bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of p130cas tyrosine phosphorylation by calyculin A
Authors: Qiu Wansong , Cobb Ronald R , Scholz Wolfgang
Published: January 2018

P130cas is a dominant tyrosine phosphorylated protein in v-src-and v-crk-transformed cells. Tyrosine phosphorylation also occurs in response to integrin-mediated cell adhesion. P130cas has a unique structure with multiple SH2 and SH3 binding sites, which makes it a candidate docking protein that might be involved in several signal transduction pathways. Little is known about how p130cas itself is regulated. In this report we present evidence that tyrosine phosphorylated p130cas was rapidly dephosphorylated in several lymphatic cell lines after treatment with calyculin A, a serine/ threonine phosphatase inhibitor. A similar result was obtained with okadaic acid, but higher concentrations and longer incubation times were required. Constitutive phosphorylation as well as receptor-cross linking-induced p130cas phosphorylation was inhibited. Furthermore, the p130cas-Crk association was disrupted by treatment of cells with calyculin A. However, the p130cas-Lyn association was not affected. These results suggest that calyculin A specifically affects SH2 domain-mediated protein-protein interactions and that Lyn does not bind to a susceptible SH2 domain. Furthermore, the data presented is consistent with the existence of a calyculin A-sensitive phosphatase or tyrosine kinase that may be a critical regulator of p130cas tyrosine phosphorylation.
Distinct adapter proteins mediate acid versus neutral sphingomyelinase activation through the p55 receptor for tumor necrosis factor
Authors: Adam-Klages Sabine , Schwandner Ralf , Adam Dieter , Kreder Dirk , Bernardo Katussevani , Krönke Martin
Published: January 2018

Ceramide, generated by the enzymatic function of sphingomyelinases (SMases) has emerged as an important signaling pathway transducing diverse biological effects of various cytokine receptors. The 55-kDa receptor for tumor necrosis factor (TNF-R55) activates two types of SMases through distinct cytoplasmic domains. The death domain that is responsible for the initiation of the apoptotic pathway also signals for the activation of an acid SMase (A-SMase). The adapter protein TRADD binds to TNF-R55 in a ligand-dependent manner and serves as anchor for the subsequent recruitment of other proteins into the signaling complex that directly lead to cell death or nuclear factor-κB (NF-κB) induction. Notably, the two proapoptotic adapter proteins TRADD and FADD are also involved in the activation of A-SMase. In contrast, the NF-κB-inducing adapters TRAF2 and RIP do not signal for A-SMase. Thus, activation of A-SMase appears to belong to signals leading to TNF-induced cell death. A second signaling domain (NSD) is located upstream of the death domain and directly links the TNF-R55 to the activation of a neutral SMase (N-SMase). A novel adapter protein, FAN, has been identified that specifically binds to the NSD. FAN contains five WD repeats at its carboxy terminus, while it shows significant sequence homology with the mouse beige protein and its human homolog, the CHS protein, in the center portion of the protein. Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, whereas truncated mutants of FAN produced dominant negative effects. FAN, however, did not interfere with any of the TNF responses signaled for by the death domain. Taken together, our data suggest that distinct cytoplasmic domains of TNF-R55 initiate independent signaling pathways by binding different adapter proteins.
Localization of eosinophil-derived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes
Authors: Sur Sanjiv , Glitz Dohn G , Kita Hirohito , Kujawa Stephen M , Peterson Ellen A , Weiler Deborah A , Kephart Gail M , Wagner Jill M , George Terry J , Gleich Gerald J , Leiferman Kristin M
Published: January 2018

Eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are generally regarded as eosinophil-specific proteins. We tested whether EDN and ECP are present in mature neutrophils. By indirect immunofluorescence, both eosinophils and neutrophils stained with antibodies to EDN and ECP. Lysates of purified (<0.1% eosinophil contamination) neutrophils contained EDN, 112 ± 4 ng/106 cells, and ECP, 163 ± 2 ng/106 cells, whereas eosinophil major basic protein (MBP) was not detectable. Electron microscopic examination of immunogold-labeled buffy coat cells stained with EDN antibody showed that EDN is localized to neutrophil granules. Finally, EDN mRNA was detected in lysates of highly purified neutrophils (0.001% eosinophil contamination) by the reverse transcription-polymerase chain reaction. We conclude that proteins that are either identical to or immunologically cross-reactive with EDN and ECP are present in neutrophils and that EDN is synthesized and localized to neutrophil granules. Thus, caution must be exercised in interpreting the presence of EDN and ECP as specific markers of eosinophil-associated inflammation in human disease.
Burn-associated Candida albicans infection caused by CD30+ type 2 T cells
Authors: Kobayashi Makiko , Kobayashi Hiroyuki , Herndon David N , Pollard Richard B , Suzuki Fujio
Published: January 2018

Burn injury is associated with the greatly increased susceptibility of thermally injured patients to infection from a variety of pathogens. In this study we investigated the role of burn-associated type 2 T cells on the increased susceptibility of burned patients to Candida albicans infection using SCID mice and peripheral blood lymphocytes (PBL) from thermally injured patients. When SCID mice that were inoculated with PBL from healthy donors were resistant to C. albicans infection, the SCID mice that were inoculated with PBL from burned patients did not show any resistance to the infection. All SCID mice exposed to the pathogen, however, survived after inoculation with patient PBL that were previously depleted of CD30+ cells. The predominance of type 2 T cell responses was demonstrated in PBLs of thermally injured patients. As burn-associated type 2 T cells, CD3+ CD8+ CD30+ IL-4 and IL-10-producing cells were demonstrated in burned patient PBL. These results suggest that burn-associated CD30+ type 2 T cells may play a role on the increased susceptibility of burned patients to C. albicans infection.
Comparative ultrastructure and expression of L-selectin on bovine αβ and γδ T cells
Authors: Leid Jeff G , Speer C A , Jutila Mark A
Published: January 2018

