Characterization of lithium-induced enzyme release from human polymorphonuclear leukocytes

Lysozyme release from purified human polymorphonuclear leukocytes was found to be uniquely enhanced by 2.5-20 mM LiCl. This effect was dose dependent and was not detected when the media was supplemented with NaCl, KCl, MgCl2, or CaCl2. The purified isotopes of Li+, 6Li, and 7Li were equally effective in enhancing lysozyme release from the cells at 10 and 20 mM, but 6Li was more effective than 7Li at 5 mM. The enhancement of enzyme release in the presence of Li+ was comparable to the enhancement observed in the presence of N-formylmethionylleucylphenylalanine (fMLP). Addition of LiCl plus fMLP did not result in lysozyme release in excess of each stimulant alone, except when the cells were incubated with 20 mM 6Li + 10(-5) M fMLP. In addition, enzyme release induced by these two agents could be further enhanced to the same degree by addition of cytochalasin D to the incubation mixtures. While similarities between enzyme release induced by LiCl and fMLP were detected, optimal stimulation of enzyme release by Li+ was much more sensitive to inhibition by pertussis toxin than was maximal fMLP stimulation. Therefore, the intracellular events altered by Li+ and the peptide may share some metabolic steps, but they differ in their sensitivity to alterations in cAMP metabolism.     

Vitamin K1 reduction in human liver. Location of the coumarin-drug-insensitive enzyme.

The protein kinase C inhibitor staurosporine influenced in different ways the functions of human neutrophils. Staurosporine prevented the enhanced protein phosphorylation in phorbol ester- and N-formylmethyionyl-leucylphenylalanine (fMLP)-stimulated cells, and was a powerful inhibitor of the respiratory burst induced by phorbol myristate acetate [IC50 (concentration causing 50% inhibition) 17 nM] and the chemotactic peptides fMLP and C5a (IC50 24 nM). It did not alter, however, the superoxide production by cell-free preparations of NADPH oxidase. Staurosporine had no effect on agonist-dependent changes in cytosolic free Ca2+ and exocytosis of specific and azurophil granules, and showed only a slight inhibition of the release of vitamin B12-binding protein induced by phorbol myristate acetate (decreased by 40% at 200 nM). On the other hand, staurosporine also exhibited neutrophil-activating properties: it induced the release of gelatinase (from secretory vesicles) and vitamin-B12-binding protein (from specific granules). These effects were protracted, concentration-dependent, insensitive to Ca2+ depletion, and strongly enhanced by cytochalasin B. Staurosporine, however, did not induce the release of beta-glucuronidase or elastase (from azurophil granules). Except for the sensitivity to cytochalasin B, these properties suggest a similarity between the exocytosis-inducing actions of staurosporine and PMA. The results obtained with staurosporine provide further evidence that different signal-transduction processes are involved in neutrophil activation, and suggest that protein phosphorylation is required for the induction of the respiratory burst, but not for exocytosis.     

Release of proteinases from stimulated polymorphonuclear leukocytes. Evidence for subclasses of the main granule types and their association with cytoskeletal components

The secretion of matrix-degrading proteinases and protein components involved in the production of cytotoxic metabolites is an important step in the sequence of defense reactions executed by polymorphonuclear leukocytes (PMNL) in response to stimulation. In the present report, we have analyzed degranulation of PMNL stimulated either with soluble synthetic peptides fLeu-Phe (fMet, formylmethionyl), or fAhx-Leu-Phe-Ahx-Tyr-Phe (Ahx, aminohexyl) which trigger chemotaxis and degranulation, or with opsonized zymosan which induces phagocytosis. The release of elastase, myeloperoxidase and lactoferrin-containing granules was not at all or only slightly (less than 6%) induced either by fAhx-Leu-Phe-Ahx-Tyr-Leu or by zymosan particles. In contrast, type-I collagenase and gelatinase were secreted in significant amounts after treatment with these agents. The disruption of microfilaments by cytochalasin B and subsequent stimulation of PMNL with the formyl-peptide led to the secretion of elastase, myeloperoxidase and lactoferrin, and enhanced the release of gelatinase. Disruption of microtubules by incubation with colcemid resulted in inhibition of fAhx-Leu-Phe-Ahx-Thyr-Leu and fAhx-Leu-Phe-Ahx-Tyr-Leu/cytochalasin-B-induced granule release. Furthermore, we found different patterns of enzyme distribution after fractionation by centrifugation: most (greater than 90%) type-I collagenase and gelatinase was measured in the supernatant whereas 60-90% of elastase, myeloperoxidase and lactoferrin had partitioned into the cytoskeleton-containing pellet. Our results suggest that the two main types of secretory vesicles identified in PMNL (specific and azurophilic granules) consist of subpopulations. The differential association of the various types of granules with cytoskeletal elements may serve to control their sequential discharge.     

