bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular Survival of Virulent Bordetella pertussis in Human Polymorphonuclear Leukocytes
Authors: Steed Lisa L , Setareh Morey , Friedman Richard L
Published: January 2018

Little is known regarding the interaction of Bordetella pertussis with polymorphonuclear leukocytes (PMNL) or the role PMNL play as an initial line of defense against B. pertussis infection. An in vitro system was developed to establish conditions for the study of phagocytosis and killing of virulent B. pertussis by human PMNL. Phagocytosis of B. pertussis strains BP504, BP165, and BP338 occurred by opsonization with anti-B. pertussis antibody, while autologous normal human sera did not induce significant phagocytosis. In PMNL bacterial killing assays virulent B. pertussis strains survived PMNL bactericidal activities while Escherichia coli controls were readily killed. Electron microscopy studies using acid phosphatase as a lysosomal marker strongly suggested that B. pertussis inhibits phagosome-lysosome fusion in PMNL. These results indicate that virulent B. pertussis strains are capable of surviving intracellularly within PMNL and that such survival may be due to inhibition of phagosome-lysosome fusion.
Activation of Human Neutrophils by Phorbol Ester Decreases the Cytoplasm Compactness and the Lactoferrin Content of the Granulocytes
Authors: Esaguy Nair , águas Artur P , Vilanova Manuel , Silva Manuel T
Published: January 2018

Human neutrophils isolated from the blood were incubated in vitro for 30 min with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, to study its effect on the fine structure of granulocytes and on the subcellular distribution of lactoferrin (Lf). Flow cytometry analysis of the human neutrophils showed that PMA induced a decrease in size and granularity of the cells. By electron microscopy, the PMA-treated cells showed numerous empty vesicles, smoothing of the cell surface, and a marked decrease in the compactness of the cytoplasmic ground substance. High-resolution immunogold ultracytochemistry showed that neutrophils lost most of their content in Lf after the PMA incubation. In thin sections, the cytoplasmic compartment of PMA-treated neutrophils contained an average of 1.03 ± 0.3 Lf-positive vesicles per μm2, whereas control granulocytes showed an average of 5.72 ± 1.49 Lf vesicles per μm2 of cytoplasm. Most of PMA-treated neutrophils kept some Lf-positive vesicles; a significant minority (10–15%) of the granulocytes showed cytological features of cell death. We conclude that the PMA-induced neutrophil activation is associated with microanatomical changes that include i) exocytosis of most, but not all, of the Lf-bearing vesicles; ii) rounding up of the cell outline; and iii) decrease in the compactness of the cytoplasmic ground substance.
Murine Eosinophil Granulocytes Bind the Murine Macrophage-Monocyte Specific Monoclonal Antibody F4/80
Authors: McGarry M P , Stewart C C
Published: January 2018

Studies designed to identify a panel of monoclonal antibodies useful for the separation of murine eosinophil myeloid progenitors revealed that F4/80, an antigen heretofore thought to be expressed only by murine monocyte/macrophage lineage cells, was expressed by eosinophil granulocytes. Eosinophils from several strains of mice stained positively with specific antibody for the epitope. A novel pathway for myeloid differentiation is proposed in which neutrophil progenitors exit a common lineage prior to a common progenitor of monocytes and eosinophils. Moreover, the results demonstrate that binding of anti-F4/80 can no longer be viewed as exclusive for mononuclear phagocytes.
Critically III Anergic Patients Demonstrate Polymorphonuclear Neutrophil Activation in the Intravascular Compartment With Decreased Cell Delivery to Inflammatory Focci
Authors: Tellado Jose M , Christou Nicolas V
Published: January 2018

Skin test anergy, the failure to produce a delayed type hypersensitivity (DTH) response, is associated with an increase in infection-related complications and death usually due to multiple organ failure (MOF). Refractory intravascular activation of polymorphonuclear neutrophils (PMNs) has been implicated in the development of MOF. We studied 20 critically ill surgical patients with life threatening infections to determine if PMN intravascular activation was present and how this affected essential PMN functions such as exudation. The 11 anergic patients had a more intense inflammatory response to their infection. Plasma lactoferrin was 6.1 ± 0.3 μg/ml in anergic patients compared to 3.9 ± 1.5 in reactives P < 0.05, accompanied by reduced total primary (3.3 ± 1.9 vs 4.7 ± 2.1 μg/106 PMN P < 0.01) and secondary (2.8 ± 0.4 vs 5.0 ± 0.9 μg/106 PMN P < 0.01) granule content, respectively. In vitro superoxide production following 100 ng/ml PMA stimulation was 0.44 ± 0.1 in anergics vs 0.36 ± 0.1 nmol/μg PMN protein in reactivities, P < 0.05. PMN chemotaxis was 8.2 ± 0.6 PMNs/HPF in anergics compared to 10.2 ± 1.6 PMNs/HPF in reactives P < 0.05, accompanied by decreased PMN delivery to skin blister windows (3.2 ± 1.4 vs 4.5 ± 1.9 × 107 PMN/ml, respectively, P < 0.05). We conclude that critically ill anergic surgical patients have increased intravascular PMN activation, which may contribute to oxygen-derived tissue damage in the vascular space, as well as a deficient delivery of effector cells in areas of bacterial invasion. This may lead to inability to clear the inflammatory signals which set up the vicious circle of MOF leading to death.
Effect of Interleukin-1, GM-CSF, Erythropoietin, and Lithium on the Toxicity Associated With 3′-Azido-3′-Deoxythymidine (AZT) In Vitro on Hematopoietic Progenitors (CFU-GM, CFU-MEG, and BFU-E) Using Murine Retrovirus-Infected Hematopoietic Cells
Authors: Gallicchio Vincent S , Hughes Nedda K , Hulette Ben C , Noblitt Lynette
Published: January 2018

