Increased iNOS activity is essential for hepatic epithelial tight junction dysfunction in endotoxemic mice

Xiaonan Han,1 Mitchell P. Fink,1,2 Takashi Uchiyama,1 Runkuan Yang,1 and Russell L. Delude1,3

Departments of 1Critical Care Medicine, 3Pathology, and 2Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Submitted 20 May 2003 ; accepted in final form 16 August 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We tested the hypothesis that increased production of nitric oxide (NO·) by inducible NO· synthase (iNOS) is a key factor responsible for alterations in the expression, localization, and function of key tight junction (TJ) proteins in mice challenged with lipopolysaccharide (LPS, endotoxin). Endotoxemia was associated with hepatobiliary epithelial barrier dysfunction, as evidenced by increased plasma-to-bile leakage of FITC-labeled dextran (relative molecular mass 40 kDa) and increased circulating levels of bile acids and conjugated bilirubin. Immunoblotting revealed decreased expression of zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin in liver after injection of C57Bl/6J mice with 2 mg/kg Escherichia coli 0111:B4 LPS. Nonidet P-40-insoluble (i.e., TJ-associated) occludin and ZO-1 were virtually undetectable 12 and 18 h after injecting LPS. Immunofluorescence microscopy also revealed deranged subcellular localization of ZO-1 and occludin in endotoxemic mice. Pharmacological inhibition of iNOS activity using l-N6-(1-iminoethyl)lysine (5 mg/kg) or genetic ablation of iNOS ameliorated LPS-induced changes in hepatobiliary barrier function, and these strategies partially preserved TJ protein expression and localization. Steady-state levels of occludin and ZO-3 transcripts decreased transiently after injecting LPS but returned toward normal by 12 and 24 h after induction of endotoxemia, respectively. These results support the view that iNOS-dependent NO· production is an important factor contributing to hepatobiliary epithelial barrier dysfunction resulting from systemic inflammation and suggest that iNOS induction may play a role in the development of cholestatic jaundice in patients with severe sepsis.

multiple organ dysfunction syndrome; jaundice; hyperbilirubinemia; endotoxemia; inducible nitric oxide synthase; lipopolysaccharide


HYPERBILIRUBINEMIA IS A COMMON occurrence in patients with extrahepatic infections, leading to the development of severe sepsis (8, 41). Persistent hyperbilirubinemia in septic patients is associated with a significantly increased risk of mortality (41). Efforts to understand the pathophysiological mechanisms responsible for cholestatic jaundice resulting from sepsis have largely focused on lipopolysaccharide (LPS)-induced alterations in the function and expression of various bile acid transporters (3, 11, 27, 35). Nevertheless, another factor that could contribute to the development of intrahepatic cholestasis is backleakage of bile from the canalicular spaces in the sinusoids (1, 36).

The primary intercellular barrier preventing the backdiffusion of bile is the one formed by the tight junctions (TJs) between adjacent hepatocytes. TJs are specialized and complex structures composed of multiple proteins that are anchored directly or indirectly to the actin-based cytoskeleton. Integral membrane proteins involved in TJ formation include occludin, members of a large class of proteins called claudins, and junction adhesion molecule (2). The former two proteins contain four transmembrane domains and are thought to be the points of cell-to-cell contact within the TJ (2). Cosedimentation assays of TJ proteins suggest that there is a strong interaction between occludin and another protein associated with TJ formation, zonula occludens (ZO)-1 (7, 25). ZO-1 has been shown to interact with the cytoplasmic tails of occludin and the claudins. In addition, ZO-1 interacts with two additional members of the membrane-associated guanylate kinase (MAGUK) family of proteins, ZO-2 and ZO-3 (10). It is thought that the integrity of TJs is maintained by this group of proteins, and thus the proper regulation of paracellular permeability is probably tightly linked to their proper expression and localization.

Recent studies suggest that inflammation is associated with structural and functional alterations of hepatic TJs (13, 15, 16, 21). Although the biochemical mechanisms responsible for these changes remain to be elucidated, important clues might come from prior in vivo and in vitro studies of the effects of LPS or proinflammatory cytokines on intestinal epithelial barrier function. It is known, for example, that induction of inducible nitric oxide synthase (iNOS) with increased production of nitric oxide (NO·) is a critical factor leading to the development of hyperpermeability when Caco-2 human enterocyte-like monolayers are stimulated with inteferon (IFN)-{gamma} alone (37, 38) or a mixture of IFN-{gamma}, tumor necrosis factor (TNF)-{alpha}, and interleukin (IL)-1{beta} (4, 12). Excessive NO· production secondary to iNOS induction also has been implicated as an important factor contributing to gut barrier dysfunction and structural derangements in intestinal epithelial TJs in mice challenged with LPS or zymosan to induce a systemic inflammatory response (5, 24, 39).

