Internalization and recycling of glycoprotein 280 in epithelial cells of yolk sac.

The luminal plasma membrane of the epithelial cells lining the visceral layer of the yolk sac and the renal proximal tubule display a well developed brush border defining numerous clathrin-coated intermicrovillar areas which are further characterized by expressing two glycoproteins, gp280 and gp330, the latter also known as the Heymann nephritis antigen. The present study analyzes the distribution, the internalization and intracellular trafficking of gp280 and gp330 in yolk sac epithelium by immunoultrastructural and cell surface labeling techniques. Immunocytochemistry revealed that gp280 and gp330 were distributed very similarly in the endocytic pathway including dense apical tubules, with the exception that gp330 was found in lysosomes to a much greater extent than gp280. To demonstrate internalization of gp280, apical cell membrane proteins of paired yolk sacs were labeled at 4 degrees C with biotin, linked via a disulfide bond cleavable under mild reducing conditions by glutathione, and either kept at 4 degrees C or incubated at 37 degrees C. These experiments showed that biotin could be cleaved from gp280 by glutathione in yolk sacs kept at 4 degrees C, whereas it became inaccessible to glutathione after incubation at 37 degrees C, suggesting internalization of gp280. Furthermore, incubation of yolk sacs in the presence of colloidal gold-labeled antibodies to gp280 demonstrated that gp280, initially expressed on the cell membrane, was translocated into endocytic vacuoles and accumulated in dense apical tubules, whereas only a small fraction reached the lysosomes. Under similar conditions, gold-labeled antibodies to gp330 were also internalized and followed a similar intracellular routing, but lysosomal accumulation was also found. Bovine serum albumin-labeled gold particles accumulated in lysosomes but were virtually absent from dense apical tubules. These observations suggest that gp280 and gp330, visualized by anti-gp280 and anti-gp330 antibodies coupled to gold particles, returned to the cell surface via dense apical tubules, whereas albumin gold particles dissociated from a potential binding protein in the early endocytic compartment and were subsequently accumulated in lysosomes. Since gp280 is internalized and apparently translocated to a recycling compartment as is gp330, our results suggest that gp280 may play a role as a receptor for endocytosis of as yet unknown ligands.

The role of natural IgM in the hyperacute rejection of discordant heart xenografts.

The mechanism of xenograft hyperacute rejection in discordant species combinations remains controversial. The purpose of this work was to study the role of natural antibodies in the hyperacute rejection of guinea pig hearts transplanted into rats, a highly discordant combination. This study was conducted in vitro, ex vivo, and in vivo. The endothelial cells of the graft being the first targets damaged in the process of hyperacute rejection, the binding of rat natural antibodies to guinea pig endothelial cells was studied by immunofluorescence. The study was carried out in vitro on guinea pig endothelial cells in culture, and ex vivo on isolated guinea pig hearts perfused with either rat serum or immunoglobulins or immunoglobulin fragments bearing the antigen-binding site. In vitro and ex vivo, rat natural IgM were found to bind specifically to guinea pig endothelial cells, since IgM fragments bearing the antigen-binding site (Fab mu and Fab' mu) could be detected on these cells. IgM fragments were able to inhibit the fixation of native IgM molecules. In contrast, rat IgG only bound to endothelial cells through Fc portions. Thus rat natural IgM might play a role in hyperacute rejection by binding to the graft endothelial cells and triggering the complement cascade activation. In order to test the role of natural IgM in vivo, isolated guinea pig hearts were first perfused with rat Fab' mu, which inhibit the binding of IgM and are unable to activate the complement cascade. These hearts were then transplanted into Lewis rats. The rejection time of Fab' mu-perfused guinea pig hearts was prolonged compared with hearts perfused with buffer or IgG F(ab')2. Therefore, in the guinea pig to rat combination, preventing the binding of the recipient's natural IgM to the graft endothelium delays the hyperacute rejection. In addition, natural IgM are likely to play a greater role than natural IgG.

Target antigens of hyperacute xenogeneic rejection in the rat to guinea pig to rat discordant combinations.

The increasing shortage in allografts has led to a renewed interest in xenogeneic transplantation. Discordant combinations are characterized by hyperacute rejection partly due to the presence of natural antixenogeneic antibodies in the recipient. The aim of this work was to characterize the target antigens, using 2 discordant models. In the rat into guinea pig model, analysis of organ homogenates by immunoblotting revealed numerous bands. Some of these bands were organ specific, whereas others, namely in the 55-kDa region, were detected in liver, heart, lung, and kidney. Using membrane extracts of liver cells or of aortic endothelial cells, only bands of 55 kDa were revealed. No band could be seen using extracts of isolated hepatocytes. Two bands of 55 kDa disappeared after preabsorption of guinea pig sera on the various rat tissue homogenates, suggesting that they represent xenoantigens common to these tissues. In order to investigate the in vivo relevance of these 55-kDa antigens, isolated rat livers were perfused with decomplemented guinea pig sera. Eluates revealed one single print of 55 kDa on rat tissue homogenates. Finally, preincubation of rat mononuclear cells with various xenogeneic sera did not inhibit the binding of mAb specific for rat class I or class II MHC antigens, suggesting that the latter are not recognized by natural xenoantibodies. In the guinea pig to rat model, the antigens detected had a molecular mass ranging from 95 to 110 kDa. Absorption and perfusion experiments also showed that these antigens were common to various tissues and involved in the binding of rat natural antibodies ex vivo. In conclusion, our results indicate that rat xenoantigens of about 55 kDa are recognized by guinea pig natural antibodies, while guinea pig xenoantigens of 95-110 kDa are bound by rat natural antibodies. These antigens are common to liver, heart, lung, and kidney, are borne by endothelial cells, and cannot be found on hepatocytes.

[Superacute xenogenic rejection: attempted treatment with anti-idiotypic antibodies]. / Rejet hyperaigu xénogénique: tentative de traitement à l'aide d'anticorps anti-idiotypiques.

Heart xenotransplantation in the guinea-pig to rat model, a highly discordant combination, is invariably followed by a hyperacute rejection of the graft. Preformed IgMs were shown to be responsible for the destruction of the graft by stimulating the complement cascade. We tried to inhibit preformed antibodies, before transplantation, by inducing anti-idiotypic antibodies using autologous Fab' mu specific for guinea-pig endothelial cells. This treatment seems to exert a neutralizing effect on xenogeneic hyperimmunization, against guinea-pig endothelial cells but not against preformed anti-guinea-pig antibodies.

The blockade of mineralocorticoid hormone signaling provokes dramatic teratogenesis in cultured rat embryos.

Although the administration of adrenocortical hormones to pregnant rats provokes only limited effect on the growth and development of the fetus, the direct influence of these steroids on cultured embryos has never been studied. The disruption of cell signaling by ZK 91587, which specifically occupies the mineralocorticoid receptor, resulted within 2 days in significant and pronounced adverse effects on the total length, the somite number, the embryo curvature, the communication between vitelline and umbilical blood vessels in the allantoid, and the vascularization of the vitelline sac, in 244-hour Wistar rat embryos in culture. The average score of 16 organs declined in a dose-dependent manner, following exposure to ZK 91587, and this was totally reversed by 10 microM aldosterone which, by itself, did not at all influence the embryonic development. The organogenesis was inhibited in the order: hind limb > fore limb > optic stalk > brain > olfactory pit > otic vesicle. ZK 91587 was completely ineffective in embryos that had attained the age of 260 hours. Similar, but less dramatic, results were obtained with the mineralocorticoid antagonist RU 26752, and with the antiglucocorticoid RU 38486. Sprague-Dawley rat embryos responded in a manner similar to the Wistar conceptuses. Thus, steroid receptor-mediated cell signaling is of critical importance to the growth and development of cultured rat embryos, which form a new model system to unravel adrenocortical hormone action.

Antibodies to the 280-kd coated pit protein, target of teratogenic antibodies, produce alterations in the traffic of internalized proteins.

Previous studies have identified two high-molecular weight (280 and 330 kd) glycoproteins expressed by coated pits of the proximal renal tubule and yolk sac and have further established that, in vivo, antibodies to gp280 but not to gp330 induce fetal malformations. In the present study, we report the effect of these antibodies on the endocytic process by yolk sac visceral epithelial cells of rat embryos explanted at day 10 of gestation. Antibodies to gp280 markedly altered development of the yolk sac and embryo, induced malformations, inhibited by 40% the uptake of [14C] sucrose and perturbed the intracellular traffic of internalized proteins. Under control conditions, rat immunoglobulin G present in the culture medium was immunolocalized in lysosomes of epithelial cells, whereas in the presence of antibody, it was detected in small vesicles scattered through the apical cytoplasm. Alterations of the endocytic pathway were confirmed by experiments analyzing the uptake of peroxidase added to the medium for 2 to 60 minutes. The initial compartments of endocytosis visualized by peroxidase were increased in size and abnormal in shape and the transfer of the internalized peroxidase to the lysosomal compartment was delayed. In contrast, antibodies to gp330 had a minimal effect on embryonic development and did not induce fetal malformations. Endocytosis was only modestly altered; uptake of [14C] sucrose was decreased by 25%, and only minor modifications of the intracellular transit of peroxidase could be detected. We suggest that the key role of anti-gp280 antibodies is via trapping of the target antigen in the early endocytic compartment thus preventing its normal function in lysosomal transfer.