Compared to most αβ T cells, bovine γδ T cells express two to five times the level of L-selectin and have a much higher capacity to roll on monolayers of endothelial cells, platelets, and leukocytes in assays done under physiological flow. To gain additional insight into the basis for these differences, scanning (SEM) and transmission (TEM) electron microscopy were used to compare the cellular ultrastructure of bovine γδ and αβ T cells and to study the expression of L-selectin on these cells. It is interesting that γδ T cells had more than twice as many microvilli and other surface projections on a per cell basis as αβ T cells. This was not due to the γδ T cell being larger; after adhesion and fixation procedures used for the EM studies, γδ T cells averaged 3.9 ± 0.01 μm in diameter, whereas αβ T cells were slightly larger (5.7 ± 0.01 μm in diameter). As previously shown for human neutrophils and lymphocytes, L-selectin was preferentially localized to the tips of the microvilli. In contrast, WC1, a lineage-specific antigen on γδ T cells, was localized on the plasmalemma between the microvilli. Our findings suggest that more effective rolling of γδ T cells in various in vitro flow assays may be due to the greater number of microvilli on γδ T cells leading to a higher number of contact sites during adhesion events. In addition, this physical parameter may explain the increased level of L-selectin expression on γδ versus αβ T cells because L-selectin is clustered at the tips of microvilli. J. Leukoc. Biol. 64: 104–107; 1998.
Blood mononuclear cell production of TNF-α and IL-8: engagement of different signal transduction pathways including the p42 MAP kinase pathway
Authors: Foreback Jami L , Sarma Vidya , Yeager Nicholas R , Younkin Ellen M , Remick Daniel G , Ward Peter A
Published: January 2018

Recent studies of human peripheral blood mononuclear cells (PBMC) stimulated with IgG subclasses have suggested that tumor necrosis factor α (TNF-α) and interleukin-8 (IL-8) production proceed along different signal transduction pathways. To investigate this possibility, inhibitors of signal transduction pathways were employed. Human PBMC were pretreated with various inhibitors before being added to IgG2-coated wells and 4-h supernatant fluids evaluated for cytokine content. The effects of various inhibitors on MAP kinase activation were determined. Inhibitors of protein tyrosine kinases, phosphatases, and phospholipase C decreased TNF-α and IL-8 production, suggesting that all three enzyme pathways are involved in cytokine generation. Inhibitors of G-proteins had differing effects: pertussis toxin inhibited IL-8 but not TNF-α production, whereas cholera toxin inhibited TNF-α but not IL-8 production. Pretreatment of PBMC with pertussis toxin resulted in reduced IgG2-induced calcium mobilization, whereas cholera toxin had no effect, correlating with the effects of pertussis toxin on IL-8 expression. Inhibitors of protein kinase C (PKC) completely blocked TNF-α generation but had no effect on IL-8 production. Gö6976, which inhibits certain isoforms of PKC, inhibited production of both IL-8 and TNF-α. Isoforms of PKC may have opposing effects on cytokine production. PD 98059, a compound that specifically inhibits the activation of mitogen-activated protein kinase kinase (MEK1), inhibited TNF-α production, but had insignificant effects on IL-8 production. Pretreatment of PBMC with either PD 98059 or genistein reduced the extent of phosphorylation of p42 MAP kinase in cells activated on contact with IgG2. These findings suggest distinct signal transduction pathways for cytokine production in PBMC stimulated with IgG2. J. Leukoc. Biol. 64: 124–133; 1998.
Giant granules of neutrophils in Chediak-Higashi syndrome are derived from azurophil granules but not from specific and gelatinase granules
Authors: Kjeldsen L , Calafat J , Borregaard N
Published: January 2018

The abnormal giant granules of Chediak-Higashi syndrome (CHS) neutrophils in humans are thought to be derived from both azurophil and specific granules, whereas the presence of gelatinase granules and their contribution to giant granule formation has not been investigated previously. We have examined the ultrastructure and mobilization of neutrophil granules from a patient with CHS by immunogold electron microscopy and exocytosis experiments of isolated leukocytes. The giant granules contained the azurophil granule components myeloperoxidase and CD63. We found no evidence of involvement of specific or gelatinase granules in the formation of giant granules because lactoferrin and gelatinase were contained in normal-appearing peroxidase-negative granules. On stimulation of leukocytes with N-formyl-methionyl-leucyl-phenylalanine and the calcium ionophore, ionomycin, there was a diminished exocytosis of myeloperoxidase in CHS compared with a healthy control, indicating a lack of mobilization of the giant granules. On the other hand, there was a normal or augmented release of lactoferrin and gelatinase in CHS neutrophils, with gelatinase granules being the most easily mobilized, as known from normal neutrophils. In conclusion, giant granules from CHS neutrophils originate from azurophil granules but not from the specific and gelatinase granules. J. Leukoc. Biol. 64: 72–77; 1998.
A soluble form of the urokinase plasminogen activator receptor (suPAR) can bind to hematopoietic cells
Authors: Mizukami Ikuko F , Todd Robert F
Published: January 2018

