bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophages/microglia and the pathophysiology of CNS injuries in AIDS
Authors: Gendelman Howard E , Tardieu Marc
Published: January 2018

How HIV-1 penetrates the central nervous system (CNS) and induces neurological disease remains poorly understood. Clinical and pathological consequences of HIV-1 associated CNS infections are the subjects of a relatively new field comprising a spectrum of many brain diseases including those that are HIV-1 associated or secondary to bacterial, fungal, protozoal, or tumor infiltration of brain. The molecular and biological approaches used to study HIV-1-mediated brain disease are interdisciplinary. A wide variety of expertise and cooperative research efforts is required between molecular virologists, cellular immunologists, and neurobiologists. Indeed, specialists studying microglial maturation, astrocyte function, neurotoxicity, HIV-1 neurovirulence, and apoptosis all play pivotal roles in examining facets of this disease complex.
Production of nitric oxide by differentiated LSTRA cells is associated with expression of macrophage-inducible nitric oxide synthase
Authors: Garcia-Welsh Adrienne , Laskin Debra L , Hwang Shiaw-min , Denhardt David T , Laskin Jeffrey D
Published: January 2018

LSTRA is a mouse lymphoma cell line overexpressing the src-related oncogene product and T cell marker p56lck. We have discovered that LSTRA cells, like HL60 and U937 promyelocytic leukemia cells, can be induced to differentiate toward macrophages by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or granulocytes by the cyclic nucleotide analogue dibutyryl cAMP (dbcAMP). One property of mature macrophages and granulocytes is the ability to produce nitric oxide, a highly reactive free radical that is important in non-specific host defense. Nitric oxide production by mature macrophages is stimulated by inflammatory mediators via an inducible form of the enzyme, nitric oxide synthase. We report that LSTRA cells acquire the ability to produce nitric oxide following differentiation induced by TPA and, to a lesser extent, by dbcAMP. Nitric oxide production by the cells is dependent on l-aiginine and blocked by several inhibitors of nitric oxide synthase, including N-monomethyl-l-arginine, l-canavanine, l-arginine benzyl ester, and l-arginine methyl ester. In macrophage-differentiated LSTRA cells, the inflammatory cytokine interferon-γ was found to be a potent inducer of nitric oxide production. This was correlated with a marked increase in nitric oxide synthase activity in the cells that was due to interferon-γ-induced expression of the macrophage-inducible form of nitric oxide synthase protein as well as mRNA. Differentiated LSTRA cells also expressed increased amounts of a constitutive form of nitric oxide synthase that was also present in undifferentiated cells. Taken together with previous findings, these results support the model that LSTRA cells have the capacity to differentiate toward mature macrophages. J. Leukoc. Biol. 56: 488–494; 1994.
Role of proteasomes modified by interferon-γ in antigen processing
Authors: Tanaka Keiji
Published: January 2018

Large multisubunit proteasomal complexes, catalyzing an extralysosomal, ATP-dependent pathway for selective degradation of unnecessary proteins, are thought to be responsible for major histocompatibility complex (MHC) class I–restricted antigen presentation. Two proteasomal polymorphic genes, LMP2 and LMP7, are localized within the MHC class II region, closely linked to the TAPI and TAP2 genes that are responsible for the ATP-dependent, size- and sequence-selective transport of antigenic peptides generated in the cytoplasm into the endoplasmic reticulum. Interferon-γ (IFN-γ) alters the peptide-degrading specificity of proteasomes and appears to produce an immunoproteasome responsible for accelerated processing of nonself endogenous antigens by inducing the replacement of subunits X and Y by LMP7 and LMP2, respectively. This IFN-γ-induced change in the subunits of proteasomes by IFN-γ is a new mechanism for an immunomodulatory action, differing from that for marked increases of TAP transporter and MHC molecules, which are possibly due to changes in transcriptional levels in cells. Based on these findings, I propose alteration of the subunit assembly of proteasomes in response to extracellular stimuli as a novel mechanism for the acquisition of their functional diversity that plays a central role in the immune response. J. Leukoc. Biol. 56: 571–575; 1994.
Identification and characterization of a bovine lipopolysaccharide-binding protein
Authors: Khemlani Lajwanti S , Yang Zhengang , Bochsler Philip N
Published: January 2018

Endogenous regulatory mechanisms exist in mammals that enable a rapid response to lipopolysaccharide (LPS, endotoxin) stemming from gram-negative bacterial infections. Serum proteins and cell surface receptors exist that bind LPS, and this interaction may either aid in nonpathogenic removal of LPS from the body or potentiate the effects of LPS. We have used a photoreactive, thiol-cleavable, radiolabeled derivative of E. coli 0111:B4 LPS [LPS-(p-azidosalicylamido)-1,3′-dithiopro-pionamide; 125I-ASD-LPS], to identify the presence of LPS-binding proteins (LBPs) in bovine serum. Ion exchange chromatography was used to fractionate bovine serum, and eluted protein was subsequently photoaffinity labeled using 125I-ASD-LPS. LBPs were identified by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several LBPs including three with apparent molecular masses of 65, 60, and 50 kDa were variably present within the chromatography pools. A 22-residue NH2-terminal amino acid sequence of the 60-kDa protein showed 77% homology with human LBP and 68% with rabbit LBP within this region. Further purification utilizing high-performance liquid chromatography yielded a protein fraction that contained the 60-kDa protein and was distinctly more active than whole bovine serum in LPS-dependent macrophage activation assays (up to 1600-fold on a weight/volume basis). The LPS-mediated macrophage activation in concert with chromatographically purified serum protein in tissue factor assays was inhibitable using anti-CD14 monoclonal antibodies. The results indicate that an LPS-binding protein exists in samples of pooled bovine serum and that this protein has features in common with human and rabbit LBP. J. Leukoc. Biol. 56: 784–791; 1994.
Influence of age on the production of Fos and Jun by influenza virus-exposed T cells
Authors: Salkind Alan R
Published: January 2018

