bluuebunny/crossref_metadata_2025 huggingface dataset:
GM-CSF-induced in vivo expansion of splenic dendritic cells and their strong costimulation activity
Authors: Hanada Ken-ichi , Tsunoda Rikiya , Hamada Hirofumi
Published: January 2018

Dendritic cells (DC), with their potent antigen-presenting and accessory activities, are involved in the stimulation of naive T cells. To examine the biological functions of DC, we developed a method for generating large numbers of murine splenic DC. First, DC were propagated in vivo by using a granulocyte-macrophage colony-stimulating factor-secreting tumor as a continuous in vivo source of the cytokine. The DC enriched in the spleen, especially in the white pulps, were purified after an overnight culture. We could reproducibly obtain 6 to 10 106 splenic DC per mouse. These DC were morphologically similar to interdigitating cells, expressed high levels of MHC class II and costimulatory molecules, and were highly allo-stimulatory in mixed lymphocyte reactions. Further analysis on T cell stimulation activity revealed that the DC had strong costimulatory activity on human T cells. Activated B cells, which express both B7-1 and B7-2, had little T cell costimulatory activity under the same assay conditions. A human histiocytic leukemia cell line, U937, that showed only weak costimulatory activity by itself, worked synergistically with DC and further intensified the T cell stimulation by DC. These findings suggest the presence of a T cell costimulation mechanism in DC, which is activated synergistically by monocytes or macrophages, and deserves further study.
Control of nitric oxide production by endogenous TGF-β1 and systemic nitric oxide in retinal pigment epithelial cells and peritoneal macrophages
Authors: Vodovotz Yoram , Letterio John J , Geiser Andrew G , Chester Louis , Roberts Anita B , Sparrow Janet
Published: January 2018

Both in vivo and in vitro experiments demonstrate that transforming growth factor-β1 (TGF-β1) suppresses expression of the inducible form of nitric oxide synthase (iNOS). In this study, we examined the effects of exogenous and endogenous TGF-β1 on retinal pigment epithelial (RPE) cells and resident peritoneal macrophages ex vivo using cells from TGF-β1 null (TGF-β1-/-) mice or age-matched wild-type (TGF-β1+/+) or heterozygous (TGF-β1+/-) littermates. RPE cells from both TGF-β1-/- mice and TGF-β1+/+ littermates produced NO and were immunocytochemically positive for iNOS protein only following treatment with interferon-γ (IFN-γ) and bacterial lipopolysaccharide (LPS); however, RPE cells from TGF-β1-/- mice produced 40% more NO than cells from TGF-β1+/+ mice. In contrast, resident peritoneal macrophages from both TGF-β1+/+ and TGF-β1-/- mice expressed iNOS protein without stimulation and in the absence of detectable production of NO. The expression of iNOS was increased by treatment with IFN-γ, resulting in detectable levels of NO. Macrophages from TGF-β1+/+ mice appeared to produce NO in a manner inversely proportional to the serum content of NO2- and NO3- of the mice from which the cells were obtained; no such correlation existed in TGF-β1+/- or TGF-β1-/- mice. Treatment of RPE cells or macrophages from both TGF-β1+/+ and TGF-β1-/- mice with exogenous TGF-β1 decreased both iNOS protein and NO production. These findings demonstrate a novel role of endogenous TGF-β1 in coupling systemic NO production to the production of NO by macrophages, and demonstrate that endogenous and exogenous TGF-β1 can act differently to suppress NO production.
Interaction of murine dendritic cells with collagen up-regulates allostimulatory capacity, surface expression of heat stable antigen, and release of cytokines
Authors: Mahnke Karsten , Bhardwaj Ranjit S , Luger Thomas A , Schwarz Thomas , Grabbe Stephan
Published: January 2018

In vivo, dendritic cells (DC) reside in direct proximity to extracellular matrix (ECM) proteins. Because ECM proteins affect morphology and function of a number of cell types, this study investigated potential effects of ECM proteins on functional properties of DC. DC were generated from murine bone marrow cultures, supplemented with granulocyte-macrophage colony-stimulating factor, and subsequently cultured on tissue culture plates coated with various ECM proteins. Among the ECM proteins tested, collagen (COL) up-regulated the T cell stimulatory capacity of DC. This effect was accompanied by sustained surface expression of the co-stimulatory molecule heat stable antigen on DC and by enhanced release of interleukin-1 and interleukin-6, respectively. Because fibronectin or solubilized COL were unable to cause similar changes in DC phenotype or function, we conclude that adherence to COL interferes specifically with DC function. These data suggest that ECM proteins may be involved in regulation of DC phenotype as well as in their functional activation.
Regulation of microglial activation by TGF-β, IL-10, and CSF-1
Authors: Lodge Patricia A , Sriram Subramaniam
Published: January 2018

Microglia are the resident macrophages of the brain and as such are active participants in immune responses in the central nervous system. Normal resting microglia express low levels of MHC class I and class II antigens and do not produce proinflammatory cytokines. However, microglial immune functions are induced in areas of infection or injury. To understand regulation of cytokines that are secreted by and act upon microglia, we examined production of interleukin (IL) -12, tumor necrosis factor-α (TNF-α), and nitric oxide (NO) by lipopolysaccharide (LPS)-stimulated microglia. We observed secretion of IL-12, TNF-α, and NO following stimulation of microglia with LPS. Addition of IL-10 suppressed TNF-α, IL-12, and NO production. Transforming growth factor-β (TGF-β) also inhibited TNF-α and NO but did not affect IL-12 secretion. IL-12 secretion became sensitive to TGF-β inhibition when microglia were cultured in the absence of CSF-1. In addition to its effect on the response to TGF-β, CSF-1 suppressed the response of microglia to LPS. These data suggest that CSF-1 may contribute to the immunologic ally privileged status of the central nervous system.
Rabbit vascular endothelial adhesion molecules: ELAM-1 is most elevated in acute inflammation, whereas VCAM-1 and ICAM-1 predominate in chronic inflammation
Authors: Abe Yasuharu , Sugisaki Katsunori , Dannenberg Arthur M
Published: January 2018

