bluuebunny/crossref_metadata_2025 huggingface dataset:
NO is not sufficient to explain maximal cytotoxicity of tumoricidal macrophages against an NO-sensitive cell line
Authors: Mateo Romeo B , Reichner Jonathan S , Albina Jorge E
Published: January 2018

Nitric oxide (NO) is a macrophage cytotoxic effector. Results presented here, however, demonstrate that NO does not fully explain macrophage cytotoxicity against NO-sensitive cells because (1) inhibition of NO production by activated macrophages reduces, not eliminates, cytotoxicity; (2) NO produced chemically in amounts equimolar to those released from macrophages fails to lyse P815 cells; and (3) macrophages isolated from wounds 10 days after injury generate NO just as tumoricidal activated macrophages but are not tumor cytotoxic. The noncytotoxic nature of these wound-derived macrophages is not explained by the release of inactive forms of NO, because they suppress lymphocyte proliferation in an NO-dependent manner, nor by the production of cytoprotective molecules, because their addition to activated macrophage–tumor cell cocultures does not quench cytotoxicity. Interestingly, cytotoxicity can be aroused in day 10 wound-derived macrophages by culture with lipopolysaccharide, and macrophages harvested earlier in the development of the wound are cytotoxic. By generating NO but not killing an NO-sensitive cell, day 10 wound-derived macrophages demonstrate that NO production is not sufficient to account for the killing of an NO-sensitive tumor by macrophages.
Role of interferon-γ in immunity to herpes simplex virus
Authors: Yu Zhiya , Manickan Elanchezhiyan , Rouse Barry T
Published: January 2018

Removal of herpes simplex virus (HSV)-infected cells from peripheral sites such as the skin is mainly an activity of T cells, particularly the CD4+ T subset. Such cells orchestrate an inflammatory response with interferon-γ (IFN-γ) appearing to play the essential role in viral clearance. In accordance with this hypothesis, we show that infection of BALB/c background mice expressing the knockout phenotype for IFN-γ (GKO mice) are significantly more susceptible to the development of cutaneous zosteriform lesions than are wild-type. However, following HSV immunization, GKO mice become solidly immune to the development of zosteriform lesions. In addition, the transfer of T cells from immune GKO mice to nude mice recipients renders them resistant to zosteriform lesions. Our results are discussed in terms of the major and compensatory mechanisms available to the body to effect immunity to viral infections.
C-C chemokine-induced eosinophil chemotaxis during allergic airway inflammation
Authors: Lukacs Nicholas W , Standiford Theodore J , Chensue Stephen W , Kunkel Robin G , Stricter Robert M , Kunkel Steven L
Published: January 2018

The production of eosinophil-specific chemotactic factors during allergic airway responses may be a pivotal event resulting in eosinophil accumulation, activation, and airway damage. Recent studies have identified specific chemokines that may play crucial roles in recruitment of eosinophils to the site of allergic reactions. In this study we have utilized an established model of schistosome egg antigen (SEA)-mediated allergic responses to examine the role of specific C-C chemokines [macrophage inflammatory protein-1α (MIP-1α), RANTES, and monocyte chemoattractant protein-1 (MCP-1)] in eosinophil recruitment. We have previously identified a role for MIP-1α in eosinophil accumulation in the lung and airway during allergic airway inflammation. We extend those studies using in vitro eosinophil chemotaxis to establish that both MIP-1α and RANTES are potent eosinophil chemotactic factors in lungs during allergic airway responses. Morphometric analysis demonstrated a peribronchial accumulation of eosinophils within the lungs beginning at 8 h, peaking at 24 h, and plateauing at 48–96 h after allergen (SEA) challenge. Utilizing whole-lung homogenates from allergen-challenged mice, in vitro eosinophil chemotactic assays demonstrated significant increases in eosinophil chemotactic activity with 8-h lung homogenates and peak activity with samples from 24-h lung homogenates. These data correlated with the morphometric analysis of peribronchial eosinophil accumulation in situ. When lung homogenates from allergen-challenged mice were preincubated in vitro with antibodies specific for MIP-1α, RANTES, or MCP-1, a significant reduction in eosinophil chemotaxis was observed with only MIP-1α and RANTES neutralization. Altogether, these studies indicate that RANTES and MIP-1α are major eosinophil chemotactic factors produced during allergic airway responses.
Surface thiols of human lymphocytes and their changes after in vitro and in vivo activation
Authors: Lawrence David A , Song Renjie , Webert Peter
Published: January 2018

Cellular thiols have long been known to play a role in cell activation and proliferation; however, the differential expression of surface thiols on the lymphoid subsets had not been described. Neither was it known whether alteration of surface thiols occurs after exposure to mitogens or infectious agents. Herein, an impermeant thiol-specific fluorescent probe was employed for flow cytometric analysis of surface thiols. Quantification of surface thiols on resting lymphocytes revealed that some subsets expressed different concentrations of surface thiols (CD19+ > CD8+ > CD4+). Furthermore, surface thiols increased on all subsets by 8 h after mitogenic activation. This increase was blocked by cycloheximide or monensin but not by actinomycin D or inhibition of glutathione synthesis by buthionine sulfoximine. In addition, bacitracin, an inhibitor of protein disulfide isomerase, inhibited the increase in surface thiols and DNA synthesis. Lymphocytes from HIV-infected individuals displayed increased surface thiols on CD19+ and CD4+ cells but not CD8+ cells. Although cellular thiols in general have been believed to play a role in protection against oxidants, signaling associated with cell growth, and apoptosis, there is now evidence that changes in exofacial thiols appear to be involved in some of these critical cell reactivities. Thus, quantitative and possibly qualitative differences in surface thiols correlate with membrane differences between lymphoid subsets and with their differential sensitivities to oxidative stress, which suggests that the mechanisms by which surface thiols are maintained and modified after activation are important cellular functions that need to be further evaluated.
Microbial killing by human neutrophil cytokineplasts: similar suppressive effects of reversible and irreversible inhibitors of nitric oxide synthase
Authors: Malawista Stephen E , Montgomery Ruth R , van Blaricom Gretchen
Published: January 2018

