Cytokines, tumor necrosis factor-alpha and interleukin-1beta, differentially regulate apoptosis in osteoarthritis cultured human chondrocytes.
OBJECTIVE
This study addresses the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on cell death in human chondrocytes.


METHODS
Osteoarthritis (OA) human chondrocytes stimulated with Actinomycin-D (ActD) were used as a cellular apoptotic model. Caspase family mRNA expression and protein synthesis were analyzed by the ribonuclease protection assay and Western-blot, respectively. Cell viability and apoptosis were evaluated using the 3-[4,5-dimethylthiazol-2yl] 2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Prostaglandin E2 (PGE2) and nitric oxide (NO) were evaluated by enzyme-linked immunosorbent assay (ELISA) and the Griess method, respectively.


RESULTS
TNF-alpha and IL-1beta differentially affected the pattern of caspase mRNA expression by human chondrocytes. TNF-alpha induced a gradual increase in caspase-1 and -8 mRNA levels that was not seen with IL-1beta. The time sequence of caspase-3 and -7 inductions by TNF-alpha differs from that induced by IL-1beta. Cell viability was not modified by TNF-alpha or IL-1beta in cultured chondrocytes. Then, we employed ActD as a model to facilitate cell death. Treatment with TNF-alpha and ActD (TNF-alpha/ActD) increased cell death induced by ActD (23%). Treatment with IL-1beta and ActD (IL-1beta/ActD) did not modulate ActD-induced cell death. Similarly, IL-1beta/ActD did not induce an increase in the activation of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP) cleavage observed by the incubation with TNF-alpha/ActD. These different effects were not due to bcl-2 or mcl-1 levels. Inhibition of PGE2 synthesis by indomethacin increased the cell death induced by IL-1beta/Act-D (59%). An inhibitor of caspase-8 significantly reduced only the TNF-alpha/ActD-induced cell death (58%).


CONCLUSIONS
TNF-alpha and IL-1beta differentially regulate the apoptotic pathway in human chondrocytes. This difference is dependent on PGE2 and caspase-8 levels.
Osteoarticular and Aging Research Unit, Rheumatology Division, C.H. Universitario Juan Canalejo, Xubias 84, 15006-A Coruña, Spain.
M J
MJ
López-Armada
B
B
Caramés
M
M
Lires-Deán
B
B
Cillero-Pastor
C
C
Ruiz-Romero
F
F
Galdo
F J
FJ
Blanco
D017209:Apoptosis / Q000187:drug effects; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism; D020169:Caspases / Q000032:analysis; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D016207:Cytokines / Q000494:pharmacology; D015232:Dinoprostone / Q000032:analysis; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D009569:Nitric Oxide / Q000032:analysis; D010003:Osteoarthritis / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D020169:Caspases; D015232:Dinoprostone