bluuebunny/crossref_metadata_2025 huggingface dataset:
Concentrations of Immunoreactive Human Tumor Necrosis Factor Alpha Produced by Human Mononuclear Cells In Vitro
Authors: van der Meer Jos W M , Endres Stefan , Lonnemann Gerhard , Cannon Joseph G , Ikejima Takashi , Okusawa Seijiro , Gelfand Jeffrey A , Dinarello Charles A
Published: January 2018

The concentrations of tumor necrosis factor (TNF) produced by human peripheral blood mononuclear cells (MNC) were measured using a radioimmunoassay (RIA) for human TNF. This was developed using a rabbit antiserum against human recombinant TNF (Hu rTNF), and Hu rTNF labeled with Na1251 by a modification of the chloramine T method. This RIA does not detect human lymphotoxin, interieukin-1 alpha or beta, interleukin 2, interleukin 6, interferon alpha or gamma, granulocyte-macrophage-colony stimulating factor, and C5a des arg. A good correlation (r = 0.89) was found between the RIA and the cytolytic bioassay for TNF. The sensitivity of the RIA is between 3 and 78 pg/ml (median 11 pg/ml).

The mean concentration of TNF in 24-h culture supernatants of human MNC exposed to different concentrations of lipopolysaccharide (LPS) was found to increase in dose-dependent fashion and then level off between 50 and 100 ng/ml. The concentrations of IL-1 beta and alpha detected by specific RIAs in these supernatants were between 0.2 and 19 ng/ml and 0.04 and 1 ng/ml, respectively. The amount ot TNF produced by human MNC in vitro was determined in a cohort of 50 normal volunteers. Without exogenous stimuli, TNF concentrations were almost always below the detection limit; with 0.5 ng/ml LPS, the median concentration of TNF was 2 ng/ml, and with PHA the median was 3.8 ng/ml. In cultures performed in the presence of indomethacin significantly (p< 0.005) more TNF was produced.

Using this RIA, we could detect TNF in the circulation of mice injected with Hu rTNF. When plasma samples of patients with febrile illnesses were added directly to the RIA, TNF was not detectable, with the exception of patients with malaria.

These studies demonstrate the range and sensitivity of LPS-induced and mitogen-induced production of immunoreactive TNF by human MNC in vitro without interference of similar cytokines in bioassays.
Measurement of Intracellular Fluorescence of Human Monocytes Relative to Oxidative Metabolism
Authors: Robinson J Paul , Bruner Leon H , Bassoe Carl-F , Hudson Jerry L , Ward Peter A , Phan Sem H
Published: January 2018

Human monocytes (MN) produce O2- and H2O2 when stimulated by agonists. Dichlorofluorescin diacetate (DCFH-DA) has been used as a substrate for measuring intracellular oxidant production in neutrophils. DCFH-DA is hydrolyzed by esterases to dichlorofluorescin (DCFH), which is trapped within the cell. This nonfluorescent molecule is then oxidized to fluorescent dichlorofluorescin (DCF) by action of cellular oxidants. DCFH-DA can not be appreciably oxidized to a fluorescent state without prior hydrolysis. We have examined the utility of DCFH-DA for the assessment of monocyte oxidative responses. The levels of intracellular fluorescence measured by flow cytometry were considerably less than expected from reported levels of O2--production or chemiluminescence assays. Compared with neutrophils, monocytes produced minimal increases in DCF fluorescence after stimulation with phorbol myristate acetate as measured by flow cytometry, but both cell types showed increases in fluorescence when bulk cell suspensions were measured by spectrofluorometry. To determine the intracellular location of the DCFH, bulk fluorescence measurements were made on both whole and sonicated cell preparations. When intact mononuclear cells were preloaded with DCFH-DA, then sonicated and oxidized with added excess H2O2, the increase in fluorescence was only 30% of the fluorescence of mononuclear cell sonicates to which DCFH-DA was added and oxidized in a similar manner. These results suggest that a portion of the DCFH-DA incorporated by intact cells, is not susceptible to oxidation by the added H2O2. Addition of NaOH to induce hydrolysis of any residual DCFH-DA in the sonicates of DCFH-DA-loaded intact mononuclear cells resulted in a further increase in fluorescence upon addition of H2O2, suggesting that a significant portion of the DCFH-DA was not hydrolyzed despite ample uptake of this dye by these cells. In contrast, no further increase in fluorescence was observed in sonicates of DCFH-DA-loaded intact neutrophils, suggesting complete hydrolysis of all incorporated DCFH-DA to DCFH. When monocytes were allowed to phagocytose DCFH-DA-loaded Staphylococcus aureus, intracellular fluorescence was measurable by flow cytometry, indicating intracellular oxidation of the fluorochromes. We therefore propose that in monocytes the mechanism of intracellular processing of these fluorochromes differs from that in neutrophils owing to differences in intracellular localization of fluorochromes, site of oxidant production, and/or accessibility of the DCFH-DA to esterolysis.
Incorporation of LPS in Liposomes Diminishes Its Ability to Induce Tumoricidal Activity and Tumor Necrosis Factor Secretion in Murine Macrophages
Authors: Dijkstra Jan , Larrick James W , Ryan John L , Szoka Francis C
Published: January 2018

