PADGEM-dependent adhesion of platelets to monocytes and neutrophils is mediated by a lineage-specific carbohydrate, LNF III (CD15).

PADGEM (platelet activation-dependent granule-external membrane protein) is a leukocyte receptor of activated platelets that mediates cellular adhesion of platelets to neutrophils and monocytes. To identify the natural ligand on neutrophils and monocytes that interacts with PADGEM, we have evaluated anti-leukocyte antibodies for their ability to block leukocyte-PADGEM binding. Only anti-CD15 antibodies were able to inhibit the binding of neutrophils, monocytes, HL60 cells, and U937 cells to platelets. Anti-CD15 antibodies inhibited the binding of U937 cells to PADGEM-expressing COS cells and to purified PADGEM incorporated into phospholipid vesicles. The CD15 antigen, lacto-N-fucopentaose III (Gal beta 1----4[Fuc alpha 1----3]NAcGlc beta 1----3Gal-beta 1----4Glc), inhibited the interaction of neutrophils or HL60 cells with platelets, whereas lacto-N-fucopentaose I did not; lacto-N-fucopentaose II demonstrated minimal inhibition. Lacto-N-fucopentaose III, and to a lesser extent lacto-N-fucopentaose II, but not lacto-N-fucopentaose I, inhibited the interaction of HL60 cells with COS cells transfected with PADGEM cDNA. CD15, lacto-N-fucopentaose III or Lex, is a component of the PADGEM ligand on neutrophils and monocytes.     

Enhancement of human neutrophil functions by a monoclonal antibody directed against a 19-kDa antigen.

A mouse IgG mAb termed P1C3 was raised against A23187-treated human peripheral blood neutrophils and has been shown to recognize an Ag with an apparent molecular mass of 19 kDa, herein named p19. This p19 Ag was weakly expressed at the cell surface of resting human peripheral blood neutrophils and monocytes, but its cell surface expression was dramatically increased upon activation of these cell types with different secretagogues, including FMLP, PMA, and the calcium ionophores A23187 and ionomycin. A large latent pool of p19 molecules became accessible by immunofluorescence flow cytometry after cell permeabilization of resting neutrophils. A practically total translocation of the intracellular pool of this p19 molecule to the plasma membrane was achieved under appropriate cell stimulation, which induced an almost total degranulation of neutrophil secretory granules. The p19 Ag was absent from platelets, PBL, as well as from the human promyelocytic cell line HL-60, the human promonocytic cell line U937, and the human lymphoid cell lines Daudi and Jurkat. The p19 Ag was also expressed by circulating and/or interstitial neutrophils and monocytes in distinct tissues examined. The mAb P1C3 was found to enhance several neutrophil responses, such as chemotaxis, cell adhesion, phagocytosis, and respiratory burst. These data indicate that the mAb P1C3 recognizes an intracellular Ag in human resting mature neutrophils and monocytes, which upon cell activation is translocated to the cell surface and is able to affect cell functionality.     

Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase

Oxidized phospholipids stimulate endothelial cells to bind monocytes, but not neutrophils, an initiating event in atherogenesis. Here, we investigate intracellular signaling events induced by oxidized phospholipids in human umbilical vein endothelial cells (HUVECs) that lead to specific monocyte adhesion. In a static adhesion assay, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and one of its components, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine, stimulated HUVECs to bind U937 cells and human peripheral blood monocytes but not HL-60 cells or blood neutrophils. Monocyte adhesion was dependent on protein kinases A and C, extracellular signal-regulated kinase 1/2, p38 mitogen activated protein kinases (MAPKs), and cytosolic phospholipase A(2) (cPLA(2)). Inhibition of 12-lipoxygenase (12-LOX), but not cyclooxygenases, blocked monocyte adhesion, and addition of 12-hydroxyeicosatetraenoic acid (12-HETE) mimicked the effects of oxidized phospholipids. Peroxisome proliferator-activated receptor alpha (PPARalpha) was excluded as a possible target for 12-HETE, because monocyte adhesion was still induced in endothelial cells from PPARalpha null mice. Together, our results suggest that oxidized phospholipids stimulate HUVECs to specifically bind monocytes involving MAPK pathways, which lead to the activation of cPLA(2) and 12-LOX. Further analysis of signaling pathways induced by oxidized phospholipids that lead to specific monocyte adhesion should ultimately lead to the development of novel therapeutic approaches against chronic inflammatory diseases.     

