bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis or necrosis
Authors: Takei Hiroshi , Araki Akemi , Watanabe Hiroshi , Ichinose Akitada , Sendo Fujiro
Published: January 2018

To elucidate the relationship between activation of neutrophils and their subsequent death, the effect of phorbol 12-myristate 13-acetate (PMA), a potent activator of neutrophils, was examined. PMA-treated neutrophils showed morphological changes quite different from those of typical apoptosis or necrosis. After fusion of the lobate nucleus, nuclear contents of chromatin uniformly decreased in compactness and soon after the nuclear envelope was broken. Even at this stage, cytoplasmic organelles did not undergo degeneration. Membrane permeability began increasing at 3 h of incubation with PMA, subsequent to nuclear change. Conventional agarose gel electrophoresis and pulsed field gel electrophoresis of DNA from PMA-treated neutrophils revealed no DNA degradation products smaller than 300 kbp. PKC inhibitors, staurosporine and H-7, prevented cytotoxicity by PMA. Furthermore, antioxidants, thiourea, dimethylthiourea, pyrrolidinethiocarbamate, and N-acetyl-L-cysteine, but not superoxide dismutase, were also active in preventing PMA cytotoxicity, suggesting that cell suicide resulting from PMA treatment is due to oxygen radicals, especially the hydroxyl radical. A certain population of neutrophils phagocytosing opsonized zymosan also showed changes similar to those observed in PMA-treated cells.
Bacterial lipopolysaccharide confers resistance to G418, doxorubicin, and taxol in the murine macrophage cell line, RAW264
Authors: Sweet Matthew J , Hume David A
Published: January 2018

Many bacterial pathogens including Salmonella and Listeria replicate within macrophages. The susceptibility of these organisms to various antibiotics is dependent on the ability of macrophages to take up, retain, and deliver the antibiotic to the correct intracellular compartment. In this context, macrophages are known to express proteins that are involved in efflux of antibiotics and cytotoxic drugs, thereby reducing intracellular accumulation of such compounds. In our studies on the action of bacterial lipopolysaccharide (LPS) on the macrophage-like cell line, RAW264 we found that LPS treatment of these cells conferred resistance to the neomycin-related aminoglycoside G418 (geneticin). This phenotype was stable and was specific to LPS since colony-stimulating factor 1 and phorbol myristate acetate had no effect on G418 resistance. We have extended this observation to show that LPS induces transient resistance to the cytotoxic drugs taxol and doxorubicin. Macrophage resistance to cytotoxic drugs and antibiotics may have a number of important clinical consequences.
Different responses of plasma ACTH and corticosterone and of plasma interleukin-1β to single and recurrent endotoxin challenges
Authors: Mekaouche Mourad , Siaud Philippe , Givalois Laurent , Barbanel Gérard , Malaval Francis , Maurel Daniel , Assenmacher Ivan , Ixart Guy
Published: January 2018

In a parallel study in 10 individual rats, three time series of plasma concentrations of ACTH, corticosterone (CORT), and interleukin-1β (IL-1β) were measured before (time 0) and at intervals between 15 and 480 min following intra-arterial (i.a.) infusions of 25μg/kg lipopolysaccharide (LPS). All LPS injections were given at 9 AM. The first time series was performed on naive rats (day 1). A sequence of six daily injections (days 3–8) of the same dose of LPS followed. The post-LPS time course of the plasma ACTH, CORT and IL-1β levels were studied on days 3 (second injection) and 8 (seventh injection). The first LPS injection induced a rapid (30 min) eightfold rise in plasma ACTH and CORT, culminating in concentrations 30 times the baseline at 60 min (ACTH) and 15 times baseline at 120 min (CORT). Both hormones receded back to the initial basal level at 480 min. On the other hand, IL-1β increased slowly to peak at 13 times baseline 120 min before declining to minimal seven- to ninefold basal levels, 480 min and even 48 h post-LPS. During the second phase of the experiment starting 48 h after the initial LPS priming sequence, the ACTH and CORT responses to daily recurrent LPS injections again differed from those of IL-1β. The post-LPS time courses of the ACTH and CORT reaction displayed a typical pattern of a progressive attenuation studied at days 3 and 8. The peak amplitudes at days 3 and 8 were reduced to 60 and 10%, respectively, for ACTH, and to 85 and 45% for CORT of those observed at the first LPS test. The duration of the response (both) was also shortened from 480 min (first LPS test) to 300 min at days 3 and 8. The post-LPS patterns of the IL-1β responses were characterized, first by basal levels seven to nine times higher than the initial baseline values (day 1), and by a rapid suppression of the post-LPS response, with only a slight (30%) increase at day 3 and no increase at day 8. Thus, after both acute and recurrent LPS administration, ACTH/CORT and IL-1β reacted differently to the endotoxin challenge. The two LPS reactive systems were not correlated. This is inconsistent with the often proposed role of increased plasma IL-1β release as an intermediary factor in the LPS-induced recruitment of the corticotropic axis in general infections.
Apoptosis of macrophages during the resolution of muscle inflammation
Authors: Tidball James G , Pierre Barbara A St
Published: January 2018