We investigated the effect of lipopolysaccharide (LPS) incorporated into phospholipid vesicles (liposomes) on the induction of macrophage-mediated tumor cytotoxicity and tumor necrosis factor (TNF) secretion. The incorporation of Salmonella minnesota rough (Re)-LPS into multilamellar or small unilamellar vesicles (liposomes) resulted in an 100-to 1,000-fold reduction in its potency to activate both the macrophage cell line RAW 264.7 and murine thioglycolate elicited peritoneal macrophages to become cytotoxic for L929 and P815 tumor cells. Liposomal LPS was also a 100- to 1,000-fold less potent inducer of TNF secretion from RAW 264.7 cells. Cytokines secreted by the activated macrophages contributed to the cytotoxic effect on the L929 cells but not the P815 cell line. Human recombinant TNF was not cytotoxic for either cell line but was cytostatic for the L929 cell line. Morphological examination of the cells after uptake of fluorescent, free, and liposomal LPS revealed that both forms were internalized by the endocytic pathway. This, together with the considerably reduced potency of liposomal LPS to induce tumor cytotoxicity and TNF secretion, suggests that the interaction of the hydrophobic part of the lipid A moiety of LPS with the macrophage plasma membrane is needed to optimally activate these cells. Incorporation of LPS into liposomes effectively abrogates this interaction.
Flow Cytometric Analysis of I-J Expression on Murine Bone Marrow-Derived Macrophages
Authors: Malley A , Stewart C C , Stewart S J , Waldbeser L , Bradley L M , Shiigi S M
Published: January 2018

Attempts to analyze bone marrow-derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I-A, I-J, and Mac-1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50–60% Mac 1-positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1-positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I-J until these cells have been in culture 3 to 4 days, and the number of cells expressing I-J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I-A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I-A, and only 20% of peritoneal cells had I-J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I-J positive (up to 70%), and only about 30% of these cells expressed I-A cell surface antigens.

The binding of anti-I-J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 μg of an IgG2a myeloma protein did not block anti-I-J antibody binding. The addition of 25-200 μg of monoclonal anti-Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti-I-Jk antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti-I-J IgM antibody.

BMDM provide a pure population of macrophages that express a significant level of cell surface I-J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I-J-positive cells parallels the increase in macrophages in these cultures. Thus, the further study of BMDM may permit resolution of questions raised by molecular biologists and immunologists regarding the nature of the I-J protein, the location of I-J genes, and the role of I-J in regulating immune responses.
Isolation and Identification of Feline Peritoneal Macrophages for In Vitro Studies of Coronavirus-Macrophage Interactions
Authors: Stoddart Cheryl A , Scott Fredric W
Published: January 2018

Feline peritoneal cells were collected by lavage with Isotonic saline without the use of irritants or need for euthanasia of the cats. Macrophages were purified by centrifugation on Percoll followed by selective adherence. Although few macrophages could be obtained from an initial lavage, a second lavage performed on the same cat 9-11 days later yielded six times as many macrophages as the first lavage, providing sufficient numbers of cells for characterization and infection experiments. Macrophages from these subsequent lavages were not more functionally activated in phagocytosis assays than the resident macrophages from the initial lavage, and they were equally susceptible to infection with feline infectious peritonitis virus (FIPV). Infected cultures produced peak titers of 105.0 TCID50 per ml, and FIPV antigen was detected in a small subset (0.1-1.0%) of cells by indirect immunofluorescence. The FlPV-infected cells were identified as macrophages by their characteristic morphology and ability to phagocytize rhodamine-labeled latex beads. The successful isolation of large numbers of unactivated feline macrophages will permit in vitro studies of feline coronavirus-macrophage interactions that otherwise would not have been possible. Such studies will undoubtedly provide valuable insights Into the pathogenesis of feline infectious peritonitis, an invariably fatal disease of domestic and exotic cats.
Prostaglandin E2 and Thromboxane B2 Release From Human Monocytes Treated With Bacterial Lipopolysaccharide
Authors: Nichols Frank C , Garrison Scott W , Davis Harold W
Published: January 2018

We investigated the capacity of counterflow-isolated human monocytes to independently synthesize thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) when stimulated with bacterial lipopolysaccharide (LPS). Independent metabolism was confirmed by establishing different specific activities (dpm/ng) of TxB2 and PGE2 released from LPS-treated cells. For metabolites released during the initial 2-hr treatment period, the specific activity of PGE2 was approximately threefold higher than that of TxB2 regardless of labeling with [3H] arachidonic acid (AA) or [14C]AA. Cells that were pulse-labeled for 2 hr with [3H]AA demonstrated a decreasing PGE2 specific activity over 24 hr, whereas the TxB2 specific activity remained unchanged. In contrast, cells continuously exposed to [14C]AA demonstrated an increasing TxB2 specific activity that approached the level of PGE2 by 24 hr. These results suggest the presence of at least 2 cyclooxygenase metabolic compartments in counterflow-isolated monocytes. Although freshly isolated monocytes have been reported to contain variable numbers of adherent platelets, additional experiments demonstrated that counterflow-isolated platelets are not capable of releasing elevated levels of TxB2 or PGE2 when treated with LPS. It is proposed from these findings that at least two subsets of monocytes exist in peripheral blood that can be distinguished on the basis of independent conversion of AA to TxB2 and PGE2.
Differentiation, Maturation, and Proliferation of Macrophages in the Mouse Yolk Sac: A Light-Microscopic, Enzyme-Cytochemical, Immunohistochemical, and Ultrastructural Study
Authors: Takahashi Kiyoshi , Yamamura Fumie , Naito Makoto
Published: January 2018

Primitive macrophages first appear in the blood islands of the mouse yolk sac on the ninth day of gestation. After the tenth day of fetal life, these cells differentiate into fetal macrophages and become mature, with the development of intracellular organelles. They appear in the mesenchymal layer and further immigrate into the extraembryonic coelom. The fetal macrophages do not show any cytochemical peroxidase or 5′-nucleotidase activity, and they possess a marked proliferative capacity. Promonocytes or monocytes that have an incomplete ultrastructure emerge in the blood islands of the yolk sac a day after the occurrence of the fetal macrophages. These events suggest that fetal macrophages differentiate from primitive macrophages before the development of promonocytes or monocytes in the mouse yolk sac; they actively proliferate and are colonized into the embryonic tissues. These results also indicate that the ontogeny of the monocyte/macrophage is different in the early embryo compared with its later developmental stages.
Lead-Induced Alterations of In Vitro Bone Marrow Cell Responses to Colony Stimulating Factor-1
Authors: Kowolenko Michael , Tracy Leigh , Lawrence David A
Published: January 2018

Bone marrow cell responsiveness to hemapoietic growth factors is an integral part of immune responsiveness. Since host resistance is often dependent on bone marrow cell responsiveness and Pb alters host resistance, the influence of Pb on bone marrow cell responsiveness to the hemapoietic growth factor CSF-1 was evaluated. Cell number, soft agar colony formation, cell cycle analysis, as well as 3H-thymidine incorporation were utilized to determine if CSF-1 driven bone marrow-derived macrophage (BMDM) proliferation in vitro is altered in the presence of PbCI2. Data obtained indicate that Pb potentiates the ability of CSF-1 to stimulate thymidine incorporation by BMDM; however, colony formation is inhibited reversibly, and the absolute number of cells in culture is adversely affected by Pb. Propagation of BMDM appears to be more sensitive to Pb (100 nM) than other immune parameters. The decrease in bone marrow cell responsiveness to CSF-1 in the presence of Pb observed in this system may contribute to the decrease in host resistance observed in Pb-exposed animals.
Selenium and the Immune Response: 1. Modulation of Alloreactive Human Lymphocyte Functions In Vitro
Authors: Petrie Howard T , Klassen Lynell W , Kay H David
Published: January 2018

A role for the dietary trace mineral element selenium in the reduction of cancer incidence has been documented in numerous epidemiological and experimental studies. The precise mechanism of this antitumor effect is not well understood, but published data suggest that both inhibition of tumor cell growth and enhancement of host immunity are likely to be involved. In this study we report that selenium at physiologic concentrations can inhibit human lymphocyte proliferation in response to irradiated tumor cells in mixed lymphocyte/tumor cell cultures (MLTC). In addition, we demonstrate that the various lymphocyte functional activities generated in these cultures exhibit different levels of sensitivity to the effects of selenium. The generation of suppressor-cell activity in MLTC was strongly inhibited by the presence of physiologic levels of selenium, while the development of cytotoxic T-lymphocyte activity in identical cultures was not affected by selenium. Production of interleukin-2 in these cultures showed an intermediate sensitivity to the effects of selenium. Thus, selenium appears to be capable of selectively regulating the generation of functional lymphocyte subsets in vitro. Such selective regulation could explain the published effects of selenium on immunity and would be consistent with a role for immunity in the observed reduction of cancer incidence associated with elevated selenium intake.
Selenium and the Immune Response: 2. Enhancement of Murine Cytotoxic T-Lymphocyte and Natural Killer Cell Cytotoxicity In Vivo
Authors: Petrie Howard T , Klassen Lynell W , Klassen Preston S , O’Dell James R , Kay H David
Published: January 2018

An inverse correlation between cancer incidence and dietary intake of the trace mineral element selenium has been well established in epidemiological and experimental studies. The mechanisms for this chemoprotective effect are unresolved. Much attention has been focused on the antiproliferative effects of selenium on various normal and neoplastic cell types. However, dietary selenium supplementation can also enhance the expression of various humoral and cellular immune responses. In examining the effects of dietary selenium on cell-mediated immunity in mice, we observed that selenium supplementation caused the enhanced expression of spontaneous natural killer (NK) cytotoxicity in spleen cells and of specific cytotoxic T-lymphocyte (CTL) cytotoxicity in peritoneal exudate cells (PEC). NK activity of spleen-cell suspensions from selenium-supplemented mice increased an average of 70% over that of the control group (basal diet). Cytotoxic activity of PEC from mice injected with tumors intraperitoneally peaked earlier in selenium-supplemented animals, and the appearance of cells staining positively for Thy 1.2 surface antigen in selenium-supplemented animals also preceded the values observed in control animals. We propose here that enhancement of in vivo cytotoxic mechanisms, is likely to act synergistically with tumor growth inhibition in the reduction of tumor incidence associated with selenium intake.
Influence of a Cytomegalovirus Infection on Functions and Arachidonic Acid Metabolism of Rat Peritoneal Macrophages
Authors: Engels Wim , Grauls Gert , Lemmens Paul J , Mullers Wil J , Bruggeman Cathrien A
Published: January 2018

An intraperitoneal (rat) cytomegalovirus (RCMV) infection in the rat caused an influx of mononuclear cells, which have been altered in functions and arachidonic acid (AA) metabolism. Phagocytosis has been increased considerably 3 days postinfection (p.l.), whereas the release of prostacyclin, thromboxane A2, 12-hydroxyheptadecatrienoic acid (HHT), 5-hydroxyeicosatetraenoic acid (5-HETE), and leukotriene B4 (LTB4) was inhibited for more than 80%. The release of superoxide anions and the chemiluminescence response (CL) upon opsonized zymosan stimulation did not differ from those observed in resident peritoneal macrophages. Additionally, the levels of cyclic nucleotides (cAMP and cGMP) were low in both resident and influx macrophages (day 3 p.i.). In contrast, peritoneal macrophages harvested on day 10 p.i. still showed a high level of phagocytosis. However, the intracellular level of cyclic AMP had decreased fivefold, whereas CL response and superoxide anion release were inhibited significantly. Moreover, the production of prostacyclin, LTB4, and 5-HETE was still suppressed in contrast to thromboxane synthesis, which has selectively been restored in these macrophages. A direct regulatory role of AA metabolites in changes in macrophage functions that were due to a RCMV infection could not be demonstrated.
Increased Production of Cytotoxic Macrophage Progenitors by Lactobacillus casei in Mice
Authors: Nanno Masanobu , Shimizu-Takeda Takako , Mike Akito , Ohwaki Makoto , Togashi Yuji , Suzuki Ryuji , Kumagai Katsuo , Mutai Masahiko
Published: January 2018

Heat-killed Lactobacillus casei YIT9018 (LC9018). when administered intravenously to normal mice, induced increase in Mac-1+ cells and Mac-2+ cells but not in Mac-3+ cells in spleen. The number of both populations changed in the same time course and was maximal 14 d after the administration. To know the effect of LC9018 on hematopoietic progenitor level, we examined the number of macrophage colony-forming cells (M-CFC), granulocyte-macrophage CFC (GM-CFC), and colony-forming units in spleen (CFU-S) in bone marrow 3 d after the administration. LC9018 stimulated the proliferation of M-CFC but not that of GM-CFC and CFU-S. LC9018-induced M-CFC were similar to normal M-CFC in dependence on macrophage colony-stimulating factor (M-CSF) and buoyant density. M-CFC-derived macrophages cultured in the presence of M-CSF expressed Mac-1 and Mac-2 but not Mac-3. They showed cytotoxic activity against syngenic tumor cells, Meth A, via direct contact, when assayed by using an in vitro colony inhibition assay or an in vivo Winn test. These results indicate that LC9018 stimulates the proliferation of cytotoxic macrophage progenitors in bone marrow and induces their differentiation in spleen. These effects may be one of the ways in which LC9018 suppresses tumor growth.
Role for Arachidonic Acid Metabolism and Protein Synthesis in Recombinant Bovine lnterferon-γ–lnduced Activation of Bovine Neutrophils
Authors: Steinbeck Maria J , Webb Deborah S A , Roth James A
Published: January 2018

Bovine neutrophils were preincubated with recombinant bovine interferon-γ (rbolFN-γ), and the molecular events leading to enhanced antibody-dependent (ADCC) and antibody-independent (AINC) neutrophil-mediated cytotoxicity and reduced random migration under agarose were investigated. Addition of α-amanitin, puromycin, or cycloheximide (RNA and protein synthesis inhibitors) during preincubation and assaying prevented the rbolFN-γ–induced AINC enhancement and migration inhibition, but did not influence the enhancement of ADCC. Treatment of neutrophils with rbolFN-γ consistently increased the synthesis of a 60 and a 94–95 kilodalton protein and of leukotriene B4 and 5-HETE. Neutrophils isolated from dexamethasone-treated cattle showed similar protein synthetic activity after rbolFN-γ treatment, but these cells failed to show enhanced AINC. Neutrophils from dexamethasone-treated cattle have been shown to have depressed arachidonic acid metabolism. Addition of lipoxygenase inhibitors (NDGA and BW755c) to neutrophils from nondexamethasone-treated cattle following preincubation with rbolFN-γ (i.e., after protein synthesis had occurred) eliminated the enhancement of AINC. Thus it was concluded that the metabolic events required for neutrophil activation by rbolFN-γ are different for the three functions measured. The enhanced AINC response required both arachidonic acid metabolism and protein synthesis, inhibition of random migration required only protein synthesis, and enhancement of ADCC required neither.
Initial Characterization of a Cytokine Which Induces Differentiation and Cytolytic Activity in HL-60 Promyelocytic Leukemia Cells: Evidence That the Cytokine Is Distinct From Other Known Differentiation-Active Cytokines
Authors: Leftwich Julie A , Hall Robert E
Published: January 2018

In this paper we report that, like dimethyl sulfoxide (DMSO), retinoic acid (RA), and conditioned medium (CM) from lectin-stimulated mononuclear leukocytes, CM from a human null cell leukemia line (Reh) induces HL-60 promyelocytic leukemia cells to respond in an enhanced manner to phorbol diester (PDE). Furthermore, Reh-CM induces PDE-resistant HL-60-1E3 cells to respond to PDE and lyse target cells. Additionally, both HL-60 and HL-60-1E3 cells exposed to Reh-CM for 3 days produce superoxide anion and express cell surface antigens present on mature mononuclear phagocytes. No colony-stimulating factor (CSF) or interferon (IFN) activity was detected in Reh-CM, and differentiation activity (DA) was not removed from Reh-CM by insolubilized anti-IFNγ. While Reh-CM is antiproliferative against a panel of cell lines, its spectrum of activity is different than tumor necrosis factor (TNF)α, and neither TNFα nor TNFβ inhibit proliferation of HL-60-1E3 or induce these cells to respond to PDE. The differentiation factor (DF) material has been partially purified by ammonium sulfate precipitation and is non-dialyzable; unstable to heat, acid, or alkali treatment; and the activity is not blocked by anti-IL-6 or anti-IFNα. The data presented in this paper suggest the presence of a differentiation-inducing factor which is distinct from CSF, IFNα or -γ, TNFα or -β, or IL-6, which may play a role in the differentiation of malignant (leukemic) and normal cells of the myelomonocytic lineage.
Difference in Changes of Membrane Fluidity of Polymorphonuclear Leukocytes Stimulated With Phorbol Myristate Acetate and Formyl-Methionyl-Leucyl-Phenylalanine: Role of Excited Oxygen Species
Authors: Masuda Midori , Komiyama Yutaka , Murakami Takashi , Murata Kenjiro , Hasui Masafumi , Hirabayashi Yoichi , Kobayashi Yohnosuke
Published: January 2018

Polymorphonuclear leukocytes (PMN) were stimulated with phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl phenylalanine (FMLP) to clarify the role of excited oxygen species in inducing changes of membrane fluidity. Membrane fluidity was assessed by the excimer-forming lipid technique using pyrenedecanoic acid and flow cytometry. Membrane fluidity of PMN decreased following stimulation with PMA, and the extent of decrease was both time- and dose-dependent. FMLP at 10-5 M induced a decrease, while FMLP at 10-7 M induced a rapid increase. On stimulation with 10-7 M FMLP as well as in a resting condition, the change of membrane fluidity of PMN from patients with chronic granulomatous disease (CGD) was similar to that of normal PMN. However, on stimulation with PMA or 10-5 M FMLP, CGD PMN did not show a significant decrease. In addition, normal PMN incubated with catalase inhibited the decrease. These findings suggest that the generation of excited oxygen species, particularly of H2O2, is important in inducing a decrease of PMN membrane fluidity.
Interleukin-4 Downregulates Interleukin-6 Production in Human Peripheral Blood Mononuclear Cells
Authors: Lee Jeffrey D , Swisher Stephen G , Minehart Elaine H , McBride William H , Economou James S
Published: January 2018

We report that recombinant human interleukin-4 (IL-4) downregulates interleukin-6 (IL-6) production by human peripheral blood mononuclear cells (PBMC). PBMC were preincubated for up to 24 hr in the presence of IL-4 (100 U/ml) and then activated with li-popolysaccharide B Escherichia coli 026:B6 (LPS, 10 μg/ml), recombinant human tumor necrosis factor-α (TNF-α, 200 U/ml), or Concanavalin A (Con A, 10 fig/ml). Although all these signals induced IL-6 production, IL-4-treated cells produced significantly reduced levels of IL-6 protein. This effect was dose and time dependent. We conclude that IL-4 is a potent downregulatory modulator of IL-6 expression in human PBMC.
Taurine and Hypotaurine Content of Human Leukocytes
Authors: Learn Douglas B , Fried Victor A , Thomas Edwin L
Published: January 2018

Taurine (T) was reported to be at high concentrations in human leukocytes. It was proposed that T is a scavenger for chlorinated oxidants produced by the myeloperoxidase system of monocytes and neutrophils. Hypotaurine (HT) would be a more effective scavenger, and HT could also detoxify products of bromide or iodide oxidation produced by the eosinophil peroxidase system. Methods previously used to measure T in leukocytes might oxidize HT to T or fail to separate T and HT. Therefore, we examined T and HT content, uptake, and biosynthesis in isolated blood cells and cultured tumor cells derived from hematopoietic/lymphoid cells. Platelets and all leukocytes including monocytes, lymphocytes, neutrophils, and eosinophils had high T levels (10–20 mM), and all except eosinophils had substantial HT levels (0.3–1 mM). Intracellular levels were 500-times higher than in plasma. Erythrocytes were the only blood cells with low levels of both T and HT. Tumor cells from lymphoid (CCRF-CEM) and myeloid (HL-60, K-562, RWLeu4, HEL) lineages took up and concentrated T and HT from the bovine calf serum in the culture medium, and intracellular levels were similar to those in leukocytes. When cells were cultured in HT-supplemented media, HT almost completely replaced T, and HT was not converted to T. Levels of T were not raised by culturing cells with possible precursors, but HT levels were raised when cysteine sulfinic acid was present. Washed tumor cells took up T and HT by way of a β-amino acid transport system, but uptake by leukocytes was very low. Therefore, leukocytes may acquire T and HT by active uptake rather than biosynthesis, and uptake may be completed during differentiation in the bone marrow. Though HT is low relative to T, HT levels may be sufficient to protect leukocytes from toxic oxidants.
Interferon-γ Induced Resistance to Legionella pneumophila in Susceptible A/J Mouse Macrophages
Authors: Klein Thomas W , Yamamoto Yoshimasa , Brown H Keith , Friedman Herman
Published: January 2018

Legionella pneumophila (Lp) grow in cultures of human, guinea pig, and mouse macrophages from A/J strain mice. Because exudate macrophages from this strain of mice have been reported deficient in responsiveness to lymphokines, we thought it of interest to document the extent of responsiveness to interferon-γ in the context of growth restriction of Lp. Peritoneal exudate macrophages were obtained from A/J mice and cultured in either the presence or absence of recombinant lnterferon-γ. These cultures were then infected with Lp and the extent of bacterial growth estimated 48 hr later by means of a colony-forming unit (CFU) assay and electron microscopy, lnterferon-γ treatment significantly restricted the number of CFUs in the culture at concentrations as low as 20 U/ml, but did not affect the uptake of bacteria by macrophages. Furthermore, treatment with interferon induced morphological changes consistent with activated macrophages. The involvement of oxygen-dependent mechanisms in phagocyte killing and growth restriction was examined by the use of inhibitors such as superoxide dismutase (SOD) and catalase. Neither one of these inhibitors of toxic oxygen metabolites affected the interferon-γ-induced suppression of Lp growth. These results suggest that although thioglycolate-induced exudate macrophages from A/J mice support the growth of Lp, these cells readily respond to the activating influence of lnterferon-γ. Furthermore, lymphokine treatment does not inhibit Lp uptake by macrophages and apparently restricts the growth of bacteria by mechanisms independent of the activity of toxic oxygen metabolites.
Tumor-Derived Products Induce Il-1 a, Il-1 b, Tnf a, and Il-6 Gene Expression in Murine Macrophages: Distinctions Between Tumor- and Bacterial Endotoxin-Induced Gene Expression
Authors: Evans Robert , Kamdar Sonya J , Duffy Theodore M
Published: January 2018

The ability of progressing tumors to regulate host physiology is an important consideration in our understanding of tumor-host relationships. Previous data indicated that several lines of murine sarcoma cells produced one or more activities that were able to regulate both Il-1 a and Il-1 b gene transcription in macrophages (Mø). We now describe an indepth analysis using Northern analysis and bioassays and show that two of these tumors produce one or more activities that when incubated with peritoneal Mø result in the transcription of the Il-a, Il-1 b, Tnf a, and Il-6 genes. Concordant with the Northern analyses was the finding that interleukin-1 (IL-1) and tumor necrosis factor (TNF) biological activities were detected in lysates of induced Mø, fixed Mø, and supernates of Mø cultures. Induction of gene expression was shown to be distinct from that induced by bacterial endotoxin or lipopolysaccharide by a number of criteria. The data suggest that tumor cell products may play an important role in regulating several host physiological processes, particularly those involving Il-1 a, Il-1  b, Tnf a, and Il-6 gene expression.
Autonomous Growth of Human T-Lymphotropic Virus Type I Infected Human Lymphocytes Treated With N-methyl-N'-nitro-N-nitrosoguanidine and Ultraviolet Rays
Authors: Maruyama Koshi , Mochizuki Shigenobu , Kawamura Kiyoko , Tamayama Chieko
Published: January 2018

Human T-cell cultures infected with human T-lymphotropic virus type I (HTLV-I) and interleukin-2 (IL-2)-dependent for their continuous growth were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then maintained in the medium containing phorbol 12-myristate 13-acetate (TPA). Cells achieved independence from IL-2 but became TPA-dependent for continuous growth. Multiple ultraviolet (UV) irradiations of TPA-dependent cells resulted in their autonomous growth. G-band karyotype analysis revealed multiple chromosomal abnormalities that were seen in cells before and after MNNG treatment and UV irradiations, and those that were only seen in autonomously growing cells. Viral expression was found to be transiently enhanced in association with emergence of certain chromosomal changes. Exposure of HTLV-I infected cells to certain mutagens may promote the occurrence of the specific rearrangement of cellular genes responsible for regulation of cellular and viral replication and may lead these cells to neoplastic transformation.
Modulation of the Oxidative Burst in Trout Myeloid Cells by Adrenocorticotropic Hormone and Catecholamines: Mechanisms of Action
Authors: Bayne Christopher J , Levy Sharon
Published: January 2018