Activation of the microvasculature is a major component of the inflammatory response. During inflammation the vascular endothelium not only becomes more permeable to plasma proteins but also develops adhesion molecules that initiate the local immigration of leukocytes. We describe herein the in vivo changes in the three major vascular adhesion molecules during the development and healing of two types of rabbit dermal inflammatory lesions: (1) acute lesions produced in rabbits by the topical application of 1% sulfur mustard (SM, the military irritant/toxicant); and (2) chronic (immune-mediated) lesions produced in rabbits by intradermal injections of Mycobacterium bovis (BCG), the vaccine strain of tubercle bacillus. In each case, frozen tissue sections were made from lesions of various ages and stained immunohistochemically for von Willebrand (vW) factor to measure the total functional microvasculature. The sections were also stained immunohistochemically for the vascular endothelial adhesion molecules ICAM-1, ELAM-1 (E-selectin), and VCAM-1, and for the leukocyte ligands for ICAM-1: LFA-1 (CDlla/CD18) and Mac-1 (CD11b/CD18). Infiltrating monocytes and lymphocytes expressed the LFA-1 ligand and infiltrating PMN expressed the MAC-1 ligand. The area of stained microvasculature per square millimeter of tissue section was determined with the use of a computerized image analyzer. Edema and cell infiltration spread apart the microvessels, changing the number of microvessels per square millimeter of tissue section. Three methods of assessing such changes are presented. In SM lesions, endothelial ICAM levels were decreased from normal by about 50% at 1 and 2 days (when the lesions reached their peak size) and returned to normal at 3 and 6 days (during the healing process). ELAM rose in peak SM lesions and remained high during healing. VCAM levels, however, were only elevated in the 6-day (almost healed) lesions. In BCG lesions the levels of endothelial ICAM and VCAM (and to a lesser extent ELAM) were increased at 9 days and remained so as the size of the lesions peaked at 23 days. During the healing phase at 37 days, the elevated ICAM and VCAM levels decreased but the slightly increased ELAM levels persisted. These findings indicate that ELAM plays a major role in acute inflammation and that VCAM and ICAM play major roles in chronic inflammation. VCAM is known to be monocyte and lymphocyte selective.
Cytotoxicity of peritoneal murine macrophages against encapsulated pancreatic rat islets: in vivo and in vitro studies
Authors: Kessler Laurence , Jesser Cathy , Lombard Yves , Karsten Véronique , Belcourt Alain , Pinget Michel , Poindron Philippe
Published: January 2018

The aim of this work was to study in vivo and in vitro the involvement of macrophages and interleukin-1β (IL-lβ) in the necrosis of encapsulated islets during xenograft and to evaluate the immuno-protective efficiency of the AN69 membrane. In vivo, 6 days after implantation, 65% of the membrane surface of the devices containing the islets was colonized with macrophages compared with only 5% of the surface of the empty control devices. The morphological aspect of implanted islets was altered and their insulin release decreased significantly compared with freshly isolated ones (265 ± 50 vs. 507 ± 81 μU/mL). In vitro, the insulin release of encapsulated islets cultured for 2 days decreased to 32 and 28%, respectively, in the presence of IL-1β and macrophages. The addition of anti-IL-1β antibody to the co-culture of macrophages and islets did not modify this loss of functional activity. Furthermore, IL-1β passed through the AN69 membrane. In conclusion, macrophages are involved in damaging encapsulated pancreatic islets and are probably partly responsible for islet transplantation failure.
Selective induction of peripheral and mucosal endothelial cell addressins with peripheral lymph nodes and Peyer's patch cell-conditioned media
Authors: Denis Véronique , Dupuis Philippe , Bizouarne Nadine , Sampaio Silvia de O , Hong Li , Lebret Marilyne , Monsigny Michel , Nakache Maurice , Kieda Claudine
Published: January 2018

Vascular endothelial cell addressins play an important role in lymphocyte homing in secondary lymphoid organs and in chronic inflammatory areas. A SV40 large T antigen-immortalized cell line from peripheral lymph nodes, HECa10 [Bizouarne et al., 1993a], was used to characterize the location of addressing with regard to environmental factors and cytokines. For this purpose, two monoclonal antibodies, MECA 79 and MECA 367, specific for peripheral lymph node vascular addressin and for mucosal addressin (Peyer's patches), respectively, were bound to unstimulated HECa10 cells. Both mucosal and peripheral addressins were detected inside the cells and in cellular extracts of the resting cells. On the cell surface, both addressins could be evidenced on the same cells at a moderate level of expression. They partly mediate the EL4/EL4IL2 lymphoma cells9 adhesion to HECa10 cells. Supernatants of cultured peripheral lymph node or Peyers' patch cells induced expression of MECA 79 or MECA 367 antigens, respectively, on the surface of HECa10 cells. Interleukins, IL-7, IL-3, and IL-8, induced the cell-surface appearance of MECA 79 but not of MECA 367 antigen. Therefore, the same cell type synthesizes both antigens, but the expression of these antigens on the cell surface is independently regulated, thus uncovering a characteristic tissue type-specific as well as environment-sensitive properties of microvascular endothelial cells.
Inhibition and stimulation of LFA-1 and Mac-1 functions by antibodies against murine CD18. Evidence that the LFA-1 binding sites for ICAM-1, -2, and -3 are distinct
Authors: Driessens Mariëtte H E , van Hulten Paula , Zuurbier Astrid , La Rivière Geertje , Roos Ed
Published: January 2018

