bluuebunny/crossref_metadata_2025 huggingface dataset:
Monocytic Origin and Postnatal Mitosis of Intravascular Macrophages in the Porcine Lung
Authors: Winkler Gian C , Cheville Norman F
Published: January 2018

Ultrastructural studies of near-term to 2-month-old pigs were done to document characteristics and developmental changes of intravascular macrophages in pulmonary capillaries. Evidence is presented that blood monocytes colonize the porcine lung perinatally, replicate within capillaries postnatally, and attach to endothelium by intercellular junctions during differentiation. Major ultrastructural features of differentiated intravascular macrophages are adhesion to capillary endothelium, pseudopods, phagosomes, and tubular structures of micropinocytosis vermiformis. Ultrastructure indicates that intravascular macrophages are cells of the mononuclear phagocyte system involved in several functions (eg, blood cell sequestration) that are usually attributed to hepatolienal macrophages. In newborn and 3-day-old pigs, the majority of cells closely apposed to endothelium consisted of few differentiated monocytes, but in 7-day-old and older animals, most cells that were joined to endothelium had characteristics of differentiated intravascular macrophages.
Selectively Eliminated Blood Monocytes and Splenic Suppressor Macrophages in Mice Depleted of Bone Marrow by Strontium 89
Authors: Shibata Yoshimi , Dempsey Walla L , Morahan Page S , Volkman Alvin
Published: January 2018

The contribution of specific activity to the effects of the bone-seeking isotope, strontium 89 on radiosensitive components of mononuclear phagocyte populations was investigated in mice. CBA/J mice received a fixed dose of 2 μCi/g body weight of 89Sr with three different specific activities, 6 Ci, 100 μCi and 20 μCi per mg Sr. The estimated radioactivity located in the bone surface was 4,200, 3,000 and 2,400 cpm/mg bone when measured 2 days after the administration of 89Sr, and was lost with an estimated biological half-life of 27, 25, and 23 days, respectively. Bone marrow suppression was assessed by quantitation of the depletion of macrophage-colony forming cells (M-CFC) grown in vitro in the presence of macrophage growth factor. The decline in M-CFC closely paralleled the level of radioactivity in the bone. These effects were clearly reflected by the depletion of monocytes in the blood, which were reduced to 14%, 14%, and 21% of control levels corresponding to SA's of 6 Ci/mg, 100 μCi/mg and 20 μCi/mg when counted on day 10. By day 30 the respective monocyte levels were 15%, 31%, and 77%. Furthermore, the induction of prostaglandin E producing suppressor macrophages (Mø) by Corynebacterium parvum administration was found to vary inversely with the effects of radioactivity in the bone, with initial impairment followed by quantitative recovery. Resident-type Mø in peritoneal cavity, however, appear to be unaffected by 89Sr-treatment. These data suggest, as before, that the monocytes and suppressor Mø are dependent on radiosensitive marrow cells. The observations also lead to the conclusion that the specific activity of 89Sr preparations is an important determinant of the degree of suppression and of the rate of recovery of bone marrow from the effects of irradiation that follow the administration of this isotope.
Modulation of Macrophage I-A Expression: Lack of Effect of Prostaglandins and Glucocorticoids on Macrophages That Continuously Express I-A
Authors: Vespa Linda , Johnson Sarah C , Aldrich Wayne A , Zwilling Bruce S
Published: January 2018

The ability of prostaglandins, glucocorticoids and lipopolysaccharide to modulate I-A expression on macrophages that continuously or transiently express I-A was compared. We found that neither prostaglandins nor glucocorticoids affected I-A expression by macrophages that continuously expressed I-A. In contrast, prostaglandin E2 (PGE2) inhibited the lymphokine-induced maintenance of I-A on macrophages that transiently expressed I-A while glucocorticoids inhibited both the induction and expression of I-A. Lipopolysaccharide caused a loss in the expression of Class II molecules on macrophages that continuously expressed l-A by a prostaglandin-independent mechanism. The lack of effect of PGE2 and glucocorticoids may be related to our observation that continuous expression of I-A does not require its continued synthesis.
Alveolar Macrophage Phagocytic Kinetics Following Pulmonary Parainfluenza-3 Virus Infection
Authors: Slauson David O , Lay John C , Castleman William L , Neilsen Nancy R
Published: January 2018

Qualitative and quantitative evaluations of the cellular components of bronchoalveolar washings of calves with experimental parainfluenza-3 virus pneumonitis and control calves were made. Calves were exposed to 109 TCID50 of PI-3 by intranasal aerosol exposure and bronchoalveolar cells obtained 7 days after infection by volume-controlled bronchopulmonary lavage. Transient tachypnea and pyrexia occurred in all infected calves, and virus was recoverable at 7 days from nasal swabs and lung tissue. Pulmonary lesions were typical of viral pneumonitis, characterized by patchy alveolitis and bronchiolitis with accumulations of cells and inflammatory debris. The mean total lavage cell yield was elevated in the virus-infected calves, and the percentage of neutrophils was elevated (P < 0.05). Increased numbers of pulmonary alveolar macrophages (PAM) were also recovered but the difference was not significant. Linear regression equations showed that a decreased proportion of PAM from virus-infected animals were phagocytic. The mean initial phagocytic rates of macrophages from calves with viral pneumonitis were significantly decreased (P < 0.05) over controls. This difference was concentration dependent and required a phagocytic stimulus in excess of 12.5 × 106beads/ml. Studies of phagocytic kinetics showed that PAM from calves with viral pneumonitis had a lower Vmax than PAM from control calves, but that Km values were comparable. No differences in PAM β-glucuronidase and acid phosphatase activity were observed. These results indicate depressed phagocytic function in PI-3 virus-inflamed lungs relative to controls. In concert with virus-induced airway lesions, such in vivo depression of PAM phagocytic functions would be expected to depress pulmonary particulate clearance and lung defense mechanisms.
The Effect of a Protein Kinase C Inhibitor, H-7, on Human Neutrophil Oxidative Burst and Degranulation
Authors: Berkow Roger L , Dodson Robert W , Kraft Andrew S
Published: January 2018

