bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Fatty Acid Metabolites in the Differentiation of the Human Monocyte-Like Cell Line U937
Authors: Bomalaski John S , Freundlich Bruce , Steiner Sheldon , Clark Mike A
Published: January 2018

The human monocyte cell line, U937, can be induced to terminally differentiate into macrophage-like cells when treated with gamma-interferon. However, if these cells were treated with gamma-interferon and esculetin, an inhibitor of the lipoxygenase pathway, or BW755C, an inhibitor of both the lipoxygenase and the cyclooxygenase pathways, a marked inhibition in cellular differentiation occurred. In contrast, inhibitors of only the cyclooxygenase pathway had no effect on differentiation. These studies suggest a role for lipoxygenase products of arachidonic acid in the differentiation of the human U937 cell line. Arachidonic acid utilized in the production of eicosanoids is derived from phospholipids by the action of phospholipase A2 and phospholipase C. When U937 cells were cultured in medium supplemented with gamma-interferon, there was a striking increase in the level of phosphatidylcholine and phosphatidylethanolamine-specific phospholipase A2 activities and phosphatidylinositol-specific phospholipase C activity as compared to control cells. Morever, although there was not a significant difference in the incorporation of labeled arachidonic acid or linoleic acid into the major phospholipids of differentiated U937 cells as compared to undifferentiated control cells, there was a marked increase in the relative amount of the labeled arachidonic acid released from the differentiated cells as lipoxygenase products compared to cyclooxygenase products. These data suggest that lipoxygenase products may be essential in the differentiation process of U937 cells and that enhanced phospholipase enzyme activities that occur during differentiation help explain how arachidonic acid becomes available to form lipoxygenase products.
Biochemical and Pharmacologic Characterization of a Phosphatidylinositol-Specific Phospholipase C in Rat Neutrophils
Authors: Mackin William M , Stevens Theresa M
Published: January 2018

The phosphatidylinositol (PI)-specific phospholipase C (PLC) activity contained in sonicates of casein-elicited (4-6 hr) rat neutrophils has been identified and characterized. With phosphatidylinositol (PI) as the substrate, PLC activity is found both in the supernate and pellet of a 100,000g spin of the neutrophil sonicate. Further fractionation of the crude sonicate by centrifugation on discontinuous sucrose gradients indicates that the PLC activity is predominantly cytosolic with lesser amounts of activity found in the plasma membrane and granule enriched fractions. Hydrolysis of PI by the sonicate PLC is linear for 15-20 min at 37° and also with respect to the amount of sonicate protein added. The enzyme shows selectivity for PI with little, if any, hydrolytic activity towards other phospholipids such as phosphatidylethanolamine (PE), phosphatidylserine (PS), or phosphatidylcholine (PC). The PLC activity has a pH optimum of 5.5-6.0, is enhanced 1.5-3-fold by the addition of deoxycholate, and is Ca+  + dependent. Kinetic analysis of the PLC hydrolysis of PI yields an apparent Km of 240 ± 85 μM and a Vmax of 34.3 ± 17.0 nmol/min/mg protein (n = 3). Similarly, when phosphatidylinositol 4,5 bisphosphate (PIP2) is used as substrate, an apparent Km of 109 ± 66 μM and a Vmax of 14.3 ± 10.4 nmol/min/mg (n = 3) protein is obtained. These data suggest that PIP2 may be a slightly better substrate for the PMN PLC relative to PI. Finally, a variety of drugs previously reported to inhibit platelet PLC activity in vitro were tested for their ability to inhibit rat PMN PLC. Of the compounds tested, none were potent (i.e., IC50 values 100 μM) inhibitors of the PMN PLC.
Functional and Biochemical Properties of Rat Kupffer Cells and Peritoneal Macrophages
Authors: Laskin Debra L , Sirak Andrea A , Pilaro Anne M , Laskin Jeffrey D
Published: January 2018

Functional and biochemical techniques were used to further characterize heterogeneity between rat Kupffer cells and peritoneal macrophages. Both macrophage cell types were found to phagocytize antibody coated sheep red blood cells in a time-dependent manner. However, Kupffer cells were two to three times more phagocytic than were peritoneal macrophages. In contrast, the peritoneal cells released significantly more superoxide anion in response to the complement cleavage product, C5a and the phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate, and produced more hydrogen peroxide than did the liver macrophages. Both cell types responded chemotactically to C5a. These results suggest that macrophages may develop specialized functions depending on the needs of their local environment. Using one and two dimensional SDS-polyacrylamide gel electrophoresis, we also compared the production of newly synthesized proteins by Kupffer cells and peritoneal macrophages. In general, the macrophages were found to produce similar types and numbers of proteins with some exceptions. These included proteins that were unique to peritoneal macrophages and other proteins observed only in Kupffer cells. The production of these proteins in liver macrophages did not appear to correlate with levels of functional activation, but may be more related to the tissue origin of the cells.
Induction of Continuous I-A Expression by Murine Peritoneal Macrophages: Lack of Correlation With T Cell Responses of Bcgr and Bcgs Mice
Authors: Kwasniewski Michael N , Zwilling Bruce S
Published: January 2018

The induction of continuous I-A expression by L3T4 and Lyt2 lymphocyte subpopulations from Bcgr and Bcgs mice was compared. We found that the induction of continuous I-A expression was independent of the Bcg geneotype of the T cells. T cells from congenie Begs BALB/c mice and C.D2Bcgr mice could not induce continuous I-A expression in vitro and were deficient in their ability to produce IFN-gamma in vitro. In contrast, T cells from other strains of Bcgr and Bcgs mice could induce continuous I-A expression. Both recombinant IL-2 and IL-4 acted synergistically with low levels of IFN-gamma to induce continuous I-A expression by macrophages from Bcgr mice.
Maintenance of Peritoneal Macrophages in the Steady State
Authors: Melnicoff Meryle J , Horan Paul K , Breslin Elizabeth W , Morahan Page S
Published: January 2018

Resident peritoneal macrophages (Mø) were labeled in situ by intraperitoneal (i.p.) injection of the green fluorescent cell tracking dye PKH-1. After immunofluorescence staining with Mø specific monoclonal antibodies (Mabs) and phycoerythrin (PE) second antibody, the resident Mø were labeled with both the green dye and red Mab label, while recruited Mø were labeled only with the red Mab tag. These populations were distinguished by two-color flow cytometry.