This study investigated age-related T cell responses after in vitro exposure to influenza A virus. Mononuclear leukocytes from young or elderly persons were sham-exposed or exposed to influenza virus for 1, 24, and 72 h. Immunofluorescent staining and flow cytometric analysis were then used to detect T cells producing the transcriptional regulating proteins Fos and Jun. Fewer virus-exposed cells from elderly donors stained for Fos and Jun at each data point compared with cells from young donors. Flow cytometric analysis also showed that at 72 h of virus exposure fewer T cells from the elderly produced interferon-gamma (IFN-γ), suggesting a link between the magnitude of the Fos and Jun and IFN-γ responses. Thus, failure of virus-exposed T cells to produce Fos and Jun could contribute to the increase in illness due to influenza virus in the elderly. J. Leukoc. Biol. 56: 817–820; 1994.
Loss of response to β-adrenoceptor agonists during the maturation of human monocytes to macrophages in vitro
Authors: Baker A J , Fuller R W
Published: January 2018

We have previously reported that human airway macrophages do not respond to the β-adrenoceptor agonist isoprenaline. The airway macrophage is known to be derived from the blood monocyte. In this study we have assessed the effect of β-adrenoceptor stimulation on human monocytes matured into macrophages in vitro, to determine whether the lack of response previously observed in the airway macrophage may be a consequence of differentiation. The release of thromboxane B2 (TXB2) from freshly isolated monocytes stimulated by opsonized zymosan (OPZ) was inhibited by 39.3 ± 5.5% in the presence of isoprenaline (10-7 M). However, the response was lost in the monocyte-derived macrophage (MDM), where isoprenaline (10-7 M) caused only 4.0 ± 9.3% inhibition of OPZ-stimulated TXB2 release. In contrast forskolin (10-5 M) inhibited MDM TXB2 release by 36.4 ± 17.3%, indicating that the adenylyl cyclase was functional. Measurement of adenylyl cyclase activity showed that there was a reduction in the basal level, 17.03 ± 4.1 to 7.9 ± 4.6 cyclic AMP pmol/min/mg protein, and NaF (10-2 M)-induced activity, 116.3 ± 32.1 to 21.9 ± 12.6 cyclic AMP pmol/min/mg protein, between freshly isolated monocytes and MDMs, respectively. In addition, there was no change in MDM basal adenylyl cyclase activity on exposure to isoprenaline. Thus we have demonstrated the loss of β-adrenoceptor function during the maturation of human monocytes to macrophages in vitro, despite a functional adenylyl cyclase system. In this respect the monocyte-derived macrophage is like the airway macrophage. J. Leukoc. Biol. 57: 395–400; 1995.
Differential inhibitory effects of interleukin-10, interleukin-4, and dexamethasone on staphylococcal enterotoxin-induced cytokine production and T cell activation
Authors: Krakauer Teresa
Published: January 2018

The cytokine profile of human peripheral blood mononuclear cells (PBMC) stimulated by staphylococcal enterotoxin (SE) A and B was examined. Production of tumor necrosis factor (TNFα), interleukin (IL)-1, IL-6, IL-2, and gamma interferon (IFN-γ) was observed. In contrast, Th2 cytokines IL-4 and IL-10 were absent from SEA- or SEB-stimulated PBMC. Moreover, adding IL-10 to SE-stimulated PBMC inhibited the production of IL-1, IL-6, TNFα, and IFNγ by 50 to 80% but had less effect (8–30%) on T cell proliferation. IL-4 was less effective than IL-10 in inhibiting cytokine production and enhanced T cell proliferation by SEA or SEB. The antiinflammatory agent, dexamethasone, was the most potent agent in controlling the SE-mediated effects as evidenced by inhibited T cell proliferation (55%) and reduced levels of IL-1, IL-6, and IFNγ (60% to 100%) and TNFα (50%). Reducing levels of toxic mediators such as TNFα, IL-1, IL-6, and IFNγ by dexamethasone in SE-induced T cell responses may be a useful therapeutic strategy to circumvent SE toxicity and pathogenesis. J. Leukoc. Biol. 57: 450–454; 1995.
Selective sensitivity of macrophages to cytotoxicity by inhibitors of macromolecular synthesis: induction of apoptosis
Authors: Lewis J G , Adams D O , Fan S
Published: January 2018

