Our studies have identified a soluble molecule in normal human plasma and serum with the characteristics of the ␣-chain of the low density lipoprotein receptorrelated protein (LRP). LRP is a large multifunctional receptor mediating the clearance of diverse ligands, including selected lipoproteins, various protease inhibitor complexes, and thrombospondin. A soluble molecule (sLRP) has been isolated from plasma using an affinity matrix coupled with methylamine-activated ␣ 2 -macroglobulin, the ligand uniquely recognized by LRP, and eluted with EDTA. This eluate contains a protein that co-migrates on SDS-polyacrylamide gel electrophoresis with authentic human placental LRP ␣-chain, is recognized by anti-LRP ␣-chain monoclonal antibodies, and binds the 39-kDa receptor-associated protein (RAP) and tissue plasminogen activator-inhibitor complexes. A similar RAP-binding molecule was detected in medium conditioned for 24 h by primary cultures of rat hepatocytes, suggesting that the liver may be the in vivo source of sLRP. In contrast, immunoprecipitation experiments failed to detect the production of sLRP by cultured HepG2 hepatoma and primary human fibroblast cells. Addition of a soluble form of LRP to cultured HepG2 cells resulted in a significant inhibition of capacity of these cells to degrade tPA, a process that has been demonstrated to be mediated by cell surface LRP. Preliminary data indicate that the concentration of sLRP is altered in the plasma of patients with liver disease. Increased levels of sLRP may antagonize the clearance of ligands by cell bound LRP perturbing diverse processes including lipid metabolism, cell migration and extracellular proteinase activity.The low density lipoprotein receptor-related protein (LRP) 1 has been previously identified as a membrane-bound endocytic receptor (1, 2). Studies have demonstrated that LRP mediates the internalization of multiple, structurally unrelated ligands, including selected lipoproteins, proteinase-inhibitor complexes, plasminogen activators, and thrombospondin (reviewed in Refs. 3 and 4). The binding of all ligands to LRP is inhibited by the receptor-associated protein (RAP), a protein that was copurified with LRP (2, 5). The range of ligands recognized by LRP suggests that it plays a role in diverse processes including lipid metabolism, cell growth, migration, and tissue invasion. LRP expression is widespread; however, it is most highly expressed in the liver, brain, and placenta. The remarkable degree of cross-species identity conserved in the LRP amino acid sequence (3) and the embryonic lethal phenotype obtained after targeted disruption of the LRP gene in the mouse (6) underscore the biological importance of this molecule.Here we report the identification of a soluble form of LRP circulating in human plasma. The characterization of this molecule, which maintains the ligand binding characteristics of cell surface LRP, introduces a new dimension to the biology of LRP. Accumulation of soluble LRP in plasma may antagonize the clearance of ligands by cell-...

Low density lipoprotein receptor-related protein/α₂-macroglobulin receptor (LRP) is a surface membrane endocytic receptor, one of whose many functions is the regulation of plasminogen activator-mediated cell migration. LRP is known to have a role in migration and invasion, but its direct involvement has been demonstrated only in non-tumour cells. We investigated six breast cancer cell lines and a normal mammary epithelial cell clone for surface and total cellular LRP expression, and confirmed that its presence corresponds to the ability to invade and migrate in vitro. We showed that LRP in the tumour cell lines is expressed at a wide range of levels: from ∼300 to ∼6,300 sites per cell. Four of the breast cancer cell lines expressed LRP at over 1,000 sites/cell and were markedly invasive in our assay, the remainder of the cell lines and the normal clone having far fewer LRP sites and lacking invasive ability. We further showed that the migratory and invasive abilities of a highly invasive breast cancer cell line are both inhibited by receptor-associated protein, a unique LRP ligand which normally has a solely intracellular distribution but which, when added to culture medium, can inhibit all other ligand interactions with this receptor.

The incidence and clinical implications of unusual patterns of expression of leucocyte differentiation antigens in acute leukaemia were assessed on 568 newly diagnosed paediatric and adult cases undergoing immunophenotyping with a panel of monoclonal antibodies at a single centre. Among patients with the precursor B (common) form of acute lymphoblastic leukaemia (ALL), the major variant seen was the group of 15 cases with expression of myeloid surface antigens. 4.5% of ALL cases tested with antibody to CD-11b were positive, 5.1% were CD-13+, and 10.8% CD-33+. All 15 patients achieved a complete remission with chemotherapy, with six of eight children and four of seven adults remaining disease free. A smaller proportion (1.5%) of precursor B ALL patients showed expression of the T lineage marker, CD-7. The only significant variant seen in the precursor T-ALL group was expression of HLA-DR antigen, which was found in five of 35 cases; although all responded to treatment, only one remains a disease-free survivor. Among patients with acute myeloid leukaemia (AML), expression of the lymphoid markers terminal transferase (TdT) and CD-7 were commonly seen (22.2% and 28.4% respectively of cases tested). Other lymphoid markers detected on AML cases were CD2 (11.1%), CD-10 (1%) and CD-19 (4.4%). These results confirm that examples of lineage infidelity are regularly seen in large series of patients with acute leukaemia. Prospective studies using uniform treatment protocols are required to establish whether these patients have significantly different disease outcomes.

