bluuebunny/crossref_metadata_2025 huggingface dataset:
Silicotic lymph node reactions in mice: genetic differences, correlation with macrophage markers, and independence from T lymphocytes
Authors: Weirich Uta , Friemann Johannes , Rehn Bernd , Henkelüdecke Uwe , Lammers Thorsten , Sorg Clemens , Bruch Joachim , Gleichmann Ernst
Published: January 2018

Quartz was injected into a hind foot of BALB/c and DBA/2 mice and on days 40, 90, and 180 the progressive response ensuing in the draining popliteal lymph node (PLN) was investigated by histopathology and immunohistopathology. The area of silicotic nodules (ASN) was measured by morphometry, and, by this parameter, strain BALB/c proved to be a high responder to quartz, and strain DBA/2 a low responder, albeit both strains showed a similar degree of reactive lymphoid hyperplasia in the draining PLN. Both strains also showed a similar quartz content in the draining PLN but in BALB/c mice quartz particles were concentrated in the ASN, whereas in DBA/2 mice they were evenly dispersed over the PLN. Because the silicotic response of athymic BALB/c nu/nu mice was even stronger than that of euthymic BALB/c mice, T cells are not required for the development of silicotic nodules. This fits the notion that quartz is not an antigen and that high and low responder strains are MHC-identical. Because quartz-treated BALB/c, but not DBA/2 mice, showed a persistent expression of the macrophage differentiation markers MRP8 and MRP14, phenotypically the observed strain difference in silicotic responsiveness seems to be expressed at the level of macrophages.
α-MSH production, receptors, and influence on neopterin in a human monocyte/macrophage cell line
Authors: Rajora N , Ceriani G , Catania A , Star R A , Murphy M T , Lipton J M
Published: January 2018

α-Melanocyte-stimulating hormone (α-MSH), a tridecapeptide derived from pro-opiomelanocortin, has potent antiinflammatory activity in laboratory animals. α-MSH inhibits nitric oxide production by murine macrophages, an influence believed to reflect activation of an autocrine circuit in these cells, one that is based on production and release of α-MSH and subsequent stimulation of melanocortin receptors. We found that THP-1 cells, human monocytic cells, produced α-MSH; this production was increased by interleukin-6, tumor necrosis factor α, or concanavalin A. These cells also expressed the gene for the human α-MSH receptor MC1. Unlike murine macrophages, THP-1 cells produced little nitrite in response to interferon-γ(IFN-γ) and lipopolysaccharide, and a-MSH inhibited this production only slightly. However, production of neopterin, a presumed primate homologue of nitric oxide in lower animals, was increased in THP-1 cells stimulated with IFN-γ plus TNF-α and α-MSH significantly inhibited this production. The evidence indicates that an autocrine regulatory circuit based on α-MSH occurs in human monocyte/macrophages much as in murine macrophages. α-MSH-induced modulation of specific inflammatory mediators/cytotoxic agents appears to differ depending on the importance of the mediators in the myelomonocytic cells of different species.
Mechanisms involved in eosinophil migration. Platelet-activating factor-induced Chemotaxis and interleukin-5-induced chemokinesis are mediated by different signals
Authors: Schweizer René C , van Kessel-Welmers Berns A C , Warringa Ruud A J , Maikoe Tjander , Raaijmakers Jan A M , Lammers Jan-Willem J , Koenderman Leo
Published: January 2018

