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Keywords:

  • corticotropin-releasing hormone;
  • enteric nerves;
  • mast cell;
  • permeability;
  • stress

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

Background  The ability to control uptake across the mucosa and protect from damage of harmful substances from the lumen is defined as intestinal barrier function. A disturbed barrier dysfunction has been described in many human diseases and animal models, for example, inflammatory bowel disease, irritable bowel syndrome, and intestinal hypersensitivity. In most diseases and models, alterations are seen both of the paracellular pathway, via the tight junctions, and of the transcellular routes, via different types of endocytosis. Recent studies of pathogenic mechanisms have demonstrated the important role of neuroimmune interaction with the epithelial cells in the regulation of barrier function. Neural impulses from extrinsic vagal and/or sympathetic efferent fibers or intrinsic enteric nerves influence mucosal barrier function via direct effects on epithelial cells or via interaction with immune cells. For example, by nerve-mediated activation by corticotropin-releasing hormone or cholinergic pathways, mucosal mast cells release a range of mediators with effects on transcellular, and/or paracellular permeability (for example, tryptase, TNF-α, nerve growth factor, and interleukins).

Purpose  In this review, we discuss current physiological and pathophysiological aspects of the intestinal barrier and, in particular, its regulation by neuroimmune factors.


Abbreviations:
ACh

acetylcholine

CD

Crohn‘s disease

COX

cyclooxygenase

CRH

corticotropin-releasing hormone

CRH-R

CRH receptor

GSNO

glial-derived s-nitrosoglutathione

HRP

horseradish peroxidase

IBD

inflammatory bowel disease

IBS

irritable bowel syndrome

JAM

junctional adhesion molecule

M cell

membranous cell

MLCK

myosin light chain kinase

NGF

nerve growth factor

PAR

protease-activated receptor

PG

prostaglandin

PI3K

phosphoinositide 3-kinase

RMCPII

rat mast cell protease II

SP

substance P

TJ

tight junction

TER

transepithelial resistance

UC

ulcerative colitis

VIP

vasoactive intestinal peptide

ZO

zonula occludens

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

The intestinal mucosa is continuously exposed to a heavy load of antigenic molecules from ingested food and microorganisms, such as resident and invading bacteria and viruses. The ability to control uptake across the mucosa and protect from damage of harmful substances from the lumen is defined as intestinal barrier function.

The normal intestinal barrier allows small amounts of antigens to pass the mucosa to interact with the innate and adaptive immune systems. If the control of the barrier function is disturbed, it can lead to enhanced antigen and bacterial passage which in turn may damage the mucosa leading to pathological conditions. A normal host can down-regulate this damage when the triggering event has resolved, whereas in a susceptible host, ongoing enhanced permeability may result in chronic inflammation. Loss of mucosal integrity is believed to play a role in the development of several clinical disorders. The diseases that have been most commonly associated with intestinal barrier dysfunction include inflammatory bowel disease (IBD), celiac disease, intestinal ischemia, food intolerance, allergy and malnutrition, rheumatoid arthritis, and more recently, diabetes type 1.1–4 Recent studies of pathogenic mechanisms have demonstrated the important role of neuroimmune interaction with the epithelial cells in the regulation of barrier function. Here, we review current physiological and pathophysiological aspects of neuroimmune regulation of intestinal barrier function.

Intestinal Barrier Function

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

The intestinal barrier, i.e. the interface between the outside world and the human internal milieu is maintained by the physical defence mechanism associated with the mucosal surface and the junctional complexes linking adjacent epithelial cells (Fig. 1). The relative importance of the barrier components varies with the chemical, physical, and immunological nature of the luminal contents. The first line of defence is the lumen itself, where bacteria and antigens are degraded by gastric acid, pancreatic, and biliary juices. Commensal bacteria inhibit the colonization of pathogens by production of antimicrobial substances (bacteriocins), pH modification of the luminal content, and competition for nutrients required for growth of pathogens. The microclimate including the unstirred water layer, the glycocalyx, and the mucus layer with secreted IgA, prevents adhesion of pathogenic bacteria to the epithelium by mucin-binding sites that compete with the epithelial binding sites, thus impeding bacterial–epithelial interaction. The epithelium, with epithelial cells connected to each other via junctional complexes, reacts to noxious stimuli with chloride secretion. The epithelial cells also secrete antimicrobial peptides, whose function is to kill microorganisms, attract monocytes, and potentiate macrophage opsonization. An important family of antimicrobial peptides is the defensins, which can be found in the Paneth cells of the crypts of the small bowel (α-defensins) and throughout the colonic epithelium (β-defensins). Finally, the lamina propria consists of cells of innate and acquired immunity secreting immunoglobulins and cytokines, the enteric nervous system and endocrine system, the myofibroblasts, the matrix components, and so on. In addition, the rapid repair process and cell turnover of the epithelial cells, and intestinal propulsive motility also have to be recognized as being important factors in gut barrier function. Electron photomicrographs with examples of normal and altered intestinal epithelial barrier are shown in Fig. 2.