We have investigated the binding of over 30 different monoclonal antibodies (MAB) belonging to three distinct clusters of differentiation (CD-11b; CD-13; CD-33; as defined by the Third International Workshop on Leucocyte Differentiation Antigens (ILWS), 1986), and which are reactive with three distinct myeloid restricted surface antigens ('gp160,95'; 'gp150'; 'gp67'). By investigating reactivity with non-haemopoietic cells, we have confirmed that CD-11b and CD-33 MAB reactivity is largely restricted to haemopoietic cells, whilst CD-13 MAB showed additional binding to a wide range of non-haemopoietic cells. Epitopic heterogeneity was also investigated within each cluster of differentiation. Tested anti-CR3 (CD-11b) MAB varied in their ability to block the binding of complement coated sheep red blood cells and zymosan particles. A more detailed analysis of MAB binding heterogeneity was performed by competitive inhibition assays. It was demonstrated that MAB from both CD-11b and CD-13 bind to several distinct epitopes (at least six and five respectively) on their respective antigen molecules. In contrast, CD-33 MAB appear to bind to only a single site on 'gp67'. These data may allow for a clearer appreciation of the disparate functional effects obtained using different MAB reagents to individual myeloid antigens, as reported by a number of workers.

Summary A subpopulation of mononuclear leucocytes was prepared from umhiHcal cord venous hlood by immunomagnetic depletion of lymphocytes and monocytes using monoclonal antihodies to CD2, CD3, CD14 and CD19 antigens, and examined for NK cell-associated phenotypic and functional properties. The depleted population was enriched for the NK markers CD16 (mean 53.6% positive) and CD56 (mean 42.7% positive). While there was considerable overlap of these two markers, approximately one-third of CD16'' cells were CD56"; in contrast, few CD56* CD16" cells were found. CD16"^/CD56* cells also co-expressed CD7 and CD45RA antigens, while a minority weakly expressed CD8. Another marker of adult NK cells, CD57, was virtually absent from CD16^/GD56* cells, as was MHG Glass 2. Freshly depleted cord cells had virtually absent natural cytotoxicity to K562 targets in a chromium release assay, but NK activity could he induced after 18 h exposure to recombinant human IL-2, without significant change in phenotype. These fmdings confirm the phenotypic differences and functional defects of NK cells in cord blood as compared to adult blood, and identify a subset of cells with unique phenotype (GD2 CD3" CD7* CD16'Ĉ D56' GD57"). The precise relationship of this subset of cells to NK lineage remains to be defmed.

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SummaryThe antigenic phenotype of neonatal lymphoid cells isolated from umbilical cord blood was investigated using monoclonal antibodies and flow cytometry. Although the majority ofcells expressed mature T or B cell differentiation antigens, small subpopulations of phenotypically immature lymphocytes were detected. A small proportion (mean 2-8%) ofcells expressed the common acute lymphoblastic leukaemia antigen (CD-10), a significantly higher figure than that detected on adult peripheral blood lymphocytes. The cortical thymocyte antigen (CD-I) was detected on a very small subset of eord lymphoid eells, but was also present on adult lymphocytes at approximately the same frequency. The nuclear enzyme terminal deoxynucleotidyl transferase (TdT). a marker of early lymphoid differentiation, was detected by immunofluoreseenee on 0 031% of mononuclear cells in cytocentrifuge preparations, representing an approximate 10-fold increase in frequency over expression in childhood or adult blood. These circulating TdT+ cells were shown in double labelling experiments to predominantly express markers of B eell differentiation (CD-24, CD-10. MHC Class 2), although occasional cells co-expressing the T lineage marker CD-2 were also seen. These findings are consistent with the circulation of B cell precursors in neonatal blood. The nature ofthe CD-I + cells is unclear, although the absence of CD-1 ^ TdT"*" double labelled cells mitigates against the possible presenee of immature thymus-processed lymphocytes in these samples.

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