Blockade of endogenous interleukin 12 results in suppression of murine streptococcal cell wall arthritis by enhancement of interleukin 10 and interleukin 1Ra

Abstract

Objective: The goal of this study was to investigate the role of endogenous interleukin 12 (IL12) in acute murine streptococcal cell wall (SCW) arthritis.

Methods: C57black/6 mice were injected intraperitoneally with rat anti-murine IL12 (C17.8), shortly before induction of arthritis by intra-articular injection of 25 microg SCW fragments into the right knee joint. Joint swelling and chondrocyte synthetic function was analysed several days after induction of SCW arthritis. Local cytokine profile was determined, protein by using ELISA and mRNA by RT-PCR technology. To confirm the findings at later time points, tissue chamber model of inflammation was used. Histology was performed to examine cell influx and cartilage damage.

Results: Suppression of joint swelling was noted at days 2 and 4, whereas no suppressive effect of anti-IL12 was found at day 1. Severe inhibition of chondrocyte proteoglycan synthesis was seen at day 1 in both arthritic control and anti-IL12 treated mice. However, chondrocyte function was restored at day 4 of arthritis in the anti-IL12 injected animals, but not in the arthritic controls. Moreover, cell influx in synovial tissue and joint cavity was reduced by anti-IL12 treatment. Neutralisation of IL12 reduced the local levels of IL1beta, IL12 and interferon gamma, when examined shortly after induction of SCW arthritis, whereas tumour necrosis factor alpha levels were not affected. In contrast, IL10 and IL1Ra protein and mRNA levels were strongly up regulated in synovial tissues after IL12 blockade. Enhancement of IL10 and IL1Ra by anti-IL12 was confirmed in a tissue chamber model with SCW induced inflammation.

Conclusions: This study indicates that IL12 is a pro-inflammatory cytokine during onset of acute SCW arthritis. Balances of proinflammatory and anti-inflammatory cytokines were strongly improved by anti-IL12 treatment.

Figure 1

SCW arthritis, joint swelling, chondrocyte proteoglycan (PG) synthesis, and cytokine profile. (A) SCW arthritis was induced by injection of 25 µg SCW fragments. Joint swelling was measured by ^99mTc uptake method and is expressed as right/left ratio. A ratio >1.10 is considered as inflammation. Inhibition of chondrocyte PG synthesis was determined by ³⁵S-sulphate incorporation. The data represent the mean (SD) of at least seven mice per group. (B) Cytokine levels were determined either by RIA or ELISA in patellae washouts at several time points. The data represent the mean cytokine level of washouts of six patellae per time point.

Figure 2

Effect of anti-IL12 treatment on joint swelling and chondrocyte PG synthesis. (A) Mice were given an intraperitoneal injection two hours before induction of arthritis with either 0.5 mg rat anti-murine IL12 or rat Igs. At days 1, 2, and 4 joint swelling was determined by ^99mTC uptake and expressed as R/L ratio. The data represent the mean (SD) of at least seven mice per group. (B) Chondrocyte PG synthesis was measured by 35S-sulphate incorporation at days 1, 2, and 4 after induction of arthritis. The data represent mean (SD) percentage chondrocyte PG synthesis of the left control patella. *p<0.05, Mann-Whitney U test, compared with rat Igs group.

Figure 3

Effect of IL12 blockade on cell influx and cartilage damage in the knee joint. (A) Arthritic nee joint of the rat Igs treated control group. Note the marked joint inflammation and cell influx in the joint cavity. Bar=80 µM. (B) Section of the control group stained for matrix proteoglycan. Note the loss of matrix PG in the cartilage layers (arrows). Bar=40 µM. (C) Knee joint of a mouse treated with anti-IL12 antibodies. Strong reduction of cell influx can be seen in synovial tissue and joint cavity. Bar=80 µM. (D) Section of anti-IL12 treated knee joint stained for PG loss. No reduction of matrix PG depletion was seen after IL12 blockade. Bar=40 µM. p= patella, f= femur, c= cartilage, js= joint space, s= synovium. (A and C) Haematoxilin and eosin staining, (B and D) safranin O staining. All specimens were taken at day 2 after induction of SCW arthritis.

Figure 4

Kinetics of IL12 neutralisation on joint swelling and chondrocyte PG synthesis. (A) Mice were treated intraperitoneally with anti-IL12 at either −2 hours, day 1 or day 2 after induction of SCW arthritis. As control we injected at the same time points rat Igs. Joint swelling was determined at day 3. The data represent the mean (SD) joint swelling of at least seven mice per group. (B) Chondrocyte PG synthesis measured at day 4. Mice were treated with either anti-IL12 or rat Igs as indicated above. The data are expressed as mean (SD) percentage of chondrocyte PG synthesis found in left control patella of at least seven patellae per group. *p< 0.05, Mann-Whitney U test, compared with rat Igs.

Figure 5

Synovial cytokine concentrations after IL12 neutralisation. Mice were given an intraperitoneal injection with either anti-IL12 or rat Igs before induction of arthritis. After either 90 minutes (TNFα) or four hours (all other cytokines), patellae were isolated and cytokine levels were determined in washouts. A, TNFα and IL1β. (B) IFNγ and IL12. C, IL10 and IL1Ra. The data represent mean (SD) cytokine level in at least six patellae washouts. *p<0.05, Mann-Whitney U test, compared with rat Igs group.

Figure 6

RT-PCR analysis of synovial tissue mRNA levels for TNFα (A), IL1β (B), IFNγ (C), IL12 (D), IL10 (E), IL1Ra (F) after intraperitoneal treatment with either anti-IL12 or rat Igs. Synovium biopsy specimens of six mice per group were pooled and RNA was extracted. Expressed is the number of PCR cycles in which gene product of interest was first detectable compared with normal synovium. Values are the mean of two experiments with SCW arthritis. The cycle curves were done by two cycle intervals. The PCR measurements of a particular cytokine was routinely repeated three times. The variations never exceeded more than two cycles. The absolute difference in mRNA levels were calculated using the difference in PCR cycles between arthritic synovium and non-inflamed synovium (two cycles mean 2² more mRNA, four cycles = 2⁴). From these values ratios for IL1Ra/IL1β (G) and IL10/TNFα (H) were calculated, using delta PCR cycles found in F/B and E/A.

Figure 7

PCR analysis of synovial tissue at days 1 and 2 after anti-IL12 treatment. Synovial biopsy specimens of six mice per experimental group were pooled, and RNA was extracted. PCR was performed as described in Methods. Animals were injected intraperitoneally with either 0.5 mg rat Igs (I) or rat anti-murine IL12 (II) before induction of SCW arthritis. Note the reduced IFNγ and IL12 and the increased IL1Ra and IL10 levels at day 2. † The number of PCR cycles.

Figure 8

Effect of anti-IL12 treatment on cytokine levels in tissue-chamber model. Tissue-chambers were implanted subcutaneously seven days before injection intraperitoneally of either anti-IL12 or rat Igs. Two hours later, 100 µl SCW fragments were injected. At days 1 and 2 200 µl tissue-chamber fluid was extracted. Levels of TNFα (A), IL1β (B), IFNγ (C), IL12 (D), IL10 (E) and IL1Ra (F) were determined by RIA or ELISA. The data are expressed as mean (SD) cytokine level of six tissue-chambers per group. *p<0.05, Mann-Whitney U test, compared with rat Igs.