The drug 3′-azido-3′-deoxythymidine (AZT), a synthetic thymidine analogue, has been used clinically in the management of acquired immune deficiency syndrome (AIDS). The drug is an effective antiviral agent due to its ability to block reverse transcriptase activity. This action of AZT was demonstrated in the Rauscher leukemia virus (RLV)-induced murine erythroleukemia model system. Unfortunately, associated with AZT has been the development of hematopoietic toxicity manifested by anemia, neutropenia, and overall bone marrow suppression. Hematopoietic growth factors (GM-CSF, erythropoietin), cytokines (interleukin-1), and agents known to potentiate hematopoiesis (lithium) have been demonstrated to modulate drug and/or radiation-induced hematopoietic toxicity. We report the results of further studies designed to investigate the ability of GM-CSF, erythropoietin, interleukin-1, and lithium to modulate AZT toxicity on murine hematopoietic granulocyte-macrophage (CFU-GM), megakaryocytic (CFU-Meg), and erythroid (BFU-E) progenitors cultured from bone marrow and spleen cells from mice infected with RLV. Hematopoietic progenitors from either normal or RLV-infected animals when exposed to AZT demonstrated concentration-dependent toxicity and differed for each progenitor with BFU-E being the most sensitive (ID50 concentration, 5 × 10-9 M) and CFU-GM the least sensitive (ID50 concentration, 5 × 10-5 M). As has been previously demonstrated using normal murine hematopoietic progenitors, when cultured with RLV-infected marrow or spleen cells, addition of GM-CSF, Meg-CSF or erythropoietin failed to inhibit AZT toxicity in vitro on CFU-GM, CFU-Meg, and BFU-E, respectively. However, in the presence of interleukin-1 (recombinant human IL-1α, 30 ngm) or lithium chloride (ultra-pure, 1.0 mM), AZT toxicity CFU-GM, CFU-Meg, and BFU-E cultured from RLV-infected marrow or spleen cells was reduced. These results further demonstrate interleukin-1 and lithium are effective in modulating the toxic action of ACT on hematopoietic progenitors and that RLV-infected animals serve as a useful viral model system to study the effect of agents capable of modulating hematopoiesis in the presence of the anti-viral drug AZT.
Role of Antibody-Dependent Cellular Cytotoxicity and Lymphokine-Activated Killer Cells in AIDS and Related Diseases
Authors: Brenner Bluma G , Gryllis Chryssa , Wainberg Mark A
Published: January 2018

This overview summarizes current knowledge on the overall efficacy and potential contribution of antibody-dependent cellular cytotoxicity (ADCC) and lymphokine-activated killer cell (LAK) activities in evoking non-major histocompatibility complex (non-MHC) cytolytic responses to human immunodeficiency virus-1 (HIV-1)-infected targets. High titers of ADCC antibodies to the HIV-1 virion are present in HIV-1-seropositive populations at all stages of disease. These antibodies are broadly reactive with a large number of HIV-1 strains and are predominantly directed against envelope determinants spanning both gp120 and gp41. However, the relative ability of natural killer (NK) effectors, derived from HIV-seropositive individuals, to evoke ADCC responses becomes increasingly impaired with disease progression. HIV-1-seropositive individuals also show marked decreases in both production of and responsiveness to interleukin-2 (IL-2). HIV-1-seropositive individuals generally have the ability to generate ex vivo propagated LAK cells; however, these cytolytic effectors are less effective than their counterparts derived from healthy controls. Increased understanding and control of non-MHC-restricted cytotoxic-responses to HIV, and their induction by lymphokines, may lead to improved treatment strategies for the management of AIDS and related diseases.
Neutrophil-induced immunoglobulin binding to erythrocytes involves proteolytic and oxidative injury
Authors: Weiss Douglas J , Aird Betsy , Murtaugh Michael P
Published: January 2018

Neutrophil-induced alterations in feline erythrocytes were studied to better understand the pathogenesis of erythrocyte destruction associated with inflammatory diseases. As in previous studies, addition of superoxide dismutase/catalase to a coculture of erythrocytes and activated neutrophils attenuated neutrophil-induced immunoglobulin G (IgG) binding. However, incubation of erythrocytes with hydrogen peroxide or neutrophil-derived anuclear cytoplasts (neutroplasts) failed to induce IgG binding. Addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to the erythrocyte-neutrophil coculture attenuated IgG binding. These observations suggest that neutrophil-derived serine protease activity is involved in IgG binding to erythrocytes. Further, incubation of erythrocytes with serine proteases, but not metalloproteases or sulfhydryl proteases, induced immunoglobulin binding. Freeze-fracture replicas of the erythrocyte membrane failed to demonstrate clustering of band 3 protein, suggesting that spatial rearrangement of band 3 protein was not the cause of the IgG binding. Neutrophil-induced IgG binding due to the combined action of proteases and oxidants may explain the accelerated destruction of erythrocytes in inflammatory diseases.
Characterization of bilirubin transport system by human peripheral blood mononuclear cells
Authors: Haga Yoshio , Tempero Margaret A , Kay H David , Zetterman Rowen K
Published: January 2018