Release of azurophilic granule contents in fMLP-stimulated neutrophils requires two activation signals, one of which is a rise in cytosolic free Ca

We have used a continuous spectrofluorimetric method to analyse the role of cytosolic free Ca²⁺ ([Ca²⁺]_i) in the lysosomal enzyme release from the azurophilic granules in human neutrophils stimulated with f-Met-Leu-Phe (fMLP) in the presence of cytochalasin B. Measurements were performed with the β-glucuronidase substrate 4-methylumbelliferyl-β- -glucuronide. We found that the transient rise in [Ca²⁺]_i induced by fMLP is a necessary signal to obtain to obtain maximal degranulation. When this Ca²⁺ transient is prevented by the Ca²⁺ chelator BAPTA, degranulation can still be induced by a stimulated Ca²⁺ influx, albeit to a lower extent. We also studied the degranulation process in the neutrophils of a patient with a generalized chemotactic defect. Release of β-glucuronidase from the patient's neutrophils could not be induced despite the occurrence of a normal Ca²⁺ response and normal degranulation of specific granules. We conclude that, besides an increase in [Ca²⁺]_i], an additional signal is required for the fusion of azurophilic granules with the plasma membrane in human neutrophils.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Synthetic leukotriene B4 is a potent chemotaxin but a weak secretagogue for human PMN

We have examined the effects of very pure (greater than 99.8%) chemically synthesized leukotriene B4 of verified structure on the chemotactic and secretory behavior of human polymorphonuclear leukocytes (PMN). The synthetic material is highly chemotactic and shows the same concentration dependence of this activity as does natural LTB4. Synthetic LTB4 is also a weak degranulating agent in cytochalasin B treated PMN. Maximally it released 11%, 17% and 26% as much N-acetyl-beta-D-glucosaminidase, myeloperoxidase and lysozyme as did N-formyl-methionine-leucine-phenylalanine (fMLP). Thus LTB4 differs significantly from other chemotaxins, such as C5a and fMLP, in that it is a poor secretagogue for enzymes of the specific and azurophilic granules of human PMN.     

Adhesomes: specific granules containing receptors for laminin, C3bi/fibrinogen, fibronectin, and vitronectin in human polymorphonuclear leukocytes and monocytes

We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co- localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.     

Synthetic leukotriene B4 is a potent chemotaxin but a weak secretagogue from human PMN

We have examined the effects of very pure (> 99.8%) chemically synthesized leukotriene B₄ of verifeid structuer on the chemotactc and secretry behavior of human polymphonuclear leukocytes (PMN). The synthetic material is highly chemotactic and shows the same concentration dependence of this activity as does natural LTB₄. Synthetic LTB₄ is also a weak degranulating agnet in cytochalasin B treated PMN. Maximally it released 11%, 17% and 26% as much N-acetyl-β-D-dlucosaminidise, myeloperoxidase and lysozyme as did N-formyl-methionine-leucine-phneylalanine (fMLP). Thus LTB₄ differs significantly from other chemotaxisn, as such as C5a and fMLP, in that it is a poor secretagogue for enzymes of the specific adn azurophilic granules of human PMN.     

