bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Maturation and Age on Lymphocyte Proliferation Induced by A23187 Through an Interleukin-Independent Pathway
Authors: Wu Wutong , Pahlavani Mohammad , Richardson Arlan , Cheung H Tak
Published: January 2018

Lymphocyte proliferation induced by lectins declines drastically with age. It has been suggested that the reduction of interleukin production by lymphocytes from old individuals is responsible for the decline in proliferation. In this study, lymphocyte proliferation was stimulated by the calcium ionophore, A23187. A23187 induced the proliferation of spleen lymphocytes from rats through an interleukin-independent pathway; depletion of spleen lymphocytes of macrophages, addition of exogenous interleukin 2 (IL 2), and addition of anti-IL 2 monoclonal antibodies had no effect on the proliferation stimulated by A23187. Spleen lymphocytes from male Fischer F344 male rats of 5, 13, 22, and 30 months of age were stimulated with either concanavalin A (Con A) or A23187. A 50% decrease in Con A- and A23187-induced proliferation was observed between 5 months and 13 months of age. A23187-induced proliferation decreased only slightly between 13 months and 30 months of age (14%), while Con A-induced proliferation decreased by 34%. This is the first report to show that the induction of lymphocyte proliferation through an interleukin-independent pathway decreases with increasing age. In addition, these results suggest that a decrease in the responsiveness of cells to calcium ions might be an important factor in the age-related decline in lymphocyte proliferation.
Phagocytosis of Unopsonized Zymosan by Human Monocyte-Derived Macrophages: Maturation and Inhibition by Mannan
Authors: Speert David P , Silverstein Samuel C
Published: January 2018

Human monocyte-derived macrophages phagocytosed zymosan in the absence of serum opsonins. The capacity to ingest zymosan developed after the cells were cultured in vitro for 3 days and was inhibited completely by mannan. We conclude that human monocyte-derived macrophages phagocytose unopsonized zymosan predominantly via mannose receptors.
Macrophage-Like Tumor Cells as Tools to Study Chemoattractive Activity
Authors: Terheggen P , Van Loveren H , Den Otter W
Published: January 2018

Macrophage-like tumor cells can be obtained in large quantities as rather homogeneous populations, making these cells useful for chemotaxis assays. Therefore, macrophage-like cells J774A, WEHI-3, P388D1, IC-21, and NCTC 1469, all of murine origin, and U937 of human origin, were tested for chemotactic activity to a number of chemoattractive agents, such as casein, an N-formyl tetrapeptide (N-formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-tyrosine), and culture supernatants of murine SL2 lymphoma cells. J774A and WEHI-3 macrophage-like cells of murine (BALB/c) origin expressed the strongest chemotactic activity to casein and N-formyl tetrapeptide, respectively. The results show that: very standardized chemotaxis assays can be performed using these cell lines; these assays require appropriate cell line–stimulus combinations; there are substantial differences among cell lines as to sensitivity to various chemoattractive substances; macrophage cell lines and functional mutants may be helpful for the study of receptors for chemotaxins and the study of transducer signals for chemotaxis.
Effect of Alkylating Agents on the Clearance of Staphylococcus aureus From Murine Lungs
Authors: Nugent Kenneth M , Onofrio John M
Published: January 2018

Alkylating agents have several effects on cellular host defense responses which could increase either the frequency or the severity of pulmonary infections. In addition, some of these agents directly injure lung parenchyma and could have effects on intrapulmonary killing processes independent of any effect on phagocyte number and function. We have used a murine model for staphylococcal clearance to evaluate the effect of cyclophosphamide and mechlorethamine on intrinsic lung defenses. Single doses of mechlorethamlne (40 μg IV) or of cyclophosphamide (150 mg/kg IP) reduce peripheral blood neutrophil counts and spleen weights on day 3 after injection; with the exception of neutrophil counts in mechlorethamine-treated mice, these parameters returned to normal by days 10-12. Both drugs reduced the number of alveolar macrophages recoverable by bronchoalveolar lavage on days 10-12 but not day 3. Mechlorethamine delayed the clearance of Staphylococcus aureus 502A from the lung on both days 3 and 12, but cyclophosphamide did not alter clearance on either day 3 or 10. The defect in clearance in mechlorenthamine-treated mice resolved by 3 wk after drug administration. These results demonstrate that alkylating agents do not have uniform effects on antibacterial processes in the murine lung. Since the mechlorethamine effect on staphylococcal elimination appears independent of its effect on macrophage numbers, these results suggest that staphylococcal clearance also depends on nonphagocytic host defense factors.
The Kinetics of Secretion From Permeabilized Human Neutrophils: Release of Elastase and Correlations With Other: Granule Constituents and Right Angle Light Scatter
Authors: Smolen James E , Stoehr Sally Jo , Traynor Alexis E , Sklar Larry A
Published: January 2018