We tested the hypothesis that apoptosis contributes to the depletion of macrophages expressing the ED1 or ED2 antigen during the resolution of rat muscle inflammation. Muscle inflammation was induced by subjecting rat soleus muscle to 10 days of unloading followed by periods of muscle reloading. Terminal deoxynucleotidyl transferase- mediated deoxyuridine triphosphate (dUTP) labeling of apoptotic nuclei showed that apoptotic inflammatory cells increase in concentration within necrotic fibers and in the connective tissue at 2 days following muscle injury caused by increased loading. Four days following injury, the apoptotic cells within damaged fibers returned to control levels, and at 7 days following injury apoptotic cells in the connective tissue returned to control concentrations. No preferential, internucleosomal cleavage of DNA from inflamed muscle was observed, although there was greater fragmentation of DNA in inflamed muscle than in controls. Double labeling studies show that cells expressing either ED1 or ED2 antigen can undergo apoptosis in vivo. The time course of apoptosis and concentration of apoptotic cells within damaged muscle fibers indicates that apoptosis contributes to returning ED1+ cells to control concentration during the resolution of inflammation. However, apoptosis of ED2+ cells during the first week following injury is not sufficient to return ED2+ cell concentrations to control values.
Adhesion mechanisms involved in C5a-induced eosinophil homotypic aggregation
Authors: Teixeira Mauro M , Rossi Adriano G , Hellewell Paul G
Published: January 2018

During the process of migration into tissues, leukocytes interact primarily with vascular endothelial cells but they have also been shown to interact with each other. In this study we investigated the adhesion mechanisms involved in guinea pig eosinophil homotypic aggregation as assessed by changes in light transmission. The anti-CD18 monoclonal antibody (mAb) 6.5E, at concentrations in excess of those previously shown to abrogate CD18-dependent eosinophil adherence to serum-coated plastic, inhibited C5a-induced eosinophil aggregation to a maximum of 49–68%. In contrast, the anti-intercellular adhesion molecule-1 (ICAM-1) mAb RR1/1, which binds to guinea pig eosinophils and has been shown to block guinea pig ICAM-1 function, had no effect on C5a-induced responses. Similarly, two functionally active anti-very late antigen-4 (VLA-4) mAbs had no effect on eosinophil aggregation and did not affect the CD18-independent component of the aggregation response. The role of L-selectin in eosinophil aggregation was investigated by using heparin, the selectin-binding polysaccharide fucoidin, and the anti-L-selectin mAb MEL-14. Heparin concentration-dependently inhibited C5a- and platelet- activating factor- (PAF) induced aggregation but C5a-induced responses were inhibited more potently. Fucoidin, but not the carbohydrate dermatan sulphate, effectively inhibited C5a-induced eosinophil aggregation. PMA- and PAF-induced responses were also inhibited by fucoidin. Moreover, fucoidin and 6.5E were additive in their ability to inhibit C5a-induced aggregation. Similarly, MEL-14 effectively inhibited C5a-induced eosinophil aggregation. In conclusion, we have demonstrated that guinea pig eosinophil homotypic aggregation is mostly dependent on CD11/CD18 and L-selectin present on the eosinophil surface. In addition, VLA-4 plays no role in mediating this aggregation response.
GM-CSF and IL-3 activities in schistosomal liver granulomas are controlled by stroma-associated heparan sulfate proteoglycans
Authors: Alvarez-Silva Marcio , Borojevic Radovan
Published: January 2018

Connective tissue cells (myofibroblasts) from liver inflammatory granulomatous reactions to schistosome eggs are able to sustain a long-term proliferation of myeloid cells, both in vivo and in vitro. We have addressed the question of the molecular mechanisms involved in control of this extramedullar stroma-dependent production of inflammatory cells. Heparan sulfate proteoglycans (HSPGs) were purified from granuloma-derived connective tissue cells and bound to plastic or collagen substrate. Their ability to bind recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), to stimulate proliferation of the FDC-P1 myeloid cell lineage, and to modify growth factor activity was monitored. The specificity of this stroma cell-derived glycosaminoglycan interaction with the myeloid growth factors was analyzed by comparing other glycosaminoglycans and sulfated polysaccharides. HSPGs could act as an artificial myelopoietic stroma; they were both required and sufficient for binding and presenting GM-CSF and IL-3 in a biologically active form. Moreover, they were able to mediate an increase in the specific growth-promoting activity of GM-CSF and IL-3. This was specific for stroma-derived heparan sulfate and heparin, since heparan sulfate derived from other cells, other glycosaminoglycans, and related molecules had no effect. These results indicate that HSPGs can stimulate and control the in situ proliferation of myeloid cells, modifying in both quantitative and qualitative terms the composition of inflammatory cell infiltrates in hepatic granulomas.
IL-15: the role of translational regulation in their expression
Authors: Bamford R N , Battiata A P , Waldmann T A
Published: January 2018

We previously reported that the human T cell lymphotropic virus type I (HTLV-I)-associated adult T cell leukemia (ATL) line HuT-102 produces a cytokine designated interleukin (IL) -T. Using anti-cytokine antibodies we demonstrated that IL-T is identical to the simultaneously described IL-15. The observation of a discordance between IL-15 message expression and IL-15 synthesis led us to examine normal and aberrant IL-15 mRNA for post-transcriptional controls that inhibit protein production at the level of RNA translation. When compared to activated monocytes, IL-15 mRNA expression was 6-to 10-fold greater in HuT-102 T cells. The predominant IL-15 message from HuT-102 is a chimeric mRNA joining a segment of the R region of the long terminal repeat of HTLV-I and the 5′ untranslated region (UTR) of IL-15. R segment introduction eliminated over 200 nucleotides of IL-15 5′ UTR including 8 of 10 upstream AUGs that are present in the normal IL-15 message. On analysis of the 5′ UTR of normal IL-15, we demonstrated that these 10 upstream AUGs interfere with IL-15 mRNA translation. Thus, IL-15 synthesis by the ATL cell line HuT-102 involves an increase in IL-15 mRNA transcription and translation secondary to the production of an HTLV-I-R fusion message that lacks many upstream AUGs.
Competition between lymphocyte function-associated antigen 1 (CD11a/CD18) and Mac-1 (CD11b/CD18) for binding to intercellular adhesion molecule-1 (CD54)
Authors: Lub Marijke , van Kooyk Yvette , Figdor Carl G
Published: January 2018