Decreased immune responses have been observed in hyperbilirubinemic patients. This study investigates bilirubin transport into human peripheral blood mononuclear cells (PBMNCs). In vitro incubation of PBMNCs at 37° with 0–12 mg/dl bilirubin in solution with a fixed bovine serum albumin (BSA) concentration (3.0 g/dl) resulted in a dose-dependent increase of intracellular bilirubin in both monocytes and lymphocytes. Bilirubin uptake in monocytes was significantly higher (up to 2.7 times) than in lymphocytes under the same culture conditions. When PBMNCs were incubated with varying concentrations of bilirubin (0–16 mg/dl) in fixed BSA (3.0 g/dl) solution or at a fixed bilirubin/albumin molar ratio (0.4), the initial velocity of uptake in both cell fractions was proportional to the free (unbound to albumin) bilirubin concentration rather than the total bilirubin concentration. Bilirubin uptake by both cell fractions was significantly inhibited by treatment with metabolic inhibitors. Bilirubin uptake by monocytes continued to increase in parallel with incubation temperature from 0° to 40°, whereas uptake by lymphocytes reached a maximal level at 20° and remained constant thereafter. These results suggest that monocytes and lymphocytes incorporate bilirubin in proportion to the free bilirubin concentration and this function may rely on different energy-dependent mechanisms.
Regulation of phospholipase A2 activation and arachidonic acid metabolism in an interleukin-3–dependent macrophage-like cell line
Authors: Shibata Yoshimi , McCaffrey Patricia G , Minowada Jun , Volkman Alvin , Oghiso Yoichi
Published: January 2018

An interleukin 3 (IL-3)–dependent macrophage-like cell line, 11-1-B3, was newly established from CBA/J mouse bone marrow cell cultures. Assay of eicosanoids in the culture supernatants of the intact and [3H]arachidonic acid (AA)–prelabeled cells showed that, after stimulation with the Ca2+ ionophore A23187, the 11-1-B3 cells synthesized and released relatively large amounts of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) but not LTC4. In addition, 11-1-B3 cells showed Ca2+-dependent and alkaline pH–optimal phospholipase A2 (PLA2) activity that preferentially hydrolyzed cleavage of sn-2-arachidonyl- but not sn-2-oleoylphosphatidylcholine. The cellular enzyme was distributed with 90% of the activity in the cytosol and 10% in the membrane fraction. Treatment of cells with A23187 for 5–10 min resulted in five- to sevenfold increases in the membrane-associated PLA2 but activity in the cytosol was unchanged. This increase in membrane-associated enzyme activity was transient, returning to the pretreatment distribution after 30 min. In sharp contrast, phorbol myristate acetate (PMA) stimulation failed to induce either eicosanoid release or PLA2 activation, although PMA induced translocation of protein kinase C (PKC) to the membrane fraction within 10 min. The data suggest that increases in cellular Ca2+ directly activate membrane-associated PLA2 and consequently initiate AA metabolism; PKC activation by PMA requires additional steps to activate PLA2, a mechanism that is apparently deficient in the IL-3–dependent Mø-like cells.
In vivo latex phagocytosis primes the Kupffer cells and hepatic neutrophils to generate superoxide anion
Authors: Bautista Abraham P , Schuler Agnes , Spolarics Zoltan , Spitzer John J
Published: January 2018

Activation of liver macrophages during clearance of endotoxins, bacteria, or other particulate materials may be accompanied by the migration of polymorphonuclear neutrophils (PMNs) into the liver and priming of the hepatic phagocytes to release toxic oxygen metabolites. In the present study we investigated the effect of in vivo administration of latex particles on the hepatic sequestration of PMNs and the release of superoxide anion (O2-) by the in situ perfused rat liver and isolated hepatic phagocytes. One hour after an intravenous injection of latex beads, a significant amount of O2- (0.7 nmol/min/g) was produced by the in situ perfused liver. Administration of latex particles into the perfused liver also elicited O2- production. Hepatic phagocytes from latex-treated rats generated large amounts of O2- (2–14 nmol/60 min/106 cells) when these cells were stimulated in vitro with opsonized zymosan or phorbol myristate acetate (PMA), whereas phagocytes from saline-treated rats released less than 0.8 nmol O2-. Intravenous infusion of superoxide dismutase or ibuprofen did not prevent the immigration of PMNs to the liver. However, ibuprofen inhibited the production of O2- by the perfused liver. Also, after addition of ibuprofen in vitro to isolated cells, there was more than 50% inhibition of O2- generation by Kupffer cells and hepatic PMNs treated with either zymosan or PMA. These observations suggest that arachidonic acid metabolites play a role in O2- release under these conditions. Thus, activation of the reticuloendothelial system by latex phagocytosis induces the migration of PMNs into the liver and enhances the production of toxic oxygen-derived radicals by these cells and the resident Kupffer cells. The toxic oxygen radicals may also contribute to hepatic injury.
Differential immunocompetence of macrophages derived using macrophage or granulocyte-macrophage colony-stimulating factor
Authors: Rutherford Mark S , Schook Lawrence B
Published: January 2018

Macrophages derived in vitro from bone marrow progenitors (bone marrow–derived macrophages, BMDMs) using either macrophage colony-stimulating factor (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF) as the myelopoietic stimulus display differential functional, morphological, and mRNA phenotypes. The data presented here demonstrate further that CSF-1– and GM-CSF–derived BMDMs differ in immunologic capacity. GM-CSF–derived BMDMs, when compared to CSF-1–derived BMDMs, showed greater cytolytic activity against tumor necrosis factor α (TNF-α)–resistant, but not TNF-α–sensitive, tumor targets. In contrast, CSF-1–derived BMDMs produced nitrite in response to lipopolysaccharide (LPS) alone, whereas GM-CSF–derived BMDMs required interferon γ plus LPS treatment. The two BMDM populations also showed differential sensitivities to LPS for secretion of TNF-α and nitrite, but the maximal inducible amounts of these factors and prostaglandin E2 were similar between the BMDM populations. Lastly, GM-CSF–derived but not CSF-1–derived BMDMs showed an L-arginine–dependent listeriacidal activity. These results show that the functional heterogeneity of CSF-1– and GM-CSF–derived macrophages is limited and appears to result largely from differences in the activational signals required by each BMDM population to elicit a given function.
Synergistic interaction of bacterial lipopolysaccharide and the monocyte-macrophage colony-stimulating factor: potential quantitative and qualitative changes in macrophage-produced cytokine bioactivity
Authors: Evans Robert , Kamdar Sonya J , Duffy Theodore M , Fuller Jane
Published: January 2018