Prompted by these findings, we hypothesized that iNOS induction and increased production of NO· might be important factors contributing to the development of structural and functional alterations in hepatic TJs as a result of systemic inflammation in vivo. To test this hypothesis, we used both pharmacological and genetic approaches to limit or ablate iNOS activity in mice challenged with a sublethal dose of LPS.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. All chemicals were purchased from Sigma-Aldrich Chemical (St. Louis, MO), unless otherwise noted. L-N6-(1-iminoethyl)lysine (L-NIL) was from A. G. Scientific (San Diego, CA). Anti-claudin-1, anti-occludin, and anti-ZO-1 polyclonal antibodies (pAb) were from Zymed Laboratories (South San Francisco, CA). Anti-actin monoclonal antibody (mAb) was from Sigma-Aldrich. Anti-{beta}-actin mAb and rabbit pAb against ZO-2 and ZO-3 were from Santa Cruz Biotechnology (Santa Cruz, CA). Unconjugated and FITC-conjugated anti-iNOS mAb was from TransLabs (Lexington, KY). All secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).

Animals. This research complied with regulations regarding animal care as published by the National Institutes of Health and was approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Male 7- to 8-wk-old C57Bl/6J mice weighing 20–25 g were from Jackson Laboratories (Bar Harbor, ME). iNOS knockout [iNOS(-/-)] mice on a C57Bl/6J background and weighing 22–25 g were generously provided by Dr. T. R. Billiar (University of Pittsburgh). Construction of the iNOS(-/-) mice was previously described (19). All animals were maintained in the University of Pittsburgh Animal Research Facility on a 12:12-h light-dark cycle with free access to standard laboratory chow and water. Animals were not fasted before experiments. Animals were anesthetized before surgical procedures by injecting pentobarbital sodium (60–90 mg/kg sc).

To induce a systemic inflammatory response, mice were injected intraperitoneally with Escherichia coli (strain O111:B4) LPS (40–50 µg/mouse; 2 mg/kg) dissolved in 1.0 ml PBS. Control animals were injected with a similar volume of PBS without LPS. Some mice were treated with two 5 mg/kg doses of L-NIL, administered by intraperitoneal injection 2 and 8 h after the injection of LPS or PBS. Some mice were treated with L-NIL according to the same schedule in the absence of prior injection of LPS. Groups of mice were anesthetized 6, 12, 18, or 24 h after injection of LPS or PBS for measuring hepatic barrier function (see below) or harvesting tissue specimens for various biochemical or histological assays.

Assessment of hepatobiliary barrier function. Serum was cleared by centrifugation and analyzed for bile acids using the colorimetric Total Bile Acid Test Kit (enzyme cycling) exactly as described by the manufacturer (Diazyme, San Diego, CA). Results are expressed as micromole of total bile acids per liter (i.e., µM), and the assay has a reported linear range of 1–180 µM. Conjugated bilirubin in plasma was measured by the University of Pittsburgh Clinical Laboratory using an automated direct bilirubin assay, and the results are expressed as milligrams conjugated bilirubin per deciliter.

Hepatic permeability was also determined by measuring the passage of fluorescent dextran with a relative molecular mass (Mr) 40 kDa (FD-40) from blood to bile using a modification of the method described by Rahner et al. (31). The femoral vein was cannulated with Intramedic polyethylene-10 tubing (Becton-Dickinson, Sparks, MD). After a midline laparotomy, the common bile duct was ligated proximal to the duodenum. The end of an 8-cm segment of polyethylene-10 tubing was inserted to within 5–8 mm from the right lobe and secured with a 4–0 silk suture. The abdomen was closed using 4–0 silk suture, and bile was collected in 0.5-ml tubes for 6 min to determine the bile flow rate by volumetric measurement. The animals then received a 0.5-ml injection of FD-40 in PBS (25 mg/ml) via the femoral cannula, and bile was collected in 2-min fractions for 8 min. Bile was diluted 1:1,000, and FITC fluorescence was measured using a Packard Fusion Microplate Reader (Boston, MA) at an excitation wavelength of 492 nm and an emission wavelength of 515 nm. Data are reported as fluorescence per microliter of bile at each time point.

Nonidet P-40-insoluble and total protein extracts. Liver tissue (150–200 mg) was homogenized on ice with a Polytron tissue homogenizer in 1 ml of Nonidet P-40 (NP-40) lysis buffer [25 mM HEPES, pH 7.4, 150 mM NaCl, 4 mM EDTA, 25 mM NaF, 1% NP-40, 1 mM Na3VO4, 1 mM 4-amidinophenylmethanesulfonyl fluoride (APMSF), 10 µg/ml leupeptin, and 10 µg/ml aprotinin]. The samples were rocked gently at 4°C for 30 min. The sample was centrifuged at 12,000 g for 30 min at 4°C, and the supernatants were discarded. The pellets were resuspended in SDS-dissolving buffer (25 mM HEPES, pH 7.5, 4 mM EDTA, 25 mM NaF, 1% SDS, and 1 mM Na3VO4) using five strokes with a Dounce homogenizer (pestle B) followed by sonication with a 0.1-Watt Fisher Scientific Sonic Dismembrator fitted with a microtip on power setting 3. Sonication continued until the precipitate was completely dissolved. These were designated the "NP-40 insoluble fractions" and are composed of insoluble cytoskeletal proteins and associated TJ proteins (32).

