bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-1 activation of endothelium supports VLA-4 (CD49d/CD29)-mediated monocyte transendothelial migration to C5a, MIP-1α, RANTES, and PAF but inhibits migration to MCP-1: a regulatory role for endothelium-derived MCP-1
Authors: Chuluyan H Eduardo , Schall Thomas J , Yoshimura Teizo , Issekutz Andrew C
Published: January 2018

We investigated the effect of interleukin-1 (IL-1) activation of human umbilical vein endothelium (HUVE) on human monocyte transendothelial migration induced by chemotactic factors. Monocyte migration across unacdvated endothelium in response to macrophage inflammatory protein-lα (MIP-1α), RANTES, platelet-activating factor (PAF), or monocyte chemoat-tractant protein-1 (MCP-1) was completely inhibited (90%) by monoclonal antibodies (mAbs; 60.3) to CD 18 of the CD11/CD18 complex on the monocyte and partially inhibited (by 75%) in response to C5a. When the HUVE was stimulated with IL-lα (5 h, 0.1 ng/ml), monocyte migration in response to C5a, MIP-lα, RANTES, or PAF was no longer inhibited by mAb to CD18. However, migration was blocked by the combination of mAb to the (α4-integrin (CD49d) chain of very late antigen-4 (CD49d(/CD29) with the mAb to CD18. In contrast to the above stimuli, activation of the HUVE with IL-1α inhibited the transendothelial migration of monocytes in response to MCP-1. mAbs to the adhesion molecules up-regulated on HUVE by IL-1, i.e., E-selectin (CD62E), intercellular adhesion molecule-1 (CD54) or vascular cell adhesion molecule-1 (CD106), did not reverse the inhibitory effect. Transendothelial migration in response to MCP-1 but not to C5a was inhibited by the treatment of monocytes with culture supernatant from IL-1α-stimulated (but not from unstimulated) HUVE. Such supernatant contained chemotactic activity for monocytes, and a mAb to MCP-1 blocked the migration inhibitory effect of IL-1 activation of the HUVE monolayer, as well as the chemotactic activity in the supernatant from IL-1-stimulated HUVE. The inhibitory effect on migration of IL-1-stimulated HUVE was specific for monocytes because polymorphonuclear leukocyte transendothelial migration in response to IL-8 (a related chemokine) was not inhibited by IL-1 activation of HUVE. These results demonstrate that: (1) C5a but not MCP-1, MIP-lα, RANTES, or PAF activates not only a CD 18-dependent but also a VLA-4-dependent mechanism on monocytes, which mediates migration across unstimulated HUVE; (2) IL-1 (tumor necrosis factor-α or lipopolysaccharide) activation of HUVE results in efficient VLA-4 (CD494/CD29)-mediated monocyte transendothelial migration in response to C5a, RANTES, MIP-1α, and PAF, in addition to the CD 18-dependent pathway, but inhibits the response to MCP-1; and (3) this selective inhibition is probably due to MCP-1 production by the activated endothelium monolayer and may be one mechanism of down-regulation by the endothelium of monocyte migration in response to extravascular MCP-1. J. Leukoc. Biol. 58: 71–79,1995.
Nitric oxide stimulates ADP ribosylation of actin in association with the inhibition of actin polymerization in human neutrophils
Authors: Clancy Robert , Leszczynska Joanna , Amin Ashok , Levartovsky David , Abramson Steven B
Published: January 2018

In these studies we provide conclusive evidence that (β/γ) actin present in human neutrophils is a substrate for nitric oxide (NO)-dependent ADP ribosylation and that this modification is associated with the inhibition of actin polymerization. A 43-kDa substrate for NO-dependent ADP ribosylation was identified as actin by four methods: (1) comigration with the botulinum C2 toxin substrate by two-dimensional gel electrophoresis (pI 5.2), (2) identity between the peptide map generated by V8 protease digestion of the NO and botulinum C2 substrates, (3) immunoprecipitation with antiactin antibodies, and (4) the ability of NO to ADP ribosylate purified neutrophil G-actin in the presence of plasma membrane cofactors. Because the ADP ribosylation of actin by the botulinum C2 toxin is known to inhibit F-actin polymerization, we examined the effect of NO on actin assembly. Flow cytometry revealed that NO inhibited formyl-methionine-leucine-phenylalanine (fMLP)-dependent (SO s at 37°) F-actin formation (108 ± 8 vs. 89 ± 6 relative fluorescence units, P < .02). These results were confirmed by quantification of F-actin formation by gel scanning (10% sodium dodecyl sulfate gel, Coomassie, and densitometry): pretreatment of polymorphonuclear leukocytes with NO resulted in a reduction of fMLP-induced, cytoskeletal-associated F-actin, which was accompanied by an increase of Triton-soluble G-actin. NO also inhibited F-actin formation, as observed by means of rhodamine phalloidin staining of neutrophils adherent to a fibronectin-coated surface. This effect was accompanied by a dose-dependent inhibition of neutrophil adherence in NO-treated cells. The data indicate that NO inhibits cytoskeletal assembly and adherence in human neutrophils in association with the ADP ribosylation of actin. J. Leukoc. Biol. 58: 196–202; 1995.
Low-molecular-weight GTP-binding proteins and leukocyte signal transduction
Authors: Quinn Mark T
Published: January 2018

