Effect of the synthetic immunomodulator, linomide, on experimental models of thyroiditis

Abstract

The drug Linomide is an immunomodulator showing marked down-regulation of several experimental autoimmune diseases. In this study, its effect on three different experimental models of thyroid disease and on spontaneous infiltration of salivary glands (sialoadenitis), was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis and sialoadenitis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti-mouse thyroglobulin antibodies and only anti-mouse thyroglobulin antibodies in the spontaneous model were completely down-modulated by the drug. One surprising fact to emerge was that Linomide-treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with Linomide. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells.

Figure 1

CBA/J mice were immunized with 50 μg MTg/CFA s.c. at the base of the tail on day 0 and boosted with the same dose s.c. in the hind footpads on day 7. Linomide was administered in the drinking water at an approximate dose of 20 mg/kg/day beginning on the days indicated. Mice were bled and thyroids taken on day 21. (a) The thyroiditis scored according to the criteria given in the methods section (P=< 0·01 for all groups compared with controls); and (b) the anti-MTg antibodies evaluated in an ELISA using goat anti-mouse poyvalent immunoglobulin conjugated to alkaline-phosphatase as second antibody (P=< 0·01 at 1/3200 and 1/6400 for all groups compared with controls). All values are the means±SE of six mice in each group.

Figure 2

CBA/J mice were immunized twice with 50 μg MTg/CFA as before ±Linomide in the drinking water. The mice were bled on day 16 and the isotype profile was examined using alkaline-phosphatase-conjugated anti-IgG1, IgG2a and IgG2b antibodies for detection.

Figure 3

Donor CBA/J mice were immunized with 50 μg MTg/CFA on days 0 and 7 as before and on day 15, the popliteal lymph nodes were removed and cultured as a single cell suspension for 3days with MTg 40 μg/ml. After washing, 2×10⁷ viable cells were transferred i.v. into normal syngeneic recipients. Linomide was either added to the culture medium at 100 μg/ml or added to the drinking water of the recipients starting 4 days before transfer. Thyroids were examined 14 days after transfer and scored as before. Values shown are the mean±SE of five mice in each group.

Figure 4

Donor CBA/J mice were immunized with 50 μg MTg/CFA on days 0 and 7±Linomide in the drinking water starting 3 days before the first immunization. Popliteal lymph nodes were removed on day 15 and cultured with MTg 40 μg/ml but without Linomide, for 3 days. After washing, 2×10⁷ viable cells were transferred i.v. to normal syngeneic recipients. The thyroids were examined after 14days and the superimposed values shown are individual scores for each mouse.

Figure 5

CBA/J mice were maintained ±Linomide and immunized on days 0 and 7 with 50 μg MTg followed 3 hr later by 20 μg LPS, both given i.v. The thyroids were taken on day 28 and the scores shown represent the means±SE of five mice in each group.

Figure 6

CBA/J mice were maintained ±Linomide and immunized with MTg/LPS as before. They were bled at day 28 and this figure shows the isotype profile for two separate experiments. The profiles were obtained using standard ELISA methods and alkaline-phosphatase-conjugated anti-IgG1, G2a and G2b.

Figure 7

Male and female NOD H-2^h4 mice were given tap water containing 0·05% iodine±Linomide. Controls were maintained on normal tap water. This regimen was continued for 6 weeks when the mice were bled and thyroids were taken for examination. Values shown are either (a) the mean±SE for females (P=0·017for NaI compared with NaI+Linomide), or (b) the individual scores for each male mouse.

Figure 8

Female mice from the experiment shown in Figure 7 were bled after 6 weeks and the anti-MTg antibodies were evaluated using standard ELISA methods. Values shown are the mean±SE for each group (of seven, eight and six mice).

Figure 9

Female NOD mice were treated between the ages of 6 and 18 weeks with Linomide in the drinking water. Controls recieved normal tap water. Three mice from each group were killed at each time-point for evaluation of salivary gland infiltration. Values shown are the average number of focal infiltrates per section in each group (P < 0·001 at 14 and 16 weeks).