Minactivin: a human monocyte product which specifically inactivates urokinase-type plasminogen activators

Culture supernatants from monolayers of human peripheral monocytes strongly inhibited colorimetric assays of urokinase in which plasmin was measured by esterolysis. This inhibitory activity of monocyte culture supernatant was enhanced after culture with muramyl dipeptide. Inhibition was specific for plasminogen activators of Mr 52 000 and 36 000, as shown by three methods: (1) inhibition of plasminogen-dependent fibrinolysis; (2) inhibition at the level of plasminogen activation in a colorimetric assay; (3) the irreversible loss of plasminogen-activating activity, as evidenced by electrophoresis, after preincubation with culture media. The factor responsible for this inactivation (which we propose to call minactivin) had an apparent Mr of 66 000 on Sephacryl S300 gel chromatography and interacted with enzyme in a biphasic manner: a rapid partial inhibition (reversible by sodium dodecyl sulphate) was followed by slow inactivation (irreversible by sodium dodecyl sulphate). It is proposed that secretion of minactivin by monocytes may contribute to regulation of extracellular proteolysis at sites of tissue injury.     

Novel properties of human monocyte plasminogen activator

Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing supernatants of human lymphocytes, could be shown to produce and secrete appreciable activities of a 52 000-Mr plasminogen activator. This enzyme was suppressed in control and stimulated cultures by dexamethasone (0.1 microM). Monocyte plasminogen activator could only be assayed under conditions of low ionic strength and had no detectable activity at 0.15 M NaCl. Intracellular enzyme was present as a proenzyme, requiring activation by preincubation with plasminogen containing traces of plasmin, before its activity could be seen on sodium dodecyl sulphate/polyacrylamide gel electrophoresis by a fibrin overlay method. Secreted enzyme was in the active form. Further incubation of lysate or supernatant plasminogen activator with plasminogen did not produce any active enzyme species of Mr 36 000, unlike incubations of urokinase with plasminogen. Moreover, comparisons with other plasminogen activators of Mr 52 000 from transformed cell lines showed that the monocyte activator was unique in its resistance to monocyte minactivin, a specific inactivator of urokinase-type plasminogen activators, and in its sensitivity to human alpha 2-macroglobulin. It was therefore concluded that human monocyte plasminogen activator, although sharing an Mr of 52 000 in common with other such activators, is not identical to the high Mr form of urokinase or the plasminogen activators of transformed cells. On present evidence it is the least likely of these enzymes to be active extracellularly under normal physiological conditions.     

Modulation by interferons of the expression of monocyte complement genes.

Interferons-alpha, -beta and -gamma (IFNs-alpha, -beta and -gamma) stimulated the synthesis of the second complement component (C2), Factor B (B) and C1 inhibitor (C1-inh) by human monocytes in vitro. The degree of increase of the secretion rates of C2, B and C1-inh was dose-dependent and proportional to increases in the abundances of their respective mRNAs. IFN-gamma was the most effective at stimulating monocyte C1-inh synthesis, whereas IFN-alpha and IFN-beta were marginally more effective at stimulating monocyte C2 and B synthesis. Kinetic studies showed that the effect of the IFNs was rapid, with maximum stimulation occurring within 1-2 h for all three proteins. After the removal of IFNs from cultures the C1-inh mRNA abundance remained elevated for over 24 h in IFN-gamma-treated monocytes but returned to control levels within 8 h in IFN-alpha-treated and IFN-beta-treated monocytes. The abundances of C2 mRNA and B mRNA also returned to basal values within 8 h after removal of any of the three cytokines from the cultures. Both IFN-alpha and IFN-beta acted synergistically with IFN-gamma to stimulate synthesis of C1-inh and B. This synergistic effect only occurred when the cytokines were present in the cultures simultaneously. The effects of IFN-gamma plus IFN-alpha or IFN-beta on C2 synthesis appeared to be additive rather than synergistic. IFN-gamma inhibited synthesis of C3 by monocytes, but IFN-alpha and IFN-beta had no effect on the synthesis of this protein. Furthermore, none of the three cytokines had any effect on the expression of actin mRNA in monocytes.     

