bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium Is Required for PMA Induced Superoxide Release From Human Neutrophils
Authors: Ishihara Y , Rosolia D L , McKenna P J , Peters S P , Albertine K H , Gee M H
Published: January 2018

Experiments were designed to reexamine the relationship between extracellular calcium and superoxide generation in phorbol myristate acetate (PMA) stimulated neutrophils exploiting a newly adapted method to measure superoxide anion (O2-) generation from adherent cells stimulated at high and low cell density. Human neutrophils were plated in microtiter wells in cell densities of either 0.2 or 2.0 million cells/well. Superoxide release was measured sequentially over 60 min by reduction of ferricytochrome c. Cells were maintained in 1 mM Ca++ or 0 mM Ca++ Hanks' buffer for 60 min prior to activation as well as during measurement of O2-. In 1 mM Ca++, 2.0 million adherent neutrophils released 10.7 ± 1.2 nmol O2- in 20 min (n=4). O2- release was not significantly different for high density cells incubated and stimulated in 0 mM Ca++. In the presence of 1 mM Ca++, 0.2 million adherent neutrophils released 6.3 ± 0.5 nmols O2- in 20 min. With cells stimulated at low density, PMA stimulated O2- release was significantly decreased (3.0 ± 0.6 nmol O2- in 20 min) as was the initial rate of secretion of O2- in the absence of extracellular calcium. Basal release of superoxide was also greater in the presence of 1 mM Ca++ (0.96 nmol/20 min) compared to basal release in 0 mM Ca++ (0.22 nmol/20 min). Additional experiments with 0.2 million cells/well showed that extracellular Ca++ was required during stimulation with PMA and that prior incubation of cells for up to 60 min in 0 mM Ca++ had no effect on O2- release measured in the presence of calcium. Furthermore, PMA stimulated O2- was independent of verapamil (10-5–10-7 M), suggesting that voltage-dependent calcium channels do not participate in this response. The planar areas for unstimulated neutrophils in 0 mM Ca++ increased after addition of PMA. Unstimulated cells in 1 mM Ca++ tended to be larger and planar areas did not increase after PMA. These studies demonstrate that PMA stimulated O2- secretion is dependent on extracellular calcium particularly when adherent neutrophils are stimulated at low cell density. Furthermore, extracellular calcium at a concentration of 1 mM primes neutrophils by increasing basal secretion of O2- and increasing superoxide release after a maximum stimulating dose of PMA.
Synergistic Effect of Interferon-γ and Tumor Necrosis Factor-α on Antiviral Activity and (2′–5′) Oligo (A) Synthetase Induction in a Myelomonocytic Cell Line
Authors: Wietzerbin Juana , Gaudelet Corinne , Catinot Liliane , Chebath Judith , Falcoff Rébéca
Published: January 2018

TNF was not observed to have an antiviral effect on either HL60 or U937 cells. However, it did significantly enhance interferon (IFN)-γ-mediated antiviral activity in U937 cells. Treatment of U937 cells with IFN-γ enhanced (2′–5′) oligo (A) synthetase activity (2.5-fold) but treatment with TNF did not Combined treatment with TNF-α + IFN-γ increased this activity dramatically (20–40-fold). This increase correlated with the very large increase in the (‘2–5’) oligo (A) synthetase mRNA level in U937 cells. No such effects were observed in HL60 cells. Furthermore, binding studies and cross-linking analysis showed that IFN-γ modulated TNF-α receptors in U937 cells without altering their properties, but did not do so in HL60 cells.
Macrophages From C3H/HeJ Mice Require an Additional Step To Produce Monokines: Synergistic Effects of Silica and Poly(I:C) in the Release of Interleukin 1
Authors: Souvannavong Vongthip , Adam Arlette
Published: January 2018

Resident macrophages from C3H/HeJ mice, in contrast with those of other strains of mice such as BDF1 mice, did not release a 35 kD m.w. factor having macrophage replacing activity (FRM) or interleukin 1 (IL-1) when, respectively, cultured alone or in the presence of silica. C3H/HeJ macrophages were nevertheless capable of producing an intracellular IL-1-like activity. In addition, after a two-step activation process, macrophages from BDF1 mice spontaneously released IL-1, whereas silica was required to induce the release of IL-1 from similarly treated C3H/HeJ macrophages. Such in vivo primed and in vitro stimulated macrophages failed to release FRM. In contrast, poly(l:C) was able to induce the release of FRM by C3H/HeJ macrophages but not that of IL-1; moreover, the addition of silica to poly(l:C)-stimulated cells led to an IL-1 release similar to that obtained with normal mice treated with silica alone. Since poly(l:C) is able to elicit the production of interferons (IFN), the involvement of IFNs was investigated in poly(l:C) activity. Neither IFN-α/β nor IFN-γ, when used alone or in the presence of silica, could induce the release of IL-1 by C3H/HeJ macrophages. In addition, antibodies to IFN-α/β and IFN-γ were unable to affect the poly(l:C) and silica induced release of IL-1. Thus, the signal provided by poly(l:C) does not appear to be mediated by IFN(s).
Tumoricidal Effector Mechanisms of Murine BCG-Activated Macrophages: Role of TNF in Conjugation-Dependent and Conjugation-Independent Pathways
Authors: Klostergaard Jim , Stoltje Paul A , Kull Frederick C
Published: January 2018

