bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence That X-Irradiation and 4-Hydroperoxycyclophosphamide Affects Different Lymphocytes That Respond to Specific Antigen In Vitro
Authors: Clark Connie , LaSota Irene , Borch Richard F
Published: January 2018

The present study examined the effect of pulse treatment with the in vitro active synthetic derivative of cyclophosphamide (CY),4-hydroperoxycyclophos- phamide (4-HPCY), and exposure to X-irradiation on the in vitro Concanavalin A (ConA), lipopolysaccharide (LPS), and antigen-specific blastogenic responses of in vivo-primed lymph node cells. Primed lymph node cells from CY- pretreated, aggregated (A) human IgG-complete Freund's adjuvant (AHGG- CFA)-immunized mice were untreated, exposed to various doses of irradiation, or pulse treated with different concentrations of 4-HPCY before being cultured in medium alone or in medium containing HGG, ConA, or LPS. The results show that HGG-responding and LPS-responding cells exhibited similar dose- inactivation profiles following exposure to irradiation or pulse treatment with 4- HPCY. More than 75% of reactivity was eliminated by exposure to 100 rads or pulse treatment with 20 4-HPCY. In contrast to preculture pulse treatment with 4-HPCY, however, when primed lymph node cells were cultured in medium containing 4-HPCY (culture treatment) LPS-responding cells were shown to be more sensitive to inactivation than HGG-responding cells. The data further show that the effect of low-dose irradiation and of culture treatment with 4-HPCY on the HGG-specific response of primed lymph node cells was additive, suggesting that these agents inactivate different cell subtypes that contribute to the HGG-specific response in vitro.
Receptor-Mediated Entry of Peroxidases Into the Parasitophorous Vacuoles of Macrophages Infected With Leishmania Mexicana Amazonensis
Authors: Rabinovitch Michel , Topper Gail , Cristello Paul , Rich Abby
Published: January 2018

Leishmania amastigotes, obligatory parasites of macrophages, lodge and multiply within long-lived phagolysosomelike “parasitophorous vacuoles” (PV). The glycoprotein horseradish peroxidase (HRP) was shown, by light and electron microscopic cytochemistry, to enter the PVs of rat in vitro-derived bone marrow macrophages infected with Leishmania mexicana amazonensis. Uptake was obtained both in preinfected macrophages incubated with HRP and in macrophages pulsed with HRP, infected, and futher incubated in ligand-free medium. Peroxidase positive and negative PVs could coexist in the same macrophages. Infected macrophages commonly displayed fewer labeled secondary lysosomes than noninfected cells. Lactoperoxidase (LP) was also shown, by light microscopy, to enter the PVs of rat macrophages. Uptake of HRP and of LP was blocked by mannan, supporting the mannose receptor mediated recognition of these ligands. Transfer of HRP to PVs was much less efficient in resident mouse peritoneal macrophages, even at 50 × higher ligand concentrations. Such macrophages expressed negligible mannose receptor function. The efficient mannan-inhibitable uptake of HRP by rat marrow macrophages was confirmed biochemically. Bulk HRP uptake in infected and noninfected cultures was found to be similar. Peroxidases should be useful in further studies of endocytosis by Leishmania-infected macrophages and in the development of lysosomotropic macrophage-targeted drug carriers.
Monitoring Human Neutrophil Granule Secretion by Flow Cytometry: Secretion and Membrane Potential Changes Assessed by Light Scatter and a Fluorescent Probe of Membrane Potential
Authors: Fletcher Mark P , Seligmann Bruce E
Published: January 2018

Purified human peripheral blood polymorphonuclear neutrophils (PMN) were incubated at 37°C with the fluorescent membrane potential sensitive cyanine dye di-O-C(5)(3) and exposed to a number of stimulatory agents (N- formylmethionylleucylphenylalanine (FMLP), cytochalasin B (cyto B) + FMLP, phorbol myristate acetate (PMA). Flow cytometry was utilized to measure changes in forward light scatter (FS), orthogonal light scatter (90°-SC), and fluorescence intensity of individual cells over time. A saturating (10 −6 M) dose of FMLP lead to a significant increase in the cells' FS without a change in 90°- SC as well as a heterogeneous loss of di-O-C(5)(3) fluorescence. PMA (100 ng/ ml) also caused an increase in FS but a uniform loss of dye fluorescence by all cells (apparent depolarization). Cyto B + FMLP produced an increase in FS, a marked loss of 90°-SC, and a uniform loss of fluorescence. Secretion experiments under identical incubation conditions indicated a significantly positive relationship between loss of enzyme markers or cell granularity and orthogonal light scatter (r = 0.959, 0.998f and 0.989 for loss of 90°-SC vs lysozyme, 0- glucuronidase, and granularity index, respectively). Sequential exposure of PMN to PMA and then cyto B + FMLP produced a stepwise shift in scatter parameters (increased FS then loss of 90°-SC). Normalization of membrane potential dye fluorescence changes for the changes in light scatter did not abrogate the heterogeneous fluorescence response of cells to stimulus, indicating that stimulus-induced scatter changes were not responsible for such fluorescence shifts. The data demonstrate that loss of 90°-SC relates closely to secretion of primary granules while changes in FS reflect alterations in cell shape and/or surface/volume ratios that accompany cell activation. Flow cytometric light scatter measurements may yield important information on the extent of prior cell degranulation or activation.
The Strain of Mouse and Assay Conditions Influence Whether MulFN- γ Primes or Activates Macrophages for Tumor Cell Killing
Authors: Pace Judith L , Varesio Luigi , Russell Stephen W , Blasi Elisabetta
Published: January 2018

