Jeyaseelan S, Kannan M S, Briggs R E, Thumbikat P, Maheswaran S K
Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108, USA.
Infect Immun. 2001 Oct;69(10):6131-9. doi: 10.1128/IAI.69.10.6131-6139.2001.
The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin (DeltaLktA) for comparison with LktA. Our results indicate that LktA induces tyrosine phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The DeltaLktA mutant does not induce TP of the CD18 tail, albeit binding to bovine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhibitor, herbimycin A; a phosphatidylinositol 3'-kinase (PI 3-kinase) inhibitor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-dependent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but not porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+]i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus, our data represent the first evidence that binding of LktA to bovine LFA-1 induces a species-specific NRTK signaling cascade involving PI 3-kinase and Src kinases and that this signaling cascade is required for LktA-induced biological effects.
溶血曼氏杆菌产生的白细胞毒素（LktA）与牛淋巴细胞功能相关抗原1（LFA-1）结合，并以细胞和物种特异性方式在牛白细胞中诱导生物学效应。我们之前已经表明，LktA也能与猪LFA-1结合，但不会引发任何效应。这些发现表明，LktA效应的特异性必定既包括与LFA-1的结合，也包括牛白细胞中存在的信号通路的激活。然而，LktA与LFA-1结合后导致生物学效应的信号通路尚未得到表征。在这种情况下，有几份报告表明配体与LFA-1结合会导致非受体酪氨酸激酶（NRTK）信号级联的激活。我们设计了以下实验目的：（i）确定LktA与LFA-1的结合是否会导致NRTK的激活，（ii）检查LktA诱导的NRTK激活是否具有靶细胞特异性，以及（iii）确定LktA诱导的NRTK激活对于生物学效应是否是必需的。我们使用了一种无生物学活性的突变白细胞毒素（DeltaLktA）与LktA进行比较。我们的结果表明，LktA在牛白细胞中诱导LFA-1的CD18尾部酪氨酸磷酸化（TP）。DeltaLktA突变体虽然与牛LFA-1结合，但不会诱导CD18尾部的TP。LktA诱导的CD18尾部TP被NRTK抑制剂赫曲霉素A、磷脂酰肌醇3'-激酶（PI 3-激酶）抑制剂渥曼青霉素和Src激酶抑制剂PP2以浓度依赖性方式减弱。此外，LktA在牛白细胞而非猪白细胞中诱导CD18尾部的TP。而且，LktA诱导的细胞内钙（[Ca2+]i）升高也被赫曲霉素A、渥曼青霉素和PP2抑制。因此，我们的数据首次证明LktA与牛LFA-1的结合诱导了一种涉及PI 3-激酶和Src激酶的物种特异性NRTK信号级联，并且这种信号级联是LktA诱导的生物学效应所必需的。