Down-regulation of surface FcRI and decrease in antibody-dependent cellular cytotoxicity of cultured monocytes. Reversal by monensin or cytochalasin-D

To investigate the mechanisms involved in regulation of antibody-dependent cellular cytotoxicity (ADCC) mediated by human monocytes, 51Cr-labeled sheep red blood cells (RBC) were used as target cells in vitro. Monocytes incubated overnight at 37 degrees C before addition of SRBC and antibody exhibited a significant decrease in ADCC activity compared with freshly isolated cells. This pattern was observed with monocytes from all donors tested, regardless of the media used for culture. Supernatants from monocyte cultures did not inhibit the cytotoxic ability of fresh monocytes and cycloheximide, a protein synthesis inhibitor, could not reverse ADCC suppression in cultured monocytes, indicating that the alteration in ADCC is probably not due to inhibitory molecules secreted or synthesized during incubation. A correlation between the decrease in the number of surface FcRI and loss in ADCC ability of cultured monocytes was found. One mechanism for the reduced FcRI expression of 1-day-old monocytes may be rapid internalization that exceeds the rate of reexpression, because cytochalasin-D or monensin, each of which inhibits receptor internalization, maintained FcR expression as well as ADCC ability of cultured monocytes. These data illustrate mechanisms whereby alteration in the number of receptors may underlie loss of receptor-mediated functions, or be involved in augmentation of their biologic activity. The findings that important monocyte functions change under conditions of storage or culture have relevance to in vitro testing of various immune functions of monocytes performed clinically to monitor or guide therapy.     

Glucocorticosteroid-induced immunoglobulin production requires intimate contact between B cells and monocytes

Glucocorticosteroid (GCS)-induced immunoglobulin (Ig) production in vitro is dependent on the functions of T cells and monocytes. T cells produce a replacing factor (TRF-S) which, with monocytes and a broad spectrum of concentrations (both above and below the physiologic range) of GCS, stimulates B cells to synthesize Ig. TRF-S is produced by T cells in cultures of mononuclear cells in the absence of stimulation over the initial 72 hr in culture. T cells, however, require the presence of monocytes or small quantities of interleukin 1 in order for the synthesis of TRF-S to occur. In addition to their role in stimulating TRF-S production, monocytes are also required in cultures of B cells responding to GCS and the cytokine. These experiments demonstrate that this monocyte function cannot be replaced by IL-1 or crude supernatants of monocyte cultures. Furthermore, exposure of TRF-S containing supernatants to oxidizing conditions does not alter the dependence of the cytokine on monocytes or GCS. Coculture of B cells and monocytes separated by a permeable membrane demonstrated that the influence of monocytes on GCS-induced Ig production is unlikely to be mediated by stable soluble factors. Thus, GCS-induced Ig production requires intimate contact between monocytes and B cells in the form of surface contact or unstable soluble mediators.     

Modulation of phenotypic and functional properties of human peripheral blood monocytes by IL-4

Highly purified peripheral blood monocytes were cultured in the presence of rIL-4. Major changes in the morphology of the monocytes were observed. After day 5 of culturing the cells acquired a macrophage-like appearance, with increased cell size and extensive processes, suggesting that IL-4 may induce monocyte-macrophage differentiation. This notion is supported by the observed increased expression of MHC class II Ag, which is thought to be associated with monocyte differentiation. Exposure of monocytes to IL-4 resulted in a dose-dependent increase of the expression of MHC class II Ag, which became apparent after only 20 h of incubation. Maximal expression was obtained after incubation for 6 days, and persisted throughout the whole culture period. Similarly, IL-4 increased the expression of R for C3bi and p150.95 Ag, two members of the leukocyte function-associated Ag 1 family, whereas the expression of the third member, leukocyte function-associated Ag 1, remained unchanged during culture. Furthermore, it was shown that IL-4 inhibited the secretion of cytostatic and chemotactic compounds. Supernatants of monocytes cultured with IL-4 were, in contrast to control cultures, much less effective in inhibiting the growth of A375 melanoma cells. In addition, these supernatants failed to direct the migration of freshly isolated monocytes in a chemotaxis assay. Further analysis revealed that these supernatants exhibited reduced IL-1 activity, as measured in a mouse thymocyte proliferation assay, which might explain the low cytostatic and chemotactic activity. Taken together these results show that IL-4 modulates monocyte phenotype and function and may induce monocyte-macrophage differentiation in vitro.     

