Thick filter neutrophil chemotaxis performed in the absence of albumin

Psychoyos, Stacy; Uziel-Fusi, Susan; Morrissey, Michael M.; Ganu, Vishwas; Smith, C. Wayne

doi:10.1016/0022-1759(91)90391-r

Cited by 10 publications

(2 citation statements)

References 14 publications

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“…This finding is in accordance with previous studies of LTB 4 -mediated responses having EC 50 values in the low nanomolar range, e.g., 2.5 nM for the chemotactic response, and 20 nM for upregulation of the integrin complement receptor type 3 in murine neutrophils [34]. For U-937 cells, HL-60 cells, and human neutrophils, EC 50 values for LTB 4 -mediated responses on endogenous receptors were observed between 0.2 nM and 30 nM [35][36][37][38][39][40][41][42][43].…”

Section: Discussionsupporting

confidence: 73%

Homologous Desensitisation of the Mouse Leukotriene B<sub>4</sub> Receptor Involves Protein Kinase C-Mediated Phosphorylation of Serine 127

Mollerup¹,

Eriksen²,

Albertsen³

et al. 2007

Cell Physiol Biochem

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Murine leukotriene B₄ (LTB₄) receptor (mBLT1) cDNA was identified by searching the EST database using human LTB₄ receptor as the query sequence. Expression of functional mBLT1 after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was demonstrated as LTB₄-evoked, Ca²⁺-activated Cl^- currents recorded by two-electrode voltage clamp. From mBLT1-expressing oocytes, a dose-dependent relationship between the Ca²⁺-activated Cl^- current and LTB₄ concentration was demonstrated with an apparent EC₅₀ of 6.7 nM. Following LTB₄ stimulation of mBLT1, we observed two transient, spatially distinct Ca²⁺-activated, inwardly directed Cl^- currents in the oocytes: a fast peak current requiring relatively high LTB₄ concentrations, and a slowly progressing Cl^- current. Nucleotides, PGE₂, 12R-hydroxy-5, 8, 14-cis-10-trans-eicosatetraenoic acid, and LTD₄ did not activate mBLT1. U75302, specifically targeting BLT1, significantly reduced LTB₄-evoked Cl^- currents. Repetitive LTB₄ administration desensitized the LTB₄-evoked currents. Activation of protein kinase C (PKC) by PMA addition completely eliminated the LTB₄-evoked currents, whereas down-regulation of PKC by prolonged PMA exposure (20 h) impaired mBLT1 desensitisation. In addition, Ser-127-Ala substitution of the PKC consensus phosphorylation site on the second intracellular loop prevented the mBLT1 desensitisation. These data indicate that PKC-mediated phosphorylation at Ser-127 leads to mBLT1 desensitisation.

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Section: Discussionsupporting

confidence: 73%

Homologous Desensitisation of the Mouse Leukotriene B<sub>4</sub> Receptor Involves Protein Kinase C-Mediated Phosphorylation of Serine 127

Mollerup¹,

Eriksen²,

Albertsen³

et al. 2007

Cell Physiol Biochem

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“…was set between the top and bottom chambers. Nitrocellulose filters were pretreated with 2% albumin (Sigma) so that neutrophil chemotaxis could take place in albumin-free medium (31). Triplicate samples were run in each experiment.…”

mentioning

confidence: 99%

A novel neutrophil-activating factor released by Trichomonas vaginalis

et al. 1992

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We have investigated the effects of a novel neutrophil-activating factor released by Trichomonas vaginalis (TV-NAF) on neutrophil chemotaxis. TV-NAF was present in the supernatant from 107 T. vaginalis (STV) cultured in 1 ml of serum-free Hanks' balanced salt solution (HBSS) at 37°C for 30 min. With a multichamber chemotactic assay, we found that there were 112 + 15 migrated neutrophils (mean ± standard deviation, n = 7) for STV and 11 ± 4 for HBSS per high-power field (x400). STV was also able to induce neutrophil actin assembly (increased 1.5-fold), enhance expression of complement receptor type 3 (increased 5-fold), and promote intracellular calcium mobilization (increased 2.5-fold). There was no chemotactic activity in the preparation of STV from killed trichomonads. The fact that heating up to 100°C or deproteinization by treatment with proteinase K at 65°C for 1 h did not abolish its chemotactic activity suggests that the TV-NAF involved was not a protein. The chemotactic activity of TV-NAF was associated with the fraction containing small molecules of less than 3,000 Da. Therefore, the possibility that eicosanoid production by trichomonads is responsible for neutrophil activation was investigated. Leukotriene B4 (LTB4; 500 pg/ml) but not thromboxane B2 (<20 pg/mi) or prostaglandin E2 (<8 pg/ml) was found in the STV by radioimmunoassay. Production ofLTB4 by trichomonads was time dependent and increased twofold when arachidonic acid (100 ,uM) was added but was not decreased when eicosanoid inhibitors were present. Evidence for the presence of LTB4 in STV was further provided by the fact that rabbit anti-LTB4 antiserum could abolish the chemotactic activity of STV. These studies suggest that the spontaneous release of TV-NAF, which is most likely LTB4, may activate neutrophils, presumably through a different arachidonate metabolic pathway than that in mammalian cells. * Corresponding author. cellular calcium mobilization. The results from these studies provide more information about the inflammatory response induced by T. vaginalis. MATERIALS AND METHODSOrganism. Seven local isolates, axenically cultivated, were maintained in a modified medium identical to the TYI-S-33 medium of Diamond et al. (7), except that 0.5% Panmede (Paines & Byrne Limited, Greenford, England) was added. The number of organisms per culture was determined with a Coulter counter (model D industrial; Coulter Electronics, Inc., Hialeah, Fla.) with a 70-,um aperture tube.Preparation of STV. Each isolate of T. vaginalis grown at 37°C in 15-ml tubes containing TYI-S-33 medium was harvested during the logarithmic growth phase after 36 h of cultivation. They were centrifugally washed three times in HBSS (GIBCO, Grand Island, N.Y.), and then viable cells were counted in a hemacytometer with trypan blue-saline. HBSS without phenol red was used as the wash buffer and diluent throughout this study. The medium-free flagellates (107) were incubated in 1 ml of HBSS at 37°C for 30 min. Motile flagellates were then removed by gentle centrifugation (500 ...

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Comparative Modeling and Evaluation of Leukotriene B4 Receptors for Selective Drug Discovery Towards the Treatment of Inflammatory Diseases

Hassan

2018

Protein J

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Thick filter neutrophil chemotaxis performed in the absence of albumin

Cited by 10 publications

References 14 publications

Homologous Desensitisation of the Mouse Leukotriene B<sub>4</sub> Receptor Involves Protein Kinase C-Mediated Phosphorylation of Serine 127

Homologous Desensitisation of the Mouse Leukotriene B<sub>4</sub> Receptor Involves Protein Kinase C-Mediated Phosphorylation of Serine 127

A novel neutrophil-activating factor released by Trichomonas vaginalis

Comparative Modeling and Evaluation of Leukotriene B4 Receptors for Selective Drug Discovery Towards the Treatment of Inflammatory Diseases

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