bluuebunny/crossref_metadata_2025_split huggingface dataset:
β2 integrins mediate protein tyrosine phosphorylation in human neutrophils
Authors: Walzog Barbara , Offermanns Stefan , Zakrzewicz Andreas , Gaehtgens Peter , Ley Klaus 
Published: January 2018

Abstract
Tyrosine kinases play a prominent role in various intracellular signal transduction pathways. In this study, β2 integrin (CD11/CD18)-mediated adhesive interactions of human neutrophils (PMN) were mimicked by antibody cross-linking of GD11/CD18. Cross-linking of CD18 induced tyrosine phosphorylation of several proteins with apparent molecular masses of ~120, 78, 72, 65, and 56 kDa, respectively, as shown by anti-phosphotyrosine immunoblotting of whole cell lysates. Cross-linking of the α-subunits of the β2 integrins demonstrated that only cross-linking of Mac-1 but not LFA-1 or gp150/95 triggered tyrosine signaling. The tyrosine phosphorylations showed a rapid and transient time course. Comparison of the CD18-mediated tyrosine phosphorylation patterns with those induced by chemoattractants gave similar results. The observed tyrosine phosphorylation was specific, since binding and non-binding irrelevant primary antibodies had no effect. Furthermore, F(ab’)2 fragments of the anti-CD18 antibody IB4 and addition of F(ab’)2 fragments of secondary antibody were sufficient to induce tyrosine phosphorylation. Inhibition of tyrosine kinases by herbimycin A resulted in partial inhibition of the CD18-mediated tyrosine phosphorylation of the 120- and 65-kDa proteins and in complete inhibition of tyrosine phosphorylation of the 78- and 56-kDa proteins. In unstimulated PMN, the tyrosine phosphatase inhibitor sodium orthovanadate had no effect on tyrosine phosphorylation of the 120-kDa protein, but induced phosphorylation of the 78-, 72-, 65-, and 56-kDa proteins. These results indicate that the β2 integrin-mediated intracellular signaling cascade involves different tyrosine phosphorylation (and dephosphorylation) events, which may play a role in the regulation of PMN functions during inflammation.
Synthesis of 5-oxo-6,8, 11, 14-eicosatetraenoic acid by human monocytes and lymphocytes
Authors: Zhang Ying , Styhler Angela , Powell William S 
Published: January 2018

Abstract
We recently demonstrated that the arachidonate metabolite 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) is converted by a highly specific dehydrogenase in human neutrophils to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), which is a potent stimulator of these cells. The objective of this study was to determine whether 5-oxo-ETE is also formed by monocytes and lymphocytes. Human monocytes (74 ± 2% pure) and lymphocytes (86 ± 1% pure) were prepared by successive centrifugations of leukocytes over Ficoll-Paque and Percoll. Both cell types converted 5-HETE to a single major product, which was identified as 5-oxo-ETE. The formation of 5-oxo-ETE was stimulated about twofold by phorbol myristate acetate (PMA; 30 nM). Dehydrogenase activity in monocyte fractions did not appear to be due to platelet contamination, since depletion of platelets did not reduce enzyme activity. The dehydrogenase was localized in membrane fractions from monocytes and required NADP+ as a cofactor. It was specific for eicosanoids containing a 5S-hydroxyl group followed by a 6-trans double bond. We also investigated the formation of 5-oxo-ETE from endogenous arachidonic acid by monocytes. 5-Oxo-ETE, 5-HETE, and leukotriene B4 (LTB4) were present in comparable amounts after incubation of these cells with A23187. PMA (EC50 ~4 nM) stimulated the formation of 5-oxo-ETE and 5-HETE and, to a lesser extent, LTB4. Although monocytes released considerably less 5-HETE and LTB4 than neutrophils, they released comparable amounts of 5-oxo-ETE. Unlike neutrophils, monocytes did not convert any of these substances to detectable amounts of -oxidation products. Although lymphocytes were capable of converting 5-HETE to 5-oxo-ETE, they released little or no 5-lipoxygenase products in response to A23187. We conclude that monocytes have a high capacity to synthesize 5-oxo-ETE and that its formation is stimulated by activation of protein kinase C.
Paradoxical effects of colchicine on the activation of human neutrophils by chemotactic factors and inflammatory microcrystals
Authors: Roberge C J , Gaudry M , Gilbert C , Malawista S E , de Médicis R , Lussier A , Poubelle P E , Naccache P H 
Published: January 2018

