bluuebunny/crossref_metadata_2025 huggingface dataset:
The nerve growth factor/tumor necrosis factor receptor family
Authors: Lotz Martin , Setareh Morey , von Kempis Johannes , Schwarz Herbert
Published: January 2018

Receptors in the nerve growth factor/tumor necrosis factor receptor family are characterized by the presence of cysteine-rich motifs of ~40 amino acids in the extracellular domain. The ligands are type II transmembrane proteins with β-strands that form a jelly-roll β-sandwich. The receptors recognize soluble or cell-surface-bound ligands and mediate diverse cellular responses. Activation of intracellular signals is mediated at least in part by the association of proteins with a RING finger motif or a death domain to the cytoplasmic domains of the receptors. In addition to cell-membrane-bound receptors soluble forms have been described for most of the receptors. Activation of intracellular signals not only occurs through ligand binding to the receptors but cross-linking of at least some members of the ligand family can regulate cell functions.
Rat eosinophils: isolation and characterization of superoxide production
Authors: Cypcar David , Sorkness Ronald , Sedgwick Julie , Nagata Makoto , Clough Jennifer , Kaplan Michael , Lemanske Robert F
Published: January 2018

Studies with isolated cells are important to the understanding of mechanisms by which eosinophils participate in allergic inflammation. Due to species variability, isolation techniques and cell biology need to be defined for each source. We developed methods to obtain rat eosinophils with purity and viability exceeding 90%, characterized the superoxide anion production of these cells in response to standard activators, and compared these results with those previously obtained in our laboratories with the use of human eosinophils. Rat eosinophils responded vigorously to phorbol myristate acetate and poorly to platelet-activating factor and to N-formyl-methionyl-leucyl-phenylalanine, parallel to the responses of human eosinophils. In contrast, rat eosinophils responded unlike human eosinophils to other activators, having a larger response to calcium ionophore A23187, a smaller response to serum-treated or serum-opsonized zymosan, and a negative rather than positive modulatory effect of cytochalasin B. We conclude that rat eosinophils can be obtained in high purity and with intact responsiveness to a number of different activators.
Constitutive expression of specific interferon isotypes in peripheral blood leukocytes from normal individuals and in promonocytic U937 cells
Authors: Lallemand Christophe , Lebon Pierre , Rizza Paola , Blanchard Brigitte , Tovey Michael G
Published: January 2018

Constitutive expression of IFN-α5 and IFN-β was detected in different lymphoid cells including peripheral blood mononuclear cells from normal individuals following amplification of IFN mRNA by reverse transcriptase–polymerase chain reaction and direct sequencing of the amplified product. The activated form of the interferon-induced transcription factor complex ISGF3 was also dectected in nuclear extracts from uninduced cells. Culture supernatants from uninduced U937 cells were also found to activate an ISRE cloned upstream of the luciferase reporter gene, indicating the presence of endogenous IFN activity equivalent to approximately 0.3 to 0.5 IU/mL. This endogenous IFN was also shown to play a role in mamtaining the basal level of expression of the major histocompatibility class I genes in lymphoid cells. These results suggest that IFN-α5 and IFN-β are produced at low levels in normal tissues and play an important role in the regulation of cell function and in the maintenance of homeostasis.
Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP-1α, MIP-1β, and RANTES
Authors: Combadiere Christophe , Ahuja Sunil K , Lee Tiffany H , Murphy Philip M
Published: January 2018

We have cloned a human cDNA for a novel CC chemokine receptor (CC CKR) designated CC CKR5 that has 48–75% amino acid identity to other CC CKRs. CC CKR5 mRNA was detected constitutively in primary adherent monocytes but not in primary neutrophils or eosinophils. Macrophage inflammatory protein-1α (MIP-1α), MIP-1β, and RANTES were all potent agonists for CC CKR5 (EC50 = 3–30 nM) when calcium flux was measured in transfected HEK 293 cells, yet the apparent binding affinities of the corresponding iodinated chemokines to intact cells expressing the receptor were low (IC50 ~100 nM). The calcium flux responses were completely blocked by treatment of transfected cells with pertussis toxin. These data suggest that CC CKR5 is a Gi-coupled receptor that may mediate monocyte responses to MIP-1α, MIP-1β, and RANTES.
Effect of spaceflight on human stem cell hematopoiesis: suppression of erythropoiesis and myelopoiesis
Authors: Davis Thomas A , Wiesmann William , Kidwell William , Cannon Tom , Kerns Laura , Serke Catherine , Delaplaine Ted , Pranger Alex , Lee Kelvin P
Published: January 2018

Humans subjected to periods of microgravity develop anemia, thrombocytopenia, and abnormalities in red blood cell structure. The causes of these abnormalities are complex and unclear. The in vitro effects of spaceflight on hematopoietic cell proliferation and differentiation were investigated during the space shuttle missions STS-63 (Discovery) and STS-69 (Endeavour). CD34+ bone marrow progenitor cells were cultured in liquid suspension culture and on hematopoietic supportive stromal cells using hollow-fiber culture modules. One set of cultures was maintained at microgravity (flight cultures) for the last 8–10 days of culture and a second control was at full gravity (ground control). Over the 11- to 13-test-day period, ground control culture total cell number increased 41.0- to 65.5-fold but flight culture total cell number increased only 10.1- to 17.6-fold (57–84% decrease). Comparing ground control cultures and microgravity cultures, respectively, for progenitor cell content, myeloid progenitor cell numbers expanded 2.6- to 17.5-fold compared with 0.9- to 7.0-fold and erythroid progenitor cell numbers expanded 2.0- to 4.1-fold in ground control cultures but actually declined at microgravity (>83% reduction). Moreover, microgravity cultures demonstrated accelerated maturation/differentiation toward the macrophage lineage. These data indicate that spaceflight has a direct effect on hematopoietic progenitor cell proliferation and differentiation and that specific aspects of in vitro hematopoiesis, particularly erythropoiesis, involve gravity-sensitive components.
Endotoxin signal transduction in macrophages
Authors: Sweet Matthew J , Hume David A
Published: January 2018

