bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential iron transport into phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169
Authors: Kuhn Donald E , Baker Beth D , Lafuse William P , Zwilling Bruce S
Published: January 2018

The transport of iron by RAW264.7 macrophage cell lines transfected with either Nramp1Gly169 (resistant) or Nramp1Asp169 (susceptible) alleles was assessed. We found no difference between resistant and susceptible cells in the rate of Fe import or export when Fe transport was measured in intact cells. In contrast, the rate of Fe import by latex-bead phagosomes isolated from resistant cells was more than double the rate by latex-bead phagosomes from susceptible cells. Similarly, phagosomes isolated from resistant cells that had been pre-labeled with 55Fe-citrate before phagocytosis contained up to four times as much Fe as the corresponding phagosomes from susceptible cells. Phagocytosis of Mycobacterium avium was accompanied by an increase in the production of hydroxyl radicals by Nramp1Gly169-transfected macrophages but not by macrophages transfected with the susceptible allele. These results are consistent with the hypothesis that Nramp1 functions to transport Fe into the bacterium-containing phagosome where it serves as a catalyst for the Haber-Weiss reaction, which accounts for the increased capacity of these cells to limit mycobacterial growth. J. Leukoc. Biol. 66: 113–119; 1999.
Urokinase plasminogen activator receptor (CD87) expression of tumor-associated macrophages in ductal carcinoma in situ, breast cancer, and resident macrophages of normal breast tissue
Authors: Hildenbrand Ralf , Wolf Georg , Böhme Beatrix , Bleyl Uwe , Steinborn Andrea
Published: January 2018

Macrophages concentrate urokinase-type plasminogen activator (uPA) at the cell surface by expressing urokinase receptors (uPAR) in order to focus the pericellular space plasminogen-dependent proteolysis important in matrix remodeling and cell movement. This study examines the uPAR levels of tumor-associated macrophages (TAM) of invasive breast carcinomas, of TAMs from ductal carcinoma in situ (DCIS) and of macrophages derived from normal (non-tumor) breast tissue. TAMs from invasive breast carcinomas (n = 30), from DCIS (n = 12), and macrophages from normal breast tissue (n = 30) were cultured and immunocytochemically phenotyped by using a panel of antibodies. Urokinase receptor levels were determined by Western blot analysis and in cell-free supernatants by enzyme-linked immunosorbent assay. Urokinase receptor cell surface fluorescence intensity was determined by FACS and by confocal laser scan microscopy. Urokinase-receptor mRNA was detected by in situ hybridization. TAMs of invasive breast carcinomas and of DCIS possess significantly elevated uPAR levels compared with macrophages derived from normal breast tissue. Conclusions: activated macrophages with elevated uPAR levels belong to inflammatory areas in close vicinity of infiltrating and non-infiltrating (DCIS) tumor cells. Blood monocytes that possess elevated uPAR-levels may be selectively recruited from the bloodstream to inflammatory sites close to carcinoma cells, and/or breast cancer and precursor lesions may induce elevated uPAR-levels in TAMs by paracrine interactions. J. Leukoc. Biol. 66: 40–49; 1999.
Mechanisms of ozone-induced inhibitory effect of bronchoalveolar lavage fluid on alveolar macrophage-mediated immunosuppressive activity in rats
Authors: Koike Eiko , Kobayashi Takahiro , Murakami Masataka , McWilliam Andrew S , Holt Patrick G
Published: January 2018

Resident alveolar macrophages (AM) play an important immunomodulatory role via suppression of lymphocyte proliferation, and nitric oxide (NO) plays a crucial role in this immunosuppression of AM. Our previous report suggested that during ozone (O3)-induced lung inflammation, bronchoalveolar lavage fluid (BALF) inhibited AM- mediated immunosuppression and concanavalin A (Con A)-induced proliferation of lymph node cells (LNC) [E. Koike et al. (1998) Toxicol. Sci. 41, 217–223]. In these studies, we investigated the mechanisms of the inhibition of BALF from O3-exposed rats (O3-BALF). We investigated whether BALF might affect (1) the interferon-γ (IFN-γ) production by Con A-stimulated LNC and IFN-γ-induced NO production by AM, and (2) the interleukin (IL)-2 production by Con A-stimulated LNC and IL-2-induced LNC proliferation. These results demonstrated that O3-BALF inhibited IFN-γ production by Con A-stimulated LNC, IFN-γ-induced NO production by AM, and IL-2-induced LNC proliferation. In addition, the major inhibitory factor against AM-mediated immunosuppression in O3-BALF may be a protein of greater than 10 kDa. J. Leukoc. Biol. 66: 75–82; 1999.
Immunobiology of DC in NOD mice
Authors: Morel Penelope A , Vasquez A Cecilia , Feili-Hariri Maryam
Published: January 2018

NOD mice spontaneously develop diabetes between 15 and 20 weeks of age, which is preceded by insulitis characterized by the infiltration of lymphocytes. Dendritic cells (DC) are among the first cells to infiltrate the islet and they have been implicated in the pathogenesis of the disease. Our work has been concerned with the detailed characterization of four distinct DC populations in NOD mice: two derived from bone marrow (BM) cells cultured in either granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4) or GM-CSF alone and two from the spleen of Flt3 ligand (Flt3L) -treated mice, isolated on the basis of CD8α expression. Phenotypic and functional differences between these DC subsets in NOD mice have been identified. In addition, we obtained a lower yield of NOD BM-derived DC and they expressed higher levels of cell-surface CD40 and IL-12 p40 mRNA than BM-derived DC from the diabetes-resistant strain, B10.BR. We have also investigated the ability of these DC populations to modulate the development and progression of diabetes in NOD mice. J. Leukoc. Biol. 66: 276–280; 1999.
Functional CD40 ligand is expressed on epidermal Langerhans cells
Authors: Salgado Claudio Guedes , Nakamura Koichiro , Sugaya Makoto , Tada Yayoi , Asahina Akihiko , Koyama Yoh-ichi , Irie Shinkichi , Tamaki Kunihiko
Published: January 2018