Abstract
Neutrophil activation by chemotactic factors and by inflammatory microcrystals is accompanied by increases in protein tyrosine phosphorylation and by the activation of the NADPH oxidase. The addition of colchicine inhibited both responses induced by triclinic monosodium urate or calcium pyrophosphate crystals. On the other hand, colchicine enhanced the tyrosine phosphorylation of specific protein in neutrophils stimulated by chemotactic factor and augmented the production of superoxide anions induced by these same agonists. The effects of colchicine were shared by other anti-microtubule agents (nocodazole and vinblastine) but not by its inactive analogue β-lumicolchicine, trimethylcolchicinic acid, indomethacin, or phenyl-butazone. Furthermore, the (enhancing as well as inhibitory) effects of colchicine on tyrosine phosphorylation and superoxide anion production were reversed by taxol. Finally, in human cytoplasts colchicine again inhibited microcrystal-stimulated tyrosine phosphorylation but did not change chemotactic factor-stimulated phosphorylation. These data strongly support the hypothesis that microtubule-related mechanisms are involved in the modulation of the tyrosine phosphorylation response in human neutrophils, and suggest that a relationship may exist between the augmentation of tyrosine phosphorylation and of the stimulation of the NADPH oxidase induced by chemotactic factors.
CD66a, CD66b, CD66c, and CD66d each Independently stimulate neutrophils
Authors: Skubitz Keith M , Campbell Kenneth D , Skubitz Amy P N 
Published: January 2018

Abstract
Four members of the earcinoembryonic antigen family, CD66a, CD66b, CD66c, and CD66d, are expressed on human neutrophils. In neutrophils these proteins are activation antigens in that their surface expression is increased following stimulation. To examine their potential role in neutrophil signaling, the effects on neutrophil adhesion to human umbilical vein endothelial cells of a panel of well-characterized CD66 mAbs was tested. CD66a, CD66b, CD66c, and CD66d antibodies each increased neutrophil adhesion to human umbilical vein endothelial cell monolayers. This increase in neutrophil adhesion caused by CD66 antibodies was blocked by a CD18 antibody and associated with up-regulation of GD11/CD18 on the neutrophil surface. This increase in neutrophil adhesion required physiological extracellular calcium concentrations at or near the time of CD66 antibody binding to the neutrophil. The incubation of CD66 antibodies with neutrophils in the absence of calcium for 10 min before repletion of calcium resulted in no increase in neutrophil adhesion. The data suggest that CD66a, CD66b, CD66c, and CD66d antibody binding to the neutrophil surface triggers a transient activation signal that requires extracellular calcium and regulates the adhesive activity of CD11/CD18. Sequential desensitization experiments indicated that CD66a, CD66b, CD66c, and CD66d can each independently transmit signals in neutrophils.
Effects of fungal β-glucan and interferon-γ on the secretory functions of murine alveolar macrophages
Authors: Sakurai Teruaki , Ohno Naohito , Yadomae Toshiro 
Published: January 2018

Abstract
We investigated the effect of a fungal component, soluble β-glucan, on secretory functions of murine alveolar macrophages (AMs) in vitro. Stimulation by β-glucan (500 μg/mL) or interferon-γ (IFN-γ; 100 U/mL) alone had a slight effect on AM functions, but when AMs were incubated together with β-glucan and IFN-γ, the production and secretion of some immune mediators, such as nitric oxide, interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α), were markedly augmented. This combined effect of β-glucan and IFN-γ was based on a priming effect of IFN-γ, because prestimulation with IFN-γ followed by β-glucan induced high nitric oxide production of AMs, but reversal of the sequence of treatments had only a slight effect. We also found that preincubation of AMs with IFN-γ enhanced the binding of fluorescein-labeled β-glucan on the AM surface, and this increased binding was abrogated to the control level by the addition of three species of soluble unlabeled (1→3)-β-d-glucans but not by soluble α-glucan. These data imply that the priming effect of IFN-γ on the AM response to β-glucan was dependent, at least in part, on the enhancement of β-glucan specific binding sites on the AM surface. It was suggested that IFN-γ is one of the principal factors controlling the pulmonary immune system against both severe fungal infection and inflammation via AM activation at the alveoli.
Interleukin-10 inhibits interleukin-2-induced tumor necrosis factor production but does not reduce toxicity in C3H/HeN mice
Authors: Lentsch Alex B , Edwards Michael J , Sims David E , Nakagawa Koji , Wellhausen Samuel R , Miller Frederick N 
Published: January 2018

