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Human neutrophils adherent to a polystyrene plastic surface are vigorously activated, whereas those adherent to fibronectin manifest only a priming response. The basis of these metabolic differences was further characterized; polystyrene-adherent cells, which were shown to spread quickly upon adhesion, exhibited an increase of cytoskeleton-associated actin (F-actin) (measured by a nitrobenzoxadiazole-phallacidin fluorescent staining assay) and a decrease of monomeric G-actin concentration (measured by a DNase inhibition assay); in contrast, fibronectin-adherent cells exhibited little spreading and decreased their F-actin, after 1.5 min of adhesion, to 33.49 +/- 6.9% (mean +/- SD, n = 5) of initial levels found in suspended cells before plating. Actin depolymerization in fibronectin-adherent cells was confirmed by measuring G-actin, which sharply increased during the first minute of adhesion, rising from 0.065 +/- 0.007 to 0.20 +/- 0.035 microgram/microgram of protein (mean +/- SEM, p less than 0.05), and then remained elevated during 5 min of observation. In contrast, soluble fibronectin induced a decrease of G-actin in suspended cells. Cells pretreated with 1 microM cytochalasin D and allowed to adhere to a plastic surface did not spread, failed to generate O2-, and exhibited elevated concentrations of G-actin (0.1 to 0.2 microgram/microgram of protein) during the 5 min of observation. Actin changes, as well as respiratory burst, in adherent cells were shown to proceed through a pertussis toxin-insensitive pathway. Fluo-3 measurements of intracellular Ca2+ concentrations ([Ca2+]i) showed a fourfold and twofold [Ca2+]i increase in polystyrene- and fibronectin-adherent cells, respectively, after 2 min. The small rise in [Ca2+]i in fibronectin-adherent cells corresponds to a primed response of these cells to subsequent activation with FMLP. Ionomycin (1 microM) added to neutrophils just before adhesion on fibronectin induced full activation, i.e., O2- production and actin polymerization. The metabolic events controlling metabolic priming and actin depolymerization are as yet uncharacterized, but fibronectin receptor-linked responses beyond the mediation of cell adhesion have now been identified, suggesting complex metabolic functions of integrin receptors.
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PMID:Comparison of actin changes and calcium metabolism in plastic- and fibronectin-adherent human neutrophils. 150 Jul 23

The potential role of platelet-activating factor (PAF)-acether and of IL-5 as an eosinophil-proliferating, activating, and/or recruiting mediator in asthma led us to study the effects of human (h) rIL-5 (hrIL-5) and PAF-acether, alone or combined, on isolated guinea pig eosinophils. Two populations of eosinophils were separated from peritoneal lavages of polymyxin B-treated guinea pigs upon a discontinuous metrizamide gradient: one of low density (between 20 and 22% of metrizamide, purity: 63 +/- 3%, n = 27) and another of normal density (between 22 and 24% of metrizamide, purity: 87 +/- 2%, n = 16). Chemotactic activity was evaluated on a micro-Boyden chamber, results being expressed as the number of migrating eosinophils (mean +/- SEM) at 40 microns through a cellulose nitrate filter (3 microns pore size) in the presence of the agonist or of the solvent alone. hrIL-5 dose-dependently stimulated normodense eosinophil chemotaxis, reaching a peak at 500 ng/ml (98 +/- 21 migrating eosinophils, n = 5, p less than 0.05). These eosinophils also responded to PAF-acether and to LTB4 and not to FMLP, hrTNF alpha, and LPS. Eosinophil preincubation with hrIL-5 increased significantly the migration by PAF-acether (173 +/- 23 migrating eosinophils with PAF-acether 10 nM after preincubation with hrIL-5 500 ng/ml vs 69 +/- 10 after preincubation with buffer alone, p less than 0.01) and failed to enhance migration by LTB4 or to uncover an activity for FMLP. Migration by PAF-acether was antagonized when the cells were preincubated with the antagonists BN 52021 and WEB 2086, which also inhibited migration by hrIL-5. Eosinophils were auto-desensitized by and to PAF-acether or LTB4, but were not cross-desensitized to each other. Eosinophils desensitized to PAF-acether failed to migrate with hrIL-5, but those desensitized to LTB4 responded to hrIL-5 as controls. hrIL-5 failed to induce the elevation of intracellular free calcium concentration and superoxide anion generation from basal values, whereas preincubation of eosinophils with hrIL-5 induced a significant increase in the rise in intracellular free calcium concentration and in superoxide anion generation by 10 nM PAF-acether but not by LTB4. In conclusion, the in vivo eosinophil migration in allergy may involve hrIL-5, particularly associated to PAF-acether.
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PMID:Activation of guinea pig eosinophils by human recombinant IL-5. Selective priming to platelet-activating factor-acether and interference of its antagonists. 165 95