Eosinophils play an important role in the pathogenesis of allergic diseases such as allergic asthma. Eosinophil migration in vitro can be divided into directed migration, or Chemotaxis, and random migration, or chemokinesis. Here, we studied intracellular signals involved in eosinophil migration in vitro induced by platelet-activating factor (PAF) and interleukin-5 (IL-5), applying a Boyden chamber assay. Migration induced by PAF (10-11-10-6 M) largely consisted of Chemotaxis with some chemokinesis, whereas IL-5 (10-12-10-8 M) induced chemokinesis only. Eosinophils were depleted from intracellular and extracellular Ca2+ to study the role of Ca2+ as a second messenger. Ca2+ depletion did not change PAF-induced Chemotaxis, however, IL-5-induced chemokinesis was inhibited. Interestingly, PAF, but not IL-5, induced changes in [Ca2+]i. This rise originated mainly from internal stores. Inhibition of protein kinase A by H-89 and protein kinase C by GF 109203X had no effect on both forms of eosinophil migration. Addition of the protein kinase inhibitor staurosporine significantly inhibited IL-5-induced chemokinesis. Inhibition of tyrosine kinases by herbimycin A completely blocked IL-5-induced chemokinesis. PAF and IL-5-induced actin polymerization was studied to compare migratory responses with a migration-associated intracellular response. Ca2+ depletion significantly enhanced PAF-induced (10-8 M) actin polymerization, whereas IL-5-induced actin polymerization was not influenced. Addition of staurosporine led to an increase in F-actin. Subsequent addition of PAF or IL-5 resulted in an additive increase in F-actin content. In summary, both forms of eosinophil migration are protein kinase A and protein kinase C independent. In contrast to PAF-induced Chemotaxis, IL-5-induced chemokinesis was found to be completely Ca2+ and tyrosine kinase dependent.
Gene transfer for cytokine functional studies in the lung: the multifunctional role of GM-CSF in pulmonary inflammation
Authors: Xing Zhou , Braciak Todd , Ohkawar Yuichi , Sallenave Jean-Michel , Foley Ronan , Sime Patricia J , Jordana Manel , Graham Frank L , Gauldie Jack
Published: January 2018

Using adenoviral-mediated gene transfer techniques, the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene is efficiently targeted to and highly expressed by the respiratory epithelium of rat lung. This lung tissue-directed expression of GM-CSF induces accumulation of both eosinophils and macrophages at early stages and an irreversible fibrotic reaction at later stages. These tissue responses to GM-CSF appear to be distinct from those induced by other proinflammatory cytokines, interleukin (IL) -5, IL-6, macrophage inflammatory protein-2 (MIP-2), or RANTES overexpressed in the lung. These findings clearly demonstrate that GM-CSF is more than a hematopoietic cytokine in the lung and may play a pivotal role in the multiple pathological processes underlying numerous respiratory illnesses, including asthma. In this overview, the differences in tissue responses induced by GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF contributes to the development of eosinophilia, macrophage granuloma, and fibrosis are discussed in conjunction with the recent findings from us and others.
Serglycin-binding proteins in activated macrophages and platelets
Authors: Kolset Svein O , Mann David M , Uhlin-Hansen Lars , Winberg Jan-Olof , Ruoslahti Erkki
Published: January 2018

The major proteoglycan in macrophages and platelets is the chondroitin sulfate proteoglycan serglycin. To study the biological role of serglycin, its binding to secreted and cell-associated proteins from macrophages and blood platelets was examined. Affinity chromatography with serglycin-Sepharose and chondroitin sulfate-Sepharose was used to isolate proteoglycan-binding proteins from macrophages and platelets. Antibodies against human macrophage inflammatory protein-1α (MIP-1α) precipitated a 14-kDa 35S-methionine-labeled protein among the chondroitin sulfate binding proteins secreted from the macrophage-like U937 cells after stimulation. Two proteins from murine macrophage J774 cells with molecular masses of ~10 and 14 kDa were precipitated by an antiserum against the murine MIP-1α. Protein sequencing of fragments obtained by trypsin digestion of a 14-kDa chondroitin sulfate-binding protein from cell extracts of stimulated U937 cells revealed 100% homology with lysozyme, a bacteriolytic enzyme. Fragment of one other protein with approximate molecular mass of 8 kDa showed high homology with bone morphogenetic protein. Inhibition studies showed that chondroitin 6-sulfate inhibited the bacteriolytic activity of lysozyme in a competitive manner more efficiently than heparin and chondroitin 4-sulfate. Amino-terminal sequencing of two proteins from platelet extracts that bound to serglycin-Sepharose revealed that they corresponded to multimeric forms of human platelet factor 4 (PF4). Chondroitin sulfate-Sepharose was shown to be equally efficient in retaining PF4 from platelet extracts as serglycin-Sepharose, indicating that the glycosaminoglycan chains mediate the binding to PF4 in the intact proteoglycan molecule. Competition experiments showed that serglycin was as efficient as heparan sulfate in blocking the binding of [3H]chondroitin sulfate to PF4, whereas heparin was one order of magnitude more efficient. Affinity measurements using fluoresceinamine-labeled glycosaminoglycans showed that the affinity of heparin for PF4 is on the order of 30 nM, whereas chondroitin sulfate has an affinity of 260 nM. Both PF4, MIP-1α, and lysozyme play important roles in different types of inflammatory reactions. The interaction with serglycin may indicate that this proteoglycan is involved in the regulation of the inflammatory response.
Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3
Authors: Schwende Heike , Fitzke Edith , Ambs Petra , Dieter Peter
Published: January 2018

