bluuebunny/crossref_metadata_2025 huggingface dataset:
Different Calcium and Oxidative Metabolic Responses in Human Blood Monocytes During Exposure to Various Agonists
Authors: Kemmerich Bernd , Pennington James E
Published: January 2018

It has become increasingly apparent that certain agonists of phagocyte cells exert their effects by using calcium as an intracellular messenger. Most studies have employed neutrophils for such analyses. We have investigated the requirement of calcium for respiratory burst activation in human blood monocytes by using three triggering agents, concanavalin A (Con A), N-formyl-methionyl-leucil-phenylalanine (FMLP), and phorbol myristate acetate (PMA). There was no overall correlation between the maximal extent of transient rise in cytosolic-free calcium (FMLP>Con A>PMA) and superoxide (O2-) release (PMA > Con A > FMLP). PMA, as the most effective agonist of respiratory burst activity, neither Increased Ionized cytosolic calcium [Ca2+]i nor plasma membrane 45Ca2+ permeability. FMLP evoked increments in [Ca2+]i occurred at lower concentrations than did respiratory burst activation, whereas with Con A a rise in [Ca2+]i and Of release were parallel. Intracellular Ca2+ release into cytosol was blocked by 8-(N,N-diethyl-amino)octyl-3,4f5-trimethoxy benzoate, HCl (TMB-8). This caused a parallel inhibition of Con A or FMLP elicited [Ca2+]i transients and Of release, while burst activity stimulated by PMA was unaffected. Our data thus suggest that a transient rise in [Ca2+]i is essential for Con A and FMLP activation In human blood monocytes. In addition to the calcium signal, other intracellular messengers are required for FMLP stimulation. Respiratory burst activation by PMA appears to act independently of [Ca2+]i transients.
Flow Cytometric Analysis of Porcine Peripheral Blood Leukocytes Infected With Pseudorabies Virus
Authors: Wang Fun-In , Pang Victor F , Hahn Edwin C
Published: January 2018

The susceptibility of fractionated porcine peripheral blood leukocytes (PBL) to pseudorabies virus (PRV) was studied by flow cytometry and defined by viral antigen expression. Viral antigens on the surface of infected cells and cell viability were evaluated by forward angle light scatter (FALS), 90-degree light scatter (90LS), green fluorescence (FITC-anti-PRV), and red fluorescence (propidium iodide). Approximately 10% of infected mononuclear cells from healthy pigs expressed cell-surface PRV antigen. Cell-surface fluorescence and cell type were confirmed by sorting live positive cells for microscopy. In sorted positive samples, the lymphocyte versus monocyte ratio was approximately 50%:50%, defined by morphology. Positive lymphocytes represent 5.75% of total mononuclear cells. When cells were stimulated with phytohemagglutinin (PHA) and lipopolysaccharide (LPS) before infection, mitogen-stimulated T-lymphoblasts showed increased susceptibility to PRV (40.7% positive) and died of infection. Monocytes, particularly adherent monocytes, were highly susceptible (40% to 71.4% positive). Granulocytes appeared to be refractory. The relative susceptibility of various PBL populations was compared by normalizing lymphocyte susceptibility to 1 as follows: resting total lymphocytes (1); B-lymphocytes (0.67); T-lymphoblasts (7.08); total monocytes (4.27); adherent cells (4.03 to 10.88); adherent monocytes (6.95 to 12.42); granulocytes (0.24). These findings suggest a possible mechanism by which PRV could have an immunosuppressive effect as well as a pathway for dissemination of PRV.
Synergy Between Tumour Necrosis Factorα and Interleukin-1 in the Induction of Polymorphonuclear Leukocyte Migration During Inflammation
Authors: Wankowicz Zbigniew , Megyeri Pal , Issekutz Andrew
Published: January 2018

Endotoxin is a potent inflammatory stimulus and induces polymorphonuclear leukocyte (PMNL) infiltration into tissues. Macrophage (MΦ) derived IL-1 has been proposed as a mediator of this response. TNFα is also produced by MΦs in response to endotoxin and both IL-1 and TNF enhance PMNL adhesion to vascular endothelium in vitro. We investigated the activity of recombinant human IL-1α, IL-1β, and TNFα in inducing PMNL infiltration into the skin of rabbits using a quantitative 51Cr labelled blood leukocyte assay. IL-1α and IL-1β induced progressive PMNL accumulation, the 50% maximal response being induced by ≈ 20 units. In comparison, TNFα even at 100,000 U, induced only mild PMNL accumulations, although IL-1α and TNFα were similarly active in inducing PMNL adherence to human umbilical vein endothelium. The human TNFα preparation was pyrogenic and induced acute, transient neutropenia in rabbits upon i.v. infusion. IL-1α, IL-1β and TNFα are often secreted simultaneously by MΦs, therefore we investigated their action in combination. The combination of IL-1α with IL-1β was nearly additive in inducing PMNL accumulation, i.e., 87% of predicted result based on the sum of the responses to individual components. The combination of TNFα with either IL-1, each in submaximal doses, resulted in 65-125% greater than the additive response. No such effect was observed when these monokines were injected in combination with PMNL chemotactic stimuli. These results indicate a complex interaction between inflammatory monokines in the regulation of PMNL accumulation in vivo.
Loss of Gelatinase in Elicited Peritoneal Neutrophils of Rats
Authors: Morton Mary E , Schneider Donald L
Published: January 2018

