bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of adenosine on the expression of β2 integrins and L-selectin of human polymorphonuclear leukocytes in vitro
Authors: Thiel M , Chambers J David , Chouker A , Fischer S , Zourelidis C , Bardenheuer Hubert J , Arfors Karl-E , Peter K
Published: January 2018

Adenosine has been shown to inhibit the adhesion of polymorphonuclear leukocytes (PMNL) to the vascular endothelium. Because the underlying molecular mechanisms have not been fully understood, the present study characterizes the effect of adenosine on the expression of adhesion molecules of human PMNL. When PMNL were activated by N-formyl-methionyl-leucyl-phenylalanine the number of cell surface β2 integrins increased fivefold, whereas L-selectin molecules were completely shed. Activation-dependent numerical up-regulation of β2 integrins and shedding of L-selectin were inhibited by exogenously applied adenosine receptor agonists in a concentration-dependent fashion. The rank or-der of potencies of adenosine receptor agonists, measured by the agonists’ half-maximal inhibitory concentrations, revealed that adenosine inhibited the numerical up-regulation of β2 integrins and shedding of L-selectin most likely via an A2(a) receptor site. When extracellular concentrations of endogenously formed adenosine were enhanced by the nucleoside uptake inhibitor dipyridamole, up-regulation of β2 integrins, and shedding of L-selectin was again inhibited. Both effects were reversed by the enzyme adenosine deaminase, which degrades active adenosine to inactive inosine, suggesting that endogenously formed adenosine may play an important role in the regulation of β2 integrins and L-selectin of human PMNL.
Priming of human peripheral blood mononuclear cells with lipopolysaccharides for enhanced arachidonic acid release and leukotriene synthesis
Authors: Surette Marc E , Nadeau Marie , Borgeat Pierre , Gosselin Jean
Published: January 2018

In a previous study, we have shown that the ability of lipopolysaccharides (LPS) to prime isolated neutrophils for enhanced leukotriene B4 (LTB4) synthesis was dependent on the presence of plasma and involved the CD14 antigen. In the present study, we have investigated the priming of human peripheral blood mononuclear cells (PBMC) with LPS for the subsequent release and metabolism of arachidonic acid. When PBMC were incubated with LPS for up to 2 h or when freshly isolated PBMC were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or with LPS alone, little or no synthesis of 5-lipoxygenase products nor arachidonic acid liberation were detected. However, the preincubation of PBMC with IPS for as little as 5 min primed cells for the subsequent synthesis of LTB4 upon stimulation with fMLP. Maximal priming was observed following a 15-min preincubation period and the priming effect was transient as cells preincubated with IPS for 90 min or more were no longer primed for leukotriene synthesis. Monocytes were found to be responsible for the enhanced response to fMLP since purified lymphocytes did not produce LTB4 nor LTC4 in contrast to monocyte-enriched suspensions. The priming for leukotriene synthesis coincided with an increased capacity for the release of free arachidonic acid as measured by mass spectrometry; IPS-primed cells released 8–15 times more arachidonic acid than unprimed cells within 1 min of stimulation with fMLP. Pruning was observed with as little as 0.001-0.01 μg LPS/mL when cells were incubated in the presence of 10% autologous plasma. Interestingly, in the absence of plasma, priming was only observed at IPS concentrations of 0.1 μg/mL or greater. Pretreatment of cells with anti-CD14 antibodies significantly decreased the priming effect observed with 0.01 μg/mL IPS but did not affect pruning with 1 μg/mL IPS. These results indicate that the priming of human PBMC with IPS for the subsequent synthesis of arachidonic acid metabolites via the 5-lipoxygenase pathway is dependent on plasma and CD14 at lower concentrations of IPS (0.001–0.01 μg/mL) but not at IPS concentrations of 0.1 μg/mL or greater.
Cloning and expression of a second human natural killer cell granule tryptase, HNK-Tryp-2/granzyme 3
Authors: Sayers Thomas J , Lloyd Andrew R , McVicar Daniel W , O’Connor Michael D , Kelly Janice M , Carter Clive R D , Wiltrout Theresa A , Wiltrout Robert H , Smyth Mark J
Published: January 2018

Cytotoxic lymphocytes possess a number of serine proteases (granzymes) usually localised in cytoplasmic granules. To date, the DNA sequences of four human granzymes have been reported. A fifth human granzyme (granzyme 3) has been biochemically purified and its N-terminal amino acid sequence has been reported. This enzyme was described as possessing tryptase activity, cleaving synthetic substrates after arginine or lysine. We recently cloned a rat granzyme tryptase (RNK-Tryp-2), and used this cDNA to screen human cDNA libraries. Isolation of cDNA fragments of a human gene could be overlapped to provide a complete cDNA sequence, which we designated HNK-Tryp-2. The N-terminal amino acid sequence deduced from HNK-Tryp-2 was identical to that reported for granzyme 3. This gene appears to be a single copy gene that is expressed in isolated natural killer cells and T cells as well as in tissues containing these cells.
Transforming growth factor-β1–deficient mice: identification of isoform-specific activities in vivo
Authors: Letterio John J , Roberts Anita B
Published: January 2018