The receptor for urokinase plasminogen activator (uPAR; CD87) is a 50- to 65-kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed by leukocytes and tumor cells where it facilitates uPA-dependent, plasmin-mediated pericellular proteolysis during cellular invasion. Because uPAR is inducibly shed into culture supernatants and human body fluids, we tested the hypothesis that soluble uPAR (suPAR) can bind to the plasma membrane of hematopoietic cells where it might modulate their invasive phenotype. As measured by flow cytometry, recombinant biotinylated-suPAR (B-suPAR) bound in a specific fashion to THP-1 leukemia cells and blood PMNs and monocytes (but not to lymphocytes). B-suPAR also demonstrated specific binding to a variety of leukemic lines, including cells that are positive or negative for membrane uPAR expression. Binding of B-suPAR to THP-1 cells was enhanced four- to sevenfold by 24-h exposure of cells to PMA or by coincubation with uPA ligand (but not its isolated catalytic and binding fragments). Conversely, binding of B-suPAR to PMNs was unaffected by brief exposure to fMLP, and was inhibited by coincubation with uPA. B-suPAR binding to PMA-differentiated THP-1 cells in the presence of uPA was further enhanced by acid washing (removing endogenous uPA) but was partially inhibited by treatment of cells with trypsin. Pretreatment of PMA-differentiated THP-1 cells and unstimulated PMNs with soluble sugars, calcium chelators, and antibodies specific for integrins or extracellular matrix proteins failed to consistently block the binding of B-suPAR. Whereas the binding of suPAR did not measurably affect cell-associated plasmin activation, suPAR did competitively inhibit the binding of exogenous uPA to membrane-associated uPAR. These observations support the hypothesis that suPAR can bind specifically to trypsin-sensitive receptors expressed by certain normal and neoplastic hematopoietic cells where its binding is variably influenced by uPA ligand. J. Leukoc. Biol. 64: 203–213; 1998.
Interleukin-18/interferon-γ-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells
Authors: Kohka Hideo , Yoshino Tadashi , Iwagaki Hiromi , Sakuma Isao , Tanimoto Tadao , Matsuo Yosinobu , Kurimoto Masashi , Orita Kunzo , Akagi Tadaatsu , Tanaka Noriaki
Published: January 2018

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a membrane glycoprotein and a member of the immunoglobulin superfamily. It plays a central role in cell to cell-mediated immune responses and is a ligand for leukocyte function-associated antigen-1 (LFA-1). We report here that a newly discovered cytokine, interferon-γ-inducing factor (IGIF) [H. Okamura et al. (1995) Nature 378, 88] recently proposed to be designated as IL-18, selectively up-regulates ICAM-1 expression in KG-1 cells, a human myelomonocytic cell line, in which IL-18 also enhances interferon-γ production. IL-18 induced heterotypic aggregation between KG-1 and Peer T cells, which was blocked by anti-ICAM-1 and/or LFA-1 antibodies. Anti-interferon-γ antibody did not block the IL-18-induced up-regulation of ICAM-1 on KG-1 cells. These results thus show that IGIF/IL-18, enhances ICAM-1 expression in KG-1 cells in an interferon-γ-independent pathway, up-regulates ICAM-1 functions, and that IL-18 might play a potential role in immunoregulation by mediating immune cell infiltration into the tissues. J. Leukoc. Biol. 64: 519–527; 1998.
The IFN-inducible nucleoprotein IFI 16 is expressed in cells of the monocyte lineage, but is rapidly and markedly down-regulated in other myeloid precursor populations
Authors: Dawson Michelle J , Elwood Ngaire J , Johnstone Ricky W , Trapani Joseph A
Published: January 2018

IFI 16 is an interferon-inducible nucleoprotein expressed by human monocytes. IFI 16 and a related mouse protein, p202, control cellular proliferation by binding and modulating the functions of cell cycle regulatory factors including p53 and the retinoblastoma gene product, pRb. In this study, we examined IFI 16 expression in myeloid precursor cells cultured in vitro in colony-forming assays using granulocyte (G-) and granulocyte-macrophage (GM-) colony-stimulating factor (CSF). IFI 16 was expressed in 100% of CD34+ cells isolated from human bone marrow. When the CD34+ cells were induced to differentiate, two subpopulations of cells were identified by two-color cytofluorography: the CD14+ (monocytoid) cells all expressed IFI 16, whereas the CD14– (polymorphonuclear precursor) cells did not. The strongest expression of IFI 16 was in the cells staining brightest for CD14, whereas depletion of CD14+ monocytoid cells from mixed monocytic/granulocytic cultures largely abolished IFI 16-stained cells. Furthermore, in eight independent colony-forming assays, the number of IFI 16+ cells correlated closely with the numbers of monocyte precursors identified morphologically (R2 = 0.99), but was unrelated to the numbers of myelocytes, promyelocytes, and metamyelocytes; nor was IFI 16 expressed by erythroid or eosinophil precursors. We conclude that IFI 16 is expressed in CD34+ and monocytoid daughter cells, but is rapidly and markedly down-regulated at the corresponding stages of polymorphonuclear and erythroid development. This differential expression of IFI 16 in myeloid precursor subpopulations and its perceived molecular properties are consistent with a possible role in regulating myelopoiesis. J. Leukoc. Biol. 64: 546–554; 1998.
Role of the mitogen-activated protein kinases and tyrosine kinases during leukotriene B4-induced eosinophil activation
Authors: Lindsay Mark A , Haddad El-Bdaoui , Rousell Jonathan , Teixeira Mauro M , Hellewell Paul G , Barnes Peter J , Giembycz Mark A
Published: January 2018

