Suppressive effects of a quinoxaline-analogue (Rob 803) on pathogenic immune mechanisms in collagen-induced arthritis

Abstract

The anti-arthritic effects of the synthetic compound 9-chloro-2,3 dimethyl-6-(N,N-dimetylamino-2-oxoethyl)-6H-indolo[2,3-b] quinoxaline (Rob 803) was evaluated by treating Dark Agouti rats with collagen-induced arthritis using three different protocols. Daily subcutaneous treatment with 40 mg/kg/day of Rob 803 from the day of immunization and 14 days forward suppressed arthritis severity significantly and delayed the onset of clinical arthritis. In contrast, similar treatment initiated when individual rats had developed clinical disease (at a score of 2 points) did not suppress disease. Oral treatment with 35 mg/kg/day of Rob 803 from the day of immunization and 21 days forward resulted in a trend towards disease suppression. In vitro analysis of rats treated subcutaneously with Rob 803 revealed an inhibition of T cell proliferation but no effect on the generation of an anti-CII immunoglobulin G response. Further in vitro analysis demonstrated that Rob 803 also inhibited the generation of nitric oxide in macrophages, although at higher concentrations than needed for inhibitory effects on T cell proliferation. Thus we report that early subcutaneous administration of the synthetic substance Rob 803 has anti-rheumatic effects that are probably mediated by affecting the proliferative capacity of lymph node T cells. Rob 803 should be considered as a new candidate substance for anti-rheumatic treatment.

Fig. 1

(a) Incidence of arthritis in rats treated subcutaneously with 9-chloro-2,3 dimethyl-6-(N,N-dimetylamino-2-oxoethyl)-6H-indolo[2,3-b] quinoxaline (Rob 803) or vehicle alone from days 0 to 14 post-injection (n = 9 in each group, from day 21 n = 7 in the treated group and n = 7 in the control group; and from day 24 n = 6 in the control group; one rat was excluded because of a weight loss greater than 15%). One of two experiments performed is depicted. (b) Arthritis severity scores for rats from the same experiment as in (a). Average values with standard deviation as error bars are presented. P < 0·05 is indicated as asterisks above the values. p.i., post-injection.

Fig. 2

(a) Incidence of arthritis in rats treated with 9-chloro-2,3 dimethyl-6-(N,N-dimetylamino-2-oxoethyl)-6H-indolo[2,3-b] quinoxaline (Rob 803) or control vehicle orally from day 0 to day 21 post-injection. (b) Arthritis severity score presented as median values median value with standard error of the mean shown as error bars (n = 10 in each treatment group). P < 0·05 is indicated as the asterisks above the value. p.i., post-injection.

Fig. 3

T cell responses, detected by in vitro proliferation of lymph node cells from four Dark Agouti rats immunized with rat collagen type II in Freund's incomplete adjuvant. The proliferation assay was performed on day 9 post-injection and the cells were stimulated with 1·5 μg concanavalin A/ml. The effect of 9-chloro-2,3 dimethyl-6-(N,N-dimetylamino-2-oxoethyl)-6H-indolo[2,3-b] quinoxaline (Rob 803) was studied by addition of this substance at different concentrations to triplicates of cell cultures.

Fig. 4

T cell responses detected by in vitro proliferation of lymph node cells from Dark Agouti rats immunized with rat collagen type II in Freund's incomplete adjuvant treated subcutaneously with 9-chloro-2,3 dimethyl-6-(N,N-dimetylamino-2-oxoethyl)-6H-indolo[2,3-b] quinoxaline (Rob 803). The proliferation assay was performed on day 9 post-injection and the cells were stimulated with concanavalin A (ConA) at 1·5 μg/ml; – denotes the average value in each group. Other concentrations of ConA were also tested: 0·5, 1·0 and 2·0 μg/ml with similar results.

Fig. 5

T cell responses detected by in vitro proliferation of lymph node cells from Dark Agouti rats immunized with rat collagen type II in Freund's incomplete adjuvant treated orally with 9-chloro-2,3 dimethyl-6-(N,N-dimetylamino-2-oxoethyl)-6H-indolo[2,3-b] quinoxaline (Rob 803). Proliferation assay was performed on day 9 post-injection and the cells were stimulated with concanavalin A 1·5 μg/ml; – denotes the average value in each group (n = 10 in each group).

Fig. 6

(a) Total α-collagen type II IgG titres in rats treated subcutaneously with 9-chloro-2,3 dimethyl-6-(N,N-dimetylamino-2-oxoethyl)-6H-indolo[2,3-b] quinoxaline (Rob 803) or vehicle for 14 days. No difference in titres between the groups was observed. Data are presented as optical density values measured at 405 nm; – denotes the average value in each group. The sera were taken during the study as the rats were killed, because of ethical reasons, from day 19 post-injection (p.i.) to the end of the experiment, day 27 p.i. (b) Total α-collagen type II IgG titres in rats treated daily and orally with Rob 803 or vehicle, from days 0 to 21 p.i., a significant statistical difference in titres being observed between the groups (P = 0·015). The sera were taken as the rats were killed, because of ethical reasons, from day 19 p.i. to the end of the experiment, day 27 p.i.

Fig. 7

The inhibitory effect of 9-chloro-2,3 dimethyl-6-(N,N-dimetylamino-2-oxoethyl)-6H-indolo[2,3-b] quinoxaline (Rob 803) on NO₂^– production by lipopolysaccharide (LPS)-stimulated macrophages. Peritoneal macrophages from Dark Agouti rats were stimulated with 5 μg/ml LPS for 72 h, with or without the addition of different concentrations of Rob 803. NO₂^– production is presented as percentage of LPS-induced NO₂^– production. Results from three individual rats are presented.