The role of C-kinase in the activation of human polymorphonuclear leukocytes has been examined using H-7, a recently described C-kinase inhibitor. We found that H-7 will inhibit PMN superoxide anion release in response to the tumor promotor phorbol myristate acetate and the calcium ionophore A23187. In contrast, no inhibition by H-7 was seen for PMN superoxide release stimulated by the chemotactic peptide FMLP. H-7 did not inhibit PMN NADPH oxidase activity or PMN degranulation by any stimulant, but it reversed a phorbol ester-induced inhibition of granule release by FMLP. The results show that H-7 differentially affects the PMN functional events of secretion and superoxide release and suggests that an H-7 inhibitable C-kinase 1) is not involved in chemotactic peptide induced activation of PMN and 2) may not regulate stimulus induced PMN degranulation.
Neutrophil Dysfunction in the Rabbit Model of Spur Cell Anemia
Authors: Lichtenstein Israel H , Zaleski Ellen M , MacGregor Rob Roy
Published: January 2018

In the rabbit model for spur cell anemia, animals fed a 5% cholesterol diet develop marked hypercholesterolemia and hemolytic spur cell anemia after several weeks on the diet. In vitro tests of granulocytes showed a 15% increase in cholesterol: phospholipid ratio, and decreased membrane fluidity measured with a fluorescent probe. Function tests revealed impairment of adherence, phagocytosis, and chemotaxis. Both a plasma factor and an intrinsic cellular defect appeared to contribute to the abnormal adherence. Bactericidal activity was normal. In vivo demargination in response to epinephrine was increased in animals on the diet, but exudation of granulocytes into sterile peritonitis fluid was diminished to 39.4% of control at 8 hours. Therefore, rabbits with experimental spur cell anemia have impaired in vitro and in vivo granulocyte function. The clinical significance of these findings for patients with spur cell anemia and less severe alcoholic liver disease is uncertain.
Effects of Adherence, Activation and Distinct Serum Proteins on the In Vitro Human Monocyte Maturation Process
Authors: Akiyama Yukio , Griffith Richard , Miller Paul , Stevenson G W , Lund Stacy , Kanapa D J , Stevenson Henry C
Published: January 2018

Elutriator-purified human monocytes were cultured in a serum-free (SF) medium, and various serum proteins and functional activating agents were assessed for their effects on the in vitro maturation of human monocytes to macrophages. Following 3 days of suspension culture in Teflon labware, 60% of the monocytes were easily recovered. When varying concentrations of human AB serum (HuAB) were employed, human monocyte maturation progressed rapidly; the kinetics of this maturation process during cell suspension culture were very similar to the pattern observed following adherence culture. In contrast, when SF medium was employed, a marked retardation of the monocyte maturation process was observed; this could not be attributed to any changes in cell recovery and/or viability. Thus, cells could be maintained in their monocytoid form for 3 days when cultured in SF medium. When HuAB was added after 3 days of culture, human monocyte maturation into macrophages proceeded at a normal rate.

We attempted to characterize certain of the serum protein(s) found in HuAB which promoted the monocyte maturation process. Human immunoglobulin G (IgG) was found to be the most potent serum protein in increasing 5′-N activity and decreasing peroxidase activity of suspension cultured monocytes. Immunoglobulin M (IgM) and albumin (Alb) were shown not to have significant monocyte maturation activity. Heat-treated human gamma globulin and IgG purified by high-performance liquid chromatography (HPLC) were shown to have patterns identical with that of untreated HGG and IgG with regard to promoting monocyte maturation; F(ab′)2 was not an active maturation promoter, indicating the need for an intact Fc portion of the IgG molecule. Fibrinogen and fibronectin also had maturation promoting activity.

Finally, addition of the potent monocyte functional activators, muramyl dipeptide (MDP), polyriboinosinic:polyribocytidilic acid (Poly I:C), and lipopolysaccharide (LPS) had no effect on the monocyte maturation process. Thus, neither cell adherence or activation appear to be critical for the monocyte to macrophage maturation process. Instead, we hypothesize that in addition to proper nutritional support, a group of serum proteins (unified mainly by their ability to interact with monocyte membrane receptors) appear to be the principal promoters of this process.
Bacterial Lipopolysaccharide Enhances Chemoattractant-Induced Elastase Secretion by Human Neutrophils
Authors: Fittschen C , Sandhaus R A , Worthen G S , Henson P M
Published: January 2018

Bacterial lipopolysaccharide (LPS) has previously been shown to enhance a number of chemoattractant-induced responses by human neutrophils. The possible role of elastase, a neutral protease with broad substrate specificity, in neutrophil-mediated vascular injury of a variety of diseases prompted us to examine a) whether or not LPS enhances the direct chemoattractant-induced secretion of elastase, b) the quantitative requirements of LPS and chemotactic factors, and c) some structural requirements of LPS for this effect. Our results show that LPS at 10 ng/ml and above, enhanced formyl-methionyl-leucyl-phenylalanine-induced neutrophil secretion of elastase, as well as secretion of myeloperoxidase and vitamin B12-binding protein. This effect was independent of cytochalasins or surface stimulation, and thus may occur during chemotactic factor stimulation in vivo. LPS also enhanced neutrophil secretory responses to the complement fragments C5a, C5a des arg, and, to a lesser degree, to leukotriene B4 and platelet-activating factor. This enhancement effect appeared to require the presence of the lipid A moiety and/or parts of the core polysaccharide but not the O-antigen portion of the LPS molecule. Our findings identify a possible LPS-dependent mechanism of neutrophil elastase-mediated tissue injury in Gram-negative infections.
Differences in the Ability of Neutrophils and Monocytes to Traverse Epithelial Occluding Junctions
Authors: Migliorisi Grace , Folkes Edmund , Cramer Eva B
Published: January 2018