Myeloperoxidase Exocytosis from Activated Neutrophils in the Presence of Heparin

Exocytosis of myeloperoxidase (MPO) from activated neutrophils has been investigated in the presence of the anionic polysaccharide heparin. The optimal concentration of heparin (0.1 U/mL), which did not cause additional activation of cells (lack of augmentation of lysozyme exocytosis from specific and azurophilic granules), was determined. After preincubation of cells with heparin (0.1 U/mL) MPO exocytosis from neutrophils was stimulated by various activators (fMLP, PMA, plant lectins CABA and PHA-L) and was higher as compared to the effects of the activators alone. Experiments performed using MPO isolated from leukocytes have shown that heparin in the range of concentrations 0.1–50 U/mL had no effect on MPO peroxidase activity. Thus, the use of heparin at a concentration of 0.1 U/mL avoids the artifact caused by the “loss” of MPO due to its binding to neutrophils and increases the accuracy of the method of registration of degranulation of neutrophil azurophilic granules based on determination of the MPO concentration or its peroxidase activity in cell supernatants.     

Effect of botulinum D toxin on human neutrophilic leukocytes and localization of its substrates

Botulinum D toxin has been shown to ADP-ribosylate 22-kD proteins in neutrophilic leukocytes, but the function of these GTP-binding proteins remains unknown. In analogy to small GTP-binding proteins like SEC4 to YPT1, it has been suggested that botulinum D toxin substrates might be involved in secretory process of myeloid cells. Three main findings lead to the opposite conclusion. First of all, in human neutrophils, botulinum D toxin does not modify the release of azurophilic and specific granules induced by a chemoattractant (a formylpeptide) or a phorbol ester. Second, botulinum D toxin ADP-ribosylates 24 to 26-kD proteins that are only present in plasma membranes of human neutrophils. The membrane location of these substrates differs largely from that of the GTP-binding proteins involved in exocytosis and located in granules. Finally, since the same quantity of the toxin substrates is present in neutrophils as in their precursors, HL60 cells (which are devoid of specific granules and characterized by immature azurophilic granules and NADPH oxidase), it is unlikely that endogenous botulinum D toxin substrates are directly involved in the secretory responses of neutrophils.     

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.     

Activation of superoxide formation and lysozyme release in human neutrophils by the synthetic lipopeptide Pam3Cys-Ser-(Lys)4. Involvement of guanine-nucleotide-binding proteins and synergism with chemotactic peptides.

Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate O2.- formation, leading to enhanced bactericidal activity.     

Influence of divalent cations upon complement-mediated enzyme release from human polymorphonuclear leukocytes.

The complement component, C5a provokes the selective release of granule-associated enzymes from the intact, viable cytochalasin B-treated human polymorphonuclear leukocytes (PMN) in the absence of phagocytosis or cellular adherence to surfaces. Consquently, in this experimental system the influence of divalent cations on these two processes can be disregarded and their effects on enzymes secretion can be studied directly. Cytochalasin B-treated PMN exposed to C5a in calcium and magnesium-free media consistently secreted significant amounts of the granule-associated enzymes, beta-glucuronidase and lysozyme. The basal secretory response was not diminished if cells were preincubated with 5.0 mM EDTA, nor was it influenced if 1.0 mm or 2.0 mM EDTA were present in the reaction mixtures. The addition of calcium (up to 1.5 to 2.0 mM) produced a concentration-dependent enhancement of beta-glucuronidase release, whereas increasing amounts of calcium (above 2.0 mM) inhibited secretion of this enzyme. Lysozyme release was similarly enhanced by the addition of calcium, but inhibition with high concentrations was not observed. Calcium per se, in the absence of C5a, provoked only the release of lysozyme from these cells. The effects of calcium upon enzyme release were not associated with alterations in the state of assembly of cytoplasmic microtubules. These findings provide another example of the role of calcium in "stimulus-secretion coupling" and provide evidence that exocytosis of various granules in human PMN is regulated by independent mechanisms involving calcium.     

