Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

Abstract

Background: Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors.

Findings: Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFalpha). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1) sandwich (342)FFGVG-G2 ELISA, (2) competition NITEGE(373)ELISA (3) sandwich G1-NITEGE(373 )ELISA (4) competition (374)ARGSV ELISA, and (5) sandwich (374)ARGSV-G2 ELISA all detecting aggrecan fragments, and (6) sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1) aggrecanase-derived aggrecan fragments are released in the early (day 2-7) and mid phase (day 9-14) into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2) the release of NITEGE(373 )neo-epitopes are delayed compared to the corresponding (374)ARGSV fragments, (3) the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4) the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21) only.

Conclusion: Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if any effect on the quantity of aggrecanase-derived fragments released from explants cultures.

Figure 1

Epitope-specificity of monoclonal antibodies binding to aggrecan fragments. Schematic representation of the monoclonal antibodies selected for incorporation into immunoassays for quantification of aggrecan fragments carrying neo-epitopes. MAb F78 recognises a repeated epitope in the G1 and G2 domain, MAb AF28 binds to the MMP-derived neo-epitope ³⁴²FFGFG, MAb 1H11 binds the neo-epitope NITEGE³⁷³, and MAb 6D6 binds to ³⁷⁴ARGSV.

Figure 2

Aggrecanase-derived aggrecan fragments released into the supernatant from cartilage explants stimulated with catabolic cytokines. Bovine articular cartilage explants were stimulated with catabolic cytokines OSM and TNFα in the absence (open circles) and presence (closed circles) of the MMP-inhibitor GM6001. For comparison, parallel cultures were incubated with vehicle (continous line, no symbols) or metabolic inactivated at start by freeze-thaw cycles (dashed line). Supernatants were collected every second or third day and tested in competition ³⁷⁴ARGSV ELISA (figure 2A), sandwich ³⁷⁴ARGSV-G2 ELISA (figure 2B), competition NITEGE³⁷³ ELISA (figure 2C) and sandwich G1-NITEGE³⁷³ ELISA (figure 2D).

Figure 3

Aggrecanase-derived aggrecan fragments released during the early, mid and late phase of cartilage explants stimulated with catabolic cytokines. As for figure 2, but release of fragments in the early (day 2-7, black bar), mid phase (day 9-14, dark grey bar) and late phase (day 16-21, light grey bar) are expressed in percentage of accumulated release during the 21 day study period.

Figure 4

Release of aggrecan- and type II collagen-derived fragments from cartilage explants stimulated with catabolic cytokines. Supernatants from the experiment described in figure 2 was investigated in the sandwich ³⁴²FFGVG-G2 ELISA (figure 4A), the sandwich CTX-II ELISA measuring C-telopeptide fragments of type II collagen (figure 4B), sGAG (figure 4C) and hydroxyproline (figure 4D).