In previous studies on the respiratory burst of neutrophils, it was observed that the small, water-soluble quinone, duroquinone, activated the respiratory burst in rat peritoneal neutrophils but not in human blood neutrophils. Here we report studies which indicate that a selective diminution of certain granule enzymes, gelatinase and azocaseinase but not elastase, occurs during elicitation in the rat. Neutrophils circulating in rat blood have about twice the amount of gelatinase and azocaseinase activity as compared to elicited peritoneal cells, and the respiratory burst of circulating cells is stimulated to only about 20% by duroquinone. The significance of these results is considered in terms of activation of the neutrophil respiratory burst.
An Assessment of the Respiratory Burst and Bactericidal Activity of Alveolar Macrophages From Adult and Senescent Mice
Authors: Esposito Anthony L , Clark Carolyn A , Poirier William J
Published: January 2018

To assess the effects of advanced age on the nonspecific antimicrobial activity of resident alveolar macrophages (AM), superoxide anion (O2-) release and the phagocytic and bactericidal capacity of cells from three genetically distinct murine strains were evaluated. In initial experiments, resident AM, obtained by bronchoalveolar lavage of pathogen-free adult female CD-1 mice and studied in suspension, were found to produce O2- spontaneously and in response to phorbol myristate acetate (PMA) snd unopsonized zymosan. Maximum quantities of O2- were released following stimulation with 1 μg/ml PMA and by a particle-to-cell ratio of 100:1 with zymosan; responses to the agonists peaked between 60 and 90 min. Resident AM obtained from pathogen and disease-free senescent (18-26-month-old) female C57BL/6, BALB/c, and DBA/2 mice released significantly more O2- in response to both PMA and zymosan than did cells secured from adult (4-8-month-old) animals. In vivo, the capacity of AM from adult and senescent animals to phagocytose Streptococcus pneumoniae (unencapsulated strain) and Staphylococcus aureus was comparable, and although the cells from the senescent mice tended to be more efficient in their ability to kill internalized bacteria, statistically significant differences between the two groups were not observed. The results of these studies indicate that the enhanced susceptibility of the senescent host to infection of the lower respiratory tract cannot be attributed to age-related changes in the nonspecific antimicrobial activity of resident AM.
LETTER TO THE EDITOR: Macrophages, Myeloperoxidase, and Mycobacterium lepraemurium
Authors: Rojas-Espinosa Oscar
Published: January 2018

In 1978, McKeever et al. [10], studying lepromatous tissues from Mycobacterium leprae-infected armadillos by cytochemical and electronmicroscopic technics, found a subpopulation of macrophages containing peroxisomes (probably monocytes), but not infected with bacilli, and a subpopulation of macrophages lacking peroxisomes and infected with bacilli. Both infected and noninfected macrophages displayed acid phosphatase activity that was more intense and less precisely localized in heavily infected and vacuolated macrophages than in moderately or noninfected cells. Then, in 1983, Gomez Estrada et al [5], looking for the presence of myeloperoxidase (MPO) in histiocytes, Kupffer cells, and PMN leukocytes of nodular lepromatous patients, found abundant M. leprae in the phagocytes of the lesions but they did not find MPO activity within these cells (except in one case of reactional leprosy). They also found that MPO activity was present in Kupffer’s cells and in PMN of patients and controls.
Production of an Eosinophil Chemotactic Lymphokine by a Monocyte-Derived Factor From Patients With
Authors: Philia Hypereosino , Muramoto K , Sakata K , Miyauchi Y , Hirashima M , Hayashi H
Published: January 2018

Peripheral OKT4-positive T lymphocytes from patients with hypereosinophilia spontaneously and selectively produced an eosinophil chemotactic factor (ECF) with chemokinetic activity. The molecular weight of the ECF was about 45,000 to 70,000. A possible mechanism of its spontaneous production by T lymphocytes was analyzed. Culture supernatants of blood monocytes from the patients showed little or no ECF activity, but they had a potency to induce the ECF production from T lymphocytes from normal donors when the cells were stimulated by the supernatants, which suggests that a monocyte-derived soluble factor (MDF) stimulated T lymphocytes to produce an ECF resembling this spontaneously produced ECF from the patients. MDF seemed to be a synthesized protein by the cells. Gel filtration indicated that molecular weight of MDF ranged between 70,000 and 100,000. MDF activity was stable at 56° for 30 min but labile at 80° for 30 min. MDF failed to show detectable IL-1 and IL-2 activity. Furthermore, supernatants of stimulated monocytes by lipopolysacchride or silica particles failed to show ECF-producing activity, whereas they showed evident lymphocyte-activation activity. Neither recombinant IL-1 nor IL-2 had ECF and ECF-producing activity. From the present experiments, it was suggested that MDF was at least partly involved in the induction of ECF production by OKT4-positive T lymphocytes in patients with hypereosinophilia.
Effects of 8-Aminoguanosine on the Toxicity of Guanosine and Deoxyguanosine for Malignant and Normal Lymphoid Cells
Authors: van der Kraan Peter M , van Zandvoort Peter M , De Abreu Ronney A , Bakkeren Jan A J M
Published: January 2018

The toxicity of guanosine and deoxyguanosine in the presence or absence of the purine nucleoside phosphorylase inhibitor, 8-aminoguanosine, for malignant lymphoid cell lines and mitogen-stimulated peripheral blood lymphocytes has been studied. Deoxyguanosine inhibited the proliferation of lymphoid cells more strongly than guanosine. Addition of 100 μM 8-aminoguanosine neither enhanced nor diminished the toxicity of guanosine to the lymphoid cells. Only the toxicity of deoxyguanosine for the leukemic T cell line, MOLT 4, and the leukemic nonBnonT cell line, KM-3, was enhanced by the addition of 100 μM 8-aminoguanosine. These data suggest a possible role of purine nucleoside phosphorylase inhibitors in the treatment of lymphoproliferative disorders of the T-acute lymphoblastic leukemia (ALL) as well as the nonBnonT-ALL subclass.
Long-Term Cultivation of Functional Human Macrophages in Teflon Dishes With Serum-Free Media
Authors: Helinski Ernest H , Bielat Kenneth L , Ovak Geraldine M , Pauly John L
Published: January 2018