Employing anucleate, granule-poor, motile fragments from human blood neutrophils (cytokineplasts; CKP), we previously provided evidence for a new staphylococcal killing pathway for human neutrophils involving reactive nitrogen intermediates: the NO synthase inhibitor Nω-monomethyl-L-argmine (NMMA), an analogue of L-arginine (L-Arg), substantially decreased the killing capacity of CKP for Staphylococcus aureus (Staph), an effect reversible by excess L-Arg but not D-Arg. We have extended these findings to two irreversible NO synthase inhibitors: the first, N-iminoethyl-L-ornithme (L-N10), is an L-Arg analogue; the other, diphenyleneiodonium (DPI), is not. After 60 min of incubation with bacteria, despite having taken up somewhat fewer staphylococci than did controls, cytoplasts treated with NO synthase inhibitors had many more live, CKP-associated bacteria: for NMMA, 6.9 times more (40.0% of the inoculum vs. 5.8%; n = 8, P = 0.003); for L-N10, 3.6 times more (25.5 vs. 7%; n = 4, P = 0.004); for DPI, 5.8 times more (37.4 vs. 6.4%; n = 7, P = 0.002). Results were similar after only 20 min of incubation. In two experiments in which the Gram-negative bacterium, Serratia marcescens, was employed instead of Staph, the results were again similar. In contrast, killing of either bacterium by intact neutrophils (PMN) was not inhibited by NMMA, by L-N10, or by DPI, a failure most likely attributable to their granule content. The irreversible inhibitors of NO synthase will be especially useful in analyzing particular effects on CKP employed in multicellular systems.
Maleylated-BSA induces hydrolysis of PIP2, fluxes of Ca2+, NF-κB binding, and transcription of the TNF-α gene in murine macrophages
Authors: Misra Uma K , Shackelford Rodney E , Florine-Casteel Kathryn , Thai Sheau-Fung , Alford Paul B , Pizzo Salvatore V , Adams Dolph O
Published: January 2018

The Interaction of altered lipids or proteins with the several scavenger receptors (SR) on macrophages can lead to disparate results in both gene expression and cell function. However, the molecular bases of signaling induced by SR ligation have remained obscure. Here we report that maleylated-bovine serum albumin (maleyl-BSA) binds a low-affinity SR, initiating POP2 hydrolysis, [Ca2+J spikes, phospholipase A2 (PLA2) activation, nuclear factor-κB (NF-κB) binding to its cognate nucleotide and tumor necrosis factor α (TNF-α) gene transcription. We recently reported that oxidized low-density lipoprotein (ox-LDL), which binds another macrophage SR, induced pertussis-toxin-sensitive hydrolysis of PIP2 and elevations in [Ca2+]i [J. Biol. Chem. 270, 3475–3478, 1995]. By contrast, maleyl-BSA-initiated events were not pertussis toxin-sensitive and produced less [Ca2+]i spiking than ox-LDL. Furthermore, maleyl-BSA led to binding of NF-κB to its cognate nucleotide and TNF-α gene transcription, whereas ox-LDL suppressed these events. Collectively, this data suggests that maleyl-BSA and ox-LDL bind to distinct SR on murine macrophages, initiate distinct signal transduction pathways, and produce different functional effects.
Monoclonal antibodies against the human mannose receptor as a specific marker in flow cytometry and immunohistochemistry for macrophages
Authors: Noorman Femke , Braat Ellen A M , Barrett-Bergshoeff Marrie , Barbé Ellis , van Leeuwen Alexander , Lindeman Jan , Rijken Dingeman C
Published: January 2018

Recently we developed mouse monoclonal antibodies (mAb) against the isolated human 175-kDa mannose receptor. In the present study we tested whether these mAb are suitable for the detection of the mannose receptor on cultured macrophages using flow cytometry and on cells in human tissues using immunohistochemistry. Human monocytes did not react with the mAb in flow cytometry. Mannose receptor expression became detectable on monocytes cultured for 3 days (macrophages), and was maximal from 4 days onward. The mannose receptor was up-regulated on dexamethasone-treated (immunosuppressed) macrophages, and down-regulated on lipopolysaccharidetreated (activated) macrophages. Immunohistochemically the staining pattern of our mAb was compared with the marker of monocytes/macrophages KP1. In a bone marrow smear, only macrophages were stained with our mAb, whereas all myeloid cells were stained with KP1. In the thymus and lymph node, mannose receptor-positive branched cells (macrophages and dendritic cells) were detected in connective tissue, thymus cortex (not medulla), and in the T cell area (not the B cell area) of lymph nodes, whereas KP1 stained branched cells in all areas. It was concluded that the mAb are useful tools in flow cytometry and immunohistochemistry for the specific detection of cells expressing mannose receptor.
Mig and IP-10: CXC chemokines that target lymphocytes
Authors: Farber Joshua M
Published: January 2018

Mig and IP-10 are related members of the CXC subfamily of the chemokine family of cytokines. The murine Mig (MuMig), human IP-10, and the mouse homologue of IP-10, Crg-2, were all identified due to the dramatic inductions of their genes in monocytic cells treated with interferon-γ (IFN-γ). Studies using recombinant (r) human proteins show that, unlike most other CXC chemokines, rHuMig and rIP-10 have no activity on neutrophils but appear to target lymphocytes specifically. rHuMig and rIP-10 are active as chemotactic factors for stimulated, but not for resting, T cells. Studies done in vitro and in vivo have shown that rHuMig and rIP-10 share additional activities, including inhibition of neovascularization, inhibition of hematopoietic progenitor cells, and anti-tumor effects. rHuMig and rIP-10 show reciprocal desensitization on activated T cells and have been demonstrated to share a receptor, CXCR3. The genes for both MuMig and Crg-2 are highly expressed in multiple tissues during experimental viral and protozoan infections in mice, but their patterns of expression differ. This suggests that the Migs and IP-10/Crg-2 may play roles in host defense and that, despite their similar activities assayed in vitro, Mig and IP-10/Crg-2 may serve non-redundant functions in vivo.
Expression of endothelial adhesion molecules and recruitment of neutrophils after traumatic brain injury in rats
Authors: Carlos T M , Clark R S B , Franicola-Higgins D , Schiding J K , Kochanek P M
Published: January 2018

Traumatic brain injury (TBI) is often accompanied by an acute inflammatory reaction mediated initially by neutrophils. Adhesion molecules expressed on vascular endothelium are requisite elements during recruitment of leukocytes at sites of inflammation. In a rat model of TBI the induction and persistent expression of E-selectin (CD62E) on cerebrovascular endothelium ipsilateral, but not contralateral, to the site of contusion was demonstrated (P < 0.05 at 4 and 48 h posttrauma). In addition, these studies confirmed up-regulation and prolonged expression of ICAM-1 (CD54) on endothelium in the traumatized hemisphere (P < 0.05 at 4, 24, 48, and 72 h posttrauma). It is of interest that increased expression of CD54 was noted on blood vessels in the contralateral, non-traumatized hemisphere 48 h posttrauma. Expression of a third endothelial adhesion molecule, PECAM-1 (CD31), was unchanged following trauma. Administration of a murine monoclonal antibody (TM-8) that inhibits the adhesive function of CD54 blocked a significant portion (37.9%) of neutrophil recruitment 24 h posttrauma (P = 0.04). Employing immunocytochemistry and a monoclonal antibody specific for rat neutrophils (RP-3), peak infiltration of neutrophils was shown to occur 48 h after trauma. In contrast to emigration of neutrophils from blood vessels within the contusion, however, entry of neutrophils occurred from the surrounding leptomeninges and choroidal vessels. These studies demonstrate the relevance of CD54 (ICAM-1) in recruitment of neutrophils following TBI. However, the majority of neutrophil influx relies on endothelial adhesion molecules other than CD54. Because emigration of neutrophils was shown to occur predominantly from vessels within the leptomeninges and choroid plexus, intrathecal delivery of agents that inhibit the adhesive interactions between neutrophils, endothelial CD54, and other endothelial adhesion molecules to be defined may offer a novel form of therapy to prevent the acute inflammatory response that follows TBI.
Establishment of an IL-12-responsive T cell clone: its characterization and utilization in the quantitation of IL-12 activity
Authors: Maruo Seiji , Ahn Hyun-Jong , Yu Wen-Gong , Tomura Michio , Wysocka Maria , Yamamoto Norihiko , Kobayashi Michiko , Hamaoka Toshiyuki , Trinchieri Giorgio , Fuijiwara Hiromi
Published: January 2018

We previously demonstrated that proliferation of terminally differentiated Thl clones depends primarily on an interleukin-12 (IL-12)-paracrme mechanism mediated by their interactions with antigen-presenting cells (APC) rather than on an IL-2-autocrine mechanism. Such a Thl clone (4-86, C57BL/6 origin) was cultured with recombinant IL-12 (rIL-12) in the absence of either antigen or APC. Some cells survived for several passages of culture with only rIL-12, and by limiting dilution, several clones highly reactive to rIL-12 alone were obtained. One of these clones, designated 2D6, was found to proliferate strongly in response to less than 1 pg/mL of rIL-12. This clone exhibited the following surface phenotypes: CD3+, T cell receptor (TCR) αβ+, Vβ11+, NK-1.1-; CD4- CD8-; LFA-1+, ICAM-1+; and CD28+, CD80+, CD86+, CTLA-4-. In accordance with high responsiveness to IL-12, 2D6 cells were also found to express IL-12 receptor (IL-12R) as detected by incubation with rIL-12 and then staining with anti-IL-12 monoclonal antibody (mAb). Stimulation of 2D6 with rIL-12 resulted in the expression of interferon-γ (IFN-γ) and IL-10 mRNAs and production of these cytokines. The 2D6 clone responded to IL-2 (vigorously), IL-7 (moderately), and IL-4 (mildly) in addition to IL-12. However, the Ab capture assay using anti-IL-12 mAb enabled us to quantify IL-12-specific activity contained in a given sample. Thus, this study describes the unique features of the IL-12-responsive T cell clone and demonstrates the utilization of this clone in the quantitation of a specific IL-12 activity.
Dendritic cell maturation is required for initiation of the immune response
Authors: Rescigno Maria , Winzler Claudia , Delia Domenico , Mutini Carmela , Lutz Manfred , Ricciardi-Castagnoli Paola
Published: January 2018

Dendritic cells (DC) are professional antigen-presenting cells (APC) whose primary function is to capture, process, and present antigens to unprimed T cells [1, 2]. They comprise a system of leukocytes widely distributed in all tissues, especially in those that provide an environmental interface (skin and mucosal surfaces) and in lymphoid organs [1].
LPS-induced blood neutrophilia is inhibited by α1-adrenoceptor antagonists: a role for catecholamines
Authors: Altenburg Sonia P , Martins Marco A , Silva Adriana R , Cordeiro Renato S B , Castro-Faria-Neto Hugo C
Published: January 2018

A role for catecholamines in the regulation of the blood neutrophilia induced by intravenous (i.v.) injection of lipopolysaccharide (LPS; 250 μg/kg) was examined in Wistar rats by means of surgical adrenalectomy or pretreatment with adrenergic and dopaminergic antagonists into naive animals. Treatment of animals with a single dose (250 μg/kg) of LPS caused a dramatic increase in the number of circulating neutrophils concomitant with a decrease in the number of these cells in the bone marrow. These effects were partially reversed when catecholamine stores were depleted with reserpine. It was found that neither adrenalectomy nor pretreatment with the dopaminergic antagonists, chlorpromazine and pimozide, affected the changes in neutrophil counts induced by LPS. The injection of the α1/α2-adrenoceptor antagonist, phentolamine, and the selective α1-adrenoceptor antagonist, prazosin, significantly decreased blood neutrophilia induced by LPS. However, neither the selective (α2-adrenoceptor antagonist, yohimbine, nor the β-adrenoceptor antagonist, propranolol, had any effect on LPS response. Taken together, these findings support the hypothesis that the catecholamine norepinephrine plays a role in the regulation of the LPS-induced neutrophilia through activation of α1-adrenoceptors. J.
Targeting antiviral nucleotide analogues to macrophages
Authors: Magnani Mauro , Rossi Luigia , Fraternale Alessandra , Casabianca Anna , Brandi Giorgio , Benatti Umberto , De Flora Antonio
Published: January 2018

Macrophages are important target cells for human immunodeficiency virus type 1 (HIV-1) infection. We have developed a drug targeting system for the selective delivery of phosphorylated nucleoside analogues to these phagocytosing cells. This system is based on the possibility of encapsulating the phosphorylated drugs into autologous erythrocytes and on the subsequent selective modification of their membranes to promote macrophage recognition and phagocytosis. Targeted delivery of phosphorylated nucleoside analogues to human, feline, and murine macrophages inhibits the infectivity of HIV-1, feline immunodeficiency virus, and LP-BM5 viruses more efficiently than the administration of the corresponding nucleoside analogues. In vivo administration of 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP) encapsulated into autologous erythrocytes to LP-BM5-infected mice was found to reduce infectivity and disease progression. Furthermore, the simultaneous administration of AZT or ddC produced additive antiviral effects. The possibility of using red cells as drug targeting systems was useful for the design, synthesis, and delivery of new antiviral nucleoside analogues. As a prototype of these new drugs, di-(thymidine-3′-azido-2′,3′-dideoxy-D-riboside)-5′-5′-p1-p2-pyrophosphate (AZTp2AZT) was prepared. Although this drug in solution has the same antiviral activity as AZT, when administered encapsulated into erythrocytes it was several times more efficient in inhibiting the infectivity of human, feline, and murine immunodeficiency viruses. Thus, the availability of a drug targeting system for the selective delivery of antivirals to macrophages offers an additional possibility for the development of new drugs and of new combination antiviral therapies.
Natural killer cells: Endothelial interactions, migration, and target cell recognition
Authors: Timonen Tuomo
Published: January 2018

Natural killer (NK) cells form a unique third group of lymphocytes that differs from T and B cells in surface phenotype, target recognition, and function. By producing cytokines and exerting cytotoxicity, NK cells participate in the resistance against microbial infections and malignant disease. The research on the molecular mechanisms of migration and target cell recognition by NK cells has recently developed rapidly. NK cells express a number of adhesion molecules common to hematopoietic lineage, bind to endothelium, extravasate, and respond to chemotactic stimuli, much resembling T cells in those respects. However, NK cells are probably capable of transmigration and infiltration merely through activation by cytokines and chemokines, as opposed to the requirement of antigen presentation in the initial activation of T cells. Target cell recognition and ensuing cytotoxicity of NK cells is a sum effect of a delicate balance between the effects of inhibitory and activating NK cell receptors. NK cells express several well-defined MHC I-recognizing receptors that inactivate their functions. In pathological alterations of MHC I expression, the inhibitory receptors do not engage and thus permit the lysis of the target cell. The receptors that trigger the cytolytic machinery of NK cells are less well known. Some candidate triggering receptors have been identified and it seems that NK cell triggering is mediated by multiple receptors, as is the inhibition of cytotoxicity. For example, NK cells clearly detect target cell-bound antibodies and thus mediate antibody-dependent cytotoxicity. They may also detect carbohydrate moieties, normal but pathologically distributed adhesion molecules, as well as ligands for a number of co-stimulatory receptors.
Agonist-specific tyrosine phosphorylation of Cbl in human neutrophils
Authors: Naccache Paul H , Gilbert Caroline , Barabé Frédéric , Al-Shami Amin , Mahana Wahib , Bourgoin Sylvain G
Published: January 2018