Initial studies designed to measure the effect of inhibiting RNA synthesis by dactinomycin on macro phage functions revealed that the cells were uniformly killed at concentrations that have been routinely used to inhibit RNA synthesis in other cell types. We, thus, deter mined the dose curve for the cytotoxicity of dactinomycin for macrophages and two other cell types, L929 cells and splenic lymphocytes Macrophages were extremely sensitive to the cytotoxicity of dactinomycin compared to the other cell types. Submicromolar concentrations that in duced 100% cytotoxicity in macrophages caused little death in L929 cells or lymphocytes. Concentrations of dactinomycin that inhibited RNA synthesis by 40% in macrophages induced almost complete cell death but in hibition of over 80% of RNA synthesis in L929 cells or lymphocytes induccd no measurable cytotoxicity. Macro phages did take up more dactinomycin than other cells but the amount was not sufficient to account for the large differences in cytotoxicity. We next tested the effects of doxorubicin and cyclohcximide and found that macro phages were also extremely sensitive to killing by these compounds, and there was a very close association be tween the amount of inhibition of protein synthesis and the amount of toxicity. The morphology of macrophages exposed to these agents was consistent with death by apoptosis. This was further supported by assays measuring membrane integrity and DNA fragmentation. These data demonstrate that inhibition of macromolecular syn thesis in macrophages, by different mechanisms, causes macrophages to undergo apoptosis. They further suggest that, in contrast to other cell types that require protein synthesis for apoptosis, macrophages require the synthe sis of certain proteins to avoid apoptosis. J. Leukoc. Biol. 57: 635–642; 1995.
Interaction of GM-CSF and IL-3 with the common β-chain of their receptors
Authors: Bagley Christopher J , Woodcock Joanna M , Hercus Timothy R , Shannon M Frances , Lopez Angel F
Published: January 2018

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL-3) are cytokines active in both normal and abnormal hemopoiesis, inflammation, and immunity. Their biological activity is mediated via receptors that comprise a ligand-specific α chain and a β chain that is common to the GM-CSF, IL-3, and IL-5 receptors. To understand the mechanism of action of GM-CSF and IL-3 in both normal and pathological conditions, we are seeking to define the structural elements required for ligand/receptor and receptor/receptor contact and their role in cellular activation. To this end we have identified a conserved motif in the first helix of GM-CSF, Glu21 that is critical for high affinity binding and biological activity. Charge-reversal mutagenesis of this residue generates a GM-CSF analogue that is devoid of biological activity and can antagonize the activity of wild-type GM-CSF. This probably results from the selective deficiency in interaction with the β chain of the receptor and suggests that similar antagonists for IL-3 and IL-5 are also feasible. Complementary mutagenesis studies on the receptor β chain have identified the putative B’-C’ loop in the membrane-proximal domain as being critical for the high affinity binding of GM-CSF but not IL-3. Characterization of the specificity of sites of interaction between the ligands and receptors may permit the design of specific or generic antagonists that may have important therapeutic implications. J. Leukoc. Biol. 57: 739–746; 1995.
Endotoxin infusion in anesthetized sheep is associated with intrapulmonary sequestration of leukocytes that immunohistochemically express tumor necrosis factor-α
Authors: Cirelli Rosemary A , Carey Lisa A , Fisher Jill K , Rosolia David L , Elsasser Ted H , Capema Thomas J , Gee Marlys H , Albertine Kurt H
Published: January 2018

Plasma levels of tumor necrosis factor-α (TNF-α) peak between 2 and 4 h during a 12-h continuous infusion of endotoxin in awake sheep. We hypothesized that a source of this TNF-α is the pool of leukocytes that accumulate in the pulmonary circulation. To test this hypothesis, we physiologically monitored six anesthetized sheep during baseline and 4-h endotoxin infusion periods (10 ng/kg min). We obtained open-lung biopsies at baseline and at 20 min and 2 and 4 h during the endotoxin infusion period for immunohistochemical localization of TNF-α. The plasma concentration of TNF-α increased from an average baseline concentration of 0.06 ± 0.03 ng/ml (mean ± SD) to a peak of 1.40 ± 0.28 ng/ml at 2 h of the endotoxin infusion. We observed increased cytoplasmic TNF-α immunoreactivity in situ among neutrophils and intravascular mononuclear phagocytes during the endotoxin infusion compared with baseline. Also, the number of immunopositive leukocytes increased in the pulmonary circulation during the continuous infusion of endotoxin. We conclude that TNF-α-producing leukocytes accumulate in the pulmonary circulation during endotoxemia. These cells probably contribute to both the rise in the circulating levels of TNF-α and the development of acute lung injury. J. Leukoc. Biol. 57: 820–826; 1995.
Elevation of rainbow trout Oncorhynchus mykiss macrophage respiratory burst activity with macrophage-derived supernatants
Authors: Jang Seon I , Hardie Laura J , Secombes Christopher J
Published: January 2018

A variety of supernatants were prepared by stimulating rainbow trout Oncorhynchus mykiss head kidney macrophages with lipopolysaccharide (LPS), tumor necrosis factor α (TNF-α), or a leucocyte-derived macrophage-activating factor (I-MAF), individually and in combination. If generated using a 12-h stimulation period, such supernatants were found to elevate significantly the respiratory burst activity of target macrophages; that is, they contained a macrophage-derived MAF (m-MAF), but supernatants generated using a shorter incubation period showed no significant activity. Combinations of these treatments were particularly effective in generating m-MAF–containing supernatants. The elevation of respiratory burst activity by supernatants generated using combined treatments could be partially inhibited by prior treatment of the target macrophages with anti-TNF-α receptor 1 (TNFR1) monoclonal antibodies (mAbs). Similarly, treatment of macrophages with combinations of 1-MAF and m-MAF generated supernatants with potent m-MAF activity and this activity was partially inhibited by prior treatment of the target cells with anti-TNFR1 mAb. In addition, the presence of anti-transforming growth factor β1 (TGF-β1) serum while generating these latter supernatants resulted in significantly increased m-MAF activity. Such data suggest that fish leukocytes secrete a variety of potent macrophage-activating (TNF-α) and -deactivating (TGF-β) factors. J. Leukoc. Biol. 57: 943–947; 1995.
Proteases and protease receptors in modulation of leukocyte effector functions
Authors: Altieri Dario C
Published: January 2018

