Tumour necrosis factor {alpha} stimulates nitric oxide production more potently than interleukin-1ß in porcine articular chondrocytes

N. J. Goodstone and T. E. Hardingham

The Wellcome Trust Centre for Cell Matrix Research, School of Biological Sciences, The University of Manchester, Manchester M139PT, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To compare the time- and concentration-dependent effects of tumour necrosis factor {alpha} (TNF-{alpha}) and interleukin 1ß (IL-1ß) 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-1ß for up to 72 h. Culture supernatants were assayed for NO production. Synthesis of HA and aggrecan was determined by radiolabelling cultures with [3H]glucosamine and/or [35S]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-1ß. It was also active at lower concentrations; rhTNF-{alpha} at 0.006 pM (100 pg/ml) was equivalent to rhIL-1ß 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-1ß 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-1ß 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.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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The function of articular cartilage in the joint is to provide a low-friction, load-bearing surface that helps distribute the stress applied to the subchondral bone [1, 2]. Hyaluronan (HA) interacts with the large aggregating chondroitin sulphate proteoglycan aggrecan to produce large multimolecular aggregates. These aggregates, together with the fibrillar collagen network, provide the essential compressive properties of the extracellular matrix of articular cartilage. The continued synthesis of HA and aggrecan by chondrocytes is necessary for the maintenance of the cartilage matrix. The progressive degradation and loss of these aggregates through accelerated proteinase-mediated matrix degradation is a characteristic feature of inflammatory joint diseases, such as rheumatoid arthritis, and of the later stages of non-inflammatory degenerative joint diseases, such as osteoarthritis [3].

The proinflammatory cytokines interleukin 1ß (IL-1ß) and tumour necrosis factor {alpha} (TNF-{alpha}) have a major catabolic effect on chondrocytes. Primary chondrocytes in vitro respond to IL-1ß with changes in gene expression that include increased expression of several forms of metalloproteinases [4] and the down-regulation of genes encoding cartilage matrix proteins [5]. Previous studies [6, 7] showed recombinant human (rh) TNF-{alpha} to have similar effects to rhIL-1ß on chondrocytes in porcine cartilage by inhibiting aggrecan synthesis and stimulating matrix degradation, but it appeared to have only ~10% of the potency. Stadler et al. [8] showed that IL-1ß-treated human articular chondrocytes expressed inducible nitric oxide synthase (iNOS) and produced large amounts of nitric oxide (NO). In the same study, other inflammatory mediators, such as interferon {gamma}, fibroblast growth factor and TNF-{alpha}, were reported not to stimulate NO production, but TNF-{alpha} and IL-1ß were synergistic. NO may contribute to cartilage catabolism by causing free radical damage in the matrix close to the chondrocytes and/or by acting intracellularly to trigger signalling events that result in further changes in gene expression [9]. There is evidence in several species that NO may mediate the inhibition of aggrecan biosynthesis [1012]. In these studies, treatment of cartilage explants with an inhibitor of NO, N-monomethyl L-arginine (L-NMA), restored aggrecan synthesis, whereas exogenous NO inhibited aggrecan synthesis. These results suggest that NO promotes matrix catabolism [9]. However, Hauselmann et al. [13] reported that IL-1 and L-NMA together were more catabolic than IL-1 alone in cultured human cartilage explants, which suggested that NO might have a protective effect.

