Green G A, Kalra V K
USC School of Medicine, Biochemistry Department, Los Angeles 90033.
Blood. 1988 Mar;71(3):636-9.
Previously we demonstrated that sickle erythrocytes sedimenting at high densities after gradient centrifugation contain higher levels of surface immunoglobulin bound in vivo in comparison to low-density erythrocytes from the same patient. The present study examines the possibility that binding of autologous IgG to sickle erythrocytes may be associated with the sickling phenomenon. In the present study we subjected low-density erythrocytes to prolonged sickling under nitrogen in the presence of platelet-poor autologous plasma with added glucose for 24 hours (37 degrees C). After reoxygenation IgG bound in vitro was quantified by a nonequilibrium 125iodinated protein A-binding assay and by flow cytometry. Results show that sickle erythrocytes incubated under nitrogen bound significantly (P less than .001) more IgG, 439 +/- 41, molecules of IgG per cell (mean +/- SD) compared with sickle cells incubated under oxygenation (227 +/- 12 molecules of IgG per red cell) or compared with 196 +/- 26 molecules IgG per cell for untreated sickle cells. In contrast, normal erythrocytes incubated in autologous plasma exhibited no detectable IgG binding in vitro under either oxygenation or deoxygenation. Flow cytometry shows that deoxygenation of sickle cells generated a two-to-sixfold increase in the subpopulation of brightly fluorescent IgG-positive cells in comparison to oxygenated sickle cells and a 13.5% +/- 3.1% (mean +/- SD) increase in median fluorescence intensity for fluorescein isothiocyanate-labeled deoxygenated sickled cells compared with labeled oxygenated sickle cells. Our studies demonstrate that prolonged sickling will induce in vitro binding of autologous IgG to sickle erythrocytes. These findings indicate that sickle erythrocytes may be unique when compared with erythrocytes from other nonimmunologic hemolytic anemias or senescent red cells in that the primary events producing surface antigens recognized by autoantibody may include the sickling process. These findings also suggest that sickling in vivo may generate membrane alterations in sickle erythrocytes that lead to cumulative binding of autoantibody in vivo.
先前我们证明，梯度离心后高密度沉降的镰状红细胞与同一患者的低密度红细胞相比，体内结合的表面免疫球蛋白水平更高。本研究探讨了自体IgG与镰状红细胞结合可能与镰变现象相关的可能性。在本研究中，我们将低密度红细胞在氮气环境下，于添加葡萄糖的无血小板自体血浆存在的条件下，在37℃下长时间诱导镰变24小时。复氧后，通过非平衡125碘标记蛋白A结合试验和流式细胞术对体外结合的IgG进行定量。结果显示，在氮气环境下孵育的镰状红细胞结合的IgG显著增多（P<0.001），每个细胞平均结合439±41个IgG分子（平均值±标准差），而在氧合条件下孵育的镰状细胞每个红细胞结合227±12个IgG分子，未处理的镰状细胞每个细胞结合196±26个IgG分子。相比之下，在自体血浆中孵育的正常红细胞在氧合或脱氧条件下体外均未检测到IgG结合。流式细胞术显示，与氧合的镰状细胞相比，镰状细胞脱氧后，明亮荧光IgG阳性细胞亚群增加了2至6倍，异硫氰酸荧光素标记的脱氧镰状细胞的中位荧光强度比标记的氧合镰状细胞增加了13.5%±3.1%（平均值±标准差）。我们的研究表明，长时间镰变会诱导自体IgG在体外与镰状红细胞结合。这些发现表明，与其他非免疫性溶血性贫血或衰老红细胞相比，镰状红细胞可能具有独特性，即产生自身抗体识别的表面抗原的主要事件可能包括镰变过程。这些发现还表明，体内镰变可能导致镰状红细胞的膜改变，从而导致体内自身抗体的累积结合。
