Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor.

Urokinase plasminogen activator (uPA) interacts with a surface receptor and with specific inhibitors, such as plasminogen activator inhibitor type 1 (PAI-1). These interactions are mediated by two functionally independent domains of the molecule: the catalytic domain (at the carboxyl terminus) and the growth factor domain (at the amino terminus). We have now investigated whether PAI-1 can bind and inhibit receptor-bound uPA. Binding of 125I-labeled ATF (amino-terminal fragment of uPA) to human U937 monocyte-like cells can be competed for by uPA-PAI-1 complexes, but not by PAI-1 alone. Performed 125I-labeled uPA-PAI-1 complexes can bind to uPA receptor with the same binding specificity as uPA. PAI-1 also binds to, and inhibits the activity of, receptor-bound uPA in U937 cells, as shown in U937 cells by a caseinolytic plaque assay. Plasminogen activator activity of these cells is dependent on exogenous uPA, is competed for by receptor-binding diisopropyl fluorophosphate-treated uPA, and is inhibited by the addition of PAI-1. In conclusion, in U937 cells the binding to the receptor does not shield uPA from the action of PAI-1. The possibility that in adherent cells a different localization of PAI-1 and uPA leads to protection of uPA from PAI-1 is to be considered.     

Gene transfer of the urokinase-type plasminogen activator receptor-targeted matrix metalloproteinase inhibitor TIMP-1.ATF suppresses neointima formation more efficiently than tissue inhibitor of metalloproteinase-1

Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Adenoviral expression of TIMP-1.ATF was used to inhibit SMC migration and neointima formation in human saphenous vein segments in vitro. SMC migration was inhibited by 65% in Ad.TIMP-1.ATF-infected cells. Infection with adenoviral vectors encoding the individual domains, Ad.TIMP-1 and Ad.ATF, reduced migration by 32% and 52%, respectively. Neointima formation in saphenous vein organ cultures infected with Ad.TIMP-1.ATF was inhibited by 72% compared with 42% reduction after Ad.TIMP-1 infection and 34% after Ad.ATF infection. These data show that binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface strongly enhances the inhibitory effect of TIMP-1 on neointima formation in human saphenous vein cultures.     

Binding of tissue plasminogen activator to human monocytes and monocytoid cells

Monocytes and monocytoid cell lines previously have been shown to express receptors for plasminogen and urokinase (u-PA). In the present study, the monocytoid cell lines, U937 and THP-1, are shown to bind tissue plasminogen activator (t-PA) in a specific, saturable, and reversible manner. These cells bound t-PA with low affinity (kd = 0.67 to 0.97 mumol/L) and high capacity (0.71 to 3.3 x 10(6) receptors/cell). Human peripheral blood monocytes bound t-PA with a kd (0.9 mumol/L) similar to that of the monocytoid cells but with a lower capacity (0.17 x 10(6) sites/cell). These binding parameters also were similar to the low-affinity interaction of t-PA with endothelial cells as measured with the cells in suspension (kd = 0.73 mumol/L and 1.1 x 10(6) sites/cell). Lysine analogues and active or diisopropylfluorophosphate-inactivated u-PA inhibited t-PA binding to monocytes, monocytoid cells, and endothelial cells with similar IC50 (concentration producing 50% inhibition) values, suggesting that the same recognition specificity mediates t-PA binding to all of these cell types. The existence of a high-affinity binding site for t-PA on monocytoid cells was also explored in detail. Unlike endothelial cells where plasminogen activator inhibitor-1 has been implicated in mediating a high-affinity interaction of t-PA with the cells, no evidence for a role of this inhibitor in ligand binding to the monocytoid cells was found. Furthermore, using both high and low 125I-t-PA concentrations, competition analyses with lysine analogues or u-PA, or treatment of the cells with carboxypeptidase B, failed to indicate the presence of distinguishable classes of t-PA binding sites. In sum, low-affinity receptors for t-PA are expressed at high density on monocytes and monocytoid cells, identifying a new element in the fibrinolytic arsenal of these cells.     

