Loitto V M, Rasmusson B, Magnusson K E
Department of Medical Microbiology, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden.
J Leukoc Biol. 2001 May;69(5):762-71.
Enrichment of chemoattractant receptors on the neutrophil surface has been difficult to assess, primarily because of limitations in sensitivity of visualization. Using an ultrasensitive, cooled charge-coupled device camera, we investigated spatial-temporal relationships between N-formyl peptide receptor distribution and directional motility of human neutrophils. Live cells were labeled with fluorescent receptor ligands, i.e., fluoresceinated tert-butyl-oxycarbonyl-Phe-(D)-Leu-Phe-(D)-Leu-Phe-OH (Boc-FLFLF) and formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK), while fixed cells were labeled with either fluorescent peptides or monoclonal antibodies. Double labeling of receptors and filamentous actin (F-actin) was done to investigate possible colocalization. N-Formyl peptide receptors on unstimulated cells were randomly distributed. However, on polarized neutrophils, the receptors accumulated toward regions involved in motility and distributed nonuniformly. In fixed neutrophils, antibody-labeled receptors colocalized with the F-actin-rich leading edge whereas peptide-labeled receptors lagged behind this region. We suggest that neutrophils use an asymmetric receptor distribution for directional sensing and sustained migration. A separation between receptors labeled with peptides and those labeled with antibodies reflects two functionally distinct receptor populations at the membrane of motile neutrophils.
中性粒细胞表面趋化因子受体的富集情况一直难以评估，主要是因为可视化灵敏度存在局限性。我们使用超灵敏的制冷电荷耦合器件相机，研究了N-甲酰基肽受体分布与人中性粒细胞定向运动之间的时空关系。活细胞用荧光受体配体标记，即荧光素化的叔丁氧羰基-Phe-(D)-Leu-Phe-(D)-Leu-Phe-OH（Boc-FLFLF）和甲酰基-Nle-Leu-Phe-Nle-Tyr-Lys（fnLLFnLYK），而固定细胞则用荧光肽或单克隆抗体标记。对受体和丝状肌动蛋白（F-肌动蛋白）进行双重标记以研究可能的共定位情况。未受刺激细胞上的N-甲酰基肽受体呈随机分布。然而，在极化的中性粒细胞上，受体向参与运动的区域聚集且分布不均匀。在固定的中性粒细胞中，抗体标记的受体与富含F-肌动蛋白的前沿共定位，而肽标记的受体则滞后于该区域。我们认为中性粒细胞利用不对称的受体分布进行定向感知和持续迁移。肽标记的受体与抗体标记的受体之间的分离反映了运动性中性粒细胞膜上两个功能不同的受体群体。
