Centre for Infection and Inflammation Research, University of New South Wales, Sydney, New South Wales 2052, Australia.
J Biol Chem. 2009 Dec 11;284(50):34839-48. doi: 10.1074/jbc.M109.035683. Epub 2009 Oct 15.
The leukocyte immunoglobulin-like receptor (LILR) B4 belongs to a family of cell surface receptors that possesses cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). LILRB4 is believed to down-regulate activation signals mediated by non-receptor tyrosine kinase cascades through the recruitment of SHP-1. However, the exact mechanisms of LILRB4-mediated inhibition are not fully elucidated. In this study, we demonstrate high level surface expression of LILRB4 on THP-1 cells and primary peripheral blood monocytes, which profoundly inhibited production of a key pro-inflammatory cytokine (TNFalpha) induced by FcgammaRI (CD64). We also report that LILRB4 aggregated to sites of activation upon co-ligation with CD64 and that this may enhance its inhibitory effects. Cross-linking of CD64 on THP-1 cells markedly increased phosphorylation of multiple proteins including tyrosine kinases and signaling molecules (Lck, Syk, LAT, and Erk), an adaptor protein that targets protein-tyrosine kinases for degradation (c-Cbl) and a protein involved in the formation of actin cytoskeletal rearrangement (alpha-actinin-4). Co-ligation of LILRB4 considerably reduced CD64-mediated phosphorylation of Lck, Syk, LAT, Erk, and c-Cbl but not alpha-actinin-4, suggesting selective inhibition of signaling molecules. Treatment of cells with a broad-spectrum phosphatase inhibitor, sodium pervanadate (SP), significantly reversed LILRB4-mediated inhibition of TNFalpha production and protein tyrosine phosphorylation. In comparison, treatment with an SHP-1 specific inhibitor, sodium stibogluconate (SS) has no effects indicating involvement of phosphatase(s) other than SHP-1 in LILRB4 signaling. Collectively, our data show LILRB4 is a potent inhibitor of monocytes activation. This may provide a new potential therapeutic strategy for inflammatory conditions characterized by excessive TNFalpha production.
白细胞免疫球蛋白样受体 (LILR) B4 属于细胞表面受体家族，具有细胞质免疫受体酪氨酸基抑制基序 (ITIM)。LILRB4 被认为通过招募 SHP-1 来下调非受体酪氨酸激酶级联介导的激活信号。然而，LILRB4 介导的抑制的确切机制尚未完全阐明。在这项研究中，我们证明了 THP-1 细胞和外周血单核细胞表面高水平表达 LILRB4，它可显著抑制 FcγRI（CD64）诱导的关键促炎细胞因子（TNFalpha）的产生。我们还报告说，LILRB4 在与 CD64 共交联时聚集到激活部位，这可能增强其抑制作用。THP-1 细胞上 CD64 的交联显著增加了包括酪氨酸激酶和信号分子（Lck、Syk、LAT 和 Erk）在内的多种蛋白质的磷酸化，该信号分子是一种将蛋白酪氨酸激酶靶向降解的衔接蛋白（c-Cbl）和一种参与肌动蛋白细胞骨架重排形成的蛋白（α-肌动蛋白-4）。LILRB4 的共交联大大降低了 CD64 介导的 Lck、Syk、LAT、Erk 和 c-Cbl 的磷酸化，但不影响α-肌动蛋白-4，表明对信号分子的选择性抑制。用广谱磷酸酶抑制剂过钒酸钠 (SP) 处理细胞可显著逆转 LILRB4 介导的 TNFalpha 产生和蛋白质酪氨酸磷酸化抑制。相比之下，用 SHP-1 特异性抑制剂葡萄糖酸锑钠 (SS) 处理没有效果，表明 LILRB4 信号通路中除 SHP-1 以外还涉及其他磷酸酶。总的来说，我们的数据表明 LILRB4 是单核细胞激活的有效抑制剂。这可能为以 TNFalpha 产生过多为特征的炎症性疾病提供一种新的潜在治疗策略。