Through its action on macrophages, bacterial lipopolysaccharide (LPS) or endotoxin can trigger responses that are protective or injurious to the host. This review examines the effects of LPS on macrophages by following events from the cell surface to the nucleus. The involvement of protein tyrosine kinases, mitogen-activated protein kinases, protein kinase C, G proteins, protein kinase A, ceramide-activated protein kinase, and microtubules in this process are reviewed. At the nuclear level, rel, C/EBP, Ets, Egr, fos, and jun family members have been implicated in activation of LPS-inducible gene expression.
β-1,2-linked oligomannosides Inhibit Candida albicans binding to murine macrophage
Authors: Fradin Chantal , Jouault Thierry , Mallet Astrid , Mallet Jean-Maurice , Camus Daniel , Sinaÿ Pierre , Poulain Daniel
Published: January 2018

Interaction of Candida albicans with cells of the macrophage lineage was examined by using heat-killed (HK) and live yeast cells. Laminarin, an analogue of the cell wall β-glucans, strongly inhibited HK yeasts adherence to J774 cell line but had no effect on live yeast binding. Phosphopeptidomannan (PPM) from Saccharomyces cerevisiae had a limited effect on the binding of both HK and live yeasts but significant inhibition was achieved by the use of C. albicans PPM. The role of β-1,2-oligomannosides was examined with regard to their exclusive presence within C. albicans PPM. PPM acid labile β-1,2-oligomannosides or a synthetic β-1,2-mannotetraose, inhibited yeasts binding in a manner comparable to the original PPM. These latter results were confirmed by using mouse peritoneal macrophages, thus suggesting a general role for β-1,2-oligomannosides in the adherence of the yeast to the macrophage membrane.
Modulation of neutrophil migration by exogenous gaseous nitric oxide
Authors: VanUffelen Bart E , de Koster Ben M , van den Broek Peter J A , VanSteveninck John , Elferink Jan G R
Published: January 2018

We studied the effect of exogenous nitric oxide (NO) on migration of rabbit peritoneal neutrophils. Exogenous NO enhanced random migration of neutrophils in a concentration-dependent way. An optimally stimulatory effect was observed with 0.5 μM NO, whereas at higher NO concentrations the enhancing effect decreased again. NO caused a rapid and transient increase in intracellular guanosine-3′,5′-cyclic monophosphate (cGMP) levels. The enhancing effect of NO on random migration was largely reversed by the inhibitors of cGMP accumulation, LY-83583 and methylene blue, and by the antagonists of cGMP-dependent protein kinase, 8-bromoguanosine-3′,5′-cycHc monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPS) and 8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS). These observations strongly suggest that the enhancement of random migration by NO is mediated by cGMP and cGMP-dependent protein kinase. The effect of NO on migration did not occur in the absence of extracellular calcium. Although NO did not induce a measurable elevation of intracellular free calcium, preincubation with the intracellular calcium chelator Fura-2/AM abolished the enhancing effect of NO. It appears therefore that a small change in the level of cytoplasmic free calcium does play a role in the enhancement of random migration by NO. High concentrations of NO were found to inhibit Chemotaxis induced by an optimal concentration of the chemotactic peptide AT-formyl-methionyl-leucyl-phenylalanine (fMLP). This inhibitory effect was also dependent on the presence of extracellular calcium. A role for cGMP in the inhibition of fMLP-induced Chemotaxis by NO is not supported by our measurements of intracellular cGMP levels. In contrast to the effects on fMLP, NO did not affect Chemotaxis induced by the phorbol ester PMA. In conclusion, we show that NO, not derived from NO donors but applied directly, may stimulate or inhibit neutrophil migration, dependent on the concentration. The enhancing effect of NO on random migration is mediated by cGMP, which emphasizes the importance of this second messenger as a modulator of neutrophil functions.
Diamide primes neutrophils for enhanced release of superoxide anion: relationship to S-thiolation of cellular proteins
Authors: Moriguchi Toshinori , Seres Tamas , Ravichandran Veerasamy , Sasada Masataka , Johnston Richard B
Published: January 2018

Stimulation of the respiratory burst in phagocytes induces the formation of mixed disulfides between sulfhydryl groups of proteins and low-molecular-weight thiols. We hypothesized that this process (S-thiolation) might be involved in turning off the respiratory burst. However, induction of S-thiolation by pretreatment of neutrophils with diamide, a direct thiol oxidizing agent, actually primed the cells for a two- to fivefold increase in total release and fourfold increase in rate of release of O2- on stimulation by f-Met-Leu-Phe. Generation of intracellular oxidants (hydroethidine fluorescence) was increased ninefold. Priming and S-thiolation were apparent at 1 min of incubation and peaked at 5–10 min. Diamide pretreatment also reduced the lag time between addition of phorbol diester and release of O2- by a mean of 23 s (41%). Dithioerythritol, a sulfhydryl-reducing agent, abolished both the S-thiolation and priming mediated by diamide. H2O2 also induced priming and S-thiolation; and these were eliminated by dithioerythritol. In contrast to the effect of endotoxin, diamide priming did not affect Ca2+ homeostasis of the neutrophils. Diamide did not significantly alter NADPH oxidase activity in a cell-free system. These findings suggest that sulfhydryl groups on one or more proteins play an important role in modulating the respiratory burst.
IL-8 release from human neutrophils by the respiratory syncytial virus is independent of viral replication
Authors: König B , Krusat T , Streckert H J , König W
Published: January 2018

