Cartilage catabolism is initiated by proteoglycan degradation followed by that of collagen fibers. Therefore, our study focused on the TNFα-dependent depletion of proteoglycans in a three-day bovine
in vitro meniscal model [
29]. TNFα induced a dose-dependent increase in GAG release supporting data from other investigations on pro-inflammatory cytokines in which IL-1 promoted GAG release in lapine and porcine meniscal tissue [
19,
21]. TNFα, therefore, appears to be another key factor in meniscal diseases.
To study the mechanisms of TNFα-dependent proteoglycan degradation we investigated the transcription of different matrix-degrading enzymes. One limitation in our study is that aggrecanases had been detected on the mRNA level only; there is no measurement of enzyme proteins, which could help to specify the degradative potencies of enzymes involved in TNFα-dependent proteoglycan degradation. A reason for the missing protein detection is that enzyme levels in the tissue are quite low compared with the large amounts of matrix proteins. We performed immunostainings in tissue sections (not shown), but differences in ADAMTS-4 expression were hard to differentiate, probably due to the fact that immunohistochemistry is not useful for the differentiation of slightly variable expression levels. We therefore mainly focus on the effect of inhibitors such as TIMPs or NO synthetase inhibitor (L-NMMA), and the cleavage products of aggrecan (NITEGE), which both suggest that aggrecanases must be involved in the early TNFα-dependent aggrecan degradation and GAG release in the meniscus (see below).
Increased concentrations of MMPs have been found in animal models of OA, in osteoarthritic human articular cartilage and in the synovial fluid of RA and OA patients [
11,
30‐
33], but only little is known about the extent to which the meniscus might be involved in the production of these enzymes. We demonstrate that the meniscus can be an additional source for MMP-3 production, especially under the influence of TNFα. Wilson and colleagues [
34] emphasise the importance of MMP activity in meniscal proteoglycan degradation after a 12-day incubation of bovine meniscal tissue from one to two-weekold calves with 20 ng/ml IL-1 and different enzyme inhibitors, but the authors do not specify the kind of MMPs. Additionally, Wilusz and colleagues [
9] found MMPs to be responsible for some of the repair inhibition by pro-inflammatory cytokines in a serum-containing porcine model. However, in our study most of the MMP-3 in the culture supernatant was in the pro-form, and it remains unclear to what extent this enzyme might have been involved in the present GAG release. But it is reasonable to believe that MMP-3 will be involved in the subsequent TNFα-dependent matrix degradation, as indicated by Wilson and colleagues [
34]. TIMP-3, but not the other TIMPs, were able to inhibit the TNFα-induced GAG release and NITEGE production. This suggests that in the early three-day phase of meniscal proteoglycan degradation, aggrecanases must be involved. TIMPs are able to inhibit the active forms of almost all MMPs by binding to the C-terminal site of these enzymes [
35]. However, TIMP-3 additionally inhibits ADAMTS-4 and -5 activity, whereas TIMP-1 and TIMP-2 have no effect on or even increase the activity of aggrecanases at concentrations of 1 μM or less [
27,
36‐
43]. According to our mRNA study, ADAMTS-4 might be one of the aggrecanases involved in TNFα-dependent proteoglycan degradation in bovine meniscal tissue, even though final evidence is still missing. This is supported by the fact that TIMP-3 inhibited, whereas TIMP-1 and -2 increased, the TNFα-dependent GAG release (in contrast to TIMP-3, TIMP-1 and -2 are known to stimulate the activity of ADAMTS-4 under certain conditions [
43]). ADAMTS-4 mRNA has also been found in degenerated human menisci [
44]. Therefore, it is likely that there might be similar effects in the human meniscus. Other studies showed that ADAMTS-5 mRNA was expressed next to ADAMTS-4 in osteoarthritic rabbit menisci [
11]. Therefore, it is possible that both aggrecanases may play a role in the degradation of meniscal tissue. However, in the present investigation there was a basal meniscal mRNA expression of ADAMTS-4 in the bovine meniscus which increased with TNFα-treatment, whereas ADAMTS-5 mRNA expression was low or not detectable.
We were able to localize the NITEGE fragment in meniscal tissue by immunostaining in TNFα-treated explants, while it was almost non-detectable in control tissue. This is another strong indicator for aggrecanase involvement, according to many articular cartilage studies [
14,
15,
45,
46]. Additionally, TNFα-dependent NITEGE-formation could be blocked by TIMP-3, while TIMP-1 and -2 had no inhibitory effects (not shown). TIMP-3 is not a specific aggrecanase inhibitor. It has to be mentioned that it also regulates the activity of members of the membrane-bound ADAM-family, sheddases (a disintegrin and metalloproteinase: ADAM-10, -12 and -17; TACE [
47‐
49]). The importance of these enzymes should therefore also be investigated in future studies.
We found a significant TNFα-dependent increase in meniscal NO production, which could be blocked completely by the common NO synthetase inhibitor L-NMMA. Although NO has been described as a meniscal product in several joint diseases and as an important mediator of meniscal tissue degradation in several studies [
4,
16‐
23], we did not see a stimulating influence of NO on the TNFα-induced GAG release or aggrecan cleavage. Our study suggests that NO is not involved in the early degradation of aggrecan in the meniscus. The slight but not significant increase in TNFα-induced GAG release after incubation with L-NMMA might reflect a protective function of endogenous NO in this context, as it has been shown previously by others [
21].