Plasminogen receptors, urokinase receptors, and their modulation on human endothelial cells

Endothelial cells are centrally involved in regulation of fibrinolysis, and receptors for plasminogen and urokinase provide a mechanism by which cells can regulate their fibrinolytic function. Therefore, the existence and characteristics of receptors for these fibrinolytic components on cultured human umbilical vein endothelial cells were examined. We verified the presence of plasminogen receptors on these cells (Kd = 2.1 +/- 1.3 mumol/L, and 1.8 +/- 1.3 x 10(7) binding sites/cell). These binding parameters and other characteristics indicate that these receptors are closely related to the plasminogen receptors on many circulating and adherent cells. Specific binding sites that interact with two-chain urokinase of mol wt 55,000 with a dissociation constant of 2.1 +/- 1.7 nmol/L, with 2.9 +/- 2.9 x 10(5) sites/cell were also identified. Single-chain urokinase of mol wt 55,000, but not the two-chain degradation product of mol wt 33,000 bound to the cells, implicating the amino-terminal aspects of the ligand in receptor recognition. When endothelial cells were stimulated with thrombin, an agent that modulates their fibrinolytic potential, both receptor types were modestly affected; urokinase binding increased 17%, whereas plasminogen binding decreased 19%. The presence and modulation of plasminogen and urokinase receptors provide a potentially important additional mechanism by which endothelial cells may regulate fibrinolysis.     

The dynamics of hepatitis C virus binding to platelets and 2 mononuclear cell lines

Hepatitis C virus (HCV) binds to platelets in chronically infected patients where free HCV constitutes only about 5% of total circulating virus. Free HCV preferentially binds to human mononuclear cell lines but free and complexed virus binds equally to platelets. The extent of free HCV binding to human Molt-4 T cells (which express CD81) and to human promonocytic U937 cells or to platelets (which do not express CD81) was similar. The binding of free HCV to the cell lines was saturated at a virus dose of 1 IU HCV RNA per cell but binding to platelets was not saturable. Human anti-HCV IgG, but not anti-CD81, markedly inhibited HCV binding to target cells in a dose-dependent manner. Human antibodies to HCV hypervariable region 1 of E2 glycoprotein partially inhibited viral binding to target cells. Recombinant E2 also inhibited viral binding to target cells in a dose-dependent manner, with the efficacy of this decreasing in the rank order of Molt-4 cells more than U937 cells more than platelets. In contrast to HCV, recombinant E2 bound to Molt-4 cells to an extent markedly greater than that apparent with U937 cells or platelets. These results suggest that the binding of HCV to blood cells is mediated by multiple cell surface receptors and that recombinant E2 binding may not be representative of the interaction of the intact virus with target cells.     

Fibrin(ogen) is internalized and degraded by activated human monocytoid cells via Mac-1 (CD11b/CD18): a nonplasmin fibrinolytic pathway

Fibrin(ogen) (FGN) is important for hemostasis and wound healing and is cleared from sites of injury primarily by the plasminogen activator system. However, there is emerging evidence in plasminogen activator- deficient transgenic mice that nonplasmin pathways may be important in fibrin(ogen)olysis, as well. Given the proximity of FGN and monocytes within the occlusive thrombus at sites of vascular injury, we considered the possibility that monocytes may play an ancillary role in the degradation and clearance of fibrin. We found that monocytes possess an alternative fibrinolytic pathway that uses the integrin Mac- 1, which directly binds and internalizes FGN, resulting in its lysosomal degradation. At 4 degrees C, FGN binds to U937 monocytoid cells in a specific and saturable manner with a kd of 1.8 mumol/L. Binding requires adenosine diphosphate stimulation and is calcium- dependent. At 37 degrees C, FGN and fibrin monomer (FM) are internalized and degraded at rates of 0.37 +/- 0.13 and 0.55 +/- 0.03 microgram/10(6) cells/h by U937 cells, 1.38 +/- 0.02 and 1.20 +/- 0.30 microgram/10(6) cells/h by THP-1 cells, and 2.10 +/- 0.20 and 2.52 +/- 0.18 micrograms/10(6) cells/h by human peripheral blood mononuclear cells, respectively. The serine protease inhibitors, PPACK and aprotinin, and the specific elastase inhibitor, AAPVCK, do not significantly inhibit degradation. However, degradation is inhibited by chloroquine, suggesting that a lysosomal pathway is involved. Factor X, a competitive ligand with FGN for the Mac-1 receptor, also blocks degradation, as does a monoclonal antibody to the alpha-subunit of Mac- 1. Autoradiography of radioiodinated, internalized FGN shows that FGN proteolysis by the pathway produces a unique degradation pattern distinct from that observed with plasmin. In a fibrin clot lysis assay, Mac-1-mediated fibrinolysis contributed significantly to total fibrinolysis. In summary, FGN is internalized and degraded by activated human monocytoid cells via Mac-1 in the absence of plasmin, thereby providing an alternative fibrinolytic pathway. Thus, in addition to the function of cell adhesion, integrins may also act as receptors that mediate the internalization and degradation of bound ligands.     