Epidermal Langerhans cells (LC) are bone-marrow-derived major histocompatibility complex (MHC) class II antigen-expressing antigen-presenting cells (APC) that comprise 1–3% of total epidermal cells (EC). LC express high levels of MHC class II antigen and augment costimulatory molecules such as B7-1, B7-2 during culture. In a previous report, using purified murine LC, we showed that freshly prepared LC (fLC) do not express CD40, whereas cLC express CD40. Tumor necrosis factor α (TNF-α) enhanced CD40 expression on LC during culture. We examined the expression of CD40L on LC and found that both fLC and cLC expressed mRNA for CD40L. FACS analysis revealed that cLC cultured for 36 h expressed CD40L but fLC did not. When we examined the cytoplasmic CD40L, however, both fLC and cLC expressed cytoplasmic CD40L. TNF-α, which up-regulated CD40 expression on LC during culture, did not modulate CD40L. Co-culture of purified LC with anti-CD40L markedly inhibited the up-regulation of B7-1 expression on LC and caused partial inhibition of B7-2 expression during culture. These results indicate that CD40L is expressed on cLC, and that CD40L on LC modulates the expression of costimulatory molecules such as B7-1 and B7-2 on LC. J. Leukoc. Biol. 66: 281–285; 1999.
Modulation of macrophage and B cell function by glycosaminoglycans
Authors: Wrenshall Lucile E , Stevens R Brian , Cerra Frank B , Platt Jeffrey L
Published: January 2018

There is increasing evidence that the behavior of antigen-presenting cells may be regulated, in part, by the surrounding microenvironment. Components of the microenvironment of solid tissues that might influence antigen-presenting cell functions include glycosaminoglycans. We previously showed that heparan sulfate glycosaminoglycans activate macrophages, leading to profound alterations in T cell responses. Here we demonstrate the functional changes that occur in murine antigen-presenting cells induced by heparan sulfate and other glycosaminoglycans, and postulate how these functional changes influence the nature of local immune responses. Heparan sulfate triggered up-regulation of ICAM-1 and I-A, caused the release by antigen-presenting cells of interleukin (IL)-1, IL-6, tumor necrosis factor, IL-12, transforming growth factor β, and prostaglandin E2 (PGE2), and (in macrophages) induced cytotoxic capability. Heparin induced IL-12 and interferon-γ production but did not promote the release of other cytokines. Chondroitin sulfate and dermatan sulfate, although not stimulating the production of cytokines or of PGE2, elicited the production by macrophages of nitric oxide. These findings support a model in which the glycosaminoglycan composition of a given tissue, which may be altered by inflammatory processes, helps to regulate the behavior of antigen-presenting cells, which in turn determines the characteristics of the immune response that ensues. J. Leukoc. Biol. 66: 391–400; 1999.
Short exposure of intestinal epithelial cells to TNF-α and histamine induces Mac-1-mediated neutrophil adhesion independent of protein synthesis
Authors: Miyata Ryukou , Iwabuchi Kazuhisa , Watanabe Sumio , Sato Nobuhiro , Nagaoka Isao
Published: January 2018

Neutrophils play an important role in intestinal inflammation by interacting with intestinal epithelial cells. In this study, we evaluated neutrophil adhesion to intestinal epithelial cells using intestinal epithelial cell line HT29 stimulated with tumor necrosis factor α (TNF-α) and histamine for a short time (30 min). The TNF-α and histamine stimulation markedly increased neutrophil adhesion. The increased adhesion was inhibited by anti-CD11b and anti-CD18 monoclonal antibodies (mAbs), but not by anti-CD11a and anti-CD54 (ICAM-1) mAbs. It is interesting that flow cytometric analysis revealed that ICAM-1 expression on HT29 cells was not changed by TNF-α and histamine stimulation. Moreover, the increased adhesion was inhibited by proteinase K treatment but not cycloheximide treatment of HT29 cells. Together these observations suggest that short exposure of HT29 cells to TNF-α and histamine induces CD11b/CD18 (Mac-1)-dependent but CD11a/CD18 (LFA-1)-independent neutrophil adhesion to intestinal epithelial cells, and ICAM-1 is not likely to be involved in the interactions. Furthermore, epithelial cell ligand(s) for neutrophils is likely protein molecule(s) that is expressed on the cell by stimulation independent protein synthesis. However, it is also possible that neutrophil activating factor(s), which stimulates neutrophils to bind with epithelial ligands via Mac-1, is expressed by epithelial cells during stimulation. J. Leukoc. Biol. 66: 437–446; 1999.
Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils
Authors: Dharmawardhane Suranganie , Brownson Delia , Lennartz Michelle , Bokoch Gary M
Published: January 2018

Leukocyte chemoattractants are known to stimulate signaling pathways that involve Rho family GTPases. Direct evidence for the regulation of the leukocyte cytoskeleton by Rho GTPases and their effector targets is limited. The p21-activated kinases (PAKs) are specific targets of activated GTP-bound Rac and Cdc42, and have been proposed as regulators of chemoattractant-driven actin cytoskeletal changes in fibroblasts. PAK1 co-localizes with F-actin to cortical actin structures in stimulated fibroblasts, and activated PAK1 mutants induce membrane ruffling and polarized cytoskeletal rearrangements. We investigated whether PAK1 was associated with remodeling ofthe actin cytoskeleton in activated human neutrophils. We monitored the redistribution of PAK1 and F-actin into the actin cytoskeleton after stimulation of human neutrophils with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the particulate stimulus, opsonized zymosan (OZ). PAK1 exhibited a similar distribution as F-actin in fMLP-stimulated leukocytes, localizing in membrane ruffles and to lamellipodia at the leading edge of polarized cells. Addition of OZ induced phagocytic uptake of this particulate stimulus, and PAK1 re-localized to the F-actin-rich pseudopodia and phagocytic cups associated with this process. Once the OZ was internalized, there was little PAK1 localized around the ingested particles, suggesting that PAK1 may be regulating the cytoskeletal extensions and events required for engulfment of bacteria, but not the subsequent steps of internalization. Localization of PAK1 and F-actin in cytoskeletal structures was abolished by the actin polymerization inhibitor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin. Our data suggest that PAK1 may regulate a subset of cytoskeletal dynamics initiated by chemoattractant and phagocytic stimuli in human neutrophils. J. Leukoc. Biol. 66: 521–527; 1999.
The actions of bacterial DNA on murine macrophages
Authors: Sester David P , Stacey Katryn J , Sweet Matthew J , Beasley Shannon J , Cronau Stephen L , Hume David A
Published: January 2018

Murine macrophages are able to distinguish bacterial from mammalian DNA. The response is mimicked by single-stranded oligonucleotides containing unmethylated CG dinucleotides (“CpG” motifs) in specific sequence contexts. The dose-response curve for activation is influenced by variation in the sequence flanking the core CpG motif. CpG or bacterial DNA activates several signaling pathways in common with bacterial lipopolysaccharide (LPS), leading to induction of cytokine genes such as tumor necrosis factor a. Pretreatment with LPS causes desensitization to subsequent activation by CpG DNA. Both stimuli also cause cell cycle arrest in macrophages proliferating in response to the macrophage growth factor colony-stimulating factor-1 (CSF-1), but prevent apoptosis caused by growth factor removal. In part, cell cycle arrest by CpG DNA and LPS may be linked to rapid down-modulation of the CSF-1 receptor from the cell surface, a response that occurs in an all-or-nothing manner. The response of macrophages to CpG DNA has aspects in common with the DNA damage response in other cell types, which may provide clues to the underlying mechanism. J. Leukoc. Biol. 66: 542–548; 1999.
Maturation decreases responsiveness of human bone marrow B lineage cells to stromal-derived factor 1 (SDF-1)
Authors: Fedyk Eric R , Ryan Daniel H , Ritterman Ion , Springer Timothy A
Published: January 2018

We compared the chemotactic responsiveness of different subsets of human B lineage cells to stromal derived factor-1 (SDF-1). High percentages (30–40% of input) of purified bone marrow progenitors including non-B lineage progenitors, pro-B cells, and pre-B cells migrated to SDF-1α, demonstrating that SDF-1 is an efficacious chemoattractant of these cells. Pro-B cells responded optimally to a lower concentration of SDF-1 than other subsets, demonstrating that SDF-1 is a more potent chemoattractant of this subset. A lower percentage (10–15% of input) of mature B lymphocytes migrated to SDF-1α than pro-B cells, demonstrating that responsiveness of B lineage cells to SDF-1 decreases during differentiation. Inhibition by anti-CXCR4 mAb demonstrated that migration of B lineage cells to SDF-1 was completely dependent on CXC chemokine receptor-4 (CXCR4). Mature B cells expressed higher levels of CXCR4 receptors than uncommitted progenitors and pro-B cells, despite differences in responsiveness to SDF-1. CXCR4 receptors expressed by unresponsive and SDF-1-responsive B cells bound SDF-1a with similar affinities (K  D= 1.7–3.3 × 10−9 M). Therefore, elements downstream from CXCR4 appear to regulate responsiveness of B cells to SDF-1. We speculate that SDF-1 and CXCR4 direct migration of progenitor cells in microenvironments that promote B lymphopoiesis. J. Leukoc. Biol. 66: 667–673; 1999.
ATP receptors and giant cell formation
Authors: Di Virgilio Francesco , Falzoni Simonetta , Chiozzi Paola , Sanz Juana M , Ferrari Davide , Buell Gary N
Published: January 2018

We have investigated the role of the purinergic P2X7 receptor in the formation of multinucleated giant cells in human monocyte/macrophage cultures stimulated with either concanavalin A or phytohemagglutinin. Macrophage fusion can be blocked by a P2X7-selective pharmacological antagonist or by a mAb directed against the extracellular P2X7 domain. Furthermore, macrophage cell clones expressing high P2X7 levels spontaneously fuse in culture, whereas macrophage clones lacking P2X7 are unable to fuse. Our findings suggest that the newly identified purinergic P2X7 receptor plays a central role in the complex chain of events leading to generation of macrophage-derived giant cells. J. Leukoc. Biol. 66: 723–726; 1999.
Differential effects of B7-1 and B7-2 on the costimulation of mouse nonspecific cytotoxic T lymphocyte development in response to anti-CD3 antibody
Authors: Makrigiannis Andrew P , Musgrave Bruce L , Hoskin David W
Published: January 2018

