bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of interferon-γ, in vitro and in vivo, on mRNA levels of phagocyte oxidase components
Authors: Weening Ron S , de Klein Angelique , de Boer Martin , Roos Dirk
Published: January 2018

Daring the international placebo-controlled trial on the efficacy of interferon-γ (IFN-γ) in chronic granulomatous disease (CGD), 19 patients entered the study via our Institute. One patient stopped treatment shortly thereafter. RNA was purified from the mononuclear cells of the remaining 18 CGD patients before and during this placebo-controlled trial. The mRNA levels for the NADPH oxidase components were subsequently analyzed. Compared with the placebo-treated CGD patients, the mRNA levels for p4 7-phox were significantly increased in the IFN-γ-treated CGD patients (P < 0.002). No significant changes were observed in the mRNA levels of the other oxidase components. These findings are in agreement with observations in vitro and indicate that IFN-γ is active on the NADPH oxidase in vivo as well. However, it remains questionable whether these effects in vivo can explain the observed reduction of infections in these patients.
ICAM-1 expression and low-molecular-weight G-protein activation of human bronchial epithelial cells (A549) infected with RSV
Authors: Arnold R , König W
Published: January 2018

The airway epithefial cell of the lower respiratory tract is the primary host target cell for respiratory syncytial virus (RSV) infection. To estimate whether infected epithelial cells contribute to the inflammatory host response observed during the acute infection phase we analyzed the cell surface expression of intercellular adhesion molecule-1 (ICAM-1, CD54) on human bronchial epithelial cells (A549) following RSV infection and cytokine priming. The epithelial cells constitutively expressed ICAM-1. The ICAM-1 surface expression was significantly up-regulated up to 72 h post RSV infection. In addition, cytokine priming with tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ), or interleukin-1α/β (IL-1α/β) induced an enhanced ICAM-1 expression on noninfected as well as on RSV-infected epithelial cells. As early as 24 h post RSV infection and cytokine priming, respectively, the maximum of ICAM-l-expressing cells was observed. In contrast, the maximal ICAM-1 up-regulation per cell was measured 24 h later, e.g., after 48 h of culture. Cytokine priming with IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) did not lead to a significant increase of ICAM-1 expression either on RSV-infected or on noninfected A549 cells up to 72 h of culture time. Performed signal transduction experiments, e.g. [α-32P]GTP-binding blot analysis, revealed that low-molecular-weight GTP-binding proteins possess an enhanced GTP-binding capacity in RSV-infected A549 cells.
Monocytic differentiation modulates apoptotic response to cytotoxic anti-Fas antibody and tumor necrosis factor α in human monoblast U937 cells
Authors: Kikuchi Hidehiko , Iizuka Ryoko , Sugiyama Shinji , Gon Gotetsu , Mori Hiroyuki , Arai Masa-aki , Mizumoto Kiyohisa , Imajoh-Ohmi Shinobu
Published: January 2018

Interferon-γ (IFN-γ), vitamin D3 (VD), and retinoic acid (RA) induce differentiation of human monoblastic leukemia U937 cells to macrophage-like cells with potential superoxide anion-generating activity upon further stimulation. Here we report that U93 7 cells thus differentiated show various responses to apoptotic induction with a cytotoxic anti-Fas antibody and tumor necrosis factor (TNF). VD-or RA-treated U937 cells acquired resistance against Fas- or TNF receptor (TNFR)-mediated apoptosis, whereas apoptotic cell death was accelerated in IFN-γ-treated cells. By flow cytometric analyses, no decrease in expression of surface Fas antigen or p55 TNFR was observed in differentiated U937 cells. Cell surface expression of CD11b was seen only when differentiation was induced with VD or RA but not with IFN-γ. The growth of VD- or RA-treated cells was retarded but IFN-γ-treated cells were prolific. These findings suggest that the differentiation state differs with the inducer and that the cellular response to apoptotic induction is closely related to the state including the cell cycle.
LPS pretreatment reprograms macrophage LPS-stimulated TNF and IL-1 release without protein tyrosine kinase activation
Authors: West Michael A , Bennett Terriel , Seatter Susan C , Clair Laurel , Bellingham Janet
Published: January 2018

Pretreatment of macrophages with low-dose endotoxin (LPSp) profoundly alters cytokine release in response to subsequent LPSa activation. These qualitative and quantitative alterations in cytokine release have been termed macrophage reprogramming. Macrophage activation by LPS is thought to occur via a mechanism involving an early protein tyrosine kinase (PTK) phosphorylation step. PTK inhibition with genistein or herbimycin A blocks LPSa-stimulated secretion of tumor necrosis factor (TNF) and interleukin-1 (IL-1). In this study we investigated whether a PTK pathway participates in LPSp pretreatment reprogramming. We show that LPSp pretreatment inhibited TNF and augmented IL-1 release in response to subsequent LPSa stimulation. Blockade of PTK activation pathways during the interval when macrophages were exposed to LPSp prevented mitogen-activated protein kinase phosphorylation, as well as LPSp-stimulated release of TNF and IL-1, but did not block LPSp reprogramming effects. We conclude that LPSp pretreatment reprogramming of macrophage cytokine production does not require PTK activation.
IgM-mediated opsonization and cytotoxicity in the shark
Authors: McKinney E C , Flajnik Martin F
Published: January 2018

