Plasminogen activator in early embryogenesis: Enzyme production by trophoblast and parietal endoderm

We have surveyed the early stages in the development and differentiation of cultured mouse embryos for plasminogen activator production. This enzyme is first detectable by the sixth equivalent gestation day. Thereafter, cultured blastocysts produce plasminogen activator with a biphasic time course: in the first phase, enzyme secretion rises to a maximum at about the eighth day and then decreases; a second phase, during which more enzyme accumulates, begins somewhat later and continues to at least the fifteenth day.By fractionating the blastocyst into its constituent cell types, we have identified the trophoblast as the cells responsible for the first phase of enzyme synthesis. The pattern of enzyme production by the trophoblast is closely correlated with the invasive period of these cells in vivo and implies that plasminogen activator is involved in embryo implantation. The second phase of plasminogen activator production is due to parietal endoderm, which initiates enzyme synthesis upon differentiation from the inner cell mass. The properties of the parietal endoderm suggest that plasminogen activator may participate in the migration of these cells and/or in the metabolism of Reichert's membrane which accompanies embryo growth.These results are consistent with the concept, developed from work on other cell types, that plasminogen activator may represent a generalized mechanism for tissue remodeling and cell migration.     

Influence of macrophage products on the release of plasminogen activator, collagenase, beta-glucuronidase and prostaglandin E2 by articular chondrocytes.

We describe the effects of products of mononuclear phagocytes on the secretory activity of chondrocytes. The primary confluent cultures of rabbit articular chondrocytes were exposed to standard medium alone or enriched with conditioned medium obtained from cultures of rabbit peritoneal macrophages, the mouse macrophage cell line P388D1 or human blood mononuclear cells. Four markers of release were assessed, the neutral proteinases plasminogen activator and collagenase, the acid hydrolase beta-glucuronidase and prostaglandin E2, and the kinetics of their changes were monitored. Chondrocytes that were cultured in standard medium secreted large amounts of plasminogen activator, some beta-glucuronidase, but no collagenase, and released only minor amounts of prostaglandin E2. The addition of conditioned medium from rabbit macrophages induced a rapid release of large quantities of prostaglandin E2 and an abundant secretion of collagenase, while abolishing or strongly decreasing plasminogen activator secretion. In addition, beta-glucuronidase secretion was markedly enhanced. The decrease in secretion of plasminogen activator appeared to reflect a diminished production, since no evidence was found for the generation of inhibitors or for an accelerated extracellular breakdown of the enzyme. Conditioned media of the mouse and human mononuclear cells influenced the secretory activities of rabbit articular chondrocytes in a similar way, suggesting that the factor (or factors) acting on chondrocytes is produced by a variety of macrophages, and that its action is not species-restricted. The time course and concentration-dependence of the effects observed indicate that the secretion of plasminogen activator and collagenase are influenced in a strictly reciprocal fashion by the macrophage products. The release of prostaglandin E2 paralleled that of collagenase.     

Plasminogen activator in the rat ovary. Production and gonadotropin regulation of the enzyme in granulosa and thecal cells

The production of plasminogen activator by ovarian granulosa cells has been previously reported to be temporally correlated with ovulation in the rat and to be under hormonal control of gonadotropins. We have examined the type of plasminogen activator produced by granulosa cells and also investigated other ovarian cell types for synthesis of this enzyme. Using antibodies specific for tissue-type or urokinase-type plasminogen activator, we have found that granulosa cells produce exclusively the tissue-type enzyme. However, in cultures of whole follicles isolated from the ovary, there is primarily synthesis of urokinase-type plasminogen activator. Examination of other isolated ovarian cell types has demonstrated that thecal cells secrete the urokinase-type plasminogen activator and that the production of this enzyme is also regulated by gonadotropins and temporally correlated with ovulation. These results suggest that ovulation requires both types of plasminogen activator and that the neighboring granulosa and thecal cells cooperate to ensure rupture of the follicle wall and unimpeded passage of the ovum into the oviduct.     

