Tight Junctions and the Intestinal Barrier

Chapter 38

Tight Junctions and the Intestinal Barrier Thomas Y. Ma, James M. Anderson, and Jerrold R. Turner

38.1  INTRODUCTION The primary function of the gastrointestinal tract is to digest and absorb nutrients. To accomplish this, it must maintain a barrier between the luminal environment, technically a space outside the body, and the internal environment of the body; and it must selectively absorb and secrete nutrients, solutes and water across the barrier. Separation of tissue spaces throughout the gastrointestinal tract is accomplished by continuous sheets of polarized columnar epithelial cells. An exception exists in the upper two-thirds of the esophagus, which is covered by a non-keratinizing squamous epithelium. Epithelial barriers are selective and capable of excluding potentially noxious luminal contents, such as gastric acid, colonic bacteria, and bacterial antigens, while at the same time capable of directional absorption and secretion of large volumes of solutes and water. Material can pass from one side of the epithelium to the other along one of two routes, either through the cell membranes or the space between them, referred to as the transcellular and paracellular pathways, respectively. The connection between individual epithelial cells is created by a series of intercellular junctions; the tight junction (TJ) is the most important for defining the characteristics of the paracellular barrier and its selectivity. The specific characteristics of epithelial barriers vary widely throughout the gastrointestinal tract, matched to each organ’s transport functions. However, in all cases, disruption of the barrier leads to a loss of normal transport and inflammation due to tissue damage or antigen exposure. In this chapter we focus primarily on the role of the tight junction in the intestinal barrier. We begin with the role of the TJ and paracellular pathway in normal transport. In recent years a large number of proteins have been identified as components of the TJ and the functions of some of these proteins are beginning to be unraveled. This allows interpretation of the barrier’s physiologic properties on a stronger cellular and molecular foundation. In this chapter, we review the latest advances in this area. The intestinal TJ barrier is highly regulated and we discuss the mechanisms and physiologic relevance for the gastrointestinal tract. Finally, we review some of the intestinal

disorders that have an associated defect in intestinal TJ barriers and the implications of TJ barrier defect in the disease pathogenesis. Since the previous edition, there have been number of important advances that better define the molecular and cellular processes that affect the TJ barrier function under both normal conditions and during pathologic states. In this edition, we have taken a comprehensive approach to cover a wide variety of topics, but not all in equal depth. Although we tried to cover as much of the relevant historical advancements, core concepts, and the latest advances in the field, due to the overwhelming amount of high-quality original publications in this area, it was not possible to cite all major advancements. Where appropriate, the reader is referred elsewhere for a more complete presentation, particularly of current controversies and unresolved issues.

38.2  INTRINSIC AND EXTRINSIC ELEMENTS OF THE BARRIER The term “epithelial barrier function” is often used to describe all the mechanisms contributing to homeostasis of the epithelial barrier. The single layer of continuous epithelial cells and their intercellular junctions constitute the intrinsic elements of the barrier. The magnitude of this barrier is most often measured as the transepithelial electrical resistance (TER) and the permeability to paracellular markers, such as mannitol and inulin. TER correlates with the ability to separate ionic charge across the epithelia, which is reflected in a transepithelial electrical potential difference or the current that creates the potential, measured experimentally as the short circuit current (Isc). Extrinsic elements include the innate and acquired mucosal immune system, protective secretion of mucus, bicarbonate, IgA, and antimicrobial peptides as well as a mechanism for epithelial repair or restitution.1 The contribution of each element varies along the gastrointestinal tract, with mucous secretion as the most constant along the entire length from mouth to anus. Our goal in this chapter is to focus predominantly on the intrinsic barrier of epithelial cells and TJs in health and disease.

Physiology of the Gastrointestinal Tract, Two Volume Set. DOI: 10.1016/B978-0-12-382026-6.00038-5 © 2012 Elsevier Inc. All rights reserved.

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38.3  THE INTESTINAL EPITHELIAL BARRIER AND TRANSCELLULAR AND PARACELLULAR TRANSPORT 38.3.1  Transport Pathways Throughout the gastrointestinal tract, transport of electrolytes, solutes, and water across epithelia occurs across both transcellular and paracellular pathways (Figure 38.1). The transcellular route for hydrophilic molecules, for example Na, Cl, and glucose, is governed by the profile of membrane pumps, carrier, and channels expressed in a particular cell type. The passive movement across the lipid component of the membrane is very limited for charged and hydrophilic molecules. For example, the electrical resistance across model lipid membrane bilayers is in the range of 106–109 Ω    cm2, whereas the resistance across real membranes in the gastrointestinal tract is 3–4 orders of magnitude less,2 reflecting facilitated conductance through protein-based channels (Table 38.1). The profile of conductance proteins differs among epithelia, which explains their unique functions. Individual transporters also show a polarized distribution to the apical or basolateral membrane surface as the basis for directional transport. For example, the apical H-K ATPase of gastric parietal cells is responsible for secreting hydrochloric acid within the stomach. Na-dependent bile acid transporters are positioned on the hepatocyte’s sinusoidal surface and the apical-luminal surface in the ileum to produce the enterohepatic circulation of bile salts. The cystic fibrosis transmembrane regulator (CFTR), a chloride channel, is positioned on the apical surface of biliary, pancreatic, and intestinal surfaces to produce luminal Cl secretion, which is followed by Na and water secretion. Mucosa

Transcellular pathway Rap

Primary transcellular transport is “active,” powered by ATP hydrolysis to move ions against an electrical or concentration gradient. The prime example is the ubiquitously expressed Na-K-ATPase, which moves three Na ions out of the basolateral surface in exchange for two K ions, and the net effect generates an inwardly directed Na and outwardly directed K gradient and negative intracellular electrical potential. The high membrane conductance for K and its exit from the cell further enhances the intracellular negative electrical potential. These electrical and chemical gradients are then used in “secondary” active transport to couple energetically unfavorable uphill movement of nutrients, such as glucose or amino acids, to the downhill movement of Na through, for example, the Na-coupled glucose cotransporter (SGLT1) of the jejunum. As a final generalization, the characteristics of transcellular transport are highly regulated by shortterm signals (e.g., hormone-stimulated bicarbonate secretion from pancreatic ducts) and long-term transcriptional control (e.g., aldosterone-stimulated expression of the

TABLE 38.1  Electrical Characteristics of Some Epithelia Epitheliuma

Species

Rcellb

Rparacellular PNa/PClc

Proximal tubule

Dog

—

6–7

1.4

Gallbladder

Rabbit

229

21

3.3

Duodenum

Rat

—

98

–

Jejunum

Rat

67

51

10.0

Ileum

Rabbit

115

100

2.5

Distal colon

Rabbit

730

385

0.6

Mouse surface

132

3,200

—

Crypt

429

—

—

Gastric fundus

Necturus

2,826

10,573

—

Urinary bladder

Rabbit

160,000

300,000

—

Caco-2

Human colon

125–250

—

3.0

LLC-PK1

Pig proximal tubule

100

—

0.6

MDCK

Dog

60–4000

—

10.0

Paracellular pathway RTJ

Cell linesd RLIS Rbl

Serosa FIGURE 38.1  Equivalent electrical circuit model of the intestinal epithelial cell layer.  Only resistive elements are shown. Series resistance across the transcellular pathway is the sum of individual resistance across the apical (Rap) and basolateral membranes (Rbl). These are in parallel with resistances of the tight junction (RTJ) plus the lateral intercellular space (RLIS). The RLIS is small, the membrane resistances are usually high, and the epithelial resistance is governed by resistance of the TJ.

a

All values can be found in Reference 16. Electrical resistance values in Ω  cm2. c Permeability ratio of Na versus Cl. PNa/PCl in free solute is 0.66. Paracellular pathways with ratios above this value are more permeable for Na than Cl, for example, cation-selective. d Values for cell lines are the personal observations of Dr. C. Van Itallie. b

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Chapter  |  38  Tight Junctions and the Intestinal Barrier

Na-K-ATPase). We outline these features of transcellular transport, before proceeding with a detailed discussion of the paracellular pathway, to highlight the sharp distinction of paracellular transport, which is passive, nonrectifying, and does not appear to be as highly regulated by physiologic stimuli.

the apical to basal axis, in transverse sections, as a series of close cell-to-cell contacts, or “kisses.” In freeze-fracture images (Figure 38.3A) the contacts are revealed as continuous rows of transmembrane protein particles. Actin filaments terminate on the plasma membrane directly at the contacts and participate in the regulation of the TJ barrier,5 and are known to bind the peripheral membrane

38.3.2  Apical Junction Complex The paracellular barrier to material movement coincides with continuous cell–cell contacts located at the apical end of their lateral surfaces (Figures 38.2 and 38.3). The earliest histological description of what we now refer to as the “apical junction complex” comes from the late nineteenth century. When sections of small intestine were stained with vital dyes a distinct intercellular density was observed between cells at the apical end of the lateral space. The English literature referred to this as the terminal bar; other names reveal an assumed role in intercellular adhesion, such as Schlussleiten, and bandelettes de fermeture. The first speculation about a barrier function is attributed to Bonnet in 1895.3 After examining several different gastrointestinal tissues obtained from an executed man, he concluded that the terminal bar was a general feature of all epithelia and might play a role in segregating the distinct fluid compositions found in different regions of the gastrointestinal tract. With the first ultrastructural images of intestinal epithelia in 1963,4 the apical junction complex was revealed as a set of morphologically distinct junction types (Figure 38.2). Each of these functions in cell–cell adhesion and signal transduction and provides links to the cytoskeleton. The TJ is invariably the most apical. It appears along

(A)

(B)

Tight

Adherens Desmosome Gap

FIGURE 38.2  Junction types within the apical junction complex between intestinal epithelial cells.  Left: Two columnar epithelial cells with apical brush border typical of the small intestine. A thick band of perijunctional actin and myosin filaments connected to the tight and adherens junctions are typical of intestinal epithelial cells. The “terminal bar” or apical junction complex when amplified reveals a series of intercellular contacts including the tight and adherens junctions, desmosomes, and gap junctions. Tight and adherens junctions are linked to the actin cytoskeleton and desmosomes to intermediate filaments. Right: Tight junction contacts magnified reveal rows of claudin strands adhering between adjacent cells to seal the paracellular space.

(C)

FIGURE 38.3  Freeze-fracture electron microscopic replica.  (A) TJ region of mouse jejunum, showing the interconnected network on claudinbased strands crossing the membrane. Continuous rows of claudins from adjacent cells adhere and seal the paracellular space. Above the barrier contact zone a few apical microvilli are visible. (B) Transmission electron micrograph of the apical junction complex region of two adjacent mouse mammary epithelial cells, rotated at 90° around a vertical axis to the image in (A). Lanthanum hydroxide (black) was added to the basolateral side; it freely diffuses through the intercellular space until it is partially blocked at the TJ from reaching the apical side. The TJ is recognized as a region of very close cell–cell apposition. Microvilli are seen on the apical surfaces. (C) Immunofluorescent microscopic localization of the TJ protein ZO-1 in mouse distal colon. The epithelial surface is at the top and two crypts are visible descending toward the bottom of the image. ZO-1 at the TJ is visible at the apical end of the lateral cell contacts, from crypt to surface. Tangential sectioning in the crypt reveals the continuous circumferential location of TJs around each cell. Bar: 10 μm. (Image C is courtesy of J. Holmes.)

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TABLE 38.2  Proteins Located at the TJ Category

Protein

Function

Transmembrane

Claudin(s)

Barrier and pore selectivity

TAMPs

Signaling scaffold, adhesion, barrier regulation

JAM(s)

Many

CAR

Coxsackie virus receptor

ZO-1

MAGUK, binds occludin, claudin, ZAK, JAM, ZAK, actin, ASIP, ZONAB

ZO-2

Binds ZO-1, actin, claudins, fos, jun, CEBP

ZO-3

Binds ZO-1, actin, claudins

MUPP-1

13 PDZs and binds claudins

ASIP/PAR-3

Atypical PKC binding protein

PAR-6

Cdc42-Par6-Par3-aPKC interaction required for polarity and junction formation

PAT-J

Aka discs lost

Pals-1

Aka Crumbs polarity protein

ZAK

Binds and phosphorylates ZO-1

aPKC

Binds polarity proteins PAR-3, PAR-6, interaction required for junction assembly

src

Occludin phosphorylation blocks ZO-1 binding

yes

Binds occluding

PTEN

Tumor suppressor binds MAGI-2 and 3

PP2A

Binds aPKC, disassembles junction

ZONAB

ErbB-2 activator

HuASH1

Drosophila ash1 homolog

PDZ-Scaffolding

Polarity

Kinases

Phosphatases

Transcription factors

CEBP GTP-binding proteins

Vesicle targeting

Other

Rab 3B

Mutants inhibit LDLR delivery

Rab 13

Mutants inhibit claudin-1 delivery

Gαi2

Binds SH3 of ZO-1

AF6

Binds to Ras, ZO-1 and actin

GEF-H1

Guanine nucleotide exchange factor influences permeability

Sec6/8

Exocyst complex

VAP33

Binds occludin and v-SNAREs

Cingulin

Binds ZO-MAGUKs, JAM-1, actin

scaffolding proteins ZO-16 and cingulin (Table 38.2).7 Below this is the adherens junction, location of cadherin, the intercellular adhesion molecule and its cytoplasmic binding partner β-catenin, and extensive attachments to a ring of perijunctional actin filaments. The importance of cadherin in adhesion and maintaining the differentiated cell phenotype is underscored by its frequent mutation as a final step facilitating metastasis of colon cancer.8

β-Catenin has a second role in the nucleus where it signals cell growth and adenoma formation unless degraded by interacting with the adenomatous polyposis coli protein (APC). Human mutations in APC leave β-catenin free to signal cell growth and transformation into adenomatous polyps.9 Below the adherens junctions are desmosomes, whose transmembrane proteins, while homologous to cadherin, are linked to intermediate filaments and not actin.

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Chapter  |  38  Tight Junctions and the Intestinal Barrier

Desmosomes serve to protect the alimentary epithelia from shear-induced damage. Gap junctions allow transfer of small signaling molecules, like Ca2 and IP3, between adjacent cells and coordinate epithelial functions like secretion and exocytosis.10 They can be positioned at any depth along the lateral surface and are often found within the TJ strands. Viewed by freeze-fracture election microscopy (Figure 38.3A) the TJ barrier coincides with a network of transmembrane strands. In unfixed tissue, the strands often appear as rows of individual particles, now known to be a family of transmembrane adhesion molecules termed claudins.11 The Latin root, claudere, means to close. Rows of claudins from each cell meet in the intercellular space forming adhesive contacts and a semipermeable seal. The complexity (number and cross-linking) of strands differs among various tissues. It was long thought the number of strands correlated with the resistance of the barrier.12 Consistent with this, in the small intestine the complexity of strands increases at the crypt-to-villus transition.13 Since discovery of the barrier-forming proteins, this structure–function correlation has been called into question; the molecular species of claudin in a particular junction appears be an important determinant. However, composition of other transmembrane TJ proteins is also likely to be important in the regulation and development of TJ barrier function. The specific proteins that affect paracellular flux of solutes of varying size need further clarification.

38.3.3  Barrier Properties: Resistance, Flux, and Permselectivity Early electron microscopic studies (Figure 38.3B) interpreted the close membrane apposition of adjacent cells at TJ contact points as membrane fusion, and even suggested convergence of the outer leaflets of the lipid bilayer.4 Supported by studies showing the inability of electrondense proteins, such as hemoglobin and colloidal lanthanum, to pass through the TJs,14 these analyses led to the popular view of the TJ as an absolute barrier to paracellular flux. Although commonly thought of as an impermeant seal, it might be more appropriate to compare TJs to sieves. However, the characteristics of the selectively permeable TJ barrier vary widely among different tissues, within different cell types of a single tissue, and in response to physiological and pathophysiological stimuli (Table 38.1). Thus, while the paracellular barrier is most often assessed by electrical conductance, transepithelial electrical resistance, or transepithelial flux of small fluid phase markers, such as mannitol or polyethylene glycol, measurement of only one or two of these parameters provides an incomplete picture of overall barrier function.