PKH-1 labeled resident peritoneal Mø were followed for 1-49 days in mice that received no further treatment (steady state). Dye labeled Mø were still detectable after 49 days in vivo, although their green fluorescence intensity had decreased steadily over time. The decrease in dye intensity was limited to Mø, as the fluorescence intensity of PKH-1 labeled peritoneal lymphocytes did not change.

Resident Mø populations were clearly separated from recruited Mø by the intensity of their staining with PKH-1 for up to 28 days. No decrease in the number of resident (dye labeled) peritoneal Mø was observed over 1-28 days. These data indicate that resident peritoneal Mø were not replaced by recruited blood monocytes in the steady state.
Photoreversal of the Ultraviolet Radiation-Induced Disappearance of ATPase-Positive Langerhans Cells in the Epidermis of Monodelphis domestica
Authors: LeVee G J , Applegate L A , Ley R D
Published: January 2018

The present study was undertaken to explore the possible causes of ultraviolet radiation (UVR)-induced disappearance of ATPase-positive, epidermal Langerhans cells (LC). Monodelphis domestica was used because it has the capacity for photoreactivation of UVR-induced pyrimidine dimers in epidermal DNA. Single, 330 J/m2 (ears) or 500 J/m2 (back) UVR exposures (FS-40 sunlamps) reduced the numbers of ATPase-positive epidermal LC in M. domestica ears to ~15% of those in unirradiated ears and ~37% of those in unirradiated dorsal skin. Immediate 90-minute exposures to photoreactivating light (PRL, 320–400 nm) post-UVR reversed the effects of the UVR, resulting in ATPase-positive LC numbers not being significantly different from controls. Exposure to PRL immediately proceeding UVR did not prevent ATPase-positive LC disappearance. The photoreactivation of UVR-induced ATPase-positive LC disappearance indicates that DNA damage (pyrimidine dimers) is involved in the loss of ATPase-positive LC.
Staphylococcus aureus Tetrapeptide With High Chemotactic Potency and Efficacy for Human Leukocytes
Authors: Rot Antal , Henderson Louis E , Sowder Raymond , Leonard Edward J
Published: January 2018

A chemotactic tetrapeptide from culture fluids of Staphylococcus aureus was purified to homogeneity by reverse-phase high-pressure liquid chromatography. The peptide comprises equimolar methionine, leucine, isoleucine, and phenylalanine. It inhibited binding of fluoresceinated fMet-Leu-Phe-Lys to human monocytes, which showed that it interacted with the formyl-methionyl peptide receptor and suggested that it was a formyl-methionyl peptide. Based on a comparison of dose-response curves for inhibition of fluoresceinated fMet-Leu-Phe-Lys binding, the relative affinity of the peptide for the receptor was comparable to that of fMet-Leu-Phe-Lys. At optimal concentrations, chemotactic efficacy (percentage of monocytes migrating to the attractant) was 53 ± 4%, in contrast to 36 ± 3% for the reference attractant fMet-Leu-Phe. Since approximately 60% of human monocytes have formyl-peptide receptors, the bacterial peptide is capable of attracting all receptor-bearing monocytes.
Macrophage Subset Repopulation in the Spleen: Differential Kinetics After Liposome-Mediated Elimination
Authors: van Rooijen Nico , Kors Nel , Kraal Georg
Published: January 2018

Different macrophage subsets can be discriminated in the well defined compartments of the mouse spleen by specialized functions and the presence of specific surface determinants. Red pulp macrophages, marginal zone macrophages, and marginal metallophilic macrophages are eliminated simultaneously within 24 hr by a single injection with liposome-entrapped dichloromethylene diphosphonate (DMDP). After such elimination, these subsets show a striking difference in their kinetics of reappearance: Red pulp macrophages are back in normal numbers after 1 week, the marginal metallophilic macrophages take 2 weeks to regain fully their position at the border of the marginal zone and periarteriolar lymphocyte sheath, but it takes over 1 month for complete reappearance of the marginal zone macrophages. Marginal zone lymphocytes, also affected by treatment with the liposome-entrapped drug, reappeared in the marginal zone within 2 weeks, indicating that marginal zone macrophages are not required for their localization and/or retention there. Approximately 2 weeks after treatment, all cells in the spleen have returned to normal numbers with the exception of marginal zone macrophages, which can be found only sporadically at that time. The results indicate that these macrophage subpopulations must have different precursor requirements. The differential reappearance of the macrophages creates the possibility of studying lineage analysis and will help to unravel the precise function of the marginal zone macrophages and marginal metallophilic macrophages in particular.
Confocal Scanning Fluorescence Microscopy: A New Method for Phagocytosis Research
Authors: Hook Gregory R , Odeyale Charles O
Published: January 2018

