Figure 1.
EAC was induced by injecting female SWXJ mice sc with 0.2 ml emulsion and 0.1 ml DDDH2O containing lyophilate from SWXJ mice with 0.1 ml of complete Freund’s adjuvant (CFA) containing 400 µg of Mycobacterium tuberculosis H37R1.
Changes in systemic CXCL9, CXCL10 and CXCL11 levels in EAC mice and CXCL10 in naive mice were measured 3 month after EAC induction (panel A). After 3 months of EAC induction, control or anti-CXCL10 Ab solutions were administered every 2 days for eight weeks. At the end of experiments, serum levels of CXCL9 (▪), CXCL10 (♦), CXCL11 (▾) in experimental and naïve mice (○) (panel B) were determined by ELISA assays. The data presented are the mean concentrations in each group. (A) Asterisks indicate statistically significant differences, i.e., p<0.01 (*), between naïve and CXCL10 groups. (B) p<0.01 (*), between CXCL10 level of EAC-induced control and anti-CXCL10 Ab-treated groups.
Figure 2.
Histopathological changes in the urinary bladders of chronic EAC mice treated with anti-CXCL10 Ab.
Bladder sections from control or anti-CXCL10 Ab groups are shown at different magnifications (A, C, E, G X30; B, D, F, H X100). Panel F shows sections from EAC-induced mice treated with control Ab to illustrate inflamed bladders characterized by differences in mucosal wall thickness, enlargement of the mucosal layer, and leukocyte infiltration. Panel H shows marked improvement after anti-CXCL10 Ab treatment.
Table 1.
Histological evaluations of mice with or without EAC-induced cystitis after control and/or anti-CXCL10 Abs treatment.
Figure 3.
Changes in CXCR3, CXCL9, CXCL10, CXCL11, TNF-α, IL-12p40 and IFN-γ mRNA expressions by EAC mice after anti-CXCL10 Ab treatment.
At the experimental end point, total RNA was isolated from the immune cells of iliac lymph nodes, or urinary bladder of the mice.RT-PCR analysis of unaffected group control Ab [white bar], Anti-CXCL10 Ab [gray bar], EAC induced group EAC+ control Ab [black bar], or EAC+ anti-CXCL10 Ab [striped bar] mRNA expression was performed. Fold increase in expression were expressed. Asterisk(s) (*) indicate statistically significant (p<0.05) increases between the EAC+ control Ab-treated group and the EAC group that received anti-CXCL10 Ab.
Figure 4.
Change in number of CD4+T-cell lymphocytes in EAC mice after anti-CXCL10 Ab treatment.
At the end of experiments, lymphocytes from the spleen, iliac lymph nodes, and urinary bladder were isolated, stained for CD4+ T cell expression, and analyzed by flow cytometry. A representative histogram is shown with the mean percentage of spleen, iliac lymph node, and urinary bladder CD4+ cells per mouse. The data presented are representative dot plots and histograms from three independent experiments involving 5 mice per group.
Figure 5.
Change in number of mast cells in EAC mice after anti-CXCL10 Ab treatment.
At the end of experiments, cells from the spleen, iliac lymph nodes, and urinary bladders were isolated, stained for CD3+ and CD117+cell (cKit- mast cells) expression, and analyzed (CD3- CD117+) by flow cytometry. Histograms show mean percentage of cKit expression. The data presented are representative dot plots and histograms from three independent experiments involving 5 mice per group.
Figure 6.
Control or anti-mouse CXCL10 Ab solutions were administered 3 months after EAC induction and every 2 days thereafter for eight weeks.
At the end of experiments, cells from the spleens, iliac lymph nodes, and urinary bladders were isolated and stained for Ly6G (neutrophils); expression was analyzed by flow cytometry. Histogram shows mean percentage in LY6G (neutrophils). The data presented are representative dot plots and histograms from three independent experiments involving 5 mice per group.