Retinal-cell-conditioned medium prevents TNF-alpha-induced apoptosis of purified ganglion cells.
OBJECTIVE
Retinal ischemic processes occurring in glaucoma or diabetic retinopathy induce the secretion of tumor necrosis factor (TNF)-alpha. This cytokine was reported to be either toxic to or protective of retinal ganglion cells (RGCs). In the present study, its effect on RGCs was analyzed in different culture conditions.


METHODS
Adult rat RGCs were prepared in mixed retinal cell cultures and in purified cultures. They were incubated in normoxic or ischemic conditions, in the presence or absence of TNFalpha and/or conditioned media isolated from rat retinal glial cell cultures and from adult mixed retinal cell cultures.


RESULTS
In mixed retinal cell culture, RGCs were insensitive to TNF-alpha, whereas it induced their degeneration in purified adult RGC cultures. This TNFalpha-elicited toxicity was suppressed by TNFalpha-R1-neutralizing antibodies or caspase 8/10 inhibitors. Analyses of mRNA and protein content in purified RGCs revealed a time-dependent reduction in the expression of the inhibitor of caspase-8, c-FLIP. c-FLIP mRNA was also undetectable after 5 days of culture in the presence of TNFalpha. The retinal cell-conditioned medium protected the RGCs from TNFalpha-induced death and prevented the decrease in c-FLIP mRNA and protein in purified cultures. This medium promoted NF-kappaB translocation in purified RGCs, whereas an NF-kappaB inhibitor induced RGC death in mixed retinal cells.


CONCLUSIONS
The results confirm that TNFalpha can induce RGC death by TNF-R1 activation. They indicate, however, that other retinal cells can release a molecule that promotes NF-kappaB translocation in RGCs, the synthesis of the anti-caspase-8, c-FLIP, and thereby prevents TNFalpha-mediated RGC death.
Fuchs
Céline
C
Laboratoire de Physiopathologie Cellulaire et Moléculaire de la Rétine, Institut National de la Santé et de la Recherche Médicale (INSERM) U592, Hôpital Saint-Antoine, Paris, France.
Forster
Valérie
V
Balse
Elise
E
Sahel
José-Alain
JA
Picaud
Serge
S
Tessier
Luc-Henri
LH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D061945:Caspase Inhibitors; D018929:Cell Culture Techniques; D002469:Cell Separation; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020318:Rats, Long-Evans; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D012160:Retina / Q000166:cytology*; D012165:Retinal Ganglion Cells / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity*
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D061945:Caspase Inhibitors; D017077:Culture Media, Conditioned; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha