one of the candidates responsible for DNA clearance [2,4]. SAP is a member of the pentraxin family of proteins and an acute phase reactant, which is produced primarily in the liver in response to infection, inflammation, and trauma [9]. SAP could recognize DNA and other ligands, activate complement, and facilitate pathogen and nuclear antigen phagocytosis, hence playing a nonredundant role in protection against autoimmune disease and in resistance against selected pathogens [10]. Furthermore, SAP shares many properties in common with IgG, including the capacity to interact with FccR and the ability to bind to ligands [11,12]. The interaction of SAP with FccR mediates several functions that are analogous or opposite to those of IgG, including modulation the response to inflammatory stimuli and opsonization of bacteria and altered or exposed self-molecules on damages cells [4,11], thus could compete with antibody and be used to treat antibody-mediated disease such as SLE. As the major DNA- and chromatin-binding protein in plasma of mice, SAP could bind to nuclear antigens that are the target of the autoantibodies of patients with SLE, as well as to damaged membranes and microbial antigens [13,14]. Furthermore, SAP2/ 2 mice spontaneously developed antinuclear autoimmunity and sever glomerulonephritis, a phenotype resembling human SLE [15], which strongly supported a role for SAP in the protection against self-DNA and chromatin-induced autoimmunity. Although the pathological relevance of SAP to autoimmune disease and the significance of self-DNA in the pathogenesis of SLE attracted much attention in recent years, whether SAP takes responsibility for self-DNA clearance and plays a protective role in self-DNA-induced SLE in a mouse model with clear genetic background remain poorly understood. In the present study, we tested the ratios of SAP to DNA and found that they decreased in ALD-DNA-induced lupus mice as compared to controls, and were negatively correlated with SLE disease. Further SAP gene administration in the early stage of SLE disease could reversed lupus nephritis via reduced pathogenic anti-dsDNA antibody production, while in the late stage of disease, SAP gene treatment alleviated proteuria and lupus nephritis via reducing the infiltration of leukocytes and the production of inflammatory markers besides decreasing the levels of anti-dsDNA antibodies. These results indicated that SAP administration would ameliorate self-DNA-induced lupus nephritis via regulating pathogenic anti- dsDNA antibody production and inflammation in lupus mice, which might provide SAP as a potential therapeutic strategy for self-antigen induced SLE and other autoimmune disease. Results ALD-DNA immunization induces SLE syndrome in non-autoimmune-prone mice Levels of serum anti-dsDNA antibodies, which represent a serological hallmark of SLE, tend to reflect disease severity for SLE patients [1]. According to our previously reported procedure, SLE murine model was generated by immunizing female BALB/c mice with ALD-DNA (Fig. 1A) [6,7]. Compared with PBS or unactivated lymphocyte-derived DNA (UnALD-DNA) injection, ALD-DNA immunization generated higher levels of anti-dsDNA IgG antibody (Fig. 1B), which was evident from week 4 and reached the maximum at week 8 after initial injection (Fig. 1B). Glomerulonephritis was also confirmed by urine protein quanti- fication, H&E staining of renal tissues, and immune complex deposition assay (Fig. 1C–G). Remarkably up-regulated urine protein (Fig. 1C), notable glomerulonephritis (Fig. 1D and E), and increased IgG deposition (Fig. 1F and G) were found in ALD- DNA-immunized lupus mice as compared to PBS- or UnALD- Reversal of Lupus Nephritis by SAP Treatment DNA-treated controls (Fig. 1C–G). These results demonstrate that SLE murine model could be established through ALD-DNA immunization. The ratios of SAP to DNA decrease in lupus mice and are negatively correlated with SLE disease To study whether SAP has a correlation to SLE disease, the levels of SAP and circulating DNA in the serum of lupus mice generated by ALD-DNA immunization were assayed. Slightly increased serum SAP levels accompanied with remarkably enhanced circulating DNA levels were found in lupus mice as compared with those in controls (Fig. 2A–D). Pearson correlation analysis showed that the serum SAP levels were closely correlated to the circulating DNA levels (Fig. 2E). However, the ratios of SAP to DNA were lower in lupus mice than in controls, which suggested that SAP protein were relatively insufficient in lupus mice (Fig. 2F). Notably, the ratios of SAP to DNA were negatively correlated with the levels of anti-dsDNA antibodies in SLE mice (Fig. 2G). Taken together, these results indicate that SAP was relatively insufficient in lupus mice. Efficiently expressed SAP protein could inhibit ALD-DNA-mediated innate immune responses in vitro Our results described above provided the basis for the hypothesis that SAP administration in vivo may modulate the immune response in SLE disease. Consequently, pcDNA3-SAP recombinant (pSAP) was constructed for expression of SAP. As shown in Fig. 3A, ELISA analysis for the expression of SAP in culture supernatants of NIH3T3 cell line transfected with pSAP shown that SAP cDNA cloned into pcDNA3 could be correctly transcripted, translated and the protein could be efficiently secreted. To detect the expression of SAP in vivo, BALB/c mice were injected intramuscularly with pSAP (100 mg/mice). Immu- nohistochemistry examination showed an obvious expression of SAP in the muscle received pSAP compared with that receiving pcDNA3 (Fig. 3B). Consistently, quantitative analysis of SAP levels in serum of mice revealed that SAP reached maximal levels at day 10 after injection and then declined (Fig. 3C). 21 days after plasmid injection, the concentration of serum SAP protein went back to the baseline level (Fig. 3C). In contrast, the levels of serum SAP protein in the mice treated with parental plasmid pcDNA3 or physiological saline were not significantly increased over the course of the experiments (Fig. 3C). To further confirm the expression of SAP in vivo, serum was collected on day10 after pSAP injection and subjected to western blot analysis using specific anti- SAP antibody. Marked immune-reactive bands were observed in the serum from mice receiving pSAP injection (Fig. 3D), indicating that SAP could be efficiently expressed in vivo. Accumulating data indicate that SAP has the capacity of binding to DNA under physiological conditions [9]. To explore the biological function of SAP purified from the serum of mice receiving pSAP injection, the binding ability of the purified SAP protein to ALD-DNA was evaluated. It was found that the purified SAP protein had the capacity to bind to ALD-DNA (Fig. 3E). Previous studies have shown that SAP can bind macrophage and opsonize the ligands for phagocytosis [11,16]. In order to investigate whether the binding of SAP to ALD-DNA had any effects on the uptake of ALD-DNA by macrophages, we performed flow cytometry to determine the uptake of ALD-DNA or the complexes of purified SAP protein and ALD-DNA (SAP plus ALD-DNA). However, the intracellular DNA did not increase in macrophages in the presence of SAP (Fig. 3F). As endocytic naked DNA was always degraded rapidly by endosomal nucleases, we used chloroquine to prevent PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22659