bluuebunny/crossref_metadata_2025 huggingface dataset:
Secretion of Mucoid Material by Lymphokine-Activated Killer Cells: Study by Light and Electron Microscopy
Authors: Ginsburg Haim , Coleman Raymond , Davidson Sima , Yahav Hava , Khoury Camellia
Published: January 2018

When lymph node cells from nude mice were grown on embryonic fibroblast monolayers together with rat interleukin-2, only one type of colonies developed. These colonies were composed of cytotoxic cells termed “granular lymphokine-activated killer mucus-secreting cells” (LAK-GM). An extensive differentiation course, in which all the cellular components were involved, ended with a population of short-lived, mature, nondividing large cells that apparently synthesized and deposited a flowing mucoid material (FMM) that stained distinctly blue with periodic acid-Schiff/alcian blue (PAS-Ab) at pH 1 and distinctly red by the naphthol AS-D-chloracetate method tor specific esterase. So far, the best monolayers to trigger the FMM synthesis were those prepared from 16- to 18-day-old whole embryos. These cells were compared with LAK cells that developed on monolayers (such as embryonic skin or adult kidney) that did not trigger FMM synthesis. They were also compared with other cell types that differentiated in colonies on the fibroblast monolayers: histiocytes (fixed macrophages), mixed granulocytes monocytes, mucosal mast cells; and with populations of mature rat T-killer cells developed on same mouse monolayers, Features distinctive to the secreting LAK-GM cells were presence of masses of membrane-limited vesicles that were strictly confined to the surface of the cells in FMM-containing colonies. All transitional forms of budding activity could be seen on the cell surface facing the masses. Within the same cells, many granules displayed varying degrees of degradation, the granular material being transformed into flocculent material that formed small pools facing each degraded surface. Other characteristics of the LAK-GM lineage were the accumulation of glycogen prior to the appearance of the FMM. the presence of several structures of a ribosome-lamella complex in the LAK-GM in colonies that did not accumulate FMM, and filopodia commonly emerging from the pole proximal to the nucleus. Of various fixation methods tried, only after treatment with absolute alcohol and subsequent drying was the FMM stained with PAS-Ab. By subsequent wetting, the capacity to be stained was irreversibly lost. After incubation of the living cultures with the enzymes hyaluronidase or chondroitinases AC or ABC, the FMM disappeared. These observations suggest a triggering mechanism by the embryonic mesenchymal fibroblastoid cells for synthesis and secretion of mucous material that is a proteoglycan of the chondroitin sulfate group.
Mononuclear Phagocytes and HSV-1 Infection: Increased Permissivity in Differentiated U937 Cells Is Mediated by Post-Transcriptional Regulation of Viral Immediate-Early Gene Expression
Authors: Kemp L M , Estridge J K , Brennan A , Katz D R , Latchman D S
Published: January 2018

Undifferentiated U937 cells are non-permissive for herpes simplex virus (HSV) infection but can be rendered permissive by treatment with phorbol myristate acetate (PMA), which causes them to differentiate to a macrophage-like phentoype. Following infection with HSV, both PMA—treated and untreated cells correctly transcribe the viral immediate-early genes at levels comparable to those observed in fully permissive cell types, but immediate-early RNA and protein are detected only in the PMA-treated cells. Hence PMA acts by relieving an early block to HSV infection caused by the rapid turnover of immediate-early RNA. This block is not caused by the production of soluble inhibitors and can also be relieved by treatment with other agents that cause macrophage differentiation such as 1, 25 dihydroxycholecalciferol. These findings therefore indicate that the non-permissivity of undifferentiated U937 cells for HSV is mediated by post-transcriptional regulation of immediate-early gene expression.
Lymphotoxin Induces Secretion of Granule Proteins From Adherent Neutrophils: Possible Role of Intracellular Free Calcium
Authors: Richter Johan
Published: January 2018

Lymphotoxin (LT) can activate human neutrophils. Using a hemolytic plaque assay to detect secretion of lactoferrin and myeloperoxidase (MPO) from single adherent neutrophils, we showed that LT induced secretion from both primary and secondary granules. Incubation of cells with cytochalasin B was required for MPO secretion, and it enhanced lactoferrin secretion. Pertussis toxin, which blocks a G-protein in the plasma membrane, inhibited LT-induced exocytosis of MPO, but not of lactoferrin. Incubation with LT did not induce any detectable changes of the cytoplasmic free [Ca2+] in neutrophils. On the other hand, secretion of granule proteins from adherent neutrophils in response to LT was blocked by loading neutrophils with quin-2 in order to increase the intracellular calcium buffering capacity. This was achieved at a concentration of quin-2, at which the secretion induced by the phorbol ester PMA and the chemotactic peptide FMLP was unaffected. Trifluoroperazine (TFP), a dual protein kinase C and calmodulin inhibitor, significantly inhibited the LT-mediated secretion of lactoferrin from adherent granulocytes. The PMA effect was unaltered by TFP under these conditions, suggesting that the inhibitory effect was on a calcium-calmodulin dependent step. The secretion induced by TNF and GM-CSF was also blocked by buffering changes in the intracellular ICa2+] and inhibited to a similar extent by TFP. Our results suggest that calmodulin and minute changes in the cytoplasmic free [Ca2+] may be involved in a common signal transduction pathway engaged in activation of adherent neutrophils by several cytokines.
Comparative Abilities of Various Metabolites of Vitamin D to Protect Cultured Human Macrophages Against Tubercle Bacilli
Authors: Crowle Alfred J , Ross Elise J
Published: January 2018

