Suppression of activation and induction of apoptosis in RAW264.7 cells by amniotic membrane extract.
OBJECTIVE
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.
He
Hua
H
TissueTech, Inc and the Ocular Surface Center, Miami, Florida 33173, USA.
Li
Wei
W
Chen
Szu-Yu
SY
Zhang
Shan
S
Chen
Ying-Ting
YT
Hayashida
Yasutaka
Y
Zhu
Ying-Ting
YT
Tseng
Scheffer C G
SC
D000199:Actins / Q000378:metabolism; D000650:Amnion / Q000166:cytology*; D000818:Animals; D017209:Apoptosis; D018122:B7-1 Antigen / Q000378:metabolism; D051940:B7-2 Antigen / Q000378:metabolism; D015153:Blotting, Western; D002457:Cell Extracts / Q000494:pharmacology*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D009000:Monocytes / Q000378:metabolism / Q000473:pathology*
D000199:Actins; D018122:B7-1 Antigen; D051940:B7-2 Antigen; D002457:Cell Extracts; D016207:Cytokines; D000949:Histocompatibility Antigens Class II; D008070:Lipopolysaccharides; D007371:Interferon-gamma