In this brief definitive report, we show that over a 6-h period and under serum-free conditions, recombinant monocyte-macrophage colony-stimulating factor 1 (rCSF-1) and lipopolysaccharide (LPS) synergize and induce macrophages to express higher levels of mRNA for interleukin 1α (IL-1α), IL-1β, tumor necrosis factor α (TNF-α), and IL-6 and to release more bioactivity than macrophages treated with LPS alone. This synergy was regulated by the amount of LPS in the culture medium. Paraformaldehyde-fixed macrophages likewise showed augmentation of IL-1 activity, but whereas all of the bioactivity associated with the fixed macrophages could be neutralized by anti-IL-1α antibody only ≍ 40% of the supernate activity could be attributed to IL-1α. Preliminary data suggest that the augmenting effect induced by CSF-1 cannot be explained solely on a quantitative basis because the addition of rIL-1α to supernates of macrophages treated with LPS alone or with the combination of LPS and CSF-1 resulted in an increase in thymocyte mitogenic activity to a level that could not be explained on an additive basis. Although the supernates contained TNF and IL-6, antibody neutralization assays made it unlikely that these were directly responsible for the augmenting effect. These results suggest that CSF-1 not only enhances basic genetic responses induced by LPS alone but also may induce a mechanism that amplifies cytokine bioactivity.
Prostaglandin E2 receptor activity and susceptibility to natural killer cells
Authors: Fulton Amy M , Chong Yen C
Published: January 2018

We have described a high-affinity receptor for prostaglandin E2 (PGE2) present on metastatic murine mammary tumor cells. Pharmacologic antagonism of this receptor increases metastatic potential. In the present study, we have asked whether the binding activity of PGE on tumor target cells plays a role in natural killer (NK)–target cell interactions. We have used three unrelated PGE-receptor antagonists, SC19220, LEO101, and AH6809, to show inhibition of [3H]PGE2 binding to YAC-1 cells and inhibition of PGE2-mediated elevation of intracellular cyclic AMP (cAMP). Addition of any of these three receptor antagonists to standard 4-h 51Cr-release assays inhibits YAC-1 lysis by NK-enriched populations from murine spleen. This is the first report that antagonism of PGE binding affects NK activity. Our studies demonstrate that these effects are mediated through inhibition of target-effector cell conjugate formation. Studies in which effector and target cells were pretreated separately indicate that the PGE-mediated effects are expressed at the target cell level.
Transcriptional inhibition of endotoxin–induced monokine synthesis following heat shock in murine peritoneal macrophages
Authors: Snyder Yvonne M , Guthrie Laura , Evans Glenn F , Zuckerman Steven H
Published: January 2018

The role of heat shock proteins (HSP) during the inflammatory response has been controversial. The effect of heat shock (HS) on the synthesis of the monokines tumor necrosis factor (TNF) and interleukin 1 (IL-1) by endotoxin–stimulated thioglycollate–elicited peritoneal macrophages was investigated. HS was deemed to have affected macrophages if a 70 kD HSP appeared on SDS gels; identity of this protein as the highly conserved HSP70 was then confirmed by immunoprecipitation. Maximal increases in HSP70 were apparent 2–5 h after HS at 45° for 12 min. Synthesis of HSP70 was no longer detected 24 h post HS. A reciprocal relationship between HSP70 and TNF was apparent in kinetic studies. TNF was not detected in culture supernatants if macrophages were endotoxin–stimulated 2 to 6 h after HS; however, the same stimulation 24 h later induced significant TNF secretion. RNA analysis of HS and non–HS macrophage cultures demonstrated a 60–fold reduction in TNF message in the HS macrophages 1 h after endotoxin stimulation. TNF mRNA levels remained depressed at 6 h while the HSP70 message had increased 30–fold. The ability of HS macrophages to ingest antibody–coated erythrocytes was not significantly affected following heat treatment. Macrophage response to HS can be said to inhibit transcription of inducible monokines while retaining other macrophage functions.
Anti-class II MHC antibody induces multinucleated giant cell formation from peripheral blood monocytes
Authors: Orentas Rimas J , Reinlib Leslie , Hildreth James EK
Published: January 2018

Multinucleated giant cells (MGCs) are an integral part of the host immune response to infectious disease and are seen in granulomas induced by pathogens and inorganic substances. We have developed a novel system for the production and study of MGCs: Peripheral blood monocytes, when cultured in the presence of anti–class II major histocompatibility complex monoclonal antibodies (MHC mAb's) and lymphocyte-conditioned medium form MGCs within 48 h. MGC formation was strictly dependent on the presence of anti–class II MHC mAb's and lymphocyte-conditioned medium. MGC formation was not induced by mAb's to other monocyte surface proteins. None of the previously identified macrophage fusion factors (calcitriol, interleukin 4, interferon-γ) were able to substitute for the lymphocyte-conditioned medium in our assay; however, the conditioned medium could be replaced by the phorbol ester phorbol 12-myristate 13-acetate. We have also demonstrated that the induction of MGCs by anti–class II MHC antibody and phorbol ester requires protein kinase C activity, because MGC formation was totally inhibited by the protein kinase C inhibitors staurosporine and H-7. In analyzing the signal induced by anti–class II MHC mAb's we have demonstrated that cross-linking of the class II MHC antigens with intact mAb's, or with F(ab')2 fragments of anti–class II MHC mAb's and F(ab')2 fragments of rabbit antimouse (RAM) immunoglobulin G, produced an intracellular calcium rise. Furthermore, using the calcium channel blocker verapamil, it was demonstrated that calcium channel activity is necessary for MGC formation. These data support the view that MGC formation is a tightly regulated differentiative pathway of peripheral blood monocytes that is dependent on protein kinase C second messenger systems and involves an inrease in intracellular calcium concentration.
Contribution of interferon γ and membrane-associated interleukin 1 to the resistance to murine typhoid of Ityr mice
Authors: Kita Eiji , Emoto Masashi , Oku Daisuke , Nishikawa Fumiko , Hamuro Akiko , Kamikaidou Noriaki , Kashiba Shuzo
Published: January 2018