Reported herein are the results of studies demonstrating the utility of a chemically defined, serum-free medium designated as AIM-V (GIBCO) for the long-term (>2 weeks) cultivation of functionally-defined human macrophages. The AIM-V medium is a mixture of HEPES-buffered Dulbecco’s Modified Eagle Medium and Ham’s Nutrient Mixture F12 that had been supplemented with purified human albumin, transferrin, insulin, and a proprietary mixture of purified factors. Nonadherent macrophages for serial studies were generated in petri dishes with a Teflon liner that had been seeded with a heterogeneous population of peripheral blood mononuclear cells of healthy human adults. For comparison, cultures were initiated with serum-free medium AIM-V and medium supplemented with AB/Rh+ serum or freshly collected autologous serum. Viability was defined by trypan blue dye, glass adherence, and phagocytosis of yeast. Macrophages were characterized by light microscopy, cytochemistry, and phenotypic analysis. Ultrastructural morphology was defined by scanning electron microscopy. These studies demonstrate that functionally defined human macrophages can be sustained in long-term culture with the use of serum-free medium that has not been augmented with mitogenic stimulants, growth-promoting lymphokines/monokines, or differentiation-inducing agents. Serum-free medium AIM-V, which has been approved for generating lymphokine, (i.e., interteukin-2; IL-2)-activated killer cells (LAK) for IL-2/LAK adoptive immunotherapy modalities, may also prove useful for studies defining and isolating regulatory proteins produced by activated monocytes and macrophages and for generating cytolytic macrophages for different antitumor regimens.
SHORT COMMUNICATION: Use of Serum-Free, Compositionally Defined Medium for Analysis of Macrophage Differentiation In Vitro
Authors: Vogel Stefanie N , Perera Pin Yu , Hogan M Michele , Majde Jeannine A
Published: January 2018

Macrophage differentiation is mediated by the action of a variety of environmental signals, such as cytokines and endotoxin. For in vitro analysis of macrophage differentiation, most studies utilize a basal tissue culture medium supplemented with fetal calf serum (FCS). As the composition of specific components within FCS varies enormously from lot to lot, one can never be certain that the differentiative effects observed in vitro are attributable solely to the exogenous signals provided to the cultures. In this study, primary macrophages were cultured in a basal medium supplemented either with FCS or a compositionally defined supplement, HL-1™, and a spectrum of differentiative functions (i.e., induction of antiviral activity, Fc receptor-mediated phagocytosis, Ia antigen expression, and tumoricidal activity) were measured following stimulation with exogenous signals. The results indicate that the use of serum-free, defined media may provide an important approach to dissect and characterize the differentiative signals which are operative in macrophage activation.
Myotonic Dystrophy: Defective Oxidative Burst of Polymorphonuclear Leukocytes
Authors: Mege J L , Pouget J , Capo C , Andre P , Benoliel A M , Serratrice G , Bongrand P
Published: January 2018

Because myotonic dystrophy (MD) is an autosomal dominant multisystemic disorder affecting plasma membrane, we have studied the oxidative burst of PMNs. The PMA and fMet-Leu-Phe-stimulated superoxide generation is defective in the patient group as compared to controls: the response is both delayed and low. The kinetic parameters of the NADPH oxidase complex are not affected. We have not found any abnormalities in the membrane potential changes. In addition, the cytosolic protein kinase C (PKC) activity of resting PMNs is similar in MD patients and controls, and the translocation of protein kinase C in response to PMA is not impaired. The decrease of the oxidative response of PMNs from MD patients may be related to an abnormality of the environment of the NADPH oxidase.
Establishment and Characterization of Long-Term Cultured Cell Lines of Murine Resident Macrophages
Authors: Lombard Yves , Bartholeyns Jacques , Chokri Mohamed , Illinger Dominique , Hartmann Dominique , Dumont Serge , Kaufmann Stefan H E , Landmann Regine , Loor Francis , Poindron Phillippe
Published: January 2018

Murine resident macrophages can proliferate in vitro when they are grown in coculture on a layer of mesothelial or endothelial type feeder cells. Resident macrophages were obtained from lung expiants of C57BI/6 Ipr/lpr mice and from spleen expiants or peritoneal washing of Balb/c mice; the cells were seeded without further washing. After 3-4 weeks of culture, the macrophages began to proliferate on a confluent layer of feeder cells. The macrophages then could be collected in the fluid phase and reseeded for permanent culture after generation of a new feeder layer. These cells were characterized as macrophages by the following criteria: 1) their morphology, ultrastructure, and adherence properties; 2) more than 90% of the macrophages phagocytized yeasts compared with less than 1% of the feeder cells; 3) the presence of functional Fc and mannose receptors, nonspecific cytoplasmic esterases, and membrane ectoenzymes such as nicotinamide adenine dinucleotide (NAD) glycohydrolase and nucleotide pyrophosphatase; 4) by cytofluorographic phenotype analysis with monoclonal antibodies, characterizing a normal macrophage population (MAC1+, Fcrec +, H-2K+, THY1 -, LYT2 -, L3T4 -). 5) by functional studies proving that the expanded macrophages could function as accessory cells in the induction of lymphocyte proliferation in response to concanavalin A (Con A), that they generated reactive oxygen radicals and that they were cytotoxic for tumor cells. During coculture, growth or activating factors such as macrophage colony-stimulating factor or gamma-interferon were released in the medium. Long-term cultured macrophages had chromosomal abnormalities. Our study suggests that tissue macrophages can proliferate in vitro and hence that it is possible to establish long-term cultured cell lines of macrophages of defined and reproducible characteristics.
Abnormal Macrophage Development in Hodgkin’s Disease: A Morphometric Study
Authors: Sokol R J , Hudson G , James N T , Wales J
Published: January 2018

