bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific Binding of Haptoglobin to Human Neutrophils and Its Functional Consequences
Authors: Oh S-K , Pavlotsky N , Tauber A I
Published: January 2018

Haptoglobin, an acute phase reactant protein, has been shown to modulate various facets of immune responses. In this paper we examined the effect of haptoglobin on human neutrophils at the molecular level. First, we found that native haptoglobin binds at two distinct sites on neutrophils. We then examined the effects of this binding at normal and pathophysiological concentrations of haptoglobin found in human serum. Of the various functional parameters assessed, neutrophil respiratory burst activity, as assessed by superoxide (O2-) production, was inhibited by native haptoglobin when the cells were stimulated with formylmethlonyl-leucylphenylalanine (FMLP), arachidonic acid (AA), and opsonized zymosan. The rise in intracellular calcium induced by FMLP stimulation was also inhibited by native haptoglobin. Since the generation of O2- was unaffected by native haptoglobin in phorbol myristate acetate (PMA)-stimulated neutrophils, the likely site of haptoglobin inhibition on neutrophil function is at a point of receptor-ligand interaction in the activation cascade. The role of haptoglobin as a modifier of the immune response has here been extended to altered neutrophil function stimulated by diverse agonists.
Priming Effect of Muramyl Peptides for Induction by Lipopolysaccharide of Tumor Necrosis Factor Production in Mice
Authors: Parant Monique , Parant Francine , Vinit Marie-Antoinette , Jupin Claude , Noso Yoshihiro , Chedid Louis
Published: January 2018

Lipopolysaccharide-induced necrosis of grafted tumors was potentiated by several hydrophilic and lipophilic muramyl dipeptide (MDP) derivatives administered a few hours prior to small amounts of lipopolysaccharide (LPS) in spite of low titers of induced circulating tumor necrosis factor (TNF). However, pretreatment with MDP derivatives did increase the level of TNF in the blood of mice challenged by a greater dose of LPS. The TNF amount in 2 h postendotoxin mouse serum reached a peak when the glycopeptide had been given 6 h before the challenge, being approximately 100-fold above that obtained in unprimed mice. The cytotoxic activity in mouse serum was inhibited by rabbit antibodies raised against recombinant mouse TNF. Although there exists a toxic synergism between BCG or MDP and endotoxin, the effect of certain MDP derivatives was not related to an increased susceptibility to the toxicity of LPS.
Hepatocyte Modulation of Kupffer Cell Prostaglandin E2 Production In Vitro
Authors: Billiar T R , Lysz T W , Curran R D , Bentz B G , Machiedo G W , Simmons R L
Published: January 2018

It is likely that dynamic interactions between hepatocytes and Kupffer cells contribute to the responses of these cell types both under normal conditions and during sepsis. In this study, we examined the influences of hepatocytes on the concentration of the inflammatory mediator PGE2 in Kupffer cell cultures. Evidence to suggest that cultured rat hepatocytes both metabolize PGE2 and produce a substance that promotes LPS-stimulated Kupffer cell PGE2 biosynthesis include the following: 1) PGE2 levels in Kupffer cell: hepatocyte coculture were lower than the levels in Kupffer cell cultures early after LPS stimulation; 2) 36 h after LPS, coculture PGE2 levels exceeded the levels in Kupffer cell cultures despite the demonstrated capacity for hepatocytes to metabolize PGE2; 3) a transferable, non-dialyzable, and heat-unstable factor in hepatocyte supernatant promoted PGE2 production when added to Kupffer cells with LPS or after LPS; 4) there was no increased PGE2 synthesis when the hepatocyte supernatant was added without LPS or if hepatocyte supernatant was preincubated with the Kupffer cells for 6 or 18 h before LPS administration; 5) there was an inability of the hepatocyte factor to promote PGE2 production in response to other macrophage-activating agents, including calcium ionophore A23187 or phorphol myristate acetate; and 6) there was no increased cell replication or protein synthesis in the Kupffer cell cultures following hepatocyte supernatant incubation. The increased Kupffer cell PGE2 production by the hepatocyte supernatant was not due to contamination of the hepatocyte supernatant by endotoxin or PGE2. These in vitro results raise the possibility that hepatocytes have the capacity to modulate local PGE2 levels by two distinct mechanisms. Hepatocytes can metabolize PGE2 as well as release factor(s) which promote LPS-induced PGE2 production by Kupffer cells.
Influenza A Virus Alters Structural and Biochemical Functions of the Neutrophil Cytoskeleton
Authors: Wheeler J Gary , Winkler Lisa S , Seeds Michael , Bass David , Abramson Jon S
Published: January 2018

Influenza A virus (IAV) has previously been shown to alter chemotactic, oxidative, and secretory functions of polymorphonuclear leukocytes (PMNL). Because of the role of cytoskeletal proteins in these processes, studies were carried out to determine if IAV altered the PMNL cytoskeleton. PMNL were incubated with buffer or IAV, stimulated with f-met-leu-phe (FMLP), fixed and stained with NBD-phallacidin (NBD-Ph) and studied by flow cytometry. Mean F-actin fluorescence was increased 18% in virus treated cells pre-FMLP stimulation and 13% 5 and 10 min post-FMLP (P <.03); no significant difference in F-actin fluorescence was noted in virus treated PMNL 15–30 s post-FMLP compared to control cells. PMNL exposed to the same conditions were solubilized and actin content was determined following SDS-PAGE of triton insoluble precipitates. Increased actin was recovered from virus treated compared to buffer treated cells before and after FMLP stimulation in the 8,000g precipitates (P <.001). Immunofluorescent microscopy studies of F-actin distribution were done in PMNL stained with NBD-Ph following FMLP stimulation for 10 min. These studies showed an increased lamellipod F-actin/uropod F-actin ratio in PMNL pre-incubated with IAV compared to controls (4.6 vs. 1.0; P <.025). Phosphorylation of specific cytoskeletal proteins was examined after immunoprecipitation. IAV alone altered phosphorylation of both vlmentin and vinculin, and in stimulated PMNL virus led to decreased phosphorylation of vimentin and vinculin. These data show distributional and biochemical effects of IAV on PMNL cytoskeletal proteins, indicating additional targets for IAV interference in the PMNL signal-transduction-function process.
Tumor Necrosis Factor–Alpha Gene Expression in Human Whole Blood
Authors: Strieter Robert M , Remick Daniel G , Ham John M , Colletti Lisa M , Lynch Joseph P , Kunkel Steven L
Published: January 2018