Cellular immune responses depend on regulated pathways of intracellular signal transduction and leukocyte activation. Although these mechanisms are coordinated by a variety of leukocyte-restricted effector molecules, recent observations have uncovered a novel role of proteases in transducing outside-in signals of leukocyte activation. Through regulated, receptor-mediated recognitions, coagulation and fibrinolytic enzymes or effector cell granular proteases influence monocyte motility and chemotaxis, modulate pleiotropic cytokine responses, contribute to mononuclear cell proliferation, or induce target cell apoptosis. Overall, these mechanisms define a novel interface between general inflammatory reactions, invariably characterized by activation of blood protease cascades, and specialized aspects of cellular immune functions. J. Leukoc. Biol. 58: 120–127; 1995.
Phorbol ester-induced priming of superoxide generation by phosphatidic acid-stimulated neutrophils and granule-free neutrophil cytoplasts
Authors: Siddiqui Rafat A , English Denis , Harvey Kevin , Cui Yi , Martin Margaret I , Wentland John , Akard Luke , Jansen Jan , Thompson James , Garcia Joe G N
Published: January 2018

This study was undertaken to examine the mechanisms involved in polymorphonuclear leukocyte superoxide release stimulated by exogenous phosphatidic acid (PA). Unlike the immediate burst of superoxide release affected by membrane-permeable dioctanoyl-glycerol (DiC8-DAG), dioctanoyl phosphatidic acid (DiC8-PA) induced superoxide release after a lag period of 5–20 min. This period was considerably reduced or eliminated when cells were primed by substimulatory levels of phorbol myristate acetate (PMA). Granule-depleted neutrophil cytoplasts also responded to DiC8-PA with a burst of superoxide generation. Activation of the cytoplast superoxide generating system in response to DiC8-PA was also significantly faster after cells had been preexposed to substimulatory levels of PMA, indicating that at least a portion of the priming mechanism was independent of PMA-induced degranulation. To further examine the potential mechanism of PMA priming of responses to PA, we evaluated the activity of neutrophil ecto-phosphatidic acid phosphohydrolase (ecto-PA phosphohydrolase), which generates diacylglycerol from exogenous PA. PMA priming had no discernable effect on the activity of this enzyme. In addition, propranolol, an inhibitor of PA phosphohydrolase, did not selectively inhibit PMA priming of neutrophil responses to DiC8-PA, indicating that priming did not result from acceleration of DiC8-PA hydrolysis. We therefore investigated the possibility that activation of protein kinase C was the basis of the primed response. Several semiselective protein kinase C inhibitors (calphostin C, H-7, and acylmethylglycerol) inhibited DiC8-DAG- and DiC8-PA-induced superoxide release as well as PMA-primed responses to approximately the same extent. These results are consistent with the hypothesis that neutrophil responses to phosphatidate are mediated by diglyceride generated by the action of ecto-PA phosphohydrolase. PMA priming does not result from increased catalytic activity of ecto-PA phosphohydrolase but rather seems to result from potentiation of an intermediate involved in the cells’ response to multiple stimuli. J. Leukoc. Biol. 58: 189–195; 1995.
Discrete pathways for arachidonic acid release from tannin versus β-glucan-stimulated rabbit alveolar macrophages
Authors: Kennedy Michael T , Bates Philip J , Wheatley Christine L , Rohrbach Michael S
Published: January 2018

Previously, we observed both tannin and β-glucan to be agonists for arachidonic acid (AA) release from rabbit alveolar macrophages. Although tannin inhibited reincorporation of exogenous AA, β-glucan had no apparent effect, suggesting separate signal transduction pathways leading to elevated AA levels. In this study alveolar macrophages were pretreated with the tyrosine phosphatase inhibitor sodium orthovanadate then stimulated with either condensed tannin or β-glucan. Vanadate exerted opposing effects on AA release. Furthermore, vanadate reversed the ability of tannin to inhibit reacylation. Additional studies using the phospholipase A probe bis-BODIPY-C11-i-PC indicated that although the known phospholipase A2 activators, calcium ionophore A23187, insoluble immune complexes, and β-glucan, generated an increase in fluorescence consistent with phospholipase A activation, tannin had no effect. These findings suggest the increase in free AA resulting from stimulation of macrophages by either tannin or β-glucan is produced via two different mechanisms. J. Leukoc. Biol. 58: 241–248; 1995.
Modulation of IgA, IgE, and IgG Fc receptor expression on human mononuclear phagocytes by 1α,25-dihydroxyvitamin D3 and cytokines
Authors: Boltz-Nitulescu George , Wiilheim Martin , Spittler Andreas , Leutmezer Fritz , Tempter Clemens , Winkler Susanne
Published: January 2018

