The Gut

M.P. Fink

Introduction

The gut probably participates in the pathogenesis of the multiple organ dysfunction syndrome (MODS) in many different ways. In order to keep this chapter focused and concise, the author will focus on three potential mechanisms: 1) alterations in intestinal epithelial permeability, leading to the systemic absorption of pro- inflammatory mediators or toxins derived from luminal microbes; 2) release into the lymphatic drainage from the gut of toxic materials; 3) release from enterocytes of pro-inflammatory cytokines, especially high mobility group box 1 (HMGB1).

Intestinal Epithelial Permeability is Increased in Critically Ill Patients

Alterations in the barrier function of the intestinal epithelium could permit the leakage of bacteria or microbial products, such as lipopolysaccharide (LPS) or exotoxin A (ETA), from the lumen of the gut into the systemic compartment, leading to the initiation or amplification of a deleterious inflammatory response and/or direct toxic effects on distant tissues. The notion that this process actually occurs in patients with MODS is supported by results from a number of clinical studies, which have documented increases in intestinal epithelial permeability in a variety of acute conditions that are associated with systemic inflammation [1–7].

Moreover, in several recent studies, increased intestinal permeability in critically ill patients has been shown to be associated with an increased risk of complications, MODS, or even mortality [3, 4, 7–9].

The strong association between increased gut mucosal permeability and MODS

and mortality in critical illness suggests the existence of a causal linkage: i. e., the

gut is the ‘motor’ that drives development of MODS [10]. There is, however, an-

other, equally plausible, possibility. According to this second notion, altered gut

mucosal permeability during critical illness is just one aspect of a more general-

ized phenomenon that affects epithelial barrier function in a variety of organs,

including the lungs, liver, and kidneys [11]. This more global view suggests the

pathophysiological mechanisms that are responsible for the derangements in gut

barrier function also pertain to epithelia in other organs. Moreover, this view

suggests that many of the clinical features of MODS, such as alveolar flooding in

the lung, cholestatic jaundice, and azotemia, can be explained – at least in part –

by a single unifying cellular mechanism: impaired expression and/or targeting of some or all of the proteins that form the tight junctions (see below) between adjacent epithelial cells.

Alterations in Intestinal Epithelial Permeability

The normal functioning of the lungs, liver, kidneys, and intestine, among other organs, depends on the establishment and maintenance of compositionally dis- tinct compartments that are lined by sheets of epithelial cells. An essential element in this process is the formation of tight junctions between adjacent cells making up the epithelial sheet. The tight junction serves as a fence that differentiates the cytosolic membrane into apical and basolateral domains. This fence function pre- serves cellular polarity and, in combination with transcellular vectorial transport processes, generates distinct internal environments in the opposing compartments that are formed by the epithelial sheet. In addition, the tight junction acts as a regu- lated semi-permeable barrier that limits the passive diffusion of solutes across the paracellular pathway between adjacent cells. Thus, the barrier function of the tight junction is necessary to prevent dissipation of the concentration gradients that exist between the two compartments defined by the epithelium. In some organs, notably the gut and the lung, this barrier function is also important to prevent systemic contamination by microbes and toxins that are present in the external environment [12].

Multiple Proteins are Necessary for the Assembly and Function of Tight Junctions

The formation of tight junction involves the assembly of at least nine different

peripheral membrane proteins and at least three different integral membrane

proteins [13]. Among the peripheral membrane proteins associated with tight

junctions are the membrane-associated guanylate kinase-like (MAGUK) proteins,

ZO-1, ZO-2, and ZO-3. The integral membrane proteins involved in tight junction

formation include, but are not limited to, occludin, and members of a large class of

proteins called claudins. Both occludin and the claudins contain four transmem-

brane domains and are thought to be the actual points of cell-cell contact within

the tight junction [14]. ZO-1 has been shown to interact with the cytoplasmic tails

of occludin and the claudins [15]. In addition, ZO-1 interacts with ZO-2 and ZO-3,

which then interact with various actin-binding proteins, such as pp120

[16,17],

thereby linking the tight junction with the cytoskeleton. Studies of mouse embryos

indicate that ZO-1 localizes in plasma membrane plaques well before occludin is

incorporated [18, 19], suggesting that ZO-1 probably plays a central role in the

assembly of mature tight junctions.

Nitric Oxide (NO) and/or Peroxynitrite (ONOO ⁻ ) Are Involved in the Regulation of Tight Junction Protein Expression and Function

We [20–22] and others [23–25] have shown that the permeability of cultured epithelial monolayers increases when the cells are incubated with various pro- inflammatory cytokines. The mechanisms responsible for cytokine-induced ep- ithelial hyperpermeability are incompletely understood. It is known, however, that compounds that spontaneously release NO increase the permeability of cultured intestinal epithelial cell monolayers [26,27]. This observation is pertinent, since in- cubating Caco-2 human enterocyte-like cells with the pro-inflammatory cytokine, interferon (IFN)- γ , or a mixture of the pro-inflammatory cytokines, IFN γ ^{+ tu-}

mor necrosis factor (TNF) and interleukin (IL)-1 β , leads to increased expression of inducible NO synthase (iNOS) and increased production of NO [21, 28, 29].