An important new method for phagocytosis research, confocal scanning fluorescence light microscopy (CSFM), is demonstrated using fluorescent microspheres ingested by murine macrophages. CSFM, in combination with Nomarski differential interference contrast microscopy (DIC). can resolve microspheres inside cells from microspheres attached to the surface of cells. Further, combined CSFM and DIC images can quantitate phagocytosis by individual cells aggregated together. No other method offers these capabilities. A comparison of CSFM and conventional epifluorescence light microscopy (EFM) images shows that CSFM produces significantly higher-resolution images of microspheres than EFM, primarily because CSFM excludes the out-of-focus light artifacts of EFM.
Macrophage Activation for Cytolysis of Virally Infected Target Cells
Authors: LeBlanc Paul A
Published: January 2018

Bone-marrow-culture-derived macrophages killed virally infected cells but not uninfected cells. This activity could be enhanced by preexposure of the macrophages to lipopolysaccharide (LPS), and/or purified interferons. The ability to kill virally infected targets was not restricted to a single cell type or virus. Comparing the ability of activators to induce activity against virally infected targets or tumor (P815) targets, it was found that much lower levels of LPS or α/β-interferon were able to induce cytolytic activity for virally infected cells than were needed for tumor targets. Further, while the antitumor activity did not change significantly with an increase in the time of exposure to activating stimuli from 4 to 24 h, the activity against virally infected cells decreased dramatically with the longer exposure to stimuli.
Receptor-Mediated Endocytosis of Carcinoembryonic Antigen by Rat Alveolar Macrophages In Vitro
Authors: Toth Carol Ann , Rapoza Alan , Zamcheck Norman , Steele Glenn , Thomas Peter
Published: January 2018

Uptake of carcinoembryonic antigen (CEA) by isolated rat alveolar cells was time, temperature, and concentration dependent (Kuptake, = 2.4 × 10-7M). Pretreatment of the alveolar cells with colchicine inhibited internalization of CEA. Uptake of 125Mabeled CEA by alveolar cells required divalent cations and was inhibited by cold CEA and nonspecific cross-reacting antigen (NCA). The carbohydrate portion of CEA was modified by neuraminidase treatment and Smith degradation. The modified glycoproteins inhibited endocytosis by the alveolar macrophages, thus excluding nonreducing terminal carbohydrate residues as the recognition site of the receptor. Endocytosis of CEA was independent of native protein conformation since performlc acid oxidized CEA and glycopeptides produced by pepsin digestion were inhibitory. Rat alveolar macrophages bound CEA with similar specificity to that of rat Kupffer cells. Alveolar macrophages, unlike Kupffer cells, did not rapidly exocytose the internalized CEA. Neither P388D1, a macrophage-like murine cell line, nor murine peritoneal exudate cells were capable of endocytosing CEA in vitro.
Induction of Proteins in Interferon-α- and Interferon-γ-Treated Polymorphonuclear Leukocytes
Authors: Rubin Berish Y , Anderson Sylvia L , Lunn Ruth M , Smith Larry J
Published: January 2018

Interferon-γ (IFN-γ) treatment of polymorphonuclear leukocytes (PMNs) results in an activation of their functions. Studying the IFN responsiveness of PMNs and using antibodies to the IFN-induced proteins, we have observed the ability of IFN-α to stimulate the production of the IFN-induced 67,000 and 56,000 dalton proteins and the ability of IFN-γ to induce the synthesis of the 67,000, 56,000, and 42,000 dalton proteins. The induction of these proteins is dependent on de novo RNA synthesis, as its induction is inhibited if the IFNs and actinomycin D are added to the cells simultaneously. The results of this study confirm the ability of PMNs to carry out gene activation and demonstrate the ability of PMNs to respond to both IFN-α and IFN-γ.
Transglutaminase Levels and Immunologic Functions of BCG-Elicited Mouse Peritoneal Macrophages Isolated by Centrifugal Elutriation
Authors: Khera Vimlarani , Mehta Kapil
Published: January 2018

BCG-elicited mouse peritoneal macrophages were separated into three subpopulations by counterflow centrifugal elutriation. The three subpopulations were characterized on the basis of the level of a protein cross-linking enzyme, tissue transglutaminase. Subpopulation-3 consisted of large cells (>95% esterase positive and >90% viable) and had at least a fivefold higher transglutaminase activity (35 ± 6 nmol/hr/mg) as compared to macrophages in subpopulation-1 (6 ± 2 nmol/hr/mg) and at least a threefold higher enzyme activity as compared to subpopulation-2 (11 ± 2 nmol/hr/mg). Subpopulation-3 also showed sevenfold higher phagocytosis of IgG-coated sheep red blood cells. The three subpopulations showed no difference in their ability to kill Listeria monocytogenes as determined by [3H]-thymidine release. Subpopulations-2 and -3 caused 90% inhibition of murine adenocarcinoma (EMT-6) tumor cell growth in the presence or absence of lipopolysaccharide. Subpopulation-1 had a poor ability to inhibit EMT-6 cell growth (29 ± 12%). However, in the presence of lipopolysaccharide, this activity increased by at least threefold (92 ± 7%). The three subpopulations showed no significant difference in their cytolytic activity against murine mastocytoma (P815) target cells in the presence or absence of lipopolysaccharide. These results suggest that tissue transglutaminase may have no significant role in bactericidal, tumoricidal, or tumoristatic function of macrophages; however, it might have some role in promoting the Fc-receptor-mediated phagocytic function of the macrophages.
Differential Induction of Macrophage GSIB4-Binding Activity
Authors: Tabor Dale R , Larry Cynthia H , Jacobs Richard F
Published: January 2018

Activated macrophages display a terminal galactopyranosyl group on their membrane surface that binds the lectin Griffonia simplicifolia–IB4 (GSIB4). Using FITC-conjugated GSIB4, we examined the induction and subsequent expression of this corresponding marker on peritoneal macrophages from normal (NMO) and LPS-treated (LPS MO) mice. Although the percentage of fresh LPS MO explants that bound GSIB4 was always higher when compared to the NMO counterparts, marker expression on the latter was readily enhanced by cufturing the cells in vitro either alone or with stimuli. Moreover, we found that an increase in this activity was promoted by either nonspecific phagocytosis of latex beads, or γ-interferon (γ-IFN) treatment. Further investigation showed that a prerequisite sequence of signal delivery to the macrophages was associated with maximal expression of the GSIB4 binding. When γ-IFN treatment preceded latex bead ingestion, maximum GSIB4 binding occurred. Data obtained from using short-term (1 hr) and long-term (24 hr) exposure to latex beads showed that metabolic processing of induction signals was required to enhance the response over time. This yielded better GSIB4-binding activity when responses to these pulses were analyzed in freshly explanted macrophages.

