bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Induction of Hepatocyte Synthesis of the Acute Phase Reactant Mouse Serum Amyloid P-Component by Macrophages and IL 1
Authors: Le Phong T , Mortensen Richard F
Published: January 2018

The in vitro culture conditions for the induction and synthesis of the mouse acute phase reactant, serum amyloid P-component (SAP), were established using isolated hepatocytes. SAP synthesis was five to eight times greater with hepatocytes isolated from mice during the acute phase of inflammation vs. hepatocytes obtained from untreated mice. The induction of SAP synthesis in normal hepatocytes for LPS-unresponsive mice was macrophage dependent. Activated macrophages provided the most “helper” activity for SAP production. Partially purified mouse IL 1 from the P388D1 macrophage line also induced SAP synthesis. Only four IL 1 units/ml were required for optimal SAP induction. The addition of IL 1 in the presence of elicited macrophages provided an additive effect on hepatocyte SAP synthesis. The SAP-inducing activity of IL 1 copurified with its thymocyte-stimulating activity and was associated with a 11 to 25-Kd MW polypeptide. Phenylglyoxal treatment of IL 1 inactivated its thymocyte stimulating activity but not its SAP inducing potential. Inhibition of m-RNA synthesis, protein synthesis, N-glycosylation, and protein secretion effectively prevented in vitro hepatocyte SAP production.
Antioxidant Effects on Cell-Mediated Immunity
Authors: Meeker Harry C , Eskew Mary Lou , Scheuchenzuber William , Scholz Richard W , Zarkower Arian
Published: January 2018

Experiments were performed to determine the effects of dietary selenium and/or vitamin E deficiency on cell-mediated cytotoxicity in the mouse. Natural killer cell-mediated cytotoxicity (NKCC) was depressed after 8 wk on diets deficient in selenium and/or vitamin E. In contrast, antibody-dependent cell-mediated cytotoxicity (ADCC) was not affected by 8 wk of dietary deficiency of selenium and/or vitamin E. T-lymphocyte-mediated cytotoxicity (TCMC) was found to be depressed by combined selenium-vitamin E deficiency after 7 weeks on diets.
1α,25 Dihydroxyvitamin D3 and Mononuclear Phagocytes: Enhancement of Mouse Macrophage and Human Monocyte Hydrogen Peroxide Production Without Alteration of Tumor Cytolysis
Authors: Gluck W Larry , Weinberg J Brice
Published: January 2018

1α,25 dihydroxyvitamin D3 (1,25 D3) is known to interact with hematopoietic cells. The purpose of this study was to determine the effect of 1,25 D3 on hydrogen peroxide (H2O2) production and tumor cell killing by mouse peritoneal macrophages and human blood monocytes. Enhanced monocyte and macrophage phorbol myristate acetate (PMA)-stimulated H2O2 production was observed at concentrations of 0.13 to 130 nM 1,25 D3 and and was maximal at 1.3 nM. At concentrations of 100 U/ml, gamma interferon (IFN-γ) alone had a similar effect but, in combination with 1,25 D3, there was no cooperative effect. At concentrations ranging from 0.13 to 130 nM, 1,25 D3 failed to augment tumor cell lysis by macrophages from peptone-injected normal or bacillus Calmette-Guerin (BCG)-infected mice, or by blood monocytes from normal humans. Our results indicate that 1,25 D3 can activate the monocyte and macrophage for H2O2 secretion without concomitant activation for tumor cell killing.
Inhibition of NK and ADCC Activity by Antibodies Against Purified Cytoplasmic Granules From Rat LGL Tumors
Authors: Reynolds Craig W , Reichardt Della , Henkart Maryanna , Millard Paul , Henkart Pierre
Published: January 2018

Highly purified preparations of cytoplasmic granules from transplantable rat large granular lymphocyte (LGL) tumor lines (rat natural killer (RNK) tumors) were used to immunize rabbits. Antibodies from these animals gave two precipitin lines with granule extracts in Ouchterlony experiments. They reacted with at least four different bands on nitrocellulose blots of SDS gels of LGL granule proteins. By immunofluorescence, specifically adsorbed antigranule antibodies did not recognize LGL or T cell surface antigens but reacted with the cytoplasmic granules in permeabilized RNK tumor cells as well as with normal rat LGL. These same antisera showed little or no reactivity with a panel of other cells, including peripheral blood T cells, thymocytes, macrophages, and EL-4 tumor cells. F(ab′)2 preparations of these antigranule antibodies completely blocked granule-mediated lysis of both SRBC and nucleated targets, while control F(ab′)2 preparations from rabbits immunized with EL-4 granules or TNP-KLH showed no significant inhibition of this cytolytic activity at the same antibody concentration. Anti-granule F(ab′)2 preparations specifically inhibited (>75%) rat natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in a dose-dependent manner but did not effect cytotoxic T cell activity. Pretreatment of either effectors or targets by these antibodies had no effect. Anti-granule F(ab′)2 preparations, at concentrations showing strong inhibition of lysis, did not inhibit the binding of LGL to YAC-1 or Ab-coated P815 targets. These results demonstrate that a granule component(s) is necessary for the lytic activity of LGL in both NK and ADCC and provide the first direct evidence that a secretory event involving these granules is part of the lytic process.
Pulmonary Interstitial Macrophages: Isolation and Flow Cytometric Comparisons With Alveolar Macrophages and Blood Monocytes
Authors: Dethloff L A , Lehnert B E
Published: January 2018

Pulmonary interstitial macrophages (IM) were isolated from rat lungs by an Fcγ receptor-based affinity technique coupled with multiparameter flow cytometry. Single cell suspensions obtained by collagenase digestion of extensively perfused and lavaged lungs were applied to carpets of opsonized sheep red blood cells (SRBC-IgG) bound to plastic tissue culture flasks. At 0-4°, optimal binding of lung cells occurred within 60 min at plating densities of 1-2 × 106 lung cells/cm2 when the SRBC substrate was opsonized with 10 μg/ml anti-SRBC IgG. Nonadherent cells were removed by gently rinsing the plates and adherent cells were recovered by lysing the SRBC-IgG substrata. By light microscopy, the mixture of adherent cells was comprised of mononuclear cells (~ 54%), many of which appeared to be macrophages, lymphocytes (~20%), polymorphonuclear leukocytes (~15%), plasma cells (~8%), eosinophils (~2%), and mast cells (~0.5%). The cells which adhered to the SRBC-IgG monolayers were further resolved into subpopulations by multiparameter flow cytometry and sorted according to their electro-optical characteristics. One subpopulation appeared morphologically to be macrophages, and >90% of these cells readily phagocytized SRBC-IgG in vitro. Peroxidase staining of this population was minimal, indicating that these cells were not blood monocytes (BM). Using a method by which alveolar macrophages (AM) were prelabeled with SRBC-IgG in situ, we demonstrated that alveolar macrophages constituted only ~5% of the total adherent cell population. We concluded from these observations that the macrophage population harvested in this manner were neither BM nor AM, but, rather, were harvested from the lung’s interstitial compartment. Flow cytometric analyses indicated that the IM exhibited electro-optical characteristics intermediate between those of BM and AM, which is consistent with the concept of the lung’s interstitium as a maturation compartment for the BM prior to migration into the alveolar compartment. However, the IM more closely resembled the BM than the AM, indicating that if the IM is in fact a precursor to the AM population, substantial maturation or differentiation must occur subsequent to its migration into the alveolar compartment. This isolation technique will be useful for harvesting highly purified IM for in vitro investigations.
Phorbol Myristate Acetate Induced Oxidation of 2′,7′-Dichlorofluorescin by Neutrophils From Patients With Chronic Granulomatous Disease
Authors: Hassan Nassef F , Campbell Donald E , Douglas Steven D
Published: January 2018

The oxidative metabolic burst of stimulated human polymorphonuclear leukocytes (PMNs) has been evaluated by the measurement of oxygen consumption, chemiluminescence, and oxygen radicals (O2-, H2O2,OH-) derived from activation of the hexose monophosphate shunt (HMPS). PMNs from patients with chronic granulomatous disease (CGD) are shown to lack functional NADPH oxidase and undetectable oxygen radical generation. However, using single cell analysis by flow cytometry and 2′,7′-dichlorofluorescin (DCFH) oxidation by H2O2, significant DCFH oxidation by the PMA stimulated CGD PMNs was observed. Furthermore, 1mM potassium cyanide enhanced DCFH oxidation by control and CGD PMNs. DCFH oxidation by cells from an obligate heterozygous mother of an X-linked CGD patient was intermediate. These observations suggest that a PMA induced oxidase enzyme is present in CGD cells.
The Human Promyelocytic Leukemia Cell (HL-60 Cell) β-Adrenergic Receptor
Authors: Sager G , Bang B E , Pedersen M , Aarbakke J
Published: January 2018

We have characterized the β-adrenergic receptor binding site and the β-adrenergic cAMP response of the HL-60 cell. The hydrophilic ligand [3H]-(–)-CGP-12177 was specifically and reversibly bound to one single class of binding sites (Kd 220 pM and Bmax 1,970 sites/cell). The adrenergic agonists inhibited the specific radioligand binding. The order of potency was isoproterenol > epinephrine > norepinephrine. The β-2 selective antagonist ICI 118551 had a binding affinity 3 orders of potency higher than the β-1 selective antagonist, atenolol. The adrenergic agonists elevated the cAMP accumulation in a concentration-dependent mode. The order of potency was isoproterenol > epinephrine > norepinephrine. Both the binding and the functional studies revealed stereospecificity and reversibility. The present data show that HL-60 cells possess β-2 adrenergic receptors functionally coupled to adenylate cyclase.
Modulation of c-fms Proto-Oncogene Expression in Human Blood Monocytes and Macrophages
Authors: Radzun Heinz J , Kreipe Hans , Heidorn Klaus , Parwaresch Mohammad R
Published: January 2018

The gene product of the c-fms proto-oncogene is a transmembrane protein with tyrosine-kinase activity that is obviously related to the receptor for the colony-stimulating-factor CSF-1. By Northern blot analysis, we investigated the expression of the cellular counterpart of v-fms in purified normal human blood mononuclear cells and different macrophage populations. The proto-oncogene c-fms expression was demonstrable in blood monocytes but not in blood lymphocytes. Short-term cultivated blood monocytes exhibited an increased expression of c-fms in comparison to freshly isolated blood monocytes, possibly due to a temporary down regulation of c-fms during the separation procedure of blood monocytes. A comparably high rate of fms-RNA expression was found in most of the analyzed samples of resident peritoneal macrophages, while resident alveolar macrophages showed a considerably lower level of c-fms expression. In this, alveolar macrophages resembled long-term cultivated adherent blood monocytes, which showed a down regulation of c-fms expression. By correlating these data obtained by Northern blot analysis with phenotypic properties of the analyzed monocyte/macrophage populations, it is concluded that different levels of c-fms expression in monocytes/macrophages correspond to their stage of differentiation and maturity.
3-Deaza-Adenosine Inhibition of Stimulus-Response Coupling in Human Polymorphonuclear Leukocytes
Authors: Fantone Joseph C , Duque Ricardo E , Davis Bruce H , Phan Sem H
Published: January 2018

In an effort to define better the functional role of S-adenosyl-methionine-mediated methylation reactions in modulating polymorphonuclear (PMN) functional responses to chemotactic stimuli, we investigated the effects of 3-deaza-adenosine (3-DZA), a known inhibitor of methylation reactions in phagocytic cells, on formyl methionyl-leucyl-phenylalanine (FMLP)-induced responses in human PMN leukocytes. Using the fluorescent cyanine dye 3,3′-dipropylthiocarbocyanine (di-S-C3-(5)) as an optical probe of membrane potential we observed that 3-DZA at concentrations that inhibit FMLP-induced O2− production does not significantly alter FMLP-induced changes in transmembrane potential. Additional studies showed an inhibitory effect of 3-DZA on FMLP-induced PMN pinocytosis and to a lesser degree on FMLP-induced degranulation. However, pretreatment of PMNs with 3-DZA did not alter FMLP-induced changes in Quin-2 fluorescence, an indicator of changes in intracellular calcium levels. These findings demonstrate a dissociation between chemotactic factor-induced cell membrane depolarization, changes in intracellular calcium, and specific neutrophil functional responses and suggest that chemotactic factor-induced changes in transmembrane potential and intracellular calcium are independent of chemotactic factor-induced methylation reactions. Furthermore, 3-DZA did not alter phorbol myristate acetate induced O2− production or fluid pinocytosis indicating a stimulus specificity for the inhibitory effects of this agent on O2− production.
Endocytic and Secretory Repertoire of the Lipid-Loaded Macrophage
Authors: Montgomery Ruth R , Cohn Zanvil A
Published: January 2018

We have studied the effects of intracellular lipid storage on macrophage function. Mouse peritoneal macrophages were lipid loaded by three regimens modeling loading through the scavenger receptor [acetylated low density lipoprotein (Ac-LDL) cells], by extracellular matrix-bound LDL [low density lipoprotein complexed with dextran sulfate (DS-LDL) cells], and by conditions of reduced cholesterol acceptors in the medium [low serum, oleic acid, Ac-LDL (LS/OI) cells]. Significantly increased cholesterol levels in all three regimens were measured by cholesterol determination and Oil Red O staining of fixed cells. Upid-laden cells were equal to control macrophages in binding and ingesting immunoglobulin-coated sheep erythrocytes, reflecting Fc-mediated endocytosis. The lipid-laden cells were compared to control cells for secretory functions of macrophages that could be important in the atherosclerotic artery. They were still capable of producing all secretory products examined, but the quantities of H2O2 and arachidonic acid metabolites were reduced in some cases, and fibrinolytic activity appeared to be increased. Western blot analysis showed a five-fold increase in the release of tumor necrosis factor by DS-LDL-loaded cells. We suggest that the location of intracellular lipid pools as well as the type of lipids (and/or lipid complexes) ingested may determine the extent of functional changes.
Phorbol Ester-Induced Loss of Membrane Sialic Acid: Implications for Tumor Cytolysis by Natural Killer Cells
Authors: Nabi Zeenat F , Zucker-Franklin Dorothea , Baten Abdul
Published: January 2018

Freeze-fracture analysis has shown that treatment of cells with phorbol myristate acetate (PMA) results in a loss of intramembranous particles (IMP) associated with the external leaflet of their plasma membranes. It has also been demonstrated that phorbol esters markedly enhance the sensitivity of tumor targets to natural killer (NK) cells, although the mechanism underlying this phenomenon has remained unexplained. Since the ability of NK cells to recognize neoplasms appears to be inversely related to the concentration of sialic acid on the target cell surface, it seemed possible that phorbols affect membrane glycoproteins which have terminal carbohydrates bearing sialic acid residues. To investigate whether phorbol treatment could be responsible for the loss of sialic acid, four tumor cell lines were examined before and after exposure to PMA. A reduction in surface sialic acid was established by four different methods: 1) standard thiobarbituric acid analysis of cell hydsrolysates, 2) metabolic labelling of cells with [3H]-mannosamine followed by treatment with neuraminidase, 3) chromatography of membrane extracts, and 4) freeze-fracture analysis of lectin-labelled intact cells. These observations suggest a mechanism whereby phorbols may facilitate NK-cell-mediated cytolysis. In addition, an entirely novel effect of these tumor-producing agents may have been uncovered.
Beta-Carotene Stimulates Human Leukocytes to Secrete a Novel Cytokine
Authors: Abril Edward R , Rybski James A , Scuderi Philip , Watson Ronald R
Published: January 2018

The effect of beta-carotene on cytokine production by human peripheral blood leukocytes was tested. Beta-carotene stimulated the secretion of a novel cytotoxic cytokine when peripheral blood cells were exposed to carotenoid concentrations between 10-6 and 10-10M. Beta-carotene–treated supernatants caused the cytolysis of four out of the six human tumor cell lines tested. Low level toxicity was also observed when normal diploid fibroblast lines were exposed to beta-carotene–treated leukocyte supernatants. The cytotoxic activity elicited by beta-carotene was found to be distinct from characterized cytokines based on both antisera neutralization and target cell specificity studies. This study demonstrates that beta-carotene can induce human leukocytes to secrete one or more cytokines that can manifest cytotoxic activity against human tumor cells in vitro.
Lead Inhibits Intracellular Killing of Leishmania Parasites and Extracellular Cytolysis of Target Cells by Macrophages Exposed to Macrophage Activating Factor
Authors: Mauël Jacques , Ransijn Adriana , Buchmüller-Rouiller Yolande
Published: January 2018

Activation of Leishmania enriettii-infected mouse macrophages in vitro by treatment with macrophage activating factor (MAF)-rich media supplemented with lipopolysaccharide (LPS) leads to rapid killing of the microorganism. When exposed to MAF + LPS in the presence of 30-100 μM lead acetate, however, macrophages failed to destroy the parasites. This effect was not due to lead toxicity for macrophages. Decreased microbicidal activity correlated with depressed respiratory burst as determined by measurements of glucose oxidation through the hexose monophosphate shunt (HMPS). Lead had little effect on Intracellular parasite killing induced by exposure of macrophages to the electron carrier methylene blue; HMPS in such cells was similarly little affected, indicating that chemical triggering of this pathway bypassed the lead-imposed blockade. Lead also abolished macrophage activation measured by the lysis of tumor target cells in vitro. The metal failed, however, to interfere with target-cell lysis by macrophages activated in lead-free medium, suggesting that lead inhibited the acquisition of the activated state rather than the functional expression of such state. Lead did not prevent the binding of radiolabelled interferon-γ to macrophages; it did, however, slow down receptor turnover and degradation of bound interferon. Lead also inhibited the LPS-triggered cytotoxicity in macrophages previously exposed to interferon-γ in lead-free medium, suggesting that depressed intracellular killing might result from an effect on both the priming (interferon or MAF-dependent) and the triggering (LPS-dependent) steps of activation.
Inhibitory Effect of SV-IV, a Major Protein Secreted From the Rat Seminal Vesicle Epithelium, on Phagocytosis and Chemotaxis of Human Polymorphonuclear Leukocytes
Authors: Metafora Salvatore , Porta Raffaele , Ravagnan Gianpietro , Peluso Gianfranco , Tufano Maria Antonietta , De Martino Luisa , lanniello Raffaele , Galdiero Francesco
Published: January 2018

The effect of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes (PMNs) has been studied. Various cytological, biochemical, metabolic, and physical correlates of both biological activities have been found to be markedly reduced by the presence in the medium of micromolar concentrations of protein SV-IV. Moreover, the Scatchard analysis of the labeled SV-IV binding to PMN cell surface has demonstrated that such binding is specific. The binding sites contain only saturable components, completely displaceable by unlabeled SV-IV. The number of the specific sites has been calculated to be 87,000/cell, with a Kd of 1.72 x 10-7 M. The molecular mechanism of the inhibitory effect is discussed along with the possible biological and clinical implications of the experimental findings.
Biologic and Immunohistochemical Analysis of Macrophage lnterleukin-1α, -1β, and Tumor Necrosis Factor Production During the Peritoneal Exudative Response
Authors: Chensue Stephen W , Shmyr-Forsch Christine , Weng Andrew , Otterness Ivan G , Kunkel Steven L
Published: January 2018

The present study examined changes in lipopolysaccharide (LPS)-induced interleukin 1 (IL-1) and tumor necrosis factor (TNF) production by murine peritoneal macrophages during the chronic exudative response to Freund’s complete adjuvant (CFA). Macrophages were isolated by peritoneal lavage and adherence at intervals over a 32 day period following i.p. injection of CFA. Optimal culture conditions for IL-1 and TNF production were predetermined, and it was found that IL-1 production was profoundly impaired at densities of above 150 cells/mm2, whereas TNF sythesis was more resistant to density effects. Using optimal conditions, we observed a sequential appearance of monokines. On day 0 there was minimal IL-1 production and no detectable TNF production. By days 4–7, IL-1 production reached maximum levels with a steady decline to baseline by day 32. TNF production steadily increased after day 2, reached maximal levels by days 16–20, and then partly declined by day 32. These findings were supported by kinetic analyses at specified days. When related to exudative events, it appeared that maximal IL-1 was associated with the recruitment stage of the reaction, whereas TNF production was associated with the established exudate. Immunohistochemical analysis revealed that TNF production could be related to the proportion of macrophages with cytoplasmic TNF expression. In contrast, IL-1α and -1β expression was comparable among populations with 85–100% of cells showing cytoplasmic expression 6 hr after LPS stimulus. Whereas cytoplasmic IL-1α persisted for the 18 hr study period, IL-1β disappeared from many adjuvant recruited cells. Our findings suggest that monokine production is orchestrated during macrophage recruitment and activation at sites of chronic inflammation.
Quantitation of the Host Cell Infiltration Kinetics of the Nonimmunogenic Colon 26 Tumor by Multiparameter Flow Cytometry
Authors: Sneed Rosie A , Stevenson Anita P , Stewart Carleton C
Published: January 2018

In this study, we examined the kinetics of host cell infiltration into the nonimmunogenic Colon 26 tumor. We found that 1 x 104 cells were required to produce tumors in 100% of mice. The in vivo doubling time was 42.5 h, and a barely palpable tumor contained 8 x 106 cells. No evidence of concomitant immunity was found. The number of host cells infiltrating the in vivo tumors increased at the same rate as the number of tumor cells, but averaged only 22% of total cells. Cycling T lymphocytes were present in the host cell infiltrate of this tumor. In addition, approximately 50% of in vivo Colon 26 cells were Thy-1.2 positive. The observed characteristic of low immunogenicity makes it a useful murine model for studying human malignant tumors.
Lipopolysaccharide Modulates Chemotactic Peptide-lnduced Actin Polymerization in Neutrophils
Authors: Howard Thomas H , Wang Danher , Berkow Roger L
Published: January 2018

To study the effect of endotoxin (LPS) on the basal and chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (fMLP) -induced alterations in neutrophil cytoskeleton, we purified (>98%) LPS-free neutrophils (LPS- < 10 pg/ml LPS), compared their cytoskeletal organization to that of circulating neutrophils, and examined the effect of LPS exposure on the basal and fMLP-induced change in the cytoskeleton as reflected by F-actin content and distribution. Shape, F-actin content and distribution were monitored by FACS analysis and fluorescence microscopy of NBDphallicidin-stained cells. The F-actin content of basal and fMLP-activated, purified LPS- cells is similar to that of circulating neutrophils (defined as cells drawn in LPS- buffers at 37°C and analyzed after <10 seconds of ex vivo manipulation). LPS- cells are round with a diffuse F-actin distribution. Exposure of LPS- cells to LPS causes cell polarization and F-actin redistribution without net gain in F-actin content. Peptide activation of the LPS- cell causes actin polymerization, which is preceded by a brief lag time. Exposure of LPS- cells to LPS (LPS+) enhances fMLP-induced actin polymerization by: 1) increasing the maximal extent of polymerization; 2) shortening the lag time preceding polymerization and increasing the rate of polymerization; and 3) lowering fMLP dose required for half maximal F-actin response. The enhancement depends on LPS dose, duration of exposure, and temperature. To examine the mechanism whereby LPS enhances fMLP-induced actin polymerization, we determined the predominant end for filament growth in LPS- and LPS+ cells, the number of actin nuclei generated in LPS- and LPS+ by fMLP activation, and the number and affinity of fMLP receptors on LPS- and LPS+ cells by 3[H]fMLP binding. Actin polymerization in both LPS- and LPS+ occurs predominantly by monomer addition to the barbed ends of nuclei, and the number of actin nuclei in basal and fMLP-activated LPS- and LPS+ cells is similar. LPS+ cells express three times more fMLP receptors than LPS- cells. The results show that LPS- cells are similar in cytoskeletal organization to circulating neutrophils, LPS causes shape change without change in F-actin content, and LPS enhances fMLP-induced actin polymerization response in neutrophils. The results suggest that LPS enhancement of actin polymerization response is associated with an increase in the number of fMLP receptors expressed on the cell surface.
Chemotactic Peptide Enhancement of Phorbol Ester-Induced Protein Kinase C Activity in Human Neutrophils
Authors: Gay James C , Stitt Ella S
Published: January 2018

Chemotactic factors and phorbol esters may act synergistically to evoke neutrophil responses, but the mechanism of such interaction is not entirely clear. We investigated the combined effects of the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) on protein kinase C (PKC) activity in human neutrophils. FMLP had little effect on the sharp decrease in cytosolic PKC activity induced by PMA. However, cells exposed to FMLP and PMA exhibited a synergistic increase in particulate PKC activity (1 ± 1 pmol 32P/107 PMNs/min in unstimulated cells, 53 ± 12 pmol 32P with 20 ng/ml PMA, 6 ± 3 pmol 32P with 10-7 M FMLP, and 191 ± 17 pmol 32P with FMLP and PMA). FMLP also markedly increased calcium/phospholipid-independent protein kinase activity in particulate fractions of control and PMA-treated cells. Enhancement of PKC activity required the presence of cytochalasin B during cell stimulation. Cellular calcium was crucial to the FMLP effect since enhancement was decreased in cells incubated with EGTA or Quin2. These results suggest that chemotactic factors and phorbol esters may mediate synergistic effects on neutrophil responses through enhancement of particulate PKC activity. The enhancing effect is probably mediated through chemoattractant-mediated increases in intracellular calcium.
Inhibition of Neutrophil and Eosinophil Chemotactic Responses to PAF by the PAF-Antagonists WEB-2086, L-652,731, and SRI-63441
Authors: Håkansson Lena , Venge Per
Published: January 2018

The influence of the three PAF-antagonists WEB-2086, L-652,731, and SRI-63441 on the chemotactic response of neutrophil and eosinophil granulocytes to PAF was investigated. When the PAF-antagonists were added to the cell suspension that was exposed to a gradient of PAF, WEB-2086 and SRI-63441 at the concentration of 10-6 mol/litre inhibited (P<.01) the neutrophil and eosinophil chemotactic response to 10-8 and 10-9 mol PAF per litre; at the concentration of 5 x 10-6 mol/litre, WEB-2086 and SRI-63441 also inhibited (P<.02) the response to 10-7 mol PAF per litre. Under the same conditions L-652,731 at the concentration of 5 x 10-6 mol/litre inhibited (P<.01) the eosinophil chemotactic response to 10-8 and 10-9 mol PAF per litre. The inhibition of the chemotactic response to PAF by the three PAF-antagonists was specific, since the chemotactic response to C5f, f-MLP, and LTB4 was not affected by WEB-2086, L-652,731, or SRI-63441, neither was the chemokinetic migration induced by albumin.
“Autoregulation” of Human Neutrophil Activation In Vitro: Regulation of Phorbol Myristate Acetate-Induced Neutrophil Activation by Cell Density
Authors: Peters Stephen P , Cerasoli Franklin , Albertine Kurt H , Gee Marlys H , Berd David , Ishihara Yoko
Published: January 2018

Phorbol myristate acetate (PMA, 10-7 M) activation of adherent neutrophils (PMNs) led to a markedly attenuated release of superoxide anion (O2-) per cell when PMNs were activated at high density (2.85 fmol O2-/PMN at 2 million in 0.1 ml) in comparison with cells activated at low cell density (12.0 fmol O2-/PMN at 250,000 in 0.1 ml). This “autoregulatory” phenomenon was not due to a defect in the superoxide anion assay employed, to a differential adherence of neutrophils at high vs. low density, or to substrate (cytochrome c) or cell stimulus (PMA) limitation. It was associated with an inhibition of apparent NADPH oxidase activity and a leftward shift (toward a lower level of activation) in the activation profile of PMNs (as determined by FACS analysis using PMNs preloaded with 2′7′-dichlorofluorescin diacetate in which H2O2 production results in the production of the fluorescent product 2′7-dichlorofluorescein intracellularly). Other aspects of the neutrophil activation response including arachidonic acid mobilization, phospholipid metabolism, and perhaps phosphatidylinositol turnover were also attenuated when PMNs were activated at high cell density. Studies with cells in solution, cells treated with cycloheximide, and cells treated with 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid suggest that PMN contact with a surface, neutrophil protein synthesis, and an increased surface expression of the heterodimer CD11b/CD18 on PMNs all were not required for autoregulation. Finally, morphometric and morphologic examination of PMNs activated at low vs. high density revealed histologic and structural correlates associated with the attenuated PMN activation response of cells triggered at high cell density. We conclude that multiple structural and functional aspects of the PMN activation response are modulated by cell density and suggest that this property is important both in the physiologic control of neutrophil activation and in the design of in vitro assays of the neutrophil activation response.
Inhibition by AZT of HIV-1 Replication in Acutely Infected U-937 Cells
Authors: Boulerice François , Wainberg Mark A
Published: January 2018

We studied the effect of AZT on the replication of HIV-1 in freshly and chronically infected U-937 monocytoid cells over a period of 2 months. Expression of viral antigens was monitored by indirect immunofluorescence, and viral replication was assessed by reverse transcriptase assay on virus pelleted from culture fluids. In U-937 cells not treated by AZT, viral antigens were expressed by 7 days after infection. The inclusion of a variety of concentrations of AZT in the culture medium was shown to retard virus replication in a dose-dependent fashion, although a complete inhibitory effect was not seen with any clinically attainable concentration of drug. Exposure of HIV-1-inoculated cells to AZT did not give rise to progeny virus possessing a drug-resistant phenotype. However, the study of clonal derivatives of U-937 cells revealed cellular variants with increased susceptibility to HIV-1, and against which AZT had reduced effectiveness in comparison with the parental line. No effect of AZT was seen on cells already infected by HIV-1, suggesting that this drug had no influence on viral replication in U-937 cells into which viral DNA had previously integrated.
Purification and Characterization of Soluble Inositol 1,4,5-Trisphosphate 5-Phosphomonoesterase From Rabbit Peritoneal Neutrophils
Authors: Kennedy Scott P , Sha'afi Ramadan I , Becker Elmer L
Published: January 2018

A specific soluble inositol phosphate 5-phosphomonoesterase has been purified approximately 2,700-fold from a 120,000g supernatant of rabbit neutrophil homogenate. The specific enzyme represented 25–50% of the total hydrolytic activity toward inositol, 1,4,5-trisphosphate (lns-1,4,5-P3) with the remaining activity hydrolyzing both lns-1,4,5-P3 as well as inositol 1,4-bisphosphate (lns-1,4-P2). However, the enzyme could not be identified on sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue, indicating that it represents a minor protein in the purified enzyme preparations. The purified enzyme has an apparent molecular mass of 43,000–47,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphomonoesterases. The enzyme hydrolyzes lns-1,4,5-P3 with an apparent Km of 18 μM and a Vmax of 1.2 μmol/min/mg. The 5-phosphomonoesterase requires Mg2+ for activity and is not affected by physiological concentrations of Ca2+ or calmodulin. The pH optimum for activity is 7.5. Inositol 1,3,4,5-tetrakisphosphate is a potent competitive inhibitor of lns-1,4,5-P3 hydrolysis (Ki = μM), whereas lns-1,4-P2 is a weak inhibitor (Ki = 173 μM). lns-1,4,5-P3 hydrolysis is relatively unaffected by monophosphorylated substrates, however, bisphosphorylated substrates are potent inhibitors. Comparisons of neutrophil 5-phosphomonoesterase characteristics with those of platelet and rat brain enzymes support the idea that each 5-phosphomonoesterase may be a unique enzyme and play a different role dependent upon the cell or tissue in which it acts.
Human Recombinant Interleukin-2 Induces Maturation and Activation Signals for Feline Eosinophils In Vivo
Authors: Tompkins Mary B , Novotney Carol , Grindem Carol B , Page Rodney , English Robert , Nelson Philip , Tompkins Wayne A F
Published: January 2018

Immunotherapy, with interleukin-2 (IL-2) or IL-2 plus lymphokine-activated killer (LAK) cells, has been used to treat cancer and acquired immunodeficiency syndrome (AIDS) in man. Similarities between feline leukemia virus (FeLV) infection in the cat and human immunodeficiency virus (HIV) infection in man have prompted immunotherapeutic studies in the cat. To develop baseline data on hematological responses to infused IL-2, cats were given daily (1–14 days) i.v. injections of 5 × 104 U/kg of recombinant human IL-2 (rHulL-2). Complete blood cell (CBC) counts were done weekly. Red blood cell (RBC), neutrophil, and lymphocyte numbers did not change appreciably over the course of the study. In contrast, rHulL-2 caused an eosinophilia in all but the 1 day treatment group. Treatment for 3 days generated a transient eosinophilia on day 7 that returned to baseline by 3 weeks. Five day and 7 day treatments generated an eosinophilia by day 7 that peaked on day 14 and returned to normal values by day 28. Treatment of cats for 14 days did not increase the magnitude or duration of the eosinophilia beyond the 5 or 7 day treatments. Bone marrow (BM) biopsies from rHulL-2-treated cats revealed a marked selective hyperplasia of eosinophil precursors. In the 5 day treatment group, all maturation stages of eosinophils were elevated by week 1 of treatment. By week 2, the early stages had returned to normal, whereas the late stage cells remained elevated, suggesting an ordered maturation response. Numbers of all eosinophil precursors approximated pretreatment numbers by weeks 3–4. Thus the BM hyperplasia preceded the blood eosinophilia by 1 week, suggesting that an enhanced maturation response of BM eosinophil precursors is a major contributor to the rHulL-2-induced blood eosinophilia. In addition to a maturation signal, rHulL-2 induces a potent activation signal for eosinophils as measured by a decrease in density and an increase in longevity in culture. The significance of the activated eosinophil in the therapeutic or toxicologic response to rHulL-2 infusion is discussed.
Sources of Antibody-Dependent Cellular Cytotoxicity Deficiency in Young Pig Leukocytes
Authors: El-Awar Faisal Y , Hahn Edwin C
Published: January 2018

Antibody-dependent cellular cytotoxicity (ADCC) activity against pseudorabies virus-infected target cells has been found to be lower in young pig peripheral blood leukocytes (PBLs) than in adults. Experiments were designed to investigate the reason(s) for low activity in the young, which are more at risk of fatal infection than adults. The percentage of polymorphonuclear leukocytes (PMNs), the major ADCC effector cell, in the whole leukocyte population did not have a bearing on the deficiency. Enrichment for PMNs did not alleviate differences in activity between young and adult pigs. Additionally, no suppressor cell(s) or factor(s) could be demonstrated to account for the ADCC deficiency. The source of the ADCC deficiency in the young was found to be related to the decreased ability of young pig effector cells to bind antibody-sensitized targets. This deficiency relative to adults was associated with decreased antibody binding to high affinity Fc receptors on young pig neutrophils.
6-Trans-Leukotriene B4 Is a Neutrophil Chemotaxin in the Guinea Pig Dermis
Authors: Fretland Donald J , Widomski Deborah L , Anglin Charles P , Walsh Robin E , Levin Stuart , Gaginella Timothy S
Published: January 2018

The products of the 5- and 12-lipoxygenase (5-LO, 12-LO) pathways of arachidonic acid metabolism are implicated as proinflammatory mediators in a number of disease states. 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] is present in large quantities in human psoriatic lesional skin and can be further metabolized by 5-LO to 5(S),12(R)-dihydroxy-(6E,8Z,10E,14Z)-eicosatetraenoic acid (6-trans-LTB4). Furthermore, leukotriene B4 (LTB4) and the sulfidopeptide leukotrienes (LTC4, LTD4) can be transformed to 6-trans-LTB4. When injected into the guinea pig dermis, 6-trans-LTB4 (1.0,10.0,20.0 μg/intradermal site) caused a significant (P < 0.02) infiltration of polymorphonuclear leukocytes (PMN) at 4 hr as assessed by histology and the levels of the PMN marker enzyme myeloperoxidase. 6-Trans-LTB4 is a more potent PMN chemoattractant than 12(R)-HETE in the guinea pig dermis but is far less potent than LTB4. Pharmacological interdiction of leukotriene production or receptor binding should take into account the proinflammatory activity of 6-trans-LTB4.
Tumor Necrosis Factor Increases In Vivo Glucose Uptake in Hepatic Nonparenchymal Cells
Authors: Spolarics Zoltán , Schuler Ágnes , Bagby Gregory J , Lang Charles H , Mészáros Károly , Spitzer John J
Published: January 2018

This study aims to elucidate the in vivo metabolic response of different liver cells following a short-term (30 min) infusion of a nonlethal dose of human recombinant tumor necrosis factor (TNF). In vivo glucose uptake of different tissues and isolated liver cells was determined by a sequential double-labeling version of the tracer 2-deoxyglucose technique. Following TNF administration glucose uptake was increased in the liver, lung, spleen, and skin while it was not changed in muscle and testis. In response to TNF infusion neutropenia developed which was sustained for 40 min. The number of lymphocytes in the blood was also decreased after the termination of TNF infusion. This short-term infusion of TNF, however, was not accompanied by marked sequestration of leukocytes into the liver. In vivo glucose uptake in response to TNF was doubled in the Kupffer cells and increased by 56% in hepatic endothelial cells. Glucose uptake of parenchymal cells was not significantly affected. The prompt increase of glucose uptake in the reticuloendothelial cells of the liver, primarily in the Kupffer cells, following TNF administration suggests that a similar metabolic response of these cells to sepsis may be mediated at least in part by TNF. It is suggested that the increased glucose uptake by the hepatic nonparenchymal cells is a reflection of the immunmodulatory effect of TNF.
Function and Regulation of the Murine Lymphocyte CD2 Receptor
Authors: Abraham D J , Bou-Gharios G , Beauchamp J R , Plater-Zyberk C , Maini R N , Olsen I
Published: January 2018

The CD2 receptor on T-lymphocytes plays a major part in mediating adhesive interactions via the LFA-3 ligand and in transducing signals for lymphocyte activation. In this study the expression, function, and internalization of the CD2 receptor was investigated in resting and activated murine T-cells. Surface iodination of intact lymphocytes showed that both types of cell expressed this antigen as a single polypeptide of 63 KDa, and flow cytometry analysis demonstrated that there was four times as much CD2 on lymphoblasts as on resting cells. Moreover, the CD2 receptor had a more prominent role in the adhesion of the activated lymphocytes to extravascular cells than in the binding of resting cells. Only activated lymphocytes internalized CD2, in the presence or absence of the anti-CD2 monoclonal antibody (mAb) 12-15, more than 80% of the 12-15/CD2 complex being removed from the cell surface within 24 hr. Application of 125l-labelled mAb 12-15 followed by subcellular fractionation on Percoll gradients showed that the complex was internalized initially into a low-density compartment and subsequently transported to heavy-density organelles, in which it was degraded. Immunogold electron microscopy revealed that immediately after the initial binding of mAb 12-15 to the lymphoblasts, the gold particles were localized in clusters exclusively at the plasma membrane. After a short period of culture, the mAb 12-15/CD2 complex was detected in small vesicles near the cell surface. Immunogold staining for a lysosomal enzyme β-glucuronidase (Gus), for the lysosomal membrane protein LAMP-1, and for the mannose 6-phosphate targetting receptor (MPR) showed that the complex was transported from the endosomal compartment to lysosomal organelles in the activated T-cell. Although mAb 12-15 bound to CD2 in resting T-lymphocytes, in these cells the complex remained associated with the plasma membrane compartment only, even after prolonged culture. These data show that activated but not resting lymphocytes endocytosed the receptor, thereby regulating the expression of this antigen at the plasma membrane. This suggests that the endocytic and lysosomal compartments of lymphocytes have major roles in immune functions, by controlling the level of receptors at the lymphocyte cell surface and thus their response to cytokines and inflammatory mediators as well as their direct interaction with other cells.
Priming of Rabbit Alveolar Macrophages for Enhanced Oxidative Responses by Herpes Simplex Virus Type 2 Infection
Authors: Giridhar G , Hayakawa H , Kucera L S , Myrvik Q N
Published: January 2018

The effect of herpes simplex virus type 2 (HSV-2) infection on the oxidative response in infant and adult rabbit alveolar macrophages (AM) was studied using either phorbol myristate acetate (0.5 μg PMA/ml) or latex (250 μg/ml) as eliciting agents in a chemiluminescence (CL) assay. Results indicated that uninfected infant AM responded to a latex-elicited but not PMA-elicited CL response. HSV-2 infection (moi=1.0) of infant AM for 2 hr at 37° did not alter the PMA or latex-elicited CL responses. In contrast, uninfected adult AM exhibited a markedly increased CL response when elicited with either PMA or latex. HSV-2 infection (moi=1) of adult AM for 2 hr further increased both PMA- and latex-elicited CL responses. Increasing the moi to 10 inhibited both PMA- and latex-elicited CL responses. Incubation of uninfected control and HSV-2 infected adult AM for 18 hr at 37° resulted in spontaneous priming of the cells for increased CL responses. In the absence of PMA HSV-2 alone failed to elicit a CL response in adult AM. Infection with heat-inactivated HSV-2 (moi=1.0 before heat inactivation) did not prime adult AM for enhanced CL responses. AM from BCG immunized adult rabbit produced a considerably higher level CL response that nonimmunized AM; however, HSV-2 infection of these cells did not further enhance the response. In summary, these data indicate that adult AM but not infant AM can be primed by active HSV-2 infection for an increased CL response elicited by either PMA or latex.
On the Mechanism of Antiinflammation Induced by Tumor Transplantation, Surgery, and Irritant Injection
Authors: Normann S , Besedovsky H , Schardt M , del Rey A
Published: January 2018

Tumor transplantation, major surgery, and injection of nonspecific irritants elicit inflammation locally while suppressing inflammation induced subsequently and at distant sites. Such systemic antiinflammation in rodents occurs via corticosterone-independent and -dependent pathways. Based upon hormone measurements and the response to adrenalectomy, antiinflammation induced by irritants and certain surgical procedures is corticosterone independent while that which follows tumor transplantation is corticosterone dependent. However, injection of tumorous ascites stimulates both pathways since it contains two antiinflammatory factors: Factor A (molecular weight <2,000) does not alter hormone balance while Factor B (molecular weight 30,000–100,000) increases corticosterone levels and is corticosterone dependent. Desensitization of systemic antiinflammation develops rapidly regardless of whether it is corticosterone dependent (Factor B) or independent (Factor A or irritants). However, tumor transplantation resists desensitization possibly by inducing an immune response since lymphocytic mitogens prevent development of and break established desensitization. Nevertheless, abolition of tumor-induced antiinflammation follows injection of tumorous ascites by a mechanism that involves Factor B suppression of the corticosterone response to the tumor while Factor A apparently raises the threshold at which physiological increases in corticosterone inhibit leukocyte emigration. We conclude that systemic antiinflammation is a general consequence of a localized inflammatory reaction and that desensitization of such antiinflammation develops rapidly. Recent evidence indicates that certain mediators of inflammation are proinflammatory when administered intradermally but antiinflammatory when given intravenously. Thus, systemic antiinflammation may arise when chemical mediators of inflammation generated by a local reaction gain access to the circulation.
Maintenance of Human Immunodeficiency Virus Type-1 Proviral DNA in Human Fetal Dorsal Root Ganglia Neural Cells Following a Nonproductive Infection
Authors: Kunsch Charles , Wigdahl Brian
Published: January 2018

Infection of the nervous system by human immunodeficiency virus type-1 (HIV-1) has been implicated in the generation of acquired immunodeficiency syndrome (AIDS)-associated neurologic dysfunction and direct infection of glia has been suggested as one of the potential mechanisms leading to deterioration of nervous system function. We have been examining the interaction of HIV-1 with the developing peripheral nervous system in vitro, and have previously shown that HIV-1 infection of primary human fetal dorsal root ganglia (DRG) neural cells resulted in HIV-1 gag antigen expression in approximately 70% of the glial cell subpopulation with little, if any, cytopathic damage to the infected cells. Accumulation of HIV-1 gag antigens and viral mRNA reached a maximum by 2–3 days postinfection and declined thereafter to minimally detectable levels in the surviving neural cell population. In addition, infection of the fetal DRG neural cells appeared to be abortive or nonproductive, with little if any, infectious progeny virus production. However, we have been able to detect HIV-1-specific proviral DNA as late as 24 days postinfection by polymerase chain reaction amplification and subsequent DNA blot hybridization. These results suggest that accumulation of HIV-1 structural proteins without the assembly and release of mature virus in HIV-1-infected human fetal DRG neural cells results in a nonproductive infection and maintenance of HIV-1 proviral DNA in the infected cell population.