The effects of lα,25-dihydroxyvitamin D3/caldtriol on the expression of Fc receptors (FcR) for IgA (FcαR), IgE (FcRII), and IgG (FcγR) on human peripheral blood monocytes and the cell lines U937, THP-1, and Mono Mac-6, in combination with various cytoldnes, was examined by flow cytometry. On both monocyte-derived macrophages and the myelomonocytic cell lines, FcαR/CD89 expression was induced by caldtriol alone and additively in combination with tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and granulocyte-macrophage colony-stimulating factor. Constitutive and mterleukin-4 (IL-4)-driven FcRD/CD23 expression was markedly diminished on caldtriol-treated U937 cells and monocytes. FcRII was also triggered by IFN-γ, TNF-α, and IL-6 on all the cell lines, an effect blocked by caldtriol. On monocytes, the basal level and IFN-γ-induced FcγRI/CD64 expression was down-regulated by caldtriol and IL-4. The expression of FcγRII/CD32 on monocytes was strongly suppressed by caldtriol. Transforming growth factor-β induced FcγRIII/CD16 on monocytes, an effect opposed by caldtriol. The ability of caldtriol-treated monocytes to phagocytose IgG-sensitized ox erythrocytes was diminished. Our results demonstrate that caldtriol, alone or in combination with cytokines, modulates FcαR, FcRII, FcγRI, and FcγRII expression on human mononudear phagocytes, as well as FcγR-mediated phagocytosis. J. Leukoc. Biol. 58: 256–262; 1995.
Ultrastructural immunogold localization of subcellular sites of TNF-α in colonic Crohn’s disease
Authors: Beil Waltraud J , Weiler Peter F , Peppercorn Mark A , Galli Stephen J , Dvorak Ann M
Published: January 2018

Tumor necrosis factor-α, a proinflammatory cytokine, might have an important role(s) in initiating, modifying, and/or sustaining chronic inflammatory processes such as those that characterize Crohn’s disease, an inflammatory bowel disease of unknown etiology. We used an immunogold ultrastructural morphometric approach to localize tumor necrosis factor-α in colonic Crohn’s disease biopsies. Tumor necrosis factor-α was present in seven cell types (fibroblasts, eosinophils, mast cells, macrophages, colonic epithelial absorptive cells, Paneth cells, neutrophils). Tumor necrosis factor-α-containing subcellular organelles included lipid bodies (fibroblasts, eosinophils, macrophages, mast cells, colonic epithelial cells, neutrophils), secretory granules (eosinophils, Paneth cells), phagolysosomes (macrophages, colonic epithelial cells), and Golgi structures and vesicle membranes (neutrophils). A gradient of extracellular tumor necrosis factor-α immunoreactivity surrounded eosinophils, mast cells, and macrophages. P values of gold counts/μm2 were significant for all cells, organelles, and extracellular spaces measured, and all positive structures significantly exceeded the background labeling density/μm2. Specificity controls (normal rabbit serum, tumor necrosis factor-α-absorbed primary antibody) either failed to label these sites or gave markedly reduced specific tumor necrosis factor-α labeling, respectively. These findings represent the first ultrastructural localization of the subcellular sites of TNF-α in vivo in seven cell lineages in human colonic tissues.
Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen
Authors: Driscoll Kevin E , Hassenbein Diana G , Howard Brian W , Isfort Robert J , Cody David , Tindal Michael H , Suchanek Maureen , Carter Janet M
Published: January 2018

Macrophage inflammatory protein-2 (MIP-2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP-2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription-polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP-2 cDNA sequence. A cDNA containing the coding region of rat MIP-2 was cloned and sequenced. Comparison to the mouse MIP-2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP-2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP-2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP-2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.
Natural resistance to infection with intracellular parasites: molecular genetics identifies Nramp1 as the Bcg/Ity/Lsh locus
Authors: Vidal Silvia , Gros Philippe , Skamene Emil
Published: January 2018

Natural resistance to infection with intracellular parasites is controlled in the mouse by the expression of a locus or group of loci on chromosome 1 alternatively named Bcg, Lsh, and Ity. Bcg affects the capacity of mature tissue macrophages to restrict the intracellular proliferation of ingested parasites in the reticuloendothelial organs of the host during the early phase of infection. This review summarizes our molecular genetic approach to the isolation and characterization of the Bcg locus. We have used a positional cloning strategy based on genetic and physical mapping, YAC cloning, and exon trapping to isolate a candidate gene for Bcg, named Nramp1, which codes for a macrophage-specific polytopic protein with 12 predicted transmembrane domains and a consensus transport motif. Sequence analysis of Nramp1 cDNA clones from 27 Bcgr and Bcgr mouse strains reveals that susceptibility to infection (Bcgr) is associated with a single nonconservative Gly to Asp substitution at position 169 within predicted transmembrane domain 4 of the Nramp protein. Cloning experiments and homology search in available databases demonstrated that the Nramp1 gene belongs to a small gene family with several members in vertebrates and in such distantly related species as yeast and plants. Nramp proteins share a remarkable degree of similarity, with strong amino acid sequence conservation in the transmembrane domains, suggesting a common transport function for the Nramp family. Finally, we generated Nramp1−/- gene knockout mice, and analysis of their phenotypic characteristics established that (1) Nramp1 plays a key role in natural defense against infection with intracellular parasites and therefore demonstrated allelism between Nramp1 and Bcg/Ity/Lsh, (2) Nramp1 functions by a novel cytocidal/cytostatic mechanism distinct from those expressed by the activated macrophage, and (3) the Nramp1Asp169 allele of Bcgr inbred strains is a null allele, pointing to a critical role of this residue in Nramp1 function.
Role of the FcγR subclasses FcγRII and FcγRIII in the activation of human neutrophils by low and high valency immune complexes
Authors: Strohmeier Gregg R , Brunkhorst Beatrice A , Seetoo Kurt F , Meshulam Tova , Bernardo John , Simons Elizabeth R
Published: January 2018

