Epithelial Barrier Dysfunction: A Unifying Theme to Explain the Pathogenesis of Multiple Organ Dysfunction at the Cellular Level
Section snippets
Is cytopathic hypoxia the underlying mechanism responsible for multiple organ dysfunction syndrome?
Previously, the authors' laboratory entertained the possibility that cellular dysfunction in sepsis and MODS is caused by an acquired intrinsic derangement in mitochondrial function leading to inadequate production of ATP, a pathologic state called cytopathic hypoxia [1], [2]. Although subsequent clinical studies have confirmed that cellular respiration is deranged in patients with sepsis [3], [4], recent data from the authors' laboratory suggest that mitochondrial dysfunction is more likely to
Increased cellular apoptosis is another mechanism that might underlie the development of organ dysfunction in patients with sepsis or other causes of multiple organ dysfunction syndrome
Certainly, massive apoptosis among lymphoid cells is a prominent feature of sepsis in both human patients and mice [12], [13], [14]. Moreover, using pharmacologic or genetic approaches to limit lymphoid cell apoptosis improves survival in mice with bacterial peritonitis [12], [14], a finding that supports the view that increased programmed cell death of lymphocytes is significant in the pathogenesis of sepsis. Intestinal epithelial apoptosis also occurs in both patients and animals with sepsis
Tight junctions maintain epithelial polarity and barrier function
The normal functioning of the lungs, liver, kidneys, and intestine, among other organs, depends on the establishment and maintenance of compositionally distinct compartments that are lined by sheets of epithelial cells. An essential element in this process is the formation of TJs between adjacent cells making up the epithelial sheet. The TJ serves as a fence that differentiates the cytosolic membrane into apical and basolateral domains. This fence function preserves cellular polarity and, in
Multiple proteins are necessary for the assembly and functioning of tight junctions
The formation of TJs involves the assembly of at least nine different peripheral membrane proteins and at least three different integral membrane proteins [17]. Among the peripheral membrane proteins associated with TJs are the membrane-associated guanylate kinase-like proteins, ZO-1, ZO-2, and ZO-3. The integral membrane proteins involved in TJ formation include, but are not limited to, occludin and members of a large class of proteins called claudins. Both occludin and the claudins contain
Organ dysfunction caused by systemic inflammation may be the aggregate macroscopic manifestation of derangements in cellular physiology that are not necessarily sufficient to cause cell death
Because many of the organs commonly affected in MODS (eg, the lungs, liver, kidneys, and gut) depend on the proper functioning of an epithelial component, it is reasonable to hypothesize that epithelial cell dysfunction is important in this syndrome. Of course, the proper functioning of epithelia depends not only on the formation of TJs, but also on the appropriate expression, localization, and activity of many other cellular constituents (eg, membrane pumps, cytoskeletal proteins, and
What are the expected physiologic and clinical manifestations of tight junction dysfunction in epithelial cells?
The proper functioning of the lungs, kidneys, liver, and gut depends on the generation and maintenance of compositionally distinct compartments.Table 1 summarizes some predicted physiologic and clinical consequences of epithelial TJ dysfunction. In the lungs, failure to maintain normal TJ formation is expected to promote alveolar flooding, and hence pulmonary edema, on the basis of backleakage of salt and water that is pumped from the apical side of the alveolar epithelium to the basolateral
Nitric oxide or peroxynitrite are involved in the regulation of tight junction protein expression and function
The authors [43], [44], [45] and others [46], [47], [48] have shown that the permeability of cultured epithelial monolayers increases when the cells are incubated with various proinflammatory cytokines. The mechanisms responsible for cytokine-induced epithelial hyperpermeability are incompletely understood. It is known, however, that compounds that spontaneously release nitric oxide (NO·) increase the permeability of cultured intestinal epithelial cell monolayers [49], [50]. This observation is
Nitric oxide–dependent changes in Na+, K+-ATPase activity can affect tight junction assembly and function
Sugi et al [57] proposed that one way that NO· might alter the expression or localization of various TJ proteins is by modulating the activity of the membrane pump, Na+ ,K+ -ATPase. In a series of studies using monolayers of T84 enterocyte-like cells, these investigators reported that intracellular sodium concentration and cell volume increase following exposure to the proinflammatory cytokine, IFN-γ. Additionally, Sugi et al [57] showed that incubating T84 cells with either NO· or IFN-γ
Functional inducible nitric oxide synthase expression is essential for lipopolysaccharide-induced alterations in intestinal permeability in mice
When Han et al [63] injected C57Bl-6J mice with a small sublethal (2 mg/kg) dose of Escherichia coli LPS, intestinal mucosal permeability to the permeability probe, fluorescein isothiocyanate-labeled dextran ([FD4] molecular mass 4 kd), increased significantly (Fig. 2A). Treatment of endotoxemic mice with L-NIL, an isoform-selective iNOS inhibitor [60], ameliorated LPS-induced ileal mucosal hyperpermeability (Fig. 2B). Basal ileal mucosal permeability in control (phosphate buffered saline
Functional inducible nitric oxide synthase expression is essential for lipopolysaccharide-induced bacterial translocation in mice
