bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Human Mononuclear Cells Using Reduced Pyridine Nucleotide Fluorescence and Flow Cytometry
Authors: Bender James G , Van Epps Dennis E , Steinkamp John A
Published: January 2018

Peripheral blood monocytes undergo an oxidative burst similar to that seen in neutrophils. The basis for this response appears to be an NAD(P)H oxidase that utilizes reduced NAD(P)H to form superoxide anion. We utilized the unique UV-stimulated fluorescence property of reduced pyridine nucleotides to analyze NAD(P)H utilization in monocytes. UV-stimulated fluorescence in mononuclear cell preparations indicated two populations of cells with the highly fluorescent cells having a Coulter volume consistent with that of monocytes. Dual laser analysis with monoclonal antibodies confirmed that these highly fluorescent cells are monocytes by showing them to be OKM1 +, Leu DR +, and anti-monocyte 0.2 +. Natural killer (NK) cells, as defined by Leu 7, were not found in this highly fluorescent population. Stimulation of mononuclear cells with phorbol myristate acetate caused a fluorescence loss indicative of NAD(P)H oxidation in monocytes but not in lymphocytes. Stimulation with suboptimal concentrations of PMA (1–5 ng/ml) resulted in a dose-dependent fluorescence loss in monocytes that occurred in an all-or-none fashion identical to the pattern observed in neutrophils. Simultaneous measurement of H2O2 production using dichlorofluorescein formation with NAD(P)H fluorescence indicates that oxidant production occurs in a graded manner. This method, then, provides a convenient way to study in single cells the metabolic events involved in depletion and replenishment of NAD(P)H during the oxidative burst and demonstrates an additional means by which to distinguish monocytes from lymphocytes using flow cytometry.
The Effect of Gamma Interferon on IL-1 Secretion of In Vitro Differentiated Human Macrophages
Authors: Haq Amin U , Rinehart John J , Maca Richard D
Published: January 2018

After being cultured overnight, human monocytes lose their ability to secrete interleukin-1 (IL-1) when stimulated by lipopolysaccharide (LPS). However, when these monocytes were cultured for up to 9 days with various concentrations of interferon-gamma (IFN-gamma), these cells were found to retain their ability to secrete appreciable amounts of IL-1 on LPS stimulation. However, the effect was observed only if the monocytes were exposed to the IFN before LPS stimulation and simultaneous addition of IFN and LPS to macrophages was ineffective. This effect of IFN-gamma was related to the concentration of IFN added to the cultures and was completely neutralized by a monoclonal antibody to IFN-gamma. In addition to inducing IL-1 secretion, IFN-gamma also appeared to increase the overall production of IL-1, since reinduction of IL-1 secretion was not associated with a decrease in intracellular IL-1 content. When these macrophages were initially cultured with IFN-gamma, washed, and further cultured with IFN free medium, these macrophages were found to progressively lose their capacity to secrete IL-1 in response to LPS. Conversely, when monocytes were initially cultured in medium free of IFN, washed, and then further cultured in new medium, but now containing IFN-gamma, these macrophages were found to regain their capacity to secrete IL-1. However, the amount of reinduced IL-1 secretion decreased as the length of the initial culture period without IFN increased, with less than optimal IL-1 secretion occurring if monocytes were allowed to mature for 6 days before IFN-gamma pretreatment. In summary, these studies suggest that IFN-gamma may be important in enhancing IL-1 production and secretion by maturing macrophages and tissue macrophages and consequently may play a role in regulating the accessory cell activity of these cells for a variety of immune responses in vivo.
Intracellular pH Changes During Neutrophil Activation: Na+ /H+ Antiport
Authors: Weisman Steven J , Punzo Amalia , Ford Charles , Sha'afi Ramadan I
Published: January 2018

Activation of the Na+ /H+ antiport mechanism was studied in human neutrophils by monitoring intracellular pH with a carboxyfluorescein derivative. N-formyl-methionylleucyl-phenylalanine (FMLP) and phospholipase C (PLC) induced biphasic pH changes. Amiloride, which inhibits the antiport, completely blocked alkalinization but enhanced acidification. Polymyxin B, which inhibits protein kinase C, only blocked alkalinization. Activation with phorbol 12-myristate 13-acetate (PMA) led to alkalinization only; this was inhibited by amiloride or polymyxin B. Thus, during polymorphonuclear leukocyte (PMN) activation, Intracellular alkalinization appears to be mediated by an amiloride-sensitive Na+ /H+ antiport. Antiport activity can also be blocked indirectly by inhibition of protein kinase C activity. Early intracellular acidification does not appear to require kinase activity but is observed when phospholipids are remodeled with PLC. The antiport was also activatable by hypertonic buffered media. This response did not appear to be mediated by protein kinase C because it was unaffected by polymyxin B. Finally, superoxide generation was investigated. It is affected by, but not soley controlled by, either antiport or protein kinase C activity.
Respiratory Burst of Normal Human Eosinophils
Authors: Petreccia David C , Nauseef William M , Clark Robert A
Published: January 2018

Oxidative metabolism of eosinophils has generally been studied in cells from patients with eosinophilia. We isolated eosinophils with purity of >95% from normal donors. Oxygen metabolism of eosinophils and neutrophils was compared using O2 production as a measure of the stimulus-induced respiratory burst. During 10 min of stimulation, using phorbol myristate acetate (PMA) or opsonized zymosan (OPZ), eosinophils produced two to three times as much O2 as did neutrophils. Continuous kinetic analysis was used to measure the latency period and maximal rate of O2 production. Eosinophils had a shorter latency period and greater rate of O2 production than did neutrophils at all concentrations of PMA. With OPZ stimulation, O2 production rates by both cells were similar. The latency periods were similar with high concentrations of OPZ but at limiting amounts of OPZ eosinophils had a longer latency period than did neutrophils. In addition, the kinetic assay demonstrated that the respiratory burst in eosinophils was more sustained than in neutrophils.

These results indicate substantial differences in the oxidative burst of eosinophils and neutrophils with respect to activation, capacity, and regulation. These distinctive features of O2 production by eosinophils may be important in host defense against metazoan parasites or in tissue injury during inflammation.
In Vitro Tumoricidal Activity of Resting and Glucan-Activated Kupffer Cells
Authors: Sherwood Edward R , Williams David L , McNamee Rose B , Jones Ernest L , Browder I William , Di Luzio Nicholas R
Published: January 2018

Kupffer cells compose 80-90% of fixed tissue macrophages and have been suggested to play an important role in hepatic antitumor resistance. In the present study, the ability of resting and activated Kupffer cells to lyse syngeneic mammary adenocarcinoma BW10232 cells was evaluated. Activated Kupffer cells were isolated from C57BI/6J mice following single of multiple intravenous (IV) injections of glucan (0.45 mg/mouse), a potent macrophage-activating agent. Mice receiving 5% (w/v) dextrose served as control. Resting Kupffer cells induced significant (P < .05) 4% and 12% specific lysis of adenocarcinoma cells at target:effector ratios of 1:10 and 1:50, respectively. Kupffer-cell-mediated tumoricidal activity was depressed on day 1 following a single IV injection of glucan. By day 3 postglucan, the antitumor activity of Kupffer cells returned to control levels and was enhanced on days 5 and 10. Following multiple IV injections of glucan on days -5, -3, and -1, Kupffer-cell-mediated cytotoxicity was elevated on days 1 and 4. These observations demonstrate that 1) resting Kupffer cells are significantly cytotoxic to adenocarcinoma cells at T:E ratios of 1:10 and 1:50 and 2) following a transient inhibition of Kupffer-cell-mediated tumoricidal activity, glucan was effective in significantly enhancing the antitumor activity of Kupffer cells.
Differential Prostaglandin Production by Unfractionated and Density-Fractionated Human Monocytes and Alveolar Macrophages
Authors: Elias Jack A , Ferro Thomas J , Rossman Milton D , Greenberg Jo Ann , Daniele Ronald P , Schreiber Alan D , Freundlich Bruce
Published: January 2018

Mononuclear phagocyte elaboration of E series prostaglandins (PGE) may be important in the regulation of inflammatory and fibrotic reactions. Mononuclear phagocytes are morphologically and functionally heterogeneous cells. To further understand the processes controlling inflammation and fibrosis, in particular that in the human lung, we characterized the ability of unfractionated and density-fractionated human alveolar macrophages and blood monocytes to elaborate PGE. Alveolar macrophages and blood monocytes constitutively elaborated small amounts of PGE, and their elaboration of PGE was increased with lipopolysaccharide (LPS) stimulation. Monocytes elaborated more PGE than autologous alveolar macrophages. In addition, denser monocytes (specific gravity > 1.055) and denser alveolar macrophages (specific gravity > 1.044) elaborated more PGE than less dense monocytes and alveolar macrophages, respectively. When monocytes were incubated in vitro, their constitutive PGE elaboration decreased with time. However, in vitro incubation did not cause monocytes to lose their capacity to elaborate PGE in response to LPS. Thus, mononuclear phagocyte populations differ in their ability to elaborate PGE. These differences can be only partially attributed to differences in cell maturation.
Shift in Subfractions of Rat Alveolar Macrophages In Vivo During Endotoxin-Induced Alveolitis
Authors: Rinaldo Jean E , Moore Susan A , Lee Robert E , Dauber James H
Published: January 2018

To elucidate changes in alveolar macrophages that accompany sepsis-induced lung injury, this study analyzed the subfractions of alveolar macrophages (AM) recovered by lung lavage during the onset of endotoxin-induced acute neutrophilic alveolar inflammation in the rat model. Centrifugation on continuous self-generated density gradients of Percoll was used to fractionate AM into subpopulations between density limits 1.012 and 1.130. Two-thirds of AM recovered from pathogen-free control rats (group C) were in a fraction with a density range of 1.058-1.078 [“normal” density fraction, (ND)]. Only 6% were located in a very low density (VLD) fraction 1.037-1.048. Neutrophils accounted for less than 1% of recovered cells and usually were found in the fraction with density range of 1.079-1.130. By contrast, if rats underwent lung lavage 15 hours after the administration of endotoxin (group E), only 38% of macrophages were recovered in the “normal” density fraction, whereas 26% of the AM recovered were in the VLD fraction. This shift in the relative sizes of the density based subpopulations coincided with the onset of acute bronchoalveolar inflammation as indicated by the recovery of neutrophils by bronchoalveolar lavage (PMN = 7 × 104 in C, vs. 9.4 × 105 in E, p < .001). The macrophages on the low density subfractions showed functional impairment: they were less viable in culture and migrated poorly in response to endotoxin-activated serum compared to macrophages in the “normal” density fraction from the endotoxin-treated animals. The rapid emergence of the low density population after endotoxin could represent an influx of new cells, but more likely indicates that injury to or previous activation of resident macrophages has caused their density to decrease. We speculate that the emergence of a population of AM in airspaces with low density and impaired function could weaken pulmonary host defense following endotoxemia.
Chemotactic Factor Binding and Functional Capacity: A Comparison Between Human Granulocytes and Differentiated HL-60 Cells
Authors: Dahlgren Claes , Andersson Tommy , Stendahl Olle
Published: January 2018

In the presence of dimethyl sulfoxide (DMSO), the leukemic promyelocytic cell line HL-60 will differentiate into mature polymorphonuclear granulocytes. In the present report, we compare chemotactic factor binding and function in HL-60 cells with that of normal human granulocytes using the chemotactic peptide N-formylmethionyl-leucyl-phenylal-anine (FMLP) as ligand. The cellular response measured as CL was changed as a result of storage or conditioning of normal peripheral blood cells. With these cells, a conditioning procedure at room temperature resulted in a pronounced increase in the CL response. The increase of the CL response was probably a result of increased expression of cryptic receptors, since the changes of the oxidative response to FMLP was accompanied by increased binding of the peptide to the cell surface. Scatchard analysis revealed that the increased binding was due to an increased number of receptors. In differentiated HL-60 cells, conditioning neither led to increased production of oxidative metabolites, nor to any increased binding of the peptide. The data thus indicate that many FMLP receptors could reside in a cryptic site that is not accessible to extracellular ligands, and that conditioning results in an increased exposure of these receptors, followed by an increased oxidative response to the ligand in normal cells but not in mature HL-60 cells.
Murine Glia Cells in Culture Can be Stimulated to Generate Reactive Oxygen
Authors: Sonderer Bruno , Wild Peter , Wyler Robert , Fontana Adriano , Peterhans Ernst , Schwyzer Martin
Published: January 2018

Induction of luminol-enhanced chemiluminescence (CL) indicative of reactive oxygen formation was studied in glia cell cultures from newborn mice. A burst of CL could be induced by phorbol myristate acetate, zymosan, and antibody-coated bovine red blood cells, whereas Sendai virus and several other agents known to induce CL in myeloid cells were ineffective. Sodium azide failed to inhibit CL, indicating a myeloperoxidase-independent mechanism of light emission. In parallel experiments we identified the cells binding antibody-coated erythrocytes as macrophages characterized by the reduction of nitroblue tetrazolium and phagocytosis of zymosan and latex particles. Brain macrophages may use reactive oxygen intermediates (ROI) as a mechanism of antimicrobial defence; and, on the other hand, ROI formed by these cells may contribute to immunopathology in the brain.
Evidence of Polymorphonuclear Neutrophils (PMN) Activation in Patients With Insulin-Dependent Diabetes Mellitus
Authors: Wierusz-Wysocka B , Wysocki H , Sieklerka H , Wykretowicz A , Szczepanik A , Klimas R
Published: January 2018

Polymorphonuclear neutrophils' chemotaxis, surface charge, superoxide anions generation, NBT (nitro blue tetrazolium) reduction and intracellular lysozyme, and β-glucuronidase content were estimated in patients with type I diabetes mellitus in a similar state of metabolic control. The chemotaxis of diabetic cells toward bacterial chemotactic factors was similar to controls, whereas migration toward complement-derived chemoattractants was significantly reduced. Polymorphonuclear neutrophils isolated from diabetic patients, when unstimulated, produced significantly greater amounts of superoxide anions and reduced NBT more efficiently. They also revealed reduced surface charge and lower intracellular content of lysozyme, whereas β-glucuronidase content was similar to controls. The results obtained seem to indicate that neutrophils in patients with insulin-dependent diabetes manifest signs of being in the activated state. The possible mechanisms of such stimulation are discussed.
The Activation of L3T4+ Helper T Cells Assisting the Generation of Anti-Tumor Lyt-2+ Cytotoxic T Lymphocytes: Requirement of 1a-Positive Antigen-Presenting Cells for Processing and Presentation of Tumor Antigens
Authors: Kosugi Atsushi , Yoshioka Takayuki , Suda Takashi , Sano Haruo , Takahama Yousuke , Fujiwara Hiromi , Hamaoka Toshiyuki
Published: January 2018

The present study investigates the mechanisms of the recognition of tumor antigens by L3T4+ helper T cells responsible for the generation of Lyt-2+ cytotoxic T lymphocytes (CTL) against a major histocompatibility complex (MHC) Class II (1a) antigen-negative syngeneic X5563 plasmacytoma. Treatment of X5563-immunized spleen cells with anti-L3T4 antibody plus complement (C) diminished the generation of Lyt-2+ anti-X5563 CTL. Since the contribution of L3T4+ cells was completely replaced by the addition of exogenous lymphokines, it was demonstrated that L3T4+ cells functioned as helper T cells assisting the generation of anti-X5563 CTL responses. Elimination of 1a-positive accessory cells (AC) from X5563-immunized spleen cells resulted in the abrogation of CTL generation, whereas the addition of exogenous lymphokines to AC-depleted X5563 immunized spleen cells restored the CTL response. The addition of anti-self la antibody to the culture also eliminated CTL responses. These observations demonstrated the requirement of 1a-positive AC for and the involvement of self la antigens in the activation of helper T cells. Moreover, use of tumor cells pretreated with paraformaldehyde to cultures of X5563-immunized spleen cells or adding back of AC pretreated with chloroquine to cultures of AC-depleted immune spleen cells failed to generate CTL responses. Finally, the addition of exogenous lymphokines to the above cultures resulted in appreciable restoration of CTL responses. Taken collectively, these results indicate that L3T4+ helper T cells are activated with tumor antigens processed and presented by 1a-positive AC.
Suppressor Alveolar Macrophages in Mice Bearing Metastatic Lewis Lung Carcinoma Tumors
Authors: Young M Rita , Endicott Roger A , Duffie G Patrick , Wepsic H Terry
Published: January 2018

The capacity of alveolar macrophages from mice injected with a metastatic Lewis lung carcinoma variant, LLC-C3, to regulate T-cell Con A blastogenesis and NK cytotoxicity was studied. During the first 5 days after subcutaneous tumor injection, alveolar macrophages were stimulatory to Con A blastogenesis of normal spleen cells. After 5 days, the alveolar macrophages shifted to become suppressive. The suppressive activity was extensive by day 11, when the primary and metastatic tumor foci were first detectable. The tumor-bearer alveolar macrophages also suppressed NK cytotoxicity. Alveolar macrophage suppressive activity was sensitive to indomethacin, suggesting a prostaglandin-dependent suppressor mechanism. Suppression was not mediated by the production of hydrogen peroxide or superoxide, as it was insensitive to catalase or superoxide dismutase. When normal alveolar macrophages were cultured with LLC-C3 supernatants for over 12 hours, suppressive activity was induced. The results of these studies show that alveolar macrophages of tumor bearers become suppressive with progressive tumor growth and might, thus, facilitate the development of pulmonary metastases.
Modulation of Human Monocyte Leukotactic Responsiveness by Thromboxane A2 and 12-Hydroxyheptadecatrienoic Acid (12-HHT)
Authors: Campbell Peter B , Tolson Timothy A
Published: January 2018

The leukotactic responsiveness of human peripheral blood monocytes is regulated by the cell-directed inhibitor of monocyte leukotaxis, CDI-MLx. The actions of CDI-MLx on normal monocytes in vitro were abrogated by co-incubation with inhibitors of cyclooxygenase and thromboxane synthetase with indomethacin and dazmegrel (UK-38,465) being most active. The actions of CDI-MLx were mimicked by the thromboxane A2 analogue, U-46619, and by 12-HHT with half-maximal inhibition observed at 10−10M; PGE2 was 1000-fold less active. SQ 29,548, a thromboxane A2 receptor antagonist, blocked the effects of CDI-MLx, U-46619, and 12-HHT. Production of PQE2 and thromboxane B2 by purified monocytes was stimulated by CDI-MLx and this effect was also blocked by indomethacin, dazmegrel, and dazoxiben. These data suggest a major regulatory role for thromboxane synthetase products in human monocyte leukotaxis.
The Role of the Na+/H+ Antiporter in Human Neutrophil NADPH-Oxidase Activation
Authors: Wright Jonathan , Maridonneau-Parini Isabelle , Cragoe Edward J , Schwartz John H , Tauber Alfred I
Published: January 2018

The Na+/H+ antiporter has been shown to regulate activation of the human neutrophil. To delineate the role of the antiporter in the stimulation cascade, superoxide ((O2−) generation was observed in PMA-stimulated neutrophils in which intracellular pH (pHI) was artificially manipulated by the use of nigericin, a K+/H+ ionophore, with and without use of the Na+/H+ antiporter inhibitor, dimethyiamiloride (DA). Decreased O2− generation was observed in a Na+-free, 140 mM K+ buffer, but addition of nigericin restored this parameter of neutrophil activation to levels found in physiologic Na+ media. Further, the inhibitory effects of DA on O2− generation by cells incubated in a 10 mM Na+, 130 mM K+ buffer were totally reversed by a similar concentration of nigericin. O2− generated by a membrane preparation of the NADPH-oxidase, made from cells incubated under the same experimental conditions, paralleled whole cell studies, but the activity of the oxidase did not vary when suspended in the various reaction mixtures. These experiments support the role of the Na+/H+ antiporter in neutrophil activation as a metabolic regulator of pHi that Influences receptor-coupled reactions proximal to expression of the NADPH-oxidase itself.
Accelerated Recovery of Hematopoiesis Following Sub-Lethal Whole Body Irradiation With Recombinant Murine Interleukin-1 (IL-1)
Authors: Gallicchio Vincent S
Published: January 2018

This communication reports the results of studies designed to investigate the ability of recombinant murine interleukin-1 (rIL-1) to enhance the recovery of hematopoiesis following administration of sub-lethal whole body irradiation (2 Gy). Mice were administered rIL-1 (100 and 500 units) i.p. Twenty-four hours later these mice were administered 2 Gy radiation. Irradiated control mice were given only phosphate buffered saline (PBS). Animals were then serially sacrificed (on days 1, 2, 4, 7, 9, and 12 following irradiation) and their peripheral blood was analyzed for indices (packed red cell volume, WBC, platelets, and differential). Femoral bone marrow was harvested and assayed for their stem cell content—erythroid (CFU-E, BFU-E), granulocyte-macrophage (CFU-GM), and megakaryocyte (CFU-MEG). Irradiated mice pretreated with rIL-1 demonstrated accelerated hematopoietic recovery as measured by higher WBC, platelets and femoral stem cell content than PBS-treated irradiated controls. These results indicate IL-1 may be an effective radioprotective agent against the hematotoxicity induced by ionizing radiation.
Alteration of Rat Kupffer Cell Function Following Mitomycin-C Administration
Authors: Bodenheimer Henry , Charland Colette , Leith John
Published: January 2018

Cancer chemotherapeutic agents modify the human immune system in diverse ways including both immunosuppression and immunostimulation. We evaluated the effects of mitomycin-C on rat Kupffer cell phagocytosis, C3b receptor binding, and lysosomal enzyme activity. Kupffer cell cultures were greater than 95% pure. Phagocytosis of IgG-coated sheep red blood cells was demonstrated by 84% of control cells but by only 25% of cells isolated two weeks following mitomycin-C administration (p < 0.0005). This depression in phagocytic ability had returned to control levels by four weeks post-treatment. Similarly, C3b receptor binding of IgM and complement coated sheep red blood cells was observed in 88% of control Kupffer cells, but declined to 47% at two weeks after drug administration (p < 0.005) and returned to normal after four weeks. Lysosomal enzyme activity was not impaired by mitomycin-C. Histologically severe ulceratlon of the colon of treated animals was seen one and two weeks after drug administration, but healed by four weeks post-mitomycin-C treatment. Depression of macrophage function as a consequence of cancer chemotherapy may have important clinical consequences in host defense against bacteria and tumor metastases.
Lung Exposure to Mineral Dusts Enhances the Capacity of Lung Inflammatory Cells to Release Superoxide
Authors: Cantin André , Dubois Frédéric , Bégin Raymond
Published: January 2018

In vitro exposure of macrophages and neutrophils to inorganic dusts can enhance their oxidative metabolism, however the effects of inorganic dust inhalation on lung-inflammatory cell-oxidative metabolism remain unknown. To address this issue, we studied the superoxide anion release from lung inflammatory cells obtained by bronchoalveolar lavage from 19 sheep exposed repeatedly to UICC chrysotile B asbestos (100 mg) over an 18 month period and 10 sheep exposed to a single exposure of quartz (100 mg) over a 6 month period. Exposure to asbestos and quartz did not induce an increase in the spontaneous release of superoxide from inflammatory cells. However lung-inflammatory cells from sheep exposed to asbestos released much higher levels of superoxide in the presence of phorbol myristate acetate (PMA) than did cells of unexposed sheep. In quartz exposed animals, superoxide release with PMA stimulation remained significantly increased up to 2 months after a single quartz infusion. These results suggest that inorganic dust inhalation primes lung inflammatory cells in vivo and markedly enhances their capacity to release toxic oxygen radicals.
In Vivo Labeling of Resident Peritoneal Macrophages
Authors: Melnicoff Meryle J , Morahan Page S , Jensen Bruce D , Breslin Elizabeth W , Horan Paul K
Published: January 2018

A novel method for labeling resident peritoneal macrophages (MO) by injection of a dye into the peritoneal cavity is described. The dye, which fluoresces green, is selectively taken up by the resident Mø. Dye labeled cells can be further characterized by labeling of cell surface antigens with monoclonal antibodies (Mabs) and phycoerythrin conjugated second antibody. After such labeling with the Mabs F4/80 or Mac 1 the resident Mø were labeled by both the green dye and the red Mab markers, while recruited Mø or neutrophils were labeled with just the red Mab; the two populations of cells were readily distinguished by two-color flow cytometry. This technique enabled identification of resident and recruited Mø in each animal without the use of radioisotopes, irradiation, or bone marrow ablation. Sufficient numbers of cells can be analyzed from each animal so that Individual animals could be evaluated.

We found no adverse effects of this labeling technique on expression of cell surface antigens or Mø mediated cytotoxicity. We did find evidence that the i.p. injection induced a mild inflammation in the peritoneal cavities of animals injected with either the dye or the balanced salt solution vehicle. Examination of the intracellular staining pattern indicated that the label rapidly sequestered in the cytoplasm of the Mø, possibly in the lysosomes. Dye solubility studies showed that the dye was partially soluble at the concentration used for in vivo labeling. We hypothesize that the Mø labeling occurred by a combination of phagocytosis of dye aggregates and endocytosis of labeled plasma membrane.
Surface Charge of Macrophages and Their Interaction With Charged Particles
Authors: Mutsaers Steven E , Papadimitriou John M
Published: January 2018

The distribution and concentration of surface ionogenic groups on murine peritoneal macrophages and the responses of their plasmalemma to charged markers were investigated by electron microscopy. Quantitation of the distribution of cationized ferritin (CF) as a marker for anionic groups on fixed cells revealed surface anions on invaginated parts of the plasmalemma to be fewer than those on flat or projecting segments. Cationic groups on the surface membrane, however, could not be labeled with anionized ferritin (AF). Interaction of viable macrophages with cationic particles at 37° resulted in their “internalization” within vesicles and coated pits and a closer apposition between many segments of plasmalemma than with neutral or anionic substances. The process occurred unaltered at 4° and was unaffected by cytochalasin B and colchicine, suggesting that this close apposition between segments of plasmalemma resulted from neutralization of surface negative electrostatic charges by the cationic material and did not reflect endocytosis.
Immunohistochemical Characterization of the Estrogen-Stimulated Leukocyte Influx in the Immature Rat Uterus
Authors: Zheng Yi , Zhou Zhao-Zong , Lyttle C Richard , Teuscher Cory
Published: January 2018

Previous studies have shown that the injection of estrogen into immature rats leads to an influx of leukocytes into the uterus. Using immunoperoxidase staining and monoclonal antibodies, we have characterized the nature of the infiltrating leukocytes in frozen sections of immature rat uteri obtained following the injection of estrogen, estrogen plus pertussigen, and the antiestrogen LY117018. Estradiol treatment for 2 days resulted in a significant increase in the number of uterine eosinophils, CD4 (W3/W25)-positive helper/inducer T lymphocytes, macrophages (MRC OX-42-positive cells), and Ia (MRC OX-6)-positive cells. In contrast, estradiol treatment failed to elicit a significant increase in the number of CD8 (MRC OX-8)-positive uterine cytotoxic/suppressor T lymphocytes and/or natural killer cells, as well as MAR 18.5- and/or MRC OX-12-positive B lymphocytes. The injection of LY117018 failed to elicit any changes in the number of cells expressing any of the phenotypes under investigation. The simultaneous injection of pertussigen, the major toxin responsible for the leukocytosis- and lymphocytosis-promoting activity of Bordetella pertussis, inhibited the estrogen-induced influx of eosinophils, macrophages (MRC OX-42-positive cells), and Ia (MRC OX-6)-positive cells but failed to prevent the influx of CD4 (W3/25) positive helper/inducer T lymphocytes. These results indicate that, in the immature rat, significant differences may exist in the susceptibility of various cell populations to the effects of estrogen, particularly with regard to uterine influx following estrogen stimulation. In addition, our observations suggest that either 1) the CD4-positive cells infiltrating the uterus following estrogen treatment may use a nonpertussigen-sensitive mechanism for chemotactic factor-receptor signal transduction or 2) a subpopulation of resident uterine cells can be induced to express the CD4 antigen following estrogen and/or estrogen plus pertussigen treatment.
SHORT COMMUNICATION: Cytokines Induce an L-Arginine-Dependent Effector System in Nonmacrophage Cells
Authors: Amber Ina J , Hibbs John B , Taintor Read R , Vavrin Zdenek
Published: January 2018

Treatment of EMT-6 mammary adenocarcinoma cells with gamma interferon (rMuIFNγ) plus tumor necrosis factor (rMuTNFα) and/or interleukin-1 (rHuIL-1α) causes release of iron-55 label, inhibition of DNA replication, and inhibition of aconitase activity. In addition, the same combinations of cytokines induce EMT-6 cells to synthesize L-citrulline, nitrite, and nitrate directly from L-arginine. Lipopolysaccharide (LPS) can act as a cofactor in the induction of these metabolic effects when added to EMT-6 cells in the presence of rMuIFNγ. The results show that increased levels of cytokines in the microenvironment can induce a novel effector pathway in somatic cells not specialized for host defense, resulting in specific metabolic effects as well as the inhibition of cellular proliferation.
Inhibition of Phagocytosis and Interleukin-1 Production in Pulmonary Macrophages From Rats With Sialodacryoadenitis Virus Infection
Authors: Boschert K R , Schoeb T R , Chandler D B , Dillehay D L
Published: January 2018

To test whether or not sialodacryoadenitis virus (SDAV) infection in rats affects pulmonary macrophage function, we intranasally inoculated pathogen-free F344 rats with SDAV and collected alveolar and interstitial macrophages 5 d later. We assessed Fc receptor-mediated attachment and phagocytosis by phase-contrast microscopic examination of monolayers of alveolar and interstitial macrophages incubated with zymosan, nonopsonized sheep erythrocytes, or erythrocytes opsonized with rabbit antisheeperythrocyte IgG. Alveolar macrophages from virus-infected rats had significantly (P ≤ .05) lower indices of attachment and phagocytosis of opsonized erythrocytes than control macrophages, but there was no difference in attachment of zymosan particles. Interstitial macrophages were not affected. Alveolar macrophages from SDAV-infected rats produced significantly less interleukin-1 than those from control rats, as assessed by testing supematants from lipopolysaccharide-stimulated macrophage cultures for induction of mouse thymocytes to take up tritiated thymidine. Effects of SDAV infection on lung macrophages could increase host susceptibility to other pathogens or complicate studies of respiratory tract immunity.
Effects of Retinoids on Macrophage Function and IL-1 Activity
Authors: Dillehay D L , Walia A S , Lamon E W
Published: January 2018

The effects of three retinoids, all-trans-retinoic acid (RA), 13-cis-retinoic acid (cRA), and N-(4-hydroxyphenyl) ratinamide (4-HPR), on macrophage function were evaluated. In vitro, RA, cRA, and 4-HPR caused a greater than twofold increase in phagocytosis of IgG-sensitized bovine erthrocytes (IgG-ORBC) by a mouse macrophage cell line (RAW). Significant increases in phagocytosis were produced by retinoid concentrations as low as 2 x 10-10 M. RA also significantly increased phagocytosis of IgG-sensitized ORBC by BALB/c peritoneal macrophages In vitro. The ability of RAW macrophages to bind IgG-ORBC was significantly increased by 10-6 to 10-14 M RA. The potentiation of myogenic responses of spleen cells to Con A and PWM by RA was relatively independent of macrophage function, i.e., splenocytes that were macrophage-depleted were responsive to the potentiating effects of RA. The effects of retinoids on T-cell-dependent B-cell mitogenesis induced by PWM appeared not to be dependent on their previously reported capacity to alter prostaglandin synthesis. Treatment of spleen cells with 10-6 M indomethacin did not abolish the potentiating effects of RA. However, RA in a dose-dependent fashion increased IL-1 activity at the level of the target T-cell. The greatest potentiation of IL-1 activity was at 10-8 M RA. These results show that retinoids can modulate macrophage function at two different levels: potentiation of phagocytosis and potentiation of IL-1 activity at the level of the T-cell.
Inhibition of Neutrophil Superoxide Anion Generation by Platelet Products: Role of Adenine Nucleotides
Authors: McGarrity S T , Stephenson A H , Hyers T M , Webster R O
Published: January 2018

Previously, we demonstrated the stimulus-specific ability of platelet lysate or release products to inhibit neutrophil functions in response to f-met-leu-phe (fMLP) or phorbol myristate acetate (PMA). The present study further characterizes these activities and identifies one of the platelet inhibitors of neutrophil superoxide anion (O) generation. Sephadex G-200 chromatography revealed two fMLP-specific inhibitory activities in platelet lysate. One component eluted with materials <13 kilodaltons (kD) molecular weight, whereas the second fractionated at the void volume (Mr > 200 kD). PMA-specific inhibitor(s) cofractionated at the void volume with the lysate’s fMLP-inhibitory activity. Release products of activated platelets contained low-molecular-weight (<13 kD), fMLP-specific inhibitor activity but failed to demonstrate PMA-specific inhibition of neutrophil O generation. Biochemical characterization identified two distinct high-molecular-weight inhibitors in platelet lysate. Pretreatment with adenosine deaminase (ADA) completely abrogated the maximum inhibitory effect (43.8 ± 7.5%) of the high-molecular-weight, fMLP-specific lysate component but failed to reduce maximum PMA-specific inhibition (93.0 ± 2.0%) of neutrophil O generation. Likewise, while the lysate’s fMLP-specific inhibitor was heat stable, heating reduced the PMA-specific inhibition 56.5 ± 10.4%. Equal sensitivity to ADA, resistance to trypsin, and heat stability were demonstrated by the low-molecular-weight inhibitor(s) in platelet lysate and release products. Neither high- nor low-molecular-weight platelet inhibitors scavenged O. High-performance liquid chromatography (HPLC) of the low-molecular-weight inhibitory activity from both platelet lysate and release products revealed physiological levels of adenine nucleotides. Addition of pure adenine nucleotides at physiological levels (1-10 μM) mimicked the effectiveness of the platelet inhibitor. We conclude that stimulus-specific limitation of neutrophil O generation by platelet adenine nucleotides may represent a mechanism for reducing inadvertent tissue damage during inflammation.
Prostaglandin E2-Mediated Inactivation of Various Killer Lineage Cells by Tumor-Bearing Host Macrophages
Authors: Parhar Ranjit S , Lala Peeyush K
Published: January 2018