38.3.3.1  Electrical Resistance Epithelia are classified as “tight” or “leaky” based on their overall electrical resistance.15,16 The small intestine, colon, and renal proximal tubule are typical examples of leaky epithelia, while gastric fundus, renal collecting duct, and urinary bladder are “tight” epithelia. In either case, paracellular ionic conductance can be measured by mounting tissue with the mucosal and serosal surfaces facing electrically isolated fluid-filled chambers with current and voltage electrodes on both sides. This is the standard Ussing chamber configuration. An equivalent circuit diagram of the epithelium can be developed to include the transcellular pathway, represented by apical and basolateral membrane resistances series, in parallel with resistance of the paracellular pathway (Figure 38.1). Because membrane resistances are generally very high, conductance by the transcellular pathway can generally be discounted and the overall resistance interpreted as representing the paracellular pathway. However, this is not the case when transcellular and paracellular resistances are similar. This may occur when plasma membrane conductance is enhanced, as can occur when large numbers of ion channels, such as CFTR, are opened, or, alternatively, when paracellular resistance is very high, as in the urinary bladder. Nevertheless, in most cases, the effects on overall transepithelial resistance reflect changes in paracellular resistance. Although the TJ and lateral intercellular space are arranged in series such that both contribute to paracellular resistance, the contribution of the lateral intercellular space is small and usually ignored. Since membrane resistances are generally high, it is the variation in TJ resistance that determined whether an epithelium is leaky or tight. Electrophysiologic studies prior to about 1960 primarily used tight epithelia, such as frog skin and urinary bladder, where the paracellular resistance is very high (more than 1500 Ω    cm2). This reinforced the misconception that the TJ is an impermeant barrier. Such studies also provided evidence that barrier function requires viable tissue, as the progressive tissue injury following devascularization correlated with decreases in overall electrical resistance. Because leaky epithelia, such as gallbladder and small intestine, always demonstrated low resistance, it was assumed that this was due to tissue fragility and damage during mounting. Despite this interpretation, the high conductance pathway across leaky epithelia displayed charge and size selectivity, which would not be expected if the “shunt” pathway was simply a result of tissue damage. The controversy was settled when the shunt pathway was localized to the paracellular space using conductance scanning methods. By passing a microelectrode over the gallbladder epithelial surface, a high conductance shunt could be demonstrated at the intercellular junctions.17 Use of smaller electron microscopic tracers, such as ionic lanthanum, also allowed ultrastructural visualization of permeation through

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the TJ. These findings led to a paradigm shift in which the idea of a selectively permeable TJ was accepted. Work over the past 40 years has characterized these permeability properties in detail and resulted in our current understanding of TJs as barriers, which contain several classes of transport channels that can be distinguished in both functional and molecular terms.18,19

38.3.3.2  Charge Selectivity Experimentally, ion selectivity is often measured as a ratio of cation to anion permeability. As sodium and chloride are the most common cation and anion, respectively, in physiological solutions, ion selectivity is most commonly reported as (PNa/PCl) (Table 38.1). However, care must be taken in interpreting PNa/PCl values, as the ratio in solution is 0.66 due to the smaller size of hydrated sodium, relative to chloride, ions.20 Thus, PNa/PCl values above 0.66 should be considered cation selective. This is true of most epithelia, although the actual ratio can vary 30-fold between anatomical sites. While this is of great physiological significance, it should be recalled that, relative to transmembrane ion channels, this level of discrimination between anions and cations is very low. Further, such transmembrane ion channels easily distinguish ions with similar size and charge densities, such as Na and K, while the ability of TJs to separate these is very poor. However, monovalent and divalent cations are treated differently by some TJs, and, as discussed in the following section, we now know that this and overall ion selectivity reflect the pattern of claudin proteins expressed. Charge selectivity is a characteristic feature of paracellular barriers and is essential for creating transepithelial gradients that direct passive paracellular transport. For example, many active transcellular absorptive processes within the intestine take advantage of the electrochemical gradient created by the Na/K ATPase. However, while the Na/K ATPase pumps Na to the basolateral space, Na-nutrient cotransport occurs at the luminal, or apical, plasma membrane. This requires that free luminal Na be sufficient to drive these processes. Thus, if there was no effective means of serosal to mucosal Na flux, Na-poor diets would result in malabsorption and osmotic diarrhea. Given that there are no defined mechanisms for transcellular Na secretion (into the lumen), it stands to reason that this transport must occur by the cation-selective paracellular route that favors Na over Cl, which is the other most abundant ion in physiological settings. Defects in paracellular Na transport may, in part, explain the abnormalities that occur when claudin-15 , which enhances paracellular Na flux,21 is knocked out in mice.22,23 Although inherited claudin mutations have not been associated with gastrointestinal disease in humans, mutations that disrupt trafficking or expression of claudins 16 and 19 in the thick ascending

SECTION  |  III  Host Defense Mechanisms

limb of the renal tubule result in failure of paracellular Ca2 and Mg2 absorption and cause the autosomal recessive disease familial hypomagnesemia with hypercalciuria and nephrocalcinosis.24–27 Nevertheless, alterations in expression and trafficking of claudin proteins are present in intestinal diseases and can be induced by specific inflammatory mediators.28–32 Whether these changes contribute to disease pathogenesis or, alternatively, represent an adaptive response, is an area in need of further study.

38.3.3.3  Size Selectivity In addition to charge selectivity, the TJ discriminates between solutes on the basis of size. This property has been recognized for over 30 years,33–39 but, for the most part, detailed analysis has not been possible due to the limited number of probes available. This obstacle has been overcome using mixtures of polyethylene glycol oligomers with hydrodynamic radii ranging from ~3 to ~7 Å.40 The relationship between size and apparent permeability of these probes changed sharply at radii of ~4 Å,40,41 indicating a size-restricted population of paracellular pores. This is similar to the ~6 Å pores reported within small intestinal villous epithelium,39 and may reflect the same structure. Notably, recent data have shown that claudin-2 expression, which enhances paracellular flux of monovalent cations, also increases flux of polyethylene glycol oligomers with radii less than 4 Å.41 Thus, it appears that a claudin-based class of paracellular channels defines overall charge selectivity as well as size selectivity of this route, which has been termed the pore pathway.18,19 Despite the cutoff at ~4 Å, larger polyethylene glycol oligomers are still able to traverse epithelial monolayers, albeit at much lower rates than smaller solutes. The magnitude of this component of paracellular flux does not vary substantially with solute radius, suggesting that these molecules traverse a size non-restricted route that is distinct from the claudin-based pore pathway.40 The route of large solute flux has been referred to as the leak pathway,18,19 and may correlate with the 50–60 Å channels described within crypt epithelium.39 Enhanced leak pathway flux is the means by which IFN-γ and TNF-α increase paracellular permeability,31,42–46 and it has, therefore, been proposed that leak pathway regulation is primarily associated with pathologic processes. While this may be true, the converse is not, as some inflammatory mediators, for example, IL-13, which enhances claudin-2 expression, increases flux across the pore pathway without affecting the leak pathway.31

38.4  PROTEIN COMPONENTS OF THE TJ Beginning in 1986 with identification of ZO-1 as the first TJ-associated protein,47 there has been continuous growth to

Chapter  |  38  Tight Junctions and the Intestinal Barrier

almost 40 distinct proteins or protein families (Table 38.2). Surprisingly, this number far exceeds the known components of other intercellular junctions, such as adherens, desmosomes, and gap junctions (Figure 38.2), and it is likely that many identified TJ proteins serve regulatory, rather than structural functions. This is certainly the case of enzymes, such as atypical protein kinase C (PKC) isoforms and myosin light chain kinase, which localize to the TJ and have been shown to regulate epithelial polarity, junction assembly, and barrier function.45,48–59 These will be discussed in Section 38.5 in the context of specific regulatory events. In contrast, it is now clear that claudin proteins define the charge, and, perhaps, size selectivity of the TJ barrier.18,27,60–69 The specific roles played by other TJ-associated proteins, such as ZO-1 and occludin, are less certain, but recent data have shed light on their functions in TJ stabilization and regulation.19,42,44,70 Through an interesting evolutionary convergence, many tight and adherens junction proteins also serve as receptors or co-receptors for pathogen entry.71–76 Finally, polarity complexes are concentrated at the TJ.51,53,54,77–79

38.4.1  Transmembrane Proteins and TJ Stability A large number of transmembrane proteins serving diverse functions are found at TJs. Given the restricted, although, presumably, friendly confines of the TJ, it has been assumed that these proteins are embedded in a densely packed array that restricts their mobility. Together with the identification of multiple sites of interaction between TJ proteins, this led to the presumption that these proteins are tightly anchored and immobile.80 While possible, this conclusion would seem at odds with data demonstrating rapid regulation of TJ structure and function57,81–85 and, potentially, with the discovery that TJ membrane microdomains are enriched in glycolipids and cholesterol (i.e., membrane rafts) in which membrane fluidity is greater than more abundant phospholipid membrane domains.86 The stability or mobility of proteins within the TJ has been studied using fluorescent-tagged TJ proteins. Importantly, these fluorescent fusion proteins were carefully validated and demonstrated to (1) colocalize and co-fractionate with their endogenous, unlabeled counterparts84; (2) not interfere with TJ function;84,87,88 and (3) complement TJ protein deficiencies in mice and in cell lines.88,89 Thus, one can conclude that their behaviors with regard to dynamic behavior are related to, and likely the same as, those of endogenous TJ proteins. Consistent with the conclusion that TJ-associated proteins are stably anchored, EGFP-claudin-1 was found to have a mobile fraction of only ~25%.90 While the hypothesis that such anchoring depends on the interaction of claudin-1 with ZO-1 (discussed in the following section), anchoring of a

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truncated EGFP-claudin construct lacking the ZO-1 binding domain was similar to that of full-length EGFP-claudin although, as also discussed in the following section , trafficking to the TJ was severely impaired.90 Such data in epithelia were also consistent with the observation that, in fibroblasts, claudin-1-EGFP, which forms TJ strand-like fibrils despite blockade of ZO-1 binding by the C-terminal EGFP, demonstrated little fluorescent recovery after photobleaching.91 In contrast to claudin-1, occludin displayed a mobile fraction of ~75%. Occludin mobility occurred by diffusion within the plasma membrane and was dependent on membrane fluidity, as it could be inhibited by cholesterol extraction, with methyl-β-cyclodextrin, or reduced temperature, but was not an energy-dependent process.90 In contrast, while the mobile fraction of the peripheral membrane or plaque protein ZO-1 was similar to that of occludin, it was recovered by energy-dependent exchange with a cytosolic ZO-1 pool and was unaffected by cholesterol extraction or reduced temperature.90 While the functional significance of these observations are poorly understood at present, the data do show that protein interactions at the TJ are labile and that, despite the densely packed nature of the TJ, the raft-like biophysical properties of TJ membranes support free diffusion of TJ proteins. Data including the observation that claudin-1-EGFP forms TJ-like strands in fibroblasts, which lack endogenous TJs, suggest that claudins form the core of TJ strands in epithelia.92 Claudins are the only junction-associated proteins that have been found to form such strands, although occludin can incorporate into claudin-based strands.92 Other transmembrane proteins, such as junction adhesion molecule 1 (JAM-1),93 CAR,74 and connexin are located around, but not incorporated into the strands.

38.4.2  PDZ Containing Scaffolding Proteins The interface between the transmembrane proteins and most cytoplasmic components is formed by a set of scaffolding proteins with multiple PDZ domains. PDZ domains, named for the proteins PSD-95, large discs, and ZO-1, are protein binding modules that recognize target sequences at the extreme C-termini of transmembrane proteins.94–96 They are well characterized for their ability to cluster signal transduction complexes at specialized membrane contacts, such as synapses and TJs.97–100 PDZproteins at the TJ include the ZO-MAGUK proteins ZO-1, ZO-2, and ZO-3; the MAGUK relatives MAGI-1, MAGI-2, and MAGI-3; a protein with 13 PDZ called MUPP-1; and several of the polarity components cited below including PAR-3, PAR-6, PALS1, PATJ, scribble, and Dlg.52,101 Most of these proteins contain several PDZ domains and bind the tails of claudins and JAMs. Some, like the ZO-MAGUKs, have other protein binding modules, including SH3 and enzymatically inactive guanylate kinase

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(GuK) domains, which are known to bind additional targets including α-catenin and occludin.6,102–111 In addition, ZO-1 has a well-defined actin binding domain6,112 as well as less well characterized U5, U6, and ZU5 domains.102 Although much work remains, several functions of ZO-1 in TJ assembly and regulation are recognized. ZO-1 recruitment to nascent adherens junctions requires the U5 motif,102 likely as a result of U5-dependent interactions with α-catenin, AVRCF, and AF-6/afadin.104,105,113,114 However, it is equally possible that ZO-1 and ZO-2 are involved in recruitment of other proteins to the nascent adherens junction. For example, mammary epithelial (Eph4) cells deficient in both ZO-1 and ZO-2 have a marked delay in adherens junction assembly,115,116 as do MDCK cells lacking only ZO-1.117 ZO-1 is able to rescue this defect.115,117 Data regarding ZO-2 are less clear, as one study showed that knockdown in MDCK cells delayed E-cadherin, ZO-1, and occludin recruitment to the plasma membrane,118 while a separate study found no effect of ZO-2 knockdown.117 Thus, while differences between labs make it difficult to precisely compare the results, these data do suggest that ZO-1 and ZO-2 have overlapping, but distinct functions. This is consistent with the observation that knockout of ZO-1 or ZO-2 results in embryonic lethality in mice119,120 as well as the association of PDZ1 domain ZO-2, but not ZO-1,with mutations of familial hypercholanemia in human patients.121 Following adherens junction assembly, ZO-1 and ZO-2 direct polymerization of claudin proteins into TJ strands.102,115,122 The interaction between claudin proteins and the first PDZ domain of ZO-1 and ZO-2 is important in the first phase of this process, as claudins are not recruited to the junctional complex in EPh4 cells that lack both ZO proteins122 and, as noted previously, claudin proteins in which the PDZ binding motif is either mutated or blocked are trafficked to TJs inefficiently.90,92,123 However, the SH3U5-GuK-U6 region is also critical to regulation of claudin polymerization.102,115,122,124 Although the means by which claudin polymerization is controlled requires further study, the U6 motif does appear to negatively regulate this process, as U6 deletion results in aberrant strand assembly.102,122,125 In addition to the accumulation of data defining a role for ZO proteins in TJ assembly, recent work has defined a role of these proteins, particularly ZO-1, in barrier regulation. ZO-1, but not ZO-2, knockdown in MDCK layers increased paracellular permeability of large solutes, such as the leak pathway.70 Although charge selectivity was not examined, ZO-1 knockdown did not affect paracellular flux of small polyethylene glycols, suggesting that pore pathway barrier function was intact.70 ZO-1-dependent leak pathway maintenance did not require protein regions beyond the U6 domain, including the actin binding region (ABR), as ZO-1 mutants truncated just after the U6 domain or in which the ABR was deleted internally were both able to restore barrier

SECTION  |  III  Host Defense Mechanisms

function.70 Thus, although further analyses are needed, the data suggest that the PDZ or SH3-U5-GuK-U6 domains support ZO-1-dependent development of leak pathway barrier function. Given the roles of these regions in supporting ZO-1 interactions with claudins (PDZ1) and occludin (SH3-U5-GuK), it is also possible that this scaffolding function that links occludin and claudins is critical and that both regions are required. While attractive, data supporting this hypothesis has only been published in abstract form,126 as discussed in the following section. In addition to linking occludin and claudins, ZO-1 is able to connect all of these to F-actin via an ABR located within the C-terminal half of the protein.6,112,127 Recent data indicate that the ABR is required for physiological, myosin light chain (MLC)-dependent barrier regulation. While the relative contributions of pore and leak components have not been described, in vitro and in vivo analyses have shown that myosin light chain kinase (MLCK) inhibition enhances TJ barrier function45,57,59,128–130 and is associated with stabilization, as assessed by fluorescence recovery of ZO-1.88 This ZO-1 stabilization required the ABR.88 Moreover, either ZO-1 knockdown or dominant negative expression of the free ABR prevented barrier function increases after MLCK inhibition.88 Together with other data, these results suggest that MLCK-dependent ZO-1 exchange and ABR-dependent ZO-1 anchoring are critical determinants of TJ barrier function.88

38.4.3  TJ-associated MARVEL Proteins Occludin, an ~65 kDa tetraspan protein, was the first TJ-associated transmembrane protein identified.131 Although this discovery was greeted with excitement,132 subsequent understanding of occludin function has been elusive. The role of occludin in TJ assembly was first called into question by the observation that occludin-deficient embryonic cells can differentiate into polarized epithelial cells with structurally and functionally intact TJs.133 As discussed in the following section, this prompted further studies that resulted in the discovery of claudins.11 Controversy over the contributions of occludin to TJs was enhanced further by the observation that occludin knockout mice are viable;134 have not been reported to have gastrointestinal or renal disease; and as assessed using Ussing chambers, have intact intestinal barrier function.134,135 Nevertheless, occludin knockout mice do have severe health defects. Males have testicular atrophy and are sterile,134,135 while females do not effectively suckle their young.135 In addition, occludin knockout mice of either gender are small and develop chronic inflammation and hyperplasia of the gastric epithelium with loss of parietal and chief cells, brain calcifications, osteopenia, and salivary gland defects.134,135 Thus, although the data indicate that occludin does have some essential functions, it does not appear to be required for intestinal epithelial TJ assembly or