Moreover, compounds that inhibit iNOS have been shown to ameliorate the de- velopment of hyperpermeability induced by exposing Caco-2 cells to IFN γ ^[21]

or ‘cytomix’ (IFN- γ + TNF + (IL)-1 β ) [29]. Similarly, L-N(6)-(1-iminoethyl)lysine (L-NIL), an isoform-selective iNOS inhibitor, blocks the development of hyper- permeability when Calu-3 (human alveolar epithelial) monolayers are incubated with cytomix [30]. Thus, IFN- γ or cytomix appear to increase intestinal epithelial permeability, at least in part, by increasing the production of NO by enterocytes.

NO reacts rapidly with superoxide (O

) to form the potent oxidizing and ni- trating species, ONOO

[31, 32]. Several lines of evidence support the view that ONOO

(or some related species) rather than NO per se is responsible for the deleterious effects of NO on intestinal epithelial barrier function. Thus, when Caco-2 monolayers are incubated with the NO donor, SNAP, permeability is sig- nificantly increased, but the magnitude of the effect is small [27]. Furthermore, the permeability of Caco-2 monolayers is not affected when the cells are incu- bated with pyrogallol, a compound that spontaneously generates O

in aqueous solutions [27]. However, if Caco-2 cells are co-incubated with both SNAP and py- rogallol, then epithelial permeability is dramatically increased [27]. SNAP-induced hyperpermeability is also markedly enhanced by co-incubating the cells with di- ethyldithiocarbamate, a compound that is known to inactivate Cu-Zn superoxide dismutase and would thereby be expected to increase the concentration of en- dogenously generated O

[27]. Taken together, these findings support the view that NO-induced hyperpermeability is enhanced by the simultaneous availability of O

; i. e., conditions favoring the formation of ONOO

. Since ONOOH is a weak acid (pKa ∼6.8) and many of the effects of ONOO

are thought to be mediated by an unstable form of the protonated species, studies from our group showing that NO-induced hyperpermeability is enhanced under mildly acidic conditions further support the notion that ONOO

/ONOOH is the responsible moiety [20,33].

The mechanism(s) responsible for NO- or ONOO

-mediated intestinal ep-

ithelial hyperpermeability remain to be elucidated. However, our laboratory re-

ported that NO generated endogenously as the result of iNOS expression induced

by incubating Caco-2 cells with cytomix, or exogenously from the NO donor

DETA-NONOate [(Z)-1-[2(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-

ium-1,2-diolate] decreased the expression and impaired proper localization of the tight junction proteins, ZO-1, ZO-3, and occludin [22]. We also showed that incubating Caco-2 cells with either DETA-NONOate or cytomix increases the ex- pression of another key tight junction protein, claudin-1, and promotes the ac- cumulation of this protein in what appear to be vesicles within the cells. These findings support the view that NO (or a related reactive species) increases epithe- lial permeability by causing derangements in the expression and/or localization of several key tight junction proteins.

NO-dependent Changes in Na ⁺ ,K ⁺ -ATPase Activity can Affect Tight Junction Assembly and Function

Sugi et al. proposed that one way that NO might alter the expression or localiza- tion of various tight junction proteins is by modulating the activity of the mem- brane pump, Na

,K

-ATPase [34]. In a series of studies using monolayers of T84 enterocyte-like cells, these investigators reported that intracellular sodium con- centration and cell volume increase following exposure to the pro-inflammatory cytokine, IFN γ . Additionally, Sugi et al. showed that incubating T84 cells with either NO or IFN γ decreases the expression and activity of Na

,K

-ATPase. Remarkably, growing the monolayers in medium with low sodium concentration inhibits the development of hyperpermeability following exposure to IFN γ and also prevents IFN γ -induced alterations in occludin expression. These findings suggest a pathway that involves the following steps: IFN γ (and/or other pro-inflammatory cytokines)

→ iNOS induction → NO production → inhibition of Na

,K

-ATPase expres- sion and function → cell swelling → altered expression and/or targeting of tight junction proteins (e. g., occludin) → hyperpermeability.

Qayyum et al. reported that treatment of rat brain membranes with ONOO

in vitro decreases the activity of the Na

,K

-ATPase, and hypothesized that this effect may be caused by nitration of the Na

,K

-ATPase [35]. However, these authors did not demonstrate that ONOO

actually modifies the Na

,K

-ATPase.

In an earlier report, the same authors also implicated lipid peroxidation in the inactivation of the Na

, K

-ATPase [36], but only showed an association between lipid peroxidation and altered Na

,K

-ATPase activity in their studies. However, these authors convincingly showed that ROS decrease the affinity of the Na

,K

- ATPase for Na

and K

, inhibiting transport of these ions [35, 36]. Taken together, these results support the notion that oxidative or nitrosative post-translational modifications of Na

,K

-ATPase can lead to decreased epithelial barrier function.

