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Keywords:

  • inflammatory bowel disease;
  • irritable bowel syndrome;
  • myosin light chain kinase;
  • paracellular permeability;
  • protease-activated receptor 2

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References

Abstract  Digestive tract proteases are best known for their proteolytic activity in the digestion of alimentary proteins. However, during the last decade, a possible role of proteases as signalling molecules has been emphasized with the discovery of a novel class of G-protein coupled receptors located on cell membranes that may be activated by proteolytic cleavage of their N-terminal extracellular domain. Type 2 protease-activated receptors (PAR-2) are cleaved by serine-proteases such as trypsin and tryptase. PAR-2 is present in many intestinal cell types and particularly on epithelial cells. Multiple functions have been demonstrated in the gut for PAR-2, including epithelial permeability, mainly the intercellular permeability that is of paramount importance in the equilibrium between the external milieu (digestive contents) and the submucosal immune system. Alterations of both tissue and luminal levels of proteases or serine-protease activity may affect gut permeability and subsequently the immune status of the mucosa. Activation of PAR-2 on epithelial cells may directly affect cytoskeleton contraction by triggering phosphorylation of myosin light chain with subsequent changes in tight junction permeability. Enhanced fecal protease level has been recently reported in both organic (ulcerative colitis) and functional (irritable bowel syndrome) intestinal disorders and may play a role in the pathogenesis of such diseases.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References

The intestinal epithelium represents the largest interface between the external environment and the internal host milieu and constitutes the major barrier through which molecules can either be absorbed or secreted. There is now substantial evidence that tight junctions (TJ) play a major role in regulating epithelial permeability by influencing paracellular flow of fluid and solutes. Tight junctions are one of the hallmarks of absorptive and secretory epithelia. Evidence now exists that TJ are dynamic rather than static structures and readily adapt to a variety of developmental, physiological and pathological circumstances. In the last 5 years, the role of this ‘porosity’ in the pathogenesis of acute viral and infectious gastroenteritis, inflammatory bowel disease (IBD) and many other gastrointestinal (GI) disease including food allergy and celiac disease has been confirmed through abundant literature. This intercellular passage comprises several different filtering structures, the TJ being the first most selective one localized at the apical level (Fig. 1A). This structure comprises junction proteins such as claudins and occludins linking together the adjacent epithelial cell membranes. These proteins are associated with internal cellular elements such as the apical ring of actinomyosin filaments through intermediary proteins like ZO1, ZO2 and ZO3 (Fig. 1B). The contraction of the cytoskeleton is driven through the phosphorylation of myosin light chain (MLC) catalyzed by the MLCKinase (MLCK) (Fig. 1C). Zonula occludens toxin (ZOT) from Vibrio cholerae was found to bind on a membrane receptor activating PKC to open TJ and zonulin was the first peptide secreted by the mucosa found to activate the ZOT receptor and favouring the activation of MLCK to open the TJ.1 More recently, attention has been paid to other membrane receptors susceptible to activate MLCK. Epithelial cells express receptors for proteases such as PAR-2 and their activation from both apical and basolateral sides increases paracellular permeability through activation of MLCK.

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Figure 1.  Structures and factors controlling paracellular permeability. Tight junction structure is the upper structure filtering the passage of ions and macromolecules (A). The intercellular structural proteins (claudins and occludins) are anchored to the cytoskeleton actinomyosin apical ring through intermediary proteins (ZO1, ZO1 ZO3, AF6). The phosphorylation of myosin light chain (contraction) is associated with opening of tight junction and junction protein deformations that favour macromolecule passage and bacterial translocation (B). The phosphorylation/dephosphorylation of myosin light chain is controlled by myosin light chain kinase (MLCK) and myosin phosphatase (MP). PAR-2 activation increases MLCK activity through external and internal Ca++ mobilization and calmodulin binding (C).

