Tumour necrosis factor alpha stimulates nitric oxide production more potently than interleukin-1beta in porcine articular chondrocytes.
OBJECTIVE
To compare the time- and concentration-dependent effects of tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) on the induction of nitric oxide synthase (NOS), the production of nitric oxide (NO) and the expression of aggrecan and hyaluronan (HA) in chondrocytes.


METHODS
Primary porcine articular chondrocytes were treated with recombinant human (rh) TNF-alpha or rhIL-1beta for up to 72 h. Culture supernatants were assayed for NO production. Synthesis of HA and aggrecan was determined by radiolabelling cultures with [(3)H]glucosamine and/or [(35)S]sulphate. Total RNA was isolated and the time courses of changes in gene expression of inducible NOS and HA synthase-2 were investigated by reverse transcriptase-polymerase chain reaction.


RESULTS
rhTNF-alpha stimulated more NO production than rhIL-1beta. It was also active at lower concentrations; rhTNF-alpha at 0.006 pM (100 pg/ml) was equivalent to rhIL-1beta at 0.29 pM (5000 pg/ml). The time course of induction was transient and slower at low concentrations. Contrary to previous reports, rhTNF-alpha and rhIL-1beta were of similar potency in the inhibition of aggrecan synthesis. In contrast, both cytokines stimulated HA synthesis, and this was correlated with the transient induction of HA synthase-2. An inhibitor of inducible NOS relieved the inhibition of aggrecan synthesis caused by both cytokines at low concentrations, but it showed little effect on HA synthesis.


CONCLUSIONS
At low concentrations, rhTNF-alpha was 50 times more potent than rhIL-1beta in stimulating NO production by chondrocytes and it was of similar potency in inhibiting aggrecan synthesis and in stimulating HA synthesis. Inhibition of inducible NOS activity relieved some of the effects on aggrecan synthesis, showing that part of the action of TNF-alpha is mediated through NO. HA synthesis was not affected.
Goodstone
N J
NJ
The Wellcome Trust Centre for Cell Matrix Research, School of Biological Sciences, The University of Manchester, Manchester M139PT, UK.
Hardingham
T E
TE
D000199:Actins / Q000235:genetics; D053674:Aggrecans; D000818:Animals; D002358:Cartilage, Articular / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000502:physiology*; D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D006801:Humans; D006820:Hyaluronic Acid / Q000096:biosynthesis; D007375:Interleukin-1 / Q000494:pharmacology*; D007700:Kinetics; D037181:Lectins, C-Type; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D018631:ras Proteins / Q000235:genetics
D000199:Actins; D053674:Aggrecans; D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D007375:Interleukin-1; D037181:Lectins, C-Type; D009573:Nitrites; D011509:Proteoglycans; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D006820:Hyaluronic Acid; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D018631:ras Proteins