Both human integrin receptors Mac-1 (CD11b/CD18, CR3) and lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) have been demonstrated to bind human intercellular adhesion molecule-1 (ICAM-1) (CD54). Here we show that LFA-1 and Mac-1 can bind to ICAM-1 in the mouse as well. Interestingly, we observed that binding of murine LFA-1 dominates over Mac-1 for binding to ICAM-1. Using three different murine macrophage cell lines that express distinct levels of LFA-1 and Mac-1 on their cell surface, we could only detect Mac-1-dependent adhesion to ICAM-1 when little or no LFA-1 is expressed on the cell surface. When LFA-1 and Mac-1 are expressed at similar levels, the LFA-1/ICAM-1 interaction dominates over Mac-1/ICAM-l interaction, indicating that there is a competition of LFA-1 and Mac-1 for ICAM-1 binding.
Platelets enhance Fcγ receptor-mediated phagocytosis and respiratory burst in neutrophils: the role of purinergic modulation and actin polymerization
Authors: Zalavary Stefan , Grenegård Magnus , Stendahl Olle , Bengtsson Torbjörn
Published: January 2018

The interaction of platelets with neutrophil granulocytes is considered to play an important role in the inflammatory process, and the present study was focused on platelet-induced modulation of Fcγy receptor-mediated functions in neutrophils. We found that phagocytosis and the respiratory burst (measured as luminol-enhanced chemiluminescence), triggered in neutrophils by immunoglobulin G (IgG)-opsonized yeast particles, were potentiated by platelets and that maximal enhancement was achieved at a physiological neutrophil/platelet ratio of about 1:50 to 1:100. Platelets both increased the intra- and extracellular generation of oxygen radicals as well as the release of myeloperoxidase from stimulated neutrophils. The presence of platelets also induced a cortical actin polymerization in neutrophils, which might explain the increased phagocytic capacity. Platelets appear to affect neutrophil function in a contact-independent manner that most likely involves ATP, indicated by the following: (1) platelet supernatants, but not fixed platelets, affected neutrophil function in the same way as viable platelets; (2) platelets raised the extracellular ATP level four- to fivefold; (3) exogenous ATP mimicked the effects of platelets on actin polymerization, phagocytosis, and the respiratory burst in neutrophils; (4) hydrolysis of extracellular ATP with apyrase or blocking of ATP receptors with suramin reversed the platelet-induced enhancement of neutrophil function. An increased accumulation of extracellular adenosine, induced by inhibiting endogenous adenosine deaminase or adding exogenous adenosine, reversed the effects of platelets. The platelet-induced potentiation of the respiratory burst was inhibited by the tyrosine kinase inhibitor genistein, suggesting that tyrosine phosphorylation is involved. However, platelets did not significantly affect the Fcγ receptor-triggered calcium response in neutrophils. In conclusion, we show that platelets, through an ATP-dependent mechanism, potentiate IgG-mediated ingestion and production of oxygen metabolites in neutrophils.
Fibronectin enhances the migration rate of human neutrophils in vitro
Authors: Everitt Elizabeth A , Malik Asrar B , Hendey Bill
Published: January 2018

Efficient polymorphonuclear neutrophil (PMN) migration depends on specific interactions between PMNs, endothelial cells, and extracellular matrix (ECM) proteins. We investigated the relationship between PMN migration and the ECM molecule fibronectin (FN). We used an in vitro migration assay system to show that human PMNs migrated across an FN-coated filter barrier toward a formyl-Met-Leu-Phe (fMLP) chemoattractant gradient in greater numbers than across (uncoated) bare filters. In 1 h of fMLP stimulation, 69 ± 6% of the PMNs had migrated across the FN-coated filters, whereas 46 ± 5% of PMNs migrated across bare filters. This effect was specific to FN; coating the filters with the ECM protein vitronectin did not enhance migration. Monoclonal antibodies against FN or against the α5 or β1 integrin subunits of the FN receptor inhibited the enhanced PMN migration response across FN-coated filters. These findings indicate that the extracellular matrix protein FN enhances PMN migration and that this response is mediated by the α5β1 FN receptor.
Differential screening identifies genetic markers of monocyte to macrophage maturation
Authors: Krause Stefan W , Rehli Michael , Kreutz Marina , Schwarzfischer Lucia , Paulauskis Joseph D , Andreesen Reinhard
Published: January 2018

Maturation of cells of the mononuclear phagocyte lineage from bone marrow precursors to tissue macrophages (MAC) via circulating blood monocytes (MO) is a multistep process only partially understood. Similarly, MAC differentiation can be observed if MO are cultured in vitro. In an attempt to further characterize molecular changes occurring during this process we carried out differential screening of a MO-derived MAC cDNA library using MO and MAC cDNA. After subcloning and confirmation by a second round of screening, partial sequencing of 41 cDNA clones was performed. In 33 clones the sequences of 7 different previously identified cDNAs were found. The mRNA expression of two of the corresponding genes (apolipoprotein E, ferritin light chain) is already known to be up-regulated during MAC maturation. For one gene (cathepsin B), a specific up-regulation of mRNA expression could be shown corresponding to previous protein data. For four genes [human cartilage glycoprotein (HC-gp39), osteopontin, type IV collagenase, and tryptophanyl-tRNA synthetase] the specific expression in MAC versus MO was previously unknown but could be confirmed by the use of Northern blot analysis. Of these genes, HC-gp39 is especially interesting because it is only expressed during the late stages of MAC differentiation.
Augmented expression of platelet-activating factor receptor gene by TNF-α through transcriptional activation in human monocytes
Authors: Dagenais Pierre , Thivierge Maryse , Parent Jean-Luc , Stankova Jana , Rola-Pleszczynski Marek
Published: January 2018