Much less is known about the effects of IL-1ß and TNF-{alpha} on HA metabolism. While the HA content of ageing tissues appears to increase, a significant decrease in HA has been observed in the cartilage of patients with osteoarthritis [14]. Recently, three distinct HA synthase (HAS) genes have been identified in vertebrates [15]. The rates of HA biosynthesis and the average polymer size both differ among the three synthases. In situ studies [16] have indicated that HAS-2 is highly expressed within epiphyseal growth plates and, in addition, is expressed within elements of the developing foetal skeleton. Furthermore, a study by Nishida et al. [17], using human articular chondrocytes, demonstrated that HAS-2 plays a crucial role in matrix assembly and retention. In the present work, we investigated the effects of rhTNF-{alpha} and rhIL-1ß on the induction of iNOS and on the synthesis of two matrix components, HA and aggrecan, in primary pig articular chondrocytes.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All chemicals and reagents were of the highest purity commercially available. Dulbeccos' Modified Eagle Medium (DMEM), L-glutamine, Hanks' balanced salt solution (HBSS), foetal calf serum (FCS), penicillin, streptomycin sulphate, FungizoneTM (amphotericin), HEPES, agarose, 1-kilobase (kb) DNA ladder and ethidium bromide were obtained from Gibco Life Technologies, Invitrogen Ltd, Paisley, UK. Tissue culture plates were from Costar Corporation, Fisher Scientific, Loughborough, UK. Cetylpyridinium chloride (CPC), L-NMA, trypan blue, papain, calf thymus DNA standard, Hoechst 33258 dye, TriReagentTM, Streptomyces hyaluronidase, type VI-S bovine testicular hyaluronidase and type IA-S collagenase were from Sigma Chemicals (Poole, UK). rhIL-1ß and rhTNF-{alpha} were from Calbiochem-Novabiochem (Nottingham, UK) and contained no detectable endotoxin (<100 pg/µg). Aliquots were stored frozen in phosphate-buffered saline at -80°C and showed consistent responses in chondrocyte preparations over several months. rhIL-1ß and rhTNF-{alpha} concentrations were standardized with QuantikineTM (Abingdon, UK) immunoassay kits from R+D Systems (UK). The Geneamp RNA polymerase chain reaction (PCR) core kit for reverse transcriptase–PCR (RT–PCR) was from Perkin-Elmer (Beaconsfield, UK). [D-6-3H]Glucosamine hydrochloride (18.5 Ci/mmol) was from Amersham (Amersham, UK). [35S]Sulphate (carrier-free) was from ICN Radiochemicals (Thame, UK). Sephacryl S-500 HR was from Pharmacia Biotech (Uppsala, Sweden). Nylon mesh (60 µm aperture) was from Gelman Sciences (Northampton, UK). Trotters from 6-month old bacon pigs were obtained from the local abattoir.

Chondrocyte isolation
Articular cartilage was dissected from the metacarpophalangeal joints of pigs (aged ~6 months) within 3 h of slaughter and transferred to culture medium comprising DMEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin sulphate, 2 mM L-glutamine, 20 mM HEPES and 2.5 ng/ml amphotericin. Articular chondrocytes were released from their extracellular matrix using a modified method [18]. Briefly, finely chopped cartilage pieces were digested sequentially in culture medium containing 1% (v/v) FCS in a shaking incubator at 37°C with testicular hyaluronidase (310 U/ml) for 1 h and then 0.25% (w/v) collagenase for 6 h. Undigested cartilage pieces were removed by sieving through sterile nylon mesh. Chondrocytes were isolated by centrifugation at 1500 g(average) for 10 min, washed once with HBSS and four times with culture medium, and finally resuspended in culture medium containing 10% (v/v) FCS. Cell viability was determined to be >95% by exclusion of trypan blue.

Primary chondrocyte culture
Chondrocyte monolayers were established by seeding 200 000 cells/well in a total volume of 200 µl of culture medium containing 10% (v/v) FCS into 96-well culture plates, and were cultured at 37°C in humidified 5% CO2. After an overnight attachment period, all experiments were initiated in triplicate by exchanging medium for serum-free culture medium containing rhIL-1ß or rhTNF-{alpha}, with or without L-NMA. Unless indicated, medium, cytokines and inhibitor were replenished daily. In chondrocyte monolayers cultured for 0, 24, 48 and 72 h, the DNA content showed that the cell number did not change significantly during the 72 h culture period [measured using Hoechst 33258 dye and a fluorometer (DyNA Quant 200; Pharmacia)]. In preliminary experiments, 48 h culture media from chondrocyte monolayers treated with either rhIL-1ß or rhTNF-{alpha} were assayed for the presence of porcine TNF-{alpha} or IL-1ß respectively. The Quantikine immunoassay kits detect porcine cytokines, but none was detected.

Glycosaminoglycan synthesis
Chondrocyte monolayers were labelled metabolically for up to 24 h with 25 µCi/ml [35S]sulphate and/or 50 µCi/ml [3H]glucosamine. After radiolabelling, the medium and cell layer fractions were separately digested with papain (200 µg in 100 µl) for 3 h at 60°C. Radiolabelled glycosaminoglycan (GAG) was then isolated as described previously by CPC precipitation [19]. The isolated GAG from each digest was redissolved in 100 µl of 0.1 M sodium acetate (pH 7.0) and the incorporation of radiolabel was determined.