image

Figure 1.  Components of the intestinal barrier. Lumen: Degradation of bacteria and antigens by bile, gastric acid, and pancreatic juice. Commensal bacteria inhibit the colonization of pathogens by production of antimicrobial substances. Microclimate: Unstirred water layer, glycocalyx, and mucus layer preventing bacterial adhesion by IgA secretion. Epithelium: Epithelial cells, connected by junctional complexes, having the ability to transport luminal content but also react to noxious stimuli by secretion of chloride and antimicrobial peptides. Lamina propria: Cells of innate and acquired immunity secreting immunoglobulins and cytokines. The endocrine- and enteric nervous system, intestinal propulsive motility (see text for more details).

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image

Figure 2.  Examples of altered intestinal epithelial barrier (A) A normal intestinal epithelium from a control rat. (B) Altered intestinal barrier in rats exposed to chronic stress. Note the abnormal morphology with multiple vacuoles suggesting increased endocytotic activity (left panel). Numerous bacteria attaching to the epithelial surface, and also internalizing (arrow heads), with actin accumulation at the contact sites (right panel).

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Passage Routes Across the Epithelium

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

There are several ways for solutes to cross the intestinal epithelium (Fig. 3). Lipid soluble and small hydrophilic compounds may pass through the epithelial cells via passive diffusion into the lipid bilayers or via small hydrophilic aqueous pores, while medium-sized (up to approximately 600 Da in vivo; up to 10 kDa in vitro in cell lines) hydrophilic molecules pass via the tight junctions (TJs) and intercellular spaces in the paracellular route.5,6 Under normal conditions, the paracellular route is believed to be impermeable to protein-sized molecules and thus constitutes an effective barrier to antigenic macromolecules.

image

Figure 3.  Passage routes across the epithelium. (A) Transcellular route (lipophilic and small hydrophilic compounds). (B) Paracellular route (larger hydrophilic compounds). (C) Transcellular route via aqueous pores (small hydrophilic compounds). (D) Active carrier-mediated absorption (nutrients). (E) Endocytosis, followed by transcytosis and exocytosis (larger peptides, proteins, and particles).

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In addition to these ‘passive,’ non-mediated permeation routes, active, energy-dependent uptake across the epithelium also takes place. Firstly, there are numerous carrier-mediated mechanisms, utilized for sugars, amino acids, vitamins, and other nutrients, and secondly, larger peptides, proteins, and particles may be endocytosed into endosomal vesicles and transported through the cells via transcytosis. A controlled protein uptake via the transcytotic route is physiologic and essential for antigen surveillance in the gastrointestinal tract.7–9 Alterations of the paracellular pathway and the transcytotic uptake of peptides are believed to be the most important in the pathophysiology of intestinal disorders.

Endo- and Transcytotic Pathways

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

Large particles and molecules, like proteins and bacterial products that cannot pass through the cell membrane or the paracellular space, can be taken up by the cell through invagination of the plasma membrane followed by vesicle formation, i.e. endocytosis. This is an essential process that mediates uptake of foreign antigens against which the body can initiate an appropriate immune response. Following endocytosis the engulfed substances are actively transported by vectorial transcytosis through the cytoplasm to their particular destination. Food proteins that are absorbed are generally transported and degraded via the lysosomal pathway of the enterocytes; those that escape lysosomal degradation appear to be a small fraction of the food antigens, but these enter the body proper and potentially induce immune responses.10 Both endocytosis and transcytosis are constantly manipulated by foreign microbes to establish an entry into the host. An intact barrier function to a large extent relies on that these processes function correctly and that the cell can eliminate the foreign substances taken up.

Endocytosis in epithelial cells can occur in different ways, depending on the nature of the substance that is taken up (Fig. 4). The first route is via clathrin-mediated endocytosis,11–13 a highly specific receptor-mediated process, utilized mainly by immunoglobulins, viruses, and growth factors from breast milk. In this special type of endocytosis, the epithelial cells synthesize apical membrane receptors and internalize molecules that have bound specifically to them.14 Clathrin-mediated endocytosis involves the formation of clathrin-coated vesicles that seldom become larger than 150 nm in diameter.15 It begins with the recruitment and assembly of clathrin as well as adaptor and endocytotic accessory proteins at the plasma membrane.16 The membrane curves into coated pits, which are sequentially severed from the plasma membrane as vesicles. Adaptor proteins are crucial for the assembly of clathrin-coated pits at the plasma membrane as well as for recognition of specific cytosolic motifs of the protein being internalized. Internalization of different plasma membrane proteins such as receptors and their ligands has been shown to occur through clathrin-mediated endocytosis. Non-signalling receptors that mediate the uptake of nutrients, like low-density lipoprotein receptors and transferrin receptors, are internalized either bound or not bound to their ligand via so called constitutive endocytosis.17,18 This suggests that clathrin-mediated endocytosis also functions in a more regulatory way to adjust the actual number of receptors present on the surface of the cell in response to environmental signals.

