Immunoregulatory cytokines in mouse placental extracts inhibit in vitro osteoclast differentiation of murine macrophages.
BACKGROUND
Previous studies showed that placental extracts (PE) alleviates arthritic symptoms in animal models of arthritis.


METHODS
To evaluate whether murine PEs obtained at embryonic days 7.5 (PE7) and 17.5 (PE18) regulate RANKL-induced osteoclast differentiation, RAW 264.7 cells were cultured with RANKL and MCSF in presence or not of PEs. Tartrate-resistant acid phosphatase (TRAP) was stained and multinucleated TRAP positive cells were visualized under a light microscope. Cathepsin K and metalloprotease expression was assessed by RT-PCR and gelatin zymography respectively. NFATc1 expression was determined by immunoblot. To analyze NFAT-dependent transcription, macrophages were transfected with a luciferase reporter plasmid. Cytokines were determined in PEs by ELISA and immunoblot. Transforming growth factor (TGF)- beta and Interleukin (IL)-10 receptor were inhibited in cell cultures with specific antibodies.


RESULTS
PE7 and PE18 inhibited RANKL-induced multinucleated TRAP positive cells, Cathepsin K expression and metalloprotease activity, as well as NFATc1 expression and activity, thereby inhibiting osteoclast differentiation of RAW cells. Inflammatory/Regulatory cytokine ratio was higher in PE7 than in PE18. Blocking TGF-beta abolished the effect of both, PE7 and PE18, on multinucleated TRAP positive cells and metalloprotease expression, whereas blocking IL-10 receptor reverted the effect of PE18 but not of PE7.


CONCLUSIONS
Inhibition of osteoclast differentiation by PEs was not unexpected, since cytokines detected in extracts were previously found to regulate osteoclast differentiation.


CONCLUSIONS
PEs inhibited osteoclast differentiation of macrophages in vitro. Downregulation of NFATc1 might be involved in this effect. Regulatory/Th2 cytokines play a role in the effect of PEs on osteoclast differentiation.
Instituto de Estudios de la Inmunidad Humoral "Profesor Ricardo A. Margni" (CONICET-UBA), Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 956 (1113), Buenos Aires, Argentina. acanell@ffyb.uba.ar
A
A
Canellada
A
A
Custidiano
F
F
Abraham
E
E
Rey
T
T
Gentile
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D056657:Cathepsin K / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis / Q000494:pharmacology*; D004622:Embryo, Mammalian; D005260:Female; D007527:Isoenzymes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects; D045726:Metalloproteases / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D050778:NFATC Transcription Factors / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010920:Placenta / Q000737:chemistry / Q000378:metabolism*; D011247:Pregnancy; D053245:RANK Ligand / Q000494:pharmacology; D000071681:Tartrate-Resistant Acid Phosphatase; D014020:Tissue Extracts / Q000032:analysis / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors
D016207:Cytokines; D007527:Isoenzymes; D050778:NFATC Transcription Factors; C494005:Nfatc1 protein, mouse; D053245:RANK Ligand; D014020:Tissue Extracts; C505689:Tnfsf11 protein, mouse; D016212:Transforming Growth Factor beta; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D045726:Metalloproteases; D056657:Cathepsin K