Regulation of ICAM-3 (CD50) membrane expression on human neutrophils through a proteolytic shedding mechanism

• Published in: European journal of immunology Vol. 24; no. 11; p. 2586
• Main Authors: del Pozo, M A, Pulido, R, Muñoz, C, Alvarez, V, Humbría, A, Campanero, M R, Sánchez-Madrid, F
• Format: Journal Article
• Language: English
• Published: Germany 01.11.1994
• Subjects: Antigens, CD; Antigens, Differentiation; Cell Adhesion Molecules - blood; Cell Adhesion Molecules - physiology; Cell Membrane - chemistry; Down-Regulation; Humans; L-Selectin; Neutrophil Activation; Neutrophils - chemistry; Renal Dialysis
• ISSN: 0014-2980
• 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.