Vitamin D metabolites have several biologic functions besides the best-known one of controlling mineral metabolism, one of which may be protection against tuberculosis. The chemical structures of the metabolites govern their functions. The most potent metabolite for regulating calcium metabolism is 1,25(OH)2-vitamin D3 (1,25-D3). This metabolite also is able to protect cultured human monocytes and macrophages (MP) against tubercle bacilli (TB). To determine whether these two functions are connected, 14 other analogs or metabolites of vitamin D were compared with 1,25-D3 for ability to protect cultured MP against TB. Blood-derived MP from 18 different donors were used in 70 experiments. The MP were infected with TB, then incubated for 7 days in medium containing 4 μg/ml of metabolite or synthesized analog. Growth of TB in the MP was measured by colony-forming unit counts from samples of lysed MP at 0, 4, and 7 days after infection. The metabolites, none of which inhibited TB in the absence of MP, varied from unprotective to bacteriostatic in the MP. Four of them were nearly as protective as 1,25-D3, and the metabolite 25S,26(OH)2-vitamin D3 was consistently more protective. An analog synthetically designed for maximum ability to promote cell differentiation was unprotective. There was no correlation between metabolite ability to protect and known ability to stimulate calcium mobilization. These results suggest that antituberculosis protection of human MP by vitamin D metabolites or analogs can be separated from their functions of inducing cell differentiation and controlling mineral metabolism.
Differential Effects of Lithocholate on Rat Neutrophil Activation
Authors: Dahm Lawrence J , Roth Robert A
Published: January 2018

Neutrophils (PMNs) may be exposed to high concentrations of biliary products during cholestasis and other hepatic disorders. We have previously reported that bile and certain bile salts enhance superoxide (O2-) release from neutrophils activated with phorbol myristate acetate (PMA) (Dahm et al.: Toxicol. Appl. Pharmacol. 95,82,1988), suggesting that PMN oxidative metabolism might be altered in toxicoses or disease states characterized by elevations in serum bile salts and other biliary products. In the present study, we characterized the priming effect of lithocholate for O2- release and also examined the effects of lithocholate on enzyme release from PMNs. PMNs preincubated with lithocholate at concentrations which did not directly stimulate O2- release (3–100 μM) and activated with PMA released greater amounts of O2- than controls exposed to PMA alone, illustrating a priming effect O2- release from lithocholate-primed PMNs rose sharply between 5 and 10 min after PMA addition and then ceased between 10 and 30 min. The priming effect of lithocholate toward PMA-activated PMNs was reduced approximately 50% by washing PMNs after lithocholate addition and was not dependent on extracellular Ca2+, although removal of Ca2+ from the incubation buffer enhanced the cytotoxicity of lithocholate toward PMNs. In Ca2+-supplemented medium, lithocholate primed PMNs for O2- release when formyl-methionyl-leucyl-phenylalanine (FMLP, 10-8-10-6 M) or calcium ionophore, A23187 (10-7 or 10-6 M), was used to activate PMNs. Lithocholate (100 μM) by Itself had only marginal effects on release of lysozyme or β-glucuronidase from PMNs. However, lithocholate (100 μM) inhibited β-glucuronidase release from FMLP-stimulated PMNs to near-baseline levels. When FMLP was added to PMNs prior to lithocholate, β-glucuronidase release was not reduced as it was when the order of addition was the reverse. Lithocholate had no effect on PMA-stimulated lysozyme release. These results indicate that lithocholate has different actions on PMN O2- release and enzyme release and suggest that lithocholate might exert its action on the PMN plasma membrane.
Differential Production of IFN-α/β by CSF-1- and GM-CSF-Derived Macrophages
Authors: Falk Lydia A , Vogel Stefanie N
Published: January 2018

Mature macrophages, derived in vitro from bone marrow progenitors under the influence of either macrophage colony stimulating factor (CSF-1) or granulocyte-macrophage (GM)-CSF, have been shown to differ morphologically and functionally. The data presented in this report demonstrate that macrophages derived from bone marrow progenitors under the influence of CSF-1 are highly resistant to infection with vesicular stomatitis virus (VSV), and that this refractoriness can be reversed by treatment of cells with anti-IFN-α/β antibody. In contrast, macrophages derived from bone marrow progenitors under the influence of GM-CSF are highly susceptible to the cytopathic effects of VSV, but can be protected by very low concentrations of exogenous IFN-α/β. These findings suggest that CSF-1 derived macrophages have a greater capacity for the production and/or utilization of IFN-α/β than GM-CSF-derived macrophages, which may account for many of the differentiative differences reported previously.
Stimulated Platelets Release Factor(s) Affecting the In Vitro Response of Human Polymorphonuclear Cells
Authors: Del Principe Domenico , Menichelli Adriana , Di Giulio Stefano , De Matteis Walter , Giordani Massimo , Pentassuglio Anna Maria , Finazzi-Agro Alessandro
Published: January 2018

The metabolic and functional responses of human polymorphonuclear cells (PMNs) to thrombin-activated platelet supernatants were studied. The incubation of PMNs with supernatants from stimulated platelets (SPS) caused a 50% decrease in both killing of Staphylococcus aureus and luminol-enhanced chemiluminescence (CL) by PMNs stimulated by opsonized-zymosan (OZ), Concanavalin A (Con A), or calcium ionophore A23187. The levels of PMN intracellular fluorescence measured by flow cytometry, using the fluorochrome dichlorofluorescin diacetate (DCF-DA), were considerably less in the presence of SPS than in resting platelet supernatants (RPS). No influence of platelet supernatant on O2 consumption and O2- generation by OZ-activated PMNs was observed. The incubation of PMNs with SPS caused a significant increase in the rate of chemotaxis and aggregation elicited by Con A, OZ, and phorbol myristate acetate (PMA). The supernatant from resting platelets did not show any of the above-reported effects. Platelets previously degranulated by thrombin were unable to inhibit CL when activated with agonists. Studies on the differential release of the granules by platelets showed that the CL-quenching activity paralleled the discharge of lysosomal content The release of myeloperoxidase (MPO) from PMNs elicited by OZ was reduced in the presence of SPS. The platelet supernatant did not affect the MPO activity if PMNs were lysed with Triton X-100. The leakage of lactate dehydrogenase (LDH) from platelets was less than 3%, and no catalase or superoxide dismutase was released. This activity withstood lyophilization, but was destroyed by 10 min heating at 100°C or by treatment with proteolytic enzymes.
Use of Colloidal Silica (Sepracell-MN) for Enrichment of Dendritic Cells From Human Peripheral Blood: Comparison With Other Methods
Authors: Chehimi Jihed , Starr Stuart E , Kawashima Hisashi , Hassan Nassef F , Douglas Steven D , Bandyopadhyay Santu
Published: January 2018

Three methods are described for enrichment of dendritic cells from human peripheral blood. In method 1, mononuclear cells were incubated in plastic tissue culture flasks for two h. Nonadherent cells were removed. Adherent cells were washed to remove floating cells and incubated for 14 h at 37°C in 5% CO2. Carbonyl iron was added, and the flasks were incubated for another 2 h. Nonadherent cells were subjected to centrifugation over metrizamide gradient Phagocytic cells containing ingested carbonyl iron, small lymphocytes, and free carbonyl iron particles passed through the metrizamide, while the interface cell population was enriched for dendritic cells. The purity and yield of enriched dendritic cells were 52.8% and 0.05%, respectively. In method 2, adherent mononuclear cells were cultured overnight, and the released cells (released adherent cells) were centrifuged over metrizamide to separate low-density cells. Monocytes from this low-density cell population were removed by panning over human gamma globulin-coated plastic Petri dishes. In this method the average purity and yield of DC were 63.8% and 0.1%, respectively. In method 3, released adherent cells were treated with anti-CD5 and anti-CD14 monoclonal antibodies plus baby rabbit complement for 15 min, washed, and centrifuged with colloidal silica (Sepracell-MN). Centrifugation with Sepracell-MN was repeated three times. Low-density cells were panned twice over human gamma globulin-coated plastic Petri dishes. Nonadherent cells were highly enriched for DC. Contamination of T cells, B cells, and NK cells was undetectable by flow cytofluorometry. Contamination of monocytes was < 2%. This method provided > 85.0% purity and 0.4% yield. This method (method 3) combines adherence, complement-dependent lysis, centrifugation with colloidal silica, and panning and provides the best yield and purity; it is therefore recommended for optimal purification of DC.
Recruitment of 99m-Technetium- or 111-Indium-Labelled Polymorphonuclear Leucocytes in Experimentally Induced Pyogranulomas in Lambs
Authors: Guilloteau Laurence , Pépin Michel , Pardon Pierre , Le Pape Alain
Published: January 2018

The recruitment of polymorphonuclear leucocytes (PMNs) during the development of experimental pyogranulomas induced by Corynebacterium pseudotuberculosis was followed in nine male lambs by scintigraphic examination. Autologous blood PMNs were labelled with 99m-technetium or 111-indium and were re-injected intravenously into infected lambs. The functional properties of the labelled cells were monitored 1) in vitro by measuring their phagocytic and bactericidal activity against C. pseudotuberculosis and their chemotaxis under agarose, and 2) in vivo by following scintigraphically their capacity to accumulate in an inflammatory focus induced by intradermal injection of latex beads coated with Salmonella abortus equi lipopolysaccharide. Following inoculation of corynebacteria into the right ear of lambs, radioactive foci were observed to be localized in the right ear and in the draining lymph nodes during the 4 days following inoculation. Histopathological examination performed 32 h after inoculation confirmed the intense accumulation of PMNs at these sites. With the exception of one animal, which presented visible foci in the neck 14 days postinoculation, no radioactive foci were observed during the later phases of experimental infection, despite the presence of multiple pyogranulomas which were confirmed by bacteriological examination after necropsy of the lambs. Histopathological examination of these lesions revealed layers of fibroblasts, lymphocytes, and macrophages surrounding a necrotic centre. The results of these studies suggest that the contribution of PMNs during the chronic phase of inflammation is considerably reduced in comparison with the acute inflammatory phase of the infectious process.
Rat and Human Natural Killers Exhibit Contrasting Immunoglobulin G Subclass Specificities in Antibody-Dependent Cellular Cytotoxicity Reflecting Differences in Their Fc Receptors (FcγR)
Authors: Song Elizabeth S , Young Katrina , Sears Duane W
Published: January 2018

Rat and human natural killers (rtNK and huNK, respectively) were compared in quantitative antibody-dependent cellular cytotoxicity (ADCC) assays for their capacity to recognize mouse and rat IgG monoclonal antibodies (MAb) of different subclasses. NK from these two species exhibit considerably different patterns of IgG subclass recognition as determined by the relative antibody concentrations required for comparable levels of target cells lysis. ADCC assays with a panel of 16 MAb revealed that the efficiency of rtNK-mediated target lysis diminished according to IgG subclass in the following order: molgG1 > rtlgG2a > molgG2b ≍ molgG2a > rtlgG2b > molgG3. By comparison, huNK recognized the same antibodies with nearly the opposite order of efficiency: rtlgG2b >> molgG2a > molgG3 > molgG2b » rtlgG2a ≍ molgG1. Only molgG2a antibodies were equally potent with rtNK and huNK. The contrasting difference in IgG subclass recognition by rat and human NK reflects the comparatively low protein sequence homology between their respective IgG Fc receptors (FcγR).
Killing of Leishmania Parasites in Activated Murine Macrophages is Based on an L-Arginine-Dependent Process That Produces Nitrogen Derivatives
Authors: Mauel Jacques , Ransijn Adriana , Buchmüller-Rouiller Yolande
Published: January 2018

The experiments described in this report were aimed at determining whether L-arginine (L-arg)-derived nitrogen oxidation products (nitric oxide, nitrous acid, nitrites) are involved in the intracellular killing of Leishmania parasites by activated murine macrophages in vitro. Peritoneal or bone marrow-derived macrophages were infected with L. enriettii or L. major, then activated by exposure to recombinant murine interferon-gamma or to macrophage activating factor (MAF)-rich media in the presence of li-popolysaccharide. Activation of macrophages in regular (i.e., arginine-containing) culture medium led to complete destruction of the microorganisms within 24 h (L. enriettii) or 48 h (L. major), concomitant with accumulation of nitrites (NO2-) in the culture fluids. When macrophage activation was carried out in L-arg-free medium, however, neither parasite killing nor NO2- production was obtained. A similar inhibition of macrophage leishmanicidal activity and of NO2- release was observed using media treated with arginase (which converts L-arg to urea and ornithine), or supplemented with NG-monomethyl-L-arg or guanidine (which inhibit the conversion of L-arg to nitrogen oxidation products). In all these situations, an excellent correlation between the levels of NO2- production by macrophages and intracellular killing of Leishmania was observed, whereas no strict correlation was detectable between leishmanicidal activity and superoxide production. Intracellular parasite killing by activated macrophages could be prevented by addition of iron salts to the incubation fluids. Incubation of free parasites with NaNO2 at acid pH (which permits the production of nitrous acid) led to immobilisation, multiplication arrest, and morphological degeneration of the microorganisms. Similarly, exposure of infected cells to NaNO2 led to killing of the intracellular parasite without affecting macrophage viability. These experiments strongly suggest that the leishmanicidal effect of activated murine macrophages involves the agency of L-arg-derived nitrogen oxidation products.
Effects of Interleukin 4 Upon Human Tumoricidal Cells Obtained From Patients Bearing Solid Tumors
Authors: Jadus Martin R , Good Robert W , Crumpacker Dina B , Yannelli John R
Published: January 2018

The use of human interleukin 4 (IL4) in the generation of tumoricidal effector cells derived from cancer patients undergoing either interleukin 2/lymphokine activated killer cells (IL2/LAK) therapy or tumor derived activated cell (TDAC) therapy was examined in the present study. Human IL4 alone did not generate LAK cells from patients undergoing IL2/LAK therapy when cultured in media containing 2% AB serum (five out of six patients). However, when the serum free medium, Aim V, was used, IL4 did generate LAK cells (eight out of nine patients), although not as cytotoxic as those activated with IL2. When cells were cultured in both IL2 and IL4 during the generation of LAK cells (3–7 days), better cell recoveries were frequently observed. The cytolytic activity of these cells against Daudi target cells was slightly reduced when compared to that response induced by IL2. This inhibition of LAK activity by IL4 was dose dependent with 1,000 U/ml IL4 producing maximal effects. This form of inhibition did not correlate with any phenotypic differences between those cells cultured in IL2 and those cells cultured in IL2 plus IL4. The IL4 mediated inhibition was also observed when the cells were cultured in Aim V medium which contains indomethacin. This inhibition induced by IL4 could not be overcome by using supra-optimal IL2. In addition to its effect during the generation of IL2 induced LAK cells, IL4 also appeared to reduce the cytolytic activity of pre-activated mature LAK cells. These results suggest that IL4 has a complex role in regulating the actions of LAK cells induced by lymphokines. When IL4 was used with IL2 during the first 5 weeks of growth of TDAC, an enhanced growth was observed when compared to the growth of TDAC when only one lymphokine was used. Besides the growth enhancement of TDAC, a better cytolytic response was observed when both lymphokines were used together. Thus, for the best growth of TDAC both IL2 and IL4 are required.
The Deficient Migration In Vitro of Lung Macrophages From Newborn Rats Normalizes After Three Days of Maturation In Situ
Authors: Kurland Geoffrey , Dauber James H
Published: January 2018

Because there are functional deficiencies in neonatal phagocytes, we investigated age-dependent differences in the directed migration of rat pulmonary macrophages (PM). Directed migration of PMs from newborn (<24 h old) rats towards partially purified rat C5a was lower than that of PMs from adults, but reached adult levels by the third post-natal day. Calculation of the dose of C5a resulting in a half-maximal migratory response (ED-50) revealed that the ED-50s for all age groups studied were similar. These results suggest that responding PMs at all times after birth have comparable sensitivity to C5a, but that a lower percentage of newborn PMs migrate toward this factor because of defects in either signal transduction or cell motility.
Characterization of the Induction of Persistent I-A Expression by Macrophages From Bcgr Mice
Authors: Faris Mary , Zwilling Bruce S
Published: January 2018

Peritoneal macrophages from mice that are resistant to Mycobacterium bovis (strain BCG) [Gros et al., J. Immunol. 127,2417, 1981] can be induced to express persistently or transiently major histocompatibility complex (MHC) class II glycoproteins. The induction of persistent expression is dependent on the dose of recombinant interferon-γ (rlFN-γ). High doses of rlFN-γ (100 U) induce persistent la expression, whereas lower closes induce only transient la expression. Once induced, the persistent expression of la does not require its continued synthesis. We show that the expression of la by macrophages that transiently express MHC class II glycoproteins is reduced by the addition of cycloheximide or monensin, whereas la expression by macrophages that persistently express la is not affected. The differential sensitivity of la expression to cycloheximide was used to study the induction of persistence that is linked to the Bcg gene. Macrophages were primed with low doses of rlFN-γ in order to induce transient, cycloheximide-sensitive la expression. The cells were then treated with high doses of rlFN-γ in order to induce persistent, cycloheximide-resistant la expression. We found that the induction of persistent la expression requires at least 3 hr of exposure to rlFN-γ. Furthermore, the addition of rlFN-γ to primed macrophages is followed by a short burst of protein synthesis that is independent of the production of new mRNA. The rapidity with which persistent la expression is induced is consistent with the rapid onset of innate resistance to Mycobacterium following injection of BCG.
Biochemical and Functional Differences Between Eosinophils From Animal Species and Man
Authors: Sun F F , Crittenden N J , Czuk C I , Taylor B M , Stout B K , Johnson H G
Published: January 2018

Eosinophils were isolated from peritoneal lavages of repeated horse serum-injected guinea pigs or rhesus monkeys and from peripheral blood of normal human donors. Eicosanoid metabolism and chemotaxis by these cells were studied by in vitro techniques. Upon calcium ionophore stimulation guinea pig eosinophils released thromboxane B2 (TXB2) and leukotriene B4 (LTB4) while monkey and human cells produced LTC4, LTB4, and 5-HETE. Guinea pig cells do not synthesize sulfidopeptide LTs, because they lack the specific LTA4 glutathione S-transferase. Guinea pig eosinophils exhibit maximal chemotactic responses to LTB4, zymosan activated plasma, and human recombinant C5a, while producing only a negligible response to platelet activating factor (PAF). Monkey and human cells responded maximally to PAF, but exhibit only a weak response to LTB4. These results suggest that the guinea pig eosinophils differ from monkey and human eosinophils in both the synthetic capacity and functional chemotaxis responses to lipid mediators.
The Ability of Polymorphonuclear Leukocyte Priming Agents to Overcome Influenza A Virus-Induced Cell Dysfunction
Authors: Abramson Jon S , Wagner M Paige , Ralston Elizabeth P , Wei Yu , Wheeler J Gary
Published: January 2018

The major mortality and morbidity resulting from influenza virus infections are due to secondary bacterial infections which occur in association with virus-induced inhibition of polymorphonuclear leukocyte (PMNL) function. The present study was undertaken to determine if compounds which prime PMNL function to subsequent stimulation with N-formylmethionyl-leucylphenylalanine (FMLP) or phorbol 12-myristate 13-acetate (PMA) can overcome influenza A virus (IAV)-induced inhibition of the PMNL chemiluminescence response to these stimuli. Granulocyte-macrophage colony stimulating factor (GM-CSF), guanosine triphosphate (GTP), and 1-oleoyl-2-acetylglycerol (OAG) were able to prime the PMNL response to FMLP and/or PMA and totally or partially overcome lAV-induced PMNL dysfunction in cells stimulated with FMLP or PMA. A direct correlation was found between the extent of PMNL priming due to GM-CSF, GTP, and OAG and the capacity of these compounds to overcome virus-induced PMNL dysfunction. The implications of these findings in regard to the mechanism by which priming agents overcome lAV-induced cell dysfunction and the potential of these compounds as therapeutic agents to treat secondary bacterial infections are discussed.
Kinetics of Phagocytosis and Phagosome-Lysosome Fusion in Hamster Lung and Peritoneal Macrophages
Authors: Bizal Colette L , Butler James P , Feldman Henry A , Valberg Peter A
Published: January 2018

The time course of phagocytosis and phagosome-lysosome fusion (PLF) in lung and peritoneal macrophages (LMs and PMs) was measured. Lysosomes in unelicited hamster LMs and PMs were labeled with lucifer yellow. Macrophages then phagocytized heat-killed Saccharomyces cerevisiae (yeast) and were evaluated at several time points for the degree to which yeast particles were adherent vs. internalized and for the presence or absence of PLF as based on the presence or absence of lucifer yellow in yeast-containing phagosomes. A three-compartment model (adherent, ingested, fused) of independent phagocytosis and PLF was developed; the number of yeast particles in each compartment was counted, and rate constants for ingestion and fusion were determined. Comparison of rate constants showed that ingestion was significantly faster in PMs (0.047 ± 0.005 min-1) than in LMs (0.016 ± 0.005 min-1) (mean ± pooled SEM; P < 0.001). Similarly, PLF was significantly faster in PMs (0.109 ± 0.013 min-1) than in LMs (0.046 ± 0.013 min-1) (P < 0.003).
Colony-Stimulating Factors Increase Resistance to Atypical Mycobacteria in Resistant Mice, Whereas They Decrease Resistance in Susceptible Strains of Mice
Authors: Denis Michel
Published: January 2018