The oxidative burst of rainbow trout (Oncorhynchus mykiss) phagocytes was previously found to be differentially modulated by adrenocorticotropic hormone (ACTH) and the catecholamine receptor agonists phenylephrine and isoproterenol. From data obtained using both luminol-enhanced chemiluminescence (LECL) and ferricytochrome C (cyt C) reduction to measure oxidative burst kinetics, we postulated that the observed modulation was mediated by affects on enzymes responsible for the production and metabolism of superoxide anion. Using exogenous superoxide dismutase (SOD) and catalase as scavengers, nitroprusside to poison endogenous SOD, and an assay for hydrogen peroxide, we have tested our postulates by exploiting the differences with which various reactive oxygen intermediates influence LECL and cyt C reduction. The ability of ACTH to potentiate both assays of the oxidative burst appears due to its enhancing influence on the production of superoxide. Phenylephrine, an α-adrenergic receptor agonist, appears to enhance the activity of endogenous SOD, whereas isoproterenol, a β-adrenergic receptor agonist, may suppress SOD activity. This work reveals how components of the natural immune system may be regulated by products of the neuroendocrine system. Also, lymphocyte-derived ACTH may provide a novel pathway for lymphoid regulation of inflammation.
Modulation of natural killer and lymphokine-activated killer cell cytotoxicity by lactoferrin
Authors: Shau Hungyi , Kim Anthony , Golub Sidney H
Published: January 2018

Natural killer (NK) and lymphokine-activated killer (LAK) cell cytotoxic functions can be strongly augmented by the iron-carrier protein lactoferrin (LF). LF significantly enhances NK and LAK activities when added at the beginning of NK or LAK cytotoxicity assays. LF is effective in augmenting cytotoxic activities at concentrations as low as 0.75 μg/ml, and higher concentrations of LF induce greater augmentation of NK and LAK. Iron does not appear to be essential for LF to increase NK and LAK, as depleting iron from LF with the chelator deferoxamine does not affect the capacity of LF to increase cytotoxicity. LF is known to have RNase enzymatic activity, and LF enhancement of NK and LAK can be blocked by RNA. However, LFs from two different sources with over 100-fold difference in RNase activity are equally effective in enhancing NK and LAK. Furthermore, purified non-LF RNase does not modulate NK or LAK activity and DNA is as effective as RNA in blocking LF augmentation of NK or LAK cytotoxicity. Therefore, the RNase activity is unlikely to be responsible for LF enhancement of the cytotoxicities. Newborn infants are known to have low NK activity and NK and LAK cells have been implicated in host defense against microbial infections. Thus, maternal milk-derived LF may have a role in boosting antimicrobial immunity in the early stages of life. In adults, LF released from neutrophils may enhance NK and LAK functions in the inflammatory process induced by microbial infections.
TNF in combination with GM-CSF enhances the differentiation of neonatal cord blood stem cells into dendritic cells and macrophages
Authors: Santiago-Schwarz Frances , Belilos Elise , Diamond Barbara , Carsons Steven E
Published: January 2018

We describe dendritic cell progenitors within the CD34+ stem cell compartment in neonatal cord blood and identify growth factors contributing to their differentiation. Granulocyte-macrophage colony-stimulating factor (GM-CSF), although mainly promoting the growth and differentiation of monocyte-macrophages (mono-mφs), also induced the differentiation of cells with the distinctiv morphological features of dendritic cells (DCs). Tumo necrosis factor (TNF) in combination with GM-CSF promoted further growth of both cell types but most notably increased the DC content. In situ analysis revealed that the cells exhibiting DC morphology were positive for class II major histocompatibility complex antigens but were CD14 negative, did not exhibit nonspecific esterase activity, and were nonphagocytic. Moreover, the mixed leukocyte reaction stimulatory capacity of cultures with the higher DC content was greater. TNF, interleukin-1 (IL-1), IL-6, or platelet-derived growth factor (PDGF) was inactive in promoting stem cell proliferation or DC morphology. IL-1 or PDGF synergized with GM-CSF to increase mono-mφ–associated cell proliferation but did not increase the DC content. The development of a common DC-monocyte precursor was suggested by the presence of colony-forming unit–like clusters containing mono-mφ8 and DCs and one sharp proliferative peak. The loss of DC morphology after 21 days, coupled with increases in mono-mφ–associated markers and a constant number of viable cells, further suggests that DC morphology may fluctuate in culture or is a transient feature acquired by certain cells of the mono-mφ lineage.
Phorbol ester reduces constitutive nuclear NFχB and inhibits HIV-1 production in mature human monocytic cells
Authors: Mufson RA , Myers Carla , Turpin Jim A , Meltzer Monte
Published: January 2018

NFχB is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocyte leukemias. There is little information available on the response of NFχB to cytokines in normal human monocytes. We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NFχB binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NFXB in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate (TPA) for 4-24 h caused the complete disappearance of NFχB from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-1. The constitutive transcription factor SPI was unaffected by addition of TPA. The disappearance of NFXB from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP-1 cells, TPA also induced a new, faster-migrating NFXB species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004, also dramatically reduced constitutive levels of nuclear NFχB. Finally, TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication, as determined by reverse transcriptase assays, in a concentration-dependent manner. These results are in striking contrast to the increase in nuclear NFχB and HIV-1 replication induced by phorbol esters in promonocyte leukemia cells U937 and HL-60, and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes.
Induction of a 26-kDa membrane-form tumor necrosis factor (TNF)-α in human alveolar macrophages
Authors: Nii Akihiko , Sone Saburo , Orino Etsuko , Ogura Takeshi
Published: January 2018