The roles of secreted and membrane-associated TNFs were investigated in activated macrophage cytolysis of L929, EMT-6, and P815 targets. While all three targets were susceptible to cytolysis in coculture, an anti-TNF antiserum blocked lysis of L929 and EMT-6 but not of the P815 targets. Of the three targets, recombinant human or mouse TNF could only lyse the L929 target; despite the fact that a role for TNF was invoked in lysis of EMT-6 targets in coculture, the latter was strongly resistant to soluble rTNF, even at concentrations 30–40-fold higher than the Ka for Its TNF-receptor. Cytolysis of the L929 target occurred when it was cocultured with BCG-activated macrophages even when these effector cells did not secrete TNF, either due to prior chemical crosslinking or to lack of exposure to a triggering level of lipopolysaccharide. Furthermore, by introduction of the anti-TNF antiserum over a dose-range, it was shown that macrophage cytolysis both of L929 and EMT-6 targets occurred in the absence of bioavailable, fluid-phase TNF. Thus, even for targets susceptible to fluid-phase TNF, TNF-dependent, direct macrophage-mediated cytolysis appears to be a function independent of secreted TNF and one that utilizes effector-target contact to express the action of a membrane form of the molecule.
Binding of the T Cell Activation Monoclonal Antibody Ta1 to Dipeptidyl Peptidase IV
Authors: Barton Randall W , Prendergast Jay , Kennedy Charles A
Published: January 2018

The monoclonal antibodies, Ta1 and IOT15, define T cell activation cell surface markers and have been assigned to the CD26 leukocyte differentiation antigen cluster. Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) is an exoaminopeptidase that, among leukocytes, is expressed almost exclusively on activated T cells. Comparative binding studies showed that the Ta1 mAb binds to DPP IV purified from human placenta as well as in extracts of the human YT lymphoid cell line and of CD3 stimulated normal human peripheral blood mononuclear cells. The mAb IOT15 did not bind to DPP IV from any source even upon repeated incubations. Western blot analysis of YT cell extracts revealed that Ta1 and IOT15 bound to distinctly different molecular weight molecules. Immunofluorescent cell surface capping experiments showed that capping of the IOT15 did not alter the surface distribution of the Ta1 fluorescence. The capping results combined with the DPP IV binding results indicate that IOT15 and Tal mAb's bind to different, apparently unassociated, molecules on the surface of T cells and that only Ta1 binds the T cell surface enzyme DPP IV.
Assessment of Chemokinetic Behavior of Inflammatory Lung Macrophages in a Linear Under-Agarose Assay
Authors: Newton-Nash Debra K , Tonellato Peter , Swiersz Melanie , Abramoff Peter
Published: January 2018

Migration of cells in response to a chemoattractant gradient is influenced by directed migration (chemotaxis) and stimulated random motility (chemokinesis). The present study quantitated the chemokinetic motility of normal and inflammatory lung macrophages by performing the linear under-agarose assay in the presence of uniform concentrations of chemoattractant. Under these conditions, cell motility can be likened to a molecular diffusion process. Mathematical analyses which describe molecular diffusion were then applied, allowing the quantitation of the parameter, μ, the cellular equivalent to the molecular diffusivity constant. Determination of changes in μ as a function of chemoattractant concentration revealed that the chemokinetic motility of alveolar macrophages recovered during the early stages of acute pulmonary inflammation was greater than that of normal alveolar macrophages and macrophages recovered later in the inflammatory response. The correlation of differences in macrophage chemokinesis with macrophage maturation and the relevance of these differences to macrophage accumulation during inflammation are discussed.
Multiple Receptors on Human Monocytes Are Involved in Adhesion to Cultured Human Endothelial Cells
Authors: Carlos Timothy M , Dobrina Aldo , Ross Russell , Harlan John M
Published: January 2018

Monocytes exhibit significant basal (unstimulated) adherence to human umbilical vein endothelium (HUVE), which is only partially inhibited by an anti-CD18 monoclonal antibody (mAb) (60.3). We examined factors modulating the residual, CD18-independent monocyte binding to HUVE by pretreating monocytes with mAb 60.3 to eliminate CD18-dependent binding. Basal adherence was reduced from 32% ± 2% to 14% ± 2% with mAb 60.3 (means ± SE of eight experiments; P < 0.01). mAb 60.3-treated monocytes were incubated with tumor necrosis factor-γ (TNF-α), interleukin-1 (IL-1), lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalamine (FMLP), or phorbol myristate acetate (PMA). Only PMA affected CD18-independent binding. Pretreatment with PMA alone reduced adherence to 21% ± 2% (mean ± SE of eight experiments; P < 0.01). In conjunction with mAb 60.3, PMA virtually eliminated monocyte adherence to HUVE (7% ± 1%, mean ± SE of eight experiments; P < 0.01). We also examined CD18-independent monocyte binding to endothelial-leukocyte adhesion molecules (E-LAMs) induced by pretreatment of HUVE with LPS. Monoclonal antibody 60.3-treated monocytes increased adherence from 14% ± 2% with unstimulated HUVE to 37% ± 2% with LPS-stimulated HUVE (mean ± SE of four experiments; P < 0.01). Monocytes pretreated with both mAb 60.3 and PMA increased adherence from 5% ± 1% with the unstimulated HUVE to 18% ± 1% with the LPS-stimulated HUVE (mean ± SE of four experiments; P < 0.01). This result implies the presence of a CD18-independent and PMA-insensttive receptor on human monocytes for an E-LAM induced by LPS. In summary, we have identified two CD18-independent mechanisms of monocyte adherence to HUVE; a PMA-sensitive mechanism mediating basal adherence and a PMA-insensitive mechanism involved in binding to E-LAMs.
Decreased Fc Receptor Expression on Macrophages Following Simple Hemorrhage as Observed by Scanning Immunoelectron Microscopy
Authors: Rana M Waheed , Ayala Alfred , Dean Richard E , Chaudry Irshad H
Published: January 2018