The overall results of this study demonstrated that macrophage binding of GSIB4 was differentially associated with stimuli induction. Moreover, select signals in the form of soluble mediators, or the mechanical events characteristic of internalization were capable of eliciting an increase in the percentage of macrophages that were positive for binding GSIB4. Thus, the enhanced affinity for binding this lectin may serve as a useful marker to determine the magnitude of macrophage responsiveness when these cells are examined following their exposure to different stimuli.
Differential Release of Mediators From Human Basophils: Differences in Arachidonic Acid Metabolism Following Activation by Unrelated Stimuli
Authors: Warner Jane A , Peters Stephen P , Lichtenstein Lawrence M , Hubbard Walter , Yancey Kim B , Stevenson Henry C , Miller Paul J , MacGlashan Donald W
Published: January 2018

We have examined the release of histamine and LTC4 from purified human basophils challenged with several different stimuli, both physiological and nonphysiological. Basophils (n=16) challenged with 0.1 μg/ml anti-IgE released 38±4% of their available histamine and 39±12 ng LTC4/109 basophils within 15-30 min. F-Met peptide (n=8) caused the release of 54±8% histamine and 42±25 ng LTC4/106 basophils within a period of 2-6 min. C5a caused the release of 22±3% histamine from selected donors but failed to initiate any LTC4 release unless combined with D2O or 5 mM extracellular calcium. The two nonphysiological stimuli A23187 and TPA caused extensive histamine release, 67±8 and 82±11%, respectively, and while A23187 initiated a large and rapid release of leukotriene, TPA failed to release any LTC4 even when combined with D2O or 2-5 mM extracellular calcium. Increased concentrations of extracellular calcium enhanced anti-IgE and f-Met peptide induced release of LTC4 but inhibited the A23187 induced release of leukotriene. A single peak of immunoreactive leukotriene C4 that comigrated with the authentic standard was identified using HPLC followed by radioimmunoassay. No LTD4 or LTE4 could be detected. Purified human basophils incubated with 0.2 μM [3H]AA incorporated 290 pmol/106 cells, or 32±5% of the available label within 60 min. The [3H]AA was taken principally into the phospholipids (73±5%), with 20±3% as neutral lipid, and only 5±2% remaining as the free acid. Three phospholipid subclasses, phosphatidylcholine, PC (24±2%), phosphatidylinositol, PI (22±1%), and phosphatidytethanolamine, PE (15±3%), accounted for the majority of the incorporated [3H]AA while the remainder of the phospholipids accounted for less than 5% of the total cpm. HPLC analysis of the lipid mediators released during stimulation with 0.1 μg/ml anti-IgE revealed [3H]LTC4 (2.4±1.0%), [3H]5HETE (1.0±0.1%), unmetabollzed [3H]AA (91±2%), and an unidentified peak (3.4±1.4%). The unknown metabolite eluted with the prostaglandins, was inhibited by indomethacin, and appeared to have a relatively high specific activity. It may thus represent an artifact of the labeling procedure rather than a novel basophil-derived prostaglandin.
Change of Membrane Fluidity of Rat Neutrophils Accompanying Escherichia coli Inoculation
Authors: Masuda Midori , Komiyama Yutaka , Nishikado Hiroyuki , Kuriki Hideki , Egawa Hiroshi , Murata Kenjiro
Published: January 2018

Membrane fluidity of rat neutrophils was studied following Escherichia coli inoculation, and characteristic changes were observed. Membrane fluidity was assessed by the excimer-forming lipid technique using pyrenedecanoic acid and flow cytometry and expressed as the fluorescence intensity ratios of excimer and monomer pyrenedecanoic acid (lE/LM ratio). High lE/lM ratios indicated high membrane fluidity. The IE/Im ratio of rat neutrophils (0.50 ± 0.048) increased after E. coli inoculation, reaching a maximum of almost 1.00 after 10-20 min and then returning to its starting value. Intravenous injection of heat-killed E.coli or E. coli-conditioned culture supernatants into rats induced a rapid increase of lE/lM ratios, which returned to initial levels after 20 min. The effect on membrane fluidity of in vitro neutrophil incubation with E. coli, heat-killed E. coli, or E. coli conditioned culture supernatants was similar to that observed in vivo. Addition of 5 mM ethylenediaminetetraacetic acid (EDTA) did not affect neutrophil membrane fluidity. Addition of either 5 μg/ml cytochalasin B or 10-5 M colchicine did not directly affect neutrophil membrane fluidity but did block the change observed following incubation with bacteria.
Interleukin-1 Inhibitor Production by Human Mononuclear Leukocytes and Leukocyte Subpopulations Exposed to Respiratory Syncytial Virus: Analysis and Comparison With the Response to Influenza Virus
Authors: McCarthy Donna O , Domurat Frank M , Nichols Joan E , Roberts Norbert J
Published: January 2018