We have previously reported that natural killer (NK) lineage cells are progressively inactivated during tumor development by prostaglandin E2 (PGE2) secreted by host macrophages; that this facilitates spontaneous tumor metastases, which can be prevented by chronic indomethacin therapy (CIT); and that CIT combined with multiple rounds of interleukin 2 (IL-2) can cure experimental metastases and activate all killer lineage cells in situ including NK cells, lymphokine-activated killer (LAK) cells, and tumoricidal macrophages. The present study tested whether PGE2 secreted by tumor-bearing host macrophages exerts pansuppressor effects against the activation of T cells, NK cells, LAK cells, and tumoricidal macrophages from normal splenic cell populations. Macrophages isolated from CBA mice bearing 21-day intraperitoneal Ehrlich ascites tumors (EAT) or C3H/HeJ mice bearing 21-day subcutaneous T58 mammary adenocarcinomas were added (± 10−5 M indomethacin, or a monoclonal anti-PGE2 ab) to syngeneic splenic lymphocytes to examine the effects on 1) polyclonal activation (3-d 3H-thymidine [3H-TdR] uptake) with concanavalin A (Con A); 2) one-way (CBA α BALB/C or C3H α BALBC) MLR (5-d 3H-TdR uptake) and subsequent CTL generation (tested against 51Cr-labeled Con A blasts of the stimulator phenotype); 3) NK activity (after 24-h co-culture) against YAC-1 targets; 4) generation of LAK cell activity (in the presence of 200 or 2,000 units recombinant IL-2 for 3 or 5 days), tested against NK-sensitive and NK-resistant targets. Similar effects were also noted on the generation of tumoricidal activity in normal splenic macrophages cultured for 3 days in the presence of LPS. Normal splenic macrophages added under the same conditions served as controls. Effects of pure PGE2 or PGF2 (10−6M) were also examined on these activation events. Results revealed that tumor-host-derived macrophages (but not normal macrophages) markedly suppressed all these activation events and this suppression was abrogated nearly totally by indomethacin and totally by anti-PGE2 ab, indicating its mediation by PGE2. This finding ran parallel with high levels of PGE2 production by tumor-host-derived but not normal splenic macrophages. Pure PGE2 but not PGF2α mimicked these suppressor effects. While tumoricidal activity was generated in normal macrophages in the presence of LPS, IL-2, or IFN-γ or their various combinations (which led to further augmentation), these agents required the presence of indomethacin to generate significant killer activity in tumor-host-derived macrophages. These results indicate that macrophages in tumor-bearing hosts exert pansuppressor effects against the activation of all tumoricidal effector lineages inclusive of T, NK, LAK cells, and killer macrophages.
Enhancement of the Expression of Activation Markers on Human Peripheral Blood Mononuclear Cells by In Vitro Culture With Retinoids and Carotenoids
Authors: Prabhala Rao H , Maxey Vada , Hicks Mary J , Watson Ronald R
Published: January 2018

Retinoids (retinol, retinal, retinoic acid, retinyl beta-glucuronide, and 13-cis retinoic acid) and carotenoids (beta-carotene and canthaxanthin) were evaluated for their immunomodulatory effects on human peripheral blood T-lymphocyte subpopulations and natural killer (NK) cells. Peripheral blood mononuclear cells (PBMC) from healthy young volunteers were isolated and incubated for 72 hours at various levels of retinoids and carotenoids including a physiological concentration (10-8 M). Expression of surface antigens for total T cells, T-helper and T-suppressor cells, and activation markers (transferrin receptor, HLA-Dr antigen, and interleukin 2 receptor) were analyzed with an EPICS V flow cytometer. Retinoic acid and 13-cis retinoic acid (13-cRA) produced significant increases in the percentage of cells with markers for total T-helper cells, with a minimal effect on percentage of lymphocytes with markers for NK cells. However, beta-carotene (BC), canthaxanthin (CTX), and retinyl beta-glucuronide (RBG) dramatically increased the percentage of PBMC with markers for NK cells and produced a smaller increase in lymphocytes with surface antigens identifying them as T-helper cells. Furthermore, retinol and retinal did not show significant change either in the percentage of lymphocytes with markers for T-helper cells or in the helper/suppressor ratio. An increase in the expression of HLA-Dr antigen and transferrin receptors was greater when cells were incubated with 13-cRA than with either BC, CTX, or RBG, while carotenoids produced a greater increase in the expression of IL-2 receptors than 13-cRA. Our study indicates that both retinoids and carotenoids might be activating different subpopulations of immune cells.
Regulation of Monokine Gene Expression: Prostaglandin E2 Suppresses Tumor Necrosis Factor but Not Interleukin-1α or β-mRNA and Cell-Associated Bioactivity
Authors: Scales W E , Chensue S W , Otterness I , Kunkel S L
Published: January 2018

