Figure 7.

Combination of TNF blockade and anti-CD40/IL-2 increases survival, decreases systemic cytokine storm, and protects from liver pathology in aged mice. (A) Percent survival of aged (12 mo) C57BL/6 mice receiving low-dose anti-CD40/IL-2 with etanercept (Etan; 1.5 mg/0.2 ml s.c.), or hIgG (1.5 mg/0.2 ml s.c.) or rIgG/PBS, n = 6–8. Survival analysis was plotted according to the Kaplan-Meier method, and statistical differences were determined with the log-rank test. (B–F) Liver (B), gut (C), and lung (D) pathology and serum ALT (F) in aged mice (12 mo), n = 3. (B–D) One representative image from each group and each organ was captured. Bars, 50 µm. (E) Multiorgan histopathology score from B–D. (G–I) Serum cytokines from groups in B–F were examined for IL-6(G), IFN-γ (H), and TNF (I; day 2) in young (2 mo) and aged (12 mo) mice. Etanercept was administered on days −1 and 1. (J–L) Aged C57BL/6 mice (12 mo old) received on day −1 either hIgG (1.5 mg/0.2 ml s.c.), or etanercept (Etan; 1.5 mg/0.2 ml s.c.), or anti–IL-6 antibody (1.0 mg/0.2ml i.p) with combination of low-dose anti-CD40/IL-2 (IT) on day 0. Control (Ctrl) group received rIgG/PBS, n = 3. (J and K) Serum cytokine levels for IL-6 (J) and TNF (K) were assessed after 2 d of treatment. (L) Liver histopathology from mice in J and K was assessed for each group. Values represent the mean ± SEM tested by one-way ANOVA with Bonferroni’s post-tests. ***, P < 0.001; **, P < 0.01; *, P < 0.05 compared with control group. n.s: not significant. Data from A–I are representative of three independent experiments and data from J–L are representative of two independent experiments.