Effects of methotrexate on the oxidative metabolism of cultured rabbit articular chondrocytes.
OBJECTIVE
The mechanism of action of methotrexate (MTX) in inflammatory joint disease is still unclear. We examined the possible interactions of MTX with the oxidative metabolism of rabbit articular chondrocytes.


METHODS
Cell cultures of articular chondrocytes enzymatically isolated from juvenile New Zealand white rabbits were incubated 24 h with either MTX (0.22 or 1.1 microM), bacterial lipopolysaccharide (LPS, 50 microg/ml), or both. Cytofluorometry was then performed using 2',7'-dichlorofluorescein diacetate (DCFH-DA), rhodamine 123 (Rh123), or propidium iodide (PI). These fluorochromes allow evaluation of cellular production of H2O2, mitochondrial membrane potential, and cell viability, respectively. In a separate experiment, we used the Griess colorimetric technique to evaluate cellular nitric oxide (NO) production.


RESULTS
Addition of MTX alone (0.22 or 1.1 microM) inhibited spontaneous production by chondrocytes of H2O2 (p < 0.01 and p < 0.001, respectively) and NO (p < 0.01 both concentrations). The LPS induced increase in H2O2, production was inhibited by MTX at 0.22 and 1.1 microM (p < 0.01 both concentrations), whereas the LPS induced increase in NO synthesis was not influenced by MTX, even at 1.1 microM. MTX did not significantly modify mitochondrial activity or cell viability.


CONCLUSIONS
MTX at therapeutic concentrations in vitro inhibits the production of H2O2 and NO by unstimulated chondrocytes, and only the H2O2 overproduction by LPS stimulated chondrocytes. These properties may contribute to the therapeutic effect of MTX in RA.
Hayem
G
G
Service de Rhumatologie, CHU Bichat-Claude Bernard and INSERM U13, Paris, France. gilles.hayem@bch.ap-hop-paris.fr
Domarle
O
O
Thuong-Guyot
M
M
Pocidalo
J J
JJ
Meyer
O
O
D000704:Analysis of Variance; D000818:Animals; D018501:Antirheumatic Agents / Q000494:pharmacology*; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008727:Methotrexate / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D011817:Rabbits
D018501:Antirheumatic Agents; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D008727:Methotrexate