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Chapter  |  38  Tight Junctions and the Intestinal Barrier

basal function. Moreover, the response of occludin knockout mice to stress has not been examined and it remains possible that other proteins can compensate for intestinal epithelial occludin deficiency. In the previous edition, it was stated that occludin is unique and, despite having a tetraspan topology similar to that of claudins, is not a member of any known protein family. The discovery of the TJ-associated protein tricellulin (so named because it is concentrated at tricellular TJs), where three cells meet, changed this idea.136 Occludin and tricellulin both contain tetraspanning MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domains137 and bind ZO-1 via their carboxyterminal cytoplasmic tail,138 although the tail is not part of the MARVEL domain. However, in contrast to occludin knockdown in MDCK cells,139 tricellulin knockdown severely compromises barrier function of Eph4 monolayers.136 Interestingly, the effects of tricellulin knockdown in Caco-2 cells are less severe.87 Tricellulin mutations have been reported in humans and linked to autosomal recessive deafness,138,140 likely as a result of defects in function of junctions between supporting and hair cells within the vestibular and cochlear epithelia. Remarkably, these tricellulin mutations, which were most often truncations that disrupted ZO-1 binding, caused deafness without creating a syndrome involving other organs.138,140 This limited distribution of disease does not reflect the absence of tricellulin expression in other tissues,136 suggesting that another protein may compensate for tricellulin loss. Because both occludin and tricellulin contain MARVEL domains, some suggested an evolutionary relationship between these proteins and a third MARVEL domain-­ containing protein, marvelD3. However, other analyses suggested that this was not the case.138 The issue was resolved by an evolutionary analysis demonstrating that human MARVEL domain-containing proteins can be segregated into four groups, one of which contains occludin, tricellulin, marvelD3, and their splice variants.87 This family has, therefore, been referred to as TJ-associated MARVEL proteins (TAMPs). MarvelD3 was found at both bicellular and tricellular TJs of Caco-2 intestinal epithelial monolayers87,141 as well as murine jejunum, hepatocytes, and renal tubules.87 However, while one study found that marvelD3 knockdown enhanced steady-state TER,141 another found that marvelD3 knockdown delayed TER development but did not affect final TER.87 This difference may relate to experimental conditions or siRNA sequences and cell lines. Although both studies used Caco-2 cells, the clones employed were clearly different, and the TERs achieved varied significantly.87,141 Further analysis showed that combined knockdown of marvelD3 and either occludin or tricellulin caused a greater delay in TER development than knockdown of either protein individually.87

Despite these analyses, defining specific functions of occludin, tricellulin, and marvelD3 has been challenging. Further, data from occludin knockout animals, patients with tricellulin mutations, and siRNA knockdown cells suggest that a simple knockout/knockdown deletional approach will not be sufficient to dissect functions of the TAMPs. However, the association of occludin internalization with barrier loss induced by actin disruption84 or TNF core family cytokines45,46,89,142–144 shows a strong link between acute occludin endocytosis and barrier loss. This was recently explored using a transgenic mouse that expresses functional EGFP-occludin, as well as endogenous occludin, within the intestinal epithelium.44 While TNF-induced occludin endocytosis depletes the protein from large regions of the intestinal epithelial TJ of wildtype mice, occludin was present throughout the intestinal epithelial TJs of transgenic mice.44 This maintenance of occludin at TJs of transgenic mice was not due to failure of occludin endocytosis, but likely a result of occludin overexpression.44 The effect of TNF-α on jejunal barrier function was also attenuated and diarrhea was entirely prevented in EGFP-occludin transgenic mice.44 In parallel with this in vivo work, an in vitro study in MDCK cells also found that occludin was required for TNF-induced barrier loss.42 Mechanisms of cytokine-induced barrier loss are discussed in further detail in the following section. The previous data therefore suggest that occludin plays a regulatory, rather than a structural role. This hypothesis could explain both the compensation in occludin knockout mice and the presence of occludin endocytosis or downregulation in pathology-associated barrier loss. However, much work remains, including analysis of protein interactions mediated by occludin and functional significance of the occludin tail hyperphosphorylation that is associated with localization at the TJ and development of barrier function.145–150 Recent studies have demonstrated that the C-terminal occludin tail associates with specific kinases and phosphatases, and that these interactions are able to regulate TJ assembly and barrier function.150,151 Moreover, it is now clear that phosphorylation of specific residues within the occludin tail modifies interactions with ZO-1.87,148,149,152,153 However, the sites identified are not within the region of occludin responsible for ZO-1 binding,110,154 suggesting that phosphorylation induces conformational changes in other regions of the tail. Definition of these structural changes as well as the means by which they impact protein interactions and barrier function remains an area in need of further study.

38.4.4  Claudins The discovery of claudins in 199892 significantly advanced our understanding of the TJ barrier. Claudins were first shown to have the ability to form strands92 and

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confer cell-to-cell adhesion.92,155 Subsequent work clearly links claudins to the selective barrier properties of the TJ.18,31,41,61–63,65,69,122,156–160 Claudins are a family of tetraspan proteins, ranging from 20 to 25 kDa characterized by a conserved amino acid motif in the first extracellular loop (W-GLW-C-C). The human and mouse genomes contain at least 24 proteins.66,161,162 The puffer fish Takifugu contains 56 claudin genes, although this animal has other examples of gene expansions that lack obvious biologic significance.163 The barrier-forming junctions of invertebrates, septate junctions, are structurally quite different from TJ, thus it was surprising to find claudin-like proteins in both flies and worms and to find that they are required to form epithelial barriers.164,165 Drosophila has six claudin sequences.165–168 Several of them are expressed at the barrier-forming septate junctions and, in addition to affecting barrier function, appear to regulate size of epithelial tubes.165–167 Interestingly, claudin-15 knockout results in mice with mega-intestine23 and zebra fish with multiple gut lumens,169 suggesting that claudins regulate epithelial tube development in vertebrates as well as flies.156 The first extracellular loops of claudins are highly conserved, range from 41 to 55 residues, and appear to make a major contribution to TJ ion selectivity and barrier function.63,65,67,158,170 Site-directed mutagenesis of specific residues within the first extracellular loop of claudins can reverse the charge selectivity of the TJ pore,63,65 while inclusion of both acidic and basic residues in that region can reduce permeability to both cations and anions.69 The second loops, which are also highly conserved, range from 10 to 21 residues and appear to be more involved in homotypic and heterotypic adhesion155,171 and are also the site of Clostridium perfringins enterotoxin binding in claudins 3 and 4.172–175 The claudin cytoplasmic tails range from 21 to 44 residues and are the least well-conserved regions. With the exception of claudin-12, all claudin cytoplasmic tails end in PDZ binding motifs, which bind PDZ domains in the cytoplasmic scaffolding proteins ZO-1 and MUPP1101,176,177 and other proteins.77 PDZ-mediated interactions of claudins with ZO-1 and ZO-2 are required for efficient delivery to the TJ,122 although this may not be universally true.124 The expression of several claudins has been documented within the mouse intestine. Each claudin shows unique expression patterns along both longitudinal (duodenum–colon) and vertical (crypt–villus) axes and is also regulated during development.178,179 These are thought to confer regional gradients of size and charge selectivity, but the mechanisms and physiological implications are incompletely defined. While much work remains, recent studies have demonstrated that claudin proteins define paracellular flux of small solutes, including ions and water.41,157,180 Claudin-2

SECTION  |  III  Host Defense Mechanisms

has been studied in the greatest detail. It forms cationselective paracellular pores, and claudin-2 expression is responsible for the differences in barrier function between MDCK cells that form leaky and tight monolayers.61,62,65 Claudin-2 pores are permeable to small cations and water,69,180 and are exquisitely size selective.41,69 While poorly characterized, the nature of the pore is beginning to be defined.68 Further details of claudin function have been discussed in recent reviews.19,66,124,156,157,181,182 Consistent with the view that claudins regulate TJ permeability, mutations in claudin-16, also known as paracellin-1, prevent paracellular absorption of divalent cations in the renal tubule.25–27,183 This results in the disease familial hypomagnesemia with hypercalciuria and nephrocalcinosis, which is also associated with claudin-19 mutations (Table 38.3).24,25,184,185 Claudin-14 mutations have also been associated with deafness,186 and claudin-1 mutations have been linked to neonatal sclerosing cholangitis and ichthyosis.187 Similarly, mice lacking claudin-1 die shortly after birth from rapid evaporative water loss from the skin.64 They do not live long enough to investigate whether they recreate the biliary pathology. However, a point mutation in the claudin-binding PDZ domain of ZO-2 is the basis of Amish familial hypercholanemia.121 While specific claudin mutations have not been linked to gastrointestinal disease, inflammatory and infectious diseases are strongly associated with changes in claudin protein expression and trafficking.29,32,188–193

38.4.5  Additional Tight-junctional Proteins and Functions In addition to proteins belonging to the ZO, TAMP, and claudin families, a vast number of other proteins are found at the tight and adherens junction. These include the JAM, proteins that appear to play a role in immune cell trafficking across the epithelium and endothelium,93,194,195 cell cycle regulation, and barrier function.196,197 In the intestine, epithelial expression of JAM-1, which is also referred to as JAM-A, is reduced in inflammatory bowel disease.196 Moreover, JAM-A knockout animals have intestinal barrier defects and enhanced cell turnover in the absence of exogenous stress as well as greater sensitivity to dextran sulfate sodium (DSS)-induced colitis.196,197 Other proteins localized to the TJ include the coxsackie virus-adenovirus receptor CAR.74 Interestingly, occludin is also required for coxsackie virus entry.73 Similarly, claudin-1 and occludin collaborate with at least two other cell surface proteins to facilitate hepatitis C virus entry.198,199 Diversity of occludin sequences confer species specificity to hepatitis C virus infection.72 Further, occludin splice variants and claudin-1 polymorphisms may impact susceptibility of some human hosts to hepatitis C virus.200,201 This theme of pathogen receptors being sequestered within the tight

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Chapter  |  38  Tight Junctions and the Intestinal Barrier

TABLE 38.3  Heritable Human Diseases of Tight Junction Proteins Gene

Disease

Pathology/Mechanism

Ref.

Cldn-1

Ichthyosis and sclerosing cholangitis

Affects skin and bile ducts

187

Cldn-8

Bartter’s with hypomagnesemia

S151P mutation, unproven pathogenesis

478

Cldn-14

Non-syndromic deafness

Cochlear hair cell degeneration

186

Cldn-14

Nephrolithiasis

GWAS found intronic SNPs

479

Cldn-16

a

Human FHHNC

Defective renal Mg

2

reabsorption

27

Bovine interstitial nephritis Defective renal Mg2 reabsorption

480

Peripheral polyneuropathies

Demyelination

481

Gene deletion

Gene duplication

Charcot-Marie-Tooth Type 1A

Dejerine-Sottas syndrome mutations

ZO-2

Familial cholanemia

Defective PDZ-claudin binding

121

Tricellulin

Non-syndromic deafness

Defective ZO-1 binding

138

Decrease transcription of claudin-1 gene

482

Cldn-19

FHHNC with ocular disease Retinal development defect

PMP22 b

HNPP

Mutations with Possible Indirect Effects in TJ Proteins P63

AECc

WNK Kinase pseudohypoaldosteronism type II

Abnl claudin-4 phosphorylation

483

Cln3 and 4 are hemizygous in patients with Williams-Beuren syndrome. Reviewed in 484. a Familial hypomagnesemia hypercalciuria with nephrocalcinosis. b Hereditary neuropathy with liability to pressure palsies. c Ankyloblepharon-ectodermal dysplasia-clefting.

and adherens junctions extends beyond coxsackie virus, adenovirus, and hepatitis C. For example, Listeria monocytogenes use E-cadherin as a receptor for infection.202 Interestingly, E-cadherin is normally inaccessible, and is only exposed and available for L. monocytogenes binding at sites of cell extrusion.76,203 The TJ is also a site of robust cell signaling,204 and is home to a large number of kinases, including c-src and c-yes, both of which induce junction disassembly.86,149,205,206 Atypical PKC isoforms, including those involved in development of epithelial polarity, are also concentrated within the tight and adherens junctions.124,207 Finally, several transcription factors are located at the junction or bind to junction proteins.208–210 Each of these topics has been extensively reviewed elsewhere.

38.5  REGULATION OF INTESTINAL EPITHELIAL TJ BARRIER During the past 30 years, a number of important advances have been made to provide new insights into the regulation

of the intestinal epithelial TJ barrier. Despite these advances, the precise intracellular mechanisms involved in the regulation of the intestinal TJ barrier remain largely undefined. Common themes that have emerged in these studies are the rapid dynamic nature of the intestinal TJ barrier regulation and the intricate interaction of cytoskeletal–TJ proteins in the regulation of the intestinal TJ barrier. Alterations in TJ protein localization and expression have also been shown to be important in the modulation of the TJ barrier function, however, the precise role of the individual proteins and the specific protein–protein interactions that regulate TJ barrier function remain mostly unknown. The involvement of claudins in the formation of TJ pore pathway has been firmly established, yet the proteins that modulate the flux through the large channel pathway have yet to be resolved.18,19 This is an important area of future investigation. The intestinal TJ barrier is rapidly regulated in response to extracellular factors that activate various intracellular signaling pathways. As the fluid composition of the luminal and serosal compartments undergo continual change during

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the various digestive and interdigestive phases and vary depending on the types and amounts of food consumed, types of medication ingested, luminal bacterial composition and the bacterial load, the type and amount of digestive and proteolytic enzymes secreted into the lumen, the inflammatory state of the intestinal mucosa and the amount of proinflammatory cytokines and inflammatory mediators present, and the ionic and solute content of the luminal and serosal fluid, the intestinal TJ barrier undergoes continual change in response to the changes in the extracellular fluid composition. As the intestinal TJ barrier is modulated by wide ranging pharmacological, physiological, microbial, and inflammatory factors, in this section we discuss those factors that have historical importance (such as the cytochalasins and the luminal osmolarity) and those that have been more extensively studied and for which the mechanisms have been supported by experimental studies. The central role of myosin light chain kinase and actin–myosin contraction in the regulation of intestinal TJ barrier has been firmly established.211 For many of the intestinal TJ barrier modulating factors, MLCK-induced perijunctional actin– myosin contraction and the subsequent cytoskeletal–TJ protein interaction (possibly mediated via the actin-ZO-1 binding interaction) appears to be a common mechanism leading to the functional and morphological opening of the intestinal TJ barrier.86 In addition, recent studies have also suggested that the alterations in TJ protein expression and endocytosis of transmembrane TJ proteins are important mechanisms leading to the increase in TJ permeability.31,44,84,212,213 Major gaps in the understanding of the intracellular processes that regulate intestinal TJ barrier remain and provide an important opportunity for future investigations in this area.

SECTION  |  III  Host Defense Mechanisms

epithelial TJ permeability.82 The dual-flux studies indicated that the increased permeation through the paracellular pathways fully accounted for the increase in mannitol and Na flux.82 The cytochalasin D increase in intestinal TJ permeability was also accompanied by condensation or aggregation of microfilaments in the perijunctional actin/myosin ring, especially in the regions of multiple cellular contacts. The condensation of the perijunctional microfilaments produced a “pulse-string” type contraction of the brush borders of surface intestinal epithelial cells with a bulging or convex appearance of the epithelial surface (Figure 38.4); a decrease in number of microvilli at the junctional contact areas; and a disturbance in distribution, decrease in number, and loss of mesh-work like organization of TJ strands (Figure 38.5).82 These findings suggested that the cytochalasin D-induced increase in intestinal TJ permeability was mediated in part by a contractile tension generated by contraction of the actin/myosin ring at the level of the TJs. The cytochalasin D-induced aggregation of perijunctional microfilaments, contraction of the perijunctional actin/myosin ring, alterations in TJ strands and increase in TJ permeability were inhibited by energy depletion induced by 2,4-dinitrophenol (DNP), indicating the energy requirement in these processes.82 Similarly, cytochalasin (B and D) treatment of filter-grown intestinal epithelial monolayers Caco-2 and T84 cells also produced an acute drop in TER and an increase in epithelial permeability to paracellular markers mannitol

38.5.1  Cytochalasins and the Intestinal TJ Barrier Cytochalasins have been widely used as pharmacologic agents to examine actin-dependent cellular functions and were among the first agents to be used to study the role of cytoskeletal elements in epithelial TJ barrier modulation.81,82,214–217 Cytochalasins disrupt actin filaments by several mechanisms including direct severing of actin filaments, inhibition of actin subunit polymerization, and by inducing reactive cellular responses.82,85,218–221 The cytochalasin disruption of actin microfilaments produces a morphologic alteration in intestinal TJs and an increase in intestinal TJ permeability in both the ex vivo and in vitro intestinal epithelial systems.82,83,85,214,222,223 The cytochalasin D treatment of Ussing chamber-mounted guinea pig small intestinal tissue produced a concentration-dependent (0.1–20 μg/ml) drop in TER.82 The drop in small intestinal tissue TER directly correlated with an increase in Na and mannitol flux, confirming an increase in intestinal

(A)

(B)

(C)

(D)

FIGURE 38.4  Scanning electron micrographs.  (A, B) Vehicle control and CD-exposed (C, D) (10 ng/ml, 60 min) mucosal sheets. The three villous ridges display smooth surfaces with intermittent linear folds. As seen in (B), control villi are covered by polygonal absorptive cells with flat apical surfaces. In contrast, CD-exposed tissues display a cobblestone-like appearance of the villous surfaces (C). Higher magnification (D) shows this cobblestone effect is due to pulse-string contraction of the brush borders of individual absorptive cells resulting in a convex apical absorptive cell surface and flaring of microvilli. Bars: 20 μm. (From 82 with permission.)