The expressions of a membrane form of TNF (m-TNF) by human alveolar macrophages (AM) and autologous blood monocytes from healthy donors were examined. Upon lipopolysaccharide (LPS) stimulation, AM produced 26-kDa TNF-α on their cell surface. We designed a bioassay for measuring m-TNF in which macrophages were fixed with paraformaldehyde after stimulation for 18 h, then m-TNF activity was assessed as cytotoxicity of fixed macrophages on L929 cells. This assay was specific to m-TNF because: 1) no soluble factors were contributed to the cytotoxicity of fixed AM, 2) anti-TNF-α monoclonal antibody completely neutralized m-TNF activity, and 3) m-TNF activity was not altered after low- pH or high-salt treatment. On LPS stimulation, AM produced significant amounts of m-TNF earlier than TNF-α secretion. AM also expressed significant amounts of m-TNF when stimulated with other bacterial components and their derivatives. Interleukin (IL)-4 suppressed both m-TNF production and TNF-α secretion. p-Toluene- sulfonyl-L-arginine methyl ester (TAME) inhibited specifically TNF-α secretion and not m-TNF expression. Although blood monocytes produced small amounts of m- TNF, monocyte-derived macrophages showed enhanced m-TNF after cultivation with GM-CSF for 10 days. These findings indicate that m-TNF is expressed as a step in the TNF-α producing system, and suggest that m-TNF may play important roles in exhibition of macrophage function in situ. J. Leukoc. Biol 53: 29–36; 1993.
Regulation by dietary essential fatty acid balance of tumor necrosis factor production in mouse macrophages
Authors: Watanabe Shiro , Onozaki Kikuo , Yamamoto Shunsuke , Okuyama Harumi
Published: January 2018

Increasing die dietary α-linolenate (18:3n - 3)/linoleate (18:2n - 6) ratio results in an increase in lipopolysaccharide (LPS)-etimulated tumor necrosis factor (TNF) production in mouse resident and casein-induced peritoneal macrophages [3]. We found that prostaglandin E2 (PGE2) production is inversely related to TNF production and that indomethacin abolishes the effect of changing the essential fatty add balance in resident macrophages. The resident macrophages enriched in n - 3 did not produce a significant amount of PGE3. Accordingly, the decreased production of PGE2 appears to be a major negative regulatory factor for enhancement of TNF production in the it - 3 enriched resident macrophages. In casein-induced macrophages the situation is more complex. Indomethadn decreased PGE2 production and increased TNF production; however, the differences in TNF production between the it - 6 enriched and it - 3 enriched macrophages were not completely abolished by indomethadn treatment. Lysosomal add phosphatase activity, a marker of activation/maturation stages, was elevated in the it - 3 enriched compared to the it - 6 enriched casein-induced macrophages but was similar in the resident macrophages of the two dietary groups. Expression of CD 14, which is a receptor for LPS, was not different in casein-induced macrophages of the two dietary groups. Thus, the differences in production of TNF between the n- 3 and n - 6 enriched resident macrophages can he accounted for mostly by a difference in the production of a negative feedback effector, PGE2. However, a significant portion of the TNF production in casein-induced macrophages is regulated by a factor(s) other than PGE2 and LPS receptor; advanced activation/maturation stages induced by the diet high in α-linolenate may underlie the enhanced TNF production in casein-induced macrophages. J. Leukoc. Biol. 53: 151–156; 1993.
Optimization of peroxide production by resident macrophages
Authors: Cook-Mills Joan M , Fraker Pamela J
Published: January 2018

Previous reports indicated that resident macrophages produced low or nondetectable quantities of H2O2 (1—10 nmol H2O2/mg resident macrophage protein) when measured by the phenol red assay described by Pick and Mizel [5]. However, modifications of the assay that included addition of calcium to assay solutions, alterations in cell-harvesting methods, increasing the concentration of cells analyzed, incubating the cells in ambient air, and enhancing the interactions between macrophages and particulate stimulants by centrifugation resulted in increased production of peroxide. Each variable was instrumental in improving the assay conditions and the combination of changes dramatically increased the amount of peroxide (30-60 nmol H2O2/mg resident macrophage protein) obtained from murine resident macrophages, making it possible to better assess the role of these cells in microbicidal killing. J. Leukoc. Biol. 53: 205–207; 1993.
GTP-binding proteins in polymorphonuclear granulocytes of severely burned patients
Authors: Brom Joachim , Köller Manfred , Müller-Lange Petra , Steinau Hans Ulrich , König Wolfgang
Published: January 2018

In order to study the biochemical mechanism underlying the cellular dysfunctions of polymorphonuclear granulocytes (PMNs) from severely burned patients, we analyzed the role of GTP-binding proteins (G-proteins) in PMNs from 11 burned patients. Our data demonstrate a significant enhancement of the basal GTPase activity within unstimulated neutrophils of severely burned patients compared to cells from healthy donors. This enhancement was significant within 4 weeks after the trauma, followed by a return to control levels. The increase in GTPase activity correlated with enhanced expression of the small G-protein Ras and the regulatory Ras-GTPase activating protein (Ras-GAP) compared to that in healthy donor cells. However, expression of the Ras-related protein Rapl, which is involved in initiation of the respiratory burst, was reduced. The observed changes in G-protein activity and expression impair the signal transduction cascade as well as bacterial killing and may lead to high susceptibility toward infections and finally to septic conditions. J. Leukoc. Biol. 53: 268–272; 1993.