Recent progress on the understanding of leukocyte signal transduction pathways indicates that many of these pathways are modulated by low-molecular-weight GTP-binding proteins (LMWGs). LMWGs are members of a growing family of GTP-binding proteins known as the Ras superfamily and act as molecular switches for regulating a wide range of signal-transduction pathways in virtually all cells. In leukocytes, LMWGs have been shown to participate in essential cellular functions such as growth control, differentiation, cytoskeletal organization, cytokine and chemoattractant-induced signaling events, reduced nicotinamide adenine dinucleotide phosphate oxidase activity, phospholipid metabolism, and intracellular vesicle transport and secretion, and many more of these functions will certainly be identified as we gain a further understanding of regulatory pathways modulating leukocyte signal transduction. This review will summarize our current understanding of this rapidly advancing area of research.
Taurolidine, an antilipopolysaccharide agent, has immunoregulatory properties that are mediated by the amino acid taurine
Authors: William R , Watson G , Redmond H Paul , Carthy Julie Mc , Bouchier-Hayes David
Published: January 2018

Taurolidine has bactericidal and antilipopolysaccharide properties. It is broken down into the amino acid taurine, which has been shown to modulate intracellular calcium activity, a critical component in the priming and activation of macrophages and polymorphonuclear leukocytes. We hypothesized that taurolidine may function to enhance immune activity in these cells. The aim of this study was to investigate the immunological effects of taurolidine and correlate findings with survival after a septic challenge in a murine model. Study 1: CD-1 mice underwent cecal ligation and puncture, were randomized to receive taurolidine (200 mg/kg body weight/i.p.) or saline control, and studied for end point survival. Study 2: CD-1 mice were randomized to receive taurolidine (200 mg/kg body weight/i.p.) or saline control. Peritoneal macrophages (PMØs) were assessed for O2-, NO, tumor necrosis factor-α (TNF-α), CD11b, phagocytosis, and PMN influx. O2-, TNF-α, CD11b expression, and phagocytosis were significantly increased in the taurolidine group. Study 3: PMØs were cultured in vitro ±0.5 mg/ml taurolidine and PMØ antimicrobial function assessed (O2-, NO, TNF-α, and phagocytosis). O2-, TNF-α, and phagocytosis were significantly increased, whereas NO was reduced. Study 4: PMØs were also cultured with taurine (0.5 mg/ml). Similar increase in O2-, TNF-α, and phagocytosis were identified. Intracellular PMØ [Ca2+] was also assessed and increases in free, unbound intracellular [Ca2+] occurred after taurine culture. Thus, in addition to its bactericidal and antilipopolysaccharide activity, taurolidine primes PMØs for enhanced antimicrobial activity and these effects appear mediated by the amino acid taurine.
Pleural fluid eosinophils suppress local IgE-mediated protein exudation in rats
Authors: Bandeira-Melo Christianne , Silva Patricia M R e , Cordeiro Renato S B , Martins Marco A
Published: January 2018

Eosinophils are supposed to play a critical role in the pathology of several allergic diseases because after activation they can release toxic and proinflammatory agents. In this study we have investigated whether IgE-mediated rat pleurisy could be affected by an ongoing pleural eosinophilic inflammatory response. IgE-passively sensitized rats were challenged with an intrapleural (i.pl.) injection of allergen (dinitrophenylated bovine serum albumin, 1 μg/cavity) and exudation assessed by measuring the amount of protein extravasated into the pleural cavity within 4 h. We have confirmed that lipopolysaccharide (LPS) stimulation (250 ng/cavity i.pl.) was followed by a marked pleural neutrophilia, apparent at 3 h, which was followed by an eosinophil accumulation noted within 48–72 h postchallenge. We have also confirmed that a boiled sample of LPS pleural washing (IPS-PW, 200 μl i.pl.) caused selective eosinophilia in recipient rats. Pleural exudation remained unaltered when the allergenic challenge was performed 3 h after IPS in a condition of intense pleural fluid neutrophilia. In contrast, this was significantly reduced (P< .001) when the challenge occurred 72 h after IPS or 24 h after IPS-PW in selective pleural fluid eosinophilia. In another series of experiments repeated daily i.pl. injections of platelet-activating factor (PAF; 1 μg/cavity) resulted in a progressive increase in eosinophil number recovered from the pleural cavity. The values were 1.2 ± 0.2, 3.0 ± 0.2, and 5.8 ± 0.5 × 106 eosinophils/cavity (mean±SEM) after 0, 1, and 4 injections, respectively. Allergen challenge performed after 0, 1, or 4 PAF stimulations led to pleural protein levels of 88.6 ± 5.7, 33.7 ± 0.7, and 19.4 ± 2.3 mg/cavity, respectively, indicating that the allergic pleurisy is inhibited in a manner dependent on the magnitude of eosinophil accumulation. Furthermore, the impairment of PAF-induced eosinophil accumulation by cetirizine (30 mg/kg i.p.) restored the exudatory response. Exudation triggered by compound 48/80 (25 μg/cavity), histamine (200 μg/cavity), or 5-hydroxytryptamine (100 μg/cavity) was not affected by four previous PAF daily injections. The findings indicate that allergen-induced exudation is selectively down-regulated in the eosinophil-enriched pleural space of rats, a suppression that increased with increasing eosinophil number and disappeared after chemical impairment of the eosinophilia.
Neutrophil functional responses depend on immune complex valency
Authors: Strohmeier Gregg R , Brunkhorst Beatrice A , Seetoo Kurt F , Bernardo John , Weil Gary J , Simons Elizabeth R
Published: January 2018