Role of cytokines in the monocyte-mediated augmentation of human natural killer cell activity

Previous work has shown that normal human monocytes can augment natural killer (NK) cell activity both when mixed with enriched null cells in the assay and when precultured with enriched null cells and removed prior to testing. The data presented here show that a 4-hr preculture period is superior to slightly longer periods (10-12 hr) for demonstrating the augmentation. The role of cytokines in the monocyte effect was then investigated using a variety of antibody and recombinant reagents. Both monoclonal and rabbit polyclonal antibodies to IL-1 and IL-2 inhibited the monocyte effect, whereas antibodies against IFN-alpha and IFN-gamma from both sources had no effect. Of these cytokines, only IL-1 could be demonstrated (using a sensitive IL-1-dependent-IL-2 synthesis assay) in the supernatants of 4-hr cultures of monocytes plus null cells or null cells only. The ability to detect IL-1 was specifically inhibited by rabbit antibody to human IL-1 at 1:20 and 1:200 dilutions, but only the greater concentration inhibited the monocyte effect on NK activity. In contrast, the detection of soluble IL-1 was not inhibited by including monoclonal anti-IL-1 (1:20 dilution) in the 4-hr culture, although the same reagent abrogated the monocyte effect under these conditions. Recombinant IL-1 (up to 100 units/ml) did not augment NK activity either when added to the assay or when precultured for 4 hr with enriched null cells, whereas either recombinant IL-2 or monocytes were effective under these conditions. These results provide the first evidence for a cellular, and potentially physiologic, basis for the regulation of NK activity by IL-1 and IL-2, which had been previously known to act at pharmacologic levels in vitro.     

Human monocytes generate basophil histamine-releasing activities

Human peripheral blood monocytes generated activities during 24-h culture that were capable of triggering histamine release from 17 of 18 human basophil donors. Monocytes and their in vitro transformed macrophages continued to elaborate these basophil histamine-releasing activities for at least 3 wk in culture. In the 18 basophil donors tested, maximum histamine release induced by monocyte supernatants was 33.8 +/- 5.9% (mean +/- SEM) of total basophil histamine content; optimum anti-IgE-induced release was 38.8 +/- 6.2%. Basophil histamine release in response to monocyte activities was optimal at 37 degrees C and at calcium concentrations of 2 to 5 mM. Release was greater than 90% complete 1 min after challenge and was inhibited by anti-allergic drugs. The mechanism of release appeared to be independent of IgE binding. Gel filtration of supernatants derived from both day 1 (monocyte stage) and day 14 (macrophage stage) cultures demonstrated activity peaks with approximate m.w. of 12,000 and 30,000. In contrast to the marked responsiveness of basophils, only 2 of 10 human lung mast cell preparations responded; release in those preparations was low: 3% and 13% histamine release, respectively. Thus, monocytes produce potent histamine-releasing activities with differential actions on basophils and mast cells.     

Production of a fibronectin-associated lymphokine by cloned mouse T cells

Azobenzenearsonate-specific cloned mouse T cells able to transfer delayed hypersensitivity reactions in vivo produced macrophage agglutination factor (MaggF) after stimulation with mitogen or antigen in vitro. Mitogen (Con A) elicited MAggF production directly from T cells. Responses to Ag were Ag-specific, required syngeneic accessory cells in addition to T cells, and were independent of T cell fine specificity for azobenzenearsonate. Mouse MAggF shared a number of biochemical and immunochemical properties with the fibronectins (FN): 1) high Mr similar to that of plasma FN; 2) binding to gelatin, heparin, and polyclonal antibodies and mAb specific for cellular and plasma FN; 3) inhibition of activity in solution by monoclonal anti-human FN directed against plasma FN gelatin-binding domain; and 4) action on peritoneal exudate macrophages mediated through a FN-receptor cross reactive with one on human monocytes. MAggF production required active protein synthesis and was associated with significant increases in gelatin-binding immunoreactive FN (Mr 440 kDa on immunoblotting) in culture supernatants and T cell lysates. Metabolically labeled peptides could be precipitated by anti-FN from culture supernatants of activated T cells. Stimulated cultures contained significantly more cells with immunohistologically demonstrable cytoplasmic FN than unstimulated control cultures. We suggest that T cell FN is a distinct species of cellular FN which may play an important role in mediating delayed hypersensitivity inflammatory reactions in vivo.     

Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.     

Stability of DNA/anti-DNA complexes.

Human monocytes in culture release small amounts of prostaglandin E (PGE) into the medium. Addition of Fc fragments of IgG to human monocyte monolayer cultures results in a marked increase in PGE release; Fab fragments, monomeric IgG, and human serum albumin have no effect. An IgG1 myeloma has no effect on PGE levels but addition of the heat aggreagted protein results in a marked increase of PGE secretion. Exposure of the cells to Con A, which binds to a specific monocyte plasma membrane receptor, also results in a large increase in PGE release. The magnitude of the increase in PGE secretion produced by exposure of the monocytes to these ligands greatly exceeds the stimulation observed after the addition of antigen-activated mononuclear cell supernatants, zymosan, Sephadex beads, or endotoxin, to monocyte cultures. Prostaglandin E2 (PGE2) accounts for approximately 70% of the total prostaglandins released by stimulated cells. After addition of Indomethacin to monocyte cultures, the stimulatory effects of the ligands on PGE release are inhibited. Addition of Con A to monocyte cultures results in an increased incorporation of [3H]-arachidonic acid into PGE2. These results suggest that this ligand stimulates synthesis as well as release of this prostaglandin.     

Regulation of nuclear antigen expression on the cell surface of human monocytes.

The expression of cell surface nuclear Ag was studied by examining the binding of anti-histone mAb to viable human peripheral blood cells. Freshly isolated cells showed no binding of these mAb. However, in vitro culture in the presence of LPS induced a dose-dependent expression of cell surface nuclear Ag on monocytes (M3+ cells). The addition of IL-1 beta to cultures also induced expression of cell surface nuclear Ag, whereas IFN-gamma was without effect. Release of nuclear material into the supernatants over time was demonstrated by using a chromatin-specific sandwich ELISA. Analysis of the DNA in the released nuclear material demonstrated banding at multiples of 190 bp, suggesting the release of polynucleosomes. Although LPS was required for cell surface nuclear Ag expression, it did not affect the release of nuclear material into the supernatants. The ability of monocytes to bind exogenous chromatin was studied by adding biotinylated-chromatin to PBL and detection with FITC-avidin. Freshly isolated PBL bound no chromatin, but when PBL were cultured in the presence of LPS, monocytes bound chromatin in a saturable manner. The LPS induction of the capacity to bind exogenous chromatin was blocked by cycloheximide. These data suggest that monocyte activation is associated with the expression of a chromatin (?nucleosome)-binding receptor and that this receptor is capable of binding nuclear material released into the cellular milieu. Monocytes may thus provide an important mechanism for the removal of extracellular nuclear material at sites of cell death and/or inflammation. The binding of nuclear Ag to cell surfaces and potential abnormalities of this binding may play a role in the induction of antinuclear antibodies and/or tissue damage in diseases such as SLE.     

Evaluation of killing, superoxide anion and nitric oxide production by Leishmania infantum-infected dog monocytes