Although it is known that macrophage (Mφ) functions such as phagocytosis and antigen presentation are depressed following hemorrhage and resuscitaion, the mechanism remains unknown. The aim of this study was to determine, using scanning immunoelectron microscopic techniques, whether there is any alteration in the Fc receptors on the Mφ after hemorrhage. To study this, male C3H/HeN mice were bled to a mean blood pressure (BP) of 35 mm Hg and maintained at that pressure for 1 hr, then resuscitated with their own blood and adequate fluids. Twenty-four hrs later, Kupffer cells from livers and splenic adherent cells were isolated, incubated for 16 hr, and then exposed to polysterene beads conjugated with antimouse IgG that specifically binds to Fc receptors. The cells were then prepared for observation by scanning electron microscopy. At least 100 cells from each animal were examined. The number of Kupffer cells from posthemorrhage mice that exhibited specific receptor labeling was significantly decreased (41.0 ± 2.6, P < 0.05) compared with control (64.2 ± 7.5). The number of splenic adherent cells from posthemorrhage mice exhibiting specific receptor labeling was also significantly decreased (35.7 ± 2.5, P < 0.01) compared with control (61.2 ± 3.9). The internalization of markers was also seen in some cells. The cause of the decrease in receptor labeling following hemorrhage may be the loss, inactivation, and/or internalization of receptors. Thus the decreased number of functional macrophages may contribute to the depression of antigen presentation and to the enhanced susceptibility to sepsis following hemorrhage.
Effects of TNFα on the Expression of Class II MHC Molecules in Macrophages Induced by IFNγ: Evidence for Suppression at the Level of Transcription
Authors: Melhus O , Koerner T J , Adams D O
Published: January 2018

Tumor necrosis factor-α (TNFα) induces surface expression of class II major histocompatibility (MHC) molecules (la molecules) in many cells, including macrophage-like cell lines. When we tested the effects of this cytokine on murine peritoneal macrophages, TNFα had little effect on surface expression of la. The strong expression of such molecules induced by interferon-γ (IFN-γ) was, however, suppressed moderately by TNFα. These effects were reflected at the level of specific messenger RNA (mRNA) as detected by Northern blot analysis. Furthermore, the locus of control appears to be transcriptional; in nuclear run-on assays, TNFα suppressed the IFNγ-induced enhancement of transcription for the murine β-chain of l-A (l-Aβ.). Taken together the data suggest that TNFα has little effect on class II MHC genes and surface expression in murine peritoneal macrophages, that TNFα is a modest suppressant of such molecules when their levels are raised by IFNγ, and that these suppressive effects are mediated at the level of transcription.
Cystatin C and Cathepsin B Production by Alveolar Macrophages From Smokers and Nonsmokers
Authors: Warfel Alwin H , Cardozo Christopher , Yoo Ok Hi , Zucker-Franklin Dorothea
Published: January 2018

The capacity of alveolar macrophages (AM) obtained from smokers and nonsmokers to secrete cathepsin B and its inhibitor cystatin C was examined because of the concept that an imbalance in the production of proteolytic enzymes and/or their inhibitors could be responsible for the lung damage seen in smokers. Quantitation of immunoprecipitates on Western blots showed that the amount of total cystatin C secreted into the culture medium by AM of smokers was significantly greater than the amount secreted by cells obtained from nonsmokers, whereas the difference between the amount of cathepsin B secreted by the AM of smokers and that from nonsmokers did not appear significant. The cystatin C found in the medium conditioned by AM of nonsmokers appeared to be more heterogeneous in molecular size, presenting either as a single band of about 14 Kd or as a high-molecular-weight triplet of about 69 Kd, 63 Kd, and 57.3 Kd. Furthermore, in some cases there were single or doublet bands at 14 Kd as well as the high-molecular-weight triplets. In contrast, smokers AM-conditioned medium uniformly possessed both the low-and the high-molecular-weight cystatin C. Cathepsin B was not detected in Western blots at its reported molecular weights but was identified at the exact area occupied by the higher molecular weight cystatin C, i.e., at bands corresponding to 69 Kd, 63 Kd, and 57.3 Kd. Therefore, it is clear that in culture media of AM, cystatin C and cathepsin B are present as proteinase–antiproteinase complexes. The observation also suggests that in smokers an excess of cystatin C may be elaborated, which, if further substantiated, would show for the first time a likely role for this proteinase inhibitor in vivo.
Modulation of Mycobacterium avium Growth in Murine Macrophages: Reversal of Unresponsiveness to Interferon-Gamma by Indomethacin or Interleukin-4
Authors: Denis Michel , Gregg Evan O
Published: January 2018

The ability of soluble factors to modulate the growth of a virulent strain of Mycobacterium avium in murine peritoneal macrophages was studied. The virulent strain, TMC 702, grew progressively in the organs of susceptible BALB/C mice. In addition, this strain of M. avium grew progressively in untreated peritoneal macrophages. Treatment of macrophage monolayers with interferon-gamma (IFN-γ) did not change significantly the intracellular growth of M. avium. Addition of indomethacin to IFN-γ-treated macrophage monolayers rendered them significantly more bacteriostatic than macrophages treated with interferon alone, suggesting a role for prostaglandins in inducing unresponsiveness to IFN-γ in infected cells. Additionally, treatment with tumour necrosis factor-alpha led to a modest increase in bacteriostasis, as compared to untreated monolayers. Further experiments with recombinant interleukins showed that interleukin-4 (IL-4), on its own, could increase bacteriostatic activity against M.avium in a reproducible fashion. Experiments with interleukin combinations showed that IFN-γ and IL-4 treatment of macrophages rendered these cells almost fully bacteriostatic against M. avium. Inclusion of scavengers of reactive oxygen species did not modify the beneficial effect of IFN-γ and IL-4. Overall, our results suggest an important role for interleukins in modulating the interaction between virulent mycobacteria and murine macrophages.
Monokines Mediate Decreased Hepatic Glucocorticoid Binding in Endotoxemia
Authors: Bengtsson Torbjörn , Dahlgren Claes , Stendahl Olle , Andersson Tommy
Published: January 2018