Functional iNOS Expression is Essential for LPS-induced Alterations in Intestinal Permeability in Mice

Several years ago, our laboratory showed that gut mucosal permeability to a flu-

orescent macromolecule, fluorescein isothiocyanate-labeled dextran (molecular

mass, 4 kDa; FD4), was increased 24 hours after injecting rats with a low dose of

LPS [37]. However, when rats were treated with either of two chemically dissimilar isoform-selective iNOS inhibitors, aminoguanidine or S-methylisothiourea, the injection of LPS failed to increase gut mucosal permeability.

In a more recent study, the biochemical basis for the increase in gut mucosal permeability was investigated by our laboratory. Specifically, C57Bl/6J mice were injected with a sublethal (2 mg / kg) dose of E. coli LPS, and ileal mucosal perme- ability to FD4 was assessed 18 hours later [38]. Although mucosal permeability increased significantly when mice were injected with LPS, treatment with L-NIL, an isoform-selective iNOS inhibitor [39], reversed this effect. Basal ileal mucosal permeability in control (PBS-treated) iNOS knockout (iNOS

^/

) mice on a C57Bl/6J background was greater than that measured in control (wild-type) iNOS

^/

mice, a finding that is consistent with reports that basal levels of NO are required for normal gut homeostasis [40, 41]. Despite a basal defect in intestinal barrier func- tion in iNOS

^/

mice, permeability to FD4 failed to increase further when these mice were challenged with LPS.

In these studies [38], we used a portion of ileal tissue to prepare total and NP-40 (detergent)-insoluble protein extracts, the latter being enriched for tight junction-associated and other cytoskeletal proteins [42]. Total protein extracts were subjected to immunoblotting. NP-40 insoluble proteins were first solubilized with detergent-containing buffer and concentrated by immunoprecipitation prior to immunoblotting. The expression of occludin in NP-40 insoluble extracts was decreased in samples obtained 6 hours after injecting mice with LPS [38]. Occludin expression in NP-40 insoluble extracts decreased still further at 12 hours, but was starting to return toward normal 18 hours after LPS challenge. In total protein extracts, changes in occludin levels were less dramatic, and the maximal decrease was observed at 12 hours. ZO-1 expression decreased slightly in total protein extracts from ileal mucosa of mice exposed to LPS. However, there was a large decrease in ZO-1 levels in the NP-40 insoluble fraction. This finding suggests that the ZO-1 that is present in the cells of endotoxemic animals is unable to assemble into tight junctions. Consistent with our observations obtained using the Caco-2 system [22], claudin-1 expression increased in total protein extracts prepared from ileal mucosa. Immunoblotting total protein extracts for actin revealed equivalent loading of the samples in these gels. As expected, iNOS protein expression increased in total protein extracts from ileal mucosa of LPS-treated mice.

Endotoxemia is Associated with Derangements in Ileal Mucosal Tight Junction Protein Localization

Immunohistochemical studies of ileal tissue from endotoxemic mice were per-

formed 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 endothe-

lium of the lamina propria from normal mice. Following injection of mice with LPS,

ZO-1 staining was maintained in the crypts, but staining progressively decreased

over the tips of the villi. In sections from endotoxemic mice, the staining patterns for ZO-1 were disrupted only in focal regions of the ileum; approximately 60% of the villi in a given section stained normally. If the endotoxemic mice were treated with L-NIL to pharmacologically block iNOS-dependent NO production, then the correct targeting of ZO-1 in the ileal mucosa was preserved. Similar findings were obtained when staining was carried out for occludin instead of ZO-1.

Parallel experiments were performed using iNOS

^/

mice. The levels of oc- cludin and ZO-1 in ileal mucosa from control iNOS

^/

mice (i. e., those not chal- lenged with LPS) were reproducibly lower than the levels of these proteins in control iNOS

^/

mice. To some extent, these basal differences in occludin and ZO- 1 expression confounded interpretation of the results obtained in LPS-challenged animals. Nevertheless, it was apparent that injecting iNOS

^/

mice with LPS failed to cause a further decrease in the expression of ZO-1 or occludin in ileal mucosa.

The localization of ZO-1 and occludin was preserved in ileal sections prepared from LPS-treated iNOS

^/

mice, being essentially unchanged from what was observed in sections from iNOS

^/

animals injected with vehicle.

Surgical Stress Leads to Systemic Absorption of Gut-derived Toxins

Dating back to the era of Jacob Fine [43, 44], investigators have recognized that the gut represents a huge reservoir of bacteria-derived products, especially LPS, and that derangements in gut barrier function might lead to systemic absorption of these agents. Although Moore et al. were unable to detect LPS in the portal venous or peripheral venous blood samples obtained from critically ill trauma victims [45], other clinical and laboratory findings provide strong support for the notion that LPS can be systemically absorbed in patients or animals with acute conditions, such as hemorrhagic shock [46], necrotizing pancreatitis [5–7], or cardiopulmonary bypass [47, 48], that are associated with derangements in gut mucosal permeability.

