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• W2034513835 abstract "Uveitis is a major and common cause of visual disability. Recent studies have shown that Th17 cells are implicated in the pathogenesis of this serious intraocular disorder. Activated T cells express an inducible costimulatory molecule called OX40, and OX40 in turn promotes the activation and proliferation of these lymphocytes. Nevertheless, it is unclear whether OX40 plays a vital role in enhancing the effector function of Th17 cells as well as the severity of uveitis. In this study, we demonstrated an increase of OX40 transcription in ovalbumin-induced uveitis, whereas anti-OX40L antibody substantially inhibited the antigen-specific ocular inflammation. Next, results from flow cytometry showed that activated Th17 cells expressed OX40, and OX40-activating antibody significantly augmented the production of Th17 cytokines in vitro. To validate the impact of OX40 in vivo, we stimulated ovalbumin-specific T cells with the OX40-activating antibody. Compared to donor cells without OX40 activation, adoptive transfer of OX40-stimulated lymphocytes elicited more severe ocular inflammation. Furthermore, an interleukin-17-neutralizing antibody attenuated OX40-mediated uveitis. In conclusion, our findings suggest that activation of OX40 augments Th17 cell function and thereby contributes to ocular inflammation. This study thus enhances our knowledge of costimulatory molecule-mediated immunopathological mechanisms of uveitis and suggests a future therapeutic strategy to treat uveitis by the targeting of OX40. Uveitis is a major and common cause of visual disability. Recent studies have shown that Th17 cells are implicated in the pathogenesis of this serious intraocular disorder. Activated T cells express an inducible costimulatory molecule called OX40, and OX40 in turn promotes the activation and proliferation of these lymphocytes. Nevertheless, it is unclear whether OX40 plays a vital role in enhancing the effector function of Th17 cells as well as the severity of uveitis. In this study, we demonstrated an increase of OX40 transcription in ovalbumin-induced uveitis, whereas anti-OX40L antibody substantially inhibited the antigen-specific ocular inflammation. Next, results from flow cytometry showed that activated Th17 cells expressed OX40, and OX40-activating antibody significantly augmented the production of Th17 cytokines in vitro. To validate the impact of OX40 in vivo, we stimulated ovalbumin-specific T cells with the OX40-activating antibody. Compared to donor cells without OX40 activation, adoptive transfer of OX40-stimulated lymphocytes elicited more severe ocular inflammation. Furthermore, an interleukin-17-neutralizing antibody attenuated OX40-mediated uveitis. In conclusion, our findings suggest that activation of OX40 augments Th17 cell function and thereby contributes to ocular inflammation. This study thus enhances our knowledge of costimulatory molecule-mediated immunopathological mechanisms of uveitis and suggests a future therapeutic strategy to treat uveitis by the targeting of OX40. Uveitis is a common and serious ophthalmologic disorder. It is comparable to diabetes as a major cause of years of visual loss.1Nussenblatt RB The natural history of uveitis.Int Ophthalmol. 1990; 14: 303-308Crossref PubMed Scopus (405) Google Scholar Many systemic diseases including sarcoidosis, ankylosing spondylitis, and juvenile idiopathic arthritis are frequently associated with uveitis. The histological changes associated with uveitis include infiltration of the intraocular space with leukocytes.2Becker MD Adamus G Davey MP Rosenbaum JT The role of T cells in autoimmune uveitis.Ocul Immunol Inflamm. 2000; 8: 93-100Crossref PubMed Google Scholar, 3Martin TM Smith JR Rosenbaum JT Anterior uveitis: current concepts of pathogenesis and interactions with the spondyloarthropathies.Curr Opin Rheumatol. 