bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of calcium on phenothiazine inhibition of neutrophil degranulation
Authors: Blackwood R Alexander , Hessler Ronald J
Published: January 2018

The phenothiazines are known to be potent inhibitors of calmodulin and have been used as probes for examining calmodulin-dependent cellular functions. We report here that the characteristics of phenothiazine inhibition of exocytosis in neutrophils more closely resemble their interaction with the annexins in vitro. Ca2+- dependent aggregation of liposomes mediated by either annexin I or annexin II was inhibited by the phenothiazines. Inhibition of liposome aggregation was not caused by interference with the binding of annexins to phospholipids. Rather, the phenothiazines increased the concentration of Ca2+ required for aggregation. Likewise, in neutrophils pepneabilized with streptolysin O, inhibition of degranulation by phenothiazines could be overcome by increasing [Ca2+]. These results suggest that inhibition by phenothiazines of neutrophil degranulation is secondary to the ability of these compounds to inhibit membrane-membrane contact promoted by the annexins. J. Leukoc. Biol. 58: 114–118; 1995.
Role of macrophage–derived nitric oxide in suppression of lymphocyte proliferation during blood–stage malaria
Authors: Ahvazi Behrouz C , Jacobs Philippe , Stevenson Mary M
Published: January 2018

Examination of the proliferative responses in vitro to mitogens (concanavalin A, phytohemagglutinin, lipopolysaccharide) of spleen cells recovered from C57B1/6 mice during blood-stage Plasmodium chabaudi AS infection revealed that the most severe suppression occurred during the first 14 days post infection, that is, during the acute phase of infection. Coincidently, inducible nitric oxide synthase gene expression was found to be up-regulated in the spleens of infected mice, and both splenic and peritoneal macrophages produced high levels of NO in vitro in response to stimulation with lipopolysaccharide (LPS). The roles of NO, a molecule recently found to mediate immunosuppression during parasitic infections, and of the well-recognized immunosuppressive molecule prostaglandin were, therefore, investigated in the suppression of proliferation to mitogens and specific antigen of spleen cells from 7- and 14-day P. chabaudi AS–infected mice. Addition of either 0.5 mM NG-monomethyl-l-arginine (L-NMMA) or 0.5 mM aminoguanidine (AG), inhibitors of NO synthase, or 10 μg/ml indomethacin (INDO), a prostaglandin inhibitor, partially but significantly abrogated the suppression in response to concanavalin A (Con A) and phytohemagglutinin (PHA). Only the addition of INDO significantly increased the responses to LPS. Addition of L-NMMA or AG in combination with INDO partially but significantly abrogated the suppression in response to Con A and completely abrogated the suppression in response to PHA. The addition of L-NMMA or AG also significantly increased proliferation in response to parasite antigen. The contribution of NO to suppression of lymphoproliferation was confirmed by adding 3-morpholino-sydnonimine-hydrochloride (SIN-1), a chemical generator of NO, to mitogen-stimulated splenocyte cultures prepared from normal mice. The mechanism of NO-mediated suppression was investigated in coculture experiments using spleen cells from normal mice and peritoneal macrophages from either normal or day 7 infected mice. The addition of 5-10 104 peritoneal macrophages from infected mice significantly and consistently suppressed Con A– or PHA-stimulated proliferation of normal splenocytes. Moreover, suppression correlated with production of NO and could be reversed by the addition of L-NMMA or AG. These results suggest that, in addition to prostaglandin, increased NO production by macrophages within the first 2 weeks after infection with P. chabaudi AS contributes to immunosuppression associated with blood-stage malaria. J. Leukoc. Biol. 58: 23–31; 1995.
CD66-dependent neutrophil activation: a possible mechanism for vascular selectin-mediated regulation of neutrophil adhesion
Authors: Stocks S Craig , Kerr Michael A , Haslett Christopher , Dransfield Ian
Published: January 2018

We have examined the role of CD66 in the modulation of neutrophil adhesion and effector function. Engagement of neutrophil CD66 with anti-carcinoembryonic antigen (anti-CEA) Ig results in activation-associated phenomena including shape change, activation of β2-integrins, and priming of the respiratory burst. Anti-CEA Ig-treated neutrophils underwent transient shape change distinct from that induced by formyl-Met-Leu-Phe (fMLP). fMLP stimulated β2-integrin up-regulation and 70% loss of L-selectin, whereas only low-level up-regulation of the β2-integrins, without loss of L-selectin, occurred with anti-CEA Ig. Anti-CEA F(ab’)2 fragments and whole Ig augmented β2-integrin-dependent adhesion. Anti-CEA Ig-induced β2-integrin activation was transient, whereas fMLP-induced activation persisted longer. Although they did not cause a significant increase in respiratory burst activity, CEA Ig and F(ab’)2 fragments of antibody primed neutrophils so that the subsequent fMLP-induced respiratory burst was significantly increased. J. Leukoc. Biol. 58: 40–48; 1995.
Defensins and other endogenous peptide antibiotics of vertebrates
Authors: Martin Edith , Ganz Tomas , Lehrer Robert I
Published: January 2018