The effects of soluble and particulate agonists on the tyrosine phosphorylation levels of the proto-oncogene Cbl in human neutrophils were examined. Experimental conditions allowing the maintenance of Cbl as well as of its tyrosine phosphorylation status were first established. Their use allowed us to observe that Cbl was tyrosine phosphorylated in response to some (FcγRII ligation, opsonized bacteria and zymosan, granulocyte-macrophage colony-stimulating factor, monosodium urate, and calcium pyrophosphate microcrystals), but not all (fMet-Leu-Phe, interleukin-8) neutrophil agonists. Cbl was also shown to account for a varying proportion of the 120-kDa phosphoprotein(s) observed in response to the above stimuli. These data establish that Cbl is present in human neutrophils and that its level of tyrosine phosphorylation is modulated by some of these cells' agonists, and in particular by phagocytic particles. Furthermore, the signaling pathways activated by chemotactic factors and the other neutrophil stimuli tested in this investigation diverge at or downstream from the tyrosine phosphorylation of Cbl.
Inhibition of LPS-mediated activation in rat Kupffer cells by N-acetylcysteine occurs subsequent to NF-κB translocation and requires protein synthesis
Authors: Fox Eben S , Leingang Karen A
Published: January 2018

Activation of the resident hepatic macrophage population, Kupffer cells, leads to production of mediators that initiate, potentiate, and modulate hepatic injury. Recent studies have shown that activation of the pluripotent transcription factor nuclear factor-κB (NF-κB) is an important step in the induction of inflammatory cytokines, chemokines, growth factors, cell adhesion proteins, and cytokine receptors, thus efforts have been focused to modulate its activity. A common observation in diverse experimental systems is that oxidant stress activates NF-κB and antioxidant drugs prevent activation and subsequent inflammatory gene transcription. However, we have recently shown that the inhibitory effect of N-acetylcysteine (NAC) is independent of its role as a substrate of glutathione synthesis and NAC can inhibit Kupffer cell activation at points beyond the initiation of activation. The goal of this study was to characterize the mechanism for NAC-mediated inhibition of Kupffer cell activation. We show for the first time that this process requires a cellular synthetic response to prevent both NF-κB and tumor necrosis factor α (TNF-α) mRNA activation. Furthermore, NAC-mediated inhibition occurs after degradation of IκB-α and nuclear translocation of NF-κB. These data suggest that inhibition of Kupffer cell activation by NAC is a nuclear event and offers a potential approach to modulate Kupffer cell activation during hepatic injury.
Mechanism of inhibition of LPS-induced IL-12p40 production by IL-10 and TGF-β in ANA-1 cells
Authors: Du Caigan , Sriram Subramaniam
Published: January 2018

IL-12, a macrophage-derived proinflammatory cytokine, consists of two polypeptide subunits (p40 and p35) encoded by two separate genes. The p35 subunit is constitutively expressed, whereas the p40 subunit is induced after activation. The bioactive interleukin-12 (IL-12; p70) influences the development of Th1 responses and is a potent activator of natural killer (NK) and T cells. In contrast to IL-12, transforming growth factor β (TGF-β) and IL-10 inhibit production of proinflammatory cytokines, including IL-12, and attenuate Th1-mediated immune response. We have examined the molecular mechanisms by which TGF-β and IL-10 inhibit production of the IL-12p40 subunit in LPS-stimulated murine macrophage cell line. We show that both IL-10 and TGF-β suppress IL-12p40 production by inhibiting the transcription of IL-12p40 gene. At equal concentrations, IL-10 was more potent than TGF-β in inhibiting IL-12p40 gene transcription. TGF-β also reduces the stability of IL-12p40 mRNA, accounting thereby to an additional mechanism of inhibition of IL-12 production. J. Leukoc. Biol. 64: 92–97; 1998.
Macrophage cytoplasmic vesicle pH gradients and vacuolar H+-ATPase activities relative to virus infection
Authors: Natale Valerie A I , McCullough Kenneth C
Published: January 2018

A number of viruses replicate in macrophages, some having an obligate requirement for a macrophage host. This raised the question concerning the role of the macrophage endosomal/lysosomal compartment during such infections. Both lysosomotropic weak bases, amantadine and chloroquine, which interfere with endosomal/lysosomal pH gradients, and the macrolide antibiotic bafilomycin A1, which interferes with vacuolar H+-ATPase, inhibited African swine fever (ASF) virus replication in porcine macrophages. This inhibition was reversible: replenishment of bafilomycin, but not amantadine or chloroquine, maintained the inhibition. The characteristics of the inhibition, and the capacity of virus to escape and re-commence replication, related to the capacity of each drug to interfere with the endosomal/lysosomal proton pump. These results demonstrate that the virus actually requires macrophage endosomal/lysosomal activity for its replication. Therein, vacuolar H+-ATPase activity is particularly critical for successful virus replication, which is interesting considering the importance of this for endosomal/lysosomal activity and macrophage function. J. Leukoc. Biol. 64: 302–310; 1998.
Interleukin-8 priming of human neutrophils is not associated with persistently altered calcium fluxes but is additive with lipopolysaccharide
Authors: van Dervort Alan L , Lam Charles , Culpepper Susan , Tuschil Andrea F , Wesley Robert A , Danner Robert L
Published: January 2018

Interleukin-8 (IL-8) priming was studied in neutrophils to examine its dependency on altered calcium fluxes and for similarity to lipopolysaccharide (LPS). IL-8 caused a rapid rise in [Ca2+]i that returned to baseline values by 20 min. Peak [Ca2+]i transients in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) were unaltered in IL-8-primed compared with unprimed cells. In comparison to LPS and tumor necrosis factor (TNF), IL-8 was a much weaker priming agent as measured by either O2– or H2O2 production. Despite their large disparity in potency, IL-8 and LPS priming were additive using fMLP, a receptor-dependent stimulator, and synergistic using the post-receptor, protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) to trigger the respiratory burst. In contrast, IL-8 and TNF priming were synergistic for fMLP (P = 0.05), but completely nonadditive when PMA was used as the neutrophil stimulant (P = 0.05 for subadditivity). Thus, lasting alterations in [Ca2+]i are not a necessary characteristic of IL-8-primed cells. IL-8 and LPS appear to prime by non-overlapping pathways, whereas IL-8 and TNF appear to share mechanisms distal to protein kinase C activation. IL-8 and LPS may independently contribute to neutrophil-mediated host defense or injury by priming through distinct pathways. J. Leukoc. Biol. 64: 511–518; 1998.
Expression of a structural domain of the β2 subunit essential for αMβ2 ligand recognition
Authors: Goodman Thomas G , DeGraaf Michael E , Fischer H David , Bajt Mary Lynn
Published: January 2018

The β2 leukocyte integrins comprise a group of closely related adhesion receptors that mediate critical events during normal and inflammatory immune responses. Central to the understanding of β2 integrin function is the basis of ligand recognition. Results from our laboratory and others indicate the presence of multiple ligand contact points in both the α and β subunit. As an approach to identify and characterize regulatory domains of the β2 subunit, we have generated two different subdomains of the β2 subunit for expression on the surface of mammalian cells through a phosphatidylinositol glycan anchor. The first subdomain contains the putative β2 MIDAS motif implicated in ligand binding [β2(LB)], whereas the second β2 subdomain contains the cysteine-rich region [β2(CR)]. Cells expressing αm and β2 constructs singly or cotransfected transiently in COS-7 cells were tested for the ability to bind to immobilized iC3b. Cells bearing the recombinant αMβ2(LB) were capable of adhering to iC3b in a manner similar to that observed with the complete αMβ2 heterodimer. In contrast, cells expressing αMβ2(CR) failed to adhere to immobilized iC3b. Moreover, cells bearing singly transfected α or β chains alone failed to adhere to immobilized iC3b. These results indicate that along with αm, the β2(LB) subdomain contains the sufficient components within the β2 subunit essential for ligand recognition. These findings support the hypothesis that the β2 subunit cooperates with site(s) within the αM subunit in a receptor/cation/ligand complex resulting in high-affinity ligand interaction. J. Leukoc. Biol. 64: 767–773; 1998.
Costimulatory molecules CD80 and CD86 in the rat; tissue distribution and expression by antigen-presenting cells
Authors: Damoiseaux Jan G M C , Yagita Hideo , Okumura Ko , van Breda Vriesman Peter J C
Published: January 2018

The CD28-CD80/CD86 costimulatory pathway provides a critical signal for T cell activation. Only recently rat CD80 and CD86 have been cloned and monoclonal antibodies have been generated. In this study we examined the expression of these molecules in lymphoid tissue and on purified subsets of antigen-presenting cells (APC). The target tissue of cyclosporin A-induced autoimmunity, i.e. the skin and tongue, were also examined for expression of CD80 and CD86. Whereas CD80 was hardly detected in the lymphoid tissues, CD86 was clearly expressed by non-lymphoid cells in the thymus, as well as in the secondary lymphoid organs. With respect to lymphoid cells, only germinal center B cells exhibited clear CD86 expression. Phenotypic analysis by flow cytometry revealed that only dendritic cells, both of thymic and splenic origin, expressed the full array of stimulatory molecules required for the proper activation of naive T cells. On development of cyclosporin A-induced autoimmunity, non-professional APC, i.e. epithelial cells, started to express MHC class II, but not the costimulatory ligands CD80 and CD86. However, CD86 staining was observed in the target tissue and was associated with Langerhans cells as well as infiltrating leukocytes. Altogether, our results show that also in the rat strong stimulatory capacity for primary immune responses is associated with the expression of the costimulatory ligands CD80 and CD86. As concluded from the in situ expression CD86 may be the predominant costimulatory ligand early in immune responses. J. Leukoc. Biol. 64: 803–809; 1998.
Lipopolysaccharide can block the potential of monocytes to differentiate into dendritic cells
Authors: Palucka Karolina A , Taquet Nicolas , Sanchez-Chapuis Francoise , Gluckman Jean Claude
Published: January 2018

We examined whether priming monocytes (MO) with lipopolysaccharide (LPS) influenced their further differentiation into either macrophages (MÕ) or dendritic cells (DC). LPS-primed MO differentiated into MÕ when cultured further with MÕ colony-stimulating factor (M-CSF) but, if cultured then with granulocyte/MÕ (GM)-CSF and IL-4 (interleukin-4), only about 30% of the cells differentiated into CD1a+CD14− DC and half became CD1a−CD14+ MÕ. Cytokines present during LPS priming could affect subsequent MO differentiation. Relative to priming with LPS alone, adding M-CSF to LPS did not modify differentiation of MO to MÕ in further culture with M-CSF, nor did it change the way of differentiation of MO into DC was altered if culture was later switched to GM-CSF/IL-4. Using GM-CSF/IL-4 plus LPS upon priming did not modify differentiation of MO to MÕ in further culture with M-CSF, as compared to priming with GM-CSF/IL-4 alone, but it counteracted the effect of LPS on the differentiation of MO to DC in further culture with GM-CSF/IL-4: about 75% of cells then became DC. Alternatively, despite activation by LPS, mature M-CSF-induced MÕ preserved the potential to differentiate into DC on subsequent culture with GM-CSF/IL-4. Thus, LPS, a bacterial product known to sustain maturation of MO/MÕ as well as of DC, may block the differentiation of MO into DC, except if signal triggering DC differentiation is delivered concomitantly, and modulate in this manner the induction of adaptive immune responses to infection. J. Leukoc. Biol. 65: 232–240; 1999.
TALL-1 is a novel member of the TNF family that is down-regulated by mitogens
Authors: Shu Hong-Bing , Hu Wen-Hui , Johnson Holly
Published: January 2018