Investigators from two different laboratories have compared several variables in the short-term macrophage-mediated cytotoxicity assays used by each group to study the role of MulFN- γin macrophage activation. The findings suggest that the capacity of MulFN- γto activate macrophages without the need for a second triggering stimulus is related to assay conditions and, most especially, the strain of mouse used to provide the macrophages.
Natural Antitumor Defense System of the Murine Liver
Authors: Cohen Stefan A , Salazar Daniel , von Muenchhausen Wittiches , Werner-Wasik Maria , Nolan James P
Published: January 2018

Murine nonparenchymal liver cells from various genetic strains isolated by collagenase digestion and differential sedimentation contain both lymphocytes and macrophages. Nonparenchymal liver cells as well as spleen cells, mononuclear blood cells, and peritoneal exudate cells from C3HeB/FeJ mice were tested for natural cytotoxicity against YAC-1 (sensitive to NK cells) and P815 (resistant to NK cells) tumor cell lines. Resident peritoneal exudate cells exerted no cytotoxicity against either tumor cell, whereas spleen and mononuclear blood cells lysed only YAC-1. In contrast, nonparenchymal liver cells lysed both YAC-1 (4 h) and P815 (18 h) tumor cells. Treatment of nonparenchymal liver cells with anti-asialo GM1 and complement abolished the antitumor activity against both tumor cell lines but not the phagocytic activity. Nonadherent nonparenchymal liver cells exerted greater cytotoxicity against YAC-1 tumor cells but little cytotoxicity against P815 tumor cells when compared with unfractionated cells. Adherent nonparenchymal liver cells (macrophages) from untreated mice exerted no antitumor activity against either tumor cell. In contrast, adherent nonparenchymal liver cells from Coryn- ebacterium parvum treated mice were directly cytotoxic to P815 tumor cells. Spleen cells that are normally not cytotoxic to P815 tumor cells (18 h) became cytotoxic when mixed with adherent nonparenchymal liver cells from untreated mice. These results indicate that the tumoricidal effector cell in nonparenchymal liver cells from untreated mice appears to be the NK cell. Apparently, murine liver marophages from untreated mice do not have tumoricidal activity per se but can “activate” NK cells to kill tumor cells normally resistant to NK cells.
Cultured Human Alveolar Macrophages From Smokers With Lung Cancer: Resolution of Factors That Stimulate Fibroblast Proliferation, Production of Collagenase, or Prostaglandin E2
Authors: Dayer J-M , Sundstrm L , Polla B S , Junod A F
Published: January 2018

Marked connective tissue remodelling involves both destruction and repair in inflammatory lung diseases. Throughout the remodelling event, it was reasoned that alveolar macrophages may release substances similar to those produced by blood monocyte-macrophages that affect fibroblast functions, ie, the interleukin 1 family of monokines (or cytokines). We have examined human alveolar macrophage cultures obtained after bronchoalveolar lavage of freshly excised lungs from heavy smokers with bronchial carcinoma. Crude culture media contained fibroblast proliferative activity and collagenase- and PGE2- production-stimulating activity. The main peak of these biological activities was located around - I8 kilodaltons (kD) on gel filtration chromatography. Resolution of this peak by high performance liquid chromatography showed the presence of three distinct peaks, with quantitative and qualitative differences in biological activities. This suggests the presence of heterogeneous factors.
Modulation of Neutrophil-Reduced Pyridine Nucleotide Content Following Stimulation With Phorbol Myristate Acetate and Chemotactic Factors
Authors: Van Epps Dennis E , Bender James G , Steinkamp John A , Chenoweth Dennis E
Published: January 2018

Modulation of NAD(P)H in human neutrophils (PMN) following stimulation with phorbol myristate acetate (PMA) or chemotactic factors was determined by flow cytometry. Stimulation of PMN with 1 μg/ml of PMA results in a time-dependent decrease in fluorescence, attributable to the oxidation of NAD(P)H. The decrease in fluorescence did not occur with PMN from a patient with chronic granulomatous disease (CGD) and was observed in only half of PMN from the mother of the patient. Loss of fluorescence in normal PMN was maximal following 7–15 min of stimulation with PMA. Simultaneous measurement of PMA-stimulated NAD(P)H oxidation and H2O2 production showed that NAD(P)H oxidation occurred as an all-or-none response while H2O2 production showed a graded response.

These data suggest that with PMA stimulation, a threshold exists beyond which constitutive NAD(P)+ reduction is suppressed and complete oxidation of NAD(P)H occurs, while H2O2 production is proportional to the concentration of PMA. PMA-stimulated oxidation of NAD(P)H was reversible, and fluorescence returned to the initial level or higher after 40–60 min. Oxidation of NAD(P)H also occurred when cytochalasin B-treated PMN were stimulated with 25 nM C5a or 100 nM formyl-methionyl-leucyl-phenylalanine (f-MLP), but occurred more rapidly, peaking at 1 to 3 min. Fluorescence also returned by 5–6 min. This response to C5a and f-MLP was graded and proportional to the concentration of chemotactic factor used. Comparative studies showed that the cytochalasin-B treatment was essential for measurement of NAD(P)H oxidation, in response to C5a and F-MLP.
Selective Recognition of Rat Follicular Dendritic Cells (Dendritic Reticulum Cells) by a New Monoclonal Antibody Ki-M4R In Vitro and In Vivo
Authors: Wacker H H , Radzun H J , Mielke V , Parwaresch M R
Published: January 2018

Using unstimulated rat peritoneal cells as immunogen a new monoclonal antibody KiM4R was produced. Ki-M4R recognizes follicular dendritic cells (dendritic reticulum cells) in germinal centers of lymphoid follicles in lymphatic tissue. In addition, sinus lining cells, endothelia of postcapillary venules, as well as mesangial cells of the renal glomerula immunoreact with Ki-M4R in vitro as well as in vivo. This antibody might be useful for studying the interaction of follicular dendritic cells and B-cell immune response.
Expression of Granulocyte Colony-Stimulating Factor by Human Cell Lines
Authors: Devlin James J , Devlin Patricia E , Myambo Kenneth , Lilly Michael B , Rado Thomas A , Warren M Kim
Published: January 2018

We have isolated and expressed a cDNA clone that encodes a human granulocyte colony-stimulating factor from the MIA PaCa-2 cell line. A genomic clone of this factor has been isolated from the CHU-2 cell line and is reported to encode two alternative transcripts [The EMBO J. 5,575, 1986]; one transcript predicts an amino acid sequence identical to that predicted by our MIA PaCa-2 cDNA clone; the other transcript predicts a similar protein containing a three amino acid residue insertion. To investigate which types of this colony-stimulating factor are produced by other cell lines, we used specific oligonucleotides to determine which types of transcripts were present in MIA PaCa-2, 5637, and LD-1 cells, all of which have been reported to produce a factor that can stimulate the growth of predominantly granulocyte colonies in human bone marrow cell cultures. Northern analysis with these probes revealed MIA PaCa-2-like transcripts in all of these cell lines and failed to detect transcripts that would encode the colony-stimulating factor that contained the three-amino-acid-residue insertion.
Independent Regulation of Leukotriene B4-Elicited Polymorphonuclear Leukocyte Exocytosis and Superoxide Generation by a Serum Factor
Authors: Mandell Brian F
Published: January 2018

Serum from a patient with inactive systemic lupus erythematosus (SLE) and ibuprofen-induced transient neutropenia was used as a probe to define further the control of human polymorphonuclear leukocyte (PMN) exocytosis and superoxide (O-2) generation. Thirty-minute preincubation of normal PMNs with 10-50H v/v of this serum, followed by washing, produced a specific dose-related suppression of leukotriene B4 (LTB4)-elicited β-glucuronidase and lysozyme release of up to 45% and 30% respectively. If cells were not washed, the inhibition increased to 60% and 40%. Superoxide production stimulated by LTB4 was unaffected. The serum had no effect on formyl-met-leu-phe (FMLP) or phorbol myristate acetate-stimulated O2- or exocytosis. O2- and β-glucuronidase release elicited by zymosan-treated serum (ZTS) were both decreased by 15%, but there was no increased inhibition seen if cells were not washed, or if the time of preincubation was increased from 7 to 30 min. In contradistinction, the serum inhibition of LTB4 exocytosis did show time dependence. Serum obtained when the patient was not leukopenic and sera from 6 normal controls, 2 patients with inactive SLE, 1 patient with SLE and chronic leukopenia, and 2 controls taking Ibuprofen did not influence any PMN function. The serum inhibition of ZTS-induced functions was qualitatively similar to that observed when PMNs were preincubated and desensitized with ZTS in vitro. Selective inhibition of LTB4 exocytosis was not seen when PMNs were desensitized with LTB4 in vitro. These observations indicate that LTB4-elicfted O2- and exocytosis can be independently and specifically regulated. The cellular site at which this serum factor acts is not clear, but the current studies strongly suggest that this inhibition is not due to in vitro deactivation by LTB4 activity.
Induction of Tissue Transglutaminase in Human Peripheral Blood Monocytes by Intracellular Delivery of Retinoids
Authors: Mehta Kapil , Turpin Jim , Lopez-Berestein Gabriel
Published: January 2018

Human peripheral blood monocytes (HPBM) from normal donors, isolated by counter-current centrifugal elutriation into two subpopulations, showed no significant difference in their ability to differentiate in vitro into macrophages as determined by induction of a protein cross-linking enzyme tissue transglutaminase (TGase). The two subpopulations were equally responsive to the augmenting effect of recombinant interferon-γ (riFN-γ) on expression of tissue TGase. In vitro maturation and treatment with rlFN-γ of HPBM were associated with increased binding of tritlated retinol. Intracellular delivery of retinol rendered this hormone active in inducing the differentiation of HPBM. The retinoid-induced expression of tissue TGase was the result of increased accumulation of the enzyme peptide and not activation of preexisting enzyme. We propose, therefore, that maturation of HPBM, induced by in vitro culture or treatment with rlFN-γ, is associated with acquisition of cell surface receptors for serum retinol-binding protein.
Requirements for Immunoglobulin Synthesis in Leukocyte Cultures Exposed to Human Cytomegalovirus
Authors: Beeler Barbara A , Hutt-Fletcher Lindsey M
Published: January 2018

T cell-depleted leukocytes from normal healthy donors lacking antibody to cytomegalovirus (CMV) were induced to secrete immunoglobulin (Ig) by exposure to inactivated CMV. The responses to two virus strains were compared. One strain had been reported to require specific T cell help to induce Ig synthesis, and the other had been reported to induce Ig synthesis in the presence of only very few cells. Both viruses induced T cell-depleted leukocytes from one group of seronegative donors to secrete Ig. However, both viruses failed to induce leukocytes from a second group of donors to secrete Ig unless B cell growth factor was added as a source of T cell help. These findings suggest that certain individuals are hyperresponsive to CMV and raise the possibility that this group may be most likely to develop mononucleosis after primary infection with CMV.
Proposal for Classification of Leukocyte-Associated Cytolytic Molecules: Denver, Colorado, September 28, 1986
Authors: Koren H , Goldfarb R , Henkart P , Bleackley C , Bonavida B , Granger G , Palladino M , Podack E , Reynolds C , Ruddle N , Ziegler-Heitbrock H W L
Published: January 2018

The continuous increase of newly described cytolytic factors and the lack of a nomenclature system to accommodate these molecules prompted the nomenclature committee of the Reticuloendothelial Society (RES) to sponsor a dry workshop focusing on this topic preceding the Annual RES Meeting, September 28, 1986, in Denver, Colorado. Similar attempts to establish a nomenclature system in other areas of immunology have been quite successful in the past. These include the HLA system, the interleukin system, B-cell stimulating factors, and the CD nomenclature for monoclonal antibodies. The workshop was attended by about 100 scientists mainly from the U.S., although representatives of other countries were present as well.
Expression of an Activation Antigen, Mo3e, Associated With the Cellular Response to Migration Inhibitory Factor by HL-60 Promyelocytes Undergoing Monocyte-Macrophage Differentiation
Authors: Todd Robert F , Bury Martin J , Liu David Y
Published: January 2018

HL-60 promyelocytic cells acquire the surface expression of the Mo3e antigenic determinant after exposure to PMA or compounds that raise intracellular concentrations of cyclic AMP (dibutyryl cyclic AMP or a combination of cholera toxin and IBMX). The expression of Mo3e by these stimulated HL-60 cells coincides with the development of features of monocyte-macrophage differentiation (characteristic morphology, nonspecific esterase activity, and respiratory burst activity). During in vitro monocyte-macrophage differentiation, HL-60 cells become responsive to migration inhibitory factor (MIF); the MIF responsiveness of differentiated HL-60 cells is blocked by anti-Mo3e monoclonal antibody. These findings further support the relationship between the expression of Mo3e and the cellular response to MIF.
Volume Dependent Polymorphonuclear Leukocytes Fractions Isolated by Counterflow Centrifugal Elutriation: PMN Volume Does Not Correlate With PMN Age
Authors: Berkow Roger L , Dodson Robert W
Published: January 2018

We have previously reported that human peripheral blood polymorphonuclear leukocytes can be separated into at least six volume dependent fractions using counterflow centrifugal elutriation (CCE). Larger volume was shown to correlate with increased superoxide release with either the chemotactic peptide F-met-leu-phe (FMLP) or the tumor promotor phorbol myristate acetate (PMA). To help explain these findings we have examined volume dependent PMN fractions (VDPF) for PMN functions felt to be correlated to PMN age, ie, alkaline phosphatase activity, phagocytosis of opsonized particles, adherence to plastic, and directed movement to FMLP and zymosan activated serum. PMN alkaline phosphatase activity was found to be low in the smallest PMNs, increasing in the PMNs of intermediate size, and then decreasing again in the largest PMN functions. A similar relationship was noted for PMN phagocytic activity. No significant difference among VDPF in adherence to plastic or Chemotaxis was found. Furthermore, no difference among VDPF was seen for receptor number or binding affinity of the ligands FMLP or phorbol dibutyrate. Total cellular activity of the NADPH dependent oxidase was, however, 200% greater in the largest compared to the smallest VDPF. Because of the lack of consistent correlation between “age” related PMN function and PMN volume, it is unlikely that PMN volume represents a manifestation of PMN age. It is likely that the increased oxidase activity seen among larger VDPF accounts for the more rapid oxidative burst previously noted.
Cells of the Human Myelomonocytic Line RC-2A Synthesize Tissue Factor-Like Procoagulant and Urokinase-Type Plasminogen Activator
Authors: Karhumäki E , Saravo L , Helin H
Published: January 2018

Cells of the myelomonocytic leukemia cell line RC-2A were studied for their ability to synthesize clotting-promoting and fibrinolytic factors. The cells were observed to generate procoagulant activity (PCA) in readily measurable quantities. Incubation of RC-2A cells with phorbol myristate acetate (PMA; 3 ng/ml) or Phytohamagglutinin (PHA, 10 μg/ ml) for 18 h resulted in a 4-5-fold increase in PCA relative to unstimulated control. The PCA of RC-2A cells was tissue factor-like in that it was dependent on factor VII but not on factors VIII or IX. RC-2A cells also produced plasminogen activator (PA). Secreted PA was approximately 70% of the PA of an identical number of human monocyte-derived macrophages; fresh isolated monocytes synthesized virtually no PA. Compared to macrophages, RC-2A cells secreted less or no PA-inhibitors. Lysates of RC-2A cells contained over three times more PA than lysed macrophages. Stimulation of the cells with lectins (PHA, concanavalin A) or PMA was followed by a modest (2-3-fold) increase in PA. Enzyme immunoassay with antibodies to urokinase (u-PA) or tissue-type PA (t-PA) identified the RC-2A plasminogen activator as being of urokinase type.
Effects of Acute Graft-vs-Host Disease on the Liver of the Brown Norway Rat
Authors: Hightower James A , Earnest David L , Martens Anton C M , Zurcher Chris , Brouwer Adriaan , Blauw Elizabeth , de Leeuw A Margreet , Hagenbeek Anton
Published: January 2018

In this study we examined the effects of acute graft-vs-host disease (aGVHD) on the Brown Norway (BN) rat liver. When clinical signs of the disease appeared, rats were inoculated with fluorescent latex beads and 30 min later nonparenchymal cells were isolated from the liver. The cells were then analyzed via flow cytometry, histochemistry, and electron microscopy. Flow cytometry demonstrated that 58% of the cells from the 80 ml/min elutriation fraction (normally rich in Kupffer cells) of the non-GVHD liver had high fluorescence intensity compared to 8% in rats with aGVHD. Determination of the cellular composition of the various fractions with electron microscopy confirmed flow cytometry observations in that only 9% of the 80 ml/min elutriation fraction of GVHD livers had peroxidase-positive rough ER and the morphological appearance of macrophages as compared to 60% in the non-GVHD liver. The low percentage of fluorescent-positive Kupffer cells in the 80 ml/min elutriation fraction of the GVHD liver is attributed to a massive lymphocytic invasion of the liver and not necessarily to a defect in the mononuclear phagocyte system.
Macrophage Migration Inhibition Induced by MDP, LPS, PMA, and MIF/MAF: Reversal by Macrophage Migration Enhancement Factor (MEF), L-Fucose, L-Fucosyl BSA, D-Mannose, and D-Mannosyl BSA
Authors: Gordon Mark R , Chida Kingo , Takata Isao , Myrvik Quentin N
Published: January 2018

Our data establish that migration inhibition factor (MIF) and migration enhancement factor (MEF) mutually neutralize the effect of each other in a concentration-dependent manner. The monosaccharides L-fucose and D-mannose were also shown to reverse MIF and additionally to stimulate alveolar macrophage (AM) migration in the absence of MIF. The specific activity of these sugars was increased 200-fold when conjugated to bovine serum albumin (BSA). Macrophage activation is usually observed concurrently with migration inhibition when macrophages are incubated with MIF preparations. Migration inhibition occurred also when AM were incubated in the presence of known metabolic activators (MDP, PMA, and LPS). It was found that L-fucose, D-mannose, L-fucosyl BSA, and D-mannosyl BSA could reverse migration inhibition caused by MIF as well as by these metabolic activators. These observations suggest that reversal of MIF by L-fucose is unexplained solely on the basis that L-fucose is functioning as a competitive inhibitor; instead, they suggest that MEF and the above sugars and their conjugates stimulate AM through a receptor system different from the MIF receptor. These observations support the concept that MEF is an important macrophage modulator in CMI responses.
Role of Colony-Stimulating Activity in Antitumor Activity of Lactobacillus casei in Mice
Authors: Shimizu Takako , Nomoto Koji , Yokokura Teruo , Mutai Masahiko
Published: January 2018

Wide-ranging differences were observed between the antitumor activities of 23 lactoba-cilli (13 species; 23 strains) and their capacities to elevate the level of serum colony-stimulating activity (CSA) by intraperitoneal administration in mice, and a good correlation existed between the two activities. The mechanism of enhanced production of CSA by administration of Lactobacillus casei YIT 9018 (LC 9018), one of the bacteria that had the strongest activities, and the role of CSA in antitumor activity of LC 9018 were studied. Colony-stimulating activity in the washing fluid from the peritoneal cavity of mice that had been administered LC 9018 intraperitoneally was elevated at 3 to 24 h after the injection, and CSA was also detected at elevated levels in the serum of the mice 6 to 12 h after injection. The cells responsible for the production of CSA after stimulation with LC 9018 seem to be the resident macrophages at the site of administration, because the resident macrophages of mice lavaged 1 h after an intraperitoneal administration of LC 9018 released CSA when they were cultured in vitro. Moreover, resident peritoneal macrophages of normal mice cultured with LC 9018 in vitro also produced CSA. Similar results were obtained with athymic nude mice, and the CSA-inducing activity of LC 9018 was diminished in the mice pretreated with carrageenan, which is selectively toxic to mature macrophages. Bone marrow cells matured to macrophages and polymorphonuclear cells by culture with the CSA induced by LC 9018 for 7 days. These matured macrophages showed strong antitumor activity both in vivo and in vitro. These results suggest that CSA plays important roles in the antitumor activity of LC 9018: it enhances not only the multiplication of committed precursor cells for macrophages and polymorphonuclear cells, but also the functional maturation of the precursor cells for macrophages which serve as potent effectors for tumor cells.
Protein Phenotypes of Mouse Macrophages Activated In Vivo for Tumor Cell Killing
Authors: MacKay Robert J , Russell Stephen W
Published: January 2018

We have shown previously that the cellular proteins 47b and 71/73 could be used to construct phenotypes that distinguish among bone marrow culture-derived (BMCD) macrophages that were either unstimulated or primed by gamma interferon or fully activated for tumor cell killing by gamma interferon in combination with lipopolysaccha-ride (LPS). In the present study we examined in vivo-derived correlates for each of these stages of macrophage activation and found that the same protein phenotypes held true: both p47b and p71/73 were expressed by cytolytic peritoneal macrophages, including macrophages from a tumoral effusion, whereas macrophages primed in vivo by the intraperitoneal injection of either concanavalin A or methyl vinyl ether copolymer II expressed p47b but lacked p71/73. Neither resident nor inflammatory macrophage populations expressed p47b, and acute inflammatory macrophages, like unstimulated BMCD macrophages, expressed little or no p71/73. By contrast, resident and thioglycollate-elicited macrophages synthesized moderate levels of p71/73. When p71/73 also was expressed, there was a quantitative relationship between p47b expression and cytolytic activity in five different in vivo-activated macrophage populations. The results suggest that, regardless of the macrophage source or stimulus, it may be possible to assess macrophage activation status by reference to protein phenotypes utilizing p47b and p71/73.
Nisoldepine Inhibits Formyl-Methionyl-Leucyl-Phenylalanine (fMet-Leu-Phe) Receptor-Coupled Calcium Transport in Human Neutrophils
Authors: Williamson Kim C , Tauber Alfred I , Navarro Javier
Published: January 2018

The formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) dependent Ca2+ uptake by human neutrophils consists of at least two components, one of which is sensitive to dihydropyridine derivatives. Inhibition by dihydropyridine derivatives showed the rank order of nisoldepine > nitrendipine > nimodepine > /Bay K 8644. The nisoldepine-sensitive calcium uptake exhibited an ID50 of 1.5 μM and maximal inhibition were observed at 5 μM. Neither calcium efflux or [3H]fMet-Leu-Phe binding was affected by nisoldepine up to 10 μM. The inhibition by nisoldepine was inversely proportional to the extracellular calcium concentration. Unlabeled nisoldepine and other dihydropyridine derivatives displaced the specific binding of [3H]PN 200-110 to human neutrophils. Our data suggest a relationship between dihydropyridine binding and the inhibition of fMet-Leu-Phe-dependent Ca2+ uptake.
Cyclophosphamide-Induced Alterations in Human Monocyte Functions
Authors: McBride W H , Hoon D B , Jung T , Naungayan J , Nizze A , Morton D L
Published: January 2018

This investigation was designed to study the effects of relatively low doses of cyclophosphamide (CY) on monocyte function in patients with surgically resected melanoma. Monocytes taken from patients 3 days after receiving 300 mg, 150 mg, or 75 mg CY/m2 had decreased interleukin-1 (IL-1) production. Production of tumor necrosis factor (TNF)-like molecules by the same monocytes appeared to be enhanced following 300 mg/m2 CY but not after 150 or 75 mg/m2 CY. In vitro studies of the direct effects of CY metabolites (mafosfamide and 4-hydroperoxycyciophosphamide) on human monocytes showed only concomitant decreases in production of IL-1 and TNF-like molecules. This occurred at concentrations that did not obviously affect viability, although monocyte spreading was inhibited. No evidence was obtained for in vitro enhancement of TNF-production. We conclude that CY can affect monocyte function. In vivo it may have both direct effects leading to decreased TNF and IL-1 production and indirect effects through lymphocytic or haematopoietic systems that activate monocytes to enhanced TNF production. The effects are dose-dependent. These CY-induced changes could be responsible in part for some of the alterations in host immunity and tumor resistance that follows administration of the drug.
Tumor Necrosis Factor-α Induces Increased Hydrogen Peroxide Production and Fc Receptor Expression, but Not Increased la Antigen Expression by Peritoneal Macrophages
Authors: Hoffman Maureane , Weinberg J Brice
Published: January 2018

It has recently been shown that tumor necrosis factor (TNF) induces increased la antigen expression on a malignant murine macrophage cell line, and that TNF is synergistic with gamma interferon (IFN) in inducing la expression. This finding raises the possibility that TNF serves as a non-interferon macrophage activating factor in vivo. Since it is known that TNF has different effects on malignant and benign cells, we chose to evaluate the effects of recombinant TNF on primary cultures of murine peritoneal macrophages (MP). Neither human nor murine TNF increased the proportion of MP which expressed la antigen, and TNF actually partially prevented the IFN-induced increase in la. However, culture with TNF activated MP for increased hydrogen peroxide production in response to phorbol myristate acetate (PMA) and antagonized the IFN-induced decrease in the proportion of the MP bearing receptors for the Fc region of IgG2b. TNF and IFN were additive in increasing peroxide production. Both human and murine TNF had the same effects on MP, and MP from C3H/HeN (endotoxin sensitive) and C3H/HeJ (endotoxin resistant) mice responded comparably to TNF and IFN. Our results support the hypothesis that TNF has some macrophage-activating activity, but its effects are selective and not identical with those of IFN.
Dissociation of the 47-Kilodalton Protein Phosphorylation From Degranulation and Superoxide Production in Neutrophils
Authors: Sha’afi R I , Molski T F P , Gomez-Cambronero J , Huang C -K
Published: January 2018

The aim for the present studies is to examine the relationship between the phosphorylation of the 47-kDa protein and some neutrophil responses such as degranulation, the synergistic effect of PMA on calcium ionophore-induced degranulation, superoxide generation, and the priming of the oxidative burst produced by the chemotactic factor fMet-Leu-Phe and phorbol 12-myristate 13-acetate (PMA).