Tumor necrosis factor-alpha (TNF) is recognized as a principal mediator of a variety of pathophysiologic and immunologic events. Lipopolysaccharide (LPS) challenge, either in vitro or in vivo, results in significant TNF production. In this study we present data demonstrating LPS-induced TNF mRNA expression and bioactivity using an in vitro tissue system of whole blood (WB). The kinetics of LPS-induced TNF production by WB was significantly accelerated as compared to isolated cultured peripheral blood monocytes (PBM). At post-LPS challenge, plasma from WB demonstrated a rapid rise in TNF bioactivity, peaking by 4 hr (1,021 units/ml/106 cells), plateauing between 4 and 8 hr, and then decreasing over the next 16 hr. In contrast, the highest measured TNF bioactivity from PBM did not occur until the 24-hr time-point (175 units/ml/106 cells). Whole blood buffy-coat TNF mRNA was assessed by Northern blot analysis, and demonstrated significant TNF mRNA accumulation at 1 hr and a peak 2 hr post-LPS challenge. By 8 hr TNF mRNA was undetectable. Concomitant administration of LPS with either prostaglandin E2 (10-6M) or Dexamethasone (10-6M) resulted in significant suppression of LPS-induced TNF production. This data supports WB as a useful in vitro medium for the molecular and cellular analyis of TNF. As specialized connective tissue, WB may provide an important environment to study the pharmacologic manipulation of TNF mRNA and bioactivity.
Quantitative Comparison of Myeloid Antigens on Five Lineages of Mature Peripheral Blood Cells
Authors: Terstappen L W M M , Hollander Z , Meiners H , Loken M R
Published: January 2018

Five-dimensional flow cytometry was used to identify the 5 lineages of peripheral blood leukocytes simultaneously in a single cell preparation. This technique was then used to compare quantitatively the distribution of cell surface antigens on each of these lineages of cells. Neutrophils, eosinophils, basophils, lymphocytes, and monocytes were uniquely identified by correlating their forward and orthogonal light scattering signals with the amount of cell surface-bound IgE. These three cellular characteristics were combined with two additional immunofluorescence labels to create a 5-dimensional space in which each leukocyte population occupied a unique position. The relative quantities of antigens on each cell type were determined for the monoclonal antibodies CD11b, CD13, CD14, CD15, CD16, CD33, CD38, CD45, CD45R, anti-HLA-DR, and anti-Leu-8 labeled with either fluorescein or phycoerythrin. The amount of antigen was described by the mean fluorescence intensity in comparison with the background fluorescence of each cell type. The distribution of the different cell surface antigens on the 5 major leukocyte populations as well as their interdonor variation were then correlated for 10 normal donors. Since none of the antigens studied was lineage specific, it was shown that the different lineages of blood cells could clearly be identified by quantitative comparison of the antigens. This study provides the basis for discrimination between mature cells and immature stages of differentiation of leukocytes and for distinction between normal and leukemic cells.
Evidence That Specific High-Mannose Oligosaccharides Can Directly Inhibit Antigen-Driven T-Cell Responses
Authors: Muchmore Andrew V , Sathyamoorthy Neeraja , Decker Jean , Sherblom Anne P
Published: January 2018

Uromodulin is an 85 Kd immunosuppressive glycoprotein originally isolated from human pregnancy urine. It is unique in that most of its biologic activity can be attributed to attached oligosaccharides. Purified immunomodulatory oligosaccharides from uromodulin have been structurally characterized using 1H-NMR spectroscopy and shown to be Man6–7GlcNAc2(M6,117). Based on these observations, we isolated high-mannose N-type oligosaccharides and glycopeptides from ovalbumin, soybean agglutinin, and yeast mannan and show that these high-mannose compounds directly inhibit in vitro antigen-driven T-cell proliferation from millimolar to nanomolar concentrations. The most active compound was a core mannose oligosaccharide derived from yeast mannan, M9(y), which acts to block early events required for normal antigen processing/presentation. These data emphasize the potential functional role of carbohydrate structure in regulating the human immune response.
Distribution of Neutral Endopeptidase Activity in Human Blood Leukocytes
Authors: Iwamoto Itsuo , Kimura Akira , Ochiai Kenichi , Tomioka Hisao , Yoshida Sho
Published: January 2018

