bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphological and Quantitative Aspects of the Development of Cytoplasmic Granules in Concanavalin A-Stimulated Normal Human Lymphocytes
Authors: McGirl Anne , Shea Giles , Melinn Mary , McLaughlin Hugh
Published: January 2018

We have studied normal human lymphocytes stimulated by concanavalin A to determine the rate and extent of development of cytoplasmic granules. Morphological studies on stimulated lymphocytes were undertaken at the light and electron microscopic level. The percentage of T lymphocytes bearing different CD antigens (CD3, CD4, and CD8) that also contained cytoplasmic granules was also assessed. Granules were identified in a high percentage of lymphocytes bearing each of the three CD markers studied.
Activated Macrophages Use Different Cytolytic Mechanisms to Lyse a Virally Infected or a Tumor Target
Authors: LeBlanc Paul A , Heath Lucie S , Um Hong-Duck
Published: January 2018

Murine bone-marrow-culture-derived-macrophages can be differentially activated to lyse either vesicular stomatitis virus infected BALB/c3T3 cells or the tumor target P815. Macrophages were activated in a manner so that they could lyse both targets. The ability of this activated population to lyse either target type was differentially inhibited by varying the assay conditions. The lysis of P815 targets was more sensitive to inhibition by the proteinase inhibitor N-p-tosyl-L-lysine chloromethyl ketone than was the lysis of virally Infected cells. On the other hand, reduction of the concentration of glucose in the assay medium, which inhibits the production of oxygen metabolytes by the hexose monophosphate shunt, or the addition of anti-tumor necrosis factor (anti-TNF) serum were able to decrease the lysis of virally infected targets but not P815 targets. Thus, the observed differences in the lysis of these two targets were due to both the activation state of the macrophages and the differential susceptibility of the targets to different effector mechanisms.
Identification of the Major Glycosyl–Phosphatidylinositol Anchored Proteins on the Surface of Human Neutrophils
Authors: Skubitz Keith M , Lakatua John D A
Published: January 2018

A novel mechanism of protein attachment to cell membranes involving the covalent linkage of the protein through an oligosaccharide to phosphatidylinositol has recently been defined. Many proteins that are anchored to the cell membrane by this mechanism can be released by treatment with phosphotidylinositol-specific phosphollpase C (PI-PLC). Monoclonal antibodies are useful as probes in the study of the roles of cell-surface components in neutrophil function. Many monoclonal antibodies that bind to human neutrophils react with the oligosaccharide lacto-N-fucopentaose III (CD15 antibodies). Human neutrophil surface proteins identified by 125l surface-labeling using lactoperoxidase were examined for PI-PLC sensitivity, to identify the major surface proteins of human neutrophils that are anchored by a glycosyl–phosphatidylinositol linkage. The major surface-labeled protein identified by lactoperoxidase-catalyzed iodination was a ~68–90-kDa protein. Three major surface proteins identified by 125I-surface labeling of ~68–80, 57, and 33-kDa were released by PI-PLC treatment Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with 125l revealed a previously unidentified 98–115-kDa protein specifically reactive with CD15 antibodies that was released from the cell by treatment with PI-PLC. The roles of these proteins in neutrophil function remain to be determined.
Activation of the Superoxide-Generating NADPH Oxidase of Macrophages by Sodium Dodecyl Sulfate in a Soluble Cell-Free System: Evidence for Involvement of a G Protein
Authors: Aharoni Iris , Pick Edgar
Published: January 2018

The superoxide (O2-)-generating NADPH oxidase of resting macrophages can be activated in a soluble cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). We demonstrate that cell-free activation is specifically enhanced by nonhydrolyzable guanosine 5′-triphosphate (GTP) analogues. Guanosine 5′-diphosphate (GDP) and guanosine 5′-O-(2-thiodiphosphate) (GDPβS) prevent cell-free oxidase activation and reverse the activated state when added to preactivated oxidase preparations. Nonhydrolyzable GTP analogues have a protective effect on the SDS-stimulated NADPH oxidase, as shown by the maintenance of more than 90% and close to 60% of enzyme activity 6 and 24 hr, respectively, after the addition of SDS to the cell-free preparation. A novel procedure is described for separating the activated NADPH oxidase from SDS and added nucleotides by gel filtration of the SDS-stimulated solubilized membrane-cytosol mixtures through a Sephadex G-25 column. By utilizing this method, it was found that the presence of micromolar concentrations of nonhydrolyzable GTP analogues during activation by SDS results in a marked increase in the recovery of SDS-independent, NADPH-dependent O2--producing activity in the excluded volume of the column. It is suggested that GTP stabilizes the SDS-induced complex between membrane and cytosolic components of the oxidase. Cell-free activation of NADPH oxidase by SDS was found to be cholera and pertussis toxin insensitive. These results serve as evidence of the participation of a G protein in the activation of the O2--generating NADPH oxidase of macrophages by SDS.
Neutrophil Accumulation and Plasma Leakage Induced In Vivo by Neutrophil-Activating Peptide-1
Authors: Colditz I G , Zwahlen R D , Baggiolini M
Published: January 2018

Neutrophil accumulation and plasma leakage induced in rabbit skin by neutrophil-activating peptide-1 (NAP-1, a 72 amino acid peptide produced by monocytes and a variety of tissue cells), E. coli endotoxin, and interleukin-1 (IL-1) were compared. Neutrophil accumulation at sites injected with NAP-1 was intense, rapid, and long-lasting; it reached a maximum rate during the first 30 min, continued at constant rate for 4–6 h, and remained detectable up to at least 8 h. In contrast, the neutrophil-attracting effect of endotoxin and IL-1 was slower in onset and more transient; it peaked in the first 2 h and declined to a very low level after 4 h. Plasma leakage induced by NAP-1 had a shorter time course than neutrophil accumulation and ceased after 6 h. Depletion of blood neutrophils by treatment with hydroxyurea prevented the plasma leakage induced by NAP-1 or endotoxin but not by histamine. Desensitization to NAP-1 was studied by restimulation of lesions. Following restimulation with NAP-1 after intervals from 6–10 h, there was diminished infiltration of neutrophils, while nearly normal responses were obtained after an interval of 24 h. Desensitization was dose dependent and affected both plasma leakage and neutrophil accumulation. In lesions initiated with NAP-1 there were normal responses following restimulation with endotoxin but marked desensitization to IL-1, suggesting that NAP-1 may contribute to inflammation induced by IL-1 but not by endotoxin.

This study indicates that NAP-1 is a potent mediator of neutrophil accumulation in vivo, with characteristics similar to those reported for C5 fragments, but with a more protracted action.
Transient Expression of Virus-Specific Promoters in Murine Resident Peritoneal Macrophages
Authors: Wu Linxian , Anaraki Farvardin , Morahan Page S , Leary Kathryn
Published: January 2018

Monocyte-macrophages (MO), being non-permissive for most viruses, play an important role in resistance to virus infection. In order to establish the mechanism of abortive infection of murine resident peritoneal MO (ResPMO) by herpes simplex virus type 1 (HSV-1), it is desirable to transfect these cells with viral promoters linked to an assayable gene, for example, the bacterial chloramphenicol acetyl transferase (CAT) gene. This will facilitate studies designed to measure levels of promoter activation or repression in these specialized cells. Transient expression of CAT in ResPMO was achieved with DEAE-dextran, but not using either calcium phosphate precipitate or lipofectin. CAT expression driven by various virus-specific promoters was less efficient in ResPMO compared with Vero cells and approximately 50% of input plasmid DNA remained in Vero cells at 48 h post transfection, but only 9% was detectable in ResPMO. However, approximately 6% of ResPMO and 9% of Vero cells contained CAT-specific DNA at 24 h post transfection. In addition, 2% of cells of either cell type contained CAT-specific polypeptide at 48 h. This is therefore the first report that the non-replicating murine ResPMO can be transfected in vitro and more importantly, that these cells express the transfected gene products.
Lysis of Autologous Human Macrophages by Lymphokine-Activated Killer Cells: Interaction of Effector Cell and Target Cell Conjugates Analyzed by Scanning Electron Microscopy
Authors: Streck Richard J , Helinski Ernest H , Ovak Geraldine M , Pauly John L
Published: January 2018

Lymphokine (i.e., interleukin 2; IL-2)-activated killer (LAK) cells derived from normal human blood are known to destroy human tumor target cells. Accordingly, immunotherapy modalities using IL-2, either alone or in combination with LAK cells, have been evaluated for eradicating metastatic cancer. In studies conducted to characterize receptors on LAK cell membrane ultrastructures, we observed that LAK cells kill autologous human monocyte-derived macrophages (Mφ). In these experiments, peripheral blood mononuclear cells of a healthy adult donor were cultured to generate LAK cells and autologous nonadherent Mφ. Thereafter, conjugates were prepared by incubating for 3 h autologous populations of LAK cells and Mφ. Examination of the conjugates by scanning electron microscopy (SEM) identified LAK cell-mediated killing of Mφ. Moreover, SEM analysis of the LAK cell membrane architecture identified microvilli-like ultrastructures that provided a physical bridge that Joined together the LAK cell and Mφ. The immunological mechanism(s) underling LAK cell killing of autologous Mφ is not known; nevertheless, these conjugates will provide a useful model to study membrane receptors on ultrastructures that mediate the initial stages of cytolysis that include target cell recognition and cell-to-cell adhesion. The results of our observations and the findings of other investigators who have also demonstrated LAK cell killing of autologous normal human leukocytes are discussed in the context of the association of IL-2 and IL-2-activated killer cells with side effects observed in ongoing clinical trials and with autoimmune disorders.
Production of Collagen Type I by Mouse Peritoneal Macrophages
Authors: Vaage Jan , Lindblad William J
Published: January 2018

The origin and identity of the cells that accumulate and produce collagen in wound healing and in granulomatous and fibrotic processes have long remained unsettled. In this study, it was found that pure cultures of peritoneal macrophages produced hydroxy-L-proline not associated with Clq and produced detectable quantities of collagenase-digestible protein. Cultured macrophages synthesized collagen while retaining their phagocytic capacity. Immuno-blot analysis identified the collagen as Type I. This shows that macrophages have collagen-forming capacity and may be productive cells in pathologic collagen deposition.
Computer Image Analysis Method for Rapid Quantitation of Macrophage Phagocytosis
Authors: Odeyale Charles O , Hook Gregory R
Published: January 2018

A new method is described for rapidly quantitating phagocytosis by adherent macrophages in culture using computer image analysis (CIA) of video light microscopic images. Ingestion of fluorescent microspheres by peritoneal murine macrophages is used to model phagocytosis. The grey levels of digital phase contrast and fluorescent microscopic images are used to quantitate the number of microspheres per cell. The method is semi-automatic, analyzes approximately 2 × 103 cells/hr, and simultaneously measures phagocytosis (microspheres/cell), cell area, and density (number of cells/mm2). CIA obtains the same microspheres/cell average as manual microscopic counting and an analytical precision of 5%. As expected, CIA found that the number of microspheres/cell linearly increases with increasing macrophage-microsphere co-culture time or increasing microsphere concentration until macrophages become saturated. CIA finds increased phagocytosis by interferon-gamma-treated cells and suppressed phagocytosis by cytochalasin B- or 4°-treated cells relative to controls, which demonstrates that CIA can resolve biological changes in macrophage phagocytosis. CIA also provides quantitative data on macrophage morphometry and density and found an increase in the cell area and density of INF treated macrophages. CIA provides significantly more phagocytic, morphometric, and density data than conventional manual microscopic counting methods or flow cytometric methods. The limitations, improvements, and future applications of this method are discussed.
Role of Adherence vs. Spreading in the Induction of Membrane-Associated Interleukin-1 on Mouse Peritoneal Macrophages
Authors: Labadia Mark , Faanes Ronald B , Rothlein Robert
Published: January 2018

Adherence of peritoneal exudate cells (PEC) to plastic has been shown to activate several PEC functions, such as tumor ceil lysis and membrane-associated interleukin-1 (mlL-1) expression. Several studies have demonstrated that leukocyte adherence is dependent on divalent cations. In this study, ethylenediaminetetrascetic acid (EDTA), a known chelator of divalent cations, was used to evaluate the role of cell attachment vs. spreading in adherence-induced mlL-1 activity on resident C57BL/6 mouse PEC. Significant inhibition of PEC spreading on plastic and mlL-1 expression was noted when PEC were cultured in the presence of 10 mM EDTA. However, PEC remained adherent in the presence of EDTA and were able to express mlL-1 activity in response to a soluble stimulus lipopolysaccharide (LPS) 1 μg/ml. These results suggest that the divalent cation-dependent spreading of PEC on plastic initiates or enhances the expression of mlL-1 activity. Additionally, adhesion and LPS stimulate mlL-1 expression by independent mechanisms.
Role of Interferon-γ in Counteracting the Suppressive Effects of Transforming Growth Factor-β2 and Glucocorticoids on the Production of Tumor Necrosis Factor-α
Authors: Dunham Diane M , Arkins Sean , Edwards Carl K , Dantzer Robert , Kelley Keith W
Published: January 2018

The multipotential cytokine, transforming growth factor-β2 (TGF-β2), is as effective as glucocorticoids in suppressing the production of tumor necrosis factor-α (TNF-α) by lipopolysaccharide (LPS)-stimulated macrophages, and this inhibition can be abrogated by exogenous interferon-γ (IFN-γ). Porcine alveolar macrophages triggered with LPS produce TNF-α as identified by complete blocking of cytotoxicity on WEHI 164 clone 13 cells in macrophage supernatants by a monoclonal antibody to human TNF-α. Platelet-derived porcine TGF-β2, at a concentration of 4 nM, inhibited LPS-induced production of TNF-α by 93%. Dexamethasone was as effective as TGF-β2, suppressing TNF-α production by 86% at a concentration of 4 nM. The natural but less potent glucocorticoid Cortisol inhibited TNF-α production by 100% at a 100-fold higher concentration (400 nM). Recombinant PoIFN-γ consistently primed LPS-triggered macrophages for increased production of TNF-α by 50-100%, and this priming was totally blocked by a polyclonal antibody to rPoIN-γ. Furthermore, the suppression in LPS-induced production of TNF-α caused by TGF-β2, dexamethasone, and Cortisol could be reversed by addition of rPoIFN-γ. These data show that alveolar macrophages can be effectively primed by rPolFN-γ even in the presence of moderately suppressive doses of TGF-β2 and antiinflammatory steroids.
Avian Inflammatory Macrophage Function: Shifts in Arachidonic Acid Metabolism, Respiratory Burst, and Cell-Surface Phenotype During the Response to Sephadex
Authors: Golemboski K A , Whelan J , Shaw S , Kinsella J E , Dietert R R
Published: January 2018

The avian inflammatory response to intraperitoneal (i.p.) Sephadex injection produces macrophages which display characteristics of an increasingly activated state over time. We examined elicited chicken peritoneal exudate cells (PECs) with respect to superoxide anion production, arachidonic acid metabolism and cell surface la and transferrin receptor (TfR) expression from 4 to 96 h after i.p. stimulation. Avian PECs showed the highest level of superoxide release when harvested just 4 h after injection, and did not produce PGE2 or 6-keto PGF1α. Early (4-h) PECs produced elevated amounts of thromboxane as compared to later (42-h) macrophages. Expression of both la and TfR increased between 4 and 24 h after Sephadex stimulation; TfR remained elevated through 96 h, but la declined after 42 h. Some aspects of chicken macrophage regulation of superoxide anion, thromboxane release, and surface antigen expression are in contrast with those reported for mouse macrophages.
Lectin Binding of Rat Bone Marrow Cells During Colony-Stimulating Factor Type 1–Induced Differentiation: Soybean Agglutinin as a Marker of Mature Rat Macrophages
Authors: Krugluger Walter , Gessl Alois , Boltz-Nitulescu George , Förster Othmar
Published: January 2018

Rat bone marrow cells (BMC) cultured in the presence of murine colony-stimulating factor type 1 (CSF-1) differentiate within 7 days into a cell population containing 96–100% macrophages (Mφ). In this study, binding of 10 different fluorescein isothiocyanate (FITC)-conjugated lectins to cultured BMC at various stages of differentiation into Mφ was investigated. Only soybean agglutinin (SBA) showed a binding pattern that was significantly correlated to Mφ differentiation. Nearly all adherent (A) cells bound SBA. Binding of SBA to nonadherent (NA) cells increased from 20% on day 0 to 80% on day 8. NA cells were separated by means of a fluorescence-activated cell sorter into SBA-, SBA±, and SBA+ fractions. On day 0 and day 2, Mφ, blast-like cells, eosinophils, and some neutrophils were found in the SBA+ population. From day 4 onwards, the SBA+ fraction contained almost exclusively Mφ. Neutrophils and some blasts were found in the SBA-population on days 0 and 2. In the SBA± fraction, mainly blasts and lymphocytes were identified. With increasing time of culture, Mφ or Mφ precursors prevailed also in the SBA-/± cell population. Cells forming colonies in soft agar in the presence of CSF-1 were highly enriched in the SBA± fraction. A large number of cells in S-G2/M phases but few colony-forming cells were found in the SBA+ population. Our data suggest that SBA is a useful additional tool to define late stages of Mφ differentiation.
Palmitate-Derivatized Antibodies Can Specifically “Arm” Macrophage Effector Cells for ADCC
Authors: Colsky Arthur S , Peacock James S
Published: January 2018

The use of palmitate-derivatized antibodies (pal-Ab) for “arming” macrophage (Mφ) effector cells for antibody-dependent cellular cytotoxicity (ADCC) is described. Pal-Ab were incorporated onto the Mφ plasma membranes by insertion of the palmitate hydrocarbon chains into the outer leaflet of the phospholipid bilayer. Mφ bearing pal-Ab specific for chicken erythrocytes (CE) mediated efficient destruction of the CE targets. Neither non-ADCC effector cell populations nor pal-Ab consisting of antibody F(ab')2 fragments effected significant CE lysis. Mφ bearing pal-Ab that were not specific for CE did not mediate CE destruction, nor did anti-CE pal-Ab-bearing Mφ lyse nonspecific human erythrocyte targets. In this system of effector cell arming, the palmitate anchor of pal-Ab allows for the incorporation of large numbers of antibodies onto the effector cell surface, where they can promote efficient target cell capture and engage preexisting or newly expressed FcR on the effector cell surface. The results in this study, together with those from previous and ongoing investigations in which F(ab')2 pal-Ab were shown to mediate Fc receptor (FcR)-independent cytotoxicity by natural killer (NK) cells and Mφ populations at appropriate states of activation, suggest that pal-Ab, by directing both ADCC and FcR-independent effector cell activity onto a specified target, offer important advantages over other methods of effector cell arming.
Inhibition of Human Neutrophil Activation by the Allergic Mediator Release Inhibitor, CI-949
Authors: Wright Clifford D , Stewart Sheila F , Kuipers Paul J , Hoffman Michael D , Thueson David O , Conroy Mary Carol
Published: January 2018

The allergic mediator release inhibitor CI-949 [5-methoxy-3-(1-methylethoxy)-1-phenyl-N-1H-tetwol-5-yl-1H-indole-2-carboxamide, L-arginine salt] was evaluated for its effects on human neutrophil functions. CI-949 (100 μM) inhibited spontaneous migration and chemotaxis toward f-met-leu-phe (FMLP) by 49.1% and 45.8%, respectively. At the same concentration, CI-949 inhibited the phagocytosis of serum-opsonized zymosan (SOZ) by 39.0%. CI-949 inhibited leukotriene B4 and thromboxane B2 release in response to SOZ with IC50S of 2.0 μM and 3.3 μM, while inhibiting the response to FMLP with IC50s of 1.7 and 2.0 μM. CI-949 also inhibited myeloperoxidase release from primary lysosomal granules in response to the following stimuli with the respective IC50s (μM): C5a (40.3); FMLP (34.4): SOZ (21.4); concanavalin A (Con A) (3.9); and calcium ionophore A23187 (91.2). In contrast, CI-949 inhibited lysozyme release from secondary granules in response to SOZ and Con A with IC50S of 99.3 and 56.1 μM, while inhibiting the response to C5a, FMLP, and A23187 by 41.2%, 52.4%, and 10.0%, respectively, at 100 μM. CI-949 (100 μM) had no inhibitory effect against lysozyme release in response to L-α-1,2 dioctanoylglycerol (DiC8), or phorbol 12-myristate 13-acetate (PMA). CI-949 inhibited superoxide anion generation stimulated by FMLP and Con A with IC50S of 33.9 and 25.8 μM, while inhibiting the response to C5a, SOZ, and A23187 by 36.6%, 24.8%, and 14.1% and having no effect on the response to DIC8 or PMA at 100 μM. These results demonstrate preferential inhibition of arachidonic acid metabolism and degranulation of primary lysosomal granules by CI-949 with selectivity for stimuli which promote intracellular calcium mobilization or calcium influx.
Intracellular pH Regulates the Production of Different Oxygen Metabolites in Neutrophils: Effects of Organic Acids Produced by Anaerobic Bacteria
Authors: Tonetti Maurizio , Cavallero Annalisa , Botta Giuseppe A , Niederman Richard , Eftimiadi Costantino
Published: January 2018

The effects of short chain carboxylic acids (SCCA), namely succinic, butyric, and iso-butyric, on neutrophil metabolic activation were assessed. SCCA induced a significant decrease in O2- recovery and chemlluminescent response in neutrophils activated with the diacylglycerol analog tetradecanoyl-phorbol-acetate (TPA). SCCA did not alter O2 consumption, H2O2 production, or the calorimetrically determined energy expenditure occurring during the metabolic burst. SCCA also induced a significant acidification of intracellular pH (pHi). These results are consistent with an increased divalent versus univalent O2 reduction performed by the NADPH oxidase at a more acidic intracellular pH.
Gelatin Sponge Model of Effector Recruitment: Tumoricidal Activity of Adherent and Non-Adherent Lymphokine-Activated Killer Cells After Culture in interleukin-2
Authors: Akporiaye Emmanuel T , Barbieri Celia A , Stewart Carteton C , Bender James G
Published: January 2018

This study examined the specific tumoricidal activity of lymphokine-activated killer (LAK) cells derived from tumor-infiltrating lymphocytes that prevent the growth of secondary tumors in animals harboring progressing primary tumors. A pre-implanted gelatin sponge was employed to capture infiltrating host effectors during the expression of concomitant tumor immunity. Additionally, this study compared the cytolytic activity of these sponge-derived cells with those of counterpart splenic lymphocytes. The cells from both sources were cultured for 4 days in IL-2 to generate LAK cells which were further expanded in IL-2-containing medium for up to 11 days. The cytotoxic activities of these cells were measured in a Chromium-51 release assay. The data revealed that the culture of splenic, or sponge-derived lymphocytes results in the emergence of non-adherent and adherent cell populations with LAK activity. The 4-day sponge-derived LAK cells (adherent and non-adherent) exhibited significant cytotysis of EMT6 cells while the spleen-derived counterparts showed minimal cytotoxicity toward these targets. Some NK activity in LAK cells derived from both sources was evident by their lysis of YAC-1 cells. LAK cells from both sources were incapable of lysing histo-compatible EL-4 (H-2b) tumor cells. The lysis of the EMT6 cells by the sponge-derived LAK cells was maintained over an 11-day period of culture in IL-2. Conversely, the spleen-derived LAK cells were unable to significantly lyse EMT6 cells during this period of in vitro culture. These results show the superior specific tumoricidal activity of LAK cells derived from lymphocytes mediating tumor rejection in vivo (sponge-derived) over that of counterpart splenic lymphocytes.
Distinct Patterns of Chemotactic Peptide-Induced Calcium Mobilization in Differentiated Myeloid Leukemia Cells and Peripheral Blood Neutrophils
Authors: Laskin Debra L , Sirak Andrea A , Robertson Fredika M , Laskin Jeffrey D
Published: January 2018

Flow cytometry was used to compare intracellular calcium mobilization in mature neutrophilic granulocytes (PMN) with HL-60 promyelocytic leukemia cells induced to differentiate with dibutyryl cyclic adenosine 3’,5'-monophosphate (dbcAMP). Using the calcium-specific probe indo-1 acetoxymethyl ester, we found that the ability of differentiating HL-60 cells to mobilize calcium in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) developed concomitantly with expression of receptors on the cells for this peptide. Mobilization of calcium in HL-60 cells, as well as in PMN, in response to fMLP was dose dependent and was related to the presence of calcium in the culture medium. In calcium-free medium, tenfold higher concentrations of fMLP were required to induce calcium mobilization than in calcium-containing medium. In HL-60 cells, calcium mobilization occurred more rapidly than in PMN and was independent of extracellular calcium. Furthermore, the response of HL-60 cells to fMLP was more prolonged than the response of PMN and was also of greater magnitude. Calcium mobilization in both HL-60 cells and PMN was partially inhibited by the calcium channel blocker, verapamil, but completely blocked by trimethylbenzoic acid 8-(diethylamino) octyl ester, an intracellular calcium antagonist. These results indicate that although both cell types mobilize calcium in response to fMLP, the characteristics of the responses are distinct. These differences may underlie distinct functional responses of PMN and differentiated HL-60 cells to fMLP.
Activation of Macrophages From Aging Mice by Detoxified Lipid A
Authors: Chen Yifang , Solem Laura , Johnson Arthur G
Published: January 2018

A detoxified derivative of endotoxic lipopolysaccharides (LPS), monophosphoryl lipid A (MPL), which is capable of inducing nonspecific resistance against several infectious organisms, was tested for its capacity to activate peritoneal macrophages (Mϕ) from young and immunodeficient aging BALB/c and C3H/HeN mice. Superoxide generation and hydrogen peroxide release by Mϕ from aging mice were elevated following intraperitoneal injection with 25 µg of LPS or MPL, although they did not reach the peak levels achieved in LPS or MPL-treated young mice. Nitroblue tetrazolium reduction (NBT) by peritoneal Mϕ from aging C3H/HeN mice treated with MPL was higher than that in control aging mice, equalling that from MPL-treated young mice. LPS, its toxic counterpart, however, failed to increase NBT reduction in either group. MPL enhanced lysozyme activity in Mϕ from both aging and young C3H/HeN mice above initial control levels. On the other hand, LPS suppressed lysozyme activity in Mϕ from young, but not aging mice. Phagocytosis of Candida albicans by Mϕ from BALB/c mice was increased in both groups when stimulated by MPL, but not LPS. Similarly, MPL enhanced the ability to kill Candida in both aging and young BALB/c mice. This effect was not seen with LPS. Thus, a detoxified derivative of LPS was found capable of activating the respiratory burst, NBT reduction, elevating lysozyme activity, as well as phagocytosis and killing of Candida in murine peritoneal Mϕ from both young and aging mice.
Respiratory Burst Capability of Polymorphonuclear Neutrophils and TNF-α Serum Levels in Relationship to the Development of Septic Syndrome in Critically III Patients
Authors: Trautinger F , Hammerle A F , Pöschl G , Micksche M
Published: January 2018

Activated polymorphonuclear neutrophils (PMN) and neutrophil activating mediators such as tumor necrosis factor-α (TNF-α) are thought to be involved in the pathophysiology of sepsis and multiple organ failure syndrome (MOFS). In critically ill patients at high risk for the development of septic syndrome (n = 17) peripheral blood PMN were assayed for O-2 and H2O2 production after stimulation with phorbol myristate acetate (PMA, 40 nM). Serum TNF-α levels were determined by ELISA. At the time of admission to the intensive care unit we found significant higher levels of TNF-α (P = 0.0001) in the serum of patients finally developing sepsis correlating to higher respiratory burst capability in comparison to nonseptic patients. Additionally we were able to demonstrate a significant (P = 0.0016) lower dismutation rate of O-2 to H2O2 in deceased patients in comparison to survivors. These results give further evidence that elevated levels of circulating TNF-α and activated PMN play a significant role in the pathogenesis of septic syndrome in critically ill patients.
Macrophage Colony-Stimulating Factor Is Required for Human Monocyte Survival and Acts as a Cofactor for Their Terminal Differentiation to Macrophages In Vitro
Authors: Brugger Wolfram , Kreutz Marina , Andreesen Reinhard
Published: January 2018

Functional competence as well as phenotype heterogeneity of macrophages depend on the completion of their maturation pathway. Differentiation of committed myeloid progenitor cells is induced by colony-stimulating factors (CSF), but no consistent data exist on which factor(s) induce the terminal maturation from the circulating blood monocyte to the mature macrophage. In vitro, monocyte to macrophage transformation occurs in the presence of serum and can be followed by the expression of the maturation-associated antigens gp65-MAX.1, gp68-MAX.3, and CD51. We describe that the differentiation-inducing activity in serum cannot be replaced by any of the known and available purified recombinant cytokines. In the absence of serum monocytes die in suspension cultures while surviving as non-differentiating cells when cultured adherent to plastic. In serum-free suspension cultures survival can be significantly improved by the addition of recombinant human macrophage (rhM)-CSF whereas other cytokines do not. At any stage of serum-free adherent culture, monocyte to macrophage differentiation can be induced rapidly by the addition of serum, whereas cytokines (rhM-CSF, recombinant human granulocyte macrophage [rhGM]-CSF, recombinant human granulocyte [rhG]-CSF, recombinant human interleukin [rhIL]-1, rhIL-3, rhIL-4, rhIL-6, tumor necrosis factor [TNF]-α, interferon [IFN]-α, IFN-γ) alone or in combination are not effective. Serum-induced maturation, however, was suppressed in the presence of neutralizing anti-M-CSF antibodies. In addition to phenotype analysis, the secretory repertoire of rhM-CSF cultured monocytes was analyzed in comparison to serum cultured monocytes which further characterized them to be immature cells, i.e., low release of maturation-associated products such as α-2-macroglobulin, neopterin, fibronectin, and TNF-α, but high IL-6 secretion, an attribute of blood monocytes. We conclude that for monocyte survival in vitro the presence of endogenous M-CSF and possibly other autocrine factors elicited by cell adherence are required for the induction of macrophage maturation; however, yet undefined additional factor(s) are necessary. They are present in serum and may act in conjunction with M-CSF but are distinct from all known cytokines. Our in vitro system may be useful in the screening and discovery of these serum factor(s).
Differential Modulation of Surface Antigens on Human Macrophages by IFN-Gamma and GM-CSF: Effect on Susceptibility to LAK Lysis
Authors: Blanchard D Kay , Djeu Julie Y
Published: January 2018

We have previously reported that cultured human monocytes are lysed by autologous lymphokine-activated killer (LAK) cells in vitro and that treatment of monocytes with interferon-gamma (IFN-gamma) decreased their sensitivity to lysis. Conversely, incubation of monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly enhanced their susceptibility to LAK-mediated cytotoxicity. To determine if certain antigens were differentially modulated on macrophages by IFN-gamma and GM-CSF, cytokine-treated and untreated monocytes were analyzed for the expression of a variety of cell surface markers by flow cytometry. Cytotoxicity assays were performed to assess the ability of antibodies to each of these markers to block LAK lysis of macrophage target cells. While several of the surface structures were differentially modulated by cytokine treatment, it was found that only monoclonal antibodies to the adhesion proteins CD11a and CD18 were capable of blocking lysis of either cytokine-treated or untreated target macrophages.
Cryopreserved Cytoplasts From Human Polymorphonuclear Leukocytes (Cytokineplasts) Are Chemotactic at Speeds Comparable to Those of Fresh Intact Cells
Authors: Malawista Stephen E , de Boisfleury-Chevance Anne
Published: January 2018

Cytokineplasts (CKP) are granule-poor cytoplasts from human polymorphonuclear leukocytes (PMN) that retain motile function, even (unlike the parent PMN) after cryopreservation. Employing time-lapse videomicroscopy, we examined the chemotactic properties of CKP after cryopreservation toward erythrocytes lysed by laser microirradiation. Paths of locomotion were plotted for six CKP in the field, and velocities were calculated at 10-sec intervals. Mean velocities of the six fragments, ranging from 9.3 to 20.8 μm/min, are of the order of fresh, intact PMN, the fastest of locomoting cells.
Purification and Characterization of Macromolecular Phagocytosis Activators Released From Platelets
Authors: Sakamoto Haruhiko , Yokoya Yukihiro
Published: January 2018

Substantial amounts of macromolecular activators of phagocytosis from platelets (MAPPs) were released in response to exposure of platelets to the specific agonists thrombin and collagen and to calcium ionophore A23187. Centrifugation of the platelets in culture medium also resulted in a release of MAPPs, but not when the platelets were frozen and thawed prior to centrifugation.

In an experiment using outdated platelet concentrates, larger and smaller MAPPs (1-MAPP and s-MAPP, respectively) were purified from the thrombin stimulated supernatant (PRPr-plasma) by sequential steps of ammonium sulfate precipitation, delipidation with ethylacetate, ConA-Sepharose affinity chromatography, MONO Q anion exchange chromatography, and Superose 12 gel filtration. This procedure yielded 59,500-fold and 63,600-fold purified 1-MAPP (0.95 mg) and s-MAPP (0.41 mg), respectively, from 1,320 ml PRPr-plasma containing 84,500 mg protein. By gradient polyacrylamide gel electrophoresis the respective molecular weights (MW) of 1-MAPP and s-MAPP were 290–320 kd and 140–160 kd; isoelectric points were 5.6 and 4.6. Both MAPPs activated neutrophil phagocytosis of IgG-SRBC at concentrations in the range of 1 pM–100 nM. Indirect enzyme-linked immunosorbent assay (ELISA) and comparisons of the concentrations required for phagocytosis activation suggested that the MAPPs were not derived from fibronectin.
Involvement of HLA-DR+ Large Granular Lymphocytes in the Induction of Tumor Necrosis Factor by Mycobacterium avium-intracellulare Complex
Authors: Michelini-Norris M Beatriz , Blanchard D Kay , Friedman Herman , Djeu Julie Y
Published: January 2018

This study shows that normal human large granular lymphocytes (LGL) secrete tumor necrosis factor (TNF) in response to Mycobacterium avium-intracellulare complex (MAI). Percoll density gradient fractionation of peripheral mononuclear cells showed TNF activity in the fractions corresponding to LGL and not T cells, even when 5% monocytes were added to the T lymphocytes for accessory function. TNF release was not abrogated by treatment of the crude LGL preparations with anti-Leu M3. -CD4. and -CD8 antibodies (Ab) plus complement (C), but was abrogated by anti-CD16 and -CD2 Ab, as expected. Interestingly, anti-HLA-DR monoclonal antibody (mAb) treatment significantly diminished TNF activity from LGL, but maintained natural killer (NK) cell function unmodified as opposed to CD2 - and CD16- cell depletion. Panning studies demonstrated that TNF secretion upon MAI stimulation resided only in the HLA-DR+ LGL and not the DR LGL population. These results indicate that normal fresh HLA-DR+ LGL, as well as monocytes, are also responsible for rapid TNF secretion during early MAI infection. These DR - cells appear to be distinct from those expressing NK function.
Polyphosphoinositide Metabolism in Polymorphonuclear Cells From Healthy and Thermally Injured Rats: Effect of the Immunomodulator RU 41740
Authors: Tissot M , Mathieu J , Mirossay L , Thuret A , Giroud JP
Published: January 2018

Burn trauma is associated with alterations of various components of host defenses, including impaired neutrophil functions. In an animal model of experimental thermal injury, we studied if the modifications of cellular reactivity result from alterations in signalling systems by comparing polyphosphoinositide breakdown, particularly the production of inositol phosphates (IP, IP2 IP3), in healthy and burned rat polymorphonuclear neutrophil leukocytes (PMNs).

Neutrophil activators such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and serum-opsonized zymosan increased in vitro production of inositol phosphates in PMNs from healthy rats. The immunomodulator RU 41740 had no effect by itself, but decreased the stimulating effect of fMLP and zymosan. In PMNs from burned rats, the stimulating effects of fMLP and zymosan were decreased, while RU 41740 stimulated inositol phosphate generation. In vivo treatment with RU 41740 inhibited the activation of phosphoinositide metabolism by fMLP or zymosan in healthy rat PMNs. Similar treatment of burned rats after injury restored the stimulating effect of fMLP and zymosan on inositol phosphate accumulation in PMNs.

Thus, RU 41740 can modulate fMLP and zymosan receptor-mediated signal transduction, inducing an attenuation of the phosphatidylinositol hydrolysis response. After burn injury, when the activating effects of fMLP and zymosan are inhibited, RU 41740 can, on the contrary, stimulate phospholipase C-mediated polyphosphoinositide turnover and the formation of intracellular messengers such as IP3.

These data show that RU 41740 has different effects on polyphosphoinositide metabolism in rat PMNs, according to the physiological and pathological state of the animals. Interestingly, it has a beneficial action on the post-burn decrease in PMN reactivity.
Granule-associated flavin adenine dinucleotide (FAD) is responsible for eosinophil autofluorescence
Authors: Mayeno Arthur N , Hamann Kimm J , Gleich Gerald J
Published: January 2018

Unstained human eosinophils exhibit marked autofluorescence in comparison to other leukocytes due to a granule-associated fluorescent substance. Fluorescence spectroscopy of granule extracts reveals excitation maxima at ~380 and ~450 nm with a single emission at ~520, characteristic of flavins. The fluorescent material from eosinophil granule extracts was characterized by fluorescence, high-performance liquid chromatographic, and enzymatic analyses. First, acidification to pH 2.6 resulted in increased fluorescence, indicative of flavin adenine dinucleotide (FAD). Second, because flavin mononucleotide (FMN) and riboflavin cannot be distinguished by acidification, high-performance liquid chromatography was performed and revealed a predominance of FAD and smaller amounts (<15%) of both FMN and riboflavin. Third, the presence of FAD was clearly demonstrated by reconstitution of the activity of d-amino acid oxidase, a FAD-dependent enzyme, when granule extracts were added to the apoenzyme. Thus, we have identified FAD as the predominant fluorophore in eosinophil granules. The small amounts of FMN and riboflavin detected may result from the hydrolysis of FAD under the acidic conditions of granule extraction. Because fluorescent material is deposited onto target cells by eosinophils, it is possible that granule-associated flavoproteins may act as a source of hydrogen peroxide and/or superoxide, which, in conjunction with eosinophil peroxidase, could yield potent cytotoxic agents.
Effects of Pseudomonas aeruginosa rhamnolipids on human monocyte-derived macrophages
Authors: McClure Carol D , Schiller Neal L
Published: January 2018

