PubMed Health⌕ Search

Biomedical subjects

Ian Dransfield

Publications and source records attributed to Ian Dransfield.

16 recordsLinked to original sources
PubMed2004

Golgi fragmentation during Fas-mediated apoptosis is associated with the rapid loss of GM130.

During apoptosis, the Golgi complex becomes fragmented and key proteins (e.g., GRASP65 and p115) are targets for caspase cleavage. GM130, an integral membrane protein, contributes to the maintenance of Golgi structure and facilitates membrane fusion with secretory vesicles. We show that GM130 levels decrease during Fas-induced apoptosis but not during staurosporine-induced apoptosis while in both models p115 levels remain unaffected. We conclude that GM130 is rapidly diminished during Fas-mediated apoptosis associated with Golgi fragmentation in contrast to previous studies which have suggested that loss of GM130 during apoptosis is a late event.

Apoptosis↗
PubMed2004

A critical 'threshold' of beta 2-integrin engagement regulates augmentation of cytokine-mediated superoxide anion release.

1. Neutrophil adhesion regulates a number of processes involved in the pathogenesis of inflammatory diseases including rheumatoid arthritis. Neutrophil destructive potential can be modulated by adhesion, allowing alteration of inflammatory cell behaviour while preserving antimicrobial defences. beta(2)-Integrin-mediated neutrophil adhesion to albumin-coated latex beads (ACLB) allows modulation of integrin clustering and ligation and analysis of the effects of adhesion on neutrophil responses. Tumour necrosis factor-alpha (TNF alpha) enhanced neutrophil binding of different diameter ACLB equally, by almost four-fold, and independently of bead size. Adhesion of neutrophils to ACLB caused a size-dependent generation and release of O(2)(-) and also potentiated TNF alpha-induced O(2)(-) release. 2. Binding of ACLB was not affected by disruption of cytoskeletal integrity with nocodazole or cytochalasin D or following blockade of tyrosine kinase activity. In contrast, tyrosine phosphorylation and an intact cytoskeleton were essential for adhesion- and cytokine-induced O(2)(-) release from neutrophils. Inhibition of adhesion- and cytokine-induced O(2)(-) release by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]pyrimidine (PP2) indicated that a Src-family tyrosine kinase was the principal regulatory pathway mediating this response in neutrophils, a distal role for p38 MAPK was revealed by use of SB203580. 3. Tyrosine phosphorylation of c-Fgr, a Src-family tyrosine kinase, occurred following ACLB adhesion and exposure to TNF alpha, and was susceptible to inhibition by PP2. We suggest that activation of the key regulatory enzyme c-Fgr is achieved following ligation of a critical threshold of integrins following binding of large (>3 microM) ACLB.

Binding Sites↗
PubMed2004

Immune complexes bind preferentially to Fc gamma RIIA (CD32) on apoptotic neutrophils, leading to augmented phagocytosis by macrophages and release of proinflammatory cytokines.

Many human inflammatory diseases are associated with tissue deposition of immune complexes and influx of neutrophils. We show that immune complexes bind preferentially to apoptotic neutrophils via FcgammaRIIA (CD32) and that increased binding is associated with clustering of immune complexes on the plasma membrane of the apoptotic cell. Phagocytosis of immune complex-opsonized apoptotic neutrophils by human macrophages was substantially enhanced (4.4-fold increase compared with control apoptotic neutrophils) and stimulated macrophages to release the proinflammatory cytokines TNF-alpha and IL-6. Immune complexes may perturb the normal pathways for clearance of apoptotic neutrophils by augmenting their clearance at the price of proinflammatory cytokine release. This represents a novel mechanism by which immune complexes may modulate the resolution of inflammation.

Animals↗
PubMed2004

Interferon gamma suppresses glucocorticoid augmentation of macrophage clearance of apoptotic cells.