Gel chromatography
A Sephacryl S-500 HR (9x600 mm) was eluted with 0.2 M sodium acetate (pH 6.8) at a flow rate of 0.4 ml/min. Papain-digested, CPC-purified GAG samples were applied to the column and fractions were collected at 2 min intervals. The excluded volume (Vo) and included volume (Vt) were determined with aggrecan monomer (Mr 2.5x106 Da) and [35S]sulphate respectively. Sample recovery of radiolabelled material was >85% of the amount applied.

Nitrite determination
To determine the amount of NO produced by the articular chondrocytes, the culture supernatants were assayed for the stable end-product of NO oxidation, nitrite, using the Greiss reaction [20] with sodium nitrite as standard. Absorbance was measured at 570 nm using a Labsystems (Helsinki, Finland) Multiskan RC plate-reader.

RNA isolation and semi-quantitative RT–PCR
Total cellular RNA was extracted from chondrocyte monolayers using TriReagent, as described in the manufacturer's instructions. RNA (1 µg) was reverse-transcribed in a 20 µl reaction volume for 1 h at 42°C and then the complementary DNA (cDNA) was amplified using Taq DNA polymerase in a 100 µl reaction volume according to the manufacturer's instructions. Expression of pig iNOS (accession number U59390) and pig HAS-2 mRNA was studied by PCR using sequence-specific primers with amplification for ß-actin as an internal control. Degenerate RT–PCR was used to identify and clone pig HAS-2 mRNA. A HAS-2-specific sense primer was combined with a degenerate antisense HAS primer, and the resulting fragment was cloned and sequenced so that pig-specific primers could be designed (data not shown). Primer sequences, primer concentrations, annealing temperatures and expected fragment size are shown in Table 1Go. Each PCR cycle (25 cycles in total) consisted of 30 s of denaturation at 94°C, 30 s of annealing at the optimized annealing temperature, 40 s of extension at 72°C, and final extension for 10 min at 72°C. The cDNA products were separated on 1.5% (w/v) agarose gels containing ethidium bromide and were visualized with ultraviolet light. The sizes of the fragments were confirmed by reference to a 1 kb DNA ladder. The identities of the cDNA products, all single bands, were all confirmed by cloning and sequencing (data not shown).


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TABLE 1.  Nucleotide primers for PCR, primer concentrations, annealing temperatures and expected product sizes for porcine iNOS, HAS-2 and ß-actin genes

 

Statistics
All cultures were radiolabelled, or assayed in triplicate, and each experiment was repeated at least three times with chondrocytes from three animals. The significances of differences between mean values were determined using a two-tailed t-test; P<0.05 was considered significant. Where appropriate, results are expressed as the mean±S.E.M.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of IL-1ß and TNF-{alpha} on aggrecan synthesis and on the production of NO in porcine chondrocytes
Experiments were performed with rhTNF-{alpha} and rhIL-1ß to determine the minimal effective concentrations for the inhibition of aggrecan synthesis (Fig. 1Go) and for the induction of NO synthesis (Fig. 2Go) over 48 h. Chondrocytes were treated with concentrations of 0–10 000 pg/ml (0–0.6 pM) of either rhTNF-{alpha} or rhIL-1ß and cultures were metabolically labelled with [35S]sulphate and [3H]glucosamine between 24 and 48 h. The concentration–response relationships for the inhibition of aggrecan synthesis were found to be broadly similar, 50% inhibition being achieved with 250 pg/ml (0.015 pM) rhTNF-{alpha} or rhIL-1ß (P>0.45). Furthermore, they did not vary when experiments were carried out with cytokines from three commercial sources: Calbiochem, Sigma and Roche (data not shown). The production of NO was measured over 0–24 and 24–48 h (Fig. 2AGo and BGo) and rhTNF-{alpha} was shown to be more potent than rhIL-1ß in stimulating NO synthesis. The threshold concentration of rhTNF-{alpha} required for NO induction was much lower than that required for rhIL-1ß, as rhTNF-{alpha} at 100 pg/ml (0.006 pM) doubled the NO concentration compared with that found in control cultures from 24 to 48 h, but this was only achieved with rhIL-1ß at 5000 pg/ml (0.29 pM).



