Oxidative inactivation of myeloperoxidase released from human neutrophils.

Within 1 min of stimulation of human neutrophils by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B, myeloperoxidase (together with other granule enzymes) was secreted and detected extracellularly. In contrast with the other granule constituents assayed (vitamin B12-binding protein and beta-glucuronidase), the activity of released myeloperoxidase rapidly decreased, so that, by 10 min after stimulation, only about 5% of the total cellular activity was detected. This inactivation was shown to be dependent on oxidant generation during the respiratory burst, since inactivation was not observed (a) after stimulation of anaerobic suspensions or (b) after release from neutrophils from a patient with chronic granulomatous disease; purified myeloperoxidase was rapidly inactivated after incubation with H2O2, presumably owing to the formation of an inactive enzyme-H2O2 complex. These results show that experiments designed to assess the role of myeloperoxidase in neutrophil functions which utilize assays based on peroxidase activity will grossly underestimate this enzyme if oxidant generation during the respiratory burst has also been activated.     

Noncytolytic extraction of soluble antigens from leukemic blast cells (L2C-EN)

Summary Lithium 3,5 diiodosalicylate (LIS), a chemical utilized for the noncytolytic extraction of cell surface antigens, was used in this study to extract glycoproteins from the cell membranes of L₂C-EN leukemic blast cells. The crude soluble antigen (LIS-L₂C) preparation was found to confer immunoprotection in syngeneic guinea pigs against a lethal challenge of L₂C-EN. Titration of the crude LIS-L₂C soluble antigen extract revealed that 1 mg antigen gave 100% protection against a 2×10⁵ viable tumor cell challenge 2 weeks after immunization and that immunizing doses of 0.1 mg, 0.25 mg, and 0.5 mg soluble antigen afforded 17%, 66%, and 83% protection, respectively. The specificity of this immune response was demonstrated by the failure of guinea pigs immunized with 1 mg LIS extract prepared from another guinea pig tumor (line 10 hepatoma) to be refractory to a similar L₂C tumor cell challenge. A cell-mediated immune response to the LIS-L₂C soluble antigen was observed in animals, based on a positive delayed hypersensitivity response to the soluble antigen 5 weeks after immunization. Similarly, in vitro testing revealed a specific blastogenic recognition of the soluble antigen by immune leukocytes.     

Characterization and partial purification of the sodium-potassium-ATPase inhibitor released from cultured rat hypothalamic cells

An inhibitor of sodium-potassium-ATPase has been partially purified from the culture medium obtained from hypothalamic cells maintained in a capillary membrane perfusion system, and some of the properties of this inhibitory factor have been investigated. Gel filtration (Sephadex G-25 Superfine) of heat-treated medium (80 degrees C for 10 min) resulted in elution of inhibitory activity in the post-salt fraction. These fractions inhibited active (i.e. sodium-potassium-ATPase-mediated) sodium transport in intact human erythrocytes, displaced [3H]ouabain from its binding site, and directly inhibited canine kidney sodium-potassium-ATPase as measured by NADH oxidation. High-performance liquid chromatography (on Hypersil ODS) of these fractions after desalting yielded one region which showed inhibitory activity on all three assays. Inhibition of sodium-potassium-ATPase was dose-related and filtered through an Amicon UM10 membrane. Incubation of this material with dispase, carboxypeptidase A, chymotrypsin, and prolidase destroyed inhibitory activity, whereas trypsin and leucine aminopeptidase were ineffective. These studies show that hypothalamic neurones release a low molecular weight heat-stable peptide which inhibits active sodium transport, ouabain binding, and sodium-potassium-ATPase.     

Effect of neuraminidase treatment on serum reactivity to autologous leukemic blast cells

Summary The sera of 35 patients with acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL) were tested for reactivity against cell surface antigens of autologous leukemic blast cells by protein A assay (PA), immune adherence assay (IA), and anti-C3 mixed hemadsorption assay (C3-MHA). Autologous serum reactivity was detectable by PA in four cases and by LA and C3-MHA in about half the patients. Autologous serum reactivity occurred more often in ALL than in ANLL. Absorption studies revealed that in one patient only the autologous reactivity was directed against a restricted antigen, which could be detected only on the individual T-ALL blast cells. All other autologous antibodies detected unspecific antigens. Neuraminidase treatment had two effects: first, it increased antibody attachment to antigens which are also present on untreated cells; secondly, after neuraminidase treatment an antigen was detectable on the cell surface which could also be demonstrated on neuraminidase-treated non-leukemic cells (e.g., erythrocytes). Neither of these two effects of neuraminidase treatment seems to be tumor-specific. Possible therapeutic effects of neuraminidase are probably caused by unspecific adjuvant effects of the enzyme.     

