Biomedical subjects

I Dransfield

Publications and source records attributed to I Dransfield.

At least 19 recordsLinked to original sources

PubMed2000

Soluble E-selectin acts in synergy with platelet-activating factor to activate neutrophil beta 2-integrins. Role of tyrosine kinases and Ca2+ mobilization.

Selectins play a critical role in neutrophil recruitment to sites of inflammation, in tethering and rolling of neutrophils on vascular endothelium, as well as triggering beta(2)-integrin-mediated adhesion. We have previously demonstrated potential pro-inflammatory effects of soluble E-selectin upon neutrophil effector functions, using a soluble recombinant molecule (E-zz), which increased beta(2)-integrin-mediated adhesion, decreased beta(2)-integrin-dependent migration, and triggered reactive oxygen species generation and release. In this study, we have examined the intracellular signals following neutrophil activation by soluble E-selectin. We show that exposure of neutrophils to E-selectin and platelet-activating factor (PAF) in combination induced a synergistic effect upon beta(2)-integrin-mediated adhesion. Although soluble E-selectin did not induce Ca(2+) mobilization in neutrophils by itself, elevation of intracellular Ca(2+) was specifically prolonged in response to PAF but not leukotriene B(4) or N-formyl-Met-Leu-Phe. The prolonged Ca(2+) mobilization observed in the presence of E-selectin was dependent on Ca(2+) influx from intracellular stores rather than influx of extracellular Ca(2+) through SKF 96365-sensitive channels. The specific alteration of Ca(2+) mobilization reported here appears not to have a role in the synergistic effects of E-selectin and PAF upon neutrophil O(2) release but may be involved in augmentation of beta(2)-integrin-mediated adhesion.

CD18 Antigens↗

PubMed2000

Molecular characterization of the surface of apoptotic neutrophils: implications for functional downregulation and recognition by phagocytes.

We have used a panel of monoclonal antibodies and lectins to examine the profile of surface molecule expression on human neutrophils that have undergone spontaneous apoptosis during in vitro culture. Neutrophil apoptosis was found to be accompanied by down-regulation of the immunoglobulin superfamily members PECAM-1 (CD31), ICAM-3 (CD50), CD66acde, and CD66b and the integrin-associated proteins CD63 and urokinase plasminogen activator receptor (CD87) that may alter the potential for adhesive interactions. Cellular interactions may be further influenced by the reduction of the expression of surface carbohydrate moieties, including sialic acid. Reduced expression of FcgammaRII (CD32), complement receptor type 1 (CD35) and receptors for pro-inflammatory mediators C5a (CD88) and TNFalpha (CD120b) associated with apoptosis might limit neutrophil responsiveness to stimuli that trigger degranulation responses. Although many of the receptors we have examined are expressed at reduced levels on apoptotic neutrophils, we found that there was differential loss of certain receptors (e.g. CD16, CD15 and CD120b) and increased expression of aminopeptidase-N (CD13). Together with our previous data showing that expression of certain molecules e.g. LFA-3 (CD58) is not altered during neutrophil apoptosis, these data are suggestive of specific changes in receptor mobilisation and shedding associated with apoptosis. Although reduced expression of CD63 (azurophilic granules) and CR1 (specific granules) indicates that granule mobilisation does not accompany apoptosis, a monoclonal antibody (BOB78), that recognises a 90 kDa antigen localised in intracellular granules, defines a subpopulation of apoptotic neutrophils that exhibit nuclear degradation yet retain intact plasma membranes. BOB78 positive neutrophils were found to bind biotinylated thrombospondin, suggesting that this mAb defines surface molecular changes associated with exposure of thrombospondin binding moieties.

Animals↗

PubMed1999

Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes.