The development of macrophages from blood monocytes from 22 patients with untreated Hodgkin’s disease and 20 normal subjects has been studied at intervals over a 6-day period of suspension culture in the presence of autologous serum and lymphocytes. Morphometric measurements were made on electron micrographs and the results subjected to multivariate and univariate analyses of variance. The cells from the Hodgkin’s group showed highly significant differences from normal. The whole-cell volume, surface area, and cell membrane excess, as well as the mitochondrial volume and surface area, showed smaller increases over the period of culture, and the normal increase in mitochondrial profile numbers was not seen. It would appear that although the patients’ monocytes transformed into macrophages in culture, their development was seriously deranged.
Comparison of Thymocyte and T Lymphocyte Gangliosides From C3H/HeN and C3H/HeJ Mice
Authors: Yohe Herbert C , Cuny Carole L , Berenson Charles S , Ryan John L
Published: January 2018

Gangliosides have been prepared from resting murine thymocytes and splenic T cells. Profoundly different two-dimensional thin layer chromatography (2D TLC) patterns were observed between these two cell types. Thymocytes contained 28–30 discrete gangliosides of which eight represented major gangliosides. Splenic T lymphocytes from both strains had much simpler patterns, with six to seven major gangliosides and 12–13 minor gangliosides. Computerized analysis of the thymocyte ganglioside patterns between LPS-responder C3H/HeN mice and lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice revealed no significant difference in the major gangliosides. However, with splenic T cell gangliosides, there is a striking difference in the relative proportion of three homologous gangliosides between the two strains. Consistent with previous observations on macrophage gangliosides, the ratio of N-acetylneuraminic acid-containing ganglioside to N-glycolylneuraminic acid-containing ganglioside was higher in both thymocytes and T-cells from the LPS-responder strain. These results show that sialic acid-containing glycolipids from thymocytes and T lymphocytes between endotoxin responder and hyporesponder strains manifest small but significant changes. These differences are present in unstimulated cell populations and may represent a manifestation of the Lps gene.
Interactions Between Endogenous Glucocorticoids and Inflammatory Responses in Normal and Tumor-Bearing Mice: Role of T Cells
Authors: Normann S , Besedovsky H , Schardt M , del Ray A
Published: January 2018

Appropriately stimulated lymphocytes and macrophages produce factors in vitro that increase serum corticosterone levels when injected in vivo. In this study, we used euthymic and congenially athymic mice on a BALB/c background to explore the role of T cells in controlling corticosterone levels and the leukocyte response to inflammation. Adult athymic mice had more intense inflammatory reactions than euthymic mice despite higher basal corticosterone levels. This latter condition may be due to interleukin-1 (IL-1) since macrophages from athymic mice when stimulated in vitro by lipopolysaccharide produced more IL-1 than macrophages from euthymic mice. In response to mitogen stimulation, however, splenocytes from athymic mice produced a factor (not IL-1), which, upon injection, increased corticosterone levels and suppressed inflammation. Production of this factor was enhanced by T cells since splenocyte supernatants from euthymic mice were more potent in eliciting both effects. Evidence for in vivo participation of T cells in regulating corticosterone levels was obtained by tumor transplantation. Injection of syngeneic tumor cells or cell-free tumorous ascites rapidly increased corticosterone levels in euthymic but not athymic mice. Anti-inflammation correlated with increased corticosterone levels but was observed also in athymic mice receiving syngeneic tumor transplants. These studies demonstrate that T cells enhance production of a lymphokine that increases corticosterone levels and are required for the corticosterone response to tumor transplantation. In addition, the data suggest two pathways of anti-inflammation in tumor-bearing hosts: a corticosterone-independent, T cell-independent mechanism and a T cell-dependent mechanism that involves a lymphokine-mediated increase in corticosterone blood levels.
Effect of Fibronectin on Antigen-Induced Lymphoproliferation and Antibody Synthesis in Rats
Authors: Rybski James A , Lause David B , Reese Andy C
Published: January 2018

Macrophages are important positive and negative regulators of both primary and secondary antibody responses, and their activity may, in turn, be controlled by soluble mediators secreted by other cells. Fibronectin is a 440,000 dalton normal constituent of plasma and extracellular membranes that acts through macrophages to inhibit mitogen-and alloantigen-stimulated lymphoproliferation. We examined the effect of Fn on the antigen-stimulated lymphoproliferative and antibody responses in cells from trinitrophenol-derivitized keyhole limpet hemocyanin (TNP-KHL) primed rats. Fn in concentrations equivalent to normal plasma levels inhibited TNP-KLH-stimulated lymphoproliferation by unseparated lymph node leukocytes. When the experiment was repeated using purified lymph node T cells and added thioglycollate-induced peritoneal exudate macrophages or splenic adherent macrophages, Fn alone and TNP-KLH alone stimulated lymphoproliferation, but in combination they were strongly inhibitory. The effect was not due to decreased lymphocyte viability in the presence of both TNP-KLH and Fn. Nor was it due to complexes between TNP-KLH and Fn or to a simple alteration in the kinetics of lymphoproliferation. Fn had to be present with the TNP-KLH within the 1st hour of incubation. If macrophages were coincubated with TNP-KLH and Fn for 24 h, washed, and added to enriched T cells, inhibition was equivalent to that seen with continuous coculture. Similarly, coculture of TNP-KLH and Fn inhibited both total immunoglobulin and TNP-KLH-specific antibody synthesis at optimal concentrations of splenic adherent cells. However, at suboptimal levels of splenic macrophages, the combination was synergistic, stimulating more total immunoglobulin synthesis than either TNP-KLH or Fn alone. These data suggest that the inhibitory effect was dependent upon the concentration and phenotype of macrophages present in culture.
Factors Mediating Lipopolysaccharide-Induced Ganglioside Expression in Murine Peritoneal Macrophages
Authors: Berenson Charles S , Yohe Herbert C , Ryan John L
Published: January 2018

