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C Haslett

Publications and source records attributed to C Haslett.

At least 109 records · Page 6Linked to original sources
PubMed1993

Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro.

Elevation of cytosolic calcium ([Ca2+]i) has been reported to induce apoptosis in a number of cell types. However, in the neutrophil, which undergoes apoptosis constitutively during aging in vitro, activation by inflammatory mediators elevates [Ca2+]i and prolongs lifespan via inhibition of apoptosis. To examine this paradox, we investigated the effects of modulation of [Ca2+]i upon apoptosis of neutrophils in vitro. Calcium ionophores (A23187, ionomycin) retarded apoptosis in neutrophil populations after 20 h (P < 0.001). Conversely, intracellular Ca(2+)-chelation, using bis-(o-aminophenoxy)-N,N,N'N'-tetraacetic acid (BAPTA) acetoxymethyl ester (AM) promoted apoptosis (P < 0.02). W-7 (an inhibitor of calmodulin) also promoted apoptosis (P < 0.05). Measurements of [Ca2+]i, using fura-2, showed (a) increased apoptosis in neutrophil populations was not associated with elevated [Ca2+]i, (b) neutrophils cultured with ionophore at concentrations inhibiting apoptosis exhibited transient (< 1 h) elevations of [Ca2+]i, to levels previously reported with receptor-mediated stimuli, and (c) BAPTA was able to prevent the elevation of [Ca2+]i and the inhibition of apoptosis produced by ionophore. Modulation of apoptosis occurred without alterations in intracellular pH. Thus, in the neutrophil, unlike lymphoid cells, elevation of [Ca2+]i exerts an inhibitory effect upon apoptosis. Furthermore, these data suggest that transient elevation of [Ca2+]i elicits signaling events leading to prolonged inhibition of apoptosis.

Apoptosis↗
PubMed1993

Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators.

Neutrophil apoptosis leads to macrophage ingestion of intact senescent neutrophils. This may represent a neutrophil removal mechanism that is important both in the control of inflammatory tissue injury and for the normal resolution processes of inflammation. Because apoptosis is likely to be a key control process in cell and tissue homeostasis, a number of inflammatory mediators were tested for their ability to modulate the rate of apoptosis in populations of neutrophils aging in culture. Endotoxic lipopolysaccharide, human recombinant complement factor 5a, and human recombinant granulocyte-macrophage colony-stimulating factor all markedly inhibited the rate of neutrophil apoptosis in a concentration-dependent fashion, without inducing necrosis (as assessed by trypan blue exclusion). This inhibitory effect on the rate of neutrophil apoptosis was shown by morphological criteria and confirmed by gel electrophoresis of extracted DNA. Inhibition of apoptosis of aging neutrophil populations was associated with prolongation of the functional life span of the population as assessed by the ability of neutrophils to spread on glass surfaces, to polarize in response to deliberate stimulation with N-formyl-Met-Leu-Phe (fMLP), and to release the granule enzyme marker myeloperoxidase on fMLP stimulation. These observations show that inflammatory mediators prolong the functional life span of neutrophils through modulation of apoptosis. Further elucidation of these mechanisms will lead to a better understanding of the processes controlling neutrophil residence and function in inflamed tissues and may provide further insights into the molecular mechanisms of apoptosis, which is of widespread importance in tissue biology.

Apoptosis↗
PubMed1992

Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells.

One of the key features associated with programmed cell death in many tissues is the phagocytosis of apoptotic bodies by macrophages. Removal of apoptotic cells occurs before their lysis, indicating that these cells, during the development of apoptosis, express specific surface changes recognized by macrophages. We have compared the mechanisms by which four different macrophage populations recognize apoptotic cells. Murine macrophages elicited into the peritoneal cavity with either of two different phlogistic agents were able to phagocytose apoptotic cells. This phagocytosis was inhibited by phosphatidylserine (PS), regardless of the species (human or murine) or type (lymphocyte or neutrophil) of the apoptotic cell. In contrast, the murine bone marrow macrophage, like the human monocyte-derived macrophage, utilized the vitronectin receptor, an alpha v beta 3 integrin, for the removal of apoptotic cells, regardless of their species or type. That human macrophages are capable, under some circumstances, of recognizing PS on apoptotic cells was suggested by the observation that PS liposomes inhibited phagocytosis by phorbol ester-treated THP-1 cells. These results suggest that the mechanism by which apoptotic cells are recognized and phagocytosed by macrophages is determined by the subpopulation of macrophages studied.

Animals↗
PubMed1992

Apoptosis in human eosinophils. Programmed cell death in the eosinophil leads to phagocytosis by macrophages and is modulated by IL-5.

