Faculty of Medicine, Institute of Microbiology and Immunology, University of Ljubljana, Ljubljana, Slovenia.
Department of Allergology, Rheumatology and Clinical Immunology, University Children's Hospital, University Medical Center Ljubljana, Ljubljana, Slovenia.
Front Immunol. 2019 Jul 16;10:1632. doi: 10.3389/fimmu.2019.01632. eCollection 2019.
Actin nucleators initiate formation of actin filaments. Among them, the Arp2/3 complex has the ability to form branched actin networks. This complex is regulated by members of the Wiscott-Aldrich syndrome protein (WASp) family. Polymerization of actin filaments can be evaluated through flow cytometry by fluorescent phalloidin staining before and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). We identified a missense mutation in the gene ARPC1B (Arp2/3 activator subunit) resulting in defective actin polymerization in four patients (three of them were related). All patients (1 male, 3 female) developed microthrombocytopenia, cellular immune deficiency, eczema, various autoimmune manifestations, recurrent skin abscesses and elevated IgE antibodies. Besides four patients with homozygous mutation in ARPC1B, we also identified six heterozygous carriers without clinical disease (3 males, 3 females) within the same family. We developed a functional test to evaluate Arp2/3 complex function, which consists of flow cytometric detection of intracellular polymerized actin after fMLP stimulation of leukocytes. Median fluorescence intensities of FITC-phalloidin stained actin were measured in monocytes, neutrophils and lymphocytes of patients, carriers, and healthy control subjects. We detected non-efficient actin polymerization in monocytes and neutrophils of homozygous patients compared to carriers or the healthy subjects. In monocytes, the increase in median fluorescence intensities was significantly lower in patients compared to carriers (104 vs. 213%; < 0.01) and healthy controls (104 vs. 289%; < 0.01). Similarly, the increase in median fluorescence intensities in neutrophils was significantly increased in the group with carriers (208%; < 0.01) and healthy controls (238%; < 0.01) and significantly decreased in the patient's group (94%). Our functional fMLP/phalloidin test can therefore be used as a practical tool to separate symptomatic patients from asymptomatic mutation associated to actin polymerization.
肌动蛋白成核因子启动肌动蛋白丝的形成。其中，Arp2/3 复合物具有形成分支肌动蛋白网络的能力。该复合物受威特综合征相关蛋白（WASp）家族成员的调节。通过用 N-甲酰基-甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）刺激前后用荧光鬼笔环肽染色通过流式细胞术可评估肌动蛋白丝的聚合。我们在四个患者（其中三个是相关的）中鉴定出 ARPC1B 基因（Arp2/3 激活亚基）中的错义突变，导致肌动蛋白聚合缺陷。所有患者（1 名男性，3 名女性）均出现血小板减少、细胞免疫缺陷、湿疹、各种自身免疫表现、反复皮肤脓肿和 IgE 抗体升高。除了四个 ARPC1B 纯合突变患者外，我们还在同一个家庭中鉴定出了六个无临床疾病的杂合携带者（3 名男性，3 名女性）。我们开发了一种功能测试来评估 Arp2/3 复合物的功能，该测试包括在白细胞用 fMLP 刺激后通过流式细胞术检测细胞内聚合的肌动蛋白。在患者、携带者和健康对照的单核细胞、中性粒细胞和淋巴细胞中测量 FITC-鬼笔环肽染色肌动蛋白的中荧光强度。与携带者或健康对照相比，纯合患者的单核细胞和中性粒细胞中的肌动蛋白聚合效率较低。在单核细胞中，与携带者（104 对 213%； < 0.01）和健康对照（104 对 289%； < 0.01）相比，患者组的中荧光强度增加明显较低。同样，在携带组（208%； < 0.01）和健康对照组（238%； < 0.01）中，中性粒细胞的中荧光强度增加明显增加，而在患者组中则明显降低（94%）。因此，我们的功能性 fMLP/鬼笔环肽测试可作为一种实用工具，将有症状的患者与无症状的肌动蛋白聚合相关突变区分开来。
