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L A Boxer

Publications and source records attributed to L A Boxer.

At least 73 records · Page 4Linked to original sources
PubMed1990

Iloprost inhibits neutrophil-induced lung injury and neutrophil adherence to endothelial monolayers.

We hypothesized that Iloprost, a long-acting prostacyclin analog, would inhibit neutrophil (PMN)-induced lung injury and decrease PMN adherence to vascular endothelium. Human PMNs infused into isolated buffer-perfused rat lungs subsequently stimulated with phorbol myristate acetate (PMA) resulted in lung injury as assessed by the accumulation of [125I]bovine serum albumin (125I-BSA) in lung parenchyma and alveolar lavage fluid. Addition of Iloprost to the lung perfusate, prior to activation of the PMNs, reduced lung injury as assessed by a decrease in the accumulation of 125I-BSA in the lung. This protective effect was not due to the vasodilatory effect of Iloprost. Protection by Iloprost was not linked to a reduction in PMA-induced PMN superoxide production since Iloprost did not reduce the amount of superoxide released into lung perfusate. In vitro, Iloprost caused a dose-dependent inhibition of PMA-stimulated PMN adherence to endothelial cells. Iloprost did not affect the number of Mo1 adhesion molecules constitutively expressed or the number of receptors expressed on the PMNs following PMA. Addition of cAMP or dibutyryl cAMP to the endothelial cells mimicked the effects of Iloprost, diminishing PMA-stimulated PMN adhesion. In separate experiments, addition of the phosphodiesterase inhibitor IBMX to Iloprost resulted in a greater inhibition of PMA-stimulated PMN adherence, while addition of an adenylate cyclase inhibitor, SQ 22,536, or cAMP antibodies with the Iloprost abolished Iloprost's inhibitory effect on PMN adhesion. Thus, Iloprost inhibits PMA-activated PMN-induced lung injury despite continued superoxide production. Iloprost inhibition of PMN adhesion is dependent on cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1990

The role of the neutrophil in inflammatory diseases of the lung.

Under certain circumstances, the neutrophil has been implicated in causing disease by damaging normal host tissue. This may occur in the adult respiratory distress syndrome (ARDS). The neutrophil has been implicated since a) substances that activate neutrophils are produced in association with the predisposing risks that lead to ARDS; b) activated neutrophils migrate into the alveolar spaces and their toxic products can be found in lung lavage fluid and in the breath of patients with ARDS; and c) the magnitude of the physiologic alterations correlate with the number of neutrophils in the alveolar space. Additionally, the neutrophils may be primed by substances which are released by activated platelets within the confines of the lung. Both platelet adenine nucleotides and the platelet-derived extracellular matrix protein (ECM), thrombospondin, can prime the neutrophil for subsequent O2- generation following activation of the cells with the chemotactic peptide, F-met-leu-phe (FMLP). Furthermore, neutrophils can be primed or O2- generation by the basement membrane ECM protein, laminin. Since neutrophils express receptors for both laminin and thrombospondin, these constituents may serve to modulate neutrophil behavior for subsequent oxidative metabolism and contribute to exacerbating pulmonary disease.

Adenine Nucleotides↗
PubMed1989

Laminin promotes the oxidative burst in human neutrophils via increased chemoattractant receptor expression.

The extracellular matrix component, laminin, enhances the chemotactic responsiveness of polymorphonuclear leukocytes (PMN) in vitro, and low doses of chemoattractant substances augment the expression of PMN cell surface receptors for laminin. This study determined whether laminin acts in concert with chemoattractants to activate PMN. Laminin (5 to 100 micrograms/ml) stimulated lysozyme release and superoxide production in response to the chemoattractant, FMLP by as much as 69%. These results could be explained by changes in cell surface chemoattractant receptor expression in that incubation of normal PMN with laminin (5 to 75 micrograms/ml) increased the binding of 19 nM FML[3H]P by 35 to 80%. This corresponded to as much as a 2.5-fold increase in the number of chemoattractant receptors/cells which had a lower average affinity. Laminin did not change the number or affinity of FML[3H]P receptors present on organelle-depleted PMN cytoplasts, and the laminin-induced increase in FML[3H]P receptors expressed on PMN from a patient with a specific granule deficiency was only 11 to 21% of that seen in normal PMN. These findings suggest that chemoattractants augment the expression of laminin receptors which mediate PMN attachment to basement membranes, followed by laminin-induced increases in the expression of cryptic chemoattractant receptors contained in intracellular granules, with resultant augmentation of the oxidative burst.