Ligand-induced cross-linking of Fcγ receptors (FcγR) on neutrophils plays a significant role in their stimulation, shown here by contrasting the responses induced by low valency immune complexes (LICs) and high valency immune complexes (HICs) and by cross-linking LICs in situ (L/Ab) after their addition to the cells. Multiparameter flow cytometry was used to measure immune complex (IC)-elicited changes in cytoplasmic Ca2+ concentration and initiation of the oxidative burst simultaneously in the same cell and to correlate these with FcγR occupancy. We have previously shown that subpopulations of neutrophils respond maximally to subsaturating concentrations of HIC; saturating dosages stimulate the entire population. This discrepancy was not due to differences in receptor occupancy. The magnitude of the transient Ca2+ increase was independent of the dose of HIC but depended on the dose when an LIC was used. As shown here, L/Ab cross-linking elicited Ca2+ responses similar to those observed in HIC-stimulated cells. In contrast, LIC elicited only minimal intracellular ΔpH and no oxidative burst or membrane potential changes at all unless FcγR was cross-linked, accomplished by HIC or by L/Ab. However, azurophilic degranulation, as determined by elastase release, was not observed in cells stimulated by the in situ cross-linking method, whereas the HIC preparation triggered azurophilic degranulation. Thus, some FcγR-mediated neutrophil effector functions such as azurophilic degranulation and oxidative burst initiation have an absolute requirement for FcγR cross-linking, whereas signaling functions such as changes in membrane potential, intracellular pH, and intracellular Ca2+ concentration can occur, albeit more slowly and to a lesser extent, if single FcγR are occupied.
Evidence for a third component in neutrophil aggregation: potential roles of O-linked glycoproteins as L-selectin counter-structures
Authors: Bennett Teresa A , Schammel Christine M G , Lynam Eric B , Guyer Deborah A , Mellors Alan , Edwards Bruce , Rogelj Snezna , Sklar Larry A
Published: January 2018

The homotypic aggregation of neutrophils requires the participation of L-selectin and the β2-integrins, but it has not been clear whether the two receptors recognize one another as counter-structures or whether other adhesion molecules are involved. We have examined aggregation of live neutrophils with target populations, manipulated to alter expression of adhesive epitopes, using flow cytometry. A target population depleted of both L-selectin and activatable β2-integrin displayed an ability to aggregate with live neutrophils, suggesting that these two molecules are not counter-structures. We also found that an O-sialoglycoprotease (GCP) from Pasteurella haemolytica is capable of inhibiting homotypic aggregation. Neutrophils treated with GCP lose O-glycosylated proteins but retain L-selectin and activatable β2-integrin. One or more of the GCP substrates appears to function in L-selectin-dependent binding but not in β2-integrin-dependent binding. Together the data suggest a mechanism of aggregation that is analogous to leukocyte-endothelial cell adhesion in which a low-affinity carbohydrate-dependent interaction precedes a high-affinity integrin-dependent adhesion.
Differential regulation of TNF-α production by listeriolysin-producing versus nonproducing strains of Listeria monocytogenes
Authors: Vazquez M A , Sicher S C , Wright W J , Proctor M L , Schmalzried S R , Stallworth K R , Crowley J C , Lu C Y
Published: January 2018

Only Listeria monocytogenes that produce listeriolysin O (LLO) elicit protective immunity. Given the importance of tumor necrosis factor α (TNF-α) in anti-Listeria immunity, we have investigated TNF-α production by macrophages after they ingested live LLO-producing compared to LLO-non-producing bacteria. We used two genetically engineered strains of Listeria that differed only in their ability (Ly+) or inability (Ly-) to produce LLO. Ly+ and Ly- caused the same kinetics of increased mRNA abundance for TNF-α during the first 90 min after phagocytosis. However, only Ly+ caused sustained transcription of TNF-α mRNA, and this may account for the increased release of TNF-α. The transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) prevented the sustained abundance of cytokine mRNA 20 h after ingestion of Ly+. In addition, nuclear run-on assays indicated sustained transcription of TNF-α genes only after ingestion of Ly+. LLO itself was not responsible for the ability of Ly+ to stimulate the sustained transcription of the TNF-α genes. Instead, LLO may allow Listeria to survive within macrophages so that other bacterial products cause sustained TNF-α gene transcription. Both Ly+ and Ly- produced molecules, isolated by 50% ammonium sulfate, that induced cytokine production. In conclusion, we now report that Ly+ causes sustained transcription of the TNF-α gene and production of TNF-α by macrophages in vitro. We speculate that the TNF-α may activate endothelium and thus allow the recruitment of T cells to sites of infection. This may contribute to the ability of only LLO-producing Listeria to induce protective immunity.
Vitamin E supplementation with interferon-γ administration retards immune dysfunction during murine retrovirus infection
Authors: Wang James Y , Liang Bailin , Watson Ronald R
Published: January 2018