Net interleukin-1 (IL-1) inhibitor activity is induced by exposure of purified human monocytes-macrophages to respiratory syncytial virus (RSV). Furthermore, IL-1 inhibitor activity was produced by monocytes-macrophages exposed to RSV in the presence of lymphocytes, that is, by unseparated mononuclear leukocytes (MNL). Purified RSV-exposed lymphocytes, as well as the lymphocytes exposed within MNL preparations, produced net IL-1 inhibitor activity. In contrast, net IL-1 activity was produced when purified monocytes-macrophages or unseparated MNL were exposed to influenza virus. The RSV-induced IL-1 inhibitors demonstrated antiproliferative effects on mitogen-stimulated human lymphocytes as well as on the mouse thymocytes used in standard assays. The results raise the possibility that such antiproliferative activity is mediated, at least in part, by monocytes-macrophages. The data also suggest that IL-1 inhibitors produced by MNL after exposure to RSV may contribute along with other factors to the recurrence of RSV infection in immune individuals.
Virus-Transformed Macrophage-Like Cell Line as Tool for Eicosanoid Research
Authors: Wessel K , Kaever V , Ostertag W , Bitter-Suermann D , Resch K
Published: January 2018

The ability of a virus-transformed murine macrophage like cell line HA 38 to produce different eicosanoid metabolites was examined. HA 38 cells release similar amounts of prostaglandins and leukotrienes as did murine peritoneal macrophages in response to both physiological and non-physiological stimuli. Enzyme systems known to be involved in the regulation of eicosanoid synthesis are expressed. HA 38 cells thus are a well defined macrophage model system and are well suited to study eicosanoid synthesis in macrophages and effects of drugs on the prostaglandin and leukotriene synthesis pathways.
Feline Hybridoma Growth Factor/lnterleukin-6 Activity
Authors: Ohashi Takashi , Goitsuka Ryo , Ono Kenichiro , Hasegawa Atsuhiko
Published: January 2018

An assay system was developed to measure feline hybridoma growth factor (HGF)/interleukin-6 (IL-6) activity in biological samples containing many kinds of cytokines by using the proliferation of the newly established mouse-rat hybridoma clone. B3B1. The proliferative response of this B3B1 clone was IL-6-specific, and could not be promoted by other cytokines including IL-1, IL-2, IL-3, and granulocyte-colony-stimulating factor (G-CSF), The anti-human B-cell stimulatory factor 2 (BSF-2)/IL-6 antiserum did not neutralize feline HGF/IL-6 activity in conditioned media prepared from feline con A-stimulated splenocytes and unstimulated alveolar macrophages, indicating antigenic differences between species. Feline HGF/IL-6 was eluted into the fractions corresponding to a molecular weight of 30,000–40,000 in gel filtration, and into the fractions at a salt concentration of 0.2–0.3 M NaCI in anion exchange chromatography. The physicochemical properties of feline HGF/IL-6 were slightly different from those of murine and human IL-6.
Killing of Mycobacterium smegmatis by Macrophages From Genetically Susceptible and Resistant Mice
Authors: Denis Michel , Forget Adrien , Pelletier Micheline , Gervais Francine , Skamene Emil
Published: January 2018

The bactericidal function of macrophages was investigated in congenic mice expressing the phenotype of susceptibility (B10.A, Bcgs) or resistance (B10.A.Bcgr) to mycobacterial infection. When splenic and peritoneal macrophages from these two mouse strains were infected in vitro with Mycobacterium smegmatis, the Bcgr macrophages were shown to inactivate M. smegmatis more efficiently than their Bcgs congenic counterparts. The mechanisms of this superior antimycobacterial activity was studied further. Addition of catalase did not abolish killing to a significant degree in either allelic type of macrophage, suggesting that hydrogen peroxide production was not involved in the killing activity controlled by the Bcg gene. Activation of Beg macrophages by exposure to crude lymphokines rendered them equally as efficient as their Bcgr counterparts in their capacity to destroy M. smegmatis. This finding suggests that both the genetically resistant and susceptible macrophages have the potential to kill M. smegmatis in vitro. This potential is expressed constitutively by the Bcgr but not Bcga macrophages and can be induced, by lymphokine treatment, in the Bcga macrophages. In a final set of experiments, the macrophage killing of M. smegmatis was evaluated as a test system to type for the Bcg gene allelic type in vitro, using a set of AXB and BXA recombinant inbred strains of mice. Results obtained show that typing of AXB/BXA recombinant inbred strains for the trait of bactericidal activity vs. M. smegmatis in vitro revealed a perfect match with the strain distribution pattern of resistance/susceptibility to Mycobacterium bovis BCG in vivo.
Priming of Polymorphonuclear Granulocytes by Lipopolysaccharides and Its Complexes With Lipopolysaccharide Binding Protein and High Density Lipoprotein
Authors: Vosbeck Klaus , Tobias Peter , Mueller Heinz , Allen Rodger A , Arfors Karl-E , Ulevitch Richard J , Sklar Larry A
Published: January 2018

Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide. by prior exposure to bacterial lipopolysaccharide (LPS). The activity of LPS and the size of its aggregates are altered by plasma constituents such as high density lipoprotein (HDL) and the recently discovered acute phase reactant lipopolysaccharide binding protein (LBP) Tobias et al.: J. Exp. Med. 164,777, 1986]. The ability of LPS, LPS–LBP, and LPS–HDL complexes to activate a number of cellular responses have been compared. LPS–LBP and LPS–HDL were prepared using LBP and HDL from rabbit serum. LPS from Salmonella minnesota Re595 and its LPS–LBP and LPS–HDL complexes differed in their ability to prime PMN O2− production in response to formyl peptide (f-Nle-Leu-Phe-Nle-Tyr-Leu [FNLPNTL]). Human PMN prepared under conditions in which O2 production is minimal (<1 nmol O2−/106 PMN/10 min) after exposure to 10-7 M FNLPNTL can be primed with 0.1–100 ng/ml LPS in a dose- and time-dependent manner to produce up to 12 nmol O2−/106 PMN/10 min. LBP complexation accelerated the priming induced by LPS, whereas HDL complexation retarded it. Priming was accompanied by a parallel two- to threefold increase in formyl peptide receptor number as determined by FACS analysis of fluoresceinated FNLPNTL binding and SDS-PAGE autoradiographic analysis of photoaffinity ligand binding. Thus binding of LPS to plasma proteins changes the response of the PMN to LPS and may represent one way in which the response of the PMN is regulated during infection. Since LBP concentrations change during an acute phase response, complexation of LPS with LBP is a mechanism that may regulate neutrophil responses in vivo during inflammation.
Differential Tumor Necrosis Factor Production by Human Monocyte Subsets
Authors: Szabo Gyongyi , Miller-Graziano Carol L , Wu Jia-Yan , Takayama Thomas , Kodys Karen
Published: January 2018

