Elucidating the molecular mechanisms of lupus-specific autoantibodies-triggering IL-10 over-production in vitro and in vivo

• Main Authors: Tai-Ping Lee, 李黛苹
• Other Authors: Kuang-Hui Sun
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/81568306074317477822

博士 === 國立陽明大學 === 醫學生物技術暨檢驗學系暨研究所 === 96 === Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease. The characteristic feature of high-titer autoantibody against DNA and ribonucleoprotien can be detected in the patients. Although changes in the titers of these autoantibodies may reflect the disease activity, especially the renal damage, their role in lupus pathogenesis remains unclear. High levels of interleukin 10 (IL-10) have been demonstrated to contribute to lupus susceptibility and severity. In order to evaluate potential role of autoantibodies in the pathogenesis of systemic lupus erythematosus, in vitro and in vivo studies were carried out using an LPS-stimulated macrophages and a transgenic mouse model, respectively. Anti-P mAb promotes IL-10 over-production in a dose- and time-dependent manner in both LPS-activated RAW 264.7 cells and primary human macrophages. Anti-P mAb enhances phosphorylation of AKT, ERK1/2, JNK1/2, and Syk, while phosphorylation of p38 remains unaltered. Furthermore, anti-P mAb decreases GSK3 activity and reduces the phosphorylation of IκBα, which results in inhibition of NFκBp65 nuclear translocation in LPS-activated macrophages. The Syk, PI3K, PKC, JNK and ERK signaling pathways involved in anti-P mAb-triggered IL-10 secretion are also confirmed using various pharmacological inhibitors. In addition, NF-κB has the negative regulatory effects on anti-P mAb-triggered IL-10 secretion. Using the reporter plasmids containing the individual nuclear factor binding site, treatment of anti-P mAb leads to activation of the corresponding factors that bind to AP-1site, SRE and CRE in the LPS-activated macrophages. Furthermore, by transfection with reporter plasmids bearing various lengths of the IL-10 promoter, AP-1 binding site, SRE and CRE are shown to be required for anti-P mAb-induced effects. These nonautoimmune transgenic mice bearing the single chain variable fragment (scFv) of 9D7 anti-double-strand-DNA antibody (soluble or transmembrane form) expressed the transgene in B cells. Autoantibody ELISA assays and Medi-Test Combi 3A were used to evaluate the concentrations of autoantibody in sera and degree of proteinuria in transgenic and wild-type mice. The 9D7-soluble transgenic mice were found to have a higher level in serum anti-dsDNA Abs or proteinuria than that of 9D7-transmembrane transgenic mice. To detect blood urea nitrogen, sera from transgenic mice (Tg) and non-transgenic littermates (NTg) were analyzed by the HITACHI 7170 auto-analyzer. The level of blood urea nitrogen was higher in 9D7-soluble transgenic mice than that of 9D7-transmembrane transgenic mice. To investigate whether the 9D7 transgenic B cells are anergic, in vitro LPS activation study was preformed to analyze the autoantibody response and cytokine profile in the splenocyte of transgenic mice. After stimulation with LPS, the splenocytes secreted more anti-dsDNA Abs, IL-10, and IFN-γ in the 9D7-soluble transgenic mice than 9D7-transmembrane transgenic mice. These findings indicate that some of the transgenic cells may be in a state of anergy. However, their activities can be recovered by mitogen stimulation. SLE is occasionally accompanied with bacterial infection. Lipopolysaccharide (LPS) released from bacteria may precipitate and exacerbate lupus nephritis. On the other hand, in vivo LPS infection experiments were also used to analyze the autoimmune response of the transgenic mice. I found that levels of BUN and proteinuria were significantly higher in the LPS-treated 9D7-soluble transgenic mice than the other mice. The body weight and survival rate were also observed to decrease in the LPS-treated 9D7-soluble transgenic mice. I also found that multiple organ have abnormal pathology and ANA test has significance of a positive peripheral antinuclear antibody test in the LPS-treated 9D7-soluble transgenic mice. In addition, the 9D7 soluble transgenic mice could be stimulated to secret more IL-10 and IFN-�� expression than that of 9D7 transmembrane transgenic mice after LPS stimulation in vivo. Moreover, there was IgG immune complexes deposition in LPS-treated 9D7-soluble transgenic mice. Furthermore, IL-10 accumulation was found in the LPS-treated 9D7-soluble transgenic mice. These findings indicate that LPS may accelerate systemic autoimmune disease in 9D7-soluble transgenic mice Collectively, this study provides a molecular model underlying the autoantibodies-triggered IL-10 over-production in LPS-activated macrophages and LPS-infected transgenic mice, which may play a role in the pathogenesis of SLE.