image

Figure 4.  Different types of endocytosis. Clathrin-mediated endocytosis: Formation of vesicles coated with clathrin, mediating uptake of receptor (R)-bound molecules. Phagocytosis: Binding of larger bacteria, viruses, and particles via Rs. Macropinocytosis: Invagination of the cell membrane mediating uptake of dissolved molecules. Caveolae-mediated endocytosis: Invagination of plasma membrane at lipid rafts coated with caveolin mediating uptake of certain enterotoxins and viruses.

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Larger (up to several micrometer in size) bacteria, viruses, and particles may be taken up via adsorptive endocytosis, or phagocytosis,19 involving binding of molecules to the cell membrane via receptors. Phagocytosis is relevant for the non-specific uptake of luminal dietary and bacterial antigens, and the process is triggered by secreted solubles from the invading bacterium.20 Phagocytosis is a more common process in the membranous (M) cell-containing follicle-associated epithelium than in the regular villus epithelium.21,22

Macropinocytosis is a non-specific process by which considerable volumes of extracellular fluid can be internalized, along with dissolved molecules as well as larger particles such as viruses, bacteria, and apoptotic cell fragments. However, macropinosomes do not usually exceed the diameter of 1 μm. The process is initiated through invagination of the cell membrane where bending of single surface lamellipodia gives rise to circular ruffles that ultimately are released in the cytoplasm as a vesicle (macropinosome).23 Several putative regulators of macropinocytosis in epithelial cells have been identified. For example, regulation via epidermal growth factor,24 dynein light chain 1 phosphorylation, Rab5,25 and phosphoinositide 3-kinase (PI3K)26 were described. Moreover, increased macropinocytosis has been observed in response to antigen stimulation in M cells and entrocytes.19,27

In recent years, attention has been paid to a fourth mechanism, referred to as lipid rafts/caveolae. This endocytotic event involves a flask-shaped invagination of cholesterol-enriched microdomains within the plasma membrane that may contain a coat protein, caveolin.28 Endocytosis via lipid rafts/caveolae is most common in endothelial cells but occurs also in enterocytes.29 Studies have shown that, for example, certain enterotoxins and viruses may be endocytosed via rafts/caveolae.

Paracellular Pathways

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

The junctional complex

The enterocytes of the epithelium are joined to each other by junctional complexes consisting of TJs, adherens junctions, desmosomes, and gap junctions.30 Tight junctions, also called zonula occludens (ZO) are located at the apical part of the lateral membrane forming a network of linking strands. Tight junctions are important in epithelial transport towards and away from the lumen (gate function), as well as in maintaining the polarity of the epithelial cells by preventing diffusion of proteins and lipids from the apical to the basolateral region in the outer cell membrane (fence function). These functions seem to be regulated in different ways.31

Adherens junctions, located below the TJs, are actin- and myosin-associated membrane structures formed by adhesion molecules of the cadherin family and their cytoplasmatic binding proteins α-, β-, and γ-catenin. It has been suggested that adherens junctions together with TJs form one single functional unit.32

Desmosomes form spot-like dense adhesions between the epithelial cells and are connected to the intermediate filaments of the cytoskeleton. Desmosomes are dispersed all over the lateral cell surfaces, however, they are frequently found to be concentrated below the adherens junctions.

Gap junctions are arrangements of cylindrical tubuli consisting of proteins called connexins. Gap junctions function as intercellular channels allowing ions and small molecules to pass between cells, thus linking the interior of adjacent cells.

TJ permeability

Tight junctions appear as focal contacts (‘kisses’) on the plasma membrane. These contacts correspond to continuous fibrils, where fibrils on one cell interact with fibrils on the adjacent cell. The fibrils consist of multiple transmembrane proteins including occludin,33 claudins,34 and members of the junctional adhesion molecule (JAM) protein family.35 The human claudin family includes at least 24 members36 and the distribution of these varies in different tissues, which explains the variable permeability seen in diverse tissues.31 A recent addition to the TJ proteins is tricellulin that is mainly located at contact points of three cells, but is expressed to a lower extent also at contacts between two cells.37 In cultured epithelial cells, tricellulin forms a central tube in tricellular junctions that allows passage of large solutes (up to 10 kDa), and the amount of expression of tricellulin regulates macromolecular permeability.6 There is a size and charge-selectivity within the TJ permeability barrier, where positively charged molecules and ions pass more easily. From recent data it seems that the permeability characteristics of small pores is defined by claudins, whereas the large pores are determined by tricellulin.38 Of the JAM members, JAM-A seems to be the one involved in regulating barrier function and inflammatory response.39 JAM-A is expressed in several cell types but is mainly abundant in epithelial and endothelial cells, where it accumulates at TJs to influence several cellular processes like regulating permeability, inflammation, and proliferation. Finally, the TJ proteins are connected to protein complexes called cytoplasmic plaques. These constitute of not only ZO-1, ZO-2, ZO-3, and ZAK, but also several peripheral proteins like cingulin, symplekin, 7H6, and p130.40