Exposure of guinea-pig eosinophils to leukotriene B4 (LTB4; 1 μM) resulted in a rapid generation of H2O2 (index of NADPH oxidase activation), stimulated [3H]arachidonic acid (AA) release (index of phospholipase A2 activity), and promoted CD18-dependent homotypic aggregation. Under similar conditions, LTB4 (1 μM) induced a rapid activation of extracellular-regulated kinases-1 and 2 (ERK-1/2) but not c-jun N-terminal kinases 46 and 54 (JNK-46/54) or p38 mitogen-activated protein kinase (p38 MAP kinase). To examine the role of ERK-1/2 in the mechanism of eosinophil activation, a selective inhibitor of MAP kinase kinase-1/2 (MEK-1/2), PD098059, was employed. However, PD 098059 at concentrations that attenuated ERK-1/2 activation had no significant affect on eosinophil activation. In contrast, a role for tyrosine kinases in LTB4-induced eosinophil activation was suggested by studies with the tyrosine kinase inhibitors, herbimycin A and lavendustin A. However, the results of those experiments implied divergent pathways for the control of eosinophil responses because the inhibitors were more effective at attenuating H2O2 generation than [3H]AA release, and had little effect on homotypic aggregation. J. Leukoc. Biol. 64: 555–562; 1998.
β2-Integrins mediate stable adhesion in collisional interactions between neutrophils and ICAM-1-expressing cells
Authors: Lynam Eric , Sklar Larry A , Taylor Andrew D , Neelamegham Sriram , Edwards Bruce S , Smith C Wayne , Simon Scott I
Published: January 2018

The aggregation of human neutrophils in suspension has features that are analogous to their attachment to activated endothelium in that both involve selectin and β2-integrin adhesion receptors. For the collisional interaction that forms neutrophil aggregates in suspension, there is a tethering step in which L-selectin on neutrophils binds PSGL-1. At relatively low shear rates (100–200 s-1) firm adhesion is mediated in equal measure by LFA-1 binding to ICAM-3, and Mac-1 binding to an as yet undefined ligand. In this report we used a mouse melanoma cell line expressing an estimated 700,000 ICAM-1 (CD54) to examine the relative roles of LFA-1 and Mac-1 over the kinetics of heterotypic cell adhesion in shear mixed suspensions. Neither heterotypic nor homotypic neutrophil aggregates formed with application of shear alone. However, the rate of aggregation peaked within seconds of chemotactic stimulation. In contrast to homotypic aggregation, neither L-selectin nor its O-glycoprotein ligands on neutrophils contributed to heterotypic adhesion. Adhesion was inhibited in a dose-dependent manner as ICAM-1 was titrated with blocking mAb. A direct interaction between LFA-1 and ICAM-1 was preferred over the first minute of stimulation, whereas at later times adhesion was supported equally by Mac-1. Activation with MnCl2 also favored participation of the constitutively expressed LFA-1. Application of defined shear in a cone and plate viscometer showed that adhesion to the ICAM-1 cells decreased from a maximum level to baseline as shear rate increased up to 400 s-1 in a manner typical of integrin adhesion alone. In contrast, homotypic aggregation supported by the transition from selectin to integrin binding exhibited an increase in efficiency up to 800 s-1. The pathophysiological significance of receptor site density and duration of contact in collisional interactions relevant to leukocyte recruitment compared to leukocyte-endothelial cell interactions on surfaces is discussed. J. Leukoc. Biol. 64: 622–630; 1998.
Macrophage/fibroblast coculture induces macrophage inflammatory protein-1α production mediated by intercellular adhesion molecule-1 and oxygen radicals
Authors: Steinhauser Matthew L , Kunkel Steven L , Hogaboam Cory M , Evanoff Holly , Strieter Robert M , Lukacs Nicholas W
Published: January 2018

This study examined the cell-to-cell interaction between fibroblasts and macrophages as a possible contributor to the chronic inflammatory state. In a coculture system, consisting of macrophages layered over confluent fibroblasts, there was a significant increase in macrophage inflammatory protein 1α (MIP-1α) compared with control cultures. ICAM-1 adhesion was identified as an important stimulus of MIP-1α production by using ICAM-1-specific monoclonal antibodies. Furthermore, fibroblasts from ICAM-1 knockout mice induced significantly less MIP-1α production from peritoneal macrophages when compared to control fibroblasts. In addition, it appeared that oxygen radicals functioned as activating molecules during cellular interaction and production of MIP-1α, as the addition of the antioxidant N-acetylcysteine (NAC) prevented MIP-1α secretion. Thus, the ICAM-1 and oxygen radical-mediated induction of MIP-1α associated with a macrophage/fibroblast coculture system provides one possible mechanism by which immune/inflammatory cell interactions may augment chemokine production and exacerbate chronic inflammatory diseases. J. Leukoc. Biol. 64: 636–641; 1998.
Glycosaminoglycan profile in macrophages exposed to Candida albicans and interleukins
Authors: Bodo Maria , Blasi Elisabetta , Becchetti Ennio , Giammarioli Monica , Conte Carmela , Bellocchio Silvia , Baroni Tiziano , Bellucci Catia , Bistoni Francesco
Published: January 2018