Despite its other name, endotoxin, LPS is not really a toxin in the true sense of the word. Rather LPS is toxic because it is capable of activating intracellular signal- ing pathways in a variety of cell types, leading to the production of a variety of po- tentially cytotoxic mediators, including ROS and cytokines, such as TNF. However, in addition to releasing LPS and other pro-inflammatory molecules, many bacteria also produce extremely potent true toxins. For example, Pseudomonas aeruginosa are known to secrete at least 19 different soluble exoproteins, including several exotoxins (A, S, U and Y), elastase, staphylolytic protease, lipase, and phospholi- pase C. ETA is distantly related to diphtheria toxin produced by Corynebacterium diphtheriae [49]. Both ETA and diphtheria toxin kill the eukaryotic cells of the host by promoting mono-ADP-ribosylation and, thereby, inactivation of a key protein, translation elongation factor 2 (eEF2), which is essential for protein synthesis [49].

ETA is an extremely potent toxin, possessing a LD50 (dose that kills 50% of treated animals) of 0.2 g / kg following intraperitoneal injection into mice [49].

Based on a remarkable series of publications from Alverdy and colleagues at

the University of Chicago as well as other investigators, data have accumulated to

support the hypothesis that surgical stress (or presumably other forms of critical illness) lead to two synergistic gut-related consequences that promote development of distant organ dysfunction and even mortality. First, P. aeruginosa within the gut lumen respond to signals from the host, such as the cytokine, IFN γ [50], other factors released by hypoxic enterocytes [51], or norepinephrine [52]. In response to these signals, the bacteria increase their expression of virulence proteins, such as the PA-I lectin, a protein that is capable of causing derangements in gut epithelial permeability. Second, when P. aeruginosa are incubated under hypoxic conditions, secretion of ETA increases substantially [53]. It is likely that IFN γ also increases ETA secretion by P. aeruginosa, although this hypothesis remains to be tested. Thus, through a variety of mechanisms, critical illness can both increase elaboration within the gut lumen of a very potent toxin (ETA) and increase the permeability of the intestinal epithelium, permitting systemic absorption of this protein. The validity of this concept has been directly verified in a series of elegant in vivo studies using mice [52].

Release of Toxic Materials into the Lymphatic Drainage from the Gut

Although many lines of investigation suggested that the gut might be the “motor of multiple organ system failure” [10], the failure in some key clinical studies to observe clear evidence of LPS in portal venous blood [45] or bacteria in mesen- teric lymph nodes [54] in trauma victims led to skepticism about this concept.

Nevertheless, two research groups, one directed by Deitch in Newark and another directed by Moore in Denver, formulated a novel hypothesis to explain the data:

rather than the portal venous system, the draining mesenteric lymphatics might be the source of key gut-derived factors that promote distant organ dysfunction in critical illness [55, 56]. Over the past few years, an impressive body of evidence, obtained mostly by studying rat models of hemorrhagic shock or burn injury, has accumulated in support of this concept. Briefly, it has been shown that post-shock (but not control) mesenteric lymph primes or activates neutrophils in vitro [56,57], and is toxic to various cell types in culture [57]. Furthermore, ligation of the main draining mesenteric lymphatic in rats ameliorates hemorrhagic shock- or burn- induced distant organ injury [55,56,58]. Finally, administration of post-shock (but not control) mesenteric lymph is capable of inducing distant organ dysfunction in rodents [55].

The precise nature of the factor (or factors) in post-shock lymph that is re-

sponsible for its deleterious actions remains elusive. Both lipophilic [59–61] and

hydrophilic [61, 62] factors have been implicated. One of the factors may be a pep-

tide, derived from the action of serine proteases on albumin [61, 63].

Release from Enterocytes of Pro-inflammatory Cytokines

The gastrointestinal tract is the largest immune organ in the body. Numerous spe- cialized lymphocytes, intraepithelial lymphocytes (IELs), reside between adjacent enterocytes within the epithelium. Plasma cells, lymphocytes, macrophages, mast cells, and neutrophils are present within the lamina propria even under basal con- ditions, and, following the induction of a local or systemic inflammatory response, many more immune cells migrate into this layer of the bowel wall as well as into the muscularis propria. These immune cells are capable of producing a wide ar- ray of cytokines and other mediators, such as platelet activating factor (PAF) and histamine.

Although they are not ‘professional’ immune cells, enterocytes making up the epithelial layer of the gut are capable of secreting chemokines [64, 65], cy- tokines [66, 67] and other pro-inflammatory mediators [68]. Recent data from our laboratory suggest that HMGB1, a recently discovered cytokine-like protein, is among the pro-inflammatory substances that are released by immunostimulated enterocytes.