Despite extensive study, the relative contribution of B7-1 and B7-2 molecules to the costimulation of cytotoxic T lymphocyte (CTL) activation remains controversial. We used blocking mAbs to B7-1 and B7-2 molecules to determine the role of these B7 family members in the in vitro induction of mouse nonspecific CTL in response to soluble anti-CD3 mAb. Optimal induction of anti-CD3-activated killer-T (AK-T) cells was found to require interactions with B7-2 on residual accessory cells in nylon wool-nonadherent spleen cell preparations during the first 12 h of culture in the presence of anti-CD3 mAb. Because B7-1 is not expressed at high enough levels on residual accessory cells in primary T cell cultures to be an effective ligand for CD28, we used LPS-stimulated B cells, which express substantial B7-1, in addition to B7-2, to determine the contribution of B7-1 to AK-T cell development. Compared with B7-2, the contribution of B7-1 to the costimulation of AK-T cells in this system was modest because anti-B7-1 mAb had only a minimal inhibitory effect on the generation of cytotoxicity, whereas anti-B7-2 mAb strongly inhibited AK-T cell development. Anti-CD3-induced cytotoxicity of T cells from CD4 knockout mice and CD4-depleted nylon wool-nonadherent spleen cells from wild-type mice was inhibited by anti-B7-2 mAb, implying that B7-2 is able to bind directly to CD28 on CD8+ T cells and costimulate their activation. B7-1 blockade, on the other hand, did not affect the costimulation of CD8+ T cells. Blockade of B7-2/CD28 interactions with anti-B7-2 mAb strongly inhibited granzyme B, but not perforin or Fas ligand gene expression, suggesting an explanation for the inhibitory effect of anti-B7-2 mAb on AK-T cell development. These data indicate that B7-2 is superior to B7-1 as a costimulator of mouse AK-T cell induction. J. Leukoc. Biol. 66: 792–802; 1999.
Modulatory effects of human herpes virus-7 on cytokine synthesis and cell proliferation in human peripheral blood mononuclear cell cultures
Authors: Atedzoé Blanche Ngola , Menezes José , D'Addario Mario , Xu Jingwu , Ongradi Joseph , Ahmad Ali
Published: January 2018

Human herpes virus-7 (HHV-7) infects cells of the immune system and thus may modulate their function. To investigate the potential immunomodulatory effects of this virus, we performed an in vitro study in which we investigated effects of HHV-7 on the synthesis of several key immunomodulatory cytokines, i.e. tumor necrosis factor α (TNF-α), interleukin-2 (IL-2), interferon-γ (IFN-γ), IL-4, IL-6, and transforming growth factor β (TGF-β). This was examined after in vitro infection of human peripheral blood mononuclear cells (PBMC) with HHV-7. We found elevated levels of TNF-α, TGF-β, and IFN-γ in the supernatants of HHV-7-infected cells. By reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, using cytokine-specific primers, we found that levels of TNF-α, TGF-β, and IFN-γ mRNA were increased in the infected cells. The HHV-7 infection also significantly (P < 0.05) decreased the production of IL-2 from activated, IL-2-producing PBMC. Furthermore, mitogen- and cytokine-induced cellular proliferative responses of human PBMC were found to be significantly (P < 0.05) down-regulated by this virus. On the other hand, HHV-7 did not affect IL-4 and IL-6 synthesis. Overall, our results indicate that HHV-7 infection causes significant immunomodulatory effects with regard to cytokine synthesis in these cells as well as inhibiting lymphocyte proliferation by various stimuli. J. Leukoc. Biol. 66: 822–828; 1999.
Phosphorylation of p40-phox during activation of neutrophil NADPH oxidase
Authors: Someya Akimasa , Nunoi Hiroyuki , Hasebe Takeshi , Nagaoka Isao
Published: January 2018

NADPH oxidase, a superoxide-producing enzyme in phagocytic cells, consists of membrane-associated cytochrome b558 and cytosolic components (p47-phox, p67-phox, p40-phox, rac 1/2). Activation of NADPH oxidase is accompanied by the phosphorylation of cytosolic components (p47-phox and p67-phox). In this study, we have examined whether p40-phox, a newly identified cytosolic component, is phosphorylated during neutrophil activation, and the relationship between p40-phox phosphorylation and NADPH oxidase activation. When 32P-labeled guinea pig neutrophils were stimulated by phorbol 12-myristate 13-acetate, p40-phox was phosphorylated as p47-phox. It is interesting that phosphorylation of p40-phox was markedly inhibited by protein kinase C inhibitor, H-7, but not by casein kinase II inhibitor, A-3, and H-7 inhibited translocation of p40-phox and activation of NADPH oxidase. Furthermore, purified protein kinase C but not casein kinase II directly phosphorylated p40-phox of p40-phox/p47-phox/p67-phox complex. Together these observations suggest that p40-phox is phosphorylated by protein kinase C during neutrophil activation, and phosphorylation of p40-phox may be important for the activation of NADPH oxidase. J. Leukoc. Biol. 66: 851–857; 1999.
Soluble TNF-α receptors bind and neutralize over-expressed transmembrane TNF-α on macrophages, but do not inhibit its processing
Authors: Watts Alan D , Hunt Nicholas H , Madigan Michele C , Chaudhri Geeta
Published: January 2018