Two types of cytotoxic reactions have been observed using cells from the nurse shark: spontaneous cytotoxicity mediated by cells of the macrophage lineage and antibody-dependent killing carried out by a different effector cell population. Previous data showed that removal of phagocytic cells using iron particles abolished macrophage-mediated killing, but not antibody-dependent reactions. The current study used single cell assays and showed that the effector of antibody-driven reactions was the neutrophil. Surprisingly, the mechanism of killing was shown to be phagocytosis mediated by both 7S and 19S immunoglobulin M (IgM). Reactions proceeded with as little as 0.01 μg of purified 19S or 7S IgM and were complete within 4–6 h. In contrast, purified immunoglobulin did not adsorb to macrophages and had no effect on target cell binding or cytotoxicity. Pretreatment of cells with cytochalasin D abolished the phagocytic reaction, but not spontaneous cytotoxicity. These data show that antibody-mediated killing results from opsonization and phagocytosis; the mechanism of macrophage killing is currently unknown. In addition, these data show that the shark neutrophil, not the macrophage lineage, carries a receptor for Fcμ.
Fas involvement in human NK cell apoptosis: lack of a requirement for CD16-mediated events
Authors: Ortaldo John R , Winkler-Pickett Robin T , Nagata Shigekazu , Ware Carl F
Published: January 2018

Propriocidal regulation of T cells refers to apoptosis induced by interleukin-2 (IL-2) activation with subsequent antigen receptor stimulation. We previously reported that natural killer (NK) cells also exhibit propriocidal death. Cell death can be induced following occupancy of the FcγRm (CD16) receptor when NK cells were pretreated with IL-2, IL-12, or IL-15. Here we show other triggering receptors on NK cells such as CD44, anti-NK-receptor antibodies, and pharmacological activation can result in the cell death signal. Requirement for cell interactions indicated that cell contact was required; however, unlike cell-mediated lysis, extracellular calcium was not required. Like T cells, the process of cell death for NK cells was receptor-induced apoptosis. Activation-induced apoptosis of T cells is mediated by members of the tumor necrosis factor (TNF) cytokine superfamily. We examined the involvement of TNF receptor family members or Fas in this rapid cell death. Antibody directed against Fas, TNFR60, TNFR80, LTBR, and LTα failed to inhibit receptor-induced death. Therefore, NK cells appear to demonstrate a rapid apoptotic episode when CD16 is cross-linked, but the mechanism of this apoptosis is quite different than was observed in T cells with CD3. The direct examination of the Fas pathway on activated NK cells revealed that susceptibility required longer treatment times and IL-2 activation. This susceptibility was paralleled by increased Fas-ligand expression. Therefore, NK cells can demonstrate an apoptotic response to CD16, CD44, NK receptors, and Fas. The enumeration of ligands capable of eliciting NK cell death and the in vivo relevance of this observation require further study.
Studies with IL-10-/- mice: an overview
Authors: Rennick Donna M , Fort Madeline M , Davidson Natalie J
Published: January 2018

Our studies have elucidated, in part, the mechanism whereby persistent stimulation by normal enteric antigens leads to the development of chronic enterocolitis in interleukin 10-deficient (IL-10-/-) mice. This disease is mediated by IL-10-/-CD4+ T cells as evidenced by their ability to transfer colitis to immunodeficient RAG-2-/-mice. Furthermore, the CD4+ T cells recovered from the affected colons of IL-10-/- mice consisted of a highly polarized Th1-like population because they produced interferon-γ (IFN-γ) but not IL-4. We found that enterocolitis could be prevented if 3-week-old mutants were treated for 6–8 weeks with either anti-IL-12 or anti-IFN-γ monoclonal antibodies (mAb). These results were consistent with the findings of in vitro studies suggesting that IFN-γ and, in particular, IL-12 direct the differentiation of naive T cells toward a Th1 phenotype. Apparently, the uncontrolled production of IL-12 and IFN-γ by accessory cells and T cells, respectively, in IL-10-/- mice ultimately resulted in the excessive generation and activation of Th1 cells, hence, immunopathology. IL-10-/- mice have also been used to evaluate the importance of IL-10 in regulating immune responses outside of the gastrointestinal (GI) tract. In these studies, IL-10-/-mice were challenged with a variety of foreign antigens using different routes of administration. In general, the results repeatedly demonstrated that a major function of IL-10 is to protect the host from the harmful side effects of an overly zealous immuneinflammatory response. However, other studies have confirmed speculations that the potent immunosuppressive activities of IL-10 may, under certain circumstances, increase the host's susceptibility to infection with certain types of pathogenic organisms.
Susceptibility of mononuclear phagocytes to influenza A virus infection and possible role in the antiviral response
Authors: Hofmann P , Sprenger H , Kaufmann A , Bender A , Hasse C , Nain M , Gemsa D
Published: January 2018

Among leukocytes, only monocytes and macrophages were found to be highly susceptible to an infection by influenza A virus. After infection, de novo viral protein synthesis was initiated but then interrupted after 4–6 h. Most macrophages died by apoptosis within 25–30 h. Before cell death, however, macrophages responded to influenza A virus with a high cytokine gene transcription and subsequent release of tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), IL-6, interferon (IFN) -α/β, and CC-chemokines. The basic mechanisms of virus-induced cytokine expression are still unknown and appear to involve transcription factors such as nuclear factor-κB and AP-1 which, however, were only activated for 2 h and declined below control values thereafter. After influenza A virus infection, only the mononuclear cell attracting CC-chemokines macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and RANTES were produced while the prototype neutrophil CXC-chemoattractants IL-8 and GRO-α were entirely suppressed. This selective induction of CC-chemokines may explain the preferential influx of mononuclear leukocytes into virus-infected tissue. Our data show that monocytes and macrophages represent a primary target for an influenza A virus infection. Thus, the mononuclear phagocyte response leads to a rapid proinflammatory reaction and an enhanced immigration of mononuclear leukocytes, which may condition the infected host for the subsequent virus antigen-specific defense.
Macrophage and interleukin-1 induced nitric oxide production and cytostasis in hamster tumor cells varying in malignant potential
Authors: Lavnikova Natasha , Burdelya Ludmila , Lakhotia Anand , Patel Naimish , Prokhorova Svetlana , Laskin Debra L
Published: January 2018