High-mobility group proteins are small DNA-binding proteins that serve an important role in transcriptional regulation [69]. One of these proteins, HMGB1, has been identified as a late-acting mediator of LPS- [70] or sepsis-induced [71]

lethality in mice. HMGB1 also has been implicated as a mediator LPS- [72] or hemorrhagic shock-induced acute lung injury (ALI) in mice [73]. Additionally, our laboratory showed that adding recombinant HMGB1 to the culture medium for Caco-2 human enterocyte-like monolayers increases the permeability of the ep- ithelial barrier to a fluorescent macromolecule, fluorescein isothiocyanate-labeled dextran with an average molecular mass of 4,000 Da (FD4) [74]. Moreover, we showed that injecting mice with HMGB1 induces gut mucosal hyperpermeability to FD4 and promotes bacterial translocation to mesenteric lymph nodes [74]. Ad- ditional studies have documented that HMGB1 is a cytokine-like molecule that can promote TNF release from mononuclear cells [75].

HMGB1 also has been implicated in the pathogenesis of human disease. In the original report describing HMGB1 as a mediator of LPS-induced lethality, Abraham et al. reported that circulating levels of this protein are increased in patients with severe sepsis [72]. Shortly thereafter, Ombrellino and co-workers described a patient with high circulating levels of HMGB1 following an episode of hemorrhagic shock [76]. More recently, increased levels of HMGB1 mRNA have been detected in whole blood samples from patients with septic shock, particularly among non-survivors [77]. Similarly, persistently high serum levels of HMGB1 protein have been detected in patients with septic shock [78]

HMGB1 is actively secreted by immunostimulated macrophages [70, 79–81],

natural killer cells [82], and pituicytes [83]. This protein is also released by necrotic,

but not apoptotic, cells [84]. Because HMGB1 has been shown to modulate in-

testinal epithelial barrier function [74], we hypothesized that active secretion by

enterocytes might be yet another source for this cytokine-like protein. Recently, we

reported that stimulating either Caco-2 cells or primary cultures of murine ente- rocytes with cytomix induced secretion of HMGB1 [85]. Additionally, we showed that addition of a neutralizing polyclonal anti-HMGB1 antibody partially blocked the increase in permeability caused by incubating Caco-2 monolayers with cy- tomix [85]. The data also indicate that HMGB1 is secreted in soluble form as well as a particulate form that is sequestered within exosomes. These data sup- port the view that enterocyte-derived HMGB1 may be an autocrine amplifier of derangements in epithelial barrier function initiated by other pro-inflammatory stimuli.

References

1. Langkamp-Henken B, Donovan TB, Pate LM, Maull CD, Kudsk KA (1995) Increased intestinal permeability following blunt and penetrating trauma. Crit Care Med 23:660–664

2. Pape H-C, Dwenger A, Regel G, et al (1994) Increased gut permeability after multiple trauma.

Br J Surg 81:850–852

3. Kompan L, Kremzar B, Gadzijev E, Prosek M (1999) Effects of early enteral nutrition on intestinal permeability and the development of multiple organ failure after multiple injury.

Intensive Care Med 25:157–161

4. Faries PL, Simon RJ, Martella AT, Lee MJ, Machiedo GW (1998) Intestinal permeability correlates with severity of injury in trauma patients. J Trauma 44:1031–1036

5. Penalva JC, Martinez J, Laveda R, et al (2004) A study of intestinal permeability in relation to the inflammatory response and plasma endocab IgM levels in patients with acute pancreatitis.

J Clin Gastroenterol 38:512–517

6. Ammori BJ, Fitzgerald P, Hawkey P, McMahon MJ (2003) The early increase in intestinal per- meability and systemic endotoxin exposure in patients with severe acute pancreatitis is not associated with systemic bacterial translocation: molecular investigation of microbial DNA in the blood. Pancreas 26:18–22

7. Ammori BJ, Leeder PC, King RFGJ, et al (1999) Early increase in intestinal permeability in patients with severe acute pancreatitis: correlation with endotoxemia, organ failure, and mortality. J Gastrointest Surg 3:252–262

8. Doig CJ, Sutherland LR, Sandham JD, Fick GH, Verhoef M, Meddings JB (1998) Increased intestinal permeability is associated with the development of multiple organ dysfunction syndrome in critically ill ICU patients. Am J Respir Crit Care Med 158:444–451

9. Oudemans-van Straaten HM, Jansen PG, Hoek FJ, et al (1996) Intestinal permeability, cir- culating endotoxin, and postoperative systemic responses in cardiac surgery patients. J Car- diothorac Vasc Anesth 10:187–194

10. Carrico CJ, Meakins JL, Marshall JC, Fry D, Maier RV (1985) Multiple-organ-failure syndrome.

Arch Surg 121:196–208

11. Fink MP, Delude RL (2005) Epithelial barrier dysfunction: a unifying theme to explain the pathogenesis of multiple organ dysfunction at the cellular level. Crit Care Clin 21:177–196 12. Stevenson BR (1999) Understanding tight junction clinical physiology at the molecular level.