Gene-encoded peptide antibiotics are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. Their de novo synthesis or release from storage sites can be induced rapidly, which makes them particularly important in the initial phases of resistance to microbial invasion. The endogenous antimicrobial peptides of animals are products of single genes and are synthesized as preproproteins. Multistep processing yields the mature peptide, which generally acts by inducing microbial membrane permeabilization. Several families of antimicrobial peptides have been identified that differ with respect to the presence of disulfide linkages, amino acid composition, structural conformation, and spectrum of activity. The arginine-rich three disulfide-containing β-βheet defensins are remarkably abundant and widely distributed in animals and plants. The antibiotic peptides of higher eukaryotes merit further study for their role in natural immunity and their potential as novel therapeutic compounds. J. Leukoc. Biol. 58: 128–136; 1995.
Associations between the neuroendocrine and immune systems
Authors: Weigent Douglas A , Blalock J Edwin
Published: January 2018

Organisms respond to infection with complex adaptations involving bidirectional communication between the immune and neuroendocrine systems. The idea of intercellular communication between the neuroendocrine and immune systems via common signal molecules has provided a conceptual framework for such crosstalk. The studies to date show that cells of the immune system contain receptors for neuroendocrine hormones and can also be considered a source of pituitary and hypothalamic peptides. The structure and pattern of synthesis of these peptides by leukocytes appear similar to neuroendocrine hormones, although some differences exist. Once secreted, these peptide hormones may function as endogenous regulators of the immune system as well as conveyors of information from the immune to the neuroendocrine system. The plasma hormone concentrations contributed by lymphocytes usually do not reach the levels required when the pituitary gland is the source, but because immune cells are mobile, they have the potential to locally deposit the hormone at the target site. Likewise, other studies show that cells of the neuroendocrine system contain receptors for cytokines and can also be considered a source of cytokines, particularly interleukin-1 (IL-1) and IL-6. In the pituitary IL-1β coexists with thyroid stimulating hormone in a subpopulation of thyrotropes, suggesting it may have a role as a pituitary paracrine factor. The cytokines, including IL-1, IL-2, EL-6, interferon-γ, and tumor necrosis factor, exert profound effects on hypothalamic pituitary axes. It is our hypothesis that the relay of information to the neuroendocrine system represents a sensory function for the immune system wherein leukocytes recognize stimuli that are not recognizable by the central and peripheral nervous systems (i.e., bacteria, tumors, viruses, and antigens). The recognition of such noncognitive stimuli by immunocytes is then converted into information and a physiological change occurs. Future studies into the physiological role that cytokines and neuroendocrine hormones have in these systems will be of considerable interest for both immunologists and endocrinologists. J. Leukoc. Biol. 57: 137–150; 1995.
Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM-1) up-regulation and IL-6 production and proliferation
Authors: Yellin Michael J , Winikoff Stephen , Fortune Sarah M , Baum David , Crow Mary K , Lederman Seth , Chess Leonard
Published: January 2018

CD40 was originally described as a functionally significant B cell surface molecule. However, CD40 is also expressed on monocytes, dendritic cells, epithelial cells, and basophils. We now report that synovial membrane (SM) or dermal fibroblasts also express cell surface CD40 in vitro. Fibroblast CD40 expression declines with increasing time in culture and recombinant interferon-γ (rINF-γ) induces fibroblast CD40 up-regulation. This effect of rINF-γ is augmented by recombinant interleukinlα or recombinant tumor necrosis factor-α. CD40 expression on fibroblasts is functionally significant because CD40L-CD40 interactions induce SM fibroblast CD54 (intercellular adhesion molecule-1) and CD106 (vascular cell adhesion molecule-1) up-regulation. Moreover, ligation of CD40 augments IL-6 production by SM fibroblasts and induces fibroblasts to proliferate. In addition, rINF-γ enhances the effect of CD40L-CD40 interactions on fibroblast proliferation. Taken together, these studies show that fibroblasts can express CD40, cytokines can regulate fibroblast CD40 expression, and CD40 ligation induces fibroblast activation and proliferation. J. Leukoc. Biol. 58: 209–216; 1995.
Supernatants from UVB radiation-exposed keratinocytes inhibit Langerhans cell presentation of tumor-associated antigens via IL-10 content
Authors: Beissert Stefan , Ullrich Stephen E , Hosoi Junichi , Granstein Richard D
Published: January 2018

Exposure of mice to midrange UV radiation (UVB) (280–320 mm) in vivo leads to suppression of the ability to induce delayed-type hypersensitivity (DTH). Systemic administration of supernatants from UVB-exposed keratinocytes (KC) similarly inhibits the ability to induce DTH and the presence of interleukin-10 (IL-10) in the supernatants has been shown to be responsible for this effect. It has been hypothesized that release of IL-10 by KC after exposure to UVB radiation in vivo may be responsible for UVB-induced inhibition of DTH and alsp for the inability of chronically UVB-irradiated mice to immunologically reject immunogenic UVB-induced skin tumors. To test directly whether supernatants from UVB-irradiated KC can inhibit presentation of tumor-associated antigens (TAA) by epidermal Langerhans cells (LC), cultures of the transformed murine KC line PAM 212 were exposed to 200 J/m2 of UVB radiation and 24 h supernatants obtained. CAF1 (H-2a/d) epidermal cells (EC) enriched for LC content were exposed to supernatants from irradiated (UV-SN) or mock-irradiated (MISN) PAM 212 cells for 3 h followed by culture for 16 h in granulocyte-macrophage colony-stimulating factor and then were pulsed with soluble TAA derived from the murine spindle cell tumor S1509a (H-2a). ECs were then washed and injected subcutaneously into naive CAF1 mice three times at weekly intervals for priming. One week after the final immunization these mice were challenged subcutaneously with live S1509a cells and tumor growth scored over time. Pretreatment of EC with UV-SN but not MI-SN inhibited the induction of effective immunity by this immunization scheme. ECs were also treated with UV-SN or MI-SN for 3 h then pulsed with TAA and injected into a hind footpad of previously immunized mice for elicitation of a DTH response. Pretreatment of EC with UV-SN but not MI-SN inhibited the ability of EC to elicit DTH. Neutralization studies with specific neutralizing antibodies to IL-10 demonstrated that the presence of IL-10 in UV-SN was responsible for the inhibition of antigen presentation both for induction and elicitation of immunity. UV-SN inhibits tumor antigen presentation by epidermal LC through the action of IL-10. J. Leukoc. Biol. 58: 234–240; 1995.
Purification and characterization of the cytotoxic factor in rat peritoneal exudate cells: its identification as the calcium binding protein complex, calprotectin
Authors: Yui Satoru , Mikami Masaaki , Yamazaki Masatoshi
Published: January 2018