Acetyl-LDL stimulates macrophage-dependent plasminogen activation and degradation of extracellular matrix

The ability of acetyl-LDL to stimulate macrophage-dependent plasminogen activation and degradation of extracellular matrix was examined. We have found that expression of plasminogen activator activity in response to the scavenger receptor ligand varied among cell populations. Exposure to acetyl-LDL stimulated plasminogen activator expression by cells which constitutively released low levels of activator. These include a virally transformed macrophage-like cell line (RAW246.7), concanavalin A and C. parvum-activated macrophages. The stimulation of plasminogen activator activity was independent of cellular lipid accumulation since nonlipoprotein inhibitors of acetyl-LDL binding to the scavenger receptor stimulated activator expression in great excess to that observed with acetyl-LDL. In contrast, acetyl-LDL was unable to induce soluble plasminogen activator activity in cells which normally do not express it. These include a macrophage-like cell line (J774A.1) and resident peritoneal macrophages. Furthermore, acetyl-LDL was unable to modulate the copious secretion of activator by inflammatory macrophages elicited with thioglycolate. When macrophages were tested for their ability to degrade smooth muscle cell derived matrix, solubilization by resident, elicited, and activated cells was variously increased in the presence of plasminogen. Furthermore, exposure to acetyl-LDL enhanced plasmin-dependent degradation by resident cells and activated cells, whereas matrix degradation by elicited cells was unaffected.     

Stimulation of macrophage plasminogen activator activity by colony-stimulating factors

Colony-stimulating factors (CSFs) stimulate granulocyte-macrophage production from single hemopoietic progenitor cells. Various preparations of purified CSFs of two different subclasses have been shown here to stimulate a plasminogen-dependent fibrinolytic (plasminogen activator) activity from resident and starch-induced mouse peritoneal macrophages. Lymphocyte supernatants also stimulate macrophage plasminogen activator (PA) activitty. Since they contain colony stimulating activity, it is possible that one or more sublcasses of CSF in these supernatants is responsible for this effect. Since both colony-stimulating and macrophage growth activities have been detected at inflammatory sites, these findings could reflect a role for CSF in inflammatory processes.     

Regulation of plasminogen activator secretion in mouse peritoneal macrophages. I. - Role of serum studied by a new spectrophotometric assay for plasminogen activators.

A chromogenic tripeptide - H-D-Val-Leu-Lys-p-nitroanilide-substrate of plasmin, can be used to follow plasminogen activation by an activator such as urokinase or the activator secreted by mouse peritoneal macrophages (thioglycolate-elicited). The acceleration of p-nitroaniline production is proportional to the initial rate of plasmin formation from plasminogen. Thus, at a given plasminogen concentration, this acceleration is proportional to the activator concentration. The acceleration can be evaluated from the spectrophotometer trace recording at 405 nm the appearance of p-nitroaniline, either by means of a computer program or by a plot of delta A405 vs.t2. The sensitivity of this assay allows detection of 0.003 CTA units of urokinase. Thioglycollate-elicited mouse peritoneal macrophages secrete plasminogen activator into the extracellular medium during in vitro cultivation only after a contact with serum.     

Opposite effects of galectin-1 on alternative metabolic pathways of L-arginine in resident,inflammatory, and activated macrophages

Recent evidence has implicated galectins and their carbohydrate ligands as master regulators of the inflammatory response. Galectin-1, a member of this family, has shown specific anti-inflammatory and immunoregulatory effects. To gain insight into the potential mechanisms involved in these effects, we investigated the effects of galectin-1 in L-arginine metabolism of peritoneal rat macrophages. Pretreatment of macrophages with galectin-1 resulted in a dose- and time-dependent inhibition of lipopolysaccharide-induced nitric oxide (NO) production, accompanied by a decrease in inducible nitric oxide synthase (iNOS) expression (the classic pathway of L-arginine). On the other hand, galectin-1 favored the balance toward activation of L-arginase, the alternative metabolic pathway of L-arginine. Inhibition of NO production was not the result of increased macrophage apoptosis because addition of this beta-galactoside-binding protein to macrophages under the same experimental conditions did not affect the apoptotic threshold of these cells. To understand how endogenous galectin-1 is regulated in macrophages under inflammatory stress, we finally explored the ultrastructural distribution, expression, and secretion of galectin-1 in resident, inflammatory, and activated macrophages. This study provides an alternative cellular mechanism based on the modulation of L-arginine metabolism to understand the molecular basis of the anti-inflammatory properties displayed by this carbohydrate-binding protein.     