Tumor necrosis factor α (TNF-α) is a cytokine produced by activated monocytes and often associated with platelet-activating factor (PAF) during the pathogenesis of many inflammatory and infectious diseases. PAFR is a G-protein-coupled receptor constitutively expressed on monocytes. TNF-α (100–400 U/mL) significantly increased PAFR mRNA expression in human monocytes. This increase was seen after 1 h of stimulation and persisted up to 24 h. Actinomycin D pretreatment studies revealed a transcriptional increase in PAFR gene expression without effect on mRNA half-life. [3H]WEB 2086 binding studies showed a significant (43%) increase in specific binding sites in 24-h-treated cells without change in receptor affinity. Increased interleukin-6 production in response to PAF was also found in 24-h TNF-α-pretreated monocytes. These observations provide new evidence for TNF-α and PAF interactions in human monocytes during inflammatory processes through up-regulation of PAFR expression by TNF-α.
Marked inhibition of the intracellular multiplication of Legionella pneumophila in monocytes isolated from carriers of human T lymphotropic virus type I
Authors: Funai Noriko , Shimamoto Yoshinori , Watanabe Masayuki , Yoshida Shin-lchi , Kohashi Osamu
Published: January 2018

Intracellular growth patterns of Legionella pneumophila were examined In monocytes obtained from carriers of human T-lymphotropic virus type I (HTLV-1) and controls who were HTLV-1 seronegative. All subjects were seronegative for antibodies against L. pneumophila. Bacterial growth was determined 0, 1, 2, and/or 3 days after infecting peripheral blood mononuclear cells (PBMCs) with the bacteria. The intracellular growth of L. pneumophila was markedly inhibited in HTLV-1 carriers compared with normal controls. When the lymphocytes were depleted from the HTLV-1 carrier PBMC cultures before infection, this inhibition was abolished. Inhibition reappeared, however, when the 72-h culture supernatants of PBMCs from HTLV-1 carriers were added to the lymphocyte-depleted cultures. Culture supernatants of infected and uninfected PBMCs from HTLV-1 carriers exhibited markedly increased levels of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) compared with the HTLV-1 seronegative controls. In the HTLV-1 carriers, IFN-γ was produced by the CD4+ lymphocytes, whereas TNF-α was secreted by the monocytes. Addition of anti-IFN-γ or anti-TNF-α antibodies to the HTLV-1 carrier PBMC cultures diminished the inhibition of intracellular growth of L. pneumophila. Results suggest that the monocytes are activated in HTLV-1 carriers. These findings may explain why an opportunistic infection by certain intracellular pathogens is rarely seen among HTLV-1 carriers.
Studies on the mechanisms involved in antigen-evoked pleural inflammation in rats: contribution of IgE and complement
Authors: Lima Marcia C R , Prouvost-Danon Annie , e Silva Patricia M R , Chagas Maria do Socorro S , Calheiros Andrea S , Cordeiro Renato S B , Latine Dominique , Bazin Hervé , Ryan Una S , Martins Marco A
Published: January 2018

Immunoglobulin E (IgE) has been shown to play a critical role in the allergic late-phase reaction, which is marked by intense leukocyte infiltration and edema. In this study we assessed the allergic pleural inflammation triggered by intrapleural (i.pl.) challenge in sensitized rats. We examined pleural effluent from actively sensitized rats following anti-IgE monoclonal antibody (mAb) (MARE-1) provocation for protein exudation, neutrophil as well as eosinophil accumulation. Inflammatory changes triggered by antigen after passive sensitization with IgE mAb was also assessed for comparison. Total serum level of IgE was found to be about threefold increased 7–8 days post-active sensitization, remaining augmented for at least 30 days. Increased levels of peritoneal leukocyte-bound IgE and serum IgE with specificity to ovalbumin were also detected. Nevertheless, the anti-IgE challenge in 14-day actively sensitized was shown to be a weak stimulus of neutrophil and eosinophil accumulation, despite being able to cause intense protein extravasation. Similarly, antigen challenge of IgE-passively sensitized rats caused protein leakage that was comparable to that induced by anti-IgE mAb in actively sensitized rats but led to a much lower neutrophil/eosinophil infiltration. Also, blockade of complement with recombinant human soluble C receptor-1 (sCR1) treatment prevented actively sensitized rats from reacting to antigen with neutrophil and eosinophil recruitment without modifying protein extravasation. These data suggest that IgE and complement-mediated mechanisms probably account for the exudation and leukocyte infiltration that is characteristic of the pleural inflammatory response observed in actively sensitized rats.
Osteoclasts, macrophages, and the molecular mechanisms of bone resorption
Authors: Teitelbaum Steven L , Tondravi M Mehrdad , Ross F Patrick
Published: January 2018

The osteoclast is a physiological polykaryon and the major if not exclusive resorptive cell of bone. It participates in bone remodeling, repair, and growth and mobilization of mineral to meet homeostatic demands. Most importantly, osteoporosis, a disease endemic in Western society and Asia, is always a reflection of enhanced osteoclastic activity relative to bone formation by osteoblasts. In fact, all forms of antiosteoporosis therapy proven successful involve inhibition of osteoclastic bone resorption. Bone resorption is regulated either by altering recruitment of osteoclast precursors into fully differentiated resorptive polykaryons or modulating the rate at which mature osteoclasts degrade bone. With this in mind, our laboratory has focused on the molecular mechanisms of osteoclast differentiation and the means by which the cell degrades bone matrix.
Defects in intracellular oxidative metabolism of neutrophils undergoing apoptosis
Authors: Narayanan P K , Ragheb K , Lawler G , Robinson J P
Published: January 2018

