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IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation
The molecular mechanisms driving specific regulation of neutrophils are not completely understood to date. In order to characterize fundamental granulocyte features on protein level, we analyzed changes in proteome composition as reaction to stress from cell activation processes. For this purpose, w...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6973177/ https://www.ncbi.nlm.nih.gov/pubmed/32010136 http://dx.doi.org/10.3389/fimmu.2019.03064 |
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author | Degroote, Roxane L. Weigand, Maria Hauck, Stefanie M. Deeg, Cornelia A. |
author_facet | Degroote, Roxane L. Weigand, Maria Hauck, Stefanie M. Deeg, Cornelia A. |
author_sort | Degroote, Roxane L. |
collection | PubMed |
description | The molecular mechanisms driving specific regulation of neutrophils are not completely understood to date. In order to characterize fundamental granulocyte features on protein level, we analyzed changes in proteome composition as reaction to stress from cell activation processes. For this purpose, we isolated primary granulocytes from equine whole blood through density gradient centrifugation followed by sodium chloride lysis and stimulated cells for 30 min with interleukin-8 (IL8) due to its role as a chemotactic factor for neutrophils. We additionally used phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), which are primarily associated to neutrophil extracellular trap formation and release of reactive oxygen species. From mass spectrometry analysis, we identified a total of 2,032 proteins describing the whole granulocyte proteome, including 245 proteins (12% of identified proteome) newly associated to in vivo expression in primary equine granulocytes (hypothetical proteins). We also found distinct and different changes in protein abundance (ratio ≥ 2) after short stimulation of cells with various stimuli, pointing to rapid and differentiated reaction pattern. IL8 stimulation resulted in increased protein abundance of 58 proteins (3% of proteome), whereas PMA induced changed protein abundance of 207 (10 % of proteome) and LPS of 46 proteins (2% of proteome). Enrichment analyses clearly showed fundamental differences between stimuli, with primary association of IL8 stimulation to processes in immune response, receptor signaling and signal transduction. Top enrichment for PMA on the other hand pointed to vesicle mediated transport and exocytosis. Stimulation with LPS did not result in any significant enrichment. Although we detected 43% overlap of enrichment categories for IL8 and PMA stimulation, indicating that activation of neutrophils with different stimuli partly induces some similar biological processes and pathways, hierarchical clustering showed clear differences in distribution and biological relevance of clusters between the chosen stimuli. Our studies provide novel information on the granulocyte proteome and offer insights into early, differentiated granulocyte reaction to stimuli, which contribute to a better understanding of molecular mechanisms involved in activation and recruitment of neutrophils, through inflammatory stimuli. |
format | Online Article Text |
id | pubmed-6973177 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-69731772020-02-01 IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation Degroote, Roxane L. Weigand, Maria Hauck, Stefanie M. Deeg, Cornelia A. Front Immunol Immunology The molecular mechanisms driving specific regulation of neutrophils are not completely understood to date. In order to characterize fundamental granulocyte features on protein level, we analyzed changes in proteome composition as reaction to stress from cell activation processes. For this purpose, we isolated primary granulocytes from equine whole blood through density gradient centrifugation followed by sodium chloride lysis and stimulated cells for 30 min with interleukin-8 (IL8) due to its role as a chemotactic factor for neutrophils. We additionally used phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), which are primarily associated to neutrophil extracellular trap formation and release of reactive oxygen species. From mass spectrometry analysis, we identified a total of 2,032 proteins describing the whole granulocyte proteome, including 245 proteins (12% of identified proteome) newly associated to in vivo expression in primary equine granulocytes (hypothetical proteins). We also found distinct and different changes in protein abundance (ratio ≥ 2) after short stimulation of cells with various stimuli, pointing to rapid and differentiated reaction pattern. IL8 stimulation resulted in increased protein abundance of 58 proteins (3% of proteome), whereas PMA induced changed protein abundance of 207 (10 % of proteome) and LPS of 46 proteins (2% of proteome). Enrichment analyses clearly showed fundamental differences between stimuli, with primary association of IL8 stimulation to processes in immune response, receptor signaling and signal transduction. Top enrichment for PMA on the other hand pointed to vesicle mediated transport and exocytosis. Stimulation with LPS did not result in any significant enrichment. Although we detected 43% overlap of enrichment categories for IL8 and PMA stimulation, indicating that activation of neutrophils with different stimuli partly induces some similar biological processes and pathways, hierarchical clustering showed clear differences in distribution and biological relevance of clusters between the chosen stimuli. Our studies provide novel information on the granulocyte proteome and offer insights into early, differentiated granulocyte reaction to stimuli, which contribute to a better understanding of molecular mechanisms involved in activation and recruitment of neutrophils, through inflammatory stimuli. Frontiers Media S.A. 2020-01-14 /pmc/articles/PMC6973177/ /pubmed/32010136 http://dx.doi.org/10.3389/fimmu.2019.03064 Text en Copyright © 2020 Degroote, Weigand, Hauck and Deeg. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Immunology Degroote, Roxane L. Weigand, Maria Hauck, Stefanie M. Deeg, Cornelia A. IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation |
title | IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation |
title_full | IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation |
title_fullStr | IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation |
title_full_unstemmed | IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation |
title_short | IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation |
title_sort | il8 and pma trigger the regulation of different biological processes in granulocyte activation |
topic | Immunology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6973177/ https://www.ncbi.nlm.nih.gov/pubmed/32010136 http://dx.doi.org/10.3389/fimmu.2019.03064 |
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