The interaction of TJs with the actin cytoskeleton has been recognized as essential and appears to be critical for TJ barrier function and regulation of paracellular permeability.41 The regulation involves different functional pathways. Rapid permeability alterations occur by cytoskeletal contraction induced by phosphorylation of myosin light chain kinase (MLCK)42 and endocytosis of TJ proteins,43,44 whereas more long-term changes include reduced transcription of TJ proteins,45 epithelial cell apoptosis,46,47 and structural epithelial ulceration.48 Regulation of paracellular permeability via endocytosis of TJ proteins may occur by at least two different mechanisms. Actin-depolymerizing drugs caused disruption of the TJs by removing occludin via caveolae-mediated endocytosis.49 Intrestingly, Ivanov et al. reported conflicting data suggesting that occludin is endocytosed by clathrin-mediated endocytosis,50 thus emphasizing the difficulty in elucidating the different endocytotic pathways.

Moreover, a series of studies by Fasano et al.51 have suggested a physiologic paracrine regulation of TJ permeability via zonulin. Recently, zonulin was identified by proteomics as prehaptoglobulin-2, the precursor of the multifunctional protein haptoglobin-2.52 Zonulin seems to exert its effects via activation of the epidermal growth factor receptor and protease-activated receptor (PAR)-2. Moreover, zonulin is overexpressed in the intestinal mucosa of patients with celiac disease, which may suggest that zonulin is involved not only in intestinal permeability, but also in intestinal mucosal immunology.

Barrier Dysfunction in Human Disease and Animal Models

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

A disturbed barrier dysfunction has been described in many human diseases and animal models, and in most cases both the paracellular and the transcellular pathways are altered. A few examples of interest from a neuroimmune perspective are given here.

Intestinal hypersensitivity

Whether abnormalities in intestinal permeability are a consequence or cause of food intolerance is yet not fully clarified.53 However, it is well known that increased intestinal permeability is a characteristic of children suffering from food hypersensitivity.54,55 The barrier dysfunction involves both trans- and paracellular pathways by mechanisms involving TNF-α.56 A series of experiments in animal models of food hypersensitivity from the McMaster group demonstrated a combined barrier dysfunction with an initial antigen-specific transcellular transport followed by a mast cell-dependent increase in paracellular permeability. In the initial studies by Berin et al.57,58 rats sensitized to horseradish peroxidase (HRP) showed enhanced HRP-uptake within the endosomal compartment of jejunal enterocytes already 2 min after antigen challenge, i.e. before mast cell activation. Later HRP was also visualized within the TJs and paracellular spaces between enterocytes. Studies with mast cell-deficient Ws/Ws rats confirmed the important role for mast cells in the second, paracellular, phase of transepithelial transport, whereas the initial transcytotic phase was unaffected by the absence of mast cells. Another study showed that IgE antibodies mediated the specific and rapid antigen uptake via interaction with the IgE receptor CD23 on epithelial cells.59 Yu et al.60 further demonstrated that IL-4 is required for the IgE/CD23-mediated enhanced HRP uptake in enterocytes of sensitized mice. An additional example of Ig-mediated transcytosis early in the pathogenesis of intestinal inflammation is the IgA-mediated transcytosis of increased amounts of gliadin through upregulation of CD71 on epithelial cell apical surfaces occurring in celiac disease.61

IBD

A disturbed intestinal barrier function is as long regarded as an important factor in the pathogenesis of Crohn’s disease (CD).62,63 This has recently been further confirmed by the development of genetic studies,64 where several of the polymorphisms that predispose to CD are related to barrier function. The barrier disturbance has proved to be a combined dysfunction of the paracellular and transcellular pathway. Early studies demonstrated increased small bowel permeability to medium-sized probes.62,65,66 Further, structural changes67 and leakage of the TJs in response to luminal stimuli68 were demonstrated in CD mucosa. Therefore, CD was suggested as a TJ disorder.69 In line with this hypothesis, Zeissig et al.70,71 showed a reduction in TJ strand number and manifestations of strand discontinuities in CD. Furthermore, western blot analysis showed decreased expression of occludin, claudin-5, and -8 in CD compared with controls, while claudin-2 was upregulated.71

Transcytosis is the most important route for protein uptake in the intestinal epithelium, and potentially contributes to inflammation and gastrointestinal disease.7,72–74 Increased transcellular uptake of protein antigens were found in microscopically normal ileum of CD,75,76 and this seemed to be mediated by mechanisms involving TNF-α.77 Mucosal mast cells are an important source of TNF-α in CD,78,79 which suggests a role of mast cell mediators in IBD (see further next section).