Initiation of plasminogen activation on the surface of monocytes expressing the type II transmembrane serine protease matriptase

uPA (urokinase-type plasminogen activator) activates plasminogen with high efficiency when bound to its cellular receptor uPAR, but only after a prolonged lag phase during which generated plasmin activates pro-uPA. How the activity of this proteolytic system might be rapidly initiated is unknown. We have now found that 2 monocytic cell lines display distinct patterns of plasminogen activation. U937 cells, but not THP-1 cells, displayed the expected lag phase, suggesting a constitutive initiation mechanism on the latter. This was shown to be due to the plasmin-independent activation of uPAR-bound pro-uPA by a cell surface-associated protease and to correlate with the expression of matriptase, a type II transmembrane serine protease that was highly expressed in THP-1 cells but undetectable in U937 cells. Kinetic analysis demonstrated that matriptase is a relatively poor activator of pro-uPA in solution, approximately 100-fold less efficient than plasmin (k(cat)/K(m) 1.16 x 10(5) M(-1)s(-1) cf 1.21 x 10(7) M(-1)s(-1)). However, down-regulation of matriptase expression in THP-1 cells by siRNA reduced the activation of cell-associated pro-uPA and the subsequent rapid initiation of plasminogen activation by 76% to 93%. Matriptase was also found to be expressed by peripheral blood monocytes and may therefore be a specific mechanism for the rapid initiation and regulation of plasminogen activation by these cells.     

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.     

Plasmodium falciparum: diversity of isolates from Malawi in their cytoadherence to melanoma cells and monocytes in vitro

We observed considerable diversity in the cytoadherence of Plasmodium falciparum isolates from Malawi to melanoma cells, U937 cells and human peripheral monocytes. Each isolate exhibited a unique cytoadherence profile for the three human cell types. These isolates generally adhered well to U937 cells and fresh monocytes, moderately to melanoma cells and poorly to TE 671, MIA-Pa-Ca, WI 38, PLC/PRF/5 and HeLa cells. An antimalarial immunoglobulin pool inhibited binding to melanoma cells by 50% or more and to U937 cells by 25% or less. There was no correlation between in vitro cytoadherence to the three cells and clinical disease. These results suggest that malarial adherence ligands exposed on the surface of infected erythrocytes vary from one isolate to another.     

Anti–citrullinated protein antibodies bind surface‐expressed citrullinated Grp78 on monocyte/macrophages and stimulate tumor necrosis factor α production

Objective

Anti–citrullinated protein antibodies (ACPAs), which are the most specific autoantibody marker in patients with rheumatoid arthritis (RA), correlate with disease activity; however, the role of ACPAs in RA pathogenesis has not been elucidated. We hypothesized that ACPAs may directly stimulate mononuclear cells to produce inflammatory cytokines. Thus, we identified cognate antigens of ACPAs on monocyte/macrophages and examined their immunopathologic roles in the pathogenesis of RA.

Methods

ACPAs were purified from pooled ACPA‐positive RA sera by cyclic citrullinated peptide–conjugated affinity column. After coculture of U937 cells with ACPAs, the tumor necrosis factor α (TNFα) production and NF‐κB DNA binding activity of the cells were measured by enzyme‐linked immunosorbent assay. The cognate antigens of ACPAs on the U937 cell surface were probed by ACPAs, and the reactive bands were examined via proteomic analysis.

Results

ACPAs specifically enhanced TNFα production and increased the DNA‐binding activity of NF‐κB in U937 cells. Proteomic analysis revealed that Grp78 protein (72 kd) was one of the cognate antigens of ACPAs. The truncated form of cell surface–expressed Grp78 (55 kd) on U937 cells contained citrulline capable of binding with ACPAs. After citrullination, glutathione S‐transferase–tagged recombinant Grp78 (97.52 kd) became a 72‐kd fragment and bound with ACPAs. ACPAs also bound to human monocytes and lymphocytes to promote TNFα production.

Conclusion

We clearly demonstrated that ACPAs enhance NF‐κB activity and TNFα production in monocyte/macrophages via binding to surface‐expressed citrullinated Grp78.