We have previously reported that human neutrophils can be permeabilized with the cholesterol-complexing agent digitonin. These permeabilized cells can be induced to secrete lysosomal constituents when exposed to micromolar levels of free Ca2+, a process that is enhanced by certain guanine nucleotides. We examined the kinetics in this system by employing both direct and indirect measures of secretion. A continuous, fluorescent assay of elastase permits real-time monitoring of secretion from azurophil granules. The kinetics of elastase release proved to be rapid, beginning within 3-10 sec and reaching a maximum at 1-2 min. Changes in the Ca2+ concentration did not affect the “lag period” for release. A comparison of the Ca2+ dose-response curves for release of the various granule constituents indicated that elastase was being secreted along with other contents of the azurophil granules. Changes in right angle light scatter (RLS), which have been shown to correlate closely with secretion, also commenced rapidly after the addition of Ca2+; when measured simultaneously, both the Ca2* dose-response characteristics for changes in RLS and elastase release were very similar. Changes in RLS could be halted within 5 sec by excess EGTA and restarted promptly by repletion with secretory concentrations of Ca2+. In addition, neomycin, a phospholipase C inhibitor, profoundly diminished degranulation as monitored by RLS and end-point techniques. A continuous assay employing 9-aminoacridine self-quenching as a measure of secretion proved far less satisfactory, but, nonetheless, produced similar kinetics and dose-response characteristics. These data thus suggest that secretion of granule constituents from permeabilized neutrophils takes place rapidly and that the kinetics are comparable to those observed with intact cells.
Candidacidal Activity of Monocyte-Derived Human Macrophages: Relationship Between Candida Killing and Oxygen Radical Generation by Human Macrophages
Authors: Sasada Masataka , Kubo Akemi , Nishimura Toshiro , Kakita Tokio , Moriguchi Toshinori , Yamamoto Kohkichi , Uchino Haruto
Published: January 2018

Freshly isolated human monocytes ingested and killed Candida albicans, and generated O-2 H2O2 and .OH efficiently. When monocytes were cultured in vitro, these cells transformed into macrophages. Cultured monocytes retained their ingestive activity but lost their candidacidal activity almost completely after day 3. The release of O-2 by monocytes decreased slightly with culture and that of .OH was markedly decreased on day 3 of culture. The activity of myeloperoxidase in the monocytes decreased with culture. These results suggested that the loss of candidacidal activity is due to the decrease of .OH generation and myeloperoxidase activity in cultured monocytes.
Receptor-Mediated Ingestion Responses by Lung Macrophages From a Canine Model of ARDS
Authors: Tabor Dale R , Kiel Deborah P , Jacobs Richard F
Published: January 2018

Receptor-mediated ingestion was examined in macrophages derived from a canine model of the adult respiratory distress syndrome (ARDS). The results showed that Fc-mediated ingestion by alveolar macrophages (AM) and macrophages from lung parenchyma (PM) was significantly diminished when compared with their respective controls. Pulsing all the experimental groups with lipopolysaccharide (LPS) for 1 hr in vitro failed to either enhance the response or return the activity to levels achieved by control cells. In parallel studies, an analysis of C3b-mediated ingestion showed that both the experimental AM and PM performed this function only at a magnitude equal to the control cells. Similar responses were observed when an LPS pulse was performed. Although there was a reduction in Fc-mediated ingestion and an apparent restraint of the C3b-mediated ingestion, both AM and PM expressed a significantly enhanced ability to spread. These results suggested that the canine model of ARDS alters at least one select macrophage function that may be important to subsequently protect the host. Such disturbances in the cellular immune response may contribute to the progression of infection and lung pathology associated with this disease process.
A High-Yield, High-Purity Elutriation Method for Preparing Human Granulocytes Demonstrating Enhanced Experimental Lifetimes
Authors: Tolley James O , Omann Geneva M , Jesaitis Algirdas J
Published: January 2018

A counterflow centrifugal elutriation procedure for preparing human granulocytes is described. This method permits isolation of 1-2 × 109 granulocytes with a yield of 70% ± 7% from a unit of whole blood in 3 h. These cells are 99% ±1% pure and 95-99% viable. When compared to cells prepared by a conventional procedure employing hypotonic lysis to remove red cells, the elutriated cells show enhanced oxidative responses resulting from enhanced longevity. Other responses including receptor-mediated uptake of chemoattractant were not significantly different from those of conventionally prepared cells after incubation at 37° prior to stimulation. The population of elutriated granulocytes, however, was significantly more homogeneous and pure than cells prepared conventionally.
Transient Suppression of the Zymosan-Induced Chemiluminescence Produced by Bone Marrow Cells During a Subcutaneous Nonspecific Inflammation in the Mouse
Authors: Herodin Francis , Benichou Gilles
Published: January 2018

The oxidative metabolism of remote phagocytes has been studied during the course of an acute nonspecific inflammation in the mouse. The bone marrow cells (BMC) from mice bearing a polyacrylamide-microbead-induced granuloma (Biogels P4 and P100) show a transient striking decrease of their chemiluminescence (CL) response to opsonized zymosan. This decreased oxidative response occurs between the third and 72nd hr, with a minimum observed at the 24th hr. Bone marrow granulocytes are assessed to represent the main chemiluminescent BMC compartment, using complement-mediated cytotoxicity assays with a monoclonal antimouse granulocyte antibody. Biogel P4-treated mice, but not Biogel P100-treated ones, show at the 24th hr a significantly decreased percentage of segmented bone marrow granulocytes (-45%, P < .001).

