Abstract: Hemorrhage and pleural effusion are prominent pathological features of systemic anthrax infection. We examined the effect of anthrax lethal toxin (LT), a major virulence factor of Bacillus anthracis, on the barrier function of primary human lung microvascular endothelial cells. We also examined the distribution patterns of cytoskeletal actin and vascular endothelial-cadherin (VE-cadherin), both of which are involved in barrier function regulation. Endothelial monolayers cultured on porous membrane inserts were treated with the LT components lethal factor (LF) and protective antigen (PA) individually, or in combination. LT induced a concentration- and time-dependent decrease in transendothelial electrical resistance that correlated with increased permeability to fluorescently labeled albumin. LT also produced a marked increase in central actin stress fibers and significantly altered VE-cadherin distribution as revealed by immunofluorescence microscopy and cell surface enzyme-linked immunosorbent assay. Treatment with LF, PA, or the combination of an inactive LF mutant and PA did not alter barrier function or the distribution of actin or VE-cadherin. LT-induced barrier dysfunction was not dependent on endothelial apoptosis or necrosis. The present findings support a possible role for LT-induced barrier dysfunction in the vascular permeability changes accompanying systemic anthrax infection. Hemorrhage and pleural effusion are prominent pathological features of systemic anthrax infection. We examined the effect of anthrax lethal toxin (LT), a major virulence factor of Bacillus anthracis, on the barrier function of primary human lung microvascular endothelial cells. We also examined the distribution patterns of cytoskeletal actin and vascular endothelial-cadherin (VE-cadherin), both of which are involved in barrier function regulation. Endothelial monolayers cultured on porous membrane inserts were treated with the LT components lethal factor (LF) and protective antigen (PA) individually, or in combination. LT induced a concentration- and time-dependent decrease in transendothelial electrical resistance that correlated with increased permeability to fluorescently labeled albumin. LT also produced a marked increase in central actin stress fibers and significantly altered VE-cadherin distribution as revealed by immunofluorescence microscopy and cell surface enzyme-linked immunosorbent assay. Treatment with LF, PA, or the combination of an inactive LF mutant and PA did not alter barrier function or the distribution of actin or VE-cadherin. LT-induced barrier dysfunction was not dependent on endothelial apoptosis or necrosis. The present findings support a possible role for LT-induced barrier dysfunction in the vascular permeability changes accompanying systemic anthrax infection. Bacillus anthracis, the causative agent of anthrax, is a spore-forming gram-positive bacterium. Anthrax toxin, the major virulence factor of B. anthracis, is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA and LF combine to form lethal toxin (LT), and EF combines with PA to form edema toxin (ET).1Leppla SH The bifactorial Bacillus anthracis lethal and oedema toxins.in: Alouf JA Freer J Comprehensive Sourcebook of Bacterial Protein Toxins. Academic Press, London1999: 243-263Google Scholar, 2Moayeri M Leppla SH The roles of anthrax toxin in pathogenesis.Curr Opin Microbiol. 2004; 7: 19-24Crossref PubMed Scopus (217) Google Scholar, 3Mock M Fouet A Anthrax.Annu Rev Microbiol. 2001; 55: 647-671Crossref PubMed Scopus (851) Google Scholar PA binds at least two identified cell surface receptors, tumor endothelial marker 8 and capillary morphogenesis protein 2.2Moayeri M Leppla SH The roles of anthrax toxin in pathogenesis.Curr Opin Microbiol. 2004; 7: 19-24Crossref PubMed Scopus (217) Google Scholar, 4Bradley KA Mogridge J Mourez M Collier RJ Young JA Identification of the cellular receptor for anthrax toxin.Nature. 