J Clin Invest 104:3–4

13. Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2:285–293

14. Tsukita S, Furuse M (2000) The structure and function of claudins, cell adhesion molecules at tight junctions. Ann NY Acad Sci 915:129–135

15. Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S (1994) Direct association

of occludin with ZO-1 and its possible involvement in the localization of occludin in tight

junctions. J Cell Biol 127:1617–1626

16. Fanning AS, Jameson BJ, Jesaitis LA, Anderson JM (1998) The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton.

J Biol Chem 273:29745–29753

17. Itoh M, Nagafuchi A, Moroi S, Tsukita S (1997) Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments. J Cell Biol 138:181–192

18. Sheth B, Fesenko I, Collins JE, et al (1997) Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1 alpha+ isoform. Development 124:2027–

2937

19. Sheth B, Moran B, Anderson JM, Fleming TP (2000) Post-translational control of occludin membrane assembly in mouse trophectoderm: a mechanism to regulate timing of tight junction biogenesis and blastocyst formation. Development 127:831–840

20. Unno N, Hodin RA, Fink MP (1999) Acidic conditions exacerbate interferon-gamma-induced intestinal epithelial hyperpermeability: role of peroxynitrous acid. Crit Care Med 27:1429–

1436

21. Unno N, Menconi MJ, Smith M, Fink MP (1995) Nitric oxide mediates interferon-gamma- induced hyperpermeability in cultured human intestinal epithelial monolayers. Crit Care Med 23:1170–1176

22. Han X, Fink MP, Delude RL (2003) Proinflammatory cytokines cause NO ·-dependent and independent changes in expression and localization of tight junction proteins in intestinal epithelial cells. Shock 19:229–237

23. Adams RB, Planchon SM, Roche JK (1993) IFN-y modulation of epithelial barrier function:

time course, reversibility, and site of cytokine binding. J Immunol 150:2356–2363

24. Planchon SM, Martins CAP, Guerrant RL, Roche JK (1994) Regulation of intestinal epithelial barrier function by TGF-b1. Evidence for its role in abrogating the effects of T cell cytokine.

J Immunol 153:5730–5739

25. Madara JL, Stafford J (1989) Interferon-y directly affects barrier function of cultured intestinal epithelial monolayers. J Clin Invest 83:724–727

26. Salzman AL, Menconi MJ, Unno N, et al (1995) Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers. Am J Physiol 268:G361–

G373

27. Menconi MJ, Unno N, Smith M, Aguirre DE, Fink MP (1998) The effect of nitric oxide donors on the permeability of cultured intestinal epithelial monolayers: role of superoxide, hydroxyl radical, and peroxynitrite. Biochem Biophys Acta 1425:189–203

28. Chavez A, Morin MJ, Unno N, Fink MP, Hodin RA (1999) Acquired interferon-y responsive- ness during Caco-2 cell differentiation: effects on iNOS gene expression. Gut 44:659–665 29. Chavez A, Menconi MJ, Hodin RA, Fink MP (1999) Cytokine-induced epithelial hyperper-

meability: role of nitric oxide. Crit Care Med 27:2246–2251

30. Han X, Fink MP, Uchiyama T, Delude RL (2004) Increased iNOS activity is essential for the development of pulmonary epithelial tight junction dysfunction in endotoxemic mice. Am J Physiol Lung Cell Mol Physiol 286:L259–L267

31. Wink DA, Mitchell JB (1998) Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radical Biol Med 25:434–456 32. Pryor WA, Squadrito GL (1995) The chemistry of peroxynitrite: a product from the reaction

of nitric oxide with superoxide. Am J Physiol 268:L699–L722

33. Unno N, Menconi MJ, Smith M, Aguirre DG, Fink MP (1997) Hyperpermeability of intestinal epithelial monolayers is induced by NO: effect of low extracellular pH. Am J Physiol 272:G923–

G934

34. Sugi K, Musch MW, Field M, Chang EB (2001) Inhibition of Na

,K

-ATPase by interferon

gamma down-regulates intestinal epithelial transport and barrier function. Gastroenterology

120:1393–1403

35. Qayyum I, Zubrow AB, Ashraf QM, Kubin J, Delivoria-Papadopoulos M, Mishra OP (2001) Nitration as a mechanism of Na+, K+-ATPase modification during hypoxia in the cerebral cortex of the guinea pig fetus. Neurochem Res 26:1163–1169

36. Mishra OP, Delivoria-Papadopoulos M, Cahillane G, Wagerle LC (1989) Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP, K+, Na+, and strophanthidin in vitro. Neurochem Res 14:845–851

37. Unno N, Wang H, Menconi MJ, et al (1997) Inhibition of inducible nitric oxide synthase ameliorates lipopolysaccharide-induced gut mucosal barrier dysfunction in rats. Gastroen- terology 113:1246–1257