For total cellular protein extracts, tissue specimens were homogenized in cold RIPA buffer [PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/ml APMSF, 1.0 mM sodium orthovanadate, and 1x mammalian protease inhibitor cocktail (Sigma-Aldrich catalog no. P 8340)] and sonicated until the sample was dissolved completely. These samples are referred to as "total protein" extracts in this study.

Immunoprecipitation. The NP-40 (100 µg)-insoluble fraction was immunoprecipitated with either occludin or ZO-1 pAb. The lysate was precleared by adding 0.25 µg normal mouse IgG, together with 20 µl suspended protein A/G agarose (Santa Cruz Biotechnology). After incubation at 4°C for 30 min, the beads were collected by centrifugation at 2,500 rpm for 5 min at 4°C. The supernatant was transferred to a fresh tube, 3 µg anti-occludin or anti-ZO-1 antibody were added, and the tube was incubated on a rocker platform for 2 h at 4°C. Resuspended agarose A/G (20 µl) was added to the tube, and the incubation was continued overnight at 4°C with gentle shaking. The agarose beads were washed five times with 1 ml NP-40 lysis buffer. Proteins were eluted by boiling in 1x Laemmli buffer (10% glycerol, 5% {beta}-mercaptoethanol, 2.5% SDS, 0.1 M Tris, pH 6.8, and 0.2% bromphenol blue) for 10 min. Equal volumes of sample were separated by gel electrophoresis followed by immunoblotting.

Immunoblotting. Equal amounts of total protein extract were mixed in 1x Laemmli buffer, boiled for 3 min, and centrifuged for 10 s. The supernatants were electrophoresed on 7.5 or 12% precast SDS-polyacrylamide gels (Bio-Rad, Hercules, CA). The proteins were electroblotted on Hybond-P polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Leicester, UK) and blocked with Blotto (1x Tris-buffered saline, 5% milk, 0.05% Tween 20, and 0.2% NaN3) or 10% normal donkey serum (ZO-2 and ZO-3 only) for 60 min. The filter was incubated at room temperature for 1 h with anti-ZO-1 or anti-{beta}-actin antibody at a 1:4,000 dilution or anti-claudin-1, anti-ZO-2, anti-ZO-3, or anti-iNOS antibody diluted 1:2,000 in PBST (PBS and 0.02% Tween 20). After being washed three times in PBST, immunoblots were exposed for 1 h to a 1:20,000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibody. After three washes in PBST and two washes in PBS, the membrane was impregnated with the Enhanced Chemiluminescence substrate (Amersham Pharmacia Biotech) and used to expose X-ray film. Autoradiographs were captured using a Hewlett-Packard (Palo Alto, CA) ScanJet 6300S. Band intensities were quantified by densitometry and expressed as mean area density using GelExpert 3.5 software (NucleoTech, San Mateo, CA). For total protein blots, mean area density is expressed relative to {beta}-actin expression.

Immunofluorescence. Frozen tissue sections (6 µm) were prefixed in cold acetone and then air-dried. The sections were fixed with 4% paraformaldehyde and then washed three times with cold PBS. The sections were blocked with 10% donkey serum. Tissue sections were incubated with primary antibodies as follows: rabbit anti-ZO-1 pAb, rabbit anti-occludin pAb, and rabbit anti-claudin-1 pAb. Some sections were incubated with PBS alone in place of primary antibody as a negative control. After a 1-h incubation at room temperature, the sections were washed three times with PBS. Tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit secondary antibody was added and incubated for 45 min at room temperature. The sections were washed three times with PBS, and the nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride (Molecular Probes, Eugene, OR). Coverslips were mounted using the Molecular Probes Antifade Kit. Images were captured using an Olympus Provis microscope or an Olympus Fluoview BX61 confocal microscope.

Hematoxylin- and eosin-stained sections. Samples of liver tissue were fixed in 10% neutral buffered formalin and embedded in paraffin. Thin sections were stained with hematoxylin and eosin (H&E) and examined using light microscopy at x200–1,000 magnification.

Serum concentrations of alanine aminotransferase and nitrite and nitrate. Blood (200 µl) was obtained by cardiac puncture and placed in a 0.5-ml centrifuge tube on ice. The samples were then centrifuged at 5,000 g for 3 min. The serum was collected and assayed for the hepatocellular enzyme, alanine aminotransferase (ALT), using an automated assay system. The end products of NO· metabolism, nitrite ()/nitrate (), were quantitated using the Bioxytech Nitric Oxide Assay Kit exactly as directed by the manufacturer (OXIS International, Portland, OR).

