REGULATION OF ICAM-3 (CD50) MEMBRANE EXPRESSION ON HUMAN NEUTROPHILS THROUGH A PROTEOLYTIC SHEDDING MECHANISM

The regulation of the cell surface expression of ICAM-3 (CD50) was inv estigated in human neutrophils. Immunofluorescence now cytometry analy sis revealed a remarkable and very rapid down-regulation of the ICAM-3 cell surface expression upon neutrophil activation with stimulating a gents such as phorbol myristate acetate (PMA) or calcium ionophore. A similar low expression of ICAM-3 was observed on neutrophils from pati ents undergoing hemodialysis with cell-activating cellulosic membranes . Internalization assays with I-125-labeled anti-ICAM-3 monoclonal ant ibody (mAb) suggested that ICAM-3-down-regulation was due to antigen r elease from the cell surface towards the outer milieu, rather than to antigen internalization. Immunoprecipitation studies confirmed this do wn-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/m l 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 PM A-induced down-regulation of ICAM-3. Functional studies showed that an ti-ICAM-3 mAb were able to trigger homotypic neutrophil aggregation bo th before and after ICAM-3 down-regulation, indicating that the fracti on of ICAM-3 molecules remaining on the neutrophil surface upon activa tion are still capable of sustaining cell adhesion. In contrast, the l oss of L-selectin (CD62L) on activated neutrophils was almost complete , thus leading to an impairment of L-selectin-mediated neutrophil-endo thelial cell adhesion. These results indicate that ICAM-3 is released to the medium upon neutrophil stimulation and that both ICAM-3 and L-s electin have a role in the neutrophil adhesive phenomena.