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FIG. 1.  Inhibition of aggrecan synthesis in chondrocyte monolayers treated with rhIL-1ß or rhTNF-{alpha}. Cultures were treated without a cytokine or with a concentration range (0–10000 pg/ml (0–0.6 pM) of either (A) rhIL-1ß (circles) or (B) rhTNF-{alpha} (triangles) for 48 h. During the last 24 h, triplicate cultures were radiolabelled with [35S]sulphate (filled symbols) or [3H]glucosamine (open symbols). Monolayers were digested with papain and GAGs were isolated by CPC precipitation. Results are expressed as total mean and S.E.M. counts per minute (cpm) per culture (n=3). Data represent the mean of three experiments.

 


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FIG. 2.  NO production in chondrocyte monolayers treated with rhIL-1ß or rhTNF-{alpha}. Cultures were untreated (squares) or treated with a concentration range (0–10 000 pg/ml, 0–0.6 pM) of either rhIL-1ß (circles) or rhTNF-{alpha} (triangles) for 48 h. Culture supernatants were removed and assayed for nitrite after (A) 24 h and (B) 48 h using sodium nitrite as standard. Nitrite accumulation at 24 h was subtracted from nitrite accumulation at 48 h. Values are total mean nitrite per culture (n=3); S.E.M. values (<5%) are not shown. *P<0.05 compared with control values (two-tailed t-test). Data represent the mean of three experiments.

 
Comparison of the production of NO in cultures stimulated with rhTNF-{alpha} during 0–24 and 24–48 h (Fig. 2AGo and BGo) suggested that, although at high concentrations it was much higher in the first 24 h, at low concentrations (<200 pg/ml, 0.01 pM) it was higher during the second day of treatment. This was substantiated in further experiments (Table 2Go), which showed that the kinetics of the rise and fall of NO production was much delayed at a low concentration of rhTNF-{alpha} (100 pg/ml, 0.006 pM). It peaked during 24–48 h and had not completely returned to control levels by 96 h. There was no detectable increase in NO production in rhIL-1ß-treated cells at this concentration.


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TABLE 2.  NO production by porcine chondrocytes treated with 100 pg/ml (0.006 pM) of either rhIL-1ß or rhTNF-{alpha}

 

Time course of the effects of TNF-{alpha} and IL-1ß on aggrecan synthesis and on the induction of NO synthesis
The time course of the effects of rhTNF-{alpha} and rhIL-1ß on aggrecan synthesis and NO production was determined up to 48 h (Fig. 3Go). rhTNF-{alpha} (5 ng/ml, 0.29 pM) and rhIL-1ß (5 ng/ml, 0.29 pM) reduced [35S]sulphate incorporation into aggrecan at 48 h to 75 and 95% of control levels respectively (Fig. 3AGo). The kinetics was steadily progressive and showed some differences for the two cytokines. rhIL-1ß caused little effect in the first 6 h but then aggrecan synthesis fell sharply, whereas there was a faster onset in rhTNF-{alpha}-treated cultures. Both cytokines thus showed strong non-transient inhibition of aggrecan synthesis.



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FIG. 3.  Time course of the effects of rhIL-1ß and rhTNF-{alpha} on the inhibition of aggrecan synthesis and NO production in chondrocytes. All cultures were radiolabelled, or assayed in triplicate, and the data represent the mean of three experiments. Chondrocyte monolayers were untreated (squares) or treated with 5 ng/ml (0.29 pM) of either rhIL-1ß (circles) or rhTNF-{alpha} (triangles) for 6 h windows. (A) Incorporation of [35S]sulphate. Results are expressed as the total mean cpm and S.E.M. (B) NO production measured at the end of each 6 h window. Results are expressed as nitrite synthesized per culture. The S.E.M. values were <5% and are not shown. *P<0.05 compared with control values (two-tailed t-test).

 
In contrast, NO synthesis (Fig. 3BGo) in the same cultures showed very different kinetics from the effects on aggrecan synthesis. In rhTNF-{alpha}- or rhIL-1ß-treated cultures, NO production peaked at 12–18 h, but by 18–24 h it had declined to a level only a little above that of the control cultures. NO production then peaked again between 36 and 42 h. Comparison of the effects of the cytokines at this concentration (5 ng/ml, 0.29 pM) showed that, during 0–6 h, similar amounts of NO were synthesized by the cells, but by 12–18 h the rhTNF-{alpha}-treated cells produced more than twice the amount of NO.

