The intestinal epithelium is the largest mucosal surface providing an interface between the external environment and the mammalian host. Under physiological circumstances the intestine represents the primary site for active transport of fluid and electrolytes from the gut lumen through the transcellular pathway. The paracellular pathway however serves as the predominant route for passive transepithelial solute flow. Healthy mature gut mucosa with its intact tj serves as the main barrier to the passage of macromolecules. Furthermore, the intestinal barrier functions as the major organ of defence against foreign antigens, toxins, and macromolecules entering the host via the oral/enteric route. During such healthy states, quantitatively small but immunologically significant fractions of antigens cross the defence barrier. These antigens are absorbed across the mucosa along two functional pathways. The vast majority of absorbed proteins (up to 90%) cross the intestinal barrier through the transcellular pathway, followed by lysosomal degradation that converts proteins into smaller non-immunogenic peptides. The remaining portion of proteins is transported as intact molecules, resulting in antigen specific immune responses. This latter phenomenon utilises the paracellular pathway that involves subtle but sophisticated regulation of intercellular tj that can contribute to antigen oral tolerance. When the integrity of the tj system is compromised such as during prematurity or exposure to radiation, chemotherapy, and/or toxins, an immune response to environmental antigens (including autoimmune diseases and food allergies) may develop.18
The specific cells that are important for the immune response lie in close proximity to the luminal antigens and account for up to 80% of all immunoglobulin producing cells in the body.19,20
Another important factor for intestinal immunological responsiveness is the major histocompatibility complex. HLA class I and class II genes are located in the major histocompatibility complex locus on chromosome 6. These genes code for glycoproteins, which bind and shuttle peptides from the cytoplasm to the cellular membrane making up the HLA-peptide complex, which is recognised by certain T cell receptors in the intestinal mucosa.21–23
Susceptibility to at least 50 diseases, including CD, has been associated with specific HLA class I or class II alleles. A common denominator of these diseases is the presence of several pre-existing conditions leading to an autoimmune process. The first component needed to develop an autoimmune process is a genetic susceptibility for the host immune system to recognise, and potentially misinterpret, an environmental antigen presented within the gastrointestinal tract. Secondly, the host must be exposed to the antigen. Finally, the antigen must be presented to the gastrointestinal mucosal immune system following its paracellular passage (normally prevented by the tj competency) from the intestinal lumen to the gut submucosa.24–26
In all cases, increased permeability appears to precede the disease and causes an abnormality in antigen delivery that triggers the multiorgan process leading to the autoimmune response.27
While our knowledge of tj ultrastructure and intracellular signalling events have significantly progressed during the past decade,11
relatively little is known about their pathophysiological regulation secondary to extracellular stimuli. Therefore, the intimate pathogenic mechanisms of diseases in which tj are affected have remained unexplored owing to our limited understanding of the extracellular signalling involved in tj regulation.13
The discovery of Vibrio cholerae
derived Zot has shed some light on the intricate mechanisms involved in the modulation of the intestinal paracellular pathway11,14,19
and has allowed us to identify an intestinal mammalian analogue that participates in tj regulation.9,10,16
This analogue, that we have named zonulin, represents a novel eukaryotic protein that reversibly opens intestinal tj.9
We have recently demonstrated that zonulin expression is increased during the early stage of CD,10
suggesting that the reported opening of tj at the early stage of the disease28,29
could be mediated by zonulin.
The studies reported in this paper were aimed at establishing the link between enterocyte gliadin exposure and zonulin release. The results of our study indicate that gliadin activates the zonulin signalling pathway in normal intestinal epithelial cells in vitro. The cellular response observed only a few minutes after gliadin incubation was characterised by significant cytoskeleton reorganisation with a redistribution of actin filaments mainly in the intracellular subcortical compartment. Spectrofluorimetry experiments revealed that such cytoskeleton reorganisation was associated with an increment in F-actin secondary to an increased rate of intracellular actin polymerisation. We can exclude the fact that the gliadin induced increment in F-actin was due to new protein synthesis as it was not affected by preincubation of cells with cycloheximide, a potent inhibitor of protein synthesis. An endocytosis dependent polymerisation of the actin filaments was also ruled out by the experiments performed at low temperature. Instead, inhibition of the effect of gliadin that was observed after pretreatment with a PKC inhibitor suggested that actin polymerisation was dependent on PKC intracellular signalling. Moreover, the experiments performed in Ussing chambers showed that addition of gliadin peptides to the intestinal mucosa in vitro caused a significant reduction in Rt within a few minutes. We have previously demonstrated30
that large proteins can cross the intestinal barrier following changes in Rt of similar magnitude (~20% decrement from baseline values) to that induced by gliadin (see fig 6). It is therefore possible to hypothesise that gliadin induced cytoskeleton reorganisation, as observed by fluorescence microscopy and spectrofluorimetry, acts on tj structural proteins causing changes in Rt and intestinal permeability to macromolecules, including gliadin.
Interestingly, the peak of actin polymerisation detected after only 60 minutes of gliadin incubation temporarily followed zonulin release by IEC-6 cells, suggesting that this event is secondary to gliadin dependent release of zonulin. We therefore elected to investigate whether the gliadin induced cytoskeleton effect was mediated by zonulin. We have previously demonstrated that following binding to its specific surface receptor, zonulin induces actin polymerisation, followed by cytoskeleton redistribution to the subcortical cell compartment.9
The cytoskeleton changes induced by zonulin are followed by tj disassembly, leading to increased intestinal paracellular permeability.9
The experiments performed with the synthetic peptide FZI/0, which can compete and block zonulin binding to its receptor,11
showed complete inhibition of the peak of F-actin increment induced by gliadin, as well as complete inhibition of the gliadin induced reduction in intestinal Rt in vitro. However, pretreatment with the synthetic peptide failed to inhibit gliadin induced zonulin release. These results suggest that FZI/0 exerts its inhibitory effect on gliadin induced actin polymerisation by blocking the zonulin receptor rather than affecting zonulin release.
At this stage we do not know whether gliadin dependent activation of the zonulin system requires interaction of gliadin with a specific enterocyte receptor(s) or is a consequence of an unspecific response. However, the observation that gliadin induces release of zonulin and changes in Rt only when added to the mucosal aspect of the enteric epithelial cells (data not shown) suggest that the gliadin polarised effect is dependent on interaction with a surface receptor present on the brush border of the enterocyte.
Considering the results of this study and our preliminary data generated using intestinal tissues from both coeliac patients in remission and healthy controls (S Drago, A Fasano, unpublished), we can hypothesise a possible gliadin mechanism of action leading to a zonulin mediated increase in actin polymerisation and intestinal permeability. Enterocytes exposed to gliadin physiologically react by secreting zonulin in the intestinal lumen. While in normal intestinal tissues this secretion is self limited in time (see fig 4), in CD gut tissues the zonulin system is chronically upregulated,10
leading to a sustained increase in intestinal permeability to macromolecules, including gliadin, from the lumen to the lamina propria. Here, gliadin is deamidated by tissue transglutaminase and then recognised by HLA-DQ2/DQ8 bearing antigen presenting cells, triggering the onset of the CD autoimmune reaction in genetically susceptible subjects. Experiments to challenge this hypothesis are currently in progress in our laboratories.