Prostaglandin E2 (PGE2)-mediated suppression of macrophage interleukin-1α,β and tumor necrosis factor-α synthesis was examined at the cellular and molecular levels. Treatment of lipopolysaccharide (LPS)-stimulated adjuvant-elicited murine macrophages with 5 × 10-7M PGE2 caused a 70% reduction in cell-associated TNF but had no suppressive effect on cell-associated interleukln-1 (IL-1) activity. Consistent with this result, Northern blot and nuclear transcription analyses demonstrated suppression of TNF mRNA but PGE2 had no effect on IL-1α and IL-1β mRNA accumulation, as compared to LPS controls. Immunoperoxidase staining for cell-associated TNFα, IL-1α, and IL-1β demonstrated that PGE2 suppressed TNF, but not IL-1α or -β expression, supporting the bioassay data. These results imply that PGE2-mediated regulation of IL-1α,β and TNFα is quite distinct. Synthesis of TNF appears to be regulated at least at the level of transcription, whereas that for IL-1α and -β is regulated post-transcriptionally.
Characterization of the Luminol-Amplified Light-Generating Reaction Induced in Human Monocytes
Authors: Johansson Agneta , Dahlgren Claes
Published: January 2018

Increased production of oxidative metabolites following interaction between mononuclear phagocytes and soluble stimuli can be measured as luminol-amplified chemiluminescence (CL). The effects of superoxide dismutase (SOD), catalase, and azide on the monocyte CL response were investigated. Azide, a myeloperoxidase (MPO) inhibitor, reduced the CL reaction by more than 80%, which indicates that the CL reaction is dependent on the granule enzyme MPO. Because SOD and catatase only partly inhibited the monocyte CL response, the authors propose that part of the monocyte CL response is of intracellular origin. This conclusion is further supported by the effects on the CL response obtained by adding extra peroxidase and the lack of correlation with techniques measuring only extracellular generated metabolites. However, it should be pointed out that the relation between extracellular and intracellular activity is stimulus dependent. Furthermore, even if quantitative differences exist between monocyte and granulocyte CL, the mechanism for the light-generating reaction seems to be the same in both cell types.
Selective Depletion of Rat Neutrophils by In Vivo Administration of a Monoclonal Antibody
Authors: Sekiya Sakae , Gotoh Seiji , Yamashita Takao , Watanabe Tadashi , Saitoh Susumu , Sendo Fujiro
Published: January 2018

A monoclonal antibody (RP-3) that depletes rat neutrophils selectively in vivo was developed by hybridization of mouse myeloma cells (P3-X63.Ag8.653) and spleen cells of BALB/c mice sensitized with peritoneal neutrophils of WKA/Hok rats. RP-3 reacted with rat neutrophils but not with lymphocytes, macrophages, natural killer cells, basophils, eosinophils, or tissues of various organs. The mitogenic responsiveness to concanavalin A (ConA), phytohemagglutinin (PHA), and lipopolysaccharide (LPS) of rats given RP-3 was not significantly different from that of normal rats.

Administration of RP-3 into the peritoneal cavity of rats that had been kept under specific pathogen-free (SPF) or clean conditions induced selective depletion of circulating neutrophils to under 100/mm3 (0.5% WBC). The numbers of monocytes, lymphocytes, and platelets were not changed. Administration of 2 ml of RP-3 reduced blood neutrophils to under 100/mm3 for approximately 24 h, and administration of 1 ml caused depletion for approximately 12 h.
Regulation of Systemic Macrophage IL-1 Gene Transcription: The Involvement of Tumor-Derived Macrophage Growth Factor, CSF-1
Authors: Evans Robert , Duffy Theodore M , Blake Sharon S , Lin Hsiu-San
Published: January 2018

Previous data indicated that progressive growth of the C57BL/6J sarcoma, MCA/76-9, was associated with a systemic increase in the level of IL-1 activity in peritoneal and tumor-associated macrophages. In this paper, the hypothesis was tested that macrophage IL-1 alpha and beta gene expression was regulated by the specific macrophage growth factor, CSF-1, produced by tumor cells. The data indicated that all of the nine C57BL/6J, BALB/cJ, and C3H/HeJ sarcomas tested transcribed the CSF-1 gene. The tumor cell culture supernates induced proliferation of bone marrow cells that differentiated into macrophages. Moreover, proliferative activity of conditioned medium and the ability to induce macrophage differentiation were abrograted by neutralization with the 5A1, rat-anti-mouse monoclonal anti-CSF-1 antibody. Conditioned medium from tumor cell cultures, as well as recombinant CSF-1 and L cell-conditioned medium, induced transcription of the IL-1 alpha and beta genes, the latter being more strongly expressed. IL-1 gene transcription was not induced after neutralization of the culture supernates by the monoclonal antibody, 5A1. The overall data would support the hypothesis that the pathway in vivo leading to the increased IL-1 expression by macrophages is regulated by at least one tumor cell product, CSF-1.
Morphological and Functional Changes During Cytomegalovirus Replication in Murine Macrophages
Authors: van Bruggen I , Price P , Robertson T A , Papadimitriou J M
Published: January 2018

Structural and functional changes were studied in murine peritoneal macrophages infected with murine cytomegalovirus by using centrifugal enhancement to achieve a high-level (> 90%) pulsed infection. During 3 d of culture the infected cells became enlarged and rounded with smooth surface contours. Transmission electron microscopy demonstrated various stages of viral maturation in the nucleus and cytoplasm. Intracellular organization was generally retained, apart from the development of large, irregular, intracytoplasmic vacuoles, in which enveloped virions and cell debris accumulated. The infected macrophages lost most surface markers tested (F4/80, Mac-1, FcR, and the receptor for gluteraldehyde-fixed sheep red blood cells), but H-2 expression was increased. Moreover, ingestion of colloidal gold or horseradish peroxidase was depressed, and the levels of acid phosphatase activity, lymphocytostatic activity, and interleukin 1 production were also decreased. The latter may explain the observed loss of accessory cell function.