2002; 14: 337-341Crossref PubMed Scopus (78) Google Scholar Although the etiology of uveitis is complex and multifactorial, T lymphocytes play an important role in the development of most forms of uveitis.4Atalla L Linker-Israeli M Steinman L Rao NA Inhibition of autoimmune uveitis by anti-CD4 antibody.Invest Ophthalmol Vis Sci. 1990; 31: 1264-1270PubMed Google Scholar, 5Kilmartin DJ Fletcher ZJ Almeida JA Liversidge J Forrester JV Dick AD CD69 expression on peripheral CD4+ T cells parallels disease activity and is reduced by mycophenolate mofetil therapy in uveitis.Invest Ophthalmol Vis Sci. 2001; 42: 1285-1292PubMed Google Scholar T cell dominant infiltration is well documented in the ocular tissue and aqueous humor in many types of uveitis, especially the ones associated with systemic inflammatory diseases.6Rothova A Buitenhuis HJ Meenken C Brinkman CJ Linssen A Alberts C Luyendijk L Kijlstra A Uveitis and systemic disease.Br J Ophthalmol. 1992; 76: 137-141Crossref PubMed Scopus (271) Google Scholar In parallel with the observation in human patients, CD4+ and other lymphocytes mediate many animal models of uveitis such as experimental autoimmune uveitis (EAU) and the antigen-specific DO11.10 uveitis model.7Yoshimura T Sonoda KH Ohguro N Ohsugi Y Ishibashi T Cua DJ Kobayashi T Yoshida H Yoshimura A Involvement of Th17 cells and the effect of anti-IL-6 therapy in autoimmune uveitis.Rheumatol. 2009; 48: 347-354Crossref PubMed Scopus (156) Google Scholar, 8Liu X Lee YS Yu CR Egwuagu CE Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases.J Immunol. 2008; 180: 6070-6076Crossref PubMed Scopus (213) Google Scholar, 9Zhang Z Zhong W Spencer D Chen H Lu H Kawaguchi T Rosenbaum JT Interleukin-17 causes neutrophil mediated inflammation in ovalbumin-induced uveitis in DO11.10 mice.Cytokine. 2009; 46: 79-91Crossref PubMed Scopus (23) Google Scholar These models greatly advance our understanding of the adaptive immune response in ocular inflammation. Under a proper cytokine milieu, naïve T cells differentiate to unique T helper (Th) subsets. Th17 cells are a recently discovered CD4+ T lymphocyte subpopulation distinct from Th1 and Th2 cells.10Bettelli E Korn T Kuchroo VK Th17: the third member of the effector T cell trilogy.Curr Opin Immunol. 2007; 19: 652-657Crossref PubMed Scopus (511) Google Scholar, 11Dong C TH17 cells in development: an updated view of their molecular identity and genetic programming.Nat Rev Immunol. 2008; 8: 337-348Crossref PubMed Scopus (865) Google Scholar, 12McGeachy MJ Cua DJ Th17 cell differentiation: the long and winding road.Immunity. 2008; 28: 445-453Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar This unique Th subset produces a repertoire of signature cytokines including interleukin (IL)-17A/F, IL-21, IL-22, and CCL20.13Ouyang W Kolls JK Zheng Y The biological functions of T helper 17 cell effector cytokines in inflammation.Immunity. 2008; 28: 454-467Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar The importance of Th17 cells is underscored by their emerging role in various autoimmune diseases (eg, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and uveitis) as well as host defense against extracellular microbes.13Ouyang W Kolls JK Zheng Y The biological functions of T helper 17 cell effector cytokines in inflammation.Immunity. 2008; 28: 454-467Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar, 14Tesmer LA Lundy SK Sarkar S Fox DA Th17 cells in human disease.Immunol Rev. 2008; 223: 87-113Crossref PubMed Scopus (830) Google Scholar, 15Abraham C Cho J Interleukin-23/Th17 pathways and inflammatory bowel disease.Inflamm Bowel Dis. 2009; 15: 1090-1100Crossref PubMed Scopus (238) Google Scholar, 16Yen D Cheung J Scheerens H Poulet F McClanahan T McKenzie B Kleinschek MA Owyang A Mattson J Blumenschein W Murphy E Sathe M Cua DJ Kastelein RA Rennick D IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6.J Clin Invest. 2006; 116: 1310-1316Crossref PubMed Scopus (1267) Google Scholar, 17Luger D Silver PB Tang J Cua D Chen Z Iwakura Y Bowman EP Sgambellone NM Chan CC Caspi RR Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category.J Exp Med. 2008; 205: 799-810Crossref PubMed Scopus (594) Google Scholar, 18Amadi-Obi A Yu CR Liu X Mahdi RM Clarke GL Nussenblatt RB Gery I Lee YS Egwuagu CE TH17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1.Nat Med. 2007; 13: 711-718Crossref PubMed Scopus (683) Google Scholar, 19Aujla SJ Dubin PJ Kolls JK Th17 cells and mucosal host defense.Semin Immunol. 