Semiquantitative RT-PCR. Total RNA was extracted from fresh liver (5 mice/group) with chloroform and TRI-Reagent (Molecular Research Center, Cincinnati, OH) according to the instructions from the manufacturer. RNA samples were treated with DNase (10 U/50 µl RNA; Ambion, Austin, TX) as directed by the manufacturer. Total RNA (1 µg) was reverse transcribed in a 40-µl reaction volume containing 0.5 µg oligo(dT)15 (Promega, Madison, WI), 1 mM of each dNTP, 15 units AMV RT (Promega), and 1 U/µl recombinant RNasin RNase inhibitor (Promega) in 5 mM MgCl2, 10 mM Tris·HCl (pH 8.0), 50 mM KCl, and 0.1% Triton X-100. The mixture was heated to 70°C for 10 min, maintained at 42°C for 30 min, and then heated to 95°C for 5 min to terminate the reaction. Reaction mixtures (50 µl) for PCR were assembled using 5 µl cDNA template, 10 units AdvanTaq Plus DNA Polymerase (Clontech, Palo Alto, CA), 200 µM of each dNTP, 1.5 mM MgCl2, and 1.0 µM of each primer in 1x AdvanTaq Plus PCR buffer. PCR reactions were performed using a model 9600 thermocycler (Perkin-Elmer, Norwalk, CT). The primer pairs employed are shown in Table 1. Amplification of cDNA was performed by denaturing at 95°C for 45 s, annealing at 60°C for 45 s, and polymerizing at 72°C for 90 s for 26 cycles. This number of PCR cycles was empirically determined to ensure that amplification was in the linear range. After the last cycle of amplification, the samples were incubated at 72°C for 3 min. Each PCR reaction (18 µl) was electrophoresed on a 1% agarose gel, scanned using a NucleoVision imaging workstation (NucleoTech), and quantified using GelExpert release 3.5 software (NucleoTech).


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Table 1. Primers for semiquantitative PCR

 

Statistical analyses. Results are presented as means ± SE. Data were analyzed using ANOVA followed by Fisher's least significant difference test. P values <0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
L-NIL inhibits LPS-induced NO· release. It is well-known that injecting mice with LPS induces iNOS expression in many organs and leads to increased circulating levels of , the end products of NO· metabolism (14). We confirmed this observation in Fig. 1. Moreover, we showed that treatment of endotoxemic mice with L-NIL, an isoform-selective iNOS inhibitor (26), significantly decreased circulating concentration, although treatment with this agent did not completely suppress LPS-induced NO· production.



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Fig. 1. Effect of lipopolysaccharide (LPS) with or without treatment with L-N6-(1-iminoethyl)lysine (L-NIL), on serum concentration of nitrite ()/nitrate (). Groups of mice were injected with PBS or LPS and killed at the indicated time points. Some mice were treated with two 5 mg/kg doses of L-NIL, administered by ip injection, 2 and 8 h after the injection of LPS. Results are means ± SE (n = 7 for each condition). *P < 0.05 vs. control (Con); {dagger}P < 0.05 vs. samples obtained 12 h after the injection of LPS. Brackets denote concentration.

 

LPS impairs hepatobiliary barrier function via an iNOS-dependent mechanism. Lora et al. (18) reported that hepatic TJ function can be assessed by measuring serum concentrations of bile acids and conjugated bilirubin (18). Compared with control values, circulating levels of both of these bile components were increased in mice injected 12 h earlier with LPS (Fig. 2, A and B). However, when endotoxemic mice were treated with L-NIL, serum levels of bile acids and conjugated bilirubin were not different from normal. Although basal serum conjugated bilirubin levels were somewhat higher in vehicle-treated iNOS(-/-) compared with iNOS(+/+) mice, LPS-induced changes in circulating bile acid and conjugated bilirubin levels were prevented by genetic ablation of iNOS function.



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Fig. 2. Effect of LPS on circulating concentrations of bile acids (A) and conjugated bilirubin (B) in mice. Total bile acids were measured in plasma samples from inducible nitric oxide synthase (iNOS)-sufficient [iNOS(+/+)] and iNOS-deficient [iNOS(-/-)] mice that were challenged with PBS (Con) or LPS 12 h earlier. In a separate experiment, groups of animals were treated with PBS (Con) or LPS. Some of the LPS-challenged animals also were treated with two doses of L-NIL (see legend for Fig. 1). Plasma samples were assayed for total bile acids 12 h after the animals were injected with PBS or LPS. Plasma concentrations of conjugated bilirubin were assayed in plasma samples from mice treated with PBS (Con) or LPS 12 h earlier. Some of the LPS-challenged animals also received L-NIL. In a separate experiment, plasma samples of conjugated bilirubin were measured in groups (n = 5) of iNOS(+/+) and iNOS(-/-) mice injected 12 h earlier with LPS. Results are means ± SE (n = 5–7 for each treatment group). *P < 0.05.

 

We employed another approach for assessing hepatobiliary tight junctional integrity. We assayed bile for the appearance of FITC-labeled dextran with Mr 40 kDa (FD-40) after intravenous injection of the tracer. In control mice, FD-40 concentration in bile increased only very slowly after injection of the tracer (Fig. 3). However, in endotoxemic mice, the concentration of FD-40 in bile increased rapidly after intravenous injection. The LPS-induced increase in biliary FD-40 concentration was prevented if the endotoxemic animals were treated with L-NIL. The rate of bile flow was ~50% lower in endotoxemic mice compared with control mice (Fig. 3, inset), a finding that is consistent with other studies (3).