Bacterial translocation from the gut lumen to mesenteric lymph nodes is another measure of in vivo mucosal barrier function. In a recent study from the authors' group, endotoxemia increased the number of bacteria that were recovered from mesenteric lymph nodes from wild-type (iNOS+/+) mice [63]. Treatment of endotoxemic iNOS+/+ mice with L-NIL to block iNOS-dependent NO· production decreased LPS-induced bacterial translocation. Similarly, LPS failed to induce bacterial translocation in iNOS−/−
Lipopolysaccharide decreases the expression of several tight junction proteins in mice
Han et al [63] used a portion of ileal tissue to prepare total and NP-40 (detergent)–insoluble protein extracts, the latter being enriched for TJ-associated and other cytoskeletal proteins. Total protein extracts were subjected to immunoblotting. NP-40–insoluble proteins were first solubilized with detergent-containing buffer and concentrated by immunoprecipitation before immunoblotting. The expression of occludin in NP-40–insoluble extracts was decreased in samples obtained 6 hours after
Endotoxemia is associated with derangements in ileal mucosal tight junction protein localization
Immunohistochemical studies of ileal tissue from endotoxemic mice were performed using samples harvested 12 hours after injection of LPS. ZO-1 formed a continuous staining pattern around the enterocyte layer near the apical region of the lateral membrane of crypt and villous cells of the epithelium and the endothelium of the lamina propria from normal mice (Fig. 3). Following injection of mice with LPS, ZO-1 staining was maintained in the crypts, but staining progressively decreased over the
Lipopolysaccharide impairs hepatobiliary barrier function by an inducible nitric oxide synthase–dependent mechanism
Lora et al [66] reported that hepatic TJ function can be assessed by measuring serum concentrations of bile acids and conjugated bilirubin. In a recent study from the authors' laboratory, Han et al [67] showed that circulating levels of both of these bile components were increased in mice injected 12 hours earlier with LPS (Fig. 4). When endotoxemic mice were treated with L-NIL, however, serum levels of bile acids and conjugated bilirubin were not different from normal. Although basal serum
Lipopolysaccharide induces hepatic inducible nitric oxide synthase expression and alterations in hepatic tight junction protein expression
In the studies performed by Han et al [67] of the effects of endotoxemia on hepatobiliary epithelial barrier function in mice, immunoreactive iNOS was not detectable by Western blotting of hepatic protein extracts from control mice. Within 6 hours after the injection of LPS, however, hepatic iNOS expression was clearly evident. Levels of iNOS protein in liver increased still further 12 and 18 hours after the injection of LPS. Following the induction of endotoxemia, occludin and ZO-1 expression
Endotoxemia is associated with derangements in hepatic tight junction protein localization
Han et al [67] found minimal evidence of hepatic inflammation or necrosis when they examined hematoxylin and eosin–stained thin sections of liver tissue from mice injected 12 hours earlier with LPS, irrespective of whether or not the animals were treated with L-NIL. In control specimens, occludin and ZO-1 were largely detected as parallel strands of staining representing the outlines of canaliculi (Fig. 6). Consistent with previously reported data [68], staining of occludin and ZO-1 in normal
A proinflammatory milieu decreases pulmonary epithelial barrier function
Prompted by the findings noted previously supporting the notion that systemic inflammation induced by injecting LPS causes alterations in epithelial TJ formation in two organs (liver and intestine), Han et al [53] from the authors' laboratory sought to extended these observations by examining the effect of LPS (2 mg/kg) on the leakage of FD4 from plasma into the alveolar space in C57Bl-6J mice. At various time points after injection of LPS (or PBS), the authors injected mice intravenously with
Lipopolysaccharide induces inducible nitric oxide synthase expression and increases nitric oxide synthase production in mice
Injecting mice with LPS significantly increased the concentration of the NO· breakdown products, NO2− and NO3−, in bronchoalveolar lavage fluid and serum (data not shown). Treatment with L-NIL only partially inhibited the accumulation of NO2− and NO3− in serum, whereas treatment with the iNOS inhibitor almost completely blocked accumulation of these NO· metabolites in bronchoalveolar lavage fluid.
Endotoxemia is associated with alterations in the expression of tight junction proteins in the lung
NP-40–insoluble occludin and ZO-1 levels decreased within 6 hours after injecting mice with a sublethal dose of LPS, and were maximally decreased at 12 hours. By 18 hours, NP-40 insoluble occludin and ZO-1 levels were starting to normalize. In lung tissue specimens from normal mice, ZO-1 was localized as a continuous line along the boundaries between neighboring bronchial and alveolar epithelial cells. The intensity of this staining was markedly reduced in lung tissue harvested from mice that
Summary
Collectively, the results from more than a decade of work by scientists in the authors' laboratory and many other laboratories around the world support the view that an inflammatory milieu leads to marked alterations in the structure and function of TJs in multiple epithelia. These data have been obtained using both reductionist in vitro models, such as Caco-2 enterocyte-like cells growing as monolayers in diffusion chambers, and in vivo models, such as mice injected with the proinflammatory
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