bluuebunny/crossref_metadata_2025 huggingface dataset:
Zymosan-Stimulated Production of Phosphatidic Acid by Macrophages: Relationship to Release of Superoxide Anion and Inhibition by Agents That Increase Intracellular Cyclic AMP
Authors: Channon Jacqueline Y , Leslie Christina C , Johnston Richard B.
Published: January 2018

Murine peritoneal macrophages (mΦ) respond to unopsonized zymosan with the production of superoxide anion (O2-). We investigated the involvement of phospholipid turnover in the transduction mechanism for this phenomenon. Zymosan-stimulated mΦ produced increased amounts of phosphatidic acid (PA); the increase was first detected at 1.5 min and continued for 10 min of incubation. Production of O2- was not detected until between 2 to 4 min after stimulation, and continued to increase through 60 min. Inhibition experiments suggested that these two processes were linked. Theophylline (theo)/ dibutyrylcyclic AMP (dbcAMP) and theo/prostaglandin E2 (PGE2) inhibited O2- production at every time point (79% and 80% inhibition, respectively, at 4 min). Corresponding inhibition of PA production was also achieved at every time point (85% by theo/dbcAMP; 67% by theo/PGE2 at 4 min). These results are compatible with a role for phospholipid remodeling in the transduction process associated with the respiratory burst. Results suggest that the phospholipid species could be phosphatidylcholine (PC) as well as phosphatidylinositol (PI).
Induction of Chemotaxis in Mouse Peritoneal Macrophages by Activators of Protein Kinase C
Authors: Laskin Debra L , Gardner Carol R , Laskin Jeffrey D
Published: January 2018

Two activators of calcium and phospholipid dependent protein kinase (protein kinase C), the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), were compared as chemotactic agents for mouse peritoneal macrophages. Both of these compounds were found to induce Chemotaxis in the macrophages to a similar extent in a time and dose dependent manner. Induction of Chemotaxis was observed in the concentration range of 10-100 nM for TPA and 25-250 μM for OAG. Two structurally related synthetic sn 1,2-diacylglycerols, 1,2-dioctanoylglycerol (diC8) and 1,2-didecanoylglycerol (diC10), were also found to be chemotactic for macrophages, while monoacylglycerol (2-monoolein) was inactive. Of the diacylglycerols, OAG was found to be the most active followed by dlC8 and diC10. In contrast to TPA, the synthetic diacylglycerols had no effect on superoxide anion release by the cells, suggesting that the mechanism of superoxide anion release by TPA in macrophages is distinct from Chemotaxis. Phorbol-12,13-diacetate, a biologically inactive phorbol ester analog that inhibits the binding of TPA to its cellular receptors, inhibited macrophage Chemotaxis induced by TPA, the synthetic diacylglycerols, and the complement fragment, C5a. Taken together, our results suggest that Chemotaxis in macrophages may be mediated by activation of protein kinase C.
Differential Protein Synthesis by Murine Peritoneal Macrophages Elicited by Various Stimuli
Authors: Tannenbaum Charles S , Nurmi-McKernan Lisa , Largen Michael T
Published: January 2018

Protein synthetic patterns of murine peritoneal macrophages were analyzed by two-dimensional Polyacrylamide gel electrophoresis (2D PAGE) of 35S methionine-labeled proteins. While the protein synthetic patterns exhibited by resident, inflammatory, and activated macrophages had numerous common features that distinguished them from the other normal non-macrophage cell types examined, unique proteins also characterized each macrophage population from the others. The accumulation by resident macrophages of proteins 23, 25, and 37 distinguished them from elicited cells, as did the former's more abundant synthesis of proteins 54 and 52. The protein synthetic patterns of inflammatory thioglycollate- and proteose peptone-elicited macrophages were strikingly similar, save for the former's greater levels of accumulation of proteins 14 and 28, and the letter's more pronounced expression of p23.5. Peritoneal macrophages elicited by treatment with heat-killed Propionibacterium acnes, the live, attenuated Mycobacterium bovis strain BCG, Listeria monocytogenes, and the protozoan flagellate Trypanosoma rhodesiense, all exhibited tumoricidal activity in 16-h or 72-h functional assays. They shared a common protein synthetic profile that differentiated them from the synthetic patterns characteristic of the non-tumoricidal resident and inflammatory macrophages. These tumoricidal macrophages were unique in synthesizing a protein(s) of approximate molecular weight 26,000 daltons. A time-course study employing P. acnes-activated peritoneal macrophages indicated that p26 accumulation decayed with tumoricidal capacity as a function of time in culture, although no direct correlation between lytic activity and p26 expression could be definitively established. Peritoneal macrophages elicited with proteose peptone were not directly tumoricidal but were rendered so upon in vitro treatment with nanogram amounts of bacterial lipopolysaccharide. The accumulation of low levels of p26 by the newly explanted proteose peptone-elicited macrophages suggests the possibility that this protein characterizes macrophage populations primed as well as triggered for tumoricidal activity.
Alveolar Macrophage/Peripheral Blood Monocyte-Derived Factors Modulate Proliferation of Primary Lines of Human Lung Fibroblasts
Authors: Jordana Manel , Newhouse Michael T , Gauldie Jack
Published: January 2018

Pulmonary fibrosis is characterized by an alteration in lung collagen synthesis and deposition, as well as by increased fibroblast proliferation. It is also characterized by an intermittent influx of immune and inflammatory cells in the lung. To investigate the nature of the target cell in this disorder, we established a series of primary lines of human adult lung fibroblasts and studied the effect of mediators released from activated normal human alveolar macrophages (AM) and peripheral blood monocytes (PBM) on the proliferation of both normal lung fibroblasts and fibroblasts established from lung tissue of patients with active fibrosis. Our data show that monocyte supernatants containing a 15-18 kD monokine from either AM or PBM inhibits growth of logarithmic phase proliferating lung fibroblasts in a dose-dependent manner. This effect can be entirely abrogated by treating the fibroblasts with indomethacin and is reconstituted by adding exogenous PGE2. A study of the kinetics of this interaction shows that exposure to monocyte supernatant for 30 min to 1 hr is sufficient to cause significant inhibition of fibroblast proliferation and that this effect can be halted, but not reversed, at any stage by incubation with indomethacin. We also show that fibroblasts derived from patients with pulmonary fibrosis are affected more quickly by exposure to the mediators, although the final extent of inhibition seen at each concentration of mediators is similar in normal and “fibrotic” fibroblasts. These studies indicate that activated AM or PBM release cytokines (including IL-1) which inhibit the growth of proliferating normal and fibrotic fibroblasts through activation of the intrinsic arachidonic acid pathway of this cell and also that this effect requires a continuous activation of this pathway to be fully expressed.
Defective Production of Reactive Oxygen Intermediates by Tumor-Associated Macrophages Exposed to Phorbol Ester
Authors: Ghezzi Pietro , Erroi Annalaura , Acero Raffaella , Salmona Mario , Mantovani Alberto
Published: January 2018

Macrophages were isolated from poorly immunogenic metastatic sarcomas (mFS6 and MN/MCA1) of C57BL/6 origin. Tumor-associated macrophages (TAM) showed little release of superoxide when exposed to phorbol myristate acetate. When exposed to a phagocytic stimulus (zymosan), TAM released appreciable amounts of superoxide. TAM had a lower number of specific binding sites for phorbol esters than resident or caseinate-elicited peritoneal macrophages, but had normal NADPH-cytochrome C reductase. The tumor environment, possibly through previously demonstrated products of neoplastic cells, may influence the functional status of in situ macrophages and, thus, impair host anti-tumor and anti-microbial defense mechanisms.
Self-Renewal of Pulmonary Alveolar Macrophages: Evidence From Radiation Chimera Studies
Authors: Tarling John D , Lin Hsiu-san , Hsu Shin
Published: January 2018

Radiation-induced chimeric mice were used to study the origin of pulmonary alveolar macrophages. Unlike in other studies, these radiation chimeras were prepared by using a special fractionated irradiation regimen to minimize the killing of alveolar macrophage colony-forming cells, putative local stem cells. For this study CBA mice with or without T6 chromosome marker were used. Under this experimental condition, the majority of alveolar macrophages in mitosis are of host origin even after 45 weeks. These data suggest that alveolar macrophages are a self-renewing population under normal steady-state conditions.
New Method for the Measurement of Eosinophil Migration
Authors: Håkansson Lena , Westerlund Dorothea , Venge Per
Published: January 2018

A new application of the Boyden chamber method for the measurement of eosinophil migration, without the need of eosinophil isolation, has been developed. A cell suspension containing a mixture of granulocytes, neutrophils to the greater part, was used. The eosinophils were identified by staining their granules with Chromotrope 2R. The method made it possible to study the migration of eosinophils from normal individuals without eosinophilia. Control experiments demonstrated that the chemotactic and chemokinetic response of eosinophils in the granulocyte mixture was in accordance with the response of isolated eosinophils from the same donor. Normal eosinophils demonstrated a significant chemotactic response to C5f, platelet-activating factor (PAF), leukotriene B4 (LTB4), and f-meth-leu-phe (f-MLP). Furthermore, PAF was demonstrated to be significantly more eosinophil chemotactic than neutrophil chemotactic.
Macrophage Cell Line B6MP102 Resembles Peritoneal Macrophages in Tumor Cell Recognition and Killing
Authors: Chapes Stephen K , Didier Elizabeth S , Tompkins Wayne A F
Published: January 2018

In this paper we describe the bone marrow-derived macrophage cell line B6MP102. We describe growth characteristics, responsiveness to biological response modifiers known to activate macrophages (MPs), and the ability of B6MP102—comparable to that of peritoneal MPs—to kill and discriminate tumor cells. We demonstrate that B6MP102 is easily maintained in culture in the presence of 15% L-M cell-conditioned media. We have found that the B6MP102 cell line is similar to in vivo-derived peritoneal MPs. These cells are responsive to biological response modifiers but are not spontaneously cytotoxic. They produce O−  2 in quantities comparable to that produced by peritoneal MPs, and they exhibit target ranges for direct killing similar to those of activated peritoneal MPs. In addition, B6MP102 is similar to MPs in mediating cytostasis. These latter two points are especially significant, suggesting that the mechanisms that MPs may use to distinguish and kill tumor cells are retained in the B6MP102 cell line. This makes the cell line a useful tool for studying MP-tumor cell interactions.
Identification of Nonadherent Mononuclear Cells in Human Cord Blood That Differentiate Into Macrophages
Authors: Santiago-Schwarz Frances , Fleit Howard B
Published: January 2018

We describe a population of nonadherent cells in neonatal cord blood that, upon in vitro cultivation, develop into monocyte-macrophages. These cells initially are negative for nonspecific esterase cytoplasmic activity, lack the monocyte marker MO.2, fall into smaller, nonmonocytic cell size areas, as determined by fluorescence-activated cell sorter (FACS)-assisted size analysis, and differentiate into macrophages under nonstimulatory culture conditions (in the absence of exogenous colony stimulating factors, <0.1 ng/ml endotoxin, and growth in suspension). In contrast to the adherent, committed macrophage precursors in cord blood, which differentiate into macrophages after 2-3 days of culture, the nonadherent precursor does not acquire monocyte-macrophage characteristics until day 14 of culture. Earlier induction is achieved by adding the monocyte-activating agents lipopolysaccharide or 1,25 dihydroxyvitamin D3 to cultures.
Differential Presentation of Tolerogenic Immunoglobulin In Vivo by Macrophages and by a Lymphoid Dendritic Cell-Like Tumor Line
Authors: Phipps Richard P , Illig Karl , Schad Victoria , Bhimani Karim
Published: January 2018

Previous work indicated that macrophages and a lymphoid dendritic cell-like tumor line, P388AD.2, possessed a differential ability to present a haptenated immunoglobulin (tolerogen) in vitro. Macrophages presented fluorescein-conjugated sheep gamma globulin (FL-SGG) and elicited B-cell unresponsiveness. In contrast, P388AD.2 presented this normally tolerogenic signal as an immunogenic one and induced augmented anti-hapten antibody responses. The objective of the present study was to determine whether differential tolerogen presentation could occur in vivo using defined accessory cells pulsed with FL-SGG. Interestingly, the intravenous (IV) injection of FL-SGG-pulsed thioglycollate-elicited macrophages, which secreted prostaglandin E2, induced hapten-specific B-cell unresponsiveness in syngeneic recipients. One thousand times as much FL-SGG in soluble form was required to produce the same degree of unresponsiveness. In contrast to macrophage-elicited negative signalling, non-prostaglandin secreting P388AD.2, when pulsed with FL-SGG, induced hapten-specific responses 2-3 times control values. Moreover, as few as 2 × 104 FL-SGG-pulsed P388AD.2 induced significant augmentation of the anti-FL antibody response. The presentation of FL-SGG in an immunogenic fashion by P388AD.2 was rapid and long lasting since increased responses were demonstrated as early as 1 day or as long as 21 days after IV injection. P388AD.2 were not simply acting as a passive carrier, nor permitting host presentation of FL-SGG, since there were requirements for P388AD.2 viability, and for syngeneic recipients in order to generate augmented anti-FL antibody responses. Moreover, inappropriate presentation of FL-SGG by P388AD.2 injected into allogeneic recipients did not elicit positive or negative signalling. In order to demonstrate that the ability of P388AD.2 to present FL-SGG in an immunogenic fashion was not simply a property of all tumor cells, the P388D1 cell line was pulsed with FL-SGG and injected. Neither tolerance nor augmentation was induced. Overall these results demonstrate that the type of antigen-presenting cell which introduces the immune system to an immunoglobulin tolerogen is critical to the induction of B-cell unresponsiveness or priming.
Protective Effects of Tumor Necrosis Factor in Experimental Legionella pneumophila Infections of Mice via Activation of PMN Function
Authors: Blanchard D Kay , Djeu Julie Y , Klein Thomas W , Friedman Herman , Stewart William E
Published: January 2018

Tumor necrosis factor (TNF) was found in the lung lavage fluids of Legionella pneumophila-infected mice within 24 hr of intratracheal (i.t.) inoculation. Since this cytokine has been reported to activate polymorphonuclear leukocyte (PMN) function, the effect of TNF on the in vitro bactericidal capacity of PMN-enriched cultures was determined. Murine thioglycollate-elicited PMN which were treated with recombinant human TNF demonstrated augmented killing of L. pneumophila bacteria in vitro. Furthermore, treatment of PMN suspensions with cytokine-containing lung lavage fluid was found to enhance the bactericidal activity of PMN. The addition of anti-cachectin/TNF antibodies partially abrogated the stimulatory effects of the lavage fluid, suggesting that in vivo activation of PMN during the course of infection was likely, and that TNF was partially responsible for the enhanced bactericidal activity. In vivo treatment of animals with TNF resulted in significant protection of the animals from mortality. Furthermore, the rate of clearance of bacteria from the lung tissues of infected mice was increased in those animals treated with TNF, and correlated with the ability of this cytokine to protect the animals. These data suggest that the induction of TNF by Legionella bacteria during infection are involved in the non-specific host defense mechanisms, and that PMN activated by the TNF may be instrumental in clearing the organism from infected lung tissues, thereby protecting the animal.
Blastogenesis of Large Granular Lymphocytes in Nonlymphoid Organs
Authors: McIntyre Kim W , Natuk Robert J , Biron Christine A , Kase Kenneth , Greenberger Joel , Welsh Raymond M
Published: January 2018

High numbers of large granular lymphocytes (LGL) accumulate in the livers and peritoneal cavities of mice during the course of viral infection. Accumulation of natural killer (NK) cells at day 3 postinfection (p.i.) was shown to be radiation-sensitive, implying that proliferation was required for this response. Accumulation occurred in splenectomized mice, indicating that the spleen, known to be an organ for mature NK cell proliferation, was not the major source for liver and peritoneal NK/LGL.

Significant percentages (> 25%) of the LGL found in the liver and peritoneal cavity following viral infection or interferon induction with poly-inosinic:pory-cytidylic acid were defined morphologically as blasts (large cells with prominent nucleoli and intensely basophilic cytoplasms containing azurophilic granules). Most blast LGL at day 3 p.i. were sensitive to administration of anti-asialo GM1 serum in vivo, were Lyt-2-, and were enriched in populations that lysed NK cell-sensitive targets in vitro, indicating that these were NK/LGL. At day 3 p.i., leukocytes from the liver and peritoneal cavity incorporated 3H-thymidine and bound to and killed NK cell-sensitive targets in single-cell cytotoxicity assays. These data suggest that NK/LGL undergo at least one round of division in the liver and peritoneal cavity during viral infection.

In contrast, blast LGL at day 7 p.i. were resistant to in vivo treatments with anti-asialo GM1 serum, were Lyt-2+, and were enriched in populations of cells that killed virus-infected histocompatible targets, indicating that they were cytotoxic T lymphocytes (CTL). These results suggest that both NK/LGL and CTL/LGL are capable of blastogenesis and presumed proliferation at sites of virus infection, providing a means for the in situ augmentation of a host’s cell-mediated antiviral defenses.
Cell Contact and Direct Transfer Between Co-Cultured Macrophages and Fibroblasts
Authors: Dean Michael F , Cooper John A , Stahl Philip
Published: January 2018

Mouse peritoneal macrophages formed attachments with β-glucuronidase deficient human fibroblasts within an hour after co-cultures were initiated. Some of these attachments were transitory, while in others macrophages remained in firm contact with fibroblasts for many hours. Attachment of one macrophage did not prevent attachment of others, since many fibroblasts made firm contact with four or five other cells. Not all macrophages, however, attached themselves to fibroblasts. Macrophages injected with Lucifer yellow did not transfer the dye to fibroblasts with which they had made contact, nor was there any reverse transfer from injected fibroblasts to macrophages. Lucifer yellow was, however, transferred rapidly from injected fibroblasts to other adjacent fibroblasts with which they had formed gap junctions. Macrophages whose lysosomes had been pre-loaded with FITC-dextran did transfer this ligand to recipient fibroblasts, where it became localised in a perinuclear pattern with many bright punctate patches adjacent to donor macrophages. Transfer of FITC-dextran was blocked when cells were separated by nucleopore membranes in an analogous manner to transfer of endogenous lysosomal β-glucurondiase.
Up-Regulation of Gamma Interferon Receptors on the Human Monocytic Cell Line U937 by 1,25-Dihydroxyvitamin D3 and Granulocyte-Macrophage Colony Stimulating Factor
Authors: Zuckerman Steven H , Schreiber Robert D
Published: January 2018

The human monoblast-like cell line U937 can be induced to differentiate by a variety of agents including phorbol esters, retinoic acid, gamma interferon (IFN γ), and 1,25-dihydroxyvitamin D3 (VD3). Increased expression of OKM1 antigen, Fc receptors, and other cell surface antigens occur with the differentiation of this cell line along the macrophage lineage. Whereas 10-8 M VD3 alone induces changes in cell surface antigens, there were no changes in the number or affinity of IFN γ receptors. Incubation of U937 with VD3 and 100 U/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in further increases in OKM1 antigen expression and an up-regulation of IFN γ receptors. The number of IFN γ receptors increased between two- and fourfold and was maximal after 48 h incubation with VD3 and GM-CSF. Scatchard analysis revealed a single class of receptors before or after differentiation, although the increase in receptor number was associated with an overall decrease in receptor-binding affinity. Incubation of U937 with VD3 plus GM-CSF and IFN γ resulted in further increases in the density of OKM1 antigen expressed per cell. This increase in OKM1 expression was greater than that observed for U937 incubated with VD3 and GM-CSF or VD3 and IFN γ alone. These results suggest that GM-CSF up-regulates IFN γ receptors on VD3-stimulated U937 and enables these cells to be induced further along the pathway of macrophage differentiation, possibly by subsequent interaction with additional cytokines such as IFN γ.
Merocyanine 540 as a Flow Cytometric Probe of Membrane Lipid Organization in Leukocytes
Authors: McEvoy Leslie , Schlegel Robert A , Williamson Patrick , Del Buono Brian J
Published: January 2018

Merocyanine 540 (MC540) is a fluorescent probe that binds preferentially to membranes with loosely packed lipids. When combined with flow cytometry, it provides a novel methodology for rapidly and quantitatively assessing lipid organization in individual leukocytes. Analysis of cells stained simultaneously with MC540 and 1-[4-trimethylam-moniumphenyl]-6-phenyl-1,3,5-hexatriene, to normalize for surface area, revealed that all leukocytes in peripheral blood bind equivalent amounts of dye per unit surface area. This result indicates that the lipids of the plasma membranes of all types of circulating cells are organized similarly. Upon activation with appropriate stimuli, lymphocytes, monocytes, and neutrophils all bound increased amounts of dye per unit surface area, indicating a change in lipid organization to a less-ordered state. Cells stained with MC540 were sorted on the basis of their fluorescence intensity yielding populations homogeneous with respect to lipid organization. Thus not only can MC540 and flow cytometry be combined for analyzing the organization of lipids in individual leukocytes, but sorted populations can be obtained for further biochemical, structural, and functional analyses.
Superoxide Anion Production in Glass-Adherent Polymorphonuclear Leukocytes and Its Relationship to Calcium Movement
Authors: Rebut-Bonneton C , Bailly S , Pasquier C
Published: January 2018

The production of superoxide anion (O) was measured in relation to 45Ca movement in glass-adherent polymorphonuclear leukocytes (PMNs), and the results were compared with those obtained by ourselves and others on PMNs in suspension. In adherent PMNs, O production stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) was of rapid onset and short duration; this was also true of PMNs in suspension. However, O production was insensitive to the concentration of extracellular calcium. Both in adherent and non-adherent PMNs, O production stimulated with phorbol myristate acetate (PMA) had a latency time and was of long duration. In adherent PMNs, pretreatment with PMA potentiated the FMLP-induced O production by lengthening its duration without changing its initial rate.

In adherent PMNs, (10-10-10-7 M) FMLP induced a fast but transient dose-dependent increase in 45Ca within 1 min, whereas PMA only released 45Ca about 5 min after its addition to the cell culture medium. Pretreatment of PMNs with 10 or 100 ng/ml PMA for 3 min before stimulation by 10-7 M FMLP reduced the 45Ca efflux observed with FMLP alone. We conclude that O production by adherent PMNs cannot simply be related to Ca2+ movement. In comparison with non-adherent cells, adherence seemed to interfere with the characteristics of both calcium and O generation, probably by modifying the cytoskeleton.
Requirements for Histoplasmin Presentation by Accessory Cells to a Histoplasma capsulatum-Reactive T-Cell Line
Authors: Harris Jeffrey E , Deepe George S
Published: January 2018

We examined the pathways involved in presentation of native histoplasmin by adherent splenocytes (as a source of accessory cells) to JC1, a Histoplasma capsulatum-reactive murine T-cell line that is CD4+. JC1 did not respond to accessory cells that had been fixed with paraformaldehyde and then exposed to histoplasmin but did proliferate to antigen-pulsed cells that were subsequently fixed. Accessory cells that were coincubated with histoplasmin and sodium azide or 2-deoxy-D-glucose failed to induce proliferation of JC1. Moreover, accessory cells exposed to the lysosomotropic agents, chloroquine and ammonium chloride, were unable to present antigen. Monensin also inhibited presentation of histoplasmin if added to accessory cells concomitant with antigen. In contrast, accessory cells that had been pulsed with antigen for 2 hr and then exposed to each inhibitor for 2 hr stimulated proliferation of JC1. The antigen-presenting capacity of accessory cells that had been pulsed with histoplasmin for 2 hr was diminished considerably by subsequent treatment with phospholipase A2. Additional studies demonstrated that cerulenin, which depresses posttranslational lipid modification of proteins, abolished presentation of histoplasmin. The reactivity of JC1 was sharply reduced by anti-L3T4 (CD4) or by anti-l-Ab monoclonal antibody. The results not only indicate that presentation of histoplasmin requires active metabolic events within accessory cells, they also delineate the pathways involved in handling this antigen.
Activation and Deactivation of Guinea Pig Peritoneal Eosinophils During Chronic Polymyxin B Stimulation
Authors: Davis W Bruce , Husney Rose M , Mohammed B Selma , Pawluk Lew J , Ackerman G Adolph , Sagone Arthur L
Published: January 2018

Eosinophils exhibit different levels of oxidative metabolism depending on their site of origin and various host factors that may influence metabolism. The present study examined the time course of eosinophil oxidative metabolism in animals undergoing chronic peritoneal stimulation. Eosinophils were purified from the peritoneal exudates of guinea pigs stimulated with weekly polymyxin B and saline peritoneal lavage. 14C-1- and 14C-6-glucose oxidation and H2O2 production were measured at week 0 and at various time points throughout 43 weeks of stimulation. Baseline oxidative metabolism of eosinophils was relatively high throughout the time course, but then declined sharply after 32 weeks. These “deactivated” cells that were recovered after 32 weeks also failed to respond to phorbol myristate acetate (PMA) or opsonized zymosan. Electron microscopy did not reveal significant differences between deactivated eosinophils and cells from earlier time points. These findings document the time course of eosinophil activation and deactivation in this model and suggest that metabolic heterogeneity of eosinophils can occur over time in response to a chronic stimulus.
Modulation of Macrophage Fibronectin Secretion by LPS
Authors: Trotter Kathleen M , Beezhold Donald H , Lause David B
Published: January 2018

The secretion of fibronectin (Fn) by rat peritoneal macrophages was found to be down-regulated by LPS. A sensitive ELISA was used to quantitate both substrate-attached and supernatant Fn. Thiogrycollate-elicited peritoneal exudate cells were observed to release a considerable amount of Fn during the adherence procedure for macrophage purification. After this procedure, macrophage Fn levels peaked within 2 hr and then declined steadily to baseline levels by 96 hr. Fn release by exudate cells during adherence purification was less affected by cycloheximide treatment than was subsequent Fn secretion by purified macrophages. Macrophages elicited with thioglycollate and P. acnes displayed enhanced Fn secretory activity when compared with resident unstimulated cells. Exposure to lipopolysaccharide (LPS) suppressed the levels of immunoreactive Fn in supernatants of elicited cells. This inhibition was shown to be dose-dependent and most significant after 24 hr of incubation. The inclusion of polymyxin B in the culture medium did not reverse the LPS-induced inhibition of Fn production but did prevent LPS stimulation of iterleukin-1 secretion in the macrophage cultures. These observations demonstrate that Fn secretion by macrophages is regulated according to the functional state of the cell.
Quantitative Model of Mononuclear Phagocyte Lineage Proliferation in Murine Bone Marrow
Authors: Novak J P , Skamene E , Gervais F
Published: January 2018