Glycosaminoglycans (GAG), are extracellular matrix macromolecules that affect the phagocytic properties of macrophages. In order to assess whether the interaction between macrophages and Candida albicans (iCa) provokes changes in the phenotype, we analyzed the GAG profiles in two macrophage lines, ANA-1 (from murine bone-marrow) and BV-2 (from murine brain). We also investigated GAG modulation by interleukin-1α (IL-1α) and interleukin-6 (IL-6). During iCa treatment and even after the addition of ILs, ANA-1 accumulated less total GAG compared to controls. IL-1 treatment, combined with iCa exposure, induced a decrease in heparan sulfate and chondroitin sulfate chains, and an increase in the hyaluronic acid percentage. IL-6 treatment, with or without iCa, decreased the hyaluronic acid/sulfated GAG ratio. The GAG pattern in BV-2 appears to be different to ANA-1 and iCa exposure does not induce any difference in total GAG. The inhibitory effect induced by ILs on GAG synthesis is less than that observed in ANA-1 and the GAG elution profile is modulated to a lesser extent by treatment with ILs and/or iCa compared to the ANA-1. We suggest that the observed changes in the expression of the individual GAG classes may be responsible for the macrophage functional heterogeneity. J. Leukoc. Biol. 64: 650–656; 1998.
Murine DEP-1, a receptor protein tyrosine phosphatase, is expressed in macrophages and is regulated by CSF-1 and LPS
Authors: Osborne Julie M , Elzen Nicole den , Lichanska Agnieszka M , Costelloe Elaine O , Yamada Toshiya , Cassady A Ian , Hume David A
Published: January 2018

The spectrum of protein tyrosine phosphatases (PTPs) expressed in bone marrow-derived murine macrophages (BMMs) was examined using reverse transcriptase-polymerase chain reaction. Ten different PTP cDNAs were isolated and in this study we focus on mDEP-1, a type III receptor PTP. Three mDEP-1 transcripts were expressed in primary macrophages and macrophage cell lines and were induced during macrophage differentiation of M1 myeloid leukemia cells. A variant mRNA was identified that encodes an alternate carboxyl-terminus and 3 UTR. The expression of mDEP-1 was down-regulated by CSF-1 (macrophage colony-stimulating factor) and up-regulated by bacterial lipopolysaccharide, an important physiological regulator of macrophage function that opposes CSF-1 action. Whole mount in situ hybridization, and immunolocalization of the protein, confirmed that mDEP-1 is expressed by a subset of embryonic macrophages in the liver and mesenchyme. mDEP-1 was also detected in the eye and peripheral nervous system of the developing embryo. Attempts to express mDEP-1 constitutively in the macrophage cell line RAW264 were unsuccessful, with results suggesting that the gene product inhibits cell proliferation. J. Leukoc. Biol. 64: 692–701; 1998.
Extrathymic T cell differentiation in vitro from human CD34+ stem cells
Authors: Pawelec Graham , Muller Robert , Rehbein Arnika , Hähnel Karin , Ziegler Benedikt L
Published: January 2018

Although it is well established that T cells are derived from CD34+ stem cells in vivo, and that T cells can develop in the absence of a functioning thymus, it has not proven possible thus far to generate human T cells in vitro from CD34+ cells in the absence of any thymic influence. We now present a limiting dilution cloning culture system that supports the differentiation of highly purified human CD34+ cells to CD3+ T cells in vitro in the complete absence of any thymic components. The culture system features the use of a serum-free medium supplemented with a cocktail of cytokines including flt-3 ligand, interleukin-3 (IL-3), stem cell factor (SCF), and IL-2. CD4+ T cell clones capable of mitogen-stimulated proliferation and response to IL-2, and expressing a varied TCR-Vβ repertoire were obtained under these conditions. This culture system therefore supports human T lymphopoiesis in the absence of any thymic influence and may prove useful for the evaluation of extrathymic T cell differentiation in vitro. J. Leukoc. Biol. 64: 733–739; 1998.
Thermal injury alters macrophage responses to prostaglandin E2: contribution to the enhancement of inducible nitric oxide synthase activity
Authors: Schwacha Martin G , Samy T S Anantha , Catania Robert A , Chaudry Irshad H
Published: January 2018

Prostaglandin E2 (PGE2) and macrophage (Mϕ) -derived reactive nitrogen intermediates (RNI) have been implicated in T cell dysfunction after thermal injury. Normally, Mϕ inducible nitric oxide synthase (iNOS) activity can be regulated by PGE2, however, it is unknown whether PGE2 modulates Mϕ iNOS activity after thermal injury. Splenic Mϕ isolated from mice 7 days after thermal injury produced higher levels of RNI than Mϕ from sham mice when stimulated with lipopolysaccharide (LPS) or interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) in combination. PGE2, when added concurrently with LPS, suppressed RNI production by Mϕ from sham mice, whereas Mϕ from injured mice were unaffected. When Mϕ were pretreated with PGE2 before LPS, RNI production was suppressed in both populations. RNI production in response to IFN-γ or IFN-γ and TNF-α in combination was enhanced by PGE2 in both populations, however, the effect was markedly greater in Mϕ from injured mice. The PGE2-mediated changes in RNI production were paralleled by similar changes in iNOS protein expression, suggesting that the effect of PGE2 was at the level of enzyme expression rather than activity. Dibutryl cAMP induced similar effects as PGE2, suggesting the response to PGE2 after thermal injury is independent of potential changes in PGE2-induced adenylate cyclase activity and is cAMP-mediated. The results indicate that Mϕ from burned mice display an altered sensitivity to PGE2, resulting in enhanced iNOS activity. Thus, PGE2, which is elevated after thermal injury and can directly suppress T cell function, may also contribute to immune dysfunction through the enhancement of Mϕ iNOS activity. J. Leukoc. Biol. 64: 740–746; 1998.
Nucleosomes inhibit phagocytosis of apoptotic thymocytes by peritoneal macrophages from MRL+/+ lupus-prone mice
Authors: Laderach Diego , Bach Jean-François , Koutouzov Sophie
Published: January 2018

The nucleosome, the basic structure of chromatin and normal product of cell apoptosis, plays a pivotal role both in the induction and the pathogenesis of systemic lupus erythematosus (SLE). Nucleosomes have been found to circulate at high levels in patients with SLE and apoptosis of lymphoid cells is increased during human and murine lupus. In this study, we examined the presence of possible defects in clearance mechanisms of apoptotic cells in murine lupus, and questioned further whether nucleosomes could compromise this phagocytic process. There did not appear to be any intrinsic functional defect of macrophages from young MRL+/+ lupus-prone mice to recognize and phagocytose apoptotic thymocytes. Nucleosomes, as a mimic of increased cell apoptotsis in vivo, induced a strong, dose-dependent, inhibition of phagocytosis of apoptotic thymocytes by young, pre-autoimmune, macrophages of MRL+/+ mice, whereas macrophages of non-autoimmune C3H mice only exhibited a trend to inhibition. The nucleosome-elicited inhibitory effect persisted during the development of the autoimmune response and appeared to be specific for the molecular mechanisms involved in macrophage phagocytosis of apoptotic cells. Our data suggest that nucleosome elicited inhibition of phagocytosis of apoptotic cells by MRL+/+ macrophages before the onset of the autoimmune response contribute, in a positive loop, to sustain and/or augment the levels of circulating (and potentially immunogenic) nucleosomes in lupus. J. Leukoc. Biol. 64: 774–780; 1998.
CD43 is associated with tyrosine kinase activity in human neutrophils
Authors: Skubitz Keith M , Campbell Kenneth D , Skubitz Amy P N
Published: January 2018

CD43 appears to be involved in signal transduction, however, the mechanism of this function is unknown. Protein kinase activity was detected in neutrophils associated with CD43. Most of the protein kinase activity associated with these antigens was tyrosine kinase activity. The src family kinases lyn and hck were found to account for much of the associated tyrosine kinase activity. The data suggest that associated tyrosine kinase activity may play a role in signal transduction via CD43 to regulate other cell functions. J. Leukoc. Biol. 64: 800–802; 1998.
CSF-1 regulation of I6 gene expression by murine macrophages: a pivotal role for GM-CSF
Authors: Evans Robert , Shultz Leonard D , Dranoff Glen , Fuller Jane A , Kamdar Sonya J
Published: January 2018

We test the hypothesis that the monocyte-macrophage colony-stimulating factor (CSF-1 or M-CSF) plays a major role in the inflammatory responses of Mϕ by acting as a priming agent that heightens their responsiveness to secondary stimulation by other mediators. We previously reported that CSF-1 induced peritoneal Mϕ (PMϕ) to transcribe several genes including interleukin-6 (I6) and granulocyte-macrophage colony-stimulating factor (Csfgm). It was reported that the I6 and Csfgm genes were individually regulated by different pathways but it was not clear to what extent the two genes interacted during Mϕ-mediated inflammatory responses. We now show that CSF-1 induces the release of bioactive GM-CSF from mouse resident PMϕ. GM-CSF induces I6 gene expression and synergizes with CSF-1 to induce the release of large amounts of IL-6. PMϕ from C57BL/6J-Csfgmnull mice were shown to release minimal IL-6 in response to CSF-1 and to express a much reduced response to the highly stimulatory combination of CSF-1 and lipopolysaccharide (LPS). Exogenous recombinant GM-CSF restored the IL-6 response of GM-CSF null PMϕ to a great extent but not completely. As controls, three other recombinant proteins were tested but of these only tumor necrosis factor α (TNF-α) was shown to synergize with both CSF-1 and GM-CSF. Using PMϕ from mice deficient in the expression of the I6 gene, it was shown that they released two- to threefold more GM-CSF in response to CSF-1 than their control counterparts. However, an exogenous supply of recombinant IL-6 had no effect on GM-CSF release. The data indicate that the pathways regulating I6 gene expression are under the control of a complex network of cytokine interactions involving at least CSF-1, GM-CSF, and TNF-ϕ, with the added possibility that IL-6 may exert modulatory activity within this network. J. Leukoc. Biol. 64: 810–816; 1998.
GM-CSF-transduced B16 melanoma cells are highly susceptible to lysis by normal murine macrophages and poorly tumorigenic in immune-compromised mice
Authors: Kumar Rakesh , Yoneda Junya , Fidler Isaiah J , Dong Zhongyun
Published: January 2018

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-transduced B16-F10 murine melanoma cells had lower tumorigenicity in both syngeneic and nude mice than parental or LacZ-transduced (control) cells. The subcutaneous (s.c.) tumors producing GM-CSF were densely infiltrated with macrophages, whereas the control tumors were not. In vitro studies showed that GM-CSF-transduced B16 cells were susceptible to lysis mediated by nonactivated murine macrophages, whereas control B16 cells were not. Macrophage-mediated cytotoxicity against GM-CSF-transduced B16 cells was independent of the presence of NO, H2O2, O2-, tumor necrosis factor α, and matrix metalloproteinase. Coculture experiments using GM-CSF-producing and -nonproducing B16 cells demonstrated that GM-CSF produced by the transduced B16 cells activated macrophages to kill the bystander non-GM-CSF-producing tumor cells. The results suggest that GM-CSF released by tumor cells can induce macrophage-mediated cytotoxicity, which in turn can inhibit the in vivo growth of GM-CSF-transduced tumor cells. J. Leukoc. Biol. 65: 102–108; 1999.
Significant expression of G-CSF-induced gene-1 (GIG-1) protein in myeloid cells and NK cells
Authors: Shimane M , Tani K , Hibino H , Setoyama M , Takahashi S , Tojo A , Yodoi J , Asano S
Published: January 2018