Several studies indicate that the actin filament system is not only involved in cell motility, but also in the regulation of other neutrophil functions. The aim of the present investigation was to examine the mechanisms by which actin filament formation participates in the control of the respiratory burst and degranulation in human neutrophils. The approach taken was to use both an inhibitor (cytochalasin B) and a potentiator (tetracaine) of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe)-induced actin polymerization. The total inhibition of fMet-Leu-Phe-induced actin polymerization in cytochalasin B-treated cells was accompanied by an impressive potentiation of both oxidase activity and degranulation (azurophilic and specific granules). However, preincubation with tetracaine, which causes an enhanced accumulation of F-actin in the periphery of fMet-Leu-Phe-stimulated cells, also augmented the rate and duration of peptide-induced superoxide anion production, but inhibited degranulation (specific granules). A likely explanation for the potentiating effects of cytochalasin B and tetracaine is provided by our observation that both of these substances reduced the acid-resistant binding of fMet-Leu-Phe (interpreted as a decrease in the internalization of fMet-Leu-Phe-receptor-complexes), resulting in an enhanced formation of second messengers (diacylglycerol). The findings that tetracaine potentiated the activity of the oxidase, whereas it inhibited degranulation (specific granules), suggest that actin polymerization per se plays a role in the latter process. Consequently, this study argues against the idea of a direct inhibitory effect of F-actin on chemotactic factor-induced oxidase activation, but supports an active role of actin filaments in the translocation and release of granule components.
Restoration of Prostaglandin Releasing Macrophage Populations in Lethally Irradiated Mice With Spleen Cells From Bone Marrow-Depleted Donors
Authors: Shibata Yoshimi , Volkman Alvin
Published: January 2018

Previous studies in mice severely depleted of bone marrow cells by 89Sr showed persistent monocytopenia and impaired expression of prostaglandin E2-releasing splenic macrophages (PGSM) despite the occurrence in the spleen of more than 10-fold increases in pluripotential stem cells and Mo colony-forming cells. To determine whether the observed deficits were due to a lack of precursors of blood monocytes and PGSM in the spleens of 89Sr-treated mice, radiation chimeras were established by iv infusion of 2 × 106spleen cells from 89Sr donor CBA/J or semisyngeneic B6CB F1 hybrid mice into lethally γ-irradiated CBA/J recipients. Blood monocyte levels were greater than normal by day 14 and PGSM induced by Corynebacterium parvum were demonstrated by day 28. These restored Mø populations expressed the donor haplotype detected in vitro with haplotypespecific monoclonal anti-H-2K plus complement. “Sr treatment of the chimeras resulted in profound depletion of monocytes and PGSM. The data indicate that the spleen of the 89Sr-treated mouse, which is an ineffective source of circulating monocytes and PGSM, contains cells which can generate both of these populations following infusion into lethally irradiated recipients. Since the bone marrow of such recipients was capable of being repopulated, the aggregate observations suggest that functional bone marrow is obligatory for the generation of blood monocytes and PGSM populations.
Influence of Lactoferrin on the Function of Human Polymorphonuclear Leukocytes and Monocytes
Authors: Gahr Manfred , Speer Christian P , Damerau Bernd , Sawatzki Günther
Published: January 2018

Polymorphonuclear leukocytes (PMN) exposed to highly purified human lactoferrin (from colostrum) exhibit an increased random motility (at least 2.5-fold) and are primed to produce more superoxide [12.1 ± 1.2 nmol O2-/min/106 PMN preincubated with lactoferrin (0.5 mg/ml) against 6.4 ± 2.3 with cells without lactoferrin after FMLP stimulation]. The action of lactoferrin seemed to be specific, because it could be abolished by simultaneous addition of antilactoferrin antibody. Addition of transferrin and iron salts to PMN was without effect. Between iron-poor and iron-saturated lactoferrin there was no difference in influence on PMN function except for a higher FMLP stimulated superoxide production by iron-saturated lactoferrin. Aggregation, degranulation (β-glucuronidase, lysozyme), and bacterial killing were not influenced by lactoferrin. Incubation of monocytes and monocyte-derived macrophages with lactoferrin did not alter their motility or their superoxide production rates. Our findings indicate that PMN become more effective after exposure to lactoferrin by having a greater motility and producing superoxide at a faster rate.
Role of Degranulation in Activation of the Respiratory Burst in Human Neutrophils
Authors: Manara F Suzi , Chin Jean , Schneider Donald L
Published: January 2018

The neutrophil response to infection and inflammation includes membrane fusion or degranulation and activation of the membranous respiratory burst oxidase. The role of degranulation in the activation of the burst was explored in resting and activated cells. Exposed membrane proteins of intact cells were labeled with impermeant reagents. Phorbol ester-activated neutrophils and enucleated cells which are granule depleted both exhibit increased labeling with [125l]lactoperoxidase over that of resting cells. The binding of antibodies to granule membranes by cells activated with phorbol ester or treated with cytochalasin B and lithium chloride were similarly increased. These data indicate that insertion of granule membrane into the cell membrane occurs during activation and enucleation of neutrophils.