     

Expression and colocalization of cytokeratin 1 and urokinase plasminogen activator receptor on endothelial cells

The cellular localization of human cytokeratin 1 (CK1), urokinase plasminogen activator receptor (uPAR), and gC1qR, high-molecular-weight kininogen (HK)-binding proteins on endothelial cells, was determined. CK1 was found on the external membrane of nonpermeabilized endothelial cells by immunoperoxidase staining, immunofluorescence, and transmission electron microscopy using immunogold. Human umbilical vein endothelial cells (HUVECs) had 7.2 +/- 0.2 x 10(4) specific CK1 membrane sites/cell by (125)I-F(ab')(2) anti-CK1 antibody binding. Flow cytometry studies confirmed the presence of CK1, uPAR, and gC1qR on HUVECs. On laser scanning confocal microscopy and transmission electron microscopy, CK1 and uPAR, but not gC1qR, colocalized on the cell surface of HUVECs. The HUVEC surface distribution of these proteins was distinctly different from that for von Willebrand factor. In competitive inhibition experiments, anti-CK1, anti-uPAR, or anti-gC1qR blocked both biotin-HK binding and prekallikrein (PK) activation on HUVECs with an inhibitory concentration of 50% (IC(50)) of 300 to 350 nM, 50 to 60 nM, or 35 to 100 nM, respectively. Also, antibodies to uPAR and gC1qR each inhibited 86% of kallikrein-mediated, 2-chain urokinase plasminogen activation, whereas antibodies to CK1 only inhibited 24% of plasminogen activation. On HUVECs, CK1 and uPAR, but not gC1qR, colocalized to be a multiprotein receptor complex for HK binding, PK activation, and 2-chain urokinase plasminogen activation.     

Plasminogen inhibits TNFalpha-induced apoptosis in monocytes

Monocytes are major mediators of inflammation, and apoptosis provides a mechanism for regulating the inflammatory response by eliminating activated macrophages. Furthermore, as a consequence of apoptosis, plasminogen binding is markedly increased on monocytoid cells. Therefore, we investigated the ability of plasminogen to modulate monocyte apoptosis. Apoptosis of monocytoid cells (human monocytes and U937 cells) was induced with either TNFalpha or cycloheximide. When apoptosis was induced in the presence of increasing concentrations of plasminogen, apoptosis was inhibited in a dose-dependent manner with full inhibition achieved at 2 microM plasminogen. Plasminogen treatment also markedly reduced internucleosomal DNA fragmentation and reduced levels of active caspase 3, caspase 8, and caspase 9 induced by TNFalpha or by cycloheximide. We examined the requirement for plasmin proteolytic activity in the cytoprotective function of plasminogen. A plasminogen active site mutant, [D(646)E]-Plg, failed to recapitulate the cytoprotective effect of wild-type plasminogen. Furthermore, antibodies against PAR1 blocked the antiapoptotic effect of plasminogen. Our results suggest that plasminogen inhibits monocyte apoptosis. The cytoprotective effect of plasminogen requires plasmin proteolytic activity and requires PAR1. Because apoptosis of monocytes plays a key role in inflammation and atherosclerosis, these results provide insight into a novel role of plasminogen in these processes.     

Role of leuCAM integrins and complement in platelet-monocyte rosette formation induced by immune complexes of human immunodeficiency virus- type 1-immune thrombocytopenic purpura patients

