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

Abstract

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. Copyright © 1994 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim