The distribution of procoagulant and plasminogen activator activities among density fractions of normal rabbit alveolar macrophages

Rabbit alveolar macrophages can directly stimulate either coagulation or fibrinolysis by producing tissue thromboplastin and plasminogen activator activities, respectively. However, it is not known whether these 2 opposed physiologic activities are expressed by the same or different cells within a population of alveolar macrophages. This study was undertaken to determine the distribution of procoagulant and plasminogen activator activities among density-defined populations of rabbit alveolar macrophages. Normal rabbit alveolar macrophages were separated into 4 density fractions on continuous gradients of Percoll, and the distribution of procoagulant and plasminogen activator activities among these fractions was determined. The procoagulant activity of the least dense cells (Fraction 1) was as much as 6 times greater than the activity displayed by denser cells (Fractions 2 to 4). By contrast, both cell-associated and secreted plasminogen activator activities were equally and uniformly distributed among all density fractions. These distributions of activities among density fractions persisted after the cells were incubated in culture medium for 24 h. After culture in vitro with lymphokine, procoagulant activity increased in the denser cells so that the activity became equal among all density fractions. We conclude that procoagulant activity distributes differently from plasminogen activator activity among fractions of normal rabbit alveolar macrophages separated according to cell density. By using density gradient fractionation, alveolar macrophages with predominantly procoagulant or plasminogen activator activities can be enriched from the lavage cell population; the dissimilar distribution of these 2 activities within the alveolar macrophage cell population does not reflect the presence of cells with fixed differences in their functional capacities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Generation of macrophage migration inhibitory activity by plasminogen activators.

Medium from cultures of simian virus 40-transformed mouse 3T3 cells (SV3T3) inhibits the migration in vitro of peritoneal exudate cells (macrophages) from guinea pigs while medium from untransformed 3T3 cultures does not [Hammond, M. E., Robbin, R. D., Dvorak, A. M., Selvaggio, S. S., Black, P. H. & Dvorak, H. F. (1974) Science 185, 955-957]. The present paper describes the generation of migration inhibitory factor (MIF)-like activity for peritoneal exudate cells from guinea pigs after incubation of a serum-free harvest fluid from SV3T3 cells with guinea pig serum. Inhibited macrophages lose a densely staining material from the cell surface coat compared with uninhibited guinea pig peritoneal exudate cells. The factor in SV3T3 harvest fluids which generates the migration inhibitory activity appears to be plasminogen activator, i.e., a serine protease, because (i) plasminogen activator activity and the factor which generates MIF-like activity copurify, and co-chromatograph on Sephadex G-200 columns, and (ii) plasminogen activator activity and capacity to generate MIF-like activity are simultaneously lost upon treatment with [3H]diisopropylfluorophosphate. In addition, a purified preparation of a known plasminogen activator, human urokinase, can also generate MIF-like activity upon reaction with guinea pig serum. Because transformation of 3T3 cells by SV40 increases their plasminogen activator secretion, enhanced secretion of plasminogen activator by SV3T3 cells may explain why formation of MIF-like activity is observed in SV3T3 but not 3T3 cultures. These results reveal a biochemical pathway whereby a product secreted by virus-transformed cells affects one function of a cell central to the host's immunological defense system.     

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.     

Modulation of plasminogen activator secretion by activated macrophages: influence of serum factors and correlation with tumoricidal potential.

A high molecular weight fraction of human serum (Fr-1) was found to both inhibit macrophage tumoricidal activity and enhance plasminogen activator activity in supernates over activated macrophages in vitro. Conversely, a 40- to 90-kilodalton serine esterase (Fr-3) also found in normal human serum and endotoxin enhanced tumoricidal potential and suppressed the supernatant plasminogen activator activity. Inactivation of either Fr-1 or Fr-3 by 2-mercaptoethanol or diisopropyl fluorophosphate, respectively, abolished both biologic effects. Examination of cell-associated and culture medium plasminogen activator activity before and after acidification to inactivate proteinase inhibitors indicated that suppression of plasminogen activator activity by Fr-3 or endotoxin most likely represents modulation of macrophage plasminogen activator secretion. The findings demonstrate that activated macrophages are capable of highly coordinated biologic responses to alterations in their microenvironment and suggest that it is in fact the high potential for such responsiveness that reliably characterizes the activated macrophage. The results also suggest that an endogenous regulatory system dependent on the interaction of serine esterases may operate to regulate the functional capabilities of activated macrophages.     

Cellular influx and activation increase macrophage cytotoxicity and interleukin-1 elaboration during pulmonary inflammation in rats

This study was undertaken to investigate the effects of pulmonary inflammation induced by bacillus Calmette-Guérin (BCG) on the density distribution of lavaged alveolar macrophages. We sought to determine macrophage cytotoxicity and interleukin-1 elaboration in density-defined subpopulations of macrophages during tissue inflammation. At all time points after intravenously administered BCG, lavaged alveolar macrophages contained increased percentages of higher density cells. Alveolar macrophage cytotoxicity against the rat sarcoma cell line XC increased maximally 2 to 6 days after intravenous administration of BCG before declining on Day 13. Macrophage interleukin-1 elaboration increased maximally 14 days after administration of BCG before declining on Day 23. Additionally, macrophage cytotoxicity and interleukin-1 elaboration were increased above normal in cells from each of five density fractions. We conclude that a subpopulation of higher density macrophages, probably recently derived from blood monocytes, accumulates in inflammatory sites. Cellular activation increases the cytotoxicity and interleukin-1 elaboration by macrophages in all density-defined subpopulations and obscures the relationship between cellular density and function that is present in normal animals.     

Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen.

Apolipoprotein(a) [apo(a)] is a glycoprotein with Mr approximately equal to 280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated domains called kringles and a trypsin-like protease domain. The amino-terminal sequence obtained for apo(a) is homologous to the beginning of kringle 4 but not the amino terminus of plasminogen. Apo(a) was subjected to limited proteolysis by trypsin or V8 protease, and fragments generated were isolated and sequenced. Sequences obtained from several of these fragments are highly (77-100%) homologous to plasminogen residues 391-421, which reside within kringle 4. Analysis of these internal apo(a) sequences revealed that apo(a) may contain at least two kringle 4-like domains. A sequence obtained from another tryptic fragment also shows homology to the end of kringle 4 and the beginning of kringle 5. Sequence data obtained from two tryptic fragments show homology with the protease domain of plasminogen. One of these sequences is homologous to the sequences surrounding the activation site of plasminogen. Plasminogen is activated by the cleavage of a specific arginine residue by urokinase and tissue plasminogen activator; however, the corresponding site in apo(a) is a serine that would not be cleaved by tissue plasminogen activator or urokinase. Using a plasmin-specific assay, no proteolytic activity could be demonstrated for lipoprotein(a) particles. These results suggest that apo(a) contains kringle-like domains and an inactive protease domain.     

Natural and induced antisera specific for pulmonary macrophages

Pulmonary macrophages of mice and hamsters but not rats have antigens on their surfaces which were absent from resident peritoneal macrophages, BCG stimulated pertioneal macrophages, and macrophages in other tissues. Antibodies to these antigens were found in the normal sera of rabbits from a variety of sources, and higher titers were induced by inoculation of rabbits with pulmonary macrophages. After absorption with peripheral blood cells and peritoneal macrophages, a highly specific anti-pulmonary macrophage serum was produced. Although usually absent in normal guinea pig sera, this antibody was induced in guinea pigs. To identify a specific antigen in mice, cell surface proteins of pulmonary and peritoneal macrophages were labelled with¹²⁵I, disassociated by the detergent NP-40, and reacted with either specific guinea pig anti-mouse pulmonary macrophage serum or normal guinea pig serum. The precipitated surface proteins were identified by SDS-polyacrylamide gel electrophoresis. Specific pulmonary macrophage antigen had a molecular weight of 110,000. It was identified with specific antibody (but not with normal guinea pig serum) only on pulmonary macrophages. It was always absent from peritoneal macrophages. Proteins which precipitated in a non-specific fashion and were common to both pulmonary and peritoneal macrophages had molecular weights of 150,000–160,000, 70,000, and 32,000–40,0000. These proteins were detected with both the specific antiserum and normal guinea pig serum. Supported in part by NIH grants HL 18339, HL 27244 and ES 0002     

Plasminogen activation by blood monocytes and alveolar macrophages in primary pulmonary hypertension.

The pathophysiology of primary pulmonary hypertension (PPH) remains poorly understood. Vascular wall remodeling and endothelial dysfunction reflected by modifications in plasma fibrinolytic proteins and von Willebrand factor have been well documented in PPH. We hypothesize that endothelial mediators, produced in excess in PPH patients, may stimulate migrating mononuclear cells and thereby modulate alveolar macrophage function; in particular, the plasminogen activation system. Components of the fibrinolytic system were therefore studied in plasma, blood monocytes and alveolar macrophages obtained from bronchoalveolar lavage in 10 patients with PPH and in four controls. Compared with controls, PPH patients had elevated plasma levels of tissue-type plasminogen activator (15.6 +/- 9.9 versus 5.5 +/- 3 ng/ml) and plasminogen activator inhibitor-1 (27.8 +/- 23 versus 16.4 +/- 12 ng/ml). In contrast, binding and activation of plasminogen by single-chain urokinase-type plasminogen activator (scu-PA) at the surface of blood monocytes and alveolar macrophages were not different from those of control values. Dissociation constants (K(d)) for binding of scu-PA and plasminogen to alveolar macrophages were similar in both PPH (4.7 +/- 1.5 and 0.88 +/- 0.3 micromol/l, respectively) and control (6.7 +/- 0.1 and 1.02 +/- 0.12 micromol/l, respectively) groups. These results indicate that in PPH patients the fibrinolytic activity of alveolar macrophages is normal, whereas endothelial fibrinolytic proteins are abnormally elevated in plasma.     

Dipyridamole stimulates urokinase production and suppresses procoagulant activity of rabbit alveolar macrophages: a possible mechanism of antithrombotic action

Dipyridamole, an inhibitor of platelet aggregation, has been shown to have beneficial effects in disorders characterized by extravascular fibrin deposition. Mononuclear phagocytes are present in extravascular sites and are capable of expressing both plasminogen activator and procoagulant activities, which suggests these cells play a central role in extravascular fibrin turnover. We therefore sought to determine whether dipyridamole affects the expression of plasminogen activator and procoagulant activities by rabbit alveolar macrophages cultured in vitro. We found that dipyridamole (10 to 100 mumol/L) caused increases in both cell-associated and released plasminogen activator activity, which reached levels of 240% (P less than .05) and 543% (P less than .01) of controls, respectively. In contrast, dipyridamole decreased the cell-associated procoagulant activity of alveolar macrophages to as little as 21.3% of controls (P less than .01). Similar effects were seen in cells cotreated with lymphokines. The procoagulant activity expressed by these cells functioned as a tissue thromboplastin. The plasminogen activator of control and treated cells was a urokinase as determined by molecular weight characteristics (50 kilodaltons) and by antibody neutralization profiles using polyclonal antibodies against human urokinase and tissue plasminogen activator. These effects of dipyridamole could not be duplicated by structurally dissimilar agents sharing some of the pharmacological actions of dipyridamole; however, two pyrimidopyrimidine compounds structurally similar to dipyridamole effectively mimicked the effects on both procoagulant and plasminogen activator activities. We conclude that dipyridamole may have antithrombotic effects by directly modulating the role of mononuclear phagocytes in fibrin turnover. Thus, dipyridamole may be useful in situations where extravascular fibrin deposition is important to the pathogenesis of tissue injury and repair.     

Plasminogen activators and cancer

Plasminogen activator (PA) is a specific serine protease which catalyses conversion of the inactive plasminogen into the broad-spectrum protease, plasmin. PA exists in two structurally related forms known as tissue-type PA (tPA) and urokinase-type PA (uPA). Conversion of normal cells into a malignant state frequently leads to increased production of uPA. There is increasing evidence that uPA is directly involved in the process of metastasis. High levels of uPA in human breast cancers is a marker for poor prognosis. Finally, uPA may be a target for anti-metastatic agents, either by inhibition of its synthesis, inhibition of its activity or its binding to receptor.     

A fibrinolytic inhibitor of human alveolar macrophages. Induction with endotoxin

Alveolar macrophages are intimately involved with fibrin during the course of acute and chronic inflammatory processes in the lung. In this study, the capability of macrophages to impede fibrinolysis was investigated. Human alveolar macrophages obtained by lavage from normal volunteers released a fibrinolytic inhibitor during the first 24 h of in vitro culture but only inconsistently and in some cases (7 of 15) not at all. Lysates of freshly lavaged cells had no activity. Endotoxin, 5 to 100 ng/ml, consistently induced intracellular accumulation and extracellular release of a fibrinolytic inhibitor by cultured macrophages. Induction required protein synthesis. The intracellular and secreted forms of the inhibitor were true plasminogen activator (PA) inhibitors, as judged by their ability to block urokinase-mediated conversion of 125I-plasminogen. On average, 10(7) endotoxin-stimulated macrophages secreted sufficient PA inhibitor during a 24-h culture in vitro to neutralize 2 picomoles urokinase (16 international units). Analysis of the interaction of the human macrophage PA inhibitor with 125I-urokinase (apparent size, 33 kilodaltons) by SDS-gel electrophoresis showed that the enzyme and inhibitor mostly dissociated in SDS, but a stable complex occurred at 60 to 65 kilodaltons and a broad band of enzyme or enzyme-inhibitor complexes between 33 and 40 kilodaltons. Either heat treatment of the inhibitor or active site inhibition of urokinase with p-nitrophenylguanidinobenzoate blocked both types of interaction. The pattern of interaction was virtually indistinguishable from that of a partially purified human placental urokinase inhibitor but different from that of serum protease inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages: A possible mechanism of inflammatory demyelination

In inflammatory demyelinating diseases such as multiple sclerosis and experimental allergic encephalomyelitis, myelin destruction occurs in the vicinity of infiltrating mononuclear cells. The observations that myelin can be altered prior to phagocytosis and in areas not contiguous with inflammatory cells suggests a common mechanism for the initial stages of demyelination. Because stimulated macrophages secrete several neutral proteases, including plasminogen activator, we have investigated the possibility that myelinolysis could be mediated directly or indirectly by these enzymes. Isolated myelin was incubated with conditioned media from cultures of thioglycollate-stimulated mouse peritoneal macrophages in the presence and absence of plasminogen. Myelin appeared to be vulnerable to attack by at least two proteolytic activities secreted by the macrophages, a plasminogen-dependent and a plasminogen-independent activity; of the major proteins in myelin, the basic protein was most susceptible. The direct myelinolytic activity of macrophage-conditioned media was abolished by EDTA, and the plasminogen-dependent hydrolysis was abolished by p-nitrophenylguanidinobenzoate, an inhibitor of plasminogen activator and plasmin. These results suggest that the plasminogen activator released by the stimulated macrophages generated plasmin which hydrolyzed basic protein in intact myelin. This interpretation was confirmed by the observation that urokinase, a plasminogen activator, in the presence of plasminogen brought about marked degradation of basic protein in myelin. We propose that the release of neutral proteases by stimulated macrophages involved in cell-mediated reactions, and its amplification by the plasminogen-plasmin system, may play a significant role in the demyelination observed in several inflammatory demyelinating diseases.     

Targeting of tissue plasminogen activator to the regulated pathway of secretion

Tissue plasminogen activator is a serine protease that plays the dominant role in removal of fibrin from the vascular tree by activating plasminogen to the primary fibrinolytic enzyme, plasmin. Tissue plasminogen activator has a widespread neuroendocrine distribution in addition to its expression by endothelial cells. Within neuroendocrine cells, secretory proteins are sorted into one of two pathways: regulated or constitutive. Proteins entering the regulated pathway are concentrated and stored in vesicles, and subsequently released upon stimulation by a secretagogue. In contrast, in the constitutive pathway, newly synthesized protein is not stored but is transported directly to the cell surface and secreted even in the absence of an extracellular signal. The focus of this article is to review recent studies demonstrating that tissue plasminogen activator is targeted to the regulated secretory pathway in neuroendocrine cells and to discuss the physiological implications of the trafficking of tissue plasminogen activator to regulated secretory vesicles.     

Minactivin expression in human monocyte and macrophage populations

Adherent monolayer cultures of human blood monocytes, peritoneal macrophages, bone marrow macrophages, and colonic mucosa macrophages were examined for their ability to produce and secrete minactivin, a specific inactivator of urokinase-type plasminogen activator. All except colonic mucosa macrophages produced and secreted appreciable amounts of minactivin, but only blood monocytes were stimulated by muramyl dipeptide (adjuvant peptide) to increase production. The minactivin from each of these populations could be shown to preferentially inhibit urokinase-type plasminogen activator and not trypsin, plasmin, or "tissue"-type plasminogen activator (HPA66). A plasminogen-activating enzyme present in monocyte cultures appeared unaffected by the presence of minactivin and could be shown to be regulated independently by dexamethasone.     

Plasminogen: a structural review.

Plasminogen is the zymogen form of plasmin, a broad specificity serine protease whose activity contributes to a variety of normal and pathological conditions, including intravascular thrombolysis and extracellular proteolysis. Plasminogen contains seven structural units or 'domains', each of which confer specific properties on the molecule. The kringle domains possess fibrin-binding functions and, together with the N-terminal peptide, regulate the ability of plasminogen to adopt at least three dissimilar conformations. These conformational forms influence the rate of formation, following activation by plasminogen activators, of the plasmin active site within its C-terminal serine protease domain. Structural and functional analogies are postulated between these plasminogen structures and the conformations of other proteins related by sequence homology.     