38. Han X, Fink MP, Yang R, Delude RL (2004) Increased iNOS activity is essential for intestinal epithelial tight junction dysfunction in endotoxemic mice. Shock 21:261–270

39. Moore WM, Webber RK, Jerome GM, Tjong FS, Misko TP, Currie MG (1994) L-N6-(1- iminoethyl)lysine: A selective inhibitor of inducible nitric oxide synthase. J Med Chem 37:3886–3888

40. Kubes P (1993) Ischemia-reperfusion in the feline small intestine: a role for nitric oxide. Am J Physiol 264:G143–G149

41. Kubes P (1992) Nitric oxide modulates epithelial permeability in the feline small intestine.

Am J Physiol 262:G1138–G1142

42. Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y, Tsukita S (1997) Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol 137:1393–1401 43. Ravin HA, Rowley D, Jenkins C, Fine J (1960) On the absorption of bacterial endotoxin from

the gastro-intestinal tract of the normal and shocked animal. J Exp Med 112:783–792 44. Fine J, Frank ED, Rutenberg SH, Schweinburg FB (1959) The bacterial factor in traumatic

shock. N Engl J Med 260:214–216

45. Moore FA, Moore EE, Poggetti R, et al (1991) Gut bacterial translocation via the portal vein:

a clinical perspective with major torso trauma. J Trauma 31:629–638

46. Luyer MD, Buurman WA, Hadfoune M, et al (2004) Pretreatment with high-fat enteral nutrition reduces endotoxin and tumor necrosis factor-alpha and preserves gut barrier function early after hemorrhagic shock. Shock 21:65–71

47. Wan S, LeClerc JL, Huynh CH, et al (1999) Does steroid pretreatment increase endotoxin release during clinical cardiopulmonary bypass? J Thorac Cardiovasc Surg 117:1004–1008 48. Martinez-Pellus AE, Merino P, Bru M, et al (1993) Can selective digestive decontamination

avoid the endotoxemia and cytokine activation promoted by cardiopulmonary bypass. Crit Care Med 21:1684–1691

49. Yates SP, Jorgensen R, Andersen GR, Merrill AR (2006) Stealth and mimicry by deadly bacterial toxins. Trends Biochem Sci 31:123–133

50. Wu L, Estrada O, Zaborina O, et al (2005) Recognition of host immune activation by Pseu- domonas aeruginosa. Science 309:774–747

51. Kohler JE, Zaborina O, Wu L, et al (2005) Components of intestinal epithelial hypoxia activate the virulence circuitry of Pseudomonas. Am J Physiol Gastrointest Liver Physiol 288:G1084–

G1054

52. Alverdy J, Holbrook C, Rocha F, et al (2000) Gut-derived sepsis occurs when the right pathogen with the right virulence genes meets the right host: evidence for In vivo virulence expression in pseudomonas aeruginosa. Ann Surg 232:480–489

53. Gaines JM, Carty NL, Colmer-Hamood JA, Hamood AN (2005) Effect of static growth and different levels of environmental oxygen on toxA and ptxR expression in the Pseudomonas aeruginosa strain PAO1. Microbiology 151:2263–2275

54. Peitzman AB, Udekwu AO, Ochoa J, Smith S (1991) Bacterial translocation in trauma patients.

J Trauma 31:1083–1087

55. Magnotti LJ, Upperman JS, Xu D-Z, Lu Q, Deitch EA (1998) Gut-derived mesenteric lymph

but not portal blood increases endothelial cell permeability and promotes lung injury after

hemorrhagic shock. Ann Surg 228:518–527

56. Zallen G, Moore EE, Johnson JL, Tamura DY, Ciesla DJ, Silliman CC (2006) Posthemorrhagic shock mesenteric lymph primes circulating neutrophils and provokes lung injury. J Surg Res 83:83–88

57. Upperman JS, Deitch EA, Guo W, Lu Q, Xu D (1998) Post-hemorrhagic shock mesenteric lymph is cytotoxic to endothelial cells and activates neutrophils. Shock 10:407–414 58. Magnotti LJ, Xu DZ, Lu Q, Deitch EA (1999) Gut-derived mesenteric lymph: a link between

burn and lung injury. Arch Surg 134:1333–1340

59. Gonzalez RJ, Moore EE, Ciesla DJ, Meng X, Biffl WL, Silliman CC (2001) Post-hemorrhagic shock mesenteric lymph lipids prime neutrophils for enhanced cytotoxicity via phospholi- pase A2. Shock 16:218–222

60. Gonzalez RJ, Moore EE, Ciesla DJ, Biffl WL, Offner PJ, Silliman CC (2001) Phospholipase A(2)–

derived neutral lipids from posthemorrhagic shock mesenteric lymph prime the neutrophil oxidative burst. Surgery 130:198–203

61. Kaiser VL, Sifri ZC, Dikdan GS, et al (2005) Trauma-hemorrhagic shock mesenteric lymph from rat contains a modified form of albumin that is implicated in endothelial cell toxicity.

