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G M Walsh

Publications and source records attributed to G M Walsh.

At least 37 records · Page 2Linked to original sources
PubMed1999

Ligation of CD45 and the isoforms CD45RA and CD45RB accelerates the rate of constitutive apoptosis in human eosinophils.

BACKGROUND: Eosinophils are important effector cells in asthma pathogenesis, and an understanding of the mechanisms involved in eosinophil apoptosis induction might thus be relevant to the resolution of asthmatic inflammation. OBJECTIVE: Our aim was to determine the role of the common leukocyte antigen CD45 and the isoforms CD45RA, CD45RB, and CD45RO in human eosinophil apoptosis induction. METHODS: Immmunostaining and flow cytometry were used to assess CD45 and CD45 isoform expression by eosinophils purified with use of density gradients and immunomagnetic negative selection. Apoptosis induction was measured by binding of fluorescein isothiocyanate-labeled annexin V to eosinophils cultured for 20 hours alone or with saturating quantities of mAb against CD45, CD45RA, CD45RB, CD45RO, CD9, CD11b, and isotype-matched controls in the presence or absence of GM-CSF. RESULTS: Freshly isolated eosinophils had high expression of CD45 and CD45RO, modest expression of CD45RB, and low expression of CD45RA. Eosinophils cultured alone for 20 hours were found to be approximately 20% to 25% apoptotic. Incubation with mAb against CD45, CD45RA, and CD45RB resulted in significant (P <.005) enhancement (>100%) of their constitutive rate of apoptosis. Incubation with CD45RO, CD11b, CD9 mAb, or isotype controls had no significant effect on the rate of eosinophil constitutive apoptosis. The addition of GM-CSF inhibited eosinophil apoptosis but did not prevent CD45, CD45RA, or CD45RB mAb-dependent apoptosis induction. CONCLUSION: These data indicate that ligation of CD45, CD45RA, or CD45RB represents a novel pathway for the induction of apoptosis in human eosinophils.

Apoptosis↗
PubMed1999

Advances in the immunobiology of eosinophils and their role in disease.

Eosinophils play a protective role in host immunity to infections by parasitic worms and, detrimentally, are involved in the pathophysiology of asthma and other allergic diseases. Airway inflammation is central to the pathology of asthma and is characterized by infiltration of the bronchial mucosa by large numbers of proinflammatory cells, amongst which the eosinophil is prominent despite being a minority constituent of circulating leukocytes. Crucial steps in eosinophilic inflammation include augmented production of eosinophils in the bone marrow, their increased release into the circulation, and their selective accumulation in the conducting airways. The eosinophil has a potent armory of proinflammatory mediators, including cytotoxic granule proteins, cytokines and lipid mediators with considerable potential to initiate and sustain an inflammatory response. Thus there is much interest in the elucidation of the mechanisms responsible for eosinophil accumulation, persistence, activation and ultimate fate. This article reviews our current understanding of the role of the eosinophil in human disease and the immunobiology of this important proinflammatory cell.

Apoptosis↗
PubMed1999

Expression of Bcl-2 and its homologues in human eosinophils. Modulation by interleukin-5.

The Bcl-2 family has been shown to be vital regulators of programmed cell death in numerous systems. To investigate the role of such proteins in the regulation of apoptosis of eosinophils, the expression of Bcl-2 and homologues Bcl-xL (death antagonists), Bax, and Bcl-xS (death agonists) were examined by immunoblot, flow cytometry, and reverse transcriptase-polymerase chain reaction analysis. Potential modulation of apoptosis-associated molecules during spontaneous apoptosis and in the presence of interleukin (IL)-5 was also investigated. Peripheral blood eosinophils were found to express constitutively Bax and Bcl-x, but Bcl-2 was absent. Analysis of mRNA revealed that the bcl-xL isoform predominated, although bcl-xS was also detectable. Spontaneous apoptosis due to culturing in the absence of cytokines for 24 h did not result in modulation of any of the Bcl-2 homologues examined. Culturing eosinophils in the presence of 100 pg/ml IL-5 for 24 h significantly reduced apoptosis (P < 0.01) to 10.7 +/- 2.6% compared with 46.8 +/- 7.4% in the absence of IL-5, and induced Bcl-2 mRNA and protein expression, with no detectable change in Bax, Bcl-x, or beta-actin as a control. This investigation indicates a specific profile of apoptotic molecules in eosinophils distinct from that of neutrophils, and indicates that survival-enhancing IL-5 modulates the expression of Bcl-2 in vitro.