The ganglioside composition of endotoxin-responsive C3H/HeN murine peritoneal macrophages is known to undergo dramatic changes in vivo in response to intraperitoneal lipopolysaccharides (LPS), unlike endotoxin-hyporesponsive C3H/HeJ macrophages. To better investigate the mechanism behind LPS-induced macrophage ganglioside changes, resident C3H/HeN peritoneal macrophages were treated in vitro with 0.1-1.0 μg/ml LPS for 6-96 hr, but showed no differences in membrane ganglioside patterns. Coincubation of macrophages with lymphocytes and treating with LPS again elicited no ganglioside changes. In contrast, interferon γ (IFN-γ)-primed macrophages showed a dramatic shift in intensity of one ganglioside when treated with LPS in vitro; an additional macrophage ganglioside appeared when IFN-γ-primed, LPS-treated macrophages were coincubated with lymphocytes. Ganglioside expression induced in vitro still did not approach the complex changes seen in vivo. However, transplanting C3H/HeN macrophages intraperitoneally into C3H/HeJ mice, followed by administration of intraperitoneal LPS, did reveal striking changes in ganglioside expression that resembled the pattern seen in vivo. Thus, LPS alone does not provide the necessary direct signal to promote macrophage ganglioside change even though it alters macrophage function. IFN-γ appears to be one important mediator; however, complex interactions involving other cytokines or migration of independent populations of mononuclear cells may be required for the full manifestation of LPS-induced ganglioside expression in macrophages.
Production and Characterization of Monoclonal Antibodies to Fcγ 2a-Binding Protein Isolated From the Detergent Lysate of a Murine Macrophagelike Cell Line, P388D1
Authors: Kagami Masaru , Funatsu Yoshiyuki , Suzuki Tsuneo
Published: January 2018

Mybridoma cell lines were produced by fusion of SP2/0 murine myeloma cell line with the spleen cells of Wister rats which were immunized with IgG2a-rbinding protein isolated from the detergent lysate of a murine macrophagelike cell line, P388D1, by affinity chromatography on IgG-Sepharose 4B. A monoclonal clone (designated as 3A2) out of a total of 13 different antibody-secreting cell lines was found to secrete IgG1 class antibodies, which inhibited more than 70% of the binding of radio-iodinated myeloma IgG2a protein to P38SD1 cells. The 3A2 Fab fragments bound specifically to P3S8D1 cells at 4° with a KD of 1.9 × 10-8 M and Bmax of 2.9 × 105 per cell. This Fab fragment also specifically bound to Fcγ 2a receptor (R)-positive T cell line (S49) with a KD of 4.4 × 10-9 M and a Bmax of 1.0 × 104 but did not bind to Fcγ 2a-negative S49 variant cell line, cyc-. The flow cytometric analysis with the use of fluorescein-isothiocyanate-tagged 3A2 F(ab’)2 also showed that this antibody binds to Fcγ 2aR-positive cells, P388D1 and S49, but not to Fcγ 2aR-negatlve cells, cyc-. Monomeric and heat-aggregated IgG2a (13-fold molar excess) inhibited the binding of the radioiodinated 3A2 F(ab’)2 to P388D1 cells by 70 and 49%, respectively, whereas the inhibition by monomelic and heat-aggregated IgG2b was 17 and 39%, respectively; 3A2 F(ab’)2 (100-fold molar excess) inhibited the binding of IgG2a and IgG2b to P38SD1 cells by 90 and 24%, respectively, whereas the inhibition of binding of these IgG to S49 cells was 79 and 49%, respectively. Western blotting analysis showed that 3A2 antibody recognizes a major protein (Mr = 100,000) and a minor component (Mr = 80,000) separated by SDS-PAGE of P388D1 or S49 cell lysates under nonreducing condition, whereas under reducing condition, this antibody recognized a major protein (Mr = 50,000) and two additional minor components (Mr = 40,000 and 35,000). Fcγ 2aR may thus exist at the cell surface as a disulfide linked dimer of a subunit of Mr of 50,000, which could be partially degraded during the isolation to smaller fragments of 40,000 and 35,000 Mr peptides which are still held together by interchain disulfide bond. Furthermore, 3A2 antibody-binding protein isolated from P388D1 cell membrane lysate was found to exhibit casein kinase activity, which was previously found to be associated with IgG2a- but not with lgG2b-binding protein isolated from a similar cell lysate.
Mononuclear Cells From Human Lung Parenchyma Support Antigen-Induced T Lymphocyte Proliferation
Authors: Nicod Laurent P , Lipscomb Mary F , Weissler Jonathan C , Toews Galen B
Published: January 2018

We have previously demonstrated that there is a subpopulation of loosely adherent pulmonary mononuclear cells that can be isolated from minced and enzyme-digested lung tissue with a potent capacity to stimulate allogeneic T lymphocyte proliferation. We now demonstrate that these cells are also capable of stimulating an autologous mixed leukocyte reaction (AMLR) and presenting antigen to autologous T lymphocytes. These loosely adherent mononuclear cells (LAM) were more effective than either alveolar macrophages or monocytes as antigen-presenting cells. Depletion of phagocytic or Fc receptor-positive cells from the LAM population enhanced the stimulation of an reaction AMLR while preserving antigen-induced T lymphocyte proliferation. These results indicate that there are nonphagocytic, Fc receptor-negative accessory cells in human lung parenchyma capable of activating resting T cells in an AMLR and supporting antigen-specific T lymphocyte proliferation. The identity of these cells is uncertain, but the data strongly suggest that the cell is not a classical monocyte-derived macrophage. These antigen-presenting cells may be critical in the initiation of immune responses within the lung.
Inhibition by Monoclonal Anticomplement Receptor Type 1 on Interactions Between Senescent Human Red Blood Cells and Monocytic-Macrophagic Cells
Authors: Gattegno L , Saffar L , Vaysse J
Published: January 2018