The murine CD18 monoclonal antibody (mAb) M18/2 was reported to inhibit lymphoma metastasis [Zahalka, M. A. et al. (1993) J. Immunol. 150, 4466]. To identify the pathways potentially blocked, we studied the effects of M18/2 compared with two new mAb against murine CD18, GAME-46, and -245. Whereas the GAME mAb blocked most Mac-l-mediated interactions, M18/2 had no effect, or even stimulated. The same was true for adhesion of LFA-1 to ICAM-1. To test effects on interactions with different ICAMs, we used L cells transfected with human IGAM-1, -2, and -3. As previously described, mouse LFA-1 does not bind to human ICAM-1 but we show here that mouse LFA-1 does bind to human ICAM-2 and -3. Again, the GAME mAb blocked completely, but M18/2 did not. These results indicate that the LFA-1 binding sites for ICAM-1 and ICAM-2 and-3, although in close vicinity, are distinct. Furthermore, effects of M18/2 on metastasis cannot be ascribed to blocking of any known β2-integrin activity.
Epinephrine attenuates down-regulation of monocyte tumor necrosis factor receptors during human endotoxemia
Authors: van der Poll Tom , Calvano Steve E , Kumar Ashwini , Coyle Susette M , Lowry Stephen F
Published: January 2018

Epinephrine inhibits lipopolysaccharide (LPS) -induced tumor necrosis factor (TNF) production by increasing intracellular cAMP concentrations. Because agents that increase cAMP levels can enhance TNF receptor expression in vitro, granulocyte and monocyte TNF receptors were determined by FACS analysis in 7 normal humans who were receiving a constant 24-h infusion of epinephrine (30 ng/kg/min), and in 15 normal subjects after intravenous injection of LPS (2 ng/kg), while they were receiving a continuous infusion of epinephrine started either 3 h (EPI-3) or 24 h (EPI-24) before LPS injection or an infusion of normal saline (LPS; n = 5 per group). Infusion of epinephrine per se did not influence TNF receptor expression. LPS induced a transient decrease in monocyte TNF receptors and a more sustained decrease in granulocyte TNF receptors (both P < 0.05). EPI-3 partly prevented LPS-induced down-modulation of monocyte TNF receptors (P < 0.05 vs. LPS only), but did not affect LPS-induced down-modulation of granulocyte TNF receptors. EPI-24 had no effect on TNF receptor expression. These data suggest that epinephrine not only influences the bioavailability of TNF by an effect on the production of this proinflammatory cytokine, but also by modulating the expression of its receptors.
CD18 adhesion blockade decreases bacterial clearance and neutrophil recruitment after intrapulmonary E. coli, but not after S. aureus
Authors: Ramamoorthy Chandra , Sasaki Steve S , Su Daniel L , Sharar Sam R , Harlan John M , Winn Robert K
Published: January 2018

Leukocyte emigration in the lung occurs by both CD18-dependent and -independent mechanisms that are stimulus specific. We examined the effect of CD18 blockade (mAb 60.3) on neutrophil (PMN) emigration into, and bacterial clearance from, the lung. After intravenous treatment with either mAb 60.3 or saline, rabbits were given an intralobar inoculation with 109 colony-forming units of either Staphylococcus aureus or Escherichia coli. Four hours after inoculation, lungs were lavaged to assess PMN emigration. CD18 blockade reduced PMN emigration to E. coli by 76% but only 45% to S. aureus. Experiments to determine bacterial recovery from the lungs at 4, 8, and 24 h after inoculation showed that CD18 blockade impaired the early (4 h) clearance of E. coli but not S. aureus. These findings suggest that PMN emigration to intrapulmonary S. aureus is largely CD18-independent. In contrast, intrapulmonary E. coli elicits CD18-mediated PMN emigration. CD18 blockade results in impaired clearance of E. coli but not S. aureus from the lung.
Differential phosphorylation of the β2 integrin CD11b/CD18 in the plasma and specific granule membranes of neutrophils
Authors: Buyon Jill P , Philips Mark R , Merrill Joan T , Slade Seth G , Leszczynska-Piziak Joanna , Abramson Steven B
Published: January 2018

Neutrophil aggregation is mediated by the β2 integrin CD11b/CD18, which has limited expression on the surface membrane of resting cells but is recruited from intracellular organelles after cell activation. We have previously found that CD11b/CD18 newly translocated to the plasma membrane does not contribute to adhesion but must be modified to be functional. Because neutrophil aggregation induced by phorbol myristate acetate (PMA) is accompanied by de novo phosphorylation of the CD18 cytoplasmic tail, we sought to determine whether CD11b/CD18 phosphorylation is separately regulated in the different cellular compartments. Accordingly, [32P]-labeled CD11b/CD18 was immunoprecipitated from purified neutrophil-specific granule or plasma membrane lysates. In plasma membrane fractions, as in whole cell lysates, CD18 became phosphorylated in cells exposed to PMA but not in untreated cells or cells treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The a chain, CD11h, was phosphorylated under all conditions. In contrast, only marginal phosphorylation of specific granule-associated CD18 or CD11b was observed. Calyculin A, an inhibitor of serine/threonine phosphatases (ppl > pp2a), induced strong phosphorylation of CD18 in the plasma membrane but not in the specific granules. Addition of intact specific granule membranes to the plasma membranes from PMA-treated neutrophils markedly decreased phosphorylation in both CD11b and CD18 subunits. These data suggest that the phosphorylation of CD11b/CD18, which accompanies neutrophil activation, is limited to plasma membrane-associated molecules. Phosphorylation, either constitutive or induced, is absent in the specific granule membranes. The difference may be due to a specific granule-associated phosphatase, probably distinct from ppl. Therefore adhesion-competent plasma membrane CD11b/CD18 and adhesion-incompetent specific granule CD11b/CD18 differ in their state of phosphorylation.
Undifferentiated U937 cells transfected with chemoattractant receptors: a model system to investigate chemotactic mechanisms and receptor structure/function relationships
Authors: Kew Richard R , Peng Tina , DiMartino Stephen J , Madhavan Deepa , Weinman Shana J , Cheng Daniel , Prossnitz Eric R
Published: January 2018