Patients with human immunodeficiency virus-type 1-immune thrombocytopenic purpura (HIV-1-ITP) have elevated polyethylene glycol (PEG)-precipitable immune complexes (ICs) composed of IgG, IgM, and complement that are threefold to sevenfold higher than in healthy control subjects. These complexes contain anti-F (ab')2 as well as anti- idiotype antibodies versus anti-HIV-1gp120. Because anti-F (ab')2 and anti-idiotype antibodies correlate with thrombocytopenia (r = .83 [J Clin Invest 77:1756, 1986] and r = .90 [J Clin Invest 89:356, 1992], respectively) we studied the binding of ICs to platelets and monocytes as well as their role in platelet-monocyte rosette formation. ICs bind to platelets in a saturation-dependent manner (optimum at 10 micrograms/mL; 0.5% of serum conc). Binding to platelets could not be inhibited with platelet saturating concentrations of aggregated IgG or with monoclonal antibody (MoAb) IV.3 versus FcR gamma II. Platelet binding could be inhibited with Fab anti-C3, anti-Clq, or anti-C4 by 57%, 40%, and 46% respectively, not with control Fab (P < .001). Monocytes from HIV-1-ITP patients form rosettes with normal platelets 16.8 +/- 5.2 rosettes/100 monocytes compared with 4.8 +/- 0.8 control monocytes plus normal platelets (P = .009). Gel-washed HIV-1-ITP platelets formed 19 +/- 2.0 rosettes with U937 cells compared to 6.3 +/- 1.0 for normal platelets (P = 0.001). Arming of U937 cells with HIV-1- ITP ICs (5 micrograms/mL) formed 36.7 +/- 2.5 rosettes compared with 10.6 +/- 1.2 for control ICs (P < .01). Rosetting of armed U937 cells could be inhibited with MoAbs versus the alpha chains of CD11a (LFA-1), 11b (Mac-1), or 11c (p150,95) by 67%, 70%, and 61%, respectively (P < .007), whereas binding of ICs to U937 cells was unaffected. Isotype- matched control as well as MoAbs versus antigens on U937 cells (CD13, CD33) or the anti-FcR gamma II receptor had no effect. However, Fab fragments of polyclonal anti-C3 inhibited rosette formation by 78% (P < .01); control Fab had no effect. Thus, platelet-monocyte rosette formation is not Fc dependent. It is complement receptor dependent and requires the cooperation of all three leuCAM integrins.     

Annexin II mediates plasminogen-dependent matrix invasion by human monocytes: enhanced expression by macrophages

Monocytes and macrophages participate in a wide variety of host defense mechanisms. Annexin II, a fibrinolytic receptor, binds plasminogen and tissue plasminogen activator (t-PA) independently at the cell surface, thereby enhancing the catalytic efficiency of plasmin production. We demonstrated previously that annexin II on the surface of both cultured monocytoid cells and monocyte-derived macrophages promotes their ability to remodel extracellular matrix. Here, we demonstrate that human peripheral blood monocytes represent the major circulating annexin II-expressing cell. Annexin II supported t-PA-dependent generation of cell surface plasmin and the matrix-penetrating activity of human monocytes. Compared to polymorphonuclear leukocytes, monocytes supported a 12.9-fold greater rate of plasmin generation in the presence of exogenous t-PA, and this activity was largely attributable to annexin II. Likewise, anti-annexin II IgG directed against the t-PA-binding tail domain inhibited plasminogen-dependent, cytokine-directed monocyte migration through extracellular matrix. On differentiation of monocytes to macrophages, there was a 2.4-fold increase in annexin II-specific mRNA, and a 7.9-fold increase in surface annexin II. Thioglycolate-elicited peritoneal macrophages, furthermore, displayed an additional 3.8-fold increase in annexin II surface expression compared with resident cells. Thus, annexin II-mediated assembly of plasminogen and t-PA on monocyte/macrophages contributes to plasmin generation, matrix remodeling, and directed migration.     

Lectin-carbohydrate interactions and infectivity of human immunodeficiency virus type 1 (HIV-1).

The aim of this study was to determine whether mannosyl-specific lectins, especially Concanavalin A (ConA), may bridge HIV-1 env glycoproteins to cell membranes to increase virus binding to its targets, and to what extent this lectin-carbohydrate interaction can modify HIV-1 infectivity for monocytic compared with lymphoid cells. Monocytic U937 and lymphoid CEM cells, which both express surface mannose, were utilized. Whether first incubated with env glycoprotein or with the cells, lectins bound both to the cells and to radiolabeled recombinant gp160 (rgp160). Thus, they enhanced rgp160 adsorption to the cells in a methyl-alpha-mannose inhibitable manner. ConA did not appear to bind to the V1 domain of CD4 at the U937 cell surface since Leu3a binding was not blocked in the presence of ConA, nor was recombinant CD4 retained on a ConA-agarose affinity matrix. Moreover, enhanced rgp160 binding to the cells was CD4 independent, since it was not modified by preincubating the cells with Leu3a. Finally, ConA did not inhibit the binding of CD4-IgG3 chimeric molecules to virions immobilized on nitrocellulose membrane, which argues against the possibility that it interferes with the interaction of gp120 and CD4. However, both when incubated with the virus or with the cells and despite mediating enhanced binding of env glycoprotein, ConA neutralized HIV-1 infectivity for monocytic U937 as well as for lymphoid CEM cells. In this respect, ConA behaves like neutralizing antibodies which do not interfere with CD4 binding of gp120 but rather with some later event that leads to virus entry. These findings obtained with plant lectins may be of relevance in vivo, inasmuch as endogenous mannosyl-binding proteins, which are known to function as opsonins, have been reported to inhibit in vitro infection by HIV-1.     