This study examines the effect of epithelial permeability on 1) the passage of the chemoattractant tritiated formyl methionyl-leucyl-phenylalanine (3H-fMLP; m.w. 437) and 2) the migration of leukocytes. In addition, it also compares the kinetics of neutrophil and monocyte transepithelial migration. As we had demonstrated with neutrophils (Milks et al.: Journal of Cell Biology 96:1241, 1983), when the permeability of the epithelium decreased, the accumulation of 3H-fMLP and the emigration of monocytes also decreased. When neutrophils and monocytes traversed epithelia with similar permeability, neutrophil accumulation was at least ninefold greater than that of monocytes at 30, 60, and 90 min. During the same time intervals, the number of neutrophil invasion sites/mm epithelium exceeded the number of monocyte invasion sites by at least fivefold, with approximately twice as many neutrophils as monocytes traversing each invasion site. These studies demonstrate that epithelial permeability affects the passage of the chemoattractant and the emigration of leukocytes. In addition, the enhanced ability with which neutrophils traverse occluding junctions compared to monocytes helps to explain, at least in part, the more rapid accumulation of neutrophils at inflammatory lesions.
Characterization of Murine Bronchoalveolar Macrophage Respiratory Burst: Comparison of Soluble and Particulate Stimuli
Authors: Sugar Alan M , Field Keith G
Published: January 2018

Stimulation of the respiratory burst of murine bronchoalveolar macrophages obtained by lung lavage was studied using four different stimuli and different assay conditions. One soluble stimulus, phorbol myristate acetate (PMA), two intracellular particles, zymosan and Blastomyces dermatitidis conida, and one extracellular particle, B. dermatitidis yeast, were incubated with either freshly obtained macrophages in suspension or 2- and 48-hour macrophage monolayers. Suspension cultures were incubated with stimuli for 90 minutes and monolayers for 10 minutes before O2− was assayed. PMA did not elicit O2−production in macrophage suspensions or 2-hour macrophage monolayers, but 48-hour macrophage monolayers exhibited a 13-fold increase above control values (P = .0001). On the other hand, zymosan elicited an increase in O2− production in both suspensions and monolayers, although monolayers incubated for 48 hours produced almost fourfold more O2− than the other systems (P = .025). Opsonization had no effect on the ability of zymosan to elicit respiratory burst B. dermatitidis conidia resulted in a two- to threefold increase in O2− production in macrophage suspensions and a five- to eightfold increase in 48-hour monolayers, representing significantly less respiratory burst stimulation than with either zymosan or PMA. Similarly, B. dermatitidis yeasts demonstrated similar submaximal stimulation of O2−, 3–4 times that over control values, and again this was less than zymosan and PMA. We conclude that 1) freshly obtained murine bronchoalveolar macrophages do not respond to PMA with an increase in O2− production, but that responsiveness is evident after 48 hours of incubation in monolayers; 2) B. dermatitidis conidia and yeasts do not stimulate respiratory burst activity to the same degree as zymosan or PMA; and 3) opsonization of zymosan is not necessary for stimulation of the murine bronchoalveolar macrophage oxidative burst, confirming previous data that functional complement receptors are not present on these cells.
Electron Spin Resonance Spectroscopy Does Not Reveal Hydroxyl Radical Production in Activated Natural Killer Lymphocytes
Authors: Gibboney James J , Haak Richard A , Kleinhans Frederick W , Brahmi Zacharie
Published: January 2018

We investigated hydroxyl radical (OH) production by human natural killer (NK) cells, using electron spin resonance (ESR) spectroscopy and 5,5 dimethyl-1-pyrroline-N-oxide (DMPO), a spin trap specific for OH production. We confirmed that hydroxyl radical scavengers, n-propyl gallate and catechin, inhibited NK cell-mediated cytotoxicity (NK-CMC) in a dose-dependent manner and demonstrated that DMPO also inhibited NK-CMC. Polymorphonuclear leukocytes (PMNL) activated by opsonized zymosan (2.4 mg/ml) and mixed with DMPO (0.14 M) showed an early increase in hydroxyl radical production, leading to a net production of free radical of almost 400 pMol/106 cells. We then mixed NK cells with K562, an NK-sensitive tumor cell, at a 1:1 ratio and added DMPO (0.14 M). We pelleted the cells to increase EC to TC binding before taking the sample readings. Activated NK cells showed no increase in OH production, leading to a net production of free radicals less than 1% that of activated PMNL. These data strongly suggest that hydroxyl radical production does not play a role in the early events of NK cell activation; they indicate a need to reevaluate the mechanism of inhibition of NK-CMC by OH scavengers.
Activated Langerhans Cells Release Tumor Necrosis Factor
Authors: Larrick James W , Morhenn Vera , Chiang Yawen L , Shi Tim
Published: January 2018

Langerhans cells act as antigen-presenting cells in immune reactions in the skin. What other roles they may play in inflammation is less well defined. We have tested whether these cells can produce TNF-alpha, an important mediator of inflammation. Resting Langerhans cells produce less than 0.1 U TNF-alpha/ml. Langerhans cells stimulated with phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) release 4-5 U TNF-alpha/ml. Specificity of the released TNF-alpha in an L929 cytotoxicity assay was confirmed by using neutralizing anti-TNF-alpha monoclonal antibodies, and the identity of TNF-alpha was further confirmed by Northern blot hybridization with an TNF-alpha oligomer DNA probe. Activated Langerhans cells may contribute to inflammation in the skin by releasing TNF-alpha, which is known to effect fibroblast growth, endothelial cell activation, and lymphocyte function.
Germinal Center B Cells and Mixed Leukocyte Reactions
Authors: Monfalcone Alan P , Kosco Marie H , Szakal Andras K , Tew John G
Published: January 2018

The present study was undertaken to determine if germinal center (GC) B cells are sufficiently activated to stimulate mixed leukocyte reactions (MLR). Percoll density fractionation and a panning technique with peanut agglutinin (PNA) were used to isolate GC B cells from the lymph nodes of immune mice. The GC B cells were treated with mitomycin C or irradiation and used to stimulate allogeneic or syngeneic splenic T cells in the MLR. Controls included high-density (HD) B cells prepared from spleens of the same mice and HD B cells activated with lipopolysaccharide (LPS) and dextran sulfate. GC B cells bound high amounts of PNA (i.e., PNAhi). Similarly, the LPS-dextran sulfate-activated B cells were PNAhl. Treatment with neuraminidase rendered the PNA HD B cells PNAhl. GC B cells and the LPS-dextran sulfate-activated HD B cells stimulated a potent MLR, while the untreated HD B cells did not. However, following neuraminidase treatment, the resulting PNAhl HD B cell population was able to induce an MLR. The PNA marker appeared to be an indicator of stimulatory activity, but incubating the cells with PNA to bind the cell surface ligand did not interfere with the MLR. GC B cells were also capable of stimulating a syngeneic MLR in most experiments although this was not consistently obtained. It appears that germinal centers represent a unique in vivo microenvironment that provides the necessary signals for B cells to become highly effective antigen-presenting cells.
Macrophage-Parasite Interactions in Leishmania Infections
Authors: Mauël Jacques
Published: January 2018

Macrophages have different roles in Leishmania infections. They function as host-cells to the parasite, they present parasite antigens to the immune system, and they also act as effector cells responsible for parasite killing in the healing stages of the infection. Protein and carbohydrate Uganda on the parasite surface, which interact with receptors on the macrophage membrane during phagocytosis, have been identified. Survival of Lelshmania in its host cell requires a metabolic adaptation to the intracellular environment The nutritional requirements of the microorganism must be satisfied and the deleterious effect of oxygen metabolites and of lysosomal hydrolases must be adequately counteracted. When a successful immune response develops, macrophages become activated and acquire the capacity to destroy the microorganism. Possible reasons why these mechanisms fail in certain individuals or experimental host-parasite combinations are discussed.
Immunolocalization of Endosomal Cathepsin D in Rabbit Alveolar Macrophages
Authors: Rodman Jane Somsel , Levy Marilyn Aach , Diment Stephanie , Stahl Philip D
Published: January 2018

Intravacuolar proteolysis appears to be an important component of antigen presentation, the activation of peptide hormones, and the conversion of biologically important mediators from inactive precursors. Cathepsin D has been identified in the endosomes of rabbit alveolar macrophages by biochemical analyses [Diment and Stahl, J. Biol. Chem. 260, 15311, 1985; Diment et al., J. Biol. Chem. 263,6901, 1988]. Using affinity-purified goat antirabbit cathepsin D IgG, we have localized cathepsin D to the endosomes of rabbit alveolar macrophages. Immunofluorescent staining of frozen sections showed labeling in lysosomes and small vesicles in the periphery of the cell. Label was not seen on the plasma membrane. With immunoperoxidase labeling at the electron microscopic level on cells containing endocytosed mannose-BSA gold, we saw labeling in endosomes and classical lysosomes. When the results were quantrtated using immunogold labeling of thin cryosections, we found that the majority of cathepsin D (62.2%) was present in lysosomes, 4.0% in large clear vacuoles, a surprisingly high percentage (29.3%) in small vesicles, 4.9% in endosomes, and none on the plasma membrane. We conclude from this study that, in addition to being present in lysosomes, cathepsin D is present in endosomes and in small peripheral vesicles.
Uncoupling of Oxidative and Non-Oxidative Mechanisms in Human Granulocyte-Mediated Cytotoxicity: Use of Cytoplasts and Cells From Chronic Granulomatous Disease Patient
Authors: van Kessel Kok P M , van Strijp Jos A G , van Kats-Renaud Henny J , Miltenburg Lia A M , Fluit Ad C , Verhoef Jan
Published: January 2018

Human polymorphonuclear leukocytes (PMN) and granule-free cytoplasts were compared for their cytotoxic capacities against red blood cells (RBC) and K562 tumor cells. Phorbol myristate acetate (PMA) stimulated PMN to efficient lysis of RBC targets, while cytotoxicity against the tumor cell line K562 was moderate. Activated cytoplasts also lysed RBC targets but were not able to kill K562 tumor cells, even at high cell numbers. Suppression of the glutathione redox cycle of the K562 tumor targets markedly increased their susceptibility to lysis by PMA-activated PMN. Despite the enhanced susceptibility of antioxidant-depleted K562 tumor cells to oxygen radical-induced damage, PMA-stimulated cytoplasts did not kill these targets. Addition of exogenous myeloperoxidase or lactoferrin to cytoplasts devoid of granule did not improve the lysis of RBC and K562 tumor cells. Coating K562 targets with specific antibodies induced efficient PMN-mediated killing in comparison to PMA-stimulated lysis of non-coated targets. Cytoplasts, however, did not kill antibody-coated K562 tumor cells; this was not improved by glutathione depletion but showed some lysis of antibody-coated RBC. PMN from a patient with chronic granulomatous disease (CGD) showed normal antibody-dependent cell-mediated cytotoxicity (ADCC) against K562 tumor cells but were not able to lyse these targets after PMA stimulation. The analysis of target cell killing by cytoplasts and PMN from a CGD patient indicated that granular constituents are important mediators in the killing of nucleated target cells and that PMN-mediated ADCC does not require the release of reactive oxygen species. Differences in the susceptibility of target cells to oxygen-mediated lysis indicates that target cell antioxidant mechanisms play an important role in the outcome of the cytotoxic response.
Prostaglandin-Dependent Desensitization of Human Monocyte cAMP Responses
Authors: Coffey Ronald G , Alberts Vicki A , Weakland Laura L
Published: January 2018

