Search

The cellular receptor for urokinase plasminogen activator (uPA-R) is a monomeric phosphatidylinositol-linked glycoprotein (gp40-65) that may contribute to the invasive capacity of tumor and inflammatory cells by focusing the activity of urokinase (uPA) in converting plasminogen to plasmin, a serine protease capable of degrading extracellular matrix proteins. The further characterization of uPA-R has been facilitated by our recent development of a monoclonal antibody, anti-Mo3f, specific for uPA-R. This mAb bound to uPA-R expressed by phorbol myristate acetate-stimulated U-937 cells and by NIH-3T3 cells permanently transfected with uPA-R cDNA. In competitive binding assays, anti-Mo3f inhibited the binding of fluorescein-conjugated uPA ligand to uPA-R expressed by U-937 cells and uPA-R transfectants; conversely, preexposure of cells to saturating quantities of exogenous uPA partially blocked the subsequent binding of anti-Mo3f mAb to uPA-R. Anti-Mo3f mAb was employed as the capture reagent in an ELISA for the quantitation of soluble forms of uPA-R (derived from U-937 cells and recombinant uPA-R) which had a sensitivity of approximately 4-12 ng/ml. Anti-Mo3f mAb was also applied as a serologic probe for the detection of uPA-R expressed by human tumor tissues. By immunoperoxidase staining, anti-Mo3f demonstrated positive tumor cell staining in 4 of 16 breast and 7 of 31 prostate carcinomas in formalin-fixed, paraffin-embedded specimens. These data indicate that the anti-M03f mAb detects an epitope proximate to or within the ligand binding domain (domain 1) of uPA-R and may be useful as a tool for the serologic detection of uPA-R in soluble form or associated with human tumors. Copyright 1994, 1999 Academic Press

The receptor for urokinase plasminogen activator (uPA-R, CD87) is a glycosylphosphatidylinositol (GPD-anchored 50 to 65 kD glycoprotein that, by regulating membrane-associated plasmin activity, may facilitate the invasion of inflammatory and malignant cells. Certain other GPI-anchored glycoproteins are shed from the cell membrane and exist as soluble products in vitro and in vivo. To determine if uPA-R undergoes a similar phenomenon, we have developed a sensitive enzyme-linked immunoabsorbent assay (ELISA) (using a rabbit antiserum as both capture and detection reagents) to measure the quantity of soluble uPA-R (suPA-R) in tissue culture supernatants and biologic fluids. Using this ELISA, we have detected suPA-R in the culture supernatants of U-937 cells and human monocytes stimulated in vitro by certain soluble inflammatory mediators (Sitrin et al. Blood84:1268, 1994; Mizukami et al., Clin Res42:115A, 1994). To determine if suPA-R exists in vivo, we have screened the plasma of 20 normal volunteers (mean ± SD, 3 ± 3 ng/mL; median, 2 ng/mL; range, 1 to 11 ng/mL [serum values slightly higher]); the plasma of 13 ICU patients with clinical sepsis syndrome (mean ± SD, 30 ± 11 ng/mL; median, 11 ng/mL; range, 4 to 221 ng/mL); and the extravascular fluids (pleural, pericardial, and peritoneal) of 84 individuals with presumed inflammatory or malignant conditions (mean ± SD, 21 ± 39 ng/mL; median, 10 ng/mL; range, 2 to 253 ng/ mL). Among the latter specimens, most were inflammatory exudates (only six were malignant by positive cytology) with the highest quantities of suPA-R associated with neutrophilic exudates. The solubility of suPA-R contained within these fluids was confirmed by reanalysis after ultracentrifu-gation to remove particulate material. When tested in a uPA ligand capture ELISA, representative specimens of extravascular body fluids and sepsis plasma contained suPA-R capable of binding uPA ligand (generally representing a small fraction of the immunoreactive material). We conclude from these data that suPA-R is immunologically detectable in vitro and in vivo with high concentrations of receptor found under conditions of inflammatory stimulation. The possibility of suPA-R's biologic activity is suggested by its partial retention of ligand binding capacity.

The receptor for urokinase plasminogen activator (uPAR; CD87) is a 50‐ to 65‐kDa glycosylphosphatidylinositol (GPI)‐anchored glycoprotein expressed by leukocytes and tumor cells where it facilitates uPA‐dependent, plasmin‐mediated pericellular proteolysis during cellular invasion. Because uPAR is inducibly shed into culture supernatants and human body fluids, we tested the hypothesis that soluble uPAR (suPAR) can bind to the plasma membrane of hematopoietic cells where it might modulate their invasive phenotype. As measured by flow cytometry, recombinant biotinylated‐suPAR (B‐suPAR) bound in a specific fashion to THP‐1 leukemia cells and blood PMNs and monocytes (but not to lymphocytes). B‐suPAR also demonstrated specific binding to a variety of leukemic lines, including cells that are positive or negative for membrane uPAR expression. Binding of B‐suPAR to THP‐1 cells was enhanced four‐ to sevenfold by 24‐h exposure of cells to PMA or by coincubation with uPA ligand (but not its isolated catalytic and binding fragments). Conversely, binding of B‐suPAR to PMNs was unaffected by brief exposure to fMLP, and was inhibited by coincubation with uPA. B‐suPAR binding to PMA‐differentiated THP‐1 cells in the presence of uPA was further enhanced by acid washing (removing endogenous uPA) but was partially inhibited by treatment of cells with trypsin. Pretreatment of PMA‐differentiated THP‐1 cells and unstimulated PMNs with soluble sugars, calcium chelators, and antibodies specific for integrins or extracellular matrix proteins failed to consistently block the binding of B‐suPAR. Whereas the binding of suPAR did not measurably affect cell‐associated plasmin activation, suPAR did competitively inhibit the binding of exogenous uPA to membrane‐associated uPAR. These observations support the hypothesis that suPAR can bind specifically to trypsin‐sensitive receptors expressed by certain normal and neoplastic hematopoietic cells where its binding is variably influenced by uPA ligand. J. Leukoc. Biol. 64: 203–213; 1998.