Aqueous Humour Suppresses LPS-induced Dendritic Cell Maturation

Purpose: The mechanisms of ocular immune privilege include the anatomical structures, such as blood-ocular barrier, and a lack of direct lymphatic drainage. In addition, there are abundant immunosuppressive mediators in aqueous humour(AH) that could influence the biological effect of immune cells. Dendritic cells(DCs), the most potent professional antigen presenting cell (APC), which reside in the iris, ciliary body and tissues lining the outflow pathways diffusely, is the key regulator for the induction of immune response, but gain less attention. Here, we mimic a inflammatory environment to analyze the inhibitory effect of aqueous humor on LPS-induced dendritic cells maturation.Methods: Fresh porcine eyes were collected from an abattoir and AH was aspirated by a 27-gauge needle limbal paracentesis within 4 h death, and stored at -80°C in a siliconized microfuge tube. Cytokine PGE₂ in AH were measured by commercial ELISA kit. Male, 6- to 8-week old, C57BL/6 mice were killed humanely. Bone-marrow-derived DCs were generated according to the previous reports. On day 7, 1μg/ml LPS was added to the culture medium, 48h later, nonadherent and loosely adherent cells were harvested as mature DCs, the purity of which was tested by flow cytometry with anti-CD11c monoclonal antibody staining. To investigate the effect of AH on the maturation of DCs, imDCs were divided into three groups: control group(cultured with 1μg/ml LPS for 48h only), experiment group1(cultured with 1μg/ml LPS and 25%AH for 48h), experiment group2(cultured with 1μg/ml LPS and 50%AH for 48h). Flow cytometry were applied to analyze the expression intensity of surface antigens and the cell endocytic ability. After depleted of red blood cells, splenic T cells, from BALB/c mice, which were purified by passing through Nylon-Wool column, were used in MLR to testify the capacity of DCs in stimulating allogeneic T cells; neutralizing anti-TGF-β₂ antibody was added into AH at a sufficient dose (20μg/ml, 10000 times of TGF-β₂ concentration according to the previous study) to neutralize the inhibitory activity of TGF-β₂ for 1 h. To examine whether PGE₂ in AH acted as a immunosuppressive factor, after AH6809 (100μM), a selective EP2 receptor antagonist, added into DCs culture medium at 37°C for 30 min, imDCs were induced to mature. Statistical evaluation was performed using SPSS 13.0.Results: 1 After cultured with RPMI-1640 supplemented with GM-CSF for 7 days, bone marrow-derived cells grown in a colony manner. The purity, analysied by flow cytometry, was greater than 70%.2 AH down-regulate the phenotypic maturation of DCs: DCs cultured with LPS alone showed a phenotypic mature state (high expression of MHC class II, CD80, CD86). The medium supplemented with AH inhibited the maturation of DCs in a dose-dependant manner, representing significantly reduced MFI of molecules.3 AH enhances the uptake of FITC-dextran by DCs and reduces the capacity of DCs in stimulating allogeneic T cells: Consistent with the phenotypic analysis, AH inhibited the down-regulation of endocytis induced by LPS in a dose-dependant manner; The purity of T cells was greater than 90%; LPS induced mature DCs displaying a high T cell stimulatory capacity, which was decreased after 48h coculture with AH. As expected, 2ml AH treated DCs exhibited a much less stimulating capacity than 1ml treated group. And at highest DC to T cell ratio, the stimulator capacity of 2ml AH treated DCs were decreased nearly to one third that of mature DCs.4 Blockade of TGF-β₂ or PGE₂ inhibits the suppression effect of AH: PGE₂, a immune mediator measured, presented in AH at a concentration of 2603.919±771.1536pg/ml (far more <10^-6mol/L). As previous study, low amount of PGE₂(<10^-5mol/L) works maily through EP2 receptor. So DCs were pretreated only with AH6809, a selective EP2 receptor antagonist, for 1h to block cell membrane signal transduction of PGE₂. Clearly, the expression of CD11c, which was not affected by various components in culture medium including AH, anti- TGF-β₂ antibody, AH6809 and DMSO, indicated the homogenicity of DCs. The high MHC class II expression induced by LPS was markedly decreased by AH, and neutralizing antibody of TGF-β₂ pretreated AH totally reversed the inhition effect. Though the inhibition effect of AH was not affected by blocking EP2 receptor on DCs, which demonstred the workless of PGE₂ in AH. Coworks of AH6809 and anti- TGF-β₂ antibody had synergistic effect on reversing AH inhibitory effect, which was testified by endocytosis analysis.Conclusions: 1 The present study, for the first time, demonstrated the immunosurpressive characteristic of AH on the process of LPS induced DCs maturation in vitro. The phenotypic and functional maturations of DCs were inhibited by AH in a dose dependant manner, including lower expressions of MHC-II molecules and costimulatory molecules CD80, CD86, a higher endocytic ability when coculture with FITC-dextran, and a weaker capability of stimulating allogeneic T cells proliferation in MLR.2 The dominant role of TGF-β₂ played in AH inhibition of LPS-induced DCs maturation represent an experiment evidence for applying TGF-β₂ in endotoxin-induced uveitis to weakeninflammatory reaction.3 PGE₂ in normal AH had no statistical inhibitory effect on the expression of MHC class II, but had synergistic effect with TGF-β₂ to enhance endocytotic ability of DCs.