Most of the data for Y. enterocolitica
invasion of cultured host cells and IL-8 induction were acquired in experiments with nonintestinal, or at least nonpolarized, cell types. Interestingly, it was demonstrated that intestinal epithelial cell differentiation inhibits apical invasion by Y. pseudotuberculosis
of the intestinal epithelial cell line Caco-2 (8
). The goal of this study was to examine Y. enterocolitica
invasion and Y. enterocolitica
-induced IL-8 secretion in a more relevant in vitro cell culture system, employing monolayers of polarized human intestinal epithelial cells (T84).
The experiments reported here show that invasion of T84 cells by pYV+ and pYV− Y. enterocolitica strains from the apical surfaces is significantly diminished during polarization of the cells. The residual number of so-called intracellular bacteria in apically infected polarized T84 cells detected by the gentamicin killing assay could be due to microdiscontinuities in T84 monolayers or to extracellular bacteria that survived the gentamicin killing rather than to actual invasion. Electron microscopic examinations of T84 monolayers infected at different time points after culture on permeable supports confirmed this assumption.
, like Y. pseudotuberculosis
, expresses three different adhesion and/or invasion factors. The invasin Inv, the major invasion factor, is maximally expressed at 27°C (27
). In contrast, the additional factors Ail and YadA are expressed at 37°C (30
). Since the contribution of each of these factors to the invasion of intestinal epithelial cells is unclear, we also investigated invasion by bacteria that were cultured at 37°C. However, Y. enterocolitica
cultured at Ail- and YadA-inducing temperatures did not enter T84 monolayers to a significant extent (data not shown).
During polarization intestinal epithelial cells acquire two distinguishable domains, apical and basolateral. Interestingly, a redistribution of β1
integrins during differentiation of Caco-2 cells in culture to the basolateral domains of the cells has been documented (56
). We have also observed a restricted localization of β1
integrins in the basolateral domains of differentiated T84 cells. It has already been suggested that this redistribution of β1
integrins accounts for the diminishment of epithelial cell invasion by Y. pseudotuberculosis
). A recent investigation reported by McCormick et al. (37
) showed that Y. pseudotuberculosis
was not able to invade polarized T84 cells. However, T84 cells became susceptible to Y. pseudotuberculosis
invasion in regions of microdiscontinuity induced by transepithelial migration of PMNs. The authors state that neutrophil transepithelial migration forces neighboring epithelial cells to separate, thereby giving yersiniae access to unmasked basolateral β1
The presence of β1
integrins on the basolateral membranes of polarized cells indicated the possibility of invasion via the basolateral route. In subsequent experiments monolayers were infected from the basolateral surface. The results showed basolateral invasion of polarized T84 cells by Y. enterocolitica
. The inability of a Y. enterocolitica inv
mutant to invade suggests that the Inv protein serves as a major invasion factor in this system. Furthermore, reduced invasion by the pYV+
strain was observed after it was cultured at 37°C. Similar observations have been reported after infection of HeLa and HEp-2 cells with Y. enterocolitica
and Y. pseudotuberculosis
. This was interpreted as a consequence of the antiphagocytic effect of YopE and YopH (18
Apical infection of polarized T84 cells with Y. enterocolitica
for 1 h or over a period of 20 h did not induce IL-8 secretion, whereas apical infection with S. typhimurium
induced substantial IL-8 secretion (data not shown). Previous reports indicated that invasion is an essential signal for induction by invasive bacteria of IL-8 secretion (15
). The fact that differentiation of epithelial cells diminishes invasion by Y. enterocolitica
could explain the lack of IL-8 secretion after infection from the apical surfaces. Consistently, infection from the basolateral surfaces not only led to invasion but also to IL-8 secretion in the physiological apical-to-basolateral direction.
Comparison of invasion and IL-8 secretion after infection with the Y. enterocolitica pYV+ strain cultured at 27 or 37°C revealed that the pYV+ strain grown at 37°C did not significantly invade but induced secretion of IL-8. This finding suggests that Yersinia invasion is not required to induce IL-8 secretion by polarized T84 cells after basolateral infection. Whether adherence-triggered IL-8 secretion is mediated by Ail or YadA is not yet clear.
Previous investigations revealed a suppressive effect of virulent pVY+ Y. enterocolitica
bacteria on infection-triggered IL-8 secretion by nonpolarized HeLa or T84 cells. This effect required the presence of an active type III secretion-translocation mechanism and at least YopB and -D (52
). However, in the present study we could not detect a suppression of IL-8 secretion after basolateral infection of polarized T84 cells. Thus, although the pYV+ Y. enterocolitica
strain cultured at 37°C did not significantly invade polarized T84 cells from the basolateral side, IL-8 secretion was comparable to that induced by the pYV−
strain, which invaded to a greater extent. This finding could be explained by the distinct differentiation of the cells employed in the two studies. However, it is more likely that this discrepancy is due to the special features of the in vitro system used in the present study. Thus, the absence of a certain host cell receptor(s) from the basolateral surfaces of polarized T84 cells may account for the lack of translocation of Yop protein(s), some of which suppresses IL-8 secretion. On the other hand, it might well be that prior to a Yop-mediated suppression residual amounts of IL-8 or other cytokines secreted by infected cells induce IL-8 secretion by a paracrine-autocrine pathway in neighboring cells (47
) which are not in contact with Y. enterocolitica
. Consequently, IL-8 production in these cells would not be suppressed by Y. enterocolitica.
Data from experiments in the mouse model suggest that yersiniae cross the intestinal epithelium by transcytosis via M cells (1
). Subsequently, Y. enterocolitica
might gain access to neighboring epithelial cells of the follicle-associated epithelium and trigger IL-8 secretion. Whether basolateral IL-8 secretion by epithelial cells and recruitment of PMNs are involved in the pathogenesis of Yersinia
-induced abscess formation in Peyer’s patch tissue remains to be shown.