Milner Y, Sagi E, Timberg R, Michel B, Me'te'zeau P, Goldberg M
Department of Biological Chemistry, Hebrew-University of Jerusalem, Israel.
J Cell Physiol. 1989 May;139(2):441-54. doi: 10.1002/jcp.1041390229.
Pemphigus is an intraepidermal autoimmune blistering disease of humans caused by circulating IgGs. We have investigated the binding mode and the fate of bound antibodies from Pemphigus sera (P-IgG) on guinea pig keratinocytes in suspension in order to find clues to the loss of cell adhesion in vivo (acantholysis). Flow cytometry, following indirect immunofluorescent labeling of the keratinocytes, and dead cells' staining with ethidium bromide, demonstrated the specific surface binding of P-IgG onto living keratinocytes only. This was shown with several Pemphigus sera or purified P-IgG. This technique, used with various Pemphigus sera, showed that the specific binding is increased when the serum titer is higher, and "Km" values for P-IgG were roughly and inversely correlated to the titers. Upon saturation the same average number of Pemphigus IgG sites per cell were found for the sera of different patients. Analysis of the specific binding of [125I]-P-IgG onto Percoll-separated (living) keratinocytes showed the existence of two classes of sites: 2 x 10(6) sites/cell high-affinity sites (Kd = 1.5 x 10(-6) M total IgG) and 25 x 10(6) sites/cell low-affinity sites (Kd = 6 x 10(-5) M total IgG). Cell sorting and flow cytometry of individual cells allowed us to correlate the light-scattering signal, the RNA content, the size and morphology, and the P-IgG binding to the cells. The results indicated that P-IgG binding is homogeneous within the living keratinocytes and increases with cell size (cell maturity). Cell-sorter analysis of cells with membrane-bound P-IgG, coupled to direct determination of P-IgG released in the medium, revealed the fate of bound P-IgG: 40-60% of the P-IgGs were released in the medium within 30 minutes at 37 degrees C. This was accompanied and followed by a much slower, metabolic energy-dependent, internalization process of the membrane-bound P-IgG. The internalization has been confirmed by electron microscopy of bound P-IgG labeled with protein A-gold. Internalized IgGs were seen in the cells in coated membranous vesicles and other endocytic compartments. Similar behavior was also observed with two other membrane ligands: i.e., concanavalin A and multispecific rabbit "antisurface" antibodies.
天疱疮是一种由循环免疫球蛋白G（IgG）引起的人类表皮内自身免疫性水疱病。我们研究了天疱疮血清（P-IgG）中结合抗体在豚鼠悬浮角质形成细胞上的结合模式及结合后的命运，以便找到体内细胞黏附丧失（棘层松解）的线索。对角质形成细胞进行间接免疫荧光标记后用流式细胞术，并用溴化乙锭对死细胞进行染色，结果表明P-IgG仅特异性地结合到活的角质形成细胞表面。几种天疱疮血清或纯化的P-IgG均显示出这种情况。用不同的天疱疮血清进行这项技术研究表明，血清滴度越高，特异性结合越强，P-IgG的“Km”值与滴度大致呈负相关。饱和后，不同患者血清中每个细胞上的天疱疮IgG位点平均数量相同。对[125I]-P-IgG在经Percoll分离的（活的）角质形成细胞上的特异性结合分析表明存在两类位点：每细胞2×10⁶个高亲和力位点（Kd = 1.5×10⁻⁶M总IgG）和每细胞25×10⁶个低亲和力位点（Kd = 6×10⁻⁵M总IgG）。对单个细胞进行细胞分选和流式细胞术使我们能够将光散射信号、RNA含量、大小和形态以及P-IgG与细胞的结合联系起来。结果表明，P-IgG在活的角质形成细胞内的结合是均匀的，并且随着细胞大小（细胞成熟度）增加。对具有膜结合P-IgG的细胞进行细胞分选分析，并结合直接测定培养基中释放的P-IgG，揭示了结合的P-IgG的命运：40% - 60%的P-IgG在37℃下30分钟内释放到培养基中。这伴随着并随后是一个慢得多的、依赖代谢能量的膜结合P-IgG内化过程。通过用蛋白A - 金标记的结合P-IgG的电子显微镜观察证实了内化。在细胞的包被膜泡和其他内吞区室中可见内化的IgG。另外两种膜配体，即伴刀豆球蛋白A和多特异性兔“抗表面”抗体，也观察到了类似的行为。