Resistance of mice to Salmonella, typhimurium in the early phase of infection is known to be controlled by the expression of chromosome 1 locus Ity. To clarify the mechanism by which the genetically resistant (Ityr) mice can overcome the first phase of salmonellosis, the early response in DBA/2 (Ityr) and BALB/c (Itys) mice was compared after a subcutaneous injection of S. typhimurium. In both strains, the growth of S. typhimurium was controlled in livers and Kupffer cells until day 3, but thereafter the bacteria multiplied rapidly in BALB/c mice. Over the first 2 days nonspecific responses (changes in levels of blood leukocytes, plasma iron, and α1-antitrypsin) were not significantly different between the strains, and the capacity of Kupffer cells isolated from infected mice of both strains to produce interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α) was of the same degree. Thereafter, only DBA/2 Kupffer cells were able to produce membrane-associated IL-1 (ma IL-1) as well as TNF-α. Moreover, only DBA/2 splenocytes were able to produce interferon γ (IFN-γ) upon stimulation with Salmonella antigens, although concanavalin A-stimulated splenocytes of both strains produced the same level of interleukin 2. Furthermore, administration of recombinant murine IFN-γ and DBA/2 Kupffer cells of day 6 to BALB/c mice 3 days after infection resulted in a significant level of protection, whereas neither of these materials alone induced protection. Injection of anti-TNF-α antibodies did not affect the resistance of DBA/2 mice. Thus, these findings suggest that the early resistance of Ityr mice is partly attributable to their capacity to produce IFN-γ and ma IL-1 after infection.
Regulation of pinocytosis in murine macrophages by colony-stimulating factors and other agents
Authors: Knight Kenneth R , Vairo Gino , Hamilton John A
Published: January 2018

Lucifer yellow (LY) accumulation was used to measure macrophage pinocytosis. The hematopoietic growth factors, macrophage colony-stimulating factor (CSF-1), granulocyte-macrophage CSF (GM-CSF), and interleukin 3, and the macrophage activators, lipopolysaccharide and zymosan, all stimulated LY uptake in both murine bone marrow–derived macrophages (BMMs) and resident peritoneal macrophages (RPMs) without affecting LY efflux. The stimulation of pinocytosis in the poorly cycling RPMs and in BMMs by nonmitogens dissociates stimulation of pinocytosis from subsequent DNA synthesis. Regulation of pinocytosis in BMMs appears to be independent of that of urokinase-type plasminogen activator expression. The increases in CSF-mediated BMM pinocytosis were not inhibited by pertussis toxin, by elevations in intracellular cAMP, or by glucocorticoids and were only partially inhibited by inhibitors of Na+/H+ antiport and Na+/K+-ATPase activities. Protein kinase C activation could be involved in regulating BMM pinocytosis because phorbol myristate acetate, oleoylacyglycerol, and exogenously added phospholipase C can all stimulate it. Ca2+ ionophores were inactive, whereas the Na+/H+ ionophore monensin potently inhibited BMM pinocytosis.
Ontogenetic development, differentiation, and phenotypic expression of macrophages in fetal rat lungs
Authors: Higashi Kenji , Naito Makoto , Takeya Motohiro , Ando Masayuki , Araki Shukuro , Takahashi Kiyoshi
Published: January 2018

Development, differentiation, and distribution of macrophages in fetal rat lungs were investigated immunohistochemically using anti-rat macrophage monoclonal antibodies. In the lung buds, RM-1+ macrophages were first detected on fetal day 13, and some showed reactivity for TRPM-2. They populated in the peribronchial mesenchyme of the lung buds, proliferated in loco, and showed no peroxidase activity in any intracellular organelles. Their immunophenotypic and ultrastructural features were consistent with those of primitive/fetal macrophages. By fetal day 16, some of them expressed ED1, but ED1+ cells were a minor subpopulation throughout the fetal period. On fetal day 18, ED2+ macrophages developed; some also were positive for RM-1, but the others were negative. Both the RM-1+ and ED2+ macrophages were major macrophage subpopulations and expressed Ki-M2R and/or TRPM-3; ED2+ and/or Ki-M2R+ cells are regarded as pulmonary interstitial resident macrophages. In organ culture, a similar expression of differentiation antigens by macrophages was confirmed. None of these macrophages cytochemically showed any peroxidase activity in vivo or in vitro. In the fetal stage, both RM-1+ and ED2+ macrophage subpopulations showed proliferative potential, suggesting their ability to proliferate and survive in vivo.
Platelet activating factor enhancement of lipopolysaccharide-induced tissue factor activity in monocytes: requirement of platelets and granulocytes
Authors: østerud Bjarne
Published: January 2018