Chapter  |  38  Tight Junctions and the Intestinal Barrier

and inulin (Figure 38.6).82,85,224 In contrast, the disintegration of Caco-2 microtubules with tubulin depolymerizing agent colchicine did not have any effect on Caco-2 TJ barrier function or junctional localization of TJ proteins, suggesting that intact microtubules are not required for the acute maintenance of the intestinal epithelial TJ barrier function.222 The cytochalasin B-induced increase in Caco-2 TJ permeability required metabolic energy and was rapidly reversible (within hours) following cytochalasin B removal,

(A)

(B)

FIGURE 38.5  Freeze-fracture replicas of villus absorptive cell occluding junctions.  (A) Control junctions are composed of a net-like mesh of cross-linked strands or grooves. Perijunctional microvilli are densely aligned above the junction. Bar: 0.1 μm. (Shadow angle, approximately left to right.) (B) Junction exposed to 10 μm/ml CD for 40 min. Junction is composed of an irregular array of strands that underlie occasional broad protrusions of the apical membrane (arrowheads). Geometric irregularities produced by such protrusions result in a fracture plane, which only focally includes the apical-most strand (straight arrows). Many perijunctional microvilli are lost and intramembrane particles penetrate into the incompletely isolated intrajunctional compartments (curved arrow). Bars: 0.1 μm, (Shadow angle, approximately left to right.) (From 82 with permission.)

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indicating that the cytochalasin effect was not due to a permanent cell damage or cell death but by a rapidly reversible process.85,222 The cytochalasin B-induced increase in Caco-2 TJ permeability also correlated with sequential changes in perijunctional actin and myosin filaments (Figure 38.7). In Caco-2 monolayers, actin and myosin filaments are localized in a belt-like manner surrounding the apical junctional area. Cytochalasin treatment produces a rapid energy-independent severing of actin filaments into small fragments (early phase response). Within seconds of cytochalasin B treatment, the perijunctional actin filaments are severed and become fragmented and are present diffusely in the cytoplasm at the level of the TJs (Figure 38.7B). This early phase fragmentation is then followed by an energydependent process in which the severed actin fragments reorganize to form large cytoskeletal aggregates containing actin and myosin filaments (late phase response).85,221 Within 15 to 30 minutes of cytochalasin B treatment, the fragmented actin and myosin filaments coalesce to form large cytoskeletal clumps or “foci” near the perijunctional areas (Figure 38.7D). It is likely that these macrocytoskeletal aggregates also contained TJ proteins; however, direct evidence for this is lacking.85 Similar effects of cytochalasin D on actin filament breakage and cytoskeletal clump formation (containing actin, myosins, and tropomyosins) were also demonstrated in the African green monkey kidney cells (BSC1cells).221 As actin–myosin contraction in smooth muscle and other cell types is mediated by MLCK activation, the possibility that the cytochalasin-induced increase in Caco-2 TJ permeability was also mediated by MLCK-activated actin–myosin interaction was considered.85 The mechanism of MLCK-induced activation of actin–myosin contraction is discussed in detail in Chapter 17. In brief, MLCK catalyzes MLC phosphorylation leading to the activation of Mg2-myosin ATPase, which hydrolyzes ATP to generate the mechanical energy needed for the actin–myosin contraction. The cytochalasin-induced alteration in perijunctional actin and myosin filaments and increase in Caco-2 TJ permeability were accompanied by an increase in MLCK activity (Figure 38.8).85 The inhibition of cytochalasin induced MLCK activation by MLCK inhibitors (ML-7, ML-9, KT-5926) prevented the late phase cytoskeletal aggregation and the increase in Caco-2 TJ permeability, but not the early phase severing of actin filaments, suggesting that MLCK activation mediated the aggregation of the cytoskeletal filaments and the subsequent increase in Caco-2 TJ permeability.85 The cytochalasin severing of actin filaments has been proposed to be the triggering event for the MLCK activation.85 Consistent with this possibility, villin-induced severing of actin filaments has been shown to induce Mg2-myosin ATPase activity and actin–myosin interaction.225 The

1056

SECTION  |  III  Host Defense Mechanisms

(A) Resistance (ohm-cm2)

380 Control 340

300

Cyto-B

260 0

2

380

6

8 10

340

300

Cyto-B

260

220

0

20

40

Cyte-B

(B) 0.008

Control

Mannitol flux (nmol/cm2)

Resistance (ohm-cm2)

4

Time (min)

60

Time (min)

0.007

0.006 Control 0.005

0.004

0

20

40 Time (min)

60

FIGURE 38.6  Effect of Cyto B (5 µg/ml) on Caco-2 epithelial resistance and paracellular permeability.  (A) Cyto B (5 µg/ml) effect on Caco-2 epithelial resistance expressed as Ω  cm2. Inset: Magnified view of the early time course. (B) Cyto B (5 µg/ml) effect on mucosal-to-serosal flux of paracellular marker mannitol expressed in nmol/cm2. Values are means   SE; n  4. (From 85 with permission.)

FIGURE 38.7  Effect of cytochalasin B (Cyto B) (5 µg/ml) on perijunctional Caco-2 actin microfilaments.  Caco-2 F-actin filaments were labeled with fluorescein-conjugated phalloidin. The sequential effect of Cyto B (5 µg/ml) on Caco-2 actin microfilaments at time 0 (A) and 1 (B), 15 (C), and 30 minutes (D) is shown in the photomicrographs (original magnification   80). By 1 minute of Cyto B exposure, perijunctional actin filaments were fragmented and present diffusely throughout the cytoplasm. By 15–30 minutes of Cyto B exposure, actin fragments coalesced to form large actin clumps or “foci” near the perijunctional areas. (From 85 with permission.)

requirement of metabolic energy and mechanical contraction of actin–myosin filaments in the TJ barrier opening was supported by the studies showing that Mg2-myosin ATPase inhibitor (2,3-butanedione monoxime) and metabolic inhibitors (DNP) inhibit the cytoskeletal clump formation (late phase response), alteration in junctional localization of ZO-1 proteins, and the increase in TJ permeability, but not the severing of the actin filaments (early phase response). Together, these studies suggested that

the cytochalasin-induced increase in intestinal TJ permeability was mediated by cytochalasin-induced activation of MLCK, which causes aggregation of perijunctional microfilaments and the contraction of the perijunctional actin/ myosin ring, which in turn leads to a pulse-string type contraction of the cell membrane and centripetal tension-generated opening of the intestinal TJ barrier.82,85 The cytochalsasin D depolymerization of actin also causes a disturbance in junctional localization of TJ protein

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Chapter  |  38  Tight Junctions and the Intestinal Barrier

20 MLC 0′

2′

5′

10′

Water flux (ml/100 cm-hr)

P

30′

FIGURE 38.8  Effect of Cyto B on Caco-2 myosin light chain kinase (MLCK) activity.  Caco-2 monolayers were exposed to Cyto B for increasing time periods (0–30 minutes). Subsequently, Caco-2 monolayers were lysed, and Caco-2 MLCK was immunoprecipitated. The activity of the immunoprecipitated MLCK was determined by in vitro kinetic measurement of MLC phosphorylation. Phosphorylated MLC (P-MLC; ~19.5 kDa) was separated by 10% SDS-PAGE, stained with Coomassie blue solution, and autoradiographed. Cyto B produced a time-dependent activation of Caco-2 MLCK with the peak activation occurring between 5 and 10 min after Cyto B exposure. (From 85 with permission.)

y = 23.0–0.05x

r = 0.98

10

0

–10 100

300 500 Luminal osmolarity (mosmol/l)

700

FIGURE 38.9  Relationship between small intestinal luminal osmolarity and water flux. (From 227 with permission.)

occludin, characterized by internalization and disassembly of occludin from junctional areas and loss of continuity in occludin junctional localization.84,226 Latrunculin A, an actin depolymerizing agent, also caused a disturbance in junctional localization of occludin and internalization of occludin in the kidney-derived cell line MDCK.84 Live cell imaging coupled with simultaneous recordings of barrier function showed that occludin internalization was temporally related to barrier loss.84 The latrunculin A-induced occludin internalization was mediated by caveolin-1 and dynamin II-dependent endocytosis, and inhibition of caveolae-mediated endocytosis prevented the latrunculin A-induced occludin internalization and drop in MDCK TER.84 These data suggested that latrunculin-induced actin depolymerization induces caveolae-medi ated endocytosis of TJ proteins, and that internalization of transmembrane TJ proteins leads to the loss of TJ barrier function. The actin depolymerization-induced activation of MLCK is presumably also involved in the endocytosis of the TJ proteins.226

38.5.2  Luminal Osmolarity and Solvent Drag Effect The relationship between luminal osmolarity, intestinal water flux, and intestinal paracellular permeability has been extensively examined in vivo by recycling perfusion of isolated rat small intestinal segment with perfusate solutions that have varying osmolarity.222,227–231 The rate of intestinal absorption of various sized paracellular markers including mannitol, PEG 400, PEG 900, and inulin was linearly related to the increasing concentration of permeability markers, consistent with a passive uptake mechanism222,227,228,230–232 In the in vivo rat intestinal perfusion studies, changing the luminal perfusate pH (from 6.0 to 7.5), varying the unstirred water layer resistance by increasing the luminal flow rate (from 1 to 3 ml/min), or disruption of the mucous layer by treatment with mucolytic agent acetylcysteine did not affect the intestinal flux rates of paracellular markers.222,227,230–232

In a leaky or low resistance epithelia such as the small intestine, the junctional or paracellular pathway is the major permeation pathway for the passive water and ionic flux.222,227,231,233 The passive water flux may be bidirectional and the direction of the net water flux may be regulated by the differences in osmotic gradient or hydrostatic pressure across the intestinal epithelial barrier. A linear relationship exists between decreasing luminal osmolarity and increasing paracellular water flux. The contribution of solvent drag on the absorption of paracellular markers may be assessed by manipulating the water flux by changing the osmolarity of the luminal perfusate solution.227,232,234 Decreasing the luminal osmolarity from 600 mOsm to 225 mOsm results in an osmotic gradient-dependent mucosal-to-serosal water flux (Figure 38.9).227,231,232 As the water moves rapidly across the paracellular or junctional pathways, hydrophilic solutes in the luminal solution are also carried along via a solvent drag.227,232,234,235 There is a direct correlation between increasing intestinal water flux and increasing flux of the hydrophilic solutes via the solvent drag (Figure 38.10). The relative contribution of diffusive and convective (or solvent drag effect) components to the passive transport of permeability probes and solvent drag reflection coefficient σf (an indicator of dependency on solvent drag effect) may be calculated using Fick’s law and modified KedemKatchalsky equation of solvent drag effect.234,235 Fick’s first law expresses the flux rate JD at which a given solute diffuses across a semipermeable membrane as diffusional coefficient, PD. 

PD = −J D /(C2 − C1 ),

where C1 and C2 are concentrations of solute on the luminal and serosal side, respectively. The solute flux across the semipermeable epithelium by solvent drag is expressed quantitatively by a modified Kedem-Katchalsky equation.235

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SECTION  |  III  Host Defense Mechanisms

Inulin flux (101 nmol/100 cm-hr)

8

y = 3.11 + 0.24x

r = 0.97

4

0 –10

0 10 Water flux (ml/100 cm-hr)

20

FIGURE 38.10  Relationship between jejunal water flux and inulin flux (n3–9 rats).  Jejunal water flux was varied by changing osmolarity of luminal perfusate. Plotted values are mean of water and inulin absorption. (From 227 with permission.)



J SD = J V (1 − σf )[(C1 + C2 ) /2],

where JSD is the solute flux rate by solvent drag, (1  σf) is the coefficient of solvent drag, JV is the solvent flow rate, and σf is the solvent drag reflection coefficient. Combining the these two equations provides an equation describing the relative contribution of diffusion and solvent drag to the net passive solute flux, JS, across a porous epithelium. 

J S = J D + J SD

 J S = −PD ∆ C + J V (1 − σf )[(C1 + C2 ) /2] Using the combined equation and the solute and solvent flux data obtained during changes in luminal osmolarity, the relative contribution of diffusion (JD) and solvent drag (JSD) on total passive solute flux (JS) may be determined.234,235 At luminal perfusate osmolarity of 300 mOsm, solvent drag accounted for approximately 60% of the total small intestinal flux for PEG 400 and mannitol.230,232 Compared to the smaller permeability markers, solvent drag contribution for inulin flux was much less and accounted for only 10–15% of the total small intestinal flux.227 The physiological contribution of solvent drag to the luminal-to-serosal solute flux is continually changing as the intestinal luminal osmolarity changes depending on the luminal osmotic load and the fluid secretory states following and in between meals. The close dependence of intestinal flux of hydrophilic solutes on solvent drag suggested that the changes in the direction and the extent of water flow could have a

significant impact on intestinal permeability. Consistent with such a possibility, exogenously added or endogenously produced secretagogues significantly affected the intestinal flux rates of the paracellular markers by altering the water flux rates.227,228,230 The addition of a known fluid secretagogue 16, 16-dimethyl PGE2 to the luminal perfusate solution causes a marked decrease in water absorption and a corresponding decrease in solute flux rates.227,228 Conversely, addition of cyclooxygenase inhibitors, which inhibit the endogenous production of prostanoids, caused an increase in water absorption and PEG 400 flux.227,228 Similarly, other fluid secretagogues including dibutyryladenosine-3, 5-cyclic monophosphate, aminophylline, taurocholic acid, and chenodeoxycholic acid also caused a net decrease in intestinal water absorption and a decrease in luminal-to-serosal flux of the paracellular markers.227,228,230,231,236 Thus, pharmacologic agents and endogenously produced water secretagogues, which affect water flux, have an important modulating affect on intestinal permeability by affecting the solvent drag effect. The potential clinical relevance of solvent drag in absorption of water-soluble drugs was suggested by studies in which the administration of atenolol and hydrochlorothiazide in high osmotic solution significantly decreased the intestinal absorption of these drugs.237 Correlating with the in vivo studies, the exposure of the mucosal surface of guinea pig jejunum mounted in Ussing chamber to a high osmotic load (600 mOsm) caused a 64% increase in TER.81 The mucosal exposure to high osmotic load caused a collapse of the paracellular spaces, focal subjunctional lateral membrane evagination, and an increase in intestinal TJ strand count and depth on freezefacture analysis (Figure 38.11).81 The observed morphological changes were consistent with the enhancement of the intestinal TJ barrier function. Similarly, in a Necturus gallbladder tissue mounted in Ussing chamber, increasing the osmotic load of the mucosal bathing solution caused a net increase in serosal-to-mucosal water flux with collapse of the intercellular spaces.238 Conversely, decreasing the mucosal osmotic load caused a net increase in mucosalto-serosal water flux with dilatations of intercellular space and focal separation of TJs.238 Increasing paracellular water absorption leads to water engorgement of intercellular spaces including the space between the TJs, presumably causing a physical separation of the TJ barrier.238,239 The precise mechanisms involved in the separation of TJ barrier during fluid absorption need further clarification. Further evidence of physiological importance of solvent drag effect on paracellular permeation of watersoluble molecules stems from the in vivo studies comparing small intestinal and colonic permeability to paracellular markers PEG 400, PEG 900, mannitol, and inulin.222,227–232 These in vivo perfusion studies have consistently shown the colon to be more permeable than the

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Chapter  |  38  Tight Junctions and the Intestinal Barrier

small intestine.222,227–232 This was a somewhat surprising observation given that the TER of the Ussing chamber-mounted colonic tissues has a higher TER than small intestinal tissues (Table 38.1), and the TJ barrier would have been expected to be less permeable in the colonic epithelium. In the in vivo perfusion studies, the water flux rates are markedly higher (about 5- to 6-fold higher) in the colon than in the small intestine under the same perfusate and osmotic conditions,222,227–232 and the contribution of solvent drag in the colonic absorption of permeability probes is also markedly greater than in the small intestine, accounting for about 90% of the PEG 400 absorption.222,227–232 Thus, these studies serve as an important example demonstrating that intestinal TJ permeability is not strictly regulated by the relative tightness of the TJ barrier as assessed by functional parameters such as TER but by other factors including solvent drag effect.

38.5.3  Na-nutrient Cotransport and Physiological Regulation

FIGURE 38.11  Freeze-fracture replicas of absorptive-cell TJs.  (A) TJ from control mucosal sheets displaying uniform depth and composition. (B and C) TJs from mucosal sheets exposed to 600 mOsm mucosal buffer for 20 minutes. Focally, junctional depth and strand counts (arrowheads) are large. In addition, the perijunctional apical membranes (asterisk) associated with these junctional areas are bulging and relatively devoid of microvilli. (From 81 with permission.)

