Article Figures & Data
Figures
- FIGURE 1.
Prevention of murine lupus erythematosus by inhibition of topoisomerase I. NZB/W F1 mice were treated from week 13 with 25 mg/kg or 50 mg/kg irinotecan three times per week every fourth week (n = 10, control mice; n = 12, both groups treated with irinotecan). A, Frequency of mice with proteinuria. Each point reflects the frequency of mice with grade 2+ (≥100 mg/dl) proteinuria at the indicated time points. B, Survival of irinotecan-treated groups was significantly better compared with saline-treated groups. p < 0.0001; Kaplan-Meier log-rank test. At week 50/51, half of the groups treated with irinotecan were sacrificed for further analysis. C, Normal development of body weight in irinotecan-treated mice versus decline of body weight in saline-treated controls owing to the onset of the lupus disease. ***p < 0.001; two-way ANOVA.
- FIGURE 2.
Irinotecan protects NZB/W F1 from the onset of lupus-associated glomerulonephritis. A and C, Representative kidney sections stained with H&E from a control mouse with end-stage lupus nephritis and from 50-wk-old mice that had been treated with either 25 mg/kg or 50 mg/kg irinotecan. The kidney score was assessed in a blinded manner according to the ISN/RPS2003 classification using paraffin sections stained with H&E, methenamine silver, and periodic acid-Schiff reagent as well as frozen sections stained for IgG. B, D, and E, Demonstration of IgG deposits in the kidneys of lupus-prone mice. The score for mesangial and subendothelial immune deposits was determined by the pathologist in a blinded manner. Original magnifications ×40 (A) and ×63 (B).
- FIGURE 3.
Induction of ssDNA and impaired renal cell apoptosis in irinotecan-treated mice. A–C, ssDNA was determined on kidney sections using the mAb F7-26. D–F, Apoptosis-induced dsDNA breaks were assessed by TUNEL staining. G, Quantification of dsDNA at a ratio of total DNA. Genomic DNA was isolated from kidneys; total DNA was measured at 260 nm and adjusted to 100 ng/ml. dsDNA was determined using the PicoGreen assay. *p < 0.05; one-way ANOVA. H, Caspase-3 activity was measured in kidney lysates using DEVD-afc as a substrate (original magnification × 63). ***p < 0.001; one-way ANOVA.
- FIGURE 4.
Recovery from established murine lupus erythematosus after treatment with irinotecan. NZB/W F1 mice with grade 3+ proteinuria (≥300 mg/dl) were treated three times per week for 2 wk with either 50 mg/kg irinotecan or saline. A third group was treated with irinotecan beginning from grade 4+ proteinuria (≥ 2000 mg/dl; n = 10, control mice; n = 12, irinotecan-treated mice [grade 3+]; n = 11, irinotecan treated mice [grade 4+]). Relapsing disease was handled similar to the initial treatment. A, Irinotecan induced a decline of proteinuria within 2 wk after initiation of the treatment. *p < 0.05; **p < 0.01; ***p < 0.001; two-way ANOVA. Data represent mean values ± SEM. B, Kaplan-Meier survival curves showed significantly increased survival of both irinotecan-treated groups compared with saline-treated controls. p < 0.0001; log-rank test.
- FIGURE 5.
Histologic changes and extent of apoptosis in NZB/W F1 mice treated with established disease. To judge the severity of the disease, kidney sections and lysates of mice (sacrificed 1 wk after completion of saline and 1 and 4 wk after completion of irinotecan treatment) were assessed. A–C, Demonstration of IgG deposits in kidneys of lupus-prone mice. The score for mesangial and subendothelial IgG was assessed in a blinded manner. D and E, The kidney score was determined in a blinded manner using the ISN/RPS2003 classification. F, Caspase-3 activity was measured in kidney lysates using DEVD-afc as a substrate. Original magnifications × 63 (A) and ×40 (D). *p < 0.05; one-way ANOVA.
