Caging environment affects the murine immune system. At the endpoint of the experiment animals were sacrificed and spleens were taken for massing (A). Splenocytes were isolated as described in Materials and Methods, and were resuspended in medium containing 5% charcoal-stripped FBS, which has minimal hormone content. Cultures were treated with LPS (1 µg/mL), anti-CD3 plus anti-CD28 (CD3/CD28) beads (1 bead/cell), or phosphate-buffered saline for 18 h. Some wells contained varying concentrations of corticosterone to assess glucocorticoid-mediated suppression of cytokine production. Data displayed are from cultures containing 0.005 µmol/L corticosterone, because this concentration is nearest to physiological levels (B-E). (A) Spleen mass for Calm, Cntl Ex and Calm Ex mice was greater than that for Cntl mice (Cntl Ex, P < 0.05; Calm, Calm Ex, P < 0.01). (B) In all groups, LPS-treated splenocytes produced comparable levels of IL-6. (C) anti-CD3/anti-CD28 stimulation of splenocytes from Calm animals exhibited slightly greater production of IL-2 than Cntl (not significant), whereas Cntl Ex and Calm Ex animals showed little difference from Cntl. (D) LPS-stimulated splenocyte production of IL-6 was positively correlated with serum corticosterone level at the time that animals were killed, but this relationship was significant only for Calm Ex animals (r2 = 0.7; P < 0.01). (E) Conversely, CD3/CD28 bead stimulation of IL-2 production was negatively correlated with serum corticosterone level at the time that animals were killed. This relationship was significant in Cntl and Calm Ex animals (Cntl, r2 = 0.41, P < 0.05; Calm Ex, r2 = 0.61, P < 0.01). Data represented as mean ± SEM. *P < 0.05, **P < 0.01.