ANIMALS

C57BL/Ola mice, 8 to 12 weeks old, were used for all experiments. The mice, originally obtained from Dr Carmeliet's laboratory (University of Leuven, Belgium), were bred in the animal facility at the Centre Hospitalier Universitaire Vaudois. The animals were fed a standard commercial diet and tap water freely. All animal procedures were done following standard procedures as required by our institution.

CYTOKINES AND INTRA-ARTICULAR INJECTIONS

Recombinant murine IL17 was kindly supplied by Dr Satwant Narula (Schering-Plough Research Institute, Kenilworth, NJ). Recombinant murine IL1β was purchased from R&D Systems (Abingdon, UK).

A total volume of 5 μl containing either recombinant IL1β or IL17 in phosphate buffered saline (PBS) was injected through the infrapatellar ligament into the left knee joint space. The right knee was injected with 5 μl of PBS as control. Briefly, mice were sedated with methoxyflurane (Metofane, Arovet, Zollikon, CH), knees shaved with scissors, and the patellar ligament visualised through a small skin incision. Knees were then injected through the infrapatellar ligament using a 30G needle.

TECHNETIUM-99M PERTECHNETATE UPTAKE MEASUREMENTS

Acute joint inflammation was determined using the technetium-99m pertechnetate (⁹⁹Tc) uptake method as originally described by Lens et al.15 Briefly, animals were sedated by intraperitoneal injection of pentobarbital (50 μg/g of body weight) and 10 mCi of ⁹⁹Tc in 0.2 ml saline injected subcutaneously in the neck region. Accumulation of the isotope was determined for both knees five minutes after the injection with an external gammacounter. Severity of inflammation was expressed as a ratio between the experimental knee, injected with recombinant IL1β or IL17, and the opposite control knee injected with PBS alone. Ratios higher than 1.1 are indicative of joint inflammation.

HISTOLOGY AND SCORING OF MURINE KNEE JOINTS

Animals were killed, knees fixed in buffered formalin for seven days, and decalcified in 50% formic acid/15% sodium formate for six additional days. After standard embedding in paraffin, 7.5 μm saggital sections were prepared and stained with a trichrome stain—safranin O, fast green, and Weighert's haematoxylin. Sections were scored in a blinded manner using an ordinal scale for inflammation from 0 to +++, where 0 is normal and +++ is extensive inflammation and infiltration. Sections were scored for loss of safranin O staining as a measure of cartilage proteoglycan depletion using a scale from 0 to +++, where 0 represents no depletion and +++ severe and mostly complete depletion of staining in the superficial layer.

CARTILAGE PROTEOGLYCAN SYNTHESIS

Cartilage proteoglycan synthesis was assayed by sulphate incorporation in explants of intact patellae.16 After intra-articular injection, mice where killed and patellae isolated by dissection. Patellae from mice in the identical experimental groups were pooled, washed in RPMI, and then pulsed for three hours with 25 μCi of ³⁵SO₄ (NEN, Le Grand-Saconnex, CH) in RPMI. The patellae were then washed thoroughly and fixed overnight in 4% non-buffered formalin. The next day, the patellae were decalcified for four hours in 5% formic acid and the cartilage dissected using a 1 mm diameter punch biopsy tool. The isolated cartilage was transferred to a scintillation vial containing 0.5 ml of Lumasolve Lumal LSD (Lumac LSC BV, Groningen, NL) as a solubiliser and incubated for eight hours at 60°C. Ten millilitres of scintillation fluid (Lipoluma Lipophilic LSC cocktail, Lumac LSC BV, Groningen, NL) was added after cooling and counts determined in a β scintillation counter. Results were expressed as a percentage of inhibition compared with PBS treated patellae.

STATISTICS

Statistical analysis for inflammation and proteoglycan depletion scores was carried out with the Kruskal-Wallis rank test and Dunnett's test for multiple comparisons between groups. Comparisons of⁹⁹Tc uptake and ³⁵SO₄ incorporation were carried out using an unpaired t test.

MATRIX DEPLETION AFTER A SINGLE INTRA-ARTICULAR INJECTION OF IL17

A single injection of 10 ng recombinant murine IL17 or IL1β for each knee produced similar effects. Histological evaluation at 24 and 48 hours after injection showed no oedema or haemorrhage and little leucocyte infiltration into the synovium (table 1). Neither IL1β nor IL17 produced detectable inflammation at six and 24 hours after the intra-articular injection as assessed by ⁹⁹Tc uptake (table2).

Proteoglycan depletion as assessed by safranin O staining was not evident at 24 hours after the intra-articular injection, but statistically significant depletion of staining was seen at 48 hours after injection, for both IL1β and IL17 (table 1). The absence of increased technetium uptake and the low level of inflammation render it unlikely that the proteoglycan loss is secondary to recruitment of extra-articular cells and most likely represents direct effects of IL17 on chondrocytes.

JOINT INFLAMMATION AND MATRIX DEPLETION AFTER REPEATED INTRA-ARTICULAR INJECTIONS OF IL17

To evaluate the effects of repeated cytokine injections, knees were injected three times at 48 hour intervals with 10 or 1 ng of recombinant murine IL17 and the effects compared with repeated injections of 10 ng of recombinant murine IL1β. Mice were killed 24 hours after the last injection.

The histology of control knees showed, as expected from the trauma of repeated injections, a slight increase in inflammation compared with the controls after a single injection (table 3). More severe inflammation and loss of safranin O staining was seen in animals with repeated as compared with single cytokine injections. The lower dose of 1 ng IL17 for each knee also induced significant effects; more severe changes were seen after injection of 10 ng for each knee (table 3). Figure 1 shows representative examples of the histological changes.

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Figure 1

Histological changes induced by intra-articular cytokine injection. Knee joints were harvested 24 hours after the last cytokine injection. Standard 7.5 μm paraffin embedded saggital knee sections were stained with safranin O, fast green, and Weighert's haematoxylin (×40). (A and C) Control knees, minimum inflammation and proteoglycan depletion; (B and D) moderate to severe inflammation with infiltration and exudation and proteoglycan depletion in an interleukin 17 injected knee.

Further dose-response studies were performed with two injections at 48 hour intervals using doses of 1, 10, 20, and 50 ng of IL17 for each knee and compared with control IL1β (10 ng/knee). At all cytokine doses tested an inflammatory infiltration of the joint and a more severe degradation of cartilage proteoglycan was seen than after a single injection. Maximum effects of IL17 were seen at 10 ng/knee. The higher IL17 doses (20 and 50 ng/knee) did not induce more severe inflammation or proteoglycan loss (four mice per group, data not shown).

IL17 HAS NO EFFECT ON PROTEOGLYCAN SYNTHESIS IN VIVO

IL1β has a longlasting suppressive effect on proteoglycan synthesis after intra-articular injection.17 As IL17 has similar effects on proteoglycan degradation after single or repeated intra-articular injections, proteoglycan synthesis in IL17 treated explant cultures of intact patellae were analysed and compared with the effects of IL1β. Firstly, patellae were incubated for 24 hours ex vivo with either IL1β or IL17 at 10 ng/ml. IL1β induced the expected decrease in proteoglycan synthesis, but no inhibition was seen with IL17. The same observation was obtained in vivo after a single intra-articular injection of 10 ng of IL17 or IL1β in each knee, where proteoglycan synthesis was decreased after injection of IL1β, but not IL17 (table 4).