Inbred strains of mice, notably the susceptible C57BL/6 and the resistant A/J strains of mice, were infected with a strain of Mycobacterium avium. The infection in the visceral organs of mice was then studied, and the effect of colony-stimulating factors, i.e., interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage colony-stimulating factor (CSF-1) on the infectious process was evaluated. Infusion of GM-CSF, CSF-1, and IL-3 led to a significant, albeit rather modest, increase in the mycobacterial resistance of A/J mice, as seen by a decrease in the number of colony-forming units (CFU) in the organs. Conversely, these CSFs dramatically increased the susceptibility of C57BL/6 mice, as seen by increased bacterial numbers in the spleens and livers. In vitro studies demonstrated that resident peritoneal macrophages from susceptible mice were more permissive than cells from resistant mice for mycobacterial growth. Application of CSFs on peritoneal macrophage monolayers led to an increased growth in both A/J and C57BL/6 monolayers for IL-3 and CSF-1 and a small microbiostatic effect for GM-CSF. Cytokine treatment did not, however, change the resistance/ susceptibility phenotype of isolated macrophages. Our results indicate that CSFs may exert beneficial or detrimental effects on resistance to mycobacteria depending on the host genetic make up.
Suppression of Human Macrophage Function In Vitro by δ9-Tetrahydrocannabinol
Authors: Specter Steven , Lancz Gerald , Goodfellow Darlene
Published: January 2018

The ability of macrophages to function in the presence of δ9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, was evaluated. THC added to macrophage cultures prepared from human peripheral blood inhibited macrophage spreading and phagocytosis of yeast. The effects of THC were concentration dependent, with inhibitory effects observed from 10 to 1 μg ml or lower. These results suggest that macrophages are more sensitive to THC than are lymphocytes because macrophage functions were inhibited by THC at concentrations that did not affect lymphocyte function. Thus, inhibition of lymphocyte function(s) by THC could be attributed to a direct effect of the drug on macrophages which indirectly results in lowered lymphoid cell activity.
Production of Granulocyte-Macrophage Colony-Stimulating Factor or GM-CSF-Like Growth Factor by Rat Thymic Epithelial Cell Line
Authors: Nanno Masanobu , Takahashi Tetsu , Sakata Tsuneaki , Horikawa Tatsuya , Sako Tomoyuki , Suzuki Ryuji , Itoh Tsunetoshi , Kumagai Katsuo
Published: January 2018

An epithelial cell line, IT-45R1, has been established from a thymus of normal Wistar rat and was found to produce growth factor which stimulated the proliferation of bone marrow cells. This growth factor induced the formation of colonies composed of macrophages, granulocytes, or both, in semi-solid medium and stimulated the proliferation of an interleukin 3 (IL3)/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent clone. Neither IL3-dependent clone nor IL6-dependent clone responded to IT-45R1 factor. Additionally, IT-45R1 factor acted on both rat and mouse bone marrow cells but not on human bone marrow cells. Molecular weight of IT-45R1 factor was 30 kD and its isoelectric point was 4.5. To determine whether IT-45R1 factor is GM-CSF or not, Northern blot analysis and neutralization with anti-mouse GM-CSF antibody were carried out. IT-45R1 expressed GM-CSF mRNA, but neither M-CSF nor IL6 transcripts. However, antiserum specific for mouse GM-CSF did not neutralize IT-45R1 factor. Taken together, a rat thymic epithelial cell line, IT-45R1, constitutively produces GM-CSF or GM-CSF-like growth factor.
Mouse T cell clones against Mycobacterium avium: identification of clones that modify resistance against atypical mycobacteria infection
Authors: Denis Michel
Published: January 2018

Mouse T cell clones against live Mycobacterium avium were generated from the spleens of BALB/c mice infected with M. avium TMC 702. Eighth clones were of the L3T4+ subset, whereas two were of Lyt2+ subset. Six of the L3T4+ T cell clones were of the TH1 subset whereas two were of the TH2 subset, judged on the profile of cytokine release. One of the Lyt2+ clones exhibited significant cytotoxicity against M. avium-infected mouse macrophages. Transfer of clones to nude BALB/c mice infected with M. avium was associated with insignificant changes in resistance for seven clones. One clone, of the L3T4+/TH2 subset, transferred significant resistance to the infection, also associated with infusion of supernatants from the clone, which was fully inhibited by neutralizing with anti–interleukin 4. By contrast, infusion of one TH1 clone and the cytolytic Lyt2+ led to increased microbial growth in the spleens and livers of infected mice, which was not apparent on infusion with supernatants. Application of clones' supernatants on infected macrophages had marginal effects on M. avium growth and was not correlated with protective or suppressive activity. Overall, these results suggest that T cells may influence M.  avium growth in vivo in a bidirectional manner and also suggest that interleukin 4 may be an important factor in host resistance to M. avium.
Differences in arachidonic acid metabolism by human myelomonocytic cell lines
Authors: Madden M C , Becker S , Koren H S , Friedman M
Published: January 2018

The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines was determined after incubation with interferon-γ (IFN-γ, 500 U/ml) or vehicle for 4 days. Cells were prelabeled with tritiated arachidonic acid, [3H]AA, for 4 h, and media supernatants were analyzed by high-performance liquid chromatography. None of the cell lines produced [3H]AA metabolites in large amounts during an unstimulated, basal release period (30 or 60 min). In response to 10 μM calcium ionophore A23187 incubation (30 min), undifferentiated and IFN-γ-differentiated HL60 cells formed both cyclooxygenase products (thromboxane and prostaglandins) and lipoxygenase products (leukotrienes and hydroxyeicosatetraenoic acids). In contrast to the HL60 cells, IFN-γ-differentiated U937 cells formed primarily cyclooxygenase products and undifferentiated and IFN-γ–differentiated ML3 cells did not form any [3H]AA metabolites in response to A23187. These results indicate the need to be careful in selecting a cell line for use in a phagocyte assay system when cyclooxygenase and/or lipoxygenase products could influence the assay results.
Steroidal nonspecific esterase metabolism of N-hydroxy-2-acetylaminofluorene: evidence for selective activation by the cellular reductant NADPH
Authors: Lund-Pero Margaretha , Pero Ronald W , Miller Daniel G
Published: January 2018

The significance of the nonspecific esterases of human mononuclear leukocytes (HMLs) in arylamine carcinogenesis is suggested by data showing that the metabolically formed hydroxamic acid derivative of 2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, is a substrate for this class of enzymes. A viable cell assay for the nonspecific esterases using α-naphthyl acetate as substrate is described, and data showing this activity to be sensitive to already known substrates for HML esterases as measured by three previously described assays are presented. All four assays of the same esterase activity are shown to be highly sensitive to up- and down-regulation by addition of NADPH or NADP to viable HML cultures. Selective activation of a purified rabbit nonspecific esterase by NADPH, but not by the other cellular reductants, NADH and glutathione, was demonstrated. Cytosols prepared from normal human tissue samples of liver, breast, colon, and brain were also activated by the presence of NADPH. These data do not indicate that steroidal nonspecific esterases are redox-modulated by the presence of mixed disulfides in their structure. Instead, they support the direct and specific influence of NADPH as a widespread activator of esterase activity by a mechanism not yet understood.
Ovine mononuclear phagocytes in situ: identification by monoclonal antibodies and involvement in experimental pyogranulomas
Authors: Pepin Michel , Cannella Dominique , Fontaine Jean-Jacques , Pittet Jean-Christophe , Le Pape Alain
Published: January 2018

In order to characterize in situ the macrophages present in experimental pyogranulomas induced in lambs with Corynebacterium pseudotuberculosis, a set of monoclonal antibodies (MAbs) was produced following immunization of BALB/c mice with alveolar macrophages from healthy sheep. Three MAbs were retained after two steps of screening using alveolar macrophages, peripheral blood lymphocytes, and polymorphonuclear leukocytes as target cells. Their reactivity was tested not only on macrophages in pyogranulomas but also on sections of various organs in steady-state conditions. One MAb, termed OM1, recognized the monocytes and the majority of cells of the mononuclear phagocyte system in lymphoid and nonlymphoid organs. The two other MAbs, OM2 and OM3, reacted with a subpopulation of alveolar macrophages and with other cell types in tissues, in particular with endothelial cells for the MAb OM2. On sections of experimental pyogranulomas that developed in lymph nodes draining the C. pseudotuberculosis-injected sites, MAb OM1 reacted with all the macrophages distributed in a palisade surrounding the necrotic center of the lesion from day 6 to day 28 postinoculation. The two other MAbs, OM2 and OM3, enabled two types of granulomas to be distinguished: one type was characterized by a large number of epithelioid cells stained by OM2; and the other was characterized by a few OM2-positive macrophages, whereas the OM3-positive cells were more numerous. These results show that macrophages are predominant cells in pyogranulomas and suggest two different histological patterns in the evolution of pyogranulomas induced by C. pseudotuberculosis, according to the immunological status of the host.
Effect of adenosine analogues and cAMP-raising agents on TNF-, GM-CSF–, and chemotactic peptide–induced degranulation in single adherent neutrophils
Authors: Richter Johan
Published: January 2018

Adenosine and adenosine analogues are potent inhibitors of the respiratory burst in neutrophils. Most investigators, however, have found little or no effect of these compounds on neutrophil degranulation from cytochalasin B–treated neutrophils in suspension. We have instead investigated the effect of adenosine and 2-chloroadenosine on degranulation in adherent neutrophils in the absence of cytochalasin B. Both adenosine and 2-chloroadenosine were effective inhibitors of lactoferrin secretion induced by the chemotactic peptide N-formyl-methionine-leucyl-phenylalanine (fMLP) [50% inhibitory concentration (IC50) of less than 10-6 M]. Secretion induced by tumor necrosis factor (TNF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was inhibited only at high concentrations (IC50 of approximately 10-4 M). In the presence of cytochalasin B no inhibitory effect of 2-chloroadenosine was seen. The effect of cAMP-raising agents on secretion from adherent neutrophils was also investigated. Dibutyryl cAMP at 0.2 mM reduced secretion in response to fMLP by 50% but did not inhibit TNF- and GM-CSF-induced degranulation. At a concentration of 2.0 mM dibutyryl cAMP also inhibited exocytosis in response to the two cytokines. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 300 μM reduced fMLP-induced degranulation, whereas a concentration of 1 mM was required to inhibit TNF- and GM-CSF-mediated secretion. The adenylate cyclase activator forskolin (50 μM) alone did not inhibit secretion in response to TNF or fMLP. However, in combination with IBMX (300 μM), forskolin (50 μM) reduced both TNF- and fMLP-induced secretion to less than 10%. PMA-induced exocytosis was unaffected by all these agents. In conclusion, adenosine appears to be an effective inhibitor of neutrophil granule protein secretion induced by fMLP but only a weak inhibitor of exocytosis in response to TNF or GM-CSF. Secretion in response to fMLP was also found to be more susceptible to a rise in cAMP than degranulation induced by TNF and GM-CSF.
In vitro development of lymphocytes that function as progenitors for mucus-secreting lymphokine-activated killer (LAK) cells
Authors: Ginsburg Haim
Published: January 2018

