Tan Emmeline, Bienzle Dorothee, Shewen Patricia, Kruth Stephen, Wood Darren
Departments of Pathobiology, Veterinary College, University of Guelph, Guelph, ON, Canada.
Vet Clin Pathol. 2012 Mar;41(1):45-55. doi: 10.1111/j.1939-165X.2011.00391.x. Epub 2012 Feb 8.
Primary immune-mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. The mechanisms underlying autoimmune reactivity remain poorly understood.
The aim of this study was to identify membrane proteins of RBCs that could be antigenic in dogs with primary IMHA.
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.
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.
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.
原发性免疫介导性溶血性贫血（IMHA）是犬发病和死亡的重要原因。自身免疫反应的潜在机制仍知之甚少。
本研究的目的是鉴定原发性IMHA犬红细胞中可能具有抗原性的膜蛋白。
用二甲苯从12只患有IMHA的犬、4只因IMHA以外原因导致贫血的犬和2只健康犬的红细胞中洗脱抗体。从17只健康犬的血液中制备合并的红细胞膜蛋白。然后通过免疫印迹分析洗脱的抗体与合并的膜蛋白和自体血浆的相互作用。在12只患有IMHA的犬中出现但在其他6只犬中未出现的条带被视为潜在的自身抗原，并通过液相色谱-串联质谱法进行鉴定。
所有18只犬的红细胞洗脱物均与带3蛋白有反应性。在患有IMHA的犬中还独特地鉴定出了针对另外6种蛋白的抗体。分别在12只患有IMHA的犬的8份样本、8份样本和4份样本中鉴定出对钙蛋白酶、补体成分3和过氧化物还原酶2的反应性，但在6只未患IMHA的犬的样本中均未检测到。
检测到针对带3蛋白的普遍免疫反应性可能表明识别了衰老的红细胞。患有IMHA的犬中被抗体独特识别的蛋白参与氧化应激和细胞凋亡（钙蛋白酶）、炎症（补体）以及活性氧的清除（过氧化物还原酶2）。这些蛋白在引发自身免疫中是否重要，或者针对这些蛋白的免疫球蛋白是否在IMHA期间产生，仍有待确定。