The granulocyte colony-stimulating factor (G-CSF)-induced gene, GIG-1, was originally cloned from G-CSF-stimulated bone marrow mononuclear cells obtained from a patient with chronic myelogenous leukemia (CML). We have characterized the GIG-1 gene and its protein product. Expression of GIG-1 mRNA was elevated by treatment with G-CSF in normal bone marrow mononuclear cells, as well as in some cases of blast cells obtained from patients with acute myelogenous leukemia (AML) and CML. Western blot analysis with antiGIG-1 peptide antiserum showed the molecular massofGIG-1 product was about 17 kDa. Immuno-staining of the hematopoietic cells demonstrated that GIG-1 product was mainly localized to the cytoplasm of both myeloid and natural killer (NK) cells. These results suggested that GIG-1 protein is an integral component that is accumulated during the differentiation of myeloid cells toward the stage of mature neutrophils. Expression of GIG-1 gene in mature neutrophils was tightly regulated and reactivation of GIG-1 gene by G-CSF in mature neutrophils may represent a compensation process for the protein lost through the activation of these cells, thus implying an important role for this protein in host defense mechanisms. J. Leukoc. Biol. 65: 109–116; 1999.
Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production
Authors: Bertholet S , Tzeng E , Felley-Bosco E , Mauël J
Published: January 2018

Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an important component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas, under similar conditions, human macrophages produce low levels or no NO at all. Although human macrophages can express iNOS mRNA and protein on activation, whether they possess the complete machinery necessary for NO synthesis remains controversial. To define the conditions necessary for human monocytes/macrophages to synthesize NO when expressing a functional iNOS, the human monocytic U937 cell line was engineered to synthesize this enzyme, following infection with a retroviral expression vector containing human hepatic iNOS (DFGiNOS). Northern blot and Western blot analysis confirmed the expression of iNOS in transfected U937 cells both at the RNA and protein levels. NOS enzymatic activity was demonstrated in cell lysates by the conversion of L-[3H]arginine into L-[3H]citrulline and the production of NO by intact cells was measured by nitrite and nitrate accumulation in culture supernatants. When expressing functional iNOS, U937 cells were capable of releasing high levels of NO. NO production was strictly dependent on supplementation of the culture medium with tetrahydrobiopterin (BH4) and was not modified by stimulation of the cells with different cytokines. These observations suggest that (1) human monocytic U937 cells contain all the cofactors necessary for NO synthesis, except BH4 and (2) the failure to detect NO in cytokine- stimulated untransfected U937 cells is not due to the presence of a NO-scavenging molecule within these cells nor to the destabilization of iNOS protein. DFGiNOS U937 cells represent a valuable human model to study the role of NO in immunity toward tumors and pathogens. J. Leukoc. Biol. 65: 50–58; 1999.
Priming of human neutrophils by peroxynitrite: potential role in enhancement of the local inflammatory response
Authors: Rohn Troy T , Nelson Laura K , Sipes Karen M , Swain Steve D , Jutila Kathryn L , Quinn Mark T
Published: January 2018

Peroxynitrite is a potent oxidant generated from the reaction of nitric oxide (NO) and superoxide anion (O2−), both of which can be produced in inflammatory tissues. In these studies, we analyzed what direct effect peroxynitrite had on neutrophil (PMN) function. We found that peroxynitrite was an effective priming agent for PMNs, as demonstrated by enhanced O2− production on subsequent activation with low doses of PMA or N-formyl-methionine-leucine-phenylalanine (fMLF), changes in the expression of PMN surface markers (L-selectin, Mac-1, flavocytochrome b, and fMLF receptor), and increased intracellular calcium levels. Analysis of the mechanism of PMN priming by peroxynitrite demonstrated that peroxynitrite resulted in minimal oxidation of protein sulfhydryl groups and subsequent protein cross-linking. In contrast, treatment of PMNs with peroxynitrite resulted in significant nitration of tyrosine residues on neutrophil proteins. In addition, inhibition of tyrosine nitration with a pyrrolopyrimidine antioxidant blocked the majority of peroxynitrite-induced priming effects, further suggesting that PMN priming was mediated primarily by nitration of tyrosine residues on PMN proteins. The ability of peroxynitrite to serve as an effective priming agent for PMNs at sites of inflammation may play a key role in modulating the host-defense process. J. Leukoc. Biol. 65: 59–70; 1999.
Identification of a chicken “C” chemokine related to lymphotactin
Authors: Rossi D , Sanchez-García J , McCormack W T , Bazan J F , Zlotnik A
Published: January 2018

In this study we describe the isolation and characterization of a new chicken (Gallus gallus) chemokine. This molecule belongs to the C or γ-chemokine family and is related to the mouse and human lymphotactin (Lptn). Mouse and human Lptn are distinguished from α and β chemokines by the absence of two cysteines (Cys 1 and 3) that form a disulfide bridge; the novel chicken chemokine shows the same cysteine pattern, but replaces a long carboxy-terminal tail found in the other Lptn proteins with a short extension rich in Arg residues. The 1-kb mRNA is mainly expressed in spleen, although weaker signals have been detected in liver and colon. It is interesting to note that the chicken chemokine seems to preferentially induce the migration of spleen B cells over T cells or B cells from the bursa of Fabricius. J. Leukoc. Biol. 64: 87–93; 1999.
Soluble complement receptor 1 is increased in patients with leukemia and after administration of granulocyte colony-stimulating factor
Authors: Sadallah Salima , Lach Estelle , Schwarz Sybille , Gratwohl Alois , Spertini Olivier , Schifferli Jürg-Alfred
Published: January 2018