It has been shown that dendritic cells may affect barrier function by extending their processes between epithelial cells into the lumen, thereby facilitating antigen uptake.80,81 In addition, dendritic cells may play a role in IBD. Indomethacin-induced ileitis in rats showed an association between increased intestinal permeability and ileal dendritic cell redistribution during inflammation.82 Moreover, a disturbed barrier to commensal bacteria in the follicle-associated epithelium of CD was demonstrated,83 leading to more pro-inflammatory subepithelial dendritic cells84 that may play a role in sustaining inflammation in CD mucosa. There is evidence of a concomitant dysfunction of trans- and paracellular pathways also in ulcerative colitis (UC).76,85 For example, upregulated claudin-2 protein expression and changes in TJ structure and function have been demonstrated,86 however, strand discontinuities were not as frequent as in CD.87 In addition, a preliminary study suggests that the increase in transcellular uptake of protein antigens in UC may involve cholinergic pathways.88

Irritable bowel syndrome (IBS)

Clinical studies have shown an increased intestinal permeability in patients with postinfectious IBS,89,90 and Marshall et al.91 demonstrated a high incidience of postinfectious IBS with increased permeability following acute bacterial gastroenteritis after a waterbourne outbreak. Further it has been shown that barrier dysfunction in the colonic mucosa of IBS is due to changes in paracellular permeability and expression of ZO-1.92 Moreover, fecal serine proteases from IBS patients caused increased colonic paracellular permeability via PAR-2 activation and phosphorylation of MLCK.93 Whether IBS also causes increased transcellular permeability is yet to be studied. However, in animal models of IBS, such as maternal separation94 and chronic stress,95 there is clear evidence that both pathways are involved (see further below).

Neuroimmune Regulation of the Intestinal Barrier

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

As obvious from the studies reviewed above, the intestinal barrier is highly regulated by immune factors. The obvious importance of mast cells and the growing evidence of nerve–mast cell interactions in human stress96,97 and IBS92,98,99 also imply the potential pathophysioloigcal importance of neural signaling related to the epithelial barrier.

Enteric and extrinsic nerves

Under both physiological and pathological conditions the enteric nervous system regulates intestinal mucosal function.100 While the myenteric plexus mainly regulates intestinal motility, the submucosal plexus together with nerve fibers in the lamina propria are involved in regulating epithelial transport functions.

These nerves form networks within the lamina propria of both crypts and villi101 with the terminal axons in close contact with the basal lamina, i.e. an ideal position not only to affect epithelial cell functions but also to detect absorbed nutrients and antigens. These substances or released mediators from epithelial cells may act on the nerve terminals to change the properties of the sensory neurons and cause peripheral sensitization. There is also functional evidence of efferent communication between the central nervous system and the gut mucosa, for example, during stress, which could occur through one or more pathways. Extrinsic efferents include the vagal nerve and pelvic parasympathetic efferents, and postganglionic sympathetic neurons. These could also act via second order connections to intrinsic enteric neurons. In other words, the requirements for a continuous cross-talk between nerves and epithelial cells are at hand. In the nerve-mediated maintenance of intestinal barrier function, an important role for direct effects of vasoactive intestinal peptide (VIP) on epithelial cells, acting on ZO-1, has been demonstrated.102

Enteroglia

In a study by Aubéet al.,103 transgenic mice expressing haemagglutinin in glia, were used as a model of glia alterations. Immunohistochemistry showed decreased levels of substance P (SP)- and VIP-positive neurons in jejunal submucosal plexus of transgenic mice compared with the non-transgenic littermates. Moreover, intestinal permeability increased in transgenic mice compared with controls. This points to that glia disruption can change the neurochemical coding of enteric neurones, which in turn might cause dysfunction in intestinal motility and permeability.

Savidge et al.104 recently showed that glial-derived s-nitrosoglutathione (GSNO) was important for maintenance of mucosal barrier function in vitro and in vivo. In mice, GSNO increased the expressions of perijunctional F-actin, ZO-1, and occludin. Furthermore, GSNO significantly restored mucosal barrier function in colonic biopsies from patients with CD, a well-described inflammatory permeability disorder associated with enteric glial-cell disruption.

Neuroimmune interaction

It is well established that close connections between nerve fibers and immune cells appear in the intestinal mucosa.105,106 Direct interaction between nerves and mast cells, eosinophils, or plasma cells often occur,107 and it has been suggested that within the intestinal mucosa more or less all inflammatory cells are structurally innervated.108 Upon neural stimulation, mast cells release compounds by a tightly regulated, selective secretion of specific mediators, which can be depicted by electron microscopy as piece-meal degranulation.109,110 It is known that SP and calcitonin gene-related peptide-immunoreactive varicosities are found adjacent to mucosal mast cells,111 suggesting the possibility of a relationship between extrinsic afferents (vagal and/or spinal) and epithelial- and immune cells of the mucosa. The close bi-directional connection between mast cells and enteric nerves, the expression of receptors for neuropeptides,110,112 together with the release of neurally active mediators (histamine, proteases, prostaglandins [PGs]), demonstrate the significance of mast cells as end effector cells of the brain–gut axis in the intestinal mucosa. The most well investigated examples of neuroimmune interaction in this respect are models of food hypersensitivity, roundworm (nematode) infections and in stress.

