Real-time PCR analysis of chemokines (CXCR3, CXCL9, CXCL10, CCR5, CCL5, CCL4, CCL3) in whole cornea (B) and corneal epithelium (C) of normal PD-L1?/? compared with normal WT mice (defined as 1, = 0.001) and a greater than twofold decrease at day 7 (= 0.049) in the transcript level of PD-L1 by the corneal epithelium of WT mice in which DED was induced compared with normal WT mice. Open in a separate Lynestrenol window Figure 2. Decreased expression of PD-L1 and increased T-cell infiltration and expression of chemokines in DED. expression of PD-L1 by corneal epithelial cells in DED and significantly increased corneal fluorescein staining score with PD-L1 functional blockade using antiCPD-L1 antibody. Conclusions. Downregulation of corneal epithelial PD-L1 amplifies dry eyeCassociated corneal inflammation and epitheliopathy by increasing the expression of chemokine ligands and receptors that promote T-cell homing to the ocular surface. Dry eye disease (DED) affects many millions of persons, in particular women, in the United States alone.1 The current literature on the immunopathogenesis of DED focuses on the inflammatory milieu of Lynestrenol the tear film or conjunctiva, whereas it is corneal inflammation that is the most clinically recognizable and important ocular manifestation of DED.2C8 There is growing evidence that CD4+ T cellCmediated inflammation plays a critical role in amplifying the pathogenesis of DED.9C15 Still, the manner by which this inflammation can induce corneal pathology remains poorly understood. PD-L1 is a member of the B7 family of receptors and has a role in regulating T cellCmediated immunity.16 In vivo studies using PD-L1?/? mice and antiCPD-L1 blocking antibody have provided evidence for the inhibitory functions of PD-L1 in both autoimmunity and alloimmunity. For example, it has been shown that tissue-specific PD-L1 expression protects against autoimmune diabetes, ocular inflammation, and corneal allograft rejection by inhibiting autoreactive and alloreactive T cells.17C20 In DED, there is increased T-cell infiltration into the conjunctiva, but, remarkably, the cornea remains relatively resistant to this infiltration. 9C13 Herein we test the hypothesis that decreased PD-L1 expression is associated with increased chemokine expression, increased T-cell infiltration, and increased corneal fluorescein staining. The purpose of the present study was to determine the effect of PD-L1 on modulating the expression of chemokine gene transcripts in the cornea. Lynestrenol Additionally, we investigated the potential role of PD-L1 in the pathogenesis of DED by inducing DED in PD-L1?/? mice and in mice treated with antiCPD-L1 blocking antibody to determine how the blockade or elimination of PD-L1 affects the expression of the principal Mouse monoclonal to CD3/CD16+56 (FITC/PE) T-cell chemokines and the clinical aspects of DED. Materials and Methods Mouse Model of Dry Eye Eight- to 12-week-old female C57BL/6 mice were obtained Lynestrenol from Taconic Farms (Germantown, NY), and Charles River Laboratory (Boston, MA). Similarly aged PD-L1?/? C57BL/6 mice were generated as previously described.16 In all the experiments performed, the mice were age-matched among the different groups. The protocol was approved by the Institutional Animal Care and Use Committee, and all animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Dry eye was induced by placement of mice in a controlled-environment chamber modified with subcutaneous administration of scopolamine to maximize ocular dryness as previously described.13C15,21,22 Age-matched mice not placed in the controlled-environment chamber were used as controls. PD-L1 Blockade To block PD-L1Cmediated signaling, five mice were treated 1 day before dry eye induction and every other day thereafter for 10 days with antimurine PD-L1 (10F.9G2, rat IgG2b; 150 g/mouse intraperitoneally) or control rat IgG (MP Biomedicals, Santa Ana, CA).17,23,24 Measurement of Corneal Fluorescein Staining Corneal fluorescein staining was performed at baseline (day 0) and then at days 2, 5, 7, and 9. One microliter of 2.5% fluorescein was applied to the lateral conjunctival sac of the mice, as previously described.13C15,21,22 Eyes were examined for fluorescein staining after 5 minutes with a slit lamp biomicroscope under cobalt blue light. Punctate staining was recorded in a masked fashion using the standard National Eye Institute grading system. Each of the five areas of the cornea was given a score from 0 to 3, with 0 denoting no Lynestrenol epitheliopathy and 3 denoting diffuse severe dry eye.25 Immunohistochemical Staining The following antibodies were used for immunohistochemical staining: purified hamster antiCmouse CD3e monoclonal antibody (T-cell marker, catalog no. 553057; isotype control purified hamster IgG1, catalog no. 11121D) with a secondary antibody Cy-3 goat antiCArmenian hamster antibody.