SAP to DNA. These results indicated that SAP was relative insufficient in ALD-DNA-induced SLE mice, which further provide the evidence that SAP defect rather than the deficiency of SAP linked genes might contribute to the pathogenesis of antinuclear autoimmunity in SAP2/2 mice [15,20–22]. Notably, the ratios of SAP to DNA were negatively correlated with the titers of anti-dsDNA antibodies in lupus mice, which verified the critical role of SAP insufficiency in ALD-DNA-induced autoimmunity, although we did not exclude other factors contributing to the pathogenesis of the SLE disease [23,24]. As SAP and IgG shared the same binding site on FccR and competed for FccR binding, SAP could be used to inhibit antibody or immune complex- mediated immune response [11]. All these results strongly support a role for SAP in the protection against self-DNA-induced autoimmunity. We thus adopted a gene therapy method using the pcDNA3- SAP plasmid (pSAP) to treat lupus nephritis. The SAP protein could be efficiently expressed and secreted into the culture supernatants when pSAP was transfected into NIH3T3 cell line, indicating that SAP cDNA cloned into pcDNA3 could be correctly transcripted, translated and the protein was efficiently secreted in vitro. Further study confirmed that the SAP protein could be efficiently expressed and secreted into the systemic circulation via intramuscular injection of pSAP, and SAP protein purified from pSAP-treated mice could promote self-DNA clearance via binding to self-DNA, indicating that efficiently expressed SAP protein could perform the biological functions. Analysis of cytokine levels of macrophages cultured with the complexes of SAP and DNA revealed that levels of inflammatory cytokines including TNF-a, IL-1b, IL-6, IL-12, and MCP-1 were notably decreased in the macrophages cultured with SAP plus ALD-DNA; however, level of IL-10 was significantly increased in the macrophages cultured with SAP plus ALD-DNA as compared with those of macrophages cultured with ALD-DNA alone, indicating that SAP inhibited ALD-DNA-mediated innate immune response in vitro. However, the mechanism of how SAP promotes the clearance of ALD-DNA needs to be further revealed. The protective effect of SAP had also been noticed by other groups and testified that the interaction of SAP with FccR is able to mediate phagocytosis of apoptotic cells, as well as mediate protective immune response [12,25]. In our study, injection of pSAP in the early stage of SLE disease with the onset of proteinuria notably reversed lupus nephritis. The beneficial effect of pSAP treatment was associated with its inhibitory effect on ALD-DNA-induced anti-dsDNA antibody production and immune complex deposition, which was consistent with the findings from other groups that anti-dsDNA antibodies promoted initiation of lupus nephritis [26]. Furthermore, injection of pSAP at the late stage of SLE disease with the established lupus nephritis remarkably reduced proteinuria and lupus nephritis, while slowly decreased levels of anti-dsDNA antibodies and immune complex deposition were noticed in these pSAP-treated mice, indicating that the protective effect of SAP treatment in the late stage of SLE disease was not exclusively attributable to the decreased levels of anti-dsDNA autoantibodies. SAP is the main acute-phase reactants in mice and its capacity to bind with DNA has been identified for more than 20 years [13,14,27,28]. However, recent emerging evidences indicate that SAP also plays a critical role in modulating cytokine production in inflammatory reactions [10,11]. The inflamed kidneys of patients with SLE, NZB/W F1 mice, and ALD-DNA-induced lupus mice contain many lymphocytes around glomeruli, blood vessels, and in the interstitium [8,29–31]. Our study verified again that CD45+ leucocytes were infiltrated into kidneys of ALD-DNA-induced lupus mice. SAP treatment in the late stage of SLE disease Reversal of Lupus Nephritis by SAP Treatment significantly decreased the numbers of CD45+ leucocytes including T cells, B cells, and macrophages but not dendritic cells in kidney tissue of ALD-DNA-induced lupus mice and the underlying mechanisms need to be elucidated in the future. Further analysis of inflammatory markers revealed that SAP treatment notably decreased systemic and local inflammatory cytokine levels of TNF-a, IL-1b, IL-6, IL-12, and MCP-1, which were closely associated with the severity of lupus nephritis [18]. However, inhibitory cytokine IL-10 was notably increased in pSAP-treated lupus mice, which might also partly contribute to the alleviation of lupus nephritis. Our results were in good agreement with previous studies, and further confirmed the immunomodulatory function and potential protective and therapeutic effect of SAP in ALD- DNA-induced lupus nephritis. In conclusion, we showed that SAP gene administration at the onset of proteinuria could reverse lupus nephritis. The main mechanism seems to be associated with the effective inhibiting the production of anti-dsDNA antibodies and immune complex deposition. While SAP gene treatment in the stage of established lupus nephritis could reduce the disease severity, which was possibly attributable to the decreased infiltration of lymphocytes and reduced levels of inflammatory markers beyond decreased anti-dsDNA autoantibody production. Our findings may provide an insight into better understanding of the underlying mechanism of ALD-DNA-induced lupus nephritis, and provide the preclinical data indicating that SAP administration can alleviate lupus nephritis. This strategy may be a clinically relevant and feasible therapeutic method for patients suffering from self-DNA-induced nephritis or other autoimmune diseases which accompany with decreased clearance of apoptotic cells. Materials and Methods Ethics statement All experiments carried out in this study were strictly performed in a manner to minimize suffering of laboratory mice. All animal procedures were performed according to the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, P.R. China, 1998) and with the ethical approval of the Shanghai Medical Laboratory Animal Care and Use Committee (Permit number: SYXK 2007-0036) as well as the Ethical Committee of Fudan University (Permit number: 2007016). Mice and plasmid Six-week-old female BALB/c mice were purchased from the Experimental Animal Center of Chinese Academy of Sciences (Shanghai, P. R. China). Mice were housed in a specific pathogen- free room under controlled temperature and humidity. The full length of SAP cDNA was amplified from total RNA of murine liver using the primers 59-CGA AGC TTG CCA CCA TGG ACA AGC TGC TGC-39 and 59-CGG AAT TCC CTC TTA CAC ATC GGC AAT C-39. 24 nucleotides encoding FLAG epitope (DYKDDDDK) were added directly at the carboxyl- terminal of SAP gene sequence by primer design. SAP cDNA with a FLAG tag was inserted into the pcDNA3 vector (Invitrogen) to generate pcDNA3-SAP plasmid (pSAP). The plasmid construct was confirmed by DNA sequencing. Generation of bone marrow-derived macrophages (BMDMs) Bone marrow (BM) cells were harvested from uninfected, normal BALB/c mice and filtered through nylon mesh. BM cells were cultured in L929 cell-conditioned medium at a density of 36105 cells/ml of medium and maintained in a 5% CO2 PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e22659