bluuebunny/crossref_metadata_2025 huggingface dataset:
Granzymes: a variety of serine protease specificities encoded by genetically distinct subfamilies
Authors: Smyth Mark J , O'Connor Michael D , Trapani Joseph A
Published: January 2018

Granzymes are a family of granule serine proteases found specifically in the cytotoxic granules of cytotoxic T lymphocytes and natural killer cells. Granzymes have features that are strongly conserved including: consensus sequences at their N-termini and around the three catalytic residues, activation from zymogenic forms, and conserved disulphide bridges. However, there is good genetic evidence to suggest that three distinct subfamilies of granzymes have coevolved. These subfamilies are most strikingly depicted by their distinct chromosomal loci and gene organization, dividing the granzyme family into subfamilies of the following: tryptases (human chromosome 5); chymotrypsinlike proteases (human chromosome 14); and a Metase amongst a cluster of elastase-like proteases (human chromosome 19). Modeling and mutational analysis has revealed that each subfamily of granzymes displays special sequence and structural features and a proteolytic specificity determined by subtle modifications to substrate binding pocket residues. It now remains of great interest to determine whether these subfamilies also possess distinct biological functions. Granzyme B has been shown to play an important role in lymphocyte-mediated target cell apoptosis and the tryptase, granzyme A, has been demonstrated to regulate the clearance of some pox virus infections. The future creation of other granzyme gene knockout mice should elucidate whether other chymotrypsin-like granzymes (C-H) also contribute to target cell apoptosis and whether the third subfamily member, natural killer cell-specific Metase, has a distinct biological function.
Infection by Candida albicans inhibits apoptosis of human monocytes and monocytic U937 cells
Authors: Heidenreich Stefan , Otte Britta , Lang Detlef , Schmidt Michael
Published: January 2018

Infectious microorganisms can differently induce or Inhibit apoptosis of immunocompetent effector and host cells. In this study we examined the influence of an infection by Candida albicans (C. albicans) on programmed cell death of monocytic U937 cells and human monocytes. Basal and tumor necrosis factor α (TNF-α)-induced DNA fragmentation of U937 cells was significantly inhibited by an infection with C. albicans. Enhanced apoptosis of U937 cells, induced by TNF-α, caused a diminished candidacidal activity of the effector cells, whereas inhibition of apoptosis by granulocyte-macrophage colony-stimulating factor (GM-CSF) was paralleled by an intensified host defense. Pretreatment of U937 cells or monocytes with the cyclooxygenase blocker indomethacin completely abolished the reduction of DNA fragmentation induced by the yeast. Studying the underlying mechanisms we found that C. albicans induced formation of prostaglandin E2 (PGE2) by U937. Exogenous administration of PGE2 down-regulated apoptosis of U937 or human monocytes to a similar extent as did fungal infection. Activation of protein kinase A by the cAMP analogue 8-bromo-cAMP inhibited U937 apoptosis, as did PGE2 On the other hand, rp-cAMP, a blocker of the cAMP-dependent signal transduction, restored and elevated DNA fragmentation levels down-regulated by C. albicans. U937 cells expressed the bcl-2 protein but the infection with fungi or PGE2 treatment did not increase proto-oncogene expression. Monocytic effector cells may therefore strengthen the defense against C. albicans by an autocrine feedback regulation via a PGE2-dependent, cAMP-transduced inhibition of apoptosis.
Dual effects of soluble CD14 on LPS priming of neutrophils
Authors: Troelstra Annet , Giepmans Ben N G , van Kessel Kok P M , Lichenstein Henri S , Verhoef Jan , van Strijp Jos A G
Published: January 2018

To evaluate the effect of soluble CD14 (sCD14) on human neutrophil response to lipopolysaccharide (LPS), we developed an LPS-priming assay that measures the chemiluminescence response to N-formyl-methionyl-leucyl-phenylalanine stimulation. Priming by 1 ng/mL rough LPS occurred in the presence of either serum or recombinant LPS-binding protein (LBP) only. Priming was completely CD14-dependent because preincubation of the neutrophils with an anti-CD14 monoclonal antibody prevented priming. We hypothesize that sCD14 enhances LPS response in neutrophils, but this response is not as effective as LPS response via membrane CD14 (mCD14). In our experiments sCD14 is present in an excess compared with mCD14. Priming of neutrophils occurs with low LBP, supposedly via sCD14-LPS complexes. With high LBP, addition of sCD14 inhibited LPS-priming of neutrophils. In that case, LPS may be transported to sCD14, preventing a more effective response via mCD14. In this study we demonstrate that the effect of sCD14 on neutrophil response to LPS is a delicate balance between activation and inhibition depending on concentration of serum or LBP.
Endothelial P-selectin and VCAM-1 each can function as primary adhesive mechanisms for T cells under conditions of flow
Authors: Konstantopoulos Konstantinos , Kukreti Sharad , Smith C Wayne , McLntire Larry V
Published: January 2018