The human monocyte (MØ) subset rosetting with anti RH-coated human erythrocytes via high-affinity, 72 kD receptors (FcRI+), contains the PGE2-producing immunosuppressive subpopulation, while the non-rosetting MØ subset (FcRI-) is the major plasminogen activator-producing and antigen-presenting MØ. This study gives additional evidence for the functional disparity of the FcRI- and FcRI+ MØ subsets. We are demonstrating that the normal human MØ subset isolated by rosetting via the FcRI receptor (FcRI+) produces greater quantities of tumor necrosis factor (TNF) than the non-rosetting (FcRI-) MØ. TNF production by the FcRI+ MØ subset is greater than that of the FcRI- MØ subset whether secreted (P < .001) or cell-associated (P < .001) TNF is assessed. The rosetting MØ subset that expresses high densities of FcRI (FcRI+) produced the majority of normal human peripheral blood MØ TNF whether the stimulation was an interferon gamma (IFNγ) prime followed by MDP or followed by interleukin-2 (IL-2). The Fc rosetting technique itself resulted in some TNF induction in the FcRI+ MØ subset accounting for some of the increased TNF production of this subset. However, increasing the stimulation level of the FcRI very-low-density (FcRI-) MØ subset did not induce it to produce TNF levels equivalent to the moderately stimulated FcRI+ MØ subset. These data, therefore, imply that only stimulation through the type I Fcγ receptor can augment or induce TNF activity. The difference in the MØ subset's TNF response remained even after the FcRI- MØ subset received a 2.5-fold increase in stimulation with the classical MØ induction regimen of IFNγ plus bacterial cell wall product. Although stimulation of the FcRI+ MØ subset via crosslinking of their FcRI receptors might represent a unique TNF stimulation pathway, this stimulation does not occur in the low-density FcRI (FcRI-) MØ subset, again indicating functional disparity between these subsets. Greater TNF production by the FcRI+ MØ subset was induced concomitant to elevation of its prostaglandin E2 production. Since both TNF and PGE2 are increased in some patient groups, a pathological shift in the FcRI+ versus FcRI- MØ ratio in these patients coupled to the functional differences in FcRI+ and FcRI+ MØ subsets could be one mechanism for the development of immunoincompetence.
Repopulation of Macrophages in Popliteal Lymph Nodes of Mice After Liposome-Mediated Depletion
Authors: Delemarre F G A , Kors N , Kraal G , van Rooijen N
Published: January 2018

Macrophages lining the subcapsular sinus (SCS) and those located in the medulla of popliteal lymph nodes (PLN) of mice were eliminated after subcutaneous (s.c.) injection of dichloromethylene diphosphonate (CI2MDP)-containing liposomes. No effect of liposome-entrapped CI2MDP could be seen on nonphagocytic cells, e.g., interdigitating cells (IDC) and B- and T-lymphocytes. One month after injection the eliminated subsets of macrophages were still absent. After 2 mo a small number of macrophages had reappeared along the SCS and in the medulla of the PLN of a few animals. Complete repopulation of the PLN with macrophages was observed only after 5 mo.

This extremely long repopulation time could be shortened drastically by local administration of Freund's complete adjuvant (FCA). A small number of macrophages reappeared along the SCS and in the medulla 1 wk after FCA and after 3 wk the repopulation of the PLN with macrophages was complete. Such rapid repopulation of macrophages was not achieved after s.c. injection of paraffin oil or paratyphoid vaccine.

These results indicate that the normal rate of influx of mononuclear phagocytes into the PLN is low, but that it can be sped up after administration of FCA.
Effects of Polymyxin B on Superoxide Anion Release and Priming in Human Polymorphonuclear Leukocytes
Authors: Aida Yoshitomi , Pabst Michael J , Rademacher Jonella M , Hatakeyama Tamiko , Aono Masao
Published: January 2018

We studied the effect of a potent inhibitor of protein kinase C, polymyxin B (PMXB), on superoxide anion (O−2 release by human polymorphonuclear leukocytes (PMNL). PMXB was compared with another inhibitor of protein kinase C, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7). Both PMXB and H-7 inhibited phorbol myristate acetate (PMA)-stimulated O−2 release. Formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated O−2 release by cytochalasin B–treated PMNL was not inhibited significantly by either PMXB or H-7. 1-Oleoyl-2-acetyl-glycerol (OAG,25–100 μM) stimulated PMNL to release O−2 with a long lag-time (8–10 min). Although H-7 inhibited OAG-stimulated O−2 release, PMXB augmented the OAG-stimulated response by increasing rate and reducing lag time. The augmenting effect of PMXB was evident only when added after stimulation by OAG, with maximum effect observed at 3 min after addition of OAG. The augmenting effect was also seen with PMXB immobilized on agarose beads. PMXB did not affect the respiratory burst response to 1,2-dioctanoylglycerol. PMXB-augmented, OAG-stimulated O−2 release was inhibited by the addition of H-7 before OAG. In contrast to the effect on O−2 release, OAG-stimulated protein phosphorylation was inhibited similarly by either PMXB or H-7, when these agents were added 3 min after stimulation by OAG. These results suggested that initial activation of protein kinase C by OAG is essential for O−2 release, but that PMXB acts in a manner independent from protein kinase C to augment OAG-stimulated O−2 release. When priming by OAG for enhanced O−2 release (as opposed to direct stimulation of O−2 release) in FMLP-stimulated PMNL was examined, PMXB inhibited O−2 release in OAG-primed PMNL, suggesting that protein kinase C is involved in priming of PMNL by OAG.
Oxidative Metabolism in the Alveolar Macrophage: Analysis by Flow Cytometry
Authors: Kobzik Lester , Godleski John J , Brain Joseph D
Published: January 2018