One of beneficial effects of glucocorticoids (GC) in inflammation may be the augmentation of macrophages' capacity for phagocytosis of apoptotic cells, a process that has a central role in resolution of inflammation. Here we define the phenotype of GC-treated monocyte-derived macrophages, comparing to IFN-gamma-treated and IL-4-treated monocyte-derived macrophages and combinatorial treatment. Our data indicate that the cytokine microenvironment at an inflammatory site will critically determine monocyte functional capacity following treatment with GC. In particular, whilst GC exert dominant regulatory effects over IFN-gamma in terms of cell surface receptor repertoire and morphology, the acquisition of a macrophage capacity for clearance of apoptotic cells is prevented by combined treatment. In terms of mechanism, GC augmentation of phagocytosis was reversed even when monocytes were pre-incubated with GC for the first 24 h of culture, a period that is critical for induction of a highly phagocytic macrophage phenotype. These findings have important implications for the effectiveness of GC in promoting acquisition of a pro-phagocytic macrophage phenotype in inflammatory diseases associated with high levels of IFN-gamma

Apoptosis↗
PubMed2003

Phagocytosis of apoptotic cells by human macrophages: analysis by multiparameter flow cytometry.

BACKGROUND: Phagocytic removal of apoptotic cells is an important regulatory event in development, tissue homoeostasis, and inflammation. There are several methodologic problems with most in vitro studies of the molecular mechanisms of apoptotic cell phagocytosis. First, cell loss occurs during rigorous washing of adherent macrophages required to ensure removal of noningested particles. Second, discrimination of adherent or internalised apoptotic cells is difficult. Third, microscopic quantification is time consuming and has the potential for significant interobserver error. Fourth, subsequent analysis of phagocyte populations is difficult. METHODS: We used a flow cytometric method that allows quantification of phagocytosis of fluorescently labelled apoptotic cells with the use of multiparameter flow cytometric analysis. RESULTS: Phagocytosis of apoptotic cells was validated by use of inhibitors (cytochalasins) or low temperature and counterstaining with cell surface markers for the phagocytic targets to exclude binding to the phagocytic surface. Populations of phagocytic macrophages were sorted, and the presence of internalized apoptotic material was validated by microscopy. CONCLUSIONS: The technique we used in this study allows observer-independent analysis of phagocytosis of apoptotic cells by macrophages. Importantly, phagocytic or nonphagocytic populations could be subjected to further characterization with the use of flow cytometry with additional fluorochrome reagents and can be re-cultured to study underlying regulatory mechanisms.

Antigens, Surface↗
PubMed2003

Specific binding of an antigen-antibody complex to apoptotic human neutrophils.

Examination of apoptotic cell surface molecules has so far failed to reveal cell type-specific membrane alterations that serve as a signal for phagocytosis. In the present study we have identified a novel murine monoclonal antibody, BOB93, which bound to the surface of apoptotic neutrophils but not to apoptotic lymphocytes. BOB93 binding to apoptotic neutrophils was dependent on the presence of the sialoglycoprotein fetuin, a constituent of bovine serum. We demonstrate that fetuin is the antigen for BOB93, and that BOB93 and fetuin form a complex in solution that is necessary and sufficient for binding to apoptotic neutrophils. Individuals who were homozygous for an adenine nucleotide at position 519 of the gene for the immune complex receptor Fc gamma RIIA exhibited markedly reduced binding of BOB93/fetuin. This report is the first to provide evidence that antigen-antibody complexes bind specifically to apoptotic neutrophils and implicates apoptosis-associated changes in Fc gamma receptor function.

Animals↗
PubMed2003

Role of leukotrienes in the regulation of human granulocyte behaviour: dissociation between agonist-induced activation and retardation of apoptosis.