2001; 414: 225-229Crossref PubMed Scopus (747) Google Scholar, 5Scobie HM Rainey GJ Bradley KA Young JA Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor.Proc Natl Acad Sci USA. 2003; 100: 5170-5174Crossref PubMed Scopus (519) Google Scholar Once formed, PA-receptor complexes facilitate the endocytosis of LF and EF. Inside cells, LF acts as a metalloprotease that cleaves all of the mitogen activated protein kinase kinases (MEKs) except MEK 5, thus disrupting the activation of major mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 MAPK, and c-Jun NH2-terminal kinases (JNK).6Bardwell AJ Abdollahi M Bardwell L Anthrax lethal factor-cleavage products of mitogen-activated protein kinase (MAPK) kinases exhibit reduced binding to their cognate MAPKs.Biochem J. 2004; 378: 569-577Crossref PubMed Scopus (84) Google Scholar, 7Duesbery NS Webb CP Leppla SH Gordon VM Klimpel KR Copeland TD Ahn NG Oskarsson MK Fukasawa K Paull KD Vande Woude GF Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.Science. 1998; 280: 734-737Crossref PubMed Scopus (884) Google Scholar, 8Kirby JE Anthrax lethal toxin induces human endothelial cell apoptosis.Infect Immun. 2004; 72: 430-439Crossref PubMed Scopus (148) Google Scholar EF acts as a Ca2+/calmodulin-dependent adenylate cyclase that causes a dramatic increase in intracellular levels of cAMP.9Leppla SH Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentration in eukaryotic cells.Proc Natl Acad Sci USA. 1982; 79: 3162-3166Crossref PubMed Scopus (756) Google Scholar, 10Ulmer TS Soelaiman S Li S Klee CB Tang WJ Bax A Calcium dependence of the interaction between calmodulin and anthrax edema factor.J Biol Chem. 2003; 278: 29261-29266Crossref PubMed Scopus (45) Google Scholar Evidence to date suggests that LT may play a more significant role than ET in the pathogenesis of systemic anthrax. In several animal models, intravascular injections of purified LT are lethal.11Moayeri M Haines D Young HA Leppla SH Bacillus anthracis lethal toxin induces TNF-α-independent hypoxia-mediated toxicity in mice.J Clin Invest. 2003; 112: 670-682Crossref PubMed Scopus (257) Google Scholar, 12Cui X Moayeri M Li Y Li X Haley M Fitz Y Correa-Araujo R Banks SM Leppla SH Eichacker PQ Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats.Am J Physiol Regul Integr Comp Physiol. 2004; 286: R699-R709Crossref PubMed Scopus (110) Google Scholar, 13Ezzell JW Ivins BE Leppla SH Immunoelectrophoretic analysis, toxicity, and kinetics of in vitro production of the protective antigen and lethal factor components of Bacillus anthracis toxin.Infect Immun. 1984; 45: 761-767Crossref PubMed Google Scholar In addition, attenuated B. anthracis strains unable to produce functional LF are 1000-fold less virulent than normal strains, whereas EF-lacking strains are 10-fold less virulent.14Pezard C Berche P Mock M Contribution of individual toxin components to virulence of Bacillus anthracis.Infect Immun. 1991; 59: 3472-3477Crossref PubMed Google ScholarVascular dysfunction or injury has long been considered a hallmark of anthrax pathogenesis.15Beall FA Dalldorf FG The pathogenesis of the lethal effect of anthrax toxin in the rat.J Infect Dis. 1966; 116: 377-389Crossref PubMed Scopus (52) Google Scholar, 16Dalldorf FG Beall FA Capillary thrombosis as a cause of death in experimental anthrax.Arch Pathol. 1967; 83: 154-161PubMed Google Scholar, 17Smith H Stoner HB Anthrax Toxic Complex.Fed Proc. 1967; 26: 1554-1557PubMed Google Scholar Prominent pathological features of systemic anthrax infection include vascular leakage, hemorrhages, and vasculitis. Guarner et al18Guarner J Jernigan JA Shieh WJ Tatti K Flannagan LM Stephens DS Popovic T Ashford DA Perkins BA Zaki SR Inhalational Anthrax Pathology Working Group: pathology and pathogenesis of bioterrorism-related inhalational anthrax.Am J Pathol. 2003; 163: 701-709Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar reported progressive and persistent pleural effusions as the main feature in inhalational anthrax patients from the 2001 bioterrorism attack in the United States. During the 1957 inhalational anthrax epidemic in New Hampshire, autopsies consistently showed edema, pleural effusion, and hemorrhages.19Plotkin SA Brachman PS Utell M Bumford FH Atchison MM An epidemic of inhalational anthrax, the first in the twentieth century: I. Clinical features.Am J Med. 2002; 112: 4-12Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Grinberg et al20Grinberg LM Abramova FA Yampolskaya OV Walker DH Smith JH Quantitative pathology of inhalational anthrax. I: quantitative microscopic findings.Mod Pathol. 