Murine retrovirus infection induces loss of vitamin E and immune dysfunction with loss of cytokine production by T-helper cells. Therefore interferon-γ (IFN-γ) was given during dietary vitamin E supplementation to effectively prevent murine retrovirus-induced immunosuppression, cytokine dysregulation, and development of murine AIDS. Administration of IFN-γ during vitamin E supplementation significantly prevented development of retrovirus-induced suppression of splenic natural killer cell activity and T cell proliferation. It also significantly slowed retrovirus-induced elevation of T helper (Th) 2 cytokine [interleukin (IL)-4, IL-5, and IL-10] production and monokine (IL-6 and tumor necrosis factor-α) secretion by splenocytes. The treatment also prevented loss of Th1 cytokine (IL-2 and IFN-γ) secretion by splenocytes from retrovirus-infected mice alleviating splenomegaly and hyper-gammaglobulinemia. The combined therapy had an additive therapeutic impact. It was more effective than IFN-γ treatment or vitamin E supplementation alone in delaying the development of retrovirus-induced immunosuppression with its cytokine dysregulation.
Interferon-γ inhibits HIV-induced invasiveness of monocytes
Authors: Dhawan Subhash , Wahl Larry M , Heredia Alonso , Zhang Yahong , Epstein Jay S , Meltzer Monte S , Hewlett Indira K
Published: January 2018

HIV-infected monocytes form highly invasive network on basement membrane matrix and secrete high levels of 92-kd metalloproteinase (MMP-9), an enzyme that degrades basement membrane proteins. In the present study, using matrigel as a model basement membrane system, we demonstrate that treatment of human immunodeficiency virus (HIV)-infected monocytes with interferon-γ at 50 U/ml inhibited the ability of infected monocytes to form an invasive network on matrigel and their invasion through the matrigel matrix. These effects were associated with a significant reduction in the levels of MMP-9 produced by HIV-infected monocytes treated with interferon-γ 1 day prior to infection with HIV as compared with that of untreated HIV-infected monocytes. Monocytes treated with interferon-γ 1 day after HIV infection showed the presence of integrated HIV sequences; however, the levels of MMP-9 were substantially lower than those produced by monocytes inoculated with live HIV, heat-inactivated HIV, or even the control uninfected monocytes. Exposure of monocytes to heat-inactivated HIV did not result in increased invasiveness or high MMP-9 production, suggesting that regulation of metalloproteinase by monocytes was independent of CD4-gp120 interactions and required active virus infection. Furthermore, addition of interferon-γ to monocytes on day 10 after infection inhibited MMP-9 production by more than threefold with no significant reduction of virus replication. These results indicate that the mechanism of interferon-γ–induced down-regulation of MMP-9 levels and reduced monocyte invasiveness may be mediated by a mechanism independent of antiviral activity of IFN-γ in monocytes. Down-regulation of MMP-9 in HIV-infected monocytes by interferon-γ may play an important role in the control of HIV pathogenesis.
New members of the chemokine receptor gene family
Authors: Raport Carol J , Schweickart Vicki L , Chantry David , Eddy Roger L , Shows Thomas B , Godiska Ronald , Gray Patrick W
Published: January 2018

Chemokines are relatively small peptides with potent chemoattractant and activation activities for leukocytes. Several chemokine receptors have been cloned and characterized and all are members of the G protein-coupled receptor superfamily. Using degenerate oligonucleotides and polymerase chain reaction, we have identified seven novel receptors with significant homology to chemokine receptors. Two of these sequences are presented here for the first time. We have shown, with gene mapping studies, that receptors with the highest sequence similarity are closely linked on human chromosomes. This close genetic association suggests a functional relationship as well.
The role of MIP-1α in Inflammation and hematopoiesis
Authors: Cook Donald N
Published: January 2018

Macrophage inflammatory protein 1α (MIP-1α) is a member of the C-C subfamily of chemokines, a large superfamily of low-molecular-weight, inducible proteins that exhibit a variety of proinflammatory activities in vitro including leukocyte Chemotaxis. MIP-1α is a particularly interesting chemokine, because in addition to its proinflammatory activities, it inhibits the proliferation of hematopoietic stem cells in vitro and in vivo. Here, the biologic properties of MIP-1α are reviewed in light of recent data on mice homozygous for a disruption of the MIP-1α gene. The MIP-1α null mice have no overt abnormalities of peripheral blood or bone marrow cells, indicating that MIP-1α is not necessary for normal hematopoiesis. However, the MIP-1α null mice have a reduced inflammatory response to influenza virus and are resistant to coxsackievirus-induced myocarditis. These data demonstrate that MIP-1α is required for a normal inflammatory response to these viruses. Agents that inhibit the action of MIP-1α may therefore prove useful for controlling inflammation in these and other settings.
β Chemokines costimulate lymphocyte cytolysis, proliferation, and lymphokine production
Authors: Taub Dennis D , Ortaldo John R , Turcovski-Corrales Susan M , Key Michael L , Longo Dan L , Murphy William J
Published: January 2018

We report here the ability of the β chemokines MIP-1α, MP-1β, RANTES, and MCP-1 to enhance some lymphocyte effector functions. Initial studies focused on the effects of chemokines on human and mouse cytotoxic T lymphocyte (CTL)–and natural killer (NK) cell–specific cytolytic responses. The results demonstrate that β chemokines are capable of augmenting mouse and human CTL and human NK–but not lymphokine-activated killer cell– or antibody-dependent cell cytotoxicity–specific cytolytic responses. Neutralisation analysis utilizing integrin-specific antibodies revealed that CTL/NK–tumor cell conjugate formation is required for chemokine-induced killing. In addition, both CTLs and NK cells incubated with various β chemokines were induced to degranulate and release granule-derived serine esterases, suggesting that chemokines may be important costimulators of CTL and NK cell degranulation and may thus augment local target cell destruction. Chemokines also modulate antigen-driven T cell proliferative responses as well as effects on lymphokine production. Many of the β chemokines were found to potentiate human and mouse antigen-specific Th1 and Th2 clone activation promoting cellular proliferation and the release of various lymphokines. This chemokine-mediated T cell proliferation was chemokine and antigen dose dependent as well as clone dependent. Chemokine pretreatment analyses with T cells and antigen-presenting cells (APCs) revealed that chemokines up-regulate both T cell and APC functions. Costimulation assays using immobilized anti-CD3 monoclonal antibody–coated plates and purified human and mouse T cells and T cell clones in the presence of various chemokines also exhibited enhanced proliferation and lymphokine secretion. This costimulation was interleukin-2 dependent and required the presence of free extracellular calcium. Examination of chemokine-treated APCs revealed that the T cell costimulatory molecule B7-1 was induced by various β chemokines. Neutralization of endogenously produced chemokines with specific antibodies during an antigen-specific T cell response blocked cellular proliferation, suggesting that the chemokines have an autocrine and/or paracrine role in antigen-induced T cell proliferative responses. Together, these results suggest that chemokines play a significant role in the activation of polyclonal as well as antigen-specific helper and cytotoxic T cells during the genesis of an immune response.
Role of yolk sac endodermal cells with special reference to the fetal macrophage differentiation
Authors: Yamashita Akira
Published: January 2018

The morphological and functional characteristics of rat yolk sac endodermal cells, with particular reference to fetal macrophage (Mφ differentiation, were studied in vivo and in vitro using DA rat embryos from 8 to 16 days of gestation. At a very early stage (day 5–6) of gestation, endodermal cells are derived from proximal endoderm as an essential and multipotent organ with various primitive functions. Based on toluidine blue staining properties and ultrastructures, we demonstrated that the endodermal cell layer of 8–16-day yolk sacs consists of two cell types, “clear” cells with clear cytoplasm (10%) and “dark” cells with dark cytoplasm (90%), and hypothesized that the endodermal cell layer is heterogeneous at both the morphological and functional levels. We produced three different monoclonal antibodies (mAbs), designated Mar 1, Mar 2, and Mar 3, that recognize rat Mφ populations. Mar 1 binds specifically to the cells constituting the mononuclear phagocyte system (MPS). Mφ Mar 3 antigen is a phagocytosis-associated molecule, and Mar 2 antigen is a differentiation antigen of the Mφ subset. Application of these mAbs in both in vivo and in vitro studies allowed the functional capability and differentiation of fetal Mφs to be assessed. The Mar 3 antigen was expressed first on proximal endodermal cells on day 6 yolk sac and continued to be presented afterward. In vitro culture study demonstrated that the adhesive, phagocytic Mar 3+ · Mar 1+ Mφs differentiate from Mφ precursors in the endodermal cell layer after the first 13 days of gestation. Based on these findings, we proposed that clear cells in the endodermal cell layer are derived from precursor dark cells, detach from the layer, move to the mesenchymal stromas, and subsequently migrate to the fetal liver, loose connective tissue, and other intraembryonic tissues, and consequently they differentiate free Mar 1+ Mφs during gestation (day 13–15). Thus, the Mφ differentiation and the peripherization of the Mφs could be almost fully developed during the prenatal period.
Alkyl-PAF and acyl-PAF human neutrophil priming for enhanced fMLP- and rC5a-induced superoxide anion production
Authors: Pinckard R Neal , Prihoda Thomas J
Published: January 2018

Alkyl-PAF induced two components of polymorphonuclear leukocyte (PMN) pruning for enhanced fMLP- and rC5a-induced superoxide anion (O2−) production. Component A priming had a shallow, linear alkyl-PAF concentration-response slope (10 pM–1 nM), and component B priming had a significantly steeper concentration-response slope (1–100 nM alkyl-PAF). Whereas the extent of component B priming decayed significantly within 5–10 min after pretreatment of PMNs with alkyl-PAF, component A priming was completely stable. WEB 2086, a specific and potent PAF receptor antagonist, abolished component A pruning when PMNs were simultaneously stimulated with alkyl-PAF and either fMLP or rC5a but only partially reduced component B priming. However, whereas WEB 2086 also obliterated component A priming when PMNs were pre-treated with alkyl-PAF for 2.5, 5, or 10 min prior to fMLP stimulation, WEB 2086 had little or no inhibitory effect on component B priming. Paradoxically, WEB 2086 significantly augmented alkyl-PAF–induced component B priming for enhanced rC5a-induced PMN O2− production yet concomitantly obliterated component A printing. PMN pruning by acyl-PAF (1 nM–1 μM) had characteristics identical to those of alkyi-PAF–induced component A priming. These studies suggest that there are at least two effector pathways modulating alkyl-PAF–induced PMN respiratory burst priming. They are also consistent with the notion that component A priming is initiated via high-affinity PAF receptors and component B priming is mediated through low-affinity PAF receptors; and whereas alkyl-PAF interacts with both high- and low-affinity PAF receptors, both acyl-PAF and WEB 2086 preferentially bind to the high-affinity PAF receptors.
Soluble CD14 and lipopolysaccharide-binding protein from bovine serum enable bacterial lipopolysaccharide-mediated cytotoxicity and activation of bovine vascular endothelial cells in vitro
Authors: Yang Zhengang , Breider Michael A , Carroll Roger C , Miller Mark S , Bochsler Philip N
Published: January 2018

