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. CONCLUSION: 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.

Differential effects of Staphylococcus aureus alpha-hemolysin on the synthesis of hyaluronan and chondroitin sulfate by rat chondrosarcoma chondrocytes.

The plasma membranes of rat chondrosarcoma chondrocytes were permeabilized by treatment with alpha-hemolysin, the major toxin produced by Staphylococcus aureus, which forms small, stable, heptameric, transmembrane pores (1-2 nm in diameter) permitting influx/efflux of low-molecular-mass molecules (< or = 2000 Da). Treated chondrocytes were permeable to entry of trypan blue and exit of ATP. We describe the effects of alpha-hemolysin on the synthesis of hyaluronan (HA) and chondroitin sulfate (CS) by chondrocytes using the simple sugar [3H]glucosamine as a metabolic precursor. Chondrocytes permeabilized with alpha-hemolysin in serum-free media decreased intracellular ATP and synthesis of CS to approximately 5% of control within 2-4 h, but synthesized HA (80% of control for 8 h; approximately 65% of control at 24 h). Adding fresh medium (with or without serum) to permeabilized cells increased ATP significantly and increased HA synthesis to near initial control values. Under the same conditions, the recovery of CS synthesis approached initial levels in control but not permeabilized cells. Our model demonstrates that the biosynthesis of HA by these cells in vitro is remarkably stable to cellular perturbations which drastically inhibit synthesis of CS on proteoglycans.

Cellular immunity to cartilage aggrecan core protein in patients with rheumatoid arthritis and non-arthritic controls.

OBJECTIVE: To identify antigen(s) among purified deglycosylated aggrecan peptides spanning the chondroitin sulphate domain that may be responsible for the initiation or perpetuation of the autoimmune responses in rheumatoid arthritis (RA). METHODS: Aggrecan was purified from human articular cartilage and deglycosylated with either bacterial glycosidases or trifluoromethanesulphonic acid (TFMS). Twelve overlapping peptides (15 residues) spanning the chondroitin sulphate domain with N-terminal residues offset by three amino acids were synthesised. T cell responses to these antigens in RA patients and age matched controls were assessed in vitro by antigen specific T cell proliferation assays. RESULTS: Enzymically deglycosylated aggrecan (EDA) stimulated proliferation of T cells isolated from the peripheral blood in a greater proportion of patients with RA than controls. In a subset (12.5%) of RA patients, the magnitude of stimulation lay outside the control range. T cell proliferative responses to TFMS treated aggrecan were greater than, but well correlated with, responses to EDA. T cells from 15 patients were also stimulated with the pooled synthetic peptides. Four of seven patients who demonstrated T cell reactivity to EDA (seven of 15) also showed enhanced T cell proliferation to synthetic peptides. CONCLUSION: These data suggest that an autoantigenic T cell epitope may lie within the chondroitin sulphate domain of aggrecan.

Cellular immunity to cartilage link protein in patients with inflammatory arthritis and non-arthritic controls.

OBJECTIVES: To determine if increased T cell responses to articular cartilage link protein have any correlation with rheumatoid arthritis (RA), and if RA patients with increased responses to link protein also respond to a 17 amino acid peptide covering the 'arthritogenic' epitope in mycobacterial hsp65 which is homologous with link protein. METHODS: The reactivity of T cells from both peripheral blood and synovial fluid, to highly purified human cartilage link protein, hsp65, the 17 amino acid peptide, and bovine type II collagen was determined in patients with RA and nonarthritic controls, by measuring the rate of mononuclear cell proliferation in the presence and absence of antigen. RESULTS: Using peripheral blood mononuclear cells (PBMC), significant reactivity (stimulation index (SI) > 1.5) to link protein was found in 12 of 46 RA patients (26%), but in only four of 44 controls (9%). A greater proportion of RA patients (eight of 17:47%) were reactive to link protein when mononuclear cells from synovial fluid were tested. SI values, however, were generally low (0.5-3.1) and only one patient showed a PBMC response above a reference range of values calculated from the logarithmic values of the normal control population. No reactivity was observed against a 17 amino acid synthetic peptide including the arthritogenic epitope from the mycobacterial hsp65 to which T cell clones isolated from rats in the adjuvant arthritis model react. However, eight of nine RA patients and all of seven controls reacted to the intact hsp65. CONCLUSION: It remains unclear if T cell responses to link protein are involved in the pathogenesis of RA, but it is unlikely that T cells specific for the sequence homologous with the arthritogenic epitope in hsp65 are present in RA patients.