Abstract
Immunotherapy with interleukin-2 (IL-2) is limited by severe side effects thought to be mediated by the activation of immune effector cells and the induction of secondary cytokines including tumor necrosis factor-alpha (TNF-α) and interferongamma (IFN-γ). In C3H/HeN mice the primary IL-2 toxicity is the production of pleural effusion with subsequent respiratory compromise. IL-10 is a cytokine that has been shown to inhibit the generation of secondary cytokines in vitro and in vivo. In this study, in C3H/HeN mice, we tested the ability of IL-10 to inhibit IL-2-induced mononuclear cell and alveolar macrophage activation and IL-2-induced increases in serum TNF-α and IFN-γ, all of which may contribute to the generation of toxicity. IL-10 was ineffective at reducing IL-2-induced pleural effusion. However, IL-10 did inhibit IL-2-induced increases in serum TNF-α, which was accompanied by a decrease in Golgi apparatus and rough endoplasmic reticulum in alveolar macrophages. In addition, ILIO combined with IL-2 increased mononuclear cell activation, which may limit the ability of IL-10 to inhibit IL-2-induced IFN-γ production and pulmonary injury.
Unidirectional heterologous receptor desensitization between both the fMLP and C5a receptor and the IL-8 receptor
Authors: Blackwood R Alexander , Hartiala Kaija T , Kwoh Ely E , Transue Amy T , Brower Ruth C 
Published: January 2018

Abstract
During inflammation neutrophils receive multiple signals that are integrated, allowing a single modified response. One mechanism for this discrimination is receptor desensitization, a process whereby ligand-receptor binding is disassociated from cell activation. We examined the effect of heterologous receptor desensitization on neutrophil Chemotaxis, calcium mobilization, and arachidonic acid production, using interleukin-8 (IL-8), C5a, and N-formyl-methionyl-leucyl-phenylalanine (fMLP). We observed reciprocal inhibition with respect to Chemotaxis. We demonstrated that homologous desensitization, with respect to the mobilization of intracellular calcium stores, lasted approximately 15 min. Heterologous desensitization between the fMLP receptor and the C5a receptor was reciprocal; either stimulant would diminish the cells9 response to stimulation by the other for approximately 3–5 min. However, we observed a unidirectional heterologous desensitization of the IL-8 receptor by both the fMLP and the C5a receptor. This unidirectional heterologous desensitization was observed with respect to both calcium mobilization and arachidonic acid production (i.e., prestimulation of the IL-8 receptor had no effect on subsequent stimulation by either fMLP or C5a).
Ex vivo model of leukocyte migration into herpes simplex virus-infected mouse corneas
Authors: Chen Wei , Tang Qizhi , Hendricks Robert L 
Published: January 2018

Abstract
We are investigating neutrophilic infiltration into herpes simplex virus type 1 (HSV-1) -infected mouse corneas. Using a novel ex vivo corneal migration assay, we demonstrated two waves of neutrophil chemotaxis in HSV-1-infected corneas. An early chemotactic gradient developed within 48 h in concert with the appearance of HSV-1 epithelial lesions, peaked by 4 days post-infection (p.i.), and degraded by 6–8 days p.i. A second chemotactic gradient appeared 10 days after infection, just before the initiation of chronic inflammation. The gradient was maintained in corneas that developed inflammation but rapidly degraded in corneas that failed to develop inflammation. The early chemotactic gradient was established in the absence of T lymphocytes, but the second chemotactic gradient was virtually abrogated by T cell depletion. We conclude that HSV-1 infection induces two temporally separated neutrophil chemotactic gradients in the mouse cornea: an early, transient, T cell-independent gradient; and a later, chronic, T cell-dependent gradient.
Human type II phospholipase A2-induced Mac-1 expression on human neutrophils
Authors: Takasaki Jun , Kawauchi Yasushi , Yasunaga Tomoe , Masuho Yasuhiko 
Published: January 2018