This study demonstrates that endothelial P-selectin and vascular cell adhesion molecule 1 (VCAM-1), but not intercellular adhesion molecule 1 (ICAM-1), are capable of supporting extensive primary adhesion of T cells under flow. To address this issue, we used human umbilical vein endothelial cells (HUVECs) stimulated with histamine, interleukin-4 (IL-4), or interferon-γ (IFN-γ) that provide essentially a P-selectin, VCAM-1, or ICAM-1 surface, respectively, in a physiologically relevant cell type. Monoclonal antibody (mAb) blockade studies were carried out to confirm the specificity of these adhesive interactions and rule out a number of other potentially important adhesion molecules. Quantitation of adhesion showed that almost all of the interacting T cells rolled on histamine-stimulated HUVECs or CHO-P cell monolayers. In contrast, 20% of the total interacting T cells with 24-h IL-4-treated HUVECs were firmly adherent. mAb blocking experiments revealed that T cell adhesion to IL-4-treated HUVECs is α4-VCAM-1 dependent. Furthermore, mAb 4B9 directed against domain 1 of VCAM-1 eliminated adhesion, suggesting that α4 integrins may not interact with either the alternatively spliced domain 4 of VCAM-1 or fibronectin in this process. At a wall shear stress of 2 dyn/cm2, the mean T cell rolling velocities were significantly lower on 24-h IL-4-activated HUVECs (10.2 ± 2.6 μm/s) compared with either CHO-P cells (15.6 ± 3.1 μm/s) or histamine-stimulated HUVECs (16.6 ± 6.1 μm/s). ICAM-1, expressed on the surface of 24-h IFN-γ-activated HUVECs pretreated with an anti-VCAM-1 mAb to eliminate any VCAM-1-dependent contribution, did not support T cell adhesion under shear conditions. Together these data indicate that T cell primary adhesion can be mediated by both endothelial P-selectin and VCAM-1 but not ICAM-1. α4 integrins are highly versatile molecules, capable of initiating T cell rolling interactions and mediating firm arrest on activated endothelium.
CD40-CD40L interactions provide “third-party” costimulation for T cell response against B7-1-transfected human breast tumor cells
Authors: Pericle Federica , Epling-Burnette P K , Podack Eckhard R , Wei Sheng , Djeu Julie Y
Published: January 2018

In this study we provide evidence that a human breast carcinoma cell line, MDA-MB-231 (MDA), can be made immunogenic following B7 transfection and that fall T cell activation is obtained through cooperation of T-B lymphocytes via CD40-CD40L interactions. Tumor cells transfected with either B7 gene (MDAB7), neomycin-resistant gene only (MDAneo), or untransfected (MDA) were used in an allogeneic mixed lymphocyte tumor culture (MLTC) to investigate their ability to stimulate T cell proliferation and generate cytotoxic T lymphocytes (CTL). MDAB7 induced moderate T cell proliferation while MDAneo or MDA did not. Substantial T cell proliferation and de novo generation of cytolytic T cells was obtained only in response to MDAB7 when B cells were present during the MLTC. CD8+-purified T + B cells proliferated to a greater extent than whole T cell populations + B or CD4+ + B in response to MDAB7. Addition of (α-B7-1 or α-CD40 in the MLTC inhibited T cell proliferation by 65 and 40%, respectively, whereas T cell proliferation and generation of CTL was completely abrogated when MLTC was performed in the presence of both antibodies. These data suggest that the engagement of CD40L on T cells with CD40 on B cells provides a costimulatory signal which, in synergism with TCR-dependent MDAB7-T cell recognition (signal 1) and B7/CD28 interactions (signal 2), leads to full T cell activation.
Evidence for an enhanced transcription-dependent de novo synthesis of C/EBPβ in the LPS activation of the chicken lysozyme gene
Authors: Goethe Ralph , Phi-van Loc
Published: January 2018

C/EBPβ has been shown to mediate the lipopolysaccharide (LPS)-induced expression of the lysozyme gene through enhanced binding to the – 6.1-kb lysozyme enhancer. In this study, we describe the LPS regulation of the C/EBPβ gene in myelomonocytic HD11 cells. Northern analysis showed that the steady state level of C/EBPβ mRNA increased in response to LPS. The half life of C/EBPβ mRNA of about 1 h in HD11 cells was not affected by exposure to LPS. Nuclear run-on transcription experiments with isolated nuclei revealed that the rate of C/EBPβ gene transcription was enhanced by LPS, demonstrating that the C/EBPβ gene in HD11 cells was regulated at the transcriptional level in response to LPS. Furthermore, the LPS-induced binding activity of C/EBPβ to the – 6.1-kb lysozyme enhancer was dependent not only on protein synthesis, but also on transcription. Thus, these results suggested that the LPS-induced binding activity to the – 6.1-kb lysozyme enhancer in HD11 cells was regulated by an enhanced transcription-dependent de novo synthesis of C/EBPβ.
Involvement of leukotrienes, TNF-α, and the LFA-1/ICAM-1 interaction in substance P-induced granulocyte infiltration
Authors: Saban Marcia R , Saban Ricardo , Bjorling Dale , Haak-Frendscho Mary
Published: January 2018

Substance P (SP) has been shown to mediate granulocyte infiltration into the mouse skin by inducing mast cell degranulation. In this study, using a variety of specific inhibitors, we investigated the cascade of events involved in the response of neutrophils and eosinophils to SP. The prostaglandin inhibitor, indomethacin, had little effect on SP-induced leukocyte migration. In contrast, pretreatment with the leukotriene (LT) synthesis inhibitor, A-64077, completely blocked neutrophil but not eosinophil migration in response to SP. Participation of tumor necrosis factor α (TNF-α) and LFA-1/ICAM-1 interaction was confirmed by inhibition of SP-induced leukocyte migration by pretreatment of mice with monoclonal antibodies to TNF-α, LFA-1, and ICAM-1. Moreover, alteration in leukocyte migration by indomethacin was found to depend on the concentration of TNF-α used. Indomethacin did not alter the number of leukocytes induced by low concentrations of TNF-α (0.1 ng), but reduced the number of cells stimulated with high TNF-α concentrations (1.0 ng). These results support the concept that SP modulates in vivo neuroinflammatory responses, as measured by granulocyte migration, initiating a cascade of events that includes LT production, TNF-α secretion, and engagement of LFA-1 and ICAM-1.
Leukocyte activation induces surface redistribution of P-selectin glycoprotein ligand-1
Authors: Bruehl Richard E , Moore Kevin L , Lorant Diane E , Borregaard Niels , Zimmerman Guy A , McEver Rodger R , Bainton Dorothy F
Published: January 2018