Human THP-1 leukemia cells differentiate along the monocytic lineage following exposure to phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (VD3). In the monocytic cell line THP-1, PMA treatment resulted in a more differentiated phenotype than VD3, according to adherence, loss of proliferation, phagocytosis of latex beads, and expression of GD11b and CD14. Both differentiating substances induced similar effects in the release of superoxide anions (O2-). VD3-differentiated cells did not release prostaglandin E2 (PGE2), in contrast to PMA-differentiated cells, and in PMA-differentiated cells phospholipase A2 (PLA2) activity and expression was increased. Lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNF-α) release was higher in PMA-treated cells. PMA- but not VD3-differentiation resulted in a translocation of protein kinase C (PKC) isoenzymes to membrane fractions. Both differentiating agents up-regulated the expression of PKC isoenzymes. Whereas VD3 elevated mainly the expression of PKC-β, PMA caused a strong increase in PKC-δ and a weak increase in PKC-α, PKC-, and PKC- expression. These results indicate that phorbol ester and the active metabolite of vitamin D induce different signal pathways, which might result in different achievement of differentiation.
Interleukin-6 stimulates neutrophil production of platelet-activating factor
Authors: Biffl Walter L , Moore Ernest E , Moore Frederick A , Barnett Carlton C , Silliman Christopher C , Peterson Verlyn M
Published: January 2018

Interleukin-6 (IL-6) is an integral mediator of the acute phase response to injury and infection; an exaggerated IL-6 response has been associated with adverse clinical events. The precise role of IL-6 is unclear, but it appears capable of modulating the functional repertoire of mature neutrophils (PMNs). Our previous work demonstrated that IL-6-stimulated PMNs are primed by lower concentrations of platelet-activating factor (PAF) than nonstimulated PMNs. Recently, we have found that IL-6 suppresses PMN apoptosis via a PAF-like mechanism. We hypothesized that IL-6 stimulates PMNs to produce PAF. PMNs isolated from healthy human donors were incubated with IL-6 (0.1–100 ng/ml) at 37°. Lipid production was measured by use of thin-layer chromatography, and PAF quantitated with a scintillation proximity assay. IL-6 (1 and 10 ng/ml) stimulated PMNs to produce increased quantities of PAF. PAF production was associated with an increase in PMN cytosolic calcium. These data may provide mechanistic insight into IL-6 regulation of PMN-mediated cytotoxicity and the role of PAF in mediating IL-6 effects on PMNs.
In vivo labeling of neutrophils using a fluorescent cell linker
Authors: Albertine Kurt H , Gee Mariys H
Published: January 2018