Tumor necrosis factor a (TNF-α) is initially synthesized as a type II integral membrane protein (transmembrane TNF-α) after macrophage activation. In this study we have investigated some aspects of the regulation of expression and biological activity of transmembrane TNF-α by both soluble TNF-α receptors (sTNF-αR) and inhibitors of TNF-α processing. We show, using the technique of receptor-mediated ligand precipitation (RMLP), that a dimeric construct of the type I sTNF-αR binds to transmembrane TNF-α, expressed on the mouse macrophage cell line RAW264.7, under cell culture conditions. This interaction between sTNF-αR and transmembrane TNF-α does not prevent processing and release of soluble TNF-α. A specific hydroxamic acid-based inhibitor of processing, BB1101 (British Biotech), was found to increase the total cellular levels of whole-cell, 26-kDa, precursor TNF-α by 2.2-fold. However, the inhibitor increased the levels of precursor TNF-α present solely on the cell surface (i.e., transmembrane TNF-α) by 5.1- to 7.5-fold. This increase in the levels of transmembrane TNF-α on the activated human monocytoid cell line mono mac 6 was associated with a similar (6.7-fold) increase in TNF-α-mediated cytotoxicity toward the human adenocarcinoma cell line Colo 205, which is sensitive only to the transmembrane form of TNF-α. Mono mac 6 cells, expressing transmembrane TNF-α, were found to be killing the Colo 205 target cells through apoptosis. This cytotoxicity could be neutralized by pre-incubating the mono mac 6 cells with either sTNF-αR or polyclonal anti-TNF-α serum. J. Leukoc. Biol. 66: 1005–1013; 1999.
P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox
Authors: Benna Jamel El , Dang Pham My-chan , Andrieu Valérie , Vergnaud Sabrina , Dewas Cedric , Cachia Odile , Fay Michèle , Morel Françoise , Chollet-Martin Sylvie , Hakim Jacques , Gougerot-Pocidalo Marie-Anne
Published: January 2018

NADPH oxidase is an O-generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67phox, p47phox, p40phox, and the G-protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67phox, p47phox, and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40phox associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40phox. When resting neutrophils were lysed in Triton X-100 or octyl glucoside buffer and separated into detergent-soluble and detergent-insoluble fractions, p40phox and p67phox were mainly associated with the detergent-insoluble fraction (defined as the cytoskeleton), whereas p47phox was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47phox translocation to the cytoskeleton but did not affect the distribution of p40phox or p67phox. Using immunofluorescence confocal microscopy, we found that p40phox colocalized with filamentous actin. In neutrophils from a p67phox-deficient patient with detectable p40phox, p40phox associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X-100 buffer, p40phox, p47phox, and p67phox were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40phox and p47phox was found in the soluble membrane fraction, but no p67phox was detected. These findings show that p40phox, like p67phox and p47phox, binds to the cytoskeleton and membrane skeleton. In addition, p40phox can dissociate from p67phox in activated membranes. J. Leukoc. Biol. 66: 1014–1020; 1999.
Retinoid X receptor suppresses transformation by the v-myb oncogene
Authors: Šmarda Jan , Zemanová Karla , Bryja Josef , Šmardová Jana , Kozubík Alois , Hofmanová Jirina , Nemajerová Alice , Ševçíková Sabina , Kohoutek Jirí , Vodiçka Petr
Published: January 2018

The v-myb oncogene of avian myeloblastosis virus causes acute monoblastic leukemia in vivo and transforms myelomonocytic cells in culture. Retinoids are potent regulators of proliferation and differentiation in various cell types, and they can initiate differentiation in certain types of leukemic cells. However, the BM2 v-myb-trans-formed chicken monoblastic cell line is resistant to retinoic acid treatment. We found that overexpression of the retinoid X receptor confers sensitivity of BM2 cells to retinoic acid, resulting in induction of growth arrest and terminal differentiation. In contrast, the frequency of apoptosis was not affected by the retinoid X receptor in this cell type. We also demonstrated that suppression of transformation by v-Myb results from the negative effect of retinoid X receptor on v-Myb transactivation function, similar to that previously described for the retinoic acid receptor. The retinoid X receptor-induced inhibition of transactivation by v-Myb seems to be enhanced by a cell type-specific factor(s), which is not required by retinoic acid receptor. J. Leukoc. Biol. 66: 1039–1048; 1999.
Trp-Lys-Tyr-Met-Val-d-Met is a chemoattractant for human phagocytic cells
Authors: Bae Yoe-Sik , Kim Youndong , Kim Yong , Kim Jae Ho , Suh Pann-Ghill , Ryu Sung Ho
Published: January 2018

Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) is a synthetic peptide that stimulates phosphoinosi-tide (PI) hydrolysis in human leukocytes. The peptide binds to a unique cell surface receptor(s). Recently we had demonstrated that human neutrophils, monocytes, and B lymphocytes express this peptide-specific receptor and that stimulation of human leukocytes with the peptide leads to activation of the oxidative respiratory system and the bactericidal activity of neutrophils or monocytes. In this study we showed that the peptide induces chemotaxis of phagocytic leukocytes and studied the signaling pathway leading to chemotaxis in human monocytes. The peptide-induced monocyte chemotaxis is pertussis toxin (PTX)-sensitive. This fact correlates with the peptide's stimulation of PI hydrolysis and intracellular Ca2+ ([Ca2+]i) release, which is also PTX-sensitive. We demonstrate that the peptide-specific receptor is different from receptors) for monocyte chemoattractant protein-1 (MCP-1). We also show that intracellular signaling of WKYMVm leading to monocyte chemotaxis is different from that of MCP-1. The peptide-medi-ated monocyte chemotaxis is insensitive to protein kinase C (PKC) inhibitor (GF109203X) and butan-1-ol, ruling out PKC and phospholipase D participation in this process. On the other hand, a tyrosine kinase inhibitor (genistein) and RhoA inhibitor (C3 transferase) curtailed the peptide-induced chemotaxis in a concentration-dependent manner, implying the involvement of tyrosine kinase and RhoA, respectively. Treatment of human monocytes with the peptide stimulates tyrosine phosphorylation of several cellular proteins, including p125FAK and Pyk2 and translocation of RhoA from the cytosol to the membrane. We conclude that WKYMVm induces chemotaxis of human phagocytic leukocytes via unique receptors and signaling. J. Leukoc. Biol. 66:915–922; 1999.
Regulation of the processing and release of tumor necrosis factor α in a human macrophage cell line
Authors: Nakada-Tsukui Kumiko , Watanabe Naoko , Kobayashi Yoshiro
Published: January 2018