Binding of P-selectin on activated endothelium to P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils mediates the initial tethering and rolling of neutrophils on the vessel wall at inflammatory sites. Upon activation of rolling cells by locally expressed signaling molecules, integrin-dependent adhesion mechanisms are engaged and transendothelial migration proceeds. P-selectin binding sites are uniformly distributed on the surface of quiescent neutrophils, but are redistributed to the uropod of activated neutrophils. It is unclear whether this activation-induced change in the surface topography of P-selectin binding sites is due to surface redistribution of PSGL-1, shedding of PSGL-1 from the lamellapod, and/or movement of PSGL-1 from an intracellular compartment to the uropod of the polarized cell. With the use of immunogold electron microscopy we previously demonstrated that PSGL-1 was localized to the tips of microvilli on neutrophils. Here we document a similar localization for PSGL-1 on eosinophils, basophils, monocytes, and lymphocytes. On quiescent neutrophils, 80% of the PSGL-1 label was on tips of microvilli, which are randomly distributed around the cell circumference. On activated, polarized neutrophils, the PSGL-1 label was restricted to a segment of 42% of the cell circumference even though total labeling decreased by only 26%. Latex microbeads coated with anti-PSGL-1 mAb bound preferentially to the uropod of activated neutrophils. Subcellular fractionation and immunogold analysis of frozen thin sections of neutrophils failed to detect PSGL-1 in any intracellular compartment. Taken together, these data indicate that the activation-induced change in the surface topography of PSGL-1 is due to surface redistribution of PSGL-1. This process may facilitate transendothelial migration by disrupting bonds between P-selectin and PSGL-1 at the leading edge of migrating cells.
Monocytic-endothelial cell interaction: regulation of prostanoid synthesis in human coculture
Authors: Koll Sylvia , Goppelt-Struebe Margarete , Hauser Ingeborg , Goerig Matthias
Published: January 2018

Coculture of a monocytic cell line (HL-60) and iliacal endothelial cells as an in vitro model of vascular inflammation was investigated for cooperative regulatory mechanisms of prostanoid synthesis under conditions of selective prestimulation. In coculture of endothelial cells and 12-O-tetradecanoylphorbol 13-acetate (TPA) -prestimulated monocytic HL-60 cells the capacity of prostanoid synthesis from arachidonic acid was strongly increased compared with monocultures. Concomitant with upregulation of specific adhesion molecules, cyclooxygenase (COX) 2 mRNA was induced in endothelial cells in coculture independent of cell contact. HL-60 cells exhibited no alterations in mRNA expression of cyclooxygenases or thromboxane synthase. Coculture of TPA-prestimulated endothelial cells with unstimulated HL-60 cells led to a selectively increased capacity of thromboxane production. Under this condition HL-60 cells upregulated COX1 and COX2 mRNA, whereas endothelial mRNA levels did not change. Our data demonstrate that the increase in prostanoid synthesis in coculture essentially depends on rapid induction of COX2 mRNA within 2 h.
Timing of prostaglandin exposure is critical for the inhibition of LPS- or IFN-γ-induced macrophage NO synthesis by PGE2
Authors: Harbrecht B G , Kim Y-M , Wirant E A , Simmons R L , Billiar T R
Published: January 2018

Macrophage nitric oxide (NO) synthesis is an integral component of the host defense system. We have previously found that NO and prostaglandins interact in a variety of ways. NO modulates Kupffer cell prostaglandin E2 (PGE2) production and we have recently described the inhibitory effects of PGE2 on NO synthesis in both Kupffer cells and hepatocytes. Activated macrophages produce a number of prostaglandins but studies regarding the capacity of prostaglandins to regulate macrophage NO synthesis have yielded conflicting results. We found that exogenous PGE2 decreased lipopolysaccharide (LPS)-induced NO synthesis in murine resident peritoneal macrophages and in the RAW 264.7 murine macrophage cell line. PGE2 also suppressed NO synthesis in response to interferon-γ (IFN-γ) alone and a combination of LPS + IFN-γ. Inhibition of endogenous PGE2 synthesis with indomethacin or ibuprofen had no effect on NO synthesis. PGE2 added with the activating stimulus was most effective. PGE2 lost the capacity to block NO synthesis if added more than 180 min after LPS. PGE2 decreased inducible NO synthase (iNOS) mRNA and immunoreactive iNOS protein, consistent with the hypothesis that exogenous PGE2 inhibits macrophage iNOS expression but that the inhibition depends on the time and concentration of prostaglandin exposure.
Primary structure of rat CD14 and characteristics of rat CD14, cytokine, and NO synthase mRNA expression in mononuclear phagocyte system cells in response to LPS
Authors: Takai Noriyuki , Kataoka Masashi , Higuchi Yasunori , Matsuura Keiko , Yamamoto Shunsuke
Published: January 2018

Rat CD14 cDNA clones were isolated. The predicted protein sequence exhibits 82,61.6, and 64% identity with the mouse, rabbit, and human CD14, respectively. The levels of rat CD14 mRNA expression in resident peritoneal macrophages (PM), alveolar macrophages (AM), and peripheral blood monocytes (BM) were constitutively high, whereas that in Kupffer cells (KC) was low. On intravenous injection with lipopolysaccharide (LPS), the expression of rat CD14 mRNA in KC increased markedly, whereas the increases in PM, AM, and BM were mild. Similar features of expression of rat CD14 in these cells were observed after stimulation with LPS in vitro. The level of tumor necrosis factor α (TNF-α) mRNA expression in KC after stimulation with LPS in vivo was comparable to that in PM, AM, and BM, whereas that of TNF-α mRNA expression in KC and PM after stimulation with LPS in vitro was lower than that in AM and BM. Interleukin (IL)-1β and iNOS mRNA expressions in KC after stimulation with LPS in vivo and in vitro were low, whereas those in PM, AM, and BM were high. Little or no expression of IL-6 was observed in KC after stimulation with LPS in vivo and in vitro, whereas higher expression was observed in PM, AM, and BM than in KC.
Cellular aspects of HIV-1 infection of macrophages leading to neuronal dysfunction in in vitro models for HIV-1 encephalitis
Authors: Nottet Hans S L M , Bär Dop R , van Hassel Hans , Verhoef Jan , Boven Leonie A
Published: January 2018

HIV-1 is a hematogenously spread virus that most likely gains entry into the brain within blood-derived macrophages. Indeed, productive viral replication selectively occurs within perivascular and parenchymal blood-derived macrophages and microglia and HIV-infected macrophages have increased potential to bind and transmigrate through the blood-brain barrier. Once inside the brain, HIV-infected macrophages secrete a variety of proinflammatory mediators that display neuromodulatory and neurotoxic activities in several in vitro models for HIV-1 encephalitis. The final outcome regarding neuronal function and cell loss is regulated through intercellular interactions between these virus-infected cells and astrocytes. In this regard, both HIV-induced intracellular events in macrophages and interactions between HIV-infected macrophages and brain cells are reviewed as factors that might lead to neuronal injury in in vitro model systems for HIV-1 encephalitis.
Inhibition of replication of HIV in primary monocyte/macrophages by different antiviral drugs and comparative efficacy in lymphocytes
Authors: Aquaro Stefano , Perno Carlo-Federico , Balestra Emanuela , Balzarini Jan , Cenci Alessandra , Francesconi Mauro , Panti Stefania , Serra Franca , Villani Nicoletta , Caliò Raffaele
Published: January 2018

Several anti-HIV drugs acting on different steps of virus replication were tested in our experimental model of primary monocyte/macrophages; the results were compared with the activity found in lymphocytes. Nucleoside analogues (AZT, ddl, ddC, d4T, PMEA, 3TC etc.) show greater activity in macrophages (M/M) than in lymphocytes. In particular, the EC50 of AZT, ddC, and ddI in M/M is 2- to 100-fold lower than that found in lymphocytes. This greater efficacy of nucleoside analogues in M/M depends on the enhancement of their chain-terminating activity by the low levels of endogenous deoxynucleoside-triphosphates (dNTP) usually found in resting cells such as M/M. Non-nucleoside reverse transcriptase inhibitors (NNRTI) do not act as chain terminators (thus their antiviral effect is not related to the intracellular concentrations of dNTP); as a consequence the activity of TSAO, HEPT, TIBO, and other NNRTI tested in M/M is similar to that found in lymphocytes. Regarding inhibitors of binding and fusion of HIV, we found that their anti-HIV activity is markedly decreased (or even nullified) when M/M are treated with cytokine activators of M/M function and enhancers of HIV replication. More relevant from a clinical standpoint, protease inhibitors are able to inhibit HIV replication in chronically infected macrophages cells carrying the proviral genome already integrated in the host genome). All other inhibitors of late stage of virus life cycle tested (antisense-rev, anti-tat, interferon-α and -γ, phosphorothioate analogues, GLQ-223, etc.) were totally inactive in chronically infected macrophages. The different effects of various classes of HIV inhibitors in lymphocytes and macrophages suggests that AIDS therapy should consider all aspects of the pathogenesis of HIV infection and must be restricted to drugs, or combinations of drugs, active against both lymphocytes and M/M in all body compartments where the virus hides and replicates.
MCP-1 and CCR2 in HIV infection: regulation of agonist and receptor expression
Authors: Sozzani Silvano , Introna Martino , Bernasconi Sergio , Polentarutti Nadia , Cinque Paola , Poli Guido , Sica Antonio , Mantovani Alberto
Published: January 2018

