Figure 3

Serum IL-1Ra from intra-colonic B. subtilis inhibits IL-1-induced thymocyte proliferation in vitro. Co-stimulation of murine thymocyte proliferation by IL-1β was assessed in cultures of C3H/HeJ thymocytes stimulated with a suboptimal concentration of PHA (1.5 μg/ml) in the absence (zero control; empty column) or in the presence of IL-1β (solid column). Left panel: Serum samples were taken from rabbits administered intra-colonically 3 h earlier with B. subtilis pSM539 (producing IL-1Ra; ●) or pSM214 (β-lactamase control; ○), and assayed by ELISA for the presence of IL-1Ra (1.99 μg/ml in the pSM539 serum; 0.00 μg/ml in the pSM214 serum). Inhibition of IL-1β activity (30 pg/ml) was assessed as decrease of thymocyte proliferation in the presence of different dilutions of the pSM539 serum (1:200, 1:2,000, 1:20,000; corresponding to 10, 1, and 0.1 ng/ml IL-1Ra) and compared to the same concentrations of human recombinant IL-1Ra (■). Right panel: Thymocyte proliferation to IL-1β (300 pg/ml) was evaluated in the presence of rabbit serum taken 6 h after intra-colon administration of pSM539 (●) or serum from the same rabbit taken before treatment (□). Serum from pSM539-treated rabbit contained 1.35 μg IL-1Ra/ml (tested by ELISA). Serum was added at dilutions (1:450, 1:1,350, 1:4,050) containing 3, 1, and 0.3 ng/ml IL-1Ra, either alone (●) or in the presence of a polyclonal antibody against human IL-1Ra (diluted 1:300; ◇). Untreated rabbit serum (negative for IL-1Ra in ELISA) was used as control at the same dilutions as pSM539 serum. Results are presented as mean ± SEM of triplicate cultures within single representative experiments. Statistical significance: p < 0.01 pSM539 serum (at 1, 3, 10 ng IL-1Ra/ml) vs. IL-1β alone and corresponding serum controls (pSM214, untreated, + anti-IL-1Ra).