The proteolytic cleavage of a variety of membrane proteins, including pro-tumor necrosis factor α (pro-TNF-α), is induced by various reagents such as phorbol 12-myristate 13-acetate (PMA). In this study we generated a human macrophage cell line that constitutively produces TNF-α, and examined how the processing and release of TNF-α are regulated. The processing and release of TNF-α were enhanced by PMA through a protein kinase C-dependent pathway. Although only hy-droxamate matrix metalloproteinase (MMP) inhibitors inhibited the basal processing and release of TNF-α, the PMA-induced processing and release of TNF-α were inhibited not only by MMP inhibitors but also by 1,10-phenanthroline, 3,4-dichloroiso-coumarin (3,4-DCI), iodoacetamide, and Nα-p-tosyl-l-phenylalanine chloromethyl ketone (TPCK). Hydroxamate MMP inhibitors and 1,10-phenanthroline inhibited the processing of TNF-α on the cell surface, whereas 3,4-DCI, iodoacetamide, and TPCK inhibited the transport of TNF-α to the cell surface. These results suggest that serine and/or cysteine protease(s) may be involved in PMA-induced processing and/or transport to the cell surface. J. Leukoc. Biol. 66: 968–973; 1999.
The individual regulation of granule protein mRNA levels during neutrophil maturation explains the heterogeneity of neutrophil granules
Authors: Cowland Jack B , Borregaard Niels
Published: January 2018

The in vivo mRNA levels for 16 granule proteins during neutrophil differentiation were determined to address the question of whether the synthesis of granule proteins is regulated individually or blockwise. RNA was extracted from peripheral blood granulocytes and three different populations of neutrophil precursors isolated from human bone marrow by Percoll density centrifugation. The mRNA levels in relation to the maturation of the cells were determined by Northern blot for the 12 matrix proteins myeloperoxidase, proteinase-3, elastase, defensin, lactoferrin, NGAL, hCAP-18, transcobalamin-I, SGP28, gelatinase, lysozyme, and serglycin and the 4 membrane proteins CD68, CD11b, N-formyl-methionyl-leucyl-phenylalanine receptor, and CD35. This panel of transcripts ensured that markers for all exocytosable organelles of the neutrophil were included in the study. A highly differentiated distribution of mRNAs for granule proteins was demonstrated that can explain the heterogeneity of the intracellular storage granules and secretory vesicles of the neutrophil. Furthermore, the individual distribution of these transcripts provides the basis for a more detailed assessment of neutrophil maturation than that obtained by morphological studies or the use of a single marker protein for azurophil, specific, and gelatinase granules. J. Leukoc. Biol. 66: 989–995; 1999.
Effect of age on human neutrophil function
Authors: Wenisch Christoph , Patruta Sanda , Daxböck Felix , Krause Robert , Hörl Walter
Published: January 2018

Neutrophil phagocytosis, reactive oxygen intermediate production (intra- and extracellular), neutrophil bactericidal activity, and chemotaxis/chemokinesis were assessed in three age groups: 21–36, 38–56, and 62–83 years. A significant age-dependent reduction in the number of phagocytized Escherichia coli per neutrophil (measured by acridine orange staining) and Staphylococcus aureus phagocytosis (measured by flow cytometry) was seen (r = 0.669 and r = 0.684, P < 0.001 for both). These findings correlated with an age-dependent increase in intracellular calcium concentrations in resting neutrophils (r = 0.698, P < 0.001) and a reduced hexose uptake (r = 0.591, P < 0.01). In addition, a significant reduction in the intracellular reactive oxygen production was seen after stimulation with S. aureus (P < 0.001) with increasing age. In contrast, no differences between the groups in reactive oxygen production was seen after stimulation with E. coli. The neutrophil bactericidal activity was impaired with increasing age (64 ± 4% of the phagocytized bacteria were killed in group 1; 66 ± 2 in group 2, and 59 ± 6 in group 3; P < 0.01). In addition, a trend toward a reduced neutrophil chemotaxis was seen with increasing age (P = 0.022). The findings suggest that increased intracellular calcium concentrations in resting neutrophils and/or a reduced hexose uptake result in reduced phagocytic ability and decreased bactericidal activity of neutrophils in the elderly. J. Leukoc. Biol. 67: 40–45; 2000.
Neonatal neutrophil interaction with P-selectin: contribution of P-selectin glycoprotein ligand-1 and sialic acid
Authors: Tcharmtchi M Hossein , Smith C Wayne , Mariscalco M Michele
Published: January 2018

Previously we had determined that neonatal neutrophils had decreased interaction with monolayers expressing P-selectin compared to adult cells. In this study we examined the function of neonatal P-selectin glycoprotein ligand-1 (PSGL-1). A rabbit polyclonal antibody directed against the amino terminus of human PSGL-1 was produced and purified (3RB-PSGL-1). Neonatal neutrophils expressed the epitope recognized by 3RB-PSGL-1 and expression was decreased compared with adult neutrophils (20%, P < 0.05). In addition neonatal neutrophils had decreased interaction with Chinese hamster ovary (CHO)-P-selectin under both shear conditions and static adhesion (P < 0.05). Treatment of both neonatal and adult neutrophils with 3RB-PSGL-1 similarly inhibited the interaction with P-selectin monolayers under shear conditions, effects similar to treatment with O-sialoglycoprotein endopeptidase (OSGE). Neuraminidase treatment of neonatal and adult cells also markedly inhibited the interaction. In a detachment assay marked differences were noted between neonatal and adult cells treated with either 3RB-PSGL-1 or neuraminidase. Such treatments had little effect on adult neutrophils until shear stress exceeded 2.8 dynes/cm2. Treated neonatal neutrophils were exquisitely sensitive to shear stress with a marked decrease in interaction noted at a shear stress as low as 0.6 dynes/cm2. Thus the adhesive mechanisms that remain after treatment with neuraminidase or 3RB-PSGL-1 have a relatively low avidity and function less well in neonatal neutrophils compared to adult neutrophils. We speculate that this may account for the less efficient adhesion of neonatal neutrophils to P-selectin under conditions of flow. J. Leukoc. Biol. 67: 73–80; 2000.
CD11b/CD18-coated microspheres attach to E-selectin under flow
Authors: Crutchfield Karen L , Patil Vivek R Shinde , Campbell Craig J , Parkos Charles A , Allport Jennifer R , Goetz Douglas J
Published: January 2018

