Transmucosal transport of phytohemagglutinin, concanavalin A and alpha-lactalbumin across rabbit ileum in vitro.

The transmucosal passage of alpha-lactalbumin (alpha-La), phytohemagglutinin (PHA), and concanavalin A (Con A) (1 mg/ml) was measured in the rabbit ileum mounted in the Ussing chamber, with and without 10(-2) M glucose or galactose. The transport of the radiolabelled proteins was assessed by radioisotopic determination, high pressure liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA). In the absence of galactose, the transmucosal transport was significantly higher for PHA (4.1 +/- 1.8 micrograms/h.cm2 mean +/- SE) than for alpha-La (2.9 +/- 1.2) and very low for Con A (0.6 +/- 0.5). HPLC analysis of the transported material revealed differential processing of the proteins. ELISA indicated that 3 percent of radiolabelled alpha-La that crossed the epithelium was in an immunoreactive form, whereas no immunoreactive forms of PHA and Con A were detected. The uptake or binding by the tissue was identical for PHA and Con A (7.8 +/- 2.9 and 5.8 +/- 2.8 micrograms/cm2, respectively), and significantly lower for alpha-La (1.5 +/- 0.31). 10(-2) M galactose did not modify the uptake or binding of alpha-La and Con A, but significantly decreased that of PHA to a level that was not significantly different from that of alpha-La. The present results indicate that the initial uptake of the proteins is most likely dependent upon their interactions with the luminal side of the epithelium. After uptake, the proteins are subjected to intracellular processing which also appeared differential. Thus, protein transport depends on the properties both of the compartment crossed (Glycocalyx, brush-border membrane, cytoplasm, basolateral membrane), and of the protein.

Transport of beta-lactoglobulin across rabbit ileum in vitro.

Intestinal transepithelial transport constitutes a major limiting step in the transfer of food protein antigens to the blood. This transport was studied in isolated rabbit ileum in Ussing chamber in vitro for the milk protein antigen beta-lactoglobulin (beta-Lg). The transepithelial passage of beta-Lg was measured by enzyme-linked immunosorbent assay (ELISA) and radiolabeled protein transfer and compared with that of the nonmetabolizable marker polyethylene glycol (PEG)-4000. When 1 mg/ml of beta-[14C]Lg or [3H]PEG was added to the mucosal side of the tissue, the total uptake, measured as the transfer of radiolabeled material across the ileum, was significantly higher for beta-Lg than for PEG (5.46 +/- 1.75 vs. 1.43 +/- 0.26 micrograms.h-1.cm-2). Measured by ELISA, 6-9% of the total amount of beta-Lg transported was absorbed in an intact antigenic form. This transport of intact beta-Lg was inhibited by the metabolic inhibitors 50 mM 2-deoxyglucose and 1 mM azide added simultaneously, was reduced by the microtubule assembly inhibitor 0.05 mM colchicine, and was enhanced by 20 mM ammonia, which inhibits lysosomal proteolytic activity. These results indicate that beta-Lg is efficiently absorbed by the intestinal mucosa of adult animals, partly in intact antigenic form and that beta-Lg transport is probably transcellular, as observed for other proteins. The finding that beta-Lg is absorbed in intact antigenic form agrees with other reports implying that beta-Lg is the main factor responsible for milk protein immunoreactivity and intolerance.

Permeability of milk protein antigens across the intestinal epithelium in vitro.

Degradations by proteolytic enzymes and intestinal epithelial permeability represent two major drawbacks to the transfer of food protein antigens to blood. These steps were studied in vitro for the milk protein antigens beta-lactoglobulin (beta-Lg), alpha-Lactalbumin (alpha-La) and beta-casein (beta-cas). Pepsin-trypsin hydrolysis and permeability in isolated rabbit ileum in Ussing chamber were suited by ELISA and radiolabelled-protein measurement. Pepsin-trypsin hydrolysis showed an increasing resistance in the order beta-cas less than alpha-La less than beta-Lg. The rate of absorption of the antigenic proteins by isolated rabbit ileum was in the same order, and the rate of absorption of the whole proteins (degraded and antigenic forms) was significantly higher for beta-Lg than for alpha-La and beta-cas. These results suggest a selective intestinal permeability for milk protein antigens. This selectivity is probably important in the mechanism of food protein sensitization via the oral route.