We investigated the distribution of neutral endopeptidase (NEP; EC 3.4.24.11) activity, a possible regulatory enzyme for neuropeptide-induced leukocyte activations, in each cell type of human blood leukocytes. The NEP activity assessed by an NEP inhibitor phosphoramidon-sensitive Met5-enkephalin degrading activity was present in neutrophils and the common acute lymphoblastic leukemia antigen (CALLA)-positive leukemic cells (59 pmol/min/106 cells and 62 pmol/min/105 cells, respectively); however, the NEP activity was virtually absent in lymphocytes, monocytes, eosinophils, basophils, CALLA-negative leukemic cells, or a promyelocytic cell line HL-60. The enzymatic activity was characterized as NEP on the basis of the values of kinetic parameters (Km = 61 μM, Kcat = 1,692 min-1, and Kcat/Km = 28 min-1/μM-1) and the values of IC50 of two NEP inhibitors phosphoramidon and thiorphan (7.4 nM and 8.4 nM, respectively). The distribution of NEP detected immunocytochemically using anti-NEP monoclonal antibodies was also found to be parallel with the distribution of NEP activity among peripheral blood leukocytes.
Neutrophil-Specific Antigen NB1 Is Anchored Via a Glycosyl-Phosphatidylinositol Linkage
Authors: Skubitz Keith M , Stroncek David F , Sun Bo
Published: January 2018

Neutrophil-Specific alloantibodies and the antigens they recognize are important in clinical medicine but little is known about the structure of these antigens. Alloimmunization to the antigen NB1 is a clinically important cause of neonatal neutropenia and leukocyte-mediated transfusion reactions. A novel mechanism of protein attachment to cell membranes involving the covalent linkage of the protein through an oligosaccharide to phosphatidylinositol has recently been defined. Many proteins which are anchored to the cell membrane by this mechanism can be released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC). The 58–64-kDa human neutrophil surface protein which contains the NB1 antigen was labeled with 125I by using lactoperoxidase and examined for PI-PLC sensitivity. The 58–64-kDa protein was specifically released from the cell by treatment with PI-PLC, and the mobility of the protein under non-denaturing conditions using non-ionic detergent was increased by treatment with PI-PLC. Surface expression of the NB1 antigen was slightly up-regulated by treatment with the chemotactic peptide f-met-leu-phe. Removal of N-linked carbohydrates with endoglycosidase-F decreased the apparent molecular weight of the protein to ~45-kDa. The data suggest that most of the 58–64-kDa protein bearing the neutrophil-specific antigen NB1 is anchored to the membrane through a glycosyl-phosphatidylinositol linkage.
Cyclic AMP Inhibits Secretion From Electroporated Human Neutrophils
Authors: Smolen James E , Stoehr Sally J , Kuczynski Barbara
Published: January 2018

It has long been known that Intracellular cAMP inhibits and cGMP enhances intact neutrophil function. However, these effects are modest and require relatively high concentrations of the cyclic nucleotides. We decided to re-examine the effects of cyclic nucleotides on Ca2+-induced secretion by electroporated cells. This system allowed us to bypass normal cell surface receptor-ligand interactions as well as to directly expose the intracellular space to native cyclic nucleotides. We found that concentrations of cAMP as low as 3 μM inhibited Ca2+-induced secretion; 30–300 μM cAMP was maximally inhibitory. cAMP was actually slightly more potent than dibutyryl cAMP, a membrane-permeant derivative. In contrast, cGMP was only slightly stimulatory at 3 μM and modestly inhibitory at 300 μM; dibutyryl cGMP was ineffective. A more detailed investigation of the effects of cAMP showed that inhibition was only obtained in the presence of Mg2+. Half-maximal inhibition by cAMP occurred at 10–30 μM. Inhibition by cAMP was achieved by shifting the Ca2+ dose-response curve for secretion to the right; this was observed for the release of both specific granules (vitamin B12 binding protein) and azurophil granules (B-glucuronidase). We previously showed that ATP could enhance Ca2+-induced secretion in the presence of Mg2+, apparently by interacting with a cell surface purine receptor. However, increasing concentrations of ATP could not overcome inhibition by cAMP; this suggested that cAMP acted at some site other than the purine receptor. Inhibition by cAMP was also less apparent in the presence of the protein kinase C agonist phorbol myristate acetate (PMA), suggesting that the cyclic nucleotide did not produce systemic desensitization of the neutrophils. In summary, these results demonstrate that low, physiologically relevant concentrations of cAMP can modulate neutrophil responsiveness.
Stimulation of Human Peripheral Blood Polymorphonuclear Cell Iodination by Lignin-Related Substances
Authors: Sakagami Hiroshi , Kawazoe Yutaka , Oh-hara Toshinari , Kitajima Ken , Inoue Yasuo , Tanuma Sei-ichi , Ichikawa Shinya , Konno Kunio
Published: January 2018

Lignin is a heterogeneous natural product composed of phenylpropane units and is usually associated with hemicellulose in its native state. Until now little attention has been paid to the potential therapeutic utility of lignified products. Natural lignified products are demonstrated in the present study to stimulate iodination significantly (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood polymorphonuclear cells (PMN). This stimulation was significantly inhibited in the presence of myeloperoxidase inhibitors. These materials were almost completely deprived of their stimulation capacity by treatment with NaCIO2, but this capacity was not affected by severe treatment with H2SO4 or trifluoroacetlc acid. Similar stimulating activity by chemically defined tannin-related polyphenolic compounds was observed. Degradation products or component units of lignin, and natural antitumor polysaccharides and their chemically modified derivatives (introduced with negatively or positively charged groups) and polysialoglycoproteins had little or no activity. The results indicate the importance of a polymerized phenolic structure for the stimulation of PMN iodination. Possible physiological relevance of the stimulation of iodination by lignified substances is discussed.
Tumor Necrosis Factor-α Is Chemokinetic for Lymphokine-Activated Killer Cells: Regulation by Cyclic Adenosine Monophosphate
Authors: Maghazachi Azzam A
Published: January 2018

