Brzezinska Agnieszka A, Johnson Jennifer L, Munafo Daniela B, Ellis Beverly A, Catz Sergio D
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.
Immunology. 2009 Jul;127(3):386-97. doi: 10.1111/j.1365-2567.2008.02980.x. Epub 2008 Nov 14.
Lipopolysaccharide (LPS) stimulates exocytosis in neutrophils. The signalling molecules involved in the regulation of this mechanism are currently unknown. Using neutrophils from interleukin-1-receptor-associated kinase (IRAK)-4- and Toll/IL-1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-deficient mice, we dissected the signalling pathways that control exocytosis. We analysed exocytosis of peroxidase-negative and azurophilic granules by following the mobilization of the beta2-integrin subunit CD11b and myeloperoxidase (MPO)-containing granules, respectively. IRAK-4-null neutrophils showed marked defects in both peroxidase-negative and azurophilic granule exocytosis in response to LPS. In contrast, the exocytic response to LPS of TRIF-deficient neutrophils was not different from that of wild-type cells. No differences were observed in the exocytosis of secretory organelles between IRAK-4-null and wild-type neutrophils when they were stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA). Electron microscopy analysis showed that no morphological abnormalities were present in the granules of IRAK-4-deficient neutrophils, suggesting that the lack of exocytic response to LPS is not attributable to developmental abnormalities. Using pharmacological inhibitors, we found that p38 mitogen-activated protein kinase (p38MAPK) is essential for the exocytosis of all neutrophil secretory organelles in response to LPS. Interestingly, we found that phosphatidylinositol 3-kinase (PI3K) is essential for azurophilic granule exocytosis but not for the mobilization of other neutrophil granules in response to LPS. Azurophilic granule exocytosis in response to Listeria monocytogenes was dependent on PI3K but not IRAK-4 activity, suggesting that alternative signalling pathways are activated in IRAK-4-deficient neutrophils exposed to whole bacteria. Our results identified IRAK-4, p38MAPK and PI3K as important regulatory components with different roles in the signalling pathways that control Toll-like receptor ligand-triggered neutrophil exocytosis.
脂多糖（LPS）可刺激中性粒细胞的胞吐作用。目前尚不清楚参与该机制调节的信号分子。利用来自白细胞介素-1受体相关激酶（IRAK）-4缺陷小鼠和含Toll/IL-1受体（TIR）结构域的接头蛋白诱导IFN-β（TRIF）缺陷小鼠的中性粒细胞，我们剖析了控制胞吐作用的信号通路。我们分别通过追踪β2整合素亚基CD11b的动员和含髓过氧化物酶（MPO）的颗粒，分析了过氧化物酶阴性颗粒和嗜天青颗粒的胞吐作用。IRAK-4基因敲除的中性粒细胞在对LPS的反应中，过氧化物酶阴性颗粒和嗜天青颗粒的胞吐作用均表现出明显缺陷。相比之下，TRIF缺陷的中性粒细胞对LPS的胞吐反应与野生型细胞无异。当用佛波酯佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）刺激时，IRAK-4基因敲除的中性粒细胞和野生型中性粒细胞在分泌细胞器的胞吐作用上未观察到差异。电子显微镜分析表明，IRAK-4缺陷的中性粒细胞的颗粒中不存在形态异常，这表明对LPS缺乏胞吐反应并非归因于发育异常。使用药理学抑制剂时，我们发现p38丝裂原活化蛋白激酶（p38MAPK）对于所有中性粒细胞分泌细胞器对LPS的胞吐作用至关重要。有趣的是，我们发现磷脂酰肌醇3激酶（PI3K）对于嗜天青颗粒的胞吐作用至关重要，但对于其他中性粒细胞颗粒对LPS的动员并非如此。对单核细胞增生李斯特菌的反应中，嗜天青颗粒的胞吐作用依赖于PI3K而非IRAK-4活性，这表明在暴露于完整细菌的IRAK-4缺陷的中性粒细胞中，替代信号通路被激活。我们的结果确定了IRAK-4、p38MAPK和PI3K是在控制Toll样受体配体触发的中性粒细胞胞吐作用的信号通路中具有不同作用的重要调节成分。