Incubation of platelet activating factor (PAF) with heparinized blood caused no induction of tissue factor activity in monocytes. However, when PAF was added in addition to weak lipopolysaccharide (LPS), it amplified the LPS effect by 80%. By using separated fresh cell populations resuspended in plasma, PAF was shown to have no enhancing effect when added to mononuclear cells incubated with platelet-rich plasma in the presence of LPS. In contrast, when granulocytes also were included, PAF caused an almost 3-fold increase in LPS-induced tissue factor activity. A PAF antagonist blocked this effect and also reduced LPS-induced tissue factor activity of monocytes in whole blood in a dose-dependent manner. In the recombined cell incubation system, the maximal inhibition by the antagonist was observed in the presence of granulocytes. These data provide evidence for an effect of PAF on granulocytes that probably generates a product that, together with platelets, enhances LPS-induced tissue factor activity in monocytes.
Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescein diacetate in the presence of a calmodulin inhibitor
Authors: Krishna Rao K Murali , Padmanabhan Jaya , Kilby Diana L , Cohen Harvey J , Currie Mark S , Weinberg J Brice
Published: January 2018

Dichlorofluorescein (DCFH) oxidation assay measures hydrogen peroxide (H2O2), which is a derivative of superoxide anion. We found that a calmodulin antagonist, W-13, which is known to inhibit superoxide anion generation enhanced the capacity of human neutrophils to oxidize DCFH. To investigate this discrepancy we studied the role of nitric oxide (NO) in DCFH oxidation. Pure NO was capable of oxidizing DCFH, and the product formed had spectral properties identical to oxidized DCFH produced by H2O2. The arginine analog, NG-monomethyl-l-arginine (NMMA), which inhibits NO production, in combination with W-13 completely inhibited the stimulus-induced increase in DCFH oxidation. We conclude that the oxidation of DCFH in human neutrophils can occur by either H2O2 or NO.
Isolation and characterization of four CSF-1-dependent placental macrophage cell lines
Authors: Boocock CA , Hayes M , Chang M-d Y , Pollard JW
Published: January 2018

By selection and cloning in the presence of colony-stimulating factor 1 (CSF-1), we have obtained from separate explants of individual 12- or 13-day mouse placentas four clonal cell lines dependent on CSF-1 for survival and growth in culture. All four cell lines show characteristics consistent with their derivation from placental macrophages. We describe the effects of CSF-1 on growth, morphology, and CSF-1 receptor phosphorylation. One cell line, JPL2A, which shows relatively complete growth arrest in the absence of CSF-1, was characterized in detail with respect to the effects of CSF-1 on DNA synthesis and protein turnover and its response to the duration of CSF-1 stimulation. In contrast to previous studies, our results suggest that extended stimulation leads to continuous recruitment of cells competent to reenter the cell cycle. These findings are discussed in terms of growth factor effects on the cell cycle and the usefulness of these cell lines for further study of growth factor signal transduction in macrophages.
Characterization and identification of interleukin 1 receptors on bovine neutrophils
Authors: Lederer James A , Czuprynski Charles J
Published: January 2018

Interleukin 1 (IL-1) has been shown to modulate various functional activities of neutrophils, presumably by first binding to cell surface IL-1 receptors. In this study, we characterized and identified IL-1 receptors on bovine neutrophils using direct and competitive receptor binding studies and affinity cross-linking analysis. The results of direct binding studies demonstrated that bovine neutrophils bound 125I-labeled bovine IL-1 by a single affinity binding site (Kd = 4.6 nM, 5600 binding sites per cell). Competitive receptor binding studies demonstrated that unlabeled recombinant bovine IL-1β, murine IL-1α, and human IL-1β competitively blocked neutrophil binding of 125I-labeled bovine IL-1β. In contrast, the IL-1 receptor antagonist (IL-1ra) demonstrated no detectable binding to bovine neutrophils as judged by direct and competitive receptor binding assays. Affinity cross-linking of 125I-labeled bovine IL-1β to neutrophils was used to identify cell surface IL-1 receptors. Two specific cross-linked products were observed with molecular sizes of 89 kd (a deduced receptor size of 71.5 kd) and more than 200 kd. The latter may indicate a complex of IL-1 receptor-associated signal-transducing components, IL-1 receptors, and IL-1. The presence of 100 nM unlabeled bovine, murine, or human IL-1 during the receptor binding and cross-linking reactions prevented the formation of cross-linked complexes of 125I-labeled bovine IL-1β and its receptor. In contrast, the IL-1ra failed to inhibit the formation of cross-linked complexes of 125I-labeled bovine IL-1β and its receptor.
Inhibition of neutrophil chemotaxis and activation following decentralization of the superior cervical ganglia
Authors: Carter Lisa , Ferrari J Kent , Davison Joseph S , Befus Dean
Published: January 2018

Recent studies have shown that bilateral decentralization (sympathectomy) of the superior cervical ganglia (SCG) of rats sensitized to the parasite Nippostrongylus brasiliensis attenuated the development of pulmonary inflammation following allergen challenge. Sympathectomy inhibited total leukocyte infiltration into lung lavage fluids, particularly neutrophil infiltration. To define the effects of decentralization of the SCG on neutrophil responses, peripheral blood neutrophils of rats were isolated and tested in in vitro chemotaxis and phagocytosis assays. Neutrophils from rats that were sympathectomized 7 days previously displayed a marked reduction in chemotaxis to N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4 compared to neutrophils from sham-operated or unoperated groups. Although the degree of chemotaxis was greater in blood neutrophils from parasite-infected rats than from uninfected rats, sympathectomy markedly reduced the chemotactic responses of both groups. In addition, neutrophils of sympathectomized rats were unresponsive to lipopolysaccharide-induced metabolic activation as assessed by in vitro phagocytosis and oxidative reduction of nitroblue tetrazolium. Thus, decentralization of the SCG of rats affects the chemotactic responses and functions of neutrophils. Understanding the role of the sympathetic nervous system in modulating the behavior of neutrophils will shed light on the interactions between the nervous and immune systems.
Soybean agglutinin–positive natural suppressor cells in mouse bone marrow inhibit interleukin 2 production and utilization in mixed lymphocyte reactions
Authors: Hoskin D W , Bowser D A , Brooks-Kaiser J C
Published: January 2018