Elevated interleukin-8 levels and a massive accumulation of neutrophils (PMN) are the hallmark of a variety of severe lung diseases. The respiratory syncytial virus (RSV), an important respiratory pathogen, induces interleukin-8 (IL-8) release from human PMN, however, the mechanism is as yet unknown. We analyzed the role of virus uptake, intracellular virus replication, virus attachment, and of virus capsid proteins for the induction of IL-8 (protein + mRNA) in human PMN. Cell supernatants were analyzed for IL-8 release via enzyme-linked immunosorbent assay; cell pellets were analyzed for IL-8-specific mRNA expression and for RSV-specific genomic and RSV-specific mRNA by reverse transcriptase-polymerase chain reaction. Stimulation of human PMN with viable, heat-inactivated, or UV-inactivated RSV [at a multiplicity of infection (m.o.i.) from 0.01 up to 10] induced IL-8 production (protein + mRNA) to a similar degree. Maximal IL-8 release was observed at a m.o.i. of 5–10 after 18–24 h. RSV-specific genomic RNA was present inside PMN up to 24 h independent of whether viable or inactivated RSV was used. Withdrawal of extracellular viable or inactivated (heat, UV) RSV after infection of PMN (2 h) abolished IL-8 mRNA expression and IL-8 release; the intracellular persistence of RSV lasted for up to 24 h. Stimulation of human PMN with purified RSV G-protein, a major capsid protein, in a concentration range from 0.1 up to 2.5 μg/5 105 PMN resulted in an increased IL-8 release from human PMN but to a significantly lesser degree compared with the intact RSV. RSV G-protein concentration above 1 μg inhibited the RSV-induced IL-8 release by up to 90%. Our data contribute to the understanding of the pathomechanisms leading to IL-8 release from human PMN.
Trichodimerol (BMS-182123) inhibits lipopolysaccharide-induced eicosanoid secretion in THP-1 human monocytic cells
Authors: Mazzucco Charles E , Warr Glenn
Published: January 2018

The fungal metabolite trichodimerol (BMS-182123) has demonstrated inhibition of lipopolysaccharide (LPS) -stimulated tumor necrosis factor-α (TNF-α) secretion in various in vitro macrophage models (human and murine) including primary and tumor cell lines. When challenged with LPS, differentiated THP-1 monocytic cells secrete elevated levels of the cyclooxygenase products prostaglandin E2 (PGE2), thromboxane B2, and prostaglandin F2α (PGF2α). Studies directed at elucidating the mechanism of action of BMS-182123 as a TNF-α inhibitor revealed that the compound has a profound inhibitory effect on prostanoid secretion in response to LPS challenge. The key enzymes in prostaglandin synthesis are the constitutive cyclooxygenase, prostaglandin H synthase-1 (PGHS-1), and the mitogen-induced cyclooxygenase (PGHS-2), which is induced upon LPS stimulation in THP-1 cells. BMS-182123 did not inhibit the cyclooxygenase activity of PGHS-1 in an in vitro assay, suggesting that inhibition is due to a blockade in synthesis of cyclooxygenase enzyme. Western blot analysis of microsomal pellets from THP-1 cells stimulated with LPS (with or without BMS-182123 pretreatment) provided convincing evidence that the inhibition of prostaglandin synthesis is a result of suppressed synthesis of PGHS-2 enzyme. Northern blot analysis of THP-1 RNA demonstrated that BMS-182123 inhibits the induction of PGHS-2 at the level of transcription.
Transient inhibition of capacitative calcium entry in human neutrophils by a monoclonal antibody directed against a 19-kDa antigen
Authors: Alonso Maria Teresa , Mollinedo Faustino , Barasoain Isabel , Alvarez Javier , Garcia-Sancho Javier
Published: January 2018

P1C3 is a monoclonal antibody that binds p19, a novel neutrophil activation antigen that translocates to the cell surface upon neutrophil activation. We find that P1C3 inhibits capacitative Ca2+ entry, induced by emptying the intracellular Ca2+ stores with thapsigargin. The effect is transient, reaching its maximum at 30–60 s, but becomes permanent upon pretreatment of the cells with the protein phosphatase inhibitor calyculin A, suggesting the involvement of protein phosphorylation. The inhibitory action is similar to the one reported previously for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), although the transduction mechanism may be different. Inhibition of Ca2+ entry by fMLP was prevented by pretreatment with pertussis toxin, whereas inhibition by P1C3 was not. Pretreatment with cholera toxin had no effect. This suggests that the effect of P1C3 may not be mediated by a heterotrimeric G protein. Tyrosine kinase inhibitors did not prevent inhibition by either fMLP or P1C3. Phospholipase C activation seems not to be involved as P1C3, contrarily to fMLP, was unable to induce Ca2+ release from the intracellular Ca2+ stores.
Concanavalin A and mistletoe lectin I differentially activate cation entry and exocytosis in human neutrophils: lectins may activate multiple subtypes of cation channels
Authors: Wenzel-Seifert Katharina , Krautwurst Dietmar , Lentzen Hans , Seifert Roland
Published: January 2018