An analysis and a synthesis of published and original experimental evidence have been performed with a view to providing a global qualitative and quantitative description of the proliferation of monocyte precursors in murine bone marrow. Two versions of a mathematical model are proposed. The first consists of two dividing generations of granulocyte-macrophage colony-forming cells (GM-CFC) and two dividing generations of macrophage colony-forming cells (M-CFC), differentiating into promonocytes (PM) and then into monocytes (MC). The second version consists of two generations of GM-CFC and three generations of M-CFC, followed by PM and MC. Both are compatible with experimental data, and further evidence is needed to elaborate a unique representation. The kinetic constants have been taken from published material whenever possible and estimated from available data and model kinetics in the case of unknown quantities. The models are applied to a pulse labeling assay, and calculated labeled subpopulations are compared with experimental values. Agreement is satisfactory for both versions in the case of MC, while a calculated PM labeling index falls more rapidly than the experimental values. However, inconsistency also exists between PM and MC experimental data.
Mitochondrial Biogenesis During the Activation of Lymphocytes by Mitogens: The Immunosuppressive Action of Tetracyclines
Authors: Van den Bogert C , Melis T E , Kroon A M
Published: January 2018

The role of mitochondrial biogenesis and function during mitogenic stimulation of rat thymocytes was investigated. The results show that mitochondrial biogenesis is required to provide the ATP for the energy-requiring processes occurring during blastogenesis. Impairment of mitochondrial biogenesis by inhibition of mitochondrial protein synthesis inhibits blast transformation. Since the tetracyclines impair mitochondrial protein synthesis, the results offer an explanation for the well-known immunosuppressive effects of these antibiotics.
Activation of Human T Lymphocytes by Crosslinking of Anti-CD3 Monoclonal Antibodies
Authors: Dixon James F P , Law Judy L , Favero Jean J
Published: January 2018

The proliferation of human T lymphocytes induced by anti-CD3 monoclonal antibodies (mAb) is used as a model for antigen-induced activation via the T cell receptor-CD3 complex. Since both systems are accessory cell (AC)-dependent, an understanding of the role of AC in anti-CD3-induced proliferation may provide an understanding of physiological activation via the T cell receptor. Previous work has implicated receptor crosslinking as an important AC function. To determine its necessity in anti-CD3-induced lymphocyte proliferation, we prepared highly purified T lymphocytes and found that these cells did not respond to the anti-CD3 mAb UCHT1, either alone or with interleukin 1 (IL1), interleukin 2 (IL2), or tetradecanoyl phorbol acetate (TPA). However, the response, as measured by appearance of IL2 receptors and proliferation, was restored by crosslinking with immobilized goat anti-mouse antibodies (GAM) and did not require the addition of IL1, IL2, or TPA. Thus, crosslinking of CD3 receptors was a sufficient signal for proliferation of these cells. Cyclosporine A (CsA) inhibited the activation induced by immobilized UCHT1. Since macrophages are the principal targets of CsA-mediated suppression of mitogen-induced proliferation, but macrophages do not participate in the response to immobilized anti-CD3, this may indicate that CsA was inhibiting crosslinking or a signal generated by it.
Rat Bone Marrow and Monocyte Cultures: Influence of Culture Time and Lymphokines on the Expression of Macrophage Differentiation Antigens
Authors: Damoiseaux J G M C , Döpp E A , Beelen R H J , Dijkstra C D
Published: January 2018

A set of seven monoclonal antibodies (moabs) has been shown to discriminate in situ between distinct subpopulations of macrophages in the rat. It is still controversial if this heterogeneity is caused by the existence of different lineages or by differentiation of a common precursor. In both cases, the differentiation process might be regulated by microenvironmental factors. The present study examines the expression of the macrophage markers recognized by the seven ED-moabs in bone marrow and monocyte cultures. Furthermore, the impact of culture time and stimulating factors on the antigen expression in these cultures was tested. The expression of the ED3 antigen is highly inducible in bone marrow cultures. Factors that might be responsible for the increased ED3 expression are investigated. This strong ED3 expression by bone marrow-derived macrophages is nearly absent by monocyte-derived macrophages. This implies that the ability to express ED3 is blocked before the macrophage precursor cells enter the circulation to become monocytes. The ED2 expression cannot be induced under the tested circumstances during culture. The expression of ED7 and ED8 is also highly inducible because bone marrow macrophages in vivo do not express these antigens. In culture, these macrophages stain positive for these markers already after the first day of culturing. The other three antigens are expressed on all macrophages under all tested circumstances.
Leukotriene B4 Modulates Phospholipid Methylation and Chemotaxis in Human Polymorphonuclear Leukocytes
Authors: Bomalaski John S , Dundee David , Brophy Lynne , Clark Mike A
Published: January 2018

Formation of phosphatidylcholine from phosphatidylethanolamine via the S-adenosylmethionine (AdoMet) pathway has been shown to be required for signal transduction of receptor–ligand interactions in a variety of cells. These interactions result in the remodeling of phospholipid pools and phospholipase activation. To extend these observations and to explore the role of the phosphatidylcholine synthesis pathway in transduction of the leukotriene B4 (LTB4) receptor–ligand response, we examined phospholipid methylation in human polymorphonuclear leukocytes (PMN) following stimulation by LTB4, a potent chemotactic agent that is a metabolite of arachidonic acid. At early time points (~3–10 min), formation of methylated phospholipids was enhanced following LTB4 stimulation. The LTB4 analogs 6-trans LTB4 as well as LTB4 epimers induced less methylation compared with LTB4, and the potencies of these analogs in inducing methylation correlated with their diminished ability to induce chemotaxis. Furthermore, the ability of these agonists to induce methylation also correlated with the binding affinity of these agents to the LTB4 receptors on these cells. Synthesis of phosphatidylcholine by the choline transferase pathway was not affected by LTB4. Inhibition of the AdoMet reaction with 3- deazaadenosine, L-homocysteine homolactone, or erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA) abrogated LTB4-induced phospholipid methylation and the chemotactic response. The potencies of these inhibitors in blocking phospholipid methylation also correlated with their ability to abrogate the LTB4-induced chemotactic response. These data suggest that phospholipid methylation and phospholipase activation play an important role in transduction of the LTB4 receptor–ligand interaction in PMN, which results in chemotaxis.
Interactions of Virulent and Avirulent Legionella pneumophila With Human Monocytes
Authors: Summersgill James T , Raff Martin J , Miller Richard D
Published: January 2018

