The effects of two anti-arthritic agents on ex vivo CD4+ cell proliferation from prearthritic collagen ii sensitised mice

Alexander Vugler, Adrian Moore, Remi Okoye, Neil Gozzard, Tim Bourne, Alastair Lawson and Roly Foulkes, Celltech R & D, 208 Bath Road, Slough, Berkshire SL1 3WE, UK.

Murine collagen II arthritis (CIA) is a chronic disease model often used in the assessment of anti arthritic drugs. To investigate this model further, drug effects were assessed on ex vivo proliferative responses of CD4+ T cells to chick collagen II (CII) at a prearthritic timepoint and the data compared with previous efficacy data on arthritis development. Male DBA/1 mice (20-25g, n=6/group) were dosed with anti IL-1β monoclonal antibody at 10mg/kg s.c. once a week), leflunomide (3mg/kg p.o.once a day) or appropriate controls . One day after treatment began, animals were sensitised by intradermal injection of 100 μg of chick collagen II in complete Freund’s adjuvant. Animals were killed 14 days later and CD4+ T cells (prepared from inguinal lymph nodes by negative selection) were plated out at 2.5x105 cells per well together with antigen presenting cells (from naïve DBA/1 spleen) at 1x10 6 per well. Denatured chick collagen was added to some wells at 50 μg/mL. Cells were incubated for 72 hours at 37 oC in 5% CO2 in RPMI 1640 media supplemented with 10% foetal calf serum, 1% penicillin/streptomycin, 0.2% 2-mercaptoethanol, 1% glutamine and 2.7% HEPES buffer. To quantify cell proliferation 1 μCi of tritiated thymidine was added to each well and 6 hours later cells were harvested and thymidine incorporation measured as counts per minute (CPM) using a β counter. Identically dosed and sensitised animals (n=14-15/group) were boosted on day 14 with 100 m g chick collagen II in incomplete Freund’s adjuvant. Isoflurane used as anaesthetic. Arthritic paws were scored daily: 1 = wrist/ankle affected. 2 = 1+ pad affected. 3 = 2+ digits affected. At termination of the experiment limbs were removed for histopathological analysis (Wooley et al., 1993). Data are mean ± s.e.m. Analysis was by Mann Whitney or one way ANOVA with Bonferronni post test. P<0.05 was considered to be statistically significant. Addition of CII to sensitised cells increased thymidine incorporation from 9446 ± 844 to 17690 ± 596 CPM (P<0.001) with anti IL-1β treatment causing complete inhibition to 9986 ± 420 CPM (P<0.001). In a separate experiment CII increased thymidine incorporation from 20914 ± 1078 to 58666 ± 2530 CPM (P<0.001) with leflunomide treatment inhibiting this increase to 34192 ± 1983 CPM (P<0.001). In the CIA model anti IL-1β caused a reduction in AUC of the clinical score from 63.61 ± 10.74 to 0.23 ± 0.20 (P<0.001) and inhibited incidence from 93% to 13%. Histopathological score was also decreased from 1.84 ± 0.17 to 0.63 ± 0.06 (P<0.001). Leflunomide caused a reduction in AUC from 40.20 ± 8.46 to 12.83 ± 5.11 (P<0.05) and inhibited incidence from 80 to 40%. Leflunomide also decreased histopathological score from 1.48 ± 0.11 to 1.08 ± 0.10 (P<0.01). Leflunomide and anti IL-1β treatment suppress symptoms of CIA and inhibit CD4+ cell proliferation. These data indicate a significant CD4+ component in CIA and represent a model in which early effects of anti arthritic drugs may be detected.

Wooley, Dutcher, and Widmer et al, 1993. Journal of Immunology. 151: 6602.