Importance, localization and functional properties of the cell-associated form of plasminogen activator in mouse peritoneal macrophages

In inflammatory macrophages, plasminogen activator exists in two active forms, a soluble form released into the extracellular medium and a cell-associated form. This communication describes some properties of the cellular form of plasminogen activator, in intact macrophages and in cell lysates. Cellular plasminogen activator is a membrane protein, associated with the outer face of the plasma membrane; in intact macrophages, it participates in the activation of exogenous plasminogen and, thus, has to be considered as an ectoenzyme. A plasminogen activator activity can be detected in cell lysates (macrophage monolayers lysed in 0.1% Triton X-100) only when plasmin production is followed by the use of small synthetic substrates because a soluble inhibitor, released during extraction, blocks plasmin fibrinolytic activity. In these lysates, plasminogen activator molecules exist as high molecular weight unstable complexes exhibiting a high affinity for plasminogen.     

Stimulation of plasminogen activator production by dimethyl sulfoxide in Chinese hamster ovary cells

The production of plasminogen activator activity in an auxotrophic mutant of the Chinese hamster ovary cell line was found be greatly stimulated by low concentrations of dimethyl sulfoxide. The production of both cell-associated and excreted plasminogen activator activities was stimulated maximally by dimethyl sulfoxide at a concentration of 2.5%. The stimulation of plasminogen activator activity production was found to be completely inhibited by actinomycin D and cycloheximide but not by mitomycin C, implying that new protein and RNA syntheses were required for this process. Using specific antibodies against plasminogen activator, the presence of a tissue-type plasminogen activator could only be detected in dimethyl sulfoxide treated cells. The dimethyl sulfoxide induced plasminogen activator production was observed only in a mutant auxotrophic for adenosine, glycine, and thymidine but not in wild-type cells. The ability of dimethyl sulfoxide to induce the synthesis of plasminogen activator was lost when the cells were hybridized with another complementary auxotrophic mutant. This implies that the ability of dimethyl sulfoxide to stimulate the production of plasminogen activator may be related to the auxotrophic mutation in this cell.     

Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells

On the basis of 125I-labeled plasminogen activator binding analysis we have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a Kd of 0.8958 x 10(-9) M [corrected]. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.     

Reciprocal upregulation of urokinase plasminogen activator and its inhibitor, PAI-2, by Borrelia burgdorferi affects bacterial penetration and host-inflammatory response

The mammalian plasminogen activation system (PAS) is a complex system involved in multiple physiological and pathological processes. Borrelia burgdorferi interacts with certain components of the PAS. Here we further investigate this interaction to determine its effect on bacterial dissemination and host cell migration in vitro. We show that stimulation of monocytic cells with B. burgdorferi induces the transient production and secretion of urokinase plasminogen activator (uPA), shortly followed by its physiological inhibitor, plasminogen activator inhibitor-2 (PAI-2). Mono Mac 6 (MM6) cells as well as peripheral blood monocytes enhanced transmigration of B. burgdorferi across a barrier coated with fibronectin mediated by uPA. Moreover, the induction of PAI-2 or the addition of recombinant PAI-2 did not have a significant effect on the uPA-potentiated transmigration of B. burgdorferi. In contrast, the induction of PAI-2 by B. burgdorferi resulted in significantly diminished invasion by monocytic cells across a reconstituted basement membrane (matrigel), which could be partially restored by treatment with purified uPA. These results show that the PAS plays a twofold role in the pathogenesis of B. burgdorferi infection, both by enhancing bacterial dissemination and by diminishing host-cell inflammatory migration.     

Plasminogen activator and MIF-like activities in Kirsten virus transformed mouse NIH culture fluids.

Kirsten virus transformed mouse NIH cells produce both a macrophage migration inhibition activity for guinea pig and mouse peritoneal exudate cells and a plasminogen activator. The migration inhibition factor activity exhibited thermal stability up to 80°C while the plasminogen activator was inactivated after 15 minutes at 70°C. Separation of these activities was achieved by absorption of the migration inhibition activity on agarose-fucosamine or high speed centrifugation.     

CD73 Activity is Dispensable for the Polarization of M2 Macrophages

The ectoenzyme CD73 catalyzes the hydrolysis of AMP, and is one of the most important producers of extracellular adenosine. On regulatory T cells, CD73 is necessary for immunosuppressive functions, and on Th17 cells CD73-generated adenosine exerts anti-inflammatory effects. However, the expression and function of CD73 in pro-inflammatory M1 and in immunosuppressive M2 macrophages is largely unknown. Here we show that CD73 expression and enzyme activity were induced in in vitro polarized pro-inflammatory human M(LPS+TNF) monocytes/macrophages, while CD73 was absent from immunosuppressive M(IL-4+M-CSF)-polarized macrophages. Inhibition of CD73 activity with the inhibitor AMPCP did not affect the polarization of human monocytes. In mice, CD73 was present on resident peritoneal macrophages. In striking contrast, elicited peritoneal macrophages remained CD73 negative regardless of their polarization towards either a pro-inflammatory M(LPS) or anti-inflammatory M(IL-4c) direction. Finally, the ability of peritoneal macrophages to polarize to pro- and anti-inflammatory cells was perfectly normal in CD73-deficient mice in vivo. These data indicate that, in contrast to other major leukocyte subpopulations, CD73 activity on macrophages does not play a major role in their polarization and that in mice host CD73 on any cell type is not required in vivo for peritoneal macrophage polarization towards either a pro- or an anti-inflammatory direction.     

Macrophages in resistance to rickettsial infection: susceptibility to lethal effects of Rickettsia akari infection in mouse strains with defective macrophage function

We have confirmed the requirement of macrophages in the antigen-induced T-lymphocyte proliferative response and in the generation of migration inhibition factor (MIF) by immune lymphocytes. Extending these observations, we have found that autologous and non-syngeneic, oil-induced peritoneal exudate macrophages were equally effective in restoring the proliferative response and MIF production by column-purified lymph node T cells. MIF activity was optimally restored when T cells were reconstituted with 1 to 40% exudate-derived macrophages whereas 10 to 30% macrophages were needed to optimally restore the T-cell proliferative response. Normal resident macrophages from the peritoneal cavity were also capable of restoring T-cell reactivity as were normal or BCG-activated pulmonary alveolar macrophages. It was also found that the addition of as few as 1.0% glycogen-elicited peritoneal exudate cells restored the production of MIF by T cells. Quantitative considerations demonstrated that the responsible cells in these preparations were polymorphonuclear cells rather than macrophages. In contrast, neither MIF production nor the proliferative response by T cells were restored by the addition of red blood cells. In these studies we were able to demonstrate that freeze-thawed macrophages could restore antigen-induced MIF production, but not antigen-induced cellular proliferation. The ability of freeze-thawed macrophages to stimulate T cells to produce MIF was apparently associated with the macrophage membranes and not with a soluble factor in the macrophage extracts. These results demonstrate that multiple sources of phagocytic cells may interact cooperatively with lymphocytes in reactions of cell-mediated immunity. Further, at least in the case of MIF production, this interaction involves a membrane-bound determinant that is effective even in the absence of viable macrophages.     

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.     

Macrophage plasminogen activator: induction by concanavalin A and phorbol myristate acetate

The synthesis and secretion of plasminogen activator by cultured macrophages can be induced and stimulated by concanavalin A and by phorbol myristate acetate, and inhibited by such agents as glucocorticoids, mitotic inhibitors and compounds affecting cAMP metabolism. By the manipulation of stimulatory and inhibitory influences, enzyme production can be modulated continuously over a 200 fold range. In the same way, the proportion of cells that secrete detectable levels of enzyme can be varied from 1–90%. No comparable modulation of lysozyme or acid hydrolase production is observed under the same conditions. These results suggest that the physiological control of macrophage plasminogen activator production is achieved by the interacting effects of mutually antagonistic stimuli; this emphasizes the utility of this enzyme for the study of regulatory phenomena, including those relating to inflammation.     

Glucocorticoid receptors and glucocorticoid-sensitive secretion of neutral proteinases in a macrophage line.

