Serosal But Not Mucosal Endotoxin Exposure Increases Intestinal Permeability in Vitro in the Rat

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  • Serosal But Not Mucosal Endotoxin Exposure Increases IntestinalPermeability in Vitro in the Rat

    N. E. OSMAN, B. WESTRO M & B. KARLSSONDept. of Animal Physiology, Lund University, Lund, Sweden

    Osman NE, Westrom B, Karlsson B. Serosal but not mucosal endotoxin exposure increases intestinalpermeability in vitro in the rat. Scand J Gastroenterol 1998;33:11701174.

    Background:Microbial endotoxins are normally present in the gut, usually without apparent harmfuleffects, whereas systemically administered endotoxin impairs the mucosal barrier function. Our aim was toinvestigate whether in vitro exposure to bacterial lipopolysaccharide (LPS) could affect the intestinalbarrier properties of the rat small intestine.Methods:Small-intestinal segments from rats were mounted inUssing diffusion chambers, and the mucosal to serosal permeation of the marker molecules bovine serumalbumin (BSA) and51Cr-ethylenediaminetetraacetic acid (EDTA) was measured after addition of LPS tothe mucosal or serosal side. Results:Mucosal exposure to LPS (0.01, 0.05, 0.25 mg/ml) had no effects onthe permeation of BSA and51Cr-EDTA, whereas when added to the serosal side at 0.05 or 0.25 mg/ml,LPS increased the marker permeation.Conclusion:Serosal LPS exposure in vitro increased the intestinalpermeability to the different-sized markers, whereas mucosal LPS did not, indicating that the mechanismsleading to intestinal barrier impairment can be initiated in the intestinal wall itself.

    Key words:Bacterial translocation; bovine serum albumin; endotoxin;51Cr-ethylenediaminetetraaceticacid; mucosal barrier; small intestine

    Borje Karlsson, Ph.D., Dept. of Animal Physiology, Lund University, Helgonavagen 3B, S-223 62 Lund,Sweden (fax:46 46 222 4539)

    The gastrointestinal mucosa, in parallel to its absorptivefunctions, constitutes a defense barrier that prevents noxiousagents contained in the gut lumen, such as bacteria andbacterial products, and antigenic macromolecules fromescaping and spreading into extraintestinal tissues and organs(13). However, small amounts of macromolecules maypermeate the gut mucosa in healthy experimental animals andhumans, and even endogenous gut bacteria can pass throughthis barrier to infect mesenteric lymph nodes and systemicorgans, a process known as bacterial translocation (3).

    Gram-negative bacteria in the gut lumen are known toconstantly shed their outer membrane fragments, and en-dotoxins are therefore produced in large amounts, surpris-ingly without obvious harmful effects (4). An increasedleakage of bacterial endotoxins from the intestinal lumen hasbeen found to occur in conjunction with a wide variety ofclinical conditions involving intestinal trauma and inflamma-tion (1, 5). Moreover, intravenous administration of endotox-in results in increased intestinal molecular permeability andbacterial translocation in humans (6), pigs (7), and rats (8).The tight junctions of the intestinal epithelium might be a sitefor leakage of microbial agents, as found by ultrastructuralanalysis of mouse ileal epithelium after parenteral endotoxinadministration (9). Bacterial endotoxins increase gut perme-ability (6, 9), alter host immune defenses (10), and arerelatively common in patients at increased risk of developingenteric infections (2).

    The aim of the present study was to experimentallyinvestigate whether mucosal (luminal) or serosal (blood-side) exposure to the bacterial endotoxin lipopolysaccharide(LPS) could affect the intestinal barrier properties. This wasdone by measuring the mucosal to serosal permeability of thetwo marker molecules51Cr-ethylenediaminetetraacetic acid(EDTA) and bovine serum albumin (BSA) in the rat smallintestine, using the Ussing diffusion chamber model.

    MATERIALS AND METHODS

    AnimalsMale rats (Rattus norvegicus; SpragueDawley strain,

    Mllegaard, Skensved, Denmark), weighing 300350 g,were kept on chopped wood bedding in polycarbonate cagesunder a 12-h daynight rhythm at 20 2C and a relativehumidity of 50% 10%. The rats had free access to rat chow(Altromin 1324, Altromin International, Lage, Germany) andtap water. The study was approved by the Lund UniversityEthical Review Committee on Animal Experiments.