To study neutrophil circulation time and trafficking in vivo requires labeling the cells so that their movement can be followed temporally and spatially. Labeling procedures used to date, however, have relied on ex vivo separation and labeling methods, an undesired consequence of which may be neutrophil activation. Moreover, the labeled cells preferentially stick in the pulmonary circulation for several hours upon reinjection into the host. Therefore, we devised an alternate labeling procedure that relied on intra-arterial infusion of the fluorescent phagocytic cell linker PKH26. We infused PKH26 (5.5 × 10-6 M for 1 h) into six awake sheep and measured systemic and pulmonary hemodynamics and lung lymph dynamics continuously for 3–4 h after the infusion. We collected simultaneous arterial and venous blood samples hourly for 4 h, and again 24 h after the infusion ended, for white blood cell and neutrophil differential counts and to identify labeled cells in fresh smears by fluorescence and differential interference contrast (DIC) microscopy. Infusion of PKH26 had minimal and transient physiologic effects on systemic and pulmonary artery pressure, lung lymph flow, and leukocyte counts. Labeled cells were present in venous blood after the infusion for at least 24 h. DIC microscopy observation of the blood smears indicated that the fluorescently labeled cells were exclusively neutrophils. Unstimulated superoxide anion release from neutrophils isolated 2 and 24 h after PKH26 infusion was not different from baseline release. Phorbol myristate acetate-stimulated release was not affected either. The labeled neutrophils responded to chemoattractants by migrating to extravascular sites. Our results indicate that neutrophils can be selectively labeled in vivo, after which trafficking of the labeled neutrophils can be determined.
β2 integrins mediate protein tyrosine phosphorylation in human neutrophils
Authors: Walzog Barbara , Offermanns Stefan , Zakrzewicz Andreas , Gaehtgens Peter , Ley Klaus
Published: January 2018

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

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

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

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

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

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

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

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

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

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

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

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 >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

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.
Internalin A can mediate phagocytosis of Listeria monocytogenes by mouse macrophage cell lines
Authors: Sawyer Richard T , Drevets Douglas A , Campbell Priscilla A , Potter Terry A
Published: January 2018

Listeria monocytogenes internalin A (InlA) is a surface protein that mediates the attachment of Listeria to, and invasion of, hepatocytes, epithelial, and endothelial cells. In this study, we tested whether InlA could also mediate phagocytosis of L. monocytogenes by the non-listericidal mouse macrophage cell lines J774A.1 and H36.12j. Recombinant InlA (rInlA) was used to derive mouse monoclonal anti-InlA antibodies (mAb) and rabbit anti-InlA antibodies. Fluorescence microscopy demonstrated that these anti-InlA antibodies reacted with wild-type L. monocytogenes, L. ivanovii, and L. innocua+, a mutant transformed with the inlAB operon that expresses surface InlA but failed to react with Bug 8, an InlA/InlB-negative transposon mutant of L. monocytogenes or with non-invasive Listeria sp. Fluorescence microscopy, radiolabeling, and flow cytometry showed that rInlA bound specifically to both macrophage cell lines. Incubation of macrophages and wild-type L. monocytogenes in the presence of rInlA or pretreatment of Listeria with anti-InlA antibodies specifically inhibited, by at least 50%, the phagocytosis of Listeria by both of these cells. By comparison, treatment with these reagents failed to affect the phagocytosis of Streptococcus pyogenes by either macrophage cell line nor did these reagents alter the ability of macrophages to internalize wild-type L. monocytogenes. We found that Bug 8, but not wild-type L. monocytogenes, failed to grow within both of these non-listericidal macrophage cell lines. In contrast to infection by wild-type L. monocytogenes, Bug 8 was rapidly eliminated from the spleens of both C57B1/6 and DBA/2 mice. Data presented here show that only invasive Listeria sp. have surface InlA and that L. monocytogenes can enter non-listericidal macrophage cell lines by binding of bacterial InlA to the macrophage cell surface.
Peroxynitrite augments fMLP-stimulated chemiluminescence by neutrophils in human whole blood
Authors: Bednar Martin M , Balazy Michael , Murphy Michael , Booth Christina , Fuller Susan P , Barton Andrew , Bingham Jemel , Golding Lisa , Gross Cordell E
Published: January 2018

