Increased vulnerability of postarthritic cartilage to a second arthritic insult: accelerated MMP activity in a flare up of arthritis

Abstract

Objective: Murine antigen induced arthritis (AIA) is a chronic, smouldering inflammation. Flares of arthritis can be induced by antigen rechallenge or exposure to inflammatory mediators like interleukin 1 (IL1). These flares are characterised by a fast and marked proteoglycan (PG) depletion if compared with the initial arthritis. This study investigated the involvement of metalloproteinases in both the initial and the flare phase of arthritis.

Methods: Murine AIA was induced and a flare up of arthritis was induced by injection of 10 ng of IL1beta. Messenger RNA levels of MMP-1 and -3 were studied by RT-PCR. MMP activity in cartilage, during both primary AIA as well as the flare up of arthritis, was studied by immunodetection of MMP specific neoepitopes in aggrecan (VDIPEN). Cartilage just before flare induction was analysed for presence of MMPs at the mRNA level as well as at the protein level by zymography.

Results: At the onset of AIA, a fast upregulation of mRNA for stromelysin and collagenase was noted. However, no VDIPEN epitopes were detected during this early phase of arthritis. They appeared when PG depletion was severe at day 7 of arthritis and disappeared when cartilage was repaired. IL1 injection into a knee joint at week 4 of AIA caused a flare up of arthritis, coinciding with a fast and marked PG degradation. This degradation was characterised by accelerated expression of VDIPEN epitopes if compared with the expression in primary AIA. Analysis of cartilage at week 4 of AIA showed still increased mRNA levels of MMP-1 and -3. Moreover, increased levels of latent MMPs were present as well, as APMA activation induced profound VDIPEN epitope. In vitro exposure to IL1 did show increased PG breakdown but no VDIPEN expression, suggesting that factors in addition to IL1 are needed to cause the in vivo VDIPEN expression.

Conclusions: The fast and marked PG depletion seen in a flare up of AIA coincides with accelarated expression of MMP induced neoepitopes compared with expression during primary AIA. This accelerated expression is probably linked to increased levels of latent enzyme, which were found to be present in the cartilage before induction of a flare up.

Figure 1

Semiquantitative mRNA analysis in cartilage during antigen induced arthritis. At several time points after arthritis induction patellar cartilage was collected and RNA was isolated. Semiquantitive mRNA analysis was done using RT-PCR. PCR products were analysed on an ethidium coloured gel. The cycle number at which the product is first detected is taken as a measure for the amount of mRNA present in the original isolated RNA. GAPDH was used to verify that equal amount of RNA were used. Amounts of cycles gene of interest−amount of cycles GAPDH=cycle difference. Control cartilage is taken as a reference point. The experiment was done two times, and each PCR reaction was carried out twice with the same material. Variation between cycle differences was low, at most one PCR cycle.

Figure 2

Proteoglycan depletion and VDIPEN expression in primary antigen induced arthritis. Adjacent frontal knee joint sections were stained with safranin O (A, C) and immunostained with anti-VDIPEN (B, D). (A), (B) day 1 of AIA, (C), (D) day 7 of AIA. P=patella, F=femur, bar represents 200 µm. The figure shows absence of VDIPEN expression at day 1 of AIA (B), and pronounced VDIPEN staining at day 7 as indicated by the arrowheads in (D).

Figure 3

PG depletion and VDIPEN expression during IL1 induced exacerbation. Ten ng of IL1α was injected into a knee joint of day 28 AIA. Frontal whole knee joint sections were stained with safranin o (A-Cc) and anti-VDIPEN (D-F). At day 28 of AIA no PG depletion and VDIPEN staining was found (A, D). IL1 injection into such a knee joint induced marked PG depletion, which is illustrated in (B) by destaining of the upper cartilage layer, and widespread VDIPEN expression ((E), arrowheads). Repeated IL1 injection into a naive joint, resulted in mild PG depletion (C) and no VDIPEN expression (F). (A), (D) Knee joint of day 28 AIA, bar represents 150 µm. (B), (E) day 1 after IL1 injection into postarthritic knee joint, bar represents 150 µm. (C), (F) day 1 after IL1 injection into a naive knee joint, bar represents 350 µm. P=patella, F=femur.

Figure 4

Increased mRNA levels in postarthritic cartilage. RNA was isolated from cartilage at week 4 of AIA (postarthritic cartilage) and semiquantitative mRNA analysis of several genes was done using RT-PCR. The cycle number at which the product is first detected is taken as a measure for the amount of mRNA present in the original isolated RNA. GAPDH was used to verify that equal amount of RNA were used. Amounts of cycles gene of interest−amount of cycles GAPDH= cycle difference. Contralateral cartilage was used as a reference point. Depicted is the mean (SD) of three separate experiments.

Figure 5

Increased gelatinolytic activity in postarthritic cartilage. Pooled extracts of five patellar cartilage layers, were subjected to zymography, using gelatin as a substrate. Postarthritic cartilage was taken at week 4 of AIA and compared with contralateral cartilage. Clear bands on the dark gel indicate gelatinolytic activities. Depicted is a representative example of five separate experiments.

Figure 6

Increased amount of latent MMPs in postarthritic cartilage. Patellas of week 4 AIA were incubated with (A) or without (B) APMA ( a known chemical activator of MMPs) in RPMI for 18 hours and cross sections were immunostained for VDIPEN. Bar represents 100 µm. C=cartilage, B=bone.

Figure 7

Increased IL1 induced proteoglycan degradation in postarthritic knee joint. Cartilage was pulse labelled with[³⁵S]-sulphate to radiolabel newly formed proteoglycans. Subsequently, labelled cartilage was incubated with 10 ng/ml IL1 for 24 hours. The amount of[³⁵S]-sulphate retained in the cartilage was measured and expressed as a percentage of the[³⁵S]-content of cartilage cultured without IL1. Values are the mean (SD) of nine separate experiments, *=p<0.05 compared with controls, by two tailed Wilcoxon rank sum test.

Figure 8

No VDIPEN expression in IL stimulated postarthritic cartilage. Patellas of day 28 AIA were incubated with (A) or without (B) IL1 for 18 hours and cross sections were immunostained for VDIPEN. Bar represents 100 µm. C=cartilage, B=bone.