Shock 23:417–425

62. Dayal SD, Hauser CJ, Feketeova E, et al (2002) Shock mesenteric lymph-induced rat polymor- phonuclear neutrophil activation and endothelial cell injury is mediated by aqueous factors.

J Trauma 52:1048–1055

63. Deitch EA, Shi HP, Lu Q, Feketeova E, Xu DZ (2003) Serine proteases are involved in the pathogenesis of trauma-hemorrhagic shock-induced gut and lung injury. Shock 19:452–456 64. Eckmann L, Jung HC, Schurer-Maly C, Panja A, Morzycka-Wroblewska E, Kagnoff MF (1993) Differential cytokine expression by human intestinal epithelial cell lines. Gastroenterology 105:1689–1697.

65. Kim JM, Kim JS, Jun HC, Oh YK, Song IS, Kim CY (2002) Differential expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cancer cell lines in response to Clostridium difficile toxin A. Microbiol Immunol 46:333–342

66. Taylor CT, Fueki N, Agah A, Hershberg RM, Colgan SP (1999) Critical role of cAMP response element binding protein expression in hypoxia-elicited induction of epithelial tumor necrosis factor-alpha. J Biol Chem 274:19447–19454

67. Jung HC, Eckmann L, Yang SK, et al (1995) A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 95:55–65

68. Eckmann L, Stenson WF, Savidge TC, et al (1997) Role of intestinal epithelial cells in the host secretory response to infection by invasive bacteria. Bacterial entry induces epithelial prostaglandin H synthase-2 expression and prostaglandin E2 and F2 production. J Clin Invest 100:296–309

69. Bustin M, Lehn DA, Landsman D (1990) Structural features of the HMG chromosomal proteins and their genes. Biochim Biophys Acta 1049:231–243

70. Wang H, Bloom O, Zhang M, et al (1999) HMG-1 as a late mediator of endotoxin lethality in mice. Science 285:248–251

71. Yang H, Ochani M, Li J, et al (2004) Reversing established sepsis with antagonists of endoge- nous HMGB1. Proc Natl Acad Sci USA 101:296–301

72. Abraham E, Arcaroli J, Carmody A, Wang H, Tracey KJ (2000) HMG-1 as a mediator of acute lung inflammation. J Immunol 165:2950–2954

73. Kim JY, Park JS, Strassheim D, et al (2005) HMGB1 contributes to the development of acute lung injury after hemorrhage. Am J Physiol Lung Cell Mol Physiol 288:L958–L965

74. Sappington PL, Yang R, Yang H, Tracey KJ, Delude RL, Fink MP (2002) HMGB1 B box increases the permeability of Caco-2 enterocytic monolayers and impairs intestinal barrier function in mice. Gastroenterology 123:790–802

75. Andersson U, Wang H, Palmblad K, et al (2001) High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes. J Exp Med 192:565–

570

76. Ombrellino M, Wang H, Ajemian MS, et al (1999) Increased serum concentrations of high- mobility-group protein 1 in haemorrhagic shock. Lancet 354:1446–1447

77. Pachot A, Monneret G, Voirin N, et al (2005) Longitudinal study of cytokine and immune transcription factor mRNA expression in septic shock. Clin Immunol 114:61–69

78. Sunden-Cullberg J, Norrby-Teglund A, Rouhianen A, et al (2005) Persistent elevation of high mobility group box-1 protein (HMGB1) in patients with severe sepsis and septic shock. Crit Care Med 33:564–573

79. Rendon-Mitchell B, Ochani M, Li J, et al (2003) IFN-gamma induces high mobility group box 1 protein release partly through a TNF-dependent mechanism. J Immunol 170:3890–3897 80. Gardella S, Andrei C, Ferrera D, et al (2002) The nuclear protein HMGB1 is secreted by

monocytes via a non-classical, vesicle-mediated secretory pathway. EMBO Rep 3:995–1001 81. Bonaldi T, Talamo F, Scaffidi P, et al (2003) Monocytic cells hyperacetylate chromatin protein

HMGB1 to redirect it towards secretion. EMBO J 22:5551–5560

82. Semino C, Angelini G, Poggi A, Rubartelli A (2005) NK/iDC interaction results in IL-18 secretion by DCs at the synaptic cleft followed by NK cell activation and release of the DC maturation factor HMGB1. Blood 106:609–616

83. Wang H, Vishnubhakat JM, Bloom O, et al (2002) Proinflammatory cytokines (tumor necrosis factor and interleukin 1) stimulate release of high mobility group protein-1 by pituicytes.

Surgery 126:389–392

84. Scaffidi P, Misteli T, Bianchi ME (2002) Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418:191–195

85. Liu S, Stolz DB, Sappington PL, et al (2006) HMGB1 is secreted by immunostimulated enterocytes and contributes to cytomix-induced hyperpermeability of Caco-2 monolayers.

Am J Physiol Cell Physiol 290:C990–999