We previously reported the existence of a growth inhibitory factor for mitogen-stimulated lymphocytes and murine tumor cell lines, MM46 and L-929, in inflammatory polymorphonuclear leukocytes. In this study, by using mouse MM46 mammary carcinoma as target, we purified the inhibitor from lysate of rat inflammatory peritoneal exudate cells by ammonium sulfate precipitation, gel filtration, isoelectrofocusing, and anion exchange chromatography. Although the in vitro inhibitory activity for MM46 growth was partitioned into three peaks in the final step, it was found that these inhibitory samples all consist of 8- and 13-kDa peptides. Analysis of amino acid sequences revealed that the partial sequences of the 8- and 13-kDa peptides completely agree with the smaller and larger components of rat calprotectin, which are predicted from cDNA, respectively, suggesting the cell growth inhibitory factor is calprotectin. In addition to MM46, the partially purified calprotectin inhibited the growth of a rat, three mice, and a human tumor cell line in similar dose-response relationships in vitro. Moreover, it exerted a cytolytic effect against all examined tumor cells. It was confirmed that the purified calprotectin induces growth inhibition and the lysis of MM46 cells and that the minimum effective concentration is between 50 and 100 μg/ml. The factor also inhibited the growth of bone marrow cells and macrophages. These results suggest that calprotectin is a negative regulatory factor for the growth and/or survival states of normal and tumor cells.
Identification of an endotoxin and IFN-inducible cDNA: possible identification of a novel protein family
Authors: Sorace James M , Johnson Robert J , Howard Deborah L , Drysdale Beth E
Published: January 2018

Abstract The response of macrophages to agents such as lipopolysaccharide (LPS) and interferon (IFN) includes the transcriptional activation of numerous genes. We have used the method of differential screening of a RAW 264.7 macrophage cell line cDNA library to isolate and characterize LPS-induced messages. One such message, LRG-47, is induced by LPS, IFN-γ, and IFN-α/β, but not by a panel of other cytokines or pharmacological activating agents. LRG-47 is homologous to two other IFN-γ-induced genes, IRG-47 and Mg21. The LRG-47 sequence is approximately 33% identical and 52% similar to both these putative protein products. All three putative proteins, particularly Mg21, bear homology to a T cell product, Tgtp, induced by T cell receptor cross-linking. The three macrophage-derived proteins share areas of homology with GTP-binding proteins, are approximately 415 amino acids in length, and have similar kinetics of induction by IFN-γ. This suggests that these genes may be members of a new family of IFN-inducible proteins.
Relationship between light scattering in flow cytometry and changes in shape, volume, and actin polymerization in human polymorphonuclear leukocytes
Authors: Keller H U , Fedier A , Rohner R
Published: January 2018

Using two different cytometers, an Epics Profile II and a FACScan, we determined the extent to which changes in forward and right angle scatter are a reliable measure for changes in polymorphonuclear leukocyte (PMN) shape, volume, and actin polymerization and whether distinct types of shape changes in PMNs can be recognized. PMN stimulation can substantially change the positions of PMNs in the scatter diagram of the FACScan but not of the Epics Profile II. Within the limits of the experiments, it has been possible to determine whether or not a shape change has taken place using the FACScan but not using the Epics Profile II. However, using either cytometer, it has not been possible to determine which type of shape change (e.g., spherical vs. polarized vs. nonpolar cells) has taken place. Furthermore, forward or right angle scatter changes are not a reliable measure for changes in cell volume or actin polymerization of human PMNs.
Leukocyte function-associated antigen 1 (LFA-1) and CD44 are signalling molecules for cytoskeleton-dependent morphological changes in activated T cells
Authors: Kelleher Dermot , Murphy Anne , Feighery Conleth , Casey Eoin B
Published: January 2018

Signaling through the leukocyte function–associated antigen 1 (LFA-1) molecule has previously been shown to induce homotypic aggregation in T cells and to induce cytoskeletal changes in T lymphoma cells. In this study we describe the induction of a dendritic phenotype associated with cytoskeletal rearrangement in activated human peripheral blood T cells stimulated with monoclonal antibody SPV-L7 to LFA-1α. Maximal expression of this phenotype required 72 h preactivation with phorbol myristate acetate and expression was abolished using the protein kinase C inhibitor staurosporine. Monoclonal antibody to CD18, the β-chain of LFA-1, did not induce this phenotype. Monoclonal antibody MEM 83 to presumably a discrete epitope on LFA-1α did not induce this phenotype but induced homotypic aggregation. However, a monoclonal antibody to CD44 induced a similar phenotype in activated lymphocytes. Induction of both homotypic aggregation and the dendritic phenotype was abolished by preincubation with soluble intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal inhibitors prevented the morphological changes in SPV-L7–activated lymphocytes. Preincubation with tyrosine kinase inhibitor, protein kinase C inhibitors, and inhibitors of new protein synthesis also prevented these morphological changes. These data suggest that discrete epitopes on LFA-1α may be capable of inducing discrete signals either for homotypic aggregation or for a dendritic phenotype. As both LFA-1 and CD44 are involved in the migration of lymphocytes through high endothelial venules, these data could suggest that these molecules transduce signals resulting in cytoskeletal modification necessary for lymphocyte transmigration.
Interleukin-6 suppression of neutrophil apoptosis is neutrophil concentration dependent
Authors: Biffl Walter L , Moore Ernest E , Moore Frederick A , Barnett Carlton C
Published: January 2018

