Metallothionein suppresses collagen-induced arthritis via induction of TGF-beta and down-regulation of proinflammatory mediators

Abstract

Metallothionein is a low molecular weight, cysteine-rich, stress response protein that can act as an antioxidant and as an immunosuppressive agent in instances of antigen-dependent adaptive immunity. In this context, we assessed the therapeutic potential and mechanisms of action of metallothionein in a collagen-induced arthritis model. Repeated administration of metallothionein-I + II during the course of disease dramatically reduced the incidence and severity of the disease. Joint tissues isolated from boostered paws of metallothionein-I + II-treated mice expressed significantly reduced levels of proinflammatory mediators, such as tumour necrosis factor (TNF)-alpha and cyclooxygenase-2, when compared with those of control-treated mice. Lymph node cells obtained from metallothionein-I + II -injected mice exhibited a significant decrease in the proliferative response and a remarkable increase in tumour growth factor (TGF)-beta production in response to type II collagen. Taken together, these results suggest that metallothionein-I + II promote the development of type II collagen-specific, TGF-beta-producing cells to antagonize the expansion of arthritogenic cells. This could lead to local suppression of inflammatory responses by inhibiting the expression of proinflammatory molecules. Thus, this study demonstrates the suppressive effects of metallothionein on collagen-induced arthritis, and indicates that there may be a potential therapeutic application for manipulation of metallothionein during the treatment of autoimmune disorders.

Fig. 1

Metallothionein decreased the clinical manifestations of established CIA. DBA/1 J mice were immunized with CII plus CFA at day 0 and followed by booster immunization with CII plus IFA at day 14. From day 21–32, mice were injected intraperitoneally with 100 μg/day of metallothionein-I + II (○) or saline (•) everyday except days 24 and 28. These figures resulted from three independent sets of experiments and represent means ± SEMs (n = 15 per group). From day 23–32, arthritic indexes from metallothionein-injected mice were significantly different from those from vehicle-treated mice at each day (P <0·01, t-test).

Fig. 2

Metallothionein reduced histopathological manifestations of CIA. On day 34 postimmunization, mice (n = 15 per group) were sacrificed and right hindpaws were removed. The specimens were fixed, decalcified, embedded in paraffin, sectioned, and stained with haematoxylin and eosin. (a) Joints of normal mice; (b) mainly normal joints from metallothionein-treated mice; (c) moderately arthritic joints from metallothionein-treated mice; (d) severely arthritic joints from vehicle-treated mice. Original magnification: ×40. Percentage of histological score per group is shown in (e). □ Normal; moderate; ▪ severe.

Fig. 3

The expression of TNF-α and Cox-2 transcripts was reduced in joints of metallothionein-injected mice. On day 34, joints of booster injected left hindpaws (+) and uninjected right hindpaws (–) were separately removed and dissected free of soft tissue. Total RNAs were extracted from joints pooled 4–5 mice per group and expression levels of (a) TNF-α and (b) Cox-2 transcripts were determined by RT-PCR. PCR products of Cox-2 were further analysed by Southern blot hybridization with the Cox-2 internal probe. The data were reproducible in two independent experiments.

Fig. 4

Metallothionein administration inhibited the proliferation of CII-specific lymphocytes upon in vitro restimulation with CII. Lymph node cells from mice sacrificed on day 34 postimmunization were stimulated in triplicate in the absence (□) or presence of 250 μg/ml CII (▪) or 5 μg/ml PHA (▪) for 72 h. The cells were cultured for another 16 h with 1 μCi/well ³H-thymidine, harvested, and counted using a scintillation counter. The data are representative of three independent experiments with similar results (* P <0·05, t-test).

Fig. 5

TGF-β production was increased in metallothionein-injected mice. Lymph node cells obtained on day 34 post mortem were cultured in RPMI medium supplemented with 1·25% FBS in the absence (□) or presence (▪) of 250 μg/ml bovine CII for 3 days. The culture supernatants were collected and analysed for TGF-β levels using cytokine sandwich ELISA. The result of a representative experiment out of three performed is shown.