The effect of different murine monoclonal antibodies (Mab) specific for the glycoprotein complement receptor type 1 (CR1), type 2 (CR2), and type 3 (CR3) on the adhesion to and on the phagocytosis of human senescent red blood cells (S-RBC) by monocytes or by monocyte-derived macrophages (Mø) was investigated.

Murine Mab anti-CR3 (anti-Leu 15 and OKM1) were found to inhibit, in the same order of magnitude, on one hand, the Fc receptors (FcR)-dependent rosetting and phagocytosis, and, on the other hand, the S-RBC rosetting and phagocytosis by adherent monocytes. Thus, the specific involvement of the CR3 epitopes recognized by Mab anti-Leu 15 or by OKM1 in the interactions between S-RBC and monocyte/macrophage could not be demonstrated.

Murine Mab anti-CR1 was found to be a significant inhibitor of binding to and of phagocytosis of S-RBC (but not of young [Y] RBC) by monocytes or Mø, whereas Mab OKM5 carrying the same isotype as Mab anti-CR1, but a different specificity, was devoid of any significant inhibitory effect Furthermore, Y-RBC or S-RBC opsonized with Mab anti-CR1 did not form FcR-dependent rosettes and were not internalized by monocytes; in addition, preincubation of phagocytes with Mab anti-CR1 did not inhibit FcR-dependent resetting and phagocytosis. These results suggest that the effect of anti-CR1 is mediated through a specific binding to CR1 and not through an FcR blockade.

As the role of specifically bound IgG on phagocytosis of human S-RBC by macrophages has previously been demonstrated by several authors, the present study suggests that rnonocyte-macrophage complement receptor type 1 may act in synergy with Fc receptors in the recognition of S-RBC by macrophages. It is shown in addition that the tripeptide Arg-Gly-Asp, identical to the region of iC3b recognized by CR3 and by several adhesion-promoting receptors that are structurally similar to CR3, such as fibronectin or vitronectin, is a significant inhibitor of the binding to and the phagocytosis of S-RBC by monocytic-macrophagic cells.
Interleukin-2 Augmentation of Interleukin-1 and Prostaglandin E2 Production
Authors: Tilden Arabella B , Dunlap Nancy E
Published: January 2018

Some of the major side effects of interleukin-2 (IL-2) therapy in the treatment of malignancies may be related to increased interleukin-1 (IL-1) and/or prostaglandin E2 (PGE2) production. We examined the effect of recombinant (rIL-2) on the in vitro production of IL-1β and PGE2 by unstimulated and LPS-activated human blood mononuclear cells (PBMC). We also compared the effect of rIL-2 on IL-1β production by adherent and nonadherent blood mononuclear cell populations. Cultures of PBMC (5 × 106/ml) were incubated for 24 hr in media only (control), 1,000 U/ml rIL-2, 2 μg/ml LPS, or both LPS and rIL-2. Supematants obtained from these cultures were analyzed for levels of IL-1β and PGE2 by radioimmunoassays. The addition of rIL-2 caused an increase in IL-1β production in 13 of 13 control PBMC cultures and in 11 of 13 LPS-stimulated cultures, which were significant increases as determined by paired f tests. When PBMC were fractionated into plastic adherent and nonadherent populations, the rIL-2 induced increases in IL-1β production were more consistent in control (six of seven cases) and LPS (seven of seven cases) cultures of plastic nonadherent cells than in control (three of seven cases) and LPS (four of seven cases) cultures of plastic adherent cells. Recombinant IL-2 did not increase PGE2 production in control PBMC cultures (none of four cases), but did so in LPS-stimulated PBMC cultures (three of four cases)). These results suggest that rIL-2 may increase IL-1 production in vivo and thus possibly account for some of the side effects of this therapy.
Guinea Pig Lung Eosinophil: Purification and Prostaglandin Production
Authors: Hirata K , Pelé J P , Robidoux C , Sirois P
Published: January 2018

Guinea pig lung eosinophils have been purified from bronchoalveolar lavage (BAL) fluid, and the production of prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-PGF1α was measured after stimulation with phorbol myrlstate acetate (PMA), platelet-activating factor (PAF), and formyl-methionyl-leucyl-phenylalanine (fMLP). A combination of plating and discontinuous Percoll gradient centrifugation was used to purify eosinophils. The purity of eosinophils in fraction E (interface between Percoll density 1.057-1.068) was around 96%, and the viability was 99%, with a mean yield of 2.7 × 106 cells per guinea pig. Similarly, in fraction D (interface between Percoll density 1.047-1.057), the mean purity of eosinophils was 76% and the viability of cells was 99%, with a mean yield of 1.1 × 106 cells per guinea pig. Purified eosinophils produced TXB2 predominantly after stimulation with PMA, fMLP, and PAF. The 6-keto-PGF1α was slightly increased in cell supematants after stimulation with PMA and fMLP, but PGE2 was not elevated with any stimulus. The highly purified eosinophils of fraction E generated amounts of TXB2 and 6-keto-PGF1α similar to the amount generated by eosinophils contaminated with some macrophages (fraction D). These results suggest a role for eosinophils in the production of TXA2 by guinea pig lung.
Downregulation of Tumor Necrosis Factor Expression in the Human Mono-Mac-6 Cell Line by Lipopolysaccharide
Authors: Haas Jürgen G , Thiel Corinna , Blömer Kathi , Weiss Elisabeth H , Riethmüller Gert , Löms Ziegler-Heitbrock H W
Published: January 2018