Neutrophils can attach to E-selectin under flow. Proposed ligands for E-selectin carry SLex-type glycans. The leukocyte β2 integrins are glycosylated with SLex. Thus, we speculated that β2 integrins could support attachment to E-selectin. To test this hypothesis, we coated 10–diameter microspheres with purified CD11b/CD18 (αMβ2) and investigated the adhesion of the resulting αMβ2 microspheres to E-selectin. Under in vitro flow conditions, the αMβ2 microspheres attached to Chinese hamster ovary cells expressing E-selectin (CHO-E) and 4-h interleukin-1 β-activated human umbilical vein endothelial cells (HUVEC). At a shear stress of 1.8 dynes/cm2, the attachment events were eliminated by pretreatment of the cellular monolayers with a mAb to E-selectin. microspheres did not attach to untransfected CHO cells or unactivated HUVEC at 1.8 dynes/cm2. Taken together, the results strongly suggest that the CD11b/CD18-E-selectin bond has sufficient biophysical properties to mediate attachment of neutro-phil-sized particles to E-selectin under flow. J. Leukoc. Biol. 67: 196–205; 2000.
Particulate adjuvants can induce macrophage survival, DNA synthesis, and a synergistic proliferative response to GM-CSF and CSF-1
Authors: Hamilton John A , Byrne Robert , Whitty Genevieve
Published: January 2018

The mode of action of immunological adjuvants is not yet completely understood. Many are particulate. Certain antigen-presenting (dendritic) cell populations belong to the monocyte/macrophage lineage and, like other members of the lineage, in some tissues appear to be short-lived. We report that many poorly degradable, particulate adjuvants, for example, aluminum hydroxide, oil-in-water emulsions, calcium phosphate, and silica, enhance murine bone marrow-derived macrophage survival; induction of DNA synthesis was even observed. No evidence could be found for a requirement for endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage-CSF (M-CSF or CSF-1). Synergy for the proliferative effects was noted in the presence of added GM-CSF or CSF-1. It is suggested from these in vitro findings that one function of certain particulate adjuvants may be to increase by enhanced survival or even proliferation the number of cells available for subsequent antigen presentation and cytokine production. J. Leukoc. Biol. 67: 226–232; 2000.
Ecto-enzymes: physiology meets pathology
Authors: Goding James W
Published: January 2018

Ecto-enzymes are catalytic membrane proteins with their active sites outside the cell. They include cholinesterase, which inactivates acetylcholine, and angiotensin-converting enzyme, which converts angiotensin I to biologically active angiotensin II, and numerous other peptidases, transpeptidases, nucleotidases, phosphodiesterases, and phosphatases. Many CD antigens of leukocytes are ecto-enzymes; some CD antigens for which no function is currently known are probably ectoenzymes. Expression is highly regulated and correlated with differentiation and activation. Some are highly restricted in distribution; others are ubiquitous. Many are shared between leukocytes and non-hematogenous cells. Biological functions appear to depend on the type and location of the cell in which expression occurs, and include recycling of nutrients, local control of response to cytokines and hormones, bone formation, cell mobility, invasion, and metastasis. Many novel regulatory functions of ecto-enzymes remain to be discovered, and may reveal new mechanisms of local extracellular control of cellular function. J. Leukoc. Biol. 67: 285–311; 2000.
Regulation of phagocytic leukocyte activities by C-reactive protein
Authors: Mortensen Richard F , Zhong Wangjian
Published: January 2018

The classic acute phase reactant C-reactive protein (CRP) is classified as an effector of innate host resistance because it activates the classical complement cascade and is opsonic. The latter action occurs via specific CRP receptors (CRP-R) that have recently been identified as both FcγRI and FcγRII on human phagocytic leukocytes. New findings also suggest an anti-inflammatory role for CRP because it modulates endotoxin shock and inhibits chemotaxis and the respiratory burst of neutrophils. CRP inhibited phorbol myristate acetate-induced superoxide (O) production more efficiently than the fMLP-triggered response. An examination of the inhibition of the protein kinase C (PKC)-dependent assembly of the NADPH oxidase complex revealed that both phosphorylation and translocation of PKC-β2 to the membrane were inhibited by a threshold acute phase dose of ∼50 μg/mL CRP. Translocation to the membrane and serine-phosphorylation of the major cytosolic p47-phox component of the NADPH oxidase complex was inhibited by CRP. CRP also inhibited membrane localization of activated Rac2, the small G protein regulator of the assembly of the oxidase components in activated neutrophils as well as the cytoskeleton during chemotactic movement. CRP-mediated regulation occurs via the CRP-R because an IgM mouse mAb to the human CRP-R mimicked CRP-induced inhibition of O production and chemotaxis. CRP may serve as an antiinflammatory regulator of activities at sites of tissue damage where it selectively accumulates and thus influences neutrophil infiltration and polymorphonuclear neutrophil activities. By contrast, CRP activates cells of the monocyte/macrophage lineage, suggesting differential regulation of these two leukocyte populations at the level of signaling. CRP appears to be a multifunctional protein with the capability of exerting both effector functions for innate host resistance, as well as exerting specific anti-inflammatory effects. J. Leukoc. Biol. 67: 495–500; 2000.
A negative autoregulatory link between Nramp 1 function and expression
Authors: Baker Stephen T , Barton C Howard , Biggs Thelma E
Published: January 2018