Bacterial endotoxin (lipopolysaccharide, LPS) has potent proinflammatory properties toward many cell types, including vascular endothelial cells. Bovine endothelial cells are often used for investigations involving the vascular endothelium in vitro, and other bovine products such as fetal bovine serum are also widely utilized in research laboratories. Evidence is presented that soluble CD14 (sCD14) is present in bovine serum and that LPS-mediated activation and cytotoxicity to bovine endothelial cells in vitro are dependent on sCD14. LPS-mediated activation of endothelial cells was quantitated by measuring tissue factor expression using an activated factor X–related chromogenic assay. Concentrations of 0.1–5.0% fetal bovine serum in the culture medium promoted LPS-induced tissue factor expression on bovine endothelial cells, and anti-CD14 monoclonal antibody (mAb) (20 μg/ml) inhibited tissue factor expression, whereas control antibodies did not. LPS-mediated damage to endothelial cells was assayed using the MTT tetrazolium assay. We found that either serum or recombinant human soluble CD14 (rsCD14, 20–2000 ng/ml) was required for LPS-related endothelial cell damage and that anti-CD14 mAb inhibited cytotoxicity. In addition, bovine LPS-binding protein (LBP, 20 ng/ml) purified from bovine serum had no effect on LPS-mediated cytotoxicity, but bovine LBP greatly enhanced the cytotoxic effect of LPS plus rsCD14. Western blot analysis performed on fractionated bovine serum samples with anti-CD14 mAb revealed immunoreactivity with a 50–55-kd protein, a size consistent with sCD14. Evidence of endothelial cell-associated CD14 was not detected using an immunofluorescence technique on cell preparations, nor by Northern blot analysis. These results indicate the existence of sCD14 in bovine serum and that soluble bovine serum factors including sCD14 and LBP facilitate presentation of LPS to receptive cells.
Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told
Authors: Lundqvist Helen , Foilin Per , Khalfan Laila , Dahlgren Claes
Published: January 2018

The paradigm for activation of the neutrophil NADPH oxidase with the protein kinase C activator phorbol myristate acetate (PMA) states that the oxidase assembles in the plasma membrane and that the metabolites generated are released extracellularly. This paradigm is challenged by the results presented. Most of the PMA-induced oxidase activity measured as chemiluminescence and dichlorofluorescein fluorescence was insensitive to scavengers of superoxide anion and hydrogen peroxide. This indicates that oxidase activity also takes place in a cellular compartment that the scavengers cannot reach. From the results obtained with granule-deficient HL-60 cells and cord blood neutrophils, we suggest that the scavenger-insensitive part of the NADPH oxidase activity in normal neutrophils re-sides in an intracellular compartment identical to or originating from granules. Our results also indicate that specific and azurophil granules have to be in very close contact to allow the generated oxygen metabolites to reach and react with myeloperoxidase.
Computerized assessment of production of multiple human cytokines at the single-cell level using image analysis
Authors: Björk Lars , Fehniger Thomas E , Andersson Ulf , Andersson Jan
Published: January 2018

A technique using a computerized image analysis system was developed for evaluating and quantifying human cytokine production. This system registered single cells as positive or negative cytokine producers based on a specific juxtanuclear staining pattern generated by accumulation of the proteins in the Golgi-endoplasmatic reticulum compartment. The characteristic morphology of the immunocytochemical staining offered the opportunity to register individual producer cells within multicomponent cell populations. A color camera was then adapted to transfer on-line images directly into the computer-controlled operating system. In this study cultured human peripheral blood mononuclear cells were polyclonally stimulated and then analyzed for interleukin-1α (IL-1α), IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor-α (TNF-α), TNF-β, interferon-γ, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor production. The image-analyzing system detected cytokine-producing cells in a sensitive and reproducible manner, which was in total congruence with enumeration by conventional microscopy. Furthermore, accurate assessments of cell distributions by signal intensity and cell area were applied at the single-cell level. The image-analyzing system allowed the detection of at least 1 in 1,000 events by using unique cytokine-associated morphometric criteria. The results of kinetic studies measuring cytokine production following activation and cell transformation provided data supporting increases in intensity of intracellular localized specific immunostaining and in cell size within the cytokine-producing cells.
Rapid secretion of intracellularly pre-stored interleukin-8 from rabbit platelets upon activation
Authors: Su Shao-Bo , Mukaida Naofumi , Matsushima Kouji
Published: January 2018

Freshly isolated rabbit platelets contained a substantial amount of intracellular interleukin-8 (IL-8) as revealed by immunohistochemistry and Western blotting analysis. Thrombin stimulation at room temperature induced platelets to secrete IL-8 rapidly, and the maximal secretion was observed within 10 min after the activation. Secreted IL-8 possessed neutrophil chemotactic activity that was neutralised by a specific neutralizing monoclonal antibody to rabbit IL-8 but not a control one. These results suggest that rabbit platelets store biologically active IL-8 intracellularly and secrete it rapidly upon activation. This study implies that platelets may be a pivotal participant in inflammatory reactions in rabbits through producing cytokines and chemokines, including IL-8.
The inflammatory response in interleukin-1β-deficient mice: comparison with other cytokine-related knock-out mice
Authors: Fantuzzi Giamila , Dinarello Charles A
Published: January 2018

Interleukin-1 (IL-1) plays a crucial role in the development of the pathophysiological responses to infection and inflammation. However, the relative contributions of IL-1α and IL-1β remain to be clarified. IL-1β-deficient mice are a powerful tool to investigate the specific role of IL-1β in various experimental conditions. In this report, we summarize the response of IL-1β-deficient mice to two different inflammatory stimuli, turpentine and endotoxin. Although IL-1β-deficient mice respond normally to the systemic administration of lipopolysaccharide (LPS), they do not develop an acute-phase response in the localized tissue damage model of turpentine injection. The results obtained using the IL-1β-deficient mice are compared here with those observed in the IL-1β-converting enzyme-deficient, IL-6-deficient, tumor necrosis factor-receptor p55-deficient, and interferon-γ-receptor-deficient mice.
The lectin jacalin triggers CD4-mediated lymphocyte signaling by binding CD4 through a protein-protein interaction
Authors: Lafont Virginie , Domand Jacques , Covassin Laurence , Liautard Jean Pierre , Favero Jean
Published: January 2018

The lectin jacalin specifically stimulates lymphocytes expressing the CD4 antigen. Recent studies have also demonstrated that this lectin interacts with CD4, inhibits in vitro HIV infection, and triggers cell signaling directly via CD4. Jacalin as a lectin was suggested to trigger CD4-mediated cell signaling by interacting with the oligosaccharide side chains of CD4 located on Asn271 and Asn300. Such a hypothesis was of importance because it implied that the glycosylated chains could represent a functional domain directly involved in CD4-related cell activation. We analyzed this possibility by studying the effect of hapten sugars on jacalin-induced CD4 cell signaling and jacalin/CD4 interaction, and by studying the binding capacities of the lectin toward glycosylated, deglycosylated, and unglycosylated CD4. The results presented in this study provide evidence that jacalin does not recognize the CD4 oligosaccharide chains and actually binds CD4 through a specific protein-protein interaction; as a consequence these results rule out the involvement of the CD4 saccharide moieties in CD4-mediated cell signaling triggered by the lectin.
The role of calcium in the regulation of apoptosis
Authors: McConkey David J , Orrenius Sten
Published: January 2018

The recognition that apoptosis is regulated by an evolutionarily conserved set of polypeptides from the nematode Caenorhabditis elegant to humans suggests that a conserved set of biochemical mechanism(s) may also be involved in the response. Work from a number of independent laboratories suggests that alterations in cytosolic Ca2+ homeostasis represent one such candidate mechanism, and molecular targets for Ca2+ are now being identified. This review will summarize what is known about the role of Ca2+ in the regulation of apoptosis and discuss how Ca2+ might interact with some of the other biochemical signals implicated in cell death.
Killing of phagocytosed Staphylococcus aureus by human neutrophils requires intracellular free calcium
Authors: Wilsson åsa , Lundqvist Helen , Gustafsson Mikael , Stendahl Olle
Published: January 2018

The mobilization of intracellular calcium plays an important role in regulating neutrophil activation. With this in mind we investigated the effect of intra- and extracellular calcium on the ability of human neutrophils to kill complement-opsonized Staphylococcus aureus. We found that a rise in intracellular calcium is necessary for efficient killing of phagocytosed S. aureus. In the presence of extracellular calcium, killing of ingested bacteria in calcium-buffered neutrophils compared with normal cells was slightly reduced. Calcium buffering had no effect on phagocytic uptake by the neutrophils, but did decrease the generation of toxic oxygen metabolites, measured as chemiluminescence (CL). In non-depleted and calcium-depleted cells, removal of extracellular calcium did not affect ingestion but did cause a marked decrease in the ability to kill the bacteria. In parallel, the CL response was substantially reduced or completely blocked. These data show that calcium is not a prerequisite for phagocytosis of S. aureus by human neutrophils, but does play a vital role in the post-ingestion killing of the bacteria by regulating the generation of toxic oxygen metabolites.
Focal effects of mononuclear leukocyte transendothelial migration: TNF-α production by migrating monocytes promotes subsequent migration of lymphocytes
Authors: de Jong Andrew L , Green David M , Trial JoAnn , Birdsall Holly H
Published: January 2018

In many inflammatory diseases, mononuclear leukocytes (MNLs) accumulate as focal infiltrates in perivascular spaces. We postulated that MNLs migrating through endothelium modify the microenvironment to promote the subsequent migration of additional MNLs into the same area. We found that as monocytes adhere to and migrate spontaneously through an endothelial monolayer, they secrete tumor necrosis fector-α (TNF-α) and interleukin-1. These cytokines stimulate endothelial cell expression of CD54 (intercellular adhesion molecule-1) and CD106 (vascular cell adhesion molecule-1). Consequently, when freshly isolated MNLs are added to that endothelial monolayer four or more hours later, significantly greater numbers of lymphocytes bind to and migrate through these endothelial monolayers. In addition to its ability to activate endothelial cell adhesion molecules, TNF-α induced directed migration of lymphocytes through collagen pads. These results illustrate a potential amplification mechanism by which MNLs moving through a vessel wall may secrete TNF-α, leading to the recruitment of additional leukocytes into the same perivascular locus.
Human tumor antigens recognized by T lymphocytes: implications for cancer therapy
Authors: Wang Rong-Fu , Rosenberg Steven A
Published: January 2018

The adoptive transfer of cytotoxic T lymphocytes (CTLs) derived from tumor-infiltrating lymphocytes (TIL) along with interleukin-2 (IL-2) into autologous patients with cancer resulted in the objective regression of tumor, indicating that these CTLs recognized cancer rejection antigens on tumor cells. In the past year, a number of such tumor antigens were isolated by the use of cDNA expression systems and biochemical approaches. The majority of tumor antigens identified to date have been found to be nonmutated, self proteins. This raises important questions regarding the mechanism of antitumor activity and autoimmune disease. Several tumor-specific mutated tumor antigens have also been recently identified, which include cell cyclin-dependent kinase 4 (CDK4) and β-catenin. For the first time, a novel human cancer antigen was recently found to be generated by the use of an alternative open reading frame of the previously identified tyrosinase-related protein-1 (TRP-1) gene. The identification of human tumor rejection antigens provides new opportunities for the development of therapeutic strategies against cancer. The potential clinical applications of these tumor antigens will be discussed.
Detection of mouse IL-8 receptor homologue expression on peripheral blood leukocytes and mature myeloid lineage cells in bone marrow
Authors: Wang Jian-bin , Zhang Yi , Kasahara Tadashi , Harada Akihisa , Matsushima Kouji , Mukaida Naofumi
Published: January 2018

Interleukin-8 (IL-8) exhibits activities on bone marrow progenitor cells such as regulation of their growth and their mobilization into peripheral blood. In order to clarify the molecular mechanism of the effects of IL-8 on mouse bone marrow cells, we examined the cellular distribution of mouse IL-8 receptor homologue by an immunofluorescence analysis. Peripheral blood Gr-1+ mature granulocytes, and a substantial portion of NK1.1+ natural killer cells in peripheral blood and spleen were stained positively with anti-mouse IL-8 receptor homologue antibody, whereas CD4+, CD8+, or B220+ lymphocytes in peripheral blood and spleen, and thymocytes were not. Moreover, a small portion of ER-MP20+ monocytes in peripheral blood but neither peritoneal resident nor bone marrow macrophages were stained with anti-IL-8 receptor homologue antibody. In bone marrow, mature granulocytes and to a lesser degree, metamyelocytes and myelocytes expressed IL-8 receptor homologue. Moreover, lineage marker (Lin)-c-kit+ bone marrow progenitor cells started to express IL-8 receptor homologue only 5 days after in vitro culture with IL-3 and stem cell factor when metamyelocytes and myelocytes appeared. These results indicated that myeloid lineage cells express a substantial number of IL-8 receptor homologues only at the stage of myelocytes.
CAP37, a neutrophil granule-derived protein stimulates protein kinase C activity in endothelial cells
Authors: Pereira H Anne , Moore Pete , Grammas Paula
Published: January 2018

CAP37 is a multifunctional protein isolated from human neutrophils with important implications in host defense and inflammation. It is antimicrobial, mediates monocyte chemotaxis, and binds endotoxin. The interaction of neutrophils with endothelial cells is a central feature in inflammation. The object of this study was to determine whether CAP37, a neutrophil-derived protein, could regulate vascular endothelial cell protein kinase C (PKC), an important signaling enzyme. We found that CAP37 stimulated endothelial PKC activity in both a time-and dose-dependent fashion. This stimulation was comparable in magnitude to that evoked by phorbol myristate acetate. A monospecific antiserum against CAP37 inhibited CAP37-induced PKC activity. To establish a structural basis for this activity, overlapping peptides, based on the sequence of native CAP37 were synthesized. Maximum PKC stimulation was evoked by a peptide corresponding to amino acids 95–122 of native CAP37. This domain was distinct from the antibiotic and endotoxin binding domain of the molecule, which resides between amino acids 20 and 44. These data demonstrate that CAP37 can alter endothelial cell PKC and suggest that CAP37 may (day a role in neutrophil-endothelial interactions.
Protein tyrosine phosphorylation in leukocyte activation through receptors for IgG
Authors: Santana Carla , Noris Gino , Espinoza Bertha , Ortega Enrique
Published: January 2018

Membrane receptors for the Fc portion of immunoglobulin G (IgG) antibodies (FcγRs) are expressed on almost every type of hematopoietic cells, where they mediate a wide variety of effector functions. A high degree of structural heterogeneity exists among FcγRs. The biological significance of such heterogeneity is unknown, since the structural diversity does not appear to be reflected in the binding specificity nor in the effector functions that each distinct receptor is able to mediate. Recent work has emphasized the essential role of protein tyrosine phosphorylation in the initiation of trans-membrane signaling by these receptors. In this article we review the role of protein tyrosine phosphorylation in signal transduction by the different types of FcγRs in order to assess to what extent the structural heterogeneity of this receptor family is related to different activation pathways utilized by each of its members.
The inhibitory effect of prostaglandin E2 on rat neutrophil aggregation
Authors: Wise Helen
Published: January 2018

Rat peritoneal neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) produce an aggregation response that can be inhibited by prostaglandin E2 (PGE2) with an IC50 value of 2.6 10-9 M. Although PGE2 can stimulate [3H]cAMP production in neutrophils (EC50 4.3 10-8 M), the anti-aggregation response cannot be significantly attenuated by inhibitors of adenylate cyclase or protein kinase A, neither can it be potentiated by inhibition of phosphodiesterase activity. Despite these observations, it still remains possible that PGE2-mediated inhibition of rat neutrophil aggregation is a cAMP-dependent response mediated by highly localized changes in neutrophil cAMP. The inhibitory effect of PGE2 does not appear to depend on effects on intracellular calcium or KATP channels. Similarities exist between PGE2 and the profile of activity of phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, suggesting that PI 3-kinase is a possible target for PGE2 action in rat neutrophils.