Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca(2+) is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca(2+) in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca(2+) by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca(2+) regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca(2+) in a concentration-dependent fashion by influx of extracellular Ca(2+) through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca(2+) even in the absence of extracellular Ca(2+). Leukotoxin-induced intracellular Ca(2+) elevation was inhibited by pertussis toxin, inhibitors of phospholipases A(2) and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca(2+) elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca(2+) signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A(2) and C and release of arachidonic acid in bovine alveolar macrophages. Ca(2+) signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca(2+) signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.

源自溶血巴斯德菌1型（Pasteurella haemolytica serotype 1）的白细胞毒素（Leukotoxin）与内毒素（endotoxin），是引发牛肺炎型巴氏杆菌病肺损伤的主要毒力因子。利用内毒素或白细胞毒素激活牛肺泡巨噬细胞（bovine alveolar macrophages），可诱导细胞因子基因表达（cytokine gene expression），且二者呈现不同的动力学特征（H. S. Yoo、S. K. Maheswaran、G. Lin、E. L. Townsend与T. R. Ames，《Infect. Immun.》63:381–388，1995；H. S. Yoo、B. S. Rajagopal、S. K. Maheswaran与T. R. Ames，《Microb. Pathog.》18:237–252，1995）。此外，白细胞毒素诱导的细胞因子基因表达需要细胞外Ca²⁺的参与。然而，Ca²⁺是否参与内毒素的作用过程，以及白细胞毒素与内毒素调控细胞内Ca²⁺的具体信号通路机制，目前仍未明确。本研究采用负载呋喃-2-乙酰氧基甲酯（fura-2-acetoxymethyl ester）的牛肺泡巨噬细胞，通过视频荧光显微镜技术研究了白细胞毒素与内毒素对细胞内Ca²⁺的调控作用。结果显示，白细胞毒素可通过细胞外Ca²⁺经电压门控通道（voltage-gated channels）内流，以浓度依赖的方式诱导细胞内Ca²⁺持续升高。在胎牛血清（fetal bovine serum）存在的条件下，即使缺乏细胞外Ca²⁺，内毒素仍可升高细胞内Ca²⁺水平。白细胞毒素诱导的细胞内Ca²⁺升高可被百日咳毒素（pertussis toxin）、磷脂酶A₂（phospholipases A₂）与磷脂酶C（phospholipases C）抑制剂，以及花生四烯酸类似物5,8,11,14-二十碳四炔酸（arachidonic acid analog 5,8,11,14-eicosatetraynoic acid）所抑制。内毒素诱导的细胞内Ca²⁺升高则可被磷脂酶C与蛋白酪氨酸激酶（protein tyrosine kinase）抑制剂所抑制，但不受百日咳毒素或该花生四烯酸类似物的影响。据我们所知，本研究首次报道了在牛肺泡巨噬细胞中，白细胞毒素通过G蛋白偶联（G-protein-coupled）机制调控Ca²⁺信号通路，该机制涉及磷脂酶A₂与磷脂酶C的激活以及花生四烯酸的释放。而内毒素介导的Ca²⁺信号通路则依赖于磷脂酶C的激活，并需要酪氨酸磷酸化（tyrosine phosphorylation）过程。二者Ca²⁺信号通路机制的差异，可能是此前报道的白细胞毒素与内毒素激活后基因表达时序差异的潜在成因。