2007; 19: 377-382Crossref PubMed Scopus (230) Google Scholar Recent studies show that certain combinations of cytokines, namely transforming growth factor-β, IL-6, and/or IL-1β, are essential for the induction of Th17 cells from naïve CD4+ lymphocytes.11Dong C TH17 cells in development: an updated view of their molecular identity and genetic programming.Nat Rev Immunol. 2008; 8: 337-348Crossref PubMed Scopus (865) Google Scholar, 12McGeachy MJ Cua DJ Th17 cell differentiation: the long and winding road.Immunity. 2008; 28: 445-453Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar Moreover, IL-21 and IL-23 receptor (IL-23R) play an important role in the proliferation and expansion of Th17 cells.11Dong C TH17 cells in development: an updated view of their molecular identity and genetic programming.Nat Rev Immunol. 2008; 8: 337-348Crossref PubMed Scopus (865) Google Scholar, 12McGeachy MJ Cua DJ Th17 cell differentiation: the long and winding road.Immunity. 2008; 28: 445-453Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar Despite recent progress, the activation mechanism of Th17 cells has not been fully defined, especially in the context of uveitis. However, one contributing event is the engagement of various costimulatory molecules at the time that the T cell receptor interacts with antigens presented by antigen-presenting cells. These costimulatory molecules provide a second signal that is crucial to the amplification and optimization of the T cell response.20Acuto O Michel F CD28-mediated co-stimulation: a quantitative support for TCR signalling.Nat Rev Immunol. 2003; 3: 939-951Crossref PubMed Scopus (544) Google Scholar, 21Redmond WL Ruby CE Weinberg AD The role of OX40-mediated co-stimulation in T-cell activation and survival.Crit Rev Immunol. 2009; 29: 187-201Crossref PubMed Google Scholar OX40 (CD134) is a costimulatory molecule that belongs to the tumor necrosis factor receptor superfamily. It is not constitutively expressed on resting naïve T cells,22Sugamura K Ishii N Weinberg AD Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40.Nat Rev Immunol. 2004; 4: 420-431Crossref PubMed Scopus (280) Google Scholar, 23Weinberg AD OX40: targeted immunotherapy–implications for tempering autoimmunity and enhancing vaccines.Trends Immunol. 2002; 23: 102-109Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 24Lane P Role of OX40 signals in coordinating CD4 T cell selection, migration, and cytokine differentiation in T helper (Th)1 and Th2 cells.J Exp Med. 2000; 191: 201-206Crossref PubMed Scopus (129) Google Scholar, 25Rogers PR Song J Gramaglia I Killeen N Croft M OX40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells.Immunity. 2001; 15: 445-455Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar, 26Nakae S Saijo S Horai R Sudo K Mori S Iwakura Y IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist.Proc Natl Acad Sci USA. 2003; 100: 5986-5990Crossref PubMed Scopus (409) Google Scholar and OX40 expression becomes evident at 24 to 72 hours following specific T cell activation. Engagement between OX40 and OX40 ligand (OX40L) on antigen-presenting cells has a critical role in the maintenance of an immune response following antigen stimulation due to its ability to enhance T cell survival and effector function.25Rogers PR Song J Gramaglia I Killeen N Croft M OX40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells.Immunity. 2001; 15: 445-455Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar Although OX40 plays an important role in the activation of Th1 and Th2 cells,21Redmond WL Ruby CE Weinberg AD The role of OX40-mediated co-stimulation in T-cell activation and survival.Crit Rev Immunol. 