Members of the tumor necrosis factor (TNF) family play important roles in modulation of immune responses. We describe the identification and cloning of a novel TNF family member that has been designated as TALL-1. TALL-1 is a 285-amino acid type II transmembrane protein. Its carboxy terminus shares ∼35% sequence identity with the recently identified APRIL and ∼20–25% with TNF, FasL, TRAIL, and lymphotoxin-α, suggesting that TALL-1 and APRIL belong to a subfamily of the TNF family of ligands. Northern blot analysis suggests that TALL-1 is expressed abundantly in peripheral blood leukocytes and weakly in spleen but is barely detectable in all other tissues examined. Reverse transcriptase-polymerase chain reaction analysis indicates that TALL-1 is specifically expressed in monocytes and macrophages but is undetectable in T and B lymphocytes. Furthermore, TALL-1 expression is dramatically down-regulated by phorbol myristate acetate/ionomycin. J. Leukoc. Biol. 65: 680–683; 1999.
Substance P primes the formation of hydrogen peroxide and nitric oxide in human neutrophils
Authors: Sterner-Kock Anja , Braun Ruedi K , van der Vliet Albert , Schrenzel Mark D , McDonald Ruth J , Kabbur Mahendra B , Vulliet P Richard , Hyde Dallas M
Published: January 2018

Substance P (SP), a neurotransmitter of the central and peripheral nervous system, has been implicated as a mediator of the pulmonary inflammatory response through its stimulatory effects on neutrophils. We investigated the role of SP in priming the production of reactive oxygen species by human neutrophils with the cytochrome c reduction assay and by flow cytometry using the intracellular oxidizable probe dichlorofluorescein. We also investigated SP-induced formation of nitrite and nitrate as an index of nitric oxide (NO) production. Our results indicate that SP primes two distinct pathways with respect to the induction of reactive oxygen species in the human neutrophil: the production of superoxide anion and hydrogen peroxide by the calmodulin-dependent NADPH oxidase, and the generation of NO by a constitutive NO synthase. Preincubation of neutrophils with inhibitors of calmodulin and NO synthase diminished the oxidative response in an additive fashion. These results give insight into distinct signal transduction pathways in the SP-primed neutrophil with respect to the formation of superoxide anion, hydrogen peroxide, and NO. J. Leukoc. Biol. 65: 834–840; 1999.
ETS transcription factors regulate an enhancer activity in the third intron of TNF-α
Authors: Tomaras Georgia D , Foster Dalton A , Burrer Christine M , Taffet Steven M
Published: January 2018

We describe an enhancer site in the third intron of tumor necrosis factor α (TNF-α). A reporter construct containing the 5′-flanking region of the mouse TNF-α gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-α to this construct resulted in an enhancement of CAT protein. This enhancement was eliminated if a conserved 20-bp sequence was removed from the intron or if a dominant-negative ets-binding factor was co-transfected with the reporter gene. Mutations of this site that destroyed potential ets transcription factor binding sites had reduced transcriptional activity. The major transcription factor that bound to the oligonucleotide was confirmed to be GABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activity was shown to be interferon-γ inducible. This may imply a role for ets transcription factors in the production of TNF-α. J. Leukoc. Biol. 66: 183–193; 1999.
Reperfusion-induced oxidative stress in diabetes: cellular and enzymatic sources
Authors: Salas A , Panés J , Elizalde J I , Granger D N , Piqué J M
Published: January 2018

Reactive oxygen metabolites (ROMs) have been implicated in the pathogenesis of the inflammatory response to ischemia/reperfusion (I/R), which is exacerbated in diabetes. This study revealed an increased (P < 0.01) ROMs production in mesenteric tissue (measured using the oxidant-sensitive fluorochrome dihydrorhodamine 123) after I/R in control and diabetic rats, with larger increments (P < 0.0001) observed in the latter group, that was associated with an increased inflammatory response measured by intravital microscopy. Either xanthine oxidase inhibition, superoxide scavenging, ICAM-1 immunoneutralization, or blockade of platelet-activating factor or leukotrienes effectively reduced leukocyte recruitment and ROMs production in control and diabetic rats. Moreover, neutrophils from diabetic rats showed an enhanced production of ROMs in vitro in basal and stimulated conditions. We conclude that the oxidative stress during reperfusion is markedly enhanced in diabetes and this appears to result from increased leukocyte recruitment and a higher capacity of diabetic leukocytes to generate ROMs in response to stimulation. J. Leukoc. Biol. 66: 59–66; 1999.
Decreased availability of GDP-l-fucose in a patient with LAD II with normal GDP-d-mannose dehydratase and FX protein activities
Authors: Körner C , Linnebank M , Koch H G , Harms E , von Figura K , Marquardt T
Published: January 2018