Although natural suppressor (NS) cells resident in bone marrow (BM) have been the subject of intensive study, the exact nature and mode of action of these potentially important immunoregulatory cells are still uncertain. Here we show that NS cells with potent inhibitory effects on mixed lymphocyte reactions (MLRs) can be isolated from BM of normal adult mice by agglutination with the plant lectin soybean agglutinin (SBA). Complement-dependent lysis of SBA receptor–bearing BM cells with antibodies to asialoGM1, Mac-1, Thy-1.2, J11d.2, and 2C1 phenotypic markers reveals the presence of at least two distinct populations of BM NS cells. Most of the SBA-binding BM cells with NS capacity have the null phenotype and resemble hematopoietic stem cells, and some inhibitory SBA+ BM cells express the 2C1 marker found on pregnancy-associated splenic NS cells and the J11d.2 antigen characteristic of B cells and immature T cells. Results of positive selection experiments confirmed these findings. The mechanism of natural suppression was also studied. Evidence is presented that SBA+ BM cells exert NS activity in MLRs by interfering with the production and utilization of interleukin 2. Indomethacin does not relieve natural suppression associated with SBA+ BM cells, indicating that prostaglandin synthesis is not a requirement for inhibitory function. However, neutralizing antibodies to transforming growth factor β (TGF-β) partially reverse the suppression mediated by SBA4 BM cells, suggesting that some BM NS cells may act through the release of an immunosuppressive molecule related to TGFβ.
Subcellular localization of CD66, CD67, and NCA in human neutrophils
Authors: Ducker Thomas P , Skubitz Keith M
Published: January 2018

CD66 and CD67 are granulocyte-specific activation antigens; their surface expression is up-regulated when neutrophils are activated. CD66 antibodies recognize an ~180-kd neutrophil surface protein that is also recognized by anti–carcinoembryonic antigen (CEA) antibodies and is therefore a nonspecific cross-reacting antigen (NCA). CD67 antibodies recognize an ~100-kd neutrophil surface protein that is attached to the membrane via a glycosyl-phosphatidylinositol anchor. To identify an intracellular pool from which CD66 and CD67 could be up-regulated, the subcellular distribution of proteins recognized by CD66 and CD67 monoclonal antibodies and polyclonal anti-CEA was studied. Neutrophil plasma membranes, granules, and cytoplasm were prepared by nitrogen cavitation and differential centrifugation and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Most of the 180-kd protein recognized by CD66 antibodies and the 100-kd protein recognized by CD67 antibodies were located in the secondary granule fraction, with lesser amounts detectable in the plasma membrane fraction. Several NCA species ranging from ~40 to 200 kd were identified, and the distribution of these NCAs was different in the primary granules, secondary granules, and plasma membrane fractions. The major NCAs in the plasma membrane fraction were of ~95 to 100 and ~180 to 200 kd; the secondary granule fraction contained major NCAs of ~42, 85, 95 to 100, and 180 to 200 kd. NCAs were also detected in the primary granule fraction, the most prominent being of ~90–100 kd; no NCA of ~180 to 200 kd was detected in the primary granules. The presence of CD66, CD67, and NCAs in the secondary granules suggests secondary granules as a likely source from which these antigens could be recruited to the cell surface with activation. The potential role for NCAs in the primary granules is unknown.
Taxol, a microtubule-stabilizing antineoplastic agent, induces expression of tumor necrosis factor α and interleukin-1 in macrophages
Authors: Bogdan Christian , Ding Aihao
Published: January 2018

Taxol, a naturally occurring diterpene with antitumor activity, induces tubulin polymerization to generate abnormally stable and nonfunctional microtubules. Previously, we showed that taxol has lipopolysaccharide (LPS)-like effects on macrophages. As LPS is a potent inducer of macrophage cytokine production, we investigated whether a similar effect is exerted by taxol. In a dose-dependent manner, LPS-free taxol induced release of biologically active tumor necrosis factor α (TNF) by inflammatory murine macrophages. Taxolinduced production of TNF was inhibitable by interleukin-10. By Northern blot, taxol (10 and 1μM) induced TNF mRNA expression to an extent similar to LPS. Induction of TNF mRNA by 10 μM taxol was detectable at 45 min of stimulation, maximal at 90 min, and evident for at least 8 h. The same low concentration of taxol also induced interleukin 1 (IL-1) α and β mRNA expression. We conclude that taxol triggers macrophages for TNF and IL-1 production. These LPS-like effects of taxol might contribute to its antitumor activity.
Inhibitory role of interleukin-6 in macrophage proliferation
Authors: Riedy MC , Stewart CC
Published: January 2018

We have shown that there are two forms of progenitor cells for macrophages. The first is characterized by a short lag period (about 1 day) before initiating the cell cycle, forms large colonies, is found in the bone marrow, and is in the nonadherent fraction. The second progenitor cell, found primarily in the adherent cell fraction of bone marrow and in peripheral tissues, forms small colonies after 14 days. We investigated the effect of combining interleukin-6 (IL-6) with colony-stimulating factor 1 (CSF-I) on macrophage proliferation. We found that IL-6 inhibited the proliferation of both types of progenitor cells, as well as more differentiated macrophages. This inhibitory effect was reversible because macrophages could initiate a proliferative response after removal of IL-6 from the culture medium. The introduction of anti–IL-6 into macrophage cultures containing IL-6 allowed proliferation, indicating that the effect was IL-6 specific. These results suggest that IL-6 may play a regulatory role in vivo by suppressing the production of bone marrow and tissue macrophages.
Specific ligation of surface α-D-galactosyl epitopes markedly affects the quantity of four major proteins secreted by macrophages
Authors: Warfel Alwin H , Zucker-Franklin Dorothea
Published: January 2018