Introduction of new genetic material into human myeloid leukemic blast stem cells by retroviral infection.

An amphotropic retroviral vector containing the bacterial neomycin phosphotransferase gene (neo) was used to infect blast cells from patients with acute myeloblastic leukemia. The infected cells acquired a G418-resistant phenotype that was stable as measured in a clonogenic assay and in long-term suspension culture. Thus, gene transfer into stem cells was accomplished by this procedure. This approach for manipulating gene expression in blast stem cells provides a means to assess the roles of a variety of genes in self-renewal, differentiation, and leukemogenesis.     

Primary structure of a proteinase inhibitor released from goat serum inter-alpha-trypsin inhibitor

An acid-resistant trypsin inhibitor was released from goat serum inter-alpha-trypsin inhibitor and isolated by affinity chromatography. The primary structure of the inhibitor was established and the inhibitory properties were estimated. The inhibitor designed gIK-14 was characterized as a serine proteinase inhibitor from the family of the double-headed Kunitz-type inhibitors as suggested.     

Ectosomes released by human neutrophils are specialized functional units.

Here we show that human polymorphonuclear leukocytes (PMN) release ectosomes independently of complement attack during their activation both in vitro and at the site of inflammation in vivo. Patterns of biotinylated proteins on the surface of PMN and on PMN-derived ectosomes indicated a specific sorting of cell surface proteins into and out of ectosomes. Ectosomes expressed clusters of complement receptor 1 (CR1), which allowed them to bind efficiently to opsonized bacteria. Myeloperoxidase and human leukocyte elastase, both stored within the azurophilic granules of PMN, were found to colocalize on ectosomes with CR1. Furthermore, myeloperoxidase colocalized with human leukocyte elastase. In contrast, not present on CR1-expressing ectosomes were CD63, a selective marker for the azurophilic granules, and CD14, which is located within the same granules and the secretory vesicles as CR1. Of the other complement regulatory proteins expressed by PMN, only CD59 colocalized with CR1, while CD55 and CD46 were almost absent. Ectosomes released by activated PMN at the site of inflammation may function as a well organized element (ecto-organelle), designed to focus antimicrobial activity onto opsonized surfaces.     

Quantitation of the ras gene product in leukemic blast cells using enzymatic staining

Few studies have focused on the significance of ras protein levels in human malignancy, in part because of the inherent difficulty in quantitation of the ras gene product. We have developed a method for the enzymatic determination of the ras gene product and have used this method for the quantitation of ras gene product levels in 19 patients with acute leukemia. This technique provides a practical means to assess p21 expression in leukemic cells ex vivo while avoiding the use of radioactive reagents. In addition, the mobility of the ras species of interest is determined. This assay should be easily modified for the use of other antibodies such as those reported to be specific for various ras species (i.e., H-, K- and N-ras), for specific ras mutations or for other nonras proteins. Because of the use of electrophoresis prior to quantitation of protein, the antibody used does not need to possess high specificity for the protein of interest.     

Pholasin chemiluminescence detects mostly superoxide anion released from activated human neutrophils

The bioluminescent oxygen metabolite indicator protein pholasin was characterized with respect to the type and location of reactive oxygen metabolites detected in suspensions of stimulated human neutrophils. Whereas pholasin detected reactive oxygen metabolites from neutrophil suspensions stimulated with soluble agents, particulate stimulants were apparently not effective triggering agents for pholasin-dependent neutrophil chemiluminescence. Neutrophils stimulated with fMet-Leu-Phe (1 to 100 nmol/l) showed maximum pholasin-dependent chemiluminescence 45 to 60s after stimulation. The time of maximum chemiluminescence was virtually independent of fMet-Leu-Phe concentration. In contrast, the time to reach maximum light emission increased from 60s with 100 nmol/l phorbol ester to 295s with 1 nmol/l phorbol ester. Significant inhibition of stimulated chemiluminescence was caused by both superoxide dismutase (20 μg/ml, 80% inhibition) and reduction of the oxygen concentration in the incubation medium to less than 0.5 μmol/l (95% inhibition). In contrast, the myeloperoxidase inhibitor sodium azide (0.1 nmol/l) afforded only 50% inhibition of the pholasin-dependent neutrophil chemiluminescence. Our results show that pholasin detects superoxide radicals released from cells stimulated by soluble stimulants but not intracellular oxidative activity elicited by particulate stimulants.     