The accurate mechanisms involved in this transient suppression of stimulus-induced CL produced by BMC remain to be elucidated.
Morphologic and Functional Heterogeneity Among Rat Alveolar Macrophage Fractions Isolated by Centrifugation on Density Gradients
Authors: Oghiso Yoichi
Published: January 2018

Lavaged rat bronchoalveolar cells were separated into four different density fractions (I—IV) by centrifugation through a discontinuous Percoll gradient. Maximum cell size was found in the lowest density fraction (I) and minimal cell size in the highest density fraction (IV), showing an inverse correlation with cell density. These fractions contained alveolar macrophages (AM) in the proportion of 97% or more by morphologic criteria, while phagocytic AM was approximately 80% in each fraction. Although the proportion of la antigen-positive AM was low in each fraction, it was elevated after incubation with supernatants from concanavalin A (Con A) stimulated spleen cell cultures, with greater la expression in higher density fractions. Differences in these fractions were also noted in several functions, including Fc receptor activity, chemotactic migration, tumoricidal activity, and interleukin 1 (IL-1) production, all of which were greater in higher density fractions (III and IV). Con A-induced T cell proliferation was, however, suppressed by these higher density fractions, whereas only an intermediate density fraction (II) enhanced T cell responses. These results indicate morphologic and functional heterogeneity among rat AM.
The Contributions of Adrenal Hormones, Hemodynamic Factors, and the Endotoxin-Related Stress Reaction to Stable Prostaglandin Analog-Induced Peripheral Lymphopenia and Neutrophilia
Authors: Ulich Thomas R , Keys Marcy , Ni Rong-Xiang , del Castillo Juan , Dakay Edwin B
Published: January 2018

Stable prostaglandin analogs are known to induce lymphopenia and neutrophilia in a dose-dependent fashion after subcutaneous injection in rats. The purpose of the present investigation is to determine whether the prostaglandin-induced changes in circulating leukocytes might be secondary to hypotension with the ensuing release of adrenal hormones. The adrenal medullary catecholamine epinephrine was found to induce neutrophilia in both intact and adrenalectomized rats, and the glucocorticosteroid analog dexamethasone induced a profound lymphopenia in rats as reported by previous investigators. A stable analog of PGF2α (15-S-15-methyl PGF2α; M-PGF2α) at the dose of 1 mg/kg induced marked systemic hypotension 1 h after injection, with lymphopenia and neutrophilia 6 h after injection. The non-prostanoid hypotensive agent captopril, at a dose of 63 mg/kg, induced a hypotension of similar magnitude and kinetics to that induced by prostaglandin. Captopril also induced lymphopenia and neutrophilia at 6 h, although the neutrophilia was of lesser magnitude than that induced by prostaglandins. The prostaglandin-induced lymphopenia was found to be mediated, at least in part, by the hypotension-induced release of adrenal hormones, as evidenced by the abrogation of lymphopenia in prostaglandin-treated adrenalectomized rats. Captopril-treated adrenalectomized rats, however, did develop a significant lymphopenia, suggesting that hypotension can result in lymphopenia even in adrenalectomized rats. The M-PGF2α-induced neutrophilia in adrenalectomized rats, by comparison to captopril-induced neutrophilia in adrenalectomized rats, was greater than the neutrophilia expected as the result of hypotension alone. Indeed, the M-PGF2α-induced neutrophilia in adrenalectomized rats was greater than the captopril-induced neutrophilia in sham-adrenalectomized rats. Thus, a portion of the neutrophilia induced by M-PGF2α in intact rats may be mediated through adrenal-independent, hemodynamic-independent mechanisms. The possibility that M-PGF2α might be inducing neutrophilia via an endotoxin-like stress reaction was investigated by examining changes in circulating white blood cells in intact and adrenalectomized C3H/HeN (endotoxin-sensitive) and C3H/HeJ (endotoxin-resistant) mice after prostaglandin administration. No quantitative differences in the prostaglandin-induced neutrophilia were noted in C3H/HeJ mice as compared to the C3H/HeN mice. In conclusion, the effects of M-PGF2α on circulating white blood cells in the intact rat are mediated in part through hemodynamic and adrenal mechanisms, and in part through adrenal-independent, hemodynamic-independent, and endotoxin-like stress reaction-independent mechanisms. Prostaglandins may contribute directly to the genesis of neutrophilia.
Mannose Inhibits the Human Neutrophil Oxidative Burst
Authors: Rest Richard F , Farrell Catherine F , Naids Fredrick L
Published: January 2018

Stimulated human neutrophils (PMNs) increase their oxygen consumption and secrete reactive oxygen species that are involved in bactericidal activity and inflammation. While studying lectin-mediated bacterial adherence, we observed that D-mannose appeared to inhibit PMN metabolism. Further studies showed that 100 mM mannose inhibited oxygen consumption by 82%, superoxide secretion by 84%, luminol-enhanced chemiluminescence (CL) by 98%, and hexose-monophosphate shunt activity by 100% when PMN were stimulated with 1 μM phorbol myristate acetate (PMA). Inhibition was also seen with 0.1 μM formyl-methionyl-leucyl-phenylalanine (fMLP), and 0.1 μM A23187, reagents thought to stimulate the respiratory burst by different transductional mechanisms. Inhibition was dose-responsive and specific since 100 mM D-galactose, alpha-D-glucose, or alpha-L-fucose only minimally affected PMN oxidative metabolism. Inhibition of PMA-induced superoxide production was seen almost immediately upon the addition of 50 mM mannose and was reversed by washing. Neutrophils remained viable as measured by trypan blue exclusion. These data suggest that mannose inhibits the neutrophil oxidative burst at the level of the hexose monophosphate shunt. Further investigation should elucidate the specific mechanism(s) of this burst inhibition as well as define uses for it as a tool to study oxidative as well as nonoxidative killing by PMN.
Enhanced Secretion of Immunoreactive Bombesin by Alveolar Macrophages Exposed to Silica
Authors: Wiedermann C J , Adamson I Y R , Pert C B , Bowden D H
Published: January 2018

Bombesin has recently been identified in alveolar macrophages (AM). Since this peptide has been shown to stimulate fibroblast growth in culture, we wished to determine whether AM exposed to the fibrogenic particle silica in vivo were capable of secreting more bombesin than AM recovered after instilling inert carbon particles to the lung. Rats received 10 mg of either carbon or silica by intratracheal injection and were killed at 3 days or 6 weeks. Both particles induced a rapid inflammatory response, and normal levels of immunoreactive bombesin were measured in lung lavage fluid and in freshly recovered macrophages from all rats. However, incubation of normal AM for 4 h in serum free medium produced a significant increase in bombesin levels measured in supernatants. Bombesin in supernatants of AM cultured after recovery from rats exposed to carbon was at the control value, while AM recovered after silica exposure in vivo secreted increased amounts of bombesin when cultured. Cells recovered 6 wk alter instilling silica to the lung and cultured for 4 h secreted 50% more bombesin than control AM. At this time, hydroxyproline measured in the silica-injected lungs was also significantly higher than in controls or carbon-injected rats. These results indicate that AM recovered from lungs after exposure to silica secrete increased amounts of bombesin during the development of pulmonary fibrosis.
Low-Density Lipoprotein Oxidized by Polymorphonuclear Leukocytes Inhibits Natural Killer Cell Activity
Authors: Tanabe Fuminori , Sato Akihiko , Ito Masahiko , Ishida Eiko , Ogata Masahiro , Shigeta Shiro
Published: January 2018

We studied the effect of low-density lipoprotein (LDL) oxidized by opsonized zymosan-stimulated polymorphonuclear leukocytes (PMN) on natural killer (NK) cell activity. Oxidized LDL inhibited NK cell activity in a dose-dependent manner, whereas normal LDL left it unaffected. However, oxidized LDL did not inhibit antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, a positive correlation was observed between the amount of thiobarbituric acid-reacting substances (TBARS) on the sample of oxidized LDL and the degree of inhibition of NK cell activity. We also showed that oxidized LDL suppressed the binding capacity of purified large granular lymphocytes (LGL) to target cells without changing the lytic activity. These results therefore suggest that activated PMN can modulate NK cell activity by oxidizing LDL.
Lectin Interactions With the Jurkat Leukemic T-Cell Line: Quantitative Binding Studies and Interleukin-2 Production
Authors: Dupuis Gilles , Bastin Bânû
Published: January 2018

Phytohemagglutinin (PHA), concanavalin A (Con A), pea lectin, and wheat germ agglutinin (WGA) have been used to investigate their binding properties to Jurkat 77 6.8 leukemic human T cells and their ability to induce these cells to produce interieukin-2 (IL-2). Binding studies showed that the Jurkat cells fixed 0.82 ± 0.11 μg pea lectin, 2.02 ± 0.17 μg Con A, 1.85 ± 0.07 μg PHA and 8.88 ± 0.61 μg WGA. Scatchard plots were linear, indicating that the binding process was homogeneous with respect to the binding constant. PHA and Con A bound with the highest affinity [K (apparent) - 9 × 109 M−1], followed by pea lectin and WGA [K, (apparent) =3 × 109 M−1]. The number of lectin binding sites was in agreement with the results of saturation experiments. We also evaluated the effect of the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the binding process. Results show that there were no gross alterations in the value of (apparent) K in the case of PHA and WGA. In contrast, the presence of TPA decreased the affinity of Con A and modified the Scatchard profile for pea lectin, which was curvilinear with a concavity turned upward. In this case, data were (apparent) K1 = 17.7 × 109 M−1 (“high-affinity” sites) and (apparent) K2 = 2.6 × 109 M−1 (“low-affinity” sites). The four lectins shared the ability to stimulate Jurkat 77 6.8 cells to secrete IL-2. Optimal lectin concentrations were 20 μg/ml (PHA) and 50 μg/ml (WGA and Con A). Pea lectin failed to display a dose-response relationship, and IL-2 production increased proportionally with lectin concentration. Con A was the most efficient stimulator (250 U/ml), followed by WGA (160 U/ml) and PHA (108 U/ml). Addition of TPA potentiated Jurkat cell response. In this case, Con A and pea lectin were the most efficient stimulators (470 U/ml), followed by PHA (316 U/ml) and WGA (271 U/ml). Here, also, pea lectin failed to show a dose-response relationship. Our data do not reveal an obvious relationship between the binding parameters and the ability of the lectins to induce IL-2 production. We suggest that the efficiency of the four lectins to stimulate IL-2 production in the case of the Jurkat 77 6.8 variant is most likely related to the nature of their specific cell surface receptor(s).
Effects of Ammonium Metavanadate Treatment Upon Macrophage Glutathione Redox Cycle Activity, Superoxide Production, and Intracellular Glutathione Status
Authors: Cohen M D , Wei C I
Published: January 2018