Hyperosmolarity, known to inhibit degranulation, also exhibited an inhibitory effect on the respiratory burst oxidase in the presence of phorbol ester or latex. Pre-treatment of cells with phorbol ester followed by an increase in osmolarity, however, still resulted in activation. Temperatures below 17°C abruptly and simultaneously abolish degranulation and activation of the respiratory burst oxidase. Pre-treatment of neutrophils with phorbol ester at 37°C, followed by measurement of oxidase activity at decreased temperatures, on the other hand, revealed a linear Arrhenius plot above and below 17°C. These results suggest that membrane fusion or degranulation is a step in activation of the respiratory burst.
Plasma Membrane Appearance of Phosphatidylethanolamine in Stimulated Macrophages
Authors: Sandra Alexander , Cai Jun
Published: January 2018

Mouse peritoneal macrophages were labeled with [1-3H]ethanolamine, and the presence of radioactive [3H]phosphatidylethanolamine (PE) at the plasma membrane was monitored by reacting the cells with trinitrobenzene sulfonic acid (TNBS) under nonpenetrating conditions. Macrophages stimulated with either the calcium ionophore A23187 or zymosan demonstrated a larger proportion of radiolabeled PE in the plasma membrane than control, nonstimulated cells. In experiments in which macrophages were labeled with ethanolamine for increasing times, appearance of membrane 3[H]PE was stimulated as early as after 2 hr of labeling. Macrophages labeled for 24 hr, then stimulated and returned to fresh medium still reflected a higher amount of membrane 3[H]PE at 2 hr after the stimulation, suggesting stimulation results in long-term alterations in plasma membrane lipids. Protease-peptone-elicited macrophages, which are not stimulated by zymosan or ionophore, did not exhibit an increase in membrane 3[H]PE upon stimulation. The size of the TNBS-accessible radiolabeled PE pool increased proportionately with a second stimulation; however, a subsequent labeling of the cells with TNBS after brief warming increased the TNBS-accessible pool in control cells only. As shown in previous studies, macrophage stimulation resulted in an increased incorporation of lipid precursors into phospholipid. The mass of plasma membrane Tnp-PE relative to mass of PE was not increased in ionophore-treated macrophages in contrast to a small (~22%) increase in zymosan-treated cells. These results are suggestive of alterations in lipid synthesis in stimulated macrophages and possible long-term changes in the structure and function of the plasma membrane of macrophages following stimulation.
Regulation of Interferon Production by Human Monocytes: Requirements for Priming for Lipopolysaccharide-Induced Production
Authors: Hayes Mark P , Enterline Joan C , Gerrard Theresa L , Zoon Kathryn C
Published: January 2018

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-α/β). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-l,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-γ (IFN-γ). The IFN secreted is of the α subtype. Monocytes primed with GM-CSF or IFN-γ also maintained LPS responses for production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-α and promoted monocyte survival. Northern analysis indicated that the induction of IFN-α by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-α by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.
Selective Inhibition of Natural Killer but not Natural Cytotoxic Activity in a Cloned Cell Line by Delta-9-Tetrahydrocannabinol
Authors: Wang Min , Richards Allen L , Friedman Herman , Djeu Julie Y
Published: January 2018

Natural killer (NK) and natural cytotoxic (NC) activities are spontaneously generated against certain tumors in vitro and their contribution to tumor immunity is being extensively investigated. We report here that the interleukin-2 (IL-2)–dependent murine cell line, NKB61A2, which we recently found to express both NK and NC functions, can be modulated selectively by 9δ-tetrahydrocannabinol (THC). THC, a major psychoactive metabolite of marijuana, could significantly inhibit NK activity without altering NC activity in NKB61A2 cells. Inhibition of NK function occurred at a post-binding stage because effector/target conjugation was unaffected by THC. With regard to NC function, neither the cytotoxic activity of the cells nor release of tumor necrosis factor was interrupted by THC. Therefore, THC may provide a useful tool for dissociating the mechanism of NK and NC activities within a single population of cells.
Metabolism of Leukotriene B4 via Reduction Into Dihydro-Leukotriene B4 in Human Monocytes, Alveolar Macrophages, and U-937 Cells
Authors: Schönfeld W , Kasimir S , Knöller J , Jablonski K , König W
Published: January 2018

The biological effects of leukotriene B4 (LTB4) within the microenvironment are controlled by rapid inactivation. In this regard human granulocytes convert LTB4 into ω-oxidated products (20-OH-LTB4 and 20-COOH-LTB4); moreover, we recently described the formation of unpolar metabolites of LTB4 in human tonsillar and lung macrophages. By means of high performance liquid chromatography (HPLC) we identified the main metabolite of LTB4 as dihydro-LTB4 (5,12-dihydroxyeicosatrienoic acid). Studies on a lymphocyte (74–78%), monocyte (19–22%), and basophil (<4%) containing cell fraction isolated from peripheral blood as well as peripheral monocytes purified by elutriation centrifugation revealed evidence that these cells metabolize LTB4 to a very low degree if incubated immediately after isolation. However, after culture for 24–72 h these cells showed a strongly increased capacity to metabolize LTB4. The pattern of metabolites in this cell fraction was identical to bronchoalveolar macrophages (purity >95%). Similarly, the LTB4-reductase was expressed in differentiated human monocytic U-937 cells almost 5–7 h after the addition of dimethylsulfoxide (1.3%) or phorbol-myristate-acetate (16 nM). The expression of this pathway was blocked in the presence of cycloheximide (10 μg/ml) whereas actinomycin (3.8 μg/ml) had no effects. Dihydro-LTB4 was further metabolized by granulocytes probably via ω-oxidation; therefore, several metabolites could be detected by radioactive high performance liquid chromatography (HPLC) after incubation of bronchoalveolar cells consisting of macrophages and granulocytes with 3H-LTB4. Our data provide evidence for a unique role of macrophages to control the level of LTB4 by generation as well as metabolism into dihydro-LTB4.
Modulation of Tumor Necrosis Factor and Gamma Interferon Production by Cocaine and Morphine in Aging Mice Infected With LP-BM5, a Murine Retrovirus
Authors: Chen Guan-jie , Watson Ronald Ross
Published: January 2018