Two different types of cultures developed when two different interleukin 2 (IL-2) preparations were introduced into cultures of lymph node cells of nulnu (nude) mice maintained on an embryonic fibroblast monolayer. In the first, human recombinant IL-2 (rIL-2) stimulated the generation of colonies of large cytotoxic cells identified as lymphokine-activated killer (LAK) cells that, when grown on mesenchyme fibroblastoid monolayers prepared from 16- to 18-day embryos, could be triggered to synthesize and secrete flowing mucoid material. In the second culture, crude supernatant from cultures of rat spleen cells stimulated by concanavalin A stimulated the appearance and multiplication of blast cells that, after 20 days, differentiated into lymphocytes. This population was homogeneously composed of “wandering” lymphocytes and could be kept in a stable resting form for at least 2 months without loss of viability. When exposed to rIL-2, this whole lymphocyte population underwent a transformation into blast cells that, on the fourth day, generated granules, became cytotoxic, and differentiated into granular mucus-secreting LAK cells. When low numbers of these transformed premitotic blast cells were plated on mitomycin C-treated embryonic fibroblast monolayers, one cell out of 15 to 20 generated a clone of LAK cells. The study demonstrates that both effector and “memory” arms of differentiation can be stimulated in vitro.
Detection and analysis of the 80-kd lipopolysaccharide receptor in macrophages derived from Lpsn and Lpsd mice
Authors: Perera Pin Yu , Chen Tai-Ying , Morrison David C , Vogel Stefanie N
Published: January 2018

An 80-kd lipopolysaccharide (LPS)-binding protein with specificity for the lipid A region has been identified on lymphocyte and macrophage membranes. In an attempt to gain insight into the hyporesponsiveness of C3H/HeJ (Lpsd) mice to LPS, an affinity-purified rabbit polyclonal IgG with specificity for this receptor was used to compare the expression and distribution of the 80-kd LPS receptor on thioglycollate-elicited peritoneal macrophages derived from normal (Lpsn) and (Lpsd) mice. By enzyme-linked immunosorbent assay, immunofluorescence microscopy, and flow cytometry, macrophages from Lpsn and Lpsd mice showed comparable expression of the 80-kd LPS receptor, although only a subpopulation of macrophages from both strains express it. Macrophages from both strains showed indistinguishable surface distribution of the 80-kd LPS receptor, as determined by confocal microscopy and analysis using an anchored cell analysis station. Treatment of macrophages with interferons, protein-rich LPS, or glucocorticoids, agents known to modulate a variety of macrophage cell surface markers and receptors, failed to alter expression of the 80-kd receptor. These findings support the hypothesis that the hyporesponsiveness of the Lpsd mouse strains to LPS is not due to an absence of receptors but rather is distal to binding of LPS and most likely attributable to a failure of these macrophages to transduce the signal derived at the cell surface to the interior of the cell.
Characterization and use of monoclonal and polyclonal antibodies against the mouse interferon-γ receptor
Authors: LeClaire Ross D , Basil Mitali , Pinson David M , Redick Mark L , Hunt Joan S , Zavodny Paul J , Pace Judith L , Russell Stephen W
Published: January 2018

To facilitate investigation of its physical and functional properties, 11 monoclonal antibodies (mAbs) and a goat polyclonal IgG specific for the mouse interferon- (IFN-γ) receptor were characterized and their potential uses studied. Eight of the mAbs interacted with epitopes on the extracellular domain of the receptor, two interacted with epitopes on the intracellular domain, and one interacted with an epitope that could not be localized definitively to either region. Of the 11 mAbs, the majority (8) were IgGs, 2 were IgMs, and 1 was an IgA. Relative avidities of the seven that could be determined ranged from 333 to 0.002 μM-1. Both the polyclonal goat IgG and mAb GR-20 (the latter specific for an epitope in the binding site for IFN-γ) blocked binding of the ligand and, as expected, prevented induction by IFN-γ of priming of macrophages for tumor cell killing. None of the other mAbs had an effect despite the fact that GR-22 partially (> 50%) blocked binding of IFN-γ. Neither the polyclonal IgG nor any of the mAbs had an agonist effect. The relative usefulness of the antibodies for immunoprecipitation, immunoblotting, immunoassay, and cell staining with and without prior fixation is described. The results of immunocytochemical staining directly confirmed that the majority of immunologically reactive receptor protein expressed by cells is intracellular. To facilitate use by other investigators, the hybridomas that produce these mAbs will be offered to the American Type Culture Collection.