Purification and characterization of human lysosomal membrane glycoproteins

Two human cell lysosomal membrane glycoproteins of approximately 120 kDa, hLAMP-1 and hLAMP-2, were identified by use of monoclonal antibodies prepared against U937 myelomonocytic leukemia cells or blood mononuclear cells. The two glycoproteins were purified by antibody affinity chromatography and each was found to be a major constituent of human spleen cells, representing approximately 0.05% of the total detergent-extractable protein. Both molecules were highly glycosylated, being synthesized as polypeptides of 40 to 45 kDa and cotranslationally modified by the addition of Asn-linked oligosaccharides. NH2-terminal sequence analysis indicated that each was approximately 50% identical to the corresponding mLAMP-1 or mLAMP-2 of mouse cells. Electron microscopic studies of human blood monocytes, HL-60, and U937 cells demonstrated that the principal location of these glycoproteins was intracellular, in vacuoles and lysosomal structures but not in the peroxidase-positive granules of monocytes. Transport of the proteins between organelles was evidenced by their marked accumulation in the membranes of phagolysosomes. A fraction of each glycoprotein was also detected on the plasma membrane of U937 and HL-60 cells but not on a variety of other tissue culture cells. This cell-surface expression may be differentiation related, since the proteins were not detected in the plasma membrane of normal blood monocytes and their expression on U937 and HL-60 cells was reduced when the cells were treated with differentiating agents. Cell-surface expression of both glycoproteins was markedly increased in blood monocytes but not in U937 cells after exposure to the lysosomotropic reagent methylamine HCl, indicating differences in LAMP-associated membrane flow in these cell types.     

Porphyromonas gingivalis fimbriae induce adhesion of monocytic cell line U937 to endothelial cells

This study used the human monocytic cell line U937 to examine whether or not Porphyromonas gingivalis fimbriae could induce the adhesion of monocytes to endothelial cells. An in vitro adhesion assay was used to investigate the effects of the fimbriae on U937 cell adhesion to human umbilical vein endothelial cells (HUVEC). The fimbriae enhanced U937 cell adhesion to HUVEC in a dose-dependent manner. U937 cells adhered better to HUVEC pretreated with the fimbriae for a minimum of 2 hr than to untreated HUVEC. The enhanced adhesion was inhibited by a monoclonal antibody against P. gingivalis 381 fimbriae. Pretreatment of U937 cells with the fimbriae for 24 hr enhanced U937 cell adhesion to HUVEC approximately 4-fold. This phenomenon was inhibited by an anti-CD11b antibody, suggesting the involvement of CD11b. These results indicate that P. gingivalis fimbriae can induce monocyte adhesion to the endothelial cell surface. They also suggest that the fimbriae may be involved in the initial event for infiltration of monocytes into the periodontal tissues of individuals with adult periodontitis.     

Downregulation of GMP-140 (CD62 or PADGEM) expression on platelets by N,N-dimethyl and N,N,N-trimethyl derivatives of sphingosine.