    Experimental protocolStarting at 09001000 h, a laparotomy was done under

    ether anesthesia, and a 50-cm-long segment of the smallintestine, ending 5 cm proximal to the ileocecal connection,was removed. The segment was further divided into 4-cm-long pieces and immediately immersed in a modified Krebs

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  • Ringer buffer, pH 7.4 (110.0 mM NaCl2, 3.0 mM CaCl2,5.5 mM KCl, 1.4 mM KH2PO4, 29.0 mM NaHCO3, 5.7 mMNa-pyruvate, 7.0 mM Na-fumarate, 5.7 mM Na-glutamate,and 13.4 mM glucose), and oxygenated with 95% O2 and 5%CO2 at room temperature. Each piece was cut along themesenteric border and mounted as a sheet, with an exposedarea of 1.78 cm2, between the mucosal and serosal reservoirsof Ussing diffusion chambers (Precision Instrument Design,Los Altos, Calif., USA), modified in accordance with Grass &Sweetana (11). The serosal and mucosal reservoirs were filledwith 5 ml KrebsRinger buffer that was continuouslyoxygenated and circulated by means of a gas lift at atemperature of 37C throughout the incubations. The experi-ments started (t = 0) within 40 min after the induction ofanesthesia, when the buffer in the mucosal reservoirs wasreplaced with 5 ml buffer containing the different-sizedmolecular markers BSA (25 mg/ml, A-4503, Sigma, Mw67,000) and51Cr-labeled EDTA (3mCi/ml, DuPont, BadHomburg, Germany, Mw 358), and the buffer in the serosalreservoirs was replaced with 5 ml fresh buffer. At the sametime LPS at different concentrations (0, 0.01, 0.05, and0.25 mg/ml, L-2630, Sigma, St. Louis, Mo., USA) was addedto either the serosal (n = 6 rats) or the mucosal reservoirs(n = 6 rats), to study its effects on the mucosal to serosalmarker permeability in the small intestine. Every 20 min for2 h, 1-ml samples were taken from the serosal reservoirs formarker analysis and replaced with 1 ml fresh buffer. Theviability of the intestinal segments throughout the experimentwas ascertained as previously reported (12).

    Marker molecule analysis51Cr-EDTA radioactivity in 1-ml samples was measured

    for 120 sec in a well-type gamma counter (LKB, Bromma,Sweden).

    BSA concentration was measured with a two-step sand-wich enzyme-linked immunosorbent assay (ELISA) (13). Inshort, a 96-microwell ELISA plate (Maxisorp, Nunc, Ros-kilde, Denmark) was coated with chicken anti-BSA (65437;ICN Biomedicals Inc., Costa Mesa, Calif., USA) diluted1:500 in 0.1 M NaHCO3, pH 8.3 (200ml/well), and incubatedovernight at4C. In another 96-microwell incubation plate(Nunc) 50ml of the samples or standardsthat is, 41000 ng/ml of BSA (A-7638, Sigma)were added to each welltogether with 100ml of specific rabbit anti-BSA antiserum(Dakopatts A/S, Glostrup, Denmark) diluted 1:10,000 in 0.01M phosphate-buffered saline (PBS), pH 7.2, plus 0.05%Tween 20, and incubated overnight at4C. After washingof the ELISA plate, 3 5 min, with 0.05% Tween in 0.9%NaCl, 50ml PBS, and 150ml mixture from the incubationplate were added to respective wells of the ELISA plate andincubated for 1 h at room temperature. After being washed,alkaline phosphatase-conjugated swine anti-rabbit immuno-globulins (D306, Dakopatts) diluted 1:1000 in PBSTweenwere added (200ml/well) and incubated for 1 h at roomtemperature. Finally, substrate, 0.8 mg/ml ofp-nitrophenyl

    phosphate (Sigma) in 0.1 M glycine buffer, pH 10.4, wasadded (200ml/well), and the color reaction followed at450 nm (iEMS reader MF, Labsystems, Helsinki, Finland).

    Calculations and statisticsThe apparent intestinal permeability coefficients (Papp) of

    the marker molecules in the intestinal epithelia werecalculated from the equation; Papp (cm/sec 10

    6) = dc/dt V/(A C0), where dc/dt is the change of the serosalconcentration during 60120 min (mol/l/sec), V is the volumein the reservoirs (cm3), C0 is the initial marker concentrationin the mucosal reservoirs (mol/l), and A is the exposedintestinal area in the chamber (cm2) (11).

    Data are presented as mean standard deviation (s).ANOVA, followed by the StudentNewmanKeuls test,was used for statistical evaluation.

    RESULTS

    Generally, the permeability of the low-molecular marker51Cr-EDTA was higher than that of the high-molecularmarker BSA (Table I). Addition of LPS in increasingconcentrations to the mucosal or serosal side in Ussingdiffusion chambers affected the mucosal to serosal passageof both marker molecules in the small intestine in differentways.

    Mucosal exposure to LPS at concentrations of 0.01, 0.05,and 0.25 mg/ml did not have any effects, compared with thecontrol, when the cumulative passage of the markers wasstudied during 2 h (Figs. 1A, 2A) or when the marker passageswere expressed as the apparent permeability coefficients(Papp) obtained between 60 and 120 min (Table I). However,when LPS was added to the serosal side, the permeabilityincreased significantly for both marker molecules, at con-centrations of 0.05 and 0.25 mg/ml, whereas at a lower

    Table I. The apparent permeability coefficient, Papp (cm/s 106),

    for the marker molecules51Cr-labeled ethylenediaminetetraaceticacid (EDTA) and bovine serum albumin (BSA) obtained afterexposure to increasing concentrations of lipopolysaccharide (LPS)(0.01, 0.05 and 0.25 mg/ml) at the mucosal or serosal side of thesmall intestine in Ussing diffusion chambers

    Groups 51Cr-EDTA BSA