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Updated: Jun 18, 2026
In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy
Published on: August 24, 2018
Neutrophil elastase-processing defect in cyclic hematopoietic dogs
Ronghua Meng1, Roger Bridgman, Maria Toivio-Kinnucan
1Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, Ala., USA.
Insights
Canine cyclic hematopoiesis (CH) involves abnormal neutrophil elastase (NE) processing. CH dogs accumulate inactive NE precursors, unlike normal dogs, impacting neutrophil function.
Area of Science:
- Hematology
- Molecular Biology
- Immunology
Background:
- Canine cyclic hematopoiesis (CH) is a model for human neutropenias.
- CH is linked to AP3B1 mutations affecting protein trafficking.
- Neutrophil elastase (NE) activity is reduced in CH.
Purpose of the Study:
- Investigate intracellular sorting and trafficking of NE in CH dogs.
- Utilize specific antibodies against canine NE.
Main Methods:
- Generated polyclonal and monoclonal antibodies to canine NE.
- Characterized antibodies using Western immunoblotting and immunocytochemistry.
- Analyzed NE precursor and mature NE localization and quantity.
Main Results:
- Developed antibodies recognizing mature NE (ELA85) and NE precursor (ELA269).
- Normal dogs show colocalization of mature NE and myeloperoxidase in primary granules.
- CH dogs accumulate inactive, higher molecular weight NE precursors, not colocalized with myeloperoxidase.
Conclusions:
- ELA85 and ELA269 are valuable tools for studying NE biosynthesis and trafficking.
- CH neutrophils exhibit abnormal accumulation of NE precursors, impacting enzyme activity.
Objective:
Canine cyclic hematopoiesis (CH), a model of human cyclic neutropenia and severe congenital neutropenia, is characterized by a periodic reduced neutrophil count and decreased neutrophil elastase (NE) enzymatic activity. Canine CH is caused by a mutation of AP3B1 encoding the beta3A subunit of adaptor protein complex-3 (AP-3). It has been proposed that trafficking of elastase is affected by AP-3. The aim of this study was to study intracellular sorting/trafficking of NE in CH dogs using antibodies specific to canine NE.
Materials And Methods:
Polyclonal and monoclonal antibodies were generated to immunogenic epitopes in the middle (aa85-98) and C-terminal (aa269-282) regions of NE. The antibodies to canine NE were characterized by Western immunoblotting and immunocytochemistry.
Results:
Antibody ELA85 (antibody to canine NE aa 85-98) specifically recognized mature 28-kD NE. Immunocytochemical analysis using ELA85 and an antibody to myeloperoxidase demonstrated colocalizaton of NE and myeloperoxidase in primary granules of normal dogs. Antibody ELA269 (antibody to canine NE aa 269-282) reacted exclusively with the 33-kD NE presumptive precursor form. Immunocytochemical analysis demonstrated that the NE precursor was not colocalized with myeloperoxidase in the primary granules of normal or CH dogs. Western immunoblotting using these antibodies demonstrated that CH dogs contained reduced mature NE, but accumulated a large amount of the NE precursor protein that was not enzymatically active.
Conclusion:
Antibodies ELA85 and ELA269 were found to be useful reagents for studying the biosynthesis, processing, and trafficking of NE during normal myelopoiesis. Neutrophils from CH dogs accumulated large amounts of higher molecular weight elastase precursors compared to normal dogs.