Histamine stimulated large increases of cyclic adenosine monophosphate (cAMP) in freshly isolated human blood monocytes in the presence of RO2-1724, a specific cAMP phosphodiesterase inhibitor. This was mediated by H2 receptors, since it was inhibited by cimetidine but not chlorpheniramine. Stimulation was attenuated in cells aged in culture 1–2 days. Indomethacin prevented the desensitization, suggesting that a cyclooxygenase product was responsible. Desensitization was heterologous, since the adenylate cyclase responses to 5′-(N-ethylcarboxamido)adenosine (A2 receptor agonist), isoproterenol (β-adrenoceptor agonist), and prostaglandin E2 (PGE2) also declined during culture. The loss of sensitivity to histamine was restored by incubating monocytes with PGE2 in the presence of indomethacin. The results indicate that, while PGE2 inhibits monocyte functions via cAMP, its accumulation paradoxically permits cells to escape this regulation through a heterologous desensitization of the cAMP response to itself and other agonists.
Differentiation of Rat Bone Marrow Cells Into Macrophages Under the Influence of Mouse L929 Cell Supernatant
Authors: Naum Chris C , Kaplan Sandra S , Basford R E
Published: January 2018

Platelets are currently thought to play a role in tissue injury and inflammatory states both directly and indirectly through their action on neutrophils (PMNs). Both stimulation and inhibition of PMN superoxide anion (O2-) production by platelets has been reported. To clarify these discrepant observations, we investigated the effects of wide ranges of platelet to PMN ratios as well as concentrations of ATP and ADP on human PMN O2- production. Platelets, at low platelet-to-PMN ratios (1:1 and 5:1), primed PMNs which were stimulated with either FMLP or PMA. However, at higher platelet-to-PMN ratios (25:1, 50:1, and 100:1), inhibition of O2- production was seen. ATP also had a biphasic effect on O2- production: low concentrations of ATP (1 x 10-6 to 3.2 x 10-4 M) increased O2 production and high concentrations of ATP (6.4 x 10-4 M and above) inhibited O2 production. ADP, when added to stimulatory concentrations of ATP, also caused inhibition of O2- production. The incubation time for platelet-neutrophil interactions in vitro was also crucial. Short incubation periods lead to priming, whereas longer periods (> 5 min) lead to inhibition. We believe that these studies help to resolve the controversy over the effects of platelets upon the production of O2- by human PMNs and lend further support to the notion that platelets may modulate injury resulting from neutrophil activation.
High-Affinity Receptor for Interferon-Gamma (IFN-γ), a Ubiquitous Protein Occurring in Different Molecular Forms on Human Cells: Blood Monocytes and Eleven Different Cell Lines Have the Same IFN-γ Receptor Protein
Authors: van Loon Adolphus P G M , Ozmen Laurence , Fountoulakis Michael , Kania Malgosia , Haiker Monika , Garotta Gianni
Published: January 2018

High-affinity receptors for human IFN-γ were analyzed using 13 different cells, including blood monocytes. Scatchard analysis showed one high-affinity binding site for each cell. One cross-linked complex between IFN-γ and the receptor was detected, although their apparent molecular masses were variable in different cells, as also confirmed in immunoblots of membrane extracts. Variations in molecular masses were abolished if N-linked glycosylation was absent. Stable tryptic fragments contained the intact binding site for IFN-γ and antibody epitopes characteristic of the extracellular domain of the IFN-γ receptor of Raji cells and were of different sizes only if glycosylated. In addition, Northern analysis showed the same mRNA encoding the high-affinity IFN-γ receptor in each cell analyzed. Thus, all cells including blood monocytes express the same high-affinity IFN-γ receptor protein. N-linked sugars may give structural stability to the IFN-γ receptor and are unlikely to be directly involved in IFN-γ binding.
Isolation and Flow Cytometric Characterization of Newborn Mouse Brain-Derived Microglia Maintained In Vitro
Authors: Hassan Nassef F , Rifat Salahaldin , Campbell Donald E , McCawley Lisa J , Douglas Steven D
Published: January 2018

Microglia have been identified in the white matter of developing and adult mouse brain using different murine macrophage markers. While several techniques for the isolation of murine microglia have been described, the small cell yields and partial purification have limited the progress of these studies. We now describe the isolation of murine microglia using a modification of McCarthy and de Vellis method. Brain tissues from 1–2 day old newborn mice were mechanically and chemically dissociated and maintained in in vitro culture for 3 weeks. In primary dissociated brain cultures, microglia are observed after 10 days migrating from small colonies. After 16–20 days, brain-derived microglia were isolated with high cell yields by continuous shaking of the cultures for 16 hr. In contrast to resident murine peritoneal macrophages, microglia express less Class II (la) antigen and a small percentage express L3T4 (CD4) antigen by flow cytometry.
Poly I:C-Induced Anti-Herpes Simplex Virus Type 1 Activity in Inflammatory Macrophages Is Mediated by Induction of Interferon-β
Authors: Pyo S , Gangemi J D , Ghaffar A , Mayer E P
Published: January 2018

