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Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack of Inhibition by Platelet-Activating Factor.
- Source :
-
Journal of immunology research [J Immunol Res] 2018 Jan 24; Vol. 2018, pp. 3178970. Date of Electronic Publication: 2018 Jan 24 (Print Publication: 2018). - Publication Year :
- 2018
-
Abstract
- N-formyl-Met-Leu-Phe (fMLF) is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF's divergent effects on FPR1 phosphorylation as well as PMN function.
- Subjects :
- Alarmins immunology
Cell Adhesion
Cells, Cultured
Humans
Isoflurophate metabolism
L-Lactate Dehydrogenase metabolism
N-Formylmethionine Leucyl-Phenylalanine immunology
Pathogen-Associated Molecular Pattern Molecules immunology
Phosphorylation
Platelet Activating Factor metabolism
Proteolysis
Neutrophils physiology
Peptide Fragments metabolism
Receptors, Formyl Peptide metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 2314-7156
- Volume :
- 2018
- Database :
- MEDLINE
- Journal :
- Journal of immunology research
- Publication Type :
- Academic Journal
- Accession number :
- 29785402
- Full Text :
- https://doi.org/10.1155/2018/3178970