Transfection of either the C5a receptor or the formyl peptide receptor into undifferentiated U937 cells generated continuously growing cell lines that stably expressed these receptors. The transfected cells displayed significant numbers of cell surface receptors that had ligand binding properties similar to fully differentiated U937 cells. Undifferentiated transfected U937 cells were capable of a ligand-specific calcium flux and showed migratory responses that were qualitatively and quantitatively similar to differentiated cells and were specific for each chemoattractant. Moreover, the chemotactic response could be desensitized by preincubating the cells in a high concentration of ligand and could be blocked by pertussis toxin. These results demonstrate that undifferentiated U937 cells possess the subcellular signaling apparatus and machinery necessary to generate a motile response and that the only missing component for chemotaxis is expression of a chemoattractant receptor. In addition, the results demonstrate that undifferentiated U937 cells transfected with chemoattractant receptors provide a defined model system to study receptor structure/function relationships and may be used to investigate receptor-mediated chemotactic responses in a relevant human myeloid cell.
MCP-1-mediated chemotaxis requires activation of non-overlapping signal transduction pathways
Authors: Yen Hua-hsing , Zhang Yujun , Penfold Sonya , Rollins Barrett J
Published: January 2018

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes. Its receptor (CCR2) is a heptahelical G-protein-coupled receptor (GPCR) whose signal transduction pathways for chemotaxis have not been completely defined. Because other GPCRs stimulate the mitogen-activated protein kinase (MAPK) cascade, we examined this pathway's activity in response to MCP-1. MCP-1 induced rapid and transient activation of MAPK in human monocytes and in Chinese hamster ovary cells expressing CCR2B. This effect was largely insensitive to pertussis toxin and wortmannin, and was protein kinase C-dependent and protein tyrosine kinase-independent. PD 098059, an inhibitor of MEK activation, not only prevented MAPK activation but also inhibited MCP-1-induced chemotaxis. Because pertussis toxin and wortmannin also efficiently inhibit chemotaxis but do not completely inhibit MAPK activation, these data may define non-overlapping signal transduction pathways that all must be activated to produce chemokine-mediated chemotaxis.
Development of laboratory and animal model systems for HIV-1 encephalitis and its associated dementia
Authors: Persidsky Yuri , Gendelman Howard E
Published: January 2018

The neuropathogenesis of HIV-1 encephalitis and its associated dementia revolves around sustained viral replication in cells of mononuclear phagocyte origin (brain macrophages, multinucleated giant cells, and microglia). Macrophage secretory factors play important roles in facilitating monocyte trafficking into the brain, in regulating productive viral replication, and in producing neurotoxic responses. To study these events, we constructed an artificial blood-brain barrier (BBB) to assay monocyte transendothelial migration and developed an animal model system for HIV-1 encephalitis to ascertain the role that virus-infected mononuclear phagocytes play in disease pathogenesis. The BBB model was composed of brain microvascular endothelial cells and astrocytes placed on opposite sides of a porous membrane. Monocyte activation, not HIV-1 infection per se, was the central event affecting monocyte BBB migration. Many of the pathological features of HIV-1 encephalitis were reproduced in SCID mice stereotactically inoculated with virus-infected monocytes. These included widespread astrogliosis, apoptosis of neurons, dendritic damage, and macrophage/microglial activation. Such laboratory and animal model systems are being used to ascertain the pathogenic potential of virus-infected macrophages in brain and ways to curb such injurious effects.
Possible participation of polymorphonuclear cells stimulated by microbial immunomodulators in the dysregulated cytokine patterns of AIDS patients
Authors: Cassone Antonio , Chiani Paola , Quinti Isabella , Torosantucci A
Published: January 2018

Macrophages and polymorphonuclear cells (PMN) play a major role as cells primarily responsive to microbial biological response modifiers (BRM). Although much attention has been given to macrophages, PMN have been relatively underinvestigated. We have recently studied the responses of PMN from HIV– and HIV+ subjects after stimulation with a powerful immunomodulatory fraction from the cell wall of Candida albicans (MP-F2) and compared this to bacterial lipopolysaccharide (LPS). Both cytokine patterns and PMN anticandidal activity were investigated. MP-F2, like LPS, was an active inducer of interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), IL-6, and IL-1β production by PMN and monocytes from all subjects. IL-12 was also produced by MP-F2-stimulated PMN in the presence of interferon-γ (IFN-γ). PMN from HIV+ subjects showed increased in vitro expression of TNF-α and IL-6 genes as determined by semiquantitative reverse transcriptase-polymerase chain reaction. In all subjects, cytokine gene expression was strongly stimulated by MP-F2 or LPS and inhibited by IL-10. Production of IL-6 and TNF-α protein (measured by ELISA) was higher in PMN from HIV+ subjects in at least one of the conditions tested (unstimulated or stimulated by LPS or MP-F2). However, the amount of the C-X-C chemokine IL-8 was equal in PMN from HIV– and HIV+ subjects. PMN from HIV+ subjects were at least as active in inhibiting candide growth as PMN from HIV– controls. In both groups PMN were equally stimulated by MP-F2 and LPS. Only in severely neutropenic subjects was there some reduction in the anticandidal activity but not in cytokine responses. When appropriately stimulated by microbial BRM, PMN are active producers of proinflammatory and immunomodulatory cytokines. This production is not only totally preserved in HIV+ subjects but may be higher than in PMN from HIV– subjects and may be coupled with an efficient anticandida activity. We suggest that during common bacterial or fungal infections PMN may contribute to the dysregulated production of inflammatory cytokines in AIDS patients.
Immune stimulation and HIV-1 viral replication
Authors: Wahl Sharon M , Orenstein Jan M
Published: January 2018

A biphasic early and late viremia is characteristic of HIV-1 infection. The first increase in circulating viral burden occurs within weeks after infection, before a host immune response, and the second, later peak emerges during the inevitable HIV-1 devastation of immune function. Recently, intermittent bouts of viremia have also been identified in HIV-1-infected individuals and found to be associated with episodes of immune challenge. Vaccinations, exposure to antigens, and infections often induce reversible increases in circulating viral levels, dependent on CD4+ T lymphocyte numbers. However, even with marked losses in CD4+ T cell counts, opportunistic infections appear to trigger a viremic response. In searching for the source of this virus, macrophages in tissues co-infected with opportunistic pathogens have been identified as prodigious producers of HIV-1. Thus, the fountain from which HIV-1 emerges may shift from CD4+ T lymphocytes in early HIV-1 infection to tissue macrophages later in the natural evolution of the disease, as the CD4+ T cells are depleted. Defining the mechanisms of this transitional event in HIV-1 infection may facilitate regulation and therapeutic control of both opportunistic infections and HIV-1.
Dexamethasone inhibits leukocyte emigration in rat mesenteric post-capillary venules: an intravital microscopy study
Authors: Tailor Anitaben , Flower Roderick J , Perretti Mauro
Published: January 2018

The effect of subcutaneous administration of dexamethasone (DEX) on interleukin-lβ (IL-lβ, 20 ng i.p., – 2 h) and platelet-activating factor (PAF, 100 nM in superfusion) -induced leukocyte interaction with the endothelium of rat mesenteric post-capillary venules was studied. DEX produced a dose-dependent inhibition of IL-lβ-induced leukocyte extravasation in the rat mesenteric vascular bed, with a calculated ED50 of 40 μg/kg and a maximal effect of 80–100% inhibition at 0.1 mg/kg. IL-lβ-induced cell adhesion to post-capillary venules was only partially inhibited by the steroid, with a calculated ED50 of 480 μg/kg and a maximal effect of 40–60% inhibition. Furthermore, the steroid inhibited leukocyte emigration, but not adhesion, caused by superfusion of the mesenteric vascular bed with PAF. A doubling of leukocyte emigration time (from 226 to 552 s) was observed after treatment of rats with DEX. Administration for 5 days of a dose of 10 μg/kg DEX (which was inactive when given as a single injection) resulted again in a selective inhibition of IL-lβ-induced leukocyte emigration, without effect on cell adhesion. These data demonstrate a preferential susceptibility of the leukocyte emigration process to the inhibitory action of DEX.
Regulation of cytosolic free Ca2+ in cultured rat alveolar macrophages (NR8383)
Authors: Zhang Guo H , Helmke Ronald J , Mörk Ann-Christin , Martinez J Ricardo
Published: January 2018

Ca2+ mobilization in the rat alveolar macrophage cell line NR8383 was examined with the Ca2+-sensitive fluorescent probe Fura-2. ATP and norepinephrine elicited a 108 and 46% increase, respectively, in cytosolic free Ca2+ concentration ([Ca2+]i). Acetylcholine, nicotine, isoproterenol, substance P, and vasoactive intestinal polypeptide did not alter [Ca2+]i. Inositol 1,4,5-trisphosphate (IP3) formation was also activated by ATP. The carbohydraterich cell wall preparation, zymosan, induced a gradual [Ca2+]i increase only in the presence of external Ca2+, but did not activate IP3 formation. This increase was abolished by laminarin and by removal of extracellular Ca2+, suggesting that the [Ca2+]i increase was activated by β-glucan receptors and mediated by Ca2+ influx. This influx was significantly reduced by SKF96365, but not by nifedipine, ω-conotoxin GVIA, ω-agatoxin IVA, or flunarizine. These results suggest that release of intracellular Ca2+ in NR8383 cells is regulated by P2-purinoceptors and that zymosan causes Ca2+ influx via a receptor-operated pathway.
Eosinophils inhibit retroviral transduction of human target cells by a ribonuclease-dependent mechanism
Authors: Domachowske Joseph B , Rosenberg Helene F
Published: January 2018

