Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
J Immunol. 2013 Sep 1;191(5):2589-603. doi: 10.4049/jimmunol.1300466. Epub 2013 Jul 31.
The role of tyrosine phosphatase Src homology region 2 domain-containing phosphatase (SHP)-1 in LPS-activated cytokine production and inflammation was investigated by determining TNF-α and IL-10 production in splenic macrophages employing SHP-1-null (me/me) mouse model. LPS-stimulated me/me splenic macrophages secreted significantly less IL-10 with concomitantly elevated levels of TNF-α compared with wild-type (WT) macrophages irrespective of LPS dose and duration of stimulation. IL-10 significantly inhibited LPS-induced TNF-α production in both me/me and WT macrophages. The critical requirement for SHP-1 in regulating LPS-induced IL-10 and TNF-α production was confirmed by interfering with SHP-1 expression in WT macrophages and by reconstituting me/me macrophages with the SHP-1 gene. To delineate the role of SHP-1 in positive regulation of LPS-induced IL-10 production, signaling proteins representing SHP-1 targets were examined. The results reveal that tyrosine kinases Src and proline-rich tyrosine kinase 2 (Pyk2) regulate SHP-1-dependent LPS-induced IL-10 production and infer that optimal LPS-induced IL-10 production requires an assembly of a protein complex consisting of SHP-1-Pyk2-Src proteins. Moreover, LPS-induced IL-10 production also requires activation of the p38 MAPK independent of SHP-1 function. Overall, to our knowledge our results show for the first time that SHP-1 acts as a positive regulator of LPS-induced IL-10 production in splenic macrophages through two distinct and independent SHP-1-Pyk2-Src and p38 MAPK pathways.
采用 SHP-1 基因敲除（me/me）小鼠模型，研究了酪氨酸磷酸酶 Src 同源区 2 结构域富含磷酸酶（SHP）-1 在脂多糖（LPS）激活的细胞因子产生和炎症中的作用，检测了脾巨噬细胞中 TNF-α和 IL-10 的产生。与野生型（WT）巨噬细胞相比，LPS 刺激的 me/me 脾巨噬细胞分泌的 IL-10 明显减少，同时 TNF-α水平升高，而与 LPS 剂量和刺激持续时间无关。IL-10 显著抑制了 me/me 和 WT 巨噬细胞中 LPS 诱导的 TNF-α产生。通过干扰 WT 巨噬细胞中 SHP-1 的表达以及用 SHP-1 基因重建 me/me 巨噬细胞，证实了 SHP-1 在调节 LPS 诱导的 IL-10 和 TNF-α产生中的关键作用。为了阐明 SHP-1 在正向调节 LPS 诱导的 IL-10 产生中的作用，检查了代表 SHP-1 靶标的信号蛋白。结果表明，酪氨酸激酶Src 和富含脯氨酸的酪氨酸激酶 2（Pyk2）调节 SHP-1 依赖性 LPS 诱导的 IL-10 产生，并推断最佳的 LPS 诱导的 IL-10 产生需要由 SHP-1-Pyk2-Src 蛋白组成的蛋白复合物的组装。此外，LPS 诱导的 IL-10 产生也需要 p38 MAPK 的激活，而不依赖于 SHP-1 的功能。总体而言，据我们所知，我们的结果首次表明，SHP-1 通过两种不同且独立的 SHP-1-Pyk2-Src 和 p38 MAPK 途径作为脾巨噬细胞中 LPS 诱导的 IL-10 产生的正调节剂发挥作用。
