and . Extracted murine retinal proteins were separated by two-dimensional electrophoresis, transferred onto nitrocellulose membranes. Western blotting was performed using sera from EAU or control mice. Representative membranes reacted with sera from complete Freund's adjuvant-treated control mice are shown in subpanel , and those reacted with sera from EAU mice are shown in subpanel . Each set of and subpanels shows the corresponding area. Arrowheads indicate the position of each candidate autoantigen on the EAU membranes. membranes.<p><b>Copyright information:</b></p><p>Taken from "Proteomic surveillance of retinal autoantigens in endogenous uveitis: implication of esterase D and brain-type creatine kinase as novel autoantigens"</p><p></p><p>Molecular Vision 2008;14():1094-1104.</p><p>Published online 12 Jun 2008</p><p>PMCID:PMC2426731.</p><p></p

Autoantibodies against the retinal autoantigens detected in experimental autoimmune uveoretinitis were tested using sera from human endogenous uveitis patients

The antibody titer was calculated as binding units according to the formula given in Methods. Statistically significant difference of positive rate between each patient group and healthy control (HC) was detected in Behcet’s disease (BD; p=0.016) and Vogt-Koyanagi-Harada disease (VKH; p=0.015) for anti-EsteD antibody, and in VKH (p=0.015) and sarcoidosis (p=0.036) for anti- brain-type creatine kinase (BB-CK).<p><b>Copyright information:</b></p><p>Taken from "Proteomic surveillance of retinal autoantigens in endogenous uveitis: implication of esterase D and brain-type creatine kinase as novel autoantigens"</p><p></p><p>Molecular Vision 2008;14():1094-1104.</p><p>Published online 12 Jun 2008</p><p>PMCID:PMC2426731.</p><p></p