In 1987, Pappenheimer and co-workers advanced a novel concept that the activation of Na-nutrient cotransport induces a physiologically regulated modulation of the intestinal TJ barrier.83,233,235 In three accompanying papers, these investigators provided the initial experimental evidence demonstrating that activation of Na-nutrient cotransport results in a physiologically regulated opening of the intestinal TJ barrier and an increase in paracellular flux of hydrophilic solutes.83,233,235 These studies formed the foundation for the subsequent studies aimed at delineating the intracellular mechanisms that mediate the physiologically regulated modulation of the intestinal TJ barrier.239–245 In these studies,83,233,235 the perfusion of isolated rat or hamster intestinal segments with perfusate solution containing glucose or amino acids (alanine, or leucine) (25 mM) produced a significant decrease in the junctional resistance, increase in paracellular permeability, and increase in water flux. It was calculated based on the rate of water absorption, clearance of the paracellular markers, and coefficient of osmotic flow (Lp) that the solvent drag through the junctional or paracellular pathways was the principle mechanism of intestinal absorption of glucose or amino acids in the small intestinal lumen, when the luminal concentrations of glucose and amino acids exceed the maximal transcellular uptake by the active transporter.83,233,235 The active transport of glucose via the transcellular pathways reaches its maximal capacity (Vmax) at luminal glucose concentration of 10–15 mM.235,240 However, intestinal absorption of glucose continues to increase with increasing luminal glucose concentration above 15 mM via the solvent drag through the paracellular pathway.240,246,247 It has been estimated that between 60 to 90% of the glucose transport in the small intestine following a normal

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meal where the luminal glucose concentration range between 50 to 300 mM may be mediated through the solvent drag.235,240,241 The true contribution of solvent drag and paracellular pathways in glucose transport remains controversial and some investigators have argued that the contribution of paracellular transport is much smaller than that proposed by Pappenheimer.248,249 The effect of luminal glucose on junctional resistance in hamster small intestine has been estimated by steadystate transepithelial impedance measurements.235 The addition of glucose or amino acids (alanine or leucine) to the luminal perfusate produced a 2- to 3-fold decrease in the junctional resistance as estimated by the impedance measurements.235 The decrease in junctional resistance was associated with an increase in junctional flux of water-soluble solutes including glucose and amino acids and paracellular markers inulin and mannitol.235 Using ferrocyanide as a non-absorbable marker, it was estimated that about 50% of fluid absorption at 25 mM luminal glucose concentration was paracellular. From these studies, it was concluded that solvent drag was a major mechanism of intestinal absorption of water-soluble nutrients (including glucose and amino acids) when the luminal glucose or amino acid concentrations were sufficiently high.235 It has been proposed by Pappenheimer and co-workers that the Na-coupled transport of solutes from the intestinal lumen to the intercellular space produces an osmotic gradient across the intestinal TJ barrier, leading to an osmotic gradient-generated bulk water flow through the junctional pathway with accompanying solvent drag of hydrophilic solutes.235,240,241 In addition to the functional changes in the intestinal TJ barrier, luminal glucose or amino acid also causes a morphologic change in the cellular structure, which correlates with the functional changes in the TJ barrier.235,240,241 The addition of glucose or amino acid to the luminal perfusate solution results in a distension of the intercellular space and formation of focal TJ dilatations.83 Accompanying the changes in the intercellular space are large protuberances or dilatations within the TJ strand meshwork and decrease in number of TJ strands, morphologically correlating with the observed decrease in TJ barrier function.83 The glucose-induced decrease in TJ barrier function and anatomic alterations in TJs were also accompanied by “condensation” of perijunctional actin/myosin ring indicating alteration in cytoskeletal components.83 It was proposed that the activation of Na-coupled nutrient transport induces a contraction of the perijunctional acto-myosin ring, resulting in a contractile tension generated by the pulling apart of the apical membrane and the TJ complex and a functional opening of the TJ barrier.83,240,241 The role of Na-glucose cotransporter activation in the luminal glucose induced increase in intestinal TJ permeability was demonstrated by the studies showing that

SECTION  |  III  Host Defense Mechanisms

a specific inhibitor of Na-glucose cotransporter phlorizin inhibits the glucose-induced increase in paracellular permeability and morphologic alterations in TJs.243,250 In addition, replacing the Na in the luminal solution with choline also prevented the increase in intestinal TJ permeability and morphologic alterations.243,250 These studies indicated that the activation of Na-nutrient cotransporter was the initiating event leading to the physiological regulation of the intestinal TJ barrier. In subsequent studies the mechanisms that mediate the Na-glucose cotransport-activated modulation of perijunctional actin/myosin filaments and intestinal TJ barrier were further delineated.57,251–255 Using an in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial cells, Turner et al. showed that the Caco-2 cells stably transfected with DNA encoding the intestinal Na-glucose cotransporter SGLT1 exhibited a physiological Na-glucose cotransport.254 The activation of Na-glucose cotransport by luminal addition of glucose (25 mM) produced a fall in transepithelial resistance in the SGLT1-transfected cells, and the addition of phlorizin (a Na-glucose cotransport inhibitor) caused an increase in TER.57 The activation of SGLT1 produced a twofold increase in MLC phosphorylation, and MLCK inhibitors ML-7 and ML-9 prevented the SGLT1-mediated drop in TER. In aggregate, these studies suggested a causal relationship between SGLT1-mediated MLCK activation and subsequent MLC phosphorylation and actin–myosin contraction-mediated increase in intestinal TJ permeability.57 Further insights into the mechanism of Na-nutrient cotransport modulation of intestinal TJ barrier were derived from enterocyte cell volume studies.256,257 Using villous epithelial cells isolated from the guinea pig jejunum, the effect of Na-nutrient on enterocyte cell volume was studied.256,257 There is an initial increase (within 30 seconds) in cell volume following activation of Naglucose transport, which is followed shortly by rapid cell shrinkage (complete in 2 minutes). Using a modest hypotonic dilution (5–7%) to induce an increase in cell volume, it was found that after an initial cell acidification, the cell shrinkage was accompanied by a rapid increase in pHi (or cell alkalinization).256,257 The alkalinization and cell shrinkage response was blocked by an inhibitor of Na-H exchanger (NHE) isoform 1 (NHE1) 5-(N-methyl-Nisobutyl) amiloride, suggesting that cell alkalinization and shrinkage required NHE activation.257 In subsequent studies, Turner and Black examined the role of Na-H exchangers in Na-glucose cotransport-induced increase in intestinal epithelial TJ permeability in SGLT1 transfected Caco-2 cells.253 The activation of Na-glucose cotransport produced an increase in pHi. The increase in pHi was inhibited by a preferential NHE3 inhibitor, S-3226, but not HOE-694 (a preferential NHE1 and NHE2 inhibitor), suggesting that NHE3 was the NHE isoform responsible for

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Chapter  |  38  Tight Junctions and the Intestinal Barrier

the alkalinization response following the initiation of Na glucose transport.253 Preferential NHE3 inhibitors were also able to inhibit Na-glucose cotransport-induced TER decreases.258 NHE3 inhibition also prevented Na-glucose cotransport-induced MLC phosphorylation, suggesting a role in the modulation of MLCK activity. These studies indicated that NHE3 activation mediates the Na-glucose cotransport modulation of cell alkalinization and shrinkage, and the subsequent activation of MLCK and the alteration in intestinal TJ barrier.251,253 Further studies have shown that Na-glucose cotransport activates Na-H exchange by inducing translocation of NHE3 from cytoplasmic vesicles to the apical membrane. P38 MAP kinase, Akt-2, and the cytoskeletal linker ezrin are all required for this process.58,255,259 Inhibition of P38 MAP kinase inhibited both ezrin phosphorylation and NHE3 activation. Expression of dominant negative ezrin also inhibited NHE3 activation but did not inhibit the P38 MAP kinase activation, suggesting that P38 MAP kinase mediated the activation of ezrin phosphorylation and ezrin activation mediated the NHE3 recruitment and activation.255 The ezrin phosphorylation appeared to be mediated by P38 MAP kinase activation of Akt-2259 as Na-glucose activation of Akt-2 was inhibited by P38 MAP kinase inhibitors. The in vitro phosphorylation studies demonstrated that the activated Akt-2 directly phosphorylates ezrin at threonine 567 in an ATP-dependent manner.259,260 These studies show that Na-glucose cotransport-induced activation of NHE3 is mediated by P38 MAP kinase activation of Akt-2, which in turn phosphorylates ezrin (a cytoskeletal linker protein), which is involved in the membrane recruitment and activation of NHE3. Na-glucose cotransport-induced NHE3 activation has now been demonstrated in murine jejunum, where both Akt activity and the adapter protein NHERF2 are required for NHE3 translocation to the brush border.261 These data suggest that SGLT1 is a master regulator, as NHE3-dependent sodium absorption adds to that mediated by SGLT1 to further enhance paracellular water absorption142,261 The clinical significance of this cannot be underestimated. For example, in the mouse model, Naglucose cotransport-induced NHE3 activation corrects cholera toxin-induced defects in sodium absorption, suggesting that this contributes to the efficacy of oral rehydration therapies.261–263 Further NHE3 has been demonstrated to provide some of the driving force for dipeptide transport.264,265 Thus, SGLT1 is able to induce both transcellular ion and nutrient transport and increase paracellular permeability, thereby enhancing both passive and active water and nutrient absorption. To demonstrate the potential clinical relevance of Naglucose transport on intestinal permeability in humans, the effect of glucose (277 mM) on intestinal permeability was assessed using creatinine as the paracellular marker.258 The

oral administration of 200 ml of 277 mM glucose (vs. mannitol) solution containing 0.8% w/v creatinine as the paracellular marker caused a significant increase in urinary recovery of creatinine (55  4% vs. 38  9% creatinine recovery for glucose vs. mannitol-containing solution, respectively), indicating that high concentrations of luminal glucose cause an increase in intestinal permeability in humans. In sum, the activation of Na-glucose cotransporter results in an enterocyte uptake of Na and glucose. This leads to MAPKAP2 and P38 MAPK activation. In turn, these activate Akt-2, which phosphorylates ezrin to drive NHERF2-mediated NHE3 translocation to the brush border. NHE3 activation is also involved in downstream activation of MLCK, which leads to MLC phosphorylation and actin–myosin contraction and contractile forcegenerated retraction of the apical membrane and the TJ complex, culminating in the opening of the intestinal TJ barrier. Na and glucose transport into the intercellular space via Na/K ATPase and by facilitated diffusion of glucose leads to an increase in Na and glucose concentration in the intercellular space. This is amplified by NHE3. Increasing Na and glucose concentrations results in an osmotic gradient across the TJ barrier and an osmotic gradient-driven mucosal-to-serosal bulk water flux and flux of hydrophilic molecules by solvent drag effect. The bulk water flux into the intercellular space causes a distention and dilatation of the intercellular space, presumably causing a mechanical separation of the TJ barrier. Thus, the Na-glucose cotransport-regulated opening of the intestinal TJ barrier appears to be mediated by the mechanical tension generated internally by MLCK-activated actin– myosin contraction and possibly by the mechanical force generated externally by the osmotic gradient-induced water influx into the intercellular space causing physical separation of the TJ seal.

38.5.4  Cytokines In addition to their known effects on modulation of the immune system, cytokines and other inflammatory mediators may also modulate intestinal inflammation by their regulatory actions on intestinal TJ barrier. Number of cytokines and inflammatory mediators including TNF-α, IFN-γ, IL-1β, IL-4, IL-6, IL-12, IL-13, insulin, insulin-like growth factor, and hepatocyte growth facor (HGF) induce an increase in intestinal TJ permeability, while some anti-inflammatory cytokines and growth factors including IL-10, TGF-β, and epidermal growth factor (EGF) appear to have a protective action in maintaining the intestinal TJ barrier function.56,212,266–314 Since the first report by Madara and Stafford showing that IFN-γ causes an increase in T84 epithelial TJ permeability,313 a number of investigators have shown that a wide variety of cytokines

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and inflammatory mediators also affect the intestinal TJ barrier.46,56,59,143,266–312,315,316 The cytokine-induced increase in intestinal TJ permeability has been proposed as an important mechanism contributing to intestinal inflammation by allowing increased paracellular permeation of luminal antigens.128,196,197,254,268,283,284,313,317 Recent studies have begun to unravel some of the intracellular mechanisms involved in cytokine modulation of the TJ barrier. However, much remains unknown and the studies have been mostly limited to just a few cytokines, namely TNFα, IFN-γ, and the IFN-γ/TNF-α combination. As most of the advances into the mechanisms of cytokine modulation of intestinal TJ barrier have been related to IFN-γ or TNFα, the focus of this section will be on the mechanisms that mediate IFN-γ or TNF-α modulation of intestinal TJ barrier. The readers are referred to other reviews for coverage of other cytokines.212,314 During the past 10 years, there have been several important advances that have provided new insight into the mechanisms involved.56,59,268,318 These studies have established transcriptional and enzymatic MLCK regulation as a central mechanism in TNFα-induced modulation of TJ barrier function.45,56,59,85,211 In addition, cytokine-induced endocytosis of transmembrane TJ proteins has been suggested to play an important role. In the initial report by Madara and Stafford,313 IFN-γ did not have an acute effect on T84 epithelial TJ permeability; instead it caused a delayed increase in T84 TJ permeability at the 72 hour time period.313 The delayed effect of IFN-γ on T84 TJ permeability suggested that the IFN-γ effect was not due to an acute intracellular signaling regulation of the T84 TJ barrier but related to an alteration in protein expression.313 Additionally, the IFN-γ-induced increase in T84 TJ permeability was not related to cell death or epithelial damage causing large epithelial gaps, but due to a reversible alteration in T84 TJ barrier function.310,313 The IFN-γ decrease in T84 TJ barrier function was associated with an accelerated degradation of TJ protein ZO-1, a decrease in ZO-1 protein synthesis, and a disturbance in the junctional localization in ZO-1 proteins.310 The alteration in ZO-1 localization was also closely linked to the perturbation in the apical actin organization, suggesting the possibility that actin alteration may be involved in the IFN-γ induced TJ barrier modulation.310 The IFN-γ-induced increase in T84 monolayer permeability was also characterized by a proportionally greater increase in flux of larger sized PEG molecules than smaller sized PEG molecules, suggesting that IFN-γ preferentially affects the flux through the non-restrictive, large channel pathway.46 Nusrat and colleagues, in a series of studies, reported that the IFN-γ increase in T84 TJ permeability was mediated by endocytosis of transmembrane TJ proteins occludin, JAM-A, and claudin-1.270,319,320 They showed that the endocytosis of TJ proteins was via macropinocytosis and

SECTION  |  III  Host Defense Mechanisms

did not involve clathrin- or caveolae-mediated endocytosis.319 The macropinosomes containing transmembrane proteins were delivered to early recycling endosomes and processed. The IFN-γ-induced endocytosis of TJ proteins was dependent on Rho-associated kinase (but not MLCK) induced phosphorylation of MLC.320 These studies suggested that the IFN-γ effect on TJ barrier was due in part to Rho-associated kinase-induced phosphorylation of MLC, leading to the macropinocytosis of transmembrane TJ proteins.320 They also reported that the IFN-γ increase in TJ permeability was not due to an increase in T84 apoptosis or cell death.270 The IFN-γ-induced increase in Caco-2 TJ permeability also correlated with an activation of PI3-kinase pathway and a decrease in occludin expression.321 Although both PI3-kinase and STAT-1 pathways were activated by IFN-γ, only PI3-kinase inhibition prevented the increase in TJ permeability.300,321,322 The IFN-γ decrease in occludin expression and increase in Caco-2 TJ permeability required PI3-kinase pathway activation of NF-κB,321 suggesting the possibility that the PI3-kinase pathway-mediated decrease in occludin expression contributed to the loss of TJ barrier function. Other recent studies have also suggested a role for AMP-activated protein kinase in IFN-γ-induced barrier regulation.323 TNF-α is a prototypical cytokine that has been shown to play a central role in intestinal inflammation of Crohn’s disease and other inflammatory conditions.318,324 The importance of TNF-α in intestinal inflammation of inflammatory bowel disease has been well validated by clinical and animal studies showing the effectiveness of anti-TNF-α antibody in the treatment of severely active Crohn’s disease and in animal models of intestinal inflammation.318,324–327 TNF-α-induced alteration of intestinal TJ barrier has been proposed as an important proinflammatory mechanism.58,89,268,283,284,324 TNF-α at physiologically relevant concentrations (1–10 ng/ml) causes an increase in intestinal epithelial TJ permeability in both in vivo and in vitro intestinal epithelial model systems (including Caco-2, T84, and HT29 intestinal epithelial cells; Figure 38.12).46,56,59,268,270,291,293,298,304,306 The TNF-αinduced increase in Caco-2 TJ permeability was shown to be associated with an increase in NF-κB activation and nuclear translocation of NF-κB p65, and the inhibition of NF-κB activation prevented the TNF-α-induced increase in Caco-2 TJ permeability. The NF-κB inhibitors also prevented the TNF-α-induced downregulation of ZO-1 protein expression and disturbance in junctional localization (Figure 38.13). These studies indicated that the TNFα-induced increase in Caco-2 TJ permeability required NF-κB activation.268 TNF-α-induced increase in intestinal TJ permeability also required prolonged exposure, suggesting that the TNF-α modulation of TJ barrier was mediated by new protein synthesis.56,59 Since MLCK has been shown to play a

Chapter  |  38  Tight Junctions and the Intestinal Barrier

central role in a wide variety of pharmacologic- and physiologic-induced alteration in intestinal TJ barrier, the role of MLCK protein expression was considered. TNF-α caused a time-dependent increase in Caco-2 MLCK protein expression, which correlated sequentially with the increase in Caco-2 paracellular permeability to mannitol and inulin and a decrease in Caco-2 TER (Figure 38.14). The increase in MLCK protein expression also correlated with an increase in MLCK activity. The inhibition of TNF-α increase in MLCK protein expression by cycloheximide

Epithelial resistance (ohm-cm2)

500

*

400

300

*

*

*

50

100

200 0

1

10

TNF-α concentration (ng/ml) FIGURE 38.12  Effect of increasing concentrations of TNF-α (0, 1, 10, 50, and 100 ng/ml) on Caco-2 epithelial resistance (Ωcm2).  Filtergrown Caco-2 monolayers were treated with TNF-α for the 48 hour experimental period. TNF-α produced a concentration-dependent decrease in Caco-2 epithelial resistance. Data represent means SE of epithelial resistance (n  4). *P  0.01 vs. control (0 hours). (From 268 with permission.)

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prevented the increase in MLCK activity and Caco-2 TJ permeability.56 Additionally, inhibition of MLCK activation by MLCK inhibitors ML-7 and ML-9 also prevented the TNF-α increase in Caco-2 TJ permeability, indicating the requirement of MLCK activation in the TNF-α increase in Caco-2 TJ permeability.56 Consistent with the role of MLCK-mediated actin–myosin contraction in the Caco-2 TJ barrier opening, Mg2-myosin ATPase and metabolic energy inhibitors also prevented the TNF-α increase in Caco-2 TJ permeability. These studies demonstrated that the upregulation of MLCK protein expression and subsequent MLCK activation mediated the TNF-α increase in Caco-2 TJ permeability.56 This process was further validated in vivo, where systemic T-cell activation induces diarrhea, in both mice and humans, by a TNF-αdependent mechanism.45,328–330 Detailed in vivo analysis of jejunal transport after T-cell activation showed that reductions in barrier function, as assessed by serum albumin leakage into the gut lumen, accompanied net fluid secretion. Both barrier loss and diarrhea could be blocked by MLCK inhibition using either a specific peptide or long MLCK knockout mice. Thus, MLCK is required for TNFα-induced barrier loss in vitro and in vivo. In subsequent studies, the molecular mechanisms involved in the TNF-α increase in Caco-2 TJ permeability were also investigated.331,332 To delineate the molecular mechanisms involved in the TNF-α-induced upregulation of MLCK protein expression, the MLCK promoter region was identified and cloned and the functional activity of the promoter validated.331,332 It was shown that the TNF-α increase in MLCK protein expression was associated with an increase in MLCK promoter activity and an increase in mRNA transcription, and inhibition of MLCK mRNA transcription (by actinomycin D) prevented the increase in Caco-2 TJ permeability.224,331,332 These studies indicated that the TNF-α-induced increase in MLCK protein expression, MLCK activity, and increase in Caco-2 TJ permeability were mediated by an increase in MLCK promoter

FIGURE 38.13  Effect of TNF-α on junctional localization of ZO-1 proteins.  The junctional localization of ZO-1 proteins in filter-grown Caco-2 monolayers was assessed by immunofluorescent antibody labeling. (A) Untreated or control Caco-2 monolayers. (B) TNF-α treatment for 48 hours. (C) Curcumin and TNF-α treatment for 48 hours. (D) Triptolide and TNF-α treatment for 48 hours. TNF-α produced a progressive disturbance in ZO-1 protein localization at the cellular borders, with disruption in ZO-1 continuity and gap-like appearance at the points of multiple cell contacts (B, arrows). Curcumin and triptolide prevented the TNF-α-induced alteration in junctional localization of ZO-1 proteins. (From 268, with permission.)

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SECTION  |  III  Host Defense Mechanisms

(A)

MLCK β-actin Time (hrs)

0.5

1

12

24

48

(c) 5

600

500

400

300

6

Mannitol flux (nmol/h-cm2)

Epithelial Resistance (ohm-cm2)

(B)

0

0

24 Time (h)

48

4

3

2

1

0

0

24 Time (h)

48

FIGURE 38.14  Time-course effect of TNF-α on Caco-2 myosin light chain kinase (MLCK) protein expression, transepithelial resistance (TER), and paracellular permeability.  Filter-grown Caco-2 monolayers were treated with TNF-α (10 ng/ml) for increasing time periods (0–48 hours). The Caco-2 MLCK protein expression was determined by Western blot analysis. The effect of TNF-α (10 ng/ml) on Caco-2 TER and mucosal-to-serosal flux of paracellular marker mannitol (10 µmol/ml) were measured sequentially over the 48 hour experimental period. (A) Time-course effect of TNF-α on Caco-2 MLCK protein expression (β-actin was used as an internal control for protein loading). (B) Time-course effect of TNF-α on Caco-2 epithelial resistance (means   SE, n  4). (C) Time-course effect of TNF-α on mucosal-to-serosal mannitol flux (means  SE, n  4). (From 56 with permission.)

activity and MLCK transcription.331,332 Using the deletion constructs generated from the full-length MLCK promoter region, the minimal promoter region and the molecular determinants responsible for the increase in MLCK activity were also identified.331,332 Ye et al. reported that there were 8 κB binding sites on the MLCK promoter region, and showed that the cis-κB site (within the minimal promoter region) was the NF-κB binding element that mediated the TNF-α-induced upregulation of the MLCK promoter activity in Caco-2 cells.332,333 Electrophilic mobility shift assay studies also confirmed that the NF-κB p65/p50 dimer binding to the cis-κB binding site upregulated the MLCK promoter activity.333 These studies suggested that the TNF-α-induced NF-κB activation mediated the increase in MLCK promoter activity and subsequent increase in MLCK transcription and translation. As discussed further in this section, other transcription factors are also involved in the cytokine modulation of MLCK promoter activity. As TNF-α is also known to induce apoptosis in various cell types, apoptosis as a possible mechanism of TNF-α increase in Caco-2 TJ permeability had been considered. In Caco-2 cells, TNF-α alone did not induce apoptosis or necrosis, indicating that apoptosis or cell death is not the mechanism responsible for the TNF-α increase in Caco-2

TJ permeability.268,304 The possibility that apoptosis may be the mechanism involved in the TNF-α-induced increase in TJ permeability in other epithelial cell types including HT29/B6 colonic epithelial cells and LLC-PK1 renal epithelial cells has been suggested by studies showing that TNF-α induces an increase in apoptosis in these cell types.293,334 While it is possible that the TNF-α increase in apoptosis may have contributed to the overall increase in TJ permeability in these cells, the low rates of apoptosis (1–4%) observed in these cell types following TNF-α treatment are unlikely to explain the increase in epithelial TJ permeability.293,334 For instance, in T84 cells, IFN-γ and TNF-α combination-induced increase in TJ permeability was also associated with about a 2- to 3-fold increase in apoptosis.270 However, the inhibition of IFN-γ/TNF-αinduced increase in apoptosis by caspase inhibitor Z-ValAla-Asp-fluoromethylketone (ZVAD-fmk) did not affect the increase in TJ permeability, indicating that the IFN-γ/ TNF-α-induced apoptosis was not the mechanism involved in the increase in intestinal epithelial TJ permeability.270 Consistent with the lack of involvement of apoptosis as a mechanism of intestinal epithelial TJ barrier disruption, it had been previously demonstrated that during normal physiological sloughing of dying intestinal epithelial cells at the villous tip, adjacent cells rapidly stretch and extend

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TJ Strands Extruding Cell (fracture face, plasma membrane)

TJ Kisses Adjacent Cell (cross-section) Terminal Web

Lysosome

FIGURE 38.15  Model of cell extrusion from intestinal epithelia. (From 335 with permission.)

to re-establish the TJ barrier as the dead cell extrudes.335 The TJ elements of adjacent epithelial cells proliferate at the point of the contact between the adjacent cells to rapidly re-establish the TJ barrier in a “zipper” like manner (Figure 38.15).203,335 As the paracellular barrier is maintained throughout the cell extrusion process, the epithelial barrier function to macromolecules is maintained.203,335 Similarly, in the in vitro model system consisting of HT-29/B6 colonic epithelial monolayers, an artificially induced single cell defect resulted in a rapid sealing of the epithelial defect (within minutes) by flattening and extension of the adjacent cells and rapid reformation of the TJ barrier.336 The reformation of the TJ barrier seal was dependent on MLCK-activated actin–myosin interaction.336 It was suggested that the TJ barrier formation may be driven by a contractile “pulse-string” mechanism in which the perijunctional ring of actin and myosin filaments contract to generate the mechanical force to induce a rapid closure of the epithelial wound and to re-establish the functional TJ barrier.336–338 The previous studies used single cytokine IFN-γ or TNF-α to examine the mechanisms involved in intestinal TJ barrier function. This reductionist approach allowed the delineation of each cytokine’s effect on the intestinal TJ barrier. However, in inflammatory conditions of the gut, multiple cytokines are activated and participate in TJ barrier regulation. Since many inflammatory cytokines including IFN-γ and TNF-α are elevated in inflammatory conditions of the gut, some investigators have also used an IFN-γ and TNF-α combination to examine their combined effects on intestinal TJ barrier function. Using such an approach, co-treatment of IFN-γ and TNF-α was found to have a synergistic effect on intestinal epithelial TJ barrier function.46,270,298 It was suggested that IFN-γ may prime the intestinal epithelial cells to respond to TNF-α.46,270 The intracellular mechanisms involved in the

combined IFN-γ/TNF-α effect mirror the effects of each cytokine alone.212 In a series of studies, it was shown that the IFN-γ/TNF-α induced increase in Caco-2 TJ permeability was mediated by an increase in MLCK expression and increase in MLCK activity.46,59,144,331 These studies showed that a highly specific peptide inhibitor of MLCK was able to reverse the IFN-γ/TNF-α-induced increase in TJ permeability.59 It was suggested that treatment with IFN-γ primes the Caco-2 cells to respond to TNF-α by inducing TNFR2 expression,144 which in turn mediates the IFN-γ/TNF-α-induced MLCK-dependent loss of TJ barrier function. In contrast to the earlier studies, showing involvement of NF-κB in TNF-α induced increase in Caco-2 TJ permeability.332,333 Wang et al. did not find NF-κB inhibitors to prevent the IFN-γ/TNF-α-induced drop in Caco-2 TER.46 In some cases, NF-κB inhibitors actually worsened the drop in Caco-2 TER, suggesting that NF-κB was not required for the IFN-γ/TNF-α-induced barrier loss.46 Thus, further studies are needed to clarify the apparent discrepancy in NF-κB involvement in the TNF-α or IFN-γ/TNF-α combination-induced alteration in intestinal TJ barrier function. In molecular studies to assess transcription factor involvement in MLCK promoter activation, it was suggested that both NF-κB and AP-1 were involved in the IFN-γ/TNF-α modulation of MLCK promoter activity.331 The analysis of NF-κB and AP-1 activation during Caco-2 cell differentiation suggested that NF-κB activation predominates early, while AP-1 plays a more significant role in well-differentiated cells.331 In a series of in vitro and in vivo studies, TNF-α or LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes entry on T-cells) also caused an internalization of occludin.45,46,89,143,339 Much like the IFN-γ/TNF-α combination studies, the Caco-2 cells used in these studies required incubation with IFN-γ to induce expression of the lymphotoxin β receptor, prior

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to LIGHT treatment.143 LIGHT caused occludin endocytosis that could be prevented by an inhibitor of caveolar endocytosis, but not by inhibitors of clathrin endocytosis or macropinocytosis.143 Caveolar endocytosis inhibitors also prevented LIGHT-induced drop in Caco-2 TER.143 Interestingly, the LIGHT-induced caveolar endocytosis was mediated by MLCK-induced MLC phosphorylation. This was in contrast to IFN-γ-induced endocytosis, which was mediated by Rho-associated kinase-induced phosphorylation of MLC and macropinocytosis.320 In addition, using in vivo imaging of transgenic mice expressing fluorescent-tagged EGFP-occludin and mRFP1-ZO-1 within the intestinal epithelium (under control of the villin promoter), it was also shown that the TNF-α modulation of intestinal TJ barrier function in vivo was also mediated by MLCK-dependent caveolar endocytosis of occludin.44 These studies also suggested that the TNF-α-induced increase in intestinal TJ permeability was also mediated in part by MLCK-dependent caveolar endocytosis of occludin.44,45,142 In addition, overexpression of occludin in the EGFP-occludin transgenic mice limited TNF-α-induced increases in intestinal TJ permeability.89 A similar requirement for occludin in TNF-α-induced increase in TJ permeability was also recently demonstrated in MDCK cell monolayers.42 Thus, although additional studies are needed to fully characterize the involvement of MLCK and occludin in barrier function, the previous studies suggest that occludin plays a critical role in TJ regulation.

38.5.5  Infectious Pathogen-induced Alteration of Intestinal Epithelial TJ Permeability Intestinal TJ barrier disruption by infectious pathogens has been postulated to play an important role in the pathophysiology of infectious diarrhea. In contrast to secretory diarrhea where the pathogen induces Cl (Na and water) secretion, disruption of the TJ could induce diarrhea by eliminating the electrochemical gradient required for absorption and by enhancing inflammation and prosecretory inflammatory mediators by permitting bacteria and antigens to breach the epithelial barrier. A number of infectious agents and toxins elaborated by the enteric pathogens have been shown to cause a disturbance of intestinal epithelial TJ barrier. In general, pathogens exert their effects directly through binding to the intestinal epithelial cell or indirectly through the actions of secreted toxins. Whether direct or indirect, their mechanisms of action can be divided into those that affect the perijunctional actomyosin ring with subsequent perturbation of the TJ barrier and those that target the activity of specific TJ proteins. The best studied infectious agents that affect the TJ barrier include enteropathogenic Escherichia coli (EPEC), Clostridium difficile, Vibrio cholerae, Bacteroides fragilis,

SECTION  |  III  Host Defense Mechanisms

C. perfringens, and rotavirus175,193,206,340–366 A summary of various effects of infectious agents and the factors secreted by the pathogens are listed in Tables 38.4 and 38.5.350 The intracellular processes involved in the EPEC modulation of intestinal TJ barrier have been among the most extensively studied using the cultured intestinal epithelial cell models.193,340–344,366,367 They involve both affects on the cytoskeleton and the specific proteins occludin and ZO-1. Infection of T84 cells with EPEC340,341 is associated with an increase in intracellular Ca2, increase in MLC phosphorylation, phosphorylation of ezrin, dephosphorylation of occludin, and an increase in transepithelial migration of PMNs.355 Phosphorylation of occludin is required for its proper TJ localization. Exposure to EPEC results in dephosphorylation of occludin, its movement to a cytoplasmic location, and a drop in barrier resistance.356 It has been speculated that the EPEC-induced increase in intracellular Ca2 is responsible for the activation of MLCK, which enzymatically phosphorylates MLC and activates Mg2-myosin ATPase. This leads to the contraction of the perijunctional actin–myosin filaments and retraction of enterocyte membrane and TJ complex, culminating in opening of the TJ barrier.343 Although this specific sequence of events has yet to be confirmed experimentally, inhibition of MLC phosphorylation by MLCK inhibition prevents the EPEC-induced increase in T84 TJ permeability, supporting the central role of actomyosin contraction in the pathophysiology.59,343 Ezrin phosphorylation may also be involved in the cytoskeletal-induced TJ retraction process and stimulation of transepithelial migration of polymorphoneutrophils.343,344 Interestingly, Helicobacter pyloriinduced barrier loss in gastric mucosa is also associated with MLCK activation and MLC phosphorylation.368 C. difficile also appears to work through altering the actomyosin cytoskeleton but by inactivating the small GTP binding proteins Rho, Rac, and Cdc42, not through activation of MLCK.206,369–371 The Rho family of small GTPases is important in regulating many aspects of actin filament dynamics, which control cell shape and cell–cell junctions. By switching between the GDP- and GTP-bound states, they control the activity of proteins, which control the location of actin attachments and filament assembly and disassembly. Both toxins A and B elaborated by C. difficile inactivate Rho family GTPs by covalently attaching glucose derived from UTP-glucose. Their inactivation leads to degradation of perijunctional actin and barrier failure.370,371 B. fragilis secretes a protease enterotoxin, also known as fragilysin, that is a 20 kDa metalloprotease.75 Exposure of human colonic cell lines and native colonic tissue to the enterotoxin resulted in disassembly of the perijunctional actin and disruption of the TJ barrier.75,351,372 Among its substrates is the adherens junction protein E-cadherin.75,373 The

