Identification of leukemia-associated inhibitory activity as acidic isoferritins. A regulatory role for acidic isoferritins in the production of granulocytes and macrophages.

Acidic isoferritins have been identified as leukemia-associated inhibitory activity (LIA), which suppresses colony and cluster formation of colony-forming unit-granulocyte macrophages from normal donors but not from patients with leukemia. LIA was detected in all ferritin preparations tested, including ferritin isolated from normal heart, spleen, liver, and placental tissues, and from the spleens of patients with chronic myelogenous leukemia and Hodgkin's disease. Purified preparations of LIA were composed almost entirely of acidic isoferritins, as determined by immunoassay, radioimmunoassay, and isoelectric focusing. The inhibitory activity in the LIA and ferritin samples was inactivated by a battery of antisera specific for ferritin, including those prepared against acidic isoferritins from normal heart and spleen tissues from patients with Hodgkin's disease, and those previously absorbed with basic isoferritins. Antisera absorbed with acidic isoferritins did not inactivate the inhibitory activity. Separation of LIA and chronic myelogenous leukemia and normal spleen ferritin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing confirmed that the regions of peak inhibitory activity corresponded in each to an apparent molecular weight of approximately 550,000 and to a pI value of 4.7. Similar physicochemical characteristics included inactivation by methods that dissociate ferritin molecules into subunits and by treatment with trypsin, chymotrypsin, pronase, and periodate. The purified preparations were extremely stable to heat treatment. The glycoprotein nature of the inhibitory activity was substantiated because it bound to concanavalin A-Sepharose and was eluted off by alpha-methyl mannose. Inhibitory activity of the activity of the acidic isoferritins was detected at concentrations as low as 10(-17)-10(-19) M and iron saturation did not appear to be necessary for its action. These results implicate acidic isoferritins in the regulation of normal myelopoiesis and suggest a role for them in the progression of leukemia.

Isolation and biochemical characterization of leukemia-associated inhibitory activity that suppresses colony and cluster formation of cells.

Leukemia-associated inhibitory activity suppresses colony and cluster formation in vitro cells derived from granulocyte-macrophage progenitor cells of normal donors. It does not inhibit these same progenitor cells from patients with leukemia and it may contribute to the proliferative advantage leukemia cells appear to possess over normal hematopoietic cells during acute leukemia. The inhibitory activity was isolated by a combination of procedures including: ultracentrifugation, Sephadex G-200, carboxymethylcellulose, SDS-polyacrylamide gel electrophoresis, thin-layer and preparative isoelectric focusing and concanavalin A-Sepharose. Leukemia-associated inhibitory activity was characterized as a glycoprotein. it was inactivated by trypsin, chymotrypsin, pronase and periodate treatment. It bound to and was eluted by alpha-methylmannose from concanavalin A-Sepharose columns and had an apparent Mr range of 450-550 000 and an isoelectric focus value between pH 4.6 and 4.9. Crude leukemia associated inhibitory activity was temperature sensitive but the more purified preparations were heat stable.

The human ENO1 gene product (recombinant human alpha-enolase) displays characteristics required for a plasminogen binding protein.

Plasminogen binds with low affinity in a lysine-dependent manner to many cell types. Previously, a 54 kDa plasminogen receptor found on the surface of U-937 cells was identified as an alpha-enolase-like molecule. The aims of this study were to determine whether recombinant alpha-enolase (r-alpha-enolase), encoded by ENO1, was a plasminogen binding protein and to generate polyclonal antibodies against this antigen. Plasminogen specifically bound r-alpha-enolase with a Kd 1.9 microM and approached saturation at 10 microM. Lysine-dependent plasminogen binding to r-alpha-enolase was demonstrated by a greater than 80% inhibition of binding by the lysine analogues epsilon-amino caproic acid and tranexamic acid, whilst only 14% inhibition occurred with the arginine analogue benzamidine. Removal of the C-terminal lysine residue of r-alpha-enolase with carboxy-peptidase B significantly reduced its plasminogen binding capacity, suggesting that binding required C-terminal lysine residue of r-alpha-enolase. Binding to r-alpha-enolase enhanced the activation rate of plasminogen by urokinase but prevented alpha 2-antiplasmin from binding plasminogen. Taken together, these data suggest that the gene product of human ENO1 encodes an authentic plasminogen binding protein.

Statin drugs and dietary isoprenoids as antithrombotic agents.

Statin drugs and various isoprenoids from plant origins inhibit mevalonic acids, cholesterol, and other isoprenoid products. Among these, reduction of farnesyl and geranylgeranyl prenylated proteins impedes signal transduction at the cellular level. The authors envision that limiting such prenylated proteins downregulates thrombin-stimulated events, including decreasing the expression and availability of protease-activated receptor-1 mitigating thrombin stimulation of cells, tissue factor preventing additional thrombin generation, and plasminogen activator inhibitor-1 allowing thrombosis. Additional processes may enhance nitric oxide production and induce other processes. Downregulation of thrombin-stimulated events should promote hypothrombotic or quiescent conditions that reduce cardiovascular disease, thus contributing to longevity.

Effect of nafenopin (SU-13,437) on liver function: influence on the hepatic transport of organic anions.

Rats treated with hypolipidemic agent, nafenopin (SU-13, 437) exhibit a higher plasma retention and a markedly reduced biliary excretion of organic anions, such as sulfobromophthalein (BSP) and its dibromo analog (DPSP), indocyaninegreen (ICG), succinylsulfathiazole (SST) and polar metabolites of bilirubin and the carcinogens 7, 12-dimethylbenzanthracene (DMBA) and 3,4 benzpyrene (BP), despite an increase in liver mass and a profound choleresis. However, taurocholate is not affected in this manner, which supports the idea of a transport mechanism for taurocholate that differs from that of other organic anions. A pharmacokinetic study was made for DBSP in vivo. After nafenopin treatment, primary hepatic uptake (k12) and transport from liver into bile (k23) are reduced in vivo. Infusion studies indicate that biliary transport maximum (Tm) for DBSP is also decreased although the calculated hepatic storage (S) is only moderately affected. In the isolated perfused liver, hepatic clearance and biliary excretion of BSP are reduced by two-thirds. The time course of anion transport inhibition and the hepato-biliary disposition of 14C-nafenopin suggest a direct effect of the drug. The extra liver mass induced by nafenopin appears to be hypo- or nonfunctional with respect to hepatic transport of organic anions.

Functional and immunologic methods for the measurement of human tissue factor pathway inhibitor.

Recent publications have addressed the topic of an important regulator of the extrinsic pathway, the tissue factor pathway inhibitor (TFPI). Tissue factor-dependent reactions are regulated by TFPI, a member of a superfamily of proteins, homologous to aprotinin (Kunitz). An indirect assay has been developed to quantitate levels of TFPI in human plasma by measuring its ability to inhibit FVIIa-TF activity (Actichrome TFPI). A sandwich ELISA (Imubind Total TFPI), which recognizes all forms of TFPI (e.g. lipid associated, carboxytruncated TFPI, as well as recombinant and native TFPI) was used to compare antigenic and functional TFPI levels. The Actichrome TFPI kit exhibited no detectable response to other clotting factors tested, in their normal and pathologic ranges. Mean circulating levels of TFPI were 55 ng/ml and 61 ng/ml using the activity assay and the ELISA, respectively. The difference might be attributed to inactive forms of TFPI that are detected by the ELISA. Intra-assay and inter-assay variation (% CV) was less than 5% for three samples of known concentration were assayed in replicates of 20. The total TFPI ELISA has a sensitivity of 0.3 ng/ml for serum and plasma samples, assayed at a 20-fold dilution. Specificities of both the capture and detection antibodies were confirmed via Western blot analysis visualizing bands at 34 kDa and 21 kDa, respectively, for full length and truncated TFPI. In conclusion, two assays which quantitate circulating levels of TFPI have been developed and optimized: the Actichrome TFPI Activity Assay and the Imubind Total TFPI ELISA.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of locomotion in lymphocyte migration.

In this study, the role of cell locomotion in lymphocyte traffic was investigated following the in vivo fate of 51Cr labelled lymph node cells pretreated in vitro with substances known to modify cell motility. The results obtained show that lymphocyte migration is an energy dependent process, as it could be impaired by sodium azide. That it requires the integrity of microfilaments, as it was modified by Cytochalasin B; and that it is altered by isoproterenol, theophylline, prostaglandin E1 and dibutyryl cyclic-AMP. The relevance of these findings to the process of lymphocyte recirculation is discussed.

Biliary excretion of 3,4-benzpyrene in nafenopin-treated rats.

When rats treated for 2 days with nafenopin are injected i.v. with 3H-3,4-benzpyrene (BP), blood disappearance rates and liver levels of the carcinogen and the rate of biliary excretion of its metabolites are, in the main, similar to those of nontreated rats. This is in accord with the observation that nafenopin does not inhibit the metabolism of BP, which is the rate-limiting step in its biliary excretion. On the other hand, when 3H-BP metabolites are injected, nafenopin pretreatment slightly retards their rate of plasma disappearance and markedly inhibits their biliary excretion, as it does other organic anions. When rats are pretreated with 3-methylcholanthrene, the rate of metabolism of 3H-BP and consequently the biliary excretion of its metabolites is greatly stimulated. In this instance, metabolism may no longer be rate-limiting in the overall biliary excretion process and inhibition by nafenopin of liver-to-bile transport of metabolites can be observed. Since nafenopin pretreatment stimulates the synthesis of new liver tissue, it is presently a matter of conjecture as to whether or not the newly formed hepatocytes have the capacity to take up and excrete BP and its metabolites or whether nafenopin inhibits transport in all liver tissue.

Transferrin, derived from an OKT8-positive subpopulation of T lymphocytes, suppresses the production of granulocyte-macrophage colony-stimulatory factors from mitogen-activated T lymphocytes.

Purified human transferrin, when saturated with iron or zinc, decreased the production of granulocyte-macrophage colony-stimulating factors (GM-CSF) by human T lymphocytes that had been stimulated by phytohemagglutin or concanavalin-A. The iron-saturated transferrin was more active than the zinc-saturated transferrin. This effect was not seen for copper-saturated transferrin or for apotransferrin, and the inhibitory effect was seen whether production of GM-CSF occurred in the absence or presence of serum. If the lymphocytes were pretreated with monoclonal antibody against transferrin receptors, no suppressive effect with transferrin was seen. Transferrin did not have a direct effect on the granulocyte-macrophage colony or cluster-forming cells (CFU-GM) or on preformed GM-CSF. Transferrin-inhibitory activity was produced and released only from a subpopulation of T lymphocytes that had the OKT8+ antigenic phenotype. Release of this activity from OKT8+ lymphocytes, into culture medium at 37 degrees C, was first detected after 6-17 hr, but the capacity of the GM-CSF-producing lymphocytes to respond to transferrin-inhibitory activity was apparent only within the first 3 hr of placing the lymphocytes at 37 degrees C. These studies demonstrate feedback interactions confined to cells of the T-lymphocyte lineage that may be of relevance to the regulation of myelopoiesis.

Lactoferrin, transferrin and acidic isoferritins: regulatory molecules with potential therapeutic value in leukemia.

In the process of evaluating roles for purified preparations of lactoferrin, transferrin and acidic isoferritins in the regulation of myelopoiesis, it was found that: (1) values reported for lactoferrin in the serum and plasma of normal donors are in most cases an over-estimation, (2) lactoferrin suppresses the production/release of granulocyte-macrophage colony stimulatory factors (GM-CSF) from monocytes in the absence of T-lymphocytes and also suppresses the production/release of acidic isoferritin-inhibitory activity from monocytes, (3) lactoferrin, transferrin and acidic isoferritins act on their specific target cells which express Ia-like antigens, (4) lactoferrin and transferrin act in vivo to suppress rebound myelopoiesis in mice recovering from sublethal dosages of Cytoxan, with preliminary observations suggesting that lactoferrin has a greater apparent effect on the bone marrow and transferrin has a greater apparent effect on the spleen, (5) active lactoferrin derives from Fc receptor positive subpopulations of PMN from patients with CML as well as from normal donors, but the percentage of Fc receptor containing PMN is lower in CML, as is the amount of active lactoferrin found in their PMN, and (6) lactoferrin, transferrin and acidic isoferritins suppress the colony formation of U937 clonogenic cells, with lactoferrin and transferrin decreasing the release of growth factors from U937 cells which are needed to stimulate U937 colony formation, and lactoferrin and acidic isoferritins suppress the colony formation of WEHI-3 cells, with lactoferrin decreasing the release of growth factors from WEHI-3 cells which are needed to stimulate WEHI-3 colony formation. Speculation on the potential usefulness of these iron binding glycoproteins to control of disease progression is given in the discussion.

Effect of nafenopin (SU-13,437) on liver functions. Hepatic uptake and biliary excretion of ouabain in the rat.

Pretreatment of rats for 2 days with the hypolipidemic drug, nafenopin, 0.5 g/kg, results in an increase in liver weight and bile flow. Despite these changes, hepatic transport of ouabain is reduced. This was demonstrated in the isolated perfused liver as well as in vivo. Although net uptake into liver is diminished in treated rats, the initial rapid phase of the plasma disappearance curve is unaffected. This suggests that the primary uptake process is unaffected by nafenopin or is altered in a manner that is not reflected in this phase of plasma disappearance. Alternatively, nafenopin may increase ouabain efflux from liver to plasma. Biliary excretion of ouabain is markedly suppressed after nafenopin pretreatment and higher liver levels of ouabain were encountered after the first 20 min in treated animals. The inference is drawn that liver to bile transport of ouabain is also suppressed by nafenopin pretreatment.

Augmentation of human monocyte chemiluminescence by iron-saturated lactoferrin.

The effect of Fe3+ bound lactoferrin (LF) on chemiluminescence (CL) generation in human monocytes in the presence and absence of opsonized zymosan was examined. Fe3+ bound LF (10(-6) or 10(-7) M) augmented the CL response in the presence but not the absence of zymosan. In contrast, Apo LF, Fe3+ bound transferrin and Fe3+ itself had no significant effect. It appears that the effect of LF on zymosan-induced chemiluminescence is dependent on iron and is receptor specific. The augmentation of CL by LF was inhibited by superoxide dismutase (SOD) and ETYA (fatty acid cyclooxygenase and lipoxygenase inhibitor), but not indomethacin (fatty acid cyclooxygenase inhibitor). Thus the enhancement of CL by LF depends on superoxide anion production and is related to the lipoxygenase pathway. The significance of this observation in terms of microbicidal or tumoricidal mechanisms remains to be determined.

A subpopulation of human polymorphonuclear neutrophils contains an active form of lactoferrin capable of binding to human monocytes and inhibiting production of granulocyte-macrophage colony stimulatory activities.

Subpopulations of human peripheral blood polymorphonuclear neutrophils (PMN) differing in functional capacity were separated by a rosetting procedure by using rabbit IgG antibody-coated sheep erythrocytes (EA). EA+ and EA- populations of PMN both contained lactoferrin (LF) as determined by radioimmunoassay. However, only LF (10(-16) to 10(-6) M) obtained from EA+ (= FC receptor (FcR)+) PMN was able to suppress the production of granulocyte-macrophage colony stimulatory activity derived from human monocytes. LF from EA- PMN was inactive at concentrations as high as 10(-5) M. Extracts of EA- PMN contained proteolytic enzymes, not apparent in FcR+ PMN, that inactivated the inhibitory activity of LF obtained from FcR+ PMN. These results were substantiated by immunofluorescence studies. LF from FcR+ PMN bound to monocytes whereas LF from EA- PMN demonstrated negligible binding to the monocytes and extracts from EA- PMN drastically decreased the capability of LF obtained from FcR+ PMN to bind to monocytes. These studies demonstrate functional heterogeneity of peripheral blood PMN and suggest a role for PMN subpopulations in the regulation of myelopoiesis.

Monocyte-macrophage-derived acidic isoferritins: normal feedback regulators of granulocyte-macrophage progenitor cells in vitro.

The recent identification of a leukemia-associated inhibitory activity (LIA) against granulocyte-macrophage progenitor cells (CFU-GM) as acidic isoferritins has now led to detection of this activity in normal bone marrow and blood cells. Detection of this activity depends on stimulation of CFU-GM by granulocyte-macrophage colony stimulatory factors (GM-CSF), and some conditioned media (CM) sources of GM-CSF (human placental and monocyte, mouse macrophage and WEHI-3) contained low levels of acidic isoferritin that lowered colony formation. Inactivation or removal of this activity increased the stimulatory capacity of the CM. CM depleted of acidic isoferritins or CM originally devoid of this activity (human GCT, 5637, Mo, lymphocytes: mouse L cells or pokeweed-mitogen-stimulated spleen cells) increased the sensitivity of the assay to detect acidic isoferritin inhibitory activity. This activity was selectively contained and released from normal monocytes and macrophages. Restriction of this activity to mononuclear phagocytes was substantiated, as only continuous cell lines of monocytes and macrophages or lines capable of induction to this lineage contained and released acidic isoferritin inhibitory activity. The cells of origin and target cells of action suggest that acidic isoferritin-inhibitory activity can be considered as a negative feedback regulator, at least in vitro.

Ferritin secretion by human mononuclear cells: association with HLA phenotype.