The ability of tumor necrosis factor-α (TNF-α) to attract lymphokine-activated killer (LAK) cells in vitro was examined. Utilizing modified Boyden chambers (BC), it was observed that TNF-α is not chemoattractant for LAK cells. On the other hand, TNF-α attracted both fresh and concanavalin A-activated T cells. However, when TNF-α was incubated in the upper compartments of BC and in the presence of LAK cells, it enhanced the random movement of these cells across the polycarbonate membranes. The effect of TNF-α was inhibited by incorporating anti-TNF-α antibody, or a high concentration (10 ng) of TFG-β1. The activity of TGF-β1 was reversed by anti-TGF-β1 antibody. Cholera toxin (CT), which is known to activate the endogenous level of cyclic adenosine monophosphate (cAMP) also inhibited TNF-α-induced LAK cell chemokinesis. The effect of CT was mimicked by the cAMP analog dibutyryl cAMP or by the phosphodiesterase inhibitors isobutyl methylxanthine or aminophylline. Measurement of the intracellular level of cAMP showed that cells incubated for 1,2, or 4 hr with TNF-α have a lower level of cAMP, whereas those incubated with a high concentration of TGF-β1 produced significantly higher levels of this messenger. cAMP level was also increased in cells incubated with TGF-β1 plus TNF-α. This level was reduced to the background when anti-TGF-β1 antibody was added to the cultures. These results suggest that cAMP negatively regulates LAK cell chemokinesis.
Translocation of CD45RA in Neutrophils
Authors: Caldwell Charles W , Patterson William P , Yesus Yohannes W
Published: January 2018

The CD45 family of high-Mr glycoprotein antigens is expressed in some molecular form on all lymphohematopoietic cells. Different cell types express various isoforms in a precisely programmed fashion. In addition to cell surface CD45 antigens, we recently demonstrated a cytoplasmic granule-associated pool of CD45RA, the highest Mr isoform, in mature neutrophils that is generally absent from the cell surface under nonstimulatory conditions. Under such conditions, the major cell surface form is CD45RO, the low-Mr isoform. In the present study, we demonstrate the ability of calcium ionophore A23187 to induce translocation of cytoplasmic CD45RA to the cell surface as well as to increase the cell surface expression of CD45RO and CD45. This process was calcium dependent and rapid, occurring within 5 min. A series of experiments using chemical antagonists of protein kinases suggest that this up-regulation may be mediated via the calmodulin system rather than via protein kinase C. Although the exact function(s) of the various isoforms of CD45 is not known, this translocation suggests a role for CD45RA in neutrophil activation.
Activation of the Neutrophil NADPH-Oxidase by Free Fatty Acids Requires the Ionized Carboxyl Group and Partitioning Into Membrane Lipid
Authors: Steinbeck Marla J , Robinson John M , Karnovsky Morris J
Published: January 2018

To investigate NADPH-oxidase activation, we studied the effects of free fatty acid (FFA), their uncharged derivatives, and calcium on membrane lipid structure and superoxide anion (O–2) release from intact neutrophils and in cell-free O–2-generating systems. This study determined that in calcium-free phosphate-buffered saline (PBS), cis-unsaturated FFA (cis FFA), trans-unsaturated FFA (trans FFA), and to a limited extent saturated FFA decreased the polarization of the membrane lipid structure probe 1,6-diphenyl-1,3,5-hexatriene (DPH), and these decreases correlated with partitioning of the FFA into the plasmalemma and the release of O–2 from intact neutrophils and in cell-free preparations. Although a decrease in DPH polarization was always observed under conditions that resulted in the release of O–2, there was not a direct correlation between the amount of decrease in DPH polarization and the release of O–2. Trans FFA did not induce a dose-dependent decrease or as dramatic a decrease in DPH polarization compared with cis FFA, yet the trans FFA stimulated a greater release of O–2 at 2.5, 5, and 10 µM concentrations. In addition, responses of the neutrophil to trans FFA, but not cis FFA, were differentially affected by the presence of calcium. When 0.1 mM calcium was added to the PBS decreases in DPH polarization in response to trans FFA were reduced by >60%, whereas O–2 was reduced by only 25–36%. The addition of 0.1 mM calcium 3 min after the trans FFA had partitioned into the membrane also reversed by 50–65% decreases in DPH polarization but did not affect the release of O–2. In the presence of 0.9 mM calcium, only the cis FFA decreased DPH polarization or stimulated the release of O–2. Calcium is known to interact more readily with the ionized carboxyl group of trans FFA, neutralizing the anionic charge through an electrostatic interaction. In support of the requirement for the ionized carboxyl group, structurally similar uncharged cis, trans, and saturated fatty alcohols; methyl esters; and aldehydes decreased DPH polarization in the absence and presence of 0.9 mM calcium; however, none of these compounds stimulated the release of O–2. These results indicate that, in addition to the partitioning of FFA into the plasmalemma, which results in calcium-modulatable decreases in DPH polarization, activation or assembly of the NADPH-oxidase requires the ionized carboxyl group.
Intracellular Localization of a Lipopeptide Macrophage Activator: Immunocytochemical Investigations and EELS Analysis on Ultrathin Cryosections of Bone Marrow-Derived Macrophages
Authors: Uhl B , Wolf B , Schwinde A , Metzger J , Jung G , Bessler W G , Hauschildt S
Published: January 2018

Synthetic lipopeptides, structurally derived from the N-terminal part of bacterial lipoprotein, constitute macrophage and B-lymphocyte activators. The molecular mechanism of macrophage activation by lipopeptides still remains unclear. The purpose of our study was to determine the route and kinetics of lipopeptide distribution in bone marrow-derived macrophages. The intracellular localization of the C-terminally biotinylated lipodipeptide Pam3Cys-Ser was investigated on ultrathin cryosections using the biotinstreptavidin-gold system. Our findings indicate that the lipopeptide penetrates the plasma membrane and can already be found within the cytoplasm, the nuclear membrane, and within the nucleus after 2 min of stimulation. The pattern of lipopeptide distribution obtained 2 min after stimulation resembles that obtained after longer incubation times (8 and 20 min). Correlating distribution patterns were observed when using the method of electron energy loss spectroscopy (EELS). These findings are a clear indication for the rapid uptake of lipopeptides into eukaryotic cells, and are of importance for further studies of the immunostimulating properties of the bacterial lipopeptides and vaccines derived therefrom.
Mononuclear Cell Adherence Induces Neutrophil Chemotactic Factor/Interleukin-8 Gene Expression
Authors: Kasahara Keita , Strieter Robert M , Chensue Stephen W , Standiford Theodore J , Kunkel Steven L
Published: January 2018

The accumulation of polymorphonuclear cells (PMN) in tissue is an essential element of the inflammatory response that is important in host defense. Adherence to endothelium constitutes the first step in PMN migration from the vascular compartment to the interstitium. We demonstrate that human peripheral blood mononuclear cells (PBMC) adherent to plastic can result in expression of interleukin-8 (IL-8), a potent PMN chemoattractant and activating cytokine. Northern blot analyses showed PBMC adherent to plastic expressed IL-8 steady-state mRNA levels by 30 min, peaked at 8 h, and then decreased over the next 16 h. In contrast, nonadherent PBMC (cultured in teflon chambers) expressed less than 25% of the maximal IL-8 steady-state mRNA levels as compared with adherent PBMC. Adherent PBMC-associated IL-8 determined by immunohistochemistry, supernatant chemotactic bioactivity, and extracellular antigenic IL-8 paralleled IL-8 mRNA expression. Antigenic and bioactive IL-8 were significantly apparent by 4–8 h, respectively, and increased significantly to maximal levels by 24 h. Furthermore, adherent PBMC IL-8 gene expression was suppressed by either concomitant treatment with actinomycin-D or cycloheximide, yet specific neutralizing antibodies directed against either IL-1β or tumor necrosis factor (TNF)-α failed to alter adherence-induced steady-state IL-8 mRNA levels. These data support the hypothesis that PBMC adherence is an important signal for the production of IL-8, and may be essential to the development of the inflammatory response through the elicrtation of PMN.
Porcine Alveolar and Pulmonary Intravascular Macrophages: Comparison of Immune Functions
Authors: Chitko-McKown C G , Chapes S K , Brown R E , Phillips R M , McKown R D , Blecha F
Published: January 2018

Although a substantial amount of information is available on pulmonary alveolar macrophages (PAMs), little is known about pulmonary intravascular macrophages (PIMs), a separate population of lung macrophages found apposed to the endothelium of pulmonary capillaries. We compared these two populations of lung immunocytes to determine their relative immunological activity. Our results suggest that PAMs are more phagocytic than PIMs; however, PIMs may be more efficient at lysing ingested bacteria than PAMs. Although similar in antibody-dependent cellular cytotoxicity, PIMs are more spontaneously cytolytic than PAMs. Depending upon the effector:target cell ratio studied, the tumoricidal activity of PIMs was less than or equal to that of PAMs. Both cell populations produced the cytokines interleukin-1 and tumor necrosis factor-α at similar concentrations. These data suggest that PIMs are immunologically active, although the degree of activity may differ between PIMs and PAMs.
Adhesion of Human Neutrophils to and Activation by Type-I Collagen Involving a β2 Integrin
Authors: Monboisse Jean-Claude , Garnotel Roselyne , Randoux Alain , Dufer Jean , Borel Jacques-Paul
Published: January 2018

We previously demonstrated that the α1(l) polypeptide chain of collagen can bind and activate polymorphonuclear neutrophils (PMN). In the present experiments, performed in culture grade 96-well plastic plates coated with collagen, fibronectin, or other proteins, adhesion was assessed by staining the adhering cells after 30 min with crystal violet and measuring absorbance at 560 nm, and activation of PMNs was assessed by measuring the amount of O2- formed. Adhesion occurred at 17 and 37°C but activation at 37°C only. Monoclonal antibody anti-CD 18 inhibited adhesion, showing that the receptor of collagen I on PMNs is a β2 integrin. On the other hand, adhesion of PMNs to fibronectin was inhibited by monoclonal antibodies to CD18 and to CD11b.
Lipoxin-induced migration of fish leukocytes
Authors: Sharp Gregory J E , Pettitt Trevor R , Rowley Andrew F , Secombes Christopher J
Published: January 2018

The migration-inducing abilities of both synthetic lipoxin A4 (LXA4) and leukotriene B4 (LTB4) for rainbow trout neutrophils were examined with an in vitro assay. LXA4 caused enhanced migration of these cells with a three- to fourfold greater response than that observed toward LTB4 at most concentrations tested. Checkerboard assays showed that synthetic LXA4 was chemokinetic for trout leukocytes, while LTB4 was chemotactic. The chemokinetic ability of LX synthesized by rainbow trout macrophages maintained in short-term culture was also determined. These cells produced both 4-and 5-series LX, namely LXA4, LXA5, 11-trans-LXA4, 11-trans-LXA5, 7-cis-11-trans-LXA4 and 6(S)-LXA4. Although greater migration was found to LXA4 than to most of the other 4-series positional isomers, at least at some concentrations, this did not exhibit the degree of stereospecificity previously reported in some studies for mammalian leukocytes. Little difference was found between the chemokinetic responses of rainbow trout leukocytes to 4- and 5-series LX. These findings suggest that LXs are important migration-inducing molecules in trout and have a relatively long evolutionary history.
Anomalous features of human neutrophil activation by influenza A virus are shared by related viruses and sialic acid–binding lectins
Authors: Hartshorn Kevan L , Daigneault David E , White Mitchell R , Tauber Alfred I
Published: January 2018

Influenza A virus (IAV) causes both activation and deactivation of the human neutrophil, which may, respectively, contribute to host defense against the virus and enhanced susceptibility to bacterial superinfection. We have shown that certain features of neutrophil activation by IAV are distinctive compared with activation by chemoattractants in terms of both the stoichiometry of the respiratory burst response and the signal transduction events that precede it. We here demonstrate that related myxoviruses as well as sialic acid–binding lectins elicit a respiratory burst response similar to that induced by IAV, in which hydrogen peroxide is formed with minimal accompanying superoxide generation. Brief preincubation of neutrophils with these agents fully inhibits subsequent activation by IAV, implying that they are binding to the same surface membrane components as IAV. Preincubation with Limax flavus agglutinin (LFA) does, in fact, substantially reduce binding of radiolabeled IAV to the neutrophil. This lectin, like IAV, both activates and deactivates the neutrophil. As in the case of IAV, LFA-induced activation (1) is mediated via stimulation of phospholipase C, (2) is pertussis toxin insensitive, and (3) entails a lesser contribution of calcium influx than is the case for chemoattractants.
Urokinase expression in mononuclear phagocytes: cytokine-specific modulation by interferon-γ and tumor necrosis factor-α
Authors: Gyetko Margaret R , Shollenberger Susan B , Sitrin Robert G
Published: January 2018

This study delineates the regulatory effects of inflammatory cytokines on mononuclear phagocyte plasminogen activator (PA) activity. The mechanisms by which mononuclear phagocytes modulate PA activity are described. Mononuclear phagocytes regulate net PA activity by the balanced expression of urokinase-type PA (uPA), in either secreted or membrane-associated forms, and a specific plasminogen activator inhibitor, PAI-2. Therefore, understanding how immunomodulators regulate macrophage PA activity requires that the comparative effects of uPA and PAI-2 be elucidated. We determined how recombinant interferon-γ (IFN) and tumor necrosis factor-α (TNF) regulate plasminogen activation in monoblast-like U937 cells and normal human monocytes. In U937 cells, both IFN and TNF induced concurrent increases in secreted PA and PA inhibitor activities. These effects were accompanied by increased immunoreactive uPA and PAI-2 in conditioned media (enzyme-linked immunosorbent assay) and steady-state levels of cellular uPA and PAI-2 mRNA (Northern analysis). To determine the relative abilities of IFN and TNF to either promote or inhibit plasmin generation, we directly compared the effects IFN and TNF, using optimal stimulating concentrations. IFN induced PA activity to 180% of the level achieved by TNF. In contrast, IFN elicited only 78% of the PA inhibitor produced by TNF stimulation. These differences in secreted activity can be explained by the shift in balance between uPA and PAI-2 proteins. Immunoreactive uPA was induced equally by IFN and TNF, but TNF generated higher levels of PAI-2. The same overall pattern of results was seen in normal human monocytes. IFN and TNF differ greatly in the ability to augment receptor-bound PA activity in U937 cells, as IFN induced a twofold increase but TNF had no effect. We conclude that IFN and TNF modulate mononuclear phagocyte proteolytic activity through coordinate regulation of secreted and receptor-bound uPA, balanced against concurrent expression of PAI-2. These effects are cytokine specific, as IFN is superior to TNF in stimulating expression of both secreted and receptor-associated PA activities. These properties suggest mechanisms by which mononuclear phagocytes control proteolysis in cytokinerich inflammatory foci.
Interferon α-induced changes in FcγR-specific mRNA expression and isotype-specific, FcγR-mediated phagocytosis in C3H/OuJ (Lpsn) and C3H/HeJ (Lpsd) macrophages
Authors: Fultz Marion J , Vogel Stefanie N
Published: January 2018

Peritoneal exudate macrophages from C3H/OuJ (Lpsn) and C3H/HeJ (Lpsd) mice were treated with interferon α (IFN-α), and isotype-specific phagocytosis and gene expression were measured. IFN-α selectively augments phagocytosis of IgG2a, with a highly significant induction of FcγRI mRNA expression, while parallel FcγRIIIα measurements show a relatively small increase.
Charcot-Leyden crystal protein in the degranulation and recovery of activated basophils
Authors: Golightly Linnie M , Thomas Larry L , Dvorak Ann M , Ackerman Steven J
Published: January 2018

The Charcot-Leyden crystal (CLC) protein, a prominent cell constituent unique to eosinophils and basophils, possesses lysophospholipase activity. This activity and the extracellular deposition and formation of CLC in tissues and body fluids in association with eosinophils suggest an extracellular function for this protein in inflammation. During degranulation, basophils release granule-derived mediators of inflammation. We postulated that CLC protein, localized in part to the basophil granule, might be released along with other mediators during this process. The extracellular release of CLC protein was studied during the degranulation of basophils stimulated by anti-immunoglobulin E (anti-IgE), N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate, eosinophil major basic protein (MBP), and calcium ionophore A23187. Histamine release was used as a marker of basophil degranulation; its release was measured utilizing the fluorometric technique. CLC protein was not released into the supernatant during this process as determined by radioimmunoassay. CLC protein in the extracellular space, either as intact crystals or aggregates, was undetectable by indirect immunofluorescent staining of basophils activated with either anti-IgE or fMLP. However, upon activation, the immunofluorescent cytoplasmic and nuclear staining pattern for CLC protein was significantly altered. Decreased cytoplasmic staining and persistent or increased nuclear staining for CLC protein were observed after activation, with recovery of the preactivation, unstimulated cellular staining pattern at 30 and 45 min after stimulation with fMLP and anti-IgE, respectively. These findings suggest that CLC protein functions intracellularly in basophils during the process of activation, degranulation, and recovery. The potential nuclear function(s) of this lysophospholipase in the basophil requires further investigation.
C5a and formyl peptide receptor regulation on human monocytes
Authors: van Epps Dennis E , Simpson Stacy J , Chenoweth Dennis E
Published: January 2018

