The main conclusion of these studies is that the staphylococcal exotoxins, SEB, SEA, and TSST-1, can rapidly cross an epithelial membrane in intact, fully functional forms. These findings have implications for the role of SEB, SEA, and similar SAg enterotoxins in food poisoning since, if the SAg activity of these toxins is important in this pathology, they must have the ability to cross the intestinal epithelium intact to gain access to T cells.
Our experiments with the human polarized enterocytic cell line Caco-2, suggest that these toxins may cross intestinal epithelium by more than one mechanism. The rate of SEA transcytosis was similar to that of a marker for water transport, HRP, suggesting a nonspecific mechanism. SEB, on the other hand, was transcytosed at three to six times the rate of HRP, suggesting a receptor-facilitated process. Correlating with this idea was the ability of Caco-2 to act as an APC for SEB, but not SEA, in stimulating T cell hybridomas bearing the appropriate Vβ element. Other evidence in support of this conclusion was the inhibition of SEB transcytosis by mutations in N23 or F44. These mutations also interfere with the SAg activity of SEB by disrupting T cell receptor and MHC class II binding, respectively. Previously, Harris et al. (37
) has shown that these mutations also interfere with SEB-induced food poisoning in monkeys. While interpreted at the time in terms of the SAg activity of SEB, our current findings suggest that the reduction in SEB transcytosis caused by these mutations may have contributed as well to the results.
Despite the appearance of a receptor-mediated mechanism for SEB transcytosis, several features usually associated with such a mechanism were not observed. The rate of transcytosis was only slightly favored in the apical to basal direction through the Caco-2 monolayer. Usually, transcytosis is biased in one direction because the distribution of the receptor involved is polarized. For example, in intestinal and mammary epithelium, transcytosis of secretory IgA from the basal to apical surface occurs via an Fc receptor (pIgR) whose distribution is polarized in favor of the basolateral surface (39
). Reciprocally, absorption of IgG from ingested milk in neonatal rats and mice is mediated by a special Fc receptor (FcRn), and polarized to the apical surface of the intestinal epithelium (44
). However, there are, in fact, examples of bidirectional transcytosis of ligands such as glutathione (46
) and glycylsarcosine (47
). Also, there is a recent description by Ellis and Luzio (48
) of a novel receptor on Caco-2 cells that can transcytose in both directions.
More importantly, in our experiments we saw no evidence for saturation of SEB transcytosis, even using SEB concentrations up to 300 μg/ml. If this were a receptormediated process, we would have expected the rate to maximize as the receptor became saturated (at concentrations of ~10 times the kD). These results suggest that the putative receptor must be of low affinity (~kD >5 × 10−5 M), a result consistent with the finding that SEB presentation to T cells by Caco-2 is much less efficient than by MHC class II+ fibroblast. (The MHC class II affinity for SEB is ~10−6 M.)
Surprisingly, we observed facilitated transcytosis of TSST-1 as well as SEB by Caco-2 cells. Again, this transcytosis occurred at a rate about fivefold greater than that of HRP and was bidirectional and could not be saturated. TSST-1 is not an enterotoxin, most likely because it is destroyed by digestive enzymes in the stomach and intestine (49
). However, TSST-1 is a major cause of toxic shock syndrome. In women, TSST-1 produced in the vagina can find its way into the blood stream, where the subsequent toxic shock is accompanied by massive stimulation of peripheral Vβ2-bearing T cells (13
). Since these cases are often associated with use of a particular type of tampon during menses, the toxin may enter the body via direct contact with blood. However, if the putative receptor mediating TSST-1 transcytosis through Caco-2 is common to other types of epithelium, then transcytosis may contribute as well to the uptake of the toxin. This notion is supported by the ability of conjuctival epithelia and endothelia to endocytose and transcytose TSST-1 (50
Previous studies have shown that there are non-MHC receptors for some of the staphylococcal toxins on intestinal epithelia, B cells, and mast cells (33
). These studies showed that human intestinal epithelial cells and mouse B cells or mast cells could present SEB, but not SEA. In contrast, lymph node cells from class II–deficient mice could present SEC1-3 and SEE, but not SEB or SEA. Some of these results are similar to those described in this paper. Perhaps, the receptors which participate in toxins binding and transcytosis are related to some of these reported molecules.
In an attempt to relate our results to a more physiological situation we documented the appearance of ingested SEA and SEB in the blood of mice. Functional toxin was detected both by the ability of serum from these animal to stimulate the appropriate T cell hybridomas in vitro and by the in vivo partial disappearance of T cells bearing the appropriate Vβ elements. These results extend those of Migita and Ochi (29
) who used Western blotting to show the appearance of intact SEB in the blood of mice fed the toxin. As in our in vitro transcytosis studies, we found that the extent of SEA uptake was less than that of SEB, although both rapidly reached functional levels in the serum. This could again reflect a passive versus active mechanism of transcytosis, but other mechanisms, such as differential degradation, are not excluded by our results.