Activated macrophages (Mφs) have terminal α-D-galactosyl (αD-Gal) residues on their membranes that are not apparent on resting cells. Ligation of these epitopes with Griffonia simplicifolia I-B4 (GSI-B4), a lectin that has specificity for α-D-Gal residues, alters selected Mφ functions. To explore the mechanism(s) that may be responsible for some of the functional changes, alterations in the secretory pattern of [35S]methionine-labeled proteins were assessed when cells were cultured with or without this ligand. The proteins were identified by Western blots and quantitated. Interestingly, α-D-Gal ligation proved to decrease the secretion of some proteins while increasing the secretion of others. Some of the most significant changes were observed in four proteins: fibronectin and transglutaminase were down-regulated by 55 and 66% respectively, while plasminogen activator inhibitor type 2 was increased by 259% and collagenase was increased 1000-fold. These observations show that the emergence of new oligosaccharide epitopes, such as α-D-Gal, concomitant with Mφ activation may serve to mediate the transduction of signals that cause quantitative changes in the elaboration of diverse Mφ products. The biologic significance of the four identified proteins has been well established. Fluctuations in their levels are likely to play a role at sites of chronic inflammation.
PMN binding to P-selectin is inhibited by sulfatide
Authors: Todderud Gordon , Alford Julie , Millsap Kathleen A , Aruffo Alejandro , Tramposch Kenneth M
Published: January 2018

The endothelial adhesion protein P-selectin binds to a ligand present on the surface of leukocytes. We have characterized the binding interaction between P-selectin and polymorphonuclear leukocytes (PMNs) in an in vitro assay. These studies have utilized a soluble chimeric protein termed receptor globulin (Rg), which consists of the lectin-EGF-CR-CR extracellular domains of P-selectin fused to a human immunoglobulin G Fc domain. The PMNs bound to immobilized Rg in a saturable and concentration-dependent manner. The binding was specific for the Rg, as preincubation of the cells with soluble Rg inhibited binding to immobilized Rg, and binding was dependent on the presence of free divalent cations. The PMNs expressed a ligand for both P-selectin and E-selectin but not for L-selectin. Previously it was shown that sulfatide is a ligand for P-selectin binding in transformed cells. We have demonstrated that the presence of sulfatide in the P-selectin–PMN adhesion assay inhibits binding in a dose-dependent manner.
Characteristics of alveolar macrophages in experimental septic lung
Authors: Goya Tomomochi , Abe Masayoshi , Shimura Hidehiko , Torisu Motomichi
Published: January 2018

We investigated the pathogenesis of lung injury in sepsis (septic adult respiratory distress syndrome) by focusing on the functional changes of alveolar macrophages (AMs). Sepsis was induced in male WK rats by cecal ligation and puncture. Histological examination of the lungs from this experimental model revealed edematous change at 24 h after the surgery. The protein and endotoxin concentrations in the bronchoalveolar lavage fluid (BALF) increased with time after the surgery. The time course studies of AM function after surgery indicated that AMs from septic rats were activated by endotoxins. Specifically, this was suggested by the finding that AM adherence to and spreading on a plastic dish had increased. On stimulation, these AMs enhanced generation of superoxide anions and increased release of lysosomal enzymes, such as β-glucuronidase. On the other hand, AMs in sepsis generated much smaller amounts of arachidonate lipoxygenase metabolites, such as leukotriene B4 (LTB4) and 12- and 5-hydroxyeicosatetraenoic acids (HETEs), on stimulation than did AMs from sham rats or untreated rats. However, the concentrations of immunoreactive LTC4 in the BALF of septic rats seemed to be higher than in untreated rats. It is suggested that the AMs of septic rats released lipoxygenase metabolites in alveoli and that these AMs could not be stimulated in vitro. These functional changes in the AMs of septic rats progressed along with the sepsis. These results implicate AMs in the development and progression of septic lung injury by releasing superoxide anions, β-glucuronidase, and arachidonate metabolites. Furthermore, we speculate that reduced production of LTB4 by septic AMs may increase host susceptibility to severe pulmonary infection during septic ARDS.
Nanomolar cyclic adenosine and guanosine monophosphates stimulate macrophage colony-stimulating factor responsiveness by murine transitional progenitors
Authors: Detrick Melinda S , Kreisberg Robert , Koontz John W , Moore Robert N
Published: January 2018

Both 3:5′ cyclic adenosine monophosphate (cAMP) and 3′:5′ cyclic guanosine monophosphate (cGMP) stimulated colony-stimulating factor 1 (CSF-1)–dependent colony formation by murine two-signal–dependent progenitors without influencing colony formation by committed CSF-1–responsive progenitors. The stimulatory effect was optimal at 10-9 M and did not diminish with increasing concentrations of the cyclic nucleotides. The membrane-permeating analogs dibutyryl cAMP and 8-Br-cGMP similarly augmented colony formation by the transitional progenitors at 10-9 M; however, with increasing concentration, enhancement diminished with eventual inhibition of total colony formation at micromolar concentrations. Stimulation by the two cyclic nucleotides was mutually incompatible. The results indicate that physiological levels of extracellular cyclic nucleotides may significantly influence myelopoiesis. Furthermore, the results introduce the interesting possibility that stimulation, unlike inhibition, may be initiated through an extracytoplasmic mechanism that does not require direct activation of cytoplasmic cyclic nucleotide–dependent protein kinases.