2001; 14: 482-495Crossref PubMed Scopus (193) Google Scholar described high-pressure and low-pressure hemorrhages, particularly in the lungs and spleens, in the inhalational anthrax victims of the Sverdlovsk incident. Rhesus and cynomolgus monkeys exposed to the fully virulent Ames strain showed edema, hemorrhages, and a variable degree of leukocyte infiltration in various tissues.21Vasconcelos D Barnewall R Babin M Hunt R Estep J Nielsen C Carnes R Carney J Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis).Lab Invest. 2003; 83: 1201-1209Crossref PubMed Scopus (121) Google Scholar, 22Fritz DL Jaax NK Lawrence WB Davis KJ Pitt ML Ezzell JW Friedlander AM Pathology of experimental inhalation anthrax in the rhesus monkey.Lab Invest. 1995; 73: 691-702PubMed Google Scholar Similar findings have recently been described in anthrax-infected wild chimpanzees.23Leendertz FH Ellerbrok H Boesch C Couacy-Hymann E Matz-Rensing K Hakenbeck R Bergmann C Abaza P Junglen S Moebius Y Vigilant L Formenty P Pauli G Anthrax kills wild chimpanzees in a tropical rainforest.Nature. 2004; 430: 451-452Crossref PubMed Scopus (126) Google ScholarStudies in mice and rats have shown that administration of purified LT, in addition to causing death, produces vascular leakage similar to that observed in systemic anthrax.11Moayeri M Haines D Young HA Leppla SH Bacillus anthracis lethal toxin induces TNF-α-independent hypoxia-mediated toxicity in mice.J Clin Invest. 2003; 112: 670-682Crossref PubMed Scopus (257) Google Scholar, 12Cui X Moayeri M Li Y Li X Haley M Fitz Y Correa-Araujo R Banks SM Leppla SH Eichacker PQ Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats.Am J Physiol Regul Integr Comp Physiol. 2004; 286: R699-R709Crossref PubMed Scopus (110) Google Scholar Although these studies support a causative role for LT in the vascular leakage pathology of anthrax, the specific events or mechanisms are not known. Vascular endothelium, which plays a central role in regulating vascular permeability, is a likely target for LT during systemic anthrax infection by virtue of its direct contact with the bloodstream. In the present study, we examined the effects of LT on endothelial barrier function using cultured primary human lung microvascular endothelial cells. The effects of LT on the cellular distribution and expression of cytoskeletal actin and vascular endothelial-cadherin (VE-cadherin) were also investigated. VE-cadherin, the major component of adherens junctions (AJs), plays a key role in regulating endothelial barrier function as evidenced by the increased permeability caused by the disruption of interendothelial AJs both in vitro and in vivo.24Gotsch U Borges E Bosse R Boggemeyer E Simon M Mossmann H Vestweber D VE-cadherin antibody accelerates neutrophil recruitment in vivo.J Cell Sci. 1997; 110: 583-588Crossref PubMed Google Scholar, 25Hordijk PL Anthony E Mul FP Rientsma R Oomen LC Roos D Vascular-endothelial-cadherin modulates endothelial monolayer permeability.J Cell Sci. 1999; 112: 1915-1923Crossref PubMed Google Scholar, 26Dudek SM Garcia JGN Cystoskeletal regulation of pulmonary vascular permeability.J Appl Physiol. 2001; 91: 1487-1500Crossref PubMed Scopus (829) Google Scholar, 27Vincent PA Xiao K Buckley KM Kowalczyk AP VE-cadherin: adhesion at arm's length.Am J Physiol Cell Physiol. 2004; 286: C987-C997Crossref PubMed Scopus (144) Google Scholar, 28Dejana E Bazzoni G Lampugnani MG Vascular endothelial (VE)-cadherin: only an intercellular glue?.Exp Cell Res. 1999; 252: 13-19Crossref PubMed Scopus (208) Google Scholar In addition, actin reorganization has been associated with compromised barrier integrity and the creation of interendothelial gaps.26Dudek SM Garcia JGN Cystoskeletal regulation of pulmonary vascular permeability.J Appl Physiol. 