Two Fcγ receptor (FcγR) subclasses on human neutrophils, FcγRII and FcγRIII, activate different cellular functions. To examine the involvement of each receptor subtype in polymorphonuclear leukocyte activation, Fab and F(ab’)2 fragments of subclass-specific monoclonal antibodies ([mAbs] mAb IV.3 against FcγRII and mAb 3G8 against FcγRIII, respectively) were used to block the binding of low valency immune complexes (LICs) and high valency immune complexes (HICs). Flow cytometry then permitted the simultaneous quantitation of antibody and ligand binding, the elicited intracellular Ca2+ concentration (Δ[Ca2+]int), initiation of the oxidative burst, and/or the phospholipase A activation in the same cell. We have previously demonstrated that subsaturating dosages of HIC bind uniformly to all the cells but elicit an “all-or-none” (i.e., dose independent) maximal Δ[Ca2+]int in a dose-dependent subpopulation of the cells. In contrast, both the proportion of cells responding and the magnitude of the Δ[Ca2+]int transient depend on the subsaturating dose of LIC, even though it too binds uniformly to all the cells, nonresponding as well as responding. These earlier findings have here been extended by single cell flow cytometric analysis to demonstrate that F(ab’)2 FcγRIII is the major FcγR involved in HIC binding (and [Ca2+]int mobilization), as well as in oxidative burst and phospholipase A activation. In contrast, both receptor subclasses must be available for LIC-elicited Δ[Ca2+]int, as blockage by either of the mAb Fab or F(ab’)2 fragments abrogates this response, even though LIC binding to the receptors is not decreased. Furthermore, LIC elicited little oxidative burst activity and failed to activate phospholipase A but cross-linking to achieve multivalency, previously shown to induce [Ca2+]int and oxidative burst responses, elicited phospholipase A activity via FcγRIII. FcγRII’s role appears to be modulation of the small, late Ca2+ influx observed at > 1 min, whereas FcγRIII modulates all the earlier larger events. Thus, simultaneous observation of receptor identity, receptor occupancy, and consequent activation parameters in the same cell by flow cytometry permits use to demonstrate that FcγRII is necessary for the small signal transduction elicited by LIC; it plays a relatively small role in polymorphonuclear leukocyte stimulation by HIC. FcγRIII is the main receptor responsible for immune complex-elicited polymorphonuclear leukocyte responses; its efficacy is greatly enhanced when the receptors are cross-linked, either by preequilibrated multivalent complexes or by in situ cross-linking of bound LIC with excess antibody.
Different patterns of deactivation of Chemotaxis and haptotaxis of human peripheral blood mononuclear leukocytes by soluble and surface-bound attractants
Authors: Wiedermann Christian J , Reinisch Norbert , Bellmann Romuald , Schratzberger Peter , Kowald Elisabeth , Kähler Christian M
Published: January 2018

Soluble mediators and inducible cell-surface and matrix-bound molecules coordinate the cascade of events giving rise to leukocyte emigration. Knowledge of the specific mechanisms underlying the attraction of cells into a local site, however, remains sketchy. In particular, it is unclear how chemoattractants cause rapidly moving immune cells to adhere to the blood vessel wall and to enter tissues. Here we show that the neuroendocrine human growth hormone, a chemoattractant for monocytes and lymphocytes in vitro, promotes haptotaxis, the migration of the cells induced by surface-bound gradients. Combination of soluble growth hormone with soluble attractants, RANTES or formyl peptide, deactivates the migratory responses, as do combinations of surface-bound growth hormone with surface-bound RANTES or formyl peptide. In contrast, exposure of mononuclear leukocytes to combinations of soluble chemotactic with surface-bound haptotactic gradients of attractants does not deactivate migration. The findings suggest that growth hormone may act as haptotactic agent, on the one hand, and that soluble attractants do not appear to affect haptotaxis when acting in concert with a surface-bound attractant, on the other. This observation may have implications for the differential regulation of leukocyte accumulation in the vessel wall at systemic and local sites.
Tumor necrosis factor triggers redistribution to a Triton X-100-insoluble, cytoskeletal fraction of β2 integrins, NADPH oxidase components, tyrosine phosphorylated proteins, and the protein tyrosine kinase p58fgr in human neutrophils adherent to fibrinogen
Authors: Yan Sen Rong , Fumagalli Laura , Dusi Stefano , Berton Giorgio
Published: January 2018

Tumor necrosis factor (TNF) triggers cell spreading, release of granule constituents, and production of toxic oxygen derivatives in human neutrophils adherent to fibrinogen. This response requires cytoskeleton reorganization and is dependent on expression of β2 integrins. We analyzed distribution of distinct proteins in Triton X-100–soluble and insoluble fractions in neutrophils adherent to fibrinogen. We found that stimulation of adherent neutrophils with TNF causes the redistribution to a Triton-insoluble fraction of α-actinin, β2 integrins, and the four components whose assembly constitutes an active NADPH oxidase: the gp91-phox, p22-phox, p47-phox, and p67-phox proteins. Redistribution of these different proteins to a Triton-insoluble fraction took relatively long times and was maximal after about 30 min of stimulation with TNF. Prevention of actin polymerization with cytochalasin B hampered the TNF-induced redistribution of these proteins from a Triton-soluble to an insoluble fraction. In addition, tyrosine phosphorylated proteins and the protein tyrosine kinase p58fgr were recovered in this Triton-insoluble fraction. These findings show that stimulated, β2 integrin–dependent adhesion of neutrophils to fibrinogen is accompanied by redistribution to cytoskeletal structures of (1) β2 integrins, that is, neutrophil receptors for fibrinogen; (2) proteins involved in neutrophil effector functions, that is, components of NADPH oxidase; and (3) tyrosine phosphorylated proteins and the protein tyrosine kinase p58fgr, molecules that are potentially involved in the formation of a submembranous signaling complex.
Taurine chloramine, a product of activated neutrophils, inhibits in vitro the generation of nitric oxide and other macrophage inflammatory mediators
Authors: Marcinkiewicz Janusz , Grabowska Agnieszka , Bereta Joanna , Stelmaszynska Teresa
Published: January 2018

Taurine (Tau) is an exceptionally abundant free amino acid in the cytosol of inflammatory cells and especially in neutrophils. Taurine protects cells from self-destruction during processes that generate oxidants. The major function of Tau in leukocytes is to trap chlorinated oxidants (HOCl). Taurine reacts with HOCl to produce the long-lived compound taurine chloramine (TauCl). Previously, we have shown that other products of the neutrophil chlorinating system are able to modify functions of macrophages. In this study, we investigated in vitro the influence of TauCl on the generation of inflammatory mediators by activated macrophages. We have found that TauCl inhibited the generation of nitric oxide, prostaglandin E2, tumor necrosis factor α, and interleukin-6, but TauCl slightly enhanced the release of IL-1α. The formation of nitrites by interferon-γ-activated macrophages was inhibited by TauCl in a dose-dependent manner. Taurine chloramine also reduced the level of inducible nitric oxide synthase (iNOS) mRNA in macrophages, in a similar concentration-dependent manner. Although our experiments do not exclude a direct effect of TauCl on enzymatic activity of iNOS, the inhibition of iNOS expression seems to be the major mechanism responsible for suppression of NO formation. Finally, we discuss the biological role of TauCl in vivo. We suggest that at the site of inflammation TauCl works as a specific signaling molecule of activated neutrophils that coordinates the generation of inflammatory mediators in macrophages.
The Duffy antigen receptor for chemokines: structural analysis and expression in the brain
Authors: Horuk R , Martin, A , Hesselgesser J , Hadley T , Lu Zhao-hai , Wang Zi-xuan , Peiper S C
Published: January 2018

The Duffy antigen receptor for chemokines (DARC) is expressed in human erythrocytes and on endothelial cells lining postcapillary venules in kidney and spleen. DARC is a promiscuous chemokine receptor and a binding protein for the malarial parasite Plasmodium vivax. The expression of DARC by subsets of endothelial cells and neurons in discrete anatomic sites in the brain suggests that this enigmatic receptor may have multiple roles in normal and pathological physiology. Conservation of this promiscuous chemokine binding function is evident from the similarity in nucleotide sequence of DARC homologues from multiple species, as well as the high-affinity binding of human chemokines to murine and avian erythrocytes. Analysis of the functional domains of DARC using chimeric receptors and monoclonal antibodies to multiple extracellular domains localized chemokine binding to structures in the amino terminal extracellular domain (E1). Scatchard analysis demonstrated that a chimeric DARC receptor, composed of the E1 domain of DARC and the predicted hydrophobic helices and loops of interleukin-8RB (IL-8RB), bound IL-8, and MGSA with K  D values almost identical to the wild type receptors and bound a repertoire of C-X-C and C-C chemokines characteristic of DARC. Although numerous reports have demonstrated that chemokines such as IL-8 are expressed in the brain, presumably by glial cells, little insight into the nature of their role in normal or pathological physiology in the nervous system has developed because the target cells that express the corresponding receptors have not yet been identified. Northern blotting experiments suggest that mRNA encoding DARC are expressed in the central nervous system, however, interpretation of this is unclear because of the ubiquitous expression of DARC lining postcapillary venules. This study provides direct evidence to localize expression of DARC in the cental nervous system. Immunohistochemical examination of human archival sections of the brain with monoclonal antibodies specific for DARC localize expression of DARC to cell bodies and processes of Purkinjie cells in the cerebellum. The immunohistochemical findings were supported by analysis of chemokine binding and radioligand crosslinking with membranes made from various brain fractions. The hierarchical expression of DARC in neurons in the cerebellum suggests that chemokines may play an important role in the modulation of neuronal activity by glial cells.
Effects of interleukin-13 on human neutrophil functions
Authors: Girard Denis , Faquin Robert , Naccache Paul H , Beaulieu André D
Published: January 2018

It was recently shown that interleukin-13 (IL-13) induces the expression and release of the IL-1 decoy receptor (type II) in human neutrophils along with the production of the IL-1 receptor antagonist. In the present study we focused on further studying the modulatory effects of IL-13 on these cells. We found that recombinant human IL-13 (rhIL-13) induces cellular morphological changes in neutrophils that are typical of activated cells. Furthermore, rhIL-13 was shown to increase tyrosine phosphorylation of several neutrophil proteins. We also demonstrate that this cytokine stimulates de novo RNA synthesis in a concentration-dependent fashion as measured by [5-3H]uridine incorporation and that rhIL-13 induces the synthesis of several neutrophil proteins according to high-resolution two-dimensional gel electrophoresis of metabolically [35S]-labeled cells. We observed that the IL-8 levels in the external milieu of IL-13-stimulated cells was almost fivefold increased when compared with control cells. Finally, we observed that rhIL-13 has no significant effect on phagocytosis and apoptosis. Taken together, our results demonstrate that IL-13 is a modulator of several human neutrophil functions. This leads us to conclude that the modulatory role of IL-13 on human neutrophils, a potentially important anti-inflammatory cytokine, is more complex than previously believed. Furthermore, because we show that the synthesis of several as yet unidentified proteins was up-regulated by IL-13, our findings open new avenues of research on the effects of this cytokine on human neutrophils.
Immunoregulation by interleukin-12
Authors: Trinchieri Giorgio , Gerosa Franca
Published: January 2018

Interleukin-12 (IL-12) is a heterodimeric cytokine produced primarily by antigen-presenting cells (monocytes, macrophages, dendritic cells, and B cells). Its production is stimulated by bacteria, bacterial products, and intracellular parasites and enhanced by priming with granulocyte-macrophage colony-stimulating factor (CM-CSF) and interferon-γ (IFN-γ) or inhibited by IL-10. The major biological activity of IL-12 is on T and natural killer (NK) cells in which it increases cytokine production, proliferation, and cytotoxicity. Its production occurs several hours after exposure to infectious agents, which induces a rapid production of IFN-γ by NK and later by T cells. This IFN-γ potentiates antigen-presenting cell functions important in clearing infectious agents (phagocytosis, oxidative burst, and production of nitrous oxide) and also increases further production of IL-12. IL-12 has been clearly demonstrated to be important in the generation of CD4 and CD8 type 1 T cells both in vivo and in vitro. Our data reveals that IL-12 primes naive T cells for high IFN-γ and IL-10 production, whereas IL-4 is required for IL-4 priming, thus suggesting that these genes and possibly others are independently regulated. IL-12 is therefore involved in the skewing of cytokine production toward a type 1 and has been implicated in being involved in selective mechanisms of established T cells. It is now becoming clear that the IL-12 acts as both a proinflammatory cytokine and an immunomodulator and therefore bridges the innate and adaptive immune responses.
IL-4-dependent proliferation of BA/F3 cells expressing a growth-negative mutant of the human IL-4 receptor is restored by enforced expression of Bcl-2
Authors: Chung Johanna , Deutsch Harald H J , Kalthoff Frank S
Published: January 2018

We have studied the regulation of growth and apoptosis in murine BA/F3 cells stably expressing cytoplasmic deletion mutants of the human interleukin-4 receptor (hIL-4R). Previously, we showed that BA/F3 cell transfectants expressing a cytoplasmic deletion mutant of the hIL-4R that lacks the region between Thr(462) and Ala(580), referred to as δR3, fails to proliferate in the presence of hIL-4. Here we report that supertransfection of δR3-expressing cells with a constitutively active murine bcl-2 gene results in prolonged survival of the δR3/bcl-2 double transfectants in the absence of cytokines. More importantly, however, the constitutive expression of Bcl-2 restored their capacity to grow permanently with hIL-4. This may provide an explanation for the discrepancy with previous reports showing growth mediation by hIL-4R truncated at position 367.
Binding affinities of the SH2 domains of ZAP-70, p56/ck and Shc to the chain ITAMs of the T-cell receptor determined by surface plasmon resonance
Authors: Labadia M E , Ingraham R H , Schembri-King J , Morelock M M , Jakes S
Published: January 2018

The chains of the T cell receptor complex play a critical role in the initiation of proximal signaling events upon T cell activation. Three pairs of potential tyrosine phosphorylation sites are located within the cytoplasmic domains of the chains. Subsequent to engagement of the T cell receptor, one or more of these tyrosine residues is phosphorylated. The phosphotyrosine residues, along with flanking amino acids, form an activation motif (and are shared by signaling subunits in the TCR, B cell receptor, and FcγRI) termed tyrosine-based activation motifs (ITAMs). ITAMs serve as binding sites for SH2 domain-containing proteins. Recent evidence suggests that the chains provide docking space for several key signal transduction molecules such as ZAP-70, p56  lck, and Shc. To determine if ZAP-70, p56  lck, and Shc bind to particular chain ITAM sequences, quantitative free-solution measurements of binding affinities (Kd) were obtained by use of surface plasmon resonance technology. The results indicate that binding affinities of distinct SH2 domains to individual and paired phosphorylation sites greatly differ, and may dictate the sequence of signal transduction events.
Heat shock protein-peptide complexes for use in vaccines
Authors: Heike Michael , Noll Bernhard , zum Büschenfelde Karl-Hermann Meyer
Published: January 2018

The heat shock proteins gp96, HSP70, and HSP90 are complexed to a diverse array of cellular proteins and peptides as a consequence of their chaperone functions. There is good experimental evidence that vaccination with these heat shock protein-peptide complexes elicit immune responses against chaperoned peptide antigens. As shown with gp96, this requires internalization of the heat shock protein-peptide complexes by macrophages and processing of the chaperoned peptides for class I restricted presentation. Via this process, primarily CD8+ antigen-specific T cells are primed by gp96 vaccination. This might represent a general mechanism for priming of MHC-class I restricted T cells by professional antigen-presenting cells. At least gp96 has been shown to be associated with peptides that are not selected by the MHC haplotype of the harboring cell. This allows for immunization with gp96-peptide complexes across MHC barriers, for example against shared tumor antigens or viral antigens.