Cellular signaling during the macrophage invasion by Trypanosoma cruzi
Cellular signaling during the macrophage invasion by Trypanosoma cruzi
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DOI：
10.1007/s00418-002-0477-0
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发表时间：
2002-12-01
影响因子：
2.3
通讯作者：
Souza, W
中科院分区：
文献类型：
--
作者：
Vieira, M;Dutra, JMF;Souza, W
我们报道了蛋白酪氨酸激酶在克氏锥虫侵入原代巨噬细胞中发挥着重要作用。在本研究中，我们使用单克隆抗磷酸酪氨酸抗体进行免疫荧光测定，以揭示寄生虫与巨噬细胞表面关联的位点酪氨酸磷酸化残基的积累，与宿主细胞富含 F-肌动蛋白的结构域共定位。对用 [S-35]L-蛋氨酸标记的巨噬细胞系 IC-21 酪氨酸磷蛋白进行 SDS-PAGE 分析，发现几种肽在与寄生虫相互作用后磷酸化水平增加。其中，检测到 140、120、112、94、73、67 和 56 kDa 的条带，这些条带与被描述为在导致单核吞噬细胞中肌动蛋白组装的事件期间被酪氨酸磷酸化的蛋白质的分子量相匹配。用酪氨酸激酶抑制剂酪氨酸磷酸化蛋白 23 预处理 IC-21 巨噬细胞可抑制锥鞭毛体的摄取，表明酪氨酸磷酸化对于寄生虫在该特定细胞系中的渗透非常重要。免疫荧光显微镜使用针对 p85（磷脂酰肌醇 3 激酶（PI 3 激酶）的调节亚基）的抗体，根据吞噬作用的报道，也将该酶置于相同的位点。我们认为，一旦克氏锥虫锥鞭毛体表面的成分被巨噬细胞受体识别，它们就会触发酪氨酸磷酸化级联的激活、PI 3激酶的募集以及肌动蛋白丝在初始细胞间接触部位的组装，类似于吞噬过程中描述的事件。这些成果支持了克氏锥虫锥鞭毛体吞噬细胞样肌动蛋白依赖性入侵巨噬细胞的模型。
We have reported that protein tyrosine kinases play an important role in the invasion of Trypanosoma cruzi into primary resident macrophages. In the present study we carry out immunofluorescence assays, using monoclonal anti-phosphotyrosine antibodies, to reveal an accumulation of tyrosine-phosphorylated residues at the site of parasite association with the macrophage surface, colocalizing with host cell F-actin-rich domains. SDS-PAGE analysis of macrophage cell line IC-21 tyrosine phosphoproteins, labeled with [S-35]L-methionine, revealed several peptides with increased levels of phosphorylation upon interaction with the parasite. Among them, were detected bands of 140, 120, 112, 94, 73, 67, and 56 kDa that match the molecular weights of proteins described as being tyrosine phosphorylated during events that lead to actin assembly in mononuclear phagocytes. The pretreatment of IC-21 macrophages with the tyrosine kinase inhibitor tyrphostin 23 inhibited trypomastigote uptake showing that tyrosine phosphorylation is important for the parasite penetration in this particular cell line. Immunofluorescence microscopy, using antibodies against p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), placed this enzyme also in the same sites, in accordance to what is reported for phagocytosis. We suggest that once the components of T cruzi trypomastigotes surface are recognized by macrophage receptors, they trigger the activation of a tyrosine phosphorylation cascade, PI 3-kinase recruitment, and assembly of actin filaments at the site of initial cell-to-cell contact, resembling the events described during phagocytosis. These achievements support the model for a phagocytic-like actin-dependent invasion mechanism for T cruzi trypomastigotes into macrophages.