TRANSCYTOSIS OF PROTEIN THROUGH THE MAMMALIAN CEREBRAL EPITHELIUM ANDENDOTHELIUM .3. RECEPTOR-MEDIATED TRANSCYTOSIS THROUGH THE BLOOD-BRAIN-BARRIER OF BLOOD-BORNE TRANSFERRIN AND ANTIBODY AGAINST THE TRANSFERRIN RECEPTOR

Diferric-transferrin (Tf; 80K mel. wt.) and the OX26 antibody (150K me l. wt.) against the transferrin receptor (TfR) were evaluated in the r at at light and ultrastructural levels as potential vehicles for the b lood to brain transcellular transfer (transcytosis) of native horserad ish peroxidase (40K mel. wt.), which by itself does not cross the bloo d-brain barrier (BBB). OX26, the Fab fragment of OX26 (50K mel. wt.), and Tf complexed to two ferric ions were conjugated to HRP irreversibl y in a 1:1 molar ratio. The indirect immunoperoxidase technique with O X26 as the monoclonal primary antibody applied to the surface of cryos tat sections or delivered intravenously to the live rat revealed TfRs on BBB capillaries, arterioles, and venules; TfRs were absent on non-B BB vessels supplying the circumventricular organs (i.e., median eminen ce, choroid plexus). OX26-HRP and OX26(Fab)-HRP delivered intravenousl y and diferric-Tf-HRP administered into the carotid artery labeled BBB vessels throughout the CNS without discernible disruption of the BBB or extravasation of the blood-borne probes into the brain parenchyma, No reaction product for the probes was observed in sites deficient in a BBB. Each of the macromolecular conjugates was endocytosed by BBB en dothelia and labeled presumptive endocytic vesicles, endosomes, and de nse bodies. OX26-HRP and Tf-HRP, but not OX26(Fab)-HRP, appeared to un dergo transcytosis through BBB endothelia for subsequent labeling of p erivascular cells. Distinct differences in the intracellular and extra cellular distributions between OX26-HRP and Tf-HRP were identified: (1 ) endocytosis and sequestration of blood-borne OX26-HRP within BBB end othelia were more prominent than those for diferric-Tf-HRP; (2) only O X26-HRP labeled the Gels complex in BBB endothelia; (3) peroxidase lab eling of CNS perivascular clefts and perivascular cells in rats receiv ing diferric-Tf-HRP was conspicuous at less than 1 h postinjection but not so in rats with blood-borne OX26-HRP at 5 min through 6 h postinj ection; and (4) peroxidase-labeled CNS neurons and glial cells were id entified readily in rats receiving diferric-Tf-HRP. The results sugges t that the receptor-mediated, transendothelial transfer of Tf-HRP from blood to brain is more efficient and direct than that of OX26-HRP. La beling of the Gels complex in BBB endothelia with blood-borne OX26-HRP implies that the transendothelial transfer of OX26-HRP follows intrae ndothelial pathways associated with the process of adsorptive transcyt osis. A diagram is provided depicting the possible intracellular and t ranscellular pathways within BBB endothelia available to blood-borne d iferric-Tf and OX26 as vectors for delivery into the CNS of non-lipid- soluble macromolecules that otherwise are denied entry by the blood-br ain fluid barriers. (C) 1996 Academic Press, Inc.