Female B6C3F1 mice were given intraperitoneal injections of ammonium metavanadate (2.5 or 10 mg V/Kg), ammonium chloride, or sodium phosphate buffer every 3 days for 6 weeks. Resident peritoneal macrophages were harvested, lysed by freeze-thawing, and the resulting cytolysate was assayed for total protein content and enzyme activities of glutathione reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. In addition, peritoneal macrophages were assayed for superoxide production using nitroblue tetrazolium reduction, as well as for intracellular levels of oxidized and reduced glutathione. Exposure of mice to vanadium resulted in a dose-trend depression in the three macrophage enzyme activities as compared with the controls. Vanadium treatment resulted in a similar decrease in the production of superoxide anion, and an increase in levels of oxidized glutathione; however, the total glutathione pool (reduced plus oxidized forms) was not affected.
Preparation and Characterization of a Monoclonal Antibody That Inhibits Human Neutrophil Elastase Activity
Authors: Skubitz Keith M , Wehner Nancy G , Gray Beulah H
Published: January 2018

Neutrophil proteases are believed to play a role in the pathogenesis of a variety of human diseases. While many studies of proteases in models of disease have focused on elastase, neutrophils contain several proteases some of which share a high degree of homology. This report describes the production and characterization of an lgG1 murine monoclonal antibody (AHN-10) that reacts with human neutrophil elastase but not with the other major neutrophil neutral proteases: cathepsin G, proteinase 3, collagenase, or the newly purified neutral protease, esterase N. AHN-10 inhibited the elastinolytic activity of purified human neutrophil elastase and could detect elastase in alcohol-fixed cytospin preparations. The epitope recognized by AHN-10 was resistant to treatment with NaIO4, suggesting that the epitope is not a carbohydrate. AHN-10 should be useful for the immunolocalization of neutrophil elastase in tissue specimens and as a stable source of characterized antibody for quantitative identification of neutrophil elastase.
Role of Activated Macrophages in Resistance to Systemic Candidosis
Authors: Baghian Abolghasem , Lee Kenneth W
Published: January 2018

To evaluate further the contribution of activated macrophages in resistance, the course of systemic candidosis was assayed in beige and NLM mice that had been previously infected with Mycobacterium bovis (BCG). Four weeks following BCG infection, mice were inoculated intravenously with 1 × 104 viable Candida albicans. At various times thereafter, the number of C. albicans colony-forming units in the livers, spleens, and kidneys was determined. The average number of CFU recovered from the kidneys of NLM mice decreased throughout the assay and was comparable in both BCG-treated and control mice. In contrast, the number of CFU cultured from the kidneys of untreated control beige mice progressively increased throughout the assay period. This profile of renal susceptibility was not appreciably altered in BCG-treated beige mice. However, fewer (10- to 100-fold) CFU were cultured from the livers and spleens of BCG-treated beige and NLM mice than from untreated controls. These results support the hypothesis that in the absence of functional polymorphonuclear leukocytes, activated macrophages represent a means to control the proliferation of C. albicans.
Biochemical Mechanisms Involved in the Priming of Neutrophils by Tumor Necrosis Factor
Authors: Berkow Roger L , Dodson Mary R
Published: January 2018

Preincubation of human neutrophils with recombinant tumor necrosis factor alpha has previously been shown by us to enhance superoxide production of neutrophils in response to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine, and the phorbol ester, phorbol myristate acetate. In this study, we further investigate the biochemical basis for this enhancement. We found that in neutrophils, TNF by itself does not induce: (1) an influx of sodium, (2) an alteration in activity or translocation of the calcium and phospholipid dependent protein kinase (C-kinase), or (3) a release of arachidonic acid from preloaded cells. TNF did, however, induce a time- and concentration-dependent increase in the phosphorylation of several neutrophil proteins, with the most dramatic concentration dependent increase in a 64,000 Da protein. Finally, the enhancement of O2 production by pretreatment of neutrophils with TNF was found to be independent of a pertussis toxin-sensitive guanine nucleotide regulatory protein.
Comparison of Methods for Assessing Chemotaxis of Monocytes and Polymorphonuclear Leukocytes Isolated From Patients With AIDS or AIDS-Related Conditions
Authors: Martin Linda S , Spira Thomas J , Orloff Sherry L , Holman Robert C
Published: January 2018

We evaluated the ability of normal human peripheral blood monocytes and polymorphonuclear leucocytes (PMNL) isolated from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related conditions (ARC) to migrate toward a chemoattractant. Migration in blind-well chambers was compared to that under agarose. Chemotaxis results obtained from both assays for PMNL were similar, however there was a difference in the results for monocyte Chemotaxis. PMNL isolated from patients with AIDS, but not ARC, exhibited decreased spontaneous and directed chemotaxis when assessed in blind-well chambers and under agarose. Spontaneous and directed Chemotaxis in blind-well chambers of AIDS patients’ monocytes was normal. Directed migration of monocytes from ARC patients was greater than that of control, but spontaneous migration was comparable. Under agarose, spontaneous migration was depressed in monocytes of AIDS patients, while migration toward the attractant was depressed in those of ARC patients.
Establishment and Characterization of Factor-Dependent Macrophage Cell Lines
Authors: Ohki Kazunori , Nagayama Ariaki
Published: January 2018