A number of different observations indicate that cells of the immune system can participate in the prevention of potential tissue toxicity from iron accumulation and that, in turn, iron and iron binding proteins have important effects on immune responses. The current studies were undertaken to examine a specific aspect of the interaction of iron with human peripheral blood mononuclear cells. A modified hemolytic plaque-forming assay was used to measure ferritin secretion in vitro by phytohemagglutinin activated or nonactivated mononuclear cells in response to stimulation by ferric citrate. Cells from 55 unrelated healthy subjects collectively representing all well-defined HLA-A, B, C, and DR antigens were studied. There were large reproducible differences in the numbers of plaques formed by different individuals, and there was a statistically significant increase in the frequency of the HLA determinant A3 among the "low" responders. Ferritin secretion measured with an antibody specific for acidic ferritin also showed a distinction between A3 and non-A3 donors. In preliminary cell mixing studies, ferritin secretion by mononuclear cells was shown to require the presence of monocytes and to be influenced by the secretion characteristics of both the monocyte and the T-cell donor. These results may provide a clue to the mechanism of development of idiopathic hemochromatosis which is an HLA-A-linked autosomal recessive disease associated with the specific HLA antigen HLA-A3.

Tissue factor regulates plasminogen binding and activation.

Tissue factor (TF) has been implicated in several important biologic processes, including fibrin formation, atherogenesis, angiogenesis, and tumor cell migration. In that plasminogen activators have been implicated in the same processes, the potential for interactions between TF and the plasminogen activator system was examined. Plasminogen was found to bind directly to the extracellular domain of TF apoprotein (amino acids 1-219) as determined by optical biosensor interaction analysis. A fragment of plasminogen containing kringles 1 through 3 also bound to TF apoprotein, whereas isolated kringle 4 and miniplasminogen did not. Expression of TF on the surface of a stably transfected Chinese hamster ovary (CHO) cell line stimulated plasminogen binding to the cells by 70% more than to control cells. Plasminogen bound to a site on the TF apoprotein that appears to be distinct from the binding site for factors VII and VIIa as judged by a combination of biosensor and cell assays. TF enhanced two-chain urokinase (tcuPA) activation of Glu-plasminogen, but not of miniplasminogen, in a dose-dependent, saturable manner (half maximal stimulation at 59 pmol/L). TF apoprotein induced an effect similar to that of relipidated TF, but a relatively higher concentration of the apoprotein was required (half maximal stimulation at 3.8 nmol/L). The stimulatory effect of TF on plasminogen activation was confirmed when plasmin formation was examined directly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In accord with this, TF inhibited fibrinolysis by approximately 74% at a concentration of 14 nmol/L and almost totally inhibited the binding of equimolar concentrations of plasminogen to human umbilical vein endothelial cells and human trophoblasts. Further, CHO cells expressing TF inhibited uPA-mediated fibrinolysis relative to a wild-type control. TF apoprotein and plasminogen were found to colocalize in atherosclerotic plaque. These data suggest that plasminogen localization and activation may be modulated at extravascular sites through a high-affinity interaction between kringles 1 through 3 of plasminogen and the extracellular domain of TF.

Interaction of single-chain urokinase with its receptor induces the appearance and disappearance of binding epitopes within the resultant complex for other cell surface proteins.

Binding of urokinase-type plasminogen activator (uPA) to its glycosylphosphatidylinositol-anchored receptor (uPAR) initiates signal transduction, adhesion, and migration in certain cell types. To determine whether some of these activities may be mediated by associations between the uPA/uPAR complex and other cell surface proteins, we studied the binding of complexes composed of recombinant, soluble uPA receptor (suPAR) and single chain uPA (scuPA) to a cell line (LM-TK- fibroblasts) that does not express glycosylphosphatidylinositol (GPI)-anchored proteins to eliminate potential competition by endogenous uPA receptors. scuPA induced the binding of suPAR to LM-TK- cells. Binding of labeled suPAR/scuPA was inhibited by unlabeled complex, but not by scuPA or suPAR added separately, indicating cellular binding sites had been formed that are not present in either component. Binding of the complex was inhibited by low molecular weight uPA (LMW-uPA) indicating exposure of an epitope found normally in the isolated B chain of two chain uPA (tcuPA), but hidden in soluble scuPA. Binding of LMW-uPA was independent of its catalytic site and was associated with retention of its enzymatic activity. Additional cell binding epitopes were generated within suPAR itself by the aminoterminal fragment of scuPA, which itself does not bind to LM-TK- cells. When scuPA bound to suPAR, a binding site for alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP) was lost, while binding sites for cell-associated vitronectin and thrombospondin were induced. In accord with this, the internalization and degradation of cell-associated tcuPA and tcuPA-PAI-1 complexes proceeded less efficiently in the presence of suPAR. Further, little degradation of suPAR was detected, suggesting that cell-bound complex dissociated during the initial stages of endocytosis. Thus, the interaction of scuPA with its receptor causes multiple functional changes within the complex including the dis-appearance of an epitope in scuPA involved in its clearance from the cell surface and the generation of novel epitopes that promote its binding to proteins involved in cell adhesion and signal transduction.