We examined the mechanism by which Polyriboinosinic:Polyribocytidylic acid (Poly l:C) augments resistance of thioglycolate-elicited inflammatory macrophages to infection with herpes simplex virus type 1 (HSV-1). We show that Poly l:C-induced antiviral activity is completely abrogated by antibodies to interferon-β (IFN-β) whereas antibodies to other interferons or to other cytokines have no effect. Furthermore, treatment of inflammatory macrophages with exogenous IFN renders them resistant to HSV-1, whereas treatment with other cytokines does not. In addition, we demonstrate that supernatants from macrophages treated with Poly l:C contain IFN-β but not IFN-α. Taken together these data indicate that the antiviral effects of Poly l:C in inflammatory macrophages are mediated solely by IFN-β, which acts in an autocrine manner to induce resistance to HSV-1.
Selective Secretion of Azurophil Granule Contents Induced by Monovalent Cation Ionophores in Human Neutrophils: Evidence for Direct Ionophore Effects on the Granule Membrane
Authors: Fittschen Claus , Henson Peter M
Published: January 2018

The study of factors contributing to secretion of neutrophil azurophil granules has previously been complicated by the inability to induce their release without concomitant exocytosis of specific granules. This publication describes the action of the first two agents, the Na-ionophore monensin and the K-ionophore nigericin which elicited only secretion of azurophil granules. Secretion depended on H+/alkali ion antiport by the ionophores since it was abolished in Na+- and K+-poor choline buffer. The secretagogue effects of both ionophores did not correlate with changes either in cytoplasmic pH or in transmembrane potential and were not associated with Ca-transients, but were closely associated with azurophil granule alkalinization suggesting that the secretory event resulted from alkali ion/H+ antiport at the granule membrane. Addition of zinc inhibited azurophil (but not specific) granule secretion in response to monensin, CB/FMLP, and zymosan, indicating that secretion induced by these agents shares a common step(s).
Induction of Tumor Necrosis Factor and Macrophage-Mediated Cytotoxicity by Horseradish Peroxidase and Other Glycosylated Proteins: The Role of Enzymatic Activity and LPS
Authors: Lefkowitz Doris L , Mills Kevin , Castro Aaron , Lefkowitz Stanley S
Published: January 2018

Recent studies by these investigators have shown that horseradish peroxidase (HRP) can cause murine thioglycollate-induced peritoneal macrophages (Mø) to produce both tumor necrosis factor (TNF) and enhance macrophage-mediated cytotoxicity (MMC) to 3T12 target cells. The present study identifies the roles of both enzymatic activity and contaminating lipopolysacharides (LPS) (≤1 ng) on these activities. The addition of 100 ng/ml of polymyxin B (PB) to enzymatically active HRP significantly reduced TNF production but did not affect MMC. Enzymatically inactive HRP (DHRP) was more effective than HRP in both TNF production and MMC but was not affected by PB. The inability of PB to modify DHRP-induced TNF suggests that LPS was not required. The induction of TNF and MMC in the absence of LPS was also corroborated by similar studies using Mø from endotoxin-resistant C3H/HeJ mice. Glycosylated proteins such as HRP, DHRP, and mannosylated bovine serum albumin (M-BSA) are known to bind to mannose receptors (mannosyl-fucosyl receptor [MFR]) on the surface of Mø. In the present studies, M-BSA behaved similarly to DHRP in that it induced both TNF secretion and MMC. These results suggest that binding to the MFR may be sufficient to induce TNF secretion and MMC. In addition, the data suggest that neither enzymatic activity nor LPS was required for DHRP-induced TNF.
Cryopreserved Cytoplasts From Human Neutrophils Migrate Across Monolayers of Human Endothelial Cells in Response to a Chemoattractant Gradient
Authors: Huang Ada J , Silverstein Samuel C , Malawista Stephen E
Published: January 2018

We have measured the capacity of two types of granule-poor anucleate cytoplasmic fragments (cytoplasts) from human polymorphonuclear leukocytes (PMN) to migrate across the barrier imposed by monolayers of human umbilical vein endothelial cells (EC) with and without chemotactic stimulation by fMLP. Cytoplasts were made by brief heating of PMN attached to surfaces (CKP) or by discontinuous gradient centrifugation (U-CYT). In the absence of chemoattractant, both types of cytoplast adhered poorly to endothelial cell monolayers, as did unstimulated intact PMN from which the cytoplasts were derived. In the presence of a transendothelial chemoattractant gradient both types of cytoplast exhibited a marked increase in adherence to, and migration across, endothelial monolayers; CKP did so to the same extent as chemoattractant-stimulated intact PMN. Since these motile cytoplasts are markedly deficient in most cytoplasmic organelles they may serve as useful tools for the dissection of cellular mechanisms that mediate PMN migration across endothelia.
Evidence for the involvement of tyrosine kinases in the locomotory responses of human neutrophils
Authors: Gaudry Murielle , Caon Adriana C , Gilbert Caroline , Lille Simonne , Naccache Paul H
Published: January 2018