We evaluated the reagents dichlorofluorescin (DCFH) and hydroethidine (HE) for use in flow cytometric analysis of the respiratory burst of alveolar macrophages and monocytes. DCFH and HE are non-fluorescent precursors which can be oxidized intracellularly to the fluorescent compounds dichlorofluorescein and ethidium.

Alveolar macrophages (AMs) loaded with either DCFH or HE were analyzed after phorbol myristate acetate (PMA) stimulation. The results, expressed as fmol/cell oxidation product (DCF or ethidium) after fluorometric standardization of the flow cytometer. show that both DCFH (273 ± 48, mean increase over control ± SE, fmol/cell, N = 9) and HE (416 ± 54, N = 11) detected the substantial respiratory burst of hamster AMs. Similar results were obtained with normal human AMs. By using multiparameter analyses, the oxidative response of AMs ingesting opsonized fluorescent latex beads was measured in subpopulations ingesting increasing numbers of particles. A graded increase in oxidation of both DCFH and HE was found in response to increasing phagocytosis. Ingestion of fluoresceinated staphylococcal bacteria caused similar changes in HE-loaded AMs. Inhibition of respiration with antimycin showed that approximately 95% of the increased oxidative metabolism of hamster AMs ingesting opsonized beads or bacteria was mitochondrial. The remaining 5% (10–40 fmol/cell) is membrane-derived oxidative activity quantitatively similar to that measured in assays of extracellular release of H2O2.

Monocytes loaded with either DCFH or HE also showed substantial increases in fluorescence after PMA stimulation (mean % increase over control ± SE at 30 ruin: 464 ± 104, DCFH, 505 ± 156, HE). While DCHF is known to measure H2O2, HE is less well characterized. Exposure of cells to an extracellular source of both superoxide anion (O2-) and H2O2, xanthine oxidase-xanthine, resulted in marked oxidation of intracellular HE. Addition of both superoxide dismutase and catalase blocked this oxidation, indicating that HE can detect both O2- and H2O2. These agents can be useful probes for precise analysis of oxidative metabolism during phagocytosis in AMs and other mononuclear phagocytes.
The Short and Happy Life of Neutrophil Activation
Authors: Becker Elmer L
Published: January 2018

This is largely a history of attempts from approximately 1960 until about 1980–81 to understand the mechanisms by which neutrophils are activated by chemotactic factors to induce chemotaxis and granule secretion. As such, it deals with the growth of our knowledge of neutrophil chemotactic factors and their receptors; the importance and role of cation fluxes, especially Ca2+, microfilaments and microtubules, membrane potential, cyclic nucleotides, and the start of our recognition of the importance of arachidonic acid and phospholipid metabolism and protein phosphorylation. In a very real sense this is a history of the origins of our present realization that reactions and functions which had been considered specific to the neutrophil are to be thought of as similar or identical to general biological and physiological processes such as muscle contraction, cellular secretion, etc.
Estimation of In Vivo Reticuloendothelial System Phagocytic Activity in Rats by Direct Blood Clearance Techniques and Nuclear Scintigraphy
Authors: Goyal Deepi G , Gutowski Karol A , Merion Robert M
Published: January 2018

There has been considerable interest in the examination off reticuloendothelial system phagocytic blockade. In this study, the kinetics of phospholipid liposome-mediated and intraperitoneal silica-mediated phagocytic blockade were examined using five methods off analysis off in vivo 99mTc-labeled albumin clearance and reticuloendothelial cell uptake. Two direct blood sampling techniques revealed significant impairment in 99mTc-labeled albumin clearance after treatment with silica (P < 0.05), while liposome treatment was not associated with such impairment. A method utilizing nuclear scintigraphy for the determination of blood clearance was incapable of detecting silica-mediated blockade but demonstrated significant impairment by liposomes at 2 hr (P < 0.001), 6 hr (P < 0.05), and 24 hr (P < 0.001). Gamma camera imaging methods for determination of hepatic uptake demonstrated significant (P < 0.05) and reversible impairment of 99mTc-labeled albumin uptake by liposomes. The most promising of these techniques utilizes deconvolutional analysis off liver region of interest time–activity curves to correct for continuously changing blood concentrations of tracer and for intracellular tracer processing and catabolism. Measurements of reticuloendothelial system phagocytic activity should include methods that take into account the observed discrepancies between blood clearance determinations and reticuloendothelial cell uptake.
Superoxide Anion Generation in the Liver During the Early Stage of Endotoxemia in Rats
Authors: Bautista Abraham P , Mészáros Károly , Bojta Julia , Spitzer John J
Published: January 2018