The mannose-specific lectin, concanavalin A (ConA), activates Ca2+ entry in human neutrophils by an as yet poorly defined mechanism. The question of whether the sugar specificity of lectins influences signal transduction is unresolved too. Therefore, we studied the effects of ConA in comparison to those of the β-galactoside-specific lectin, mistletoe lectin I (MLI), on cation entry and exocytosis in human neutrophils. ConA- and MLI-activated influx of Ca2+, Mn2+, Ba2+, Sr2+, and Na+. Lectin-induced cation influxes were inhibited by 1-{β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl} -1H-imidazole hydrochloride (SK&F 96365) and Gd3+. There were differences in the effectiveness of lectins to activate cation entry and of SK&F 96365, Gd3+, and modulators of protein phosphorylation to block entry. MLI but not ConA inhibited thapsigargin-induced Ca2+ entry. Under whole-cell voltage-clamp conditions, MLI activated an inward current that was substantially reduced by removal of extracellular Na+. ConA and MLI synergistically activated Ca2+ entry and lysozyme release. SK&F 96365 and removal of extracellular Ca2+ and Na+ partially inhibited exocytosis. Our data show the following: (1) ConA and MLI activate monovalent and divalent cation entry in human neutrophils by a SK&F 96365- and Gd3+-sensitive pathway, presumably nonselective cation channels. (2) Ca2+ and Na+ entry are involved in the activation of exocytosis by lectins. (3) The differential and/or synergistic effects of ConA and MLI on cation entry and exocytosis may be attributable to mannose- and β-galactoside-specific activation of signal transduction pathways, i.e., activation of multiple and differentially regulated subtypes of nonselective cation channels.
Fcγ receptor I (CD64)-negative human monocytes are potent accessory cells in viral antigen-induced T cell activation and exhibit high IFN-α-producing capacity
Authors: Grage-Griebenow Evelin , Flad Hans-Dieter , Ernst Martin
Published: January 2018

Blood monocytes represent a heterogeneous cell population with respect to phenotype and function. We have previously described that a minor subset of Fcγ receptor I-negative (CD64−) monocytes, comprising < 10% of all monocytes, exhibits a significantly higher accessory capacity in allogeneic or purified protein derivative (PPD) of tuberculin-induced T cell activation than the majority of CD64-expressing (CD64+) monocytes. CD64− monocytes were also found to represent the major source of Newcastle disease virus (NDV)-induced interferon (IFN)-α within human monocytes. In the present study we demonstrate that CD64− monocytes are also the main producers of IFN-α in response to Herpes simplex virus type 1 (HSV-1) or influenza (type A) antigens. The virus-induced IFN-α release by monocytes, but mainly that by CD64− monocytes, can be further increased by co-culture with autologous T cells, which alone do not produce significant amounts of IFN-α in response to virus. In addition, CD64− and CD64+ monocytes also differ in their accessory capacity in virus-induced T cell responses. CD64− monocytes exposed to influenza antigens induced higher IFN-γ release and proliferation by the responding autologous T cells than virus-exposed CD64+ monocytes. In virus-stimulated monocyte/T cell co-cultures, CD64− monoyctes also induced larger size cell clusters than CD64+ monocytes, indicating direct cell-to-cell interaction with a higher number of T cells, which represent the main component of these clusters.
Osteopontin inhibits nitric oxide production and cytotoxicity by activated RAW264.7 macrophages
Authors: Rollo Ellen E , Laskin Debra L , Denhardt David T
Published: January 2018

Osteopontin (OPN), a secreted acidic phosphoglyeoprotein found in many tissues and body fluids, is produced in increased amounts in response to certain infections and after malignant transformation. In this study we examined the action of OPN on macrophage cytotoxicity and nitric oxide (NO) synthesis. A human OPN cDNA was cloned into the bacteriophage T7-based vector, pET8C, and the encoded protein purified from an induced culture of Escherichia coli carrying the plasmid. Recombinant OPN inhibited NO production by macrophage-like RAW264.7 cells stimulated with lipopolysaccharide plus interferon-γ. OPN also inhibited the cytolytic activity of the activated macrophages toward NO-sensitive P815 mastocytoma cells, an action that was blocked by the NO synthase inhibitor, NG-monomethyl-l-arginine. Inhibition of NO production correlated with an OPN-dependent decrease in the abundance of inducible NO synthase mRNA. The shape of the dose-response curve, with a maximal effect over a narrow range of OPN concentrations, suggested a complex interaction of OPN with cell surface receptors. Our data support the hypothesis that tumor-cell-derived OPN functions to protect the tumor cells from macrophage-mediated destruction.
Prenylation of an interferon-γ-induced GTP-binding protein: the human guanylate binding protein, huGBP1
Authors: Nantais David E , Schwemmle Martin , Stickney John T , Vestal Deborah J , Buss Janice E
Published: January 2018