Apoptosis↗
PubMed1998

Initial cytokine exposure determines function of macrophages and renders them unresponsive to other cytokines.

The functional properties of infiltrating macrophages (Mphi) must be tightly regulated to facilitate appropriate responses to complex conditions in an inflammatory focus. This study was designed to ascertain whether uncommitted Mphi that have been exposed to combinations of cytokines with opposing functions develop properties dictated by one cytokine or by cytokine mixtures. Uncommitted rat bone marrow-derived Mphi (BMDMs) were incubated with IFN-gamma, TNF-alpha, TGF-beta, IL-4, IL-6, and IL-10 alone or sequentially in combinations. After 48 h, function was assessed by nitric oxide (NO) generation, uptake of apoptotic neutrophils, and beta-glucuronidase expression. IFN-gamma followed 4 h later by TNF-induced NO generation. The pretreatment of BMDMs before IFN-gamma priming with TNF, TGF-beta, and IL-4 suppressed NO generation by 87%, 92%, and 85%, respectively; IL-10 had no effect. The same cytokines administered at 4 h after IFN priming had no effect on NO generation. The uptake of apoptotic polymorphonuclear leukocytes was augmented by TNF (40% vs 29% controls; p < 0.05) and decreased by IFN-gamma, IL-10, and IL-4. The TNF response was unaffected by subsequent treatment with IFN-gamma, IL-4, or IL-10. Similarly, the decreased polymorphonuclear leukocyte uptake induced by IFN-gamma, IL-4, or IL-10 was unaffected by the subsequent addition of TNF. Beta-glucuronidase expression was increased by TGF-beta and decreased by IFN-gamma. These responses were not modified by cytokines with the opposing function. Thus, the functional response of BMDMs to complex mixtures of cytokines was determined by the first cytokine to which they were exposed. Once activated, BMDMs become unresponsive to alternative activating signals, a finding which has obvious implications for Mphi function in vivo.

Animals↗
PubMed1998

A comparative study of different methods for the assessment of apoptosis and necrosis in human eosinophils.

Eosinophils, prominent cells in asthmatic inflammation, undergo apoptosis or programmed cell death following deprivation of contact with survival-promoting cytokines such as IL-5 and GM-CSF. The aim of this study was to assess a number of techniques for the quantification of apoptosis in human eosinophils cultured with or without IL-5 or GM-CSF and following staurosporine treatment. The relationship between apoptosis and necrosis in eosinophils was also determined. Eosinophils 'aged' in vitro for 48 h exhibited endonuclease DNA degradation, apoptotic morphology, increased red autofluorescence and externalisation of phosphatidylserine (PS) as assessed by binding of FITC-labelled annexin V. Annexin V-FITC binding was first detectable in eosinophils maintained at 37 degrees C for 5 h post-purification. This method proved to be the most sensitive marker of apoptosis. Morphological assessment of wet preparations of eosinophils by Kimura staining was found to be the next most-sensitive marker followed by increased red autofluorescence. The latter was a relatively insensitive method for the detection of apoptosis. At 5, 20 and 24 h of culture trypan blue exclusion indicated that eosinophil viability was high (85-90% viable cells). However, propidium iodide (PI) staining and flow cytometry revealed that, by 24 h, approximately 75% of cells had compromised membrane integrity. Eosinophils maintained in IL-5 or GM-CSF exhibited a non-apoptotic morphology and levels of annexin V-FITC binding and PI uptake similar to that of freshly isolated cells. Staurosporine (10(-5) M) treatment of eosinophils maintained in IL-5 or GM-CSF resulted in significant levels of apoptotic morphology at 2 h (23.8% +/- 6.9, p < 0.025) which was associated with negligible annexin binding. At 6 h post-staurosporine treatment significant annexin-FITC binding (38% +/- 1.5, p < 0.025) was observed compared with 93% +/- 1.2 of eosinophils displaying apoptotic morphology. Exclusion of PI demonstrated membrane integrity at all time points up to 6 h. Thus, eosinophils aged in vitro in the absence of viability-promoting cytokines exhibit evidence of both apoptosis and necrosis simultaneously. In contrast, staurosporine-treated eosinophils exhibited both membrane integrity and rapid apoptosis-associated morphological changes detected by single step Kimura staining which preceded externalisation of PS.

Annexin A5↗
PubMed1998

Effects of a novel purine nucleoside phosphorylase inhibitor, BCX-34, on activation and proliferation of normal human lymphoid cells.

The antiproliferative effect of BCX-34 was tested in normal human peripheral blood mononuclear cells (PBMCs) induced to proliferate with OKT3, tetanus toxoid, the mixed lymphocyte reaction, or IL-2. In the case of OKT3, tetanus toxoid, or the MLR the IC50s ranged between 0.7 and 4 microM. With IL-2, the IC50 was 14.6 microM. In T-cells purified by rosetting the IC50 with IL-2 was 0.62 microM. In CD4 or CD8 cells obtained by magnetic activated cell sorting the IC50s with IL-2 were 0.24 and 0.62 microM, respectively. BCX-34 inhibition of proliferation in human PBMCs may not depend entirely upon the accumulation of intracellular dGTP because tetanus toxoid-induced proliferation was inhibited in the absence of deoxyguanosine and was not reversed by deoxycytidine. BCX-34 did not inhibit IL-2 release from PBMCs and did not alter PBMC viability. The results of these studies show that BCX-34 is a potent inhibitor of normal human T-cell proliferation induced by antigenic or IL-2 stimulation. BCX-34 in normal human T-cells has a deoxyguanosine-independent mechanism to suppress in vitro proliferation. BCX-34 appears to have little effect on T-cell viability. The data suggest that BCX-34 may be useful in the treatment of T-cell proliferative disorders.

Cell Division↗
PubMed1998

Generation and characterization of a mutant of influenza A virus selected with the neuraminidase inhibitor BCX-140.

Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.

Animals↗
PubMed1997

Design and synthesis of benzoic acid derivatives as influenza neuraminidase inhibitors using structure-based drug design.

A series of 94 benzoic acid derivatives was synthesized and tested for its ability to inhibit influenza neuraminidase. The enzyme-inhibitor complex structure was determined by X-ray crystallographic analysis for compounds which inhibited the enzyme. The most potent compound tested in vitro, 5 (4-acetylamino)-3-guanidinobenzoic acid), had an IC50 = 2.5 x 10(-6) M against N9 neuraminidase. Compound 5 was oriented in the active site of the neuraminidase in a manner that was not predicted from the reported active site binding of GANA (4) with neuraminidase. In a mouse model of influenza, 5 did not protect the mice from weight loss due to the influenza virus when dosed intranasally.

Animals↗
PubMed1997

High-performance liquid chromatographic determination of 9-(3-pyridylmethyl)-9-deazaguanine (BCX-34) in biological fluids.

9-(3-Pyridylmethyl)-9-deazaguanine (BCX-34), a new purine nucleoside phosphorylase inhibitor, has selective immunosuppressive activity with potential therapeutic value in T-cell-mediated disease. We now report a sensitive, specific and reproducible method for measurement of 9-(3-pyridylmethyl)-9-deazaguanine in biological fluids using high-performance liquid chromatography (HPLC). 9-(3-Pyridylmethyl)-9-deazaguanine was extracted from plasma using perchloric acid precipitation followed by passage through Sep-Pak C18 cartridges (average extraction efficiency, 64.6%). Standard curves were linear over the range of interest (28-1120 ng/ml in plasma and 200-4000 ng/ml in urine, r2 > 0.999). Within-day and between-day coefficients of variation were less than 8%. The limit of quantitation was 28 ng/ml in plasma and 200 ng/ml in urine. This HPLC method should be useful in future clinical studies with this drug.

Administration, Oral↗
PubMed1997

Dexamethasone inhibits prolonged survival and autocrine granulocyte-macrophage colony-stimulating factor production by human eosinophils cultured on laminin or tissue fibronectin.

BACKGROUND: Eosinophil autocrine generation of viability-promoting cytokines resulting from interaction with extracellular matrix (ECM) proteins may be important in promoting their persistence in allergic inflammation. OBJECTIVE: We examined eosinophil interaction with fibronectin, laminin, collagen, fibrinogen, hyaluronate, or vitronectin in terms of adhesion and for their ability to promote prolonged eosinophil survival by autocrine generation of IL-3, IL-5, or granulocyte-macrophage colony-stimulating factor (GM-CSF). The effect of dexamethasone was examined because it may inhibit integrin-dependent autocrine generation of viability-enhancing cytokines. METHODS: Microtiter wells were coated with different ECM proteins, and assays were performed to determine their levels of eosinophil adhesion, and tests were performed to determine their prolonged survival in culture. The receptors involved in these processes, the cytokines generated, and the effect of a dose range of dexamethasone were all examined. RESULTS: Eosinophils cultured for 3 days on laminin or tissue fibronectin exhibited dose-dependent prolonged survival, which was not seen with any other ECM protein. Unstimulated eosinophils adhered to wells coated with laminin or fibronectin but not to wells coated with any of the other ECM proteins that were tested. Laminin-dependent eosinophil adhesion and prolonged survival were inhibited by specific monoclonal antibodies to beta1 and alpha6beta1. Laminin-dependent survival was inhibited by a neutralizing antibody to GM-CSF, an anti-IL-5 antibody achieved a measurable but insignificant inhibition of survival, and an antibody to IL-3 had no effect. Increasing concentrations of dexamethasone (maximal at 10(-7) mol/L) inhibited both laminin- and fibronectin-dependent enhanced eosinophil viability at 3 days. Eosinophils cultured on either laminin or fibronectin for 24 or 72 hours generated picogram quantities of GM-CSF, which were inhibited by the addition of dexamethasone (10(-7) mol/L). CONCLUSION: We have confirmed that eosinophils adhere to laminin in an alpha6beta1-dependent manner, and this interaction results in the autocrine generation of GM-CSF, which enhances eosinophil survival in culture. Both laminin and tissue fibronectin-dependent autocrine GM-CSF generation were inhibited by physiologically relevant concentrations of dexamethasone. This may represent an important mechanism by which glucocorticoids reduce the tissue eosinophilia in allergic disease.

Anti-Inflammatory Agents↗
PubMed1997

Mechanisms of human eosinophil survival and apoptosis.

Eosinophils, with their potentially harmful armoury of toxic products, have available a mechanism by which they can be cleared from the tissues while still intact in the absence of an undesirable pro-inflammatory response. However, our understanding of the induction and control of apoptosis in eosinophils is still incomplete and a great deal of work in this area remains to be done. In other cell types, important regulators of apoptosis have been well characterized. These include the proto-oncogene bcl-2 and related molecules, the family of proteases which share homology with IL-1 beta converting enzyme (ICE) and another protein family which interact with products of the proto-oncogene c-myc. These regulators act at various points on the apoptosis pathway and it appears their interactions are vital in determining whether a cell will survive or die. In particular, the ICE-like proteases have been implicated as a universal apoptosis effector which is associated with the downstream events of programmed cell death [62]. Whether these molecules are involved in the cell death pathway(s) of eosinophils remains to be determined. Much of eosinophilic inflammation might be a result of defects in pathways controlling eosinophil apoptosis and/or their clearance by phagocytes resulting in necrosis and the release of toxic products. A greater understanding of the processes involved in the induction and control of eosinophil apoptosis might provide novel approaches for therapeutic intervention.

Apoptosis↗
PubMed1996

Adhesion interactions involved in eosinophil migration through vascular endothelium.

Considerable progress has been made in our understanding of the molecular mechanisms involved in eosinophil migration into sites of allergic inflammation. A number of differences between eosinophils and neutrophils have been observed in their pattern of adhesion interactions. These include expression of alpha 4 beta 1, alpha 4 beta 7, and alpha 6 beta 1 by eosinophils but not neutrophils and structural differences between eosinophil and neutrophil PSGL-1 associated with increased eosinophil binding to P-selectin. On the basis of current evidence the various receptors and mediators involved are summarized in FIGURE 1. Once in the tissues eosinophils may persist for several days or weeks, surviving under the influence of locally generated cytokines and this persistence may also partly explain selective tissue accumulation of eosinophils. Understanding the molecular mechanisms involved in leucocyte migration offers the possibility of selective and effective antagonists to treat allergic disease by preventing cell migration. Results in a number of animal models already offer the hope that this approach may be successful. The development of drugs that can be tested in the clinic are awaited with considerable interest.

Animals↗
PubMed1996

Functional and structural characterization of the eosinophil P-selectin ligand.

Our recent studies have indicated an important role for P-selectin in eosinophil adhesion. We have therefore compared eosinophil and neutrophil binding with nasal polyp endothelium as well as purified P-selectin. We have also compared the structure and expression of the eosinophil and neutrophil P-selectin ligands. Using the frozen section assay, eosinophils bound to 2-fold more blood vessels within the nasal polyp tissue than neutrophils. Up to 10-fold more eosinophils than neutrophils bound per unit length of endothelium. Neutrophil and eosinophil binding was inhibited by a mAb against P-selectin and a P-selectin chimera which binds to the P-selectin ligand. Eosinophils bound with approximately 2-fold greater avidity to purified P-selectin under flow conditions. Using SDS-PAGE we characterized the eosinophil P-selectin ligand as a sialylated, homodimeric glycoprotein consistent with the known structure of PSGL-1. However, expression of PSGL-1 by eosinophils was significantly greater than on neutrophils. The eosinophil ligand had a calculated molecular mass by SDS-PAGE of approximately 10 kDa greater than the neutrophil ligand, which was not due to differences in N-glycosylation. Eosinophils expressed the 15-decapeptide repeat form of PSGL-1 compared with neutrophils that have the 16-decapeptide repeat form. The increased binding of eosinophils, compared with neutrophils, to P-selectin in both an ex-vivo and in vitro assay suggests that P-selectin may have a role directing the specific migration of eosinophils in diseases such as asthma. The increased avidity may be due to increased expression of PSGL-1 by eosinophils, differences in the peptide backbone, or post-translational modifications.

Antibodies, Monoclonal↗
PubMed1996

Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor.

Peripheral blood (PB) eosinophils rapidly undergo apoptosis and cell death in vitro unless cultured in the presence of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) in which their survival is prolonged for up to 10 days. CD69 is a type II membrane antigen expressed by cytokine-activated, but not freshly isolated, PB human eosinophils. We have examined the effect of ligation of CD69 by specific monoclonal antibody (MoAb) on the viability of human eosinophils cultured with recombinant human (rh)GM-CSF. Eosinophils were purified by immunomagnetic selection and cultured with GM-CSF (10(-10) mol/L). Eighteen hours after the start of culture, a panel of CD69 MoAb or controls (anti-CR3 or isotype-matched control MoAb) were added. Viability was assessed by trypan blue exclusion and apoptosis by morphologic assessment, DNA laddering, and flow cytometric analysis of eosinophil red autofluorescence. Up to 50% of the eosinophils had undergone apoptosis 48 hours after addition of anti-CD69 MoAb compared with less than 10% apoptosis for CR3 or the isotype matched control. The majority of apoptotic eosinophils excluded trypan blue at 48 hours post CD69 ligation. More apoptotic eosinophils were observed at later time-points and this was associated with loss of viability. At 120 hours post-addition of the anti-CD69 MoAb MLR3, 24% +/- 10.6% eosinophils were viable compared with 84% +/- 3.4% for the CR3 control (P < .001). A F(ab)2 fragment of CD69 MoAb P8, also induced apoptosis in GM-CSF cultured eosinophils. A more rapid induction of eosinophil apoptosis was obtained with CD69 MoAb immobilized via their Fc portions on protein-A coated plastic 96 well plates. Ligation of CD69 or CR3 resulted in the release of comparable quantities of eosinophil peroxidase at 48 hours post-ligation. These levels of EPO were consistent with the viability of these cells at 48 hours as assessed by exclusion of trypan blue. Finally, a neutralizing MoAb to TGF beta 1 had no effect on CD69-dependent apoptosis induction nor were there detectable quantities of TGF beta 1 in supernatants from GM-CSF--cultured eosinophils ligated with CD69 or control MoAb. These results suggest that eosinophils cultured with GM-CSF can be induced to undergo apoptosis as a result of cell signalling mediated by perturbation of CD69. This may represent an important physiologic mechanism for eosinophil removal in vivo.

Antigens, CD↗
PubMed1996

In vivo and in vitro pharmacologic activity of the purine nucleoside phosphorylase inhibitor BCX-34: the role of GTP and dGTP.

BCX-34 inhibits RBC PNP in vitro from humans, rats, and mice with IC50S ranging from 5 to 36 nM. BCX-34 also, in the presence but not in the absence of deoxyguanosine, inhibits human CCRF-CEM T-cell proliferation with an IC50 of 0.57 microM but not rat or mouse T-cell proliferation up to 30 microM. Inhibition of human T-cell proliferation is accompanied by an accumulation of intracellular dGTP with an associated reduction in GTP. These nucleotide changes do not occur in BC16A mouse T-cells and explain why proliferation is not inhibited by PNP inhibitors in this case. Reduction in intracellular GTP is not essential for the antiproliferative action of BCX-34. Oral bioavailability of BCX-34 in rats is 76%. BCX-34 is orally active in elevating plasma inosine in rats (2-fold at 30 mg/kg), in suppressing ex vivo RBC PNP activity in rats (98% at 3 h. 100 mg/kg), and in suppressing ex vivo skin PNP in mice (39% at 3 h, 100 mg/kg). The results demonstrate that BCX-34 inhibits human PNP and T-cell proliferation, is orally bioavailable in rodents, and pharmacologically active in vivo in rodents after oral dosing with no apparent side effects or toxicity. BCX-34 may, therefore, be useful in treating human T-cell proliferative inflammatory disorders.

Animals↗