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D English

Publications and source records attributed to D English.

At least 91 records · Page 5Linked to original sources
PubMed1994

Contributions of capillary pathway size and neutrophil deformability to neutrophil transit through rabbit lungs.

Neutrophil margination within the pulmonary capillary is due to a delay in their transit compared with that of red blood cells (RBC). This delay has been attributed to the large fraction of capillary segments that are narrower than spherical neutrophils and differences between the time required for deformation of neutrophils and that required for deformation of RBC. This study investigated the characteristics of neutrophil deformation in vivo and the perfusion patterns of segments within capillary pathways. Studies comparing the extraction of neutrophils with that of nondeformable microspheres in one transit through the pulmonary circulation suggest that neutrophils can undergo a rapid deformation from 6.4 to 5.0-5.1 microns, whereas larger deformations require a delay. Effective diameters of the perfused capillary pathways were larger than expected for a random distribution of capillary segment diameters within these pathways. The longer transit times of neutrophils in the upper regions of the lung were associated with a greater fraction of pathways containing narrow segments. These studies suggest that neutrophil deformability and capillary pathway diameters are important in determining the size of the marginated pool of neutrophils within the pulmonary capillaries.

Animals↗
PubMed1994

Quantitation of neutrophil migration in acute bacterial pneumonia in rabbits.

The circulating neutrophils must slow down, adhere to the vessel walls, and migrate out of the microvasculature into the tissue and air spaces to defend the lung against microorganisms. The present study was designed to provide quantitative information about each of these steps. Streptococcus pneumoniae was instilled into the left lower lobe of New Zealand White rabbits to induce a pneumonia, and this lobe was compared with the same region of the opposite lung. The distribution of blood flow was determined by using radiolabeled macroaggregated albumin, and the patterns of perfusion within the capillary bed were quantitated using Monastral blue. The number of neutrophils delivered to the pneumonic site was determined by multiplying the circulating neutrophil count and blood flow. The results show that retention of 51Cr-labeled neutrophils was increased in the pneumonic region 2, 4, and 8 h after instillation of the organisms. The number of intracapillary neutrophils was increased in the pneumonic regions at all time points and in the control regions at 1 and 4 h. Neutrophil migration occurred in the pneumonic site, but only 1-2% of the total neutrophils delivered to the region migrated out of the pulmonary vessels into the air space. We conclude that the circulating neutrophils undergo a generalized response that increases their margination throughout the lung, that increased margination in the pneumonic site changes the distribution of capillary flow, and that the majority of neutrophils delivered to a pneumonic site are returned to the circulation without migrating into the air space.

Acute Disease↗
PubMed1994

A non-radioisotopic method for tracing neutrophils in vivo using 5'-bromo-2'-deoxyuridine.

Polymorphonuclear leukocytes (PMN) labeled in vivo with 5'-bromo-2'-deoxyuridine (BrdU) in donor animals were transferred to recipients to determine the rate of clearance of labeled PMN from the circulation, their margination within the vascular space, and migration into Streptococcus pneumoniae-induced inflammatory sites. The donor animals received intravenous infusions at 25 mg/kg/day of BrdU for 7 days when cytospins of leukocyte-rich plasma (LRP) showed that 80 +/- 2.3% PMN were labeled. The BrdU labeled cells were then transfused to serum-compatible recipients as either whole blood, leukocyte-rich plasma, or PMN purified from an equal volume of whole blood. Twenty-four hours after transfer, the distribution of BrdU-labeled PMN in the lung, liver, spleen, bone marrow, and gut was determined morphometrically and by Southern blot analysis of DNA extracted from these organs. BrdU-labeled PMN transfused as either LRP or purified PMN provided no advantage over transfusing whole blood. The half-life of BrdU-labeled PMN in the recipient circulation after transfusing whole blood was 270.4 min (95% confidence intervals, 248.5 to 296.4 min). The majority of the BrdU-labeled DNA was found in the spleen, where DNA analysis showed that the white blood cells underwent programmed cell death by apoptosis. Four hours after infection with S. pneumoniae and 1 h after transfusion of labeled whole blood, BrdU-labeled PMN had migrated into the infected sites. We conclude that transfer of BrdU PMN in whole blood provides a simple, effective method of tracing labeled PMN in vivo.