Nramp 1 (natural resistance-associated macrophage protein) controls resistance to infection by intracellular pathogens in mice. Nramp 1 regulates the microenvironment of the invading pathogen by increasing the luminal iron that participates in the Haber-Weiss reaction, producing radicals that attack the pathogen. We have studied the effect of inflammatory stimuli, iron, and sodium nitroprusside on Nramp 1 expression in bone marrow macrophages. Investigations show all three up-regulate Nramp 1 expression with a parallel increase in immunoreactivity to an amino-terminal antibody and Nramp 1 mRNA. Growth rates are reduced in macrophage cell lines expressing Nramp 1. This is through a decrease in iron availability, shown by an increase in IRP2 activity and a reciprocal decrease in conventional protein kinase Cβ-1 expression. We propose that Nramp 1 activity may control its own expression via a negative autoregulatory loop that is important for iron homeostasis and maintenance of low cytoplasmic redox active iron levels in the macrophage. J. Leukoc. Biol. 67: 501–507; 2000.
The critical role of CD40/CD40L in the CD4-dependent generation of CD8+ T cell immunity
Authors: Clarke Sally R McK
Published: January 2018

Control of virus infections and eradication of tumors usually involves the lytic activity of CD8+ cytotoxic T lymphocytes (CTLs). The induction of effective CTL immunity relies on several factors, one of the most important of which is CD4+ T cell help. Numerous studies have demonstrated the dependence of CTL priming on the presence of CD4+ T cells, but until recently little was known of the mechanisms regulating this process. Based on reports that CD4+ and CD8+ T cells must recognize antigen on the same antigen-presenting cell (APC), help was originally thought to be provided through the delivery of short-range, CD4+ T cell-secreted cytokines. However, the results of subsequent studies favor an alternative mechanism, whereby CD4+ T cells modify the APC, converting it into a stimulatory cell for CD8+ T cell priming. It is important that CD40 and its ligand, CD40L, have been implicated in the provision of this help and, in particular, the generation of long-lasting CTL memory. J. Leukoc. Biol. 67: 607–614; 2000.
Induction of cyclooxygenase-2 by human monocytes exposed to group B streptococci
Authors: Maloney Christopher G , Thompson Samuel D , Hill Harry R , Bohnsack John F , McIntyre Thomas M , Zimmerman Guy A
Published: January 2018

Group B streptococcal (GBS) infections are associated with high morbidity and mortality. The molecular pathways mediating the pathophysiological events in GBS infection are not fully delineated. Cyclooxygenases (COX) are the enzymes that convert arachidonate to active eicosanoids. To identify the effects of GBS on eicosanoid metabolism and regulatory mechanisms, we exposed human monocytes to GBS and found that they secreted prostaglandin E2, prostacyclin, and thromboxane A2. Exposure to GBS caused monocytes to express COX-2 mRNA and protein in both a time- and concentration-dependent manner that correlated with eicosanoid production. COX-1 protein was unchanged. Addition of the anti-inflammatory cytokines interleukin (IL)-4 or IL-10 markedly attenuated GBS-induced COX-2 protein accumulation after GBS exposure, as did inhibition of p38 MAPK. Our experiments are the first to show that exposure of monocytes to a gram-positive bacterium (GBS) results in induction of functional COX-2, suggesting that eicosanoids may play important roles in the pathogenesis of GBS infections. J. Leukoc. Biol. 67: 615–621; 2000.
Migration of dendritic cells within 3-D collagen lattices is dependent on tissue origin, state of maturation, and matrix structure and is maintained by proinflammatory cytokines
Authors: Gunzer Matthias , Friedl Peter , Niggemann Bernd , Bröcker Eva-B , Kämpgen Eckhart , Zänker Kurt S
Published: January 2018

The function of dendritic cells (DC) depends on active migration through three-dimensional (3-D) extracellular matrices. We have analyzed the migration of murine DC from different tissue origins within 3-D collagen lattices through the use of time-lapse videomicroscopy and single-cell tracking. Directly after incorporation, 50–90% of DC from the spleen (spDC) and Langerhans cells freshly isolated from the epidermis (fLC) displayed active motility in these matrices. Whereas mature spDC showed multilateral pseudopod dynamics as well as fast and heterogeneous migration, immature fLC displayed a spherical shape with faint membrane processes and very homogenous, slow migration characteristics. In the absence of external stimuli, migration of both, spDC and fLC, vanished after >36 h due to cell death. Maintaining fLC viability by external granulocyte-macrophage colony-stimulating factor or tumor necrosis factor  a  prolonged migration up to 5 days. During this period fLC transformed into mature cells with large dendrites, thereby developing a heterogeneous migration pattern more similar to spDC. In randomly polymerized collagen matrices cell paths were without preferential orientation. In contrast, in artificially aligned lattices directional paths in accordance with the forced fiber orientation were observed. Thus, migration is an inherent property of DC, largely influenced by tissue origin, degree of maturity, and the 3-D structure of the environment. J. Leukoc. Biol. 67: 622–629; 2000.