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Paola Kammrath Betancor, Antonia Hildebrand, Florian Emmerich, Thomas Reinhard, Daniel Boehringer, Gunther R Schlunck, Thabo Lapp; An in vitro model to study antigen-induced macrophage activation and cytokine production in corneal allograft rejection. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
To establish an in vitro system for assessment of corneal antigen detection and processing, cell activation, and cytokine production of human monocyte-derived macrophages.
Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into monocyte-derived macrophages (MDM). A protocol was established to standardize the fragmentation of human corneal tissue. MDMs were stimulated with processed human corneal material. Lipopolysaccharide (LPS) or interferon-gamma (IFNγ) served as controls for MDM activation. Chemokines were detected in MDM supernatants using a Luminex bead-based multiplex assay for 37 cytokines and compared to data from clinical aqueous humour samples. Transwell migration experiments, cell viability assays, and fluorescence-activated cell sorting were used to assess cell recruitment, immunogenicity of corneal endothelium (CEC), and monocyte survival.
Various inflammatory and chemotactic cytokines were released by MDMs after stimulation with human corneal antigen (paired t-test after 48h stimulation: MIP-1β [p<0.001], MCP-1 [p<0.001], MDC [p<0.001], IP-10 [p<0.001]). These in vitro samples shared distinct similarities with human samples of clinical rejections. The presence of CEC material in the stimulation fragments attenuated the upregulation of distinct pro-inflammatory chemokines, (3-way ANOVA: FGF-2 [p<0.01], VEGF [p<0.001]). Medium conditioned by stimulated MDMs induced a selective recruitment of monocytes in a transwell migration model (monocytes versus lymphocytes; 3-way ANOVA with neat supernatant [p<0.001] and with a 1:10 dilution of the MDM supernatant [p<0.001]). Antigen detection and cell activation are not changing MDM cell morphology, but the supernatant modulates immune cell viability and attracts further immune cells.
Our data indicate that distinct aspects of corneal transplant rejection can be successfully modeled in vitro. The observations further support the hypothesis that macrophages play a significant role in the initiation of transplant rejection. Macrophages process allogenic corneal tissue fragments and generate an inflammatory milieu to recruit further immunocompetent cells and modulate cell survival and differentiation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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