Protozoa of the genus Leishmania infect reticuloendothelial cells of several mammalian species, including dogs, in which they often give rise to a chronic, not self-healing visceral disease. The parasitocidal mechanism of peripheral blood monocytes towards Leishmania in the dog has not been investigated in detail. Consequently, Leishmania infantum-infected monocyte cultures of healthy dogs were evaluated using the following parameters: (1) phagocytosis and killing capacities; (2) oxidative burst, in terms of superoxide anion (O2-) release, and (3) nitric oxide (NO) activity, in terms of nitrite (NO2-) production in the presence or absence of the NO synthase inhibitor NG-monomethyl-L-arginine (NGMMLA). Parallel experiments were performed on monocytes stimulated with supernatants of concanavalin A-activated PBMC and on unstimulated monocytes. The amount of IFN-gamma in PBMC supernatants used for monocyte activation was determined by a biological assay on a canine Madin Darby cell line. Results demonstrated that phagocytosis, killing capacity and O2- production significantly increased in monocytes stimulated with supernatants, in comparison with unstimulated cells. A positive correlation was observed between the killing capacity, the O2- production and the amount of IFN-gamma in PBMC supernatants employed for monocyte activation. No significant differences were observed in NO production between unstimulated and stimulated cultures, or between the same cultures with and without NGMMLA. Finally, the killing percentage was similar in the presence or absence of NGMMLA, suggesting that in this experimental model peripheral blood dog monocytes lack NO-mediated killing.     

Production and characterization of cytostatic protein factors released from human monocytes during exposure to lipopolysaccharide and muramyl dipeptide

The effect of activating human monocytes in vitro with lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on the production of cytostatic protein factor(s) (CF) has been investigated, and an antiserum against CF has been raised and tested. Upon incubation for 7 hr with LPS, in vitro differentiated human monocytes released CF. During LPS exposure, the presence of the protein synthesis inhibitor cycloheximide, at concentrations which reduced the overall protein synthesis by 60 and 80%, reduced the amount of CF released by only 20 and 40%, respectively. This indicates that the released CF was to a large extent already present in the monocytes before exposure to LPS. Compared to LPS, MDP induced only modest CF release. However, when lymphokine-activated monocytes were exposed to MDP, an increased CF release was observed. By immunizing a rabbit with CF purified by ion-exchange chromatography, chromatofocusing, and gel filtration, an antiserum was raised which neutralized the cytostatic activity released from monocytes exposed to LPS or lymphokines/LPS in sequence on the fourth day of culture. The cytostatic activity obtained by incubating freshly isolated monocytes with LPS was inhibited by the antiserum to a lesser extent, indicating the presence of other cytotoxins or cytotoxic cellular products in addition to CF in supernatants from freshly isolated monocytes. Various CF preparations were tested for IL-1 activity; no correlation between IL-1 activity and cytostatic activity was observed. Moreover, upon gel filtration the CF and IL-1 activities could be separated from each other and are consequently associated with different proteins.     

Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

This study examines the role of L-selectin in monocyte adhesion to arterial endothelium, a key pathogenic event of atherosclerosis. Using a nonstatic (rotation) adhesion assay, we observed that monocyte binding to bovine aortic endothelium at 4°C increased four to nine times upon endothelium activation with tumor necrosis factor (TNF)-α. mAb-blocking experiments demonstrated that L-selectin mediates a major part (64 ± 18%) of monocyte attachment. Videomicroscopy experiments performed under flow indicated that monocytes abruptly halted on 8-h TNF-α–activated aortic endothelium, ~80% of monocyte attachment being mediated by L-selectin. Flow cytometric studies with a L-selectin/IgM heavy chain chimeric protein showed calcium-dependent L-selectin binding to cytokine-activated and, unexpectedly, unactivated aortic cells. Soluble L-selectin binding was completely inhibited by anti–L-selectin mAb or by aortic cell exposure to trypsin. Experiments with cycloheximide, chlorate, or neuraminidase showed that protein synthesis and sulfate groups, but not sialic acid residues, were essential for L-selectin counterreceptor function. Moreover, heparin lyases partially inhibited soluble L-selectin binding to cytokine-activated aortic cells, whereas a stronger inhibition was seen with unstimulated endothelial cells, suggesting that cytokine activation could induce the expression of additional ligand(s) for L-selectin, distinct from heparan sulfate proteoglycans. Under flow, endothelial cell treatment with heparinase inhibited by ~80% monocyte attachment to TNF-α–activated aortic endothelium, indicating a major role for heparan sulfate proteoglycans in monocyte–endothelial interactions. Thus, L-selectin mediates monocyte attachment to activated aortic endothelium, and heparan sulfate proteoglycans serve as arterial ligands for monocyte L-selectin.