2001; 91: 1487-1500Crossref PubMed Scopus (829) Google Scholar In the present study, we report that LT causes endothelial barrier dysfunction in vitro, supporting a potential role for LT in the vascular permeability changes accompanying systemic anthrax infection.Materials and MethodsMaterialsPhosphate-buffered saline (PBS), Hank's balanced salt solutions with calcium and magnesium (HBSS+) or without (HBSS−), and l-alanyl-l-glutamine (GlutaMAX I) were obtained from Invitrogen (Carlsbad, CA). Bovine serum albumin (BSA), propidium iodide (PI), Triton X-100, Hoechst 33342, and fluorescein isothiocyanate-labeled human serum albumin (FITC-HSA) were obtained from Sigma Chemical Co. (St. Louis, MO). Enzyme-linked immunosorbent assay (ELISA) detection reagents (hydrogen peroxide and 3,3′,5,5′ tetramethylbenzidine), FITC-labeled annexin V and a mouse IgG1 primary monoclonal antibody that binds to the extracellular domain of VE-cadherin were purchased from BD Biosciences (San Diego, CA). Alexa Fluor 555-labeled goat anti-mouse IgG1 secondary antibody and Alexa Fluor 488-labeled phalloidin were purchased from Molecular Probes (Eugene, OR). Peroxidase-conjugated goat anti-mouse IgG (H+L) secondary antibody was obtained from Jackson ImmunoResearch (West Grove, PA). U0126, an inhibitor of MEK1/2; SB230580, an inhibitor of p38 MAPK; and SP600125 (JNK inhibitor II), an inhibitor of JNK, were purchased from CalBiochem (San Diego, CA). Caspase inhibitor, z-VAD-fmk (zVAD), was obtained from Enzyme Systems Products (Livermore, CA). LF, PA, and mutant LFE687C were prepared as previously described and kindly provided by Dr. Stephen H. Leppla (National Institutes of Health, Bethesda, MD).29Park S Leppla SH Optimized production and purification of Bacillus anthracis lethal factor.Protein Expr Purif. 2000; 18: 293-302Crossref PubMed Scopus (117) Google Scholar, 30Ramirez DM Leppla SH Schneerson R Shiloach J Production, recovery and immunogenicity of the protective antigen from a recombinant strain of Bacillus anthracis.J Ind Microbiol Biotechnol. 2002; 28: 232-238Crossref PubMed Scopus (80) Google Scholar Toxin proteins were diluted in sterile PBS before cell treatment.Endothelial Cell CulturePrimary human lung microvascular endothelial cells were obtained from Cambrex (Walkersville, MD). Cells were grown in phenol red-free MCDB 131 medium (Hyclone, Logan, UT) supplemented with 10 mmol/L l-alanyl-l-glutamine, human epidermal growth factor, hydrocortisone, gentamicin, amphotericin-B, vascular endothelial growth factor, human fetal growth factor-B, recombinant growth factor-1 (R3-IGF-1), ascorbic acid, and 5% fetal bovine serum (Cambrex). Cells were cultured in 100-mm dishes at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were passaged 1:3 when cultures reached 90 to 95% confluence. For harvesting, cells were washed with HEPES-buffered saline and incubated with 0.025% trypsin/0.01% EDTA for 7 minutes at 37°C. After cell detachment, trypsin-neutralizing agent was added, and cells were centrifuged at 220 × g for 5 minutes at 4°C. Cell counts were obtained using a Z1 dual threshold Coulter counter (Beckman-Coulter, Hileah, FL). Experimental data were obtained from cells in their third to seventh passages.Transendothelial Electrical Resistance (TEER) MeasurementCells were grown to confluence on porous polyester membrane inserts (12 mm diameter, 0.4 μm pore size; Transwell, Corning, Cambridge, MA). The upper and lower compartments contained 0.5 and 1.5 ml of media, respectively. For experimental treatments, LF (1 to 1000 ng/ml), PA (1 to 1000 ng/ml), or both (LT) were added to the upper compartment. Alternatively, catalytically inactive mutant LFE687C (1 μg/ml) was added in the presence of 1 μg/ml PA. TEER measurements were performed using an EVOM volt-ohmmeter connected to a 12-mm Endohm unit (World Precision Instruments, Sarasota, FL). To measure TEER, culture inserts were transferred to the Endohm chamber containing 1.8 ml of HBSS+. The Endohm unit was washed with sterile PBS between measurements to avoid treatment cross-contamination. At the indicated time intervals, resistance readings (ohms) were obtained from each insert and multiplied by the membrane area (ohms × square centimeters). The resistance value of an empty culture insert (no cells) was subtracted. A decrease in TEER indicates an increase in monolayer permeability, whereas an increase in TEER signifies an increase in monolayer integrity. Data were collected from duplicate inserts per treatment in each experiment. Values were reported as the percentage of basal TEER obtained by dividing the resistance values of each treated monolayer by the resistance value of the control monolayer at each given time point.Albumin Permeability AssayCells grown to confluence on porous membrane inserts (12 mm diameter, 0.4 μm pore size) were treated as described in the previous section. After 72 hours, 50 μl of culture medium from the upper chamber was replaced with an equal amount of medium containing 5 mg/ml FITC-HSA (final concentration 500 μg/ml). After 2 hours, 20 μl samples were drawn from the lower chamber and diluted 10-fold. Data were collected from duplicate inserts per treatment in each experiment. Fluorescence measurements were obtained using a microplate reader (Genios, Tecan, Research Triangle Park, NC) with excitation and emission filters of 485 and 535 nm, respectively. FITC-HSA concentrations were calculated using a FITC-HSA standard curve. To quantify the trans-membrane flux (micrograms per hour per square centimeter), the FITC-HSA concentration was multiplied by the volume of the lower chamber and divided by the membrane area and the FITC-HSA incubation time.Immunofluorescence MicroscopyCells grown to confluence in 24-well dishes were treated with LF, PA, or both (LT). After treatment, the monolayers were washed once with fully supplemented media containing 5% fetal bovine serum and then fixed with 3.2% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) for 10 minutes. The cells were then washed twice with HBSS+ supplemented with 0.1% BSA and permeabilized with 0.1% Triton X-100 for 5 minutes. After two washes with 0.1% BSA, the cells were incubated for 1 hour with a primary monoclonal antibody to VE-cadherin (1 μg/ml, 1:200). The cells were then washed twice with 0.1% BSA and subsequently incubated for 1 hour with an Alexa Fluor 555-labeled secondary antibody (2 μg/ml, 1:800). Forty minutes into the second incubation, Alexa Fluor 488-labeled phalloidin (0.2 μg/ml) was added to stain F-actin, and Hoechst 33342 (10 μg/ml) was added to visualize the nuclei. All incubations were done at room temperature. After staining, the cells were washed three times with 0.1% BSA. Photomicrographs were obtained using an Olympus IX71 inverted microscope (Olympus America, Melville, NY) equipped with an Olympus DP70 digital camera connected to a Pentium 4 computer. Excitation light from the mercury arc lamp was passed through a triple band filter set (U-M61000v2; Chroma Technology Corp., Brattleboro, VT) to capture all three fluorochromes simultaneously or through a second filter set (41002C) for VE-cadherin alone. Standardized microscope and software settings were applied during image capture and postprocessing.VE-Cadherin Cell Surface ELISACells grown to confluence in 24-well dishes were treated with LF, PA, or both (LT). After treatment, the monolayers were washed once with fully supplemented media containing 5% fetal bovine serum and then fixed with 1% paraformaldehyde in HBSS+ for 20 minutes. After two washes with HBSS+ supplemented with 0.1% BSA, the cells were incubated for 1 hour with the primary monoclonal VE-cadherin antibody (1 μg/ml, 1:200). The cells were then washed three times with 0.1% BSA and incubated for 1 hour with a peroxidase-conjugated secondary antibody (1:10,000 dilution). The monolayers were then rinsed four times with 0.1% BSA, followed by one wash with HBSS+. For detection, equal parts of the substrate reagents hydrogen peroxide and 3,3′,5,5′ tetramethylbenzidine were added to each well. After color development, 1 N HCl was added to stop the reaction. Absorbance was measured at 450 nm using the previously described microplate reader.Analysis of Monolayer Cell DensityMonolayer cell density was assessed by counting the number of adherent cells with normal nuclear morphology