The Intestinal Mucosal Barrier: Mechanisms of Injury & Repair
ACVIM 2008
Vanessa L. Cook, VetMB, MS, DACVS, DACVECC
Raleigh, NC, USA

Introduction

The mucosal epithelium consists of a single layer of columnar epithelial cells. The principal function of these cells is to absorb nutrients, water and electrolytes, and secrete fluid. However, the epithelium is also critical to providing a barrier between bacteria and toxic substances in the lumen, and the tissues. Injury of the epithelium can occur due to ischemia/reperfusion, and contact with bile or acid. This allows pathogens and toxins to invade the lamina propria and initiate the inflammatory cascade. Therefore, recovery of the epithelium must occur rapidly after it is damaged, to minimize exposure to the luminal contents. This presentation will review the structure of the mucosal epithelium, the mechanisms by which its integrity is lost, and the steps involved in repair.

The Mucosal Epithelium

The single layer of epithelium that lines the intestinal tract has an extremely rapid turnover rate. New cells arise from stem cells in the crypt and migrate upwards along the villi, differentiating as they do so. Cells eventually undergo apoptosis at the villus tip and are shed into the lumen, with the entire process taking approximately 3 days. As there are no villi in the colonic epithelium, cells migrate from the crypts to the intracryptal tables. The production of new cells is much slower in the colon than in the small intestine resulting in a much longer turn over time. The epithelial cells prevent luminal contents from contacting the laminar propria and limit the interaction of bacteria and toxins to the surface of the epithelial cell.

The epithelial barrier is comprised not only of the epithelial cells themselves, but also the tight junctions between them. The apical tight junctions prevent toxins and bacteria from passing between the cells but must still allow passive absorption and secretion of solutes and water. The tight junction is composed of three basic proteins, occludin, claudins and junctional adhesion molecule. These are regulated by intracellular second messengers which cause the cellular cytoskeleton to contract or relax and hence open or close the tight junction. In general the tight junctions are more closed at the villus than the crypt, so absorption of amino acids and glucose must occur through active transporters. Tight junctions are also tighter in the colon to prevent passive loss of water that has been reabsorbed.

The intestinal epithelial cells also act as sentinels for the presence of microbial substances. The binding of conserved bacterial and viral motifs to receptors, such as Toll-like receptors, on the epithelial cell, leads to proinflammatory gene expression and hence activation of resident mast cells, dendritic cells and macrophages in the lamina propria. This triggers the influx of inflammatory cells such as neutrophils, to the site of inflammation in order to control the infection.

Injury to the Mucosal Epithelium

The epithelial cells are continually exposed to noxious substances and organisms in the lumen of the intestine which can cause cellular injury. Damage and loss of epithelial cells leaves the underlying basement membrane exposed and allows infectious agents to penetrate the body, while active absorption of nutrients is no longer possible.

Ischemic Injury

Interruption of blood flow to the intestine causes damage to the epithelial cells as a result of oxygen and nutrient deprivation. The intestinal epithelial cells are particularly susceptible to ischemia due to their high metabolic rate.1 In horses, ischemic intestinal injury is usually due to a strangulating intestinal obstruction resulting in occlusion of the venous drainage while arterial supply is maintained. This hemorrhagic strangulating obstruction causes edema and hemorrhage in the intestine. Complete obstruction of both the arterial and venous supply to form an ischemic strangulating obstruction can occur with a tight strangulation, but is much less common. Occlusion of the arterial blood supply can occasionally occur from an embolus or thrombus causing an intestinal infarction. Nonocclusive ischemia can occur after any situation that causes reduced cardiac output, such as severe hypovolemia, hemorrhage and shock. Ischemic injury can also occur after a simple intestinal obstruction if the intralumenal pressure exceeds the capillary blood pressure.

During ischemic injury, epithelial cells are lost progressively from the tip of the villous down into the crypt, resulting in an increasing grade of mucosal damage. Cells at the villous tip are affected first due to two reasons. The fountain arrangement of the villous blood supply allows a counter current exchange mechanism to shunt oxygen from the central arteriole into the venules, and short circuit oxygen from the villous tip. Therefore oxygen tension is low at the villous tip even in during normal circulation. Additionally, cells near the villous tip are highly differentiated, have the highest metabolic rate, and are therefore most sensitive to ischemia.2

Additional injury when blood flow and oxygen supply is restored is called reperfusion injury. Classically this is caused by the generation of reactive oxygen metabolites by xanthine oxidase. However, there is little xanthine oxidase in the equine colon so the importance of this enzyme in equine reperfusion injury is unknown. Influx of neutrophils subsequent to ischemic injury is more likely to result in further injury due to the release of their granular contents and physical disruption of the epithelial cells as the neutrophils migrate.