Cathepsin G is a strong platelet agonist released by neutrophils.

The present studies were undertaken to characterize a serine protease released by N-formyl-L-Met-L-Leu-L-Phe (fMet-Leu-Phe)-stimulated neutrophils that rapidly induces platelet calcium mobilization, secretion and aggregation. The biological activity associated with this protease was unaffected by leupeptin, was only weakly diminished by N-p-tosyl-L-Lys-chloromethane, but was strongly inhibited by alpha 1-antitrypsin, soyabean trypsin inhibitor, N-tosyl-L-Phe-chloromethane and benzoyloxycarbonyl-Gly-Leu-Phe-chloromethane (Z-Gly-Leu-PheCH2Cl). These observations indicated that the biological activity of neutrophil supernatants could be attributed to a chymotrypsin-like enzyme such as cathepsin G. Furthermore, platelet aggregation and 5-hydroxytryptamine release induced by cell-free supernatants from fMet-Leu-Phe-stimulated neutrophils were found to be blocked by antiserum to cathepsin G in a concentration-dependent manner but were unaffected by antiserum to elastase. The biological activity present in neutrophil supernatants co-purified with enzymic activity for cathepsin G during sequential Aprotinin-Sepharose affinity chromatography and carboxymethyl-Sephadex chromatography. SDS/polyacrylamide-gel electrophoresis of the reduced, purified protein, demonstrated three polypeptides with apparent Mr values of 31,500, 29,000 and 28,000 and four polypeptides were resolved on acid-gel electrophoresis. Purified cathepsin G from neutrophils cross-reacted with anti-(cathepsin G) serum in a double immunodiffusion assay and elicited platelet calcium mobilization, 5-hydroxytryptamine secretion and aggregation. Calcium mobilization and secretion induced by low concentrations of cathepsin G were partially dependent on arachidonic acid metabolites and ADP, while stimulation by higher enzyme concentrations was independent of amplification pathways, indicating that cathepsin G is a strong platelet agonist. These results suggest that pathological processes which stimulate neutrophils and release cathepsin G can in turn result in the recruitment and activation of platelets.     

Tumor necrosis factor-alpha enhances platelet activation via cathepsin G released from neutrophils

In this paper we show that TNF-alpha enhances platelet activation. Experiments were performed on a human polymorphonuclear neutrophil (PMN)-platelet cooperation system in which PMN, stimulated by FMLP, release cathepsin G (Cat.G), a serine proteinase responsible for the activation of nearby platelets. Pretreatment of the mixed cell suspension with 5 ng/ml TNF-alpha resulted in a strong platelet activation (37.7 +/- 3.2% aggregation; 46.0 +/- 14.4% serotonin release) in response to a weak concentration of FMLP (1.25 x 10(-8) M) inducing by itself only 7.7 +/- 4.0% of aggregation and 3.8 +/- 4.1% of serotonin release (mean +/- SD; n = 10). This effect was concentration dependent (maximum between 5 and 10 ng/ml) and was optimal for a brief preincubation time (5 min). Under these experimental conditions the target of TNF-alpha was PMN, as shown by beta-glucuronidase release. The observed potentiation was modified neither by 0.1 mM acetyl salicylic acid (a cyclo-oxygenase inhibitor) nor by 0.1 mM BN 52021 (a platelet-activating factor antagonist), while such a phenomenon was fully inhibited by 20 micrograms/ml eglin C, a strong and specific inhibitor of the human granulocytic proteinases, elastase and Cat.G. In fact, full inhibition was also observed with 300 nM alpha-1-antichymotrypsin, a specific inhibitor of Cat.G. This clear-cut evidence of Cat.G involvement was substantiated by the enhancement of Cat.G release from FMLP-activated PMN primed with TNF-alpha. These results demonstrate that the priming of PMN by TNF-alpha may modulate the activation of other inflammatory cells, particularly of platelets. It is hypothesized that this phenomenon could contribute to pulmonary pathologies, and more specifically to the adult respiratory distress syndrome, a disease for which PMN, platelet and TNF-alpha involvement has been proposed.     