Nitric oxide has been shown to contribute to cytotoxicity in mouse and rat tumor cells. In these studies we examined the role of nitric oxide in cytostasis in hamster tumor cells varying in their malignant potential. Spontaneously transformed hamster embryonic fibroblasts (STHE cells) with low metastatic activity produced significantly greater amounts of nitric oxide in response to interleukin-1 (IL-1) or lipopolysaccharide (LPS) -activated hamster alveolar macrophages (HAM) than did tumor cell lines with high experimental metastatic activity (HET-SR, HET-SR1, STHE-83/20 cells). HET-SR cells, which exhibit low spontaneous metastastic activity, also produced relatively high levels of nitric oxide in response to IL-1, whereas the response of the spontaneously metastatic lines, HET-SR1 and STHE-83/20 cells, was low. IL-1 and HAM also induced cytostasis in nitric oxide-producing STHE and HET-SR cells. However, the nitric oxide synthase inhibitor, NG-monomethyl-l-arginine (l-NMMA), had no effect on this activity. These findings, together with the observation that anti-tumor necrosis factor α antibody prevented HAM-mediated cytostasis in all of the tumor cell lines demonstrate that nitric oxide is not involved in hamster macrophage-induced tumor cell growth suppression. In contrast to HAM, rat alveolar macrophages, which produced nitric oxide in response to LPS, exerted similar levels of cytostasis toward all of the hamster tumor cell variants, an action that was blocked by l-NMMA in HETSR, HET-SR1, and STHE-83/20 cells. Thus production of nitric oxide by hamster tumor cells is inversely correlated with their malignant potential. However, nitric oxide does not appear to be involved in IL-1-or HAM-mediated cytostasis toward hamster tumor cells.
Characterization of clonally derived, spontaneously transformed bone marrow macrophage cell lines from lipopolysaccharide hyporesponsive LPS(d) and normal LPS(n) mice
Authors: Monner David A , Denker Brigitte
Published: January 2018

Six macrophage cell lines, each derived from a bone marrow macrophage colony grown in soft agar, were established by expansion of the macrophage clones in liquid culture until spontaneous transformation occurred. Four lines originated from the LPS(d) nonresponder mouse strain C3H/HeJ and two from the LPS(n) responder strain CBA/J. The cell lines adhered to plastic and glass surfaces and displayed typical macrophage functions such as phagocytosis and nonspecific esterase activity. Flow cytometry analyses showed that the lines expressed the macrophage surface markers CD11b, CD13, CD32/16, F4/80, and BM8 constitutively. A moderate expression of the adhesion receptor CD11a, but only a very low expression of its ligand CD54, was observed. A minor fraction of the cells in each line constitutively expressed MHC class II antigen, and its expression could be up-regulated in each cell line by treatment with interferon-γ (IFN-γ). Secretion of the inflammatory mediators nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) after induction by three bacterial derivatives, heat-killed Salmonella typhimurium (HKS), lipopolysaccharide (LPS), and the Mycoplasma fermentans-derived amphiphilic lipid MDHM, were examined in detail. Not only did the lines differ in the amounts of mediators secreted in response to any one stimulus, but the doses of MDHM or LPS required for 50% maximal induction of NO varied up to 10-fold among the four LPS(d) cell lines, suggesting considerable functional heterogeneity between the clones. Secretion of large amounts of TNF-α was induced in all the cell lines by HKS. Although it could be shown that exogenously added TNF-α acted synergistically with IFN-γ to induce NO release from the cell lines, an autocrine role for TNF-α during HKS-IFN-γ induction of NO synthesis could not be substantiated. Neutralization of TNF-α with a specific antibody completely blocked NO induction by exogenous TNF-α but did not abrogate NO release either by HKS-IFN-γ-induced cells or by macrophages treated with supernatant from an HKS-IFN-γ-activated cell line. These results indicate that the clones are arrested in distinct stages of differentiation and retain some properties of normal untransformed macrophages. They should be helpful tools for investigations into macrophage function.
Secretory leukocyte proteinase inhibitor is a major leukocyte elastase inhibitor in human neutrophils
Authors: Sallenave Jean-Michel , Si-Ta har Mustapha , Cox Gerard , Chignard Michel , Gauldie Jack
Published: January 2018

Secretory leukocyte proteinase inhibitor (SLPI) is the main neutrophil elastase (HLE) inhibitor found in the upper airways during pulmonary inflammation. It has been shown to be synthesized and secreted in vitro by epithelial cells and has been localized in tracheal glands and bronchiolar epithelial cells by immunocytochemistry. In this study, using immunodetection and immunopurification techniques with specific anti-SLPI immunoglobulin G (IgG), we show that SLPI is present as a native 14-kDa molecule in neutrophil cytosol. In addition, we demonstrate that SLPI is the major inhibitor of HLE present in neutrophil cytosol because pre-incubation with specific anti-SLPI IgG was able to inhibit completely the anti-HLE activity of the cytosol. SLPI can be secreted (probably in an inactive form) by neutrophils and its secretion is enhanced when the cells are stimulated with phorbol myristate acetate (PMA). Elafin, an elastase-specific inhibitor, is also present in minute amounts in neutrophil cytosol and its secretion can be upregulated. The presence of SLPI in the cytosol of neutrophils may serve as a protective screen against proteinases spilling from azurophilic granules. An alternative or supplementary role may be the maintenance of a differentiated phenotype.
Complex regulation of human neutrophil activation by actin filaments: dihydrocytochalasin B and botulinum C2 toxin uncover the existence of multiple cation entry pathways
Authors: Wenzel-Seifert Katharina , Lentzen Hans , Aktories Klaus , Seifert Roland
Published: January 2018