Interferons (IFN) and lipopolysaccharide (LPS) cause multiple changes in isoprenoid-modified proteins in murine macrophages, the most dramatic being the expression of a prenyl protein of 65 kDa. The guanylate binding proteins (GBPs) are IFN-inducible GTP-binding proteins of ~65 kDa that possess a CaaX motif at their C-terminus, indicating that they might be substrates for prenyltransferases. The human GBP1 protein, when expressed in transfected COS-1 cells, incorporates radioactivity from the isoprenoid precursor [3H]mevalonate. In addition, huGBPs expressed from the endogenous genes in IFN-γ-treated human fibroblasts or monocytic cells were also found to be isoprenoid modified. IFN-γ-induced huGBPs in HL-60 cells were not labeled by the specific C20 isoprenoid, [3H]geranylgeraniol, but did show decreased isoprenoid incorporation in cells treated with the farnesyl transferase inhibitor BZA5B, indicating that huGBPs in HL-60 cells are probably modified by a C15 farnesyl rather than the more common C20lipid. Differentiated HL-60 cells treated with IFN-γ/LPS showed no change in the profile of constitutive isoprenylated proteins and the IFN-γ/LPS-induced huGBPs remained prenylated. Despite being prenylated, huGBP1 in COS cells and endogenous huGBPs in HL-60 cells were primarily (~85%) cytosolic. Human GBPs are thus among the select group of prenyl proteins whose synthesis is tightly regulated by a cytokine. HuGBP1 is an abundant protein whose prenylation may be vulnerable to farnesyl transferase inhibitors that are designed to prevent farnesylation of Ras proteins.
Eosinophil viability during immunoglobulin-induced degranulation
Authors: Weiler Catherine R , Kita Hirohito , Hukee Margaret , Gleich Gerald J
Published: January 2018

Eosinophil adhesion and degranulation appear to be associated with cell death. Eosinophils bound avidly and degranulated with secretory immunoglobulin A (slgA)- and IgG-coupled Sepharose 4B beads but bound poorly and did not degranulate with ovalbumin beads. Through the use of dye staining, we found that about 50% of the bound eosinophils were dead by 4 h, regardless of the protein coating. Colchicine and reduced calcium concentration inhibited binding to beads and eosinophil degranulation in a concentration-dependent manner but did not decrease the percentages of dead bound eosinophils. Electron microscopy showed that eosinophils bound to and spread over bead surfaces. Typical granule exocytosis with release of membrane-free granules occurred in about 20% of bound eosinophils. Eosinophil degeneration and lysis with release of membrane-coated granules occurred in about 50% of bound eosinophils; often only membrane-bound granules were present. Therefore, bound eosinophils degranulate both by classical exocytosis and by release after cytolytic degeneration. By increasing the numbers of bound cells, both IgG and sIgA increase the numbers of dying cells.
Relative role of chloramines, hypochlorous acid, and proteases in the activation of human polymorphonuclear leukocyte collagenase
Authors: Claesson Rolf , Karlsson Magnus , Zhang You Yi , Carlsson Jan
Published: January 2018

The activation of collagenase released by polymorphonuclear leukocytes (PMNs) has been extensively studied in vitro, but the activation of the enzyme in vivo is not fully understood. For further evaluation of the relative role of oxidative and proteolytic mechanisms in the activation of collagenase, PMNs were stimulated by serum-opsonized zymosan under both aerobic and anaerobic conditions. The results showed that similar amounts of collagenase were released by the PMNs under aerobic and anaerobic conditions, but the activity of the released collagenase was twice as high under aerobic conditions as under anaerobic conditions. Under aerobic conditions the enzyme was rapidly activated by hypochlorous acid and chloramines, which are products of the myeloperoxidase-H2O2-chloride system of the PMNs. There was also a slow proteolytic activation of the enzyme, which could be ascribed to cathepsin G and possibly to some other serine proteases of PMNs. When extrapolating these findings to in vivo conditions, it seems probable that the oxidative activation of collagenase will proceed mainly by chloramines, which are more long-lived in the tissue than hypochlorous acid. In poorly oxygenated tissues, collagenase may be mainly activated by proteolytic mechanisms.
Interleukin-4 rapidly down-modulates the macrophage colony-stimulating factor receptor in murine macrophages
Authors: Sbarba Persio Dello , Rovida Elisabetta , Caciagli Barbara , Nencioni Lucia , Labardi Danilo , Paccagnini Alessandro , Savini Luca , Cipolleschi Maria Grazia
Published: January 2018

The activation of macrophages interferes with their response to macrophage colony-stimulating factor (M-CSF), the main growth and differentiation factor for mononuclear phagocytes. We tested the rapid effects of interleukin-4 (IL-4), the alternative macrophage activator produced by Th2 helper lymphocytes, on the receptor for M-CSF (M-CSFR) expressed on the cell surface of murine macrophages. IL-4 rapidly down-modulated M-CSFR in a dose-dependent fashion. This effect was unique to IL-4 among a number of Th2-produced cytokines, none of which, with the exception of IL-4 itself, is able to activate macrophages. The down-modulation of M-CSFR by IL-4 was partially prevented by the inhibition of the activity of phospholipase C or protein kinase C. The data are consistent with the hypothesis that the down-modulation of M-CSFR is a property common to, and exclusive of, macrophage activators, and is driven by different activators via a common mechanism.
Transforming growth factor-β1 stimulates degranulation and oxidant release by adherent human neutrophils
Authors: Balazovich Kenneth J , Fernandez Rosemarie , Hinkovska-Galcheva Vania , Suchard Suzanne J , Boxer Laurence A
Published: January 2018