Stress and barrier dysfunction

There is an increasing body of evidence that acute and chronic stress affects mucosal barrier dysfunction113 and several studies have confirmed the importance of stress in gastrointestinal disease in humans. Barclay and Turnberg showed in the late 1980’s that psychological stress, induced by dichotomous listening114 or cold-induced hand pain,115 reduced mean net water absorption and transformed net absorption of sodium and chloride to secretion. These effects were inhibited by atropine, suggesting the involvement of cholinergic neurons, acetylcholine (ACh). In later studies, Santos et al.96 extended this model and found that jejunal water secretion induced by cold pain stress was associated with luminal release of typical mast cell mediators, histamine, and tryptase. These findings pointed to an interaction between the intestinal mucosa, mast cells, and the central nervous system via cholinergic pathways during stress in humans. The neuro-endocrine factors involved in changes in mucosal function during stress have not been fully clarified. The main mediators include ACh116 and corticotropin-releasing hormone (CRH),117 but neurotensin117 and SP118 have also been implicated.

ACh  Studies in rodents of mechanisms involved in the mucosal stress response confirmed the findings in humans.114,115 Jejunal paracellular permeability to 51Cr-EDTA was inhibited by pretreating the rats with atropine or atropine methyl nitrate (does not pass blood–brain barrier), but not with hexamethonium.116 The increase in transcellular endosomal uptake and flux of HRP was also inhibited by atropine119,120 as well as by an antagonist of the muscarinic M3 receptor.121 Similar findings were recently found in the colon in models of acute and chronic stress.122,123 Taken together these results suggest that ACh can mediate stress-induced increase in paracellular permeability and transcellular uptake of proteins via muscarinic receptors located in the small and large intestine.

CRH  Corticotropin-releasing hormone-mediated effects in the intestinal mucosa of rats are associated with secretion of rat mast cell protease (RMCP) II and mast cell activation, and these effects are inhibited by mast cell stabilizers.124 By stressing adult rats that earlier had been subjected to neonatal maternal deprivation colonic paracellular permeability increased as a result from mast cell release of nerve growth factor (NGF) via activation of the CRH receptor (R)-1.125 This was in line with Santos et al. who showed that CRH modulates barrier function in rat colon by acting on mucosal/submucosal CRH-R-bearing cells, through mast cell-dependent pathways.120,124 Moreover, in a recent study,126 early weaned pigs were given selective CRH-R antagonists. Results showed that CRH-R1 activation is mediating barrier dysfunction and hypersecretion, whereas CRH-R2 activation may be responsible for novel protective properties in the porcine intestine in response to early life stress. Thus, animal studies together suggest that acute stress-induced intestinal barrier dysfunction to a large extent is mediated by CRH operating via activation of mucosal mast cells. Recently, Wallon et al. demonstrated that CRH regulates transcytosis of HRP in human colon via CRH-R1 and on subepithelial mast cells.110 This suggests that the stress-CRH-mast cell pathway may be present also in human intestinal mucosa. The role of CRH in intestinal diseases is unclear, however, increased expression of CRH was found by immunstaining in the colon of UC,127 and the clinical course of UC has been found to be affected by chronic stress.128,129

Mast cells  Several studies have confirmed the role of mast cells in stress-related changes of intestinal mucosal function. For example, studies have shown increased levels of RMCPII during stress,96,130 inhibition of stress-induced changes by adding mast cell stabilizers,120,131,132 and ultrastructural mast cell activation in combination with stress-induced barrier dysfunction.119,133 Moeser et al.134 showed that the intestinal mucosa of early weaned piglets showed elevated mucosal mast cell tryptase levels and enhanced mast cell degranulation. Pretreatment with the mast cell stabilizer cromolyn abolished these early weaning-induced intestinal barrier disturbances. Furthermore, mast cell-deficient (Wv/Wv) mice and (Ws/Ws) rats do not respond with stress-induced changes of barrier function.109,135,136 In humans, acute psychological stress affects jejunal ion secretion,114 and neuroimmune regulation of intestinal ion transport via mast cells does occur in humans.96,137 In addition, Alonso et al.97 recently showed that acute stress in women was accompanied by increased secretion of tryptase and albumin into the jejunal lumen.