Investigation of the induction of iNOS mRNA (Fig. 4Go) in cytokine-treated chondrocytes showed that it was rapid and transient and followed a pattern similar to that of the production of NO. It was maximal at 6 h but had declined at 24 h and was also low at 48 h. Expression of iNOS mRNA was not detected in control cells at any of the time points examined.



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FIG. 4.  Induction of iNOS mRNA in chondrocyte monolayers treated with rhIL-1ß or rhTNF-{alpha}. Cultures were untreated or treated with either (A) rhIL-1ß (5 ng/ml, 0.29 pM) or (B) rhTNF-{alpha} (5 ng/ml, 0.29 pM) for 48 h. RNA was isolated at 0, 6, 24 and 48 h, reverse-transcribed and amplified using primers specific for porcine iNOS. Representative data from one experiment are shown.

 

Effects of L-NMA, an NOS inhibitor, on aggrecan synthesis in the presence of TNF-{alpha} or IL-1ß
Incubation of the chondrocytes with cytokines in the presence of an inhibitor of NOS, L-NMA (1 mM), showed that the inhibitor was quite effective in relieving the inhibition of aggrecan synthesis (Fig. 5Go). Below 150 pg/ml (0.009 pM) of either cytokine, the inhibitor completely restored aggrecan synthesis, and even at higher concentrations it produced significant partial recovery of aggrecan synthesis. At a cytokine concentration of 2.5 ng/ml (0.147 pM), L-NMA had a greater effect on the recovery of aggrecan synthesis in rhIL-1ß-treated chondrocytes (80%) than in rhTNF-{alpha}-treated chondrocytes (55%).



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FIG. 5.  Effect of L-NMA on the inhibition of aggrecan synthesis by rhIL-1ß- or rhTNF-{alpha}-treated chondrocytes. Monolayer cultures were treated with a concentration range (0–5 ng/ml, 0–0.29 pM) of either rhIL-1ß (circles) or rhTNF-{alpha} (triangles) for 48 h. Incorporation of [35S]sulphate from 24 to 48 h in cytokine-treated cultures with L-NMA (1 mM) was divided by [35S]sulphate incorporation for control cultures without L-NMA and the results are shown as percentages. Inhibition was suppressed up to 300 pg/ml (0.018 pM) and partially relieved at 2.5 ng/ml (0.147 pM). Data represent the mean of three experiments.

 

Effects of TNF-{alpha} and IL-1ß on aggrecan and HA synthesis
The total amount of GAG radiolabelled between 24 and 48 h from control cultures and cultures treated with rhTNF-{alpha} (5 ng/ml, 0.29 pM) or rhIL-1ß (5 ng/ml, 0.29 pM) was analysed by Sephacryl S-500 chromatography (Fig. 6Go). The first peak from both treated and untreated chondrocyte monolayers contained only 3H activity (Fig. 6CGo and DGo) and was sensitive to digestion with Streptomyces hyaluronidase (data not shown), showing it to represent HA. rhIL-1ß and rhTNF-{alpha} appeared to stimulate HA synthesis by 60 and 40% respectively. At similar concentrations of cytokine, L-NMA (1 mM) caused no significant inhibition of HA synthesis (data not shown).



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FIG. 6.  Sephacryl S-500 HR chromatography of GAGs. Macromolecules labelled with 35S (A and B, open symbols) or 3H (panels C and D, filled symbols) were isolated from monolayer cultures radiolabelled in the absence of cytokines (squares) or in the presence of rhIL-1ß (circles) or rhTNF-{alpha} (triangles) between 24 and 48 h. Papain-digested, CPC-precipitated GAGs were analysed by Sephacryl S-500 chromatography (as described in Materials and methods). The position of the eluted HA is illustrated in the inset panels E and F. Representative data from one experiment are shown.

 
For both treated and untreated cultures, a GAG peak (Kd=0.85) was observed which contained both 3H (Fig. 6CGo and DGo) and 35S (Fig. 6AGo and BGo) activity. This peak, which contained all of the 35S activity, contained the chondroitin sulphate peptides generated by the papain digestion. The rhIL-1ß-treated cells showed a decrease in incorporation of 3H and 35S of 45 and 36% respectively. In comparison, rhTNF-{alpha} had a slightly weaker effect, decreasing incorporation of 3H and 35S by 23 and 30% respectively. Furthermore, for both cytokines the ratio of 3H to 35S activities in the GAG peak (Kd=0.85) was similar, suggesting that the decrease in aggrecan synthesis was not due to selective effects on sulphation, and the average length of the chains also showed no change.