The signal transduction pathways that are activated by cytokines and growth factors binding to their receptors on human neutrophils (PMN) are poorly understood. When PMN in suspension encounter many of these agonists they are not activated, but rather are primed for subsequent activation. We and others reported that when PMN are plated onto fibrinogen and stimulated with cytokines or with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) they respond by releasing hydrogen peroxide (H2O2) and the specific granule component lactoferrin. Transforming growth factor-β1 (TGF-β1) is released by many cells including PMN. It has been reported that TGF-β1 stimulates chemotaxis but not exocytosis or superoxide production by cells in suspension. We hypothesized that TGF-β1 would activate PMN to release H2O2 when they were adherent to fibrinogen, a response mediated by β2 integrin receptors. In this study, we determined whether TGF-β1 stimulated H2O2 and lactoferrin release by PMN adherent to fibrinogen. TGF-β1 stimulated H2O2 and lactoferrin release from adherent PMN in a concentration-dependent manner, with effects seen in the range of 0.1 to 100 pg/mL. Both H2O2 and lactoferrin release were detected by 60 min and continued for at least 180 min. Adhesion and spreading of PMN paralleled H2O2 and lactoferrin release. Ethanol (200 mM) blocked both H2O2 and lactoferrin release, suggesting the involvement of the phospholipase D pathway. In PMN labeled with lyso[3H]phoephatidylcholine, we observed that TGF-β1 treatment caused an increase in [3H]phoephatidate. Propranolol (150 μM), an inhibitor of phosphatidate phosphohydrolase, blocked both H2O2 and lactoferrin release, suggesting that the conversion of phosphatidic acid to diradylglycerol is an important step in PMN activation by TGF-β1. Overall, these results are similar to those reported for fMLP activation of adherent PMN and suggest that a common pathway is involved in both chemoattractant and cytokine activation.
Monocyte chemoattractant protein-1: receptor interactions and calcium signaling mechanisms
Authors: Needham M , Sturgess N , Cerillo G , Green I , Warburton H , Wilson R , Martin L , Barratt D , Anderson M , Reilly C , Hollis M
Published: January 2018

Monocyte chemoattractant protein-1 (MCP-1) is a member of the Cys-Cys chemokine family. Two related MCP-1 receptors have been identified (CC-CKR2A and CC-CKR2B), although the precise kinetics of ligand binding and calcium signaling of these receptors has yet to be investigated, To examine this more closely, the human MCP-1 receptors were cloned and expressed in Chinese hamster ovary (CHO) cells. Membranes prepared from cells expressing CC-CKR2B bind MCP-1 selectively and with high affinity (Kd = 120 pM). MCP-1 stimulation of recombinant CHO cells expressing CC-CKR2B induces a rapid increase in intracellular Ca2+ through both receptor-operated Ca2+ channels and mobilization of Ca2+ from intracellular stores, and leads to a rapid temperature-dependent internalization of the ligand/receptor complexes. In contrast, recombinant CHO cells expressing CC-CKR2A, and membranes prepared from these cells, fail to bind detectable levels of MCP-1. However, MCP-1 stimulation of cells expressing CC-CKR2A induces a small but significant increase in intracellular Ca2+. Repeated stimulation of these cells with MCP-1 leads to a potentiation of the response to a level comparable to that seen in cells expressing CC-CKR2B. These observations suggest that the levels of cell surface CC-CKR2A are controlled by novel mechanisms.
Characterization of the processing and granular targeting of human proteinase 3 after transfection to the rat RBL or the murine 32D leukemic cell lines
Authors: Garwicz Daniel , Lindmark Anders , Hellmark Thomas , Gladh Mathias , Jögi Jonas , Gullberg Urban
Published: January 2018

Proteinase 3 (PR3) is a neutrophil serine protease stored in the azurophil granules of the promyelocyte and its successors. The protease has been identified as an autoantigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) occurring in patients with Wegener's granulomatosis. To characterize the biosynthesis and processing of human PR3 in a transgenic cellular model, cDNA encoding human pre-proproteinase 3 cloned from U-937 cells was transfected to the rat basophilic/mast cell line RBL-1 and the murine myeloblast-like cell line 32D cl3. The stable expression of transgenic proteinase 3 was characterized by biosynthetic labeling, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. After pulse labeling for 30 min two proforms of PR3 (32 and 35 kDa), differing in carbohydrate content but with protein cores of identical size, were demonstrated. Chase of the label resulted in a processed 32-kDa form clearly visible in RBL, but only faintly in 32D cells, probably indicating delayed intracellular transfer in the latter cell line. Partial digestion with N-glycosidase F showed that both potential N-glycosylation sites on PR3 were occupied and conversion of the oligosaccharide side chains into complex forms was demonstrated by acquisition of resistance to endoglycosidase H. Translocation of PR3 to granules was shown by subcellular fractionation and immunocytochemistry. Enzymatic activation of PR3 was suggested by affinity to diisopropylfluorophosphate and removal of an amino-terminal propeptide. Cells transfected with PR3 showed positive immunofluorescence for ANCA-containing sera from patients with Wegener's granulomatosis. Our results show that human PR3 transfected to RBL or 32D cells is synthesized as a 29-kDa protein core glycosylated on two distinct sites. Oligosaccharide trimming and proteolytic processing occur and the protein is targeted for granular storage in a form antigenic for ANCA.
Alteration of deacylated lipopolysaccharide antagonistic properties by interaction with plasma factors
Authors: Mey Anne , Hmama Zakaria , Normier Gérard , Revillard Jean-Pierre
Published: January 2018

The acyl poly(l,3)galactoside (APG) from Klebsiella pneumoniae is a bis-acylated lipopolysaccharide (LPS) devoid of ester-linked fatty acids. APG interacts with CD14 and CD11b/CD18 on monocytes. This study addressed the role of serum proteins in the binding and functional properties of APG as a candidate LPS antagonist. In the absence of serum, APG did not induce tumor necrosis factor α (TNF-α) synthesis by human mononuclear cells and dose-dependently inhibited their activation induced by different LPS. Conversely, in the presence of 5% autologous plasma, APG activated cells and did not antagonize LPS. Serum decreased APG but not LPS binding to monocytes. Binding competition experiments indicated that APG and LPS competed for the same receptors in serum-free conditions but bound to different receptors in the presence of plasma. The data indicate that serum-dependent LPS receptors do contribute to LPS activation of monocytes but do not recognize deacylated LPS analogues.
Elevated levels of NO in both unchallenged and LPS-challenged C. parvum-primed mice are attributable to the activity of a cytokine-inducible isoform of iNOS
Authors: Smith Sidney R , Manfra Denise , Davies Liza , Terminelli Carol , Denhardt Georgette , Donkin Jennifer
Published: January 2018