Regulation of C5a and formyl-methionine-leucine-phenylalanine-lysine (fMLPL) receptors on human monocytes has been studied using fluorescein-conjugated derivatives and flow cytometry. Monocytes have receptors for each of these ligands, as evidenced by their ability to bind specifically biologically active fluorescein derivatives of these ligands. Quenching experiments showed that bound fluoresceinated C5a and fMLPL are rapidly internalized at 37°C. Once internalized, monocytes are able to reexpress these receptors, returning to control levels within approximately 90 min. This contrasts with rate differences seen in polymorphonuclear neutrophils (PMNs), where fMLPL receptors return more rapidly (~ 30 min) than do C5a receptors (~ 100 min). Monensin inhibited the reexpression of C5a but not fMLPL receptors, suggesting that a receptor recycling process is necessary to replenish C5a receptors on the monocyte surface. Similar although less efficient inhibition of C5a receptor reexpression was observed with NH4Cl treatment. Reexpression of both G5a and fMLPL receptors was independent of extracellular Ca2+. Treatment with various agents known to stimulate monocytes and PMNs increased the expression of fMLPL receptors in both cell types but either had no effect on or reduced the level of C5a receptor expression. This would indicate that monocytes, like PMNs, have intracellular pools of preformed fMLPL receptors, available for reexpression. These studies show that, like PMNs, monocytes modulate C5a and fMLPL receptors through different mechanisms. Furthermore, monocytes are capable of reexpressing these receptors following exposure to ligand, a theoretical requirement for chemotaxis.
Effect of circulating neutrophil depletion on lung injury induced by inhaled silica particles
Authors: Gavett Stephen H , Carakostas Michael C , Belcher Leigh A , Warheit David B
Published: January 2018

Polymorphonuclear leukocytes (PMNs) recruited into the alveolar region during inflammation may injure the lung parenchyma by releasing cytotoxic oxygen radicals and proteases. Because brief exposures to crystalline silica elicit recruitment of PMNs into the alveolar region, which is strongly correlated with parameters of cytotoxicity, increased alveolar epithelial permeability, and lysosomal enzyme release, we sought to evaluate the potential role of PMNs in silica-induced lung injury. Rats were depleted of PMNs by administration of an anti-rat PMN antiserum prior to exposure to silica. Pulmonary inflammatory responses to silica in this group were compared to responses in normal silica-exposed rats as well as sham-exposed normal or PMN-depleted rats. Bronchoalveolar lavage fluids from normal, silica-exposed rats contained 9.7 × 106 PMNs immediately after exposure for 3 days, compared to 0.01 × 106 PMNs for both normal or PMN-depleted, sham-exposed rats. Bronchoalveolar lavage fluids from successfully PMN-depleted, exposed rats contained significantly fewer (0.7 × 106) PMNs compared to normal silica-exposed rats. In both groups of silica-exposed rats, a variety of biochemical indicators of lung injury were increased significantly compared to measurements from both sham-exposed groups, but there were no differences between PMN-depleted and normal silica-exposed groups. The results suggest that recruitment of PMNs into the alveolar region is not a necessary prerequisite for the observed increases in biochemical indicators of silica-induced acute lung injury.
Chondroitin sulfate proteoglycan and heparan sulfate proteoglycan production by cultured pigeon peritoneal macrophages
Authors: Owens Rick T , Wagner William D
Published: January 2018

Proteoglycan production was examined in cultures of thioglycollate-elicited peritoneal macrophages obtained from White Carneau and Show Racer pigeons. Following a 24-h incubation in the presence of [35S]sulfate and [3H]serine, total production and distribution of 35S-labeled proteoglycan into media (60–65%), pericellular (21–27%), and intracellular (13–14%) compartments was similar in White Carneau and Show Racer macrophage cultures. Media proteoglycans consisted of high-molecular-weight chondroitin sulfate proteoglycan, low-molecular-weight chondroitin sulfate proteoglycan, and heparan sulfate proteoglycan. High-molecular-weight chondroitin sulfate proteoglycan was predominantly 6-sulfated (80%) and contained a core protein larger than 200 kd, whereas low-molecular-weight chondroitin sulfate proteoglycan was 4-sulfated and contained a 28-kd core protein. Pericellular proteoglycan was similar in size to low-molecular-weight proteoglycan and consisted of a predominantly 6-sulfated (75%) chondroitin sulfate proteoglycan and heparan sulfate proteoglycan. Intracellular 35S-labeled chondroitin sulfate and heparan sulfate were smaller than media and pericellular proteoglycans, suggestive of intracellular degradative processing.
Listericidal and nonlistericidal mouse macrophages differ in complement receptor type 3-mediated phagocytosis of L. monocytogenes and in preventing escape of the bacteria into the cytoplasm
Authors: Drevets Douglas A , Canono Beth P , Campbell Priscilla A
Published: January 2018