Effects of TNF-{alpha} and IL-1ß on the expression of pig HAS-2
Specific primers for pig HAS-2 were designed, tested and used to study the effects of rhTNF-{alpha} and rhIL-1ß on the levels of expression of mRNA transcripts in primary chondrocytes (Fig. 7Go). In comparison with the effects on iNOS mRNA, the induction of pig HAS-2 expression was again transient, but it was much slower and reached a maximum at 24 h and was low at 48 h. The increase in HA synthesis was thus correlated with an increase in the level of HAS-2 message (Fig. 6EGo and FGo).



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FIG. 7.  Induction of pig HAS-2 mRNA in chondrocyte monolayers treated with rhIL-1ß or rhTNF-{alpha}. Cultures were untreated or treated with either (A) rhIL-1ß (5 ng/ml, 0.29 pM) or (B) rhTNF-{alpha} (5 ng/ml, 0.3 pM) for 48 h. RNA was isolated at 0, 6, 24 and 48 h, reverse-transcribed and amplified using primers specific for porcine HAS-2. Representative data from one experiment are shown.

 


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, a comparison of the actions of rhTNF-{alpha} and rhIL-1ß on porcine chondrocytes showed that rhTNF-{alpha} is much more potent than was reported previously. Although this study used human cytokines and porcine cartilage, the first studies reporting the effects of these cytokines on cartilage also used human cytokines on porcine cartilage [6, 7] and suggested that rhTNF-{alpha} was only 10% as potent as rhIL-1ß in inhibiting aggrecan synthesis. Subsequent work with human cartilage confirmed these observations [21], but suggested that human cartilage was rather less responsive than cartilage from young animals. From these earlier studies, it was concluded that IL-1 was potentially more damaging to articular cartilage in joint disease than TNF-{alpha}. However, there have been few recent studies directly comparing the actions of rhTNF-{alpha} and rhIL-1ß on articular cartilage of animal or human origin. In the present study, the similar potency of rhTNF-{alpha} and rhIL-1ß in inhibiting aggrecan synthesis was unexpected, but it was found consistently and did not vary when cytokines from three commercial sources were used. The difference between the present and previous results may be the consequence of improved methods for the expression and purification of recombinant proteins, which may retain more of their bioactive properties. In addition, there are new and improved immunoassays, such as the Quantikine kits (R+D Systems), which are available to quantitate and standardize rhIL-1ß and rhTNF-{alpha} accurately.

Having established that rhTNF-{alpha} had similar potency to rhIL-1ß in the inhibition of aggrecan synthesis, it was then most interesting to discover that it was much more potent than rhIL-1ß in the induction of NO synthesis. rhTNF-{alpha} stimulated up to three times more NO production than rhIL-1ß at similar concentrations, and again this was found consistently with rhTNF-{alpha} from three commercial suppliers. The potency (and transient time course) of the action of rhTNF-{alpha} on iNOS in experiments with pig articular cartilage explants was similar to that in isolated cells (data not shown). Although the potency differed, the kinetics of the induction of iNOS mRNA was similar for the two cytokines. The onset occurred within 6 h, but the iNOS gene was expressed only transiently and levels were much lower by 24 h. Interestingly, the kinetics was much slower at lower concentrations, which raises the question of whether chronic low exposure to TNF-{alpha} induces a lasting low level of expression of NO by articular chondrocytes, which may cause accumulative damage in the tissue.

Previous studies using human [20] and rabbit [10] chondrocytes in alginate and monolayer cultures have suggested that the inhibition by IL-1ß of aggrecan synthesis is mediated by NO, although in different cartilage systems the reported effect of iNOS inhibitors on IL-1ß-inhibited aggrecan synthesis has varied from no effect [22] to full relief [12]. The present results, comparing the effects of rhTNF-{alpha} and rhIL-1ß, clearly show no direct linear correlation between NO synthesis and the inhibition of aggrecan synthesis. However, the effect of L-NMA showed that inhibiting NO synthesis was able to provide some relief of the inhibition of aggrecan synthesis by both cytokines. This suggests that signal transduction via NO may contribute to the inhibition of aggrecan synthesis. The effects with different cytokine concentrations showed that, with lower concentrations (<150 pg/ml, 0.009 pM), there was complete recovery of aggrecan synthesis, but at higher concentrations of cytokine (>150 pg/ml, 0.009 pM), L-NMA restored aggrecan synthesis only partially, even though it appeared to block NO production completely. These results suggest that, at low cytokine concentrations, the inhibition of aggrecan synthesis is caused by NO, but at higher cytokine concentrations there are additional NO-independent signalling mechanisms.

