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Originally published In Press as doi:10.1167/iovs.08-1781 on July 9, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:4468-4475.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.08-1781
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Suppression of Activation and Induction of Apoptosis in RAW264.7 Cells by Amniotic Membrane Extract

Hua He,1 Wei Li,1,2 Szu-Yu Chen,1 Shan Zhang,1 Ying-Ting Chen,1 Yasutaka Hayashida,1 Ying-Ting Zhu,1 and Scheffer C. G. Tseng1

1From TissueTech, Inc. and the Ocular Surface Center, Miami, Florida.

PURPOSE. Macrophages play a pivotal role in initiating, maintaining, and resolving host inflammatory/immune responses but may cause recalcitrant inflammation and tissue damage if not controlled. Clinically, amniotic membrane (AM) transplantation suppresses inflammation in ocular surface reconstruction. Experimentally, the authors and others have reported that AM facilitates macrophage apoptosis. However, it remains unclear whether such anti-inflammatory activity is retained in AM extract (AME).

METHODS. Herein the authors demonstrate in resting and activated (by interferon [IFN]-{gamma}, lipopolysaccharide [LPS], or IFN-{gamma}/LPS) murine monocyte/macrophage RAW264.7 cells that AME suppresses cell spreading and reduces actin filaments determined by phalloidin staining and Western blotting of Triton X-100 extracted cell lysate.

RESULTS. Western blot and immunocytochemistry staining showed AME downregulates the expression of such cell surface markers as CD80, CD86, and major histocompatibility complex class 2 antigen. Cell growth/viability is inhibited whereas cell apoptosis is enhanced by AME. Accordingly, secreted proinflammatory cytokines such as TNF-{alpha} and IL-6 are reduced, but anti-inflammatory cytokine IL-10 is upregulated.

CONCLUSIONS. Collectively, these results suggest that, similar to amniotic membrane, AME retains anti-inflammatory activities and does so by downregulating activation and inducing apoptosis in macrophages.






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