Tables
- Table I. Immunology of NZB/W F1 mice treated from week 13 with irinotecan compared with saline-treated controls
Saline 25 mg/kg Irinotecan 50 mg/kg Irinotecan Spleen cell populations B220+ cells (%) 32.89 ± 3.31 22.17 ± 2.32* 20.95 ± 3.89*** IgMlowIgDlow B cells (%) 9.79 ± 3.72 14.25 ± 4.65 11.13 ± 3.59 IgG-producing B cells (ELISPOTs per 106 cells) 5497 ± 3813 1787 ± 1703** 479 ± 602** CD4+/CD8+ ratio 4.79 ± 2.2 2.34 ± 0.43** 2.09 ± 0.25** CD69+ CD4 cells (%) 6.01 ± 1.56 4.03 ± 1.13** 3.13 ± 0.77*** CD11b+ cells (%) 1.73 ± 048 2.64 ± 1.47 2.24 ± 0.32 Neutrophils from peripheral blood (103/mm3) 0.40 ± 0.37 0.42 ± 0.21 0.34 ± 0.17 IgG anti-ds DNA (A405–490 nm) 2.01 ± 0.67 2.31 ± 0.50 1.29 ± 0.63 Total IgG (mg/ml) 9.87 ± 4.82 13.99 ± 5.39 12.72 ± 6.77 Saline-treated control mice were sacrificed when they had reached proteinuria grade 4+ and weight loss >25% calculated from the beginning of the disease (n = 6). The mean age of saline-treated animals was 39 wk. Irinotecan-treated animals were sacrificed at week 50/51 (n = 6, both groups).
↵* p < 0.05; **p < 0.01; ***p < 0.001.
- Table II. Immunology of NZB/W F1 mice with established lupus nephritis treated with irinotecan compared with saline-treated controls.
Treatment from Proteinuria Grade 3+ (Time to Sacrifice after Completion of the Treatment) 1 wk after Saline 1 wk after Irinotecan 4 wk after Irinotecan Spleen cell populations B220+ cells (%) 32.19 ± 7.34 24.04 ± 5.20 31.78 ± 6.81 IgMlowIgDlow B cells (%) 20.59 ± 5.94 13.67 ± 2.52* 23.68 ± 3.77 IgG-producing B cells (ELISPOTs per 106 cells) 12011 ± 5952 1387 ± 1120** 11093 ± 1577 CD4+/CD8+ ratio 5.91 ± 2.98 3.92 ± 1.34 6.16 ± 1.88 CD69+ CD4 cells (%) 5.79 ± 3.28 4.63 ± 1.52 9.66 ± 1.95* Neutrophils from peripheral blood (103/mm3) 0.33 ± 0.12 0.65 ± 0.72 0.33 ± 0.15 IgG anti-ds DNA (A405–490 nm) 2.17 ± 0.63 1.68 ± 0.90 2.37 ± 0.50 Total IgG (mg/ml) 11.72 ± 8.22 8.62 ± 5.62 25.78 ± 10.60* Two groups of NZB/W F1 mice were treated from grade 3+ proteinuria with irinotecan (50 mg/kg, a total of six applications within 2 wk) or saline (10 ml/kg), and sacrificed 1 wk after completion of the treatment. A third group of irinotecan-treated mice was sacrificed 4 wk after the last application.
↵* p < 0.05; **p < 0.001.
- Table III. Comparison of different studies demonstrating survival and remission rates after treatment of established lupus nephritis in NZB/W F1 mice
Median Survival in Weeks (Median Time to Death after Onset of Proteinuria in Weeks) Grade 3+ Grade 4+ Grade 3+ and 4+ Remission of Proteinuria Grade 3+ (4+) (%) Median Time to Relapse Grade 3+ (4+) in Weeks CTX (11) — — — 10a — Azathioprine (12) — — — 18a — CTX + azathioprine + methylprednisolone (14) — — 44 0 — C-reactive protein (16) — 46 — 100 — CTX + CTLA4Ig and CTX + CTLA4Ig + α-CD154 (15) — — — 60–80b,c — Irinotecan 70 (35) 76 (39) 73 (35) 75 (55)b,c 9 (12)
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