Our previous studies have shown that purified C. difficile
toxin A induces cell rounding, detachment from the basement membrane, and apoptosis in primary human colonic epithelial cells (17
). In vivo, loss of the surface epithelial cells would expose cells in the underlying lamina propria to C. difficile
toxins. A large number of T cells, which are phenotypically and functionally distinct from peripheral blood cells (10
), are resident in the normal intestinal lamina propria. In addition to eosinophils, there is also a significant heterogenous population of macrophages that is especially prominent below the surface epithelium and the basement membrane (16
). Following the loss of the injured epithelium, lamina propria T cells, macrophages, and eosinophils would be exposed to C. difficile
toxin A via pores in the basement membrane (18
). In this study, we have investigated the responses of these resident colonic lamina propria cells to purified C. difficile
Culture with toxin A led to a loss of viability of isolated colonic lamina propria cells in a dose- and time-dependent fashion. Subsequent phenotypic studies (on cytospin preparations) using high concentrations of toxin A showed that macrophages were more sensitive than T cells and eosinophils. In studies confirmed by electron microscopy, no macrophages were seen following exposure to the toxin for 72 h. Since they are a small proportion of the isolated lamina propria cell population, it has not been possible to determine the type of cell death occurring in the toxin-exposed macrophages. By contrast, our studies convincingly demonstrate that toxin A-induced T-cell and eosinophil cell death occurs by apoptosis. In ultrastructural studies, the retention of characteristic granules in the cytoplasm allowed the identification of apoptotic eosinophils. C. difficile toxin A-induced apoptosis of colonic T cells was confirmed by FACS analysis using dual staining with monoclonal antibodies to Apo 2.7 and CD3. Studies by electron microscopy and FACS analysis of cells expressing annexin V and Apo 2.7 showed that the number of T cells undergoing apoptosis progressively increased with the duration of exposure to toxin A. In studies performed after each 24-h period of exposure to toxin A, there was a modest but significant increase in the proportion of T cells expressing the interleukin 2 receptor (CD25). It is possible that toxin A-induced activation of T cells preceded cell death by apoptosis.
Previous studies have shown that antigen-induced apoptosis of mature T cells occurs in distinct phases (4
). Following activation and expression of growth factors and their receptors, there is a proliferation of T cells before programmed cell death. Staphylococcal enterotoxin B binds to major histocompatibility complex class II molecules and the Vβ region of a T-cell receptor to induce activation and proliferation of T lymphocytes (9
) and subsequent cell death by apoptosis (11
). However, in our studies there was a significant reduction in the incorporation of [3
H]thymidine by colonic lamina propria cells exposed to C. difficile
toxin A, consistent with cell death in the absence of proliferation.
Following exposure to toxin A for 24 h, there was induction of TNF-α production by the isolated lamina propria cells. However, after culture with toxin A for 48 h, the amount TNF-α produced was not significantly different from the amount produced by control cells. The likely explanation for this is the loss of TNF-α-producing macrophages after culture with the toxin for 48 h. Since TNF-α may induce apoptosis in T cells (6
), studies were performed to investigate its role in toxin A-induced cell death. However, exposure to toxin A induced apoptosis in colonic T cells despite the presence of the specific neutralizing anti-TNF-α antibody. Another mechanism by which apoptosis may occur in T cells is by the interaction of Fas with the Fas ligand (12
). In our studies, a majority (>90%) of the control and toxin A-exposed colonic lamina propria T cells expressed Fas but only 0.7 to 2.3% of the cells expressed the Fas ligand (unpublished observations).
We have shown that C. difficile
toxin A also induced apoptosis in purified T cells. This implies that C. difficile
toxin A induces cell death following a direct interaction with T lymphocytes. After this toxin binds to specific receptors, its biological effects would be expected to occur following internalization. Recent studies with other cells have shown that subtypes of the Rho family of GTP binding proteins, which regulate the cytoskeleton, are substrates for C. difficile
toxin A (1
). The role of the Rho family of GTP binding proteins in T-cell apoptosis remains to be characterized. One recent study has shown that transient expression (using a Sindbis virus-based transient gene expression system) of Clostridium botulinum
exoenzyme C3, which ADP ribosylates Rho, induced apoptosis in EL4 murine lymphoma cells (21
). The authors of this study postulated that apoptosis may have resulted from the combined effects of inactivation of Rho and the stress induced by viral replication (21
). It can be postulated from our studies that the inactivation of Rho is sufficient to induce T-cell apoptosis.
We have previously shown that C. difficile
toxin A induces apoptosis in colonic epithelial cells (17
). The epithelial cell apoptosis was preceded by cell rounding and detachment from the basement membrane. However, detachment of epithelial cells from the basement membrane, in the absence of the toxin, also induced apoptosis (17
). We therefore postulated in that study that toxin A induced programmed cell death by causing a loss of epithelial cell adherence to extracellular matrix components of the basement membrane. However, our current studies of T cells (which are not dependent upon adhesion to the extracellular matrix for survival) suggest that C. difficile
toxin A can itself directly induce apoptosis.
There is increasing interest in apoptosis in host-microbial interactions (31
), but the significance to the host or the bacterium of C. difficile
toxin A-induced programmed cell death in the colonic mucosa remains to be determined. Macrophages and T cells are important components of the colonic mucosal immune system (10
). C. difficile
toxin A-induced early loss of macrophages and T cells would be expected to lead to suppression of the host mucosal immune system. Our studies suggest that such impairment of the mucosal immune system would be dependent upon the concentration of toxin A to which the colonic mucosa is exposed. Such a dose-dependent effect of C. difficile
toxin A has also been seen with intestinal epithelial cells (17
) and may determine the severity of clinical disease.
In the present study, we also show that a small population of isolated colonic lamina propria T cells spontaneously undergo apoptosis in culture. An increased level of apoptosis in unstimulated human intestinal T cells (isolated by collagenase digestion), compared to the level in peripheral blood T cells, has also been reported recently (3
). One explanation for this spontaneous apoptosis of intestinal T cells could be that they represent lymphocytes that were destined to die in the lamina propria.
In conclusion, our study has shown for the first time that C. difficile toxin A induces a rapid loss of resident colonic lamina propria macrophages, followed by T cells and eosinophils. For the last two cell types, cell death occurred by apoptosis. Exposure of normal colonic lamina propria cells to toxin A for 24 h induces the release of TNF-α, but its neutralization did not influence toxin-induced apoptosis in T cells. In addition, our studies with purified cells suggest that toxin A induces apoptosis after interacting directly with the T cells. C. difficile toxin A-induced loss of lamina propria macrophages and T cells in vivo may suppress the colonic mucosal immune system and lead to severe disease.