Erbstatin, a recently described inhibitor of tyrosine kinases, has been used to examine the potential role of tyrosine phosphorylation in human neutrophil locomotion. Preincubation of human neutrophils with erbstatin inhibited both spontaneous and directed migration induced by chemotactic factors such as formyl-methionylleucylphenylalanine (fMet-Leu-Phe) and leukotriene B4. The decreased migratory responses were correlated with an inhibition of adherence of neutrophils to serum-coated surfaces. Erbstatin did not, however, affect the adherence of human neutrophils to uncoated surfaces. These results indicated that the inhibitory effects of erbstatin were specific and not due to a generalized alteration of the surface of human neutrophils. To elucidate the mechanism of the inhibitory effect of erbstatin on adherence properties, the expression of the leukocyte integrin Mo1 was studied. Erbstatin induced a small but significant increase in the expression of Mo1, but decreased the stimulation of the expression of Mo1 elicited by fMet-Leu-Phe. These results suggest that mechanisms in addition to alteration of the number of surface integrins are involved in the inhibition of neutrophil adherence and locomotion by erbstatin. J. Leukoc. Biol. 51: 103–108; 1992.
Effects of in vivo T lymphocyte subset depletion on mycobacterial infections in mice
Authors: Flory Craig M , Hubbard Randall D , Collins Frank M
Published: January 2018

The relative importance of CD4+ and CD8+ T cell subsets in the expression of acquired resistance to systemic infection by Mycobacterium kansasii was determined. T cell subsets were depleted in thymectomized C57BL/6 mice by the intravenous administration of monoclonal antibodies directed against the relevant T cell determinants. Depletion of the CD4+ subset exacerbated the severity of the infection in intravenously challenged mice. This effect was apparent in the first 2 weeks of the infection and persisted throughout the 12 weeks of the study. On the other hand, depletion of the CD8+ cells had no apparent effect on the growth curves. Infections by Mycobacterium tuberculosis Erdman or bacille Calmette-Guérin (BCG) Pasteur were also substantially enhanced by CD4 depletion, but not by the depletion of CD8+ cells. The effect of subset depletion on infections by M. tuberculosis and BCG was examined in both innately susceptible C57BL/6 mice and innately resistant B6D2 mice.
The timing of exposure of mononuclear phagocytes to recombinant interferon γ and recombinant tumor necrosis factor α alters interactions with Nocardia asteroides
Authors: Beaman LoVelle , Beaman Blaine
Published: January 2018

Nocardia asteroides modulates phagocyte function and grows within macrophages. Because interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α) have been shown to activate macrophages to kill a variety of microorganisms, the effects of IFN-γ and TNF-α on the activation of murine macrophages and human monocytes to kill nocardiae were studied. It was found that macrophages or monocytes treated with either IFN-γ, TNF-α, or lipopolysaccharide as a secondary signal did not demonstrate increased microbicidal activity against N. asteroides even though these phagocytes were effective at killing the fungus Coccidioides immitis and the protozoan Toxoplasma gondii. Preincubation of phagocytes for 24 h with these compounds resulted in an enhancement of nocardial growth. In contrast, coincubation of these factors with the nocardiae and mononuclear cells during phagocytosis resulted in inhibition of nocardial growth even though this bacterium was not killed. Therefore, the specific timing of the exposure of the phagocyte in vitro to IFN-γ or TNF-α has a significant effect on its ability to alter nocardial growth.
Mouse macrophages contain a truncated CD4 transcript
Authors: Moore Steven C , Anderson Stephen J , Walker William S
Published: January 2018

Mouse macrophages do not express CD4 on their surfaces. We used the polymerase chain reaction to investigate CD4 gene transcription in individual clones of primary mouse splenic macrophages and cell lines of spleen and bone marrow macrophages. The results show only the presence of CD4 mRNA transcripts that are truncated in the 3′ coding sequence, thus explaining the lack of expression of a mature CD4 gene product by these cells.
Interleukin-6 in mouse hypersensitivity pneumonitis: changes in lung free cells following depletion of endogenous IL-6 or direct administration of IL-6
Authors: Denis Michel
Published: January 2018

In this study, we examined the role of interleukin-6 (IL-6) in the development of chronic lung inflammatory conditions, using a mouse model of hypersensitivity pneumonitis established by intranasal instillation of the thermophilic actinomycete Faeni rectivirgula. Challenged mice developed an early neutrophilic response at 24 h, followed by a macrophage/lymphocyte recruitment. The impact of IL-6 on the development of the inflammatory response was assessed by giving infusions of a monoclonal antibody against IL-6 so as to deplete endogenous levels of this cytokine or by giving exogenous IL-6 to challenged mice. Mice challenged in-tranasally with the actinomycete and given the anti–IL-6 antibody developed a strong, sustained neutrophilic response, with a significantly higher lung free cell number than control mice. Assessment of fibrosis by measuring lung hydroxyproline levels showed that challenged mice given anti–IL-6 developed more significant fibrosis than control mice. Conversely, infusions with IL-6 diminished F. rectivirgula–induccd cell recruitments and the fibrotic response in the lungs. Moreover, alveolar macrophages from mice given 2 weeks of F. rectivirgula treatment released high levels of tumor necrosis factor a (TNF-α) bioactivity upon in vitro lipopolysaccharide challenge, compared to mice instilled with saline only. This TNF-α activity produced by macrophages was decreased by in vivo IL-6 treatment and enhanced by in vivo neutralization with anti-IL-6. These observations suggest that IL-6 may play a role in regulating the cellular recruitment in the lungs during an inflammatory response, with dramatic consequences for the cellular profile in the bronchoalveolar lavage and the subsequent fibrosis.
Heterogeneity of rat liver and spleen macrophages in gadolinium chloride–induced elimination and repopulation
Authors: Hardonk MJ , Dijkhuis FWJ , Hulstaert CE , Koudstaal J
Published: January 2018