Four pairs of virulent/avirulent strains of Legionella pneumophila were examined for their adherence/uptake and activation of human monocytes. Oxidative metabolic responses of monocytes were quantitated by measuring intracellular hydrogen peroxide generation using flow cytometry and by assessment of superoxide dismutase-inhibitable superoxide anion generation. All L pneumophila strains induced less of a response than did Escherichia coll. Within each pair of isolates, virulent strains of L. pneumophila stimulated the oxidative response of monocytes less than avirulent variants. To determine effects of complement fixation by each strain on their adherence to monocytes, a phagocytic index (PI) was determined under various conditions. In autologous donor serum (AS), all L. pneumophila strains had a PI in the range of 2.1–3.1 bacteria per monocyte, with E. coll having a PI of 9.1. No significant differences were observed between virulent L pneumophila strains and their avirulent variants. In the presence of heat-inactivated AS, all PI fell to 0.13–0.20 for the L pneumophila strains, and to 2.16 for E. coll. Using heat-inactivated AS reconstituted with exogenous human complement as a source of opsonization, levels of PI were indistinguishable from their respective levels in AS. This suggests that complement fixation plays an important role in the adherence of virulent and avirulent L pneumophila to human monocytes.
An Endotoxin-lnduced Factor Distinct From lnterleukin-1 and Tumour Necrosis Factor α Produced by the THP-1 Human Macrophage Line Stimulates Polymorphonuclear Leukocyte Infiltration In Vivo
Authors: Megyeri Pal , Issekutz Thomas B , Issekutz Andrew C
Published: January 2018

Endotoxin and gram-negative bacteria induce vigorous inflammatory reactions. Our previous work showed that rabbit macrophages (M⊘) incubated with endotoxin produce a 45,000 darton protein that recruited polymorphonuclear leukocytes (PMNL) into the skin of rabbits. This factor was separated from lnterleukin-1 (IL-1) but could not be unequivocally distinguished from rabbit tumour necrosis factor (TNFα). Here we have examined the human Mo cell line, THP-1, for the production of an analogous protein. After exposure to phorbol diester the THP-1 cells assumed the characteristic M⊘ phenotype and function. During 6 hours of culture with LPS these Mo released a factor(s) that caused PMNL recruitment into the skin of rabbits when injected intradermally, measured using 51Cr-labelled blood leukocytes. This activity, referred to as PMNL recruiting activity (PRA), was heat labile, and its production was blocked by cycloheximide, suggesting that this is most likely a de novo synthesized protein. Sephadex-G 100 and Superose-12 FPLC chromatography indicated a molecular weight in the 45,000–65,000 dalton range. The active fractions were free of IL-1 activity (< 0.2 U/ml), and Superose-12 chromatography separated the peak of PRA, which eluted around 45,000 daltons, from TNFα eluting at 20,000 daltons. The peak PRA was not neutralized by antiserum to IL-1α, IL-1β TNFα, IL-6, and granulocyte-macrophage colony-stimulating factor (GMCSF), indicating that it was distinct immunologically from these cytokines. The major PRA did not induce migration of rabbit or human PMNLs in vitro in a Boyden chamber chemotaxis assay, although peaks of chemotactic activity and weak PMNL recruitment in vivo were detected in fractions eluting around 15,000 daltons and 800 daltons. The generation of PRA by a human Me cell line is analogous to that reported previously with rabbit Me. Here we extend these observations to a human Me system and confirm that this molecule is distinct from several other Me cytokines and Me chemotactic factors with inflammatory properties.
Interferons Exhibit Temporally Distinct Regulation of Two Bovine Macrophage Fc Receptors
Authors: Pontzer Carol H , Russell Stephen W
Published: January 2018

Interferon (IFN)-induced modulation of two distinct types of Fc receptors (FcR) on bovine bone marrow culture-derived macrophages was quantified by flow cytometry. We have established the presence of separate FcR for monomeric and aggregated IgG on these cells, equivalent to the FcRI and FcRII of mice, respectively. These two kinds of FcR differed in protease sensitivity, hierarchy of preferential binding of immunoglobulin subclass, and cross-inhibition. Treatment of macrophages with either recombinant bovine IFN-gamma (rBolFNγ) or rBolFNα1 produced a dose-dependent increase in the expression of both types of FcR; however, expression of the FcR for aggregated IgG was increased a full 24 h prior to that for monomeric antibody. Further, expression of the FcRII-equivalent declined substantially by 48 h of IFN exposure, while the presence of the FcRI-equivalent receptor was still enhanced. Finally, low doses (1 unit/ml) of either rBolFNγ or rBolFNα1 failed to alter FcR expression, yet mixtures of IFNs at this concentration were able to potentiate FcR expression. This is the first time that a differential time course of induction of FcR types by IFNs and the effects of mixtures of IFNs on FcR expression has been described in any species.
Simultaneous Flow Cytometric Measurements of Cytoplasmic Ca++ and Membrane Potential Changes Upon FMLP Exposure as HL-60 Cells Mature Into Granulocytes: Using [Ca++]in as an Indicator of Granulocyte Maturity
Authors: Bernardo John , Newburger Peter E , Brennan Lisa , Brink Holly F , Bresnick Sandra A , Weil Gary , Simons Elizabeth R
Published: January 2018

