APICAL-TO-BASOLATERAL TRANSEPITHELIAL TRANSPORT OF HUMAN LACTOFERRIN IN THE INTESTINAL-CELL LINE HT-29CL.19A

The role of human lactoferrin (hLf) in alimentary iron absorption acro ss intestinal cells was explored using a human differentiated colon ca rcinoma cell line, HT-29cl.19A. The apical surface of HT-29cl.19A cell monolayers exhibited 1.5 x 10(6) specific binding sites for hLf per c ell, with a dissociation constant of 8.3 x 10(-7) M. The apical-to-bas olateral transport of I-125-labeled hLf (I-125-hLf) or I-125-Fe-59-lab eled hLf (I-125-[Fe-59]hLf) was investigated using filter-grown HT-29c l.19A cell monolayers mounted in Ussing chambers. Transport of total ( intact plus degraded) hLf, measured by the I-125 flux, was not saturab le up to an apical hLf concentration of 12.5 mu M, whereas immunoreact ive hLf transport measured by enzyme-linked immunoabsorbent assay was saturable at 3.75 mu M. Lowering the temperature to 4 degrees C reduce d the total and immunoreactive hLf fluxes by 77% and 90% and colchicin e (500 mu M) reduced these fluxes by 30% and > 97%, respectively. Thes e results indicate that both types of transport are transcellular. Ele ctrophoretic analysis revealed that I-125-hLf transported to the basal compartment consisted of largely degraded fragments (< 2 and 10-20 kD a) and a small portion of intact hLf. At an apical concentration of 3. 75 mu M diferric I-125-[Fe-59]hLf, the immunoreactive hLf flux (64.6 /- 14.3 ng.h(-1).cm(-2)) constituted similar to 12% of the total hLf f lux (552.0 +/- 61.6 ng.h(-1).cm(-2)) and was similar to the [Fe-59]hLf -equivalent flux (77.0 +/- 12.3 ng.h(-1).cm(-2)). In addition, 88% of the transported Fe-59 was bound to immobilized anti-hLf immunoglobulin G, indicating that iron is mainly transported across the cell monolay ers by immunoreactive (probably intact) hLf. These results suggest tha t hLf may be transported across the intestinal epithelial cells via tw o functional intracellular pathways: a major degradative pathway trans porting similar to 90% that leads to iron release from hLf and the tra nscellular transport of degraded fragments, and a minor pathway transp orting similar to 10% that allows the passage of immunoreactive hLf an d its bound iron.