Osteoarthritic synovial fibroblasts possess an increased level of tumor necrosis factor-receptor 55 (TNF-R55) that mediates biological activation by TNF-alpha.
OBJECTIVE
To investigate the presence, number, and level of expression of tumor necrosis factor receptors (TNF-R) in normal and osteoarthritic (OA) human synovial fibroblasts; to examine which receptor isotype mediates the biological response of these cells to TNF-alpha; and to study homologous regulatory mechanisms of TNF-R by TNF-alpha.


METHODS
We used radioligand binding assay with [125I]TNF-alpha and flow cytometric analysis with specific antireceptor antibodies to characterize receptor populations, densities, and ligand induced internalization of TNF-R. Inducible cyclooxygenase (COX-2) synthesis, prostaglandin E2 (PGE2) release, and TNF-R shedding (soluble receptors, TNF-sR) were measured after incubation with TNF-alpha the presence or absence of receptor specific blocking antibodies.


RESULTS
Although radioligand binding assays showed no difference in the density or affinity of TNF-R in OA synovial fibroblasts compared with normal cells, flow cytometric analysis revealed that OA cells express a significantly higher level of TNF-R55 (p < 0.04) than normal cells. The TNF-R55 was found to be the major receptor species responsible for ligand binding activity, such as COX-2 induction and PGE2 synthesis, since a specific antireceptor TNF-R55 blocking antibody inhibited about 76% of TNF-alpha binding and TNF-alpha stimulated COX-2 induction and PGE2 production. Further experiments revealed that TNF-R55 was the only receptor type internalized after binding TFN-alpha, whereas TNF-R75 was concomitantly shed. Moreover, reducing the shedding of TNF-sR, particularly the TNF-sR75, with a synthetic inhibitor decreased TNF-alpha induced PGE2 production.


CONCLUSIONS
TNF-R55 is the major receptor isoform transducing PGE2 and COX-2 responses to TNF-alpha in OA synovial fibroblasts; soluble receptors could be involved in facilitating the binding of TNF-alpha to its receptor.
Alaaeddine
N
N
Osteoarthritis Research Unit, Louis-Charles Simard Research Centre, Notre-Dame Hospital, University of Montreal, Canada.
DiBattista
J A
JA
Pelletier
J P
JP
Cloutier
J M
JM
Kiansa
K
K
Dupuis
M
M
Martel-Pelletier
J
J
D000368:Aged; D019138:Antibodies, Blocking / Q000494:pharmacology; D015703:Antigens, CD / Q000378:metabolism* / Q000502:physiology; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis; D008565:Membrane Proteins; D008875:Middle Aged; D010003:Osteoarthritis / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D011869:Radioligand Assay; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism* / Q000502:physiology; D047888:Receptors, Tumor Necrosis Factor, Type I; D013583:Synovial Membrane / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D019138:Antibodies, Blocking; D015703:Antigens, CD; D007527:Isoenzymes; D008565:Membrane Proteins; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone