ULTRASTRUCTURAL ALTERATIONS CAUSED BY IMMUNOLOGICAL REACTIONS AFTER INTRACARDIAC INJECTION OF ALLOGENEIC ANTIBODIES AGAINST BLOOD-GROUP ANTIGENS - AN EXPERIMENTAL-STUDY USING THE IN-VITRO WHOLE-RAT EMBRYO CULTURE

The effects of intracardiac injection of 0.5 mu l allospecific hemolyz ing rat-antirat antibodies, directed against the blood group antigens, on the endothelium of the dorsal aortae were studied in 9-14 somite-s taged Wistar and RIV:Tox rat embryos, using both transmission electron microscopy (TEM) and immunoelectron microscopy (IEM). In a TEM study ii was further investigated if either apoptosis or cell necrosis occur red as a result of the forementioned intracardiac injection. The resul ts were compared to ultrastructural findings of the dorsal aortae in s ham- and noninjected rat embryos of the same gestational age. In the c ontrol rat embryos, the aortic vascular wall consisted of a single con tinuous layer of endothelial cells. No clear basal lamina was present in TEM. Furthermore, no immunoreactivity against the endothelium or th e intravascular blood cells was noted. Embryos injected with hemolyzin g rat-antirat antibodies displayed clefts or pores, and diaphragmatic fenestrations of the endothelial lining of the dorsal aortae after 2 h r. Alterations resembled those induced by vasoactive mediators such as histamine, serotonin, bradykinin, and prostaglandins. The above chang es had disappeared 4 and 6 hr after injection with complete restoratio n of the endothelial lining. Immunogold staining demonstrated Ig depos itions along the luminal side of the endothelium, in the vicinity of t he intercellular spaces, and in the subendothelial space of the dorsal aortae. Numerous particles were seen located inside intracytoplasmati c vesicles, indicating Involvement of transcytoplasmatic transport as well as intracytoplasmatic phagocytosis. Similar depositions were obse rved in and around intravascular embryonic blood cells. Apoptosis, or programmed cell death, an important component in immunological reactio ns, occurred in rot embryos injected with hemolyzing rat-antirat antib odies. The excessive amount of apoptosis seen in this study is in acco rdance with the pathogenetic cell degeneration found in our earlier st udies. Cell necrosis was not observed. The results from this study ind icate that the endothelium of the dorsal aortae and intravascular bloo d cells only display a transient reaction following injection with hem olyzing rat-antirat (RAR) antibodies. The temporary reaction is presum ably due to the release of vasoactive mediators. The smaller vessels a nd capillaries are still in an earlier stage of development, displayin g fenestration, making them more susceptible for injury after immunolo gical interaction. The results are indicative that the pathogenetic ef fect of the immunological reaction after intracardiac injection takes place at the level of the microcirculation by ''switching on'' apoptos is. Programmed cell death is essential in embryogenesis and developmen t. Therefore excessive apoptosis, i.e., inappropriate apoptosis, will eventually induce congenital malformations. (C) 1995 Wiley-Liss, Inc.