In human neutrophils, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenalalanine (fMLP), the Ca2+-ATPase inhibitor, thapsigargin, and the lectins, concanavalin A (Con A) and mistletoe lectin I (ML I), stimulate the entry of Ca2+ and Na+ with subsequent activation of exocytosis and superoxide anion (O2–) formation. We studied the role of actin in neutrophil activation. The actin filament-disrupting substances, dihydrocytochalasin B (dhCB) and botulinum C2 toxin (C2 toxin) potentiated fMLP- and lectin-stimulated Ca2+- and Na+ entry. Lectin-induced Mn2+ entry was enhanced by actin disruption, whereas fMLP-triggered Mn2+ entry was unaffected. dhCB and C2 toxin inhibited fMLP- and lectin-stimulated Ba2+ influx. The actin disrupters also inhibited fMLP- and ML I-induced Sr2+ influx, whereas Con A-stimulated Sr2+ entry was not influenced by dhCB and C2 toxin. Thapsigargin-stimulated cation entry was not altered by actin disruption. DhCB and botulinum C2 toxin potentiated lysozyme release induced by all four stimuli. Con A and ML I per se activated O2 – formation only in the presence and not in the absence of dhCB. Con A potentiated the stimulatory effects of ML I on O2 – formation in the presence of dhCB and primed neutrophils to respond to ML I in the absence of dhCB. Our data indicate the following: (1) dhCB and C2 toxin uncover the existence of multiple cation entry pathways in neutrophils; (2) actin disruption facilitates exocytosis and O2 – formation by enhancement of Ca2+- and Na+ entry and by altering the function of proteins involved in activation of secretion and O2 – formation; and (3) Con A and ML I, which possess different sugar specificities, activate different signaling pathways in neutrophils.
Quantitation of surface CD14 on human monocytes and neutrophils
Authors: Antal-Szalmas Peter , van Strijp Jos A G , Weersink Annemarie J L , Verhoef Jan , van Kessel Kok P M
Published: January 2018

The absolute number of membrane-expressed CD14, the most important endotoxin receptor, on human monocytes and neutrophils shows remarkable variation in the literature. To quantify these numbers two fluorescence methods using fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (mAb) were applied. A commercially available set of standard beads was used in flow cytometry to quantitate CD14 with eight different mAbs. Independent from their isotype the various mAbs showed minor differences and indicated that peripheral blood monocytes expressed 99,500–134,600 (115,400 ± 10,600) and neutrophils 1,900–4,400 (3,300 ± 800) CD14 receptors. There was no significant difference in CD14 expression on leukocytes in unprocessed freshly obtained whole blood and after a Ficoll isolation procedure. However, a short temperature shift resulted in a 1.3- to 1.6-fold upregulation of CD14. The results obtained with the reference beads were verified with fluorescence Scatchard analysis and spectrofluorometry using mAb 26ic-FITC and showed 109,500 CD14 per monocyte and 6,700 CD14 per neutrophil. For comparison the number of CD14 on the monocytic THP-1 cells and Fcγ-receptors on human leukocytes were determined using the reference beads and flow cytometry and gave results comparable to published data. Our data indicate that resting human monocytes express roughly 110,000 CD14 molecules on their surface using a simple fluorometric assay. Correct determination of the number of CD14 and other cell surface receptors is of importance in the monitoring of septic patients.
Desensitization of the fMLP-induced NADPH-oxidase response in human neutrophils is lacking in okadaic acid-treated cells
Authors: Harbecke Olle , Liu Li , Karlsson Anna , Dahlgren Claes
Published: January 2018

The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) interacts with neutrophils, generating signals that induce activation of the superoxide anion/hydrogen peroxide-producing NADPH-oxidase. Low temperature binding of fMLP to its neutrophil surface receptors is associated with a desensitization of the cells with respect to activation of the oxidase. Other stimuli can still activate the oxidase (in fact even induce a primed response), indicating that the observed phenomenon is stimulus specific and could not be accounted for by an effect on the oxidase itself. Furthermore, no desensitization is obtained in the presence of cytochalasin B, suggesting that the cytoskeleton is involved in the process leading to desensitization. Okadaic acid is a toxin produced by dinoflagellates and exerts its effects by an inhibition of cellular phosphatases. To investigate the role of phosphorylation/dephosphorylation events in the desensitization process we used okadaic acid as a scientific tool. We show that neutrophils treated with okadaic acid are primed with respect to the fMLP-induced production of superoxide anion, and that no desensitization is obtained in toxin-treated cells. Because the recovery of ligand-receptor complexes in a Triton X-100-insoluble fraction is very low in the cells treated with okadaic acid, we suggest that protein dephosphorylation is required to obtain binding to the cytoskeleton of occupied fMLP receptors; binding of the occupied receptors to the cell cytoskeleton being the mechanism behind desensitization.
Significance of macrophage tropism of SIV in the macaque model of HIV disease
Authors: Stephens Edward B , Galbreath David , Liu Zhen Qian , Sahni Manisha , Li Zhuang , Lamb-Wharton Rebecca , Foresman Larry , Joag Sanjay V , Narayan Opendra
Published: January 2018

