Elsner J, Roesler J, Emmendörffer A, Lohmann-Matthes M L, Welte K
Fraunhofer Institute ITA, Department of Immunobiology, Hannover, Germany.
Exp Hematol. 1993 Jan;21(1):38-46.
Severe congenital neutropenia (SCN) can be corrected in vivo by treatment with pharmacological dosages of recombinant human granulocyte colony-stimulating factor (rhG-CSF). In order to analyze the decreased chemotaxis of neutrophils from SCN patients receiving rhG-CSF, neutrophil functions essential for chemotaxis were investigated. The mobilization of cytosolic calcium ([Ca2+]i) and the functional state of cytoskeletal proteins in neutrophils from SCN patients were compared with either neutrophils from healthy donors (or, in selected experiments, from patients with cyclic neutropenia) and neutrophils from patients with chemotherapy-induced neutropenia also receiving rhG-CSF. Using flow cytometric analysis, two neutrophil subpopulations were detected in SCN patients in response to N-formylmethionine leucyl-phenylalanine (FMLP) (10(-9) M to 10(-7) M), one of which was unable to respond to this stimulus with an increase in [Ca2+]i. Whereas a homogeneous increase in [Ca2+]i in normal neutrophils occurred at 10(-9) M FMLP, neutrophils from SCN patients required 10(-6) M FMLP to respond homogeneously with an increase in [Ca2+]i. In contrast, G-CSF induced neutrophils from patients with cyclic neutropenia and from patients with chemotherapy-induced neutropenia showed a normal increase in [Ca2+]i after stimulation. The [Ca2+]i-dependent superoxide anion (O2-) generation in response to FMLP was also significantly diminished in neutrophils from SCN patients compared to normal neutrophils. However, O2- generation elicited by phorbolester (PMA), which directly activates protein kinase C (PKC), was not affected in SCN neutrophils. The total immunoreactive actin content and basal F-actin content in neutrophils from SCN patients were elevated as compared to normal neutrophils and neutrophils from patients with chemotherapy-induced neutropenia. The increase in F-actin content following FMLP activation was much lower in neutrophils from SCN patients as compared with normal neutrophils. These data suggest a defect in the signal transduction pathway in neutrophils from SCN patients between FMLP ligand-receptor interaction and Ca2+ mobilization, whereas upstream of PKC, triggered events seem to be unaffected. Therefore, [Ca2+]i-dependent neutrophil function in response to FMLP, such as actin disassembly, chemotaxis and O2- generation are diminished in SCN neutrophils. The pathomechanism responsible for the defective [Ca2+]i increase might be an initial step in understanding the underlying pathophysiology of SCN.
严重先天性中性粒细胞减少症（SCN）可通过药理剂量的重组人粒细胞集落刺激因子（rhG-CSF）进行体内纠正。为了分析接受rhG-CSF的SCN患者中性粒细胞趋化性降低的情况，对趋化性所必需的中性粒细胞功能进行了研究。将SCN患者中性粒细胞的胞质钙（[Ca2+]i）动员和细胞骨架蛋白的功能状态与健康供者的中性粒细胞（或在选定实验中与周期性中性粒细胞减少症患者的中性粒细胞）以及同样接受rhG-CSF的化疗诱导性中性粒细胞减少症患者的中性粒细胞进行了比较。使用流式细胞术分析，在SCN患者中检测到两个中性粒细胞亚群对N-甲酰甲硫氨酰亮氨酰苯丙氨酸（FMLP）（10^(-9) M至10^(-7) M）有反应，其中一个亚群在[Ca2+]i增加方面对该刺激无反应。正常中性粒细胞在10^(-9) M FMLP时[Ca2+]i出现均匀增加，而SCN患者的中性粒细胞需要10^(-6) M FMLP才能均匀地出现[Ca2+]i增加。相比之下，周期性中性粒细胞减少症患者和化疗诱导性中性粒细胞减少症患者的G-CSF诱导中性粒细胞在刺激后[Ca2+]i出现正常增加。与正常中性粒细胞相比，SCN患者中性粒细胞对FMLP的[Ca2+]i依赖性超氧阴离子（O2-）生成也显著减少。然而，佛波酯（PMA）直接激活蛋白激酶C（PKC）所引发的O2-生成在SCN中性粒细胞中未受影响。与正常中性粒细胞和化疗诱导性中性粒细胞减少症患者的中性粒细胞相比，SCN患者中性粒细胞中的总免疫反应性肌动蛋白含量和基础F-肌动蛋白含量升高。与正常中性粒细胞相比，SCN患者中性粒细胞在FMLP激活后F-肌动蛋白含量的增加要低得多。这些数据表明，SCN患者中性粒细胞在FMLP配体-受体相互作用和Ca2+动员之间的信号转导途径存在缺陷，而在PKC上游，触发事件似乎未受影响。因此，SCN中性粒细胞中对FMLP的[Ca2+]i依赖性中性粒细胞功能，如肌动蛋白解聚、趋化性和O2-生成均减少。[Ca2+]i增加缺陷的发病机制可能是理解SCN潜在病理生理学的第一步。