Adult↗
PubMed1989

Abnormal peroxidase-positive granules in "specific granule" deficiency.

"Specific granule" deficiency (SGD) has been previously associated with lactoferrin deficiency. The antimicrobial peptides termed defensins, comprising 30% of normal primary granule proteins, have also been shown to be markedly deficient in SGD. The present study was undertaken to correlate these findings with ultrastructural morphometric analysis and peroxidase cytochemistry. Peroxidase-positive, rim-stained, large, defensin-rich dense granules, previously described as a subpopulation of azurophil or primary granules in normal neutrophils, were markedly decreased in a patient with SGD. Morphometric studies of peroxidase-positive granules indicated an average peroxidase-positive granule area (all profiles) in the patient of 0.019 +/- 0.017 micron 2 (mean +/- SD, n = 941) compared to control values from normal neutrophils of two volunteers of 0.049 +/- 0.033 micron 2 (n = 896) and 0.050 +/- 0.039 micron 2 (n = 873) (P less than 0.001 between patient and control samples). Granule histograms showed a single peak of small peroxidase-positive granules, whereas control samples contained more prominent subpopulations of larger peroxidase-positive granules. The total number of peroxidase-positive granules per 100 micron 2 of cytoplasm in the patient was 255 +/- 124 (mean +/- SD, n = 15 cell profiles), which was similar to control values of 266 +/- 63 and 212 +/- 109. Thus, the defensin deficiency in SGD is associated with a decrease in size rather than number of peroxidase-positive granules; suggesting that defensins contribute to normal peroxidase-positive granule size and that SGD is a more global granule deficiency than originally thought.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Proteins↗
PubMed1989

Sequential expression of cell surface C3bi receptors during neutrophil locomotion.

Fluorescence microscopy has been used to study the cell surface distribution of the complement receptor for C3bi (CR3) on human neutrophils during locomotion. CR3 is an integral membrane protein that participates in cell attachment phenomena including chemotaxis. Fluorescein- and rhodamine-conjugated monoclonal IgG or Fab fragments were used to label CR3. We have previously shown that CR3 is uniformly distributed on unstimulated cells. During cell locomotion the fluorescent labels redistribute to the uropod and retraction fibers. To better understand the role of CR3 in chemotaxis, we have performed sequential two-color labeling experiments in conjunction with fluorescence microscopy. Double-labeling experiments were conducted by labeling adherent neutrophils with fluorescein-conjugated anti-CR3 followed by chemotaxis in a gradient of FMLP (10(-7) M). The cells were then labeled again with rhodamine-conjugated anti-CR3. The uropod and distal training filopodia were labeled with fluorescein, whereas the cell body and occasionally proximal filopodia near the uropod were labeled with rhodamine. When neutrophils were fixed and permeabilized prior to the second CR3 labeling, the second fluorescent label was localized to a granule-like compartment(s), often near the lamellipodium. The results suggest a flow of CR3 from intracellular granules----lamellipodia and cell body----uropod----trailing filopodia during chemotaxis.

Cell Compartmentation↗
PubMed1989

Cytoplasts and Mo1-deficient neutrophils pretreated with plasma and LPS induce lung injury.

