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D. Bauer, S. Wasmuth, M. Hennig, H. Baehler, K.-P. Steuhl, A. Heiligenhaus; Influence of Amniotic Membrane on Macrophage Functions in the Absence of Interferon-Gamma. Invest. Ophthalmol. Vis. Sci. 2007;48(13):729.
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© ARVO (1962-2015); The Authors (2016-present)
Corneas with ulcerative herpetic stromal keratitis (HSK) rapidly improve after amniotic membrane transplantation (AMT). As apoptosis could previously be induced in macrophages that were cocultured with AM together with interferon(IFN)-g, we determined the modifications of macrophage functions in the presence or absence of LPS, IFN-g or AM in vitro.
Mice with necrotizing HSK were treated with or without AMT, and were followed for clinical signs of keratitis. Corneal cytokine content was quantified by ELISA. Bone-marrow derived or peritoneal macrophages were isolated and cocultured with AM. In some experiments, macrophages were activated with LPS, IFN-g or both. The proliferative responses were determined by 3H-thymidine uptake. IL-12, IL-6 and TNF-a in cell culture supernatants were evaluated. Activation phenotype of macrophages was analyzed by flow cytometry for CD25, CD69 and MHCII. Apoptosis and viability of cells were determined by Hoechst staining and MTT-assay respectively. Macrophages cocultured with UV-HSV-1 with or without AM were used as antigen presenting cells to induce proliferation and cytokine release by HSV-1 specific T-cells.
Two days after AMT, keratitis severity and macrophage infiltration (p<0.01), and expression of IL-12, IL-6, and TNF-a (each, p<0.05) were reduced. Macrophages cocultured with AM showed a decreased proliferative response, a lower IL-12, IL-6 and TNF-a production, and a decreased expression of CD25+, CD69+ and MHC II+. The MTT assay disclosed that viability was comparable in LPS groups without any sign for apoptosis, while macrophage apoptosis could be found after activation with IFN-g and AM. AM treated macrophages led to a reduced proliferative response and IFN-g and IL-2 production in HSV-1-specific lymphocytes.
The data suggest that AMT decreases innate immune responses in HSK corneas. Macrophage functions like antigen presentation, regulatory function with cytokines and accessory function to T-cells were reduced after cocultivation with AM. AM can modulate macrophages functions independent from IFN-gamma and AM-induced apoptosis.
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