identified by dual staining with the membrane-permeable DNA fluorochrome Hoechst 33342 and PI. This staining method distinguishes between normal, apoptotic, or necrotic cells. Normal cells were defined as those with no PI staining and without evidence of nuclear condensation. Apoptotic cells were defined as those with condensed or fragmented nuclei without PI staining. Necrotic cells were defined as those visibly stained by PI indicative of cell membrane lysis. Briefly, cells grown in 12- or 24-well dishes were treated with varying concentrations of LF (1 to 1000 ng/ml), 1 μg/ml PA, or both (LT). At the indicated time intervals, cultures were incubated with 1 μg/ml Hoechst 33342 and 1 μg/ml PI. After a 20-minute incubation, monolayers were washed once with complete medium. Photomicrographs of five separate fields per well (top, bottom, right, left, and center) were obtained using the microscope and digital camera system described above. Digital images were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD). For each experiment, monolayer density was calculated as the percentage of adherent cells with normal nuclear morphology relative to untreated cultures performed in parallel.Annexin V/Propidium Iodide AssayCells grown to confluence in 12-well dishes were treated with varying concentrations of LF (1 to 1000 ng/ml), PA (1 μg/ml), or both (LT). Nonadherent and adherent cells, harvested by trypsinization, were pooled, washed twice in HBSS−, and resuspended in 150 μl of assay buffer containing 10 mmol/L HEPES/NaOH, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2, pH 7.4. Annexin V-FITC (5 μl of 2 μg/ml) and PI (10 μl of 5 μg/ml) were added to a 100-μl aliquot of this cell suspension. After a 15-minute incubation in the dark at room temperature, stained cells were diluted with 200 μl of assay buffer and analyzed using a FACScan flow cytometer (Becton Dickinson Biosciences, San Jose, CA). Data were analyzed from a minimum of 10,000 cells per sample using WinMDI software (version 2.8). Cells stained annexin V positive and PI negative (annexin V+, PI−) were defined as early apoptotic cells. Cells stained annexin V positive and PI positive (annexin V+, PI+) represented late apoptotic or necrotic cells and included any cells damaged nonspecifically during the harvesting procedure.Cell Redox ActivityCell redox activity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based colorimetric assay (CellTiter 96, AQueous One Solution Reagent; Promega, Mannheim, Germany). The MTS reagent is bioreduced mainly by mitochondrial enzymes and electron carriers present in metabolically active cells. Briefly, confluent cultures grown in 96-well dishes were treated with 200 μl of medium alone or medium containing LF (1 to 1000 ng/ml), 1 μg/ml PA, or both (LT). At the indicated time intervals, treatment medium was replaced with 200 μl of phenol red-free MCDB 131 medium supplemented with 10 mmol/L l-alanyl-l-glutamine and 0.1% BSA containing 10 μl of MTS reagent. After a 2-hour incubation, absorbance was read at 492 nm using the previously described microplate reader. Background absorbance readings generated in cell-free wells containing medium and MTS were subtracted from experimental readings. Experiments were run in triplicate or quadruplicate per treatment, and values were reported as the percent MTS reduction relative to untreated wells.Statistical AnalysisData are represented as means ± SE for replicate experiments. Statistical analysis was performed by the unpaired two-tailed Student's t-test using the JMP (v. 5.1) software (SAS Institute Inc, Cary, NC). P < 0.05 was considered statistically significant.ResultsLT Increases Endothelial Monolayer PermeabilityThe barrier function of endothelial monolayers grown on porous membrane inserts was assessed by the measurement of TEER and the permeability to FITC-HSA. TEER readings were obtained between 0 and 72 hours from monolayers treated with LF, PA, or both (LT). Figure 1 shows that LT produced a progressive decrease in TEER, with statistically significant differences at 12, 24, 48, and 72 hours. LF or PA alone did not produce any changes