The actions of retroviral infections, aging, and cocaine and morphine injection on cytokine production were investigated in C57BL/6 female mice. Retroviral infection with LP-BM5 murine leukemia virus was further developed as a model of murine acquired immunodeficiency syndrome (AIDS). The effects of cocaine and morphine on gammainterferon (IFN) and tumor necrosis factor (TNF) production in vivo and with isolated spleen cells were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) method. Serum IFN was generally not detected in any group except mice injected with saline and young mice infected with LP-BM5 virus. Splenocytes from mice with murine AIDS produced less IFN when stimulated in vitro by ConA. In aged mice, IFN production by spleen cells was severely suppressed by retroviral infection. Cocaine had a tendency to suppress IFN production by stimulated cells in vitro. Morphine tended to reduce IFN production by spleen cells from retrovirally infected animals.

The serum TNF level in mice with murine AIDS was elevated creating higher levels in morphine and morphine plus cocaine treated uninfected mice while cocaine injection eliminated serum TNF. When stimulated in vitro by lipopolysaccharides (LPS), splenocytes from mice with murine AIDS also produced more TNF than uninfected controls. TNF production in vitro and in vivo was significantly increased by retroviral infection. Therefore, results indicate that cocaine and retroviral infection modulate TNF and IFN production.
Murine Peritoneal Macrophage Gangliosides Inhibit Lymphocyte Proliferation
Authors: Berenson Charles S , Ryan John L
Published: January 2018

Gangliosides have been shown to act as immunoregulatory agents by altering proliferative responses of lymphocytes to both antigens and mitogens. Most early studies have utilized brain gangliosides and have required high concentrations. The role of endogenous gangliosides from macrophages has remained unexplored. In this study, thioglycollate-elicited murine peritoneal macrophage gangliosides were purified and added to cultures of murine lymphocytes. Nanogram amounts caused a profound inhibition of LPS-induced DNA synthesis of splenocytes and of purified B lymphocytes, without demonstrable cellular toxicity. No effect was seen using asialo-GM1. This effect was present across a wide range of lipopolyssacharide (LPS) doses. Nanogram amounts of macrophage gangliosides also inhibited concanavalin A (ConA)-mediated lymphocyte proliferation. Inhibition of LPS-induced mitogenesis was present even if gangliosides were removed from the extracellular environment after 15–60 min of incubation prior to the addition of LPS. This inhibition was reversible with incubation of ganglioside pre-treated lymphocytes in medium containing serum. These inhibitory properties of macrophage gangliosides are distinct from those found in studies using brain gangliosides, and support a potential role for macrophage gangliosides as negative modulators of lymphocyte proliferation.
The Liver and the Hematolymphoid System: I. The Regulation of Nylon-Passed Spleen Cell Proliferation by Active Factors Released From Syngeneic Nonparenchymal Liver Cells
Authors: Mabuchi Ayako , Komuro Toshiro , Saizawa Takashi , Sakamoto Toshiki , Watari Eiji , Yokomuro Kozo
Published: January 2018

Nylon-passed spleen cells were found to proliferate when cultured with syngeneic nonparenchymal adherent liver cells and their culture supernatants. The supernatants contained IL-1, IL-6, GM-CSF, and IFN (α + β) activities but not IL-2 and IL-3 activities. The IFN level was higher in early culture sup (2–24 hr) than in later culture sup (48–72 hr). Proliferation was greatly increased by anti-IFN (α + β) serum in the spleen cells cultured in the earlier sup. This antiserum increased the spleen cell proliferation only slightly in the later culture sup. This suggests that nonparenchymal liver cells produce two factors, one having a suppressor, and the other an enhancer action, with IFN being one of the suppressor factors. With culture time, DNA synthesis of spleen cells increased and IL-2 and IL-3 activities were generated in the culture sup. Cells proliferated during culture were found to be morphologically lymphocytes, granulocytes, and macrophages. The mechanisms by which nonparenchymal liver cells regulate the hematolymphoid system are discussed based on our observations.
Involvement of Fibronectin and Its Receptor in Human Lymphocyte Proliferation
Authors: Klingemann Hans G , Kohn Fred R
Published: January 2018