2009; 29: 187-201Crossref PubMed Google Scholar it is unclear whether ligation of OX40 could propagate Th17-mediated effects. According to a recent study, IL-17 does not induce OX40 expression.26Nakae S Saijo S Horai R Sudo K Mori S Iwakura Y IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist.Proc Natl Acad Sci USA. 2003; 100: 5986-5990Crossref PubMed Scopus (409) Google Scholar Nevertheless, activation of T cells through OX40 ligation enhances IL-17 production.26Nakae S Saijo S Horai R Sudo K Mori S Iwakura Y IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist.Proc Natl Acad Sci USA. 2003; 100: 5986-5990Crossref PubMed Scopus (409) Google Scholar This indicates that the expression of OX40 by Th17 cells and signaling through OX40 also contributes to Th17 response. Lastly, it is well documented that OX40 is implicated in many T cell-mediated diseases.27Croft M So T Duan W Soroosh P The significance of OX40 and OX40L to T-cell biology and immune disease.Immunol Rev. 2009; 229: 173-191Crossref PubMed Scopus (363) Google Scholar, 28Huang MC Liao JJ Bonasera S Longo DL Goetzl EJ Nuclear factor-kappaB-dependent reversal of aging-induced alterations in T cell cytokines.FASEB J. 2008; 22: 2142-2150Crossref PubMed Scopus (57) Google Scholar, 29Weinberg AD Bourdette DN Sullivan TJ Lemon M Wallin JJ Maziarz R Davey M Palida F Godfrey W Engleman E Fulton RJ Offner H Vandenbark AA Selective depletion of myelin-reactive T cells with the anti-OX-40 antibody ameliorates autoimmune encephalomyelitis.Nat Med. 1996; 2: 183-189Crossref PubMed Scopus (138) Google Scholar, 30Nohara C Akiba H Nakajima A Inoue A Koh CS Ohshima H Yagita H Mizuno Y Okumura K Amelioration of experimental autoimmune encephalomyelitis with anti-OX40 ligand monoclonal antibody: a critical role for OX40 ligand in migration, but not development, of pathogenic T cells.J Immunol. 2001; 166: 2108-2115Crossref PubMed Scopus (108) Google Scholar Nevertheless, little is known of OX40 during the activation of uveitogenic lymphocytes and the development of uveitis. In this study, we aimed to examine the impact of OX40 activation on Th17-related effects and the role of OX40 in uveitis. Here, we demonstrate an up-regulation of OX40 in ovalbumin (OVA)-induced uveitis, and blocking OX40 signaling by anti-OX40L antibody inhibited the ocular inflammation. In addition, activation of OX40 augmented the expression of IL-17, IL-21, and IL-23R. Furthermore, IL-17 neutralizing antibody attenuated OX40-enhanced uveitis. Collectively, these results indicate that the OX40-mediated costimulatory signal is an important step in the activation of Th17 cells as well as the development of ocular inflammation. Six- to 8-week-old BALB/c, DO11.10 mice on a BALB/c background, and B10.RIII mice (Jackson Laboratory, Bar Harbor, ME) were used for the experiments. The animal experimental protocols are in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and have been approved by our institutional animal care and use committee. BALB/c mice that express transgenic dsRedII under the control of the CD4 promoter were kindly provided by Dr. Ulrich H. von Andrian (Harvard University). For OVA-induced uveitis experiment, these mice were further crossed to DO11.10 mice for 10 generations such that the mice express dsRedII on a CD4 promoter but carry OVA-specific CD4+ T cells. After DO11.10 mice were sacrificed, their spleens were removed. Single cell suspensions were prepared by passing the tissue through a 70-μm cell strainer (BD Biosciences, Mountain View, CA). Red blood cells were lysed with 1X red blood cell lysis buffer (Sigma, St. Louis, MO) at room temperature for 5 minutes. The cell suspension was washed twice with RPMI 1640 and then cultured in RPMI 1640 with 10% fetal bovine serum in an atmosphere of 95% air and 5% CO2 at 37°C. Sorted naïve CD4+ T cells (2 × 105/200 μl) were cultured in the presence of OVA323-339 peptide (Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg) (AnaSpec, Fremont, CA) with irradiated (3300 rad) BALB/c splenocytes (2 × 106/200 μl) as antigen-presenting cells. We used irradiated “feeder” cells from naïve nontransgenic BALB/c mice instead of DO11.10 mice to eliminate the possible contamination of any radiation-surviving OVA-reactive T cells. Some DO11.10 cells were further stimulated with and without OX40-activating antibody (clone OX86). The antibody was produced in Dr. Weinberg's laboratory from hybridomas and affinity purified over protein G columns. For adoptive transfer, the lymphocytes were further purified using Lympholyte-M (Cedar Lane Laboratories, Burlington, NC) according to the manufacturer's instructions. After the purification, 85 to 90% of DO11.10 lymphocytes