in TEER. To further assess endothelial barrier sensitivity to LT, monolayers were treated with varying concentrations of either LF or PA in the presence of 1 μg/ml PA or LF, respectively. After 72 hours, a significant decrease in TEER was measured in cultures treated with 1, 10, 100, and 1000 ng/ml LF (Figure 2A). In PA dose-range experiments, a statistically significant reduction in TEER was observed with ≥10 ng/ml PA (Figure 2B). To determine whether the changes in TEER correlated with an increase in permeability to macromolecules, the flux of FITC-HSA across monolayers was also analyzed. Figure 3A shows that LF concentrations ranging from 1 to 1000 ng/ml in the presence of 1 μg/ml PA enhanced permeability to FITC-HSA. In PA dose-range experiments, significant increases in permeability were observed with PA concentrations ≥100 ng/ml in the presence of 1 μg/ml LF (Figure 3B). The minor increase with 10 ng/ml PA did not reach statistical significance. LF or PA alone did not alter permeability to FITC-HSA (Figure 3, A and B). Moreover, cultures treated with a catalytically inactive mutant LFE687C and PA did not alter TEER or FITC-HSA permeability, indicating that LT-induced barrier dysfunction was dependent on enzymatically active LF (Figure 2, Figure 3). Taken together, these two established indices of barrier function provide direct evidence that LT causes endothelial barrier dysfunction in vitro.Figure 2Concentration-dependent effects of LT on TEER. A: Monolayers grown on porous membrane inserts were incubated with medium alone or medium containing 1 μg/ml LF, 1 μg/ml LFE687C and PA, or varying amounts of LF in the presence of 1 μg/ml PA. TEER readings were obtained after 72 hours and reported as the means ± SE for a minimum of three independent experiments. *P < 0.001, †P < 0.01 versus medium alone. B: Monolayers were treated with medium alone, medium containing 1 μg/ml PA, or varying amounts of PA in the presence of 1 μg/ml LF. TEER readings were obtained after 72 hours and reported as the means ± SE for a minimum of three independent experiments. *P < 0.001 versus medium alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Concentration-dependent effects of LT on albumin permeability. A: Monolayers were grown and treated as in Figure 2A. After 72 hours, FITC-HSA was added to the upper compartment of the insert. After 2 hours, the amount of FITC-HSA in the bottom compartment was measured using a fluorescent microplate reader. Values were calculated as the micrograms of FITC-HSA per hour per square centimeter and reported as the means ± SE for a minimum of three independent experiments. *P < 0.001, †P < 0.01 versus medium alone. B: Monolayers were grown and treated as in Figure 2B. Data were collected as in Figure 3A and reported as the means ± SE for a minimum of three independent experiments. *P < 0.001, †P < 0.01 versus medium alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT)LT Induces Elongated Morphology, Actin Stress Fiber Formation, and VE-Cadherin RedistributionTo assess whether LT-induced barrier dysfunction correlated with changes in endothelial cell morphology, cultures were examined by phase contrast and immunofluorescence microscopy. LF- or PA-treated monolayers were morphologically indistinguishable from control cultures (Figure 4). LT-treated monolayers remained essentially intact relative to untreated monolayers despite a slight LT-induced elevation in the nonadherent cell population over the course of 72 hours. Notably, LT-treated cells exhibited an elongated morphology compared with untreated cells (Figure 4J). This elongated morphology was observed in random regions of the monolayer by 24 hours and became progressively more evident thereafter. By 48 and 72 hours, LT-treated monolayers also contained small interendothelial gaps detectable under high-power magnification (Figure 4J).Figure 4Phase contrast morphology and immunofluorescence visualization of F-actin, VE-cadherin, and nuclei. Cells were incubated with medium alone (A to C) or medium containing 1 μg/ml LF (D to F), 1 μg/ml PA (G to I), or both (J to L). After 72 hours, monolayers were wa