The neutrophil respiratory burst was examined by the technique of luminol-dependent chemiluminescence (LDCL) triggered by submaximal concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP) in diluted whole blood. We sought to identify the chemical species responsible for LDCL in whole blood, to examine the role of leukotriene B4 (LTB4) and other arachidonic acid metabolites as mediators of the fMLP signaling pathway, and to investigate the effect of peroxynitrite on this response. Both sodium azide and taurine significantly inhibited LDCL (93% inhibition with 100 μM azide, 52% inhibition with 10 mM taurine). More modest inhibition was seen with superoxide dismutase (SOD), catalase, the nitric oxide synthase inhibitor monomethyl-l-arginine (l-NMMA), and with inhibitors of the cyclooxygenase (indomethacin), lipoxygenase (AA-861; no effect), and cytochrome P-450 (SKF 525-A) pathways of arachidonic acid metabolism. The nitric oxide donor SIN-1 (1–100 μM) and peroxynitrite (10–300 μM) also augmented fMLP-induced LDCL. The augmentation seen with peroxynitrite and SIN-1 was attenuated by SOD. Despite the increase in LDCL, peroxynitrite caused a dose-related inhibition of fMLP-stimulated LTB4 release. In summary, our results indicate that (1) LDCL elicited by fMLP in diluted whole blood appears primarily mediated by hypo-chlorous acid derived from myeloperoxidase; (2) pretreatment with the nitric oxide donor SIN-1 or with peroxynitrite augments LDCL; and (3) LTB4 release does not contribute to fMLP-stimulated LDCL or in the modulation of LDCL by SIN-1 or peroxynitrite.
Mechanisms involved in sodium arsenite-induced apoptosis of human neutrophils
Authors: William R , Watson G , Redmond H Paul , Wang Jiang Huai , Bouchier-Hayes David
Published: January 2018

Apoptosis is a distinct mechanism by which eukaryotic cells die. Factors governing the induction of polymorphonuclear leukocyte (PMN) apoptosis should be important in understanding resolution of acute inflammation. The mechanisms for induction of PMN apoptosis remain uncertain; however, oxidative stress has been suggested. The aims of this study were to determine whether reactive oxygen intermediates play a role in PMN apoptosis and to investigate inhibition of this process by selective use of antioxidants. PMN were isolated from 10 healthy volunteers. PMN (1 106 PMN/mL) were cultured in 40, 80, and 160 μM of arsenite for 2, 6, 12, 18, and 24 h. Apoptosis was assessed qualitatively by morphology and gel electrophoresis and quantitatively by CD16 receptor expression and propidium iodide DNA staining. There was a significant (P < 0.05) increase in the rate of apoptosis on incubation with arsenite (80 and 160 μM). To investigate the mechanism of this process, intracellular respiratory burst activity was measured following arsenite culture. We found that arsenite-induced PMN apoptosis correlated with an increase in intracellular respiratory burst. To further investigate the role of oxidative injury in inducing apoptosis, the antioxidants catalase, dimethyl sulfoxide (DMSO), glutathione (GSH), N-acetylcysteine (NAC), and taurine were investigated and we demonstrated that GSH, NAC, and taurine were significantly protective against arsenite-induced apoptosis. However, catalase and DMSO failed to induce protection. This study demonstrates that arsenite induces PMN apoptosis through an oxygen-dependent mechanism that can be prevented through selective antioxidants.
Endotoxin-inducible granulocyte-mediated hepatocytotoxicity requires adhesion and serine protease release
Authors: Sauer Achim , Hartung Thomas , Aigner Joachim , Wendel Albrecht
Published: January 2018