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TABLE 38.4  Enteric Pathogens That Modify Epithelial Barrier Function Organism

Cell line

Pathophysiological outcome

Structural modification of TJ

Clinical relevance

Diffusely adhering Caco-2/TC7 Escherichia coli

No change Rt (up to 5 hours); increased paracellular permeability*

Disorganization of occludin and ZO-1 but not E-cadherin†

Associated in some studies with diarrheal disease

Enterohemorrhagic T84 E. coli‡

Decrease in Rt (onset at 12 hours)

ZO-1 staining diminished and disrupted, but E-cadherin unaffected; minor disruption of F-actin

Leading cause of infectious hemorrhagic colitis; foodborne pathogen; associated with HUS

Enteropathogenic E. coli

Decrease in Rt (onset at 2 hours or later in all studied cell lines)

Intact apical actin filaments and ZO-1 staining (MDCK)

Serious cause of acute and persistent diarrhea in very young children ( 3 years old)

T84 MDCK I

Caco-2

No gross TJ change, but F-actin disorganized and, after 18 hours, ZO-1 staining focally diffused (T84; by TEM)

Rotavirus

Caco-2

No change Rt (up to 24 hours); increased paracellular permeability*

Disorganization of occludin but not E-cadherin†

Major cause of infantile diarrhea globally

Salmonella typhimurium

MDCK II; Caco-2; T84

Decrease in Rt by 15 minutes in MDCK II cells; decrease in Rt by 1 hour in Caco-2 cells; no change in Rt for up to 3 hours in T84 cells

Apical pole contraction with clustering of F-actin, E-cadherin, and ZO-1 at sites of bacterial attachment (MDCK II cells)

Major cause of food-borne disease; Salmonella spp. may infect up to 1% of population annually§

No gross TJ change (Caco-2; by TEM) 350

From with permission. TJ, tight junction; Rt, transepithelial electrical resistance; HUS, hemolytic uremic syndrome; TEM, transmission electron microscopy; MDCK I and II, MadinDarby canine kidney cells forming high-(I) and low-(II) resistance monolayers; * demonstrated with [3H] mannitol. † Using the actin stabilizer jasplakinolide, disruption of the normal occludin and ZO-1 structure was shown to be independent of the F-actin. ‡ Purified Shiga toxins 1 or 2 do not decrease Rt. In addition, Shiga toxin-negative enterohemorrhagic E. coli (EHEC) strains do not differ from wild-type EHEC strains in their effects on T84 monolayer permeability, indicating that Shiga toxins do not play a role in the decreased Rt caused by these strains. § S. typhimurium is predicted to account for ~25% of food-borne Salmonella infections. Both S. choleraesuis and S. enteritidis have been reported to decrease Rt by 2–4 hours in MDCK I monolayers without gross TJ disruption by TEM or change in E-cadherin staining.

inflammation induced has also been implicated in carcinogenesis.75 Because the integrity of TJs is dependent on proper adherens junction function, loss of the key adherens junction adhesion molecule is presumed to affect the TJ barrier. C. perfringens represents an interesting example where the toxin does not specifically target the TJ but uses a TJ protein as its cell surface receptor on a path to inducing cytolysis. The C. perfringens enterotoxin (CPE) is a common cause of diarrheal food poisoning and less commonly antibioticassociated diarrhea. The toxin is a 35 kDa polypeptide with a C-terminal domain that binds to several different claudins, notably 3 and 4, and an N-terminal domain required for toxicity.172,174,175,374,375 Binding results in formation of a large protein complex, which includes claudins and occludin and a coincident increase in ionic permeability across the plasma membrane.173,360 It is hypothesized that the toxin induces claudins to form a transmembrane pore and allow cytotoxic

levels of calcium to enter. There has been speculation that CPE could be used as a chemotherapeutic agent for GI malignancies that overexpress claudin-4, such as pancreatic cancer.363 This seems doubtful given the wide distribution of claudin-4 throughout the body. Rotaviruses infect epithelial cells of the small intestine and induce diarrhea without obvious histological tissue damage at early stages of infection. Rotavirus infection of cultured Caco-2 cells causes a drop in TJ barrier function coincident with redistribution of claudin-1, occludin, and ZO-1. The mechanism of TJ disruption is unclear, but it has been speculated to result from decreased ATP levels.345 In vivo, the rotavirus-induced diarrhea was also associated with an increased intestinal permeability to lactulose in infants.347,349 The defective TJ barrier function may contribute to diarrhea by allowing paracellular leakage of intestinal fluid into the lumen (or allowing increased penetration).

TABLE 38.5  Enteric Pathogen Virulence Factors That Alter TJ Proteins and/or Modify Epithelial Barrier Function Organism

Virulence factor

Cell line/tissue studied

Proposed mechanism

Pathophysiology and TJ structure

Clinical relevance of pathogen

Bacteroides fragilis

Metalloprotease toxin

T84, Caco-2, HT-29, HT-29/C1

Cleavage of E-cadherin

Decrease in Rt (onset in ~15 minutes)

Associated with communityacquired diarrheal disease in children and adults

Absent E-cadherin, dissociation of occludin and ZO-1 from TJ

MDCK Human colon

Clostridium difficile

Toxin A Toxin B

T84, Human colon, Rabbit ileum T84, Caco-2, human colon

Gross separation of some TJ and zonula adherens junctions Monoglucosylation of Rho at threonine 37

Decrease in Rt (toxin A and B onset in Only clearly documented cause 2 and 4 hours, respectively, in T84 of infectious nosocomial diarrhea monolayers)* Loss (toxin A) or flocculation (toxin B) of apical F-actin with intact TJ (EM)

Clostridium perfringens

Enterotoxin

Escherichia coli

MDCK I

Cleavage of claudins 3 and 4 but not 1 and 2

Decrease in Rt (onset by 4 hours) Loss of claudins and TJ strands (freeze-fracture EM)

Food-borne toxin-mediated noninflammatory diarrheal disease

Cytotoxic necrotizing Caco-2 factor 1

Deamidation of Rho at glutamine 63

Decrease in Rt (onset after 1 hours) Enhanced actin filament formation†

Limited case reports of clinical illnesses, including diarrheal disease

Helicobacter pylori

Vacuolating toxin

T84 MDCK I

Unknown

Decrease in Rt (onset by 1 hour) No changes in occludin, ZO-1, cingulin, or E-cadherin observed

Associated with gastritis, gastroduodenal ulcer, gastric adenocarcinoma, and MALT lymphoma

Listeria monocytogenes

Internalin

Caco-2

Binds to E-cadherin to mediate Unknown bacterial cellular invasion Proline 16 of E-cadherin is critical to E-cadherin-internalin interaction

Food-borne systemic disease (sepsis, meningitis) in immunocompromised hosts with 25% mortality; shortlived febrile diarrheal illness in immunocompetent hosts

Vibrio cholera

Zonula occludens toxin Hemagglutinin/ protease‡

Rabbit ileum MDCK I

Activation of protein kinase C Cleavage of occludin

Classic epidemic secretory diarrheal disease Oral zonula occludins toxin treatment has been proposed as a strategy to enhance drug absorption (e.g., insulin)

Decrease in Rt (onset by 1 hour) Loss of TJ strands (freeze-fracture EM) Decrease in Rt (onset after 1 hour) Occludin degradation; ZO-1 and F-actin reorganization

From 350, with permission. *Toxin B reduced Caco-2 monolayer Rt more rapidly; EM, electron microscopy. † No specific information on TJ proteins reported; MALT, mucosal-associated lymphoid tissue. ‡ Data derived from studies of crude culture supernatants of a mutant V. cholerae strain, CVD 110, which is positive for hemagglutinin/proteinase. Studies using purified hemagglutinin/proteinase are not available.

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38.6  CLINICAL DISORDERS OF INTESTINAL TJ BARRIER DEFECT A number of intestinal disorders have been shown to have an associated defect in intestinal TJ barrier as evidenced by an increase in intestinal permeability. An important question that remains to be resolved is whether the intestinal TJ barrier defect represents a primary pathogenic factor of the disease leading to intestinal inflammation or whether the defect is secondary to epithelial damage resulting from intestinal inflammation. In both cases, the defect in intestinal TJ barrier is likely to contribute to intestinal and systemic inflammation by allowing increased paracellular permeation of toxic luminal antigens and other harmful luminal agents that are normally excluded.284,376–380 In this section, we discuss the intestinal TJ barrier defect and the possible implications of this defect in those permeability disorders that have been studied the most, including celiac disease, Crohn’s disease, and non-steroidal anti-inflammatory drug (NSIAD)-associated enteritis.

38.6.1  Clinical Assessment of Intestinal TJ Barrier Defect Intestinal epithelium serves a dual role.377,378,381,382 Intestinal epithelial cells regulate the uptake of nutrients and fluid while at the same time act as a barrier to the permeation of potentially harmful substances present in the intestinal lumen. This selective barrier function has been referred to as “intestinal permeability.” In clinical studies, the term intestinal permeability refers to intestinal barrier function to passively absorbed water-soluble markers. Intestinal permeability has been defined as “the ability of medium and large sized water-soluble compounds to passively traverse the intestinal epithelial layer through paracellular tight-junctional areas.”377–380 Intestinal permeability studies assess the relative leakiness of the intestinal TJ barrier or paracellular pathways to passive permeation of water-soluble probes. For usage in clinical studies, permeability markers must be hydrophilic and passively absorbed, inert and non-toxic, not metabolized or endogenously produced, rapidly and completely excreted in urine, and should be easily measured.378,380,382,383 The most commonly used permeability probes to assess the small intestinal permeability in humans include PEG 400, mannitol, rhamnose, lactulose, cellobiose, and 51 Cr-EDTA.378–380 Less commonly, creatinine and inulin have also been used. Because of their hydrophilicity, permeability probes permeate poorly across the bi-lipid enterocyte membrane, but they are able to permeate across through the aqueous TJ or paracellular pathways. The intestinal permeation rates of the probes are directly related to their molecular size and the functional pore size of the TJs.378,380,382 Because molecular radius of commonly used

permeability probes are 4 Å, these probes do not permeate across the claudin-dependent pore pathway but through a large channel or leak pathway,40 and presumably measure the flux rates through a large channel pathway. All of the commonly used probes are relatively inert and non-toxic. Of the permeability probes mentioned, only PEG 400 and 51 Cr-EDTA are not metabolized or endogenously produced. The sugar probes including mannitol, rhamnose, lactulose, cellobiose, and inulin are degraded by intestinal bacteria and creatinine is produced endogenously. The permeability probes are excreted in a variable manner depending on their body distribution and urinary excretion. All of the probes are measured with relative ease in urine. The intestinal permeability studies are intended to assess the “leakiness” of the intestinal TJ barrier in humans. Typically, in clinical studies, permeability markers are ingested orally following an overnight fast and urine is collected for varying time periods (between 6 and 24 hours). Previous studies have demonstrated no significant difference in the reliability of the permeability test, whether 6 or 24 h urinary collection period was used. Currently, the most commonly used collection period in clinical studies is 6 hours. The amount of permeability probe excreted in the urine is then used as an indirect measurement of intestinal TJ permeability.

38.6.2  Permeability Index and Celiac Disease A permeability index (PI), lactulose/mannitol or large probe/small probe urinary excretion ratio (PI  % urinary excretion of larger probe/% urinary excretion of smaller probe), has been used by investigators as an index of intestinal permeability in clinical studies.384–388 For this purpose, most commonly used large probes are disaccharides lactulose and cellobiose and 51Cr-EDTA and small probes are monosaccharides mannitol, rhamnose, and D-xylose. Historically, the usage of large probe to small probe urinary excretion ratio (including cellobiose/mannitol, lactulose/mannitol, or 51Cr-EDTA/mannitol excretion ratio) has been shown to be more sensitive in the detection of intestinal permeability disorders than when the excretion rates of probes were used alone.384–386,388–393 Celiac disease, also referred to as gluten-sensitive enteropathy, is an immune-mediated inflammation of the small intestine in response to dietary gluten in genetically predisposed individuals.394 Gluten-containing food products induce immune-mediated small intestinal inflammation, characterized by destruction of villous surface, villous atrophy, and crypt hyperplasia. The intestinal immune response is triggered by the gliadin component of gluten, and elimination of gluten from diet leads to the resolution of the disease. Active celiac disease is characterized by an increase in intestinal permeability.394 In celiac disease

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SECTION  |  III  Host Defense Mechanisms

patients, intestinal permeability to smaller probes including mannitol and L-rhamnose is decreased, correlating with the decrease in the net intestinal absorptive surface area from immune-mediated destruction of the villous surface.388,391–393,395 The decrease in intestinal permeability to mannitol and L-rhamnose is present during active exacerbations of celiac disease and the permeability changes resolve with improvement of the disease following removal of gluten from the diet.377,396–400 Paradoxically, intestinal permeability of larger probes including lactulose, cellobiose, and 51Cr-EDTA increases with increasing mucosal damage and villous atrophy.388,391–393,395 Comparison of sensitivity of the permeability index versus intestinal permeability measurements of individual permeability markers in the detection of celiac disease indicated that the permeability index was more sensitive than individual permeability measurements.384–386,390,400 The higher sensitivity of the permeability index is due to the inclusion of mannitol (or small probe) permeation rates into the equation, which provides an indirect accounting of the decrease in mucosal absorptive surface area.378 As discussed in previous sections in this chapter, in normal healthy intestinal epithelium, the effective pore size of the intestinal TJs is such that the smaller sized probes (4 Å in molecular radius) readily diffuse across the TJ pores. In healthy human intestinal epithelium, the intestinal permeation rates of larger probes (lactulose and Cr-EDTA) are about 50- to 100-fold lower than the smaller probes (mannitol, PEG 400, and L-rhamnose). As the villous absorptive surface is damaged in celiac patients, due to gluten-induced intestinal inflammation, the absorptive

surface area markedly decreases. The inflammationinduced damage of the mucosal surface also causes morphological and functional disturbance in TJ barrier.401 The differences in intestinal permeability to large-sized versus small-sized permeability probes in celiac disease (and also in Crohn’s disease; CD) may be explained by the increasing functional size of paracellular channels during the disease states and the simultaneous decrease in the absorptive surface area resulting from the mucosal damage (Figure 38.16).378 The smaller sized probes (rhamnose, mannitol, and PEG 400) have smaller cross-sectional diameters and more readily permeate across the normal intestinal epithelium and have higher rates of urinary recovery (range between 10 and 30% urinary recovery per 6 hour collection period) in healthy individuals.378 In contrast, the larger sized probes (lactulose and 51Cr-EDTA) are mostly excluded by the healthy intestinal epithelium and have lower rates of urinary recovery in healthy individuals (range between 0.2 and 0.7% urinary recovery per 6 hour collection period). There is about a 50- to 100-fold difference in the relative intestinal permeability of smallsized versus large-sized permeability probes in humans.378 As the smaller sized probes readily permeate across the intestinal TJ barrier in the healthy intestinal epithelium, the increase in functional TJ pore size during the disease states has relatively minor impact in the TJ flux rates of the smaller sized probes compared to larger probes. In active celiac disease, there is extensive destruction of the absorptive villous surface and almost complete denudation of the villous surface resulting in a marked decrease in the overall absorptive surface area. Concurrently, the functional

(A)

Large Probe

Paracellular pore

Small Probe

(B)

Paracellular pore

FIGURE 38.16  Proposed model of alteration of paracellular pathways or “pores” in celiac and CD.  In normal healthy intestine (A), absorptive surface is relatively large and the TJ pores are moderate in size. The smaller probes (e.g., mannitol and PEG 400) readily permeate (urinary recovery rate of 10–30%/6 hours), whereas larger probes (e.g., lactulose) are mostly excluded (urinary recovery rate of 0.2–0.4%/6 hours) from permeation across the TJ pores in the healthy intestinal epithelia. In diseased states (B), the overall surface area of absorptive surface is diminished due to mucosal damage, but the TJ pores are large either as a primary defect or secondary to inflammation. The enlarged TJ pores allow penetration of larger probes. The permeation of smaller probes are also increased, but to a much smaller extent. The decrease in absorptive surface area has greater impact on small probe permeation than the increase in the TJ pore size. (From 378 with permission.)