Elevated levels of nitric oxide (NO2-/NO3-) were detected in the serum of mice 3–7 days after priming with Corynebacterium parvum (Propiombacterium acnes). The serum NO2-/NO3- response was completely inhibited when C. parvum-primed (C. parvum) mice were treated with NG-monomethyl-l-arginine (l-NMMA) or aminoguanidine (AG) on days 6 and 7 post priming. The response was also inhibited when the mice were treated with interleukin-10 (IL-10) and the cytokine was most effective when given in multiple doses beginning on the day of priming. In contrast to l-NMMA and AG, IL-10 had no effect on the serum NO2-/NO3- response when administered to the mice on days 6 and 7 post priming. The inducible isoform of NOS (iNOS) appeared to be responsible for the elevated NO2-/NO3- response in C. parvum mice because iNOS transcripts were readily detected in their livers. Moreover, these transcripts as well as the circulating levels of NO2-/NO3- were dramatically reduced when the mice were treated with anti-tumor necrosis factor α (anti-TNF-α) or antiinterferon-γ (anti-IFN-γ) monoclonal antibodies (mAbs) during the priming interval. There was a modest increase (less than twofold) in the serum NO2-/NO3-response following a lipopolysaccharide (LPS) challenge to C. parvum mice (C. parvum/LPS mice). LPS had a more dramatic stimulatory effect if the levels of NO2-/NO3- preexisting in C. parvum/LPS mice were reduced by treatment with l-NMMA, AG, or IL-10 before the challenge. Thus the levels of NO2-/NO3-that preexisted in C. parvum/LPS mice appeared to influence their ability to mount a NO2-/NO3- response subsequent to the LPS challenge. The NO2-/ NO3- response did not contribute to lethality in C. parvum/LPS mice because anti-TNF-α and anti-IFN-γ mAbs were protective but had no effect on serum NO2-/NO3- levels when administered to mice 24 h before the LPS challenge.
Growth-inhibitory and apoptosis-inducing activities of calprotectin derived from inflammatory exudate cells on normal fibroblasts: regulation by metal ions
Authors: Yui Satoru , Mikami Masaaki , Tsurumaki Kazusa , Yamazaki Masatoshi
Published: January 2018

We have shown previously that a calcium-binding protein complex, calprotectin, derived from polymorphonuclear leukocytes exerts a cytostatic and cytolytic effect against a very broad range of tumor cell lines. We also described that calprotectin is an apoptosis-inducing factor for certain tumor cells and that zinc ion attenuates the calprotectin activities. The titers of the factor in body fluids are known to increase greatly in various types of inflammation. In this study, to learn the role of calprotectin in inflammation, the growth-inhibitory and the apoptosis-inducing activities of the factor against normal fibroblasts were examined because fibroblasts are a cell type constituting a local inflammatory site. Bat calprotectin inhibited the growth of murine embryonic as well as human dermal fibroblasts. Although calprotectin induced apoptotic morphology in both fibroblasts, the reaction was slower and less efficient than cases using tumor cells as targets. The activities were significantly abrogated by 10–50 μM Zn2+, Cu2+, Mn2+, or Fe2+, respectively, whereas the trivalent cations Al3+ and Fe3+ had no effect. The dose-response curves of the calprotectin effects were shifted to about 10-fold lower concentration ranges in the divalent metal ion-depleted medium. These results suggest that calprotectin extracellularly affects the inflammatory processes by modulating the growth and survival states of normal fibroblasts, and that the effects are physiologically controlled by several metal ions.
Preactivation exposure of RAW 264.7 cells to taurine chloramine attenuates subsequent production of nitric oxide and expression of iNOS mRNA
Authors: Park Eunkyue , Schuller-Levis Georgia , Jia Jun-Hua , Quinn Michael R
Published: January 2018

Recent studies demonstrate that taurine chloramine (Thu-Cl) inhibits production of nitric oxide (NO) and other proinflammatory mediators in cultured macrophages when added to the media at the time of activation. Because Thu-Cl may react with various media constituents and it is difficult to measure Thu-Cl in complex solutions, we designed experiments to more carefully control cell exposure to various chloramines and NaOCl. RAW 264.7 cells were exposed to 1 mM of NaOCl, Thu-Cl, or chloramine preparations of the following amino acids: l-alanine (l-Ala-Cl), β-alanine (β-Ala-Cl), serine (Ser-Cl), or glycine (Gly-Cl) in Hanks' balanced salt solution (HBSS) for up to 2 h (37°C, 5%CO2). The HBSS solution was then replaced with complete media containing interferon-γ (IFN-γ) and lipopolysaccharide (LPS) for an additional 24 h before measuring cell viability. The chemical stability of NaOCl and each chloramine was evaluated after various times of preactivation exposure by measuring retention of each solution's UV absorption spectra and ability to oxidize KI. Cytotoxicity of each solution was evaluated by the maintained ability of RAW 264.7 cells to reduce MIT. Whereas Thu-Cl, β-Ala-Cl, and Gly-Cl were stable chloramines, only Thu-Cl was not cytotoxic. l-Ala-Cl, Ser-Cl, and the highly reactive oxidant NaOCl were unstable and toxic. In further studies RAW 264.7 cells were exposed to Thu-Cl in HBSS for 2 h and the solution was then replaced with complete media containing IFN-γ and LPS, taxol, lipoarabinomannan, or interleukin-2. Production of NO was measured 24 h later and was inhibited in activated cells that were previously exposed to Thu-Cl. Inhibition of NO production was dependent on Thu-Cl concentration and was accounted for by reduced expression of inducible nitric oxide synthase mRNA, regardless of activator combinations. These results support the idea that Thu-Cl has the potential to function as an inhibitory modulator of inflammation.
Dexamethasone-induced suppression of apoptosis in human neutrophils requires continuous stimulation of new protein synthesis
Authors: Cox Gerard , Austin Richard C
Published: January 2018