Treatment of human leukemic HL-60 cells with N,N-dimethylformamide (DMF) induces them to mature until they reach granulocytoid morphology 3–6 d later. We have reported a maturation-dependent ability of these cells to respond to phorbol myristate acetate (PMA), as evaluated by membrane depolarization and by oxidative burst product formation (Newburger et al.: J. Biol. Chem. 259, 3771, 1984). More recently we have attempted to develop techniques for simultaneous evaluation of these parameters during HL-60 cell maturation. Here, we compare the cytoplasmic [Ca+ +] and membrane potential changes elicited by the chemotactic peptide fMLP via simultaneous measurement of individual cells in a fluorescence-activated cell sorter (FACS), as done previously for mature granulocytes (Lazzari et al.: J. Biol. Chem. 261,9710, 1986). The stimulus-induced [Ca++]in changes are detected with the fluorescent probe lndo-1 and reproducibly increase in magnitude for a subpopulation of cells as the cells mature into granulocytes. Ca+ + responsiveness to formyl peptide is restricted to a subpopulation of HL-60 granulocytes which expresses receptors for chemotactic peptide and consistently increases in magnitude (in response to the same concentration of agonist) with maturation. In contrast, there is less consistency in the direction or magnitude of membrane potential changes elicited under the same circumstances from the same maturing HL-60 cells.
Interaction of Immune and Connective Tissue Cells: I. The Effect of Lymphokines and Monokines on Fibroblast Growth
Authors: Thornton Stephen C , Por Suzanne B , Walsh Bradley J , Penny Ronald , Breit Samuel N
Published: January 2018

In order to determine if mononuclear cells may be secreting factors capable of modulating fibroblast growth, the in vitro proliferative response of fibroblasts to cytokines known to be secreted by mononuclear cells was measured, using both growth arrested and proliferating cells. Of the cytokines tested, which included interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), platelet derived growth factor (PDGF), and tumor necrosis factor-alpha (TNF-alpha), only TNF-alpha and PDGF had demonstrable growth factor activity. Neither IL-1 alpha nor beta showed any true growth factor activity but were able to enhance the replication of already proliferating cells. No inhibition of proliferation was noted by any of the cytokines with the exception of TNF-alpha and TGF-beta. TNF-alpha in doses greater than 500 ng/ml caused fibroblast death, probably by a prostaglandin related mechanism as fibroblasts remained viable, although non proliferative, when assayed in the presence of indomethacin, a known inhibitor of prostaglandin E2 (PGE2) synthesis. TGF-beta was inhibitory to proliferation at doses greater than 100 ng/ml, while fibroblasts remained viable. This effect was not influenced by indomethacin and hence is unlikely to be PGE2 related.
The Effects of 16,16-Dimethyl PGE2 and Phosphodiesterase Inhibitors on Con A Blastogenic Responses and NK Cytotoxic Activity of Mouse Spleen Cells
Authors: Ellis N K , Duffie G P , Young M R , Wepsic H T
Published: January 2018

Prostaglandins function as down regulators of immune responses probably by increasing the concentration of intracellular cAMP. Phosphodiesterase inhibitors, which prevent the breakdown of cAMP, also increase the intracellular levels of cAMP. Prostaglandins and phosphodiesterase inhibitors have both been shown to suppress immune responses in vitro. In this study 16,16-dimethyl PGE2 (dm-PGE2), added in vitro, suppressed the mouse spleen cell concanavalin A (Con A) response by 38% and natural killer (NK) activity by 53%. Addition of the phosphodiesterase inhibitors, theophylline, RO20-1724, or dipyridamole, decreased both the Con A response and NK activity by at least an additional 30%. We also demonstrate that treatment with dm-PGE2 and theophylline in vivo is more effective than either compound alone in inhibiting NK activity of both untreated mice and mice treated with polyinosinic-polycytidylic acid. These studies support the hypothesis that the immunosuppressive effect of dm-PGE2 is mediated by cAMP and suggest that treatment with a combination of dm-PGE2 and phosphodiesterase inhibitors can augment this immunosuppressive effect.
Comparison of Stimulation by Chemotactic Formyl Peptide Analogs Between GTPase Activity in Neutrophil Plasma Membranes and Granule Enzyme Release From Intact Neutrophils
Authors: Kanaho Yasunori , Kermode John C , Becker Elmer L
Published: January 2018

Stimulation by fMet-Leu-Phe analogs of GTPase activity in plasma membranes from rabbit neutrophils was compared with the stimulation of degranulation in intact neutrophils. All four formyl peptides examined (fMet-Leu-Phe-Phe, fMet-Leu-Phe, fNle-Leu-Phe, and fVal-Leu-Phe) were full agonists for both responses. Their ED50 values for the two responses correlated well, although those for GTPase stimulation were uniformly about tenfold greater. The specific antagonist tBoc-Phe-Leu-Phe-Leu-Phe competitively inhibited both GTPase activity and degranulation stimulated by fMet-Leu-Phe; its K values were similar for the two responses. Pertussis toxin treatment, in contrast, inhibited the maximal stimulation of both responses by fMet-Leu-Phe with minimal shift in ED50. The inhibitory actions of tBoc-Phe-Leu-Phe-Leu-Phe and pertussis toxin on GTPase activity thus paralleled the effects on degranulation. These observations substantiate the hypothesis that a guanine nucleotide-binding protein that is a pertussis toxin substrate couples the formyl peptide receptors to physiological function in the neutrophil.