Monocyte chemotactic protein-1 (MCP-1) interacts with the chemokine receptor CCR2. Two CCR2 cDNAs have been described. Sequence analysis as well as Northern blotting and RNase protection with different probes revealed that the CCR2 gene is expressed in activated natural killer (NK) cells and mononuclear phagocytes as a predominant long transcript (3.4 kb) consisting of CCR2B followed by a novel sequence (X), corresponding to an intron in the genome, and by a CCR2A specific portion. The predominant long transcript is polyadenylated and present in the cytoplasm. We found that bacterial products and cytokines affect CCR2 expression. Interleukin-2 (IL-2) augmented CCR2 mRNA in monocytes and NK cells. The augmented migratory capacity of Reactivated versus resting NK cells was associated with increased CCR2 transcript levels. Lipopolysaccharide (LPS) and other microbial agents caused a rapid and drastic reduction of CCR2 mRNA levels. The rate of nuclear transcription of CCR2 was not affected by LPS, whereas the mRNA half life was reduced. These results suggest that regulation of receptor expression, in addition to agonist production, is probably a crucial point in the regulation of the chemokine system. Down-regulation of chemokine receptor expression may play a role in the modulation of HIV infection in macrophages by LPS. Levels of MCP-1 were markedly elevated in the cerebrospinal fluid (CSF) but not in blood of HIV-infected patients with cytomegalovirus (CMV) encephalitis. The CSF levels of MCP-1 in CMV encephalitis were markedly higher than those found in the CSF of HIV-infected patients with or without unrelated neurological diseases. IL-8, the prototype of C-X-C chemokines and RANTES and macrophage inflammatory protein-1α (C-C chemokines) were not substantially increased in the liquor of CMV encephalitis patients. High levels of MCP-1 may underlie monocyte recruitment and tissue damage in CMV encephalitis and may represent a rapid and useful tool in the diagnostic armamentarium for neurological disorders associated with HIV.
Polyribonucleotides induce nitric oxide production by human monocyte-derived macrophages
Authors: Snell Julie C , Chernyshev Olga , Gilbert Daniel L , Colton Carol A
Published: January 2018

Cytokine-mediated activation of inducible nitric oxide synthase (iNOS) in monocytes or macrophages is species specific. In contrast to rat or mouse, human macrophages do not produce measurable levels of nitric oxide (NO) when induced by inflammatory mediators. Exposure to noncytokine mediators such as tumor cells or viruses, however, has recently been shown to activate human iNOS. NO production in response to these mediators is much lower than that seen for rat or mouse cells and often requires several days of stimulation. We have found that the synthetic, double-stranded polyribonucleotide polyinosinic-polycytidilic acid (Poly I:C), commonly used to mimic viral exposure, activated iNOS in human monocyte-derived macrophages (MDM). The production of NO, measured by nitrite accumulation, was detected after 24 h of stimulation with Poly I:C. The single-stranded polyribonucleotide Poly I, but not Poly C, also increased NO production. Nitrite production was enhanced when the MDM were primed (pretreated) with γ or α interferon or other immune mediators such as IL-4 and was reduced by the iNOS inhibitor, N-methyl-L-arginine (L-NMMA). The use of Poly I:C to initiate NO production in human macrophages provides a useful tool to study the differences between the commonly used animal models and human cells and may provide insight into the pathophysiological significance of these differences.
Liver granulomas in schistosomiasis: mast cell-dependent induction of SCF expression in hepatic stellate cells is mediated by TNF-α
Authors: Brito José M , Borojevic Radovan
Published: January 2018

Mast cell proliferation, survival, and differentiation are under control of the surrounding stroma and are largely mediated by the stem cell factor (SCF). Mast cells are abundant in liver fibrosis and are proposed to be causally related with its persistence and intensity through secretion of fibrogenic cytokines. In normal adult liver, local connective tissue cells (stellate cells) do not constitutively express SCF. We studied primary cultures of stellate cells obtained from fibro-granulomatous reactions elicited in mouse liver by schistosomal infection. We have shown that the SCF expression can be induced in vitro by coculture with mast cells and this induction was dependent on the release of tumor necrosis factor α (TNF-α). Because SCF induces in mast cells proliferation and release of both fibrogenic factors and TNF-α, the described interaction between liver stroma and mast cells represents an auto-stimulatory loop, which may explain the progressive and persistent character of liver fibrosis associated with chronic inflammations or infections. Granulomatous reactions in liver elicited by chronic schistosomiasis sustain a local production of inflammatory cells, and this extramedular myelopoiesis is potentially also dependent on the induction of SCF expression in the liver stellate cells.
Immunomodulatory action of G-CSF In a rat model of endotoxin-induced liver injury: an intravital microscopic analysis of Kupffer cell and leukocyte response
Authors: Vollmar Brigitte , Messner Sven , Wanner Guido A , Hartung Thomas , Menger Michael D
Published: January 2018

In contrast to the anticipation that in sepsis granulocyte colony-stimulating factor (G-CSF) would overactivate the nonspecific immune system by recruiting and priming leukocytes with consequent aggravation of inflammatory tissue lesions, recombinant (r) G-CSF pretreatment was protective in various experimental non-neutropenic models of inflammation. The mechanisms of protection, however, are not fully understood. Using intravital fluorescence microscopy, we show that rG-CSF enhances leukocyte endothelial cell interaction within the microvasculature of normal rat livers, whereas rG-CSF pretreatment of animals exposed to lipopolysaccharide (LPS) attenuates the LPS-induced leukocytic response, including stasis in sinusoids as well as rolling and adherence in postsinusoidal venules with subsequent tissue infiltration. Moreover, rG-CSF, which did not affect Kupffer cell activity in normal rat livers, reduced the immediate activation of Kupffer cells on LPS exposure, as indicated in vivo by the delayed adherence/phagocytosis of intra-arterially administered latex particles associated with attenuation of proinflammatory cytokine release (tumor necrosis factor α and interleukin-6). Finally, rG-CSF reduced LPS-induced nutritive perfusion failure and hepatocellular excretory dys function. This study provides evidence for a distinct, possibly tumor necrosis factor α-dependent modulation of LPS-induced cellular response within the liver by rG-CSF, thereby achieving protection against microcirculatory perfusion failure and hepatic dysfunction. J.
Innate resistance to Listeria monocytogenes in tumor-bearing mice
Authors: Gahan Cormac G M , Barrett John R , O'Brien Michael G , O'Sullivan Gerard C , Shanahan Fergus , Collins John K
Published: January 2018

We have previously described the isolation, cloning, and characterization of a tumorigenic murine fibrosarcoma, designated JBS. Growth of JBS tumors in syngeneic mice initiates an antitumor immune response that initially manifests as progressive splenic hyperplasia and an increased proliferative ability in cultured splenocytes. In animals with tumors progressing beyond the 2 cm stage there is a reduction in spleen size and a gradual decrease in splenocyte proliferative abilities, leading to anergy at heavy tumor burdens (>3.5 cm). During the phase of immune hyperresponsiveness in tumor-bearing mice clearance of Listeria monocytogenesby components of the innate immune system is increased. This heightened resistance to infection is most likely macrophage-mediated because these mice demonstrate an increased ability to recruit macrophages to the peritoneal cavity during Listeriainfection. In addition, these macrophages are highly activated in vivo as evidenced by an elevated capacity to express class II MHC (Ia) molecules. This increase in macrophage activation status is coincident with an increased capacity of splenocytes from tumor-bearing mice to secrete IFN-γ. In mice with much heavier tumor burdens (>3.5 cm), down-regulation of the immune response leads to a reduction in peritoneal macrophage numbers, decreased macrophage Ia expression, and diminished splenic clearance of L. monocytogenes. Our data demonstrate that activation of macrophages distal to the tumor site occurs as an initial consequence of tumor growth. It is only in mice with very heavy tumor burdens that functionality of macrophages is sufficiently suppressed to allow increased splenic growth of L. monocytogenes.
Desensitization of IgE-mediated IL-4 release from human basophils
Authors: MacGlashan Donald
Published: January 2018

Previous studies demonstrated marked differences in the time course of interleukin-4 (IL-4) and histamine release, implying different mechanisms of desensitization. To explore this possibility basophils were desensitized with anti-IgE antibody by treating the cells for various periods of time in the absence of extracellular calcium (Cae). The basophil response following addition of Cae was assessed by measuring IL-4 or histamine release by measuring the cytosolic calcium response ([Ca2+]i). The kinetics of desensitization for IL-4 and histamine release did not differ; 50% desensitization occurred at 37 ± 8 and 29 ± 16 min for IL-4 and histmaine release, respectively. The kinetics of desensitization as assessed by the [Ca2+]i response was found to be biphasic; an initial enhancement of the [Ca2+]i response after 5–15 min of desensitization was followed by a rapid decrease so that by 30 min the amount of desensitization was equivalent to that observed for histamine release. Other than this initial period of enhanced calcium response, desensitization kinetics were unexpectedly similar for all measured endpoints. These results suggest that, during stimulation in the absence of Cae, there is a down-regulation process that influences all events on a similar time scale. Because the time scales for secretion of histamine, LTC4 or IL-4 or the [Ca2+]iresponse markedly differ (i.e., stimulation in the presence of Cae), these results also indicate that down-regulatory processes differ depending on the presence or absence of Cae.
Two distinct Ca2+ storage and release sites in human neutrophils
Authors: Pettit Elizabeth J , Hallett Maurice B
Published: January 2018

It is well established that changes in cytosolic free Ca2+ play a role in a number of neutrophil responses such as cell shape change and oxidase activation. The release of intracellular stored Ca2+ occurs with stimuli that either act by occupancy of seven membrane-spanning domain receptors or those which act by receptor cross-linking. Here, two distinct Ca2+ storage and release sites have been identified in neutrophils. Using chlortetracycline fluorescence as an indicator of Ca2+ storage, two separate Ca2+ storage sites have been identified. One site was located peripherally under the plasma membrane and the other was in the juxtanuclear space. Confocal imaging of Fluo3-loaded neutrophils demonstrated that the central Ca2+ storage site released Ca2+ in response to N-formyl-methionyl-leucyl-phenylalanine, whereas engagement and clustering of CD11b/CD18 integrins causes Ca2+ release from the peripheral stores. The release sites also correlated with organelles that stained with DiOC6(3). Localized phototoxicity generated by DiOC6(3) excitation resulted in inhibition of the release of stored Ca2+, which was selective for the stimulus used. The presence of two distinct cellular locations for these Ca2+ stores and their independent release raises the possibility that separate intracellular messengers for their release are generated.
Pattern of inheritance in hereditary myeloperoxidase deficiency associated with the R569W missense mutation
Authors: Nauseef W M , Cogley M , Bock S , Petrides P E
Published: January 2018

Myeloperoxidase (MPO) is an essential component of the oxygen-dependent microbicidal system of neutrophils and monocytes. Hereditary deficiency of MPO occurs in 1 in 2,000 to 4,000 individuals in the general population and has been generally considered an autosomal recessive trait. Previous studies have used the peroxidase activity of blood leukocytes to assess the phenotype of affected family members. Eosinophil peroxidase (EPO) also contributes to the peroxidase activity of blood leukocytes. Because EPO expression is normal in MPO-deficient subjects, eosinophil contamination can significantly contribute to peroxidase activity in leukocytes from family members of an MPO-deficient subject and thereby undermine correct interpretation of the inheritance pattern. To avoid this potential problem, we used cytochemical, immunochemical, and genetic techniques to assess the inheritance pattern of MPO deficiency in sixteen individuals from five unrelated kindreds. Each kindred had an index case with MPO deficiency and the R569W missense mutation, a genotype that causes MPO deficiency. Our analysis demonstrated that MPO deficiency was not inherited as a simple autosomal recessive trait. Most subjects were compound heterozygotes with respect to the R569W mutation and demonstrated a spectrum of phenotypes. Our data demonstrate the broad phenotypic impact of compound heterozygosity on the expression and function of a multimeric protein such as MPO.
Identification of glyceraldehyde-3-phosphate dehydrogenase as a Ca2+-dependent fusogen in human neutrophil cytosol
Authors: Hessler Ronald J , Blackwood R Alexander , Brock Thomas G , Francis Joseph W , Harsh Donna M , Smolen James E
Published: January 2018

The membrane fusion events observed during neutrophil degranulation are important aspects of the immunoregulatory system. In an attempt to understand the regulation of granule-plasma membrane fusion, we have begun characterizing human neutrophil cytosol for fusion activity, finding that 50% of the fusogenic activity could be attributed to members of the annexin family of proteins. The major non-annexin fusion activity (25% of the total cytosolic activity) was enriched by ion exchange chromatography after depletion of annexins by Ca2+-dependent phospholipid affinity chromatography. The fusion activity co-purified with a 10,14-kDa dimer identified as leukocyte L1 (which was non-fusogenic), along with an approximately 36-kDa protein. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by amino-terminal sequencing, and the fusion activity was verified using commercially available GAPDH. GAPDH may play an important role in degranulation because it is as potent as annexin I on a mass basis and may constitute up to 25% of the total cytosolic fusion activity of the neutrophil.
Interleukin-5 binds to heparin/heparan sulfate. A model for an interaction with extracellular matrix
Authors: Lipscombe Richard J , Nakhoul Anne-Marie , Sanderson Colin J , Coombe Deirdre R
Published: January 2018

Interleukin-5 (IL-5) is the major cytokine regulating eosinophil production. In allergic disease tissue damage is primarily caused by eosinophils. Heparan sulfate proteoglycans are components of the bone marrow stroma, which supports hemopoietic cell differentiation and proliferation. We show that at low IL-5 concentrations heparan sulfate enhances the proliferation of an IL-5-dependent cell line. To investigate a mechanism for this effect we used an artificial proteoglycan to establish an enzyme-linked immunosorbent assay for the binding of heparin to proteins. Using this assay we demonstrate that IL-5 binds to heparin. The IL-5/heparin interaction is inhibited by ethylenediaminetetraacetate and enhanced by low concentrations of zinc ions. IL-5 interacts with iduronic acid containing glycosaminoglycans, and heparan sulfate preparations that have numerous N-sulfated domains per chain are especially efficient at inhibiting heparin binding. Both IL-5/ heparin binding and the synergistic effect of IL-5 and heparan sulfate on cell proliferation were inhibited by an anti-IL-5 monoclonal antibody. These data suggest that the binding of IL-5 to heparan sulfate modulates IL-5 activity.
Role of chemokines and cytokines in a reactivation model of arthritis in rats induced by injection with streptococcal cell walls
Authors: Schrier D J , Schimmer R C , Flory C M , Tung D K-L , Ward P A
Published: January 2018

Intraarticular injection of streptococcal cell wall (SCW) antigen followed by intravenous challenge results in a T cell-mediated monoarticular arthritis in female Lewis rats. Initial studies showed that this reactivation response to intravenous SCW antigen is dependent on the presence of interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) and that the early phase of swelling is neutrophil-dependent. Neutrophil depletion or passive immunization with antibodies to P-selectin or macrophage inflammatory protein-2 reduced the intensity of ankle edema and the influx of neutrophils. After the first few days, however, the arthritic response is mediated primarily by mononuclear cells. Joint tissues showed up-regulation of mRNA for monocyte chemotactic protein-1 (MCP-1), which could be inhibited in part by anti-IL-4; treatment of rats with antibodies to IL-4 or MCP-1 significantly suppressed development of ankle edema and histopathological evidence of inflammation. Antibodies to interferon-γ or IL-10 had no effect. Treatment with anti-MCP-1 also suppressed influx of 111In-labeled T cells into the ankle joint. These data suggest that the late, mononuclear-dependent phase of SCW-induced arthritis in female Lewis rats requires cytokines that up-regulate MCP-1, which in turn may facilitate recruitment and extravasation of mononuclear cells into the joint.
Synergistic induction of MCP-1 and -2 by IL-1β and interferons in fibroblasts and epithelial cells
Authors: Struyf Sofie , van Coillie Els , Paemen Liesbet , Put Willy , Lenaerts Jean-Pierre , Proost Paul , Opdenakker Ghislain , van Damme Jo
Published: January 2018

Monocyte chemotactic protein (MCP)-1 and MCP-2, two closely related CC chemokines, are important mediators of monocyte and lymphocyte migration. These chemokines are secreted by various normal cell types, including fibroblasts, epithelial cells, and leukocytes, as well as by tumor cells. After stimulation with different cytokines and cytokine inducers the MCP-2 production levels are always lower than those of MCP-1. In human diploid fibroblasts cytokines differentially regulate chemokine induction, interleukin (IL)-1β and interferon (IFN)-γ being potent stimuli of MCP-1 and MCP-2, respectively. Co-stimulation of fibroblasts by 10 U/mL IL-1β and 20 ng/mL IFN-γ resulted in a synergistic induction of MCP-2, whereas the combined effect on MCP-1 and IL-6 production was rather additive. These findings were confirmed at the mRNA level by Northern blot analysis. In contrast, in human MG-63 fibroblastoid cells and HEp-2 epithelial cells, selected for their poor responsiveness to IL-1β and IFN-γ, MCP-2 as well as MCP-1 and IL-6 were synergistically induced, yielding protein levels that were increased 3- to 30-fold above the additive levels. When IFN-β was used as a co-stimulant of IL-1β, a similar synergistic induction of MCP-1 and MCP-2 was measured both at the protein and the mRNA level. It can be concluded that, when synergy occurred, the MCP-1 and MCP-2 expression levels reached a comparable maximum, indicative for an equal contribution of these chemokines in normal and pathological conditions.
Transcription factors that regulate monocyte/macrophage differentiation
Authors: Valledor Annabel F , Borràs Francesc E , Cullell-Young Martin , Celada Antonio
Published: January 2018

Although all the cells in an organism contain the same genetic information, differences in the cell phenotype arise from the expression of lineage-specific genes. During myelopoiesis, external differentiating signals regulate the expression of a set of transcription factors. The combined action of these transcription factors subsequently determines the expression of myeloid-specific genes and the generation of monocytes and macrophages. In particular, the transcription factor PU.1 has a critical role in this process. We review the contribution of several transcription factors to the control of macrophage development.
The role of CD40/CD154 interactions in the priming, differentiation, and effector function of helper and cytotoxic T cells
Authors: Mackey Matthew F , Barth Richard J , Noelle Randolph J
Published: January 2018

This review focuses on the emerging body of literature suggesting a critical role for CD40/CD154 interactions in antigen-presenting cell (APC) activation, CD4+ and CD8+ T cell priming, and effector T cell maturation. In this context effective antigen presentation involves not only T cell expansion and long-term survival but also the ability of the APC to guide the T cell response toward the Th1 (interferon-γ producing) or the Th2 (interleukin-4 producing) phenotype. We suggest a model to explain why CD40/CD154 interactions are critical for some helper and cytotoxic T cell responses, whereas others occur independently of this receptor/ligand pair. In addition, we will discuss the potential role for CD40/CD154 interactions in effector T cell maturation and cytokine production.
Endotoxemia, pentose cycle, and the oxidant/antioxidant balance in the hepatic sinusoid
Authors: Spolarics Zoltán
Published: January 2018

During the innate immune response, excessive release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver and other tissues. The consequence of oxidative stress is determined by the status and adaptive changes of antioxidant pathways. In this review, we present evidence that the synchronized response of hepatic sinusoidal endothelial cells, the primary sites of phagocyte attachment, plays an important role in defense against phagocyte-derived ROS. An essential component of the metabolic adaptation of hepatic sinusoidal cells to lipopolysaccharide (LPS)-induced oxidative stress is the stimulated expression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the pentose cycle (hexose monophosphate shunt, HMS). All major ROS-metabolic enzymes, i.e., glutathione peroxidase, glutathione reductase, catalase, superoxide dismutases, NADPH oxidase, and nitric oxide synthase, directly or indirectly depend on NADPH, which is produced in the HMS in these cells. The functional significance of up-regulated HMS within a particular cell type depends on the accompanying adaptive changes in ROS-metabolizing enzymes. In LPS-activated Kupffer cells, the elevated expression of glucose transporter GLUT1 and G6PD mainly serves primed production of superoxide anion, hydrogen peroxide, and nitric oxide. In sinusoidal endothelial cells, the LPS-induced response pattern of glucose- and ROS-metabolizing enzymes results in elevated ROS detoxifying capacity. The described studies also suggest the existence of an intercellular oxidant balance between prooxidant Kupffer cells and antioxidant endothelial cells in the hepatic micro-environment. Maintenance of the intercellular oxidant/antioxidant balance between phagocytes and endothelial cells may represent an important mechanism protecting the hepatic parenchyma against exogenous oxidative stress during the inflammatory response.
Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide modulate endotoxin-induced IL-6 production by murine peritoneal macrophages
Authors: Martínez Carmen , Delgado Mario , Pozo David , Leceta Javier , Calvo Juan R , Ganea Doina , Gomariz Rosa P
Published: January 2018

Vasoactive intestinal peptide (VIP) is a neuropeptide synthesized by immune cells that can modulate several immune aspects, including the function of cells involved in the inflammatory response, such as macrophages and monocytes. Production and release of cytokines by activated mononuclear phagocytes is an important event in the pathogenesis of ischemia-reperfusion injury. VIP has been shown to attenuate the deleterious consequences of this pathologic phenomenon. We have investigated the effects of VIP and PACAP38 on the production of interleukin-6 (IL-6), a proinflammatory cytokine, by endotoxin-activated murine macrophages. Both neuropeptides exhibit a dual effect on the IL-6 production by peritoneal macrophages. Whereas VIP and PACAP inhibit with similar dose-response curves the release of IL-6 from macrophages stimulated with a LPS dose range from 100 pg/mL to 10 μg/mL, both neuropeptides enhance IL-6 secretion in unstimulated macrophages and in macrophages stimulated with very low LPS concentrations (1-10 pg/mL). The inhibition on LPS-induced IL-6 production is specific, presumably mediated through a subtype of the PACAP-R. VIP and PACAP regulate the production of IL-6 at a transcriptional level. These results were correlated with an inhibition on both IL-6 expression and release in endotoxemic mice in vivo. These findings support the idea that in the absence of stimulation or in the presence of low doses of LPS, VIP and PACAP could play a role in immune system homeostasis. However, under toxicity conditions associated with high LPS doses, VIP and PACAP could act as protective mediators that regulate the excessive release of IL-6 in order to reduce inflammation or shock.