Mono-Mac-6 cells, but not U937 cells, can be induced to rapidly express tumor necrosis factor (TNF) mRNA and protein when triggered with lipopolysaccharide (LPS) at 1 μg/ml. Preincubation of the cells for 3 d with low amounts of LPS (10 ng/ml) results in nearly complete suppression of TNF secretion. This downregulation appears to occur at the pretranslational level since specific mRNA is virtually undetectable under these conditions. By contrast, the same preincubation with 10 ng/ml LPS results in enhanced phagocytosis (28.6-67.2% for Staphylococcus aureus), demonstrating that not all monocyte functions are suppressed. While these results show that only stringent exclusion of LPS from culture media allows for induction of TNF in the Mono-Mac-6 cell line, the pronounced effect of LPS preincubation may also provide a suitable model with which to study the mechanisms of LPS-induced desensitization.
Human Sera and Culture Supernatants From Human Tumors and Diploid Fetal Fibroblasts Suppress Tumor Necrosis Factor Secretion In Vitro
Authors: Scuderi Philip , Rippe Richard A , Raitano Arthur B , Rybski James
Published: January 2018

Human sera and culture supernatants from human tumors and diploid fetal fibroblasts suppressed peripheral blood leukocyte secretion of tumor necrosis factor (TNF). The suppressive activities of all three fluids had similar characteristics: each was heat and acid stable, removed by adsorption on immobilized lectins, and abrogated the stimulatory effect of interferon-γ. Inhibition of leukocyte TNF secretion was observed only when either serum or conditioned medium was added to leukocytes at the initiation of culture; delaying the addition by 2 h failed to suppress cytokine secretion. Suppression by all fluids was also found to be reversible by washing cells free of suppressive activity. Although serum, tumor, and fibroblast culture supernatants inhibited cytokine secretion, they failed to alter the cytotoxic activity of recombinant human TNF on murine L929 cells. This study suggests that factors which can inhibit TNF secretion are present in human blood and are secreted by both fibroblasts and tumor cells. These suppressive factors may play an important role in the regulation of TNF secretion and cytokine homeostasis.
Effect of Granulocyte-Macrophage Colony-Stimulating Factor on Superoxide Production in Cytoplasts and Intact Human Neutrophils: Role of Protein Kinase and G-Proteins
Authors: Mege Jean-Louis , Gomez-Cambronero Julian , Molski Thaddeus F P , Becker Elmer L , Sha'afi Ramadan I
Published: January 2018

Granulocyte-macrophage colony-stimulating factor, GM-CSF, potentiates superoxide generation produced by human neutrophils stimulated with fMet-Leu-Phe and platelet-activating factor, PAF, but not by phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan. The potentiation is greatest in fMet-Leu-Phe-stimulated cells. This indicates that the actions of only certain receptors are potentiated by GM-CSF. Incubation of the cells with the protein kinase inhibitor H-7 or with the protein synthesis inhibitor cyclohexamide before the addition of GM-CSF does not affect the observed potentiation. The rationales behind these studies are to examine the roles of protein kinase C and protein synthesis in the action of GM-CSF. The data suggest that neither protein kinase C nor protein synthesis is necessary for GM-CSF action. On the other hand, no potentiation can be seen in the presence of cytochalasin B. Unlike intact cells, GM-CSF does not enhance superoxide production by cytoplasts stimulated with fMet-Leu-Phe. The rationale behind the use of cytoplasts is to examine the role of granules and/or nucleus in GM-CSF action, and the data indicate that one or more of these two components is necessary for the priming effect of GM-CSF. The amount of actin associated with the cytoskeleton under control or fMet-Leu-Phe-stimulated condition is the same in normal and GM-CSF-treated human neutrophils. Botulinum D toxin ADP-ribosylates a protein with a moleuclar weight of 22 kDa This ribosylation is reduced in homogenates obtained from cells pretreated with botulinum D toxin or GM-CSF. Botulinum D toxin does not affect the basal or the fMet-Leu-Phe-induced rise in the intracellular concentration of free calcium in human neutrophils. GM-CSF also increases the rise in intracellular concentration of free calcium in human neutrophils stimulated with PAF or fMet-Leu-Phe. The increases are inhibited by petussis toxin, Several important conclusion can be drawn from these data. 1) GM-CSF potentiates the rise in Ca2+, produced by PAF and fMet-Leu-Phe, and these potentiations are inhibited in pertussis-toxin-treated cells. 2) GM-CSF does not prime cytoplasts to stimulation by fMet-Leu-Phe. This suggests that the granules and/or nucleus are necessary for the priming action. 3) The priming by GM-CSF is not mediated by the H-7-sensitive protein kinase C, botulinum D-sensitive G-protein, or protein synthesis.
Monoclonal Antibodies (G1.4, D7.5) Induce Secretion of Lytic Factors From Natural Killer and T Lymphocytes
Authors: Stein-Streilein Joan , Guffee Judy
Published: January 2018

