To our knowledge, this is the first report of a stress induced increase in transcellular permeability of FAE. There was a greater than threefold increase in permeability to the antigenic protein HRP in FAE after chronic stress. Moreover, macromolecular uptake was more than three times higher in FAE than in VE of the same stressed animal. Enhanced uptake in FAE was further emphasised by increased passage of chemically killed E coli in rats exposed to chronic stress.
Under normal conditions, the HRP molecule passes through epithelial cells via pinocytosis,1,25
although after chronic restraint stress and in sensitised animals HRP can also permeate between cells.18,26
In the present study, paracellular permeability to ions (conductance) was similarly affected in VE and FAE after stress. After chronic stress, permeability to the paracellular marker molecule 51
was increased in VE but almost unaffected in FAE. In contrast, stress induced changes in permeability to proteins was significantly more pronounced in FAE, with an almost fourfold increase in HRP flux. Electron microscopy revealed that HRP was transported in endosomes via the transcellular pathway whereas no HRP staining was found in the paracellular spaces. Taken together, these findings suggest that chronic stress, in the FAE, mainly affects transcellular antigen uptake. Ultrastructural examination further revealed penetrating bacteria in FAE of stressed rats which hinted at increased uptake of bacteria. Subsequent Ussing chamber studies demonstrated that permeability to E coli
was more than 30 times higher in the FAE of stressed rats compared with controls, and six times higher in FAE than in VE. Although the E coli
strain used in our study was chemically killed, lost motility, and could not replicate, they preserved their antigenicity. These particles have been used for phagocytosis studies27
and should be a good model of bacterial invasion. With confocal microscopy, it was seen that after 45 minutes, bacteria were present on the epithelial surface, and after 120 minutes, bacteria had passed through the epithelium. Increased uptake of bacteria by the FAE reflects a region made permeable to normally non-invasive bacteria. It may be speculated that a significant part of the bacterial translocation that takes part in various stress reactions may be due to invasion via the FAE, as revealed by our studies. As the earliest signs of CD are aphthoid ulcers in the FAE, barrier dysfunction of the FAE may be involved in the early stages of this disease.
In VE, conductance was, as in previous studies,20,21,28
enhanced by both acute and chronic stress. In contrast, permeability to the paracellular probe 51
was increased only by chronic stress. This finding suggests partially different mechanisms involved in the paracellular permeability defect induced by acute and chronic stress. Recently, Ferrier et al29
found that in vivo permeability to 51
in the mouse colon was unaffected by a single stress episode whereas repeated stress over 3–4 days induced a pronounced increase in paracellular permeability. This barrier defect was mediated by T cells and interferon γ. It could be speculated that this T cell activation, leading to interferon γ production and eventually increased paracellular permeability to macromolecules, is initiated by a WAS induced increase in transcellular permeability to antigens. This increase in transcellular permeability may involve mast cells. The importance of mast cells in stress induced mucosal abnormalities have been demonstrated previously.14,16,20,21
In the present study, we found a threefold increase in the number of mucosal mast cells and signs of mast cell activation after chronic stress. The interaction between mast cells and nerves has been widely discussed, and it is known that in response to stress, mast cells secrete mediators that affect nerves and immune cells.30
Direct contact between gastrointestinal mast cells and nerves has also been reported in the small intestine,31
but whether the altered FAE function seen in the present study involves nerve-mast cell interaction remains to be elucidated. Nevertheless, the responsiveness of FAE to acute stress suggests neurally mediated effects on FAE permeability. However, another possibility could be corticosterone mediated effects on barrier function which has been shown in vivo in severe acute stress.32
The present study clearly demonstrates the importance of FAE in the uptake of luminal antigens. Its superior uptake capacity compared with VE is partly due to M cells that constitute approximately 10% of rat FAE.33
M cells lack a developed microvillus border and glycocalyx coat which facilitates access for luminal pathogens.2,33
Previous studies showed that M cell numbers were altered in bacterial eneritis,34,35
indomethacin induced ileitis,36
and in patients with spondylarthropy.37
These studies indicate that M cells may play a role in the development of intestinal inflammation. The results of the present study may suggest that stress alters the function of M cells in the FAE. M cells meeting the criteria for identification1,24
were found in the FAE by electron microscopy. The close interaction between antigen, M cells, and immune cells was visualised and most likely this interaction is of major importance in the alteration of FAE function. However, more analyses are needed to determine whether the alteration involves changes in M cell number or activity, a problem superseded by the lack of reliable rat M cell markers. Cytokeratin 8 (clone 4.1.18) has been suggested but earlier studies have shown conflicting results36,38,39
and we have yet to obtain a specific staining. In a recent study40
it was shown that dendritic cells could extend their processes through the epithelial tight junctions and thereby sample both pathogenic and non-pathogenic luminal microbes directly, independent of M cells and enterocytes. Stress induced changes in dendritic cells and other immune functions in lymph follicles are important issues that need to be addressed in the continuation of this study.
In conclusion, we found that chronic stress modulated the barrier function of the FAE, allowing uptake of normally non-invasive luminal microorganisms. Enhanced bacterial uptake may lead to increased antigen load and presentation in the dome area of Peyer’s patches. Thus a local inflammatory reaction could be initiated, eventually leading to intestinal inflammation. This may have implications for stress related events in IBD.