Infiltration of polymorphonuclear neutrophils (PMN) in the rat liver 3 hr after an intravenous (IV) injection of a sublethal dose of Escherichia coli lipopolysaccharide (LPS) was observed without any significant alteration in the total number of Kupffer and endothelial cells. Since previous studies have demonstrated that phagocytic cells in the liver were in a state of metabolic activation under similar experimental conditions, we investigated the in vitro generation of superoxide anion (O2-) by this cell type following the administration of LPS. Kupffer cells from normal rats did not release O2-, in contrast to those obtained from LPS-treated rats. The generation of O2- by Kupffer cells from endotoxic rats was elevated from 3.0 ± 1.9 nmol/106 cells/60 min (mean ± SD) in the absence of macrophage (Mφ) activators, to 5.0 ± 2.36, 11.33 ± 5.40, and 4.33 ± 0.90 in the presence of opsonized zymosan, phorbol myristate acetate (PMA), and the calcium ionophore A23187, respectively. Hepatocytes from normal or endotoxic rats did not produce detectable O2-. Endothelial cells from LPS-treated rats generated < 0.8 nmol/106 cells in the presence of zymosan. PMN that accumulated in the livers of endotoxic rats released O2- only in the presence of zymosan (8.12 ± 5.40), PMA (15.43 ± 5.84), or A23187 (1.70 ± 0.12). The O2- generation by blood monocytes and PMN increased significantly after endotoxin administration and in the presence of activators. These results suggest that the hypermetabolic state of phagocytic cells in the liver shortly after LPS treatment may be correlated with the increased generation of O2-. The latter may subsequently contribute to the induction of hepatic injury in endotoxemia.
Surface Characteristics, Morphology, and Ultrastructure of Human Adherent Lymphokine-Activated Killer Cells
Authors: Melder Robert J , Walker Elizabeth R , Herberman Ronald B , Whiteside Theresa L
Published: January 2018

Human adherent lymphokine-activated killer (A-LAK) cells represent a population of highly cytotoxic, interleukin-2 (IL-2)-activated peripheral blood lymphocytes that have large granular lymphocyte (LGL) morphology and display natural killer (NK) cell-associated surface markers (CD3-CD56+). The ultrastructure of A-LAK cells also is consistent with that of highly activated NK cells. After their initial isolation, continued culture of A-LAK cells in the presence of IL-2 resulted in cyclic shifts between adherent and nonadherent phases with about 90% of the cells floating and non-adherent. All of these A-LAK cells were spheroidal with numerous villi and pseudopodia. In the adherent phase, A-LAK cells were motile, moving along the solid surface at a speed of about 1 μm per minute, and polar, with a ruffled leading edge at one end of the cell and a terminal tuft of villi at the opposite end. Transmission electron microscopy of these cells also demonstrated a high degree of Internal polarity, with the nucleus at the leading edge of the cell and richly granulated cytoplasm at the terminal end. Extensive cytoskeletal structures, multivesicular bodies, and an active Golgi complex characterized these cells. A-LAK cells in the adherent phase were found to express numerous point contacts (podosomes) and larger adherence structures containing polymerized actin, which appear to be important for interactions of these cells with the substrate. Increased expression of adhesion molecules in association with surface structures mediating adherence is also responsible for effective binding of A-LAK cells to solid surfaces.
Dependence on Ca2+ of Lipoprotein Lipase Stability
Authors: Goldman Rachel
Published: January 2018

Macrophages continuously secrete lipoprotein lipase (LPL) into the culture medium. When LPL was collected from thioglycollate-elicited peritoneal macrophages (Tg-Mø) or J774.1 cells over a 4 h period in Ca2+ and Mg2+-free Dulbecco's modified Eagle's medium (d-DMEM) the activity in the collection medium was reduced by 40–62% and 23%, respectively, as compared to that expressed in full medium (DMEM). Ca2+ supplementation during the collection period in d-DMEM augmented LPL activity in the medium; about 1 mM Ca2+ was required for attainment of activity comparable to that expressed in DMEM. Addition of Ca2+ during the assay did not enhance LPL activity collected into d-DMEM. Addition of EGTA to the assay mixture reduced LPL activity by 34–60% and when present in the collection medium, EGTA led to a reduction in enzyme activity greater than 90%. A 4 h incubation of Tg-Mø in 3 mM EGTA led to an almost complete loss of intracellular Ca2+ (measured by efflux of 45Ca2+ from preloaded cells), yet there was no change in the overall synthesis and secretion of proteins and in the phagocytic capability of the cells. LPL activity in the enzyme collection medium after its removal from cell monolayers was stable at least up to 4 h at 0°C and at 23°C. Activity was progressively lost with increased temperatures: up to 40% loss at 37°C in 4 h. Addition of EGTA to the above medium led to an enhanced rate of irreversible enzyme inactivation: 76–86% loss of activity in 4 h at 37°C. No inactivation was observed at 0°C and at 23°C in the presence of EGTA. The results indicate a critical role for Ca2+ in enzyme stabilization.
Suppression of Late Phase Enhanced Vascular Permeability in Rats by Selective Depletion of Neutrophils With a Monoclonal Antibody
Authors: Sekiya Sakae , Yamashita Takao , Sendo Fujiro
Published: January 2018

We investigated the role of neutrophils in increased vascular permeability by a selective reduction of neutrophils using a monoclonal antibody, RP-3. An intraperitoneal injection of RP-3 not only selectively depleted peripheral blood neutrophils, but prevented the neutrophil infiltration to the tissues. Proteose peptone, zymosan, and BCG induced three different types of inflammatory edema, showing the early phase only, early plus late phase, and the late phase only, respectively. Only the late phase response of zymosan and BCG was inhibited by a depletion of neutrophils by RP-3, though the early phase response induced by proteose peptone and zymosan was not affected. Reconstitution of neutrophil-depleted rats by in situ infection of these cells restored the inflammatory edema induced by BCG, depending upon the number of neutrophils injected.