GMP-140 (CD62 or PADGEM), a member of the selectin family, is a membrane glycoprotein in secretory granules of platelets and endothelial cells. When these cells are activated by agonists such as thrombin or AMP, GMP-140 is rapidly redistributed to the cell surface. The carbohydrate epitope defined by GMP-140 was identified as sialosyl-Le(x) (as for ELAM-1), which may play an essential role in adhesion of leukocytes or tumor cells on endothelial cells, through aggregation with platelets. Redistribution of GMP-140 from alpha-granules of platelets to the cell surface, induced by thrombin and PMA, was strongly inhibited by preincubation of platelets with N,N-dimethylsphingosine (DMS) or N,N,N-trimethylsphingosine (TMS) at 10-20 microM concentration for a brief period (5 min). Inhibition of GMP-140 redistribution to the cell surface by DMS or TMS was also detected by a cell adhesion assay using HL60 cells, which highly express sialosyl-Le(x); i.e., HL60 cells adhered on platelets activated by thrombin or PMA but not on platelets which were briefly preincubated with DMS or TMS followed by activation. The inhibitory effect of DMS or TMS on GMP-140 redistribution is not due to cytotoxicity, since the TMS-treated platelets were fully capable of aggregating in the presence of ristocetin. Sphingosine (SPN) and protein kinase C inhibitors such as H-7 and calphostin C showed weaker inhibitory activity than DMS and TMS. Our results indicate that both DMS and TMS could be useful reagents to inhibit cell surface expression of crucial selectins which promote adhesion of Le(x-) or sialosyl-Le(x)-expressing cells with platelets and endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific activation by concanavalin A of the superoxide anion generation capacity during U937 differentiation

Phorbol myristate acetate (PMA) induces changes in the human monocyte-macrophage-like cell line U937 which reflect cellular differentiation. PMA prompted the expression of the superoxide anion (O2-) generating capacity in U937 upon appropriate stimulation. A highly specific stimulation by Concanavalin A (Con A) of O2- release was observed in PMA-differentiated U937 cells, which exceeded in 10-20 times that obtained with Con A-stimulated monocytes and neutrophils. These results indicate that a highly specific machinery required for Con A stimulation, practically absent in mature monocytes and neutrophils, is synthesized during PMA-induced U937 differentiation. A novel cytochrome b putatively involved in O2- generation was detected in U937 cells. This cytochrome b content was increased during PMA-induced cell differentiation, although no linear correlation was found between capability to produce O2- by macrophage-like U937 cells and their content of cytochrome b.     

Identification of histone H2B as a regulated plasminogen receptor

Tethering of plasminogen to cell surfaces controls plasmin formation and, thereby, influences pericellular proteolysis and cell migration. Modulation of cellular plasminogen binding sites provides a mechanism for regulation of these events. In this study, two distinct models, phorbol ester-stimulated adhesion of U937 monocytoid cells and culturing of peripheral blood neutrophils, treatments which modulate plasminogen binding sites, have been examined to determine the molecular basis for the upregulation of plasminogen receptors. Membranes were isolated from cell populations, with and without upregulated plasminogen binding capacities, and analyzed by [(125)I]plasminogen ligand blotting of gel transfers. Approximately 15 different [(125)I]plasminogen-binding proteins were discerned in the membrane fractions, and only relatively minor differences in the intensities of individual bands were noted in the different cell populations. The notable exception was the presence of a 17 kDa band, which was selectively and markedly enhanced in the membranes from cells with enhanced plasminogen binding capacities. The 17 kDa protein was isolated from both cell types, and amino acid sequencing of peptide fragments identified the same protein, histone H2B. Increased expression of histone H2B was observed on stimulated U937 cells and cultured neutrophils by confocal microscopy with an antibody raised to the carboxy-terminal octopeptide sequence of histone H2B. This antibody or its Fab fragments substantially decreased the level of binding of plasminogen to these cultured neutrophils and stimulated U937 cells that exhibited elevated levels of binding but not to nonstimulated cells. Thus, histone H2B represents a regulated plasminogen receptor, which contributes significantly to the plasminogen binding capacity of cells.     

The cellular location and specificity of bacterial cytochrome c peroxidases.