As indicated above, mast cells play a major role in stress-induced changes of intestinal permeability. Although, it has been shown that the neural effects on intestinal permeability are not always mediated by mast cells.138 In rat jejunum, deoxycholic acid increased fluid transport via involvement of enteric nerves, while RMCPII was unchanged. This indicates that enteric neural effects on intestinal paracellular permeability function may also occur independently of mast cells. This was also recently confirmed by Demaude et al. in a model of acute stress in mice.122

Mast cell mediators with effects on intestinal barrier function

Activated mast cells have the ability to release a wide variety of bioactive mediators. These include preformed mediators stored in the granules (e.g. histamine and tryptase), and newly synthesized mediators (e.g. PGs, leukotrienes, and cytokines). Several of these mediators have effects on mucosal barrier function, for example, IFN-γ, TNF-α, and tryptase.

IFN-γ

It is well-known that IFN-γ has direct effects on TJ permeability. For example, it reduced the transepithelial resistance (TER), ZO-1 levels and disrupted apical actin in T84 cells.139,140 Several further studies of human epithelial cells have confirmed the effects of IFN-γ not only on monolayer paracellular permeability,50,141,142 but also on transcellular permeability.143,144 In addition, in vivo studies have revealed an important role for IFN-γ in gut permeability evoked by stress and inflammation.143,145,146 For example, repeated stress in mice induced an overexpression of colonic IFN-γ147 and increased permeability in murine colon involved an overproduction of IFN-γ.132

TNF-α

It is well known that TNF-α affects the barrier in several ways. Experiments in cell lines have shown that TNF-α decrease monolayer integrity and diminish the promoter activity of occludin.45,87 TNF-α treatment also resulted in increased paracellular permeability by decrease of junctional strands and a reduction in the depth of the TJs.87,148 Moreover, TNF-α can upregulate claudin-2,71 and induce epithelial cell apoptosis,148 resulting in increased paracellular permeability. It has also been shown that the elevated levels of TNF-α following T-cell activation in mice causes MLCK phosphorylation and thereby increased permeability.149

Tumor necrosis factor-α expression and secretion is increased in mucosal immune cells of CD, including mast cells,78,79 and has for a long time been considered a key mediator driving the disease. Isolated lamina propria mononuclear cells from CD patients secrete TNF-α that reduces TER in T84 monolayers,150 and the increased transcytosis of protein antigens across the ileal mucosa of CD is mediated by TNF-α.77 Moreover, there is a TNF-α-induced epithelial cell apoptosis in areas with barrier defects in IBD,70,86 which can be restored by anti-TNF-α (infliximab) treatment in patients with CD.70,151

Tryptase and PAR-2

Upon activation mast cells release tryptase,152 which is preformed and stored in granules. Tryptase and other proteases can affect epithelial permeability directly via PAR-2 expressed on the basolateral and apical side of epithelial cells, but also indirectly via PAR-2 on enteric nerves and on mast cells themselves.152–155 This gives the opportunity for intricate regulation of epithelial function via PAR-2 in a para- and autocrine fashion (see Bueno & Fioramonti,155 NGM 2008 for review). In brief, it has been shown that peptides that are specific agonists of PAR-2 (for example, SLIGRL) increase paracellular permeability and bacterial translocation in the colon of rats.146,156 These effects were mimicked by mast cell supernatant and abolished by tryptase inhibitor.157 The PAR-2-mediated effects on permeability in rats were inhibited by treatment with antibiotics that reduced expression of PAR-2 on epithelial cells.158 On the other hand, PAR-2 expression and effects on permeability were unaffected by dexamethasone.159 These data suggest that bacterial factors are important in regulating PAR-2-mediated effects on epithelial function.

IL-1β, IL-4, and IL-13

Several interleukins have effects on gut permeability. For example, IL-1β is a pro-inflammatory cytokine known to play an important role in the pathogenesis of intestinal inflammation in IBD.160 A recent study in Caco-2 cells revealed that IL-1β causes a drop in TER which correlated with increased epithelial TJ permeability.161 Studies on the effects of IL-4, a cytokine increased in food allergy and in early CD, demonstrated increased permeability across human intestinal epithelial cells.162,163 In T84 monolayers IL-4 affected TER, flux of 3H-formyl-methionyl-leucyl-phenylalanine and HRP,163 as well as transepithelial permeability to Dextran 4000.164 The IL-4-induced increase in T84 paracellular permeability was associated with an increase in the expression of claudin-2, and was prevented by PI3-kinase inhibitors,165 suggesting a mechanistic role for this signal transduction pathway. IL-13 may be a key effector cytokine in barrier defect and epithelial apoptosis in UC.86 Studies of IL-13 in epithelial cell lines have shown increased paracellular permeability in combination with upregulated claudin-2 expression, while only limited effects on claudin-1, 3, and 4 were seen.86,166 Mechanistic studies have further shown that IL-13 increases paracellular permeability in T84 cells via PI3K transduction pathways.165,166

Prostaglandins

Stress induced by weaning in piglets was shown to cause increased short-circuit current, reduced TER and increased intestinal permeability to mannitol134 that was sensitive to treatment with tetrodotoxin and the cyclooxygenase (COX) inhibitor, indomethacin, suggesting activation of enteric nerves and PG synthesis. A connection between nerves and COX was also described by Nylander et al.167 showing that COX inhibition excites enteric nerves to induce motility, increase alkaline secretion, and decrease paracellular permeability in rat duodenum. Intestinal mucosal mast cells produce and release PGs, in particular PGD2 and PGE2, in response to various activating stimuli.120,168,169 Both PGD2 and E2 are considered mainly to have protective and immunomodulatory, functions in the mucosa. The PGD2 metabolite 15d-PGJ2 is an important endogenous agonist of the anti-inflammatory transcription factor, PPAR-γ, which is involved in barrier regulation in stress and inflammation.170 PGE2-induced closure of the paracellular pathways has been shown to be an important first step in epithelial repair,171 and mast cell-derived PGs reduce epithelial hypersecretion induced by infection and neural stimulation.172 On the other hand, it was also shown that prolonged release of PGD2 after resolved TNBS colitis leads to altered mucosal barrier function with increased bacterial translocation173 and may predispose to chemically induced cancer in rats.174 Further studies are therefore needed to fully understand the role of mast cell-derived PGs in intestinal barrier function.

NGF

A CRH-mediated release of NGF from colonic mast cells was recently described.125 This study showed that adult rats that had been exposed to neonatal maternal deprivation early in life released NGF in response to activation of CRH-R1, which induced increase in paracellular permeability. Neonatal maternal deprivation also induced a closer association of colonic mast cells with enteric nerves, and this effect was abolished by anti-NGF therapy.175 Thus, mast cell-derived NGF may play an important role in mucosal dysfunction induced by chronic stress.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

To summarize, mucosal barrier function plays an essential role in innate defence by keeping the body proper separated from the external environment. A small, but controlled uptake of antigens takes place for immunosurveillance, to maintain the defence systems on alert. However, derangement of barrier integrity, with increased antigen load to the lamina propria, is believed to be important in the pathophysiology of several intestinal disorders. In most of the disease states where barrier function is disturbed, a concomitant alteration of the paracellular pathway (via TJs) and the transcytotic uptake route for antigens is seen. Neuroimmune modulation of the mucosal barrier is important in controlling innate defence, but can also lead to barrier dysfunction that potentially causes disease. One of the most well studied neuroimmune signalling pathways in gastrointestinal pathophysiology is nerve-mediated activation of mast cells, summarized in Fig. 5. Upon activation by CRH-containing neurons or via cholinergic pathways, the mucosal mast cells release a range of mediators (for example, tryptase, TNF-α, NGF, and interleukins-1β, -4, -13), which can have effects both on trans- and paracellular permeability. This may lead to an inappropriate immune activation that in the predisposed host goes on to mucosal inflammation. The therapeutic effect of anti-TNF in IBD has during the last decade become well established. Interfering with other mediators of nerve–mast cell–epithelial interaction has shown promising effects in animal studies, but important species differences in these pathways are present. We therefore need to learn more about the activating signals and mediators in the human intestinal mucosa in health and disease before we will be able to balance the neuroimmune response in a way that benefits our patients.

image

Figure 5.  A simplified schema of potential mechanisms involved in neuroimmune modulation of intestinal mucosal barrier function. Neural impulses from extrinsic vagal and/or sympathetic efferent fibers or intrinsic enteric nerves influence mucosal barrier function via direct effects on epithelial cells (e.g. acetylcholine [ACh]- or vasoactive intestinal peptide-mediated effects) or via interaction with immune cells (e.g. mast cells or plasma cells). For example, corticotropin-releasing hormone (CRH) induces piece-meal degranulation of mast cells to release various mediators that affect epithelial cells. Mast cell mediators such as TNF-α, tryptase (via PAR-2), nerve growth factor (NGF), and interleukins may affect paracellular permeability (e.g. by altering expression of various claudins in the tight junctions or the transcellular uptake route (e.g. by increasing macropinocytosis) to disrupt the barrier to antigens and bacteria. In most intestinal disorders, there is a concomitant disturbance of the paracellular and the transcellular pathway.

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Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References

JD Söderholm’s stress research is supported by the Swedish Research Council (VR-M), The Ihre Foundation of the Swedish Society of Medicine, and Medical Research Council of Southeast Sweden (FORSS).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Intestinal Barrier Function
  5. Passage Routes Across the Epithelium
  6. Endo- and Transcytotic Pathways
  7. Paracellular Pathways
  8. Barrier Dysfunction in Human Disease and Animal Models
  9. Neuroimmune Regulation of the Intestinal Barrier
  10. Conclusions
  11. Acknowledgments
  12. Disclosures
  13. References