Acid Injury

The stomach and proximal duodenum are usually physically protected from the acidic luminal contents by a thick layer of mucous adjacent to the epithelium. Additionally, epithelial cells secrete bicarbonate into the lumen which becomes trapped in the adjacent mucous layer. Therefore a pH gradient is generated at the epithelial surface so that the epithelial cells are not directly exposed to a low pH. The stratified squamous epithelium of the stomach is particularly vulnerable to damage as it lacks the bicarbonate mucous layer. In certain situations the local environment is altered and epithelial cells are exposed to high concentrations of acid. This occurs when horses are fasted as the pH of the stomach drops to extremely low levels. Feeding readily fermentable carbohydrate can also cause epithelial damage as this results in high concentrations of short chain fatty acids which diffuse into epithelial cells and then dissociate causing intracellular acidification and cell death.3,4

Bile Injury

Bile salts secreted into the duodenum can regurgitate into the stomach and contact the stratified squamous epithelium in horses with duodenogastric reflux.5 At the low pH found in the stomach, bile salts become unionized and can be absorbed into the surface epithelium causing cell death. Bile salts are usually actively reabsorbed in the ileum but can contact the colonic mucosa after a jejunocecostomy has been performed, potentially damaging the colonic epithelium and causing diarrhea.

Repair of the Mucosal Epithelium

There are several steps involved in restoring the continuity, integrity and function of the epithelium after injury. Initial restoration of the mucosal barrier occurs at the expense of the absorptive and secretory properties of the epithelial cells, which are then restored later.

Villous Contraction

In the small intestine, the most rapid and immediate response to mucosal injury is contraction of the villi. This reduces the surface area of the defect that must be repaired, and possibly also protects the epithelium from further injury. The initial immediate contraction of the villous tip is triggered by enteric nerves synapsing onto the subepithelial network of myofibroblasts underneath the denuded basement membrane. Villous contraction can be inhibited by denervation or ATP depletion, and inhibition of contraction reduces the rate at which the epithelium recovers.6

Following this immediate villous contraction, is a sustained contraction of smooth muscle cells in the core of the villous resulting in a reduction in villous height. This is again mediated by enteric nerves and is stimulated by prostaglandin E2. However, inhibition of this step does not reduce the overall rate of repair.

Epithelial Restitution

Migration of preexisting surrounding epithelial cells to cover the denuded basement membrane is termed restitution. The neighboring cells are initially signaled to migrate by growth factors such as transforming growth factor-β (TGF-β), insulin like growth factor-1, hepatocyte growth factor and platelet derived growth factor. TGF-β is the central peptide involved in migration and the majority of other growth factors ultimately stimulate restitution through a common pathway by triggering its activation. Having received the chemotactic signal to migrate, the enterocyte becomes flattened and the microvilli are disassembled to provide lamellipodia for the leading edge of the migrating cell.7 Integrins provide contact between the cytoskeleton of the cell and the denuded basement membrane. The signaling protein focal adhesion kinase (FAK) is phosphorylated when integrins bind to the extracellular matrix and triggers a cascade of intracellular signals that stimulate cell migration and growth. Forward progress is driven by treadmilling actin monomers from the back to the front edge of the lamellipodia, followed by detachment of the rear of the cell, and contraction of the cytoskeleton to pull the trailing edge forward.

Growth factors that stimulate cell migration also stimulate cell synthesis of nitric oxide (NO). NO has an anti adhesive effect that allows migration by reducing cell adhesion to the extra-cellular matrix. Trefoil peptides are also involved in restitution. These peptides are secreted by goblet cells into the intestinal lumen. They appear to promote migration by disrupting the cell-cell adhesion of adjacent cells to allow motility. Another peptide family, polyamines, are highly charged cations that can bind to the negative backbone of DNA and regulate expression of key genes, such as TGF-β. They also play a central role in cytoskeleton cross linking during migration.

Closure of Intercellular Tight Junctions

The final step in reestablishing barrier integrity is closure of the intercellular tight junctions. This is achieved by recruiting the tight junction proteins ZO-1 and occludin, and closing the paracellular space. Prostaglandins play a key role in this step through their effects on electrolyte transport. Specifically, they stimulate Cl- secretion in the crypt which drains the intercellular space of water and collapses the paracellular space. Prostaglandins also inhibit the absorption of Na+ on the villous tip, which prevents water from being pulled in through the intercellular space, allowing the tight junction to close.

Epithelial Proliferation and Differentiation

Once barrier integrity is restored, the final process is to restore the number and function of the epithelial cells. Epithelial proliferation by the stem cells in the base of the crypts is accelerated by growth factors and nutrients in the lumen, and allows the villous architecture to return to normal. Finally, the digestive and absorptive functions of the epithelial cells are restored as the newly formed cells migrate towards the villous tip.

References

1.  Mallick IH, et al. Dig Dis Sci 2004;49:1359-1377.

2.  Hinnebusch BF, et al. J Gastrointest Surg 2002;6:403-409.

3.  Nadeau JA, et al. Am J Vet Res. 2003 Apr;64(4):404-12.

4.  Nadeau JA, et al. Am J Vet Res. 2000 Jul;61(7):784-90

5.  Berschneider HM, et al. Equine Vet J Suppl. 1999 Apr;(29):24-9.

6.  Moore R, et al. Am J Physiol. 1989 Aug;257(2 Pt 1):G274-83

7.  Albers TM, et al. Lab Invest. 1995 Jul;73(1):139-48

Speaker Information
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Vanessa Cook, VetMB, MS, DACVS, DACVECC
North Carolina State University
Raleigh, NC


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