Microglia, alveolar macrophages, and Langerhans cells are representatives of cells of macrophage lineage that are susceptible to infection with HIV-1 and they play important roles in the pathogenesis of AIDS dementia, lymphoid interstitial pneumonia, and systemic viral invasion from mucosal surfaces, respectively. In contrast, elimination of CD4+ T cells with resultant development of immunosuppression and AIDS is thought to be reflective of the exclusive tropism of the virus for CD4+ T cells. Examination of these concepts in macaques infected with molecularly cloned strains of SIVmac suggested that all strains of the virus are both macrophage- and lymphocyte-tropic and that all aspects of pathogenesis including loss of CD4+ T cells are dependent on infection in both cell types. However, viral clones that caused productive lytic infection in macrophages were less virulent than those which caused persistent nonproductive infection. The former caused subclinical and even immunizing infections, whereas the latter caused activation and productive infection in CD4+ T cells, AIDS, and systemic infection, even after inoculation of the virus on mucosal surfaces. If these findings on SIVmac are relevant to HIV-1 disease, then demonstration that HIV-1 isolates are macrophage-tropic probably does not necessarily correlate with their pathogenic potential.
GM-CSF and its effects on replication of HIV-1 in cells of macrophage lineage
Authors: Crowe Suzanne M , Lopez Angel
Published: January 2018

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hemopoetic growth factor that is a member of the four-helix bundle family of cytokines and growth factors. It regulates the proliferation and differentiation of granulocytes and cells of macrophage lineage from bone marrow progenitor cells, mediating these activities through binding to its receptor. Most studies examining the effects of GM-CSF on HIV-1 replication in primary monocytes and macrophages, and in related cell lines, have demonstrated augmentation of HIV-1 expression in vitro, although some reports have been at variance with these findings. These laboratory-based observations have been confirmed in limited clinical trials. This review outlines the details of these studies and considers mechanisms by which GM-CSF may exert its effects on cells of this lineage.
Intracellular GSH content and HIV replication in human macrophages
Authors: Garaci Enrico , Palamara Anna T , Ciriolo Maria R , D'Agostini Cartesio , Ab del-Latif Mohamed S , Aquaro Stefano , Lafavia Emanuela , Rotilio Giuseppe
Published: January 2018

In vitro HIV-1 infection induced a significant decrease in intracellular reduced glutathione (GSH) in human macrophages. Such a decrease was observed at the time of infection corresponding to maximum release of virus from infected cells and was not related to cell cytotoxicity. GSH loss was not related to its oxidation or leakage through the cell membrane. Inhibition of intracellular GSH synthesis by buthionine sulfoximine (BSO) did not further decrease GSH levels with respect to the decrease caused by HIV alone. However, treatment of macrophages with BSO significantly increased the HIV yield in the supernatant. Exogenous GSH strongly suppressed the production of p24 gag protein as well as the virus infectivity. Previous observations with other RNA and DNA viruses consistently showed that GSH antiviral effect occurred at late stages of virus replication and was related to the selective decrease of specific glycoproteins, such as gp120, which are particularly rich in disulfide bonds.
Monoclonal antibody to ICAM-1 preserves postischemic blood flow and reduces infarct size after ischemia-reperfusion in rabbit
Authors: Zhao Zhi-Qing , Lefer David J , Sato Hiroki , Hart K K , Jefforda R R , Vinten-Johansen J
Published: January 2018

Neutrophils are pivotal in the pathogenesis of reperfusion injury leading to myocardial infarction. Firm adhesion of PMN to endothelium may be initiated by the interaction between constitutively expressed intercellular adhesion molecule-1 (ICAM-1) on endothelium and β2 integrin (CDllb/CD18) on neutrophils. We tested the hypothesis that a monoclonal antibody (mAb RR1/1) against ICAM-1 would preserve postischemic myocardial blood flow and attenuate myocardial injury in an anesthetized rabbit model of coronary occlusion and reperfusion. Either mAb RR1/1 or isotypematched control mAb (R3.1) was injected 10 min before reperfusion. Postischemic myocardial blood flow in the area at risk (Ar) and necrotic area was significantly improved with mAb RR1/1 treatment compared with vehicle and mAb R3.1 during the reperfusion period. RR1/1 had no effect on nonischemic zone blood flow. The Ar as a percent of left ventricle was comparable between groups. Infarct size (TTC) as a percent of Ar was significantly reduced by mAb RR1/1 compared with saline vehicle and mAb R3.1. Plasma creatine kinase activity confirmed the reduction of infarct size in mAb RR1/1 group. In in vitro studies, 40 μg/mL mAb RR1/1, which approximates the plasma concentration of 2 mg/kg mAb RR1/1, markedly inhibited platelet-activating factor-stimulated neutrophil adherence to rabbit aortic endothelium. We conclude that blockade of ICAM-1 during reperfusion reduces postischemic perfusion defects and attenuates the progression of myocardial injury leading to necrosis. This cardioprotection by mAb RR1/1 may be due to inhibition of neutrophil adhesion to the coronary endothelium.
Apoptosis and in vivo distribution and clearance of eosinophils in normal and Trichinella spiralis-infected rats
Authors: Gon Songi , Saito Susumu , Takeda Yuji , Miyata Hiroaki , Takatsu Kiyoshi , Sendo Fujiro
Published: January 2018

We examined apoptosis of eosinophils obtained from normal rats and rats infected with Trichinella spiralis (T. spiralis). A considerable percentage of normal rats underwent apoptosis within a few hours, however, the apoptosis rate of infected rats was significantly lower. This rate began decreasing only 12 h after infection, reached its lowest level on day 3, and was restored to normal by day 40. We also studied the effects on eosinophil apoptosis of culture supernatants from Concanavalin A-stimulated spleen cells from infected rats and found that supernatants from days 3, 7, and 18 of infection inhibited apoptosis eosinophils from normal rats. We then examined the distribution and clearance of eosinophils in vivo. In normal rats, the largest numbers of eosinophils were found in the spleen, liver, and bone marrow. In the infected rats, however, by day 3, by which time the worms had begun to migrate, there was a striking increase in these cells in the intestines. In summary, normal eosinophils are unexpectedly sensitive to apoptosis, whereas infected eosinophils become resistant to this form of cell death.
Modulation of nitric oxide production in RAW 264.7 cells by transforming growth factor-beta and interleukin-10: differential effects on free and encapsulated immunomodulator
Authors: Seyler Isabelle , Appel Martine , Devissaguet Jean-Philippe , Legrand Philippe , Barratt Gillian
Published: January 2018

The mouse macrophage cell line RAW 264.7 can be stimulated to produce nitric oxide (NO) by muramyltripeptide cholesterol included within biodegradable poly(D,L-lactide) nanocapsules (NC MTP-Chol). The aim of this work was to determine whether one or both of the cytokines transforming growth factor-beta (TGF-β) and interleukin-10 (IL-10) could be responsible for feedback control seen at high concentrations. Activated RAW 264.7 cells produced TGF-β1. When exogenous TGF-β1 was added during stimulation, a dose-dependent inhibition of NO production was observed when NC MTP-Chol was used, whereas activation by the soluble muramyl dipeptide (MDP) was not affected. Furthermore, addition of a blocking antibody to TGF-β arrested the fall in NO production seen at high concentrations of NC MTP-Chol. Addition of IL-10 during RAW 264.7 cell activation also reduced NO production; however, in this case, both NC MTP-Chol and MDP were equally affected. The presence of anti-IL-10 antibody during activation significantly increased NO production.
Effect of alterations of blood cholesterol levels on macrophages in the myocardium of New Zealand White rabbits
Authors: Kinscherf Ralf , Kamencic Huse , Deigner Hans-Peter , Pill Johannes , Schmiedt Walther , Schrader Michael , Metz Jurgen
Published: January 2018

We investigated the effect of alterations of blood cholesterol levels on macrophages (mΦ) in the myocardium of New Zealand White (NZW) rabbits. Three groups of NZW rabbits were used: controls, rabbits fed a 0.5% cholesterol-enriched diet (CH-D) for 96 days, and rabbits fed a 0.5% CH-D for 96 days followed by normal chow for 4 months. Immunohistochemical analysis by mAbs directed against mΦ (RAM-11) and Mn superoxide dismutase (MnSOD) were quantified by computer-assisted morphometry. Using cultured human and rabbit mΦ, a cross-reaction of the human MnSOD mAbs was found as well as the predominant localization of MnSOD-immunoreactivity (IR) in mitochondria, In group 1, only a very few RAM-11-immunoreactive (ir) mΦ occurred in the interstitial space of the myocardium. In group II blood cholesterol levels significantly increased in parallel with the numbers of mΦ, which often contained lipid droplets (foam cells). Although blood cholesterol concentrations regressed about 10-fold in group III, mΦ in the myocardium were found to be reduced only about 20%. Most mΦ were also MnSOD-ir. In atherosclerotic coronary arteries RAM-11-IR was located in mΦ and also extracellularly, whereas MnSOD-IR was found only in mΦ. Drastically induced MnSOD in the mitochondria of mΦ is suggested as an indicator of increased oxidative stress caused by in vitro conditions or by phagocytosis of low-density lipoprotein in vivo. Elevation of the cholesterol level leads to a long-term increase and its regression results in a delayed reduction of such mΦ, which seem to play a key role in the atherogenesis of the coronary arteries as well.
Effects of acute ethanol exposure on cellular immune responses in a murine model of thermal injury
Authors: Faunce Douglas E , Gregory Meredith S , Kovacs Elizabeth J
Published: January 2018

To test the effects of acute ethanol exposure on immune function after thermal injury, mice with blood alcohol levels of 100 mg/dL were given a 15% total body surface area dorsal scald or sham injury. Bacterial challenge resulted in 100 and 40% mortality in burn + ethanol- and burn + vehicle-treated mice, respectively. Delayed-type hypersensitivity responses were also significantly suppressed in burn + ethanol-treated mice. At 1 and 4 days post-burn, concanavalin A (ConA) -induced total splenocyte proliferation in burn + ethanol-treated groups was significantly decreased (P < 0.01) compared with burn + vehicle- or sham-treated animals. This decrease was not observed in total splenocytes cultured with anti-CD3ε or among adherence-depleted splenocytes given ConA or anti-CD3ε. FACS analyses revealed no changes in splenocyte sub-type ratios in burn + ethanol mice. The data herein demonstrate that acute ethanol exposure before thermal injury results in enhanced susceptibility to bacterial infection and markedly suppressed cellular immunity, which appears to be macrophage dependent.
Signal transduction in Jurkat T lymphocytes. Evidence for early Ca2+ movements between the cytoplasm and the nucleoplasm in activated cells
Authors: Gagné Daniféle , Ahnadi Charaf E , Martel Julie , Payet Marcel D , Dupuis Gilles
Published: January 2018