Incubation of neutrophils with the protein kinase inhibitor 1-(5-isoquinoline-sulfonyl)-2-methylplperazine (H-7) inhibits the phosphorylation of the 47-kDa protein produced by PMA and fMet-Leu-Phe but does not affect lysozyme release induced by the same stimuli or fMet-Leu-Phe-induced N-acetyl-β-glucosaminidase release. Furthermore, the synergistic effect of PMA on the A23187-induced degranulation is not inhibited by H-7. Also, pretreatment of the cells with H-7 inhibits superoxide production produced by PMA but not by fMet-Leu-Phe. The inhibitory effect of H-7 is more pronounced on the rate than on the extent of PMA-induced superoxide release. On the other hand, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, HA-1004, a less potent protein kinase inhibitor, has no Inhibitory effect on superoxide generation produced by fMet-Leu-Phe or PMA. In the case of superoxide production, the addition of low concentrations of PMA to rabbit neutrophils primes these cells to the subsequent stimulation by fMet-Leu-Phe, dramatically increasing the effect. Conversely, low concentrations of fMet-Leu-Phe prime the cells to PMA stimulation. The stimulation by PMA of cells primed with fMet-Leu-Phe is inhibited by H-7. Moreover, the priming effect by PMA is also inhibited by H-7. This inhibition is less pronounced at a low concentration of PMA. On the other hand, in the case of human neutrophils, the priming effect of PMA is not inhibited by H-7. These results suggest several points. First, phosphorylation of the protein identified on two-dimensional gel electrophoresis as having a molecular weight of 47 kDa and PI of 4.9 is not necessary for degranulation produced by either PMA or fMet-Leu-Phe. Second, the phosphorylation of the 47-kDa protein is not necessary for superoxide generation, at least in the case of fMet-Leu-Phe and possibly for other stimuli. Third, in rabbit neutrophils, both the priming and stimulation of superoxide production with PMA are inhibited by H-7. In human neutrophils, the priming by PMA is not affected by H-7. In the case of human neutrophils, it is reasonable to conclude that the priming of the oxidative burst by PMA does not appear to be mediated by the phosphorylation of the 47-kDa protein.
Hepatocyte Injury During Post-Operative Sepsis: Activated Neutrophils as Potential Mediators
Authors: Holman John M , Saba Thomas M
Published: January 2018

We evaluated the concept that the vascular entrance of both bacterial and nonbacterial participate material could lead to hepatic parenchyma) cell injury, either due to post-phagocytic Kupffer cell activity or the margination of activated leukocytes in the liver. Injection of denatured, collagen-coated particles as well as heat-killed bacteria were used as particulate challenges. Hepatic parenchymal cell injury in vivo during postoperative sepsis was evaluated by plasma aspartate aminotransferase (AST) and ornithine carbamyl transferase (OCT) enzyme levels over 3-72 h. AST and OCT levels elevated following either laparotomy plus cecal ligation (mild sepsis) or laparotomy plus cecal ligation with puncture (severe sepsis), with the peak level at 24 h. In addition, the direct intravenous injection of either nonbacterial foreign particles or heat-killed Pseudomonas aeruginosa into normal rats also produced a dose-dependent elevation of AST and OCT. The plasma level of either AST or OCT actually increased 350-400% after injection of the non-bacterial particles. A similar dose related elevation in enzymes followed the intravenous injection of heat-killed Pseudomonas. To differentiate the potential contribution of activated hepatic Kupffer celts versus activated marginated neutrophils to the in vivo hepatic injury, we determined the release of the hepatic specific enzyme OCT by cultured hepatic parenchymal cells when they were exposed to isolated Kupffer cells or isolated PMNs that were activated by exposure to dead bacteria. Bacteria alone when added to cultured hepatocytes did not induce significant OCT release. In contrast, activated PMNs but not Kupffer cells induced a significant (p < 0.05) release of OCT from parenchymal cells into the culture media. Thus, in vivo transient hepatic parenchymal cell injury with post-operative sepsis may be mediated by the margination of activated PMNs in the liver.
Characterization of the Natural Suppressor Cell Population in Adult Rat Bone Marrow
Authors: Noga Stephen J , Wagner John E , Horwitz Louis R , Donnenberg Albert D , Santos George W , Hess Allan D
Published: January 2018

Natural suppressor cell activity (NSCA) has been ascribed to a subset of cells present in human and murine hematopoietic tissues which can suppress a variety of lymphocyte responses without MHC restriction. We investigated NSCA in lymphocyte-depleted rat bone marrow (BM) which is used as a model for prevention of graft vs host disease (GVHD) following allogeneic BM transplantation (BMT). The T-cell depleted fraction obtained after elutriation contained higher levels of NSCA than the unseparated BM. Further separation of this graft fraction by discontinuous Percoll gradient centrifugation revealed high levels of radiosensitive NSCA in the low density (< 1.070) fraction which represented 0.5% of the original BM population. These cells were of blast morphology, stained intensely with a dansylated derivative of cyclosporine A (dans CsA) and weakly expressed macrophage/granulocyte antigens and non-specific esterase (NSE). These cells were initially non-adherent but proliferated in culture to produce intensely NSE positive, adherent, phagocytic cells of macrophage morphology. We conclude that the highly suppresses, radiosensitive cell present in rat BM may be of early progenitor or monocyte lineage. The grafting of natural suppressor (NS) cells and progenitor cells may affect graft/host immunoregulation and their characterization may provide insight into GVH biology and graft rejection.
Effect of Lead on Macrophage Function
Authors: Kowolenko Michael , Tracy Leigh , Mudzinski Stanley , Lawrence David A
Published: January 2018

Lead (Pb) has been shown to alter various parameters of immune function such as host resistance and antibody formation. In addition, various heavy metals have been implicated as inducers of autoimmunity. In these experiments, macrophages, isolated from the peritoneal cavity of mice exposed to various doses of lead in vivo as well as cells exposed in vitro were tested for the following immunologic parameters: phagocytosis, antigen presentation, interleukin 1 production, and their ability to stimulate the autologous mixed lymphocyte reaction (AMLR). The results obtained indicate that Pb appears to alter the ability of macrophages to present antigen by enhancing the AMLR while having no effect on phagocytosis or IL-1 production. These data suggest that Pb may interfere with antigen-specific interactions between macrophages and T cells.
An Abnormal Calcium Uptake Pump in Chediak-Higashi Neutrophil Lysosomes
Authors: Styrt Barbara , Pollack Charles R , Klempner Mark S
Published: January 2018

