FIGURE 10.

Adoptive transfer of macrophages stably expressing shDAI alleviates lupus nephritis. Macrophages stably expressing shDAI (shDAI Mϕ) were retrieved and 2.5 × 10⁶ injected (i.v.) into mice at weeks 0, 2, and 4 after the initial ALD-DNA immunization. Mice injected with macrophages expressing shControl (shControl Mϕ) were used as control. A, real-time PCR analysis of the DAI mRNA level in renal macrophages purified from shDAI macrophage-treated lupus mice or shControl macrophage-treated lupus mice at week 12 after the initial immunization. B, the renal macrophages purified from lupus mice adoptively transferred with shDAI-macrophages were infected with 0.1 μg of pNF-κB-Luc or pIRF3-Luc for 36 h. The cells were stimulated with ALD-DNA (1 μg/ml), UnALD-DNA (1 μg/ml), or PBS for another 12 h. Luciferase activities were measured and normalized to Renilla luciferase activities. C, at week 12 after the initial immunization, mRNA levels of TNF-α, IL-6, IL-10, and MCP-1 in the renal macrophages purified from shDAI macrophage-treated lupus mice or shControl macrophage-treated lupus mice were evaluated by real-time PCR. D, at week 12 after the initial immunization, the purified renal macrophages from the shDAI macrophage-treated lupus mice or shControl macrophage-treated lupus mice were cocultured with CD4⁺ T cells and CD19⁺ B cells from the SLE mice, then stimulated with ALD-DNA for 6 days. Levels of anti-dsDNA IgG in the culture supernatants were analyzed by ELISA. E, at week 12, levels of TNF-α, IL-6, IL-10, and MCP-1 in serum of the mice were determined by ELISA. F, at week 12, kidney tissues were collected and homogenized, the expression of TNF-α, IL-6, IL-10, and MCP-1 were determined by ELISA. G, serum anti-dsDNA Ab level every 2 weeks were measured by ELISA. H, urine protein levels of mice were assessed by the BCA Protein Assay kit. Data in A-H are mean ± S.E. of three independent experiments, n = 8. I, 12 weeks after the initial immunization, glomerular immune deposition was detected by direct immunofluorescence for IgG in frozen kidney sections of mice. Representative images (magnification ×200) of 10 mice are shown for each group. J, mean glomerular fluorescence intensity (arbitrary units) was determined for IgG in shDAI macrophage-treated lupus mice or shControl macrophage-treated lupus mice at week 12 after the initial immunization, n = 10. **, p < 0.01. K, 12 weeks after the initial immunization, nephritic pathology was evaluated by H&E staining of renal tissues. Images (magnification ×200) are representative of at least 10 mice in each group. L, the kidney score was assessed using paraffin sections stained with H&E in J. ***, p < 0.001.