bluuebunny/crossref_metadata_2025 huggingface dataset:
The Relationship Between Intracellular Cyclic AMP Concentrations and the In Vitro Growth of Macrophages
Authors: Inouye L K , Wharton Walker
Published: January 2018

The addition of cholera toxin, prostaglandins, or one of a series of xanthine phosphodiesterase inhibitors to bone marrow-derived macrophages maintained in liquid culture caused a dose-dependent decrease in colony formation measured 7–10 days following seeding. The growth inhibitory effects of xanthines were in the same order of potency (caffeine < theophylline < isobutylmethylxanthine) as their reported ability to inhibit cyclic AMP phosphodiesterase. The relationship between the magnitude of the increases in intracellular concentrations of cyclic AMP observed following the addition of the drugs and the degree of growth inhibition was complex. Combinations of cholera toxin and phosphodiesterase inhibitors caused synergistic elevations in cyclic AMP levels after a lag of approximately 3 days. However, the growth rate was decreased immediately following the addition of the combination of drugs, and thus seemed to be independent of the nucleotide levels. A cyclic AMP-resistant variant of a cloned nontransformed macrophage cell line was found to be also resistant to the growth inhibitory actions of both cholera toxin and prostaglandins. However, resistance to the inhibitory effects of cyclic AMP did not render the cells resistant to a xanthine-induced growth inhibition.
Heterogeneity in Chronic Granulomatous Disease Detected With an Improved Nitroblue Tetrazolium Slide Test
Authors: Meerhof Louis J , Roos Dirk
Published: January 2018

The microscopic nitroblue tetrazolium (NBT) slide test, used to score the ability of individual phagocytic leukocytes to produce superoxide, was improved according to the following procedure. Purified granulocyte suspensions are incubated with NBT and fixed in suspension, thereafter centrifuged on microscope slides and stained with nuclear fast red. This method precludes stimulation and selection of cells by adherence and washing. The number of formazan grains per cell can be judged in a semiquantitative way. In parallel incubations, the cells are stained with May-Grünwald/Giemsa, allowing identification of formazan-positive and -negative cells. The test discriminates well between cells from normal individuals, cells from patients with chronic granulomatous disease (CGD), and cells from heterozygotes for the X-linked form of CGD. Several patients and heterozygotes with an autosomal or variant form of CGD were detected with decreased NBT-reducing activity in their neutrophils and/or eosinophils. The relation between NBT-reductase activity of the phagocytes and the clinical situation is discussed.
Therapeutic effect of Lipoxin A4 in malaria-induced acute lung injury
Authors: Pádua Tatiana A , Torres Natalia D , Candéa André L P , Costa Maria Fernanda Souza , Silva Johnatas D , Silva-Filho João Luiz , Costa Fabio T M , Rocco Patricia R M , Souza Mariana C , Henriques Maria G
Published: January 2018

Acute lung injury (ALI) models are characterized by neutrophil accumulation, tissue damage, alteration of the alveolar capillary membrane, and physiological dysfunction. Lipoxin A4 (LXA4) is an anti-inflammatory eicosanoid that was demonstrated to attenuate lipopolysaccharide-induced ALI. Experimental models of severe malaria can be associated with lung injury. However, to date, a putative effect of LXA4 on malaria (M)-induced ALI has not been addressed. In this study, we evaluated whether LXA4 exerts an effect on M-ALI. Male C57BL/6 mice were randomly assigned to the following five groups: noninfected; saline-treated Plasmodium berghei-infected; LXA4-pretreated P. berghei-infected (LXA4 administered 1 h before infection and daily, from days 0 to 5 postinfection), LXA4- and LXA4 receptor antagonist BOC-2-pretreated P. berghei-infected; and LXA4-posttreated P. berghei-infected (LXA4 administered from days 3 to 5 postinfection). By day 6, pretreatment or posttreatment with LXA4 ameliorate lung mechanic dysfunction reduced alveolar collapse, thickening and interstitial edema; impaired neutrophil accumulation in the pulmonary tissue and blood; and reduced the systemic production of CXCL1. Additionally, in vitro treatment with LXA4 prevented neutrophils from migrating toward plasma collected from P. berghei-infected mice. LXA4 also impaired neutrophil cytoskeleton remodeling by inhibiting F-actin polarization. Ex vivo analysis showed that neutrophils from pretreated and posttreated mice were unable to migrate. In conclusion, we demonstrated that LXA4 exerted therapeutic effects in malaria-induced ALI by inhibiting lung dysfunction, tissue injury, and neutrophil accumulation in lung as well as in peripheral blood. Furthermore, LXA4 impaired the migratory ability of P. berghei-infected mice neutrophils.
Effect of Human Recombinant Tumor Necrosis Factor on the Growth of Different Human and Mouse Long-Term Hematopoietic Cell Lines
Authors: Hahn Cathy J , Ovak Geraldine M , Donovan Renea M , Pauly John L
Published: January 2018

Previous studies have demonstrated that one of the most salient features of tumor necrosis factor (TNF) is its ability to induce tumor necrosis in vivo, and the specificity of its cytotoxic/cytostatic activity for tumor cells has been demonstrated in in vitro studies in which this lymphokine has been shown to kill cultured cells of malignant lines and to have no effect on cells of normal diploid lines. Studies described herein defined the effect of highly purified human recombinant TNF on cells of 34 different human and murine hematopoietic cell lines, particularly human leukemic T and B cells of long-term lymphoblastoid cultures. Results of these studies demonstrated that TNF at concentrations of 3,600 U/ml had no significant effect on the growth of these cells as defined by cytotoxicity, measured with the use of the trypan blue dye-exclusion assay and as defined by cytostasis, assayed by the enumeration of cells and the uptake of [3H]-thymidine and -uridine. In contrast, positive control cultures of TNF-sensitive cells from a murine tumor (L-M/clone L-929, connective tissue) displayed at 50% (LD50) reduction in growth by TNF at ~ 5 U/ml. Likewise, human tumors (MCF-7, breast, and HT-29, colon) were also highly sensitive (LD50 < 100 U/ml). These studies demonstrate that T and B cells of lymphoblastoid lines as well as cells of other hematopoietic lines display little or no sensitivity to TNF.
Ganglioside Identification on Human Monocyte Membrane With Clostridium perfringens Delta-Toxin
Authors: Cavaillon J M , Jolivet-Reynaud C , Fitting C , David B , Alouf J E
Published: January 2018

Clostridium perfringens delta-toxin was first described as a hemolysin with a restricted lytic spectrum. A selective cytotoxicity of the delta-toxin was then found on rabbit leukocytes: peritoneal and alveolar macrophages were uniformly killed, whereas thymocytes were essentially resistant. The toxin was shown to be specific for GM2 ganglioside or a GM2-like structure. In the present study we report the interaction of delta-toxin with human monocytes. A specific, saturable, and irreversible binding of 125I-delta-toxin was demonstrated. Binding was inhibited by preincubation of the radiolabeled toxin with GM2 and with high amount of GM1 ganglioside. As judged by dye exclusion, no cytotoxicity was observed on freshly isolated monocytes, but when added at the beginning of a culture of human adherent cells, the cytotoxic effect was detected after 48 hours of culture. Taken together, these data indicate the presence of monosialoganglioside(s) at the surface of human monocytes, and suggest a possible reorganisation of such structure into the cell membrane when monocytes mature in vitro toward macrophage-like cells.
Gender-Related Variations and Interaction of Human Neutrophil Cyclooxygenase and Oxidative Burst Metabolites
Authors: Mallery Susan R , Zeligs Barbara J , Ramwell Peter W , Bellanti Joseph A
Published: January 2018

Gender-related variations in human neutrophil membrane bound oxidative metabolism were evaluated employing the calcium ionophore A23187. These included the measurement of cyclooxygenase metabolites of arachidonate by specific RIA determination of thromboxane B2 (TxB2), prostaglandin E2 (PGE2), and 6-Keto PGF1 (6KPGF) as well as the initiation of the oxidative burst by the quantitative evaluation of superoxide (O2−) reduction of nitroblue tetrazolium (NBT). Neutrophils from women generated 30% less TxB2 and PGE2 than those obtained from men. In contrast, the neutrophils from women demonstrated relatively higher O2− production with a cyclic pattern of both TxB2 and O2− which correlated with their menstrual cycle. The elevated O2− generation appeared to inversely correlate with TxB2 production. Further, introduction of an intracellular oxygen centered radical (OCR) scavenger, sodium benzoate, for the hydroxyl (OH) radical was observed to affect cyclooxygenase metabolism in a dose-response manner. At higher concentrations of sodium benzoate, i.e., 10-2M, TxB2 production was inhibited; in contrast, 10-3M sodium benzoate enhanced neutrophil TxB2 generation which was particularly marked during times of increased oxidative burst activity, i.e. O2− production. We conclude that the decreased production of cyclooxygenase metabolites observed in neutrophils from women in part derive from an increased oxidative burst activity. This suggests that a regulatory mechanism may exist between the neutrophil membrane bound oxidative system(s) involving oxygen centered radicals generated during both the oxidative burst and prostaglandin cyclic endoperoxide reduction. Further, these gender-related differences may be partially attributable to variations in circulating endogenous sex steroids.
Monocyte-Derived Growth Factors for Human Tumor Clonogenic Cells
Authors: Hamburger Anne W , White Christine P , Lurie Karin , Kaplan Richard
Published: January 2018

Human peripheral blood monocytes secrete a soluble factor that enhances the ability of human epithelial tumor cell lines to clone in soft agar. Monocytes increased colony growth in a concentration-dependent manner, although inhibition of cell growth was observed in the presence of high concentrations of monocytes. Addition of indomethacin to monocytes did not abrogate this inhibition. Exposure of monocytes to endotoxin increased their ability to secrete stimulatory factors. Nonadherent lymphocytes were unable to support colony growth. Conditioned media from unstimulated monocytes also increased colony growth. Growth-promoting activity was detected in the media within the first 24 hr of culture, reaching a peak at 72 hr. Activity was not observed in monocyte lysates. Monocytes released this activity when cultured in the presence of both serum-free and serum-containing media. The activity was nondialyzable, relatively heat stable, and failed to adhere to CM-Sephadex. The demonstration of a monocyte-derived factor that enhances growth of epithelial tumor colonies supports findings indicating that inflammatory products may enhance tumor cell growth in vitro.
Tumor Growth Stimulatory Macrophages Induced by a Subclinical Bacterial Infection In Vivo
Authors: De Groot Jan W , Compier-Spies Philome I , Wilbrink Bert , Dullens Hub F J , De Weger Roel A , Rademakers Louk H P M
Published: January 2018

Nonelicited peritoneal macrophages obtained from normal mice from our animal house unexpectedly expressed a strong tumor growth stimulatory effect in vitro. Macrophages expressing this stimulatory effect had an aberrant morphology compared to the morphology of normal macrophages as observed by electron microscopy.

The results of immunization of these affected mice with tumour cells led to the usual lymphocyte sensitization. No external symptoms were observed, and the mice looked healthy.

Treatment of the affected macrophage donors with antibiotics resulted in the abolishment of the tumor growth stimulatory effect by the macrophages. Thus, this tumor cell growth stimulation by marcrophages was probably due to a subclinical infection of the mice.
Biochemical Events Associated With the Stimulation of Rabbit Neutrophils by Platelet-Activating Factor
Authors: Naccache Paul H , Molski Marshall M , Volpi Mario , Shefcyk Jean , Molski Thaddeus F P , Loew Leslie , Becker Elmer L , Sha'afi Ramadan I
Published: January 2018

The functional and biochemical responses evoked by the addition of platelet-activating factor (PAF) to a suspension of rabbit neutrophils have been characterized in an effort to define the mode of action of this lipid mediator. PAF was found to elicit a secretory response and to stimulate a rapid breakdown of the polyphosphoinositides, an increase in the cytoplasmic level of free calcium (as monitored by quin2), a decrease in the fluorescence of cell-associated chlortetracycline, an enhanced activity of the sodium/hydrogen antiport, a transient depolarization, and an increase in the level of cytoskeletal actin. The quin2 response to PAF was found to be detectable at concentrations as low as 0.01 nM, to be very dependent on the presence of extracellular calcium, and to be sensitive to inhibition by phorbol esters. On the other hand, the increase in free calcium induced by PAF in the presence of extracellular calcium was essentially unaffected by pertussis toxin. PAF-induced neutrophil degranulation was similarly extracellular calcium dependent and phorbol ester sensitive. The secretory activity of PAF was evident only at concentrations in excess of 1 nM. All of the other effects of PAF were found to be independent of the presence of external calcium and to be demonstrable only at concentrations larger than 1 nM. In addition, all neutrophil responses to PAF (with the above noted exception of quin2) were potently inhibited by pertussis toxin. These results are interpreted in terms of the possible existence of two functionally distinct populations of receptors. The occupation of one set (of apparent high affinity) induces an increase in permeability to calcium in a phorbol-ester-, but not pertussis-toxin-, sensitive manner. The activation of the other set of receptors at higher concentrations of PAF stimulates the polyphosphoinositide-specific phospholipase C and induces the attendant biochemical responses. These latter responses appear to be mediated by a guanine-nucleotide-binding regulatory protein.
Effects of Diethylstilbestrol on Propionibacterium acnes Immunomodulation: Inhibition of Macrophage Activation and Antitumor Activity
Authors: Ackermann Michael F , Mark Raymond , Chakraborty Prasanta R , Morahan Page S
Published: January 2018

Exposure of mice to diethylstilbestrol (DES) inhibited Propionibacterium acnes-induced antitumor activity in vivo against the B16F10 subcutaneous tumor. The inhibitory effect was associated with inhibition by DES of the characteristic P. acnes induced splenomegaly and changes in splenic and peritoneal macrophages (Mφ) cell populations. The characteristic P. acnes induced reductions in Mφ alkaline phosphodiesterase I (APD) ectoenzyme activity and in total RNA synthesis, proposed biochemical markers of tumoricidal Mφ, were partially or completely reversed in DES-treated mice. As predicted from these in vivo and in vitro results, DES treatment significantly decreased P. acnes activation of Mφ antitumor activity in vitro against B16F10 melanoma and Lewis lung carcinoma cells. These data suggest a macrophage activation defect may be involved in the reduced resistance that DES-treated animals exhibit to a variety of neoplastic and microbial challenges.
Role of Pulmonary Macrophages in Resistance to Experimental Metastasis
Authors: Jackson R A , Lill P H , Gangemi J D
Published: January 2018

Intranasal inoculation of C3H/HeN mice with Propionibacterium acnes activates pulmonary macrophages but not splenic or peritoneal macrophages. When mice so treated were injected IV with tumor cells, no protection against the challenge was seen. Conversely, inoculation of C3H/HeN mice with P. acnes by the IP route activated splenic and peritoneal macrophages but not pulmonary macrophages. When mice with activated splenic and peritoneal macrophages were challenged with an IV injection of tumor cells, the mice demonstrated an increased resistance to the formation of pulmonary tumors. Thus, activation of splenic and peritoneal macrophages correlated better with protection against IV tumor challenge than activation of pulmonary macrophages. Experiments were done that demonstrated that the effect was not due to the augmentation of NK cell activity.

The data are consistent with the conclusion that activated pulmonary macrophages alone are not effective in conferring resistance to pulmonary tumor nodule formation in this tumor model.
Biochemical Analysis and Biological Activities of Immunoaffinity Purified Natural Murine Gamma Interferon (MuIFN-γ)
Authors: Greenfield Robert S , Kiener Peter A , Chin David P , Curry Robert C , Spitalny George L
Published: January 2018

A rapid one-step procedure for the purification of natural murine gamma interferon (MuIFN-γ) by immunoaffinity chromatography has been developed. Crude MuIFN-γ was passed through a column containing immobilized anti-MuIFN-γ monoclonal antibody, and the column was washed with high salt and detergent. Active MuIFN-γ was subsequently eluted with ammonium thiocyanate/glycerol (yields of 20-40%). Analysis of the immunopurified interferon by sodium dodecyl sulfate Polyacrylamide gel electrophoresis under reducing conditions revealed biologically active bands of about 19.5, and 14 kd. Under nonreducing conditions, three active bands at 35, 19.5, and 14 kd could be seen. Gel filtration of the immunopurified MuIFN-γ under reducing or nonreducing conditions showed a single major peak of antiviral activity corresponding to a molecular weight of 43 kd. The protein eluted from each band from the SDS-gel, in addition to possessing antiviral activity, was shown to activate macrophages in three assays: a) macrophage tumoricidal activity, b) hydrogen peroxide secretion, and c) expression of la antigen on the surface of exudate macrophages.
Two-Dimensional and Three-Dimensional Movement of Human Polymorphonuclear Leukocytes: Two Fundamentally Different Mechanisms of Location
Authors: Schmalstieg Frank C , Rudloff Helen E , Hiliman Gilbert R , Anderson Donald C
Published: January 2018

Patients with an inherited deficiency of the adherence glycoproteins LFA-1, Mac-1, and p150,95 are unable to mobilize polymorphonuclear leukocytes (PMNLs) to peripheral sites of inflammation. LFA-1/Mac-1/p150,95-deficient PMNL exhibited profoundly impaired movement stimulated by chemotactic factors when the cells were required to move over two-dimensional surfaces. Less impairment of movement was demonstrated in three-dimensional movement through cellulose filters. A possible explanation for this difference in cell translational mobility is that movement in cellulose filters is less adherence dependent than movement over a two-dimensional plastic surface. Movement of PMNL in collagen gels is known to be relatively independent of adherence. No deficiency of translational mobility of PMNL from LFA-1/Mac-1/p 150,95-deficient patients was observed in collagen gels. Antibodies against the common beta subunit effectively blocked two-dimensional movement but had little effect on three-dimensional movement through cellulose filters or collagen gel matrices. HL-60 cells were employed as a model to investigate the effects of adherence on cell movement. Treatment of HL-60 cells with phorbol myristate acetate resulted in the appearance of Mac-1 and p150,95 on the cell surface. Concurrently, the cells exhibited increased adherence to glass and plastic. In spite of increased adherence, HL-60 cells showed no translational movement, indicating factors other than the ability to adhere were important in cell motility. These experiments implied that PMNLs undergo two fundamentally different kinds of motion, one adherence dependent (two-dimensional movement) and the other largely adherence independent (three-dimensional movement). These findings are consistent with the view that egress of PMNLs from the vascular space is adherence dependent. Movement through extravascular tissues may be adherence independent.
Comparison of Five Short-Term Assays That Measure Nonspecific Cytotoxicity Mediated to Tumor Cells by Activated Macrophages
Authors: Russell Stephen W , Pace Judith L , Varesio Luigi , Akporiaye Emmanuel , Blasi Elisabetta , Celado Antonio , Schreiber Robert D , Schultz Richard M , Stevenson Anita P , Stewart Carleton C , Stewart Sigrid J
Published: January 2018

Five different short term assays (<48 h) used to measure macrophage-mediated, nonspecific cytotoxicity were compared under similar conditions in the same laboratory using the same reagents. The purpose was to determine the extent to which results were comparable. Three of the assays were dependent on the release of a radioisotope to measure cytotoxicity, one was dependent on cell counting, and the last was dependent on flow cytometric quantification of remaining viable tumor target cells after they had been exposed to macrophages. The variables examined were the following: a) three different populations of macrophages; b) four different kinds of target cells; c) two types of radioisotopes; and d) two different agents that trigger the expression of cytolytic activity by primed macrophages. Recombinant gamma interferon was used as the priming agent in all the experiments. There was unexpectedly good agreement between the results of the various assays. No differences were found among the different macrophage populations, the isotopes or the triggering agents. Perhaps the most important finding was that differences in target cell susceptibility to killing by activated macrophages, which were ap-
Polyanionic Agents Do Not Inhibit Phagosome-Lysosome Fusion in Cultured Macrophages
Authors: Goren Mayer B , Vatter Albert E , Fiscus Judith , Friedrich O A F
Published: January 2018

The survival of some intracellular pathogens within macrophages may be aided by an ability of the organism to antagonize, from within the entrapping phagosome, its fusion with lysosomes. On the other hand, certain polyanionic agents have been implicated in imposing a similar block to fusion from the lysosomal domain—because the transfer of various foreign markers from lysosomes to newly formed phagosomes is remarkably inhibited in these polyanion-containing cells. Based on an analysis of various observations and our own recent data, we propose that the polyanionics do not, in fact, prevent phagosome-lysosome fusion but, instead, physically entrap the usual markers in a gelatinous matrix within the lysosomes. This view accounts for many paradoxical consequences of polyanionic accumulation and for the curiously normal behavior of macrophages that are presumed to be suffering from such a crucial Intracellular dysfunction.

“This continuous search for truth can be a painful and punishing process. Sometimes it seems that all of education consists of first learning things and then learning that they are not true.”
Peripheral Blood Mononuclear Leukocytes Release a Mediator(s) That Induces Phagocytosis of C-Reactive Protein-Coated Cells by Polymorphonuclear Leukocytes
Authors: Kilpatrick J Michael , Gresham Hattie D , Griffin Frank M , Volanakis John E
Published: January 2018

We studied the phagocytosis by human polymorphonuclear leukocytes (PMN) of sheep erythrocytes (E) passively sensitized with pneumococcal C-polyseccharidc (E-PnC), E-PnC coated with C-resctive protein (E-PnC-CRP), and E coated with rabbit antisheep E IgG (E-lgG). PMN isolated from the blood of normal individuals failed to ingest either E-PnC or E-PnC-CRP; however, after incubation with supernatents from stimulated peripheral blood mononuclear leukocytes, glass adherent PMN ingested E-PnC-CRP with a mean phagocytic index of 47.8 ± 14.9 (means ± SD, n = 9) and E-PnC to a lesser extent with a phagocytic index of 9.6 ± 2.9 (mean ± SD, n = 4). We also observed a statistically significant increase in the ingestion of E-lgG by lymphokine-stimulated PMN with phagocytic indices of 85.2 ± 21.2 (mean ± SD, n = 12) for unstimulated PMN and 158 ± 37.1 (mean ± SO, n = 9) for stimulated PMN. The best conditions for stimulating release of this phagocytosis-promoting mediator included exposure to Phytohemagglutinin (PHA) in the presence of monocytes that had ingested lgG-coated sheep erythrocytes. The induction of phagocytosis of E-PnC-CRP was rapid, reaching a maximal level after stimulation of the adherent PMN with the conditioned media for 30 min. The factor(s) responsible for the induction of the ingestion of E-PnC-CRP was lees than 10,000 dations and was heat stable (56°C for 45 min). These data are similar to earlier results obtained with PMN activated by 12-0-tetradecanoyt-phoboi-13-acetale (PMA), an important contrast being that in the current studies PMN were activated by a soluble factor released from stimulated mononuclear cells under conditions simulating those in vivo.
Migration of Neutrophils From Lung to Tracheobronchial Lymph Node
Authors: Harmsen Allen G , Mason Margaret J , Muggenburg Bruce A , Gillett Nancy A , Jarpe Michael A , Bice David E
Published: January 2018

Translocation of particulate antigen deposited in the lung to tracheobronchial lymph nodes (TBLN) is important in the induction of pulmonary immune responses. We have previously shown that alveolar macrophages can contribute to particle translocation to TBLN, but whether neutrophils can ateo contribute to this process is not known. To determine if neutrophils can carry particles to the TBLN, dog neutrophils were elicited by instillation of red or green fluorescent microspheres into individual lung lobes. Autologous neutrophils that had phagocytized fluorescent microspheres were then Instilled Into an unexposed lobe of the same dog's lung. After 24 hr, the TBLN of instilled dogs had numerous neutrophils, 99% of which contained either red or green fluorescent microspheres but not both. Use of the two different colored microspheres as labels precluded the possibility that neutrophils had phagocytized the microspheres in the TBLN. In a second experiment, dogs were depleted of peripheral blood neutrophils by injections of hydroxyurea. Hydroxyurea treated and normal dogs were instilled with fluorescent microspheres and killed after 40 h. Hydroxyurea treatment reduced neutrophil accumulation in the lung by 79% and reduced particle translocation to the TBLN by 80%. Results of these experiments indicate that neutrophils are similar to pulmonary alveolar macrophages (PAM) In their ability to phagocytize particles in the lung and then migrate to the TBLN.
Receptor Binding and Activation of Polymorphonuclear Neutrophils by Tumor Necrosis Factor-Alpha
Authors: Shalaby M R , Palladino M A , Hirabayashi S E , Eessalu T E , Lewis G D , Shepard H M , Aggarwal B B
Published: January 2018

The interaction of highly purified recombinant human tumor necrosis factor-alpha (rTNF-α) with human polymorphonuclear neutrophils (PMNs) was investigated. Binding of 125l-rTNF-α to PMN reached maximum levels in 30 min at 37°C and in 2 h at 4°C. Scatchard analysis of competitive binding data indicated approximately 6000 receptor sites per cell and a Kd of 1.37 nM. Binding data at 37°C indicated a rapid internalization of rTNF-α. Following this receptor-mediated interaction, recombinant TNF-α was found to inhibit the migration of PMNs under agarose and to enhance PMN production of superoxide anion (O-2) in a dose-dependent manner. Furthermore, rTNF-α-activated PMNs caused a marked disruption of human umbilical-vein-derived endothelial cell monolayers and caused inhibition of their proliferative activities. These data substantiate the role of TNF-α as an activator of PMN functions and indicate that PMN/TNF-α/endothelial cell interactions may play a major role in inflammatory reactions.
Suppressive Effect of Interferon-β on Development of Tumoricidal Activity in Mouse Macrophages
Authors: Pace Judith L , MacKay Robert J , Hayes Mark P
Published: January 2018

The suppressive effect of IFN-alpha and IFN-beta on the induction of tumoricidal activity in mouse bone marrow-derived macrophages was investigated. Macrophages incubated for 24 hr with IFN-beta developed lower levels of cytolytic activity when stimulated with IFN-gamma and LPS, in comparison with macrophages pretreated with medium. The suppressive effect was dependent on the pretreatment dose of IFN-beta over a concentration range of 1 to 1,000 U/ml. Analysis of IFN-gamma dose response curves of IFN-beta treated macrophages showed that these cells were less sensitive to IFN-gamma. The suppressive effects were fully neutralized by an antiserum to IFN-alpha/beta. Prostaglandins were apparently not involved in this process since the addition of indomethacin to IFN-beta treated macrophages did not prevent the loss of responsiveness to activating stimuli. In contrast to the results obtained with IFN-alpha and IFN-beta, macrophages pretreated with IFN-gamma did not develop lower levels of cytolytic activity when again stimulated with IFN-gamma and LPS. These observations provide evidence for a potentially important negative regulatory role for IFN-alpha and IFN-beta in macrophage activation for tumor cell killing.
Monocytes from mobilized stem cells inhibit T cell function
Authors: Ino Kazuhiko , Singh Rakesh K , Talmadge James E
Published: January 2018

Granulocyte-macrophage colony-stimulating factor, mobilized peripheral blood stem cell (PSC) products, and peripheral blood leukocytes posttransplantation contain cells that cause allogeneic and autologous T cell apoptosis. Isolation and characterization of these cells demonstrated that they were low-density (Percoll fractionation) CD14+ monocytes. T cells in PSC products have a depressed phytohemagglutinin (PHA) mitogenic response; however, purified CD4+ or CD8+ T cells exhibit a statistically normal mitogenic function. Furthermore, no T cell inhibitory activity was observed in CD14+, CD4+, and CD8+ cell-depleted fractions enriched in CD4–CD8–TCRα/β+ T cells. Inhibition of T cell function by CD14+ monocytes required cell-cell contact, and the analyses of DNA fragmentation by Southern and TUNEL analysis demonstrates an activation-induced T cell apoptosis in the presence of CD14+ monocytes. Reverse-transcriptase polymerase chain reaction studies suggested that high levels of interleukin-10 or tumor necrosis factor gene transcripts in the PSC products may contribute to the inhibition of T cell function.
Phenotypic and functional differences between human dendritic cells derived in vitro from hematopoietic progenitors and from monocytes/macrophages
Authors: Triozzi Pierre L , Aldrich Wayne
Published: January 2018

We compared dendritic cells (DC) derived from CD34+ hematopoietic progenitor cells with tumor necrosis factor α and granulocyte-macrophage colony-stimulating factor (GM-CSF) to DC derived from monocytes/macrophages with interleukin-4 (IL-4) and GM-CSF. Monocyte/macrophage-derived DC demonstrated higher levels of CD1a, lower levels of CD14, greater stimulatory activity in mixed lymphocyte reactions, and greater capacity to present soluble protein antigen than CD34+ cell-derived DC. Lymphocytes stimulated with antigen-pulsed, monocyte/macrophage-derived DC produced more IL-10 than those stimulated with antigen-pulsed, CD34+-derived DC. Whereas CD1a+ DC could be derived from CD34+ cells in serum-free- and human-sera-containing cultures, the derivation of CD1a+ DC from monocytes/macrophages required the presence of fetal calf serum. The spectrum of cytokine mRNA expression, the presentation of peptide antigen, and the sensitivity to human immunodeficiency virus-1 infection of CD34+- and monocyte/ macrophage-derived DC were comparable. Although cells derived by both methods are potent antigen-presenting cells, there are differences between DC derived in vitro from hematopoietic progenitors and from monocytes/macrophages that may influence their in vivo activity.
Chemoattractant properties of PR-39, a neutrophil antibacterial peptide
Authors: Huang Hsuan-Jen , Ross Christopher R , Blecha Frank
Published: January 2018

The proline-arginine (PR) -rich antibacterial peptide, PR-39, kills bacteria by a non-pore-forming mechanism. Because this neutrophil peptide possesses several distinct functional properties and because other antimicrobial peptides are chemoattractants, we sought to determine whether PR-39 was a chemoattractant for porcine leukocytes. The peptide was synthesized by the solid-phase method using t-Boc chemistry and purified by reversed-phase high-performance liquid chromatography. Leukocyte migration was assessed with the use of a 48-well microchemotaxis chamber. PR-39 induced the directed migration of neutrophils. The peak chemotaxis response occurred at 0.5–2 μM, which was slightly lower than the minimal inhibitory and bactericidal concentrations for PR-39. However, the peptide was not a chemoattractant for mononuclear cells. Truncation of PR-39 suggested that the neutrophil chemoattractant domain may be contained within the first 26 amino acid residues. Intracellular Ca2+ fluxes in response to PR-39 were monitored by flow cytometry and showed a transient increase that peaked at approximately 40 s and approached basal values by 4 min. However, in a Ca2+-free environment, the PR-39-induced Ca2+ increase was abrogated. Furthermore, PR-39 did not induce neutrophil chemotaxis in the absence of extracellular Ca2+, and pertussis toxin inhibited both neutrophil chemotaxis and Ca2+ mobilization. Taken together, these data suggest that PR-39 is a Ca2+-dependent chemoattractant of neutrophils. The finding of a neutrophil antibacterial peptide that is also a neutrophil chemoattractant is intriguing and may indicate an important role for PR-39 in inflammation.
Cell-specific and inducible Nramp1 gene expression in mouse macrophages in vitro and in vivo
Authors: Govoni Gregory , Gauthier Susan , Billia Filio , Iscove Norman N , Gros Philippe
Published: January 2018

Mutations in the Nrampl gene abolish natural resistance to infections with many unrelated intracellular parasites in vivo. Global cDNA amplification was used to analyze Nrampl mRNA expression in bone marrow-derived cell colonies corresponding to either undifferentiated progenitors or to mature lymphoid, erythroid, and myeloid lineages. Nramp1 mRNA was detected in mature myeloid colonies expressing molecular markers for either the monocyte/macrophage or granulocytic lineages. Having established constitutive expression of Nramp1 in phagocytic cells, the parameters of inducible Nramp1 expression by cytokines and lipopolysaccharide (LPS) were studied in the mouse macrophage cell line RAW 264.7. LPS caused upregulation of Nramp1 expression in a time- and dose-dependent fashion. This induction required de novo protein synthesis and was abrogated by treatment with cycloheximide. Treatment with interferon-γ (IFN-γ) also caused a modest but reproducible twofold induction of Nramp1 mRNA expression. In addition, maximum Nramp1 mRNA induction in RAW 264.7 cells was observed after pretreatment with IFN-γ followed by LPS exposure. In vivo, Nramp1 mRNA expression could be upregulated in macrophage populations by intraperitoneal injection of either LPS or thioglycollate. Together these results indicate that Nramp1 is expressed in professional phagocytes of the myeloid lineage and can be further upregulated during macrophage activation in response to infectious, inflammatory, or cytokine stimuli. Finally, the patterns of constitutive and inducible expression of Nramp1 have been compared to those of the inducible nitric oxide synthase (iNOS) gene in the same cells.
Attenuation of monosodium urate crystal-induced arthritis in rabbits by a neutralizing antibody against interleukin-8
Authors: Nishimura Akito , Akahoshi Tohru , Takahashi Michio , Takagishi Kenji , Itoman Moritoshi , Kondo Hirobumi , Takahashi Yuichi , Yokoi Kenji , Mukaida Naofumi , Matsushima Kouji
Published: January 2018

Accumulating evidence implicates interleukin-8 (IL-8) as an essential mediator in neutrophil-mediated acute inflammation. Neutrophils have also been shown to have a crucial role in the pathogenesis of acute gouty arthritis. Thus, we investigate the pathophysiological role of IL-8 in an experimental model of acute gout, monosodium urate (MSU) crystal-induced arthritis in rabbits. The injection of MSU crystals into knee joints caused a marked swelling of joints. Concomitantly, the infiltration of leukocytes, mostly neutrophils, was observed in synovial membrane and synovial fluids. The injection of MSU crystals also induced an elevation in synovial fluid IL-8 levels preceding neutrophil infiltration into synovial fluids, without an accompanying increase in plasma IL-8 levels. Immunoreactive IL-8 protein was detected in synovial lining cells at 12–24 h after the injection. IL-8 protein was also observed in infiltrated leukocytes in synovium as early as 3–24 h after the injection. Finally, the intraarticular injection of a neutralizing anti-IL-8 antibody significantly attenuated the crystal-induced joint swelling that occurred at 12 h, and neutrophil infiltration into arthritic joints at 12 and 24 h after the induction. These results provide evidence on the pathogenic roles of locally produced IL-8 in MSU crystal-induced gouty arthritis.
The role of shear forces in ischemia/reperfusion-induced neutrophil rolling and adhesion
Authors: Kubes Paul
Published: January 2018

Intravital microscopy was used to examine the role of reduced shear forces on neutrophilendothelium interactions in single 25–40 μm diameter vessels in the feline postischemic mesenteric microvasculature. Neutrophil rolling, neutrophil adhesion, and microvascular permeability alterations were determined in vessels exposed to ischemia/reperfusion in untreated animals and in cats that received feline plasma directly into the superior mesenteric artery to increase intestinal blood flow and venular shear rates. Ischemia, followed by reperfusion, caused a profound increase in venular shear forces during the initial hyperemic response, which then decreased to below control values by 10 min of reperfusion and to less than 50% of control by 60 min of reperfusion. Associated with the decrease in blood flow was a profound increase in neutrophil rolling and neutrophil adhesion and an increase in microvascular permeability. Also, leukocyte rolling velocity decreased dramatically to 10–20% of control values during the first 10 min of reperfusion. Infusion of autologous plasma into the intestinal vasculature to maintain the shear forces at control levels during reperfusion did not affect the flux of rolling neutrophils. The rolling velocity of the neutrophils was not increased despite dramatically improved shear rates. Improved shear rates did reduce the number of adherent cells, resulting in less vascular dysfunction. The reduction in shear forces through inflamed microvessels does not contribute to the increased leukocyte rolling flux but is an essentential, permissive component for neutrophil adhesion and subsequent vascular dysfunction in postischemic microvasculature.
Monocytes incubated with surfactant: a model for human alveolar macrophages?
Authors: Geertsma Minke F , Van Furth Ralph , Nibbering Peter H
Published: January 2018

Monocytes migrate to the lungs and enter the alveoli where they come into contact with surfactant and differentiate into alveolar macrophages. This study focused on the question of the extent to which monocytes and monocyte-derived macrophages (MDM) incubated with surfactant resemble alveolar macrophages. Surfactant-incubated monocytes shared with alveolar macrophages the intracellular presence of surfactant, efficient phagocytosis of opsonized Staphylococcus aureus, and poor intracellular killing of ingested bacteria. The suppressive effect of surfactant on bactericidal activities of monocytes could not be attributed to either the surfactant lipid fraction or surfactant protein A. Monocytes incubated with surfactant differed from alveolar macrophages with respect to expression of various Fc and complement receptors involved in intracellular killing of bacteria. Surfactant-incubated monocytes produced significantly more H2O2 upon stimulation with phorbol ester than alveolar macrophages, but significantly less than control monocytes. Together, monocytes and MDM incubated with surfactant, although similar to alveolar macrophages in some aspects, are not an adequate model for alveolar macrophages. Most likely, factors other than surfactant in the microenvironment of the alveoli, such as oxygen tension, play a role in the differentiation of monocytes to alveolar macrophages as well.
Granulocyte chemotactic protein-2 and related CXC chemokines: from gene regulation to receptor usage
Authors: Van Damme Jo , Wuyts Anja , Froyen Guy , Van Coillie Els , Struyf Sofie , Billiau Alfons , Proost Paul , Wang Ji Ming , Opdenakker Ghislain
Published: January 2018