We have found that an anti-CD11c monoclonal antibody (MAb) inhibits the respiratory burst induced in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells as well as in human peripheral blood monocytes and neutrophils upon cell stimulation with concanavalin A. The MAb had no effect, however, when the added stimulus was fMet-Leu-Phe or PMA. Flow cytometry analyses indicated that concanavalin A was able to interact with CD11c. The anti-CD11c MAb inhibited significantly concanavalin A binding to differentiated U937 cells, and concanavalin A blocked binding of anti-CD11c MAb to the cells. Binding of labelled concanavalin A to membrane proteins which were separated by PAGE and transferred to nitrocellulose paper indicated that proteins with apparent molecular masses similar to those of CD11c (150 kDa) and CD18 (95 kDa) molecules were the main concanavalin A-binding proteins in differentiated U937 cells as well as in mature neutrophils. Similar experiments carried out in the presence of the anti-CD11c MAb showed a specific and significant inhibition of concanavalin A binding to the CD11c molecule. These results indicate that concanavalin A binds to the CD11c molecule and this binding is responsible for the concanavalin A-induced respiratory burst in PMA-differentiated U937 cells as well as in human mature monocytes and neutrophils.     

Leukophysin: a 28-kDa granule membrane protein of leukocytes.

A membrane glycoprotein of human platelet dense granules, called granulophysin, with serologic homology to synaptophysin has recently been identified. To determine if this protein was present in granulated leukocytes, we examined several cell types for the presence of the protein by indirect immunofluorescence. Antigranulophysin mAb staining was detected in a granular pattern in the cytoplasm of permeabilized IL-2-stimulated CD3+ peripheral lymphocytes, neutrophils, U937 monocytes, and mast cells. Immunohistochemistry of human lymph nodes showed cytoplasmic staining of macrophages, neutrophils, and some dendritic cells. Induction of granule exocytosis in granulated CD3+ lymphocytes after stimulation with PMA and calcium ionophore A23187 resulted in a redistribution of the reactive epitope from the cytoplasm to the plasma membrane. Subcellular fractions contained two peaks of reactivity; the first peak coincided with N-benzyloxycarbonyl-L-lysine thiobenzyl ester-esterase activity in dense granules whereas the second peak was present in lighter fractions. The affinity purified protein from both peaks was identical in Western blot analysis and had a molecular mass of 28 kDa under reducing conditions. The protein could only be solubilized in detergent suggesting that it was an integral membrane protein. We have named this protein leukophysin to differentiate it from the 40-kDa granulophysin of platelets. Monocytes contained a protein with identical m.w. to leukophysin, whereas a protein of a slightly higher m.w. was detected in neutrophils. We propose that leukophysin is a common granule membrane protein of leukocytes.     

Study of the interaction of the C-reactive protein monomer with the U937 monocyte

C-reactive protein (CRP) has two structurally distinct isoforms, the CRP pentamer and the CRP monomer. A role for the CRP monomer in atherosclerosis is emerging, but the underlying mechanisms are only beginning to be understood. Monocytes are an important contributor to atherosclerosis, and foam cell formation is the hallmark of atherogenesis. However, whether the CRP monomer can directly interact with the monocytes and modulate their responses remains unknown. Furthermore, although FcγRIII (CD16) has been identified as the receptor for the CRP monomer on neutrophils, its role in mediating the CRP monomer’s biological effects in other cell types has been questioned. In this study, we investigated the interaction of the CRP monomer with the monocytes using the U937 monocytic cell line. The CRP monomer specifically binds to U937 cells. This binding is unique in that it is independent of FcγRs and insensitive to protease digestion of the cell surface proteins. Further assays revealed that the CRP monomer directly incorporates into the plasma membrane. Interestingly, the presence of the CRP monomer efficiently retards oxidized low-density lipoprotein-induced foam cell formation of PMA-differentiated U937 macrophages and peripheral blood monocytic cell-derived macrophages. These findings provide additional evidence for the notion that the CRP monomer is an active CRP isoform that plays a role in atherogenesis via the direct modulation of the behavior of the monocytes.     