Complement receptor type 1 is expressed by erythrocytes and most leukocytes. A soluble form is shed from the leukocytes and found in plasma (sCR1). sCR1 is a powerful inhibitor of complement. We report an increased sCR1 in the plasma of leukemia patients, up to levels producing measurable complement inhibition. Half of the 180 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) had sCR1 levels above the normal range. The highest levels were observed in T-ALL (17 patients). The complement function of a T-ALL serum was improved by blocking sCR1 with a specific mAb (3D9). Measurements in 16 peripheral stem cell donors before and after granulocyte colony-stimulating factor (G-CSF) administration showed an increase in sCR1 (before, 43.8 ± 15.4; at day 5, 118.3 ± 44.7 ng/mL; P < 0.0001). This increase paralleled the increase in total leukocyte counts and was concomitant with de novo leukocyte mRNA CR1 expression in all three individuals tested. Whether pharmacological intervention may be used to up-regulate sCR1 so as to inhibit complement in vivo should be further investigated. J. Leukoc. Biol. 65: 94–101; 1999.
T cell independence of bleomycin-induced pulmonary fibrosis
Authors: Helene M , Lake-Bullock V , Zhu J , Hao H , Cohen D A , Kaplan A M
Published: January 2018

The role of T cells and cytokines in bleomycin (BLM)-induced fibrosis was evaluated in susceptible and resistant strains of normal and SCID mice. Histology and hydroxyproline analysis showed that BLM induced pulmonary fibrosis in C57BL/6 and (C57BL/6 × BALB/c)F1 mice, whereas BALB/c mice were resistant to the disease. To test whether lymphocytes were required for the induction of BLM-induced pulmonary fibrosis, SCID mice were injected intratracheally with BLM and evaluated for the development of pulmonary inflammation and fibrosis. Similar morphological changes and increases in hydroxyproline were observed in both C57BL/6 SCID and (C57BL/6 × CB.17)F1 SCID animals compared to those seen in wild-type C57BL/6 and (C57BL/6 × BALB/c)F1 mice. In contrast, CB.17 SCID mice, which are genetically similar to BALB/c mice, were resistant to disease induction. Analysis of the cellular infiltrate in BLM-treated C57Bl/6 SCID mice confirmed a lack of T cells in the lungs of SCID mice and demonstrated a pronounced accumulation of eosinophils in areas of developing pulmonary fibrosis. NK cells were significantly elevated in untreated SCID mice and did not increase further after BLM treatment. Analysis of selected cytokines 1 day after initiation of BLM-induced pulmonary fibrosis indicated that the levels of TNF-α and IFN-γ appeared to segregate with fibrosis in both the SCID and wild-type mice. The data demonstrate that T cells are not required for the induction of fibrosis by BLM and suggest that responses by non-lymphoid cells may be sufficient for the induction of fibrosis. J. Leukoc. Biol. 65: 187–195; 1999.
Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils
Authors: Lundqvist-Gustafsson Helen , Bengtsson Torbjörn
Published: January 2018

Oxidative stress induces apoptosis in many types of cells, including human neutrophils. Our objective was to determine whether reactive oxygen species (ROS) produced by activated neutrophils are associated with accelerated apoptosis. Exposing neutrophils to ionomycin or phorbol myristate acetate (PMA) induced intracellular H2O2 production and rapid onset of apoptosis, measured as condensed chromatin, cellular shrinkage, and DNA fragmentation. Neutrophils activated with formyl-methionyl-leucyl-phenylalanine (fMLP) generated mainly extracellular H2O2 and did not undergo apoptosis. Exogenously added H2O2, together with the catalase blocker sodium azide, induced apoptosis to the same extent and with similar kinetics as PMA and ionomycin. Adenosine inhibited ionomycin-induced intracellular H2O2 production and apoptosis. Neither PMA nor ionomycin caused apoptosis in dimethyl sulfoxide-differentiated HL-60 cells, which are incapable of intracellular H2O2 production, whereas H2O2 induced apoptosis more efficiently in these cells than in neutrophils. We propose that activated neutrophils use intracellularly formed H2O2 to commit suicide. J. Leukoc. Biol. 65: 196–204; 1999.
Functions of Bruton's tyrosine kinase in mast and B cells
Authors: Kawakami Yuko , Kitaura Jiro , Hata Daisuke , Yao Libo , Kawakami Toshiaki
Published: January 2018

Bruton's tyrosine kinase (Btk) plays crucial roles in B cell differentiation as well as mast cell activation through the high-affinity IgE receptor (Fc∊RI). Defects in the btk gene lead to agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Mast cells from xid and btk null mice exhibit mild defects in degranulation and severe impairments in the production of proinflammatory cytokines upon Fc∊RI cross-linking. Recent studies demonstrated the role of Btk in a sustained increase in intracellular calcium concentrations in response to antigen receptor stimulation. Btk is also involved in the activation of stress-activated protein kinases, JNK/SAPK1/2, and thereby regulates c-Jun and other transcription factors that are important in cytokine gene activation. Regulation of the JNK/SAPK activation pathway by Btk may be related to the proapoptotic function of Btk in the programmed cell death in these hematopoietic cells. J. Leukoc. Biol. 65: 286–290; 1999.