Leukocyte adhesion deficiency type II (LAD II) is caused by a disorder in the metabolism of GDP-l-fucose, which causes hypofucosylation of glycoconjugates. This study analyzes a newly identified LAD II patient who shows the same severe hypofucosylation of glycoconjugates as the other described patients. However, in vitro assays of cytosolic extracts from leukocytes and fibroblasts of the patient demonstrated a normal GDP-l-fucose biosynthesis from GDP-d-mannose. Analysis of the two enzymes involved in the pathway, GDP-d-mannose 4,6-dehydratase and FX protein, revealed normal numbers of transcripts without any detectable mutations within the coding regions of either gene. In contrast to previously published observations [Sturla et al. (1998) FEBS Lett. 429, 274–278], the major pathway of GDP-l-fucose synthesis can be normal in LAD II. J. Leukoc. Biol. 66: 95–98; 1999.
Myeloid dendritic cells
Authors: Steinman Ralph M , Inaba Kayo
Published: January 2018

There is considerable but as yet incomplete evidence for two developmental lineages of dendritic cells: a myeloid lineage shared with phagocytes and a lymphoid lineage shared with T cells. The two corresponding functional states, which may not require the existence of two formal lineages, are that myeloid dendritic cells capture antigens in the periphery and then migrate to the lymphoid organs to initiate immunity, whereas lymphoid dendritic cells are found in the thymic medulla and lymph node T cell areas and are responsible for tolerance. The latter may occur through immune regulation and/or deletion. Myeloid dendritic cells undergo many activities that contribute to the initiation of immunity. These are summarized here and include mobilization from progenitors and precursors in the blood and marrow, maturation from immature cells in the skin and interstitial spaces, formation of MHC-peptide complexes in MHC class II compartments or MIICs, migration to the T cell areas, and finally, mortality. The death of migratory dendritic cells seems to be accompanied by their phagocytosis and processing by other dendritic cells in the T area. We speculate that this transfer of antigens, including self-peptides captured by the uptake of apoptotic cells in peripheral tissues, is an important prelude to the regulatory function of resident or lymphoid dendritic cells in the T cell area. These features of dendritic cell biology provide targets to manipulate the immune response in vivo. J. Leukoc. Biol. 66: 205–208; 1999.
Donor dendritic cell persistence in organ allograft recipients in the absence of immunosuppression
Authors: O'Connell Peta J , Burlingham William J
Published: January 2018

Donor-derived leukocytes are known to persist in the peripheral blood of organ allograft recipients after withdrawal of all immunosuppressive drug therapy and can exert a donor-specific veto effect. Antigen-presenting cells (APC), in particular dendritic cells (DC), have been proposed as a candidate for this veto leukocyte. Myeloid DC were derived from the peripheral blood of two non-compliant organ transplant recipients: D. S., a heart transplant recipient, and J. M., a liver transplant recipient. Donor-specific signal was enriched in the cultured DC fraction relative to whole blood for both patients. The clinical outcome in each patient was different: D. S. lost his heart allograft due to biopsy-proven acute and chronic rejection 2.5 years after discontinuing anti-rejection medication; J. M. continues to maintain adequate liver function. The results have important implications for the planned withdrawal of immunosuppression in tolerance protocols as DC may play a role either in the maintenance of tolerance or immune activation. J. Leukoc. Biol. 66: 301–305; 1999.
Annexins VII and XI are present in a human macrophage-like cell line. Differential translocation on FcR-mediated phagocytosis
Authors: Pittis M G , Garcia R C
Published: January 2018

We have studied the divalent cation-dependent association of proteins to subcellular fractions of human macrophage-like cells before and after FcR-mediated phagocytosis. Among these proteins we have identified annexins VII and XI for the first time in these cells, along with annexins I, III, and VI. Although all of these annexins are present in the cytosolic fraction, the extent of their association to membrane and phagosome fractions from resting and stimulated cells is variable. Annexin VII translocates from cytosolic to membrane fractions after phagocytic stimulation, along with annexin I, III, and VI. Annexins VII and XI are found associated with purified phagosomes along with I, III, and VI, and this association is greater after a 24-h chase period. Our results show differences in the intracellular distribution of different annexins in macrophage-like cells on phagocytosis. Annexins VII, VI, III, and I respond to particle ingestion by translocating to phagosomes and other cell membrane structures, whereas annexin XI translocates predominantly to phagosomes, suggesting dissimilarities in their function. J. Leukoc. Biol. 66: 845–850; 1999.
Role of IFN-γ on dissociation between nitric oxide and TNF/IL-6 production by murine peritoneal cells after restimulation with bacterial lipopolysaccharide
Authors: Tominaga Kaoru , Saito Shinji , Matsuura Motohiro , Funatogawa Keiji , Matsumura Haruo , Nakano Masayasu
Published: January 2018

Murine peritoneal exudate cells (PEC) pre-exposed to bacterial lipopolysaccharide (LPS) show augmented nitric oxide (NO) production by LPS restimulation, in contrast to LPS tolerance with reduced production of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6). Significant amounts of interferon-γ (IFN-γ) were detected in the PEC cultures on LPS stimulation, and anti-IFN-γ antibody suppressed the LPS-induced NO, but not TNF-α and IL-6, production. Addition of anti-IFN-γ antibody to the cultures in the LPS pre-exposure step strongly suppressed the augmented NO production on LPS restimulation. Anti-IL-12 antibody, which suppressed the LPS-induced IFN-γ production, also suppressed the augmented NO production, as did anti-IFN-γ antibody. Taken together, we propose the following mechanisms: (1) T and NK cells in PEC produce IFN-γ by the action of IL-12, which is derived from LPS-stimulated macrophages, and (2) the de novo-produced IFN-γ activates macrophages to augment NO production on LPS restimulation. J. Leukoc. Biol. 66: 974–980; 1999.