Human immunoglobulin G potentiates superoxide production induced by chemotactic peptides and causes degranulation in isolated human neutrophils

Neutrophils are major cellular mediators of host defense and inflammation. They can be activated to produce superoxide and to release the contents of their granules to the extracellular space. We observed that monomeric human immunoglobulin G (IgG) sensitizes these cells to the chemotactic peptide N-formylmethionylleucylphenylalanine (fMLP). In cells submaximally stimulated by fMLP this enhancement was especially prominent. With saturating fMLP concentrations, the rate of O2- production was still about twice that in the control. No synergy with other activators (phorbol myristate acetate, concanavalin A) was observed. Binding of fMLP to the cells was decreased by IgG, resembling the effect of cytochalasin B. IgG did not induce O2- production on its own, but it stimulated degranulation of the neutrophils.     

PGBX, a prostagandin derivative, mimics the action of the calcium ionophore A23187 on human neutrophils

The interactions have been studied of a water-soluble, polymeric derivative of prostaglandin B1, PGBX, with human polymorphonuclear leukocytes (PMN). PGBX, which is a potent ionophore of divalent cations, provoked superoxide anion (O2.-) generation and lysosomal enzyme release in cytochalasin B-treated PMN in the presence of extracellular divalent cations (Ca2+, Sr2+, Mg2+, Mn2+, Ba2+). Kinetic and dose-response studies showed that PGBX mimicked te action of ionophore A23187 in PMN. Both ionophores induced superoxide generation and release of enzymes from specific and azurophil granules (lysozyme > beta-glucuronidase) without provoking release of the cytoplasmic marker enzyme lactic dehydrogenase. In contrast, the precursor of PGBX, prostaglandin B1 (PGB1), and arachidonate did not mimic ionophore-induced stimulation of PMN. PGBX induced enzyme release both in the presence of extracellular Ca2+ and Ba2+ (both of which it translocates in model liposomes), whereas A23187 showed specificity for Ca2+ (which it translocates preferentially over Ba2+). These studies indicate that the actions of a water-soluble polymer (PGBX) derived from a naturally occurring prostaglandin (PGB1) on human neutrophils resemble those of a classical ionophore (A23187). Moreover, they provide additional evidence that increments in the intracellular levels of divalent cations may signal stimulus-secretion coupling in human neutrophils.     

A serine proteinase inhibitor isolated from Tamarindus indica seeds and its effects on the release of human neutrophil elastase

Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.     

Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A, B, C, D and E. Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of beta-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 0.2 microM cytochalasin A, C greater than 2 microM cytochalasin B greater than or equal to 4-5 microM cytochalasin D, E. While maximal rates of O2- release and extents of exocytosis require extracellular calcium (1-2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na+, K+ or choline inhibit either cytochalasin B- or E-stimulated O2- production with IC50 values of 5-10 mM and inhibition occurs whether Cl-, NO3- or SCN- is the anion added with Na+ or K+. Release of beta-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl(IC50 approximately 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K+ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of beta-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O2- or beta-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

Data mining of plasma peptide chromatograms for biomarkers of air contaminant exposures

Background

Monocytes can be primed in vitro by lipopolysaccharide (LPS) for release of cytokines, for enhanced killing of cancer cells, and for enhanced release of microbicidal oxygen radicals like superoxide and peroxide. We investigated the proteins involved in regulating priming, using 2D gel proteomics.

Results

Monocytes from 4 normal donors were cultured for 16 h in chemically defined medium in Teflon bags ± LPS and ± 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor. LPS-primed monocytes released inflammatory cytokines, and produced increased amounts of superoxide. AEBSF blocked priming for enhanced superoxide, but did not affect cytokine release, showing that AEBSF was not toxic. After staining large-format 2D gels with Sypro ruby, we compared the monocyte proteome under the four conditions for each donor. We found 30 protein spots that differed significantly in response to LPS or AEBSF, and these proteins were identified by ion trap mass spectrometry.

Conclusion

We identified 19 separate proteins that changed in response to LPS or AEBSF, including ATP synthase, coagulation factor XIII, ferritin, coronin, HN ribonuclear proteins, integrin alpha IIb, pyruvate kinase, ras suppressor protein, superoxide dismutase, transketolase, tropomyosin, vimentin, and others. Interestingly, in response to LPS, precursor proteins for interleukin-1β appeared; and in response to AEBSF, there was an increase in elastase inhibitor. The increase in elastase inhibitor provides support for our hypothesis that priming requires an endogenous serine protease.