Animals↗
PubMed1994

Parallel up-regulation of CD11B and CD45 on neutrophilic leukocytes exposed to soluble factors of oral pathogens.

We tested culture supernatants from a battery of oral bacterial strains for their ability to influence the expression of CD11b and CD45 on the neutrophil plasma membrane. Several bacterial extracts stimulated the up-regulation of both CD11b and CD45 simultaneously. Two supernatants in particular (a clinical isolate of A. actinomycetemcomitans and F. nucleatum ATCC25586) potently stimulated the deployment of CD11b and CD45 from their intracellular storage site to the plasma membrane. Both supernatants inhibited superoxide release stimulated by exposure of neutrophils to formyl methionyl leucyl phenylalanine (FMLP) but had variable effects on superoxide release stimulated by phorbol myristate acetate (PMA). The ability of products of oral bacteria to modulate neutrophil plasma membrane antigen composition may regulate functional reactivity and thus be an important factor in the pathogenesis of periodontal infection and inflammation.

Antigens, CD↗
PubMed1993

Characterization of phosphatidic acid phosphohydrolase in neutrophil subcellular fractions.

This investigation was designed to confirm the presence of PA phosphohydrolase in human neutrophils and to determine the distribution and characteristics of the enzyme in soluble and particulate subcellular fractions of disrupted neutrophils. Enzyme activity was detected in unseparated extracts of sonicated neutrophils. The majority of the recovered activity was recovered in a particulate fraction rich in neutrophil plasma-membrane markers; moderate levels (20%) of the total activity were recovered in the cytosol. While Mg2+ markedly potentiated the cytosolic but not the particulate activity, Ca2+ moderately inhibited both the cytosolic and particulate enzymes. The plasma-membrane-associated activity was absolutely dependent on detergent (0.5% Triton X-100) and displayed an apparent Km of 62 microM for phosphatidic acid. Enzyme activity was markedly inhibited by NaF, not influenced by excess glycerophosphate and slightly attenuated by propranolol, an inhibitor of PA phosphohydrolase in other systems. Preincubation of plasma membranes with N-ethylmaleimide at concentrations up to 25 mM had little effect on enzyme activity. However, activity in cytosolic and microsomal fractions of neutrophils were completely abolished by preincubation with N-ethylmaleimide at concentrations of less than 5 mM. We conclude that neutrophils possess a potent PA phosphohydrolase localized in their plasma membranes. Metabolism of cellular second-messengers by this enzyme may exert a profound effect on the functions of stimulated neutrophils.

Calcium↗
PubMed1993

Thrombin receptor activating peptides induce Ca2+ mobilization, barrier dysfunction, prostaglandin synthesis, and platelet-derived growth factor mRNA expression in cultured endothelium.

Endothelial cell activation by thrombin is a key event in wound healing, inflammation, and hemostasis. To better define thrombin-endothelial cell interactions we synthesized several peptides of varying length corresponding to the initial 14 amino acid sequence of the cloned human platelet thrombin receptor after cleavage at an arginine41 site (R/SFLLRNPNDKYEPF). Thrombin receptor activating peptides (TRAPs) as short as 5 amino acids induced significant levels of PGI2 synthesis and expression of PDGF mRNA in human endothelium and produced dose-dependent cellular contraction and permeability of confluent human umbilical vein and bovine pulmonary artery endothelial monolayers. To explore whether TRAPs utilized similar signal transducing pathways as alpha-thrombin to accomplish endothelial cell activation, phospholipase C production of the Ca2+ secretagogue IP3 was measured and detected 10 seconds after either TRAP 7 or alpha-thrombin. Furthermore, TRAPs ranging from 5-14 residues induced significant dose-dependent increases in Fura-2 fluorescence indicative of Ca2+(1) mobilization. These results indicate that thrombin-mediated proteolytic cleavage of the human and bovine thrombin receptor initiates stimulus/coupling responses such phospholipase C activation, Ca2+ mobilization, and protein kinase C activation. The functional consequence of this cellular activation via the cleaved receptor is enhanced cellular contraction, barrier dysfunction, PGI2 synthesis, and expression of PDGF mRNA.

Amino Acid Sequence↗
PubMed1993

Inositol trisphosphate is involved in norepinephrine- but not in hypoxia-induced pulmonary arterial contraction.

The role that second messengers play in pulmonary vasoconstriction is not understood. The purpose of this study was to directly measure inositol phosphates in isolated pulmonary arterial preparations before and during norepinephrine (NE) stimulation and acute hypoxia. Rat main pulmonary arteries were isolated and incubated with myo-[3H]-inositol. After incubation, control tissue was stimulated with 0.5 microM NE or 30 mM KCl. Test preparations were precontracted with 30 mM KCl and then exposed to hypoxia. Samples were homogenized and applied to a high-pressure liquid chromatography column for analysis of inositol phosphates. Results show that inositol trisphosphate (IP3) increases twofold at 5 s following NE stimulation. Thirty micromolars of KCl results in a slight but significant increase in IP3 formation at 5 min following the stimulation. Phentolamine inhibits the KCl-induced increase in IP3 formation, whereas A23187 has no effect on IP3 levels. Hypoxia caused a biphasic contraction in the precontracted isolated rat pulmonary artery. IP3 levels did not change during the hypoxic period. In conclusion, NE causes a rapid increase in IP3 formation consistent with the time course of production of an excitation-contraction coupling second messenger. However, inositol trisphosphate is not involved in the signal transduction pathway leading to pulmonary arterial contraction induced by hypoxia.

Acute Disease↗
PubMed1993

Effect of positive end-expiratory pressure on leukocyte transit in rabbit lungs.

Previous experiments in humans and animals have shown that neutrophils (PMN) are delayed in the lung by the inhalation of cigarette smoke. Others have shown that cigarette smoking raises airway resistance, and it follows that this will increase the expiratory time constant of the airways. When the expiratory time constant of the airways exceeds that of the chest wall, alveolar pressure will rise and compress alveolar capillaries. To determine the effect of alveolar compression on PMN retention, we measured the arteriovenous (A-V) difference for leukocytes across the lung and determined the retention of 51Cr-labeled PMN in the lungs of anesthetized ventilated rabbits. The results show that the application of positive end-expiratory airway pressure (PEEP) produced an immediate difference for PMN across the lung, which disappeared in approximately 3 min when PEEP was continuously applied. This effect was attributed to alveolar compression rather than reduced cardiac output, because a similar A-V difference for PMN was observed in separate experiments in which cardiac output was maintained by vascular expansion during PEEP. The results also show that, when PEEP was continuously applied for approximately 30 min, it failed to increase the percent retention of either inactivated (PEEP = 14 +/- 2% vs. non-PEEP 16 +/- 3%) or activated (PEEP = 62 +/- 7% vs. non-PEEP 63 +/- 6%) 51Cr-PMN. We conclude that the application of PEEP traps PMN in compressed alveolar capillaries, creating an immediate A-V difference that disappears as blood flow is redistributed to vessels that are not compressed by PEEP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Simultaneous mobilization of Mac-1 (CD11b/CD18) and formyl peptide chemoattractant receptors in human neutrophils.

Mobilization of a distinct subset of specific granules provides a physiologically important mechanism to recruit Mac-1 (CD11b/CD18) from an intracellular pool to the external surface of the neutrophil plasma membrane, where the functionally active heterodimer mediates several adherence-dependent processes that are crucial for adequate host defense and cellular inflammatory responses. We observed similar characteristics for translocation of Mac-1 and neutrophil formyl peptide receptors (FPR) and hypothesize that the readily accessible pools of both Mac-1 and FPR are colocalized within this specific granule subset. Plasma membrane levels of both FPR (assessed with 3H-FMLP) and Mac-1 (assessed by fluorescence-activated cell sorter analysis of fluorescein isothiocyanate [FITC]-Mo-1-labeled cells) were markedly downregulated in cells prepared at low temperature from blood cooled to 4 degrees C immediately after removal from the circulation. Levels of both FPR and Mac-1 remained low on cells held at 4 degrees C. Upon warming, spontaneous upregulation of Mac-1 and FPR occurred with similar kinetics and temperature dependency. Translocation of both Mac-1 and FPR was markedly potentiated by exposure of cells to either fluoride ion (which has been shown by others to specifically elicit exocytosis of gelatinase-rich and vitamin B-12 binding protein-poor granules) or granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that markedly potentiates the neutrophils' host defense capabilities. Levels of both FPR and Mac-1 on F-- or GM-CSF-treated neutrophils exceeded those present on cells incubated at 37 degrees C for extended time intervals, indicating that stimulated translocation may involve mobilization of an additional granule subset. Scatchard analysis showed that only low-affinity FPR were translocated during spontaneous and stimulus-dependent upregulation. To directly compare FPR levels on the surface of cells displaying varying levels of Mac-1 within a single cell suspension, cells were labeled with FITC-Mo-1 and sorted into subpopulations based on fluorescence intensity. After sorting, the individual populations were held at 4 degrees C to prevent further spontaneous upregulation, concentrated by centrifugation, and assayed for FPR levels. Under a variety of conditions, FPR levels correlated with Mac-1 (CD11b) expression on cell populations selected on the basis of CD11b fluorescence intensity. Analysis of subcellular fractions obtained from disrupted neutrophils before and after upregulation provided additional support for the hypothesis that Mac-1 and FPR are colocalized within a readily accessible subset of neutrophil granules.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Membrane↗
PubMed1992

Effect of zymosan-activated plasma on the deformability of rabbit polymorphonuclear leukocytes.

Intravascular infusion of inflammatory mediators causes a sudden neutropenia due to the sequestration of polymorphonuclear leukocytes (PMN) within the microvasculature of the lung and other organs. This sequestration could be due to a decrease in the ability of PMN to deform and pass through the narrow capillary bed. The purpose of this study was to determine if the complement fragments present in zymosan-activated plasma (ZAP) caused a rapid stiffening of PMN. The PMN deformability was determined by measuring the pressure required to pass PMN through a polycarbonate filter containing 5-micron pores at a constant flow rate as well as the extraction of PMN compared with red blood cells and 125I-labeled albumin by the filter. The role of the cytoskeleton in PMN deformation was examined in studies where F-actin formation was inhibited using cytochalasin B or microtubule assembly was inhibited using colchicine. The results showed that treatment with ZAP induced a rapid decrease in PMN deformability. Inhibiting the formation of F-actin made the unstimulated PMN more deformable and reduced the stiffening induced by ZAP. In contrast, inhibition of microtubule reassembly did not alter either normal deformability or the ZAP-induced decrease in deformability. In vivo, colchicine increased normal PMN margination but did not inhibit the rapid sequestration of PMN induced by infusion of ZAP. These studies indicate that ZAP induces a rapid decrease in PMN deformability that is mediated through the cytoskeleton. They suggest that this decrease is due to the polymerization of F-actin.

Actins↗
PubMed1992

Late effects of endotoxin on the accumulation and function of monocytes in rabbit lungs.

Recent studies from our laboratory show that the lung contains a marginated pool of monocytes. The present study was designed to investigate monocyte accumulation in this pool 4 to 28 h after a single dose of endotoxin when the endotoxin had disappeared from the circulation. This was accomplished by administering a single intravenous dose of endotoxin (Escherichia coli, 50 micrograms/rabbit) to unanesthetized animals (n = 6) and saline to controls (n = 5) at time 0. Four hours after this dose of endotoxin, 111In-monocytes (93.5% pure) isolated from donors were injected intravenously, and, at 27 h, the rabbits were anesthetized and colloidal carbon (CC, 1 ml/kg body weight) was injected intraarterially to provide a phagocytic stimulus. The animals were sacrificed at 28 h, and the lungs were fixed in situ with glutaraldehyde. The data show that lungs from the endotoxin-treated rabbits contained 4.8 times more 111In-monocytes than controls, that 92% of these radiolabeled monocytes were in the alveolar capillaries, and that 72% of these labeled cells had phagocytosed CC. The histologic studies of unlabeled cells confirmed that this endotoxin treatment caused a 3-5-fold increase in unlabeled mononuclear cells and neutrophils (PMN) in the microvasculature and that many of the unlabeled monocytes in the endotoxin-treated group had also phagocytosed colloidal carbon. The behavior of the donor monocytes injected after the endotoxin had time to disappear from the circulation suggests that they accumulate in the lung in response to the indirect effects of endotoxin on endothelial cells.

Animals↗
PubMed1992

Gp-regulated phosphoinositide hydrolysis in turkey and human erythrocytes exposed to fluoride ion: relationship to calcium influx.

Previous studies have demonstrated that although both mammalian and avian erythrocytes express an inducible inositol bisphosphate-specific phospholipase C, only the latter possess the guanine nucleotide-binding protein (Gp) that regulates this activity. In confirmation of previous reports, turkey erythrocyte plasma membranes responded to guanosine 5'-0-(3-thio)triphosphate (GTP-gamma-S) and fluoroaluminates with hydrolysis of phosphoinositides, release of inositol phosphates, and generation of diacylglycerol, whereas human erythrocyte plasma membranes exhibited no such changes when incubated with known activators of guanine nucleotide regulatory proteins. We next contrasted responses of intact turkey and human erythrocytes to fluoroaluminates to develop a model to investigate the cellular effects of Gp activation. When turkey erythrocytes were exposed to fluoroaluminates, cellular levels of diacylglycerol and phosphatidic acid rapidly increased as phosphoinositides were hydrolyzed. The alterations in the lipid composition of turkey erythrocytes effected by fluoroaluminates were remarkable; phosphatidic acid levels increased over 30-fold, whereas levels of polyphosphoinositides were decreased to less than 10% of those present before stimulation. In contrast, fluoroaluminates caused only minor alterations in the diacylglycerol and phospholipid content of intact human erythrocytes. To define the role of inositol-specific phospholipase C activation in the transmembrane conveyance of extracellular Ca++, we compared the influx of extracellular Ca++ in human and turkey erythrocytes exposed to fluoroaluminates. Fluoroaluminates initiated a sustained influx of extracellular 45Ca++ into turkey, but not human, erythrocytes. These results provide strong support for the hypothesis that Gp activation results in an influx of calcium into stimulated cells. Moreover, the data demonstrate that comparison of responses of human and turkey erythrocytes to fluoroaluminates provides a well-defined method for investigating the mechanisms and consequences of Gp activation in intact cells.

Animals↗
PubMed1992

Humoral and cell surface interactions during gamma-irradiation leukemogenesis in vitro.

Gamma-irradiation of plateau phase cultures of the clonal murine bone marrow stromal cell line D2XRII followed by cocultivation of a clonal interleukin 3 (IL-3) (granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent hematopoietic progenitor cell line FDC-P1JL26 results in a significant increase in "cobblestone islands" of attachment and emergence of subclonal factor-independent malignant sublines. Biochemical purification of conditioned medium from irradiated D2XRII cells yielded a 75,000-dalton glycoprotein termed leukemogenic stromal factor (LSF) that was neutralized by a polyclonal antiserum to murine macrophage colony-stimulating factor (M-CSF). A monoclonal antibody to the murine M-CSF receptor (c-fms) neutralized the biological activity of this molecule in a manner comparable to its effect on recombinant human or murine M-CSF. FDC-P1JL26 parent cells were positive for Ly5, MEL-14, mGR, VLA-4, PGP-1 (CD44), and Thy1.2. After culture in LSF, Thy1.2, MEL-14, and mGR became undetectable; however, significant cell surface MAC-1 antigen and c-fms (M-CSF receptor) were expressed. Neither line was positive for Ly6, Ly22, I-CAM-1, or B220 antigen. LSF-precultured FDC-P1JL26 cells transferred as single cells to microwell culture with 5000-cGy-irradiated D2XRII cells revealed a 60-fold increase in frequency of cobblestone island formation and evolution of factor-independent subclones compared to the parent line. Both parent and LSF-precultured cells became factor independent at a 100-fold lower frequency if kept in suspension in LSF in the absence of stromal cells. Antiserum to M-CSF or monoclonal antibody to the murine M-CSF receptor (c-fms) did not inhibit or displace cobblestone island formation by either clone of FDC-P1 on irradiated stromal cells indicating a mechanism of binding not involving the M-CSF receptor. However, anti-serum to the M-CSF receptor inhibited growth of one factor-independent subclone. In separate studies, a subclone of IL-3-dependent 32Dc13 cells, expressing the transfected murine c-fms protooncogene but not the parent 32Dc13 cell line or another subclone expressing the transfected gene for the human M-CSF receptor, showed adherence and became factor independent when cocultivated with irradiated D2XRII stromal cells. Thus, irradiated stromal cells bind M-CSF receptor-positive hematopoietic progenitor cells and induce c-fms-dependent factor-independent tumorigenic subclones. The cellular interactions in this model may be relevant to gamma-irradiation leukemogenesis in vivo.

Animals↗
PubMed1991

Diacylglycerol generation in fluoride-treated neutrophils: involvement of phospholipase D.

Neutrophils exposed to fluoride ion (F-) respond with a delayed and sustained burst of superoxide anion release that is both preceded by and dependent on the influx of Ca2+ from the extracellular medium. The results of this study demonstrate a similarly delayed and sustained generation of 1,2-diglyceride in F(-)-treated neutrophils, over 90% of which was 1,2-diacylglycerol. Diacylglycerol generation was not dependent on the presence of extracellular Ca2+. Conversely, in contrast to results obtained with other agonists, removal of extracellular Ca2+ markedly potentiated synthesis of diacylglycerol in F(-)-treated neutrophils. This effect was accompanied by a corresponding decrease in the recovery of phosphatidic acid. In either the presence or absence of extracellular Ca2+, phosphatidic acid accumulated before diacylglycerol in F(-)-treated cells, suggesting the latter was derived from the former. Consistent with this hypothesis, the phosphatidic acid phosphohydrolase inhibitor, propranolol, suppressed generation of diacylglycerol as it potentiated the accumulation of phosphatidic acid in F(-)-treated neutrophils. This effect was observed both in the presence and absence of extracellular Ca2+. Moreover, high levels of propranolol (160 mumol/L) effected complete inhibition of diacylglycerol generation in F(-)-treated neutrophils with a corresponding increase in phosphatidic acid generation. Phosphatidylethanol accumulated in neutrophils stimulated with F- in the presence of ethanol. The extent of phosphatidylethanol accumulation at all time points after addition of F- corresponded to decreased levels of both phosphatidic acid and diacylglycerol, indicating that phosphatidylethanol was derived from the phospholipase D-catalysed transphosphatidylation reaction. The results indicate that F- activates a Ca(2+)-independent phospholipase D, which appears to be the major, if not sole, catalyst for both phosphatidic acid and diacylglycerol generation in F(-)-treated neutrophils. Ca2+, mobilized as a result of F- stimulation and possibly as a consequence of phospholipase D activation, exerts a profound effect on cellular second messenger levels by modulating the conversion of phosphatidic acid to diacylglycerol.

Calcium↗