Diazepam, which binds both central (neuronal) and peripheral (non-neuronal) benzodiazepine binding sites, and Ro5-4864, a ligand selective for benzodiazepine peripheral binding sites (PBS), both inhibited the FMLP induced chemotaxis in human neutrophils at concentrations as low as 10(-8) M. A selective peripheral benzodiazepine antagonist, PK-11195 (10(-5) M), partially reversed the benzodiazepine inhibition of chemotaxis. Diazepam also inhibited the superoxide production induced by FMLP, NaF, and A23187, but not that induced by PMA whose stimulant action was insensitive even to 10(-4) M diazepam. The FMLP-induced superoxide production was most sensitive to diazepam inhibition (ID50 = 2.25 x 10(-6) M diazepam); the effect of NaF was slightly less sensitive (ID50 = 1.34 x 10(-5) M diazepam); and the effect of A23187 was least sensitive as it was suppressed only at 10(-4) M diazepam concentrations. Like diazepam, Ro5-4864 inhibited the FMLP-induced superoxide production, and PK-11195 (10(-5) M) significantly antagonized both diazepam and Ro5-4864 inhibition. Binding studies showed the presence of a saturable benzodiazepine 'peripheral' type binding site (PBS) on human neutrophils with a Kd of 1.2 +/- 0.06 x 10(-8) M (+/- SEM), and a Bmax of 1028 +/- 86.2 fmol/10(6) cells (+/- SEM) for [3H]Ro5-4864; the binding was displaceable by PK-11195, Ro5-4864 and diazepam but not by clonazepam.
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PMID:Benzodiazepines inhibit neutrophil chemotaxis and superoxide production in a stimulus dependent manner; PK-11195 antagonizes these effects. 166 97

A number of natural and recombinant human cytokines have been tested for their ability to activate basophil and neutrophil adhesiveness for human umbilical vein endothelial cells in vitro. Coincubation of basophils and endothelial cell monolayers for 10 min with biologically relevant concentrations of rIL-1, natural IL-2, rIL-4, rIL-5, rIL-6, rIL-8, rGM-CSF, and rIFN-gamma had no effect on basophil adhesiveness. In contrast, rIL-3 induced basophil adhesiveness for endothelial cells (optimal at 1 ng/ml: 144 +/- 18% of control adherence (mean +/- SEM); control basophil binding, 13 +/- 3%, n = 9, p less than or equal to 0.05). This increase in adhesiveness was similar in magnitude to that induced by an optimal concentration of a known potent inducer of basophil adhesiveness (1 microM FMLP, 164 +/- 15% of control adherence, n = 9). Under these experimental conditions, the effects of rIL-3 occurred at concentrations of 0.1 to 30 ng/ml, were partially dependent on calcium, and were not accompanied by histamine release. Fixation experiments demonstrated that the effect of rIL-3 was directed against the basophil rather than the endothelial cell. Neither rIL-3 nor the other cytokines tested had any effect on the adherence of 51Cr-labeled neutrophils, even when tested simultaneously on cells from the same donors. Under experimental conditions that permitted histamine release, no correlation was seen between the ability of rIL-3 (0.3 to 300 ng/ml) to induce histamine release or enhance adhesiveness (n = 8). mAb blocking experiments demonstrated a role for both CD11 and CD18 adherence glycoproteins in basophil adherence induced by rIL-3, and indirect immunofluorescence and flow cytometric analysis revealed that rIL-3 treatment led to rapid and sustained increases in cell surface expression of CD11b antigens on basophils but not neutrophils (e.g., after 10 min: 217 +/- 29 vs 91 +/- 11% of control mean fluorescence intensity, p less than 0.05). However, no correlation was seen between the magnitude of changes in CD11b expression and changes in adhesion when tested simultaneously. These results suggest that local production of IL-3 during allergic reactions in vivo may selectively promote basophil activation, adhesion to endothelium, and recruitment to extravascular sites of inflammation.
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PMID:IL-3 augments adhesiveness for endothelium and CD11b expression in human basophils but not neutrophils. 169 10

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.
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PMID:Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology. 171 65

Mononuclear phagocytes infected with Leishmania have been shown to have defective responses to extracellular stimuli. To investigate the potential relationship of these findings to alterations in calcium-dependent signaling pathways, the regulation of [Ca2+]i concentrations was examined in human peripheral blood monocytes infected with amastigotes of Leishmania donovani. Measurements of [Ca2+]i in fura-2-loaded monocytes were made at the single cell level by microfluorimetry. In normal monocytes, resting [Ca2+]i was 56 +/- 2 nM (mean +/- SEM). In contrast, in monocytes infected with Leishmania there was an approximately twofold increase in basal [Ca2+]i (122 +/- 5 nM, p less than 0.01 vs control). Treatment of cells with pertussis toxin before infection did not abrogate infection-induced increases in basal [Ca2+]i, suggesting that this effect was not mediated via the activation of a G protein coupled to phospholipase C. However, elevated resting [Ca2+]i did correlate with increased rates of 45Ca2+ uptake by infected monocytes. As expected, in response to treatment with 10(-7) M FMLP, control monocytes showed rapid net increases in [Ca2+]i of 303 +/- 19 nM. In contrast, net transients of [Ca2+]i in infected monocytes in response to FMLP were attenuated to only 137 +/- 9 nM (p less than 0.01 vs control). This result was not related to excess buffering of [Ca2+]i in infected cells as both control and infected monocytes showed equivalent transients of [Ca2+]i in response to the calcium ionophore A23187. Rather, inhibition of agonist-induced calcium release in infected cells appeared related to defective generation of second messenger because compared to control cells labeled with myo-[2-3H]inositol, little accumulation of inositol 1,4,5-trisphosphate was detected in infected monocytes. Attenuation of inositol phosphate accumulation and calcium release in response to chemotactic peptide correlated with decreased FMLP-induced superoxide and hydrogen peroxide production by infected monocytes. These results provide direct evidence for defective regulation of [Ca2+]i and calcium-dependent signaling in Leishmania-infected monocytes and provide a basis for understanding abnormalities in activation-related responses that involve signaling through Ca(2+)-regulated pathways.
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PMID:Stimulus-response coupling in monocytes infected with Leishmania. Attenuation of calcium transients is related to defective agonist-induced accumulation of inositol phosphates. 173 35

Upon engagement of chemoattractant receptors, neutrophils generate inositol trisphosphate and diacylglycerol (DG) by means of a phosphatidylinositol-specific phospholipase C (PI-PLC) which is regulated by a GTP-binding protein(s). We have previously reported (Reibman, J., H. M. Korchak, L. B. Vosshall, K. A. Haines, A. M. Rich, and G. Weissmann. 1988. J. Biol. Chem. 263:6322-6328) a biphasic rise in DG after exposure of neutrophils to the chemoattractant FMLP: a rapid (less than or equal to 15 s) phase ("triggering") and a slow (greater than or equal to 30 s) phase ("activation"). These derive from distinct intracellular lipid pools. To study the source of rapid and slow DG, we have used a unique probe, protein I, a porin that is the major outer membrane protein of Neisseria gonorrhoeae. Treatment of neutrophils with protein I inhibits exocytosis and homotypic cell adhesion provoked by FMLP without inhibiting assembly of the NADPH oxidase responsible for O2-. generation. DG turnover in PMN labeled with [3H]arachidonate and [14C]glycerol was profoundly altered by protein I. Whereas the rapid peak of DG was only modestly diminished (FMLP vs. FMLP plus protein I = DG labeled with [3H]arachidonic acid (3H-a.a.-DG): 142 +/- 14% SEM vs. 125 +/- 22%; DG labeled with the glycerol backbone with [14C]glycerol (D-14C-G): 125 +/- 10% SEM vs. 107 +/- 8.5% SEM), the slow rise in both 3H-a.a.-DG and D-14C-G was essentially abolished. Moreover, treatment of neutrophils with 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which, like protein I, inhibits exocytosis without affecting O2-. generation also inhibited slow DG. However, protein phosphorylation and dephosphorylation (47phox, 66phox) were unaffected in the absence of slow DG. To determine the source of the slow DG, we have analyzed radiolabeled phospholipid (PL) turnover after FMLP +/- protein I (P.I.). Treatment of PMN with FMLP (0.1 microM) resulted in breakdown of phosphatidylcholine (PC), beginning at 30 s, and reaching a nadir at 60 s (3H-PC = 59 +/- 10.2% SEM of resting, 14C-PC = 57 +/- 6.4%). Protein I (0.25 microM) significantly inhibited PC turnover after FMLP ([3H]PC = 95 +/- 5.6% and [14C]PC = 86 +/- 8.4% of resting at 60 s), but failed to alter the metabolism of 3H- or 14C-phosphatidylinositol after FMLP (91 +/- 19.6 and 88 +/- 16.5% vs. 92 +/- 9.2 and 91 +/- 16% at 60 s).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of protein I of Neisseria gonorrhoeae on neutrophil activation: generation of diacylglycerol from phosphatidylcholine via a specific phospholipase C is associated with exocytosis. 190 86

A simple semiquantitative cytofluorometric method has been developed for measuring neutrophil respiratory burst activity in whole blood samples. This technique avoids the introduction of laboratory artefacts which modulate neutrophil function. In addition, flow cytometric analysis allows the response to be studied in individual cells. We show here that neutrophils examined freshly ex vivo, exhibit only weak respiratory burst activity in response to stimulation with the chemotactic peptide FMLP (10(-6) M). Prior incubation with rhGM-CSF results in an increase in the number of responding cells from 13.5 +/- 2.36% (mean +/- SEM) to 46.7 +/- 6.3% (P less than 0.0001) with an increase in total respiratory burst activity of 567% (P = 0.001). The majority of neutrophils in whole blood (67.1 +/- 8.1%) exhibit respiratory burst activity in response to stimulation with phorbol ester (1 micrograms/ml of TPA), and this response is also significantly primed by rhGM-CSF (P = 0.004). The enhancement of respiratory burst activity induced by rhGM-CSF is due to both recruitment of previously unresponsive neutrophils, and to intensification of the response of the responding cells. In vivo administration of rhGM-CSF also results in priming of the respiratory burst in response to FMLP, although the enhancement of activity is not as great as that obtained when pre-infusion blood samples are incubated with rhGM-CSF in vitro.
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PMID:The effects of rhGM-CSF on the neutrophil respiratory burst when studied in whole blood. 219 31

The effect of the cytoprotective bile acid tauroursodeoxycholic acid (TUDCA) on basal cytosolic free Ca++ (Ca++)i and receptor-mediated (Ca++)i increase was studied in human polymorphonuclear neutrophils using the fluorescent dye quin2. Basal levels of (Ca++)i were 96 +/- 6 nmol/l (mean +/- SEM, n = 48). TUDCA and its cytotoxic epimer taurochenodeoxycholic acid (TCDCA) at 500 mumols/l increased (Ca++)i by 31 +/- 12 and 27 +/- 7 nmol/l, respectively (n = 6, p less than 0.05). Stimulation of neutrophils with the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10(-7) mol/l) induced a (Ca++)i increase of 200 +/- 32 nmol/l which was inhibited after preincubation with TUDCA (500 mumols/l) or TUDCA + TCDCA (500 mumols/l, each) by 60.1% and 59.5%, respectively, but not with TCDCA (500 mumols/l) alone. The inhibitory effect of TUDCA on FMLP-induced (Ca++)i increase was strongly concentration-dependent and was nearly complete at 1000 mumols/l. Since (Ca++)i is discussed as a mediator of cellular injury we hypothesize that TUDCA may exert its protective effects at least partly via inhibition of (Ca++)i-mediated cytotoxic processes.
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PMID:Tauroursodeoxycholic acid inhibits the cytosolic Ca++ increase in human neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine. 222 31

Neutrophils (PMN) preincubated with recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) for 2 h and then stimulated with the chemotactic factors, C5a or FMLP, produce substantial amounts of the lipoxygenase products 5-Hete, LTB4, and omega-oxidised LTB4 metabolites (4.36 +/- 0.95 (SEM) pM (n = 21) LTB4 and LTB4 metabolites/10(6) PMN). No lipoxygenase metabolites are detected by HPLC and RIA if purified PMN are stimulated by either GM-CSF or chemotactic factors in the absence of exogenous arachidonate. The priming effect of GM-CSF upon chemotactic factor induced generation of lipid mediators is a relatively slow process, clearly evident after 1 h and optimal after 2 h. Leukotriene generation is measurable with 0.8 U GM-CSF/10(6) PMN and is maximal with 80 U (10(-11)-10(-9) M). Upon activation of primed PMN with chemotactic factors, leukotriene synthesis is induced very rapidly. Already 2.5 min after activation the major lipoxygenase metabolites present are 20-OH LTB4 and 20-COOH LTB4. Our study shows that the synthesis of lipoxygenase metabolites from endogeneous AA can be initiated in PMN through receptor mediated processes by the appropriately timed combination of biological soluble inflammatory mediator peptides. Furthermore, these results indicate that GM-CSF not only enhances effector cell functions but can qualitatively change the mediator profile formed after activation with a second triggering signal. Such a mechanism might be important in amplifying inflammatory responses. Alternatively, lipid mediators formed might also have an intracellular or autocoid role and be responsible for the enhancement of other PMN functions like oxygen radical release.
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PMID:Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony-stimulating factor and stimulated with the complement component C5A and FMLP as second signals. 283 56


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