We examined the mechanisms of corticosteroid inhibition of cell death by apoptosis in human neutrophils. Suppression of apoptosis by dexameth-asone was abolished by co-treatment with the protein synthesis inhibitor cycloheximide. At doses of 1 μg/mL cycloheximide did not reduce basal survival of neutrophils but effectively inhibited dexamethasone-induced increases in 24-h survival (24.4 ± 8.7 vs. 49.6 ± 10%, P < 0.01). Similar results were obtained with actinomycin D, an inhibitor of mRNA synthesis. The factor(s) responsible for mediating increased survival following dexamethasone treatment is not active extracellularly because dexamethasone-treated neutrophil-conditioned medium (CM) had no effect on the survival of naive neutrophils when the direct effects of dexamethasone were neutralized with the steroid antagonist RU-486. In contrast, LPS-treated neutrophil CM significantly increased neutrophil survival even after addition of polymyxin b. The survival effect of dexamethasone required the continuous presence of the agonist because addition of RU-486 caused prompt development of apoptosis in dexamethasone-treated cells. When naive and dexamethasone-treated cells were examined by mRNA differential display, a limited number of cDNA bands were consistently and reproducibly detected that were increased in intensity, indicating up-regulation by dexamethasone. Thus, corticosteroid regulation of neutrophil apoptosis is a specific effect that depends on continuous stimulation of synthesis of a (protein) survival factor.
Secretion and inactivation of myeloperoxidase by isolated neutrophils
Authors: King Charles C , Jefferson M Margaret , Thomas Edwin L
Published: January 2018

Neutrophils prevent infection by ingesting and killing microorganisms but oxidants and proteases released by neutrophils damage host tissues. Our aim was to identify factors that regulate oxidant production by the enzyme myeloperoxidase (MPO) following secretion of MPO into the medium. Cells stimulated with phorbol myristate acetate (PMA) or opsonized zymosan particles secreted MPO and released superoxide free radicals (·O2-). Dismutation of ·O2- produced hydrogen peroxide (H2O2) and MPO catalyzed the oxidation of chloride ion by H2O2 to produce the toxic oxidant hypochlorous acid (HOCl). Adding the enzyme superoxide dismutase (SOD) to increase the rate of conversion of ·O2- to H2O2 had pH-dependent effects on HOCl production. From pH 6.0 to 7.4, SOD promoted HOCl production by up to 500% but SOD had no effect at pH 7.6 and inhibited by 40 ± 10% at pH 7.8. In further experiments at pH 7.0, MPO activity in the cells decreased by 25 ± 2 and 44 ± 4% during 1-h incubations with PMA and zymosan. Only 1 ± 0 and 3 ± 1% of the total activity was found in the medium, indicating that most of the secreted MPO was inactivated. Loss of activity was not accompanied by proteolytic destruction of the MPO protein, which was measured with anti-MPO antibodies. SOD raised the amount of active MPO in the medium two- to sevenfold, but adding deferoxamine to chelate iron or adding ferric ion had no effect. The ionophore A23187 was as effective as zymosan as a stimulus for MPO secretion but ·O2- production by ionophore-stimulated cells was less than 4% of that of PMA- or zymosan-stimulated cells and most of the secreted MPO was found active in the medium. When PMA-stimulated cells were incubated with purified MPO, the added MPO activity was lost from the medium. Binding or proteolysis did not account for loss of activity as indicated by recovery of added radioiodinated MPO from the medium. The visible absorption spectrum of MPO was lost, indicating destruction of the iron-containing prosthetic group. Loss of activity and loss of the MPO spectrum were blocked by SOD but not by deferoxamine or catalase. The results indicate that, in the physiological pH range, inactivation of MPO in the medium suppressed HOCl production. Inactivation required ·O2- but not HOCl, H2O2, or free iron. Inactivation of secreted MPO may limit MPO-mediated damage to host tissues by stimulated neutrophils.
Immunoelectron microscopy shows a clustered distribution of NADPH oxidase components in the human neutrophil plasma membrane
Authors: Wientjes Frans B , Segal Anthony W , Hartwig John H
Published: January 2018

The NADPH oxidase that produces superoxide in professional phagocytic cells is a flavocytochrome b electron transport chain in the membrane, a heterodimer of gp91phox and p22phox, that is activated by a number of cytosolic proteins, including p47phox p67phox, and the small GTP-binding protein p21rac, which translocate to the membrane and attach to the flavocytochrome on activation. The components of this oxidase were localized on the cytoplasmic surface of the plasma membrane of adherent unroofed neutrophils by immunolabeling. Components of the NADPH oxidase and p21rac were found together in punctate clusters occupying 0.03–0.1 μm2 of the cytoplasmic surface of the plasma membrane where the density of labeling of the cytosolic components was increased after stimulation with phorbol myristate acetate.