title: 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.
authors.affiliation: Servicios de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain.
authors.forename: M A
authors.initials: MA
authors.lastname: del Pozo
authors.forename: R
authors.initials: R
authors.lastname: Pulido
authors.forename: C
authors.initials: C
authors.lastname: Muñoz
authors.forename: V
authors.initials: V
authors.lastname: Alvarez
authors.forename: A
authors.initials: A
authors.lastname: Humbría
authors.forename: M R
authors.initials: MR
authors.lastname: Campanero
authors.forename: F
authors.initials: F
authors.lastname: Sánchez-Madrid
mesh_terms: D015703:Antigens, CD; D000943:Antigens, Differentiation; D015815:Cell Adhesion Molecules / Q000097:blood* / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D015536:Down-Regulation; D006801:Humans; D019041:L-Selectin; D018375:Neutrophil Activation; D009504:Neutrophils / Q000737:chemistry*; D006435:Renal Dialysis
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D015815:Cell Adhesion Molecules; C483616:ICAM3 protein, human; D019041:L-Selectin