A dialyzable protein synthesis inhibitor released by mammalian cells in vitro

Macrophages, lymphocytes, and HeLa cells when incubated $\text{in vitro}$ at 37° in Krebs-Ringer phosphate buffer release a dialyzable, heat-stable, ninhydrin-reacting factor which inhibits protein synthesis by intact cells and isolated ribosomes. Release of the inhibitor appears to be dependent on metabolism. Partial purification of the inhibitor by Sephadex G-10 chromatography suggests it has a molecular weight of 400–600.     

The high molecular weight proteins released from cultured cells.

Studies have been performed with the serum-free culture medium taken from several fibroblast monolayer culture lines. A high molecular weight protein fraction was separated from the concentrated medium by sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis was used to assess the degree of purification obtained. In the electron microscope the negatively stained high molecular weight proteins were found to closely resemble the alpha2-macroglobulins. The suggestion that these proteins from cultured cells resemble the cylindrical protein complex isolated from mammalian erythrocyte ghosts is not supported by this study. The results are discussed in the light of the extensive literature now available on the electron microscopy of high molecular weight proteins.     

Plasminogen activator inhibitor 2 (PAI-2) is not inactivated by exposure to oxidants which can be released from activated neutrophils

Purified recombinant human monocyte plasminogen activator inhibitor 2 (PAI-2) retained inhibitory activity after exposure to a number of oxidants, including hypochlorite anion (OCl-), chloramine-T (CT) and hydrogen peroxide (H2O2). Analysis of PAI-2 exposed to oxidants by gel filtration chromatography and SDS-PAGE indicated that although the protein could no longer be detected by silver staining, this was not due to fragmentation of the PAI-2 molecule. The sensitivity of a number of serine protease inhibitors (serpins), (eg. alpha 1 proteinase inhibitor (alpha 1PI) and plasminogen activator inhibitor 1 (PAI-1] to oxidative inactivation has been attributed to oxidation of reactive site methionine residues and/or tertiary structural modifications. The relevance of these phenomena and the potential for PAI-2 to be used as a therapeutic inhibitor of urokinase (uPA)-dependent proteolysis during inflammation and tumour metastasis is discussed.     

Modulation of macrophage phenotype by soluble product(s) released from neutrophils

The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.     

Complement inhibitor(s) released from leukocytes. II. Evidence that lymphocytes release and produce an inhibitor of C2 activation.

We have previously reported that mouse spleen and thymus cells in short term culture release an hitherto undescribed complement inhibitor that prevents the activation of C2. Here it is shown that the inhibitor is released by B lymphocytes and may be produced by lymphocytes. Release of the inhibitor takes place at 37 °C and in the presence of metabolic inhibitors, but not at 4 °C. Thus the release of the inhibitor could be related to the shedding of structures located on the lymphocyte membrane.     

Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils

Human neutrophils release vesicles when activated in vitro and in vivo, in local and systemic inflammation. We have suggested that the presence of these vesicles is due to ectocytosis, defined as the release of rightside-out oriented vesicles expressing a select set of membrane proteins. Herein we have characterised the vesicles released by neutrophils to be ectosomes with specific properties. They contained cytosolic F-actin indicating their outside-out orientation. They bound Annexin V, suggesting that they expose phosphatidylserine, similarly to platelet microparticles. They expressed a subset of cell surface proteins (selectins and integrins, complement regulators, HLA-1, FcgammaRIII, and CD66b, but not CD14, FcgammaRII, and CD87). There was no specificity for transmembrane or glycosyl-phosphatidylinositol-linked proteins and, unexpectedly, L-selectin, known to be cleaved from the surface of activated neutrophils, was present. Ectosomes exposed active enzymes released by neutrophils upon degranulation (matrix metalloproteinase-9, myeloperoxidase, proteinase 3, and elastase). In particular, released myeloperoxidase was able to bind back to ectosomes. The purified complement protein C1q and C1q from serum bound to ectosomes as well. Another aspect of ectosomes was that they became specifically adherent to monocytic and endothelial cells. These observations suggest that neutrophil-derived ectosomes have unique characteristics that make them candidates for playing roles in inflammation and cell signaling.