GMP-140 binding to neutrophils is inhibited by sulfated glycans.

GMP-140 is a 140-kDa granule membrane glycoprotein localized to the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells. Expression of GMP-140 on the activated cell surface has been shown to mediate the adhesion of thrombin-activated platelets to neutrophils and monocytes and the transient adhesion of neutrophils to endothelium. In contrast, fluid-phase GMP-140 strongly inhibits the CD18-dependent adhesion of tumor necrosis factor alpha-activated neutrophils to endothelium suggesting that GMP-140 can also serve an anti-adhesive function. In the present report, it is demonstrated that fluid-phase GMP-140 which exists predominantly as a tetramer binds to a single class of high affinity receptor on neutrophils and HL60 cells. Binding of 125I-labeled GMP-140 to neutrophils and HL60 cells and the rosetting of neutrophils and HL60 cells by thrombin-activated platelets were inhibited by EDTA, excess unlabeled fluid-phase GMP-140, Fab fragments of an affinity-purified rabbit anti-GMP-140 antibody, and by the murine anti-GMP-140 monoclonal antibody, AK 4. Both neutrophil and HL60 GMP-140 binding and platelet rosetting were strongly inhibited by heparin, fucoidin, and dextran sulfate 500,000, were partially inhibited by dextran sulfate 5,000 and lambda- and kappa-carrageenan, but were not inhibited by chondroitins 4- and 6-sulfate. Since this sulfated glycan specificity is identical to that previously reported by us for GMP-140, the present results suggest that the sulfated glycan binding site and the neutrophil receptor binding site on GMP-140 are either identical or proximal.     

Enhancement by IL-1 beta and IFN-gamma of platelet activation: adhesion to leukocytes via GMP-140/PADGEM protein (CD62).

We have examined the effect of inflammatory cytokines on the platelet activation. IL-1 beta and IFN-gamma were found to enhance the adhesion of thrombin-treated platelets to monocytic leukemia cells (U937), when the adhesion was assayed by platelet-mediated cell agglutination. The agglutination was inhibited by a monoclonal anti-GMP140 antibody or EDTA, suggesting that the enhanced platelet adhesion to the leukemic cells was mediated by GMP140. In addition, these cytokines also increased the release of 5-HT from platelets in the presence of a low concentration of thrombin. These data suggest that platelet functions are regulated by the cytokines and that activated platelets participate in inflammatory process.     

Influence of cellular arachidonic acid levels on phospholipid remodeling and CoA-independent transacylase activity in human monocytes and U937 cells

The availability of free arachidonic acid (AA) constitutes a limiting step in the synthesis of biologically active eicosanoids. Free AA levels in cells are regulated by a deacylation/reacylation cycle of membrane phospholipids, the so-called Lands cycle, as well as by further remodeling reactions catalyzed by CoA-independent transacylase. In this work, we have comparatively investigated the process of AA incorporation into and remodeling between the various phospholipid classes of human monocytes and monocyte-like U937 cells. AA incorporation into phospholipids was similar in both cell types, but a marked difference in the rate of remodeling was appreciated. U937 cells remodeled AA at a much faster rate than human monocytes. This difference was found not to be related to the differentiation state of the U937 cells, but rather to the low levels of esterified arachidonate found in U937 cells compared to human monocytes. Incubating the U937 cells in AA-rich media increased the cellular content of this fatty acid and led to a substantial decrease of the rate of phospholipid AA remodeling, which was due to reduced CoA-independent transacylase activity. Collectively, these findings provide the first evidence that cellular AA levels determine the amount of CoA-independent transacylase activity expressed by cells and provide support to the notion that CoA-IT is a major regulator of AA metabolism in human monocytes.     