Spatial analyses of the distribution of Ca2+in resting and activated T and B lymphocytes have shown that the bulk of increased [Ca2+]i appears to be associated with the nuclear region. These observations suggest that Ca2+ is released from the perinuclear space or that it diffuses to the nucleoplasm, or both. We have used laser scanning confocal microscopy to assess whether cytoplasmic diffusion of Ca2+ could contribute to the rise in nuclear Ca2+. We found that the activation of individual Jurkat cells by use of an anti-Ti (β-subunit) mAb induced a nucleus-associated increase in [Ca2+]i. In cells loaded with the InsP3 receptor antagonist heparin, the nuclear Ca2+ response was abolished but not the response to thapsigargin. Evidence for a cytoplasmic Ca2+ response was obtained by loading Jurkat cells with a cytoplasm-restricted Ca2+ probe (Calcium Green-1-Dextran). These observations suggested that a process of diffusion of cytoplasmic Ca2+ contributed to the rise of nuclear Ca2+ in Jurkat T cells. This interpretation was supported by the findings (1) that rapid scanning of thapsigargin-released Ca2+ showed an inverse relationship between the levels of cytoplasmic and nuclear Ca2+ and (2) that modulation of the external concentration of Ca2+ in thapsigargin-treated Jurkat cells showed a time-dependent decrease of fluorescence from the nucleoplasm that was reversed by raising the concentration of external Ca2+. We conclude that Ca2+can rapidly diffuse between the cytoplasm and the nucleoplasm in activated Jurkat T lymphocytes and that hydrophilic Ca2+ probes largely partition to the nucleoplasm, thus giving rise to distorted nucleus-to-cytoplasm fluorescence ratios.
Arginine-specific ADP-ribosyltransferases in leukocytes
Authors: Saxty Barbara A , Kefalas Panagiotis , Yadollahi-Farsani Masoud , MacDermot John
Published: January 2018

The posttranslational modification of proteins by the addition of an ADP-ribose group is mediated by ADP-ribosyltransferases, which are expressed widely in many eukaryotic tissues, including leukocytes. DNA encoding arginine-specific ADP-ribosyltransferases has been cloned from both polymorphonuclear neutrophil leukocytes and lymphocytes, and their primary structures are widely conserved, particularly in those domains of the enzyme implicated in NAD+ binding and catalysis. In most cases the enzymes are tethered to the outer aspect of the cell surface or are released directly from the cell surface. The protein substrates of some of the ADP-ribosyltransferases have been identified and the catalytic activity of these enzymes has been implicated in several immune responses as well as white cell chemotaxis. This review describes recent significant advances in this field, and it seems likely that additional leukocyte functions, most particularly those linked to the activity of surface integrins, will be assigned to this class of enzymes.
Subcellular localization of glycosphingolipids in human neutrophils
Authors: Kniep Bernhard , Skubitz Keith M
Published: January 2018

The subcellular distribution of five major glycosphingolipids (GSLs) in human neutrophils was analyzed. The neutrophils were isolated from the blood of six donors and subdivided in three fractions containing the cell membranes, and the primary and the secondary granules, respectively. The separation was confirmed with antibodies detecting established subcellular fraction-specific molecules. The two main neutral GSLs Galβ 1-4Glcβ 1-1 ‘Cer (lactosylceramide, LacCer) and nLc4Cer (paragloboside, PG) and the three gangliosides IV3NeuAcnLc4Cer (2-3SPG), IV6NeuAcnLc4Cer (2-6SPG), and VI3NeuAcnLc6Cer (2-3SnHC) were quantitated using the immunochemical digoxigenin (DIG) staining procedure. Secondary granules contained the highest amount of these GSLs. They are followed by the primary granules and the cell membranes. Based on this quantitation, we conclude that the majority of the GSLs of neutrophils occur intracellularly. These findings are in striking contrast to the general assumption of GSLs being mainly concentrated in the cell membrane.
Novel monoclonal antibodies against membrane structures that are preferentially expressed on IL-2-activated rat NK cells
Authors: Giezeman-Smits Katinka M , Jonges Liesbeth E , Chambers William H , Brisette-Storkus Cynthia S , van Vlierberghe Ronald L P , van Eendenburg Jaap D H , Eggermont Alexander M M , Fleuren Gert Jan , Kuppen Peter J K
Published: January 2018

Interleukin-2 (IL-2)-activated natural killer (NK) cells are known to mediate specific functions such as cytolytic activity and tumor infiltration more efficiently than nonactivated NK cells. To investigate whether these characteristics are associated with induction or up-regulation of expression of membrane structures after IL-2 activation, we selected four hybridomas (mAbs ANK44, ANK66, ANK7, and ANK123) derived from mice immunized with rat IL-2-activated NK cells and compared the expression of the epitopes recognized on IL-2-activated NK cells versus unstimulated NK cells. We found that ANK44-expression was induced after activation with IL-2. The antigens recognized by ANK66, ANK7, and ANK123 were expressed selectively on subsets of nonactivated NK cells. After activation with IL-2 the level of expression of these antigens was increased. Moreover, the majority of NK cells then expressed these antigens after IL-2 activation. Biochemical characterization of the membrane structures recognized on IL-2-activated NK cells showed that they have not previously been described. The precise function of these membrane structures is not yet known, however, the selective nature of their expression implies their involvement in the specific functions of IL-2-activated NK cells.
Salmonella infections in the absence of the major histocompatibility complex II
Authors: Chapes Stephen K , Beharka Alison A
Published: January 2018

