Abstract
Purpose::
Activated macrophages play a pivotal role in initiating, maintaining and resolving host inflammatory responses, but they also exert deleterious effects on the host by producing excessive free radicals, lytic enzymes and inflammatory cytokines, which could cause tissue damages and chronic inflammation. Previously we reported that cryopreserved amniotic membrane facilitates macrophage apoptosis. We would like to explore deeper how amniotic membrane may exert an anti-inflammatory activity through suppression of activated macrophages.
Methods::
RAW264.7 cells, a mouse macrophage cell line, were cultured on plastic in DMEM with 10% FBS and stimulated with or without 200U/ml IFN-γ or/and 1ug/ml LPS. Amniotic membrane powder (AMP) was prepared from cryopreserved amniotic membrane and added at different concentrations. In the conditioned media, levels of nitric oxide (NO) and those of PGE2 and PGD2 were measured by Griess reagent and ELISAs, respectively. TGF-ß signaling was determined by adenoviral transfection of a promoter construct and measurement of the enzyme activity of the reporter gene, luciferase, while corrected by that of ß-galactosidase.
Results::
Addition of AMP induced rapid morphological rounding and growth inhibition and up-regulation of NO production by RAW264.7 when stimulated by IFN-γ, LPS or both. However, NO up-regulation was more acute and transient at 24 h than during 24-72 h period. The ratio between PGD2 and PGE2, an index of anti-inflammatory action, was significantly higher when cells were cultured on amniotic membrane stromal surface as compared to those cultured on plastic. The above anti-inflammatory action was correlated with the activation of TGF-ß signaling as judged by the up-regulation of TGF-ß1 promoter activity in transfected RAW264.7.
Conclusions::
Therefore, one plausible mechanism that amniotic membrane may exert its anti-inflammatory effect is to facilitate the resolution of the inflammation caused by activated macrophages.
Keywords: inflammation • nitric oxide • immunomodulation/immunoregulation