Apoptosis permits neutrophil recognition by macrophages, thereby not only limiting potential cytotoxicity but also promoting resolution of inflammation. A direct relationship between apoptosis and intracellular hydrogen peroxide (H2O2) production was observed in phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils aged in culture. A significant decrease in intracellular H2O2 production was observed in aging neutrophils at 12,24, and 48 h. However, intracellular superoxide anion production in response to PMA stimulation was preserved up to 24 h, implying retention of intracellular signaling pathways leading to NADPH oxidase stimulation. A significant decrease in the cytoplasmic content and activity of Cu,Zn superoxide dismutase was responsible for the observed decline in intracellular H2O2 production in apoptotic neutrophils. Intracellular glutathione content also decreased concomitantly with H2O2 production. These observations indicate that onset of apoptosis in neutrophils is in part mediated by oxidative stress resulting from the down-regulation of key antioxidant defense systems of the cell, namely superoxide dismutase and glutathione.
Random migration of polymorphonuclear leukocytes induced by GM-CSF involving a signal transduction pathway different from that of fMLP
Authors: Harakawa Nari , Sasada Masataka , Maeda Akinori , Asagoe Kohsuke , Nohgawa Masaharu , Takano Kuniko , Matsuda Yuzuru , Yamamoto Kokichi , Okuma Minoru
Published: January 2018

Granulocyte-macrophage colony-stimulating factor (GM-CSF) Induced random migration of human polymorphonuclear leukocytes (PMNs) but not chemotaxis. Chemoattractants such as N-formylmethionyl-leucyl-phenylalanine (fMLP), leukotriene B4 (LTB4), and interleukin-8 (IL-8) induced both random migration and chemotaxis. Other inflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF), interleukin 1α (IL-1α), and tumor necrosis factor α (TNF-α), did not induce either movement. One-minute exposure of PMNs to GM-CSF was sufficient for the induction of random migration, whereas fMLP-induced random migration required continued presence of fMLP. Inhibitors of phosphatidylinositol 3-kinase (PI3-K), protein kinase C (PKC), and protein tyrosine kinase (PTK) had no effect on random migration induced by GM-CSF, whereas fMLP-induced movements were partially inhibited by PTK inhibitors but not by inhibitors of PI3-K inhibitors nor PKC inhibitors. Myosin light chain kinase inhibitors inhibited movements of PMNs induced by both GM-CSF and fMLP. These findings also imply that some aspects of the signal transduction pathway of GM-CSF leading to random migration is different from that of fMLP. Our findings suggest that cell movements are controlled through diverse signal transduction systems.
Generation of multinucleated giant cells by culture of monocyte-derived macrophages with IL-4
Authors: Dugast Catherine , Gaudin Annette , Toujas Louis
Published: January 2018

Culturing human monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) has been reported to provoke the formation of multinucleated giant cells (GCs). In the present work, GCs were generated in a two-step procedure in which macrophages were first differentiated from monocytes before being fused into GCs. The two cytokines used acted sequentially. GM-CSF was required for monocyte differentiation and IL-4 for macrophage fusion. Macrophages were purified from cultures of blood mononuclear cells maintained for 7 days in plastic bags. When seeded in conventional plastic-ware in the presence of IL-4, these macrophages showed an increased motility, spread in thin cytoplasmic lamellae, regrouped in clusters, and within 1–3 weeks, differentiated into GCs. Multinucleated cells also appeared in IL-4-untreated macrophage cultures but the number of nuclei did not exceed 2 or 3, compared with more than 30 in the presence of IL-4. Scanning electron microscopy of GCs showed highly developed pseudopods. GCs reacted with anti-CD11b, -CD54, -CD68, -HLA-ABC, and -HLA-DR monoclonal antibodies and AMH-152 but were CD14- and CD64-negative. Both untreated and IL-4-treated macrophages conserved pinocytic and phagocytic activity. Thus, IL-4 induced a differentiation process in which macrophages lost markers like CD14 and CD64, acquired an enhanced membrane motility, and fused in multinucleated GCs.
Mechanisms of neutrophil-induced parenchymal cell injury
Authors: Jaeschke Hartmut , Smith C Wayne
Published: January 2018

Neutrophils are involved in organ damage induced by an excessive acute inflammatory response after ischemia-reperfusion, trauma, and sepsis. In addition to causing vascular injury, neutrophils can transmigrate and attack parenchymal cells. This review summarizes recent advances in our understanding of neutrophil-induced parenchymal cell injury using the liver as an example. Reviewed are the mechanisms of neutrophil sequestration in the hepatic vasculature, transendothelial migration, adherence to hepatic parenchymal cells, and mechanisms of cytotoxicity. Discussed are the involvement of various adhesion molecules in these processes, the role of cytokines and chemokines in the pathophysiology, as well as the effects of proteases and reactive oxygen species released by neutrophils. The emerging understanding of the basic mechanisms of neutrophil-induced organ damage is critical for the development of therapeutic strategies to attenuate excessive acute inflammatory responses without compromising essential host defense mechanisms.
Increased expression of an endopeptidase (γ-EGE/IDE) hydrolyzing β-endorphin during differentiation and maturation of bone marrow macrophages
Authors: Sarada Boppana , Thiele Dwain , Dang Tuyet , Safavi Afshin , Hersh Louis B , Cottam Gene L
Published: January 2018

The presence and regulated expression of peptidase activity is a powerful mechanism with the potential to terminate or alter receptor recognition, cell membrane signal transduction, and physiological responses of immune cells to exogenous opioid peptides. In this study, the expression of an endopeptidase that hydrolyzes β-endorphin to γ-endorphin and other peptide products was investigated during in vitro differentiation and maturation of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) -derived, bone marrow-derived macrophages. In freshly isolated intact isolated mouse bone marrow cells the rate of β-endorphin hydrolysis is undetectable (<0.1 nmol β-endorphin hydrolyzed/h/106 cells). However, total intracellular β-endorphin hydrolytic activity was increased significantly to 20.0 ± 1.7 nmol/ h/106 cells in the mature mouse macrophages derived in vitro by culture with rGM-CSF. rGM-CSF-derived macrophages expressed significantly higher levels of both protein and mRNA for the major β-endorphin endopeptidase, γ-endorphin-generating enzyme/insulin-degrading enzyme (γ-EGE/IDE). Moreover, this enzymatic activity appears to be responsible for cleavage of exogenous β-endorphin by intact rGM-CSF-derived macrophages or peritoneal macrophages to generate γ-endorphin and other peptide products.
Phagocytosis of Gram-negative bacteria by a unique CD14-dependent mechanism
Authors: Schiff Deborah E , Kline Lois , Soldau Katrin , Lee J D , Pugin Jerome , Tobias Peter S , Ulevitch Richard J
Published: January 2018

THP-l-derived cell lines were stably transfected with constructs encoding glycophosphatidylinositol (GPI) -anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytosis of serum (heat-inactivated) -opsonized Escherichia coli(opEc). Both the CPI-anchored and transmembrane forms of CD14 supported phagocytosis of opEc equally well. Lipopolysaccharide-binding protein (LBP) played a role in CD14-dependent phagocytosis as evidenced by inhibition of CD14-dependent phagocytosis of opEc with anti-LBP monoclonal antibody (mAb) and by enhanced phagocytosis of E. coliopsonized with purified LBP. CD14-dependent phagocytosis was inhibited by a phosphatidylinositol (PI) 3-kinase inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrphostin 23) but not a protein kinase C inhibitor (bisindolylmaleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti-LBP mAb 18G4 and anti-CD 14 mAb 18E12 were used to differentiate between the pathways involved in CD14-dependent phagocytosis and CD14-dependent cell activation. F(ab′)2 fragments of 18G4, a mAb to LBP that does not block cell activation, inhibited ingestion of opEc by THP1-wtCD14 cells. 18E12 (an anti-CD14 mAb that does not block LPS binding to CD14 but does inhibit CD14-dependent cell activation) did not inhibit phagocytosis of LBP-opEc by THP1-wtCD14 cells. Furthermore, CD14-dependent phagocytosis was not inhibited by anti-CD 18 (CR3 and CR4 β-chain) or anti-Fcγ receptor mAb.
Epithelioid cells from foreign-body granuloma selectively express the calcium-binding protein MRP-14, a novel down-regulatory molecule of macrophage activation
Authors: Aguiar-Passeti T , Postal E , Sorg C , Mariano M
Published: January 2018

The S-100 proteins MRP-8 and MRP-14 are expressed by cells of the myelomonocytic lineage, either alone or simultaneously during certain stages of cellular differentiation. We demonstrate that MRP-14 but not MRP-8 was detected by immunostaining in the cytoplasm of epithelioid cells on the surface of round coverslips implanted for 14 days into the subcutaneous tissue of mice. Using this experimental model, our laboratory has previously shown that epithelioid macrophages are poor phagocytic cells that release a macrophage-deactivating factor (MDF) in short-term cultures. The full chemical characterization of MDF has not been achieved so far. We provide evidence that the calcium-binding protein MRP-14 was also released by epithelioid macrophages in short-term cultures and that its neutralization from the culture medium after addition of monoclonal antibody anti-MRP-14 abolished the MDF activity of the conditioned medium. Purified or recombinant human MRP-14 but not MRP-8 inhibits the respiratory burst of BCG-activated macrophages. Recombinant mouse MRP-14 also down-regulate macrophage activation in vitro, and PMA does not revert the inhibitory effect induced by MRP-14. It is thus concluded that epithelioid cells from foreign-body granuloma express and release MRP-14 in short-term cultures and that this molecule is endowed with MDF activity.
Anti-inflammatory activities of cAMP-elevating agents: enhancement of IL-10 synthesis and concurrent suppression of TNF production
Authors: Eigler Andreas , Siegmund Britta , Emmerich Uta , Baumann Klaus H , Hartmann Gunther , Endres Stefan
Published: January 2018