Three macrophage cell lines from bone marrow cells of C3H/HeN mice were isolated by successive transfer of the cells in culture with L-cell-conditioned medium (LCM) or WEHI-3 cell-conditioned medium (WEHI-3CM). These cell lines, which express Fc receptors, are involved in Fc-mediated phagocytosis and possess nonspecific esterase activity. Two (BDM-1 and BDM-2) of three cell lines show dependency for growth on either macrophage colony-stimulating factor (M-CSF) (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and do not respond to interleukin 3 (IL-3). The third clone (BDM-3) proliferates in response to IL-3 as well as to GM-CSF and weakly responds to M-CSF and to interleukin 4 (IL-4). GM-CSF, in combination with the suboptimal concentration of M-CSF, acted synergistically on the proliferation of BDM-1 cells. The tumor-promoting phorbol diester, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) also acted synergistically with the three CSFs (IL-3, GM-CSF, and M-CSF) to stimulate the proliferation of BDM-1 cells. The synergistic effect was observed when cells were pretreated with TPA and subsequently stimulated with IL-3. The calcium ionophore A23187 enhanced the proliferation of BDM-1 cells costimulated with TPA and IL-3. These factor-dependent macrophage cell lines should be useful for studying signal transduction mechanisms in the regulation of cell growth.
Inflammatory Cells Degrade Inter-α Inhibitor to Liberate Urinary Proteinase Inhibitors
Authors: Pratt Charlotte W , Swaim Mark W , Pizzo Salvatore V
Published: January 2018

The relationship between inter-α inhibitor (IαI) and urinary proteinase inhibitor (UPI) was examined by comparing purified UPI with a proteolytic fragment of IαI (l’), and by demonstrating that inflammatory cells produce similar fragments under physiologic conditions. Purified I’, derived by chymotrypsin digestion of IαI, was similar to UPI in apparent molecular weight (68,000-69,000), amino acid composition, immunoreactivity, and inhibitory activity against trypsin, chymotrypsin, and neutrophil elastase. The production of similar inhibitory fragments by murine peritoneal macrophages, human neutrophils, and a murine mast cell line was quantified. Neutrophils were most efficient at proteolyzing IαI. Comparison of the pattern of IαI degradation by neutrophil preparations with that by pure enzymes, suggested that both elastase and cathepsin G mediate neutrophil proteolysis of IαI. These proteinases may thus be responsible for inflammation-related increases in UPl-like inhibitor levels in vivo.
Biosynthesis of Proteochondroitin Sulfate by HL-60 Human Promyelocytic Cells
Authors: Bentley Stuart A , Kirby Suzanne L
Published: January 2018

Human promyelocytic cells (HL-60) were labeled with 35S-sulfate and either 3H-glucosamine or 3H-serine as precursors. Accumulation of 35S-labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/105 viable cells reached a plateau level after 24 h. Virtually none of the cell-associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE-Sephacel chromatography, isopyknic CsCI gradient centrifugation, and gel filtration chromatography. HL-60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL-4B and Kav = 0.32 on Sepharose CL-6B, recovered primarily from the high-density fractions of a dissociative CsCI gradient (ρ > 1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr = 14.5 kD, yielding virtually 100% 4-sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL-60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in 35S-sulfate incorporation to 45% of control values or 32%, expressed as activity/105 cells. Proteoglycans synthesized by DMSO-treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.
Polyclonal Anti-ldiotypes Influence Macrophage Chemotaxis in Coxsackievirus-lnduced Murine Myocarditis
Authors: Paque Ronald E , Miller Ruth
Published: January 2018

Investigation into the rnechanism(s) whereby monocytes-macrophages are mobilized to the myocardium of coxsackievirus (CVB3)-infected mice was approached by assessing the role of anti-idiotypic antibodies against specific antiviral IgG molecules. Balb/c mice preinocuiated with polyclonal anti-idiotypic, syngeneic antibodies resulted in significantly decreased myocarditis after virus challenge. On the other hand, oil-induced peritoneal exudate cells obtained from virus-exposed animals demonstrated increased chemotaxis in the presence of polyclonal anti-Id preparations, whereas peritoneal exudate cells from normal animals did not Specificity of the anti-Ids as confirmed by the binding of the syngeneic anti-anti-idiotypic antibodies (antibody 3) to solubilized viral antigen(s) and Fab fragments prepared from the anti-idiotypic antibodies. Paradoxic biological effects of anti-Id, demonstrated in vitro and in vivo, suggest possible and subtle immunoregulatton of the macrophage effector arm by anti-ldiotypes during the course of virus infection.
Stimulation of Granulocytic Cell Iodination by Pine Cone Antitumor Substances
Authors: Unten Senwa , Sakagami Hiroshi , Konno Kunio
Published: January 2018