We hypothesized that neutrophil adhesion and lung injury could occur independent of the surface receptor glycoprotein, Mo1 (C3bi receptor). We investigated whether preincubation of human neutrophil-derived cytoplasts (cell fragments that lack nuclei and granules and have a fixed number of surface Mo1 receptors) with plasma and lipopolysaccharide (LPS) would augment the cytoplasts' ability to cause lung injury when activated. We also investigated whether preincubating normal human neutrophils treated with anti-Mo1 antibody with plasma and LPS would increase the neutrophils' ability to adhere and cause lung injury. Human neutrophils infused into isolated salt-perfused rat lungs subsequently stimulated with phorbol 12-myristate 13-acetate (PMA) resulted in lung injury as assessed by the accumulation of 125I-bovine serum albumin in the lung parenchyma. The infusion of cytoplasts resulted in significantly less injury. Cytoplasts preincubated in 20% human plasma and LPS caused an increase in lung injury. Similarly, neutrophils treated with plasma, LPS, and anti-Mo1 antibody or neutrophils congenitally deficient in the Mo1 surface receptor and treated with plasma and LPS augmented lung injury. Plasma and LPS preincubation also increased anti-Mo1 antibody-treated neutrophil adhesion to endothelial cell monolayers after activation by PMA. Thus, plasma and LPS increase adhesion and lung injury caused by neutrophils or neutrophil fragments that share defects in Mo1 receptor expression.

Animals↗
PubMed1989

Selective defect in myeloid cell lactoferrin gene expression in neutrophil specific granule deficiency.

Neutrophil specific granule deficiency (SGD) is a congenital disorder associated with an impaired inflammatory response and a deficiency of several granule proteins. The underlying abnormality causing the deficiencies is unknown. We examined mRNA transcription and protein synthesis of two neutrophil granule proteins, lactoferrin and myeloperoxidase in SGD. Metabolically labeled SGD nucleated marrow cells produced normal amounts of myeloperoxidase, but there was no detectable synthesis of lactoferrin. Transcripts of the expected size for lactoferrin were detectable in the nucleated marrow cells of two SGD patients, but were markedly diminished in abundance when compared with normal nucleated marrow cell RNA. Because lactoferrin is secreted by the glandular epithelia of several tissues, we also assessed lactoferrin in the nasal secretions of one SGD patient by ELISA and immunoblotting. Nasal secretory lactoferrin was the same molecular weight as neutrophil lactoferrin and was secreted in normal amounts. From these data, we conclude that lactoferrin deficiency in SGD neutrophils is tissue specific and is secondary to an abnormality of RNA production. We speculate that the deficiency of several granule proteins is due to a common defect in regulation of transcription that is responsible for the abnormal myeloid differentiation seen in SGD patients.

Blotting, Northern↗
PubMed1989

Characterization and cytoskeletal association of a major cell surface glycoprotein, GP 140, in human neutrophils.

The binding of specific ligands to neutrophil cell surface receptors and the association of these receptors with the cytoskeleton may represent an essential step in activation. To identify surface proteins that are linked to the cytoskeleton during activation, neutrophil 125I-surface labeled plasma membranes were extracted with Triton X-100, and the soluble and insoluble (cytoskeleton) fractions analyzed by SDS-PAGE and autoradiography. The major cell surface proteins recruited to the cytoskeleton after activation with Con A, FMLP, zymosan-activated serum, or immune complexes possessed a relative molecular mass in the range of 80 to 13 kD. In addition to these proteins, WGA stimulates the recruitment of a 140-kD protein (GP 140) to the cytoskeletal fraction. That GP 140 is a WGA-binding protein was verified by Western blotting and WGA-Sepharose affinity chromatography. The Coomassie blue staining pattern of the WGA cytoskeletal fraction revealed major protein bands at apparent molecular weights of greater than 200 (approximately 250, 240, 235), 200, 115, 82/78 (a doublet), 56, 43, 36, and 18 kD. Labeling cells with 32PO4 before WGA treatment indicated that the cytoskeletal proteins with molecular weights of 115, 82/78, and 72 kD, and a 40-kD detergent soluble protein, are phosphorylated during activation. The 78 kD cytoskeletal phosphoprotein co-migrates with the lower subunit of erythrocyte (RBC) band 4.1 and shows strong cross-reactivity with RBC anti-band 4.1 antibody. Phosphorylation of cytoskeletal proteins like 4.1 may be involved in the regulation of interactions between GP 140 and the actin-containing cytoskeleton. Unlike the C3bi receptor, GP 140 is a major surface component of unactivated PMNs, has no stoichiometrically related 95-kD subunit, and has two isoforms with pIs in the range of 6.4 to 6.6. Under conditions that result in an increased expression of the C3bi receptor (such as treatment with the Ca2+ ionophore A23187), the amount of GP 140 on the PMN cell surface appears to be significantly reduced. The interaction of GP 140 with the cytoskeleton during activation suggests that GP 140 may play an important role in neutrophil functional responses.

Actins↗
PubMed1988

Human neutrophil protein kinase C: calcium-induced changes in the solubility of the enzyme do not always correlate with enzymatic activity.

We hypothesized that calcium and 1,2-diacylglycerols stimulated human neutrophil (PMN) protein kinase C (EC 2.7.1.37) in a two-step mechanism. The proposed mechanism entails (1) increased insoluble protein kinase C activity and (2) endogenous protein phosphorylation, events which have not been biochemically dissociated. PMN which were treated with 100 nM ionomycin shifted protein kinase C activity from being mostly soluble to insoluble. Concentrations of ionomycin greater than 300 nM stimulated a doubling of total cellular (soluble + insoluble) protein kinase activity and stimulated increased endogenous phosphorylation of PMN proteins. Intracellular calcium (measured with fura-2) increased from 65 nM (basal) to 680 nM using 500 nM ionomycin; calcium increases were dose-dependent. The anti-inflammatory agents acetylsalicylic acid and sodium salicylate (but not ibuprophen, indomethacin or acetaminophen) inhibited ionomycin-induced protein kinase C activation and protein phosphorylation in a dose-dependent manner by inhibiting the production of diacylglycerols. 1-Oleoyl-2-acetylglycerol reversed the inhibitory effect of salicylates. In contrast to the effect of acetylsalicylates on protein kinase C functional activity the distribution of phorbol receptors was unaffected in acetylsalicylate-treated, ionomycin-stimulated PMN using a phorbol-binding assay. Our results show that ionomycin increased intracellular diacylglycerol levels 3.5-fold over those present in control PMN, while acetylsalicylate decreased diacylglycerol production in ionomycin-stimulated PMN below baseline values. These results support the hypothesis that increased intracellular calcium activated protein kinase C leading to protein phosphorylation in two distinct dissociable events: (1) increased intracellular calcium; and (2) increased 1,2-diacylglycerol levels.

Algorithms↗
PubMed1988

Stimulation of human neutrophil adhesive properties by adenine nucleotides.

Inasmuch as adenine nucleotides may be secreted by platelets during inflammation, we sought to determine whether ATP and related compounds could serve as stimuli of neutrophil (polymorphonuclear cells, PMN) activation as manifested by an increase in their adhesive properties. Exposure of isolated human PMN to ATP or its nonhydrolyzable analog, adenosine 5'-O-(3-thiotriphosphate) did indeed stimulate an increase in cellular adhesive function as assessed by an increase in the surface expression of the leukocyte adhesion-promoting glycoprotein, Mo1 (CD11b/CD18), the initiation of PMN aggregation, and (in the case of ATP) the attachment of increased numbers of albumin-coated polystyrene latex beads. However, this increase in PMN adhesive function was not accompanied by the generation of products of the respiratory burst. These in vitro data suggest the possible influence of secreted adenine nucleotides in promoting neutrophil adhesion-dependent interactions at inflammatory sites in vivo.

Adenosine Triphosphate↗
PubMed1988

Optical microscopy of antibody-dependent phagocytosis and lysis of erythrocytes by living normal and chronic granulomatous disease neutrophils: a role of superoxide anions in extra- and intra-cellular lysis.

Novel optical microscopic techniques have been developed to observe neutrophil-mediated effector functions at the level of individual cells. Conventional absorption spectrophotometry has shown that exposure of hemoglobin to superoxide anions decreases the intensity of the Soret band and shifts it to lower wavelengths. This oxidative event can be visualized within intact erythrocytes using bright-field microscopy in conjunction with violet illumination at 430 nm. The sequential oxidation of IgG-opsonized sheep erythrocytes bound to normal human neutrophils can be observed. Chronic granulomatous disease (CGD) neutrophils which do not generate superoxide anions were not capable of influencing target absorption at 430 nm. Cytolytic events were visualized by fluorescence microscopy. Cytosolic or membrane compartments of sheep erythrocytes were labeled with eosin Y or fluorescein isothiocyanate, respectively. Time-dependent studies of erythrolysis show that targets are lysed extra- and intra-cellularly. The fluorescent diffusion gradient generated at the site of membrane rupture suggests that a pore of approximately 30 nm in diameter is formed in the target membrane. The site of pore formation is not found at the target-effector cell interface. CGD neutrophils did not display these cytolytic phenomena. Furthermore, the cytosolic label eosin Y could be followed into an associated granule compartment; we suggest that the phenomenon of piranhalysis may participate in antibody-dependent effector mechanisms. Phagocytosis can also be observed using fluorescently-labeled erythrocytes. Determinations of phagocytic index are more reliable with this approach. These microscopical methods are both simple and efficient. To our knowledge, these are the first direct microscopic studies of effector cell-mediated target cell oxidation and cytolysis. These experiments provide a fresh approach to the study of phagocyte effector functions at the cellular level and illuminate the importance of superoxide anions in antibody-dependent erythrolysis.

Animals↗
PubMed1988

Microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: Chediak-Higashi syndrome and "specific" granule deficiency.

Although several genetic defects are known to impair oxidative microbicidal/cytotoxic mechanisms in human PMN, no deficiencies of PMN granule components that mediate oxygen-independent microbicidal activity have yet been reported. We analyzed PMN from patients with various granulocyte disorders for their content of two azurophil granule constituents, defensins and cathepsin G, that exert microbicidal/cytotoxic activity in vitro, and one component, elastase, that has ancillary microbicidal/cytotoxic activity. PMN from two (of two) patients with specific granule deficiency (SGD) displayed an almost complete deficiency of defensins, which in normal cells constitute greater than 30% of the protein content of azurophil granules. The SGD PMN contained normal or mildly decreased amounts of cathepsin G and elastase. Conversely, the PMN of three (of three) patients with Chediak-Higashi syndrome (CHS) substantially lacked cathepsin G and elastase, but their defensin content was normal or mildly decreased. Both CHS and SGD patients suffer from frequent and severe bacterial infections, and CHS patients frequently develop an atypical lymphoproliferative syndrome. The profound deficiency of PMN components with microbicidal/cytotoxic activity in SGD and CHS may contribute to the clinical manifestations of these disorders.

Blood Bactericidal Activity↗
PubMed1988

Deficiency in immune complex uptake by chronic granulomatous disease neutrophils.

Receptor-mediated binding and endocytosis by normal and chronic granulomatous disease (CGD) polymorphonuclear (PMN) leucocytes have been investigated. Our results suggest that activation of the respiratory burst is a requisite for normal immune complex internalization. Cell-associated immune complexes were detected by fluorescence microscopy, electron microscopy and flow cytometric techniques. These measurements indicate that immune complex uptake is defective in CGD patients, but not in normal subjects. Antibody-dependent binding and complement-dependent binding and endocytosis were not affected. The functional association of the respiratory burst and antibody-dependent endocytosis was further explored using rhodamine-labelled glucose oxidase:anti-glucose oxidase immune complexes. These complexes generate H2O2 in the presence of glucose. When treated with these complexes, CGD PMNs showed similar glucose-dose-dependent increases in both H2O2 production and cell-associated fluorescence. Therefore: (1) respiratory burst and immune complex uptake are coexisting deficiencies that may be a manifestation of a single defect; and (2) the respiratory burst may be one participant in immune complex endocytic triggering.

Antigen-Antibody Complex↗
PubMed1988

The role of calcium in the age-related decline of neutrophil function.

Upon activation by formyl-methionyl-leucyl-phenylalanine (FMLP), either in the presence of absence of cytochalasin B, neutrophils from old subjects generated significantly less superoxide than did neutrophils from the young. This reduction in activity was associated with a significant decrease in the basal cytosolic calcium concentration and a diminished flux of calcium to the cytosol after activation. At all concentrations of FMLP tested, cytosolic calcium remained significantly lower in neutrophils from the old as compared with the young, whereas permeability to extracellular calcium and efflux of calcium from the cell were also significantly diminished. Pretreatment of the cell with the ionophore ionomycin elevated the cytosolic calcium concentration and significantly improved function in old neutrophils. These findings demonstrate that aging results in alterations in neutrophil calcium homeostasis that may play a role in the age-related decline in neutrophil function.

Adult↗
PubMed1988

Neutrophil granule constituents and their release in health and disease.

In this review of secretion by neutrophils, we have given a detailed description of the characteristics of the exocytotic responses of these cells to a variety of stimuli. Secretion of the contents of the two types of granules follows different rules. Azurophil granules are discharged only when neutrophils interact with phagocytizable particles, whereas specific granules are discharged on interaction of the cells with both particulate and soluble stimuli. In the latter case, specific granule constituents are released directly into the extracellular environment, demonstrating that these granules can function as secretory organelles. Studies of patients with specific granule deficiency have indicated the role of specific granules in modulating the inflammatory response. Additionally, studies of one of these patients has provided a better understanding of basic processes and pathways contributing to priming of neutrophils for subsequent activation by soluble stimuli. It appears that elevated intracellular calcium levels, rather than the mere translocation of protein kinase C to the plasma membrane, can account for the exaggerated response of specific granule-deficient cells to subsequent stimulation. Further studies are needed to unravel the biochemical mechanisms underlying the abnormal fusion of granules in the Chédiak-Higashi syndrome. It is possible that further studies of Chédiak-Higashi syndrome will result in a better understanding of the mechanisms by which granules normally fuse with phagosomes. Studies of the biochemical properties of novel secretory organelles containing either gelatinase or alkaline phosphatase and of their discharge on stimulation will also provide much needed information on the role of secretory events in the early stages of neutrophil adhesion, diapedesis, and movement within tissues.

Animals↗
PubMed1987

Changes in protein kinase C activity are associated with the differentiation of Friend erythroleukemia cells.

We investigated the activity and cellular distribution of protein kinase C during the dimethylsulfoxide (DMSO) and hypoxanthine-induced differentiation of Friend murine erythroleukemia cells. Most of the cellular protein kinase C activity was found in the soluble fraction of unstimulated Friend cells. Within 15 min of the addition of DMSO or hypoxanthine, protein kinase C underwent a dramatic and prolonged reversal of this distribution which was accompanied by a gradual decline in total cellular protein kinase C activity over the ensuing 5 days. The loss of total activity was found to be dose dependent although maximal translocation from soluble to insoluble components occurred at even lower concentrations of the inducers tested. Two clones of Friend cells, selected for their failure to differentiate in response to DMSO, showed alterations in protein kinase C activity and/or distribution following DMSO addition when compared to wild-type Friend cells. These data show that different inducers of Friend cell differentiation have similar effects on cellular protein kinase C, that the protein kinase C changes accompanying this process are immediate but prolonged, and that changes in protein kinase C activity and distribution are associated with Friend cell differentiation.

Animals↗
PubMed1987

Human neutrophil laminin receptors: activation-dependent receptor expression.

Activated human polymorphonuclear leukocytes (PMN) isolated from peripheral blood specifically bind 125I-laminin after stimulation with phorbol 12-myristate 13-acetate (PMA) or f-Met-Leu-Phe (FMLP) at 37 degrees C. Changes in laminin receptor expression are stimulus dose dependent at both chemotactic (10(-10) M to 10(-6) M) concentrations of FMLP, and secretory (greater than 5 ng/ml) levels of PMA. In the presence of cytochalasin B (5 micrograms/ml), 10(-7) M FMLP activation stimulates specific laminin binding, with an apparent Kd = 3.9 X 10(-9) M and 6.47 X 10(5) binding sites/cell, reaching equilibrium within 10 min at 4 degrees C. This observed activation-dependent change in laminin receptor expression is not due to interference by endogenous laminin, because no fluorescein-visualized anti-laminin antibody bound to cells without added glycoprotein, regardless of the level of activation. Levels of neutrophil lysozyme release, which show a PMA dose dependence similar to that of receptor binding activity, suggest that granule-plasma membrane fusion may be significant during increases in receptor expression. A lack of receptor stimulation by PMA from a granule-deficient patient or in granule-depleted cytoplasts from normal donors additionally supports this hypothesis. Electroblot transfer and autoradiography of subcellular fractions from unstimulated PMN reveals the presence of a 68,000 dalton laminin-binding component in the secondary/tertiary granule (beta) fraction, which may represent an intracellular laminin receptor pool.

Cell Compartmentation↗