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The subcellular distribution of myeloid-related protein 8 (MRP8) and MRP14 in human neutrophils
David F Stroncek1, Raji A Shankar, Keith M Skubitz
1Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA. dstroncek@cc.nih.gov
Background:
Myeloid-related protein 8 (MRP8) and MRP14 are S100 family calcium binding proteins that form a heterodimer known as calprotectin or MRP8/14 that is present in the cytosol of neutrophils and monocytes. MRP8/14 becomes associated with endothelium at sites of monocyte and neutrophil adhesion and transmigration and induces a thrombogenic and inflammatory response by increasing the endothelial transcription of proinflammatory chemokines and adhesion molecules. The distribution of MRP8/MRP14 among neutrophil granules and plasma membranes is unclear and was investigated to better understand the role of this molecule in acute inflammation.
Study Design:
Three monoclonal antibodies specific for MRP8 and MRP14 were characterized and used in immunoblotting assays of neutrophil whole cell extracts, and isolated plasma membranes, primary granules, secondary granules and cytosol.
Results:
MRP8 and MRP14 were detected in neutrophil cytosol, plasma membrane, primary granule and secondary granule fractions. MRP8/14 demonstrated a calcium-dependent adherence to plasma membranes and primary granules and could be removed by washing with EGTA in a high ionic strength buffer. In contrast, MRP8/14 was found within the contents of the secondary granules. Activated neutrophils released secondary granules and MRP8/14.
Conclusion:
MRP8/14 is located in neutrophil cytosol and secondary granule fractions and is loosely associated with plasma membranes. MRP8/14 released with secondary granules by activated neutrophils likely binds to endothelium and plays an important role in acute inflammation.
Insights
Myeloid-related protein 8/14 (MRP8/14) is found in neutrophil cytosol and secondary granules, loosely associating with plasma membranes. Activated neutrophils release MRP8/14, contributing to inflammation.
Area of Science:
- Immunology
- Cell Biology
- Inflammation Research
Background:
- Myeloid-related protein 8 (MRP8) and MRP14 form the calprotectin (MRP8/14) heterodimer, a calcium-binding protein in neutrophils and monocytes.
- MRP8/14 associates with endothelium, promoting thrombotic and inflammatory responses by upregulating endothelial chemokines and adhesion molecules.
- The precise intracellular localization of MRP8/14 within neutrophils, particularly its distribution in granules and plasma membranes, remained unclear.
Purpose of the Study:
- To investigate the distribution of MRP8/14 within neutrophil subcellular compartments.
- To elucidate the role of MRP8/14 localization in neutrophil function and inflammatory processes.
Main Methods:
- Characterization of three monoclonal antibodies specific for MRP8 and MRP14.
- Immunoblotting assays were performed on neutrophil whole cell extracts, isolated plasma membranes, primary granules, secondary granules, and cytosol.
Main Results:
- MRP8 and MRP14 were detected in neutrophil cytosol, plasma membrane, primary granule, and secondary granule fractions.
- MRP8/14 exhibited calcium-dependent adherence to plasma membranes and primary granules, removable by EGTA.
- MRP8/14 was localized within the secondary granules, which are released by activated neutrophils.
Conclusions:
- MRP8/14 is present in neutrophil cytosol and secondary granules, with loose association to plasma membranes.
- Release of MRP8/14 with secondary granules from activated neutrophils suggests a key role in endothelial binding and acute inflammation.