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Fig. 3. Effect of LPS on the diffusion of FD-40 from plasma into bile. The appearance of FD-40 in bile was measured over an 8-min period after the injection of iv bolus of FD-40 in groups of mice 12 h after injection with PBS (Con) or LPS. Some of the LPS-challenged animals also received L-NIL. The main graph shows the mean fluorescence ± SE of bile. The inset shows bile flow rate. Results are means ± SE (n = 5–7 for each treatment group). *P < 0.05 vs. LPS and LPS + L-NIL groups.

 

LPS-induced increases in serum ALT concentration are dependent on NO· production. Circulating levels of ALT are commonly used as a measure of liver damage from a variety of insults. The mean plasma ALT concentration increased gradually after injection of LPS (Fig. 4A). Treatment of mice with L-NIL significantly ameliorated the LPS-induced increase in circulating ALT (Fig. 4B). When these studies were performed in iNOS(-/-) mice, we measured relatively high serum ALT concentrations, even in control (nonendotoxemic) mice (Fig. 4C). When iNOS(-/-) mice were challenged with LPS, however, their circulating ALT levels did not change significantly.



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Fig. 4. LPS-induced changes in serum alanine aminotransferase (ALT) levels are nitric oxide (NO·) dependent. A: appearance of ALT in serum was determined at the times indicated after injecting mice with LPS (n = 5–7/group). L-NIL treatment (B) or an iNOS(-/-) genetic background (C) prevented the LPS-induced increase in serum ALT. *P < 0.05; **P < 0.01.

 

LPS induces hepatic iNOS expression and alterations in hepatic TJ protein expression. Immunoreactive iNOS was not detectable by Western blotting of hepatic protein extracts from control mice (Fig. 5). However, within 6 h after the injection of LPS, hepatic iNOS expression was clearly evident. iNOS levels further increased by 12 h and decreased slightly at the 24-h time point. After the induction of endotoxemia, occludin and ZO-1 levels decreased in NP-40-insoluble extracts (cytoskeletal fraction with associated TJ proteins) of hepatic tissue. Decreased expression of these TJ proteins was also observed in total protein extracts, but the change in occludin expression occurred more gradually. ZO-2 and ZO-3 levels also decreased in total protein extracts. Total cellular occludin and ZO-1 levels returned to normal within 24 h of injecting LPS, but ZO-2 and ZO-3 expression remained lower than baseline levels. Claudin-1 expression did not change reproducibly in total protein extracts. {beta}-Actin immunoblots confirmed equal loading of total protein extracts.



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Fig. 5. Effect of LPS on the expression of iNOS and several tight junction (TJ) proteins in mice. Mice were injected ip with LPS (2 mg/kg) or PBS (Con), and sections of liver were removed at the times indicated. Total protein and Nonidet P-40 (NP-40)-insoluble protein extracts were subjected to immunoblotting. The results are each representative of blots performed on samples from 4–6 different mice. OCC, occludin; ZO, zonula occludens; Cldn, claudin-1; L6, L12, L18, and L24, treatment with LPS for 6, 12, 18, and 24 h, respectively.

 

Endotoxemia is associated with derangements in hepatic TJ protein localization. There was minimal evidence of hepatic inflammation or necrosis on H&E-stained thin sections of liver tissue from mice injected 12 h earlier with LPS, irrespective of whether the animals were treated with L-NIL or not (Fig. 6). In control specimens, occludin and ZO-1 were largely detected in longitudinal sections of canaliculi as parallel strands of staining representing the outlines of canaliculi (Fig. 7). Consistent with previously reported observations reported by Notterpek et al. (30), staining of occludin and ZO-1 in normal liver tissue was predominantly limited to focal regions of hepatocyte-hepatocyte contact and endothelial cell-cell junctions of blood vessels and bile ducts. Hepatic tissue from endotoxemic mice showed a widespread decrease in occludin staining (Fig. 7). The remaining areas of occludin staining were tortuous and discontinuous. Similarly, ZO-1 staining was reduced greatly after injection of LPS, and the residual ZO-1 staining was distorted. In contrast to the dramatic decrease of immunostaining for ZO-1 and occludin in hepatocytes of the LPS group, there was obvious preservation of occludin and ZO-1 staining along the outlines of canaliculi in endotoxemic mice treated with L-NIL (Fig. 7). Similar protection against endotoxin-induced alterations in ZO-1 staining patterns were observed when hepatic sections from LPS-challenged iNOS(+/+) and iNOS(-/-) mice were compared (Fig. 8).



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Fig. 6. Hematoxylin- and eosin (H&E)-stained sections of liver tissue from control mice (Con; A), mice injected with LPS 12 h earlier (LPS; B), and endotoxemic mice that were treated with L-NIL (LPS + L-NIL; C). The original magnification was x600, and the bar represents 50 µm.

 


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Fig. 7. Indirect immunofluorescence confocal images of liver from control mice (Con), mice exposed to LPS for 12 (LPS), and endotoxemic mice that were treated with L-NIL (LPS + L-NIL). In samples of hepatic tissue from control mice, occludin and ZO-1 immunostaining flanked the margins of the canaliculi. Depending on the scanning direction, staining for occludin and ZO-1 appeared as continuous parallel lines or channels (arrows). In contrast, staining for ZO-1 and occludin was decreased dramatically in mice treated with LPS. The normal pattern of staining was disrupted (arrowheads). A much more normal staining pattern for occludin and ZO-1 was apparent in liver tissue samples from endotoxemic mice treated with L-NIL. The original magnification was x600 for the main panels and x1,000 for the insets. Bar = 50 µm.

 


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Fig. 8. Indirect immunofluorescence images of liver samples obtained from control iNOS knockout mice (A) or iNOS knockout mice 12 h after injection with LPS (B). The original magnification was x600.

 

Pharmacological or genetic blockade of iNOS activity modulates the decrease in hepatic ZO-1 and occludin expression in endotoxemic mice. Pharmacological inhibition of iNOS activity with L-NIL blocked the decreased expression of occludin and ZO-1 measured in NP-40-insoluble protein extracts 12 h after injection with LPS (Fig. 9). Similarly, iNOS(-/-) mice were protected from LPS-induced changes in hepatic occludin and ZO-1 expression. However, increased levels of both occludin and ZO-1 were present in NP-40-insoluble liver extracts from vehicle-treated iNOS(-/-) compared with vehicle-treated iNOS(+/+) mice. L-NIL also preserved the localization of both proteins in the liver.



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Fig. 9. Effect of pharmacological inhibition of iNOS activity or genetic ablation of functional iNOS expression on hepatic expression of TJ proteins in endotoxemic mice. Mice were injected ip with LPS or PBS (Con), and sections of liver were removed 12 h later. NP-40-insoluble protein extracts were subjected to immunoblotting. The results are representative of blots prepared from specimens from 4–6 different mice.

 

Endotoxemia is associated with alterations in steady-state hepatic TJ mRNA levels. Occludin, ZO-3, and to a lesser extent ZO-2 mRNA levels decreased within 6 h of injecting LPS (Fig. 10). ZO-2 mRNA increased above control levels within 12 h of LPS injection. Occludin mRNA levels started to return toward normal at 12 h and continued to increase until reaching control levels at 24 h. In contrast to these findings, endotoxemia was not associated with detectable alterations in steady-state levels of transcripts for either ZO-1 or claudin-1. Indeed, claudin-1 mRNA expression tended to increase at 12 h after injection of LPS.



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Fig. 10. Effect of LPS on steady-state hepatic mRNA levels of TJ proteins. The images depict representative results of RT-PCR analyses on total RNA isolated from control mice or mice injected with LPS for the times indicated. Densitometry data are means ± SE (n = 4–6/condition).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we used three different assays (measurement of circulating bile acid concentration, measurement of circulating conjugated bilirubin concentration, and measurement of plasma-to-bile flux of FD-40) to document that injecting mice with LPS impaired epithelial TJ function in the hepatobiliary system. Immunoblotting of total protein extracts showed decreased levels of ZO-1, ZO-2, ZO-3, and occludin. Decreased total occludin and ZO-1 expression was apparent at 6 h and continued to be low compared with controls until 24 h after LPS injection. Decreased expression of ZO-2 and ZO-3 was not apparent until 12 h after LPS injection. Unlike occludin and ZO-1, these proteins did not return to normal, even at 24 h after LPS injection. The decreases in occludin and ZO-1 content in NP-40-insoluble complexes were more profound than were the decreases of these same proteins in total protein extracts. This observation suggests that these proteins were synthesized in the liver after the injection of LPS but were not properly targeted to or incorporated into TJ strands. Decreased expression of occludin and ZO-1 was confirmed using immunofluorescence microscopy of thin sections of hepatic tissue.

Although we observed a transient decrease in the expression of occludin mRNA 6 h after injecting mice with LPS, steady-state levels of this transcript recovered significantly by 12 h. However, normalization of occludin mRNA expression was not accompanied by increased expression of occludin in total protein extracts at the 18-h time point. ZO-2 mRNA levels were greater than baseline at 12 h, but ZO-2 protein levels were decreased during the period from 12–24 h after injection of LPS. ZO-3 transcript levels were decreased from 6 to 18 h after injecting LPS, suggesting that decreased expression of this protein might relate to changes in the rate of transcription of the ZO-3 gene and/or changes in the stability of ZO-3 mRNA in immunostimulated hepatocytes. Accordingly, our data suggest that LPS-induced inflammation alters the expression of several TJ proteins via both transcriptional and posttranscriptional mechanisms.