Antitumor substances (Fractions VI and VII) prepared from the NaOH extract of pine cone significantly stimulated the iodination (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood adherent mononuclear cells, polymorphonuclear cells (PMN), and human promyelocytic leukemic HL-60 cells. In contrast, these fractions did not significantly increase the iodination of nonadherent mononuclear cells, red blood cells, other human leukemic cell lines (U-937, THP-1, K-562), human diploid fibroblast (UT20Lu), or mouse cell lines (L-929, J774.1). iodination of HL-60 cells, which were induced to differentiate by treatment with either retinoic acid or tumor necrosis factor, were stimulated less than untreated cells. The stimulation of iodination of both PMN and HL-60 cells required the continuous presence of these fractions and was almost completely abolished by the presence of myeloperoxidase inhibitors. The stimulation activity of these fractions was generally higher than that of various other immunopotentiators. Possible mechanisms of extract stimulation of myeloperoxidase-containing cell iodination are discussed.
Regulation of Phagolysosome pH in Bovine and Human Neutrophils: The Role of NADPH Oxidase Activity and an Na+/H+ Antiporter
Authors: Mayer Susan J , Keen Peter M , Craven Neil , Bourne F John
Published: January 2018

Fluorescein-labelled Staphylococcus aureus were used to follow changes in phagolysosome (PL) pH of bovine and human neutrophils following phagocytosis. Under aerobic conditions there was an alkalinisation of the PL followed by a slow decline. Under anaerobic conditions no alkalinisation of the PL was seen, and pharmacological inhibition of the NADPH oxidase with diphenyleneiodonium (DPI) resulted in a rapid acidification of the PL following phagocytosis.

The inclusion of amiloride, an inhibitor of Na+/H+ antiporter activity, produced a more rapid alkalinisation phase following phagocytosis under aerobic conditions and reduced, but did not abolish, the acidification phases seen under anaerobic conditions or following treatment of neutrophils with DPI. The results suggest that PL pH is influenced by NADPH oxidase activity and to a lesser extent by a Na+/H+ antiporter. The antibacterial effectiveness of neutrophil granule proteins may be affected under conditions that influence the functioning of these two systems.
Study of Mitochondrial Organization in Living Resident and Activated Macrophages Using the Laser Dye Rhodamine 123
Authors: de Carvalho Técia U , de Souza Wanderley
Published: January 2018

Rhodamine 123, a fluorescent laser dye that labels metabolically active mitochondria of living cells, was used to analyse the pattern of distribution of mitochondria in resident and activated mouse peritonal macrophages kept in culture for 4 or 24 hr. Activated macrophages kept for 4 hr in culture showed abundant small mitochondria distributed throughout the cell. This pattern changes to a situation in which the mitochondria are filamentous and radiate from the perinuclear region when these cells are kept in culture for 24 hr, acquiring a pattern similar to that observed in resident macrophages. After treatment with phorbol myristate acetate, resident macrophages presented a mitochondrial distribution similar to that observed in activated macrophages.
The Calcium Ionophore Ionomycin can Prime, but not Activate, the Reactive Oxygen Generating System in Differentiated HL-60 Cells
Authors: Dahlgren Claes
Published: January 2018

Both the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) and the calcium lonophore ionomycin induced a metabolic response in normal neutrophils. However, the presence of azide, a potent inhibitor of the hydrogen peroxide-consuming enzymes catalase and myeloperoxidase, was required to detect any release of hydrogen peroxide induced by ionomycin. In differentiated HL-60 cells, only FMLP stimulation was associated with any notable metabolic activation. The response to FMLP proceeds with a rate and time course similar to that seen in normal cells. The use of ionomycin as a stimulating agent did not result in any detectable activation of the system that generates reactive oxygen metabolites, even if azide was present in the measuring system. Raising the concentration of cytoplasmic free Ca2+ is therefore not sufficient to activate the system responsible for the generation of reactive oxygen metabolites in HL-60 cells. However, preincubation with ionomycin primed HL-60 cells to an increased response during stimulation with the chemotactic peptide FMLP and the phorbol ester PMA. Since HL-60 cells lack specific granules but have an intact ligand-receptor coupling mechanism, a role for the subcellular granule is proposed, in the generation of reactive oxygen species in normal granulocytes, and analysis of the data presented leads to two conclusions: 1) FMLP, which acts through cells surface receptors, causes the cells to produce oxygen radicals, which to a large extent are released from the cells, a process that is not dependent on the specific granule content of the cells, whereas 2) ionomycin, which bypasses cell-surface receptors, is also capable of stimulating an oxygen-radical formation that is granule dependent and retained inside the cells. Furthermore, the results suggest that an increase in intracellular Ca2+ is not sufficient to initiate activation of the plasma membrane–bound system that generates reactive oxygen metabolites, but the results support a role for Ca2+ in the priming event.
Characterization of Arachidonic Acid Metabolism, Superoxide Production, and Bacterial Killing in Bovine Circulating Neutrophils and Elicited Alveolar Neutrophils
Authors: Heidel Jerry R , Taylor Stephen M , Laegreid William W , Silflow Ron M , Liggitt H Denny , Leid R Wes
Published: January 2018