Preferential binding of platelets to monocytes over neutrophils under flow

This study was undertaken to systematically investigate the binding kinetics of platelet recruitment by monocytes relative to neutrophils in bulk suspensions subjected to shear as well as the molecular requirements of leukocyte-platelet binding. Hydrodynamic shear-induced collisions augment the proportion of monocytes with adherent platelets more drastically than that of neutrophils with bound platelets. These heterotypic interactions are further potentiated by platelet activation with thrombin or to a lesser extent by monocyte stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Monocyte-platelet heteroaggregation increases with increasing shear rate and shear exposure time. Platelet P-selectin binding to monocyte P-selectin-glycoprotein-ligand-1 is solely responsible for maximal platelet adhesion to unstimulated monocytes in shear flow. However, the enhanced platelet binding to fMLP-treated monocytes involves a sequential two-step process, wherein P-selectin-PSGL-1 interactions are stabilized by CD18-integrin involvement. Blocking platelet alpha(IIb)beta(3) or monocyte beta(1)-integrin function had no effect. This study underscores the preferential recruitment of platelets by monocytes relative to neutrophils in shear flow, and demonstrates that the shear environment of the vasculature coupled to the state of cell activation modulates the dynamics and molecular constituents mediating monocyte-platelet adhesion.     

Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion

Mesangial expansion, the principal glomerular lesion in diabetic nephropathy, is preceded by a phenotypic activation and transient proliferation of the glomerular mesangial cells and by a prominent glomerular infiltration of monocytes and macrophages. Because this infiltration seems to play a key role in the subsequent mesangial matrix expansion, we tested the response of cultures of rat mesangial cells (RMCs) for monocyte adhesion in response to hyperglycemia. Increasing the medium glucose concentration from 5.6 mm (normal) to 25.6 mm (hyperglycemic) significantly increased hyaluronan in the cell matrix, with a concurrent 3- to 4-fold increase in adhesion of U937 monocytic leukemic cells to cultures of near confluent RMCs. These responses were attributed directly to the high glucose concentration and not to increased extracellular osmolality. The monocytes primarily bind directly to hyaluronan-based structures in vitro. Abnormal deposits of hyaluronan were found in glomeruli of kidney sections from diabetic rats 1 week after streptozotocin treatment, often with closely associated monocytes/macrophages, suggesting that similar structures are relevant in vivo. The monocyte adhesion response to high glucose concentration required growth stimulation of RMCs by serum and activation of protein kinase C, and was inhibited by prior passage of the RMCs in the presence of heparin. These results suggest that the response may be cell growth state and protein kinase C-dependent. When incubated with the viral mimetic, poly I:C, in the presence of normal glucose, heparin-passaged RMCs still increased cell-associated hyaluronan and exhibited hyaluronan-mediated adhesion of monocytes, indicating that the two stimuli, high glucose and viral mimetic, induce the production of the hyaluronan structures that promote monocyte adhesion by distinctly different intracellular signaling mechanisms.     

Human monocytes and U937 cells bear two distinct Fc receptors for IgG

Several convergent lines of evidence have led us to propose that human monocytes and the related cell line U937 possess a second class of IgG Fc receptor (FcR) in addition to the 72-Kd high affinity FcR previously described. IgG affinity purification from detergent lysates of surface radiolabeled U937 cells has yielded both a 40-Kd IgG-binding membrane protein (p40) and the 72-Kd FcR protein. By the same procedure, only the p40 was isolated from the erythroblast cell line K562 and from the B cell lines, Daudi and Raji. Serologic cross-reactivity between the 40-Kd FcR on U937 and Daudi cells was demonstrated using a goat anti-FcR antiserum. A murine (m) monoclonal antibody, raised against the FcR of K562 cells, precipitated the 40-Kd FcR from lysates of U937 and K562 cells but not from Daudi or Raji cells. This antibody, referred to as anti-p40 (IV.3), selectively inhibited the binding of murine IgG1-coated erythrocytes to U937 cells, whereas monomeric human IgG selectively inhibited binding of human anti-Rh(D)-coated erythrocytes to U937 cells. Both Daudi and U937 cells mediated mIgG1 anti-T3 (Leu-4)-induced stimulation of T lymphocytes. In contrast, mIgG2a anti-T3 (OKT3)-induced stimulation was supported effectively by U937 cells but only modestly by Daudi cells. Intact IgG or Fab fragments of anti-p40 (IV.3) blocked mIgG1 anti-T3 (Leu-4) stimulation but not mIgG2a anti-T3 (OKT3) stimulation of T cells; monomeric human IgG blocked only OKT3-induced stimulation. The simplest interpretation of these results is that human monocytes and U937 cells bear two classes of IgG FcR, one of 72 Kd and the other, as described above, of 40 Kd. We propose that the 72-Kd FcR mediates rosette formation with red cells coated by human anti-Rh IgG as well as T cell stimulation by mIgG2a anti-T3 (OKT3) and that the 40-Kd FcR mediates rosette formation with erythrocytes bearing mIgG1 as well as T cell stimulation by mIgG1 anti-T3 (Leu-4). Furthermore, we suggest that these two FcR are the human homologues of the murine macrophage FcRI (binding mIgG2a) and FcRII (binding mIgG2b/1).     

