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W. Li, H. He, T. Kawakita, S.C. G. Tseng; Amniotic Membrane Stroma Induces Cell Death of Interferon– Activated Macrophages In Vitro . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4517.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Amniotic membrane (AM) used as a temporary or permanent graft for ocular surface reconstruction has demonstrated a potent anti–inflammatory effect. To further explore the mechanism, we investigated whether AM can induce apoptosis of interferon–γ (IFN–γ) activated macrophages in vitro, and modulate their nitric oxide (NO) production. Methods: Mouse macrophages, 0.5 x 106 Raw 264.7 cells/well, were cultured on AM stroma or plastic in a serum–free DMEM containing insulin, transferring and selenium with or without 200 U/ml IFN–γ. Cells were stained by 0.4% Trypan blue at 24, 48, 72, and 96 hours of cultivation, while their media were harvest for the concentration of nitrite (as an index for NO) measured by Griess assay. Results: Non–activated macrophages cultured on AM and plastic did not undergo cell death even after 96 hours of cultivation. In contrast, macrophages activated by IFN–γ exhibited cell death as early as 24 hours and a nearly total cell death at 48 hours when cultured on AM stroma. However, macrophages activated by IFN–γ continued to show no sign of cell death even after 96 hours on plastic. As compared to the non–activated counterparts, IFN–γ–activated macrophages produced a much higher NO both on AM and plastic. Under IFN–γ activation, plastic cultures continuously produced a high level of NO for 96 hours, while AM cultures ceased to produce NO after 48 hours. Conclusion: AM stroma induces rapid cell death of IFN–γ activated, but not resting, macrophages and precludes continuous production of NO in vitro. This effect may partly explain the anti–inflammatory action of AM transplantation in vivo. The nature of such induced cell death will be further defined by immunostaining to annexin V, TUNEL labeling assay, and DNA gel electrophoresis.
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