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Functional gastrointestinal disorders (FGID), and particularly irritable bowel syndrome (IBS), are associated with a micro-inflammation of the mucosa and increased TJ permeability but the link between them is still under debate. However, several pathways may be experimentally highlighted to explain the cause–effect relationships between inflammation, permeability and symptoms in which PAR-2 activation may have a crucial role (Fig. 2).

image

Figure 2.  Consequences of PAR-2 induced alteration of permeability on local immune system and visceral pain. PAR-2 induced phosphorylation of MLC is a short lasting phenomenom (15–120 min) but restoration of permeability linked to re-synthesis-migration of new junction proteins may last more than 12 h during which there is an activation of the resident immune system and attraction of new immunocytes. Mediators released during this micro-inflammation by both T cells (IFN-γ) and mast cells (tryptase) may prolong MLCK activation. The micro-inflammation sensitizes nerve terminals that may in turn favour a neurogenic inflammation and maintain a long-term hypersensitivity to mechanical stimuli.

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Importance of gut permeability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References

Both transcellular and paracellular permeability are crucial events within the gut not only for nutritional aspects but also to maintain the immune balance between the internal and external milieu. While the transcellular pathway often requires an active transport and is therefore elective, the paracellular pathway acts as a filter and is the major way of non-specific macromolecule transport. Many human digestive pathologies are associated with alterations of gut paracellular permeability including celiac disease,2,3 Crohn’s disease,4 ulcerative colitis (UC)5 and IBS.6 In all these digestive pathologies, increased permeability is found associated with activation of the mucosal immune system. Abnormal gut paracellular permeability is considered as a primary event in the genesis of diseases such as necrotizing enterocolitis7 and to some extent celiac disease.8 In other diseases such as Crohn’s disease, there is no direct proof of a pivotal role; however, increased permeability was often present in familial relatives not developing the disease and was found associated with NOD2 variants in both familial and sporadic Crohn’s disease9 suggesting that altered permeability is both a genetic and a non-genetic factor favouring the occurrence of the disease. Furthermore, it is presently considered that altered permeability may greatly contribute to the occurrence of relapses.10 Increased permeability precedes symptoms with a correlation between clinical disease activity index and intestinal/colonic TJ leakage.11

Recently, the role of altered permeability in the genesis of IBS symptoms has been raised.12 Indeed, during the last 4–5 years, altered intestinal and/or colonic permeability has been identified in all IBS patients according to ROME I criteria6 or in subsets of either diarrhoea-predominant (IBS-D) or postinfectious IBS patients.13,14 The relationships with symptoms have not been established in humans, likely because different markers such as sugars or Cr-EDTA have been used to measure intestinal permeability.11 However, animal studies have demonstrated that factors which enhance colonic permeability such as stress, lipopolysaccharides, bile salts or the activation of PAR receptors, initiate a long-term hypersensitivity of the gut to distension15 and conversely, pharmacological blockade of increased permeability prevents hypersensitivity to colorectal distension.16 Animal studies also confirm that increase of TJ permeability is associated with mucosal micro-inflammation17 and bacterial translocation.18

These data provide a rationale to investigate the possible endogenous and luminal factors that may be responsible for the increased TJ permeability.

Factors controlling gut permeability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References

Gut paracellular permeability mainly depends upon the configuration of TJ proteins such as occludins and claudins. Among them, claudins 2 and 4 are the most important filtering molecules with a high degree of selective filtering not only for ions but also for small non-ionic molecules.19 Tight junction proteins are anchored to the apical actinomyosin ring of the cytoskeleton through specific proteins such as ZO1, ZO2 and ZO3. The contraction of epithelial cell cytoskeleton is linked to either an activation of MLC kinase that phosphorylates MYC or an inhibition of myosin phosphatase that dephosphorylates MLC which in both cases result in an increased permeability.20 The reversible contraction of epithelial cell cytoskeleton may be considered as the most common mechanism involved in TJ opening. In most cases, MLC phosphorylation does not last more than 2 h but this short duration is enough to trigger long-term (24 h) alterations of the TJ protein configuration and associated increased permeability. The TJ permeability is influenced by both endogenous and luminal factors. Indeed, both acute and chronic stress increase TJ permeability, an effect associated with bacterial translocation.18,21 Mast cells mediators released under stressful stimuli, such as rat mast cell protease II (RMCPII) are responsible for enhanced gut macromolecular passage.22 The stress-induced permeability changes involve local activation of corticotrophin-releasing factor (CRF) receptors.23 Neonatally stressed rats also present long-term alterations of colonic permeability under the dual control of nerve growth factor and CRF.24,25 In mice, the stress-induced increase in paracellular permeability depends upon the presence of activated T cells in the lamina propria and the subsequent release on interferon (IFN)-γ responsible for the final MLCK-dependent cytoskeleton contraction.18 Many toxins from pathogenic bacteria such as EPEC, shigella, or salmonella have been shown to alter intestinal paracellular permeability through MLCK activation but the role of commensal flora or the nature of luminal factors responsible for such control remains unclear and unexplored. However, based on the use of oral antibiotics, commensal flora has also been shown to play an important role in the control of basal and stress-induced increased permeability.26

