特发性血小板减少性紫癜患者脾脏CD5+和CD5-B细胞共同参与血小板自身抗体的产生

目的：研究慢性特发性血小板减少性紫癜（ITP）患者脾脏CD5^ B细胞水平的变化及CD5^ 和CD5^-B细胞与血小板膜糖蛋白（GP）特异性自身抗体产生的关系，以识别致病B细胞亚群。方法：应用双色流式细胞仪检测8例慢性ITP患者脾脏CD5^ B细胞水平。选择4例血浆抗GPⅡb/Ⅲa和抗GP Ⅰb/Ⅸ抗体双阳性ITP切脾患者，应用Ficoll密度梯度离心及花环形成分离法分离脾脏B淋巴细胞，继而采用镝产珠分选法分选、纯化CD5^ B细胞和CD5^-B细胞，并分别进行体外培养，应用改良MAIPA法检测血浆和细胞培养上清液的血小板特异性抗体。结果：ITP患者脾脏CD5^ B细胞水平圈晨自身免疫性疾病患者略有增高，二者之间差异无统计学意义。CD5^ B细胞水平与患者血小板计数无相关性。4例血浆抗GPⅡb/Ⅲa抗体和抗GPⅠb/Ⅸ抗体双阳性。另外1例CD5^ B细胞培养液抗GPⅡb/Ⅲa抗体阴性，抗GPⅠb/Ⅸ抗体阳性;CD5^-B细胞培养液抗GPⅡb/Ⅲa抗体和抗GPⅠb/Ⅸ抗体双阳性。结论：脾脏CD5^ 和CD5^-B细胞 均可产生血小板GP特异性自身抗体，抗体产生种类和滴度无明显差异。提示二者共同参与了ITP的发病过程。

Participation of both splenic CD5+ and CD5-B lymphocytes in production of platelet glycoprotein-specific autoantibodies in chronic ITP

OBJECTIVE: To investigate the percentage of splenic CD(5)(+) B lymphocytes in chronic idiopathic thrombocytopenic purpura (IT) and the impact of splenic CD(5)(+) and CD(5)(-) B lymphocytes on the production of platelet glycoprotein (GP)-specific autoantibodies. METHODS: Splenic CD(5)(+) B lymphocytes were identified by two-color flow cytometric analysis in eight patients. Four of the eight patients displayed plasma autoantibodies against both GPIIb/IIIa and GPIb/IX, and their splenic B lymphocytes were separated by Ficoll-Hypaque density gradient and sheep erythrocyte, and further purified by magnetic activate cell separation (MACS). Purified CD(5)(+) and CD(5)(-) B lymphocytes were cultured separately with or without staphylococcus aureus cowan I (SAC). GP specific autoantibodies in culture supernatants were measured by modified monoclonal antibody immobilization of platelet antigen assay (MAIPA). RESULTS: The percentage of splenic CD(5)(+) B lymphocytes in ITP patients was slightly higher than that in control with no statistical significance. MACS purified splenic CD(5)(+) and CD(5)(-) B lymphocytes from three out of four ITP patients produced high levels of anti-GPIIb/IIIa and anti-GPIb/IX antibodies. Culture supernatants of CD(5)(+) B lymphocytes from the other patient showed positive reaction only in GPIb/IX MAIPA. Culture supernatant of CD(5)(-)B lymphocytes from the same patient were double positive in both GPIIb/IIIa and GPIb/IX MAIPA. CONCLUSIONS: Both splenic CD(5)(+) and CD(5)(-) B lymphocytes produce platelet GP-specific autoantibodies in chronic ITP with similar antibody spectrum and titer, and may all play a role in the autoimmune pathogenesis of ITP.