were CD4+ T cells. To generate uveitis in direct response to antigen stimulation, DO11.10 mice were injected with 100 μg of OVA (Sigma) in 2 μl of PBS or irrelevant antigen bovine serum albumin into the vitreous chamber of each eye. The injections were performed with ultrathin, pulled borosilicate glass needles (outer diameter about 50 μm) and Hamilton syringes under direct visualization through a surgical microscope. Some mice also simultaneously received intravenous (10 μg per mouse) and intravitreal (1 μg per eye) anti-OX40 ligand (OX40L) antibody (clone 18260, R&D Systems, Minneapolis, MN) during intravitreal OVA challenge. For the adoptive transfer model of uveitis, OVA-activated DO11.10 lymphocytes with and without OX40-activating antibody priming were injected into naive BALB/c mice (1.5 × 107 cells/animal) via the tail vein. Then, these mice were challenged intravitreally with 250 ng of Escherichia coli strain 055:B5 lipopolysaccharide (Sigma) plus 100 μg of OVA in PBS. Twenty-four or 48 hours after the OVA challenge, uveitis was evaluated by intravital microscopy. For EAU, B10.RIII mice received subcutaneous immunization (near the base of the tail) of 50 μg of interphotoreceptor retinoid-binding protein (IRBP)161-180 peptide (Ser-Gly-Ile-Pro-Tyr-Ile-Ile-Ser-Tyr-Leu-His-Pro-Gly-Asn-Thr- Ile-Leu-His-Val-Asp) (AnaSpec) in 200 μl of complete Freund's adjuvant (Sigma) with Mycobacterium tuberculosis strain H37RA. Some B10.RIII mice were also treated with anti-OX40L antibody (10 μg per mouse) via tail vein injection on days 0, 3, 7, and 14 after IRBP immunization. On day 21, the eyes were harvested, and the severity of EAU was examined by histology and graded on a four-point scale based on inflammatory cell infiltration, retinal folding, and destruction.31Caspi RR Roberge FG Chan CC Wiggert B Chader GJ Rozenszajn LA Lando Z Nussenblatt RB A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens.J Immunol. 1988; 140: 1490-1495PubMed Google Scholar For DO11.10 mice that did not express fluorescent protein under the CD4 promoter, 150 μl of rhodamine (0.2% in PBS) was administered intraperitoneally into the mice to label intravascular leukocytes right before intravital microscopy as we have previously described.32Dullforce PA Seitz GW Garman KL Michael JA Crespo SM Fleischman RJ Planck SR Parker DC Rosenbaum JT Antigen-specific accumulation of naive, memory and effector CD4 T cells during anterior uveitis monitored by intravital microscopy.Cell Immunol. 2006; 239: 49-60Crossref PubMed Scopus (18) Google Scholar, 33Becker MD Crespo S Martin TM Planck SR Naramura M Rosenbaum JT Intraocular in vivo imaging of activated T-lymphocytes expressing green-fluorescent protein after stimulation with endotoxin.Graefes Arch Clin Exp Ophthalmol. 2001; 239: 609-612Crossref PubMed Scopus (14) Google Scholar Labeled inflammatory cells in the iris and ciliary/limbal region were observed by intravital epifluorescence videomicroscopy. This imaging system comprised of a modified DM-LFS microscope (Leica, Bannockburn, IL) and a CF 84/NIR B&W camera from Kappa (Gleichen, Germany), or a color Optronics DEI-750CE camera (Optronics International, Chelmsford, MA). This technique has been reported in detail previously.32Dullforce PA Seitz GW Garman KL Michael JA Crespo SM Fleischman RJ Planck SR Parker DC Rosenbaum JT Antigen-specific accumulation of naive, memory and effector CD4 T cells during anterior uveitis monitored by intravital microscopy.Cell Immunol. 2006; 239: 49-60Crossref PubMed Scopus (18) Google Scholar, 33Becker MD Crespo S Martin TM Planck SR Naramura M Rosenbaum JT Intraocular in vivo imaging of activated T-lymphocytes expressing green-fluorescent protein after stimulation with endotoxin.Graefes Arch Clin Exp Ophthalmol. 2001; 239: 609-612Crossref PubMed Scopus (14) Google Scholar Real-time videos were recorded in NTSC format for 10 seconds each. Both rolling and adherent leukocytes in the iris vessels were identified as a marker for anterior chamber uveitis.32Dullforce PA Seitz GW Garman KL Michael JA Crespo SM Fleischman RJ Planck SR Parker DC Rosenbaum JT Antigen-specific accumulation of naive, memory and effector CD4 T cells during anterior uveitis monitored by intravital microscopy.Cell Immunol. 