A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase

The secretion of plasminogen activators has been implicated in the controlled extracellular proteolysis that accompanies cell migration and tissue remodeling. We found that the human plasminogen activator urokinase (Uk) (Mr 55,000 form) binds rapidly, specifically, and with high affinity to fresh human blood monocytes and to cells of the monocyte line U937. Upon binding Mr 55,000 Uk was observed to confer high plasminogen activator activity to the cells. Binding of the enzyme did not require a functional catalytic site (located on the B chain of the protein) but did require the noncatalytic A chain of Mr 55,000 Uk, since Mr 33,000 Uk did not bind. These results demonstrate the presence of a membrane receptor for Uk on monocytes and show a hitherto unknown function for the A chain of Uk: binding of secreted enzyme to its receptor results in Uk acting as a membrane protease. This localizes plasminogen activation near the cell surface, an optimal site to facilitate cell migration.     

Morphine inhibits indolactam V-induced U937 cell adhesion and gelatinase secretion

We demonstrate that indolactam V, a non-phorbol protein kinase C activator, promotes U937 cell attachment to fibronectin, type IV collagen and laminin. In the absence of indolactam V, 2-4% of U937 cells attach to all test substrates, however, in the presence of 100 nM indolactam V, 25, 16 and 11% of U937 cells attach to fibronectin, type IV collagen and laminin, respectively. When added concomitantly, 90 microM H-7, a protein kinase C inhibitor, reduces indolactam V-induced U937 cell adhesion to fibronectin by 91%. Monoclonal antibodies directed against both the beta1 and alpha 5 integrin subunits inhibit indolactam V-induced U937 cell adhesion to fibronectin by 62 and 52%, respectively. Indolactam V also promotes homotypic aggregation in U937 cells, which is blocked with either anti-ICAM or anti-LFA-1 antibodies. In addition, indolactam V promotes U937 cell secretion of a 92 kDa gelatinase as demonstrated by zymography. In the presence of low levels of morphine (10 nM-1.0 microM), the U937 cell attachment to matrix proteins was not significantly affected. However, in the presence of 10 microM morphine, the indolactam V treated cells exhibit a 71-74% reduction in cell adhesion to the matrix proteins. Further, 10 microM morphine also blocks indolactam V-induced homotypic aggregation and gelatinase secretion. The inhibitory effect of morphine on cell-matrix adhesion and gelatinase secretion was not inhibited by the opiate receptor antagonist naloxone (1 microM). While 10 microM naloxone did partially counteract the effect of 10 microM morphine on U937 cell attachment, this effect was likely non-specific since 10 microM naloxone alone increased cell adhesion. Supporting this conclusion, PCR analysis revealed that U937 cells do not express the mu high affinity morphine receptor. Also, indolactam V did not induce mu receptor expression, suggesting that morphine acts on U937 cells in a non-specific fashion.