Listeria monocytogenes is a facultative intracellular bacterium that escapes phagocytic vesicles and replicates in the cytoplasm, where it becomes coated with F-actin. Macrophages, important anti-Listeria effector cells, are heterogeneous in their ability to kill Listeria. Complement receptor type 3 (CR3) mediates most phagocytosis of Listeria by listericidal macrophages. Experiments described here tested whether nonlistericidal macrophages also phagocytosed Listeria through CR3 and whether the ability of Listeria to escape into the cytoplasm correlated with lack of listericidal activity. We show here that CR3 mediated an average of 66% of the phagocytosis of serum-opsonized Listeria by listericidal peptone-elicited macrophages but only 35% by nonlistericidal thioglycolate-elicited macrophages. In thioglycolate-elicited macrophages, most Listeria were cytoplasmic and actin coated, whereas in peptone-elicited macrophages most were retained in the phagosome. These results indicate that listericidal and nonlistericidal macrophages phagocytose Listeria through different receptors and that nonlistericidal macrophages allow Listeria to escape into the cytoplasm.
Effects of phenobarbital on leukocyte activation: membrane potential, actin polymerization, chemotaxis, respiratory burst, cytokine production, and lymphocyte proliferation
Authors: Yang Kuender D , Liou Wen-Yu , Lee Chung-Shinn , Chu Meng-Ling , Shaio Men-Fang
Published: January 2018

Leukocyte activation is known to involve cell membrane potential changes. Phenobarbital, an anesthetic and anticonvulsant that can inhibit neuronal membrane depolarization, may also affect leukocyte activation. Measuring membrane potential, actin polymerization, chemotaxis, superoxide production, lymphocyte proliferation, intracellular calcium concentration, and cytokine production, we found that phenobarbital at a concentration of 15–30 μg/ml, which is considered a therapeutic serum level for controlling seizures, did not affect polymorphonuclear neutrophil (PMN) activation. At levels higher than 100 μg/ml, phenobarbital significantly suppressed formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. Concentrations greater than 300 μg/ml also inhibited phorbol myristate acetate–stimulated membrane potential change. In contrast, 30 fig/ml phenobarbital significantly inhibited lymphocyte proliferation stimulated by phytohemagglutinin (PHA) and pokeweed mitogen. This concentration of phenobarbital also suppressed the increase of intracellular free calcium induced by PHA. However, only a higher concentration of phenobarbital (300 μg/ml) was able to inhibit PHA-induced interleukin-2 (IL-2) production and suppress the proliferation of PHA-induced IL-2 receptor–bearing lymphocytes. These results suggest that concentrations of phenobarbital associated with anticonvulsive levels do not affect PMN activation but suppress lymphocyte activation, possibly by affecting intracellular signal transduction.
Interferon-γ and Interleukin-4 down-regulate soluble CD14 release in human monocytes and macrophages
Authors: Landmann Regine , Fisscher Ada E , Obrecht Jean-Paul
Published: January 2018

CD14 is a 53-kd glycoprotein that is mainly expressed in myeloid cells and exists in two forms. The membrane-bound form represents the receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein. The function and regulation of the soluble form are unknown. In the present study we investigated the release of soluble CD14 (sCD14) in cultures of human mononuclear leukocytes, elutriated monocytes, and monocyte-derived macrophages. The release of sCD14 into the medium of the cells cultured for 15 and 45 h was investigated in the absence or presence of selected cytokines. sCD14 release occurred constitutively and correlated with cell number. In monocytes differentiating into macrophages, cumulative release of sCD14 was linear from day 1 to day 7. Spontaneous sCD14 release after 15 h of culture (2 × 106 cells/ml) was higher in the supernatant from monocytes (314 ± 58 ng/ml) than that from mononuclear leukocytes (68 ± 10 ng/ml) and similar to that from macrophages (469 ± 79 ng/ml). Cycloheximide and actinomycin D inhibited sCD14 release. Recombinant interferon-γ (rIFN-γ) and recombinant interleukin-4 (rIL-4) directly decreased sCD14 release in mononuclear leukocyte, monocyte, and macrophage cultures. rIL-2 and rIFN-α reduced sCD14 release into the supernatants of mononuclear leukocytes only. Use of anti-IFN-γ antibodies indicated that the down-regulation of sCD14 release by rIL-2 and rIFN-α was partially due to induction of endogenous IFN-γ. The down-regulation of sCD14 release by all four cytokines was both time and dose dependent. rIFN-γ and rIL-4 added simultaneously had a synergistic effect on sCD14 down-regulation. In conclusion, sCD14 release may have an immunomodulatory role in circulating monocytes, is apparently not related to the process of macrophage differentiation, and is selectively down-regulated during an immune response when levels of IFN-γ and IL-4 are high.
Mepacrine inhibits fMLP-induced activation of human neutrophil granulocytes, leukotriene B4 formation, and fMLP binding
Authors: Földes-Filep Éva , Filep János G
Published: January 2018

Pulmonary sequestration and activation of polymorphonuclear leukocytes (PMNLs) are characteristic of many forms of acute lung injury. The present experiments were designed to study the effects of mepacrine on human neutrophils challenged with N-formylmethionyl- leucyl-phenylalanine (fMLP). Mepacrine inhibited fMLP-induced superoxide production and degranulation in a dose-dependent manner with Kd values of 2.3 ± 0.5 × 10−7 M and 5.7 ± 1.3 × 10−6 M, respectively. Stimulation of PMNLs by 10−6 M fMLP provoked the formation of barely detectable amounts of leukotriene B4 (LTB4) (< 5 pg/107 cells). Pretreatment of the cells with cytochalasin B augmented generation of LTB4 in response to fMLP (339 ± 79 pg/107 cells). LTB4 formation was also inhibited by mepacrine (50% inhibitory concentration 1.0 ± 0.5 × 10−6 M). Furthermore, mepacrine inhibited the specific binding of [3H]fMLP to neutrophils with a Ki value of 1.4 ± 0.4 ˟ 10”5 M. Mepacrine decreased the receptor binding affinity without altering the number of receptors. These findings demonstrate that the inhibitory effect of mepacrine is response dependent and suggest that this action of mepacrine could, in part, be attributed to a decrease in fMLP receptor affinity.