Phagocyte recognition, uptake, and nonphlogistic degradation of neutrophils and other leukocytes undergoing apoptosis promote the resolution of inflammation. This study assessed the effects of anti-inflammatory glucocorticoids on this leukocyte clearance mechanism. Pretreatment of "semimature" 5-day human monocyte-derived macrophages (M phi) for 24 h with methylprednisolone, dexamethasone, and hydrocortisone, but not the nonglucocorticoid steroids aldosterone, estradiol, and progesterone, potentiated phagocytosis of apoptotic neutrophils. These effects were specific in that the potentiated phagocytosis of apoptotic neutrophils was completely blocked by the glucocorticoid receptor antagonist RU38486, and glucocorticoids did not promote 5-day M phi ingestion of opsonized erythrocytes. Similar glucocorticoid-mediated potentiation was observed with 5-day M phi uptake of alternative apoptotic "targets" (eosinophils and Jurkat T cells) and in uptake of apoptotic neutrophils by alternative phagocytes (human glomerular mesangial cells and murine M phi elicited into the peritoneum or derived from bone marrow). Importantly, methylprednisolone-mediated enhancement of the uptake of apoptotic neutrophils did not trigger the release of the chemokines IL-8 and monocyte chemoattractant protein-1. Furthermore, longer-term potentiation by methylprednisolone was observed in maturing human monocyte-derived M phi, with greater increases in 5-day M phi uptake of apoptotic cells being observed the earlier glucocorticoids were added during monocyte maturation into M phi. We conclude that potentiation of nonphlogistic clearance of apoptotic leukocytes by phagocytes is a hitherto unrecognized property of glucocorticoids that has potential implications for therapies aimed at promoting the resolution of inflammatory diseases.

Animals↗

PubMed1999

Pharmacological manipulation of granulocyte apoptosis: potential therapeutic targets.

Resolution of inflammation involves the clearance of excess or effete inflammatory cells by a process of physiological programmed cell death (apoptosis) and the subsequent recognition and removal of apoptotic cells by phagocytes. The therapeutic induction of apoptosis for the resolution of chronic inflammation and the general pharmacology of apoptosis have become subjects of increasing interest. In this article, some of the unique and important differences in the control of apoptosis of various inflammatory cells (particularly neutrophil and eosinophil granulocytes) are highlighted. It is suggested that apoptosis can be specifically regulated pharmacologically and could be exploited to develop new drug therapies.

Apoptosis↗

PubMed1999

Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo.

Resistance to chemotherapy is a principal problem in the treatment of small cell lung cancer (SCLC). We show here that SCLC is surrounded by an extensive stroma of extracellular matrix (ECM) at both primary and metastatic sites. Adhesion of SCLC cells to ECM enhances tumorigenicity and confers resistance to chemotherapeutic agents as a result of beta1 integrin-stimulated tyrosine kinase activation suppressing chemotherapy-induced apoptosis. SCLC may create a specialized microenvironment, and the survival of cells bound to ECM could explain the partial responses and local recurrence of SCLC often seen clinically after chemotherapy. Strategies based on blocking beta1 integrin-mediated survival signals may represent a new therapeutic approach to improve the response to chemotherapy in SCLC.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Differential effects of bacterial lipopolysaccharides upon neutrophil function.

Lipopolysaccharide (LPS) is a potent inflammatory agent which augments neutrophil sensitivity to subsequent inflammatory stimuli. In this study, the effects of structurally different LPS types upon neutrophil effector functions were examined. Rough LPS types, which have lost the O-polysaccharide moiety, were found to act more rapidly than smooth LPS types in stimulating neutrophil beta2 integrin activity and fMLP-induced respiratory burst. These findings suggest an involvement of the O-polysaccharide region of LPS in regulating neutrophil responsiveness to different LPS chemotypes with important implications for the mechanisms underlying regulation of the inflammatory response in conditions associated with elevation of LPS in plasma, e.g. septic shock or acute respiratory distress syndrome.

CD18 Antigens↗

PubMed1998

Requirement for in vivo production of IL-4, but not IL-10, in the induction of proliferative suppression by filarial parasites.

Loss of T lymphocyte proliferation and the emergence of a host response that is dominated by a Th2-type profile are well-established features of human filariasis. We have previously reported that adherent peritoneal exudate cells (PEC) from mice transplanted with adult Brugia malayi parasites suppress the proliferation of lymphocytes without blocking Ag-cytokine production in vitro. We now show that infection of mice with the infective larval (L3) stage of B. malayi generates a similar population of PEC. Suppressive cells are generated within 7 days of infection and mediate their effects through a nitric oxide-independent pathway. Both L3 and adult infection elicit high levels of host IL-4 whereas the microfilarial stage of the parasite induces IFN-gamma production and does not generate a similar form of suppression. Production of host IL-4 was necessary to allow the generation of suppressive PEC, given that IL-4-deficient mice implanted with adult parasites failed to induce proliferative block. However, IL-10-deficient mice implanted with adult parasites resulted in T cell suppression, indicating that IL-10 is not essential for the induction of hyporesponsiveness. Neither IL-4 nor IL-10 were directly responsible for ablating cellular proliferation in vitro, as the addition of neutralizing Ab to either cytokine did not reverse the proliferative block. Thus, IL-4 produced in vivo in response to filarial L3 and adult parasites is essential for the induction of proliferative suppression but is not itself the suppressive factor.

Animals↗

PubMed1998

Regulation of macrophage phagocytosis of apoptotic cells by cAMP.

Regulation of macrophage capacity to remove apoptotic cells may control the balance of apoptotic and necrotic leukocytes at inflamed foci and the extent of leukocyte-mediated tissue damage. Although the molecules involved in the phagocytic process are beginning to be defined, little is known about the underlying regulatory and signaling mechanisms controlling this process. In this paper, we have investigated the effects of treatment of human monocyte-derived macrophages with PGs and other agents that elevate intracellular cAMP on phagocytosis. PGE2 and PGD2 specifically reduced the proportion of macrophages that phagocytosed apoptotic cells. Similar results were obtained with the membrane-permeable cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP but not with the cGMP analogue dibutyryl-GMP. Consistent with the observation that phagocytosis was inhibited by cAMP elevation, treatment of monocyte-derived macrophages with PGE2 resulted in rapid, transient increase in levels of intracellular cAMP. These effects were not due to nonspecific inhibition of monocyte-derived macrophage phagocytosis given that ingestion of Ig-opsonized erythrocytes was unaffected. Elevation of cAMP induced morphologic alterations indicative of changes in the adhesive status of the macrophage, including cell rounding and disassembly of structures that represent points of contact with substrate containing actin and talin. These results strongly suggest that rapid activation of cAMP signaling pathways by inflammatory mediators regulates processes that limit tissue injury and that modulation of cAMP levels represents an additional therapeutic target in the control of resolution of inflammation.

Apoptosis↗

PubMed1998

Requirement for in vivo production of IL-4, but not IL-10, in the induction of proliferative suppression by filarial parasites.

Animals↗

PubMed1998

Constitutive neutrophil apoptosis in culture is modulated by cell density independently of beta2 integrin-mediated adhesion.

Although inflammatory mediators modulate the rate of constitutive neutrophil apoptosis in vitro the effects of micro-environmental conditions have not been fully investigated. In this study, we demonstrate that the rate of constitutive neutrophil apoptosis is affected by the number of cells per unit surface area, with enhanced survival at high cell density. Furthermore, the presence of protein or serum in the culture medium also enhances neutrophil survival. These effects were independent of beta2 integrin-mediated adhesion and were not influenced by specific adhesion to extracellular matrix components. Thus, the rate of neutrophil apoptosis is fundamentally influenced by microenvironmental conditions and indicates that factors such as cell density and extracellular protein concentration must be considered when investigating mechanisms regulating inflammatory cell apoptosis in vitro.

Animals↗

PubMed1998

Potential pro-inflammatory effects of soluble E-selectin upon neutrophil function.

Appropriate recruitment of neutrophils to sites of infection or tissue injury is a key event in the inflammatory response. A number of studies have shown the critical role of selectins in tethering and rolling of neutrophils on vascular endothelium, as well as a more complex regulatory role, since they have the potential to alter leukocyte recruitment by triggering beta2 integrin-mediated adhesion. In this study, we report that in contrast to patients "at risk" of developing acute respiratory disease syndrome (ARDS), elevated plasma levels of soluble E-selectin are found in patients with established disease. Since neutrophil granulocytes are implicated in ARDS pathogenesis, we have investigated the possibility of a link between elevated soluble plasma E-selectin levels and disease progression by examining the effects of soluble recombinant E-selectin (E-zz) upon neutrophil function. In this paper, we describe the novel finding that exposure of neutrophils to E-zz potentiates a number of neutrophil functions which may act to drive inflammatory processes. Although neutrophil deformability, an important parameter determining retention within the lung microvasculature, was not affected by E-zz, neutrophil polarization was observed. In addition, neutrophil beta2 integrin-mediated adhesion was found to be augmented by E-zz without alteration in levels of surface expression of alphaMbeta2 or the "activation" reporter epitope defined by monoclonal antibody 24. Concomitantly with increased beta2 integrin-mediated adhesion, we observed an inhibition of formyl-Met-Leu-Phe-directed chemotaxis. Together with an augmentation of neutrophil reactive oxidant species production and release of superoxide anions, these data raise the possibility that soluble E-selectin exerts pro-inflammatory effects upon neutrophil function at sites of inflammation, thereby exacerbating disease processes.

CD18 Antigens↗

PubMed1998

Regulation of macrophage phagocytosis of apoptotic neutrophils by adhesion to fibronectin.

The potential for leukocyte-mediated host tissue damage during resolution of inflammatory responses is influenced by the rate at which extravasated apoptotic leukocytes are cleared from inflammatory sites. Regulation of macrophage capacity for clearance of apoptotic granulocytes is likely to be an important factor determining whether inflammation ultimately resolves or progresses to a chronic state. In this study we have investigated the molecular basis for rapid augmentation of macrophage phagocytosis of apoptotic neutrophils, which was observed following macrophage adhesion to fibronectin. We used a combination of monoclonal antibodies, blocking peptides, and recombinant fibronectin fragments to investigate the role of beta1 integrins in mediating the fibronectin effects. Blockade of alpha5beta1 or alpha4beta1 alone did not attenuate fibronectin-augmentation of phagocytosis. In addition, adhesion of macrophages to recombinant fibronectins lacking alpha4beta1 recognition motifs failed to promote phagocytosis of apoptotic neutrophils. Our results would be consistent with a model in which multiple fibronectin receptors, including beta1 integrins, act co-operatively to augment macrophage phagocytic responses. Together, these data suggest that the extracellular matrix environment of macrophages may provide regulatory signals that act indirectly to rapidly alter the potential for removal of apoptotic cells and influence the process of resolution of inflammation.

Antibodies, Blocking↗

PubMed1998

You can lead a macrophage to an inflammatory site but can you make it eat?

Explore the source record for details and available documents.

Cell Adhesion Molecules↗

PubMed1997

Regulation of neutrophil apoptosis by tumor necrosis factor-alpha: requirement for TNFR55 and TNFR75 for induction of apoptosis in vitro.

Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-alpha (TNF-alpha), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-alpha inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4, and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-alpha was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-alpha neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-alpha to accelerate apoptosis was lost if neutrophils were primed with 1 micromol/L PAF or aged for 6 hours before TNF-alpha addition. The TNFR55-selective TNF-alpha mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-alpha. Although the TNFR75-selective mutant (D143F) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-alpha-stimulated apoptosis. Hence, TNF-alpha has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.

Antibodies, Monoclonal↗

PubMed1997

CD44 regulates phagocytosis of apoptotic neutrophil granulocytes, but not apoptotic lymphocytes, by human macrophages.

Phagocytosis of apoptotic neutrophil granulocytes by macrophages at inflammatory sites is an important determinant of the process by which inflammation resolves. We demonstrate that phagocytosis of apoptotic neutrophils, but not apoptotic lymphocytes, by human monocyte-derived macrophages is augmented rapidly following ligation of CD44 by bivalent Abs in vitro. Previously defined inhibitors of apoptotic cell recognition did not affect CD44-augmented phagocytosis of apoptotic neutrophils, suggesting that unique molecular recognition pathways are involved. These observations, together with the lack of effect of CD44 Abs upon macrophage phagocytosis of zymosan or Ig-opsonized erythrocytes, imply that CD44 may regulate the differential clearance of apoptotic leukocytes during evolution of inflammatory responses. This represents a novel role for CD44 in inflammation and tissue repair.

Apoptosis↗

PubMed1997

Activation of neutrophil function via CD66: differential effects upon beta 2 integrin mediated adhesion.

To further define the role of CD66 glycoproteins in the regulation of neutrophil function, we analysed the effects of a CD66 monoclonal antibody, Kat4c, which recognizes an epitope present on AB domains of CD66a, CD66b and CD66c. Intact Kat4c and F(ab')2 fragments were found to augment fMLP-induced oxidation of 1,2,3-dihydrorhodamine (oxidant species production) and beta 2 integrin-mediated adhesion to fibrinogen but did not promote beta 2 integrin-mediated binding of albumin coated latex beads. Since the latter assay is a sensitive indicator of neutrophil CD11b/CD18 functional activation, these results imply CD66 may exert differential effects upon beta 2 integrin activity. Neutrophil oxidant species production and spreading on fibrinogen substrates were further potentiated by cross-linking of Kat4c F(ab')2, in keeping with the suggestion that ligation of CD66 regulates neutrophil function. However, although intact Kat4c promoted beta 2 integrin-dependent homotypic neutrophil adhesion, F(ab')2 fragments were without effect, implying a role for Fc receptors in this effect which has previously been attributed to CD66. Together these data define more clearly the role of CD66 in regulation of neutrophil function and further suggest that augmented beta 2 integrin-mediated adhesion following CD66 ligation occurs independently of affinity regulation.

Antibodies, Monoclonal↗

PubMed1996

Recognition of apoptotic cells by phagocytes.

Effective removal of dying cells is crucial to a variety of processes in health and disease. Cells undergoing apoptosis are recognized and ingested intact by phagocytes, which are not stimulated to release inflammatory mediators. The alternative uncontrolled form of cell death, necrosis, is associated with release of cell contents with the potential to cause tissue damage and inflammation. Four distinct molecular mechanisms have been identified to date which mediate recognition by phagocytes of mammalian cells undergoing apoptosis, but further mechanisms remain to be discovered. The capacity for phagocyte removal of cells undergoing apoptosis may be closely regulated, for example by local cytokines.

Animals↗

PubMed1996

Inhibition of neuropeptide-stimulated tyrosine phosphorylation and tyrosine kinase activity stimulates apoptosis in small cell lung cancer cells.

Small cell lung cancer (SCLC) cell growth is sustained by multiple autocrine and paracrine growth loops involving neuropeptides. The bombesin family of peptides are autocrine growth factors in H345 SCLC cells and provide a paradigm for the study of growth factors and mitogenic signaling in SCLC cells. We show that bombesin (and other neuropeptides) stimulates protein tyrosine phosphorylation (particularly focal adhesion kinase) and protein tyrosine kinase (PTK) activity in intact SCLC cells. Furthermore, the broad spectrum neuropeptide receptor antagonist [D-Arg, D = Phe, D-Trp, Leu11]substance P inhibits all neuropeptide-mediated signals (including PTK activation), SCLC cell growth in vivo and in vitro, and also increases the natural rate of apoptosis seen in growing SCLC cell lines. Hence the effect of selective PTK inhibition on SCLC cell growth and apoptosis was examined. We show that selective inhibition of PTK activity, with genistein and (3,4,5-tri-hydroxyphenyl)-methylene(-propanedinitrile) tyrphostin-25 inhibits basal and neuropeptide-stimulated SCLC cell growth. Genistein and tyrphostin-25 also stimulate apoptosis in SCLC cells. Inhibition of proliferation in these cells is intimately linke to apoptosis, because these changes occurred without any effect on SCLC cell cycle kinetics, suggesting that apoptosis occurs independently of the cell cycle and that failure to progress through the cell cycle results in apoptosis. Because tyrphostin-25 fails to influence p53 or Bcl-2 expression in these cells, this mode of programmed cell death appears to be via a p53- and Bcl-2-independent mechanism. These results provide evidence that tyrosine phosphorylation is a mitogenic signal in SCLC cells and suggest that regulation of the level of protein tyrosine phosphorylation represents a critical determinant of whether SCLC cells survive and proliferate or die by apoptosis. Thus PTK inhibition may provide a novel therapeutic option in SCLC that has become resistant to conventional chemotherapeutic agents.

Apoptosis↗