Pseudomonas aeruginosa, a major opportunistic gram-negative pathogen, produces and secretes two heat-stable hemolytic glycolipids, a monorhamnolipid and a dirhamnolipid. In this paper a simplified method for the isolation of these rhamnolipids is described. The effect of these two rhamnolipids, both together and individually, on the viability and structural morphology of human monocyte-derived macrophages (MDMs) was examined. These cells were found to be very susceptible to the cytolytic activity of the rhamnolipids, particularly the dirhamnolipid. The monorhamnolipid, although not as cytolytic as the dirhamnolipid, caused extensive blebbing of the MDM plasma membrane. Comparison studies with several detergents confirmed the different yet distinct detergent-like activity of each rhamnolipid form. At sublethal doses, the rhamnolipids produced marked cellular distortions of the MDMs and inhibited the ability of these cells to bind and/or ingest preopsonized bacteria. The potential mechanism of action of these rhamnolipids on the MDM membranes is discussed, as well as the possible significance of these extracellular bacterial glycolipids as a virulence factor in the pathogenesis of P. aeruginosa.
Simultaneous assay for oxidative metabolism and adhesion of human neutrophils: evidence for correlations and dissociations of the two responses
Authors: Bellavite P , Chirumbolo S , Mansoldo C , Gandini G , Dri P
Published: January 2018

An assay method for the simultaneous evaluation of the oxidative metabolism and adherence of human neutrophils is described, together with certain specific applications. Incubations were performed in serum-coated microtiter plates, where oxidative metabolism was measured as O−2 release and, after washing out the nonadherent cells, the adhesion was measured as activity of acid phosphatase. Three agonists tested in this system –- opsonized zymosan, concanavalin A, and N-formyl-methionyl-leucyl-phenylalanine –- induced both activation of O−2 release and cell adhesion, but the two functions had time course and dose dependence patterns that varied depending on the stimulant. Particularly with concanavalin A, O−2 release and adhesion response were markedly dissociated; this lectin at low doses increased neutrophil adherence without triggering any O−2 production, whereas at high doses it increased both O−2 production and adherence. Anti-integrin monoclonal antibodies did not affect adhesion induced by low-dose concanavalin A but inhibited the adhesion induced by the other tested agonists. Adhesion and O−2 production were also found to be differentially affected by the NADPH oxidase inhibitor diphenylene iodonium, the sulfhydryl reagent N-ethylmaleimide and the A2 agonist adenosine, indicating that these neutrophil responses have various transductional pathways that also depend on the type of stimulus.
A monoclonal antibody specific for immunoglobulin A receptor triggers polymorphonuclear neutrophil superoxide release
Authors: Shen Li
Published: January 2018

An immunoglobulin M (IgM) monoclonal antibody, My43, specific for IgA Fc receptor (FcαR) on human monocytes, bound to human polymorphonuclear neutrophils (PMNs) and inhibited their ability to bind IgA but not IgG. It was observed that the PMN oxidative burst was induced by both polymeric IgA and aggregated IgG, whereas IgM was without effect. The IgG-mediated oxidative burst was inhibited by anti-FcγRII Fab and anti-FcγRIII F(ab')2 but not by My43. Conversely, the IgA-mediated oxidative burst was inhibited by My43 but not by anti-FcγRII or anti-FcγRIII. When anti-Fc receptor monoclonal antibodies (mAbs) were used directly as ligands, it was observed that both anti-FcγRII Fab and anti-FcγRII F(ab')2 promoted the oxidative burst when cross-linked. Moreover, My43, when cross-linked with F(ab')2 antimouse IgM, also triggered the oxidative burst, whereas an IgM anti-GDI5 mAb, PM81, did not stimulate function. This demonstrates that IgA receptors on PMNs are function-triggering molecules and that an anti-IgA receptor mAb may be substituted as a ligand.
Recognition of Histoplasma capsulatum yeast-cell antigens by human lymphocytes and human T-cell clones
Authors: Henderson Harold M , Deepe George S
Published: January 2018

We have prepared a detergent extract from the cell wall and cell membrane of Histoplasma capsulatum yeast cells that is recognized by T cells from mice immunized with viable organisms or with the extract. A 62-kd antigen from this extract has been isolated and has been shown to be antigenic and to confer protective immunity in mice. In this study, we examined the in vitro proliferative response by human lymphocytes and human T-cell clones to both the extract from the cell wall and cell membrane and the 62-kd antigen, termed HIS-62. Seven healthy individuals were identified whose peripheral blood mononuclear cells responded to the cell wall and cell membrane extract from H. capsulatum yeast cells. Mononuclear cells from all seven individuals recognized histoplasmin. T-cell clones were generated from peripheral blood mononuclear cells from a single person using the extract from cell wall and cell membrane as the antigenic preparation. Nineteen clones were propagated; all expressed the surface phenotype CD3+, CD4+, T-cell receptor α/β+. The clones were antigen-specific. Of 17 clones studied, none responded to extracts from Blastomyces dermatitidis or Coccidioides immitis or to tetanus toxoid. Sixteen of 19 clones recognized HIS-62 in vitro. Additional analysis revealed that cells from each of the seven individuals who responded to the extract mounted a proliferative response to HIS-62. Thus, the extract from the cell wall and cell membrane and HIS-62 are targets of the human cell-mediated immune response to H. capsulatum. Moreover, HIS-62 appears to be an immunodominant antigen.