Human eosinophils contain a number of granule proteins for which specific physiological roles remain unclear. The combined ribonucleolytic and membrane disruptive properties of the eosinophil-derived neurotoxin and eosinophil cationic protein, respectively, suggest the possibility that eosinophils might participate in host defense against enveloped single-stranded RNA viruses. To test this hypothesis, stocks of a replication-defective retrovirus encoding the reporter gene β-galactosidase were pretreated with isolated human eosinophils, then used to transduce human erythroleukemia (K-562) target cells. Histochemical staining for β-galactosidase activity was used to detect and quantitate the transduced cells. Co-incubation of retrovirus with eosinophils (0.4 X 106/mL) before target cell transduction resulted in a marked decrease in transduction efficiency corresponding to an ~ 20-fold dilution of viral stock (P < 0.01), an effect that was directly proportional to the concentration of eosinophils, and that was reversed in the presence of ribonuclease inhibitor. Reverse transcriptase-polymerase chain reaction analysis demonstrated loss of the retroviral RNA genome as a result of eosinophil pretreatment, indicating that eosinophils are capable of mediating direct ribonucleolytic destruction of the isolated retroviral particles. Our results demonstrate that eosinophils function as effective anti-retroviral agents in vitro via the actions of their secreted ribonucleases, and suggest that eosinophils may represent an unrecognized arm of host defense against enveloped single-stranded RNA viral pathogens.
The Ly-49 family: regulation of cytokine production in murine NK cells
Authors: Ortaldo J R , Mason L H , Gregorio T A , Stoll J , Winkler-Pickett R T
Published: January 2018

Most proteins encoded by members of the Ly-49 gene family are class I-recognizing receptors on murine natural killer (NK) cells. Class I recognition by Ly-49 receptors usually results in inhibition of NK cell lysis of target cells. However, NK cells function not only in a lytic capacity, but also can mediate cytokine production. In this report we have demonstrated the ability of Ly-49A and Ly-49G2 to inhibit production of cytokines by NK cells by showing that specific antibodies against these gene products stimulate cytokine production. Murine NK cells were cultured in the presence of P815 (H2-Dd), and supernatants were analyzed for the production of interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF). NK cell populations were sorted for Ly-49A+ or Ly-49G+ subsets, and these subsets were analyzed for their ability to alter cytokine induction by target cell interaction. In the presence of target cells expressing the appropriate class I molecules, Ly-49A and G2 were found to inhibit cytokine induction by NK cells. Examination of mRNA for IFN-γ and GM-CSF indicated that Ly-49 receptors increased mRNA levels of NK cells. These results demonstrate that class I binding of these NK receptors can inhibit production of important physiological cytokines, in addition to the regulation of cytotoxic activity.
Effects of dexamethasone treatment on IL-1 receptor mRNA levels in vivo
Authors: Yu Pei-Wen , Schuler L A , Kehrli M , Mattocks L , Nonnecke B J , Czuprynski C J
Published: January 2018

Using semiquantitative reverse transcriptase-polymerase chain reaction and Northern analysis, we observed in vivo up-regulation of interleukin-1 (IL-1) RI and IL-1RII mRNA levels in peripheral blood mononuclear cells (PBMCs) and neutrophils (PMNs) from Holstein cattle injected with dexamethasone (0.04 mg/kg). Baseline levels of IL-1RI mRNA were greater than IL-1RII mRNA levels in PBMCs and PMNs before dexamethasone treatment. This is in contrast with the previously reported predominance of IL-1RII in unstimulated human PMNs. IL-1RII mRNA was strongly induced in both bovine PBMCs and PMNs at 24 h and returned to baseline levels by 72 h, after dexamethasone injection. Conversely, the greatest increase in IL-1RI mRNA in PBMCs and PMNs was not detected until 72 h after dexamethasone injection. These data provide evidence for sequential in vivo up-regulation of first IL-IRII mRNA and later IL-1RI mRNA by dexamethasone that is consistent with the anti-inflammatory activity of glucocorticoids.
Effector molecules In expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids
Authors: Akaki Tatsuya , Sato Katsumasa , Shimizu Toshiaki , Sano Chiaki , Kajitani Hiroko , Dekio Satoshi , Tomioka Haruaki
Published: January 2018

We studied microbicidal activities of reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) against Mycobacterium aviumcomplex (MAC) and the mode of macrophage (mΦ) production of these effectors. (1) Intracellular growth of MAC in murine peritoneal mΦs was accelerated by scavengers for ROI or RNI and inhibitors of nitric oxide synthase or phospholipase A2, indicating roles of ROI, RNI, and FFA in mΦ anti-MAC functions. (2) Acidified NaNO2-derived RNI, FFA (linolenic and arachidonic acids), and the H2O2-mediated halogenation system exhibited a significant anti-MAC bactericidal activity. The combination of RNI with FFA showed a synergistic effect However, the H2O2-halogenation system in combination with either RNI or FFA showed an antagonism. When Listeria monocytogenes(Lm) was used as a target organism, the combinations of RNI + FFA and RNI + H2O2-halogenation gave a synergistic effect, whereas FFA + H2O2-halogenation showed an antagonism in exerting bactericidal activity. In addition, when ROI generated by the xanthine oxidase-acetaldehyde system was combined with RNI, anti-Lm but not anti-MAC activity was potentiated. (3) ROI production by murine peritoneal mΦs was observed immediately after contact with MAC organisms (MAC stimulation) and ceased within 2 h. FFA release was seen 1-24 h after MAC stimulation. RNI production was initiated from 3 h and increased during the first 36 h and continued at least for 4 days. These findings suggest that RNI and FFA rather than ROI are important effectors of anti-MAC functions of mΦs, and the collaborating action of RNI with FFA temporarily participates in mΦ-mediated killing of MAC in the relatively early phase after MAC stimulation.
Bacterial LPS and IFN-γ trigger the tyrosine phosphorylation of vav in macrophages: evidence for involvement of the hck tyrosine kinase
Authors: English B Keith , Orlicek Shari L , Mei Zhuyong , Meals Elizabeth A
Published: January 2018

We and others have previously reported that tyrosine kinases play key roles in the activation of macrophages by both bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ). However, little is known regarding the substrates of tyrosine phosphorylation that mediate macrophage activation and the resultant production of inflammatory mediators. In lymphocytes and other hematopoietic lineages, tyrosine phosphorylation of the proto-oncogene vavappears to be an essential component of cell activation. In this study, we demonstrate that both LPS and rIFN-γ trigger the prompt, dose-dependent tyrosine phosphorylation of vavin murine RAW 264.7 macrophages. In addition, vavis physically associated with the src-related kinase hckin murine macrophages, and antisense oligonucleotides specific for murine hckblock both LPS and rIFN-γ-mediated vavphosphorylation. These findings suggest that hckprobably mediates vavtyrosine phosphorylation during macrophage activation and that LPS and rIFN-γ-mediated signaling pathways partially overlap.
The cytoplasmic domains of complement regulatory protein CD46 interact with multiple kinases in macrophages
Authors: Wong Timothy C , Yant Stephen , Harder Brandon J , Korte-Sarfaty Jennifer , Hirano Akiko
Published: January 2018

Membrane cofactor protein (CD46), which normally protects autologous cells from complement lysis, is the human cell receptor for measles virus (MV). Interaction between MV and CD46 on monocytes can lead to suppression of monocyte activation. We have investigated the interaction between the cytoplasmic sequences of CD46 and kinases in a mouse macrophage cell line. Glutathione-S-transferase (GST) fusion proteins bearing the Cyt1 or Cyt2 alternative cytoplasmic domain of CD46 associate with macrophage kinase activity, which phosphorylates multiple proteins co-purified with the GST fusion proteins. Association with the macrophage kinase activity correlates with tyrosine phosphorylation of the CD46 cytoplasmic domains. Removing the CD46 sequences or introducing a frame-shift mutation abrogates the association with macrophage kinase activity. Renaturation studies reveal multiple kinases with apparent molecular mass of 82, 79, 58, and 50/49 kDa, which associate specifically with both CD46 cytoplasmic domains. Alanine substitutions at a juxtamembrane Tyr-X-X-Leu motif in the Cyt1 domain completely abrogate the association with macrophage kinases and tyrosine phosphorylation of Cyt1; but similar substitutions in the Cyt2 domain only partially reduce the association with kinases and tyrosine phosphorylation of Cyt2. These results reveal a specific interaction between complement regulatory protein CD46 and macrophage kinases. These findings may provide an important clue for understanding immune modulation by MV.
Selenium and vitamin E deficiency impair transferrin receptor internalization but not IL-2, IL-2 receptor, or transferrin receptor expression
Authors: Pighetti Gina M , Eskew Mary L , Reddy C Channa , Sordillo Lorraine M
Published: January 2018

Vitamin E and Se deficiency increase the risk of disease by impairing the immune response. To aid in the understanding of how vitamin E and Se deficiency reduce immune competence, this study examined several mechanisms necessary for lymphocyte proliferation. Weanling rats were fed a vitamin E-deficient, selenium-deficient, or control diet for 8 weeks. At this time splenic mononuclear cells were isolated and stimulated with concanavalin A for 48 h. Although the percentage of lymphocytes and monocytes capable of proliferating were consistent among the dietary groups, lymphocyte proliferation was decreased significantly in vitamin E- and selenium-deficient rats. This decrease in proliferation was not associated with alterations in interleukin-2, interleukin-2 receptor, or transferrin receptor expression. However, stimulated cells from vitamin E- and Sedeficient rats internalized few if any transferrin receptors. Reduced transferrin receptor internalization may limit lymphocyte expansion by depleting the intracellular iron stores needed for cellular function and proliferation.
Myeloid-specific gene expression
Authors: Clarke Sandra , Gordon Siamon
Published: January 2018

The mononuclear phagocytes are recruited from bone marrow precursors to most tissues of the body, particularly during inflammation or immune stimulation. This combination of accessibility as stem cells and heterogenity of tissue locations makes the myeloid cell potentially important as a carrier of therapeutic agents. Understanding the regulation of transcription in myeloid cells is necessary for any future design of tissue-specific gene targeting vectors, particularly if there are inherent size limitations. Identified members of the C/EBP, Runt/PEBP2/CBF, and Ets families of transcription factors are critical for myeloid-specific gene expression and may have myeloid-restricted expression or myeloid-specific regulation in the hematopoietic system. AP-1, Sp1, and Myb appear to be important for myeloid-restricted expression in some cases. In addition, factors involved in the up-regulation of the level of gene expression when macrophages are activated by agents such as interferon-γ and bacterial products have been identified. Some of the sequences to which these transcription factors bind in myeloid-restricted genes have been tested in cell lines and in transgenic mice and it is now possible to make an attempt to describe the characteristics of a myeloid-specific promoter.
CD10 inhibitors increase f-Met-Leu-Phe-induced neutrophil transmigration
Authors: Hofman Paul , Selva Eric , Le Negrate Gaëlle , d'Andrea Lisanne , Guérin Sandrine , Rossi Bernard , Auberger Patrick
Published: January 2018

A variety of bacterial enterocolitis in their active stages are characterized by the migration of polymorphonuclear leukocytes (PMNs) across epithelial surfaces. These mechanisms could explain some effects of enterotoxins observed in the intestinal mucosae. Here, using specific inhibitors, we investigated the potential role of CD10 (E.C. 3.4.24.11), present at the surface of human neutrophils, on formyl-Met-Leu-Phe (fMLP)-induced PMN migration across cultured monolayers of the human intestinal cell line T84. Transmigration of human neutrophils across T84 epithelial cells was observed for concentrations of fMLP as low as 10–9 M, whereas maximal effect was achieved at 10–7 M as determined by transepithelial resistances and PMN myeloperoxidase assays. RB25, a CD10 inhibitor, reduced by two orders of magnitude the concentration of fMLP required to obtain full neutrophil transmigration across T84 epithelial cell line. RB25 response was concentration dependent with half-maximal and maximal effect occurring at 10–9 and 10–7 M, respectively. These concentrations of RB25 corresponded exactly to the half-maximal and maximal inhibition of endopeptidase 24.11 at the neutrophil cell surface. However, the effect of CD10 inhibitors on PMN transmigration cannot be accounted for by a direct action on T84 epithelial cells, since these cells fail to express any detectable endopeptidase 24.11 activity. Moreover, blocking of CD10 enzymatic activity by various and selective inhibitors potentiated the effect of low concentrations of fMLP on PMN transmigration. Finally, RB25 failed to affect interleukin-8 (IL-8)-induced PMN transmigration across T84 epithelial cells, in agreement with the preference of CD10 for small peptidic substrates. Taken together, these results demonstrate that inhibition of CD10 significantly reduced the concentration of fMLP needed for eliciting transmigration of PMN across intestinal epithelia.
IL-8 is an essential mediator of the increased delayed-phase vascular permeability in LPS-induced rabbit pleurisy
Authors: Fukumoto Takumi , Matsukawa Akihiro , Yoshimura Teizo , Edamitsu Sunao , Ohkawara Susumu , Takagi Katsumasa , Yoshinaga Masaru
Published: January 2018

We investigated the involvement of IL-8 in the delayed vascular permeability (VP) in rabbit lipopolysaccharide (LPS)-pleurisy. Maximal level of interleukin-8 (IL-8) was detected in pleural fluid at 2 h after LPS injection and anti-IL-8 inhibited the delayed VP by 90%. Injection of homologous IL-8 induced VP, the time-course of which preceded that of LPS-induced delayed VP. Production of IL-8 in LPS-pleurisy was inhibited with anti-tumor necrosis factor α (TNF-α), whereas the production of TNF-α was not affected with anti-IL-8. Injection of IL-8 did not induce TNF-α production and anti-TNF-α had no effect on IL-8-induced VP. Injection of homologous TNF-α induced IL-8 production and VP, and TNF-α-induced delayed VP was blocked with anti-IL-8. These results indicate important roles of IL-8 in LPS-induced delayed VP and that TNF-α causes the delayed VP through the production of IL-8.
Leukocytes of the intestinal muscularis: their phenotype and isolation
Authors: Kalff Jörg C , Schwarz Nicolas T , Walgenbach Klaus J , Schraut Wolfgang H , Bauer Anthony J
Published: January 2018

The basal presence of immunologically potent cells within the intestinal muscularis externa and their functional significance is unclear. Our aim was to investigate the basal distribution of various leukocyte populations within the rat jejunal muscularis. In addition, we sought to immunohistochemically phenotype the muscularis macrophage in jejunal whole-mounts, isolate these cells in primary culture, and investigate their ontogenesis. Macrophages form a regularly distributed network that expresses major histocompatibility complex class II, CD14 receptors, and a low level of CD11/ CD18. The macrophages are activated by dissection and are present in fetal animals. Enriched macrophage cultures show a normal resident phenotype and remain present for weeks in dissociated muscularis cultures. The results also demonstrate the presence of neutrophils, monocytes, mast cells, and lymphocytes within the muscularis and suggest that the dense network of muscularis macrophages may be a potent resident trigger for inflammation in response to tissue injury or bacterial translocation.
Activation of cytokine production, tumoricidal properties, and tyrosine phosphorylation of MAPKs in human monocytes by a new synthetic lipopeptide, JBT3002
Authors: Dong Zhongyun , Killion Jerald J , Kumar Rakesh , Eue Ines , Yang Xiulan , Lu Weixin , Su Bing , Fidler Isaiah J
Published: January 2018

We investigated the expression of cytokine genes and tumoricidal properties in human blood monocytes in response to a new synthetic immunomodulating lipopeptide, JBT3002. Incubation of peripheral blood monocytes with free-form JBT3002 or JBT3002 encapsulated in multilamellar phospholipid vesicles (liposomes, MLV-JBT3002) induced tumoricidal properties in a dose-dependent manner. Both MLV-JBT3002 and free-form JBT3002 induced production of tumor necrosis factor α, interleukin-1β, and interleukin-6 in a dose-dependent manner with similar kinetics. Treatment of monocytes with interferon-γ did not significantly alter the expression of cytokine genes but increased the expression of cytokines induced by MLV-JBT3002 and free-form JBT3002. In contrast to monocyte activation by lipopolysaccharide (LPS), activation by JBT3002 was independent of serum and was not inhibited by CD14-neutralizing antibody. Incubation of monocytes with JBT3002 induced a rapid increase in tyrosine phosphorylation of proteins with apparent molecular masses of 42 and 38 kDa, a migration band shift of c-Jun NH2-terminal kinase 1 (JNK1), and activation of extracellular signaling regulated kinases. Consistent with its effect on cytokine expression, stimulation of these intracellular signaling pathways by JBT3002 was not inhibited in serum-free conditions. Collectively, the data indicate that the synthetic lipopeptide JBT3002 is a potent monocyte activator that modulates monocyte function by mechanisms similar to LPS but by a distinct receptor.