Release of ciliary neurotrophic factor from cultured astrocytes and its modulation by cytokines.
CNTF rescues various types of lesioned neurons in vivo, and it needs to be released from astrocytes into the extracellular space to have the effect. However, direct evidence for CNTF release has not been unequivocally demonstrated. We hypothesized that the rapid sequestration by CNTF receptor present on cultured astrocytes might be the cause of the inability to detect CNTF released into astrocyte-conditioned medium (ACM). Therefore, we measured CNTF immunoreactivity in medium conditioned by astrocytes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) which was used to prevent released CNTF from binding to the CNTF receptor, since PI-PLC cleaves glycosyl-phosphatidylinositol anchor of CNTFR alpha, the unique component involved in CNTF binding. CNTF was not detectable in untreated ACM, but was detectable in PI-PLC-treated ACM. These results together with the evidence that PI-PLC treatment did not have a toxic effect on astrocytes prove the fact that CNTF can be released from astrocytes without cell lysis. Subsequently, the effect of cytokines such as IL-1 beta, TNF-alpha, and EGF on CNTF release was examined. These cytokines increased CNTF protein levels in ACMs without increasing CNTF protein levels in astrocyte-extracts, indicating that they enhanced CNTF release from astrocytes.
Kamiguchi
H
H
Department of Neurosurgery, Keio University School of Medicine, Tokyo, Japan.
Yoshida
K
K
Sagoh
M
M
Sasaki
H
H
Inaba
M
M
Wakamoto
H
H
Otani
M
M
Toya
S
S
D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor; D017077:Culture Media, Conditioned; D016207:Cytokines / Q000494:pharmacology*; D009414:Nerve Growth Factors / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar
D002457:Cell Extracts; D020934:Ciliary Neurotrophic Factor; D017077:Culture Media, Conditioned; D016207:Cytokines; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase