bluuebunny/crossref_metadata_2025 huggingface dataset:
Ultrastructural morphology of immature mast cells in sequential suspension cultures of human cord blood cells supplemented with c-kit ligand; distinction from mature basophilic leukocytes undergoing secretion in the same cultures
Authors: Dvorak Ann M , Mitsui Hideki , Ishizaka Teruko
Published: January 2018

The ability to culture human mast cells and, thus, to determine their ontogeny and possible relationships to other lineages has been facilitated by new studies using cocultures of cord blood cells with mouse fibroblasts and recombinant human or murine c-kit ligand- supplemented suspension cultures of cord blood cells. In this study, we examined c-kit ligand-supplemented cord blood cell suspension cultures designed so that the effects of growth factor source, individual cord sample, and culture time (3-17 weeks) on the developing mast cell lineage could be individually evaluated. We found that human mast cells, basophils, neutrophils, eosinophils, macrophages, megakaryocytes, and endothelial cells were present in these cultures. The numbers of mast cells and their granules increased with culture time; mature basophils, present in quantity in 3-week cultures, decreased in number and released granule contents with increased culture times. The mast cell lineage developed similarly, regardless of which factor preparation was added to cultures, but considerable variability existed among individual donors from whom cord bloods were obtained. Unlike the mature, crystal-containing mast cells that regularly developed in fibroblast cord blood cocultures (Furitsu et al. [1989] Proc. Natl. Acad. Sci. USA 86, 10039-10043), human mast cells failed to attain full maturity in the suspension cultures examined here, regardless of individual cord sample, added growth factor, or culture time. Furthermore, unlike cells of the basophil lineage in which granule content release was regularly observed, morphologic evidence of secretion from human mast cells was absent. Instead, these cells were actively undergoing granule building as determined by the increasing numbers of granules and filling of these containers over culture time. Crystal granules never developed, even at the maximum culture time of 17 weeks. We conclude that fibroblasts are necessary and sufficient for the differentiation and maturation of human mast cells in vitro from their agranular precursors in cord blood but that soluble c-kit ligand is not.
Selective inhibition of antigen-specific T lymphocyte proliferation by acute ethanol exposure: the role of impaired monocyte antigen presentation capacity and mediator production
Authors: Szabo Gyongyi , Verma Bikash , Catalano Donna
Published: January 2018

Ethanol consumption is associated with impaired immunity. Our data demonstrate that even a single dose of a biologically relevant concentration (25-150 mM) of ethanol can down-regulate antigen-specific T lymphocyte proliferation. In contrast, ethanol augmented mitogen-induced T cell proliferation, suggesting that its inhibitory effect on antigen-specific T cell proliferation was due to its effects on monocytes (mφs) rather than on T cells. The immunodepressive effects of ethanol on mφ antigen-presenting cell (APC) capacity were manifested whether alcohol treatment was limited to the antigen uptake-processing period only or was present during the entire period of antigen presentation. These inhibitory effects of ethanol were also evident on both the high- antigen-presenting, FcγRI-negative (-31 ± 17%), and low-antigen-presenting, FcγRI-positive (-42 ± 15%) mφ subpopulations. Further analysis demonstrated that ethanol inhibits the production of interleukin-1 β (IL-lβ) and induces transforming growth factor β (TGF-β) and prostaglandin E2 (PGE2), monocyte-derived mediators that can affect T cell proliferation. Ethanol resulted in a dose-dependent down-regulation of secreted and cell- associated IL-lβ protein as well as IL-lβ mRNA levels induced by adherence or bacterial stimulation. The causal relationship between decreased mφ IL-lβ production, elevated TGF-β levels, and the decreased mφ APC capacity was further substantiated when exogenous IL-lβ protein or anti-TGF-β neutralizing antibody prevented the down-regulatory effect of ethanol on antigen-specific T cell proliferation. Utilizing a cyclooxygenase inhibitor, we also demonstrated that the ethanol-induced decrease in mφ APCs is not mediated by enhanced PGE2 production.
Complement and tumor necrosis factor-α contribute to Mac-1 (CD11b/CD18) up-regulation and systemic neutrophil activation during endotoxemia in vivo
Authors: Witthaut Rochus , Farhood Anwar , Smith C Wayne , Jaeschke Hartmut
Published: January 2018

The increased expression of Mac-1 (CD11b/CD18) adhesion glycoproteins on neutrophils was studied using flow cytometry in male Fischer 344 rats treated with 5 mg/kg Salmonella cnteritidis endotoxin. A rapid and sustained threefold increase of Mac-1 expression was observed after endotoxin injection. Inhibition of complement activation with the soluble complement receptor type 1 (sCRl) completely suppressed the initial up-regulation of Mac-1 (≤ 15 min) but did not prevent the activation during the later phase (30–90 min). During that time period, Mac-1 expression increased in parallel with the concentration of tumor necrosis factor α (TNF-α) in plasma and could be significantly attenuated with TNF antiserum. To verify the results, isolated human neutrophils were incubated with rat plasma obtained at various times after endotoxin injection. Using shape change as indicator of neutrophil activation, complement and TNF-α could be identified as responsible mediators for neutrophil activation during endotoxemia in vivo. In contrast, the massive neutrophil accumulation in the liver after endotoxin was only slightly reduced by sCRl and unaffected by TNF antiserum. It is concluded that Mac-1 up-regulation on neutrophils after endotoxin injection in vivo may have limited relevance for hepatic neutrophil infiltration but may be important for the pathogenesis of endotoxin-induced liver injury by facilitating adherence-dependent neutrophil cytotoxicity. J. Leukoc. Biol. 55: 105–111; 1994.
Inhibition of crystal-induced neutrophil activation by a protein tyrosine kinase inhibitor
Authors: Burt Helen M , Jackson John K , Salari Hassan
Published: January 2018

The objective of this work was to investigate the role of tyrosine kinase in monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) crystal-induced neutrophil activation using the tyrosine kinase inhibitor lavendustin C (LVC). Human neutrophils pretreated with LVC at concentrations between 10 and 150 μM or control neutrophils were stimulated by plasma-coated CPPD or uncoated MSUM, and chemiluminescence, superoxide generation, intracellular calcium concentration, and degranulation (myeloperoxidase and lysozyme release) were monitored with time. LVC strongly inhibited chemiluminescence, superoxide anion generation, myeloperoxidase and lysozyme release, and calcium mobilization. After 1-min crystal-neutrophil incubations, neutrophil cytosolic fractions showed extensive inhibition of tyrosine kinase activity by LVC. We conclude that the inhibition of neutrophil responses to crystal stimulation, by the protein tyrosine kinase inhibitor LVC, provides evidence that supports the involvement of tyrosine kinases in crystal-induced neutrophil activation. J. Leukoc. Biol. 55: 112–119; 1994.
gp120 HIV envelope glycoprotein increases the production of nitric oxide in human monocyte-derived macrophages
Authors: Pietraforte Donatella , Tritarelli Elena , Testa Ugo , Minetti Maurizio
Published: January 2018

The effect of recombinant gp120 HIV envelope glycoprotein on the generation of free radicals by monocyte-derived macrophages (MDM) was measured by EPR spin trapping with 5,5 -dimethyl-1-pyrroline-N-oxide (DMPO). After 1 day in culture, MDM produced a spin trap adduct of DMPO with hyperfine splitting constants superimposable on those of DMPO-OH. The addition of gp120 to MDM increased the production of DMPO-OH and after 1 h, the amount of DMPO-OH produced by 40 μg/ml gp120 was about 300% that of untreated MDM. The use of selective inhibitors suggested the participation of the nitric oxide/l-arginine oxidative pathway, but did not provide evidence for trapping of hydroxyl radical or other oxygen free radicals. The specificity of gp120 was proven by two different anti-gp120 antibodies that either in-hibited (polyclonal) or increased (monoclonal) the production of free radicals. Dexamethasone inhibited the effect of gp120, suggesting the possible involvement of an inducible nitric oxide (NO·) synthase. Moreover, treatment of MDM with gp120 for 15 h increased in a dose-dependent manner the production of NO2-, a stable end product of NO·. Soluble CD4 did not modify the intensity of the DMPO-OH adduct, whereas yeast mannan and Ca2+-chelators abolished the increase in the DMPO-OH signal induced by gp120. These data suggest the possible involvement of mannose-specific endocytotic lectin of MDM. The reaction of DMPO with sodium nitroprusside, an organic nitrate that releases NO·, also produced DMPO-OH. Our findings indicate that gpl20 increases free radical production from MDM as detected by spin-trapping methods, and that the spin trap adduct results from a reaction involving NO· or closely related oxidized derivatives. J. Leukoc. Biol. 55: 175–182; 1994.
Comparison of leukotriene B4 and D4 effects on human eosinophil and neutrophil motility in vitro
Authors: Spada C S , Nieves A L , Krauss A H-P , Woodward D F
Published: January 2018

The motility of isolated normal human peripheral blood eosinophils and neutrophils in response to exogenous leukotrienes B4 and D4 was examined by means of a modified under-agarose technique and a novel quantitative sampling strategy. Leukotriene D4 was a potent chemoattractant for eosinophils, with a significant threshold chemotactic effect evident at 10-10 M. The abolition of eosinophil chemotaxis by the potent and selective peptide-leukotriene-antagonist SK&F 104353 indicated the pharmacological specificity of the leukotriene D4-induced response. The chemokinetic response of eosinophils to leukotriene D4 generally did not differ significantly from spontaneous migratory activity of unstimulated cells. Leukotriene D4 did not, however, alter directed neutrophil motility until a very high concentration (10-5 M) was achieved, although significant neutrophil chemokinesis relative to unstimulated movement was observed over the tested concentration range. Directional emigration of both eosinophils and neutrophils was induced by leukotriene B4 at concentrations as low as 10-8 M. Analysis of leukocyte orientations provided evidence that chemokinetic responses were not being interpreted as indications of chemotactic behavior. These studies suggest that leukotriene D4 may behave as a potent and selective chemoattractant for human eosinophils at physiologically relevant concentrations. J. Leukoc. Biol. 55: 183–191; 1994.
Cytokine induction in mice by the immunomodulator imiquimod
Authors: Reiter Michael J , Tester man Traci L , Miller Richard L , Weeks Charles E , Tomai Mark A
Published: January 2018

Imiquimod has been identified as a potent antiviral and antitumor agent in animal models. The biological activity associated with imiquimod has been attributed to its induction of interferon (IFN)-α. The present studies evaluated imiquimod administered orally for its ability to stimulate production of IFN and other cytokines in mice. The cytokine profile induced by imiquimod was compared with other known immunomodulators. Imiquimod was found to stimulate increased serum IFN in mice. Daily dosing of imiquimod for five consecutive days led to diminished production of IFN in mice as measured after the final dose. Elevated levels of serum tumor necrosis factor (TNF)-α and interleukin (IL)-6· but not IL-1α were found in serum from mice treated with imiquimod. Imiquimod produced significantly higher levels of IFN but lower levels of TNF and IL-6 and IL-1α than lipopolysaccharide. Polyinosinic acid:polycytidylic acid induced significantly higher amounts of IFN but lower levels of TNF and IL-6 than imiquimod. Imiquimod stimulated significantly higher levels of IFN when compared with 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidi-none (ABPP) and similar levels of IFN when compared with tilorone. Neither ABPP nor tilorone induced TNF or IL-6. Finally, imiquimod stimulated TNF, IFN, and IL-6 production in cultures of mouse spleen and bone marrow cells. These studies demonstrate that imiquimod induces not only IFN but other cytokines as well, all of which may contribute to its biological activity. J. Leukoc. Biol. 55: 234–240; 1994.
The monoclonal antibody MAC387 detects an epitope on the calcium-binding protein MRP14
Authors: Goebeler Matthias , Roth Johannes , Teigelkamp Stefan , Sorg Clemens
Published: January 2018

The present study was initiated to identify the antigen recognized by the monoclonal antibody (MAb) MAC387 which is widely used for phenotypical characterization of myelomonocytic cells in situ. MAC387 has been described to show a similar reaction pattern as antisera to a complex formed by the calcium-binding proteins MRP8 and MRP14. However, the exact nature of the molecule recognized by MAC387 has been controversial. Using Western blot analysis, MAC387 was found to detect a single protein band of 14 kDa in lysates of human monocytes and granulocytes. Transfection of embryonic lung fibroblasts L132 with either MRP8 or MRP14 cDNA revealed that MAC387 reacts with MRP14 but not MRP8. This finding was confirmed by dot blot analysis of recombinant MRP8 and MRP14. Our data thus provide unequivocal evidence that MAC387 is a monoclonal antibody directed against the calcium-binding protein MRP14. J. Leukoc. Biol. 55: 259–261; 1994.
The role of tyrosine kinases and phosphotyrosine-containing recognition motifs in regulation of the T cell-antigen receptor-mediated signal transduction pathway
Authors: Isakov Noah , Wange Ronald L , Samelson Lawrence E
Published: January 2018

T cell-mediated immune responses are initiated by interaction of antigen bound to a glycoprotein encoded by the major histocompatibility complex with the T cell antigen receptor (TCR). These recognition and binding steps are followed by multiple intracellular biochemical events. The earliest event detected is an increase in intracellular protein tyrosine phosphorylation that involves a complex interaction of tyrosine kinases and phosphatases. Subsequently, one observes an increase in protein serine/threonine phosphorylation, phospholipid hydrolysis, and changes in intracellular Ca2+ levels. These and other biochemical changes lead to cell proliferation, differentiation, and acquisition of effector functions. While binding of extracellular growth factors to receptors containing cytoplasmic protein tyrosine kinase (PTK) domains induces direct activation of their kinase activity, the multichain TCR lacks an intrinsic kinase domain and therefore represents a distinct type of receptor. It transduces signals via the interaction with, and activation of, non-receptor PTKs. Recent efforts directed at defining the TCR-linked signaling pathways have provided insight into the regulatory role of three PTKs, and the functional importance of some unique protein motifs in both TCR subunits and PTKs, which mediate critical protein-protein interactions in this pathway. J. Leukoc. Biol. 55: 265–271; 1994.
Inhibitory effect of a dendritic epidermal T cell line on K1735 melanoma cells in vivo and in vitro
Authors: Love-Schimenti Cheryl D , Kripke Margaret L
Published: January 2018

Murine epidermis contains a resident population of dendritic epidermal T cells (DETCs) with unknown function. We tested the ability of a cultured line of these cells to inhibit the growth of murine melanoma cells in vitro and in vivo. The interleukin-2 (IL-2)-dependent T cell line AU16, derived from the epidermis of C3H mice, was used as the source of DETCs. AU16 cells were cytotoxic to syngeneic K1735 and CM3205 C3H melanoma cell lines, as well as to a chemically induced cutaneous fibrosarcoma, in an in vitro 51Cr release assay. Mixing viable AU16 cells, but not splenic T cells, with K1735 melanoma cells immediately before intradermal injection into the flank of mice prevented or delayed the outgrowth of melanomas. AU16 cells killed by freezing and thawing and supernatants from cultures of AU16 cells did not inhibit melanoma growth in vivo or in vitro. Injection of viable AU16 cells intravenously or at a separate intradermal site had no effect on melanoma growth. These studies suggest that IL-2-activated DETCs have the potential to inhibit the growth of melanoma cells in vivo and that their cytotoxic activity may be responsible for this effect. J. Leukoc. Biol. 55: 379–384; 1994.
Histaminergic regulation of antibody-dependent cellular cytotoxicity of granulocytes, monocytes, and natural killer cells
Authors: Hellstrand Kristoffer , Asea Alexander , Hermodsson Svante
Published: January 2018

We investigated effects of the biogenic diamine histamine on antibody-dependent cellular cytotoxicity (ADCC) against autologous anti-D-coated red blood cells mediated by human granulocytes, monocytes, and natural killer (NK) cells. Effector cells were separated from peripheral blood by countercurrent centrifugal elutriation. ADCC of monocytes and neutrophilic granulocytes was suppressed by histamine. ADCC of enriched CD3-/56+ NK cells was unchanged by histamine. ADCC of NK cells was effectively inhibited by elutriated monocytes or neutrophils. Histamine completely reversed the inhibition of NK cell-mediated ADCC induced by monocytes and partly reversed the inhibition induced by neutrophils; thereby, histamine augmented ADCC of NK cells in the presence of monocytes or neutrophils. The indirect effect of histamine on ADCC of NK cells and the effect of histamine on ADCC of monocytes/neutrophils were completely antagonized by the specific H2 receptor (H2R) blocker ranitidine. We conclude that activation of H2R suppresses ADCC reactivity of monocytes/neutrophils and, concomitantly, promotes ADCC reactivity of NK cells by abrogating a phagocyte-derived, suppressive signal. J. Leukoc. Biol. 55: 392–397; 1994.
Selective regulation of human neutrophil functions by the cell activation inhibitor CI-959
Authors: Wright Clifford D , Stewart Sheila F , Kuipers Paul J , Hoffman Michael D , Devall Larry J , Kennedy John A , Ferin Mark A , Thueson David O , Conroy Mary Carol
Published: January 2018

The cell activation inhibitor CI-959 [5-methoxy-3-(1-methylethoxy)-N-1H-tetrazol-5-ylbenzo[b]thiophene-2-carboxamide, monosodium salt] was evaluated for its effects on human neutrophil functions. CI-959 inhibited spontaneous migration and chemotaxis toward N-formyl–methionyl-l-leucyl-l-phenylalanine (fMLP) with 50% inhibition (IC50) values of 3.6 and 3.1 μM, respectively. CI-959 also inhibited superoxide anion generation in response to C5a, fMLP, serum-opsonized zymosan (SOZ), concanavalin A (Con A), and calcium ionophore A23187 with IC50 values of 2.5, 4.7, 14.5, 5.4, and 14.8 μM, respectively. In comparison, CI-959 inhibited myeloperoxidase release in response to C5a, fMLP, SOZ, and Con A with IC50 values of 11.6, 16.1, 7.5, and <1.0 μM, respectively, while inhibiting the response to A23187 by only 5.5% at 100 μM. At concentrations up to 100 μM, CI-959 had no effect on the respiratory burst or degranulation in response to L-α-1,2-dioctanoylglycerol (DiC8) or phorbol 12-myristate 13-acetate (PMA). In addition, the compound inhibited leukotriene B4 release stimulated by fMLP and SOZ (IC50 values 4.0 and 2.5 μM, respectively), while having less activity against the A23187-stimulated response (IC50>100 μM). These results demonstrate that CI-959 inhibits cellular responses to stimuli that mobilize intracellular calcium. For cellular responses to ionophore-mediated calcium influx, only oxygen radical production was inhibited by CI-959. CI-959 was further evaluated for its effects on neutrophil stimulus-response coupling. At 100 μM, CI-959 had no effect on human neutrophil phospholipase C or protein kinase C. CI-959 inhibited fMLP-stimulated intracellular calcium mobilization and calcium influx with IC50 values of 16.7 and 3.1 μM, respectively, and exhibited less potent calmodulin antagonist activity (IC50 = 90.5 μM). These results indicate that CI-959 may exert its stimulus- and response-specific inhibitory effects on neutrophil functions, in part, through inhibition of calcium-regulated signalling mechanisms. J. Leukoc. Biol. 55: 443–451; 1994.
Serum components enhance bacterial lipopolysaccharide-induced tissue factor expression and tumor necrosis factor-α secretion by bovine alveolar macrophages in vitro
Authors: Yang Zhengang , Khemlani Lajwanti S , Dean David F , Carter Candace D , Slauson David O , Bochsler Philip N
Published: January 2018

We have compared the effect of bacterial lipopolysaccharide (LPS) in combination with normal adult bovine serum (NBS), fetal bovine serum (FBS), or a bovine serum fraction on tissue factor expression and tumor necrosis factor α (TNF-α) secretion by bovine alveolar macrophages. At a concentration of 1 ng/ml, bacterial LPS alone failed to induce measurable tissue factor expression by the macrophages, but the presence of FBS, NBS, or a fraction of normal pooled bovine serum isolated by ion-exchange chromatography (fraction 2) markedly potentiated the effect of LPS. A protein concentration of 64 μg/ml NBS, 192 μg/ml FBS, and only 640 ng/ml fraction 2 was required to induce maximal tissue factor expression on the macrophages in combination with 1 ng/ml LPS. Comparison of quantities of added serum protein required to induce maximal potentiating effects indicated that fraction 2 was 100 times more potent than1 whole NBS and 300 times more potent than whole FBS. We similarly found that TNF-α secretion by macrophages exposed to LPS was responsive to serum and was highly responsive to fraction 2. LPS alone (1 ng/ml) induced a relatively low level of TNF-α secretion by the macrophages, and the presence of FBS, NBS, or fraction 2 potentiated the effect of LPS. A concentration of 64.0 μg/ml NBS, 320.0 μg/ml FBS, and 3.2 μg/ml fraction 2 serum protein induced near-maximal TNF-α secretion by the macrophages. Comparison of the concentration of serum protein required to induce these potentiating effects indicated that fraction 2 was approximately 20 times more potent than whole NBS and 100 times more potent than whole FBS. The stimulatory effect of LPS plus fraction 2 serum proteins was dependent on the CD14 receptor, as monoclonal antibodies directed against CD14 (My4, 60bd; 10 μg/ml) inhibited tissue factor expression and TNF-α secretion by the macrophages. J. Leukoc. Biol. 55: 483–488; 1994.
Regulation of hepatic endothelial cell and macrophage proliferation and nitric oxide production by GM-CSF, M-CSF, and IL-1β following acute endotoxemia
Authors: Feder Lisa S , Laskin Debra L
Published: January 2018

Treatment of rats with bacterially derived lipopolysaccharide (LPS), a condition that mimics acute endotoxemia, results in a significant increase in the number of endothelial cells and macrophages in the liver. This is correlated with the release of proinflammatory and cytotoxic mediators that induce liver damage. In the present studies, we analyzed the effects of various inflammatory mediators released during the pathogenesis of hepatic injury on proliferation of liver nonparenchymal cells. To induce acute endotoxemia female Sprague-Dawley rats were injected intravenously with 5 mg/kg LPS. Endothelial cells and macrophages were isolated 48 h later by combined collagenase and pronase perfusion of the liver followed by centrifugal elutriation. Interleukin-1α (IL-1α), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) had no effect on proliferation of either endothelial cells or macrophages. In contrast, whereas interleukin-1β (IL-1β) inhibited the proliferation of endothelial cells from untreated rats, this cytokine stimulated the growth of cells from endotoxemic rats. The colony-stimulating factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-GSF), also markedly enhanced the proliferation of endothelial cells, as well as macrophages from endotoxemic rats. Macrophages from endotoxemic rats were more sensitive to the colony-stimulating factors than cells from untreated rats. In contrast, the inflammatory mediators LPS and interferon-γ (IFN-γ) inhibited endothelial cell and macrophage growth, an effect that was partially blocked in endothelial cells by the nitric oxide synthase inhibitor NG-monomethyl-l-aiginine (L-NMMA). This suggests that growth inhibition in these cells is mediated, in part, by nitric oxide. Interestingly, in both endothelial cells and macrophages from endotoxemic rats, GM-CSF, M-CSF, and IL-1β synergized with LPS and IFN-γ to induce nitric oxide production. This was correlated with a further inhibition of proliferation that was partially reversed by L-NMMA in endothelial cells but not macrophages. Taken together these data demonstrate that endothelial cell and macrophage proliferation in the liver is controlled by a variety of mediators released during endotoxemia; however, the mechanisms regulating growth in the two cell types are distinct. J. Leukoc. Biol. 55: 507–513; 1994.
Use of the 5′ -flanking region of the mouse perforin gene to express human Fcγ receptor I in cytotoxic T lymphocytes
Authors: Smyth Mark J , Kershaw Michael H , Hulett Mark D , McKenzie Ian F C , Trapani Joseph A
Published: January 2018

Expression of the gene encoding the cytolytic granule protein perforin is restricted to cytotoxic lymphocytes. To undertake a functional analysis of the immediate 5′-promoter region of the mouse perforin gene, we transiently transfected mouse perforin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs into cytotoxic T, T lymphoid, B-lymphoid, and nonlymphoid cell lines. The transcriptional activity of the perforin promoter was restricted to cytotoxic lymphocytes. The perforin promoter was controlled by several positive (in perforin-positive cells) and negative (in perforin-negative cells) cis-acting regions, spread over at least 1.1 kilobases. The most specific expression of the CAT reporter gene in the interleukin-2-dependent cytotoxic T cell line CTLL-R8 was obtained with the mouse perforin promoter encompassing positions -1104 to +1 in relation to the RNA cap site. This construct expressed 65- to 70-fold higher CAT activity than the promoterless CAT construct in perforin-expressing cells but only 1- to 5-fold higher CAT activity than the promoterless construct in nonlymphoid cells. On the basis of these data, we used this most specifically active mouse perforin promoter, -1104 to +1, to express in CTLL-R8, a chimeric human receptor comprising the extracellular domains of human FcγRI and the transmembrane and intracellular domains of TCR . Selection in G418-containing medium produced CTLL-R8 transfectant clones that (1) expressed high levels of human FcγRI mRNA; (2) expressed cell surface FcγRI as demonstrated by immunoprecipitation and their ability to bind the Fc portion of human and mouse monoclonal antibodies (mAbs) in an isotype-specific manner, and (3) bound RBC expressing mucin-1 (Muc-1) peptide in the presence of a chimeric mouse-human anti-Muc-1 mAb. Activation of CTLL-R8 transfectants upon engagement of the human FcγRI was evidenced by their ability to lyse tumor target cells in an mAb isotype-dependent manner. The successful expression of a functional chimeric gene in CTLL-R8 suggests that the mouse perforin promoter represents a novel reagent for expressing exogenous genes in cytotoxic T lymphocytes. J. Leukoc. Biol. 55: 514–522; 1994.
Gestation-related expression of the interferon-γ receptor gene in mouse uterine and embryonic hematopoietic cells
Authors: Chen Hua-Lin , Kamath Ravi , Pace Judith L , Russell Stephen W , Hunt Joan S
Published: January 2018

Macrophages and natural killer (NK)–like cells are the major hematopoietic cell populations in the cycling and pregnant mouse uterus and are also found in the embryo. In order to evaluate potential receptivity of these cells to interferon-γ (IFN-γ), tissues taken from cycling and pregnant mice were tested for IFN-γ receptor (IFN-γR) mRNA and protein. Macrophages were identified immunohistochemically by using the specific monoclonal antibody F4/80. NK cells were identified by their large size, distinctive intracellular granules, and binding of a monoclonal antibody to the common leukocyte antigen. In cycling uteri, the abundance of IFN-γR mRNA relative to an invariant message (glyceraldehyde-3-phosphate dehydrogenase) increased during progression of the hormonally regulated estrous cycle. IFN-γR mRNA in situ hybridization signals were slightly higher in macrophage-like than in other types of endometrial stromal cells. In pregnant uteri, the highest proportions of IFN-γR mRNA were observed at gestation day (g.d.) 16. Specific message and protein were present in uterine macrophages by g.d. 7 and in NK cells by g.d. 9. IFN-γR expression in both lineages remained stable through the balance of pregnancy. In embryos, IFN-γR mRNA increased between g.d. 14 and 16. Specific transcripts were present in many cells at g.d. 14, but none were detected in embryonic liver macrophages until g.d. 16. The results of this study suggest relationships between IFN-γR expression and ovarian hormones as well as cell maturation and support the postulate that IFN-γ receptor-ligand interactions may improve the ability of uterine and embryonic hematopoietic cells to perform specific tasks during gestation. J. Leukoc. Biol. 55: 617–625; 1994.
Cytokines and tuberculosis
Authors: Wallis Robert S , Ellnor Jerrold J
Published: January 2018

The evolution of Mycobacterium tuberculosis as an intracellular pathogen has led to a complex relationship between it and its host, the human mononuclear phagocyte. The products of M. tuberculosis–specific T lymphocytes are essential for macrophage activation for intracellular mycobacterial killing. However, some cytokines, including products of both lymphocytes and phagocytic cells, may contribute to enhanced mycobacterial survival and replication. In human immunodeficiency virus-associated tuberculosis, cytokine products may mediate enhanced susceptibility to tuberculosis as well as accelerated progression to AIDS. Better understanding of these interactions will allow the development of increasingly specific immune-based interventions for prevention and treatment of tuberculosis. J. Leukoc. Biol. 55: 676–681; 1994.
Temporal down-regulation of FcγRIII expression and Fcγ receptor-mediated phagocytosis in human monocyte-derived macrophages induced by TNF-α and IL-1β
Authors: Liao Guojuan , Simon Sanford R
Published: January 2018

Effects of tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) on the expression of Fcγ receptors (FcγRs) and on FcγR-mediated phagocytosis in human monocyte-derived macrophages (MDMs) have been determined. Treatment of MDMs with TNF-a (400 pg/ml) or IL-1β (400 pg/ml) for 80 min decreased cell surface FcγRIII expression, as measured by flow cytometry (FAGS), while the levels of surface FcγRI and FcγRII remained unchanged. Surface FcγRIII expression remained depressed for 7 h after exposure of MDMs to either cytokine but then rose progressively until, by 24 h of stimulation, the levels were at least as high as those on unstimulated cells. In parallel with cell surface FcγRII I levels, phagocytosis of immunoglobulin G-opsonized fluorescent beads, as measured by FAGS, decreased and then increased over time after stimulation of MDMs with TNF-α while the opsonin-independent phagocytic activity was unchanged, suggesting that among the three classes of Fcγ receptors on macrophages, FcγRIII may play a critical role in regulating opsonic phagocytosis. After treatment with IL-1β, both opsonic and nonopsonic phagocytic activities of MDMs were significantly decreased, regardless of FcγRIII levels. FAGS analysis of rhodamine-phalloidin-stained MDMs showed that after incubation with IL-1β, but not TNF-α, the content of Factin declined in MDMs, coinciding with inhibition of particle phagocytosis. Rapid down-regulation and subsequent recovery of macrophage FcγRIII expression following cytokine stimulation could serve as a novel regulatory mechanism employed by inflammatory cells in host defensc. J. Leukoc. Biol. 55: 702–710; 1994.
Synthetic polysulfated hyaluronic acid is a potent inhibitor for tumor necrosis factor production
Authors: Chang Nan-Shan , Intrieri Catherine , Mattison Jeffery , Armand Gerard
Published: January 2018

Based on the premise that naturally occurring glycosaminoglycans could serve as building blocks for synthesizing nontoxic drugs for suppression of tumor necrosis factor (TNF) production by inflammatory cells, we have chemically modified hyaluronic acid (HA) and tested its effects in blocking TNF-α and TNF-β production in vitro. HA was chosen mainly for its structural simplicity, nonimmunogenicity, and readiness for chemical modifications. When HA was chemically polysulfated to a sulfate/hexosamine molar ratio of 3.9, the sulfated HA, was shown to be a potent inhibitor of TNF-α production in lipopolysaccharide (LPS)- or interferon-γ-activated THP-1 cells. For example, a concentration of HAs as low as 10 ng/ml reduced TNF-α production in LPS-activated THP-1 cells more than 50%, whereas achieving a similar extent of reduction required 50 μg/ml native HA. By decreasing the extent of polysulfation, the inhibitory effect of HAS on TNF-α production was diminished. Other chemical modifications, including deacetylation, thiolation, or reduction of the carboxylic groups, could not increase the efficacy of HA in suppression of TNF-α production. Naturally polysulfated glycosaminoglycans, such as chondroitin sulfates, keratan sulfate, heparan sulfate, and heparin, failed to inhibit TNF-α production. HA, also restricted TNF-β (lymphotoxin) secretion in an Epstein-Barr virus-transformed B cell line, Roha-9, which constitutively produces TNF-β. HA, had no inhibitory effect on the proliferation of THP-1 or Roha-9 cells, which would account for the reduced TNF-α or TNF-β production. Furthermore, time-course metabolic labeling studies revealed that HA, could not restrict overall protein synthesis and secretion in THP-1 cells. However, HA, increased complement Clq secretion in THP-1 in a dose-dependent manner, but it had no effect on biosynthesis of complement Cl inhibitor, factor D, and Fcγ receptor type II (FcγRII). These results indicate that HA, selectively restricts the production of TNF-α, TNF-SbT, and probably several other protein species. J. Leukoc. Biol. 55: 778–784; 1994.
Cytokine induction of granule protein synthesis in an eosinophil-inducible human myeloid cell line, AML14
Authors: Paul Cassandra C , Ackerman Steven J , Mahrer Susan , Tolbert Marshall , Dvorak Ann M , Baumann Michael A
Published: January 2018

Study of eosinophil growth and differentiation has been hampered by the difficulty of obtaining adequate numbers of highly purified eosinophil progenitors or mature eosinophils for analysis. The AML14 myeloid leukemia cell line has the unusual ability to exhibit eosinophilic differentiation in response to stimulation by combinations of the eosinophil-active cytokines interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5. We now demonstrate that AML14 cells can be stimulated by a combination of these cytokines to produce mRNA encoding all the eosinophil granule proteins, including major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and the Charcot-Leyden crystal (CLC) protein (eosinophil lysophospholipase). The production of the mature proteins was demonstrated by Western blotting, and ultrastructural analysis demonstrated the presence of immature secondary granules in cells that had been induced to differentiate to eosinophils. These findings demonstrate the utility of the AML14 cell line as a model for the study of cytokine induction of eosinophil growth and differentiation. J. Leukoc. Biol. 56: 74–79; 1994.
Role of diradylglycerol formation in H2O2 and lactoferrin release in adherent human polymorphonuclear leukocytes
Authors: Nakamura Tadashi , Suchard Suzanne J , Abe Akira , Shayman James A , Boxer Laurence A
Published: January 2018

Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in the release of H2O2 in response to fMLP. Previously, we demonstrated that H2O2 release in adherent PMNs coincides with the exocytosis of lactoferrin-containing specific granules and activation of phospholipase D (PLD). We also found that chelation of intracellular calcium blocked both lactoferrin and H2O2 release in stimulated PMNs in spite of the fact that adhesion and spreading remained normal. Since diradylglycerol (DRG) formation has been implicated in PMN secretion and oxidant release, we determined the effect of intracellular calcium chelation on PLD activation and DRG formation to ascertain whether DRG formation was coupled to lactoferrin and H2O2 release. We observed that chelation of intracellular calcium with bis-(O-aminophenoxy)-ethanol-N,N;N'- tetraacetic add (BAPTA) prevented PLD activation as monitored by inhibition of phosphatidylethanol formation. Formation of DRG derived from phosphatidic acid (PA) was also inhibited in the presence of BAPTA. Following the addition of the calcium ionophore ionomycin to the BAPTA-treated PMNs, lactoferrin and H2O2 release was coincident with the onset of DRG formation. Also the addition of sn-1,2-didecanoylglycerol to the BAPTA-treated PMNs stimulated them to release H2O2. Our studies support the hypothesis that DRG derived from PLD activation is required for degranulation of specific granules and associated H2O2 release from adherent PMNs. J. Leukoc. Biol. 56: 105–109; 1994.
Use of suicide genes in gene therapy
Authors: Tiberghien Pierre
Published: January 2018

Gene therapy encompasses a broad range of treatment modalities that may eventually be applied to a variety of genetic as well as acquired diseases. In addition to compensating for a defective gene—and enhancing a cellular function—gene transfer can allow for a conditional negative selection of target cells. Indeed, the transfer of a gene encoding a susceptibility factor can make a cell specifically sensitive to a drug. After genomic integration, such a potentially destructive gene is endogenously expressed and has therefore been coined a suicide gene. This review describes current experimental approaches and prospects for using suicide genes for the treatment of human diseases. J. Leukoc. Biol. 56: 203–209; 1994.
Sulfated polyanions prevent HIV infection of lymphocytes by disruption of the CD4-gp120 interaction, but do not inhibit monocyte infection
Authors: Lynch G , Low L , Li S , Sloane A , Adams S , Parish C , Kemp B , Cunningham A L
Published: January 2018

Sulfated polyanions (SPs) bind variably to lymphocyte-expressed CD4 and inhibit binding of monoclonal antibodies to the first two domains of CD4. To further define this interaction, soluble recombinant CD4 (sCD4; four extracellular domains), its truncated amino-terminal two-domain derivative, and three linear peptide analogues spanning residues 6–60 (6–24, 20–40, 41–60) in the first domain were investigated for SP binding. Dextran sulfate (DXS) (500 kDa), polyvinyl sulfate, fucoidan, and carrageenan-kappa, each immobilized on carboxymethyl cellulose fibers, bound strongly to both the two-domain and four-domain recombinant CD4 molecules (similar to that observed with native CD4), whereas dextran sulfate (5 kDa), chondroitin 6-sulfate, and pentosan sulfate bound relatively poorly. No peptide binding to SPs was observed. Recombinant gp120 bound poorly (<10%) to all of the immobilized polyanions, except pentosan sulfate (17%), for which some binding was noted. Binding of radiolabeled V3 loop peptide to SPs was slightly greater, with 20-30% binding to polyvinyl sulfate, dextran sulfate (500 kDa), and pentosan sulfate. Competitive binding studies demonstrated the predominance of sCD4 rather than rgp120 binding to SPs and supported previous data demonstrating a binding site for DXS (500 kDa) on the first domain of CD4 adjacent to the gp120 binding site and recognized by OKT4C and E monoclonal antibodies. Hence disruption of the CD4-gp120 interaction is probably responsible for most of the observed antiviral activity of SPs toward HIV infection of lymphocytes. However, HIV infection and gp120 binding to monocytes was unaffected by SPs, probably because SPs were unable to block the CD4-gp 120 interaction in monocytes. J. Leukoc. Biol. 56: 266–272; 1994.
Expression of CD11/CD18 and ICAM-1 on monocytes and lymphocytes of HIV-1-infected individuals
Authors: Stent Gianna , Cameron Paul U , Crowe Suzanne M
Published: January 2018

The role of the leukocyte integrins in the infection of cells by HIV-1 or in the progression of AIDS-related disease is of continuing interest. Using a duallabeling flow cytometric method, we determined the level of expression of the leukocyte integrins and intercellular adhesion molecule 1 (ICAM-1) on monocytes and lymphocytes from HIV-1-infected and uninfected individuals. The mean fluorescence of CD18 on lymphocytes and CD11c on monocytes of HIV-1-infected subjects was significantly higher than for the control group (P=.008 and.014, respectively). There was a trend toward higher fluorescence of CD11a on lymphocytes from HIV-1-infected subjects compared with cells from the control group (P=.089). Integrin expression on lymphocytes or monocytes from HIV-infected individuals did not correlate with their CD4 lymphocyte number. However, the mean fluorescence associated with ICAM-1 on monocytes and CD11a and CD18 expression on lymphocytes was related to clinical stage of disease. J. Leukoc. Biol. 56: 304–309; 1994.
Role of endogenous interferon-β in the restriction of HIV replication in human monocyte/macrophages
Authors: Gessani Sandra , Puddu Patrizia , Varano Barbara , Borghi Paola , Conti Lucia , Fantuzzi Laura , Gherardi Giovanni , Belardelli Filippo
Published: January 2018

In vitro culture of human monocytes results in a time-dependent differentiation into macrophages. Monocyte/macrophages were infected with HIV-1 Ba-L at different times after isolation and subsequent culture. When 7-day macrophages were infected in the presence of antibodies to interferon-β (IFN-β), a significant increase in HIV-1 p24 release was observed. This effect was not detected in 1-day monocytes. Treatment of 7-day cultured macrophages with HIV-1 rgp120 resulted in resistance to vesicular stomatitis virus infection. This rgp120-induced antiviral state was neutralized in the presence of antibodies to IFN-β. The overall results indicate that the infection of monocyte/macrophages with HIV-1 results in the induction of IFN-β, which, in turn, inhibits HIV-1 expression in macrophages. The finding that HIV-1 itself (possibly through its gp120) can induce a potent antiviral factor (IFN-β) in macrophages underlines the complex physiological function of these cells in maintaining normal homeostasis in vivo in response to virus infection. J. Leukoc. Biol. 56: 358–361; 1994.
In vitro activity of inhibitors of late stages of the replication of HIV in chronically infected macrophages
Authors: Perno Carlo-Federico , Aquaro Stefano , Rosenwirth Brigitte , Balestra Emanuela , Peichl Peter , Billich Andreas , Villani Nicoletta , Calio Raffaele
Published: January 2018

Because of the importance of macrophages in the pathogenesis of the disease caused by HIV, we investigated the efficacy of various anti-HIV drugs in human primary macrophages acutely or chronically infected by this virus. The results obtained for acutely infected macrophages show that dideoxynucleosides (AZT, ddI, and ddC), interferon-α and -γ, mismatched double-stranded RNA, Tat inhibitor, phosphorothioate antisense, and inhibitors of HIV protease, all significantly inhibit virus replication at concentrations far below those toxic for the cells. However, in macrophages in which proviral DNA is already integrated (chronically infected macrophages), only the three inhibitors of HIV protease induced significant virus inhibition at concentrations 100 or more times higher than those effective in acutely infected macrophages. Treatment of macrophages with macrophage colony-stimulating factor does not affect the anti-HIV efficacy of protease inhibitors. These results suggest that therapeutic strategies with activity for macrophages, including inhibitors of HIV protease, are worth pursuing in patients with HIV infection. J. Leukoc. Biol. 56: 381–386; 1994.
Presentation of endogenous tumor antigens to CD4+ T lymphocytes by murine melanoma cells transfected with major histocompatibility complex class II genes
Authors: Chen Peter W , Ullrich Stephen E , Ananthaswamy Honnavara N
Published: January 2018

In a previous study, we demonstrated that K1735 transfectants expressing either Kk or Ak antigens alone produced tumors in syngeneic mice, whereas transfectants that expressed both antigens were rejected. In this study, we investigated whether K1735 transfectants expressing Ak molecules can present endogenous tumor antigens to CD4+ T lymphocytes in the absence of normal accessory cells. Our results indicate that K1735 transfectants expressing Kk and Ak molecules presented antigen to both CD4+ and CD8+ T lymphocytes, whereas K1735 transfectants expressing only the Ak or the Kk antigen preferentially stimulated either CD4+ or CD8+ T cells. Analogous to endogenous antigens, K1735 transfectants expressing Ak molecules also presented exogenous hen egg lysozyme (HEL) to HEL-specific 3A9 hybridomas in the absence of normal accessory cells. These results demonstrate that K1735 murine melanoma cells expressing Ak molecules can function as antigen-presenting cells and that the generation of an effective antitumor immune response by K1735 melanoma cells expressing Kk and Ak antigens is due to their ability to present endogenous tumor antigens to both helper and cytotoxic T cells. J. Leukoc. Biol. 56: 469–474; 1994.
Characterization of the eosinophil granule proteins recognized by the activation-specific antibody EG2
Authors: Rosenberg Helene F , Lee Tiffany H
Published: January 2018

Monoclonal antibodies EG1 and EG2 have the unique ability to distinguish the storage from the secreted forms of the eosinophil cationic protein (ECP). EG2 has been used extensively as a marker for activated, secreting eosinophils, despite the fact that no biochemical differences between the storage and secreted forms of ECP have been identified. We have determined that the activation-specific EG2 detects only one of three distinct glycosylated forms of ECP (18 kDa); in contrast, both EG1 and polyclonal anti-ECP antiserum can detect three glycosylated forms of this protein (18, 20, and 22 kDa). We have also determined that EG2 detects fully deglycosylated ECP as well as fully deglycosylated eosinophil-derived neurotoxin. Our results indicate that activation-specific EG2 recognizes a polypeptide epitope that is masked in the higher-molecular-weight, more heavily glycosylated forms of ECP. These findings suggest that deglycosylation may occur in conjunction with activation and secretion; alternatively, the 18-kDa form of ECP may be present in the storage granule of resting eosinophils but may remain undetected in an inaccessible location or conformation. J. Leukoc. Biol. 56: 502–506; 1994.