A remarkable range of activities has been ascribed to the family of proteins known as transforming growth factor-β (TGF-β). Each plays an important role in development and homeostasis, influencing mesenchymal-epithelial interactions, regulating cellular differentiation, and maintaining control of cell proliferation. Although in vitro comparisons of activity demonstrate a high degree of functional similarity, recent studies of mice with a targeted deletion of the TGF-β1 gene reveal that true isoform-specific activities do exist in vivo and that the three mammalian isoforms are not functionally redundant. This approach has defined a unique role for TGF-β1 in the establishment and maintenance of normal immune function, shed new light on the relevance of endogenous TGF-β1 to the normal wound healing process, and expanded the list of known mechanisms of TGF-β1 activity to include endocrine functions. Thus, the TGF-β1-deficient mouse allows the definition of isoform-specific activities, providing an invaluable window through which to view the principal functions of TGF-β1 in vivo.
Soluble versus cell-bound CD4, CD8 from bronchoalveolar lavage: correlation with pulmonary diagnoses in human immunodeficiency virus-infected individuals
Authors: Willsie Sandra K , Herndon Betty L , Miller Leslie , Dew Michelle
Published: January 2018

Identification and assessment of cell populations in bronchoalveolar lavage (BAL) specimens may be used to follow the course of a disease state or response to specific therapy. Beyond cellular assessment, there are indications that the presence and quantity of soluble surface antigens released from activated cells may lead to improved understanding and facilitated diagnosis of a number of disease states. This study evaluated soluble markers (sCD4 and sCD8) in BAL and serum from HIV-infected individuals undergoing diagnostic bronchoscopy, and compared these values to flow cytometry-quantified BAL and peripheral blood cell CD4 and CD8. Patient pulmonary diagnosis (based on cytology and microbiology) was compared with patient blood and BAL-soluble and cell-bound CD4 and CD8 to determine the relationship of these markers to disease states in this population. Serum sCD8 in patients with fungal infections was significantly elevated above sCD8 in patients with Pneumocystis carinii or pulmonary bacterial infections, p = 0.0001. BAL sCD4/sCD8 ratio was also significantly different in patients with bacterial vs. fungal pulmonary infections, p = 0.01. These findings suggest that soluble markers, particularly elevated sCD8, may be an important indication of pulmonary disease progression in these HIV+ patients with fungal infections.
Nitric oxide production by peritoneal macrophages of Mycobacterium bovis BCG-infected or non-infected mice: regulatory roles of T lymphocytes and cytokines
Authors: Saito Shinji , Nakano Masayasu
Published: January 2018

Infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG) confers mice with strong abilities to produce nitric oxide (NO) and cytokines. Because the peritoneal macrophages taken from the mice immunized with live or heat-killed BCG can produce NO without any accessory cells and stimulants, it is difficult to clarify the immune regulation on NO production by manipulating the macrophages. Therefore, we investigated the participation of immune T cells and cytokines in NO production by using in vitro co-cultures of macrophages from non-immune mice with T cells prepared from BCG-infected mice in the presence or absence of a mycobacterial antigen, purified protein derivative (PPD). Although the non-immune thioglycollate (TGB)-elicited macrophages could not produce any detectable NO in the presence of PPD, supplementation of the macrophage cultures with CD4+ T cells prepared from BCG-infected mice enabled the macrophages to produce NO. Immunocytostaining showed that the macrophages, but not the immune T cells, expressed inducible NO synthase (iNOS), indicating that they were NO producers. PPD could only induce NO production if there was cell-cell contact of the CD4+ T cells in the immune cells and antigen-presenting macrophages were required for the NO production in response to PPD; this interaction led to the production of soluble mediators that induced NO production by the TGB macrophages. NO production by the co-cultured cells was abrogated by adding either anti-interferon-γ(IFN-γ) or anti-tumor necrosis factor α (TNF-α) antibody. Furthermore, the roles of immune T cells and PPD could be replaced by adding recombinant IFN-γ together with TNF-α to the macrophage cultures, but neither alone was sufficient to induce NO production by the macrophages. Our present data indicate that TNF-α produced by PPD-stimulated macrophages and IFN-γ produced by cell-cell interaction of BCG-immune T cells and antigen-engulfed macrophages together activate the macrophages to produce NO.
Recombinant natural killer enhancing factor augments natural killer cytotoxicity
Authors: Sauri Humberto , Ashjian Peter H , Kim Anthony T , Shau Hungyi
Published: January 2018

Natural killer enhancing factor (NKEF) was originally identified and studied because of its ability to enhance NK cytotoxicity in vitro. After cloning the two genes responsible for NKEF proteins, NKEF-A and -B, we found that they belong to a newly described and highly conserved antioxidant gene family. We have now produced recombinant proteins of both genes and used them to test for their ability to promote NK cytotoxicity. Although recombinant NKEF (rNKEF)-A and -B have similar levels of antioxidant function, only the reduced form of rNKEF-A can enhance NK cytotoxicity. These results indicate that both the antioxidant and NK-enhancing functions of rNKEF-A and -B probably involve the cysteine residues of the proteins but are mediated by separate domains of the molecules. We pretreated both effector cells and target cells to investigate which population was influenced by rNKEF-A, and determined that the protein must be present during the cytotoxicity assay to enhance the activity. Despite the similarities between NK cytotoxicity and lymphokine-activated killer (LAK) cytotoxicity, rNKEF-A is not effective in augmenting LAK cytotoxicity. Therefore, rNKEFs can be useful tools in not only protecting cells from oxidative damage, but also in selectively promoting NK cytotoxicity against certain tumor cells.
Effects of spaceflight on rat peripheral blood leukocytes and bone marrow progenitor cells
Authors: Ichiki A T , Gibson L A , Jago T L , Strickland K M , Johnson D L , Lange R D , Allebban Z
Published: January 2018