Monoclonal antibodies were made by standard procedures using mononuclear cells isolated from hamster lung tissue to immunize the mice. The Moabs (G1.4, D7.5), secreted by the selected hybridomas, were isotyped as IgG2a Kappa and were lytic in the presence of complement for 20% lymphocytes from lymph nodes or lung tissue; 40-50% of lymphocytes from spleen or bone marrow; and 70-80% of thymocytes or nylon wool nonadherent lung or lymph node lymphocytes. Functional studies showed that prior incubation of either Moab with enriched natural killer (NK) and T lymphocyte subpopulations did not interfere with effector: target conjugate formation but did block the ability of the effector cells to lyse NK-sensitive targets. We conclude that the Moabs bind to a molecule(s) on NK and T lymphocytes. Further, the preincubation medium acquired the capacity to lyse preferentially NK-sensitive targets but not NK-lnsensitive targets. Interestingly, the Moabs were able to induce secretion of lytic factors from thymocytes and to induce resting peripheral T lymphocytes to acquire NK activity and release lytic factors. The ability of the Moab to signal release of lytic factors was retained even when monovalent Fab fragments were used; therefore, the Moabs initiate secretion by mechanisms that do not apparently involve crosslinking or Fc receptor interaction. G1.4 and D7.5 Moabs were functionally and antigenically crossreactive with human, rat, and hamster lymphocytes but not with mouse lymphocytes. Biochemical and immunochemical analysis showed that the G1.4 and D7.5 antigen on thymus cells and lung mononuclear cells has an apparent molecular weight of 27 kD. Although it is unlikely that there is an universal mechanism for secretion used by cells in general, it is interesting that Moab treatment of three different subpopulation of lymphocytes induced secretion of lytic factors and suggests that under physiologic conditions a common molecule might participate in induction of the secretion pathway. These studies lead to the hypotheses that 1) NK and T lymphocytes share a cell surface molecule that participates in signals that initiate secretion and that 2) the expression of the G1.4, D7.5 antigens precedes thymic differentiation.
Dexamethasone Modulation of In Vivo Effects of Endotoxin, Tumor Necrosis Factor, and Interleukin-1 on Liver Cytochrome P-450, Plasma Fibrinogen, and Serum Iron
Authors: Bertini Riccardo , Bianchi Marina , Erroi Annalaura , Villa Pia , Ghezzi Pietro
Published: January 2018

Treatment of mice with endotoxin (lipopolysaccharide, LPS) and the two LPS-induced monokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), caused a depression of liver cytochrome P-450 and related drug-metabolizing enzymes, as well as other acute-phase changes including increase in plasma fibrinogen levels and hypoferremia. However, only IL-1, not TNF or LPS, depressed cytochrome P-450 in cultured hepatocytes, suggesting that the effect of TNF in vivo might be mediated by a second mediator. TNF-or LPS-stimulated monocytes released a factor capable of depressing cytochrome P-450 in cultured hepatocytes. This factor was inhibited by anti-IL-1 antiserum, and its synthesis, like that of IL-1, was inhibited by dexamethasone (DEX). Pretreatment of mice with DEX protected against the depression of liver cytochrome P-450 by LPS or TNF but not by IL-1, suggesting that IL-1 directly depresses cytochrome P-450 and that DEX acts by inhibiting IL-1 synthesis in vivo induced by LPS or TNF. However, DEX did not inhibit two other effects of LPS and TNF in vivo: increase of plasma fibrinogen levels and decrease of plasma iron, suggesting that these might not be mediated by IL-1. Therefore, the effect of DEX in vivo, although supporting the hypothesis that depression of liver cytochrome P-450 by LPS and TNF is mediated by IL-1, indicates the existence of IL-1-independent pathways in the acute-phase response.
Phagocytosis and Killing of Cryptococcus neoformans by Rat Alveolar Macrophages in the Absence of Serum
Authors: Bolaños Benjamin , Mitchell Thomas G
Published: January 2018

The in vitro interaction between yeast cells of Cryptococcus neoformans and Lewis rat alveolar macrophages (AMÕ) was studied in the absence of serum. AMÕ were harvested by lung lavage, and monolayers of adherent cells were established in wells of microtiter plates. Radiolabeled yeast cells were added to fresh AMÕ monolayers, the plates were incubated at 37°C under 5% CO2, nonadherent yeasts were removed, and phagocytosis (i.e., attachment or ingestion) was determined by measuring adherent radioactivity. AMÕ were able to bind or ingest, and kill, encapsulated strains of C. neoformans in the absence of serum. Serum-free phagocytosis was suboptimal by comparison with phagocytosis in the presence of serum. The mechanism of serum-free phagocytosis Involves a receptor on the AMÕ with affinity for mannose-rich determinants present on the yeast cell walls and unrelated to the capsular polysaccharide. Opsonin-independent phagocytosis was only detected with nonencapsulated, small, and medium encapsulated strains of C. neoformans. Large encapsulated strains were not taken up without serum. Serum-free phagocytosis could be of critical importance in the alveolar spaces, where only marginal concentrations of serum opsonins are initially present.
Characterization of the Interaction Between Recombinant Human lnterferon-γ and Its Receptor on Human Polymorphonuclear Leukocytes
Authors: Hansen Brian D , Finbloom David S
Published: January 2018

The interaction of human recombinant interferon-γ (rlFN-γ) with human polymorphonuclear cells (PMN) was investigated. Bolton-Hunter radioiodinated rlFN-γ bound to PMN in a specific and saturable manner. Eleven hundred binding sites were observed with a Ka of 0.56 x 1010 M-1. Binding to PMN was rapid with a K1 of 9 x 105 M-1 sec-1 at 4°C. At 37°C binding was complete within 6 min. About 50% of bound ligand was internalized within 30 min at 37°C. The receptor demonstrated moderate lability at 37°C in culture. After 1 h at 37°C, PMN lost 80% of their 125l-rlFN-γ binding sites. This loss was reversed in part by the presence of interleukin-1 in the culture, but not tumor necrosis factor. These studies provide a framework for further investigation into the signalling process of rlFN-γ on PMN.