Calcium is mobilized from intracellular stores during phagocyte activation and appears to be involved in stimulus-response coupling in these and other cell types. Because the lysosome is a calcium-sequestering organelle in the neutrophil and abnormal lysosome morphology is associated with defective neutrophil function in Chediak-Higashi syndrome (CHS), we examined ATP-dependent calcium uptake in neutrophil lysosomes from the beige mouse model of CHS. We present findings indicating that CHS lysosomes have an enhanced capacity for ATP-dependent calcium uptake relative to control lysosomes. Kinetic analysis showed differences in Vmax and in the Km for both ATP and calcium, suggesting that both the number of lysosomal calcium uptake pumps and their substrate affinity may be altered in CHS. We conclude that a genetically determined abnormality of a subcellular calcium transport system may contribute to the structural and functional defects of CHS cells.
An Improved Method for the Isolation of Eosinophilic Granulocytes From Peripheral Blood of Normal Individuals
Authors: Koenderman L , Kok P T M , Hamelink M L , Verhoeven A J , Bruijnzeel P L B
Published: January 2018

A simple and improved procedure is described for the isolation of human eosinophils from normal individuals with about 2% eosinophils in their peripheral blood. This method comprises a preincubation of a mixed granulocyte preparation with 10 nM fMLP for 10 min at 37° followed by a one-step density centrifugation on isotonic Percoll. The recovery of eosinophils is 49 ± 4% at 89 ± 4% purity. Because of the relatively high rate of recovery, it is now possible to isolate eosinophils from blood samples as small as 20 ml.

Because treatment with fMLP may alter the functional activity of the eosinophils, the following metabolic functions were tested: changes in cytosolic free Ca2+, oxygen consumption, chemiluminescence, chemotaxis, and leukotriene C4 formation. We found that 10 nM fMLP does not activate eosinophils in these assays, whereas 1 μM fMLP does (with the exception of chemotaxis). Furthermore, pretreatment of eosinophils with 10 nM fMLP did not influence the response to other stimuli in these assays. The usefulness of this method was evaluated by comparing it with three other previously described procedures. In our hands, only the method presented here enabled us to isolate eosinophils from normal individuals with about 2% eosinophils in their peripheral blood.
Inhibition of Neutrophil Functions by Platelets and Platelet-Derived Products: Description of Multiple Inhibitory Properties
Authors: McGarrity S T , Hyers T M , Webster R O
Published: January 2018

The effect of platelets and their products on in vitro responses of human neutrophils to f-met-leu-phe (fMLP) and phorbol myristate acetate (PMA) was studied. Platelets produced a concentration-dependent inhibition of neutrophil superoxide anion (O2-) generation with maximum inhibition of the response to fMLP approaching 42.2 ± 5.4% and that to PMA approaching 85.9 ± 3.4%. Supernates of thrombin-activated platelets produced maximal inhibition of neutrophil O2- generation in response to fMLP (66.3 ± 4.2%) at a ratio of 10 platelet equivalents (PEQ) to 1 neutrophil but failed to affect the cells’ response to PMA. In contrast, lysate of sonicated platelets Inhibited neutrophil O2- generation in response to both fMLP (59.6 ± 2.0%) and PMA (90.1 ± 1.3 %). Biochemical characterization of platelet lysate identified at least two stimulus-specific inhibitory activities which differ in size, thermal stability and enzyme sensitivity. Platelet lysate also maximally inhibited neutrophil degranulation and chemotaxis at a ratio of 20 PEQ to 1 neutrophil. Our findings indicate that physiologically achievable levels of platelets or their products modulate the response of neutrophils in a stimulus-specific manner and are thereby capable of potentially limiting tissue damage which accompanies neutrophil activation during inflammation.
Induction of Adenosine Deaminase and 5′ Nucleotidase Activity in Cultured Human Blood Monocytes and Monocytic Leukemia (THP-1) Cells by Differentiating Agents
Authors: Murray James L , Mehta Kapil , Lopez-Berestein Gabriel
Published: January 2018

The in vitro effect of short-term culture as well as the effect of retinol (ROH), retinoic acid (RA), muramyl dipeptide ([Abu']MDP), lipopolysaccharide (LPS), and gamma interferon (IFN-γ) on the induction of the purine metabolic enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5′nucleotidase (5NT) in human peripheral blood monocytes (HPBM) was examined. HPBM isolated by centrifugal elutriation were cultured for up to 96 h. Following an initial time lag of 24 h, mean ADA activity from seven separate experiments as measured in nmoles/106 cells/h increased from a baseline of 31.3 ± 9.3 to 57.8 ± 16.4 (P < 0.005) at 72 h and to 72 ± 21.5 (P < .025) by 96 h. 5NT activity increased from a baseline of 2.2 ± 0.9 to a maximum of 44 ± 10.1 by 72 h and then declined to 29 ± 18 (P < 0.005) by 96 h, while no significant change in PNP activity was observed. HPBM incubated for 3 d with optimal concentrations of LPS, RA, and IFN-γ had increases in ADA and 5NT activity ranging from three- to 10-fold compared to HPBM cultured in media alone, whereas no effect was observed with ROH and [Abu']MDP. RA, but not ROH, significantly enhanced ADA activity in a monocytic leukemia cell (THP-1) line. Addition of RA or the tumor promoter, phorbol 12-myristic 13-acetate (PMA), to HPBM or THP-1 cells resulted in significant increases in 5NT activity with opposite effects on ADA activity. These findings suggest that the biological mechanisms associated with differentiation in normal and malignant monocytes seem to be related and that the sequence and degree to which the various differentiation agents induce the enzyme elevations are also related to the mechanisms of activation/differentiation.