The white blood cell (WBC) elements and the bone marrow myeloid progenitor cell populations were analyzed to ascertain adaptation to microgravity and subsequent readaptation to 1 G in rats flown on the 14-day Spacelab Life Sciences-2 (SLS-2) mission. Bone marrow cells were harvested from one group of rats killed inflight (FD13) and blood was drawn from three other groups at various times. The WBC level was normal on FD14 with the exception of neutrophilia. On FD13, numbers of colony-forming units-granulocyte (CFU-G), CFU-GM, and CFU-M from flight animals were decreased compared with ground controls when incubated with recombinant rat interleukin-3 (rrIL-3) alone or in combination with recombinant human erythropoietin (rhEpo). On recovery (R + 0), flight rats had decreased numbers of total leukocytes and absolute numbers of lymphocytes and monocytes with elevated neutrophils compared with control rats. They had lower numbers of CD4, CD8, CD2, CD3, and B cells in the peripheral blood but no differences in spleen lymphocytes.
Apoptotic cell death in activated monocytes following incorporation of clodronate-liposomes
Authors: Schmidt-Weber Carsten B , Rittig Michael , Buchner Eberhard , Hauser Ingeborg , Schmidt Irina , Palombo-Kinne Ernesta , Emmrich Frank , Kinne Raimund W
Published: January 2018

The present study was performed to elucidate whether sterically stabilized liposomes laden with clodronate, which lead to depletion of macrophages (Mɸs) and amelioration of experimental autoimmune arthritis in vivo, selectively affect cells of the mɸ lineage in vitro. The rates of incorporation of drug-free, fluorescent liposomes and the rates of cell death following exposure to clodronate-liposomes were assessed in human peripheral blood monocytes, as well as in polymorphonuclear leukocytes (PMNs), T cells, endothelial cells, and fibroblasts, both at rest and following activation. Gel electrophoresis of nuclear extracts and ultrastructural analyses were performed to identify the modality of cell death. Monocytes, particularly upon activation, were more efficient in incorporating sterically stabilized liposomes than all other cells except PMNs. Twenty percent of resting monocytes and up to 65% of activated monocytes died within 24 h of exposure to clodronate-liposomes, whereas the other cell types, including PMNs, remained unaffected. Activated monocytes exposed to clodronate-liposomes, but not resting or activated monocytes exposed to drug-free liposomes, showed clear signs of apoptotic cell death. In most of the assays, sterically stabilized liposomes were more efficient than conventional phosphatidylcholine-liposomes. Sterically stabilized clodronate-liposomes preferentially affect cells of the mɸ lineage, particularly if activated. Selective elimination of activated Mɸs by apoptosis may explain both therapeutic efficacy and safety of clodronate-liposomes in experimental models of autoimmunity.
Cloning of the murine eosinophil peroxidase gene (mEPO): characterization of a conserved subgroup of mammalian hematopoietic peroxidases
Authors: Horton Margaret A , Larson Kirsten A , Lee James J , Lee Nancy A
Published: January 2018

The mouse eosinophil peroxidase (mEPO) gene was cloned by screening a random-primed bone marrow cDNA library at reduced criteria using a hEPO cDNA. An mEPO cDNA was subsequently used to isolate the mEPO gene from a λ-genomic library. The mEPO gene displays a high degree of conservation with its human homologue: the transcription units are approximately the same size, conserve the relative size and position of the 12 exons associated with each gene, and at a nucleotide level the mouse and human EPO genes are 86% identical in the protein coding regions and 66% identical in the 3'-untranslated trailer regions. This strong conservation extends to the encoded proteins which show ~90% amino acid identity. Expression of the mEPO gene is restricted to tissues containing eosinophil progenitor cells (e.g., bone marrow and spleen), a pattern similar to the expression of another murine eosinophil granule protein, major basic protein.
HIN-200: a novel family of IFN-inducible nuclear proteins expressed in leukocytes
Authors: Dawson Michelle J , Trapani Joseph A
Published: January 2018

In recent years a novel family of Interferon (IFN)-inducible nuclear proteins has been identified in both mouse and humans and implicated in the control of myeloid differentiation. Recent evidence suggests that this role may be mediated by an effect on cell cycle regulation through interaction with known transcriptional regulators. Our laboratory first identified a nuclear protein designated IFI 16, which is strongly inducible with IFN-α and -γ in myeloid cells. IFI 16 shares homology with at least six IFN-inducible mouse genes, designated the Gene 200 cluster, and also has close similarity with the human myeloid cell nuclear differentiation antigen, whose expression has also been associated with later stages of normal myelopoiesis and some myeloid malignancies. The structural hallmark of each protein in this family of hemopoietic IFN-inducible nuclear proteins is a 200-amino acid motif (hence HIN-200), present singly or as a tandem repeat. The restricted expression of these proteins, their ability to bind to nucleic acid, and their expression in response to a variety of stimuli eliciting myeloid differentiation strongly suggest that they play a role in hemopoietic maturation.
Migration of rat skin dendritic cells
Authors: Richters C D , van Pelt A M , van Geldrop E , Hoekstra M J , van Baare J , du Pont J S , Kamperdijk E W A
Published: January 2018