Human neutrophil Fc gamma receptor distribution and structure.

Fc receptors (FcR) for IgG on human cells were analyzed with two monoclonal antibodies, 3G8 and 4F7. Fab fragments of both hybridoma IgGs inhibited binding to neutrophils of soluble rabbit IgG complexes and sheep erythrocytes (E) coated with rabbit IgG. The number of sites for 3G8 Fab was 135,000 per neutrophil, roughly equivalent to the number of sites for rabbit IgG in immune complexes (112,000 per cell). We did not observe human IgG1 binding sites on neutrophils, although binding of IgG1 to FcR-bearing lines U937 and HL-60 was readily demonstrated. Other cell types bearing 3G8 binding sites were mature chronic myelogenous leukemia cells, eosinophils, 6% of E-rosetting lymphocytes, and 15% of Ig-bearing peripheral lymphocytes. No binding of 3G8 Fab was observed on the FcR-bearing cell lines U937, HL-60 Raji, Daudi, and K562 or on blood monocytes. However, 15% of monocytes cultured for 7 days and 60% of lung macrophages expressed 3G8 antigen. When analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the neutrophil FcR immunoprecipitated with 3G8 or 4F7 Fab-Sepharose displayed a broad band extending from Mr 73,000 to 51,000; in many experiments this band was resolved into two poorly separated components, centered at Mr 66,000 and 53,000. These results show that human neutrophil FcR for IgG is different from that on monocytes, with respect to both antigenic composition and binding of monomeric IgG1.     

Fluorescent probes as tools to assess the receptor for the urokinase-type plasminogen activator on tumor cells.

Flow cytofluorometric protocols (FACScan) are described for the rapid and quantitative real-time analysis of binding of FITC-pro-u-PA to cell surface receptors (u-PAR) on living, resting, and also on PMA-stimulated human monocytic U937 cells. Binding of pro-u-PA was visualized by CLSM. This fairly new technique is superior over conventional fluorescence microscopy and is an alternative to electron microscopic approaches. Both flow cytofluorometry and confocal laser scanning microscopy allow the analysis quantitatively and with high-sensitivity binding of FITC-pro-u-PA to single suspended or adherent cells. By CLSM u-PA/u-PAR were found to be located in heterogeneously distributed discrete patches at the cell surface on U937 and not inside the cell. This is in agreement with previous studies by Hansen et al, who applied radioiodinated u-PA and electron microscopy to locate u-PAR on microvilli of fixed U937 cells. By flow cytofluorometry, it was possible to quantify the time-dependent and temperature-dependent binding of FITC-pro-u-PA to living single U937. Apparent saturation of u-PAR was achieved at 5 nM FITC-pro-u-PA for both nonstimulated and PMA-stimulated U937 cells. Half saturation of u-PAR was also determined. Nonstimulated U937 was 0.7 nM, and PMA-stimulated U937 was 1.1 nM of FITC-pro-u-PA. This increase in half-saturation concentration in PMA-stimulated cells is paralleled by a steep increase in binding sites (3.6-fold). The use of fluoresceinated reference beads is recommended to verify changes in affinity and binding sites. Using CLSM or flow cytofluorometry, it is also possible to study the structure relationship of u-PA/u-PAR in the presence of competitive binding analogues or inhibitors. Fluorescence techniques will also permit the identification of u-PAR-positive cells in blood, ascitic fluid, or biopsies obtained from cancer patients.     

Correlation between nuclear melatonin receptor expression and enhanced cytokine production in human lymphocytic and monocytic cell lines

The report shows that melatonin enhances IL-2 and IL-6 production by two human lymphocytic (Jurkat) and monocytic (U937) cell lines via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZRalpha, RORalpha1 and RORalpha2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mtl receptor. In the absence of the membrane receptor, melatonin still activates IL-2 production. U937 cells express only the mtl receptor. Although melatonin binds to both U937 nuclei and membranes, CGP 52608, a ligand of the nuclear receptor for melatonin, does not inhibit melatonin binding to U937 nuclei, suggesting that a protein other than the RZR/RORalpha receptor was involved in the process. In U937 cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus LPS (lipopolysaccharide), a treatment that downregulates the expression of the mtl receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the RORgamma1 and RORalpha2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.