The findings we obtained by using semiquanatitative RT-PCR to estimate hepatic changes in the steady-state levels of occludin transcripts when mice are rendered acutely endotoxemic are consistent with previously reported data showing that occludin mRNA levels decrease in HT-29 intestinal epithelial cells exposed to TNF-{alpha} or IFN-{gamma} (20) and astrocytes exposed to TNF-{alpha} (42). In contrast, Youakim and Ahdieh (43) reported that ZO-1 mRNA levels decrease when T84 intestinal epithelial cells are incubated with IFN-{gamma}, but steady-state levels of occludin transcripts do not change under these conditions. Similarly, our laboratory reported that levels of ZO-1 mRNA decrease when Caco-2 human enterocyte-like cells are incubated with a cocktail of proinflammatory cytokines, but occludin mRNA levels are not affected by this perturbation (12). We can offer no explanation to account for the differential effects of proinflammatory cytokines on occludin mRNA expression in various cell types or hepatic tissue from mice with systemic inflammation induced by LPS. However, taken together, these studies suggest that both changes in mRNA levels and changes in posttranscriptional processes are important factors contributing to the downregulation of TJ protein expression in immunostimulated epithelial cells.

Endotoxemia causes increased expression of iNOS and consequent NO· production in hepatocytes and Kupffer cells in mice (33). Using immunofluorescence microscopy, we also found that iNOS is expressed by hepatocytes in endotoxemic mice (Han, Fink, and Delude, unpublished results). By using a combination of genetic and pharmacological approaches, we obtained evidence that the LPS-induced changes in hepatobiliary TJ function were iNOS dependent. Injection of iNOS(+/+) mice with LPS increased circulating levels of bile acids and conjugated bilirubin. These changes were not observed in either endotoxemic iNOS(-/-) mice or endotoxemic wild-type mice treated with L-NIL, an isoform-selective iNOS inhibitor (26). The appearance in bile of the macromolecular tracer, FD-40, after intravenous injection was significantly greater in wild-type mice previously challenged with LPS compared with nonendotoxemic controls. However, if endotoxemic mice were treated with L-NIL to inhibit iNOS-dependent NO· production, diffusion of FD-40 from plasma into bile was not significantly different from that observed in nonendotoxemic controls.

Basal circulating levels of conjugated bilirubin, but not bile salts, were significantly higher in control iNOS(-/-) mice compared with control iNOS(+/+) mice. In another study, we similarly observed that basal ileal mucosal permeability to FITC-dextran (Mr 4 kDa) is greater in iNOS(-/-) compared with iNOS(+/+) mice (Han, Fink, and Delude, unpublished results). Although we can only speculate about the mechanism responsible for these observations, the possibility exists that a low background rate of NO· production from constitutively expressed iNOS is necessary for the proper assembly of TJs. In support of this notion, we cite data reported a number of years ago by Kubes (17), who showed that mucosal permeability is increased when autoperfused segments of cat ileum are infused intra-arterially with the isoform-nonselective NOS inhibitor, NG-nitro-L-arginine methyl ester.

In our studies, bile flow rate was significantly lower in endotoxemic mice compared with control mice, a finding that is consistent with previously reported observations (3, 40). Although a decreased bile flow rate would tend to increase the measured concentration in bile of a marker like FD-40, the ~50% decrease in bile flow rate observed in the endotoxemic mice is insufficient to account for the ~10-fold increase in FD-40 concentration in bile that was detected in LPS-challenged compared with control animals. Accordingly, we feel confident that the marked increase in biliary FD-40 concentration that we observed was evidence of deranged hepatobiliary TJ function.

The reason for decreased bile flow in endotoxemic mice is unclear. Studies that were performed a number of years ago by Utili et al. (40) suggest that it is the bile salt-independent secretion of bile (i.e., the basal component) that is decreased by endotoxemia. Nathanson et al. (29) suggested that decreased expression of the gap junction proteins, connexin (Cx) 26 and Cx32, may be one mechanism underlying the changes in bile flow. However, Gonzalez et al. (9) reported that inflammation-induced decreases in the expression of Cx26 and Cx32 in hepatocytes are not dependent on NO· production. Our data show that the decreased flow of bile in endotoxemic animals is an NO·-dependent event, as it was ameliorated by L-NIL administration. Thus our findings raise the possibility that changes in bile flow in this model of systemic inflammation are the result of changes in the expression and targeting of TJ proteins. This idea is consistent with a model proposed by Nathanson et al. (28), who suggested that decreased bile flow can result from increased paracellular permeability.

The functional protection afforded by pharmacological or genetic inhibition of iNOS activity was paralleled by protection against LPS-induced changes in the expression and localization of key TJ proteins. Although treatment with L-NIL did not completely preserve normal levels of occludin or ZO-1 expression, partial protection was obvious and reproducible (see Fig. 8). Whereas LPS injection caused a marked decrease in ZO-1 expression in iNOS(+/+) mice, there was no apparent change in ZO-1 expression in iNOS(-/-) mice. Interestingly, much higher levels of occludin expression were apparent in control iNOS(-/-) compared with control iNOS(+/+) mice, suggesting the possibility that basal iNOS activity might play a role in regulating the hepatic expression of this protein in normal animals. Although basal levels of occludin were abnormally high in iNOS(-/-) mice, there was no change in expression of this protein when these animals were challenged with LPS.