In primary cultures of Kupffer cells and hepatocytes, human granulocytes potentiated toxicity of endotoxin about 1000-fold. Granulocyte elastase activity was found to correlate with toxicity. The serine protease inhibitors α1-antitrypsin, eglin C, and aprotinin protected against toxicity. Tumor necrosis factor-α (TNF-α) induced cytotoxicity and elastase release, whereas neutralization of TNF-α blocked both events. We conclude that TNF-α formed by Kupffer cells activates granulocytes. Experiments in cultures where cells were separated by membranes permeable to mediators indicated that cell contact is needed for toxicity. Scanning electron microscopy showed granulocytes adhering to and interdigitating with hepatocytes. Using liver cells from ICAM-1-deficient mice had no effect on toxicity. However, neutralizing CD31 inhibited toxicity and elastase release but not granulocyte adhesion. Our findings demonstrate that adhesion of granulocytes is a necessary but not sufficient condition for the synergistic interaction of endotoxin-stimulated liver macrophages and granulocytes in the proteolytic killing of hepatocytes.
Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leukocytes
Authors: Su Shao-bo , Mukaida Naofumi , Wang Jian-bin , Nomura Hideki , Matsushima Kouji
Published: January 2018

cDNA cloning has revealed the presence of at least three distinct human receptors for macrophage inflammatory protein-1α (MIP-1α) and RANTES: C-C chemokine receptor (CCR) 1, 4, and 5. To clarify the physiological role of CCR1, we prepared specific antibodies to CCR1 by immunizing rabbits with recombinant glutathione-S-transferase (GST) fused with its NH2-terminal portion. The resultant antibodies stained positively 293 cells transfected with CCR1 cDNA but neither parental cells nor cells transfected with CXCR1 [interleukin-8 (IL-8) receptor type A] cDNA, confirming its specificity. Immunofluorescence analysis revealed that peripheral blood lymphocytes and monocytes but not neutrophils express CCR1. Positive staining of transfectants, monocytes, and lymphocytes was inhibited by the GST protein fused with the NH2-terminal portion of CCR1, further indicating that this antibody recognized the NH2-terminal portion of CC CKR1. A majority of CD3+, CD4+, CD8+, or CD16+ peripheral blood lymphocytes but not CD19+ lymphocytes expressed CCR1. Among CD4+ peripheral blood lymphocytes, CD45RO+ cells expressed a larger number of CCR1 compared with CD45RO-. Moreover, CD34+ cells in human bone marrow as well as cord blood were uniformly stained with this antibody. Furthermore, the antibody inhibited calcium mobilization in CCR1 transfectants stimulated with human rMIP-1α, suggesting that its NH2-terminal portion is critically involved in ligand binding or signaling. Finally, the antibody partially inhibited monocyte chemotactic activities of human rMIP-1α, suggesting that CCR1 is a functional receptor for MIP-1α on human peripheral blood monocytes.
Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins
Authors: DeLeo Frank R , Quinn Mark T
Published: January 2018

Phagocytes such as neutrophils play a key role in the body's innate immune response to infection. These cells travel throughout the body in search of pathogens and are rapidly mobilized to sites of inflammation where they phagocytose these pathogens and subsequently release a variety of toxic oxygen radical species and proteolytic enzymes to directly destroy the engulfed particle. The generation of microbicidal oxidants by neutrophils results from the action of a multi-protein enzymatic complex known as the NADPH oxidase. Altogether, there are currently seven proteins reported to be associated with the NADPH oxidase assembly. In resting neutrophils, these NADPH oxidase protein components are segregated into cytoplasmic and plasma membrane compartments. However, during assembly and activation of the NADPH oxidase, the cytosolic protein components translocate to the plasma membrane or phagosomal membrane where they assemble around a central membrane-bound protein known as flavocytochrome b. This assembly process is highly regulated and involves multiple binding interactions between the individual NADPH oxidase proteins, resulting in an active oxidase complex. Over the past few years, a number of these sites of binding interaction between the oxidase proteins have been identified, leading to a clearer understanding of the intermolecular interactions occurring among protein components during the assembly process. In addition, this information has contributed to our understanding of the roles played by each protein during the activation and assembly process. In this review, we describe the key features of each NADPH oxidase protein and then summarize our current understanding of the specific molecular interactions occurring between these proteins, focusing on the role these protein:protein binding interactions play in the NADPH oxidase assembly process.