Chemokines contribute to the inflammatory response by selective attraction of various leukocytic cell types. Human GCP-2 was originally identified by amino acid sequence analysis as a CXC chemokine co-produced with IL-8 by osteosarcoma cells. Furthermore, the complete coding domain of human GCP-2 was disclosed by means of RT-PCR. Similarly, mouse GCP-2 was isolated from fibroblastoid and epithelial cells and completely identified by sequence analysis. Human and mouse GCP-2 share 61% identical amino acids. Both chemokines occur as multiple NH2-terminally truncated forms. The shorter forms of mouse, but not those of human, GCP-2 showed a higher neutrophil chemotactic potency and gelatinase B releasing capacity. Mouse GCP-2 was a more potent neutrophil activator than human GCP-2, natural mouse KC, and MIP-2. Human GCP-2 was not chemotactic for monocytes, lymphocytes, or eosinophils. Quantitative studies of mRNA expression in diploid fibroblasts revealed GCP-2 induction by IL-1β. Human GCP-2 induced [Ca2+]i increase in neutrophils, which was reciprocally desensitized by IL-8, GROα, and ENA-78. Human GCP-2 induced [Ca2+]i increases and chemotactic responses in both CXCR1- and CXCR2-transfected cells. Finally, GCP-2 provoked neutrophil accumulation and plasma extravasation in rabbit skin. In humans, GCP-2 complements the activity of IL-8 as neutrophil chemoattractant and activator but it constitutes a major neutrophil chemokine in the mouse. GCP-2 induces neutrophil chemotaxis and activation but it might also contribute to detrimental tissue damage in sepsis, acute respiratory distress syndrome, acute hypersensitivity reactions, and autoimmune diseases. It might also influence the invasive capacity of GCP-2-secreting tumor cells.
Pivotal role of interleukin-8 in the acute respiratory distress syndrome and cerebral reperfusion injury
Authors: Matsumoto Tetsuya , Yokoi Kenji , Mukaida Naofumi , Harada Akihisa , Yamashita Junkoh , Watanabe Yoh , Matsushima Kouji
Published: January 2018

Neutrophil recruitment is one of the hallmarks of acute inflammation. A potent neutrophil chemotactic and activating factor, interleukin-8 (IL-8), has been demonstrated to be elevated in body fluids in various human diseases and experimental animal models. Recent investigations on animal disease models using blocking antibodies to IL-8 have revealed the essential involvement of IL-8 in acute inflammation. We previously reported that the administration of a neutralizing antibody against IL-8 prevented the neutrophil infiltration and neutrophil-mediated tissue injury in several animal studies. In addition, we have recently demonstrated that anti-IL-8 treatment is also effective in prevention of two models that are very relevant to clinical situations: cerebral reperfusion injury and endotoxemia-induced acute respiratory distress syndrome-like lung injury. These results further support the hypothesis that IL-8 has a pivotal role and is a novel target for therapeutic intervention in neutrophil-mediated injury.
Sequence similarities of a subgroup of CXC chemokines related to murine LIX: implications for the interpretation of evolutionary relationships among chemokines
Authors: Smith Jeffrey B , Rovai Leonor E , Herschman Harvey R
Published: January 2018

The murine CXC chemokine LIX has distinctive sequence features that suggest it is a novel chemokine. Among known human chemokines, ENA-78 and GCP-2 are the two most closely related to LIX. We have recently cloned the human GCP-2 gene. Phylogenetic analysis shows that the LIX coding region is more distant from both human GCP-2 and ENA-78 than is porcine AMCF-II, the chemokine with greatest sequence similarity to LIX. Human GCP-2 and ENA-78 have very high nucleotide similarity in non-coding as well as coding sequences, which suggests that these genes are the result of an evolutionarily recent gene duplication event. If this duplication occurred during primate evolution, then non-primate species may have only a single chemokine corresponding to this pair of human genes. This example shows that a one-to-one genetic correspondence does not necessarily exist between all the chemokine genes in two different species. These observations may have important implications for other chemokines that belong to clusters of closely related genes.
Effect of mast cell deficiency and leukotriene inhibition on the influx of eosinophils induced by eotaxin
Authors: Harris R R , Komater V A , Marett R A , Wilcox D M , Bell R L
Published: January 2018

Eosinophils are believed to be important cells in the pathogenesis of asthma and allergic disease. Mast cells and leukotrienes may play a role in eosinophil recruitment. Eotaxin was recently described as a specific chemoattractant for eosinophils. Therefore we examined the effects of eotaxin on eosinophil flux in the mast cell-deficient WBB6F1/J-Kitw/ Kitw-v (W/Wv) mice and in mice treated with zileuton or ABT-761, specific 5-lipoxygenase inhibitors. Mice were injected intraperitoneally with eotaxin and at various times later the peritoneal cavities were lavaged and cell populations determined. Murine recombinant eotaxin induced a dose-dependent increase in eosinophils, which reached a maximum at 1-2 h and subsided at 4 h in both BALB/c and W/Wv littermate control mice (no other cell population was altered). However, in eotaxin-injected W/Wv mice, the peak of eosinophil influx was delayed, peaking at 2 h, and a lower number of eosinophils was seen. The specific lipoxygenase inhibitors zileuton and ABT-761 given 30 min before eotaxin caused a 63-79% reduction in the level of eosinophils seen in the lavage fluid. These data suggest that eotaxin may either be activating eosinophils to release leukotrienes or making them more responsive to leukotrienes. In addition, mast cells may be playing a role in the amplification of the eotaxin effect.
Quantitative CrAssphage real‐time PCR assay derived from data of multiple geographically distant populations
Authors: Cinek Ondrej , Mazankova Karla , Kramna Lenka , Odeh Rasha , Alassaf Abeer , Ibekwe MaryAnn U. , Ahmadov Gunduz , Mekki Hanan , Abdullah Mohammed A. , Elmahi Bashir M. E. , Hyöty Heikki , Rainetova Petra
Published: January 2018

After its computational inference from human stool metagenomes, the CrAssphage has proven to be the most prevalent phage in the human gut, with presumably very wide geographic distribution. The currently available molecular assays do not sufficiently reflect the CrAssphage sequence variability. Here, we report a novel real‐time PCR assay whose primers and probes are derived from data of multiple CrAssphage strains obtained from gut viral metagenomes of European, Asian, and African subjects. This assay can be useful in analyses of putative bacterial host co‐occurence, and in association studies of non‐infectious diseases where the phage may modify the content of gut bacteriomes.