As the synthesis of NO is transient, rising and falling to low levels within 24 h, it is interesting that the inhibition of aggrecan synthesis is not transient, but sustained. In a previous study [23] using porcine cartilage explants, it was shown that the recovery of aggrecan synthesis after the removal of rhIL-1 following 24 h of treatment was extremely slow, taking several days to return to the control level. The recovery of aggrecan synthesis was improved by treatment with the growth factors insulin-like growth factor 1 and transforming growth factor ß, but still did not return to control levels for 2–3 days. This suggests that the suppression of aggrecan synthesis is caused by changes in transcription that do not depend on sustained exposure to cytokine action, and transient NO synthesis could therefore be responsible for initiating this signalling. Although NO may mediate some of the effects of cytokines on the inhibition of aggrecan synthesis, our data suggest that both cytokines suppress aggrecan synthesis through NO-dependent and NO-independent mechanisms.

Studies on the treatment of human cartilage explants with IL-1{alpha} suggested that NO induction alters the sulphation pattern of newly synthesized GAG chains and mediates the inhibition of sulphate incorporation [24]. However, in our study with porcine cartilage neither rhIL-1ß nor rhTNF-{alpha} altered the ratio of [3H] to [35S] in newly synthesized GAG chains. This result suggests that the decrease in aggrecan synthesis demonstrated in our porcine culture model was not due to selective effects on sulphation, and this is supported by other studies using porcine tissue [6, 25].

Investigating the effects of rhTNF-{alpha} and rhIL-1ß on HA synthesis by chondrocytes showed that both cytokines caused an increase in synthesis, and this correlated with the induction of HAS-2 gene expression, but in contrast to the inhibitory and sustained effects on aggrecan synthesis, the effect on HAS-2 was transient stimulation. Recently published data [26] on human chondrocytes have also shown that HAS-2 gene activity and HA biosynthesis are up-regulated following IL-1{alpha} treatment. In our study, although rhTNF-{alpha} stimulated more NO production than rhIL-1ß at similar concentrations, it did not appear to be more potent in the stimulation of HA synthesis. Moreover, L-NMA did not inhibit the stimulation of HA significantly following cytokine treatment (data not shown) and the changes in HAS-2 expression therefore appeared to be independent of NO production. The effects on HA and aggrecan synthesis of TNF-{alpha} and IL-1ß may cause changes to the matrix properties of articular cartilage that eventually compromise its function.

These data clearly illustrate that rhTNF-{alpha} is not only a much more potent inducer of NO synthesis than rhIL-1ß, but also has a more potent action on aggrecan synthesis in primary chondrocytes than was thought previously. If these results with porcine articular chondrocytes were recapitulated in human articular chondrocytes, this would suggest that TNF-{alpha} contributes to cartilage damage in joint disease. Furthermore, TNF-{alpha} has been found at significant levels in joint fluids in patients with rheumatoid arthritis and following joint trauma [27]. Treatment of patients with rheumatoid arthritis with neutralizing TNF-{alpha} antibodies has been shown to provide significant reversal of clinical symptoms [28]. Even if only low levels of TNF-{alpha} are present chronically, it may act on articular chondrocytes to induce NO synthase, and this could contribute to the chronic nature of joint disease and help initiate and perpetuate a slow degenerative process within articular cartilage.


    Acknowledgments
 
The authors wish to acknowledge the helpful comments and advice of Dr Andrew Spicer (University of California Davis, USA) on the degenerate RT–PCR and the subsequent identification and cloning of pig HAS-2 mRNA. This work was supported by The Wellcome Trust.


    Notes
 
Correspondence to: N. J. Goodstone, The Arthritis Research Centre, The Robert Jones & Agnes Hunt Orthopaedic Hospital, Oswestry, Shropshire SY107AG, UK. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 21 August 2001; Accepted 28 February 2002