We examined the pathogenesis of the facultative intracellular bacterium, Salmonella typhimurium in MHCII-/-, C2D knock-out mice, and wild-type C57BL/6J mice. The MHCII knock-out shortened the kinetics of animal death and reduced the dose of S. typhimurium needed to kill mice. We measured the physiological and cytokine responses of both mouse strains after S. typhimurium injection. Animal weight loss, spleen weights, liver weights, thymus weights, and serum corticosterone concentrations were comparable after injection with several doses of bacteria. The only physiological differences observed between the two strains were observed 3 days after injection of the highest dose of bacteria tested. Serum concentrations of tumor necrosis factor α, interleukin-2, and interleukin-6 increased in a dose-dependent fashion irrespective of mouse MHCII expression. Therefore, even in the absence of MHCII, mice are able to mount relatively normal physiological and immunological responses. Consistent with these normal responses, an increased percentage of MHCII-/-mice, primed with a low dose of bacteria 13 days earlier, were able to survive a lethal challenge of Salmonella compared with unprimed controls. Lastly, C2D mice had significantly higher serum interleukin-10 concentrations than C57BL/6J mice 48 h after infection with all doses of S. typhimurium. C2D macrophages also secreted significantly more IL-10 and less NO and O2–after lipopolysaccharide or phorbol ester stimulation in vitro than wild-type macrophages.
Cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of NADPH oxidase activation
Authors: Tal Tal , Michaela Sharabani , Irit Aviram
Published: January 2018

We have previously reported inhibition of cell-free activation of the neutrophil superoxidegenerating NADPH oxidase by a soluble cationic protein of neutrophil granules and by low concentrations of human defensin. Subcellular fractionation carried out in the current study indicated that the inhibitory substance was derived from azurophilic granules, was released into the medium on cell stimulation, and was resistant to phenylmethylsulfonyl fluoride (PMSF). Phorbol ester was the most effective stimulus for the release of the blocking activity. The possibility was raised that granule protein(s) act in vivo as negative modulators of superoxide production. Gel filtration of granule extract revealed a markedly retarded protein peak exhibiting oxidase-blocking activity and containing lysozyme as the main protein. Because lysozyme did not exert inhibitory effects on oxidase activation, association of the inhibitory protein with lysozyme was assumed. Indeed a column of immobilized lysozyme retained a fraction of the granule extract's oxidase-blocking activity. Elution with a low-pH buffer recovered a component capable of inhibition of the NADPH oxidase in stimulated neutrophils and in the cell-free system. The main 29-kDa protein band in the eluted fraction was identified as proteinase 3, a serine protease of azurophilic granules. Enzymatically active as well as PMSF-blocked conventionally purified proteinase 3 interfered with phorbol myristate acetate-induced superoxide release. These findings support the hypothesis that exocytosed granule constituents may prevent excessive activation of the NADPH oxidase.
Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils
Authors: Brazil Timothy J , Rossi Adriano G , Haslett Christopher , McGorum Bruce , Dixon Paddy M , Chilvers Edwin R
Published: January 2018

The synthetic formylpeptide fMLP is widely used as a model chemoattractant and secretagogue for mammalian neutrophils. Despite possessing fMLP receptors, equine neutrophils do not produce superoxide anions in response to fMLP and there is no inflammatory reaction in the horse when fMLP is injected intradermally. The functional capability of these receptors was investigated after pretreatment with recognized priming agents. Purified neutrophils were pretreated with lipopolysaccharide (LPS), platelet-activating factor (PAF), or tumor necrosis factor α (TNF-α) and superoxide anion generation and shape change quantified by lucigenin-dependent chemiluminescence (LDCL) and flow cytometry, respectively. LPS, TNF-α, and PAF pretreatment induced significant LDCL in response to fMLP; similarly LPS pretreatment was a prerequisite for fMLP-stimulated neutrophil polarization in response to fMLP. However, LPS failed to induce fMLP-mediated chemotaxis of equine neutrophils. These data indicate that equine neutrophil fMLP receptors are not vestigial as previously thought but can trigger both respiratory burst activity and cell polarization responses after priming.
Ligation of CR1 attenuates Fc receptor-mediated myeloperoxidase release and HOCl production by neutrophils
Authors: Sambandam Thiagarajan , Chatham W Winn
Published: January 2018

Surface adherent immunoglobulins are potent stimuli for inducing neutrophil release of myeloperoxidase and production of hypochlorous acid (HOCl), an oxidant that promotes activity of neutrophil proteases. Opsonization of surface adherent IgG (SAIgG) by complement results in attenuation of these responses, despite augmenting neutrophil production of superoxide and release of specific granule enzymes. The role of complement receptor ligation in modulating Fc receptor-triggered myeloperoxidase release and HOCl production by neutrophils was determined by incubating neutrophils with SAIgG in the presence of complement receptor ligating antibody reagents. Ligation of CR1 by F(ab)2 derived from CR1 specific monoclonal antibody (mAb 543) resulted in significant attenuation of surface-associated IgG (SAIgG)-induced release of myeloperoxidase and HOCl production but did not result in attenuation of SAIgG-induced superoxide or hydrogen peroxide production; ligation of CR1 by mAb 543 F(ab)2 also attenuated surface adherent IgA-induced myeloperoxidase release and HOCl production. HOCl production was not significantly attenuated when neutrophils were activated with SAIgG in the presence of surface adherent C1q or when CR3 was ligated by F(ab)2 derived from mAb having specificity for the CD11b (mAb M1/70) or CD18 (mAb TS1) subunits of CR3. These results indicate that ligation of CR1 on neutrophils by C3b fixed to IgG may alter signal transduction events linking ligation of neutrophil Fc receptors to cellular events required for release of myeloperoxidase and generation of HOCl.