■CD11b/CD18表达在嗜中性粒细胞效应功能中的重要性是众所周知的。除了参与诱导高亲和力结合CD11b/CD18构象的KINDLIN3和TALIN1之外,协调这种反应的信号通路仍未完全理解。
■我们通过生物素鉴定(BioID)进行了无偏筛选方法,用于蛋白质选择,并研究了KINDLIN3相互作用组。我们使用液相色谱和串联质谱作为强大的分析工具。使用CRISPR-Cas9技术实现NB4CD18,KINDLIN3或SKAP2敲除中性粒细胞的生成,并使用流式细胞术检查细胞的效应子功能,活细胞成像,显微镜,附着力,或抗体依赖性细胞毒性(ADCC)。
■在显著富集的325种蛋白质中,我们鉴定了Src激酶相关磷蛋白2(SKAP2),参与肌动蛋白聚合和整合素介导的外内信号传导的蛋白质。在原代或NB4嗜中性粒细胞中的CD18免疫沉淀表明在稳定状态下在CD11b/CD18复合物中存在SKAP2。在这种情况下,对塑料的附着力,在不存在SKAP2的情况下观察到ICAM-1或纤连蛋白,可以通过用latrunculinB阻断肌动蛋白重排来消除。与纤连蛋白的粘附增强,而CD18聚集大大减少。这种反应与显著受损的CD11b/CD18依赖性NADPH氧化酶活性相对应,吞噬作用,和对肿瘤细胞的细胞毒性。
■我们的结果表明SKAP2具有双重作用。它可能仅在静息条件下限制CD11b/CD18介导的粘附,但它的主要贡献在于调节动态CD11b/CD18介导的肌动蛋白重排和聚集,这是人类中性粒细胞细胞效应功能所必需的。
The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood.
We performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3, or SKAP2 knockout neutrophils was achieved using CRISPR-Cas9 technology, and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion, or antibody-dependent cellular cytotoxicity (ADCC).
Among the 325 proteins significantly enriched, we identified Src kinase-associated phosphoprotein 2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at a steady state. Under this condition, adhesion to plastic, ICAM-1, or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin rearrangements with latrunculin B. Upon stimulation of NB4 SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis, and cytotoxicity against tumor cells.
Our results suggest that SKAP2 has a dual role. It may restrict CD11b/CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin rearrangements and clustering as required for cellular effector functions of human neutrophils.