Abstract
To investigate the effect of type II phospholipase A2 (PLA2-H) on neutrophil function, we assessed the Mac-1 (CD11b/CD18) expression on human neutrophils by flow cytometry after incubation of the cells with human PLA2-IL PLA2-II at a concentration of 10μg/mL increased the Mac-1 expression by 150% compared with unstimulated cells at 30 min and after. Under these conditions PLA2-II increased the exocytosis from secretory vesicles but not from azurophilic, specific, or gelatinase granules. The results suggest that PLA2-II induces translocation of Mac-1 from the secretory vesicles to the plasma membrane. The Mac-1 induction mediated by PLA2-II was inhibited by an anti-PLA2-II antibody, which was able to inhibit the catalytic activity. However, the Mac-1 induction by PLA2-II was not inhibited by a 5-lipoxygenase, cyclooxygenase inhibitor, or a platelet-activating factor antagonist. Thus, we examined the effects of fatty acids and lysophospholipids on Mac-1 expression. Only arachidonic acid induced Mac-1 expression. These results imply that PLA2-II induces Mac-1 expression on neutrophils via production of arachidonic acid.
Enhanced aggregation of human neutrophils by MnCl2 or DTT differentiates the roles of L-selectin and β2-integrins
Authors: Lynam Eric B , Rogelj Snezna , Edwards Bruce S , Sklar Larry A 
Published: January 2018

Abstract
MnCl2 and dithiothreitol (DTT) enhance the adhesive functions of β2-integrins. We have used these agents and flow cytometry to distinguish the contributions of β2-integrins and L-selectin to neutrophil aggregation. Although neither compound induced aggregation, they prolonged N-formylmethionyl-leucyl-phenylalanine-induced aggregation and produced larger aggregates. Because activated polymorphonuclear granulocytes (PMN) shed L-selectin in the presence of MnCl2, but not DTT, we could evaluate the role of L-selectin in the early and late stages of aggregation. Blocking L-selectin sites with DREG200 Fab and/or β2-integrin sites with IB4 Fab indicated that aggregation under all conditions remained β2-integrin- and L-selectin-dependent. Disaggregation was integrin-dependent whether L-selectin was present or shed. The disaggregation kinetics suggested that integrin bonds turned over at a slower rate in MnCl2-treated cells. Enhanced aggregation due to DTT and MnCl2 required sustained energy output, suggesting intracellular rather than strictly conformational control. These results provide evidence that PMN aggregation, like leukocyte-endothelial cell adhesion, utilizes L-selectin to form intercellular contacts that are maintained through activated integrins.
M-CSF and GM-CSF promote alveolar macrophage differentiation into multinucleated giant cells with distinct phenotypes
Authors: Lemaire Irma , Yang Huixin , Lauzon Wallace , Gendron Nathalie 
Published: January 2018

Abstract
Multinucleated giant cells (MGC) are a hallmark of granulomatous reactions but the mechanisms that regulate their formation are unknown. To address this issue, we cultured resident alveolar macrophages (AM) from rat lung and examined the effects of defined cytokines on AM differentiation and MGC formation. The presence of MGC was found after 3 days in culture with maximal numbers obtained at 7 days and thereafter (up to 21 days). Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (25–75 U/mL) stimulated the formation of MGC (up to 4-fold), whereas interleukin (IL) -3, IL-10, and interferon-γ (IFN-γ) had no stimulatory effect. Interestingly, MGC with distinct phenotypes were observed in AM cultures: (1) spherical MGC with 3–16 nuclei, dense cytoplasm, and lower expression of β3 integrin (Type 1) and (2) irregular MGC with 3–30 nuclei, thin and vacuolated cytoplasm, and higher expression of βsintegrin (Type 2). Furthermore, the actions of M-CSF and GM-CSF on AM were found to he different. GM-CSF promoted, in AM cultures, the appearance of an elongated fibroblastoid phenotype and stimulated mostly the formation of Type 2 MGC. In contrast, M-CSF did not cause significant change in the general morphology of regular AM but stimulated the appearance of both Type 1 and Type 2 MGC. Reverse transcriptase-polymerase chain reaction analysis demonstrated that, under these conditions, M-CSF induced GM-CSF gene expression in AM. In addition, neutralizing antibodies against M-CSF selectively decreased the formation of Type 1 MGC, whereas neutralizing anti-GM-CSF inhibited Type 2 formation. These data suggest that M-CSF promotes AM differentiation into Type 1 MGC, whereas GM-CSF stimulates the formation of Type 2 and that M-CSF and GM-CSF may selectively regulate in an autocrine fashion AM differentiation into distinct MGC.
Interferon-γ reduces Ia+ dendritic cell traffic to the lung
Authors: Suda Takafumi , Callahan Ronald J , Wilkenson Robert A , van Rooijen Nico , Schneeberger Eveline E 
Published: January 2018