Apoptosis of polymorphonuclear leukocytes (PMNs) is a critical step in the resolution of tissue inflammation. PMN apoptosis has been studied extensively in vitro, and diverse inflammatory mediators have been shown to modulate the process. The reported effects of interleukin-6 (IL-6) on PMN apoptosis are inconsistent; however, analysis of published studies reveals at least one discriminating factor–the use of varied concentrations of PMNs in the experimental design. Consequently, we hypothesized that the in vitro effects of IL-6 on PMN apoptosis varied with the concentration of PMNs in culture. PMNs isolated from healthy human donors were cultured at concentrations from 1 to 20 × 106/mL, and incubated with IL-6 doses from 1 to 100 ng/mL. PMNs cultured at 1–5 × 106/mL were unaffected by IL-6; in contrast, IL-6 inhibited apoptosis in PMNs cultured at 10–20 × 106/mL, compared with untreated similarly concentrated PMNs. These data suggest caution in interpreting in vitro studies of apoptosis; on the other hand, appropriately designed experiments may help elucidate the regulation of apoptosis in vivo.
Activation of cytokine production and adhesion molecule expression on THP-1 myelomonocytic cells by macrophage colony-stimulating factor in combination with interferon-γ
Authors: Kimball Edward S , Kovacs Elizabeth , Clark M Christina , Schneider Craig R
Published: January 2018

THP-1 myelomonocytic leukemia cells cultured with either macrophage colony-stimulating factor (M-CSF) or interferon-γ (IFN-γ) alone produce, at best, only low levels of interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α). However, combinations of the two factors resulted in at least 3- to 20-fold greater amounts of IL-1β and TNF-α than would have been predicted by additive mechanisms. This enhanced cytokine production was observed when M-CSF and IFN-γ were added simultaneously or when M-CSF was added 24 h after addition of IFN-γ to the cells. Similar results were obtained with fresh human peripheral blood cells treated with IFN-γ + M-CSF. Cycloheximide treatment of the cultures containing M-CSF and IFN-γ inhibited the production of IL-1β and TNF-α. Northern blotting studies revealed no effect of IFN-γ alone on IL-1β or TNF-α mRNA production. IL-1β and TNF-α mRNA expression was observed at 2 and 6 h after treatment with M-CSF or IFN-γ + M-CSF. Higher TNF-α mRNA expression was observed at 2 and 6 h after treatment with IFN-γ + M-CSF, and higher IL-1β mRNA expression was observed at 2 h after treatment with IFN-γ + M-CSF compared with mRNA levels observed for cells cultured only with M-CSF. These results suggest that the augmented cytokine production resulting from treatments with combinations of M-CSF and IFN-γ occurs due to increased cytokine mRNA and increased cytokine protein synthesis. In addition to up-regulating cytokines, combinations of IFN-γ and M-CSF resulted in augmented cell surface expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. This was accompanied by morphological and functional changes that included plastic adherence, extensive homotypic aggregation, and a macrophage-like appearance. These phenotypic changes and enhancements in cytokine expression and cell surface molecule expression may be related to activation of monocytic cells to become cytotoxic effectors by M-CSF and IFN-γ combinations. In vitro cytotoxicity against A-375 melanoma cells was greatest for cultures that contained M-CSF and IFN-γ in combination.
Some aspects of IL-8 pathophysiology III: chemokine interaction with endothelial cells
Authors: Rot Antal , Hub Elin , Middleton James , Pons Françoise , Rabeck Christa , Thierer Kamillo , Wintle Jonathan , Wolff Barbara , Zsak Marion , Dukor Peter
Published: January 2018

Chemolrines have been convincingly implicated in driving leukocyte emigration in different inflammatory reactions. However, the cellular and molecular mechanisms of chemokine involvement in leukocyte emigration are not yet clear. We and others suggested that leukocyte adhesion to the endothelium and transmigration are induced by chemokines immobilised on the endothelial cell surface. This would require the presence of specific chemokine binding sites in this microanatomical location. Using an in situ binding assay we demonstrated the presence of binding sites for interleukin-8 (IL-8) and RANTES, but not monocyte inflammatory protein-1α on the endothelium of postcapillary venules and small veins in human skin. In contrast, venules and veins in various anatomical locations showed dramatically differing IL-8 binding patterns. The subcellular distribution of IL-8 in the venular endothelial cells following its in vivo and ex vivo injections was studied by use of electron microscopy. Our results suggest that IL-8 was internalized by the endothelial cells, transported transcellularly via plasmalemmal vesicles, and released onto the luminal surface where it appeared located preferentially on tips of membrane protrusions. We were unable to study the endothelial IL-8 binding or transport in vitro because all the in vitro propagated endothelial cell lines and primary endothelial cells tested lacked IL-8 binding sites. This includes human umbilical vein endothelial cells (HUVECs), which also did not bind IL-8 in situ. However, HUVECs provided a satisfactory in vitro system to study the secretion of IL-8 by the endothelial cells. Two possible alternative pathways were described: secretion directly from the Golgi apparatus or following storage in Weibel-Palade bodies.
Role of eosinophil-chemotactic C-C chemokines in cutaneous inflammation
Authors: Schröder Jens-M , Noso Norio , Sticherling Michael , Christophers Enno
Published: January 2018

In the dermal sites of atopic skin, eosinophil (Eo) granule protein or more rarely intact Eos represent a characteristic histological feature. We addressed the question of whether lesional scales of patients with various eosinophilic skin disorders contain Eo attractant and tried to characterize it biochemically. In scales of a patient with drug reaction, heparin-binding Eo attractants could be identified. High-performance liquid chromatographic analyses together with specific ELISA and Western blot analyses revealed identity with RANTES. No other heparin-binding Eo chemotaxin could be identified. HPLC analysis of pooled lesional scale extracts of patients with atopic dermatitis showed fractions containing only weak heparin-binding Eo-chemotactic activity, which, however, showed RANTES immunoreactivity. In experiments to elucidate the putative cellular origin of Eo-attracting chemokines in human skin we investigated supernatants of atopic skin–derived T lymphocytes as well as supernatants of stimulated dermal fibroblasts for Eo-chemotactic factors. Unexpectedly, we did not find any heparin-bound Eo attractants in supernatants of stimulated cultured atopic skin–derived T lymphocyte clones, whereas fibroblasts produced RANTES as well as granulocyte-macrophage colony-stimulating factor. Therefore, fibroblasts are a likely source of eosinophil attractant cells, which could contribute to the Eo infiltrate. Selectivity of the infiltrate might come from selective induction of RANTES and/or induction of other as yet unidentified Eo-specific chemokines.
Effects of dexamethasone* on bovine circulating T lymphocyte populations
Authors: Burton Jeanne L , Kehrli Marcus E
Published: January 2018

In cattle, γδ T cells represent a higher proportion of circulating T cells than in humans. Bovine γδ T cells can be recognized by expression of γδ T cell receptor (γδ TCR) determinants or by a 215/230-kDa surface antigen (WC1). WC1 is expressed on 90% or more of circulating bovine γδ T cells. The effects of dexamethasone on this and other subsets (CD3, CD2, CD4, CD8) of peripheral blood T lymphocytes were determined by flow cytometric analysis. Twelve 15-month old bulls were injected with dexamethasone (0.04 mg/kg/day) for 3 consecutive days and four bulls were untreated controls. Blood samples were collected daily for 3 days before dexamethasone injections and for an additional 7 days starting on the third injection day. Data were recorded as percent positive cells and as mean fluorescent intensity (MFI) of positive cells. Initially, CD3+ cells represented 65–73% of all peripheral blood mononuclear cells (PBMC). Dexamethasone reduced CD3+ cells to 30%, and these recovered to 50% positive cells by 9 days after the last dexamethasone injection. Loss of CD3+ cells was not due to reductions in αβ T cells because dexamethasone did not influence the percent CD2+, CD4+, or CD8+ cells. However, percent WC1+ cells rapidly declined from a baseline of 26.4% of PBMC to < 6% by the final injection. During injections, the MFI of WC1 increased. The MFI of WC1 returned to control values 7 days after the last injection of dexamethasone, but the percent γδ T cells recovered to only 14% WC1 + PBMC by the final day of the study. During its maximum effects on WC1, dexamethasone also caused a profound decrease of L-selectin MFI on remaining PBMC (mostly αβ T cells and monocyte/macrophages). In a second trial, two-color analyses determined that dexamethasone did not increase apoptosis in WC1 + cells and did not reduce L-selectin MFI on either CD2+ or WC1 + cells. The cumulative results suggested that dexamethasone promoted γδ T cell migration out of peripheral blood via an L-selectin-independent mechanism and that dexamethasone did not alter αβ T cell migration kinetics.
Myocardial protection by N,N,N-trimethylsphingosine in ischemia reperfusion injury is mediated by inhibition of P-selectin
Authors: Scalia Rosario , Murohara Toyoaki , Delyani John A , Nossuli Tareck O , Lefer Allan M
Published: January 2018

Polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia/reperfusion (MI/R) injury. We examined the cardioprotective effects of N,N,N-trimethylsphingosine (TMS) in a murine model of MI (20 min) and R (24 h) injury in vivo, focusing on leukocyte-endothelial interactions. TMS is a synthetic N-methylated sphingosine derivative that has protein kinase C inhibitory activity and has been shown to prevent leukocyte activation. TMS (18 μg/kg), administered intravenously 1 min prior to reperfusion, significantly attenuated myocardial necrotic injury assessed by myocardial creatine kinase loss compared with MI/R rats receiving only vehicle (P< 0.001). Cardiac myeloperoxidase activity, an index of PMN accumulation in the ischemic myocardium, was also significantly attenuated by TMS compared with rats receiving vehicle (P< 0.001). We further examined whether TMS can attenuate leukocyte-endothelial interaction by intravital microscopy. TMS significantly attenuated NG-nitro-l-arginine–methyl ester (l-NAME)–stimulated PMN rolling and adherence to the rat microvascular endothelium. This action of TMS appears to be mediated by reduction of P-selectin expression because immunohistochemical analysis demonstrated that TMS significantly attenuated endothelial P-selectin expression in the l-NAME-superfused rat mesenteric microvasculature. Similarly, TMS markedly attenuated rapid P-selectin expression in rat platelets stimulated with either thrombin or l-NAME assessed by flow cytometry. In conclusion, TMS seems to be an effective cardioprotective agent by inhibiting early leukocyte-endothelial interaction, thus preventing leukocyte accumulation in the ischemic reperfused myocardium.
Macrophage dysfunction following the phagocytosis of IgG-coated erythrocytes: production of lipid peroxidation products
Authors: Loegering Daniel J , Raley Michael J , Reho Thomas A , Eaton John W
Published: January 2018

The phagocytosis of erythrocytes may contribute to the increased susceptibility to life-threatening infections in patients with burn injury, sickle cell anemia, and malaria. The phagocytosis of Immunoglobulin G–coated erythrocytes (EIgG) is followed by a transient depression of several macrophage functions including phagocytosis, respiratory burst capacity, and killing of bacteria. The present study suggests the possibility that after erythrophagocytosis hemoglobin-derived iron conspires with reactive oxygen products of the macrophage respiratory burst to cause oxidant damage to the phagocyte. Challenge of elicited peritoneal macrophages with EIgG phagocytosis was followed by an increase in lipid peroxidation as assessed by thiobarbituric acid–reactive substances (TBARS). Doses of EIgG associated with increased TBARS also caused a depression of Fc receptor–mediated phagocytosis and phorbol myristate acetate (PMA)–stimulated hydrogen peroxide production. Time course experiments demonstrated that the increase in TBARS coincided with the depression of macrophage function. There was no increase in TBARS following the phagocytosis of IgG-coated erythrocyte ghosts, suggesting that hemoglobin iron is involved in the generation of TBARS. The phagocytosis of erythrocyte ghosts did not depress macrophage function. Since complement receptor–mediated phagocytosis does not stimulate the respiratory burst, the role of the respiratory burst in causing lipid peroxidation was assessed using the phagocytosis of complement-coated erythrocytes. Phagocytic challenge with complement-coated erythrocytes caused neither an increase in TBARS nor a depression of macrophage function. However, there was an increase in TBARS when the respiratory burst was stimulated with PMA following complement receptor–mediated phagocytosis of erythrocytes. These results suggest that hemoglobin iron and phagocyte-generated oxidants collaborate to cause the depression of macrophage function following EIgG phagocytosis.
Respiratory syncytial virus G-protein modulates cytokine release from human peripheral blood mononuclear cells
Authors: König B , Streckert H-J , Krusat T , König W
Published: January 2018

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children and adults. In vivo the host has to cope with intact replicative virus, with non-replicative virus, and/or with viral structural proteins including the outer membrane G-protein. We analyzed the role of purified RSV G-protein with regard to its modulatory efficacy for interleukin (IL) -10, IL-12, and tumor necrosis factor-α (TNF-α) release from human peripheral blood mononuclear cells (PBMC). These cytokines seem to contribute to the deleterious effect in viral infections. Treatment of PBMC with RSV at a multiplicity of infection of 10 down to 0.001 induced the release of TNF-α, IL-10, and IL-12; also time kinetics and dose-responses differed markedly. Stimulation of PBMC with purified RSV G-protein (from 0.001 up to 10 μg/106 PBMC) led only to a pronounced increase in IL-10 within a concentration range from 0.01 up to 0.5 μg/106 PBMC with a maximum between 12 and 18 h of incubation. At later time points (24, 48, and 72 h) G-protein concentrations above 1 μg/106 PBMC suppressed IL-10, TNF-α, and IL-12 release from human PBMC. Coating of PBMC with RSV G-protein suppressed IL-10, TNF-α, and IL-12 release after subsequent stimulation with RSV. Our data indicate a regulatory role of RSV G-protein for immune responses toward viral infection.
IL-8 induces T cell Chemotaxis, suppresses IL-4, and up-regulates IL-8 production by CD4+ T cells
Authors: Gesser Borbala , Lund Marianne , Lohse Nicolai , Vestergaard Christian , Matsushima Kouji , Sindet-Pedersen Steen , Jensen Steen Lindkaer , Thestrup-Pedersen Kristian , Larsen Christian Grønhøj
Published: January 2018

Interleukin-8 (IL-8), a neutrophil-activating cytokine, also activates certain T cell functions such as Chemotaxis. We additionally find (n = 6) that recombinant (rIL-8; 1–100 ng/ml), when added to 24 h culture of human CD4+ T cells, suppressed the spontaneous production of IL-4 (50–85%). Steady state production of IL-4 was typically around 30 pg/ml, determined by use of a solid- phase immunoabsorbant assay. De novo synthesis of IL-4 from CD4+ T cells cultured for 3 days was also evaluated by use of detection of [35S]methionine incorporation, as visualized by autoradiography of 2-D gels, and showed that IL-8 suppressed IL-4 production. This suppression of IL-4 production was confirmed in the cytosol fraction by use of Western blotting. The effect of IL-8 (100 ng/ml) was comparable to that of 10 ng/ml recombinant interferon-γ, both strongly suppressing IL-4 production. The regulatory effect of IL-8 on IL-4 production was also indicated by the fact that addition of a neutralizing monoclonal anti-IL-8 antibody (WS.4) enhanced the spontaneous IL-4 production when added to the cultures of CD4+ T cells, thereby probably inactivating the effect of IL-8 originating from the cutured T cells. Also, we observed that IL-4 mRNA expression was down-regulated when the CD4+ T cells were cultured for 12 h in the presence of 100 ng/ml IL-8. The suppression of IL-4 mRNA expression could be prevented by adding anti-IL-8 (20 μg/ml) or IL-10 (100 ng/ml) 1 h before adding rIL-8. Thus, IL-8 may be an important regulator of CD4+ T cell-derived IL-4, thereby possibly regulating the balance between humoral and cellular T cell-dependent responses.
Progesterone inhibits inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages
Authors: Miller Lance , Alley Evan W , Murphy William J , Russell Stephen W , Hunt Joan S
Published: January 2018

The purpose of this study was to determine whether the female hormones estradiol-17β (E2) and progesterone (P4) influence inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) by interferon-γ(IFN-γ)- and lipopolysaccharide (LPS)-activated mouse macrophages. Treatment with P4 alone caused a time- and dose-dependent inhibition of NO production by macrophage cell lines (RAW 264.7, J774) and mouse bone marrow culture-derived macrophages as assessed by nitrite accumulation. RAW 264.7 cells transiently transfected with an iNOS gene promoter/luciferase reportergene construct that were stimulated with IFN-γ/LPS in the presence of P4 displayed reduced luciferase activity and NO production. Analysis of RAW 264.7 cells by Northern blot hybridization revealed concurrent P4-mediated reduction in iNOS mRNA. These observations suggest that P4-mediated inhibition of NO may be an important gender-based difference within females and males that relates to macrophage-mediated host defense.
Neutrophil oxidative metabolism and killing of P. brasiliensis after air pouch infection of susceptible and resistant mice
Authors: Meloni-Bruneri Lucia Helena , Campa Ana , Abdalla Dulcineia S P , Calich Vera Lucia Garcia , Lenzi Henrique L , Burger Eva
Published: January 2018

The oxidative burst of polymorphonuclear neutrophils (PMN) and their ability to inhibit Paracoccidioides brasiliensis growth was studied in susceptible (B10.A) and resistant (A/J) mice. The cells were obtained after subcutaneous inoculation in air pouches, yielding highly pure PMN preparations; the number of cells was similar for both strains at 24 h and five times higher in the resistant strain at 15 days. The oxidative metabolism of these PMN was evaluated by the luminol and lucigen-enhanced chemiluminescence upon stimulation with PMA or killed P. brasiliensis (Pb). At 24 h of infection PMN from both strains showed similar responses. However, at 15 days a great enhancement of the Pb-stimulated luminol-enhanced chemiluminescence was observed only in PMN from resistant mice. Such increase was markedly inhibited by the addition of catalase. Independent of the mouse strain or time of infection the lucigenin-enhanced chemiluminescence showed the same intensity. The lucigenin-enhanced chemiluminescence of PMN without stimuli from resistant mice did not change with the time of infection, however, after 15 days of infection a significantly lower chemiluminescence was detected with PMN from susceptible mice. At 15 days of infection the PMN from B10.A were unable to kill P. brasiliensis yeast cells in vitro. Because the lucigenin- and luminol-enhanced chemiluminescence detects, respectively, the O2- production and the myeloperoxidase/hydrogen peroxide halide system, the present data show parallels between deficiency in the production of oxygen-reactive species by PMN and lower fungicidal activity.
The secretion of preformed granules by macrophages and neutrophils
Authors: Tapper Hans
Published: January 2018

The ability of macrophages and neutrophils to defend tissue homeostasis and participate in inflammatory responses depends on their ability to mobilize granule-membrane proteins and granule content into their external milieu and into phagosomes by regulated secretory processes. Many laboratories have invested much time and effort into furthering our understanding of vesicular transport and secretion. A surge of interest in phagocytosis and phagosomal maturation is also apparent (e.g., the March 1995 issue of Trends in Cell Biology was entirely devoted to phagocytosis). The signaling and the regulation of the secretory response are most likely different for secretion into phagosomes than for secretion into the external milieu. However, these differentially targeted secretory processes rely both upon proteins in vesicular membranes, plasma membrane/phagosomal membrane, and cytosol and upon their interactions with cytoskeletal structures. It is the complex molecular interactions between these components that form the basis for regulation and control of secretion. In the following, the signaling role of granular and cytosolic pH in phagocyte lysosomal secretion is discussed and the current literature on regulated secretion by macrophages and neutrophils is reviewed.
Interleukin-12 protects thermally injured mice from herpes simplex virus type 1 infection
Authors: Matsyo Ryuichi , Kobayashi Makiko , Herndon David N , Pollard Richard B , Suzuki Fujio
Published: January 2018

Severe burn injury is associated with increased susceptibility to severe herpesvirus infections. Type 2 cytokines [interleukin (IL)-4 and IL-10] released from burn-associated CD8+ type 2 T cells (BA-type 2 T cells) have been shown to play a role in the increased susceptibility of thermally injured mice (TI-mice) to herpes simplex virus type 1 (HSV-1) infection. Because IL-12 has been shown to inhibit the generation of type 2 T cells, murine rIL-12 was injected into TI-mice exposed to HSV-1 to determine whether IL-12 could influence HSV-1 infections in individuals bearing type 2 T cells. rIL-12 improved the resistance of TI-mice or mice inoculated with T6S cells (a BA-type 2 T cell clone) against HSV-1 infection. Type 2 cytokines were detected in sera of TI-mice or mice inoculated with T6S cells (T6S-mice). However, treatment of TI-mice or T6S-mice with rIL-12 inhibited type 2 cytokine production in the sera of these mice. All TI-mice exposed to a lethal dose of HSV-1 survived when they were treated with a mixture of monoclonal antibodies (mAbs) against type 2 cytokines. Staphylococcal enterotoxin A [an interferon-γ (IFN-γ) inducer] stimulated serum IFN-γ production in TI-mice and T6S-mice treated with rIL-12, whereas no IFN-γ was produced in mice treated with saline. These results suggest that IL-12 has the potential to protect TI-mice infected with a lethal dose of HSV-1 via a shift to type 1 T cell responses from type 2 T cell responses.
Activation of nitric oxide synthase gene for inhibition of cancer metastasis
Authors: Xie Keping , Dong Zhongyun , Fidler Isaiah J
Published: January 2018

The process of cancer metastasis consists of multiple sequential and highly selective steps. The vast majority of tumor cells that enter the circulation die rapidly and only a few survive and proliferate to form distant metastases. This survival is not random. Metastases are clonal in origin and are produced by specialized subpopulations of cells that preexist in a heterogeneous primary tumor. Metastatic cells of the murine K-1735 melanoma survive in the circulation to produce experimental lung metasteses, whereas nonmetastatic cells do not. After incubation with different cytokines or LPS, nonmetastatic cells exhibit a high level of inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) production, whereas metastatic cells do not. To provide direct evidence for the inverse correlation between the production of endogenous NO and the ability of K-1735 cells to produce metastasis in syngeneic mice, highly metastatic clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive mutated iNOS (C4.S2), or neomycin resistance (C4.Neo) genes in medium containing 3 mM NMA. C4.P, C4.Neo3, and C4.S2.3 cells were highly metastatic, whereas C4.L8.5 cells were not. Moreover, C4.L8.5 cells produced slow-growing subcutaneous tumors in nude mice, whereas the other three cell lines produced fast-growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells was associated with apoptosis. Multiple intravenous injections of liposomes containing a synthetic lipopeptide up-regulated iNOS expression in murine M5076 reticulum sarcoma cells growing as hepatic metastases. The induction of iNOS was associated with the complete regression of the lesions. Collectively, these data demonstrate that the expression of iNOS in tumor cells is associated with apoptosis, suppression of tumorigenicity, abrogation of metastasis, and regression of established hepatic metastases.
Diamine oxidase-gold ultrastructural localization of histamine in isolated human lung mast cells stimulated to undergo anaphylactic degranulation and recovery in vitro
Authors: Dvorak Ann M , Morgan Ellen S , Schleimer Robert P , Lichtenstein Lawrence M
Published: January 2018

A new enzyme-affinity-gold ultrastructural method makes use of the affinity of the enzyme, diamine oxidase coupled to gold, for its substrate, histamine, for localization of histamine in isolated human lung mast cells (HLMCs). The method works with routinely prepared ultrastructural samples, thereby allowing precise identification of ultrastructural structures that contain histamine. We used this method to identify the release of histamine from granule stores in anti-immunoglobulin-E (IgE)-stimulated HLMCs and the replacement of histamine in secretory granules of HLMCs during recovery from anaphylactic degranulation in vitro. The findings show that electron-dense granules in unstimulated HLMCs, maintained up to 24 h in culture, and in responding anti-IgE-stimulated HLMCs, over the same time period in vitro, contained histamine. Alteration of granules, resulting in decreased electron density of their contents, was associated with decreased label for histamine. Electron-lucent intracytoplasmic degranulation chambers were devoid of histamine. Recovering HLMCs developed new granule stores of histamine by a mixture of conservation, synthetic, and endocytotic mechanisms.
Inhibition of apoptosis in polymorphonuclear neutrophils from burn patients
Authors: Chitnis Dhanajay , Dickerson Camille , Munster Andrew M , Winchurch Richard A
Published: January 2018

Normal human polymorphonuclear neutrophils (PMN) undergo rapid apoptosis during in vitro culture. In contrast, apoptosis is inhibited in PMN from patients with severe burns. This inhibition is not an inherent property of the cells but is caused by thermolabile factors present in the plasma. Endotoxin and the proinflammatory cytokines interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) do not appear to be directly responsible. The ability of burn plasma to inhibit apoptosis was reduced by neutralizing antibodies to human granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF levels could not be detected in the burn plasma. However, the incubation of burn-derived or normal leukocyte populations consisting primarily of PMN in burn plasma induced the production of GM-CSF. The results suggest that activation of GM-CSF synthesis by factor(s) in burn plasma may play a role in regulating inflammation by the inhibition of apoptosis.
Cooperation between CR1 (CD35) and CR3 (CD11b/CD18) in the binding of complement-opsonized particles
Authors: Sutterwala Fayyaz S , Rosenthal Louis A , Mosser David M
Published: January 2018

We analyzed the binding of sheep erythrocytes bearing C3b (EC3b) to cells transfected with human complement receptors. EC3b bound avidly to cells expressing CR1 but failed to bind to cells expressing CR3. In the presence of factor I, the binding of EC3b to CR1 was transient. Primary monocytes and cotransfected cells expressing both CR1 and CR3 mediated a stable rosetting of EC3b, even in the prolonged presence of factor I. This stable adhesion was dependent on the presence of CR3, because blocking CR3 with mAb resulted in the factor I-dependent release of erythrocytes from these cells. A model is proposed in which these two complement receptors cooperate in a unique manner. These results suggest that the stable adhesion of complement-opsonized particles to cells expressing CR1 and CR3 is actually a dynamic molecular process in which an important function of leukocyte CR1 is to generate the liganda for CR3.