Interleukin-10 (IL-10) and tumor necrosis factor (TNF) exert key roles in some acute and chronic inflammatory diseases. In this study we investigated (1) the potency of different cAMP-elevating agents in enhancing IL-10 synthesis, (2) the involvement of protein kinase A in this enhancement, and (3) the mutual dependence of cAMP-enhanced IL-10 formation and cAMP-suppressed TNF synthesis. Rolipram, a specific phosphodiesterase inhibitor and cicaprost, a prostacyclin analogue, were applied as cAMP-elevating agents. The stable cAMP antagonist (Rp)-cAMPS was used to abrogate activation of protein kinase A. Human peripheral blood mononuclear cells were stimulated with lipopolysaccharide (LPS). TNF was quantified by radioimmunoassay, IL-10 by enzyme-linked immunosorbent assay, and mRNA by reverse transcriptase-polymerase chain reaction. After LPS stimulation alone 253 ± 45 pg/mL IL-10 was synthesized, which increased to 644 ± 117 pg/mL in the presence of 1 μM rolipram. (Rp)-cAMPS reversed this increase of IL-10 formation. In the same samples, the LPS-stimulated production of TNF was markedly attenuated by rolipram or cicaprost. A kinetic analysis revealed a significant increase in TNF production before IL-10 formation was detectable. These results demonstrate that (1) cAMP-elevating agents enhance IL-10 synthesis and suppress TNF production; (2) these regulative functions of cAMP-elevating agents are mediated by activation of protein kinases A; (3) suppression of TNF synthesis by cAMP in the early phase is not mediated by endogenous IL-10. Taken together, rolipram and cicaprost exert a dual regulatory function by enhancing IL-10 formation and attenuating TNF synthesis.
Endotoxin-induced abortion in mice is mediated by activated fetal macrophages
Authors: Wang Yu-Ying , Tawfik Ossama , Wood Gary W
Published: January 2018

Leukocyte numbers and function were assessed in uterus, placenta, and fetus during endotoxin-induced abortion. Tissues initially contained high numbers of macrophages but no granulocytes or lymphocytes. Endotoxin treatment resulted in rapid, transient neutrophil accumulation in uterus and placenta. Moderate increases in macrophage numbers occurred in the uterus but there were no changes in tissue distribution. Myometrial, endothelial, and epithelial cells were unaffected by endotoxin but proliferating, differentiated fibroblasts that made up the primary decidua disappeared. As abortion progressed, the proportional representation of macrophages in placenta and embryo increased until, during the late stages of fetal resorption, they constituted nearly all viable fetal cells. At the same time, overall expression of class II major histocompatibility complex gene products increased in maternal and fetal tissue. Leukocyte distribution and macrophage activation data suggested that endotoxin-induced fetal failure is mediated by products of activated maternal and fetal macrophages acting in concert to destroy actively proliferating maternal and fetal cells.
Activated polymorphonuclear leukocytes affect red blood cell aggregability
Authors: Baskurt Oguz K , Meiselman Herbert J
Published: January 2018

Activated leukocytes can affect adjacent cells by generating oxygen free radicals and secreting proteolytic enzymes, and red blood cells (RBC) exposed to such agents should be susceptible to their effects. This study was thus designed to investigate the effects of activated polymorphonuclear leukocytes (PMN) on RBC aggregability (i.e., on intrinsic RBC aggregation characteristics). PMN were isolated from human blood by density separation and suspended in glucose-enriched buffer with RBC isolated from the same blood sample (RBC/PMN ratio of 150:1). PMN were then activated in this suspension by adding 1 ng/mL tumor necrosis factor α (TNF-α) and 10–7 M N-formyl-methionyl-leucyl-phenylalanine (fMLP) or fMLP alone. After incubation for 2 h at 37°, RBC aggregation behavior in autologous plasma was assessed; RBC deformability and partition coefficients were also measured. RBC aggregation was significantly increased after incubation and deformability and partitioning were decreased; these effects were prevented by phenylmethylsulfonyl fluoride (1 mM) or superoxide dismutase (20 μg/mL) plus catalase (40 μg/mL). TNF-α and fMLP alone had no effect on RBC aggregation if PMN were not present. Activated PMN can thus markedly affect RBC aggregability, apparently via both proteolytic enzymes and oxygen free radicals; enhanced aggregation seen in clinical states associated with PMN activation or observed during in vivo RBC aging may also involve such PMN-RBC interactions.
The EGF-TM7 family: unusual structures at the leukocyte surface
Authors: McKnight Andrew J , Gordon Siamon
Published: January 2018

The isolation of cDNA clones encoding mouse F4/80, human epidermal growth factor (EGF) module-containing mucin-like hormone receptor 1 (EMR1) and human CD97 has resulted in the description of a novel family of seven-transmembrane spanning cell surface molecules. These members of the EGF-TM7 family are characterized by a variable number of NH2-terminal EGF domains and seven transmembrane-spanning hydrophobic regions, which demonstrate a degree of sequence homology to the corresponding region in members of the G-protein-coupled peptide hormone receptor family. The F4/80 molecule is macrophage-restricted, as determined by immunohistochemical analysis of a wide range mouse tissues, while mRNA transcripts encoding EMR1, the predicted human F4/80 homologue, also appear to be abundantly expressed by cells of the myelomonocytic lineage. CD97, for which a cellular ligand has been identified (CD55), is expressed on a diverse array of leukocytes and is markedly upregulated on activated T and B cells. The regulation of expression of the EGF-TM7 genes, the physiologic function(s) of these novel receptors and the further identification of their ligands is the subject of both intense study and this review.
CD40L-CD40 interactions regulate endothelial cell surface tissue factor and thrombomodulin expression
Authors: Miller Debra L , Yaron Renat , Yellin Michael J
Published: January 2018

During immune responses, activated endothelial cells down-regulate thrombomodulin and up-regulate tissue factor expression leading to the development of a procoagulant surface. CD4+ T cells are known to promote endothelial cell procoagulant activity, however, the molecular interactions that mediate this effect are not completely known. CD40L is an activation-induced CD4+ T cell surface molecule that functionally interacts with CD40 expressed on endothelial cells. In this study we ask if CD40L-CD40 interactions modulate endothelial cell surface tissue factor or thrombomodulin expression in vitro. Human umbilical vein endothelial cells (HUVEC) were cocultured with control cells or CD40L+ Jurkat T cells in the presence or absence of anti-CD40L mAb. By two-color FACS analysis we demonstrated that CD40 ligation induces HUVEC tissue factor expression and thrombomodulin down-regulation. Utilizing neutralizing antibodies, we show that CD40L-mediated tissue factor and thrombomodulin modulation, as well as E-selectin and VCAM-1 up-regulation, is independent of tumor necrosis factor α, interleukin-1α, or interleukin-1β production. Together these data suggest that CD40L-CD40 interactions may directly regulate endothelial cell procoagulant activity during inflammatory responses.
Processing of C5a by human polymorphonuclear leukocytes
Authors: Hetland Geir , Pfeifer Philippe H , Hugli Tony E
Published: January 2018

Uptake of human C5a by neutrophils was monitored in vitro using both 125I-labeled and unlabeled C5a. The ligand was internalized by the cells in a dose-dependent manner and maximal binding/uptake was observed after 5 min of incubation. Neutrophils were incubated with labeled C5a and the cytosol and supernatant were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. C5a degradation products were primarily observed in the supernatant, whereas most of the protein remained intact in the cytosol even after 60 min of incubation. Cytosol from neutrophils incubated for 20 min with unlabeled C5a was examined by radioimmunoassay and found to contain antigenically intact C5a and retained the ability to induce a neutrophil (shape change) response. The functional activity of C5a recovered from the cytosol was inhibited by antibodies to either C5a or the C5a receptor (CD88). This data supports our hypothesis that although C5a is internalized it remains antigenically intact and functionally active inside the cell and is primarily degraded extracellularly.
Role of the liver in T cell differentiation—generation of CD3–CD4+/CD8+TCRβ– cells and CD3–4–8–TCRβ+ cells from CD4–8–TCRβ– athymic nude bone marrow cells by culture with parenchymal liver cells
Authors: Mabuchi Ayako , Kodaira Yuzo , Norose Yoshihiko , Saizawa Mitsuyoshi , Kitajima Masumi , Yokomuro Kozo
Published: January 2018

To investigate the influence of the liver on differentiation of hematopoietic stem cells/ pro-T cells, TN-NWP-BMC (athymic nude bone marrow cells that were treated with anti-TCRβ, anti-CD4, and anti-CD8 Abs plus complement and then passed through a nylon wool column) were cultured on parenchymal liver cells. After culture for 2.5 days, CD3–4–8–TCRβ+ cells and CD3–CD4+/CD8+TCRβ– cells were developed from TN-NWP-BMC. TCRVβ8+ cells comprised 19.9% of CD3–4–8–TCRβ+ cells, and Vβ8 mRNA was detected in the CD3–4–8–TCRβ+ cells by reverse transcriptase-polymerase chain reaction. The CD3–CD4+/CD8+ TCRβ– cells contained not only single-positive cells but also CD4+8+ double-positive cells. The CD8 protein consisted of 88.9% CD8α+β–, 10.1% CD8α+β+, and 1% CD8α–β+ molecules. From these results and the finding of co-expressed antigens, CD3–4–8–TCRβ+ cells and CD3–CD4+/CD8+TCRβ– cells appear to be immature cells not committed to a certain cell lineage.
Construction and expression of a soluble form of human CD30 ligand with functional activity
Authors: Powell Isabelle F , Li Tianhong , Jäck Hans-Martin , Ellis Thomas M
Published: January 2018

CD30 engagement in human lymphoid cells induces pleiotropic cellular responses that affect cellular viability and proliferation, cytokine production, and nuclear factor κB (NF-κB) nuclear translocation. Studies examining the molecular basis for this pleiotropism thus far have relied on the use of antibodies and cells transfected with CD30L to trigger CD30, two methods of receptor induction that present important limitations: antibodies are not physiological receptor-triggering molecules and CD30L transfectants induce high background intracellular signaling in the cells under study. We have generated and expressed a functional soluble human CD30L molecule (sCD30L/CD8α) comprised of the extracellular domain of human CD30L fused to the extracellular domain of the human CD8α chain. Immunoprecipitation and Western blot analysis of sCD30L/CD8α revealed the existence of at least two forms of sCD30L/CD8α, which exhibited molecular sizes consistent with the existence of monomeric and trimeric forms of the molecule. Binding analyses performed using a soluble CD30 fusion protein (sCD30/γ1) confirmed the ability of sCD30L/CD8α to bind to CD30. Functionally, immobilized sCD30L/CD8α-induced cell death in the CD30-expressing lines Karpas-299 and HDLM-2 and reduced proliferative levels in Karpas-299; these effects were inhibitable by the addition of sCD30/γ1. These studies demonstrate the utility of sCD30L/CD8α in characterizing the normal function of CD30L and CD30 and indicate the natural ability of soluble forms of CD30L to trimerize.
IL-10 suppresses LPS-induced KC mRNA expression via a translation-dependent decrease in mRNA stability
Authors: Kim Hee Sun , Armstrong David , Hamilton Thomas A , Tebo Julie M
Published: January 2018

This study examines the mechanism of interleukin-10 (IL-10)-mediated suppression of KC chemokine gene expression in mouse macrophages. Suppression of KC mRNA levels by IL-10 occurred late in the time course of response to lipopolysaccharide (LPS). Equivalent IL-10-mediated suppression was observed when the agent was added 1 h before, simultaneous with, or 1 h after LPS. IL-10 did not inhibit KC gene transcription but rather produced a decrease in the stability of KC mRNA. The suppressive action of IL-10 was prevented in macrophages that were also treated with inhibitors of protein synthesis even when added 2 h after LPS and IL-10. These results suggest that IL-10 acts to destabilize LPS-induced KC mRNA through a process that depends on coincident KC mRNA translation. J. Leukoc. Biol. 64: 33–39; 1998.