Adhesion between lymphocytes and antigen-presenting cells is necessary for the development of certain immune reactions. We have previously shown that fibronectin (FN) added to mixed lymphocyte cultures (MLC) can restore a decreased lymphocyte proliferation in immunocompromised individuals. Using highly purified cell populations from peripheral blood for depletion and adding back experiments we show here that exogenous FN enhanced proliferation only when allogeneic monocytes were co-cultured with responder lymphocytes. Although lymphocyte proliferation in MLC was augmented by FN, there was no preferential proliferation of any particular major lymphocyte subpopulation in cultures supplemented with FN as compared to control cultures lacking its addition. Antibody against the FN receptor (FN-R) of the β1 integrin family, as well as Arg-Gly-Asp containing peptide, could inhibit alloantigen-induced lymphocyte proliferation in a concentration-dependent manner. Anti–CD3-lnduced proliferation was inhibited by anti–FN-R antibody but not Arg-Gly-Asp peptide whereas no inhibition was seen with the phytohemagglutinin (PHA)-induced lymphocyte proliferation. This study presents further evidence that FN and its receptor (α5β1) are involved in the augmentation of T-cell responsiveness to proliferative stimuli.
Involvement of Cytokines in Determining Resistance and Acquired Immunity in Murine Tuberculosis
Authors: Denis Michel
Published: January 2018

Herein we demonstrate that continuous infusion of either TNF-α or IFN-γ (104 U/day) via osmotic micropumps leads to an increased resistance of mice infected with a lethal dose (107) of M. tuberculosis H37Rv, associated with a decreased microbial growth in all target organs. This result was reinforced by the finding that infusion of antibodies against TNF-α or IFN-γ greatly enhanced susceptibility of naive mice to tuberculosis. In a final set of experiments, using neutralizing antibodies, we show that IFN-γ, not TNF-α is involved in determining acquired resistance against murine tuberculosis, as seen by the fact that acquired immunity is resistant to anti-TNF-α antibodies, yet sensitive to anti-IFN-γ antibodies. This suggests a role for both IFN-γ and TNF-α in determining innate resistance whereas IFN-γ may be the mediator of the anamnestic response.
Fibronectin fragments stimulate tumor necrosis factor secretion by human monocytes
Authors: Beezhold Donald H , Personius Christine
Published: January 2018

Fibronectin (Fn) is a high molecular-weight glycoprotein that can influence many aspects of monocyte function. The purpose of this study was to determine whether Fn could stimulate monocyte tumor necrosis factor (TNF) secretion. Monocytes were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation and adherence to plastic (2 h). Plasma Fn was purified from the blood by gelatin-Sepharose affinity chromatography. Monocytes were stimulated with Fn for 18 h and the supernatants were assayed for TNF activity using the L929 bioassay. Intact Fn stimulated the secretion of TNF in a dose-dependent manner. Intact Fn–induced TNF secretion by monocytes was inhibited (50%) but not eliminated by the addition of the R-G-D–containing peptide GRGDSP. Limited proteolysis of the Fn molecule using insoluble chymotrypsin resulted in a fragment preparation that was dramatically more stimulatory than the intact Fn preparation. High-performance liquid chromatographic (HPLC) purification of the fragments demonstrated that at least two fragments were capable of stimulating TNF secretion. Further purification by affinity chromatography and HPLC localized the stimulatory activity to the 120-kd cell-binding fragment. The possibility that the stimulatory activity was the result of endotoxin contamination was ruled out using macrophages from C3H/Hej mice. These results suggest that Fn fragments are potentially important molecules for activation of monocytes and may stimulate monocyte cytotoxic activity.
In vivo testing confirms a blunting of the human cell-mediated immune mechanism during space flight
Authors: Taylor Gerald R , Janney Robert P
Published: January 2018

The cell-mediated immune (CMI) mechanism was evaluated in 10 space shuttle astronauts by measuring their delayed-type hypersensitivity response to seven common recall antigens. The Multitest CMI test system was used to administer antigens of tetanus, diphtheria, Streptococcus, Proteus, old tuberculin, Candida, and Trichophyton to the forearm 46 h before nominal mission termination; readings were conducted 2 h after landing. The mean number of reactions was reduced from 4.5 preflight to 3.0 inflight, and the mean reaction score was reduced from 21.4 to 13.7 mm inflight. The data presented suggest that the CMI system is still being degraded by space flight conditions on day 4 and that between day 5 and day 10, the depression maximizes and the system begins to adjust to the new conditions. The relation of these in vivo findings to previously reported in vitro results is discussed. J. Leukoc. Biol 51: 129–132; 1992.
Fc receptor expression, concanavalin A capping, and enzyme content of bovine neonatal neutrophils: a comparative study with adult cattle
Authors: Zwahlen Roland D , Wyder-Walther Marianne , Roth Daniel R
Published: January 2018

The increased susceptibility of newborns to infection may in part be related to impaired in vitro functions of neonatal polymorphonuclear neutrophils (PMNs). To evaluate early steps in the activation cycle of bovine PMNs we determined the expression of Fc receptors (FcRs) with an erythrocyte rosetting assay utilizing bovine anti-sheep immunoglobulin G2 IgG2 and the accumulation of ligand receptor complexes or “caps” with fluorochrome-coupled concanavalin A (Con A caps) on neutrophils from adult (A-PMN) and newborn (N-PMN) bovines. In addition, the levels of myeloperoxidase (MPO) and alkaline phosphatase (AP) were determined. FcR expression is reduced in N-PMNs (P < .001), in contrast to results observed with human N-PMNs. Basal capping of Con A binding sites is reduced (P < .05) in N-PMNs but is enhanced (P < .001) upon pretreatment with colchicine (0.5, 5.0, and 50.0 μm). These findings are again contrary to results observed with human N-PMNs. Consistent with findings in human neonates, however, are reduced levels of cellular MPO (P < .05) and elevated cellular AP (P < .001) in the neonate. The functional significance of elevated AP levels and altered Con A capping in N-PMNs is unclear. However, diminished expression of FcR could potentially contribute to impaired adherence and phagocytosis of bacteria, and reduced activity of neutrophil MPO could indicate weaker microbicidal capacity of neonatal cells. The demonstrated impairment of N-PMN functions could potentially contribute to reducing the effectiveness of the cellular host defense system in neonatal calves.
A neutrophil-derived proteolytic inactive elastase homologue (hHBP) mediates reversible contraction of fibroblasts and endothelial cell monolayers and stimulates monocyte survival and thrombospondin secretion
Authors: Østergaard Erik , Flodgaard Hans
Published: January 2018

