Potentially antigenic RBC membrane proteins in dogs with primary immune-mediated hemolytic anemia.
BACKGROUND
Primary immune-mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. The mechanisms underlying autoimmune reactivity remain poorly understood.


OBJECTIVE
The aim of this study was to identify membrane proteins of RBCs that could be antigenic in dogs with primary IMHA.


METHODS
Antibodies were eluted with xylene from RBCs of 12 dogs with IMHA, 4 dogs with anemia due to causes other than IMHA, and 2 healthy dogs. Pooled RBC membrane proteins were prepared from blood of 17 healthy dogs. The eluted antibodies were then analyzed by immunoblotting for interactions with the pooled membrane proteins and autologous plasma. Bands present in the 12 dogs with IMHA but not in the 6 other dogs were considered potential autoantigens and were identified by liquid chromatography followed by tandem mass spectrometry.


RESULTS
RBC eluates from all 18 dogs had reactivity against band 3 protein. Antibodies to 6 additional proteins were uniquely identified in dogs with IMHA. Reactivity to calpain, complement component 3, and peroxiredoxin 2 was identified in 8, 8, and 4 of the 12 samples, respectively, from dogs with IMHA, but in none of the samples from the 6 dogs without IMHA.


CONCLUSIONS
Detection of universal immune reactivity against band 3 protein probably indicates recognition of senescent RBC. Proteins uniquely recognized by antibodies in dogs with IMHA are involved in oxidative stress and apoptosis (calpain), inflammation (complement), and scavenging of reactive oxygen species (peroxiredoxin 2). It remains to be determined if these proteins are important in initiating autoimmunity or if immunoglobulins targeting these proteins develop during IMHA.
Departments of Pathobiology, Veterinary College, University of Guelph, Guelph, ON, Canada. etan@uoguelph.ca
Emmeline
E
Tan
Dorothee
D
Bienzle
Patricia
P
Shewen
Stephen
S
Kruth
Darren
D
Wood
D000744:Anemia, Hemolytic, Autoimmune / Q000209:etiology / Q000276:immunology / Q000662:veterinary*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001797:Blood Protein Electrophoresis / Q000662:veterinary; D001798:Blood Proteins / Q000276:immunology* / Q000378:metabolism; D016022:Case-Control Studies; D004283:Dog Diseases / Q000097:blood / Q000209:etiology / Q000276:immunology*; D004285:Dogs; D004910:Erythrocyte Membrane / Q000276:immunology* / Q000378:metabolism; D005260:Female; D015151:Immunoblotting / Q000662:veterinary; D008297:Male; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D018384:Oxidative Stress
D000906:Antibodies; D001798:Blood Proteins; D008565:Membrane Proteins