This study examines the in vivo migration of rat skin dendritic cells (including Langerhans cells) after skin transplantation. As donor animals, PVG-RT7b rats were used. The leukocytes of these rats bear an epitope of the leukocyte common antigen that can be recognized by use of the antibody His 41. The cells of allogeneic (ACI) recipient strains do not label with this antibody. Four days after transplantation of PVG-RT7b skin on allogeneic recipients, His 41+ cells showing a dendritic morphology were present in the T cell area of the draining lymph nodes. During culture of rat skin explants, dendritic cells migrated spontaneously into the medium. These in vitro migrated cells showed a high capacity to stimulate allogeneic T cells. When these cells, obtained from PVG-RT7b skin, were injected into the hind footpads of allogeneic recipients, they migrated to the same compartments of the draining lymph node. These data indicate that the cells that migrate from a transplanted allogeneic skin graft are the same cells that migrate in vitro from explants. Most probably, they initiate graft rejection in the draining lymph nodes of the recipient.
Liposome-encapsulated dichloromethylene diphosphonate induces macrophage apoptosis in vivo and in vitro
Authors: Naito Makoto , Nagai Hirotaka , Kawano Sunao , Umezu Hajime , Zhu Hong , Moriyama Hiroshi , Yamamoto Takashi , Takatsuka Hisakazu , Takei Yoshiyuki
Published: January 2018

Dichloromethylene diphosphonate (MDPCl2) encapsulated in multilamellar liposomes was selectively incorporated by macrophages, immediately transferred to lysosomes, then released from liposomes into lysosomes by enzymatic digestion of the liposomal lipid layers. From 4 h after ingesting liposome-encapsulated MDPCl2 murine macrophages in vivo and in vitro acquired the ultrastructural features of apoptosis, such as condensed nuclear chromatin, nuclear fragmentation, cell shrinkage, and blebbing of the plasma membrane. Murine peritoneal macrophages and isolated rat Kupffer cells incubated in the medium containing liposome-encapsulated MDPCl2 increased DNA fragmentation in a dose-dependent manner. Electrophoretic analysis of extracted DNA from the isolated Kupffer cells showed DNA fragmentation. Another diphosphonate, Alendronate (4-amino-1-hydroxybutylidene-1,1-diphosphonate) had less potent macrophage cytotoxicity. However, MDPCl2, Alendronate, and gadolinium chloride in solution were not cytotoxic to macrophages. These results implied that the intralysosomal accumulation of MDPCl2 generates signals to induce macrophage apoptosis.
Signaling by IL-2 and related cytokines: JAKs, STATs, and relationship to immunodeficiency
Authors: Johnston James A , Bacon Chris M , Riedy M C , O'Shea John J
Published: January 2018

Cytokines that bind to the interleukin-2 (IL-2) receptor common gamma chain (γc), including IL-2, IL-4, IL-7, IL-9, and IL-15, are important for the growth and differentiation of T and B lymphocytes, natural killer cells, macrophages, and monoctyes. These cytokines have overlapping biological effects that in part result from the use of the shared receptor subunit γc. Recently it has become clear that these cytokines activate a number of important intracellular signaling molecules, including the Janus kinases JAK1 and JAK3 and members of the transcription factor family of signal transducers and activators of transcription (STATs). The discovery of these signaling pathways has led to important new insights into their role in lymphocyte maturation, as it has emerged that mutations in the genes encoding both γc and JAK3 result in similar forms of severe combined immunodeficiency (SCID). In this review we examine the structure and function of cytokine receptors and the signaling pathways involved in their regulation of gene expression. Furthermore, we discuss recent advances that have led to a better understanding of how cytokines elicit intracellular responses, as well as their role in normal lymphoid development.
Elevated TNF-α and inducible nitric oxide production by alveolar macrophages after exposure to a nitrite inhalant
Authors: Soderberg Lee S F , Chang Louis W , Barnett John B
Published: January 2018

Abuse of nitrite inhalants, widespread among male homosexuals, has been identified by epidemiological studies as an independent risk factor for AIDS and for Kaposi's sarcoma. Subchronic exposure of mice to inhaled isobutyl nitrite was previously found to impair the tumoricidal activity of peritoneal macrophages. Because inhalants would be expected to have the greatest effects on cells in the lung, alveolar macrophages from exposed mice were examined in this study. Mice were exposed to 900 ppm isobutyl nitrite in an inhalation chamber for 45 min/day for 14 days. Following this treatment, the lungs of exposed mice had large increases in ccllularity, both in the alveolar septa and within the alveoli. Broncho alveolar lavages also contained increased numbers of cells. Alveolar macrophages collected from treated mice had increased tumoricidal activity compared with controls and produced higher levels of inducible nitric oxide and tumor necrosis factor-α (TNF-α), The frequency of alveolar cells secreting TNF-α was increased ninefold in mice exposed to the inhalant. Cell influx into the lung, as indicated by the presence of red blood cells in lung lavages, was evident after only a single 45-min exposure to inhaled isobutyl nitrite at doses as low as 300 ppm.
Gadolinium chloride inhibits Kupffer cell nitric oxide synthase (iNOS) induction
Authors: Roland Claude R , Naziruddin Bashoo , Mohanakumar T , Flye M Wayne
Published: January 2018

Kupffer cells (KC) are the phagocytic macrophages of the liver. The rare earth metal, gadolinium (GdCl3), is a lanthanide, which, after phagocytosis by the KC, has been found to alter various aspects of KC physiology. In this study, we describe for the first time that the in vivo administration of GdCl3 to rate decreases the release of NO by isolated rat KC in response to lipopolysaccharide. Western blot analysis shows decreased expression of both imlucible nitric oxide synthase as well as total cellular calmodulin after GdCl3 treatment. Possible mechanisms for this phenomenon are suggested.
Regulation of cytokine and cytokine receptor expression by glucocorticoids
Authors: Almawi Wassim Y , Beyhum Hayfa N , Rahme Amal A , Rieder Michael J
Published: January 2018