Protease-activated receptors in the gut

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References

Protease-activated receptors belong to a family of seven transmembrane domain G-protein-coupled receptors that are activated by cleavage of their N-terminal domain by a proteolytic enzyme.27 The unmasked new N-terminal sequence acts as a tethered ligand that binds and activates the receptor. Four types of PAR have been described: PAR-1, PAR-3 and PAR-4 are cleaved by thrombin, PAR-2 and PAR-4 are cleaved by trypsin and tryptase, while PAR-4 is also cleaved by cathepsin G.28 PARs are expressed throughout the GI tract on several cell types, such as enterocytes, mast cells, smooth muscle cells, myenteric neurons and endothelial cells. Immunohistochemical studies indicate that PAR-2 are localized on both baso-lateral and apical sites of colonic epithelial cells.29 Activation of PAR-2 from luminal site triggers intestinal water secretion through a PGE2-dependent mechanism in rats27 or by a direct cellular mechanism in humans.30 At the gastric level, PAR-2 activation plays a protective role by stimulating mucus secretion through a mechanism involving capsaicin-sensitive neurons,31 increases gastric mucosal blood flow,32 and prevents gastric acid secretion evoked by carbachol or pentagastrin.33 Activation of both PAR-1 and PAR-2 has been found able to modulate GI motility in different ways. For example, activation of PAR-1 and PAR-2 contracts rat or mouse gastric smooth muscle while, it reduces contractility of both circular and longitudinal colonic smooth muscle.34 It accelerates GI transit, at least in mice.35In vivo, intracolonic activation of PAR-2 leads to delayed colonic inflammation in mice suggesting an indirect inflammatory mechanism36 involving both capsaicin sensory neurons, tachykinins and nitric oxide.37 Bacterial translocation to peritoneal organs is also one of the features observed after colonic PAR-2 activation from luminal site suggesting an altered permeability that may trigger at least in part the long-term colonic inflammatory reactions.17

PAR-2 activation-induced altered permeability and its consequences

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References

In the healthy gut in vivo, PAR-2 are present on both apical and basolateral membrane of the enterocytes. Many experiments have been performed in vitro, and it is not clearly established whether in vivo one way of activation (apical vs basolateral) predominates over the other. Moreover, the expression of PAR-2 on cultured tumoural cell lines may be different from in vivo.38

Supernatant from degranulated mast cells increased intracellular Ca2+ in colonocytes, an increase which was blocked by a tryptase inhibitor or prevented by a previous activation of PAR-2 by a PAR-2 agonist. When applied to the basolateral surface of colonocytes, PAR-2 agonists and mast cell supernatant decreased transepithelial resistance, increased transepithelial flux of macromolecules, and induced redistribution of TJ ZO-1, occludins and perijunctional F-actin. When mast cells were co-cultured with colonocytes, mast cell degranulation increases paracellular permeability of colonocytes, an effect prevented by a tryptase inhibitor.38 Extracellular signal-regulated kinases (ERK1/2 and beta-arrestins) which recruit ERK1/2 to PAR-2 in endosomes and retain ERK1/2 in the cytosol, have been implicated in PAR-2-mediated alterations of permeability. For instance, an ERK1/2 inhibitor abolishes the effects of PAR-2 agonist on permeability and redistribution of F-actin. In addition, downregulation of beta-arrestins with small interfering RNA inhibits PAR-2-induced activation of ERK1/2 and suppressed PAR-2-induced changes in permeability.39 Thus, mast cells signal to colonocytes in a paracrine manner by release of tryptase and activation of PAR-2. PAR-2 couples to beta-arrestin-dependent activation of ERK1/2, which regulates the reorganization of perijunctional F-actin to increase epithelial permeability. These mechanisms may explain the increased epithelial permeability of the intestine during stress and inflammation. In vivo the apical expression of PAR-2 has been shown to fluctuate during a chronic oral treatment (12 days) with a mixture of non-absorbable antibiotics.26

In 2002, Coelho et al.40 showed for the first time in rats that, intracolonic infusion of the PAR-2 activating peptide SLIGRL triggers a dose-related increase in colonic paracellular permeability and that this effect cannot be reproduced by similar doses injected intraperitoneally. In mice, a dose of 5 μg of SLIGRL infused intracolonically increases colonic permeability while a 10 times higher dose is required to induce inflammatory reaction of the mucosa. Moreover, while afferent nerve integrity and IFN-γ are required to initiate mucosal MPO increases, only ML-7, a MLCK selective inhibitor can block the response to low doses of SLIGRL.17

This in vivo increase was also found for higher doses, which were associated with mucosal inflammatory reaction, and bacterial translocation into peritoneal organs.24 The local site of action on the apical side was confirmed in colonic strips in Ussing chambers using either trypsin or the PAR-2-activating peptide SLIGRL. However, a basolateral site of action remains questionable as the mast cell degranulator C48/80 also releases proteases able to increase permeability.26 Two levels have to be considered in the action of PAR-2 from luminal site: at low doses, SLIGRL only affects permeability without subsequent colonic inflammation.17 This effect corresponds to a direct Ca2+/calmodulin dependent activation of MLCK controlling the cytoskeleton-mediated contraction of mouse epithelial cells (Fig. 1C). Interestingly, this effect is not prevented by corticoid treatment.41 At high doses, SLIGRL induces increased permeability and a local inflammatory reaction that involves mast cells, sensory nerves and nitric oxide (Fig. 2). It was also demonstrated using SCID mice and IFN-γ deficient mice that the inflammatory reaction depends upon the presence of T-lymphocytes and IFN-γ. Moreover, the pro-inflammatory action of PAR-2 activation depends upon the pathophysiological state of the mucosa. It has been confirmed in rats that SLIGRL infused into the duodenum causes mucosal inflammation, while it dramatically reduces the inflammatory damage induced by ischaemia/reperfusion.42 Among the PAR family, the activation of PAR-1 by the selective activating peptide TFLLR also increases paracellular permeability in mice.43 This effect is also mediated through an activation of MLCK, but is associated with apoptosis of epithelial cells, an effect never described for PAR-2 activation.

Luminal factors responsible for PAR-2-dependent regulation of permeability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References

Proteases are present in great amount in the GI tract. In addition to their digestive role in protein degradation, they play a role as signalling molecules regulating cell functions by cleaving PARs. Protease-activated receptors are activated by a variety of proteases, such as digestive enzymes (trypsin and trypsinogen), proteases released from mast cells and neutrophils, and by proteases of the coagulation cascade. Bacteria may degrade these enzymes but are also able to release serine-proteases into the lumen. The impact of the microflora on the colonic luminal level of proteases was evidenced by an antibiotic treatment in rats. Indeed, a reduction in luminal serine-protease activity was seen after 2 weeks of oral treatment with a mixture of ampicillin and neomycin associated with a reduction in basal colonic permeability and a downregulation of PAR-2.26 Such decrease in permeability is also observed after intracolonic infusion of a cocktail of serine-protease inhibitors.26

Luminal proteases, PAR-2 and IBS

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References

All these data lead us to hypothesize that bacterial proteases, or even luminal proteases of endogenous origin, may act on PARs present on apical site of colonocytes. Activation of PAR-2 from luminal site affects colonic paracellular permeability by modulating the degree of cytoskeleton contraction. In mice, the colonic inflammation induced by intraluminal infusion of PAR-2 agonist depends upon sensory neuron activation, substance P and calcitonin gene-related peptide release.36,37 In animals, oral antibiotic treatment is associated with a downregulation of PAR-2 receptors expressed by epithelial cells associated with a reduced basal permeability and response to luminal activating factors.26 Over-expression of PAR-2 was observed in biopsies from IBD patients, suggesting a pathophysiological role of PAR-2 in the development of colonic inflammation.44 A recent study has highlighted the high level of serine-proteases found in supernatant from feces of both IBS and IBD patients. A subgroup analysis revealed that this increase was selective of diarrhoea-predominant IBS patients and not observed in constipated and alternating IBS patients.45 This increase which is similar to that observed in patients with UC, is not observed in feces from patients suffering from infectious diarrhoea. Contrasting with fecal supernatants from UC patients, those of IBS-D patients are not associated with any significant elevation of inflammatory markers such as MPO and calprotectin. The origin of this increase of fecal serine-protease activity has not been clearly established. Pancreatic elastase-1 concentration has been found similar in feces from IBS-D and controls. Likewise, mast cell tryptase activity and secretory leucocyte protease inhibitor concentration were normal. Consequently, the source of fecal serine-protease activity found increased in IBS-D does not seem to be from pancreas, mast cells or immune cells. The hypothesis of a bacterial origin for such increased protease activity is indeed possible, as it is well established that colonic commensal bacteria release considerable amounts of proteases that are partly degraded by host proteases and bacterial peptide hydrolases.46

The pathophysiological significance of the increased colonic serine protease activity found in IBS-D patients has been investigated in mice both in vitro and in vivo. Interestingly, diluted fecal supernatants from IBS-D patients trigger an increase in paracellular permeability when applied to mucosal side of mice colonic mucosa in Ussing chambers.47 This increase is suppressed after previous incubation with a cocktail of serine-protease inhibitors and is not observed in PAR-2 deficient mice. These supernatants infused intracolonically in mice also produce both a delayed colonic hypersensitivity to distension and change in the expression of colonic TJ proteins with activation of MLC phosphorylation.48 Even though until now, no correlation has been established between fecal level of serine-protease activity and the intensity/frequency of symptoms, it is tempting to speculate, based on animal data, that such activity contributes to the genesis of symptoms and particularly abdominal pain in these patients. Interestingly, a decreased colonic potential difference, corresponding to an increase in permeability, has been found in IBS-D49 and UC50 patients compared with healthy subjects. We can speculate that this decreased potential difference is linked to the elevated protease activity found in these patients through an activation of PAR-2.

Cenac et al.51 have recently shown that the colonic mucosa of IBS patients contains high levels of proteases with a high expression of tryptase able to activate PAR-2. When infused intraluminally to mice, supernatants from colonic biopsies of IBS patients trigger an exaggerated abdominal response to colorectal distension. Stress has been shown to increase colonic permeability through activation of mast cell and release of tryptase.17,22 However, stress-induced increase in permeability is associated with a high luminal concentration of trypsin-like activity resulting from stress-induced stimulation of exocrine pancreatic secretion and particularly trypsin a selective activator of PAR-2 that may reach the colonic lumen.52 All these data suggest a cascade where activation of PAR-2 on apical membrane by luminal stimuli triggers a mucosal immune activation, involving mast cells and tryptase, which maintains a high degree of gut ‘porosity’.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References

The role of gut permeability and particularly paracellular permeability in the pathogenesis of organic and functional GI disorders has recently been emphasized. Increased intestinal and colonic permeability are associated with the passage of luminal antigens, toxins and bacteria that activate the mucosal immune system with immunocytes recruitment and release of inflammatory mediators. Activation of PAR-2 receptors from both luminal and internal sites plays a crucial role in altered gut permeability and several pathways have been identified in such activation (Fig. 2). The fact that luminal proteases, found at elevated level in colonic contents of from IBS and UC patients, are able to activate PAR-2 to promote increased permeability suggests that PAR-2 are relevant new targets in the therapeutic approach of digestive diseases.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Importance of gut permeability
  5. Factors controlling gut permeability
  6. Protease-activated receptors in the gut
  7. PAR-2 activation-induced altered permeability and its consequences
  8. Luminal factors responsible for PAR-2-dependent regulation of permeability
  9. Luminal proteases, PAR-2 and IBS
  10. Conclusions
  11. Acknowledgments
  12. Competing interest
  13. References