Blockade of phagocytosis and selective elimination of macrophages (mφs) are generally accepted procedures for gaining knowledge about the function of mφs in vivo. This study demonstrates that intravenous injection of gadolinium chloride (GdCl3) not only blocks phagocytosis by rat liver mφs (Kupffer cells) but also selectively eliminates the large mφs situated in the periportal zone of the liver acinus. Repopulation of mφs starts at 4 days after injection. During repopulation, mφs are less vulnerable to GdCl3. When repopulation is complete, the new mφs show the same vulnerability as the original ones. Splenic mφs are less vulnerable to GdCl3 because only some of the red pulp mφs transiently disappear. The white pulp mφs are not affected. Repopulation occurs sooner than in liver. These results indicate that administration of GdCl3 is a suitable approach to studying the in vivo function of large Kupffer cells.
Activation of tumoricidal properties in macrophages by lipopolysaccharide requires protein-tyrosine kinase activity
Authors: Dong Zhongyun , O’Brian Catherine A , Fidler Isaiah J
Published: January 2018

The purpose of these studies was to determine whether triggering murine peritoneal macrophages to a tumoricidal state by lipopolysaccharide (LPS) requires protein-tyrosine phosphorylation. The LPS-triggered activation of mouse macrophages to lyse syngeneic B16 melanoma cells was significantly inhibited in a dose- dependent manner by the protein-tyrosine kinase (PTK) inhibitors genistein, herbimycin A, and tyrphostin. Genistein was effective only when added to macrophages prior to or simultaneously with LPS. Genistein potently inhibited the productive interaction of macrophages with LPS but had only a minor effect on the action of interferon- γ. The effects of genistein on LPS-triggered macrophage activation were not due to nonspecific changes in macrophage metabolism or toxicity because genistein did not prevent lysis of tumor cells by activated macrophages, nor did it reduce the capacity of macrophages to phagocytose antibody-opsonized sheep erythrocytes. Western blot analysis with antiphosphotyrosine monoclonal antibody revealed that incubation of macrophages with LPS produced a rapid increase in tyrosine phosphorylation of several proteins and that the induced phosphorylation could be inhibited by effective concentrations of genistein, herbimycin A, or tyrphostin. Taken together, these data indicate that protein-tyrosine phosphorylation plays an important role in LPS-induced tumoricidal activation of macrophages. J. Leukoc. Biol. 53: 53–60; 1993.
Inflammatory fluids regulate TNF-α, but not IL-1β, production by human peritoneal macrophages. A study of patients on continuous ambulatory peritoneal dialysis with peritonitis
Authors: Hart Prue H , Jones Catherine A , Finlay-Jones John J
Published: January 2018

To gain insight into the factors regulating an inflammatory response in vivo, the activities of peritoneal macrophages and the influence of the inflammatory fluids from which they were harvested were studied in continuous ambulatory peritoneal dialysis (GAPD) patients with peritonitis. Specifically, the production of tumor necrosis factor a (TNF-α), interleukin-lβ (EL-lβ), and prostaglandin E2 (PGE2) by lipopolysaccharide-stimu- lated human peritoneal macrophages from GAPD patients with peritonitis was examined in the presence of the inflammatory dialysates from which they were isolated. When tested at a dilution of 20%, the dialysates inhibited production of TNF-α, a key monokine in the orchestration of the inflammatory response. In contrast to the effect on TNF-α, the dialysates did not affect IL-Iβ production by stimulated macrophages. The activity inhibitory for TNF-α synthesis was not fully characterized, but a number of known inhibitors were shown not to be responsible for the suppressive activity. The inhibitory activity was detected in cases of noninfective peritonitis and excluded the possibility that a bacterial product was responsible. Hyperosmolality, pH, protein levels, glucose, uremic molecules, cortisol, heparin, and antibiotics were not responsible for the inhibitory activity. The activity had some similarity to the reported actions of α-globulins (which are acute phase proteins), PGE2, TNF-α soluble receptors, and EL-6, but there was no evidence for their involvement. Whether the selective suppression of TNF-α production is a general finding in inflammatory responses will require additional studies. This study nevertheless illustrates the potential for the host to regulate tightly the development of an excessive inflammatory response and thus to limit tissue pathology. However, it is unknown whether an appropriate host defense is compromised. J. Leukoc. Biol. 53: 309–319; 1993.
Cytotoxic cell proteinase gene expression and cytolytic activity by anti-CD3-activated cytotoxic T lymphocytes is sensitive to cyclosporin A but is not dependent on interleukin-2 synthesis
Authors: Kaiser Michael , Brooks-Kaiser Jane , Fitzpatrick Lyn , Bleackley R Chris , Hoskin David W
Published: January 2018

We have examined the role of interleukin (IL) 2 in the expression of cytotoxic cell proteinases (CCP) 1 and 2, as well as in the induction of major histocompatibility complex (MHC)-unrestricted cytotoxic activity in murine T cell cultures following stimulation with anti- CD3 monoclonal antibody. A dramatic reduction in CCP-1 and CCP-2 gene expression and near absence of cytolytic activity was shown to occur in these cultures when the expression of IL-2 was inhibited by 10−6 M cyclosporin A (CsA). The inhibitory effect of CsA could not be eliminated by the addition to culture of recombinant IL-2 at concentrations typically present in anti- GD3-stimulated T cell culture supernatants. Furthermore, when endogenous IL-2 (45-60 U/ml) present in anti-CD3-stimulated T cell cultures was neutralized with anti-mouse IL-2 antibody there was no effect on CCP-1 and CCP-2 mRNA expression and only a slight decrease in cytolytic activity. The expression of CCP-1 and CCP-2 gene products and the induction of MHC-unrestricted cytotoxic activitv in anti-CD3-stimulated T cell cultures therefore occur independently of IL-2 synthesis but are regulated by a CsA-sensitive mechanism.