Abstract
MHC class II+ lung dendritic cells (DC) increase in number following treatment of animals with interferon-γ (IFN-γ) [kradin et al. (1991) Am. J. Resp. Mol. Biol. 4, 210; Gong et al. (1992) J. Exp. Med. 175, 797]. To test whether this is due to increased sequestration and/or trafficking of DC to the lung, bone marrow DC from BALB/c mice were obtained by culturing bone marrow with granulocyte-macrophage colony-stimulating factor (GM-CSF). Recipient BALB/c mice were injected intraperitoneally (i.p.) for 4 days with one of the following: IFN-γ, dexamethasone (Dex), or phosphate-buffered saline (PBS). Twenty-four hours after the last dose, they were injected intravenously (i.v.) with carboxyfluorescein (FI) -labeled DC (1 106/mouse) and killed 4 h later. DC, double immunostained for Ia and Fl, were quantified by morphometry in frozen sections of lung. The number of injected dual-labeled DC/cm2 was reduced by 90% in IFN-γ-treated mice. By contrast, there was no significant difference between Dex- and PBS-treated animals in the number of double-labeled DC retained in pulmonary capillaries. Biodistribution and imaging studies were conducted on IFN-γ- and PBS-treated mice using 111In-labeled DC. Reduced radioactivity in the lung was accounted for by an equivalent increase in the liver of IFN-γ-treated mice; imaging studies confirmed these observations. Removal of &gt;80% of alveolar macrophages (AM) by pretreatment with intratracheally administered chlodronate-loaded liposomes did not change the biodistribution of DC in IFN-γ- and PBS-injected mice. Serum levels of tumor necrosis factor-α (TNF-α) and nitrite/nitrate in IFN-γ-treated mice were similar to those of controls. Immunostaining for inducible nitric oxide synthase (iNOS), however, revealed a 1.5-and 6-fold increase in the number of positively stained cells in the lung and liver, respectively, of IFN-γ-treated mice; the number of iNOS-expressing cells was markedly reduced in Dex-treated animals relative to controls. To test whether the systemic treatment with IFN-γstimulated the cytotoxic activity of Kupffer cells, mice were injected with chlodronate liposomes 5 days before death. After treating the mice in the ensuing 4 days with IFN-γ or PBS, bio distribution and imaging studies with 111In-labeled DC were conducted on the 5th day. After administration of chlodronate liposomes, there was a significant increase in the radioactivity detected in the lungs of IFN-γ-injected mice but not in those of PBS-injected controls, a finding confirmed by imaging studies. We conclude that IFN-γ treatment augmented Kupffer cell cytotoxic activity, which, in turn, effectively reduced the number of injected DC in circulation, with the result that fewer of these cells were retained in the lung vasculature. We further conclude that IFN-γincreases the number of Ia+ lung DC by up-regulating Ia expression of resident Ia- DC precursors and not by promoting the migration of circulating DC to the lung.
Increased plasma GlyCAM-1, a mouse L-selectin ligand, in response to an inflammatory stimulus
Authors: Suguri Takayasu , Kikuta Akio , Iwagaki Hiromi , Yoshino Tadashi , Tanaka Noriaki , Orita Kunzo 
Published: January 2018

Abstract
GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) is one of the sialomucin-like ligands for L-selectin, which is a member of the selectin family and mediates initial adhesion of leukocytes to specialized high endothelial venules in lymph nodes and venules at sites of inflammation. GlyCAM-1, lacking a transmembrane domain, is supposed to be secreted into the blood, To understand the functional role of secreted GlyCAM-1, we performed sandwich enzyme-linked immunosorbent assay to measure GlyGAM-1 plasma levels after inflammatory stimulus. BALB/c mice were injected with complete Freund's adjuvant (CFA) in the hind footpads; serum levels of GlyCAM-1 and L-selectin bound to GlyCAM-1 and several inflammatory cytokines, including interleukin-6 (IL-6), were measured at various intervals. IL-6 showed a significant increase 3 h after CFA stimulation. GlyGAM-1 was increased at 3 h, reached peak levels at 12 h, and gradually decreased thereafter. Levels of L-selectin bound to the plasma GlyCAM-1 changed over a similar time course, reached peak at 12 h after, and then began to decrease. The binding of L-selectin to plasma GlyCAM-1 was completely eliminated with the presence of ethyleneglycol-bis(β-aminoethylether)-N,N'-tetraacetic acid, showing the calcium dependency of this binding. These findings show that GlyGAM-1 release is enhanced by inflammatory stimulation and also suggest that released plasma GlyGAM-1 may trap, at least in part, soluble L-selectin shed from stimulated leukocytes to neutralize each other.