A continuous line of mouse macrophages (P388D1) has been shown to secrete elastase, collagenase, and plasminogen activator at activities comparable to those of macrophages elicited by an inflammatory stimulus in vivo. At physiologic concentrations anti-inflammatory glucocorticoids selectively and reversibly inhibited secretion of the three proteinases but did not inhibit secretion of lysozyme, a constitutive enzyme produced by the P388D1 cells. The secretion of the neutral proteinases was inhibited 50% by 2 to 10 nM dexamethasone. Proliferation of the macrophages was also glucocorticoid sensitive. The P388D1 macrophages contained about 4000 saturable glucocorticoid-binding sites per cell. Concentrations of hormone saturating the high affinity receptor site (for dexamethasone the dissociation constant for steroid-receptor binding, Kd, was 4 nM) correlated well with concentrations inhibiting secretion of the proteinases. Only glucocorticoids and progesterone competed for binding to the specific receptors. Temperature-sensitive translocation of hormone-receptor complexes from "cytoplasm" to nucleus similar to that found with rat thymocytes was demonstrated. Thus, the interaction between glucocorticoids and the P388D1 cell line provides a model for the regulation of macrophage secretion of neutral proteinases under normal and stress conditions.     

Extracellular matrix-induced cyclooxygenase-2 regulates macrophage proteinase expression

Chronic inflammatory diseases are characterized by the persistent presence of macrophages and other mononuclear cells, tissue destruction, cell proliferation, and the deposition of extracellular matrix (ECM). The tissue degradation is mediated, in part, by enhanced proteinase expression by macrophages. It has been demonstrated recently that macrophage proteinase expression can be stimulated or inhibited by purified ECM components. However, in an intact ECM the biologically active domains of matrix components may be masked either by tertiary conformation or by complex association with other matrix molecules. In an effort to determine whether a complex ECM produced by vascular smooth muscle cells (SMC) regulates macrophage degradative phenotype, we prepared insoluble SMC matrices and examined their ability to regulate proteinase expression by RAW264.7 and thioglycollate-elicited peritoneal macrophages. Here we demonstrate that macrophage engagement of SMC-ECM triggers PKC-dependent activation of MAPK(erk1/2) leading to increased expression of cyclooxygenase (COX)-2 and prostaglandin (PG) E(2) synthesis. The addition of PGE(2) to macrophage cultures stimulates their expression of both urokinase-type plasminogen activator and MMP-9, and the selective COX-2 inhibitor NS-398 blocks ECM-induced proteinase expression. Moreover, ECM-induced PGE(2) and MMP-9 expression by elicited COX-2(-/-) macrophages is markedly reduced when compared with the response of either COX-2(+/-) or COX-2(+/+) macrophages. These data clearly demonstrate that SMC-ECM exerts a regulatory role on the degradative phenotype of macrophages via enhanced urokinase-type plasminogen activator and MMP-9 expression, and identify COX-2 as a targetable component of the signaling pathway leading to increased proteinase expression.     

Plasminogen activator production by parietal endoderm cells: Stimulation by a protein from pituitary

It has been shown previously that dibutyryl cyclic AMP increases the production of plasminogen activator in mouse parietal endoderm cells. This fact suggested that the production of plasminogen activator by parietal endoderm cells may be under the control of a hormone acting via adenylate cyclase. We have cultured rat parietal endoderm cells in the absence of serum and show that they respond to dibutyryl cyclic AMP with an increase in plasminogen activator production and a change in morphology. We describe the existence of a compound from pituitary which is capable of stimulating plasminogen activator secretion in these cells. Relatively impure preparations of ovine and bovine TSH contain significant amounts of activity, whereas more highly purified preparations of TSH, and all other pituitary hormones tested, are inactive, indicating that the factor is not a known pituitary hormone. The active compound was characterized using ovine and bovine TSH as a source, and it is macromolecular and proteinaceous, and depends on protein synthesis for its effect. The stimulation is enhanced by methylisobutylxanthine, a phosphodiesterase inhibitor, suggesting that the event is mediated by cyclic AMP. This observation leads to the prediction that the coaddition of dibutyryl cAMP and the active compound at nonsaturating concentrations should be additive. Instead, the stimulation is synergistic, and depends on the addition of dibutyryl cyclic AMP first when the compounds are added sequentially. Finally, we show that mouse teratocarcinoma cells chemically induced to differentiate to a cell type indistinguishable from parietal endoderm respond to a source of the compound by increasing plasminogen activator production.     

Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA–/–) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1) developed a novel methodology to detect pericellular proteolytic function, (2) demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3) demonstrated its usefulness in real-time studies of cell migration, and (4) showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis.