Miranda M E, Tseng C E, Rashbaum W, Ochs R L, Casiano C A, Di Donato F, Chan E K, Buyon J P
Hospital for Joint Diseases, New York University School of Medicine, NY 10003, USA.
J Immunol. 1998 Nov 1;161(9):5061-9.
Access of intracellular Ags SSA/Ro and SSB/La to cognate maternal autoantibodies is unexplained despite their strong association with congenital heart block. To investigate the hypothesis that apoptosis facilitates surface accessibility of these Ags, human fetal cardiac myocytes from 16- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusing the aorta with collagenase. Confirmation of cardiac myocytes included positive staining with antisarcomeric alpha-actinin and contractility induced by 1.8 mM calcium. Incubation with 0.5 microM staurosporine or 0.3 mM 2,3-dimethoxy-1,4-naphthoquinone induced the characteristic morphologic and biochemical changes of apoptosis. The cellular topology of Ro and La was evaluated with confocal microscopy and determined in nonapoptotic and apoptotic cardiocytes by indirect immunofluorescence. In permeabilized nonapoptotic cardiocytes, Ro and La were predominantly nuclear, and propidium iodide (PI) stained the nucleus. In early apoptotic cardiocytes, condensation of the PI- and Ro- or La-stained nucleus was observed, accompanied by Ro/La fluorescence around the cell periphery. In later stages of apoptosis, nuclear Ro and La staining became weaker, and PI demonstrated nuclear fragmentation. Ro/La-stained blebs emerged from the cell membrane, a finding observed in nonpermeabilized cells, supporting an Ab-Ag interaction at the cell surface. In summary, induction of apoptosis in cultured cardiocytes results in surface translocation of Ro/La and recognition by Abs. Although apoptotic cells are programmed to die and do not characteristically evoke inflammation, binding of maternal Abs and subsequent influx of leukocytes could damage surrounding healthy fetal cardiocytes.
尽管细胞内抗原SSA/Ro和SSB/La与先天性心脏传导阻滞密切相关，但其与同源母体自身抗体的接触情况仍无法解释。为了研究细胞凋亡促进这些抗原表面可及性的假说，采用一种新技术对16至22周流产胎儿的人胎儿心肌细胞进行培养，该技术是在用胶原酶灌注主动脉后分离细胞。心肌细胞的确认包括用抗肌节α-肌动蛋白进行阳性染色以及由1.8 mM钙诱导的收缩性。用0.5 microM星形孢菌素或0.3 mM 2,3-二甲氧基-1,4-萘醌孵育可诱导细胞凋亡的特征性形态学和生化变化。用共聚焦显微镜评估Ro和La的细胞拓扑结构，并通过间接免疫荧光在非凋亡和凋亡心肌细胞中进行测定。在通透的非凋亡心肌细胞中，Ro和La主要位于细胞核内，碘化丙啶（PI）染细胞核。在早期凋亡心肌细胞中，观察到PI和Ro或La染色的细胞核凝聚，同时细胞周边出现Ro/La荧光。在凋亡后期，细胞核Ro和La染色变弱，PI显示细胞核碎片化。Ro/La染色的小泡从细胞膜出现，这一发现也在非通透细胞中观察到，支持细胞表面的抗体-抗原相互作用。总之，培养的心肌细胞中诱导细胞凋亡会导致Ro/La的表面易位并被抗体识别。尽管凋亡细胞被编程死亡且通常不会引发炎症，但母体抗体的结合以及随后白细胞的流入可能会损害周围健康的胎儿心肌细胞。
