del Pozo M A, Pulido R, Muñoz C, Alvarez V, Humbría A, Campanero M R, Sánchez-Madrid F
Servicios de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain.
Eur J Immunol. 1994 Nov;24(11):2586-94. doi: 10.1002/eji.1830241104.
The regulation of the cell surface expression of ICAM-3 (CD50) was investigated in human neutrophils. Immunofluorescence flow cytometry analysis revealed a remarkable and very rapid down-regulation of the ICAM-3 cell surface expression upon neutrophil activation with stimulating agents such as phorbol myristate acetate (PMA) or calcium ionophore. A similar low expression of ICAM-3 was observed on neutrophils from patients undergoing hemodialysis with cell-activating cellulosic membranes. Internalization assays with 125I-labeled anti-ICAM-3 monoclonal antibody (mAb) suggested that ICAM-3-down-regulation was due to antigen release from the cell surface towards the outer milieu, rather than to antigen internalization. Immunoprecipitation studies confirmed this down-regulatory effect, and revealed the presence of ICAM-3 in cell-free supernatants from activated neutrophils. Furthermore, the presence of a soluble form of ICAM-3 with a range of concentrations of 0-296 ng/ml in the plasma from healthy human volunteers was detected by using a two-site mAb radioimmunoassay. A proteolytic mechanism likely accounts for this process since protease inhibitors virtually abrogated the PMA-induced down-regulation of ICAM-3. Functional studies showed that anti-ICAM-3 mAb were able to trigger homotypic neutrophil aggregation both before and after ICAM-3 down-regulation, indicating that the fraction of ICAM-3 molecules remaining on the neutrophil surface upon activation are still capable of sustaining cell adhesion. In contrast, the loss of L-selectin (CD62L) on activated neutrophils was almost complete, thus leading to an impairment of L-selectin-mediated neutrophil-endothelial cell adhesion. These results indicate that ICAM-3 is released to the medium upon neutrophil stimulation and that both ICAM-3 and L-selectin have a role in the neutrophil adhesive phenomena.
在人中性粒细胞中研究了细胞间黏附分子-3（ICAM-3，即CD50）的细胞表面表达调控。免疫荧光流式细胞术分析显示，在用佛波酯肉豆蔻酸酯乙酸酯（PMA）或钙离子载体等刺激剂激活中性粒细胞后，ICAM-3细胞表面表达显著且非常迅速地下调。在用具有细胞激活作用的纤维素膜进行血液透析的患者的中性粒细胞上，也观察到了类似的ICAM-3低表达。用125I标记的抗ICAM-3单克隆抗体（mAb）进行的内化试验表明，ICAM-3下调是由于抗原从细胞表面释放到细胞外环境中，而不是由于抗原内化。免疫沉淀研究证实了这种下调作用，并揭示了在活化中性粒细胞的无细胞上清液中存在ICAM-3。此外，通过使用双位点mAb放射免疫分析法，在健康人类志愿者的血浆中检测到了浓度范围为0 - 296 ng/ml的可溶性ICAM-3。由于蛋白酶抑制剂几乎消除了PMA诱导的ICAM-3下调，因此蛋白酶解机制可能参与了这一过程。功能研究表明，抗ICAM-3 mAb在ICAM-3下调之前和之后都能够引发同型中性粒细胞聚集，这表明活化后残留在中性粒细胞表面的ICAM-3分子部分仍然能够维持细胞黏附。相反，活化中性粒细胞上L-选择素（CD62L）的丢失几乎是完全的，从而导致L-选择素介导的中性粒细胞与内皮细胞黏附受损。这些结果表明，中性粒细胞受刺激后ICAM-3释放到培养基中，并且ICAM-3和L-选择素在中性粒细胞黏附现象中都起作用。