In previous studies, our laboratory (39) and others (23) have obtained evidence that LPS-induced derangements in the barrier function of another epithelial tissue, the intestinal mucosa, are also iNOS dependent. Additionally, in a series of experiments, we have obtained evidence that pulmonary epithelial barrier function is altered when mice are challenged with LPS, but these changes are significantly ameliorated if the animals are treated with L-NIL to inhibit iNOS-dependent NO· production (Han, Fink, and Delude, unpublished observations). In addition, Cuzzocrea et al. (5) showed that the localization of the TJ proteins, ZO-1 and occludin, was disrupted in the ileal epithelium of iNOS(+/+) but not iNOS(-/-) mice after the animals were injected intraperitoneally with zymosan to induce a systemic inflammatory response. Taken together with the findings reported here, these data suggest that systemic inflammation, whether induced by a bacterial product like LPS or a fungal product like zymosan, leads to alterations in the structure and function of the epithelia in multiple organs. The organs involved with this process in LPS- or zymosan-challenged mice (i.e., the liver, lungs, and gut) include those that are commonly involved in a syndrome, the multiple organ dysfunction syndrome (MODS; see Ref. 6), that affects many patients with severe sepsis or other causes of poorly controlled systemic inflammation. Accordingly, it seems reasonable to hypothesize that iNOS-dependent derangements in epithelial TJ structure and function in multiple organs may play a role in the pathogenesis of MODS.

The notion, advanced herein, that iNOS-dependent NO· production is a key factor leading to alterations in hepatobiliary TJ structure and function is further supported by a series of in vitro experiments recently reported by our laboratory (12). In this study, we used a cocktail of proinflammatory cytokines ("cytomix") to induce iNOS expression in Caco-2 human enterocyte-like cells and increase the permeability of Caco-2 monolayers. The cytomix-induced increase in epithelial permeability was accompanied by derangements in the expression and localization of TJ proteins that are very reminiscent of the changes observed here in liver tissue after the injection of mice with LPS. Incubation of the Caco-2 cells with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO· scavenger, ameliorated the changes in TJ protein expression and localization as well as the functional readout (i.e., permeability of the monolayers to FD-4) in a fashion similar to what was observed in the present studies when the endotoxemic mice were treated with L-NIL to inhibit iNOS-dependent NO· production. These data are consistent with the view that the mechanism(s) responsible for the alterations in TJ protein expression and localization were similar in the two model systems.

Prior in vitro studies have shown that the permeability of Caco-2 enterocytic monolayers is increased when the cells are incubated with various NO· donors, such as S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (22, 23). This effect does not appear to be mediated by NO· directly, but rather by ONOO-, a potent oxidizing and nitrosating agent that is formed when NO· reacts with . Support for this view comes from studies showing that SNAP-induced hyperpermeability is augmented by addition of diethyldithiocarbamate, a superoxide dismutase inhibitor, or pyrogallol, an generator (22). Furthermore, SNAP-induced hyperpermeability is blocked by Tiron, an agent that scavenges , as well as various ONOO- scavengers, such as urate and deferoxamine (22). When Caco-2 monolayers are incubated with IFN-{gamma} or a combination of IFN-{gamma}, TNF-{alpha}, and IL-1{beta}, iNOS expression is induced and permeability is increased (4, 37, 38). The increase in permeability can be blocked by inhibiting NO· production or scavenging ONOO- (4, 37, 38). These findings support the view that cytokine-induced intestinal epithelial hyperpermeability is mediated, at least in part, by the formation of ONOO-. Further studies will be required to determine whether ONOO-, or some other reactive nitrogen intermediate, is the actual moiety responsible for the deleterious effects of NO· on TJ protein expression that we observed in the livers of endotoxemic animals.

We have focused herein on the importance of TJs in maintaining the proper compartmentalization of bile constituents, a concept that is supported by results from recent studies using experimental models of colitis or bile duct ligation (16, 18, 21, 31, 34). We recognize, however, that alterations in the function or expression of key transporters are also likely to be important factors contributing to the development of cholestatic jaundice in patients with severe sepsis or endotoxemic animals (3, 11, 27, 35). Because pharmacological inhibition of iNOS activity or genetic ablation of functional iNOS expression prevented hyperbilirubinemia in endotoxemic mice, we are tempted to speculate that some of the previously identified alterations in hepatobiliary transport function also might be iNOS dependent.


    ACKNOWLEDGMENTS
 
We thank Meaghan E. Killeen and Peter S. Williams for technical assistance and Dr. Simon S. Watkins and Sean Alber for assistance with the immunohistochemical staining and imaging.

GRANTS

This work was supported by General Medical Sciences Grants GM-58484 and GM-37631.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. L. Delude, Dept. of Critical Care Medicine, 616 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (E-mail: deluder{at}ccm.upmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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