Eosinophils are believed to injure tissues in a variety of allergic disease by virtue of their highly histotoxic contents and metabolites. They are readily observed in tissues during the allergic response yet the mechanisms governing the duration of tissue residence and route of removal remain obscure. We have previously reported in vitro and in vivo evidence that neutrophils undergo apoptosis (programmed cell death) and are recognized and ingested as intact cells by macrophages. We report that eosinophils, purified from the peripheral blood of asymptomatic healthy atopics, undergo apoptosis in vitro. After 72 to 96 h in culture, 57.0 +/- 6.2% (mean +/- SE) of the eosinophil population showed characteristic morphologic changes of apoptosis. Electrophoresis of the DNA from these cells demonstrated the typical "ladder" pattern of internucleosomal DNA cleavage, the hallmark of apoptosis-associated endonuclease activation. The rate of eosinophil apoptosis, slower than that reported for neutrophils, was delayed (by 80 +/- 6 h) in the presence of recombinant human IL-5, a cytokine previously reported to prolong eosinophil life in vitro but not known to modulate apoptosis. Aged, apoptotic eosinophils, but not fresh or aged preapoptotic eosinophils, were recognized and ingested as intact cells by macrophages. Apoptosis and ingestion by macrophages may represent a mechanism whereby the tissue longevity and removal of eosinophils is controlled.

Cell Death↗
PubMed1992

Resolution of acute inflammation and the role of apoptosis in the tissue fate of granulocytes.

We have identified a pathway for granulocyte removal in tissues which is controlled by apoptosis. This process can be modulated by agents in the inflammatory microenvironment and leads to loss of neutrophil secretory function and its phagocytosis by macrophages which utilize a novel recognition mechanism such that the macrophage does not release pro-inflammatory mediators. It is hypothesized that this represents an alternative fate to necrosis, and one which would tend to limit tissue injury and promote inflammatory resolution. This is not to suggest that granulocyte necrosis does not occur; even at sites of 'beneficial inflammation' some necrotic neutrophils may be seen. However, since the development of inflammatory disease is currently thought to result from a multifactoral, quantitative imbalance between potentially injurious inflammatory influences and tissue defences, it is reasonable to suggest that the balance between neutrophil apoptosis and necrosis could represent one of several possible pivotal points in the control of inflammation. Finally, as the specific trigger and induction processes which permit closely related cells to undergo apoptosis at markedly different rates are uncovered, it may be possible to design new anti-inflammatory therapeutic strategies directed towards causing specific cell types to 'commit suicide' and to be cleared by the mechanisms which 'nature intended'. Thus we have begun to dissect the cellular events occurring in the resolution of acute inflammation. We believe that the rapid cessation of neutrophil emigration which occurs remarkably early in the evolution of the acute inflammatory response represents one of the earliest events in the resolution process. Clearly there is much work to be done on the underlying mechanisms. These are likely to be more accessible with the recent molecular characterization of chemotactic cytokines and surface molecules involved in neutrophil-endothelial adhesion transmigration events. It is also clear that apoptosis, which represents an alternative tissue injury-limiting fate to necrosis in situ, may be important in limiting tissue injury and determining whether inflammation persists or resolves. It is also possible that there are circumstances in which the macrophage recognition and clearance of apoptotic neutrophils is impaired. However, a second glance at the events which must occur during the resolution of even the simplest model of acute inflammation (Fig. 1) is sufficient reminder that the 'surface has barely been scratched'. There remain many obscure areas, not the least of which is the fate of the inflammatory macrophage after it has completed its scavenging functions.

Animals↗
PubMed1992

Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis.

We have investigated the cell surface recognition mechanisms used by human monocyte-derived macrophages (M phi) in phagocytosis of intact aging human neutrophils (PMNs) undergoing apoptosis. This study shows that the adhesive protein thrombospondin (TSP) was present in the interaction, both associated with the M phi surface and in solution at a mean concentration of 0.59 micrograms/ml. The interaction was inhibited by treatment of M phi (but not aged PMN) with cycloheximide, but could be "rescued" by replenishment with exogenous TSP. Under control conditions, M phi recognition of aged PMNs was specifically potentiated by purified platelet TSP at 5 micrograms/ml, present either in the interaction or if preincubated with either cell type, suggesting that TSP might act as a "molecular bridge" between the two cell types. In support, both aged PMN and M phi were found to adhere to TSP, and phagocytosis of aged PMN was specifically inhibited by (a) excess soluble TSP; (b) antibodies to TSP that also inhibit TSP-mediated adhesion to aged PMN; and (c) down-regulation of M phi receptors for TSP by plating M phi on TSP-coated surfaces. Furthermore, inhibition with mAbs/Arg-Gly-Asp-Ser peptide of the candidate M phi receptors for TSP, CD36, and alpha v beta 3 exerted synergistic effects on both M phi recognition of aged PMN and M phi adhesion to TSP, indicating that "two point" adhesion of TSP to these M phi structures is involved in phagocytosis of aged PMN. Our findings indicate newly defined roles for TSP and CD36 in phagocytic clearance of senescent neutrophils, which may limit inflammatory tissue injury and promote resolution.

Antibodies, Monoclonal↗
PubMed1992

Phagocytosis of apoptotic neutrophils does not induce macrophage release of thromboxane B2.

Senescent human neutrophils undergo programmed cell death (apoptosis), leading to their recognition and phagocytosis by mature macrophages. At inflamed sites in vivo these processes may represent a neutrophil removal mechanism with the potential to limit the histotoxic capacity of these cells. Phagocytosis can provoke marked proinflammatory responses by macrophages. A macrophage proinflammatory response to the ingestion of apoptotic neutrophils would limit the efficacy of this neutrophil removal mechanism as a component of inflammatory resolution. In the present study we examined two macrophage proinflammatory responses; secretion of the granule enzyme N-acetyl-beta-D-glucosaminidase (NAG) and release of the membrane lipid-derived inflammatory mediator thromboxane A2 (TxA2, measured as TxB2). By contrast with the marked release of NAG and TxB2 elicited by phagocytosis of control particles (opsonised zymosan and immunoglobulin G-coated erythrocytes), macrophage ingestion of apoptotic neutrophils resulted in minimal release of NAG and no release of TxB2; indeed, there was a small depression of TxB2 release that was not due to a toxic effect of neutrophil uptake because macrophages ingesting apoptotic neutrophils retained marked TxB2 responses to subsequent stimulation with opsonised zymosan. Furthermore, there was significant TxB2 release in response to macrophage phagocytosis of apoptotic neutrophils that had been coated with opsonic serum, demonstrating that the lack of macrophage response was determined by the mechanism of recognition rather than the properties of the apoptotic particle itself. These observations are consistent with the hypothesis that macrophage clearance of senscent neutrophils undergoing apoptosis is an injury-limiting mechanism that favors resolution rather than persistence of the inflammatory response and are consistent with observations that the waves of apoptotic cell removal seen in embryological removal and thymic involution do not trigger an inflammatory response.

Acetylglucosaminidase↗
PubMed1991

Neutrophil apoptosis and clearance from neonatal lungs.

8 newborn babies with airways inflammation, who were mechanically ventilated, underwent bronchoalveolar lavage to examine the fate of neutrophils in the inflamed airways. Light microscopy and electronmicroscopy showed evidence of neutrophil apoptosis and ingestion of intact neutrophils by macrophages in specimens from all 8 infants. Neutrophil apoptosis, without the local release of intracellular contents that promote inflammation, might represent a mechanism by which tissue injury is reduced during the resolution of neonatal pulmonary inflammation.

Bronchoalveolar Lavage Fluid↗
PubMed1991

Single pulses of cytoplasmic calcium associated with phagocytosis of individual zymosan particles by macrophages.

We have measured cytosolic free calcium levels ([Ca++]i) in individual macrophages during the phagocytosis of single zymosan particles. We report here that the contact of a macrophage with zymosan results in a rapid transient elevation of [Ca++]i. Each [Ca++]i pulse is symmetrical lasting for up to 30 seconds. In contrast, macrophage spreading is associated with repetitive [Ca++]i spiking occurring in salvos of up to four smaller spikes, each lasting for between 8 and 18 seconds. These qualitative and kinetic differences might suggest that the role of [Ca++]i in phagocytosis is distinct from its role in spreading.

Animals↗
PubMed1991

A 19-year-old man with leucocyte adhesion deficiency. In vitro and in vivo studies of leucocyte function.

We describe a male patient with leucocyte adhesion molecule deficiency (LAD) of moderate phenotype. Although diagnosis was made only 2 years before his death, the patient survived until 19 years of age. This enabled us to perform a number of novel investigation, both in vivo and in vitro, relating to his leucocyte biology. Monocytes cultured in vitro matured into morphologically normal, phagocytically capable macrophages, which were able to recognize aged 'apoptotic' neutrophils. By injection of radiolabelled autologous neutrophils we demonstrated a prolonged neutrophil half-life, but normal margination, de-margination on exercise, and splenic pooling. Neutrophil adherence in vitro to vascular endothelium was normal. Histological examination of the patient's lungs at post-mortem showed intravascular aggregation of polymorphonuclear leucocytes but a paucity of cells in the interstitium and alveolar spaces. These findings indicate that the peripheral blood leucocytosis commonly observed in these patients may be due to prolonged intravascular neutrophil survival, and suggest that CD11/18 molecules have an important role in facilitating neutrophil emigration from blood vessels at sites of inflammation.

Adult↗
PubMed1990

Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis.

Phagocyte recognition of cells that have undergone apoptosis (programmed cell death) is an event of broad biological significance. Characterized by endogenous endonuclease activation, which results in chromatin fragmentation and nuclear condensation, apoptosis leads to swift ingestion of intact but 'senescent' or 'unwanted' cells by phagocytes in processes as diverse as the physiological involution of organs, the remodelling of embryonic tissues, and metamorphosis. The cell-surface mechanisms by which macrophages recognize apoptotic cells as 'senescent-self' have remained obscure. Here we report that macrophage recognition of apoptotic cells (both neutrophils and lymphocytes) is mediated by the vitronectin receptor, a heterodimer belonging to the beta 3 or cytoadhesin family of the integrins. Previously, the functions of the vitronectin receptor were believed to be limited to cell anchorage, but our findings indicate that the receptor has a novel and direct role in self-senescent-self intercellular recognition leading to macrophage phagocytosis of cells undergoing apoptosis.

Humans↗
PubMed1990

Interaction between chemoattractants and bacterial lipopolysaccharide in the induction and enhancement of neutrophil adhesion.

Chemotactic peptides in the circulation stimulate neutrophils to become sequestered in the pulmonary vasculature, and low concentrations of bacterial lipopolysaccharide (LPS) enhance and prolong this effect. This interaction of neutrophils with the vascular endothelium is thought to involve, in part, the increase in adhesiveness induced in neutrophils by such stimuli. In this study, the binding of albumin-coated latex beads to neutrophils was used to determine whether the enhancement seen with LPS results from an increase in the number of adhesive cells, from the enhancement of the adhesiveness of individual neutrophils, or both. Chemotactic peptides alone and LPS alone induced an increase both in the adhesive population and in the number of beads bound per individual neutrophil. The number of beads bound per cell increased over a very wide range of stimulus concentrations, showing that the degree of adhesiveness of an individual cell in the population varies over a considerable range. Trace concentrations of LPS (10 ng/ml or less), i.e., levels close to those measurable in vivo, had little effect on the proportion of neutrophils that were stimulated by chemotactic factor to become adhesive but did significantly enhance the number of beads bound to each individual neutrophil. The enhancement may require the presence of the CD11/18 glycoprotein complex, but was not further upregulated by LPS. No evidence could be obtained to suggest that the effect of LPS involved release of tumor necrosis factor (TNF) from the numbers of monocytes in the preparation, and the observations are consistent with a direct effect of LPS on the neutrophils. It is suggested that this increase in adhesive sites on the cell could explain the persistence of the sequestration of neutrophils in the microvasculature seen in the presence of both chemoattractants and LPS by enhancing the "strength" of the adhesion to endothelial cells. The increased adhesion may also set the stage for enhanced endothelial injury.

Antigens, CD↗
PubMed1989

Cessation of neutrophil influx in C5a-induced acute experimental arthritis is associated with loss of chemoattractant activity from the joint space.

Neutrophil emigration is a critical component of the inflammatory process and is generally thought to play a role in host defense as well as in the tissue injury that often accompanies inflammation. Most inflammatory reactions exhibit a sequence of emigrating cell types, thus clearly demonstrating that the neutrophil influx eventually ceases and that the neutrophils are then removed from the lesion. It has been our premise that in order to understand the processes that lead to the progressive inflammatory reactions that underly so many disease processes, it is important to determine the mechanism by which the "normal" inflammatory response resolves. The purpose of this study was to identify the time of cessation of neutrophil influx in experimental arthritis induced by the injection of C5 fragments (C5f) and to investigate mechanisms underlying the cessation process. The migration of i.v. delivery pulses to inflamed joints was assessed by lavage of the joint space and by external scintigraphy. We found no evidence for the development of inhibitory systems against chemotactic factors or "desensitization" of the inflamed site, because a second injection of C5f into joints which had been injected previously with C5f resulted in enhancement rather than inhibition of migration. Neither was evidence found for altered tissue barriers to migration or for desensitization of neutrophils as possible explanations for cessation of influx. The major mechanism appeared to be a loss of chemoattractant activity in the joint space between 2 h and 6 h after C5f injection which was detected by transfer into a fresh joint. Radiolabeled C5a des-Arg had a t1/2 of disappearance from the joint of less than 1 h, which suggested that the transferred chemoattractant must, in part, have been due to the generation of new chemotaxins by C5f injection. These observations suggest that continued generation of chemoattractants or failure of their subsequent removal may be mechanisms leading to persistent neutrophil influx in chronic inflammation.

Acute Disease↗