Glucocorticoids (GCS) profoundly inhibit several aspects of T cell immunity largely through inhibition of cytokine expression at the transcriptional and posttranscriptional levels. GCS were also reported to act indirectly by inducing transforming growth factor-β expression, which in turn blocks T cell immunity. In exerting their antiproliferative effects, GCS diffuse into target cells where they bind their cytoplasmic receptor, which in turn translocates to the nucleus where it inhibits transcription of cytokine genes through direct binding to the glucocorticoid response elements (GRE), which are located in the promoter region of cytokine genes or, alternatively, through antagonism of the action of transcription factors required for optimal transcriptional activation. In contrast to their inhibitory effects on cytokine expression, GCS upregulate cytokine receptor expression that correlates with enhanced cytokine effects on target cells. In this review, we summarize the current state of knowledge of the mechanism of action of GCS, including the phenomenon of steroid-induced rebound, which ensues upon GCS withdrawal.
Requirement for CD44 in proliferation and homing of hematopoietic precursor cells
Authors: Khaldoyanidi Sophia , Denzel Angela , Zöller Margot
Published: January 2018

The hematopoietic form of the adhesion molecule CD44 is known to be involved in lymphocyte maturation and homing. To define lineage and stage of maturation, which requires expression of CD44, murine long-term bone marrow cultures were established. Stroma formation and proliferation of early as well as committed erythroid, myeloid, and lymphoid progenitors were evaluated under the influence of monoclonal anti-CD44 antibodies. Although the formation of stromal elements was not affected, formation of cobblestone areas was completely abolished. [3H]thymidine suicide confirmed that anti-CD44 treatment interfered with the proliferation of early and committed hematopoietic progenitor cells. In addition, homing and seeding of bone marrow cells was impaired by anti-CD44. The data are indicative of a dual functional mode of CD44 in adhesion and proliferation of hematopoietic progenitors and confirm an essential requirement for the molecule in early stages of hematopoiesis.
N-acetyl-L-cysteine-induced up-regulation of HIV-1 gene expression in monocyte-derived macrophages correlates with increased NF-κB DNA binding activity
Authors: Nottet Hans S L M , Moelans Inge I M D , de Vos N Machiel , de Graaf Loek , Visser Maarten R , Verhoef Jan
Published: January 2018

Nuclear factor κB (NF-κB) is an important cellular regulator of human immunodeficiency virus (HIV) gene expression. In T cells, N-acetyl-l-cysteine (NAC) inhibits the induction of NF-κB and transcription of HIV-1. However, NAC up-regulates HIV-1 replication in monocyte-derived macrophages (MDM). In this study we demonstrate that NAC treatment of MDM transfected with a chloramphenicol acetyltransferase (CAT) construct under transcriptional control of the HIV-1 long terminal repeat resulted in an up-regulation of CAT activity. Furthermore, MDM transfected with a HIV-1-NF-κB-CAT construct also produced increased CAT activity after NAC treatment. In addition, electrophoretic mobility shift assays revealed that nuclei of NAC-treated MDM contained increased binding activity to wild-type, but not mutant, κB oligonucleotides. Components of the binding activity were identified with antibodies as the NF-κB subunits p50 and p65. These data indicate that NAC-induced enhancement of HTV-1 replication in MDM is regulated at the level of viral gene expression and mediated by NF-κB.
Immunoreceptor tyrosine-based activation motif (ITAM), a unique module linking antigen and Fc receptors to their signaling cascades
Authors: Isakov Noah
Published: January 2018

Signal transduction by the T cell and B cell antigen receptors and by receptors for a variety of immunoglobulins' Fc region is strictly dependent on a receptor subunit cytoplasmic module termed immunoreceptor tyrosine-based activation motif (FE4M). Hris module exists in one or more copies in each of the receptor-associated signal-transducing molecules and it possesses two repeats of the consensus sequence Tyr-X-X-Leu/IIe spaced by six to eight amino acids. Receptor engagement is followed by a rapid and transient phosphorylation of tyrosine residues within their ITAMs, thereby creating temporary binding sites for Src homology 2 (SH2)-containing signaling molecules operating downstream of the activated receptor. The purpose of this review is to discuss recent findings on the functional role of ITAMs in antigen and Fc receptor-mediated signal transduction, with a particular emphasis on kinases operating upstream and downstream of the ITAMs.
Enhancement of immune response to naked DNA vaccine by immunization with transfected dendritic cells
Authors: Manickan Eianchezhiyan , Kanangat Sivadasan , Rouse Richard J D , Yu Zhiya , Rouse Barry T
Published: January 2018

Immunization with plasmid DNA encoding various proteins promises to be a valuable vaccine approach especially if its immunogenicity could be optimized. In this study we show that the intramuscular delivery in dendritic cells (DC) of naked plasmid DNA encoding two proteins of herpes simplex virus (HSV) leads to the induction of significantly enhanced levels of resistance to viral challenge. Whereas DC transfected in vitro with DNA induced enhanced immunity, similarly transfected macrophage (Mɸ) populations lacked immunogenicity even though plasmid expression occurred in vitro. The enhanced immunity induced by DC-delivered DNA appeared to be associated mainly with an increased Thl CD4+ T cell response. Our results add evidence that DC are the essential antigen-presenting cell types involved in immune responses to intramuscularly administered DNA vaccines.
γδ T cell activation or anergy during infections: the role of nonpeptidic TCR ligands and HLA class I molecules
Authors: Poccia Fabrizio , Malkovsky Miroslav , Gougeon Marie Lise , Bonneville Marc , Lopez-Botet Miguel , Fournié Jean Jacques , Colizzi Vittorio
Published: January 2018