2006; 239: 49-60Crossref PubMed Scopus (18) Google Scholar, 33Becker MD Crespo S Martin TM Planck SR Naramura M Rosenbaum JT Intraocular in vivo imaging of activated T-lymphocytes expressing green-fluorescent protein after stimulation with endotoxin.Graefes Arch Clin Exp Ophthalmol. 2001; 239: 609-612Crossref PubMed Scopus (14) Google Scholar These cells were quantified to assess the severity of the ocular inflammation.32Dullforce PA Seitz GW Garman KL Michael JA Crespo SM Fleischman RJ Planck SR Parker DC Rosenbaum JT Antigen-specific accumulation of naive, memory and effector CD4 T cells during anterior uveitis monitored by intravital microscopy.Cell Immunol. 2006; 239: 49-60Crossref PubMed Scopus (18) Google Scholar, 33Becker MD Crespo S Martin TM Planck SR Naramura M Rosenbaum JT Intraocular in vivo imaging of activated T-lymphocytes expressing green-fluorescent protein after stimulation with endotoxin.Graefes Arch Clin Exp Ophthalmol. 2001; 239: 609-612Crossref PubMed Scopus (14) Google Scholar For further histological evaluation, the eyes were fixed in 3% paraformaldehyde. Then, the tissues were embedded in paraffin, sectioned, and stained with H&E. Ocular inflammation was assessed by light microscopy. Naïve DO11.10 CD4+ T cells (2 × 105/200 μl) were co-cultured with the irradiated BALB/c splenocytes (2 × 106/200 μl) in the presence of 1 μg/ml OVA323-339 peptide. Th17-polarizing conditions were 1 ng/ml transforming growth factor-β, 30 ng/ml IL-6, 10 ng/ml IL-1β, 10 ng/ml tumor necrosis factor-α, 20 ng/ml IL-23, 20 μg/ml anti-interferon-γ and anti-IL-4 antibodies. After 4 days of incubation, Th17 polarizing media were replaced with regular RPMI containing 10% fetal bovine serum for 12 hours. This allowed differentiated lymphocytes to rest before further real-time PCR analysis and intracellular staining of IL-17. DO11.10 splenocytes were suspended in PBS containing 2% fetal bovine serum and 0.1% sodium azide. Anti-CD4 (clone RM4-5) and anti-OX40 antibodies conjugated with different fluorescent colors were used to label these cell surface markers. For IL-17 staining, the cells were stimulated with phorbol myristate acetate (50 ng/ml) and ionomycin (1 μg/ml) for 5 hours. Then, brefeldin A (1:1000) was added for 2 hours. The cells were collected and stained with fluorescein isothiocyanate-labeled anti-mouse CD4 antibody for 30 minute. After PBS wash, the cells were fixed and permeabilized overnight with 1X fixation/permeabilization solution (eBioscience, San Diego, CA) at 4°C. Then these cells were stained intracellularly with allophycocyanin-conjugated monoclonal antibody against IL-17 (clone eBio17B7) (eBioscience) for 1 hour at 4°C. Data acquisition was performed on a FACSCalibur flow cytometer, and data were analyzed using CellQuest software. The culture media of DO11.10 splenocytes and naïve CD4+ T cells from various experimental groups were collected for enzyme-linked immunosorbent assay to measure the IL-17 and IL-21 levels according to the manufacturer's protocols (R&D Systems). DO11.10 lymphocytes treated with or without OX40-activating antibody were collected in 1X LDS lysis buffer (Invitrogen) on ice. The lysates were then centrifuged at 12,000 × g for 10 minutes. Thirty microliters of total protein from each group were separated by electrophoresis through a 4 to 12% gradient Tris-glycine SDS gel and then transferred to nitrocellulose membrane using an Xcell SureLock Mini Cell (Novex, San Diego, CA). After milk blocking, the nitrocellulose membrane was incubated with polyclonal antibody against IL-23R (R&D Systems) or β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), followed by horseradish peroxidase-conjugated secondary antibody. The signals of IL-23R and β-actin were detected by enhanced chemiluminescence luminol reagent. Total RNA from cultured CD4+ cells was isolated with RNAeasy Mini Kit (Qiagen, Valencia, CA). First-strand cDNA synthesis was accomplished with an oligo(dT)-primed Omniscript reverse transcriptase kit (Qiagen, Valencia, CA). Gene-specific cDNA was amplified by PCR using mouse specific primer pairs (IL-17A sense: 5′-GTGGCG GCTACAGTGAAGGCA-3′ and