Mechanism of dexamethasone inhibition of plasminogen activator in rat hepatoma cells.

Glucocorticoids rapidly and completely inhibit intracellular plasminogen activator activity in rat hepatoma cells, as assayed by the solubilization of 125I-labeled fibrin. Experiments in which extracts of dexamethasone-treated cells are mixed with extracts of control cells demonstrate an inhibitor of protease activity. Plasminogen activator is found primarily in the particulate fraction of cell lysates, whereas the inhibitor is localized in the soluble fraction. Variant cells have been isolated previously that are fully resistant to the dexamethoasone inhibition of plasminogen activator activity. These variants have no demonstrable inhibitor activity, whereas plasminogen activator in these cells is fully sensitive to inhibitor from wild-type cells. Thus, the basis for hormone resistance appears to be the failure of dexamethasone to induce an inhibitor.     

Characteristics of alveolar macrophages in an animal model of resolving pulmonary inflammation

Little is known about the functions of alveolar macrophages during the later resolving phases of pulmonary inflammation. We have used an animal model of resolving pulmonary inflammation to obtain inflammatory macrophages (IMs) and have compared several IM functions with those of resident macrophages (RMs). IMs were frequently peroxidase positive and contained large amounts of myeloperoxidase activity. IMs also contained significant amounts of a serine protease type of elastase. The procoagulant activity of IMs was less than that of RMs, and IMs exhibited increased plasminogen activator activity when incubated on fibrin matrices. IMs also degraded fibrin directly, without plasminogen, and this activity was due to two different enzymes of molecular weights 39 and 63 kD that were present in IM granules and plasma membranes. These results suggest that, in vivo, IMs take up PMN enzymes and alter their procoagulant and fibrinolytic activity to maximize fibrin removal. These IM functions may be important for successful resolution of inflammatory injury.     

卡介苗诱导小鼠产生保护性免疫的机制研究

目的　为了探索结核感染保护性免疫的产生机制及主要抗原成分。方法 我们调查了小鼠PEC细胞分别经活卡介苗和热杀死卡介苗刺激后,所产生的IL-12p40,IL-18和γ-干扰素的水平。调查卡介苗菌体裂解液和培养过滤液诱导细胞因子能力。结果 活的卡介苗可以激活巨噬细胞,诱导出IL-12p40和IL-18,而死的卡介苗却不能。活的和死的卡介苗菌体裂解物都可以诱导细胞产生IL-12p40。活的卡介苗的培养过滤液可以诱导出IL-12p40。而热杀死卡介苗过滤液却不能产生IL-12p40。结论 活的卡介苗分泌到培养上清液中的热稳定物质可以诱导细胞因子的大量产生。     

Synthesis of alpha 1-protease inhibitor by resident and activated mouse alveolar macrophages

Alpha 1-protease inhibitor (alpha 1Pi), an acute-phase reactant, is the major inhibitor of neutral proteases causing lung tissue injury, such as elastase. While examining the acute-phase reaction to the nematode parasite Nippostrongylus brasiliensis, we noticed that the alveolar macrophage was closely associated with alpha 1Pi when the larvae were present in the lung. Histologic examination revealed marked edema and hemorrhage with numerous alveolar macrophages that stain intensely for intracellular alpha 1Pi. Isolation of these cells by bronchoalveolar lavage showed the macrophage to be activated. Cultured alveolar macrophages from normal and infected animals synthesized and secreted alpha 1Pi, as revealed by [35S]-methionine incorporation, but the amounts were insignificant compared with that synthesized by hepatocytes. There was, however, no apparent difference in alpha 1Pi synthetic activity between normal and activated macrophages. The presence of demonstrable intracellular alpha 1Pi in the parasite-activated alveolar macrophage likely represents endocytosis as host protease- and/or parasite protease-antiprotease complexes. Although alpha 1Pi is synthesized primarily by hepatocytes, synthesis by alveolar macrophages may provide immediate local protection in the microenvironment of the lung during an acute inflammatory response.     

Protease content and fibrinolytic activity of human leukocytes

The fibrinolytic activity of human leukocytes was studied by the fibrin platemethod and by the histochemical fibrin slide method, using plasminogen-richand plasminogen-free fibrin substrates. Lysis is caused by a protease. Plasminogen activator is absent. The slide method showed the effect of leukocytes tobe weak in comparison to that produced by the plasminogen activator incapillary endothelial cells invading fibrin deposits in the body.

Submitted on April 26, 1966 Accepted on June 26, 1966