Vγ9Vδ2-encoded T cell receptors (TCR) expressed by most human peripheral blood γδ T cells mediate the recognition of nonpeptidic phosphoantigens from various pathogens without any known requirement for HLA molecules. Functionally mature Vγ9Vδ2 T cells display a potent natural killer (NK) -like cytotoxic activity, share with NK cells the expression of inhibitory receptors for HLA class I molecules, and release a plethora of cytokines, most notably interferon-γ and tumor necrosis factor α. Hence, through local activation, the early recruitment and stimulation of Vγ9Vδ2 T cells may promote efficient anti-infectious immunity. However, a chronic overactivation of this T cell subset may result in immunopathology. The meeting held in St. Vincent, Val d'Aosta, Italy (symposium on γδ T cells in natural immunity to infections: a rationale for vaccine development organized by the World Foundation for AIDS Research and Prevention, the UNESCO, and the Italian National Research Council, December 2–4, 1996) focused on the importance of γδ T cell activation and anergy for the pathogenesis of tuberculosis, malaria, and HIV infections.
Transendothelial migration induces rapid expression on neutrophils of granule-release VLA6 used for tissue infiltration
Authors: Roussel Eugène , Gingras Marie-Claude
Published: January 2018

Little is known of the mechanisms allowing neutrophils to infiltrate tissue after transendothelial migration. We postulated that VLA6 might be involved in neutrophil infiltration because it revealed as the most expressed β1 integrins among VLA5, VLA4, and VLA3, which also appeared to define subsets within the blood neutrophil population. Transendothelial migration up-regulated by threefold (5,000 to 15,000 receptors) VLA6 expression on neutrophils. VLA6 up-regulation was transient, peaking in 6 min and returning to baseline in 1 h when tested in response to N-formyl-methionyl-leucyl-phenylalanine. Neutrophil degranulation experiments revealed a steady correlation between expression of VLA6 and granule content release, notably β-glucuronidase, indicating that VLA6 molecules were preformed and stored mostly in azurophilic granules. Migration across fibroblast monolayers of neutrophils preactivated for VLA6 up-regulation was blocked when they were preincubated with anti-VLA6. However, anti-VLA6 had no effect on transfibroblast migration of non-preactivated neutrophils. These results indicate that VLA6 was functional only on activated neutrophils that used their up-regulated VLA6 to cross fibroblasts. Activation of neutrophils by transendothelial migration induces rapid expression of granule release VLA6 that appears to be a key adhesion mechanism used by a subset of neutrophils to infiltrate tissue.
Rat monocytes up-regulate NKR-P1A and down-modulate CD4 and CD43 during activation in vivo: monocyte subpopulations in normal and IFN-γ-treated rats
Authors: Scriba Andreas , Schneider Michael , Grau Veronika , van der Meide Peter H , Steiniger Birte
Published: January 2018

LEW rats were treated intravenously with recombinant rat interferon-γ (IFN-γ) for 3 days to achieve intravascular accumulation, proliferation, and activation of monocytes. Monocytes, defined by their expression of the ED1, ED9, and Ox41 antigens, were recovered from the vasculature by perfusion with PBS/EDTA, subsequently depleted of erythrocytes and granulocytes by Percoll density gradient centrifugation, and analyzed by flow cytometry and immunocytology. In untreated and control-infused specified pathogen-free (SPF) rats, lymphocytes and monocytes formed overlapping cell populations with respect to size and internal granularity. At least two intravascular monocyte subsets, probably central and marginating cells, were distinguished by their size and differential expression of CD43, CD4, CD11a, CD18, and L-selectin. It is interesting to note that a fraction of the monocytes in normal and control-infused animals carried the NKR-P1A molecule. IFN-γ treatment provoked a duplication of monocyte size and granularity. Both the number of positive monocytes and the level of expression of NKR-P1A strongly increased after IFN-γ infusion, whereas CD43 (leukosialin) and CD4 were impressively down-regulated. NKR-P1A+ L-selectin+CD43low CD4- monocytes also occur in the vasculature of rats during immune reactions in vivo. We speculate that these cells are involved in organ damage and that their number is controlled by activation-induced cell death within the vessels.
Maintenance and down-regulation of primed neutrophil chemiluminescence activity in human whole blood
Authors: Brown Glenn E , Reiff Joel , Allen Robert C , Silver Geoffrey M , Fink Mitchell R
Published: January 2018

Priming of polymorphonuclear neutrophils (PMN) in whole blood (by tumor necrosis factor α and interleukin-8 for enhancement of luminoldependent chemiluminescence induced by human complement-opsonized zymosan) was stable for 120 min. In contrast, priming of isolated PMN in plasma-free suspension for responses to opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, and phorbol myristate acetate was markedly less stable. Decay of priming was not due to irreversible inactivation of the terminal CL production machinery because PMN could be reprimed by platelet-activating factor or leukotriene B4. The tumor necrosis factor-α-primed state of isolated PMN was stabilized by host plasma in a concentration-dependent fashion. We conclude that PMN priming results in a dynamic state that is reversible. Our findings suggest the existence of blood-borne components that may act to stabilize or modify PMN priming.
PGG-Glucan activates NF-κB-like and NF-IL-6-like transcription factor complexes in a murine monocytic cell line
Authors: Adams David S , Pero Stephanie C , Petro J Benjamin , Nathans Robin , Mackin William M , Wakshull Eric
Published: January 2018

PGG-Glucan (Betafectin®) is a novel soluble β-glucan immunomodulator that enhances leukocyte microbicidal activities without inducing inflammatory cytokines. Although several different receptors for soluble and particulate β-glucans have been described, the signal transduction pathway(s) used by soluble β-glucans have not been elucidated. We report that in a murine monocytic cell line (BMC2.3) PGG-Glucan activates nuclear factor-κB (NF-κB)-like and NF-interleukin-6 (IL-6)-like transcription factors. Electrophoretic mobility shift assays showed that PGG-Glucan activation of the factors is time- and concentration-dependent. The NF-κB-like complex includes subunit p65 (rel-A) as one of its components, but apparently not p50 (κB1), p52 (κB2), p68 (rel-B), or p75 (C-rel) family members. The NF-IL-6-like complex contains subunit C/EBP-β (NF-IL-6α) as one of its components, but apparently not C/EBP-α or C/EBP-δ (NF-IL-6β). As expected, lipopolysaccharide (LPS) activated p65/p50 NF-κB and C/EBP-β NF-IL-6 complexes, increased the nuclear titer of p65 and p50 antigens, and increased cytokine (IL-1β, tumor necrosis factor α) mRNA production. In contrast, PGG-Glucan increased the nuclear titer of p65, but apparently not p50, and did not induce cytokine mRNA production, These data demonstrate that PGG-Glucan utilizes signal transduction pathways different from those used by LPS. The data suggest that activation of the PGG-Glucan-stimulated factors is not sufficient to stimulate cytokine mRNA transcription.
Induction of endotoxin tolerance depletes nuclear factor-κB and suppresses its activation in rat alveolar macrophages
Authors: Blackwell Timothy S , Blackwell Thomas R , Christman John W
Published: January 2018

To investigate the mechanism of endotoxin tolerance in macrophages, a rat alveolar macrophage cell line (NR8383) was rendered endotoxin tolerant by treatment with endotoxin at 40 ng/mL for 48 h. This treatment induced a state of tolerance such that subsequent exposure to high-dose endotoxin (5 μg/mL) resulted in decreased production of macrophage inflammatory protein-2, tumor necrosis factor α, and nitric oxide compared to endotoxin-sensitive cells. Suppressed mediator production by endotoxin-tolerant cells was associated with impaired activation of nuclear factor-κB (NF-κB) in response to treatment with 5 μg/mL of endotoxin. This impairment of NF-κB activation was found to be associated with depletion of latent NF-κB (both RelA and p50) in the cytoplasm of endotoxin-tolerant cells. These data suggest that a mechanism of endotoxin tolerance is depletion of RelA/ p50, which could limit the amount of NF-κB available for activation by subsequent stimuli and thereby inhibit transcription of NF-κB-dependent genes. Limiting NF-κB-dependent inflammatory gene transcription by inducing endotoxin tolerance is a potential therapeutic strategy for diseases where excessive production of inflammatory mediators leads to tissue injury.
Selectins and mechanisms of signal transduction
Authors: Crockett-Torabi Elahe
Published: January 2018

Leukocyte emigration is essential in both lymphocyte homing, as a central part of immune surveillance, and in leukocyte invasion at sites of inflammation. The emigration of leukocytes requires the interplay of adhesion molecules of the selectin and integrin families and chemokines. Selectindependent cell-cell interaction is essential in localizing leukocytes within tissues by promoting the rolling of leukocytes along the endothelial cell surface before development of tight adhesion and subsequent transendothelial migration. Selectins also play an active role in the initiation of intracellular signaling pathways and regulation of cell-cell interactions involving monocytes, lymphocytes, platelets, and endothelial cells. This review focuses mainly on the emerging evidence of biochemical signaling mechanisms involved in the regulation of selectin-dependent leukocyte activation and adhesion, as well as the critical role played by selectins as leukocyte stimulatory molecules. This evidence has serious implications regarding the development of immune and inflammatory responses. This article will also review key structural features of the selectin receptors. A summary is provided of our current understanding of the specific molecular interaction occurring between these adhesion molecules and their counter-receptors, focusing on the critical roles they may play in the regulation of functional responses.
Endogenous monocyte chemoattractant protein-1 recruits monocytes in the zymosan peritonitis model
Authors: Ajuebor Maureen N , Flower Roderick J , Hannon Robert , Christie Mark , Bowers Keith , Verity Anne , Perretti Mauro
Published: January 2018

The role of monocyte chemoattractant protein-1 (MCP-1) in the recruitment of blood-derived monocytes in a model of zymosan peritoneal inflammation was investigated. After zymosan injection (1 mg) a rapid influx of polymorphonuclear leukocytes (PMN) and monocytes into the peritoneal cavity associated with mouse MCP-1 (JE) gene activation and protein secretion in the exudates occurred. MCP-1 production (maximal at 4 h) preceded the accumulation of monocytes (F4/80-positive cells, maximally recovered between 16 and 24 h). Treatment of mice with a single injection of anti-mouse MCP-1 antibody inhibited 16-h monocyte accumulation by ~40%, however, a significant decrease in the number of PMN was also measured. Finally, intraperitoneal injection of murine recombinant MCP-1 (1 μg) produced a selective accumulation of monocytes (F4/80-positive cells) into the peritoneal cavity. In conclusion, we show the novel existence of a strict relationship between MCP-1 production and leukocyte accumulation in this model of acute inflammation.
Thermal injury-induced immunosuppression in mice: the role of macrophage-derived reactive nitrogen intermediates
Authors: Schwacha Martin G , Somers Scott D
Published: January 2018

Macrophages (MΦ) have been implicated in the suppression of lymphocyte function following thermal injury. Splenocytes isolated from C57BL/ 6NCR female mice 4–7 days after thermal injury displayed suppressed proliferative responses to Concanavalin A (ConA) and lipopolysaccharide (LPS) and high levels of reactive nitrogen intermediate (RNI) production. Inhibition of nitric oxide synthase activity with NG-monomethyl-L-arginine restored ConA responses but not LPS responses. Surprisingly, ConA-stimulated interferon-γ (IFN-γ) production was increased in splenocytes from injured mice. IFN-γ contributed to the RNI-mediated immunosuppression as antibodies against IFN-γ reduced RNI production and immunosuppression. ConA-stimulated co-cultures of splenic MΦ from injured mice and normal splenocytes produced high levels of RNI only under conditions of cellular contact and splenic mΦ from injured mice were capable of suppressing normal splenocytes responses in co-culture. These results indicate that MΦ activity and specifically RNI production contribute to the suppression of T lymphocyte function after thermal injury.
Protective effect of plasma in polymorphonuclear neutrophil-mediated cytotoxicity of endothelial cells in the systemic inflammatory response syndrome
Authors: Chen Xuwu , Christou Nicolas V
Published: January 2018

Polymorphonuclear neutrophil (PMN)-mediated endothelial cell (EC) cytotoxicity is well described in many in vitro systems. These observations have been extended in vivo and suggest that circulating PMNs adherent to endothelial cells can damage these endothelial cells and produce the capillary leak that is central to the evolution of a systemic inflammatory response to multiple organ failure. However, most PMC-EC interactions in the circulation must occur in the presence of plasma, therefore we studied PMN-induced endothelial cell cytotoxicity in the absence and presence of autologous plasma from healthy human volunteers and patients with the systemic inflammatory response syndrome (SIRS) requiring admission to an intensive care unit. PMNs from patients with SIRS had increased endothelial cell adherence compared with controls, but equivalent endothelial cell cytotoxicity. Endothelial cell activation with TNF-α and IL-1β markedly increased PMN adherence to endothelial cells. Plasma had a minimal effect on the adherence of PMNs to endothelial cells at baseline or after endothelial cell activation. In contrast, plasma provided endothelial cells with almost complete protection from PMN-induced cytotoxicity at baseline, as well as after endothelial cell activation in both human volunteers and patients with SIRS. The data suggest that PMNs may cause cytotoxic damage in end organs such as the lung only after they diapedese through the endothelial barrier into the extracellular matrix removed from the protective effect of circulating plasma.
Fas or ceramide induce apoptosis by Ras-regulated phosphoinositide-3-kinase activation
Authors: Gulbins Erich , Brenner Birgit , Koppenhoefer Ursula , Linderkamp Otwin , Lang Florian
Published: January 2018

We demonstrate a rapid and transient activation of phosphoinositide-3-kinase (PI-3-K) by Fas receptor triggering or cellular treatment with synthetic C6-ceramide. The stimulation of PI-3-K is critical for Fas or C6-ceramide-induced programmed cell death because transfection with a transdominant inhibitory PI-3-K construct or pretreatment with the PI-3-K inhibitor wortmannin almost completely prevented Fas or C6-ceramide-mediated apoptosis. Treatment with the caspase inhibitor Ac-YVAD-cmk or cellular transfection with transdominant inhibitory N17Ras prevented PI-3-K stimulation by Fas, suggesting that Fas activates PI-3-K via caspases and Ras. N17Ras expression also prevented C6-ceramide-initiated PI-3-K stimulation. The notion of a PI-3-K regulation by Ras upon Fas receptor ligation or ceramide treatment is supported by co-immunoprecipitation experiments revealing an activation-dependent association of PI-3-K and Ras.
Trypanosoma brucei infection elicits nitric oxide-dependent and nitric oxide-independent suppressive mechanisms
Authors: Beschin Alain , Brys Lea , Magez Stefan , Radwanska Magda , De Baetselier Patrick
Published: January 2018

During murine Trypanosoma brucei infection, macrophages contribute significantly to the inhibition of T cell responses. Although nitric oxide (NO) was shown to play a central role in macrophage-mediated splenic suppression, macrophage-mediated lymph node suppression occurred in an interferon-γ (IFN-γ)-dependent manner. In this study, using NO inhibitor NG-monomethyl-L-arginine and anti-IFN-γ antibodies, the relative contribution of NO and IFN-γ to the active inhibition of ex vivo concanavalin A-induced T cell proliferation taking place in the spleen and the lymph nodes of T. brucei-infected mice was investigated. NO contributes to the suppressive activity of spleen and lymph node cells only during early-stage infection. The existence of NO-independent suppressive pathway was further evidenced in IFN-γ-/--infected mice. Spleen cells from such animals do not produce NO but exert significant suppressive activity during the whole course of infection. In contrast in the lymph nodes, no suppressive activity is recorded at any moment of infection. Moreover, addition of exogenous IFN-γ to cultures containing lymph node cells from IFN-γ-/--infected mice does not impair proliferation despite NO production in such cultures. Thus during late-stage infection, an IFN-γ-independent suppressive mechanism is elicited in the spleen, whereas in the lymph nodes, IFN-γ is required yet not sufficient to inhibit T cell proliferation.