Human heparin-binding protein (hHBP) is a recently discovered proteolytically inactive neutrophil elastase homologue with sequence identity to azurocidin and CAP37. The protein has antibacterial properties and chemotactic activity toward monocytes. In the present work, we show that monocytes, cultured under serum-free conditions, developed morphological changes and formed multicellular aggregates 4 h after the addition of hHBP at a concentration of 10 μg/ml. However, after prolonged incubation (11 days) with unchanged medium, the cells spread again. The hHBP-treated cells had a two- to threefold increase in survival compared to control cells, measured using trypan blue as an indicator of living cells. Differentiation of the alive cells to macrophages was detected by changes in morphology, a threefold increase in protein content, and a three- to fourfold increase in acid phosphatase activity. When monocytes in parallel experiments were labelled with [35S]methionine de novo synthesis and secretion of thrombospondin in a dose-dependent manner was observed after 16 h, with half-maximal secretion at 2 μg hHBP/ml and a maximal 12-fold increase in secretion with respect to controls at 16 μg/ml. Supplementary labeling with [35S]sulfate revealed that the same monocytes down-regulated the secretion of a large proteoglycan (300–400 kd), apparently also with a half-maximal decrease rate at 2 μg/ml hHBP. Exposure of confluent fibroblast and endothelial cell monolayers to hHPB (10 μg/ml) in the absence of fetal calf serum resulted in cell contraction leaving gaps between cells, the phenomenon being recognizable within 4 h after addition of hHBP. Addition of fetal calf serum to a concentration of 10% completely restored the monolayers. A unique role of hHBP in host defense involving recruitment of monocytes and a key funciton of hHBP in neutrophil extravasation in response to inflammatory chemotactic signals such as leukotriene B4, complement peptide G5a, and N-formyl-methionyl-leucyl-phenylalanine are suggested.
Pulmonary surfactant phospholipids modulate priming of rabbit alveolar macrophages for oxidative responses
Authors: Hayakawa Hiroshi , Giridhar Girish , Myrvik Quentin N , Kucera Louis
Published: January 2018

We investigated the effect of individual phospholipids contained in pulmonary surfactant (PS) on the macrophage-activating factor (MAF)-induced priming of rabbit alveolar macrophages (AMs) for oxidative responses elicited by phorbol myristate acetate (PMA) or opsonized zymosan (Op-Zym). AMs were incubated with MAF with or without phospholipids for 18 h. After incubation, oxidative responses were elicited with PMA (0.5 μg/ml) or Op-Zym (250 μg/ml) and monitored by chemiluminescence (GL) assays. The data indicate that natural surfactant inhibited MAF-induced priming of rabbit AMs for PMA- or Op-Zym-elicited oxidative responses. Artificial surfactant inhibited PMA-elicited CL responses but enhanced Op-Zym-elicited GL responses. Individual phospholipids differed in modulative activities. Dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylglycerol (DPPG), and phosphatidylinositol (PI) inhibited MAF-induced priming when the oxidative responses were elicited with PMA. Whereas DPPG inhibited Op-Zym-elicited oxidative responses, dipalmitoyl phosphatidylcholine (DPPC) and DOPC primed AMs for increased Op-Zym-elicited oxidative responses. DOPC did not affect the binding of phorbol dibutyrate to AMs, which suggests that reduced cell binding of phorbol ester was not responsible for the inhibition of PMA-elicited oxidative responses in AMs treated with DOPC. Similarly, DPPC, DOPC, and DPPG did not affect the number of zymosan particles phagocytosed by AMs compared to the control, which suggested that enhanced or reduced Op-Zym-elicited oxidative responses by phospholipids were not due to altered phagocytic activity of AMs. In conclusion, our data indicate that individual surfactant phospholipid differently modulates priming of AMs for oxidative responses, and the effect of individual phospholipids does not account for the effect of complete PS on priming of AMs.
Monocyte adherence to fibronectin: role of CD11/CD18 integrins and relationship to other monocyte functions
Authors: Owen Caroline A , Campbell Edward J , Stockley Robert A
Published: January 2018

Adherence of monocytes to extracellular matrix components is critical for their accumulation at sites of infection. To gain insight into the factors that regulate monocyte recruitment, we have studied monocyte adherence with regard to the regulatory effects of bacterial lipopolysaccharide (LPS) and the mechanisms involved; moreover, we have contrasted the phenotypes of adherent and nonadherent cells. Our results show that only a minor subpopulation of monocytes (20–25%) adhere spontaneously to fibronectin and that LPS stimulated a threefold increase in the proportion of adherent cells. Basal adherence and LPS-stimulated adherence of monocytes to fibronectin were substantially mediated by CD11/CD18 integrins. Further studies revealed that spontaneously adherent monocytes were 14-fold more actively phagocytic, released 1.6-fold more superoxide anion, and contained 20-fold more peroxidase activity than nonadherent cells, whereas LPS-adherent cells had an intermediate phenotype. These results indicate that LPS may enhance the accumulation of monocytes with an antimicrobial phenotype and thereby promote resolution of tissue infection.