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pore size of the TJs in the remaining epithelial surface is increased (Figure 38.16). Presumably, the permeation rates of smaller probes are much more affected by the decrease in the absorptive surface area than by the increase in the TJ pore size, thus the net decrease in the permeation rates of the smaller sized probes. In contrast, as the larger sized probes are mostly excluded by the normal intestinal TJ barrier, their permeation rates are much more affected by the increases in TJ pore size during disease states than by the decrease in absorptive surface area, and thus the net increase in the intestinal permeability to the larger probes (Figure 38.16). The alterations in intestinal TJ permeability in celiac disease closely correlate with the extent of the mucosal damage and villous atrophy induced by the gluten ingestion.387,398 The alteration in intestinal TJ permeability normalizes with the removal of gluten from the diet and subsequent villous regeneration.387,393,395,398 Following gluten withdrawal-induced normalization of intestinal permeability, an exposure to a single oral dose of gluten causes an increase in intestinal permeability.395 Studies from Fasano and co-workers have provided new insight into the mechanisms involved in intestinal TJ barrier disruption in celiac disease. These studies suggested that zonulin, a 47 kDa human intestinal homolog of zonula occludins toxin (an enterotoxin elaborated by V. cholerae, which causes intestinal TJ barrier disruption),402 plays a central role in mediating the TJ barrier defect in celiac disease.403,404 They showed that zonulin expression was increased in intestinal tissue from active celiac disease patients compared to controls and patients in clinical remission.362 When purified zonulin was added to the mucosal surface of Rhesus monkey intestine mounted in an Ussing chamber, zonulin caused a drop in TER of jejunal and ileal tissue but not colonic tissue.405 The serosal treatment of zonulin did not affect the intestinal tissue TER, suggesting that the zonulin effect was mediated by interaction with an apically located membrane receptor. The role of gliadin in zonulin secretion was confirmed by studies showing that gliadin addition to the mucosal surface induces secretion of zonulin by intestinal epithelial cells and intestinal tissue.406,407 Only apical (but not basolateral) compartment addition of gliadin to celiac disease intestinal tissue caused an increase in zonulin secretion, and the increase in zonulin secretion was only on the apical bathing solution.407 There was only a transitory zonulin secretion in intestinal tissue from control patients. It was also shown that zonulin inhibitor prevented the gliadin-induced drop in TER.406 Together, these data suggested that the gliadin-induced increase in intestinal TJ permeability was mediated by apical secretion of zonulin. Subsequent studies have also shown that gliadin-induced enterocyte secretion of zonulin was mediated by gliadin binding to chemokine receptor CXCR3 on the apical membrane surface via a

MyD88-dependent process.408 Together, these studies suggested that the increase in intestinal permeability observed in celiac disease was mediated in part by gliadin-induced secretion of zonulin from intestinal epithelial cells. Fasano and co-workers have proposed that gliadin-induced increase in zonulin secretion may be a key pathogenic mechanism responsible for the loss of intestinal TJ barrier in celiac disease; and that the loss of TJ barrier with a resultant increase in intestinal permeation of luminal antigens in genetically susceptible individuals leads to the clinical manifestation of inflammatory disease.409 Consistent with the potential importance of zonulin as a central factor in celiac disease pathophysiology, clinical studies have been initiated to assess the therapeutic effect of zonulin antagonist AT-1001 in the treatment of celiac disease.

38.6.3  CD and Intestinal TJ Barrier Defect CD is a chronic inflammatory bowel disease of unknown etiology characterized by recurrent bouts of exacerbations.410 Clinical presentation during acute exacerbations includes abdominal pain, diarrhea, weight loss, and bloody stool. Patients with Crohn’s disease as a group have an increase in intestinal permeability.378–380,389,411,412 As early as 1963, Gryboski et al. found CD patients to have an abnormal increase in intestinal permeability to lactulose and sucrose.413 Subsequently, numerous other investigators have shown CD patients to have an increase in intestinal TJ permeability to a wide variety of permeability probes including 51Cr-EDTA, lactulose, cellobiose, and PEG 400.236,389,411,412,414–419 Similar to celiac disease, clinical studies have consistently shown CD patients to have an increase in intestinal permeability to larger permeability probes and either a decrease or no change in intestinal permeability to smaller probes.378 As discussed in Section 38.6.2, the probe size-related differences in intestinal permeability in CD also appear to be related to the increase in the pore size of the TJ barrier having a greater contribution to the increased TJ permeation of larger probes and the decrease in the intestinal absorptive surface (as a result of intestinal inflammation) having a greater contribution to the decrease in the absorption of smaller probes (Figure 38.16).378 Although it is well established that CD patients have an abnormal increase in intestinal TJ permeability, the significance of the defective intestinal TJ barrier in the pathogenesis of intestinal inflammation of CD remains unclear. It had been proposed by a number of investigators that the defect in intestinal TJ barrier may represent the genetic disorder of CD, which predisposes susceptible individuals to develop CD later in life when exposed to the appropriate environmental factors.284,376,378,381,414,420,421 Another possibility may be that the increase in intestinal TJ permeability is secondary to intestinal inflammation and plays a secondary role in promoting intestinal inflammation by

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allowing increased antigenic penetration.378–380 In support of the first possibility, clinical studies have consistently shown that the healthy first degree relatives of CD patients (an at-risk group to develop CD) have a pre-existing intestinal permeability defect prior to any clinical evidence of CD.378–380,382,414 The first degree relatives of CD patients have ~30–100 times greater risk of developing CD compared to the normal population and have an ~10% life-time risk of developing CD.410 Intestinal permeability studies in healthy first degree relatives of CD patients have consistently shown 10–30% of the relatives to have abnormal increase in intestinal permeability,389,410–412,414,417,421–425 suggesting the possibility that the first degree relatives with an increase in intestinal TJ permeability may be the ones that are genetically predisposed to develop CD later in life when exposed to appropriate environmental stresses. Consistent with such a possibility, oral administration of a known intestinal permeability stressing agent, aspirin (325 mg), caused a significantly greater increase in intestinal permeability in the healthy first degree relatives compared to the normal controls.420,426,427 A causal relationship between increased intestinal permeability and immune activation was suggested in a study by Yacyshyn and Meddings428 in which they showed that all clinically healthy first degree relatives with increased intestinal permeability had elevated CD45RO expression in B-cells377 (an indicator of immune activation),429 while none of the first degree relatives with normal intestinal permeability had elevated CD45RO expression. This suggested that the increase in intestinal permeability was responsible for the immune activation. In addition to the functional abnormality in intestinal TJ barrier function, CD patients also have morphological alterations in intestinal TJ complexes.430–432 The examination of normal, uninvolved areas of small intestinal tissue obtained from CD patients revealed alteration in TJs characterized by irregular distribution, fragmentation, and aberrant TJ strands.430–433 The role of defective intestinal TJ barrier in the intestinal inflammation of CD is further supported by studies showing that the relapse of CD is often preceded by drugs or conditions that affect the intestinal TJ barrier, such as NSAID usage, acute gastroenteritis, and alcohol binges.434–437 Together, these studies form the basis for the “leaky gut” hypothesis that the defect in the intestinal TJ barrier in CD patients may predate the development of CD and may be an important etiologic factor contributing to the pathogenesis of CD (Figure 38.17).377–380,414,421 More investigations are required to further define whether the defective intestinal TJ barrier is truly a primary etiologic factor of CD. An important long-term study would be to follow those first degree relatives of CD patients that have an abnormal increase in intestinal permeability to determine whether these individuals eventually develop CD. Consistent with such a possibility, there is a case report of a first degree relative of a CD patient who developed CD 8 years after having

SECTION  |  III  Host Defense Mechanisms

been found to have an increase in intestinal permeability.438 In support of a primary etiologic role, previous studies in animal models of immune-mediated intestinal inflammation have shown that the preservation of the intestinal TJ barrier prevents the development of intestinal inflammation.439 A commonly used mouse model of CD is the IL-10deficient mice.440 IL-10-deficient mice, when exposed to a normal bacterial environment, develop enterocolitis. The development of enterocolitis is preceded by an increase in intestinal permeability by several weeks.297 Oral administration of the TJ barrier-enhancing agent AT-1001 (zonulin antagonist) to IL-10-deficient mice preserved the intestinal TJ barrier and prevented the development of intestinal inflammation, suggesting that the defective intestinal TJ barrier was required for the subsequent development of intestinal inflammation.439 In another approach, an intestinal TJ barrier defect was produced in mice by targeted overexpression of constitutively active MLCK within the intestinal epithelium.128 The overexpression of MLCK in mouse intestinal epithelium resulted in an increase in MLC phosphorylation and intestinal permeability. The MLCK overexpressing

Defective intestinal epithelial barrier

Exposure to environmental or endogenous permeability stressing factors

Increase in intestinal permeability “Leaky gut”

Intestinal penetration of luminal antigens

Activation of immune response

Intestinal inflammation FIGURE 38.17  Proposed model of “leaky gut” hypothesis.  The proposed primary defect of intestinal TJ barrier (increased paracellular permeability) allows increased mucosal penetration by toxic luminal antigens (including infectious agents). The antigenic penetration activates immunologic and inflammatory responses, which produce epithelial injury and further damage of the intestinal TJ barrier. Exposure to environmental or endogenous factors that increase intestinal permeability, such as NSAIDs, may act as a trigger for the onset or exacerbation of CD in a susceptible population. (From 378 with permission.)

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Chapter  |  38  Tight Junctions and the Intestinal Barrier

transgenic mice did not have overt clinical disease but did have evidence of mild inflammatory changes. Subsequent challenge with CD4 CD45Rbhi lymphocytes (an adoptive transfer model of colitis) induced much more severe intestinal inflammation and higher rates of mortality in the transgenic mice than in wild-type mice, suggesting that the presence of an intestinal TJ barrier defect predisposes the transgenic mice to more severe colitis when challenged by immune activation.128 Similarly, it is possible those first degree relatives of CD patients that have an increase in intestinal permeability, when exposed to environmental or intrinsic factors that induce immune activation, have more exaggerated inflammatory response leading to a more severe and prolonged intestinal inflammation. Recently, an association between polymorphisms of TJ-associated protein membrane-associated guanylate kinase inverted 2 (MAGI2) and CD was also demonstrated,441 suggesting the possibility that MAGI2 polymorphism may have a primary role in CD pathogenesis. In support of the possibility that the increase in intestinal TJ permeability in CD is secondary to intestinal inflammation, a number of proinflammatory cytokines and inflammatory mediators that are produced during active inflammation of CD have been shown to cause an increase in intestinal TJ permeability, as discussed in previous sections of this chapter. Increase in MLCK expression and activity were important mechanisms mediating the cytokine modulation of intestinal TJ barrier. A direct correlation exists between the degree of active intestinal inflammation and increase in intestinal permeability; and improvement of intestinal inflammation in response to medical treatment is associated with normalization of intestinal permeability.442–444 Additionally, an increase in intestinal permeability has been shown to be an excellent predictor of clinical relapse.442–444 Examination of intestinal tissue expression of MLCK and MLC phosphorylation in intestinal biopsy specimens obtained from patients with CD and ulcerative colitis also show a correlation between the increase in MLCK expression and MLC phosphorylation and the increase in disease activity,445 suggesting that the severity of intestinal inflammation influences the level of MLCK expression. These studies suggest that the active inflammatory process in CD leads to an increase in MLCK expression and activity, and MLCK induced an increase in intestinal TJ permeability. Thus, intestinal inflammation in CD also appears to be an important factor affecting the intestinal TJ barrier function.

38.6.4  NSAIDs and Intestinal Permeability NSAIDs are widely used as therapeutic agents for antiinflammatory purposes and for pain control. Important adverse effects of NSAIDs are related to its damaging effects on the gastrointestinal mucosal surface leading to ulcers and gastrointestinal bleeding.446–448 Chronic and

acute NSAID therapy is associated with a high prevalence of small intestinal inflammation and ulcers.447,449 In autopsied patients, 10% of the NSAID users (chronic and occasional users) had small intestinal ulcers.449 NSAIDs are known to cause an acute increase in intestinal permeability in humans and in animals.435,436,447,448 Acute ingestion of NSAIDs (including aspirin, indomethacin, and ibuprofen) causes a significant increase in small intestinal permeability in humans.436,447 This increase in small intestinal permeability appears to be mediated by systemic effects, as rectal administration also causes an increase in intestinal permeability.436,447 As the NSAID-induced increase in intestinal TJ permeability precedes any detectable injury to the intestinal mucosal surface, the increase in intestinal TJ permeability does not appear to be related to the damage to the mucosal surface.447,448,450 Since the increase in intestinal permeability precedes the damage to the mucosal surface, it was proposed that the increase in intestinal TJ permeability may be a pathogenic factor leading to the NSAID-induced small intestinal inflammation.382,450 The mechanisms that mediate the effect of the NSAIDs on intestinal permeability remain unclear; however, it appears that its effect on cyclooxygenase inhibition is important, as the potency of the NSAID effect on intestinal permeability was directly related to the drug potency to inhibit cyclooxygenase,447 and the NSAID effect was attenuated by prostaglandin analogs.435 As discussed earlier in this chapter, it has been shown in rat intestinal perfusion studies that the NSAID effect on increasing intestinal permeability was related in part to an increase in the solvent drag effect.228,451 The effect of NSAIDs on intestinal permeability is likely to be due to a combination of effects including modulation of intestinal fluid fluxes (increase net fluid absorption) and the disruption of intestinal TJs. The possibility that the NSAID action on the intestinal TJ barrier may be mediated by inhibition of metabolic energy (or ATP depletion) has been hypothesized, but direct experimental evidence is lacking.452,453 Because a single dose of NSAIDs causes an acute increase in intestinal permeability in humans, NSAIDs have been used as a permeability stressing agent in clinical studies.420,426,427,436,447 As an example, administration of aspirin as a permeability stressing agent in CD patients and their healthy relatives caused a significantly greater increase in intestinal permeability in CD patients and their relatives than in normal controls.420,426,427 Thus, NSAIDs are useful as a permeability stressing agent to study the susceptibility of the intestinal TJ barrier to external stressing agent in clinical studies.

38.6.5  Other Intestinal Permeability Disorders A number of other clinical conditions have been shown to have an associated increase in intestinal TJ permeability

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including sepsis, critical illness, burn injury, heat stress, alcoholic liver disease, irritable bowel syndrome, and various infectious diarrheal syndromes.271,454–473 In each of these clinical conditions, the breach in the intestinal TJ barrier has been proposed to play an essential pathogenic role in promoting intestinal or systemic inflammation. For example, in alcoholic liver disease, the defective intestinal TJ barrier has been proposed as the primary pathogenic factor allowing epithelial penetration of bacterial endotoxins, which induce inflammatory reaction in the liver.454,455,457,474,475 Similarly, in burn injury and in the critically ill, the leaky intestinal TJ barrier allows bacterial translocation and antigenic penetration leading to sepsis and systemic inflammation.436,447,458,470 Recent studies have also suggested that the stress-induced alteration of the intestinal TJ barrier may play a prominent role in immune activation in irritable bowel syndrome.420,443 While the precise mechanisms that modulate intestinal TJ barrier in these clinical conditions remain to be further elucidated, the rapidly expanding investigations into these areas are likely to provide important new insights into the intracellular and molecular mechanisms involved in the modulation of the intestinal TJ barrier, establish a causal relationship between intestinal TJ barrier defect and development of various diseases, and identify novel therapeutic approaches to induce re-tightening of the intestinal TJ barrier in intestinal permeability disorders.

38.7  CONCLUDING REMARKS The intestinal epithelial TJs have an integral role in the regulation of paracellular barrier function. They are relatively leaky and continually undergo change in barrier function in response to a constantly changing intestinal luminal milieu. The composition of the luminal solution is a crucial factor that affects the intestinal epithelial TJ barrier function, and a variety of luminal factors including luminal osmolarity, nutrient content, bacterial composition, and the presence of pharmacological agents affect the barrier function. Accumulating data have identified claudins as an important component of TJ pores that allow flux of solutes 4 Å in radius. Other transmembrane proteins occludin and JAM proteins also appear to be important in barrier function, but their precise role remains to be further defined. The cytoplasmic PDZ-proteins including ZO-1 also have an important role in the regulation of TJ barrier and, through their direct binding interactions, appear to be important in the recruitment and assembly of transmembrane TJ proteins and in regulating cytoskeletal-dependent modulation of intestinal TJ barrier function. The central role of MLCK in both physiological and pathological regulation of the intestinal TJ barrier has been firmly established. The increases in MLCK protein expression and activity are a key component of both Na-nutrient cotransport and cytokine-induced

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increase in intestinal TJ permeability, and signal-transduction cascade modulation of MLCK gene is an important mechanism of TJ barrier regulation. The defective intestinal TJ barrier has important clinical implications and appears to be an important pathogenic factor contributing to the development of various inflammatory conditions of the gut and systemic inflammatory conditions. Recent advances have suggested the importance of zonulin and MLCK in intestinal barrier dysfunction of celiac disease and CD. The importance of maintenance or preservation of intestinal TJ barrier in preventing development of intestinal inflammation in CD and in animal models of inflammatory bowel disease has also been well established. These studies suggest that pharmacologic preservation of intestinal TJ barrier may be an important therapeutic approach to prevent the recurrence or development of intestinal inflammation. There have been a number of important scientific advances that have established the importance of the defective intestinal TJ barrier in the pathogenesis of various clinical diseases and have clarified some of the cellular and molecular processes that regulate intestinal TJ barrier function in health and disease. However, much remains unknown and exciting opportunities exist to further delineate the precise molecular interactions and mechanisms that regulate intestinal TJ barrier in physiological and pathological states and to develop novel therapeutic agents that enhance intestinal TJ barrier and perhaps prevent the development of clinical disease with an associated intestinal TJ barrier defect.

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