The in vitro generation and release of 5-lipoxygenase metabolites of arachidonic acid by bovine peripheral blood neutrophils and alveolar neutrophils elicited with either a heat-killed bacterium, Haemophilus somnus, or platelet-activating factor, were compared. After stimulation with calcium lonophore A23187 for 2.5-60 min, up to 4.5 ± 0.7 (mean ± SEM) ng of LTB4 per 106 cells was released into the media by circulating neutrophils. LTB4 release by alveolar neutrophils was significantly less (P<.05) than that of peripheral blood neutrophils from the same animal; 5-HETE release by circulating neutrophils was maximal after 5 min stimulation by lonophore (1.2 ± 0.1 ng/106 cells) but was not identified in cell culture media after 20 min. Alveolar neutrophils released similar amounts of 5-HETE when compared to circulating neutrophils, and release of 5-HETE by alveolar neutrophils was maximal after 5 min of stimulation. However, the 5-HETE released into the culture media persisted throughout the 60 min time period at levels which were maximal (1.5 ± 0.2 to 1.8 ± 0.3 ng/106 cells). Bacterial killing and the release of superoxide anion were not different between the two cell populations.
Preparation and Characterization of Monoclonal Antibodies to Human Neutrophil Cathepsin G, Lactoferrin, Eosinophil Peroxidase, and Eosinophil Major Basic Protein
Authors: Skubitz Keith M , Christiansen Neal P , Mendiola John R
Published: January 2018

This report describes the production and characterization of five murine monoclonal antibodies that react with granule proteins of human granulocytes. Monoclonal antibody AHN-11 (lgG2a) reacted specifically with neutrophil cathepsin G; no reactivity with the homologous neutrophil neutral proteases, elastase, proteinase 3, or esterase N was detected. Antibodies AHN-9 (lgG1) and AHN-9.1 (lgG2b) each reacted with different epitopes on human lactoferrin, but not with the homologous protein transferrin. Two lgG1 antibodies, AHE-1 and AHE-2, reacted specifically with eosinophils; AHE-1 reacted strongly with eosinophil peroxidase but not eosinophil major basic protein while AHE-2 recognized eosinophil major basic protein but not eosinophil peroxidase. All five antibodies could detect their respective antigens in alcohol-fixed cytospin preparations. These antibodies should be useful for immunolocalization and quantification of their respective antigens as well as for other studies of the roles of these proteins in granulocyte function and differentiation.
Arachidonic Acid Metabolism in Guinea Pig Eosinophils: Synthesis of Thromboxane B2 and Leukotriene B4 in Response to Soluble or Particulate Activators
Authors: Sun Frank F , Czuk Christine I , Taylor Bruce M
Published: January 2018

The arachidonic acid metabolism of guinea pig eosinophils isolated from either peritoneal cavity or bronchoalveolar lavages was studied by reverse-phase high-performance liquid chromatography. The purified eosinophils (95-100%) from either source released thromboxane B2 (TxB2), luekotriene B4 (LTB4) and 5-hydroxy eicosatetraenoic acid (5-HETE) following calcium lonophore A23187 stimulation. Quantification by radioimmunoassay indicated that maximal mediator output from the stimulated peritoneal cells was reached at 3 min after stimulation. The increase in production of TxB2 and LTB4 was correlated to increasing calcium ionophore A23187 concentration up to 1.0 μg/ml. In addition to calcium ionophore, the guinea pig peritoneal cells were also activated by f-met-leu-phe, phorbol 12-myristate, 13-acetate (PMA), and to lesser extent platelet-activating factor (PAF) to produce TxB2. LTB4 synthesis was stimulated by calcium ionophore, by f-met-leu-phe, as well as by unopsonized glucan, a particulate phagocytotic stimulus. The guinea pig eosinophils do not synthesize sulfidopeptide leukotrienes because of the absence of the specific LTA4 glutathione S-transferase. These results suggest that the guinea pig eosinophils differ from the human circulating eosinophils in the synthetic capacity of lipid mediators derived from arachidonic acid metabolism. This difference may be important in the understanding of the role of the eosinophils in inflammatory reactions such as that which occurs in the bronchial tissues of asthmatics.
Differentiation of Human Hematopoietic Cells Increases Expression of a Gene Transferred by a Retroviral Vector
Authors: Holland Christie A , Chada Sunil , Wright Jocyndra , Whitney Constance , Harigaya Kenichi , Greenberger Joel S , Newburger Peter E
Published: January 2018

To study expression of a retroviral vector in human hematopoietic lineages, two established human hematopoietic cell lines (HL60 and K562) and a human adherent stromal cell line (KM101) were infected with the vector pZIP-SV(X). Expression of the transferred neomycin resistance gene (neo) of pZlP-SV(X) was evaluated as the ability of the cells to form colonies (>50 cells) in an agar assay in the presence of the neomycin analogue, G418. After infection, all three cell lines produced colonies resistant to G418. The level of neo mRNA in separate colonies was analyzed by Northern blot analysis. The neo gene transferred by the vector pZIP-SV(X) was expressed in both human hematopoietic and stromal cell lines. In addition, primary adherent human stromal cells infected with pZIP-SV(X) grew in the presence of G418. To determine if differentiation of hematopoietic cells affects expression of the retroviral vector, HL60 cells infected with pZIP-SV(X) were induced to differentiate, and the level of neo mRNA measured. The amount of neo mRNA increased when HL60 cells were induced to differentiate along the granulocytic pathway. Conversely, when HL60 cells were induced toward monocytoid differentiation (TPA), the level of neo mRNA did not significantly increase. We conclude that the neo gene transferred by a retroviral vector, pZIP-SV(X), is functionally expressed. In addition, expression of the transferred neo gene is regulated during myeloid differentiation of HL80 cells.