Biomedical subjects

Anthony W Segal

Publications and source records attributed to Anthony W Segal.

15 recordsLinked to original sources

PubMed2007

Mice lacking neutrophil elastase are resistant to bleomycin-induced pulmonary fibrosis.

Neutrophil elastase is a serine protease stored in the azurophilic granules of leukocytes. It has been implicated in the pathology of several lung diseases and is generally presumed to contribute to the tissue destruction and extracellular matrix damage associated with these conditions. To delineate the role of neutrophil elastase in pulmonary inflammation and fibrosis, neutrophil elastase-null mice were intratracheally instilled with bleomycin. In neutrophil elastase-null mice, biochemical and morphological characteristics of pulmonary fibrosis were attenuated for at least 60 days after bleomycin administration despite a typical response to bleomycin as evidenced by assessment of indices of DNA and cell damage. Neutrophil burden of bleomycin-treated wild-type and neutrophil elastase-null mice was comparable, and marked neutrophilic alveolitis was manifest in bleomycin-treated neutrophil elastase-null mice. An absence of immunostaining for active transforming growth factor (TGF)-beta in lung tissue from bleomycin-treated neutrophil elastase-null mice suggested a defect in TGF-beta activation, which was confirmed by biochemical assessment of TGF-beta levels in bronchoalveolar lavage fluid and lung tissue. These data point to novel and unexpected fibrogenic consequences of neutrophil elastase activity in the inflamed lung.

Animals↗

PubMed2006

The role of grancalcin in adhesion of neutrophils.

Grancalcin is a protein specifically expressed in neutrophils and monocytes/macrophages. The function of grancalcin has not been identified. Grancalcin-deficient neutrophils were previously demonstrated to exert normal recruitment to the inflamed site, NADPH oxidase activation, extracellular release of secondary granules, apoptosis and activation-induced Ca2+ flux. In this study we analyzed granule numbers in resting and activated grancalcin-deficient neutrophils, their phagocytic activity and adherence to extracellular matrix proteins. Results revealed normal phagocytosis and degranulation of grancalcin-deficient neutrophils, while their adhesion to fibronectin was decreased by 60%. Consistently, the processes associated with neutrophil adhesion, such as formation of focal adhesion complexes and spreading, were also impaired in grancalcin-deficient neutrophils by 89 and 38%, respectively. In contrast, adherence to other extracellular matrix proteins: collagen, laminin and vitronectin, was not significantly altered. We thus report for the first time a function of grancalcin.

Animals↗

PubMed2006

Defective acute inflammation in Crohn's disease: a clinical investigation.

BACKGROUND: The cause of Crohn's disease has not been mechanistically proven. We tested the hypothesis that the disease is a form of immunodeficiency caused by impaired innate immunity. METHODS: We investigated inflammatory responses in patients and controls by quantifying neutrophil recruitment and cytokine production after acute trauma, interleukin 8 secretion by cultured monocyte-derived macrophages after exposure to inflammatory mediators, and local inflammatory and vascular changes in response to subcutaneous injection of heat-killed Escherichia coli. FINDINGS: In patients with Crohn's disease, trauma to rectum, ileum, or skin led to abnormally low neutrophil accumulation (differences from healthy individuals of 79%, n=8, p=0.0003; 57%, n=3, p=0.05; 50%, n=13, p<0.0001, respectively) and lower production of proinflammatory interleukin 8 (63%, n=7, p=0.003; 63%, n=3, p=0.05; 45%, n=8, p<0.0001) and interleukin 1beta (50%, n=8, p=0.0005). Interleukin 8 secretion by cultured macrophages was reduced after exposure to acute wound fluid (38%, n=50, p<0.0001), C5a (48%, n=41, p=0.0005), or tumour necrosis factor alpha (52%, n=27, p<0.0001). Local inflammatory reaction to inoculation with E coli was attenuated, as quantified by changes in bloodflow (ileal disease 50%, n=6, p=0.01; colonic disease 77%, n=6, p=0.0003). This response was mediated by nitric oxide in controls, was increased by sildenafil in patients, and was not related to CARD15 genotype. INTERPRETATION: In Crohn's disease, a constitutionally weak immune response predisposes to accumulation of intestinal contents that breach the mucosal barrier of the bowel wall, resulting in granuloma formation and chronic inflammation. Polymorphisms in CARD15 do not underlie this phenotype, but incapacitate the NOD2 pathway that can compensate for impairment of innate inflammation. Current treatment of secondary chronic inflammation might exaggerate the underlying lesion and promote chronic disease.

Case-Control Studies↗

PubMed2005

How neutrophils kill microbes.

Neutrophils provide the first line of defense of the innate immune system by phagocytosing, killing, and digesting bacteria and fungi. Killing was previously believed to be accomplished by oxygen free radicals and other reactive oxygen species generated by the NADPH oxidase, and by oxidized halides produced by myeloperoxidase. We now know this is incorrect. The oxidase pumps electrons into the phagocytic vacuole, thereby inducing a charge across the membrane that must be compensated. The movement of compensating ions produces conditions in the vacuole conducive to microbial killing and digestion by enzymes released into the vacuole from the cytoplasmic granules.

Animals↗

PubMed2004

The NADPH oxidase of professional phagocytes--prototype of the NOX electron transport chain systems.

The NADPH oxidase is an electron transport chain in "professional" phagocytic cells that transfers electrons from NADPH in the cytoplasm, across the wall of the phagocytic vacuole, to form superoxide. The electron transporting flavocytochrome b is activated by the integrated function of four cytoplasmic proteins. The antimicrobial function of this system involves pumping K+ into the vacuole through BKCa channels, the effect of which is to elevate the vacuolar pH and activate neutral proteases. A number of homologous systems have been discovered in plants and lower animals as well as in man. Their function remains to be established.

Animals↗

PubMed2004

The large-conductance Ca2+-activated K+ channel is essential for innate immunity.

Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BK(Ca)) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BK(Ca) channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the alpha-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BK(Ca) channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.

Calcium↗

PubMed2004

N-Formyl peptide receptor subtypes in human neutrophils activate L-plastin phosphorylation through different signal transduction intermediates.

We investigated the coupling of the fMLP (N -formyl-L-methionyl-L-leucyl-L-phenylalanine; 'chemotactic peptide') receptor with phosphorylation of the actin-binding protein L-plastin in neutrophils. Using two-dimensional IEF (isoelectric focusing)/PAGE and MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight)-MS, L-plastin was identified as a major phosphoprotein in fMLP-stimulated neutrophils whose phosphorylation was dependent on phosphoinositide 3-kinase, PLD (phospholipase D) and PKC (protein kinase C) activity. Two fMLP receptor subtypes were identified in neutrophils, characterized by a distinct sensitivity to fMLP and antagonistic peptides. Both receptor subtypes induced the phosphorylation of L-plastin. L-plastin phosphorylation induced by low-affinity fMLP receptors involves an action of phosphoinositide 3-kinase, PLD and PKC isotypes. In contrast, none of these intermediates are utilized by high-affinity fMLP receptors in the phosphorylation of L-plastin. However, the PKC inhibitor Ro-31-8220 inhibits L-plastin phosphorylation induced by the high-affinity fMLP receptor. Thus, an as yet unknown Ro-31-8220-sensitive kinase regulates L-plastin phosphorylation in response to the high-affinity fMLP receptor. The results suggest a model in which receptor subtypes induce a similar endpoint event through different signal-transduction intermediates. This may be relevant in the context of cell migration in which one receptor subpopulation may become desensitized in a concentration gradient of chemoattractant.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2003

Lipid rafts determine efficiency of NADPH oxidase activation in neutrophils.

We have investigated the contribution of lipid rafts to activation of the NADPH oxidase enzyme system in neutrophils. Membrane-bound NADPH oxidase subunits are present in the lipid raft compartment of neutrophils. Cytosolic NADPH oxidase components are mainly absent from but are recruited to rafts upon Fcgamma receptor activation. In parallel, protein kinase C isotypes are recruited to the rafts. Kinetic analysis of NADPH oxidase activation revealed that rafts determine the onset but not the maximal rate of enzyme activity. Thus lipid rafts serve to physically juxtapose the NADPH oxidase effector, protein kinase C and Fcgamma receptor, resulting in efficient coupling.

Cells, Cultured↗

PubMed2003

PX domain takes shape.

In recent years, a number of protein domains have been identified that bind phosphoinositides and direct proteins to membrane targets. A recent addition to this group is the Phox homology or PX domain, a 120-amino acid domain conserved from yeast to humans, which is present in proteins involved in cell signaling, protein sorting, vesicle fusion, and the assembly of components of the superoxide generating system of neutrophils. These domains have varying affinities for phosphatidylinositol-3-phosphate (PI(3)P), and PI(3,4) and (4,5) bisphosphates, which couple the PI kinase and phosphatase signaling networks to the assembly of proteins at membrane surfaces. These PX domains also contain a PXXP motif, allowing them to bind to proteins containing Src homology 3 (SH3) domains.

Animals↗

PubMed2003

Reassessment of the microbicidal activity of reactive oxygen species and hypochlorous acid with reference to the phagocytic vacuole of the neutrophil granulocyte.

During phagocytosis, neutrophils undergo a burst of respiration in which oxygen is reduced to superoxide (O(-)(2)), which dismutates to form H(2)O(2). Myeloperoxidase (MPO) is discharged from the cytoplasmic granules into the phagosome following particle ingestion. It is thought to utilize H(2)O(2) to oxidize halides, which then react with and kill ingested microbes. Recent studies have provided new information as to the concentration of O(-)(2) and proteins, and the pH, within the vacuole. This study was conducted to examine the antimicrobial effect of O(-)(2), H(2)O(2) and hypochlorous acid under these conditions and it was found that the previously described bactericidal effect of these agents was reversed in the presence of granule proteins or MPO. To establish which cellular proteins were iodinated by MPO, cellular proteins and bacterial proteins, iodinated in neutrophils phagocytosing bacteria in the presence of (125)I, were separated by 2D gel electrophoresis. Iodinated spots were detected by autoradiography and the oxidized proteins were identified by MS. The targets of these iodination reactions were largely those of the host cell rather than those of the engulfed microbe.

Cells, Cultured↗

PubMed2003

Granulocyte function in grancalcin-deficient mice.

Grancalcin, one of the penta-EF-hand Ca(2+) binding proteins, is expressed at high levels in polymorphonuclear granulocytes (neutrophils). EF-hand proteins are implicated in the regulation of diverse processes including cell migration, apoptosis, and mobilization of neutrophil effector functions. To determine the role of grancalcin in vivo, we inactivated the gene encoding grancalcin (Gca) in embryonic stem cells and generated grancalcin-deficient mice. Homozygous Gca mutants appeared healthy and reproduced normally. Leukocyte recruitment into the peritoneal cavity upon induction of inflammation was not significantly affected by the absence of grancalcin. The mutants also resisted systemic fungal infection similarly to wild-type mice, and in vitro killing of Staphylococcus aureus by inflammatory cells was not significantly impaired. While marginally increased survival rates of mutants faced with endotoxic shock may indicate a contribution of grancalcin to immunopathogenesis, it is not essential for vital leukocyte effector functions required to control microbial infections.

Animals↗

PubMed2002

Catalase negative Staphylococcus aureus retain virulence in mouse model of chronic granulomatous disease.

Myeloperoxidase-mediated chlorination is thought to be a necessary microbicidal mechanism. The H2O2 required for this process is generated by the NADPH oxidase. Staphylococcus aureus can also produce H2O2, which is not broken down by catalase negative organisms. It has been thought that this bacterial H2O2 can substitute for cellular H2O2 in the halogenation reaction in chronic granulomatous disease (CGD) where neutrophils are lacking the NADPH oxidase. We have readdressed this issue in a mouse model of CGD using clinical isolates of catalase positive and negative strains of S. aureus. The results showed these organisms to be equally virulent and that the H2O2 they produced is insufficient to cause significant iodination, a marker for chlorination, thereby contradicting the accepted views on this subject.

Animals↗

PubMed2002

Involvement of protein kinase D in Fc gamma-receptor activation of the NADPH oxidase in neutrophils.

Protein kinases involved in the activation of the NADPH oxidase by Fc gamma receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70-80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fc gamma receptors. Down-regulation of PKC isotypes resulted in inhibition of Fc gamma-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-mu (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fc gamma-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40(phox) and p47(phox). Taken together, these data indicate that Fc gamma receptors engage PKD in the regulation of the NADPH oxidase.

Carbazoles↗

PubMed2002

Killing activity of neutrophils is mediated through activation of proteases by K+ flux.

According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.

Animals↗

PubMed2001

Ym1 is a neutrophil granule protein that crystallizes in p47phox-deficient mice.

Crystals were discovered within the aged lung and at sites of chronic inflammation in a mouse model of chronic granulomatous disease. Following re-crystallization at neutral pH, the crystals were identified as the chitinase-like protein Ym1, expressed in organs of the lymphoreticular system, the lung, and distal stomach. Ym1 was shown to be a neutrophil granule protein and to have weak beta-N-acetylglucosaminidase activity, indicating that it might contribute to the digestion of glycosaminoglycans. Crystal formation is likely to be a function of excess neutrophil turnover at sites of inflammation in the chronic granulomatous disease mouse. Failure to remove subcutaneous Ym1 crystals injected into knockout mice indicates that a failure of digestion may also contribute to crystallization.

Animals↗