Loss of transforming growth factor counteraction on interleukin 1 mediated effects in cartilage of old mice

Abstract

Objective: To investigate if a difference exists between young and old mice in the response of articular cartilage to interleukin 1 (IL1) and transforming growth factor beta (TGFbeta) alone or in combination.

Methods: The interaction of IL1 and TGFbeta was studied in cartilage of young (three months) and old mice (18 months) both in vivo and in vitro. Therefore, IL1, TGFbeta, or IL1 together with TGFbeta was injected into the knee joints of mice on days 1, 3, and 5 before harvest of the patellae on day 6. Alternatively, isolated patellae were stimulated with IL1, TGFbeta, or IL1 together with TGFbeta in culture for 48 hours. Proteoglycan (PG) synthesis and nitric oxide (NO) production were measured.

Results: IL1 inhibited PG synthesis and increased NO production in cartilage of both young and old mice. On the other hand, TGFbeta stimulated PG synthesis and reduced NO production in both age groups. Importantly, TGFbeta was able to counteract IL1 mediated effects on PG synthesis and NO production in young but not in old mice.

Conclusions: Contrary to the findings in young mice, the cartilage of old animals does not antagonise IL1 effects via TGFbeta. This loss of responsiveness to the pivotal cytokine TGFbeta on effects of IL1 can be important in the initiation and progression of osteoarthritis (OA).

Figure 1

Synthesis of PG in patellar cartilage in old and young mice. Right knee joints of young mice (three months) and old mice (18 months) were injected on days 1,3, and 5 with TGFß (0.1 µg), IL1α (1 ng), or IL1 + TGFß. Left knee joints were injected with vehicle and served as internal controls (=100%). On day 6, patellae were isolated and PG synthesis was measured ex vivo by ³⁵S-sulphate incorporation (two hours, 37°C). Each value represents the mean PG synthesis of five patellae. Results were statistically analysed by ANOVA; ** p<0.001.

Figure 2

In vitro PG synthesis in patellar cartilage of young (three to five months) and old mice (12–21 months). Patellae were isolated and cultured in RPMI 1640 medium. Patellae were treated for 48 hours with IL1α (10 ng/ml), TGFß₁ (10 ng/ml), a combination of IL1 + TGFß₁, or were not treated. The tissue culture medium was changed every 24 hours. Synthesis of PG was measured via ³⁵S-sulphate incorporation (two hours, 37°C). Incorporation levels of ³⁵S were transformed to percentage compared with control treatment (=100%). Results were statistically analysed by ANOVA; **p<0.001.

Figure 3

In vitro NO production after TGFß treatment by patellar cartilage of young (three to five months) and old mice (12 to 21 months). Patellae were isolated, placed in RPMI 1640 medium, and treated for 48 hours with TGFß (10 ng/ml) or were not treated. The tissue culture medium was changed every 24 hours. NO₂- in the medium was measured after 24 and 48 hours by the Griess reaction. Production of NO by control treated patellae was stated as 100%. Results were statistically analysed by ANOVA; *p<0.01; ** p<0.001, significantly different from control treatment.