Neutrophil elastase–processing defect in cyclic hematopoietic dogs
Received 13 October 2008; received in revised form 25 September 2009; accepted 30 September 2009. published online 26 November 2009.
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 β3A 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.
aScott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, Ala., USA
bAuburn University Hybridoma Facility, Auburn University, Auburn, Ala., USA
cDepartment of Pathology, College of Veterinary Medicine, Auburn University, Auburn, Ala., USA
dDepartment of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Ala., USA
eSchool of Veterinary Medicine, University of California, Davis, Calif., USA
Offprint requests to: Clinton D. Lothrop, Jr., Ph.D., D.V.M., Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 404 MCLM Building, 1918 University Avenue, Birmingham, AL 35294-0024
ABSTRACT