IL-17A antisense: 5′-GACAATCGAGGCCACGCAGGT-3′; IL-21 sense: 5′-ACCAGACCAAGGCCCTGTC-3′ and IL-21 anti-sense: 5′-TGGGCTCTTGTTGAGTTGAGATT-3′; IL-22 sense: 5′-TCAGACAGGTTCCAGCC-3′ and IL-22 antisense: 5′-TCCAGTTCCCCAATCGCC-3′; RORγt sense: 5′-ACCTCTTTTCACGGGAGGA-3′ and RORγt antisense: 5′-TCCCACATCTCCCACATTG-3′; β-actin sense, 5′-ATGCCAACACAGTGCTGTCT-3′, and β-actin antisense, 5′-AAGCACTTGCGGTGCACGAT-3′). OX40 primers were commercially purchased from SABiosciences (Frederick, MD). The real-time PCR was performed using a RT2 Realtime PCR Master Mix (SABiosciences) and running for 40 cycles at 95°C for 15 seconds and 55°C for 40 seconds. The mRNA levels of Th17-related genes in each sample was normalized to β-actin mRNA and quantified using a formula: 2 [(Ct/β-actin – Ct/gene of testing gene)]. The result was expressed as fold difference in the cells stimulated with both OVA and OX40-activating antibody compared to the group treated with OVA alone. Data are expressed as the average ± SEM, and a representative experiment is shown for each figure. For EAU scoring, median difference between control and experimental groups was compared using exact Wilcoxon two-sample test. Other statistical probabilities were evaluated by Student's t-test, with a value of P < 0.05 considered significant. Recently, we have generated a novel uveitis model by direct administration of OVA into the vitreous chamber of DO11.10 mice. These mice develop a rapid onset anterior uveitis due to the specific T cell receptor response to OVA. The OVA-induced uveitis is CD4+ T cell dependent and mainly mediated by IL-17.9Zhang Z Zhong W Spencer D Chen H Lu H Kawaguchi T Rosenbaum JT Interleukin-17 causes neutrophil mediated inflammation in ovalbumin-induced uveitis in DO11.10 mice.Cytokine. 2009; 46: 79-91Crossref PubMed Scopus (23) Google Scholar Since activated T cells express OX40, we investigated OX40 induction in this T cell-dependent uveitis model. DO11.10 mice were intravitreally challenged with 100 μg OVA or bovine serum albumin as a control. Twenty-four hours later, the eyes were harvested to examine OX40 expression. Real-time PCR revealed a mean 3.3-fold increase (SD = 0.8) of OX40 transcripts in the ocular samples after intravitreal OVA challenge compared to non-antigenic bovine serum albumin injection. To further confirm the role of OX40 signaling in uveitis, we compared the severity of ocular inflammation between the mice treated with anti-OX40L monoclonal antibody and rat IgG2a as an isotype control. We first used DO11.10 mice that express transgenic dsRedII under the control of the CD4 promoter.34Mempel TR Pittet MJ Khazaie K Weninger W Weissleder R von Boehmer H von Andrian UH Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation.Immunity. 2006; 25: 129-141Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar Anti-OX40L antibody was administered simultaneously along with OVA on the induction of uveitis. DsRedII-labeled T cell infiltration in the eye was monitored by intravital microscopy at 48 hours after the intraocular antigen challenge. As shown in Figure 1A, a marked infiltration of red T cells was observed in the extravascular area of the iris. No infiltrating T cells were observed in the eyes at 0 hour before OVA injection or in the control animals that received bovine serum albumin (data not shown). Compared to OVA stimulation alone, anti-OX40L monoclonal antibody significantly inhibited the lymphocyte infiltration in the iris (Figure 1, A and B). To examine inflammatory cell migration in the eye, we administered OVA intravitreally to regular DO11.10 mice. Forty-eight hours later, the circulating leukocytes in these mice were labeled with rhodamine by intraperitoneal injection, and uveitis was monitored by intravital microscopy. The intravitreal challenge of OVA resulted in a marked ocular influx of leukocytes, whereas anti-OX40L an" @default.
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• W2034513835 date "2010-12-01" @default.
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• W2034513835 title "Activation of OX40 Augments Th17 Cytokine Expression and Antigen-Specific Uveitis" @default.
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