1 Since most inflammatory mediators that stimulate granulocyte responsiveness also delay apoptosis, it is often assumed that activation and longevity are causally related. Using isolated human peripheral blood neutrophils and eosinophils, we examined this association by exploiting the proinflammatory lipid mediators, the leukotrienes (LTs), and investigated granulocyte function and apoptosis. 2 LTB(4) induced elevation of intracellular free Ca(2+) concentration ([Ca(2+)](i)), cell polarisation and retardation of neutrophil apoptosis, although the antiapoptotic effect occurred only at concentrations > or =300 nM. LTB(4)-induced activation was attenuated by CP-105,696, a BLT1-specific antagonist suggesting classical LTB(4) receptor BLT1 involvement. 3 Despite demonstrating the presence of the neutrophil intracellular LTB(4) receptor peroxisome-proliferator activator receptor-alpha (PPARalpha) in neutrophils, the selective PPARalpha agonist WY-14,643 did not mimic LTB(4)-induced prosurvival effects. 4 LTB(4)-induced survival, however, also appeared to be mediated by BLT1 since CP-105,696 inhibited the LTB(4)-mediated antiapoptotic effect. Furthermore, based on studies with CP-105,696 and 5-lipoxygenase inhibitors, lipopolysaccharide (LPS)-, granulocyte-macrophage colony-stimulating factor (GM-CSF)-, dexamethasone- and dibutyryl-cAMP (db-cAMP)-induced delay of neutrophil apoptosis did not involve autocrine production of LTB(4). 5 Although LTB(4) and LTD(4) induced human eosinophil [Ca(2+)](i) elevation and polarization, these LTs did not influence eosinophil apoptosis. Furthermore, LTB(4)- and LTD(4)-induced eosinophil activation was attenuated by CP-105,696 and the Cys-LT(1) receptor antagonist montelukast, respectively, highlighting specific receptor dependency. 6 Thus, mediator-triggered granulocyte activation and antiapoptotic pathways are distinct events that can be differentially regulated.

Acetates↗
PubMed2003

Apoptosis: future perspectives.

The field of apoptosis has expanded to include research in almost all subject areas of cell biology. Despite this growth, there are still many unanswered questions, some of which are fundamental to the development of therapeutic strategies. Most importantly, the underlying mechanisms involved in conferring specificity of apoptotic signals must be determined. This will allow the development of treatments to delete injured or transformed cells and minimize damage to other tissues. Such information is also necessary to combat degenerative diseases, to prevent critical loss of cells. In addition, it would be of therapeutic benefit to selectively induce apoptosis in specific cells where removal of unwanted cells is required (e.g. tumour cells in malignant tissue or inflammatory cells responsible for tissue damage in chronic inflammatory disease). Therefore we have to understand how the full complement of signalling pathways, and survival and apoptotic proteins determine the end effect of an apoptotic signal. Apoptotic cells are known to functionally influence phagocytes that ingest them, but little is known of the effect of apoptosis on neighbouring cells or of signals generated during the apoptotic process itself. Although the processes of mitosis and apoptosis would appear to be diametrically opposed, cell-cycle proteins are involved in apoptosis, and may be able to influence cell-death mechanisms; conversely, proliferation may be caspase-dependent in some cells. Further study is necessary to discover how these pathways are interlinked since such knowledge would be particularly useful in cancer treatment. The link between pathogen-survival strategies and apoptotic mechanisms will also reward further work. Viruses, in particular, exploit cellular survival and death pathways for replication purposes and may represent a targeting approach for specific treatments.

Apoptosis↗
PubMed2003

Fetuin/alpha2-HS glycoprotein enhances phagocytosis of apoptotic cells and macropinocytosis by human macrophages.

Inflammatory diseases are associated with reduced serum concentrations of alpha(2)-HS glycoprotein (the human homologue of bovine fetuin), but the role of fetuin in inflammation is poorly understood. We hypothesized that fetuin may influence the resolution of inflammation by modulating the phagocytosis of apoptotic cells by macrophages. Using an in vitro flow cytometry-based phagocytosis assay, we investigated the role of fetuin in apoptotic cell clearance. Bovine fetuin and human alpha(2)-HS glycoprotein significantly augmented the phagocytosis of apoptotic cells by human peripheral blood monocyte-derived macrophages, whereas the control proteins BSA, sialylated BSA and asialofetuin were ineffective. The enhancement of phagocytosis was concentration-dependent, and required the presence of intact fetuin at the time of interaction between macrophages and apoptotic cells. Fetuin also substantially increased the uptake of labelled dextran 70000 by macrophages, which occurs by macropinocytosis, suggesting that this may be one of the mechanisms utilized for apoptotic cell uptake.

Apoptosis↗
PubMed2002

Cytokine-dependent inflammatory cell recruitment patterns in the peritoneal cavity of mice exposed to the parasitic nematode Brugia malayi.

Mice exposed intraperitoneally to either adult or first larval stage (microfilaria) of the human nematode parasite Brugia malayi display polarized cytokine responses. We have used this model to investigate the impact of altered cytokine profiles on inflammatory cell recruitment patterns in vivo. Here we demonstrate that Th2-inducing adult parasites drive the recruitment of eosinophils and macrophages after implant into the murine peritoneal cavity whereas Th1-inducing microfilaria do not. The underlying mechanism of recruitment was further defined by use of mice deficient in the key Th2 cytokines IL-4 or IL5 and mice that lack T cells (nude mice). Recruitment dynamics differed in IL-4 and IL-5 deficient mice, showing reduced or absent eosinophilia. These data emphasize the pivotal role of these cytokines in shaping the cellular profile of inflammatory responses. Surprisingly, the absence of T cells failed to influence inflammatory cell recruitment indicating that recruitment signals are provided by other cell types.

Animals↗
PubMed2002

Prostaglandin D2 and its metabolites induce caspase-dependent granulocyte apoptosis that is mediated via inhibition of I kappa B alpha degradation using a peroxisome proliferator-activated receptor-gamma-independent mechanism.

Many inflammatory mediators retard granulocyte apoptosis. Most natural PGs studied herein (e.g., PGE(2), PGA(2), PGA(1), PGF(2 alpha)) either delayed apoptosis or had no effect, whereas PGD(2) and its metabolite PGJ(2) selectively induced eosinophil, but not neutrophil apoptosis. This novel proapoptotic effect does not appear to be mediated via classical PG receptor ligation or by elevation of intracellular cAMP or Ca(2+). Intriguingly, the sequential metabolites Delta(12)PGJ(2) and 15-deoxy-Delta(12,) Delta(14)-PGJ(2) (15dPGJ(2)) induced caspase-dependent apoptosis in both granulocytes, an effect that did not involve de novo protein synthesis. Despite the fact that Delta(12)PGJ(2) and 15dPGJ(2) are peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activators, apoptosis was not mimicked by synthetic PPAR-gamma and PPAR-alpha ligands or blocked by an irreversible PPAR-gamma antagonist. Furthermore, Delta(12)PGJ(2) and 15dPGJ(2) inhibited LPS-induced I kappa B alpha degradation and subsequent inhibition of neutrophil apoptosis, suggesting that apoptosis is mediated via PPAR-gamma-independent inhibition of NF-kappa B activation. In addition, we show that TNF-alpha-mediated loss of cytoplasmic I kappa B alpha in eosinophils is inhibited by 15dPGJ(2) in a concentration-dependent manner. The selective induction of eosinophil apoptosis by PGD(2) and PGJ(2) may help define novel therapeutic pathways in diseases in which it would be desirable to specifically remove eosinophils but retain neutrophils for antibacterial host defense. The powerful proapoptotic effects of Delta(12)PGJ(2) and 15dPGJ(2) in both granulocyte types suggest that these natural products control the longevity of key inflammatory cells and may be relevant to understanding the control and resolution of inflammation.

Apoptosis↗
PubMed2002

Increased platelet binding to circulating monocytes in acute coronary syndromes.

BACKGROUND: Present therapies for acute coronary syndromes aim toward limiting platelet-platelet adhesion and aggregation processes. However, platelet-leukocyte interactions may contribute importantly to disease progression in the arterial wall. Recent studies suggest that prevention of platelet-leukocyte binding via P-selectin glycoprotein ligand-1 (PSGL-1) may be beneficial in animal models of vascular injury. METHODS AND RESULTS: P-selectin-PSGL-1 interactions were found to account for most platelet-monocyte binding observed in peripheral blood samples from healthy donors. However, a significant component of observed adhesion was calcium independent, involving neither PSGL-1 nor P-selectin. Platelet-monocyte interactions were examined in 52 patients admitted within 14 hours of symptom onset, with acute coronary syndromes defined as unstable angina (n=12) and acute myocardial infarction (n=13) or noncardiac chest pain (n=27). When compared with patients with noncardiac chest pain, significantly elevated levels of platelet-monocyte binding were found in patients with acute myocardial infarction (70.1+/-15.4% versus 45.4+/-23.3%; P<0.01) and unstable angina (67.4+/-12.9% versus 45.4+/-23.3%; P>0.01). Calcium-independent platelet-monocyte binding was significantly elevated in myocardial infarction patients alone (14.7+/-7.7% versus 6.1+/-5.96%; P<0.001). CONCLUSIONS: There is evidence for a significant P-selectin-independent molecular component to the platelet-monocyte conjugation observed in peripheral blood. Patients with myocardial infarction and unstable angina demonstrate increased total binding of platelets to monocytes. Additionally, calcium-independent adhesion was significantly elevated in patients with evidence of myocardial infarction. These findings demonstrate that novel cation-independent adhesion mechanisms may mediate platelet-monocyte binding, representing a new therapeutic target after vascular injury associated with myocardial infarction.

Adult↗
PubMed2002

Inhibition of nuclear factor-kappaB activation un-masks the ability of TNF-alpha to induce human eosinophil apoptosis.

Apoptosis renders eosinophils functionally effete and marks them for "silent" removal from inflamed sites by macrophages. We show, for the first time, that eosinophils exposed to TNF-alpha rapidly lose their cytoplasmic levels of IkappaBalpha, the inhibitory subunit of NF-kappaB. Consequently, TNF-alpha triggers NF-kappaB mobilization from the cytoplasm to the nucleus, as determined by tracking the NF-kappaB subunit p65 by immunofluorescence and Western blot analysis. Inhibition of TNF-alpha-mediated IkappaBalpha degradation and NF-kappaB activation by gliotoxin or the proteasome inhibitor MG-132 un-masks the caspase-dependent pro-apoptotic properties of TNF-alpha. In addition, cycloheximide similarly renders TNF-alpha pro-apoptotic, suggesting that NF-kappaB activation controls the production of a protein(s) that protects eosinophils from the cytotoxic effects of TNF-alpha. Evidence is presented suggesting that TNF-alpha triggered apoptosis is more susceptible to NF-kappaB inhibition than constitutive apoptosis, leading to the possibility of the specific targeting of apoptosis in eosinophil sub-populations. Prior to morphological signs of apoptosis, TNF-alpha-induced IL-8 synthesis is abrogated by inhibition of NF-kappaB. We propose that NF-kappaB activation plays a critical role in controlling eosinophil responsiveness and apoptosis, and speculate that selective inhibitors of eosinophil NF-kappaB activation may ultimately provide alternative therapeutic agents for the treatment of eosinophilic diseases, including asthma and allergic rhinitis.

Apoptosis↗
PubMed2002

A blast from the past: clearance of apoptotic cells regulates immune responses.

Apoptosis, which is a programmed and physiological form of cell death, is known to shape the immune system by regulating populations of effector lymphocytes. However, the binding and ingestion of dying cells by monocytes/macrophages and dendritic cells can also influence immune responses markedly by enhancing or suppressing inflammation. Therefore, dead cells, which are a reflection of an organism's immediate past, can control its immunological future.

Animals↗
PubMed2002

Role of macrophage CD44 in the disposal of inflammatory cell corpses.

Understanding the cellular and molecular mechanisms that determine whether inflammation resolves or progresses to scarring and tissue destruction should lead to the development of effective therapeutic strategies for inflammatory diseases. Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation, providing a mechanism for down-regulation of function and triggering clearance by macrophages without inducing a pro-inflammatory response. However, if the rate of cell death by apoptosis is such that the macrophage clearance capacity is exceeded, apoptotic cells may progress to secondary necrosis, resulting in the release of harmful cellular contents and in damage to the surrounding tissue. There are many possible ways in which the rate and capacity of the macrophage-mediated clearance of apoptotic cells may be enhanced or suppressed. Ligation of human macrophage surface CD44 by bivalent monoclonal antibodies rapidly and profoundly augments the capacity of macrophages to phagocytose apoptotic neutrophils in vitro. The molecular mechanism behind this effect and its potential significance in vivo is a current focus of research.

Antibodies, Bispecific↗